Article

Journal of
Biomedical Nanotechnology
Copyright © 2015 American Scientific Publishers
All rights reserved Vol. 11, 351–361, 2015
Printed in the United States of America www.aspbs.com/jbn

Solid Lipid Nanoparticles Formulated for

Transdermal Aconitine Administration and
Evaluated In Vitro and In Vivo
Zhang Yong-Tai† , Han Meng-Qing† , Shen Li-Na, Zhao Ji-Hui, and Feng Nian-Ping∗
Department of Pharmaceutical Sciences, School of Pharmacy, Shanghai University of Traditional Chinese Medicine,
Pudong New District, Shanghai 201203, P. R. China

In this study, solid lipid nanoparticles were formulated for transdermal delivery of aconitine to improve its safety and
permeability. Aconitine-loaded solid lipid nanoparticles were formulated as an oil-in-water microemulsion. Drug encapsu-
lation efficiencies for these formulations were higher than 85%, and correlated positively with levels of surfactant and oil
matrix. The size of the solid lipid nanoparticles was increased with an increase of the oil matrix, and reduction of the
surfactant levels. Compared with an ethanol tincture, all the tested solid lipid nanoparticle formulations achieved improved
transdermal fluxes and drug deposition in skin in vitro. Real-time monitoring of drug distribution in rat dermis using in vivo
microdialysis showed that aconitine concentration was markedly higher following application of solid lipid nanoparticles,
compared to tincture, throughout Delivered
the experimental period. ATechnology
by Publishing regional comparison of rat skin found that application of
to: xing liwei
IP: 166.111.120.71
solid lipid nanoparticles to the scapular On:in Tue,
region resulted 20AUC
higher Jan0−t2015
and 16:48:29
Cmax , compared to those achieved with
application to the abdomen or chest (p Copyright: American
< 0
05). In contrast, Scientific Publishers
the application to the chest resulted in the lowest AUC0−t
and Cmax . Together with findings of a structural study of the skin, these results indicated that the drug accumulated more
readily in thicker skin regions, and to a lesser extent in well-perfused skin, because of drug transfer to capillaries. The
superior transdermal permeability of aconitine-loaded solid lipid nanoparticles contributed to stronger anti-inflammatory
and analgesic effects on mouse in vivo models of pain than the tincture (p < 0
05). In vitro and in vivo studies indicated
that smaller particle sizes of solid lipid nanoparticles enhanced the transdermal permeability of aconitine, which can
promote drug efficacy, reduce administration time, and improve medication safety.

KEYWORDS: Encapsulation Efficiency, Microdialysis, Permeability, Topical Delivery, Transdermal.

INTRODUCTION conventional formulations, such as topical tinctures and

Aconitum carmichaelii Debeaux, known as Chuanwu in
China, is a traditional herb with a long history of com-
mon use in traditional Chinese medicine. It is usually used
to treat intractable pain and has been officially listed in
the Pharmacopoeia of the People’s Republic of China.1
2
The major active ingredient isolated from this herb is
aconitine, which has strong analgesic effects, and does not
cause addiction.3 However, the therapeutic range of aconi-
tine is narrow and oral administration has a poor safety
profile.4 Previous studies have investigated the efficacy of
∗
Author to whom correspondence should be addressed.
Email: npfeng@hotmail.com
†
These two authors contributed equally to this work.
Received: 23 August 2013
Revised/Accepted: 4 January 2014
poultices containing aconitine, in the treatment of vari-
ous types of pain including those induced by arthritis and
cancer.5 Although transdermal administration improves the
clinical safety and effectiveness of aconitine, these advan-
tages are reduced in conventional formulations that require
frequent dosing due to poor transdermal permeability.6
Solid lipid nanoparticles (SLN) provide a novel nanocar-
rier for drug delivery. Drug loaded in SLN can be
released in a sustained manner, effectively improving its
bioavailability.7 Over the past decade, many reports have
focused on the advantages of SLN formulated for transder-
mal delivery. The SLN can greatly enhance drug transder-
mal permeability, and effectively increase sustained drug
release from the epidermis into deep skin tissue, and
onwards into the systemic circulation.8 Previous findings
showed that if drug was efficiently entrapped in SLN, they

