Novel Vitamin and Gold-Loaded Nanofiber Facial Mask for Topical Delivery

Abstract

L-ascorbic acid has been widely used in cosmetic and dermatological products because of its ability to scavenge free radicals and destroy oxidizing agents. However, it is chemically unstable and can easily be oxidized. The current cosmetic facial masks available in the market are pre-moistened, which means that the aqueous fluid content of the mask may oxidize some of the unstable active ingredients such as ascorbic acid. This work presents an anti-wrinkle nanofiber face mask containing ascorbic acid, retinoic acid, gold nanoparticles, and collagen. This novel face mask will only be wetted when applied to the skin, thus enhancing product stability. Once moistened, the content of the mask will gradually dissolve and release the active ingredients and ensure maximum skin penetration. The high surface area-to-volume ratio of the nanofiber mask will ensure maximum contact with the skin surface and help to enhance the skin permeation to restore its healthy appearance. Electrospun fiber mats may provide an attractive alternative to the commercial facial cotton masks.

Full text

Research Article
Novel Vitamin and Gold-Loaded Nanofiber Facial Mask for Topical Delivery
Anahita Fathi-Azarbayjani,
1
Lin Qun,
1
Yew Weng Chan,
2
and Sui Yung Chan
1,3
Received 17 July 2009; accepted 22 June 2010; published online 27 July,2010
Abstract. L-ascorbic acid has been widely used in cosmetic and dermatological products because of its
ability to scavenge free radicals and destroy oxidizing agents. However, it is chemically unstable and can
easily be oxidized. The current cosmetic facial masks available in the market are pre-moistened, which
means that the aqueous fluid content of the mask may oxidize some of the unstable active ingredients
such as ascorbic acid. This work presents an anti-wrinkle nanofiber face mask containing ascorbic acid,
retinoic acid, gold nanoparticles, and collagen. This novel face mask will only be wetted when applied to
the skin, thus enhancing product stability. Once moistened, the content of the mask will gradually
dissolve and release the active ingredients and ensure maximum skin penetration. The high surface area-
to-volume ratio of the nanofiber mask will ensure maximum contact with the skin surface and help to
enhance the skin permeation to restore its healthy appearance. Electrospun fiber mats may provide an
attractive alternative to the commercial facial cotton masks.
KEY WORDS: ascorbic acid; cis-retinoic acid; collagen; gold nanoparticle; topical facial mask.
INTRODUCTION
Many marketing strategies include the incorporation of
antioxidants and other skin nutrients into cosmetic products.
Collagen is important for the strength and function of the skin
and is an important factor in skin rejuvenation and wrinkle
reversal effect. The amount of collagen in the skin tends to
decline with age; therefore, it is widely used as a moisturizer
in cosmetic creams and products. Vitamin C (L-ascorbic acid)
has been widely used in cosmetic and dermatological
products because of its photoprotective effect and the ability
