ArticlePDF Available

Abstract and Figures

Vitamin C is the exogenous compound necessary for a variety of metabolic processes; therefore, the efficient delivery is critical for the maintenance of body homeostasis. Vitamin C pharmacokinetics and low quantities in processed foodstuff, necessitates its continuous supplementation. In the paper, we present the new liposomal formulation of vitamin C free of harmful organic solvents. The formulation was quantitatively characterized with respect to its chemically composition and nano-structuring. The vitamin C accessibility to cells from the formulation was evaluated using evidence derived from experiments performed on cell cultures. Finally, the enhanced bioavailability of vitamin C from the formulation was demonstrated in the medical experiment.
Content may be subject to copyright.
Full Terms & Conditions of access and use can be found at
Journal of Liposome Research
ISSN: 0898-2104 (Print) 1532-2394 (Online) Journal homepage:
New oral liposomal vitamin C formulation:
properties and bioavailability
Maciej Łukawski, Paulina Dałek, Tomasz Borowik, Aleksander Foryś, Marek
Langner, Wojciech Witkiewicz & Magdalena Przybyło
To cite this article: Maciej Łukawski, Paulina Dałek, Tomasz Borowik, Aleksander Foryś, Marek
Langner, Wojciech Witkiewicz & Magdalena Przybyło (2019): New oral liposomal vitamin C
formulation: properties and bioavailability, Journal of Liposome Research
To link to this article:
View supplementary material
Published online: 02 Jul 2019.
Submit your article to this journal
View Crossmark data
New oral liposomal vitamin C formulation: properties and bioavailability
Maciej Łukawski
, Paulina Dałek
, Tomasz Borowik
, Aleksander Fory
, Marek Langner
Wojciech Witkiewicz
and Magdalena Przybyło
Department of Biomedical Engineering, Wroclaw University of Science and Technology, Wrocław, Poland;
Lipid Systems Ltd, Wrocław,
Centre of Polymer and Carbon Materials, Polish Academy of Sciences, Zabrze, Poland;
Research and Development Centre,
Specialized Hospital in Wrocław, Wrocław, Poland
Vitamin C is the exogenous compound necessary for a variety of metabolic processes; therefore, the
efficient delivery is critical for the maintenance of body homeostasis. Vitamin C pharmacokinetics and
low quantities in processed foodstuff, necessitates its continuous supplementation. In the paper, we
present the new liposomal formulation of vitamin C free of harmful organic solvents. The formulation
was quantitatively characterized with respect to its chemically composition and nano-structuring. The
vitamin C accessibility to cells from the formulation was evaluated using evidence derived from experi-
ments performed on cell cultures. Finally, the enhanced bioavailability of vitamin C from the formula-
tion was demonstrated in the medical experiment.
Received 20 December 2018
Revised 10 April 2019
Accepted 22 April 2019
Liposome; vitamin C;
efficacy; bioavailabil-
ity; stability
1. Introduction
Vitamin C is a compound essential for maintaining cellular
homeostasis in mammals. However, the endogenous adjust-
ments of vitamin C in humans are not possible because of a
mutation that eliminated its endogenous availability.
Consequently, it has to be acquired from the diet. The lack
of direct feedback between the body requirement and the
intrinsic supply of ascorbic acid causes a large proportion of
the human population to be in a state of various levels of
permanent or transient deficiencies (Padayatty et al. 2004,
Lindblad et al. 2013, Padayatty and Levine 2016). As vitamin
C is directly involved in many vital processes, such as protec-
tion against excess of reactive oxygen species, maintenance
of iron homeostasis and gene expression control, therefore
even moderate deficiency can have serious health conse-
quences (Salganik 2001, Fraga and Oteiza 2002, Babaev 2010,
Finck et al. 2014, Lane and Richardson 2014, Young et al.
2015). This can be easily prevented by the diet adjustment
and/or additional supplementation. The supplementation is
safe since kidneys remove any excess of vitamin C prevent-
ing overdose. The recommended daily intake of vitamin C
for a healthy person has been determined to be 500 mg/day
(Savini et al. 2005, Harrison et al. 2014, Paschalis et al. 2016).
The recommendation does not account for the elevated
demand during physical or psychological stress (May and Qu
2005, Nair et al. 2016). For example, it has been demon-
strated that it is beneficial to increase the dose of vitamin C
in the course of oncological therapies (Du et al. 2012, Fritz
et al. 2014), during the treatment of skin diseases (May and
Qu 2005, Stamford 2012, Kishimoto et al. 2013), in the reduc-
tion of stroke effects (Spector 2016) or in the elimination of
certain disorders of the digestive system (Aditi and
Graham 2012).
The delivery of vitamin C, using the oral delivery route,
requires reduction of the rate of its degradation in the gut
and facilitation of its absorption. Vitamin C is usually admin-
istered orally in the crystalline form or as a solution, which
makes it susceptible to degradation in the gastrointestinal
tract, especially in the presence of metal ions (Michels and
Frei 2013). Degradation of vitamin C can be effectively
reduced by its association with the hydrophilichydrophobic
interface, which can be provided by lipid aggregates (Nagle
and Tristram-Nagle 2000, Pastoriza-Gallego et al. 2012,
Wechtersbach et al. 2012).
Lipid aggregates, such as liposomes, are well suited for
this purpose. They reduce the vitamin C degradation in the
gastrointestinal tract, slow down its release and enhance
absorption (Hickey et al. 2008, Wechtersbach et al. 2012).
Liposomes also mitigate possible perturbances of gastro-
intestinal tract functioning, what enables application of ele-
vated doses of vitamin C for extended periods of time. In
addition, it is not to be overlooked that lipids, phosphatidyl-
cholines in particular, are an important component of a bal-
anced diet with documented positive effects on the patient
overall wellbeing (Alvarez and Rodriguez 2000, Keller 2001,
Kullenberg et al. 2012, Blesso 2015, Garcia and Aguero 2015,
van der Veen et al. 2017). All that have stimulated numerous
works leading to the development of liposomal formulations
of vitamin C for varieties of applications (Hickey et al. 2008,
CONTACT Paulina Dałek Department of Biomedical Engineering, Wroclaw University of Science and Technology, Plac
Grunwaldzki 13, Wrocław 50-377, Poland
Supplemental data for this article is available online at
ß2019 Informa UK Limited, trading as Taylor & Francis Group
Xie and Ji 2008, Marsanasco et al. 2011). The manufacturing
of liposomal preparations on an industrial scale requires
strict process control of both chemical and physicochemical
parameters, which make the production very challenging
(van Nieuwenhuyzen and Szuhaj 1998, van Nieuwenhuyzen
and Tomas 2008). It is essential, not only to ensure the
proper and stable chemical composition of the preparation
but also its nano-scale structuring. This means, that the ana-
lysis of liposomal preparations requires the application of
advanced measurement methods such as: high-performance
liquid chromatography (HPLC), dynamic light scattering
(DLS), and transmission electron cryomicroscopy. The add-
itional challenge is the elimination of organic solvents from
the formulation and preferably from the production process
as well. In the paper, we present new liposomal formulation
of uniform lipid vesicle population without application of
any undesirable organic solvent, with long-term stability. The
developed new formulation significantly enhances vitamin C
bioavailability and maintains its efficacy on the cellular level.