J. Biomed. Nanotechnol. 2015, Vol. 11, No. 2 1550-7033/2015/11/351/011 doi:10.1166/jbn.2015.1902 351

Solid Lipid Nanoparticles Formulated for Transdermal Aconitine Administration and Evaluated In Vitro and In Vivo
could accumulate in the epidermis and form a drug reser-
voir, resulting in superior sustained drug release.9
10 SLN
produced more effective sustained release than nanostruc-
tured lipid carriers (NLC).11 This enhanced percutaneous
drug absorption from SLN may be due to the nanopar-
ticles tendency to form a dense lipid film adhered to
the skin surface. This promotes hydration of the stratum
corneum, facilitating its penetration by SLN. The perme-
ability of SLN can be influenced by nanoparticle size dis-
tribution and crystallinity. SLN with small particle size
and high crystallinity can form the lipid film more effec-
tively, enhancing adhesion on the skin surface and increas-
ing drug permeability.12 Lipophilic drugs entrapped within
SLN can show sustained release after topical administra-
tion, producing a continuous supply of drug to the dermis
and markedly enhancing drug transdermal permeability.13
SLN systems may provide a viable approach to control-
ling dermal drug penetration, which is particularly valu-
able for drugs with narrow therapeutic ranges.14 SLN
were successfully used for transdermal administration of
podophyllotoxin and triptolide, demonstrating their suit-
ability for this application.15
16 These features indicated
that SLN represented a promising transdermal aconitine
carrier.
The present study aimed to improve aconitine safety and
permeability by establishing a novel transdermal delivery
system using SLN as the vehicle,Delivered
prepared using the hot Technology to: xing liwei
by Publishing
microemulsion method.17 In vitroIP:transdermal permeation
166.111.120.71 On: Tue,A20
studies were carried out to investigateCopyright:
the effect American
of SLN Scientific Publishers 17
composition on permeability, compared with a tincture.
In vivo dermal microdialysis was also used to provide a
more realistic measure of aconitine release from SLN into
the dermis in a range of skin regions.
MATERIALS AND METHODS
Materials
Diethylene glycol single ethyl ether (Transcotol P) and
®
glyceryl behenate (Compritol 888 ATO) were pur-
chased from Gattefossé (Lyon, France). Polyethylene gly-
col (PEG)-35 castor oil (Cremophor® EL) was obtained
from BASF (Ludwigshafen, Germany). Ethyl oleate was
purchased from the Shanghai Yunhong Chemical Prepa-
ration Auxiliary Technology Co., Ltd. (Shanghai, China).
Aconitine (isolated from aconite), with a purity of not
less than 98.0%, was supplied by Ze-lang BioScience
(Nanjing, China). Votalin (diclofenac diethylamine emul-
gel, drug content 1% [w/w]) was purchased from Novar-
tis AG (Basel, Switzerland). All other chemicals were
obtained from the Sinopharm Chemical Reagent Co., Ltd.
(Shanghai, China) and were of HPLC or analytical grade.
Animals
Male Sprague-Dawley rats of clean grade, weighing 300 ±
20 g with about 2 months of age, and Konmin mice weigh-
ing 20 ± 2 g, were used. The animal study was approved
352
Yong-Tai et al.
by the Animal Ethical Committee, Shanghai University of
Traditional Chinese Medicine. Animals were kept in an
agreeable environment with free access to rodent diet and
water, and were acclimatized for at least 1 week before
the start of the study.
High Performance Liquid Chromatography
(HPLC) Assay
The concentrations of aconitine in samples were deter-
mined by HPLC using a LC-2010A HT Liquid Chro-
matograph system (Shimadzu Corporation, Kyoto, Japan).
A diamonsil C18 reverse phase column was used
(5 m, 4.6 mm inner diameter × 25 cm; Welch Mate-
rials, Inc., Shanghai, China) with a mobile phase was
of methanol:water:trichloromethane:triethylamine (70:30:
2:0.1, v/v) and a flow rate of 1 mL/min. The column tem-
perature was constant at 35  C and the detection wave-
length was 235 nm. The HPLC method was validated for
accuracy, precision, limit of detection and limit of quantita-
tion for aconitine. Samples from in vitro experiments were
filtered through a nylon 0.45 m pore disposable syringe
filter (diameter: 13 mm [Welch Materials, Inc., Shanghai,
China]) before automatic injection into the HPLC. Sam-
ples from in vivo microdialysis were directly assayed in a
timely manner without any handling.
Preparation of SLNs and Tincture
Jan 2015 16:48:29
microemulsion precursor method was employed for the
preparation of SLNs. A mixture was prepared by melting
oil (Compritol® 888 ATO, the core matrix of the SLNs),
surfactant (Cremophor® EL) and co-surfactant (Transco-
tol P) at 80  C. Aconitine was dissolved in this mixture
prior to adding water dropwise at 80  C under mag-
netic stirring (magnetic stirrer, IKA Works GmbH & Co.,
Staufen, Germany) at 300 rpm until a uniform microemul-
sion was formed. The microemulsion (SLN suspension)
was refrigerated quickly at − 20  C for 2 h and then stored
at 4  C. The amount of aconitine loaded in all the SLN
suspensions was 0.25 mg/mL (0.25‰ w/v), as shown in
Table I.
A tincture was prepared with the same aconitine con-
centration as the SLNs (0.25‰ w/v) in 70% ethanol
water (v/v).
Characterization of SLNs
The mean particle size of the prepared SLNs was mea-
sured using dynamic light scattering (DLS). The SLN sus-
pensions were diluted with 8 times (v/v) with purified
water before DLS measurements. A computerized Zeta-
sizer Nano ZS90 (Malvern Instruments Ltd., Malvern, UK)
was employed to detect particle size and zeta potential of
the SLNs. All measurements were performed in triplicate.
The appearance of the SLN formulation was also exam-
ined by transmission electron microscopy (TEM, Philips
Tecnai 12; Philips, Amsterdam, The Netherlands). TEM
J. Biomed. Nanotechnol. 11, 351–361, 2015
Yong-Tai et al. Solid Lipid Nanoparticles Formulated for Transdermal Aconitine Administration and Evaluated In Vitro and In Vivo

Table I. Compositions and drug loading of solid lipid nanoparticle formulations.

