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Journal of Liposome Research
ISSN: 0898-2104 (Print) 1532-2394 (Online) Journal homepage: https://www.tandfonline.com/loi/ilpr20
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: https://doi.org/10.1080/08982104.2019.1630642
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Published online: 02 Jul 2019.
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ARTICLE
New oral liposomal vitamin C formulation: properties and bioavailability
Maciej Łukawski
a
, Paulina Dałek
a,b
, Tomasz Borowik
b
, Aleksander Fory
s
c
, Marek Langner
a,b
,
Wojciech Witkiewicz
d
and Magdalena Przybyło
a,b
a
Department of Biomedical Engineering, Wroclaw University of Science and Technology, Wrocław, Poland;
b
Lipid Systems Ltd, Wrocław,
Poland;
c
Centre of Polymer and Carbon Materials, Polish Academy of Sciences, Zabrze, Poland;
d
Research and Development Centre,
Specialized Hospital in Wrocław, Wrocław, Poland
ABSTRACT
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.
ARTICLE HISTORY
Received 20 December 2018
Revised 10 April 2019
Accepted 22 April 2019
KEYWORDS
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 hydrophilic–hydrophobic
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 paulina.dalek@pwr.edu.pl 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 https://doi.org/10.1080/08982104.2019.1630642.
ß2019 Informa UK Limited, trading as Taylor & Francis Group
JOURNAL OF LIPOSOME RESEARCH
https://doi.org/10.1080/08982104.2019.1630642
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 Bligh–Dyer 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
measurement.
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
2
) 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
mm
2
) and the measurement was made using a hydrophobic
phase gradient of n-hexane–isopropanol 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
2 M. ŁUKAWSKI ET AL.
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
determination
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
ðÞ
100
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
(0.4–5 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
2
. 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%
trypsin–EDTA 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 31–65) 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
JOURNAL OF LIPOSOME RESEARCH 3
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
max
,C
max
, and T
1/2
using
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 Bligh–Dyer
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
4 M. ŁUKAWSKI ET AL.
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
max
reaches
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
max
) is longer by approximately 1 h when
compared to the free form (T
max
¼180 min vs. 96 min). The
increased half-life (t
1/2
>6 h vs. t
1/2
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)
(c)
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.
JOURNAL OF LIPOSOME RESEARCH 5
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
(a)
(c)
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)
l¼k_
cn1:The determined value of nequals to 0.8 indicates that liposomal
gel is a pseudoplastic system (Tadros 2004).
6 M. ŁUKAWSKI ET AL.
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.
Funding
This work was supported by Error! Hyperlink reference not valid.; the
National Centre for Research and Development under Grant number
UOD-DEM-1-027/001.
ORCID
Paulina Dałek http://orcid.org/0000-0002-3554-5532
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).
JOURNAL OF LIPOSOME RESEARCH 7
References
Aditi, A. and Graham, D.Y., 2012. Vitamin C, gastritis, and gastric disease:
a historical review and update. Digestive diseases and sciences, 57(10),
2504–2515.
Aguilera, O., 2016. Vitamin C uncouples the Warburg metabolic switch in
KRAS mutant colon cancer. Oncotarget, 7(30), 47954–47965.
Alvarez, A.M.R. and Rodriguez, M.L.G., 2000. Lipids in pharmaceutical and
cosmetic preparations. Grasas Y aceites, 51(1–2), 74–96.
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), 1751–1757.
Beilstein, F., et al., 2016. Characteristics and functions of lipid droplets
and associated proteins in enterocytes. Experimental cell research,
340(2), 172–179.
Blesso, C.N., 2015. Egg phospholipids and cardiovascular health.
Nutrients, 7(4), 2731–2747.
Bligh, E. and Dyer, W., 1959. A rapid method of total lipid extraction and
purification. Canadian journal of biochemistry and physiology, 37(8),
911–917.
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), 13604–13609.
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), 25–30.
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), 443–457.
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), 557–570.
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), 268–283.
Fraga, C.G. and Oteiza, P.I., 2002. Iron toxicity and antioxidant nutrients.
Toxicology, 180(1), 23–32.
Fritz, H., et al., 2014. Intravenous vitamin C and cancer. Integrative cancer
therapies, 13(4), 280–300.
Garcia, J.T. and Aguero, S.D., 2015. Phospholipids: properties and health
effects. Nutricion hospitalaria, 31(1), 76–83.
Harrison, F., Bowman, G., and Polidori, M., 2014. Ascorbic acid and the
brain: rationale for the use against cognitive decline. Nutrients, 6(4),
1752–1781.
Hickey, S., Roberts, H.J., and Miller, N.J., 2008. Pharmacokinetics of oral
vitamin C. Journal of nutritional & environmental medicine, 17(3),
169–177.
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(1–2),
132–138.
Keller, B.C., 2001. Liposomes in nutrition. Trends in food science & technol-
ogy, 12(1), 25–31.
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), 579–584.
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, 69–83.
Letter, W.S., 1992. A rapid method for phospholipid separation by HPLC
using a light-scattering detector. Journal of liquid chromatography,
15(2), 253–266.
Lindblad, M., Tveden-Nyborg, P., and Lykkesfeldt, J., 2013. Regulation of
vitamin C homeostasis during deficiency. Nutrients, 5(8), 2860–2879.
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), 3039–3046.
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), 178–186.
Michels, A. and Frei, B., 2013. Myths, artifacts, and fatal flaws: identifying
limitations and opportunities in vitamin C research. Nutrients, 5(12),
5161–5192.
Nagle, J.F. and Tristram-Nagle, S., 2000. Structure of lipid bilayers.
Biochimica et biophysica acta, 1469(3), 159–195.
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), 2119–2131.
Padayatty, S.J. and Levine, M., 2016. Vitamin C: the known and the
unknown and Goldilocks. Oral diseases, 22(6), 463–493.
Padayatty, S.J., et al., 2004. Vitamin C pharmacokinetics: implications for
oral and intravenous use. Annals of internal medicine, 140(7), 533–537.
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), 45–53.
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), 908–915.
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), 464–472.
Savini, I., et al., 2005. Vitamin C homeostasis in skeletal muscle cells. Free
radical biology and medicine, 38(7), 898–907.
Spector, R., 2016. Dehydroascorbic acid for the treatment of acute ische-
mic stroke. Medical hypotheses, 89, 32–36.
Stamford, N.P.J., 2012. Stability, transdermal penetration, and cutaneous
effects of ascorbic acid and its derivatives. Journal of cosmetic derma-
tology, 11(4), 310–317.
Tadros, T., 2004. Application of rheology for assessment and prediction
of the long-term physical stability of emulsions. Advances in colloid
and interface science, 108–109, 227–258.
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), 1558–1572.
van Nieuwenhuyzen, W. and Szuhaj, B.F., 1998. Effects of lecithins and
proteins on the stability of emulsions. Lipid –fett, 100(7), 282–291.
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), 472–486.
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), 43–49.
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),
22–37.
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), 766–781.
Young, J.I., Zuchner, S., and Wang, G.F., 2015. Regulation of the epige-
nome by vitamin C. Annual review of nutrition, 35, 545–564.
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