to scavenge free radicals and destroy oxidizing agents. It can
also induce collagen synthesis and suppress the pigmentation
of the skin while reducing signs of photoaging. However, it is
chemically unstable, and it can easily be oxidized; therefore,
more stable derivatives of ascorbic acid such as ascorbyl
palmitate, ascorbyl tetraisopalmitate, and magnesium
ascorbyl phosphate are widely used in pharmaceutical
industry (1,2). These derivatives can easily be converted to
the active compound, ascorbic acid, after ingestion. However,
topical applications of these derivatives are not able to
efficiently increase the skin levels of this antioxidant (3). A
formulation strategy to improve the stability of ascorbic acid
is to deliver using emulsions. The oil phase may partially
protect the vitamin from oxidative degradation caused in
aqueous solutions (4–6).
Retinoic acid enhances the repair of ultraviolet (UV)-
damaged skin and can reduce wrinkles caused by photoaging.
It can also be used in the treatment of acne (7–11). Due to its
lipophilic structure, it is practically insoluble in aqueous
solution, which decreases its bioavailability (12–14).
Gold nanoparticles have been studied as potential
vaccine carriers and in transdermal delivery (15–18). Gold
facial masks are now being used in beauty clinics and saloons.
They help to improve blood circulation, skin elasticity, and
can rejuvenate the skin and reduce the formation of wrinkles.
Skin permeation studies demonstrate that spherical gold
nanoparticles are not inherently toxic to human skin cells
(15).
Cyclodextrins (CDs) are a group of cyclic oligosacchar-
ides. They are able to form inclusion complexes with
lipophilic guest molecules which have been shown to improve
aqueous solubility, dissolution rate, and bioavailability of such
drugs (19,20). Due to the solubility, hygroscopicity, and
toxicity concerns of CDs, they were modified to randomly
methylated β-CD (RM β-CDs). Methylated β-CD has been
assessed in cultures of human skin fibroblasts without
reported toxicity (21).
Polyvinyl alcohol (PVA) is a biodegradable, hydrophilic
polymer with distinct properties such as high degree of
swelling, inherent non-toxicity, and good biocompatibility
(22,23).
Electrospinning is a remarkably simple and effective
method for fabricating polymeric nanoscale fibers with high
surface area-to-volume ratio and porosity. The collected
nonwoven fabric may be applied to different areas of research
such as drug delivery (24,25).
Conventional beauty face masks available in the market
are cotton masks that are pre-moistened with skin nutrients.
The aqueous phase of the pre-moistened mask can increase
1
Department of Pharmacy, National University of Singapore, Block
S4, level 2, Science Drive 4, 117543, Singapore, Singapore.
2
Department of Anesthesiology, Singapore General Hospital,
169608, Singapore, Singapore.
3
To whom correspondence should be addressed. (e-mail: phacsy@
nus.edu.sg)
AAPS PharmSciTech, Vol. 11, No. 3, September 2010 ( #2010)
DOI: 10.1208/s12249-010-9475-z
1530-9932/10/0300-1164/0 #2010 American Association of Pharmaceutical Scientists 1164