Formulation Km S-Cos/oil matrix (w/w) S (%, w/v) Cos (%, w/v) Oil matrix (%, w/v) Aconitine (‰ w/v) Water (%, w/v) EE (%)

SLN1 1:1 9:1 18
00 18
00 4
00 0.25 60.00 89.90 ± 5.30
SLN2 1:1 8:2 16
00 16
00 8
00 0.25 60.00 89.52 ± 3.44
SLN3 1:1 7:3 14
00 14
00 12
00 0.25 60.00 88.79 ± 6.48
SLN4 1:2 9:1 12
00 24
00 4
00 0.25 60.00 88.64 ± 6.00
SLN5 1:2 8:2 10
67 21
33 8
00 0.25 60.00 87.92 ± 5.70
SLN6 1:3 9:1 9
00 27
00 4
00 0.25 60.00 87.87 ± 4.02
SLN7 1:3 8:2 8
00 24
00 8
00 0.25 60.00 87.81 ± 7.36

Notes: Compared with each other for EE, p > 0
05. (For EE detection, n = 3); Abbreviations: SLN, solid lipid nanoparticles; Km, ratio of surfactant to cosurfactant
(w/w); S, surfactant (Cremophor® EL); Cos, cosurfactant (Transcotol P); S-Cos, total content of surfactant and cosurfactant; oil matrix (Compritol® 888 ATO);
EE, encapsulation efficiency.

samples were prepared for negative staining by placing
copper nets carrying formvar-supporting film (200 mesh,
Zhong Jing Ke Yi Technology Inc., Beijing, China) onto a
stencil plate and dropping SLNs gently onto the film. The
film was allowed to dry for about 20 min before a drop
of 2% uranyl acetate was added and allowed to dry for
10 min prior to imaging the film using TEM.
Entrapment Efficacy of SLNs
Centrifugal ultrafiltration was used to investigate entrap-
ment efficacy (EE). The aconitine-loaded SLN suspension
was sucked up precisely with a pipettor (Eppendorf AG,
Hamburg, Germany), transferred into a centrifugal ultra-
filtration tube with a molecular-weight cutoff
Delivered (MWCO) Technology to: xing liwei
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of 10 kDa (Pall Corporation, New York, USA), and cen-
IP: 166.111.120.71
On: Tue,
trifuged at 12 000 rpm (× 11913 g)Copyright:
for 20 minAmerican
in a Scientific
Sigma 2-16K centrifuge (Sigma Laborzentrifugen GmbH,
Osterode am Harz, Germany). It was demonstrated no
free aconitine crystals dispersed in the prepared SLN sus-
pensions by using scanning calorimetry and polarization
microscope in our previous studies. The concentration of
aconitine in the ultrafiltrate was determined directly by
HPLC, as described above. The EE was calculated using
the following formula (Eq. (1)):
EE% = Wt − Wf /Wt × 100 (1) for 10 min. The supernatants were filtered through a
where Wt was the amount of aconitine loaded in the SLN
suspension and Wf was the amount of aconitine in the
ultrafiltrate.17
In Vitro Skin Permeation Studies
A rat was humanely sacrificed, its abdominal fur was
shaved off with a razor, and the skin was excised care-
fully to keep undamaged, and washed with normal saline.
The permeation of aconitine delivered by SLN or alcohol
tincture was compared in vitro using a Franz diffusion cell
(Fulansi Electronic Science and Trade Co., Ltd., Tianjin,
China) fitted with excised rat skin. Each donor compart-
ment had a diffusion area of 1.77 cm2 and 2 mL of SLN
suspension or tincture was added to each compartment
under non-occlusive conditions. Each receptor compart-
ment had a volume of 16 mL and was filled with freshly
prepared 20% (v/v) polyethylene glycol 400 (PEG 400)
aqueous solution magnetically stirred (300 rpm) and main-
tained at 32 ± 0.5  C to provide sink conditions. Each
experiment was performed in triplicate. At predetermined
time points, 1 mL samples were removed from each recep-
tor compartment and replaced with an equal volume of
receptor fluid equilibrated to 32 ± 0.5  C. The concentra-
tion of aconitine in the obtained samples was determined
using HPLC, as described above. The permeation profile
for aconitine was obtained by plotting the mean cumulative
permeation per cm2 of rat skin against time. Linear regres-
sion analysis was performed to determine the transdermal
flux of aconitine from SLN preparations, as compared to
tincture.
Determination
of Skin Aconitine Deposition
20 Jan 2015 16:48:29
At the endPublishers
of the in vitro skin permeation studies (24 h),
the skins were removed from the diffusion cell, and
the residual aconitine preparations on their surfaces were
wiped off carefully with cotton wool balls, then washed
3 times with 3 mL fresh receptor fluid per time, which
assured that the adherent drug on skin surface was com-
pletely removed. The skins were cut into small pieces and
homogenized in manual glass homogenizers with 1 mL
fresh receptor fluid before centrifuging at 10,000 rpm
0.45-m filter membrane, and assayed for aconitine using
HPLC. The skin aconitine deposition was shown as: skin
deposition = Wa /Sd , where Wa means total amount of
aconitine deposited in per rat skin, and Sd is the effective
diffusion area (1.77 cm2 ).
Stability Study
SLN stability was determined by investigating size distri-
bution and EE after being stored for 0 day and 30 days
at 4  C. The change rates of the particle size or EE (size
change rate, SCR; EE change rate, ECR) were calculated
using Eq. (2):
S − S0  E − E0 
SCR = 30 × 100 ECR = 30 × 100 (2)
S 0 E0
where S0 or E0 means particle size or EE determined at
the day SLNs were prepared (0 days), and S30 or E30 were
the corresponding values after storage for 30 days.