2. Materials and methods
2.1. Materials
Soybean phosphatidylcholine (Phospholipon 90G) was pur-
chased from Lipoid GmbH (Ludwigshafen, Germany) and
rapeseed lecithin from Somar (Wa˛chock, Poland). Sodium
ascorbate was of pharmaceutical grade and was obtained
from Brenntag (KeRdzierzyn-Ko
zle, Poland) and glycerine was
obtained from TechlandLab (Tarnobrzeg, Poland). Water in all
solutions was highly purified with a conductivity of 0.056 uS/
cm app. 17.86 MX(AquaEngineering, Warszawa, Poland).
Ammonium formate and organic solvents such as n-hexane,
2-propanol, and acetonitrile were of HPLC grade and were
purchased from VWR International (Radnor, PA, USA).
2.2. Preparation of liposomal formulation
Liposomal formulation of vitamin C was prepared using the
pharmacologically accepted glycerine, as a solvent for lipids.
The other unique feature of the formulation is high content
of lipids, i.e. more than 20% w/w. The high lipid content
results in the high encapsulation efficiency. Liposomes were
formulated by mixing two solvents: glycerine containing lip-
ids (1:1 w/w) and the aqueous solution containing sodium
ascorbate, with the API concentration equals 20% w/w. The
uniform population of liposomes were formed spontaneously
upon mixing. The high content of lipids (more than 20%) in
the mixture results with structured aqueous phase, character-
ized by high viscosity and gel-like consistency. For control
studies, the aqueous solution of sodium ascorbate was
exchanged for pure water. Liposomes for HPLC method val-
idation were prepared by hydration with 20 mM ammonium
formate, pH 3.2 and 33.3% (w/w) of sodium ascorbate. After
each preparation of liposome suspension, the size and poly-
dispersity was measured to confirm the creation of monodis-
perse liposome population.
2.3. Samples preparation for HPLC analysis
The hydrophobic and hydrophilic components of the formu-
lation were separated by the BlighDyer method (Bligh and
Dyer 1959) with some critical modifications. The volume of
the aqueous phase was highly increased to prevent the crys-
tallization of ascorbic acid induced by methanol. Specifically,
the sample and methanol were mixed in the volume ratio
1:1. Two volumes of chloroform were added and the sample
was intensively shaken. Next, the sample was 19 times
diluted in 20 mM ammonium formate buffer (pH 3.2) and
vortexed again. Finally, the sample was centrifuged at 2500
rpm for 10 min to speed up the separation of phases. The
upper aqueous phase with vitamin C was then taken for the
measurement. With this protocol, method recovery was more
than 99%, which was established in the separate experiment.
2.4. HPLC analysis
HPLC analyses were performed with Knauer system (Knauer
GmbH, Berlin, Germany) consisting of Knauer Azura Pumps,
P2.1 ceramic head, Optimas Autosampler with 96 positions
and Knauer Azura CT2.1 thermostat. All reagents were fil-
tered through 220 nm pore size filter and degassed prior the
2.4.1. Determination of vitamin C concentration
in liposomes
Vitamin C content in liposomes was determined by HPLC
equipped with a UV-VIS detector Knauer Azura UVD2.1S. A
Knauer LiChrospher 100-5 Diol column (125 4mm
) was
used to separate the compound from other components.
The column temperature was set to 20 C. The mobile phase
consisted of a 10% (v/v) solution of 20 mM ammonium for-
mate acidified with formic acid to pH 3.2 and 90% (v/v)
acetonitrile. The flow during analysis was 1 mL/min.
Standard samples for calibration curves were prepared by
dissolving the appropriate amount of sodium ascorbate in 20
mM ammonium formate buffer (pH 3.2).
2.4.2. Determination of lipid concentration in liposomes
Analysis of phosphatidylcholine content was performed by
HPLC equipped with evaporative light scattering detector
(HPLC-ELSD) (Letter 1992) and was carried out according to
the method L-M-HPLC-SPC-3E/01 provided by Lipoid GmbH
(Ludwigshafen, Germany) (Lipoid GmBH, 2006). The method
utilizes Knauer LiChrospher 100-5 Diol column (125 4
) and the measurement was made using a hydrophobic
phase gradient of n-hexaneisopropanol in the aqueous
phase containing acetic acid and triethylamine. The method
was adjusted to the ELSD Alltech 3300 detector (Buchi,
Flawil, Switzerland), what improved signal quality. This modi-
fication involved the elimination of phase modifiers, namely
acetic acid and triethylamine. Modifications did not affect
parameters of the recorded signals, so the full width at half
maximum (FWHM) was 0.1020 ± 0.0034 (relative standard
deviation (RSD)¼3.3%) and the measured signal height
equals to 352 ± 14 (RSD ¼4.1%). The measurement was car-
ried out in a mobile phase gradient consisting of two com-
ponents. Phase A consists of n-hexane and 2-propanol mixed
in the volume ratio of 830:170 and phase B consists of 2-pro-
panol and water mixed in the volume ratio of 340:55.3. The
mobile phase compositions are shown in supplementary
data Table S1.
2.5. Liposome size distribution and zeta potential
The DLS technique allows the measurement of zeta potential
and the size distribution of aggregates in the aqueous sus-
pension. For that purpose, highly concentrated liposomal
vitamin C samples were diluted 60 times with isosmotic solu-
tion prior to the analysis. Measurement was performed in 1
cm polystyrene cuvettes. Size measurements were performed
using ZetaSizer Nano ZS (Malvern, UK).
2.6. Determination of the vitamin C
encapsulation efficiency
The encapsulation efficiency (%EE) of vitamin C in liposomes
was determined using the ultrafiltration through membranes
with 50 kDa cutoff mass (SpectrumLabs, Rancho Dominguez,
CA) followed by HPLC UV-VIS determination of vitamin C
content in the permeate. The stability of liposomes during
ultrafiltration was controlled with DLS measurements.
Vitamin C encapsulation efficiency in percentiles was calcu-
lated according to the formula:
EEvitC ¼1mfree=mtotal
where mfree is the mass of vitamin C in the permeate and
mtotal is the total mass of vitamin C in the sample.
2.7. Cryogenic transmission electron
microscopy imaging
Cryogenic transmission electron microscopy (cryo-TEM)
images were obtained using a Tecnai F20 TWIN microscope
(FEI Company, Hillsboro, OR, USA) equipped with a field
emission gun, operating at an acceleration voltage of 200 kV.
Images were recorded on the Eagle 4k HS camera (FEI
Company, Hillsboro, OR, USA) and processed with TIA soft-
ware (FEI Company, Hillsboro, OR, USA). Specimen prepar-
ation was done by verification of the aqueous solutions on
grids with a holey carbon film (Quantifoil R 2/2; Quantifoil
Micro Tools GmbH, Großl
obichau, Germany). Prior to use, the
grids were activated for 15 s in oxygen plasma using a
Femto plasma cleaner (Diener Electronic, Ebhausen,
Germany). Cryo samples were prepared by applying a drop-
let (3 mL) of the solution to the grid, blotting with filter
paper and rapid freezing in liquid ethane using a fully auto-
mated blotting device Vitrobot Mark IV (FEI Company,
Hillsboro, OR, USA). After preparation, the vitrified specimens
were kept under liquid nitrogen until they were inserted into
a cryo-TEM-holder Gatan 626 (Gatan Inc., Pleasanton, CA,
USA) and analysed in the TEM at 178 C.
2.8. Rheology studies
The rheological characteristic of the novel liposomal formula-
tion was measured using Brookfield rheometer DV2T of the
cone-plate type, with cone type CPA-51Z. The measurement
chamber was thermostated at 25 C by circulating water
bath (VWR).