the degradation rate of the unstable ingredients such as
ascorbic acid.
The objective of this work is to develop a novel
polymeric nanofiber face mask (from PVA and RM β-CD)
that can accommodate several skin nutrients such as ascorbic
acid, retinoic acid, gold, and collagen. These face masks were
developed and characterized using field emission scanning
electron microscope and X-ray elemental analysis. Skin
permeation of ascorbic acid and retinoic acid from these
nanofiber face masks was tested using human epidermis.
METHOD
Materials and Methods
L-ascorbic acid, chloroauric acid, PVA, trisodium citrate,
and collagen were obtained from Sigma, Singapore. 13-cis-
Retinoic acid was obtained from Toronto Research Chemicals.
RM β-CD (degree of substitution of about 1.8) was a gift from
Wacker (Burghausen, Germany).
Electrospinning
Gold nanoparticles were prepared by trisodium citrate
reduction of HAuCl
4
in PVA ethanol solution (30% v/v)
(26,27). Briefly, 2 ml o f 2% w/vtrisodium citrate was added to
the PVA solution mixture and stirred at 95–100°C, and then
0.7 ml 1% w/vchloroauric acid aqueous solution was added
to this mixture where the color of the solution changed to
blood red. The mixture was cooled to room temperature
while stirring using a magnetic stirrer at 700 rpm. RM β-CD,
ascorbic acid, retinoic acid, or collagen was dissolved in the
mixture prior electrospinning. The face mask is produced
from electrospinning the aqueous phase containing the
polymer and the active ingredients shown in Table I. The
electrostatic spinner used for the experiments was equipped
with an adjustable DC power supply (RP50-1.25 R/230
DDPM, Gamma high voltage Research, USA) and a syringe
pump (KD-100, KD Scientific, Inc., USA) on which a 3-ml
syringe was connected to a blunt 27-G stainless steel needle.
The applied voltage was set at 25 kV. A constant flow rate of
2 ml/h was applied to all formulations. The depositions were
performed at room temperature. The nonwoven electrostati-
cally spun fabric was removed from the collector and was
dried under vacuum for a week at room temperature to
remove water prior to usage.
Field Emission Scanning Electron Microscopy (FESEM)
and Energy Dispersive X-Ray Spectroscopy (EDS) Analysis
The surface topography of the electrospun fibers was
assessed using a field emission scanning electron microscopy
(FESEM; Jeol JSM-6701F, Japan). Electrospun fibers were
placed at the center of an aluminum stud using carbon tape.
A very thin layer of platinum was applied to the fibers by a
sputtering unit (Jeol JFC-1600 auto fine coater, Japan).
Coated fibers were placed in the microscope chamber at high
vacuum. Surface morphological features were obtained under
5 kV accelerating voltage. The diameter distribution of the
electrospun fibers was derived from a random sample of at
least 20 fibers.
Energy dispersive X-ray spectroscopy (EDS) measure-
ments were carried out by means of a FESEM equipped with
an energy dispersive X-ray source to identify the presence of
gold in the fibers. After coating with platinum, samples were
analyzed at 15 kV voltage. The area to be analyzed was
selected, and the electron beam scanned and identified the
intensity of characteristic X-ray energies of specific elements.
Fourier Transform Infrared Measurements (FTIR)
Fourier transform infrared (FTIR) spectra of the poly-
meric nanofiber mats were taken with a Perkin Elmer
(Spectrum 100) in the wavelength region 500–4,000 cm
−1
at
room temperature. Samples were placed on KBr holder and
mounted in the enclosed sample chamber, away from
moisture to get their spectra.
UV–Vis Spectroscopy
UV absorption measurements were carried out at room
temperature on a UV–Vis spectrophotometer (U-1800 spec-
trophotometer, Hitachi, Japan) at 261 and 349 nm for ascorbic
acid and retinoic acid, respectively. Standard solutions of
ascorbic acid and retinoic acid (0.05 to 2.00 μg/ml) were
prepared in water and water ethanol (60% v/v)solutions,and
the R
2
value of the calibration curves for the standard solutions
were 0.9994 and 0.9992, respectively.
Skin Preparation and Permeation Studies
Abdominal skin from an adult was obtained with patient
consent and ethics approval post-plastic surgery. Subcutane-
ous fat was carefully separated from the epidermis after
immersing the whole skin in distilled water at 60 ± 5°C for
Table I. The Compositions of the Face Mask Formulations
Formulations
Ingredient
Ascorbic acid (1% w/v)cis-Retinoic acid (0.1% w/v) Collagen (0.01% w/v) Gold (0.1% w/v)
A√–––
B√––√
C–√––
D√√√–
E√√√√
All formulations contain 10% w/vpolyvinyl alcohol and 20% w/vrandomly methylated β-cyclodextrin
1165Novel Vitamin and Gold-loaded Nanofiber Facial Mask