J. Biomed. Nanotechnol. 11, 351–361, 2015 353

Solid Lipid Nanoparticles Formulated for Transdermal Aconitine Administration and Evaluated In Vitro and In Vivo Yong-Tai et al.

Microdialysis System STD solutions were used as perfusates at a flow rate of

The microdialysis system consisted of a WZ-50C6 micro
infusion pump (Smiths Medical, Norwell, MA) with a
20 mL plastic syringe and a linear microdialysis probe.
Spectra/Por® microdialysis hollow fibers (Spectrum Labo-
ratories, Inc., Chicago, USA) were prepared using regen-
erated cellulose (200 m inner diameter, 280 m outer
diameter, 13 kDa MWCO). The fibers were glued to quartz
capillary tubing (Welch Materials, Inc., Shanghai, China)
with cyanoacrylate adhesive (Mxbon® Super Glue; Cartell
Chemical Co. Ltd., Chia-Yi Hsien, Taiwan) in order to cre-
ate linear microdialysis probes, which were connected to
the microinjection pump using polyethylene tubing. In all
experiments, the length of the membrane was 20 mm,
and the perfusate flow rate was 3.33 L/min. Cannulae
were used as insertion guides, and vials were used to col-
lect the dialysate samples. Standard solutions of aconitine
(STD) were prepared by dissolving pure aconitine in 20%
PEG 400.
Recovery Validation In Vitro and
Correction In Vivo
In vitro recovery was estimated prior to microdialysis
studies to ensure that the probes used would provide
reproducible and efficient sampling. A linear microdialy-
sis probe was placed in a 50 mL beaker containing dif-
ferent concentrations of aconitineDelivered by (Table
in sequence Publishing
II), Technology to: xing liwei
and the dialysis membrane portion IP:of
166.111.120.71 On: Tue,istration
the probe was com-
Copyright: American
Scientific
pletely immersed in the solution at room temperature.
The probe was perfused with 20% PEG 400 at a flow
rate of 3.33 L/min. After an equilibration period of
30 min, the dialysate was collected into a small vial for
30 min and analyzed using HPLC to determine the aconi-
tine concentration. Relative recovery (RR) was calculated
as the slope of the linear regression of drug concentration
in the dialysate (Cd ) as a function of drug concentration
in the medium (Cm ; Eq. (3)).
RR = Cd /Cm × 100 (3)
For the retrodialysis studies, the probe was perfused
with aconitine STD solutions (Table II). A linear probe
was placed in a 50 mL beaker with the membrane por-
tion completely immersed in 20% PEG 400 at 25  C.
3.33 L/min. After equilibration for 30 min, a dialysate
sample was collected into a vial for another 30 min. The
diffusive loss of aconitine from perfusates was determined,
and RR (also known as delivery rate) was calculated using
Eq. (4), where Cp was the drug concentration in the per-
fusate.
RR = Cp − Cd /Cp × 100 (4)
In vivo RR was determined using the retrodialysis-by-
drug method, which relies on the assumption that net drug
transport from the perfusate into the surrounding tissues
through the microdialysis membrane is equal to the net
drug transport from the tissues into the perfusate. A rat
was anesthetized with urethane aqueous solution (1.3 g/kg,
intraperitoneal), and anesthesia was maintained throughout
the experiment. The ambient temperature was kept con-
stant at 25  C. A linear microdialysis probe was inserted
into the dermis of the rat’s abdominal skin. After perfusing
with 20% PEG 400 for 1 h, STD aconitine was used as
the perfusate. HPLC assays were carried out to determine
the loss of aconitine from the STD solutions, and RR was
calculated using Eq. (4).
In Vivo Microdialysis Studies
A rat was anesthetized with urethane aqueous solution
prior to administration of aconitine, the fur at the admin-
20 Jan sites
2015 on each rat was manually shaved. The
16:48:29
administration
sites (abdomen, chest, and scapularis) were
Publishers
located with the bellybutton, the intermediate point of two
front paws and the shoulder blade as the centers, respec-
tively. A microdialysis probe was implanted in the der-
mis, with the active dialysis window placed immediately
below the site of topical drug administration. The probe
was continuously perfused with 20% PEG 400. The skin
was allowed to equilibrate for 1 h before a blank sam-
ple was taken from the microdialysis probe. At 1.5 h after
the start of perfusion, the drug was applied. A flat cylin-
drical plastic cover about 1 cm high, 1.5 cm in diame-
ter, and with an edge width of 2 mm was glued above
the administration site using cyanoacrylate adhesive. 1 mL
of SLN suspension or tincture was applied to the skin
within the cover. During the experiment, the administra-
tion site and the probe were kept level. Dialysate samples