2.9. Comparative studies of liposomal vs. free vitamin C
cell cytotoxicity
The experiments were performed on two breast cancer cell
lines SKBR3, MCF7 and the healthy BJ cell line as a control.
All cell cultures were purchased from the American Type
Culture Collection (Manassas, VA, USA). Survival of the cells
in the presence of high concentrations of sodium ascorbate
(0.45 mM) in both free and liposome form was determined
with protocols described elsewhere (Karlsen et al. 2005). In
short, cells were cultured in media containing 10% FBS
obtained from Thermo Fisher Scientific (Waltham, MA, USA)
at 37 C under 5% CO
. Sodium ascorbate and liposomal for-
mulation of sodium ascorbate were diluted in culture
medium prior to the addition to cells. Cell cultures were
maintained using following reagents: RPMI medium, DMEM
medium, penicillin, streptomycin, L-glutamine 200 mM, 0.25%
trypsinEDTA purchased from Thermo Fisher Scientific
(Waltham, MA, USA) and PBS CaMg purchased from Corning
(Corning, NY, USA).
The cell survival was determined with the MTT test, per-
formed according to ISO EN ISO 10993-5. 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
was purchased from Sigma Aldrich (St. Louis, MO, USA). Cells
were grown on 96-well plates for 48 h then the sodium
ascorbate was added (the final concentration was in the
range from 0.4 mM to 5 mM). After one-hour incubation, the
medium was withdrawn and the culture was supplemented
with a 100 lL of fresh medium. The cells were incubated for
another 22 h, followed by the addition of the MTT solution
in PBS without magnesium and calcium ions. Concentrations
of MTT in tested cultures were 330 lg/mL. After 2.5 h incu-
bation, MTT was removed; the cells were supplemented with
100 lL DMSO (POCH, Gliwice, Poland) and stirred to dissolve
the formazan crystals. After the dissolution of crystals, the
formazan absorbance was measured with SPECTROStar Nano
spectrometer (BMG Labtech, Cary, NC, USA).
2.10. Bioavailability studies of liposomal and free
vitamin C formulations
The aim of the study was to compare the profiles of vitamin
C serum concentration in healthy volunteers after the single
oral administration either in a liposomal suspension or as an
aqueous solution. After obtaining written consent, 20 healthy
volunteers (10 women and 10 men aged 3165) were
included in the study. Volunteers under 18 years of age,
pregnant women, with the history of renal failure and gastro-
intestinal disorders were excluded from the study. Volunteers
were preconditioned by fasting for 12 h before the test. Each
participant was equipped with a peripheral venous catheter,
which allowed multiple blood withdrawals within the time-
frame of the study. Prior to the main study, the blood sam-
ples were drawn to determine the initial vitamin C
concentration. Each participant was administered 10 g of
vitamin C, either in the free or in the liposomal form, in one
single dose, dissolved/suspended in 250 mL of water.
Following vitamin C administration, blood samples were
taken at 30 min, 60 min, 90 min, 120 min, 180 min, 240 min,
and 360 min. Each time 4 mL of blood was transferred to
the test tube, containing heparin as an anticoagulant, as rec-
ommended by Karlsen et al. (2005). Vitamin C concentrations
in blood samples were determined within 48 h after collec-
tion using HPLC equipped with UV/VIS detector. Blood sam-
ples were stored at 4 C before analysis. Studies were carried
out by the Research and Development Centre at the
Specialized Hospital in Wrocław.
2.11. Determination of vitamin C concentrations in
blood samples
Determination of vitamin C concentrations in blood samples
was performed according to the method described by
Karlsen et al. (2005). Specifically, blood samples were centri-
fuged at 15 000 rpm for 5 min. 500 lL of serum was then
taken and mixed with a 10% TCA solution in a 1:1 ratio fol-
lowed by centrifugation to remove precipitated proteins.
Next, the supernatant was passed through a syringe filter
with the pore diameter of 220 nm. Resulting solutions were
analysed for vitamin C content. The calibration curves were
generated individually for each patient by adding the prede-
termined amounts of sodium ascorbate solution to 1 mL of
the whole blood followed by the sample treatment as
described above. The recovery rate was determined in the
separate experiment using samples prepared by the addition
of a known amount of vitamin C to the plasma using the
procedure described above. The following parameters were
calculated from the raw data: AUC, T
, and T
Qtiplot data analysis program.
2.12. Statistical analysis
Data are expressed as the mean ± standard deviation (SD).
Differences between the groups of cells in toxicity tests were
analysed by one-way ANOVA, bioavailability data were ana-
lysed by independent t-test. A value of p<0.05 was consid-
ered significant. For statistical analysis, Qtiplot program
was used.
2.13. Ethical approvals for the study
All procedures involving human subjects/patients were
approved by the Bioethical Commission at the Research and
Development Centre at the Specialized Hospital in Wrocław
number: KB/5/2016. Written informed consent was obtained
from all subjects. Liposomal vitamin C, classified as a dietary
supplement, was manufactured by Lipid Systems Ltd.
(Wrocław, Poland) under conditions meeting HACCP
requirements in accordance with the European Parliament
Regulation No. 852/2004 from April 29 2004 (Journal of Laws
EU Office of 2004 as amended) and the Act on Food and
Nutrition Safety from August 25 2006 (Journal of Laws from
2015, item 594).
3. Results
3.1. Stability of liposomal vitamin C
For liposomal vitamin C stability determination, the following
parameters were monitored: liposome size distribution and
zeta potential, vitamin C and phospholipid contents, vitamin
C encapsulation efficiency and rheology. Stability of the lipo-
somal vitamin C was determined for six series of samples
and a summary of analytical data is presented in Table S2
(Supplementary data). Figure 1(A) shows the set of typical
distributions of liposome sizes. The quality of the correlation
function fitting to the experimental data is shown in Figure
1(B). Samples for stability testing were stored under inter-
mediate stability storage conditions, namely at temperature
T¼30 C±2C and relative humidity RH ¼65 ± 5%.
Measurements were performed once a month. Since the DLS
is an indirect method of the particle size distribution deter-
mination, the liposomal vitamin C formulation was visualized
also with cryo-TEM technique. Based on the obtained
images, the size distribution and morphology of liposomes
were determined. Examples of cryo-TEM images obtained for
the liposomal vitamin C suspension are shown in Figure 1(C).
For quantitative analysis, 20 different cryo-TEM images were
analysed with Image J program and the mean liposomal
diameter was determined as 180 ± 30 nm. This result is in a
good agreement with the value of 168 ± 25 nm as deter-
mined with DLS technique.
The physicochemical characterization of the liposomal
vitamin C included also the quantitative determination of the
sodium ascorbate and phosphatidylcholine content. Since
the liposomal formulation cannot be considered as a simple
solution, the quantitative determination of vitamin C in lipo-
somal formulations was based on the modified BlighDyer
separation method (Bligh and Dyer 1959). Dilution of the
hydrophilic phase with 20 mM ammonium formate at pH
3.2, following the lipid extraction step, resulted in recovery
levels reaching 99.21%±0.52%. Examples of chromatograms
and resulting calibration curve along with the recovery levels
are presented in Figure 2. Concentrations of sodium ascor-
bate were determined as areas under the peak obtained by
HPLC analysis.