2 min. Samples were stored at −80°C until use. Prior to
permeation studies, the skin samples with stratum corneum
(SC) side facing upwards were equilibrated for 2 h in 0.9% w/v
sodium chloride solutions containing 1% v/vantibacterial
antimycotic solution (28).
Permeation studies were performed using a flow-through
diffusion cell apparatus. The donor compartment was filled
with 25 mg of vitamin-loaded mat and hydrated with 300 μlof
water. The skin permeation studies were only performed from
formulations containing single ingredient (A, B, and C), and
the results were compared to cotton masks that contained the
same concentration of the active ingredient. This was done in
order to prevent the UV interference from multi-ingredients.
However, it is assumed that presence of all the active
ingredients in one formulation should not have a significant
impact on the release pattern of the molecules. Loading
efficiencies of the fiber masks were calculated by fully
dissolving a certain amount of mask in 20% v/vethanol and
measuring its concentration with UV spectrophotometer.
The receptor compartment was phosphate buffer saline
with pH adjusted to 5.5. Control samples were cotton sheet
pre-moistened with vitamin solutions. The exposed surface
Fig. 1. Field emission scanning electron microscopy images of nanofibers formulation and a macrographic image of a typical
nanofiber mat
1166 Fathi-Azarbayjani et al.

area of the skin for the permeation of the drug was 0.785 cm
2
.
Samples from the receptor compartment were collected at
predetermined time points over a 4-h period, and the
amounts of ascorbic acid and retinoic acid permeated were
analyzed by UV spectrometer. Cell temperature was kept at
37± 0.5°C throughout the experiment.
Skin Histology
Skin samples used in diffusion studies were processed for
light microscopy. Samples were soaked overnight in 85 ml of
80% ethanol, 10 ml formaldehyde, and 5 ml acetic acid (29).
After a series of dehydration, samples were embedded in
paraffin, and semi-thin sections were stained with hematoxylin
and eosin prior being examined with a light microscope (Leica
EC 3, USA).
Statistical Analysis
Results were expressed as the mean± SD of at least three
experiments. Analysis of variance was carried out (Graph
Pad Prism, version 2.0) followed by the Tukey post hoc and
Student's ttest to determine the differences between treat-
ment groups. A value of p<0.05 was considered statistically
significant.
RESULTS
Fiber Morphology and EDS Analysis
Continuous fibers without beads or sputtering of the
solution were obtained with diameter ranging from ~100 nm
to 2 μm (Fig. 1). There was a decrease in fiber diameter of
electrospun solutions containing gold. This may be due to the
increase in the charge density of the solution due to the
presence of gold (30). Figure 2illustrates the spectra of gold
present on the fibers.
FTIR Studies
Figure 3indicates FTIR analysis of PVA, PVA–RM β-
CD, and PVA–RM β-CD loaded with the active ingredients.
For pure PVA, a broad band around 3,369 cm
−1
is attributed
to the O–H stretching vibration of the hydroxyl group. The
vibrational bands at 2,932 and 1,455 cm
−1
represent the –CH
stretching. The sharp peak band at 1,090 cm
−1
corresponds to
C–O–C symmetrical stretching present on the PVA backbone
(31–33). With the addition of RM-β-CD, the band
corresponding to O–H stretch of PVA showed a decrease in
intensity. This suggested that RM-β-CD formed hydrogen
bond with PVA, which could explain the enhanced solubility
of the fibers when RM-β-CD was added to the PVA. There
was a decrease in the intensity of the O–H group when
collagen was added to the solution. The addition of gold to
PVA/RM-β-CD fiber caused a shift of O–H band to a higher
frequency of 3,389 cm
−1
. This suggests that PVA stabilized the
gold particles (34).
Ascorbic acid can rapidly decompose in aqueous
solutions and produce many degradation products. Studies
show that CD has minor effect on the degradation of ascorbic
acid, and the inclusion of ascorbic acid into the CD cavity is
negligible (35). Stability of collagen has been studied, and
results show that blending collagen with PVA increases the
thermal and photochemical stability of collagen (36). PVA
acts as a good stabilizer to protect gold nanoparticles (34).
The stability of retinoic acid from nanofiber has been tested,
and it was found that the retinoic acid was stable in the
medium (25).
0
001
0013 0 µm30 µm
30 µm
30 µm
3
3
0
0
µ
µ
m
m
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
keV
001
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
Counts
C-K
O-K
Na-K
Au-M
Au-M
Au-M Au-L Au-L
005
005
10 µm
10 µm
10 µm
10 µm
1
1
0
0
µ
µ
m
m
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
keV
005
0
100
200
300
400
500
600
700
800
900
1000
Counts
C-K
O-K
Na-K
Au-M
Au-M
Au-M
Au-L Au-L
006
006
10 µm10 µm
10 µm
10 µm
1
1
0
0
µ
µ
m
m
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
keV
006
0
100
200
300
400
500
600
700
800
900
1000
Counts
C-K
O-K
Na-K
Au-M
Au-M
Au-M
Au-L Au-L
ab c
Fig. 2. X-ray energy spectrum of gold-loaded nanofiber sheet, demonstrating the presence of the mineral-specific signals. aArea analysis and b,
cspot analysis of the nanofiber surface. C and O signal is due to the backbone of the polymers, and Na signal is due to the addition of sodium
citrate used in the reduction of gold. Au signals represent the presence of gold on the fiber structure
1167Novel Vitamin and Gold-loaded Nanofiber Facial Mask