Table II. Recovery of aconitine for microdialysis probe. (In vitro: n = 3; In vivo: n = 5).

Cm (Cp ) (g/mL) a
Cd (g/mL) a
RR (%) b
Cp − Cd (g/mL) b
RR (%) c
Cp − Cd (g/mL) c
RR (%)

1.00 0.34 ± 0.01 34.28 ± 4.06 0.35 ± 0.04 35.04 ± 3.83 0.33 ± 0.03 32.67 ± 2.65
5.00 1.68 ± 0.25 33.50 ± 2.37 1.72 ± 0.35 34.37 ± 4.03 1.57 ± 0.21 31.42 ± 3.18
10.00 3.23 ± 0.67 32.31 ± 3.50 3.46 ± 0.65 34.61 ± 3.12 3.10 ± 0.45 31.03 ± 3.28
25.00 8.32 ± 0.88 33.27 ± 3.33 8.43 ± 0.74 33.72 ± 2.94 8.14 ± 0.78 32.56 ± 2.02
50.00 17.04 ± 2.45 34.08 ± 4.58 17.33 ± 3.19 34.66 ± 3.00 15.79 ± 1.99 31.58 ± 1.73

Note. (a) Recovery of different aconitine standard solutions in vitro determined using Eq. (3) in text; (b) Recovery of different aconitine standard solutions
with retrodialysis-by-drug method in vitro determined by using Eq. (4) in text; (c) Recovery of different aconitine standard solutions with retrodialysis-by-drug
method in vivo determined by using Eq. (4) in text; a RR compared with b RR, p > 0
05.