The new liposomal formulation shows gel-like behaviour
without addition any gelling substance. The dependence of
liposome gel viscosity (log l) on the share rate (log _
c) is pre-
sented in Figure 3. The viscosity decrease with share rate
indicates that the gel behaves as a thinning system, which
can be quantitated using the Ostwald model (Tadros 2004).
The model states that l¼k_
cn1;where kand nare consist-
ency index and shear thinning index, respectively. Fitting
experimental data to the model shows that n¼0.8 as
expected for pseudoplastic material. The rheological
properties of the new liposome preparation are unique and
not possible to obtain using other preparation procedures.
3.2. Cytotoxicity studies
In order to determine the sodium ascorbate efficacy on the
cellular level the toxicity was used as a quantitative measure.
MTT tests in the cell cultures exposed to sodium ascorbate
aqueous solutions and its liposomal formulation were per-
formed. The relationship between the concentration of
sodium ascorbate, incubation time and the type of cell line
and cytotoxicity for sodium ascorbate aqueous solution, its
liposomal formulation and liposomes without sodium ascor-
bate are presented in Figure 4. The experiment shows that
there is no difference between the two formulations with
the respect to effect on tested cells. Following one-hour
incubation, both sodium ascorbate formulations were not
toxic to the reference cell line (BJ) at concentrations reaching
5 mM, whereas the effect on cancerous cell lines (SKBR3 and
MCF3) was already significant at concentration higher than 1
mM. This result is in good agreement with data presented
elsewhere (Chen et al. 2005, Aguilera 2016). In addition, it
has been demonstrated that the new liposome formulation
alone is not toxic and that it does not interfere with the
ascorbic acid activity. When the exposure time was extended
to 3 h, the effect on all cells types was stronger regardless
on formulation used.
3.3. Bioavailability studies
Figure 5 shows the dependence of sodium ascorbate con-
centration in blood on time, determined for two groups of
persons, following the oral intake of 10 g of sodium ascor-
bate as an aqueous solution or as a liposome formulation. In
the liposomal vitamin C treatment arm, vitamin C
higher values than in the aqueous solution of vitamin C
treatment arm (303 ll vs. 180 ll). In addition, for liposomal
formulation, the delay time of the maximum vitamin C blood
concentration (T
) is longer by approximately 1 h when
compared to the free form (T
¼180 min vs. 96 min). The
increased half-life (t
>6 h vs. t
¼4 h) and elevated AUC
(81 570 llmin vs. 45 330 llmin) indicate that the pres-
ence of liposomes enhances bioavailability of vitamin C.
4. Discussion
Effective vitamin C homeostasis requires two opposing fluxes
(supply and elimination), which ensure the maintenance of
its concentration at various body locations within physio-
logical ranges. In humans, any excess of vitamin C is effi-
ciently eliminated by kidneys. However, since there is no
endogenous source of vitamin C, the necessary supply for
the metabolic consumption and/or any temporally elevated
demand can only be compensated by diet adjustments.
When the excessive concentration of vitamin C is used for
therapeutic purposes, the dose should exceed the excretion
(a) (b)
Figure 1. Examples of size distributions of liposomes loaded with sodium ascorbate as measured with the dynamic light scattering (DLS) technique and deter-
mined from images acquired with cryo-TEM microscopy. Panel a shows size distributions (expressed as hydrodynamic diameters) of liposomes with vitamin C.
Panel b shows correlation curves calculated for samples presented in panel a. Experimental points were fitted with the correlation curve calculated for a single-
population liposome suspension (continuous line). Panel c shows examples of cryo-TEM images using low (left image) and high (right image) resolutions.
capacity of the renal system. This is the reason why the
therapeutic vitamin C is delivered intravenously, so the high
serum concentration can be reached and maintained for an
extended period of time (Padayatty et al. 2004, Wilson et al.
2014). The inability to produce endogenous vitamin C,
changing demand and uncertain intake from the diet may
produce deficiencies, which can be easily eliminated by the
effective supplementation (Padayatty and Levine 2016). The
vitamin C blood concentration, in addition to supply and
elimination, also depends on redistribution. The majority of
vitamin C in the human body is inside cells (over 97%),
whereas only a small fraction of it is found in extracellular
fluids. High concentrations of vitamin C inside cells are pos-
sible thanks to specific sodium-dependent vitamin C trans-
porters (SVCT1 and SVCT2). The intricate system of ascorbic
acid fluxes in the human body prevents excessive fluctuation
of its concentrations inside cells as required by metabolic
processes. In order to affect the homeostatic balance, using
a convenient oral delivery route, the vitamin C intake should
be sufficiently high and preferable extended in time. To this
end, the high concentration of vitamin C in the digestive
tract should be maintained for extended periods of time so
it would be available for absorption and would not require
Figure 2. HPLC analysis of vitamin C concentration in serum. Panel a shows chromatograms obtained for a series of calibration samples containing sodium ascor-
bate encapsulated in liposomes after Blight-Dyer extraction procedure. A plot of areas under peaks, for series of sodium ascorbate concentrations constructed from
chromatograms from panel a, is presented in panel b. Panel c shows the dependence of areas under the peaks derived from chromatograms obtained for two sets
of experimental data used for the determination of the sodium ascorbate recovery rate; values determined for sodium ascorbate from simple solution (circles) and
from liposomal formulation (squares) extracted using modified Blight-Dyer protocol. Samples after Blight-Dyer extraction contained the same amount of sodium
ascorbate but were diluted 20-fold with ammonium formate at pH 3.2.
Figure 3. The dependence of the liposomal gel viscosity on the shear rate.
Experimental points were fitted with the power law model (continuous line)
cn1:The determined value of nequals to 0.8 indicates that liposomal
gel is a pseudoplastic system (Tadros 2004).
frequent dosing. Maintaining high levels of vitamin C in the
digestive tract is dependent mainly on the rate of its
hydrolysis. The vitamin C degradation can be significantly
reduced by its association with lipid interfaces, which are
abundant in the liposomal formulation (Wechtersbach et al.
2012). The effectiveness of the liposomal formulation
depends, in addition to vitamin C content, on quality of lipo-
somes quantitated by their size distributions (Ensign et al.
2012, Beilstein et al. 2016). The presented method of lipo-
some containing vitamin C preparation produces a single
population of vesicles, as demonstrated using DLS and elec-
tron microscopy techniques (Figure 1). The formulation is
stable in time with respect to both, chemical composition
and physicochemical properties, as required for any mar-
keted product. When evaluating the formulation efficacy, it
has been shown that lipid vesicles do not interfere with the
vitamin C accessibility to cells, regardless of their type
(Figure 2). This implies that, if a certain concentration of
ascorbic acid in digestive tract is maintained for a sufficient
time period, the high absorption level will be achieved and
maintained. The other important aspect of the liposome for-
mulation is possibility to deliver large doses of vitamin C for
an extended period of time, since the lipid capsule mitigates
the irritation of gastrointestinal tract typically accompanying
large oral doses of ascorbate. In addition, as described else-
where lipids by themselves have beneficial physiological
effects (Davis et al. 2016). Subsequently, the formulation bio-
availability has been demonstrated in the medical experi-
ment on healthy volunteers, where liposomal vitamin C
outperforms the traditional form with the respect to the
maximal concentration as well as the halftime in serum
(Figure 3). The other important feature of the presented for-
mulation is that the process of liposome formation does not
require toxic organic solvents. Pharmacologically acceptable
glycerine has been used instead.