Skin Permeation
Results of the skin permeation are shown in Fig. 4. It can
be seen that nanofiber-loaded vitamins slightly increased the
skin permeation of ascorbic acid and retinoic acid when
compared to the vitamin-loaded cotton face mask (p>0.05).
The addition of a penetration enhancer to the nanofiber
formula may result in significantly higher skin permeation
rate.
Gold did not cause any significant change in the skin
permeation of ascorbic acid (p>0.05). This effect might be
due to the limited concentration of gold present in the
formulations.
Microscopic appearance of the skin treated with the
gold-loaded nanofiber mask is shown in Fig. 5. In the control,
skin samples treated with nanofiber mask containing PVA
and RM β-CD, a clearly defined SC, could be seen (Fig. 5a),
but after treatment with gold-loaded nanofiber, slight detach-
ment of the SC layer occurred. The SC layer was fragmented,
and enlargement of inter-keratinocyte spaces was observed,
while the other epidermis layers were more compact. There is
a need to further study the effect of gold and gold nano-
particles on the skin surface and to investigate its toxicological
effects.
These novel facial masks have high surface area-to-
volume ratio that can increase the contact area of the mask
with the skin surface. Compared to conventional pre-mois-
tened cotton masks, these nanofiber masks have a dry nature
that increases the stability of the nutrients and minimizes the
oxidation of the antioxidants. Incorporation of RM β-CD to
the PVA solution decreased the degradation rate of the
resulting fiber mat.
Selection of the type of CD and its addition to the
polymeric solution prior to electrospinning can increase the
disintegration rate of the nanofibers and help it dissolve on
the skin surface within a shorter period of time. PVA can
produce fibers that have a disintegration time of a few weeks
(23). Addition of RM β-CD to PVA solution can produce
fibers that dissolve within minutes upon contact with an
aqueous medium. This may be due to the –OH bonding
between PVA and RM β-CD as observed on the FTIR
7001400210028003500
Wavenumber (cm-1)
Transmittance
PVA
PVA-RM β-CD
A
B
C
D
E
Fig. 3. Fourier transform infrared analysis of nanofiber mats
containing pure polyvinyl alcohol (PVA), PVA and randomly
methylated β-cyclodextrin, and formulations A–E as listed in Table I
0
3
6
9
12
0
Time (mins)
Cumulative RA (µg/cm
2
)
control
Electrospun mat-RA
0
10
20
30
40
50
Time (mins)
Cumulative AA (µg/cm
2
)
Electrospun mat-AA
Electrospun mat-Au-AA
control
25020015010050
0 25020015010050
Fig. 4. Permeation profile of ascorbic acid and retinoic acid across
human epidermis (n=3)
Fig. 5. Morphology of human epidermis: histological skin examina-
tions were performed in vitro 24 h after skin permeation studies
(×400). aSkin samples treated with nanofiber mask containing
polyvinyl alcohol (PVA) and randomly methylated β-cyclodextrin
(RM β-CD). bSkin samples treated with gold-loaded nanofiber
containing PVA and RM β-CD. The nucleated cells of the epidermis
have been stained blue; unsaturated lipids, including fatty acids and
esters, have been stained red
1168 Fathi-Azarbayjani et al.

studies mentioned in the earlier section (37). In other words,
RM β-CD can decrease the dissolution time of PVA fibers.
This rapid dissolution of the face mask will provide fast
release of the active ingredients that are incorporated within
the polymeric fiber mat. Therefore, once the dry face mask is
placed on the skin surface and wetted with water, it will
dissolve in less than 15 min and release its entire active
components thus ensuring maximum skin penetration of the
active molecules.
Angberg et al evaluated degradation reaction of ascorbic
acid in aqueous solution. It was found that oxidation rate of
Ascorbic Acid was dependent on the availability of oxygen
and its dissolution in the head space of the vial. Therefore,
the amount of oxidation was higher at the beginning when
oxygen content was high and directly available in the
solution, and the percentage decrease in ascorbic acid was
greater in the first 5 h (38). Although in our work the
experiment was carried out for 4 h, however, as with
commercial facial masks, the nanofiber mat formulation is
intended to be placed on the skin surface for no more than
15 min, which will decrease the oxidation rate of ascorbic
acid.
These properties of the electrospun mat make it an
attractive alternative facial mask.
CONCLUSION
An innovative nanofiber facial mask composed of
ascorbic acid, collagen, retinoic acid, and gold nanoparticle
was developed. The face mask is dry in nature and will only
be moistened with water prior to application. The wetted
mask will then gradually dissolve and release the ingredients
condensed on it. The dry nature of the face mask increases
the stability of the antioxidants and its shelf life when
compared to pre-moistened cotton masks available in the
market. Electrospun fiber mats may provide an attractive
alternative to the commercial facial cotton masks.
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