354 J. Biomed. Nanotechnol. 11, 351–361, 2015

Yong-Tai et al. Solid Lipid Nanoparticles Formulated for Transdermal Aconitine Administration and Evaluated In Vitro and In Vivo
were collected into small vials, which were replaced every
30 min for a maximum of 10 h.
Detection of Skin Blood Flow
A rat was anesthetized and fur was shaved from its
abdomen, chest and scapularis regions, as described for
the in vivo microdialysis study. After being anesthetized
for 1 h, a surface probe (XP200, low-profile service probe)
for laser doppler flowmetry (OxyLab LDF™ and OxyFlo™ ,
Oxford Optronix, Oxford, UK) was closely pasted to the
skin surface with a special adhesive ring plate. The doppler
bandwidth was 780 ±10 nm, the blood flow reflection time
was 0.2 s, and recordings were made using a unipolar
lead and multi-channel physiology recorder with Chart 5.0
software for Windows (Powerlab 8/30, ADInstruments Pty
Ltd., New South Wales, Australia). Data were expressed
as the mean value for 1 h.
Regional Skin Structure
A rat was humanely sacrificed before excising skin sam-
ples of approximately 1 × 1 cm from the abdomen, chest
and scapularis regions and fixing in 4% paraformaldehyde
solution (w/v). After 24 h, the skins were embedded in
paraffin and sliced transversely. The sections were dehy-
drated stepwise with alcohol, and stained with hematoxylin
and eosin (HE). The sections were observed using an opti-
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Copyright: American Scientific Publishers
Anti-Inflammatory and Analgesic Effects
Four frequently used mouse models of inflammation
and pain were employed to evaluate the pharmacolog-
ical effects of aconitine-loaded SLN, compared with
the tincture. These were the acetic acid, formaldehyde
or dimethylbenzene induced inflammatory pain models
(respectively abbreviated as AAIPM, FIPM, and DIPM),
and the hot water-induced pain model (HWPM). Ani-
mals were administered 20 L of 0.6% acetic acid (v/v)
by intraperitoneal injection (AAIPM); 2.5% formaldehyde
(w/v) aqueous solution by subcutaneous injection at the
back of the right rear paws (FIPM); or pure dimethyl-
benzene, daubed on the inside and outside surfaces of
the right auricles (DIPM). For the HWPM, half of the
mouse’s tail was immersed into hot water at 50  C.
Except for the DIPM, 1 × 1 cm of fur was shaved from
the dorsal region. For every pharmacological experiment,
70 mice (50% male, 50% female) were divided randomly
into 7 groups as follows: normal saline group (NSG,
with 20 L normal saline application to per mouse);
positive medicine control group (PCG, with 20 mg of
Votalin [Diclofenac Diethylamine Emulgel] application to
per mouse); blank based group (BBG, with 20 L SLN
suspension without aconitine application to per mouse);
aconitine-loaded tincture group (TTG, with 20 L appli-
cation to per mouse); and 3 SLN groups with low, medium
J. Biomed. Nanotechnol. 11, 351–361, 2015
or high aconitine-loaded aconitine SLN suspension vol-
umes application to per mouse (SLN-LDG, with 10 L;
SLN-MDG, with 15 L; and SLN-HDG, with 20 L). The
test substances were applied evenly on the dorsal region.
After 2 h topical administration, the inflammation and
pain models were established and relevant behaviors were
immediately recorded. For AAIPM, body twisting within
15 min was recorded. The latency before beginning to lick
the affected paw and the number of licks within 5 min
(I phase) as well as repeat licking within 1 h (II phase)
were recorded for the FIPM. The weight gain of the left
(control) auricle as compared to the right, after removal
from the mice and shaving, was used to assess the DIPM
and the time required for mice to lift their tails out of the
water bath was recorded for the HWPM, as a measure of
pain threshold.
Statistical Analysis
The results are expressed as mean ± standard deviation.
Statistical data were analyzed by one-way analysis of vari-
ance (ANOVA), and comparisons were made with least-
significant difference (LSD) using SPSS software (v 13.0;
SPSS, Inc., Chicago, USA). P -values less than 5% were
considered to be significant. The cutaneous pharmacoki-
netic parameters of aconitine were calculated using Win-
NonLin (v 5.2; Pharsight Corporation, Sunnyvale, USA)
and noncompartmental
to: xing liweianalysis.
2015 16:48:29
RESULTS AND DISCUSSION
SLN Preparation and Characterization
To investigate the appropriate concentration range of com-
ponents for microemulsion formation, the weight ratios
of surfactant and co-surfactant (Km) were set at 1:1, 1:2,
1:3, and the weight ratios of oil in a mixture of surfactant
and co-surfactant were set at 1:9, 2:8, 3:7, 4:6, 5:5, 6:4,
7:3, 8:2, and 9:1. After equilibration, the mixtures were
visually assessed to determine whether they had formed
microemulsions or not. Seven formulations that readily
formed uniform microemulsions were then selected and
used to form SLNs (SLN1 to SLN7). The components of
these SLNs are listed in Table I.
Previous reports indicated that use of Compritol® 888
ATO as the SLN lipid core could be conducive to form-
ing stable dispersions with smaller particle sizes, and this
was borne out by the present study, where particle size
distributions ranged from 34.78 ± 3.75 nm to 131.90 ±
24.65 nm.18–20 The mean particle sizes were increased
by reducing the Km in the presence of a constant ratio
of S-Cos (surfactant [Cremophor® EL] and cosurfactant
[Transcotol P]) versus oil matrix (Compritol® 888 ATO).
This indicated that SLN size increased as surfactant con-
centration decreased. Previous studies have reported a sim-
ilar relationship between mean particle size and surfactant
concentration.21
22 At a constant Km value, the particle
sizes decreased with an increasing level of oil matrix
355
Solid Lipid Nanoparticles Formulated for Transdermal Aconitine Administration and Evaluated In Vitro and In Vivo Yong-Tai et al.