In summary, encapsulation of vitamin C in new types of
liposomes causes the enhancement of vitamin C bioavailabil-
ity on a physiological level, without compromising its
potency on the cellular level. The liposomal formulation of
vitamin C, in addition to its high activity, as ensured by ele-
vated bioavailability, should also satisfy strict regulatory
requirements regarding the content of potentially harmful
compounds, stability and reproducibility of production proc-
esses. In the paper, it has been demonstrated that the new
liposomal preparation can be produced with consistently sta-
ble critical parameters such as: chemical composition and
homogeneity of the liposome population. In addition, the
glycerine-based production process overcomes the major
obstacle, common in other production processes, the need
for application of pharmacological undesirable organic sol-
vents such as ethanol.
Disclosure statement
PD, MP, and ML are employed by the Lipid Systems Ltd.
This work was supported by Error! Hyperlink reference not valid.; the
National Centre for Research and Development under Grant number
Paulina Dałek
Data availability
The data that support the findings of this study are available from the
corresponding author, PD, upon reasonable request.
Figure 4. Effect of the sodium ascorbate concentration in the aqueous solution
(white and grey) and in liposomes (light grey and black) on the survival rate of
MCF7 cancer cell line and healthy BJ cells. Panel a shows data obtained after
one-hour incubation whereas panel b after three-hours incubation. Panel c
shows the survival rate of SKBR3 cancer cell line and healthy BJ cells after one-
hour incubation.
Values are significantly different (p<0.05).
Figure 5. Averaged concentration profiles of sodium ascorbate in serum deter-
mined for two groups of persons, following the oral intake of 10 g of sodium
ascorbate in the form of the aqueous solution (squares) and encapsulated in
liposomes (circles). The lines between points were drawn arbitrarily to guide
the eye. Values of plasma concentrations measured for specific time points for
liposomal and non-liposomal dosages are significantly different (p<0.05) for
three last points (180, 240, and 360 min).
Aditi, A. and Graham, D.Y., 2012. Vitamin C, gastritis, and gastric disease:
a historical review and update. Digestive diseases and sciences, 57(10),
Aguilera, O., 2016. Vitamin C uncouples the Warburg metabolic switch in
KRAS mutant colon cancer. Oncotarget, 7(30), 4795447965.
Alvarez, A.M.R. and Rodriguez, M.L.G., 2000. Lipids in pharmaceutical and
cosmetic preparations. Grasas Y aceites, 51(12), 7496.
Babaev, V.R., 2010. Combined vitamin C and vitamin E deficiency wor-
sens early atherosclerosis in apolipoprotein E-deficient mice.
Arteriosclerosis, thrombosis, and vascular biology, 30(9), 17511757.
Beilstein, F., et al., 2016. Characteristics and functions of lipid droplets
and associated proteins in enterocytes. Experimental cell research,
340(2), 172179.
Blesso, C.N., 2015. Egg phospholipids and cardiovascular health.
Nutrients, 7(4), 27312747.
Bligh, E. and Dyer, W., 1959. A rapid method of total lipid extraction and
purification. Canadian journal of biochemistry and physiology, 37(8),
Chen, Q., et al., 2005. Pharmacologic ascorbic acid concentrations select-
ively kill cancer cells: action as a pro-drug to deliver hydrogen perox-
ide to tissues. Proceedings of the national academy of sciences of the
United States of America, 102(38), 1360413609.
Davis, J.L., et al., 2016. Liposomal-encapsulated ascorbic acid: influence
on vitamin C bioavailability and capacity to protect against ischemia-
reperfusion injury. Nutrition and metabolic insights, 2016(9), 2530.
Du, J., Cullen, J.J., and Buettner, G.R., 2012. Ascorbic acid: chemistry, biol-
ogy and the treatment of cancer. Biochimica et biophysica acta (BBA)
reviews on cancer, 1826(2), 443457.
Ensign, L.M., Cone, R., and Hanes, J., 2012. Oral drug delivery with poly-
meric nanoparticles: the gastrointestinal mucus barriers. Advanced
drug delivery reviews, 64(6), 557570.
Finck, H., et al., 2014. Is there a role for vitamin C in preventing osteo-
porosis and fractures? A review of the potential underlying mecha-
nisms and current epidemiological evidence. Nutrition research
reviews, 27(2), 268283.
Fraga, C.G. and Oteiza, P.I., 2002. Iron toxicity and antioxidant nutrients.
Toxicology, 180(1), 2332.
Fritz, H., et al., 2014. Intravenous vitamin C and cancer. Integrative cancer
therapies, 13(4), 280300.
Garcia, J.T. and Aguero, S.D., 2015. Phospholipids: properties and health
effects. Nutricion hospitalaria, 31(1), 7683.
Harrison, F., Bowman, G., and Polidori, M., 2014. Ascorbic acid and the
brain: rationale for the use against cognitive decline. Nutrients, 6(4),
Hickey, S., Roberts, H.J., and Miller, N.J., 2008. Pharmacokinetics of oral
vitamin C. Journal of nutritional & environmental medicine, 17(3),
Karlsen, A., Blomhoff, R., and Gundersen, T.E., 2005. High-throughput
analysis of vitamin C in human plasma with the use of HPLC with
monolithic column and UV-detection. Journal of chromatography B-
analytical technologies in the biomedical life sciences, 824(12),
Keller, B.C., 2001. Liposomes in nutrition. Trends in food science & technol-
ogy, 12(1), 2531.
Kishimoto, Y., et al., 2013. Ascorbic acid enhances the expression of type
1 and type 4 collagen and SVCT2 in cultured human skin fibroblasts.
Biochemical and biophysical research communications, 430(2), 579584.
Kullenberg, D., et al., 2012. Health effects of dietary phospholipids. Lipids
in health and disease, 11, 3.
Lane, D.J.R. and Richardson, D.R., 2014. The active role of vitamin C in
mammalian iron metabolism: much more than just enhanced iron
absorption! Free radical biology and medicine, 75, 6983.
Letter, W.S., 1992. A rapid method for phospholipid separation by HPLC
using a light-scattering detector. Journal of liquid chromatography,
15(2), 253266.
Lindblad, M., Tveden-Nyborg, P., and Lykkesfeldt, J., 2013. Regulation of
vitamin C homeostasis during deficiency. Nutrients, 5(8), 28602879.
Marsanasco, M., et al., 2011. Liposomes as vehicles for vitamins E and C:
an alternative to fortify orange juice and offer vitamin C protection
after heat treatment. Food research international, 44(9), 30393046.
May, J.M. and Qu, Z.C., 2005. Transport and intracellular accumulation of
vitamin C in endothelial cells: relevance to collagen synthesis.
Archives of biochemistry and biophysics, 434(1), 178186.
Michels, A. and Frei, B., 2013. Myths, artifacts, and fatal flaws: identifying
limitations and opportunities in vitamin C research. Nutrients, 5(12),
Nagle, J.F. and Tristram-Nagle, S., 2000. Structure of lipid bilayers.
Biochimica et biophysica acta, 1469(3), 159195.
Nair, V.S., Song, M.H., and Oh, K.I., 2016. Vitamin C facilitates demethyla-
tion of the Foxp3 enhancer in a Tet-dependent manner. The journal
of immunology, 196(5), 21192131.
Padayatty, S.J. and Levine, M., 2016. Vitamin C: the known and the
unknown and Goldilocks. Oral diseases, 22(6), 463493.
Padayatty, S.J., et al., 2004. Vitamin C pharmacokinetics: implications for
oral and intravenous use. Annals of internal medicine, 140(7), 533537.