content of surfactant (Table I). The augmented entrapment

of aconitine with increased surfactant concentration sug-
gested that aconitine might be embedded in the surfac-
tant layer, rather than incorporated into the lipid matrix.22
As a highly lipophilic drug, aconitine can be efficiently
solubilized in the oil phase (composed of oil, surfactant
and cosurfactant) of the microemulsion system. In addi-
tion, the contents of aconitine were only 0.25‰ (w/v) in
every SLN formulations, so that all SLNs achieved high
EE, with no significant differences between the 7 formu-
lations (p > 005).
Figure 1. Mean particle sizes and zeta potentials of the
aconitine-loaded solid lipid nanoparticle formulations (S-Cos:
In Vitro Permeation Studies
oil matrix [w/w] = 1:9 [
], 2:8 [
], 3:7 [•]). Abbreviations: SLN,An enhanced transdermal release of aconitine was
solid lipid nanoparticles; Km, ratio of surfactant to cosurfac- achieved by using SLNs as the vehicle for topical adminis-
tant (w/w); S, surfactant (Cremophor® EL); Cos, cosurfactant tration, compared with the tincture. Transdermal fluxes of
(Transcotol P); S-Cos, total content of surfactant and cosur-
aconitine from the SLN formulations ranged from 1.15 ±
factant; oil matrix (Compritol® 888 ATO). (n = 3).
0.20 g/cm2 /h to 11.80 ± 0.76 g/cm2 /h, and were all
(Fig. 1). A higher level of oil matrix may decrease the vis- higher than the tincture (Figs. 3 and 4). Although the high
cosity of the melted droplets, increasing the likelihood that ethanol concentration in tinctures can promote transdermal
23
they will disperse and result in smaller particle sizes. The delivery, the permeability provided is limited and erratic,
zeta potentials of the SLNs were distributed from − 3.42 ± due to evaporation.
0.45 to − 26.97 ±2.31 mv. Zeta potentials were lower with At a constant Km value, aconitine transdermal flux
increased use of oil matrix at a constant Km value. They increased with an increased level of oil matrix (Fig. 4),
were also lower at reduced Km value with a constant ratio while the SLN particle sizes were reduced with increased
of S-Cos to oil matrix, indicating that the level of surfac- oil matrix. Reduced surfactant level led to increased trans-
Delivered by Publishing
tant could affect the zeta potential. These zeta potential dermal
Technology flux,
to:and decreased
xing liwei particle size, at a constant ratio
IP: 166.111.120.71
results suggested that the SLNs had a negatively charged On: Tue,
of 20 Jan
S-Cos 2015
to oil 16:48:29
matrix (w/w). A small particle size can per-
Copyright: American Scientific
mit close Publishers
contact of SLNs with the stratum corneum and
surface, which may be provided by the surfactant molecu-
®
lar chain of Cremophor EL surrounding the edge of the further improve drug permeation into skin. After applica-
nanoparticle. 22–24
Thereby, larger amounts of Cremophor ® tion onto the skin, the small SLNs can form a close lipid
EL led to increased negative charge on the SLN surface, film on the stratum corneum, and drug molecules can be
resulting in higher particle zeta potential. expulsed from SLNs, which can form a higher concen-
SLN7 was found to be spherical in shape when imaged tration gradient towards the skin, further enhanced more
25
using TEM (Fig. 2). The nanoparticles appeared sepa- effectively the transdermal delivery of drug, compared
rately from each other, and showed no change in shape
before and after 8-fold dilution with purified water.

SLN Entrapment Efficacies

The EE of SLN1 to SLN7 varied between 87.81 ± 7.36%
and 89.90 ± 5.30%, and was raised slightly with increasing

(a) (b)

Figure 3. In vitro rat skin permeation profiles of aconitine

Figure 2. Transmission electron microscopy of aconitine-
loaded solid lipid nanoparticles (SLN7). ((a) 10,000× magnifi-
cation; (b) 100,000× magnification).
from the aconitine-loaded solid lipid nanoparticle formula-
tions and a tincture (S-Cos:Oil matrix [w/w] = 1:9 [
], 2:8 [
],
3:7 [•]). Abbreviations: SLN, solid lipid nanoparticles; TT, tinc-
ture; Km, ratio of surfactant to cosurfactant (w/w); S, sur-
factant (Cremophor® EL); Cos, cosurfactant (Transcotol P);
S-Cos, total content of surfactant and cosurfactant; oil matrix
(Compritol® 888 ATO). (n = 3).

356 J. Biomed. Nanotechnol. 11, 351–361, 2015

Yong-Tai et al. Solid Lipid Nanoparticles Formulated for Transdermal Aconitine Administration and Evaluated In Vitro and In Vivo