Paschalis, V., et al., 2016. Low vitamin C values are linked with decreased
physical performance and increased oxidative stress: reversal by vita-
min C supplementation. European journal of nutrition, 55(1), 4553.
Pastoriza-Gallego, M.J., Losada-Barreiro, S., and Bravo-D
ıaz, C., 2012.
Effects of acidity and emulsifier concentration on the distribution of
vitamin C in a model food emulsion. Journal of physical organic chem-
istry, 25(11), 908915.
Salganik, R.I., 2001. The benefits and hazards of antioxidants: controlling
apoptosis and other protective mechanisms in cancer patients and
the human population. Journal of the American college of nutrition,
20(5), 464472.
Savini, I., et al., 2005. Vitamin C homeostasis in skeletal muscle cells. Free
radical biology and medicine, 38(7), 898907.
Spector, R., 2016. Dehydroascorbic acid for the treatment of acute ische-
mic stroke. Medical hypotheses, 89, 3236.
Stamford, N.P.J., 2012. Stability, transdermal penetration, and cutaneous
effects of ascorbic acid and its derivatives. Journal of cosmetic derma-
tology, 11(4), 310317.
Tadros, T., 2004. Application of rheology for assessment and prediction
of the long-term physical stability of emulsions. Advances in colloid
and interface science, 108109, 227258.
van der Veen, J.N., et al., 2017. The critical role of phosphatidylcholine
and phosphatidylethanolamine metabolism in health and disease.
Biochimica et biophysica acta-biomembranes, 1859(9), 15581572.
van Nieuwenhuyzen, W. and Szuhaj, B.F., 1998. Effects of lecithins and
proteins on the stability of emulsions. Lipid fett, 100(7), 282291.
van Nieuwenhuyzen, W. and Tomas, M.C., 2008. Update on vegetable
lecithin and phospholipid technologies. European journal of lipid sci-
ence and technology, 110(5), 472486.
Wechtersbach, L., Poklar Ulrih, N., and Cigi
c, B., 2012. Liposomal stabiliza-
tion of ascorbic acid in model systems and in food matrices. LWT-
food science and technology, 45(1), 4349.
Wilson, M.K., et al., 2014. Review of high-dose intravenous vitamin C as
an anticancer agent. Asia-Pacific journal of clinical oncology, 10(1),
Xie, W.L. and Ji, J.M., 2008. Antioxidant activities of vitamins E and C in a
novel liposome system. Journal of food biochemistry, 32(6), 766781.
Young, J.I., Zuchner, S., and Wang, G.F., 2015. Regulation of the epige-
nome by vitamin C. Annual review of nutrition, 35, 545564.
... This shows that the distribution of ascorbate among cells is fundamental for the survival of the organism. As described elsewhere, the level of intracellular ascorbate is maintained by the balance of two fluxes, both powered by genetically controlled active transport [6], where the passive transport dissipates the ascorbate concentration gradient generated by the active transport across the plasma membrane. Being a critical element of body homeostasis, the level of ascorbate in extracellular fluids is tightly controlled and the excess is rapidly excreted [7]. ...
... Ascorbate is generally considered nontoxic, however, when added to tissue cultures in concentrations of a few mM causes the death of cancer cells. These experiments indicate that cancerous cells are sensitive to elevated concentrations of ascorbate [6]. Based on such data, the treatment of various cancers has been proposed in which a large amount of ascorbate is administered intravenously [8][9][10]. ...
... The only ways to reduce the ascorbate concentration inside the cell is its passive flow across the plasma membrane or its oxidation to DHA, which subsequently diffuses out of the cell through GLUT channels or is rapidly reduced back to ascorbate by metabolic processes inside the cell. Therefore, the oxidation of ascorbate will not affect significantly its cytoplasmic concentration [6]. The excessive level of ascorbate in the cytosol may trigger ferroptosis, leading to cell death [21]. ...
Full-text available
Ascorbate is an important element of a variety of cellular processes including the control of reactive oxygen species levels. Since reactive oxygen species are implicated as a key factor in tumorigenesis and antitumor therapy, the injection of a large amount of ascorbate is considered beneficial in cancer therapy. Recent studies have shown that ascorbate can cross the plasma membrane through passive diffusion. In contrast to absorption by active transport, which is facilitated by transport proteins (SVCT1 and SVCT2). The passive diffusion of a weak acid across membranes depends on the electrostatic potential and the pH gradients. This has been used to construct a new theoretical model capable of providing steady-state ascorbate concentration in the intracellular space and evaluating the time needed to reach it. The main conclusion of the analysis is that the steady-state intracellular ascorbate concentration weakly depends on its serum concentration but requires days of exposure to saturate. Based on these findings, it can be hypothesized that extended oral ascorbate delivery is possibly more effective than a short intravenous infusion of high ascorbate quantities.
... The encapsulation of drug into vesicular delivery systems has been widely used to enhance the solubility, stability, therapeutic efficacy and bioavailability of poorly soluble drugs [21][22][23]. Liposomes are widely used as a delivery system for hydrophilic and hydrophobic drugs [24,25]. Liposomes contain a lipid bilayer that is similar to the cell membrane. ...
Full-text available
The present research work is designed to prepare and evaluate piperine liposomes and piperine–chitosan-coated liposomes for oral delivery. Piperine (PPN) is a water-insoluble bioactive compound used for different diseases. The prepared formulations were evaluated for physicochemical study, mucoadhesive study, permeation study and in vitro cytotoxic study using the MCF7 breast cancer cell line. Piperine-loaded liposomes (PLF) were prepared by the thin-film evaporation method. The selected liposomes were coated with chitosan (PLFC) by electrostatic deposition to enhance the mucoadhesive property and in vitro therapeutic efficacy. Based on the findings of the study, the prepared PPN liposomes (PLF3) and chitosan coated PPN liposomes (PLF3C1) showed a nanometric size range of 165.7 ± 7.4 to 243.4 ± 7.5, a narrow polydispersity index (>0.3) and zeta potential (−7.1 to 29.8 mV). The average encapsulation efficiency was found to be between 60 and 80% for all prepared formulations. The drug release and permeation study profile showed biphasic release behavior and enhanced PPN permeation. The in vitro antioxidant study results showed a comparable antioxidant activity with pure PPN. The anticancer study depicted that the cell viability assay of tested PLF3C2 has significantly (p < 0.001)) reduced the IC50 when compared with pure PPN. The study revealed that oral chitosan-coated liposomes are a promising delivery system for the PPN and can increase the therapeutic efficacy against the breast cancer cell line.
Liposomes, enclosed phospholipid vesicles with bilayered membrane structures, are effective and widely used encapsulation systems in the pharmaceutical and dietary supplement industries such as nutritional supplements. These encapsulating materials have attracted great attention due to their bioavailability, biodegradability, absence of toxicity, ability to deliver a wide variety of bioactive compounds, increasing ingredients solubility, and enhanced stability against a range of environmental, enzymatic, and chemical stresses. There has been rapid development of liposomes to carry different functional compounds known as supplements needed for a healthy life. In this chapter, the application of liposomes as emerging 236carrier vehicles of supplements such as minerals, vitamins, herbal extracts, essential fatty acids, and antioxidant compounds is discussed in detail.
Citrus peel polymethoxyflavonoids (PMFs), which exhibited a significant anti-lipase effect in functional foods, were widely used in diet therapy. However, its biological application was limited by the low content in citrus peel and poor solubility in water. To enhance the solubility and anti-lipase activity of citrus peel PMFs, the citrus peel PMFs extract was enriched by reflux extraction. Then, liposomes loaded with citrus peel PMFs extract (namely, PLS) were prepared by thin film hydration-high pressure homogenization method. Citrus peel PMFs extract, mainly including eight PMFs compounds such as nobiletin and tangeretin, were enriched from citrus peel with purity of 82.76%. The PLS prepared were spherical vesicles under transmission electron microscope (TEM), and they exhibited small particle size (70.94 ± 0.82 nm), high encapsulation efficiency (87.59 ± 2.55%), sustained release and good stability in characteristics. Furthermore, PLS showed significantly better solubility and stronger anti-lipase effect compared with free PMFs. Overall, PLS might be a potential anti-lipase inhibitor for obesity, which might allow the effective valorization of citrus peel and provide a potential value-added product used in dietary supplement.
Full-text available
The antioxidant, anti-inflammatory, immunomodulating, anti-thrombotic, and antiviral effects along with its protective effects against respiratory infections have generated a great interest in vitamin C (vitC) as an attractive functional/nutraceutical ingredient for the management of COVID-19. However, the oral bioavailability and pharmacokinetics of vitC have been shown to be complex and exhibit dose-dependent non-linear kinetics. Though sustained-release forms and liquid liposomal formulations have been developed, only marginal enhancement was observed in bioavailability. Here we report a novel surface-engineered liposomal formulation of calcium ascorbate (CAAS), using fenugreek galactomannan hydrogel in powder form, and its pharmacokinetics following a randomized, double-blinded, single-dose, 3-way crossover study on healthy human volunteers (n = 14). The physicochemical characterization and in vitro release studies revealed the uniform impregnation of CAAS liposomes within the pockets created by the sterically hindered galactomannan network as multilaminar liposomal vesicles with good encapsulation efficiency (>90%) and their stability and sustained-release under gastrointestinal pH conditions. Further human studies demonstrated >7-fold enhancement in the oral bioavailability of ascorbate with a significant improvement in pharmacokinetic properties (C max, T max, T 1/2, and AUC), compared to the unformulated counterpart (UF-CAAS) when supplemented at an equivalent dose of 400 mg of CAAS as tablets and capsules.
Full-text available
Lipid vesicles (liposomes) are a unique and fascinating type of polymolecular aggregates, obtained from bilayer-forming amphiphiles—or mixtures of amphiphiles—in an aqueous medium. Unilamellar vesicles consist of one single self-closed bilayer membrane, constituted by the amphiphiles and an internal volume which is trapped by this bilayer, whereby the vesicle often is spherical with a typical desired average diameter of either about 100 nm or tens of micrometers. Functionalization of the external vesicle surface, basically achievable at will, and the possibilities of entrapping hydrophilic molecules inside the vesicles or/and embedding hydrophobic compounds within the membrane, resulted in various applications in different fields. This review highlights a few of the basic studies on the phase behavior of polar lipids, on some of the concepts for the controlled formation of lipid vesicles as dispersed lamellar phase, on some of the properties of vesicles, and on the challenges of efficiently loading them with hydrophilic or hydrophobic compounds for use as delivery systems, as nutraceuticals, for bioassays, or as cell-like compartments. Many of the large number of basic studies have laid a solid ground for various applications of polymolecular aggregates of amphiphilic lipids, including, for example, cubosomes, bicelles or—recently most successfully—nucleic acids-containing lipid nanoparticles. All this highlights the continued importance of fundamental studies. The life-saving application of mRNA lipid nanoparticle COVID-19 vaccines is in part based on year-long fundamental studies on the formation and properties of lipid vesicles. It is a fascinating example, which illustrates the importance of considering (i) details of the chemical structure of the different molecules involved, as well as (ii) physical, (iii) engineering, (iv) biological, (v) pharmacological, and (vii) economic aspects. Moreover, the strong demand for interdisciplinary collaboration in the field of lipid vesicles and related aggregates is also an excellent and convincing example for teaching students in the field of complex molecular systems.
Nowadays the importance of vitamins is clear for everyone. However, many patients are suffering from insufficient intake of vitamins. Incomplete intake of different vitamins from food sources due to their destruction during food processing or decrease in their bioavailability when mixing with other food materials, are factors resulting in vitamin deficiency in the body. Therefore, various lipid based nanocarriers such as nanoliposomes were developed to increase the bioavailability of bioactive compounds. Since the function of nanoliposomes containing vitamins on the body has a direct relationship with the quality of produced nanoliposomes, this review study was planned to investigate the several aspects of liposomal characteristics such as size, polydispersity index, zeta potential, and encapsulation efficiency on the quality of synthesized vitamin-loaded nanoliposomes.
Full-text available
KRAS mutation is often present in many hard-to-treat tumors such as colon and pancreatic cancer and it is tightly linked to serious alterations in the normal cell metabolism and clinical resistance to chemotherapy. In 1931, the winner of the Nobel Prize in Medicine, Otto Warburg, stated that cancer was primarily caused by altered metabolism interfering with energy processing in the normal cell. Increased cell glycolytic rates even in the presence of oxygen is fully recognized as a hallmark in cancer and known as the Warburg effect. In the late 1970′s, Linus Pauling and Ewan Cameron reported that vitamin C may have positive effects in cancer treatment, although deep mechanistic knowledge about this activity is still scarce. We describe a novel antitumoral mechanism of vitamin C in KRAS mutant colorectal cancer that involves the Warburg metabolic disruption through downregulation of key metabolic checkpoints in KRAS mutant cancer cells and tumors without killing human immortalized colonocytes. Vitamin C induces RAS detachment from the cell membrane inhibiting ERK 1/2 and PKM2 phosphorylation. As a consequence of this activity, strong downregulation of the glucose transporter (GLUT-1) and pyruvate kinase M2 (PKM2)-PTB dependent protein expression are observed causing a major blockage of the Warburg effect and therefore energetic stress. We propose a combination of conventional chemotherapy with metabolic strategies, including vitamin C and/or other molecules targeting pivotal key players involved in the Warburg effect which may constitute a new horizon in anti-cancer therapies.
Full-text available
Intravenous administration of vitamin C has been shown to decrease oxidative stress and, in some instances, improve physiological function in adult humans. Oral vitamin C administration is typically less effective than intravenous, due in part to inferior vitamin C bioavailability. The purpose of this study was to determine the efficacy of oral delivery of vitamin C encapsulated in liposomes. On 4 separate randomly ordered occasions, 11 men and women were administered an oral placebo, or 4 g of vitamin C via oral, oral liposomal, or intravenous delivery. The data indicate that oral delivery of 4 g of vitamin C encapsulated in liposomes (1) produces circulating concentrations of vitamin C that are greater than unencapsulated oral but less than intravenous administration and (2) provides protection from ischemia-reperfusion-mediated oxidative stress that is similar to the protection provided by unencapsulated oral and intravenous administrations.
Full-text available
Cytosolic lipid droplets (LDs) are observed in enterocytes of jejunum during lipid absorption. One important function of the intestine is to secrete chylomicrons, which provide dietary lipids throughout the body, from digested lipids in meals. The current hypothesis is that cytosolic LDs in enterocytes constitute a transient pool of stored lipids that provides lipids during interprandial period while lowering chylomicron production during the post-prandial phase. This smoothens the magnitude of peaks of hypertriglyceridemia. Here, we review the composition and functions of lipids and associated proteins of enterocyte LDs, the known physiological functions of LDs as well as the role of LDs in pathological processes in the context of the intestine.
Full-text available
Eggs are a major source of phospholipids (PL) in the Western diet. Dietary PL have emerged as a potential source of bioactive lipids that may have widespread effects on pathways related to inflammation, cholesterol metabolism, and high-density lipoprotein (HDL) function. Based on pre-clinical studies, egg phosphatidylcholine (PC) and sphingomyelin appear to regulate cholesterol absorption and inflammation. In clinical studies, egg PL intake is associated with beneficial changes in biomarkers related to HDL reverse cholesterol transport. Recently, egg PC was shown to be a substrate for the generation of trimethylamine N-oxide (TMAO), a gut microbe-dependent metabolite associated with increased cardiovascular disease (CVD) risk. More research is warranted to examine potential serum TMAO responses with chronic egg ingestion and in different populations, such as diabetics. In this review, the recent basic science, clinical, and epidemiological findings examining egg PL intake and risk of CVD are summarized.
Full-text available
Phospholipids are amphipathic lipids, which are found in all the cell membranes, organized as a lipid bilayer. They belong to the glycerol-derived lipids, showing a similar structure as triglycerides. The current interest of them comes from its effectiveness to incorporate different fatty acids in the cell membrane, as they exhibit better absorption and utilization than triglycerides. In this paper, the bibliographical data published about the benefits of the phospholipids in inflammatory processes, cancer, cardiovascular diseases, neurological disorders, liver disease and as an antioxidants transporter is reviewed. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.
Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most abundant phospholipids in all mammalian cell membranes. In the 1950s, Eugene Kennedy and co-workers performed groundbreaking research that established the general outline of many of the pathways of phospholipid biosynthesis. In recent years, the importance of phospholipid metabolism in regulating lipid, lipoprotein and whole-body energy metabolism has been demonstrated in numerous dietary studies and knockout animal models. The purpose of this review is to highlight the unappreciated impact of phospholipid metabolism on health and disease. Abnormally high, and abnormally low, cellular PC/PE molar ratios in various tissues can influence energy metabolism and have been linked to disease progression. For example, inhibition of hepatic PC synthesis impairs very low density lipoprotein secretion and changes in hepatic phospholipid composition have been linked to fatty liver disease and impaired liver regeneration after surgery. The relative abundance of PC and PE regulates the size and dynamics of lipid droplets. In mitochondria, changes in the PC/PE molar ratio affect energy production. We highlight data showing that changes in the PC and/or PE content of various tissues are implicated in metabolic disorders such as atherosclerosis, insulin resistance and obesity. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo Escríba-Ruíz.
In animal models of acute ischemic stroke, intravenous dehydroascorbic acid (DHAA), unlike ascorbic acid (AA), readily enters brain and is converted in both normal and ischemic brain into protective ascorbic acid. When given parenterally DHAA minimizes infarct volume and facilitates functional recovery. I hypothesize the same effect will occur in humans with acute ischemic stroke. Efficacy in reducing infarct volume is demonstrable in mice and rats even when DHAA is infused three hours after the experimental infarct. Moreover, there is five-fold mechanistic rational for DHA beside excellent pharmacokinetics and rapid penetration of brain and conversion to protective AA: 1) in ischemic brain, there is a precipitous decline in AA which can be reversed by intravenous DHAA; 2) after reduction of DHAA to AA in both normal and ischemic brain, AA can reduce oxidized vitamin E and glutathione, other protectors of brain against damaging reactive oxygen species which build up in ischemic brain; 3) AA itself can protect brain against damaging reactive oxygen species; 4) AA is an essential cofactor for several enzymes in brain including ten-eleven translocase-2 which upregulates production of protective molecules like brain-derived neurotrophic factor; and 5) DHAA after conversion to AA prevents both lipid oxidation and presumably oxidation of other labile substances (e.g., dopamine) in ischemic brain. In terms of safety, based on all available animal information, DHAA is safe in the proposed dosing regimen. For human clinical trials, the methodology for conducting the proposed animal safety, clinical pharmacology and phase II efficacy studies is straightforward. Finally, if DHAA preserved brain substance and function in humans, it could be employed in pre-hospital stroke patients.
Demethylation of CpG motifs in the Foxp3 intronic element, conserved noncoding sequence 2 (CNS2), is indispensable for the stable expression of Foxp3 in regulatory T cells (Tregs). In this study, we found that vitamin C induces CNS2 demethylation in Tregs in a ten-eleven-translocation 2 (Tet2)-dependent manner. The CpG motifs of CNS2 in Tregs generated in vitro by TGF-β (iTregs), which were methylated originally, became demethylated after vitamin C treatment. The conversion of 5-methylcytosin into 5-hydroxymethylcytosin was more efficient, and the methyl group from the CpG motifs of Foxp3 CNS2 was erased rapidly in iTregs treated with vitamin C. The effect of vitamin C disappeared in Tet2(-/-) iTregs. Furthermore, CNS2 in peripheral Tregs in vivo, which were demethylated originally, became methylated after treatment with a sodium-dependent vitamin C transporter inhibitor, sulfinpyrazone. Finally, CNS2 demethylation in thymic Tregs was also impaired in Tet2(-/-) mice, but not in wild type mice, when they were treated with sulfinpyrazone. Collectively, vitamin C was required for the CNS2 demethylation mediated by Tet proteins, which was essential for Foxp3 expression. Our findings indicate that environmental factors, such as nutrients, could bring about changes in immune homeostasis through epigenetic mechanisms.
Vitamin C (Ascorbic acid, abbreviated as AA; the terms vitamin C and ascorbic acid are used interchangeably) is synthesized by all plants and most animals (Smirnoff et al., 2001). It is a vitamin for humans because the gene for gulonolactone oxidase, the terminal enzyme in the AA synthesis pathway has undergone mutations that make it non-functional (Linster & Van Schaftingen, 2007). Animals that have lost the ability to synthesize ascorbic acid do not have a phylogenetic relationship with each other. These animals include non-human primates, guinea pigs, capybara and some birds and fish (Chaudhuri & Chatterjee, 1969, Chatterjee, 1973, Cueto et al., 2000). Deficiency of ascorbic acid produces the fatal disease scurvy, which can be cured only by the administration of vitamin C. This article is protected by copyright. All rights reserved.
Emerging evidence suggests that ascorbate, the dominant form of vitamin C under physiological pH conditions, influences activity of the genome via regulating epigenomic processes. Ascorbate serves as a cofactor for Ten-eleven translocation (TET) dioxygenases that catalyze the oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), and further to 5-formylcytosine (5fC) and to 5-carboxylcytosine (5caC), which are ultimately replaced by unmodified cytosine. The Jumonji C (JmjC)-domaincontaining histone demethylases also require ascorbate as a cofactor for histone demethylation. Thus, by primarily participating in the demethylation of both DNA and histones, ascorbate appears to be a mediator of the interface between the genome and environment. Furthermore, redox status has a profound impact on the bioavailability of ascorbate in the nucleus. In order to bridge the gap between redox biology and genomics, we suggest an interdisciplinary research field that can be termed redox genomics to study dynamic redox processes in health and diseases. This review examines the evidence and potential molecular mechanism of ascorbate in the demethylation of the genome, and it highlights potential epigenetic roles of ascorbate in various diseases. Expected final online publication date for the Annual Review of Nutrition Volume 35 is July 17, 2015. Please see for revised estimates.