Figure 5. Perfusate concentration of aconitine (Cp ) as deter-

Figure 4. In vitro transdermal fluxes and skin deposition of mined by the retrodialysis-by-drug method in vitro and in vivo
aconitine from solid lipid nanoparticle formulations or tinc- (Cp : aconitine concentration in perfusates, Cd : aconitine con-
ture through excised rat skin (S-Cos:oil matrix [w/w] = 1:9 [
], centration in dialysate sample).
2:8 [
], 3:7 [•]). Abbreviations: SLN, solid lipid nanoparticles;
TT, tincture; Km, ratio of surfactant to cosurfactant (w/w); constant Km value and a constant ratio of S-Cos to oil
S, surfactant (Cremophor® EL); Cos, cosurfactant (Transco-
tol P); S-Cos, total content of surfactant and cosurfactant; oil
matrix, the transdermal flux and skin deposition of aconi-
matrix (Compritol® 888 ATO). Compared with the tincture, † p < tine from SLNs were increased as the particle zeta poten-
0
05 for skin deposition, # p < 0
05 for transdermal flux. (n = 3).
tial decreased. The repulsion between negatively charged
cell membranes and nanoparticles was weakened when the
zeta potential value was reduced, promoting SLN perme-
with the SLNs possess larger particle sizes.26–28 In addi- ability and drug deposition in the epidermis.37
38
tion, they may enhance transdermal drug delivery via hair Results for the in vitro permeation studies showed that
follicles.29
30 transdermal flux followed the same trend as skin depo-
SLNs have been reported to increase epidermal drug sition of aconitine from the SLN formulations. Previous
deposition.8
31 In this study, the skin deposition of aconi- studies reported that SLN strongly targeted the epidermis,
tine from SLNs ranged from 18.19 ± 234 g/cm2 (SLN1) leading to ato:high drug concentration gradient between the
Delivered by Publishing Technology xing liwei
to 66.62 ± 5.11 g/cm2 (SLN7, IP: Fig.166.111.120.71
4). Aconitine depo-
On: Tue, 20 Jan 2015
epidermis 16:48:29
and deep skin layers, promoting drug perme-
sition by all the SLNs were 1.5- to Copyright:
5.6-fold thatAmerican
of the Scientific Publishers
ation through skin and contributing to a high transdermal
tincture. Drug was delivered by tincture with poor per- flux. 8
13
meability into the skin directly, and immediately trans- The formulation SLN7 showed the highest transdermal
ferred to the microcirculation under the epidermis. This flux and skin deposition of aconitine and was selected for
may result in the lowest drug deposition from the tinc- further studies.
ture. In contrast, lipid carriers such as SLN can attach to
the skin surface and allow lipid exchange with the out-
Stability of SLN7
ermost layers of the stratum corneum, thus providing a
After being stored for 30 days at 4  C, SLN7 particle
higher drug concentration in the skin.32 In addition, the
size distribution was increased and EE was reduced, but
lipid matrix of the SLN oil core, formed by solid crystal-
neither change was statistically significant (p > 005, data
lization, may transition to a more ordered structure super-
not shown). The SCR and ECR were 6.81 ± 0.63% and
saturated with drug after being applied to the skin surface,
3.95 ± 0.19%, respectively. Using Compritol® 888 ATO as
leading to drug expulsion and accumulation as a drug
reservoir in the upper skin layers and prolonging skin res-
idence time.33
34 These factors may account for the higher
skin deposition of aconitine from SLN, as compared to
tincture.
The amounts of aconitine deposited in skin were
improved with an increased level of oil matrix at a constant
Km value, while they were decreased with increased sur-
factant at a constant ratio of S-Cos to oil matrix (Fig. 4).
Increased oil matrix and decreased surfactant produced
smaller particles, leading to stronger skin hydration and
transdermal permeability of SLNs, which promoted lipid
exchange with epidermal lipids and increased drug skin
Figure 6. Ten-hour time course of aconitine concentration
deposition.35
36 after application of drug-containing solid lipid nanoparticles
The present studies discovered that SLN surface charge (SLN7) or tincture to the abdominal skin of Sprague-Dawley
also affected skin permeability (Figs. 2 and 4). At a rats in vivo. (n = 5).

J. Biomed. Nanotechnol. 11, 351–361, 2015 357

Solid Lipid Nanoparticles Formulated for Transdermal Aconitine Administration and Evaluated In Vitro and In Vivo Yong-Tai et al.

Table III. Pharmacokinetic parameters of aconitine loaded the perfusate (in vitro: Cp − Cd  = 0345Cp − 0003, r 2 =
in SLN and the tincture via microdialysis after applied on 0999; in vivo: Cp − Cd  = 0317 Cp + 0015, r 2 = 0999;
abdomen of rat skin in vivo. (n = 5). Fig. 5). This correlation implied that retrodialysis recovery
Parameter Unit SLN Tincture was independent of perfusate concentration both in vitro
and in vivo, supporting the feasibility of detecting the true
Tmax min 300.00 ± 00.00 60.00 ± 00.00†
Cmax g/mL 1.21 ± 0.11 0.34 ± 0.14†
unbound extracellular level of aconitine using the micro-
AUC0−t min/g/mL 346.31 ± 25.21 152.94 ± 28.95 † dialysis technique.
MRT0−t min 314.92 ± 16.35 313.71 ± 26.23 The aconitine concentrations in rat abdominal skin
after topical application of SLN or tincture are shown
Note: Compared with the pharmacokinetic parameters from the SLN, † p <
0
05; Abbreviations: SLN, solid lipid nanoparticle; Tmax , time to peak concen- in Figure 6 and Table III. Skin aconitine levels were
tration; Cmax , peak concentration; AUC, area under the concentration-time higher with SLN than with tincture throughout the experi-
curve; MRT, mean residence time.
ments. The peak concentration (Cmax ) and AUC0−t for SLN
were 3.56-fold and 2.26-fold higher than the correspond-
ing measures using tincture, respectively. These findings
demonstrated that use of SLN increased aconitine levels in
the dermis, indicating improved transdermal permeability
compared to tincture.
The drug concentration profile in skin exposed to SLN
rose initially and then decreased. The Cmax occurred
300 min after SLN application. This reflected accumula-
tion of SLN-borne drug molecules in the epidermis prior
to their release and transfer to capillaries. For the ini-
tial 300 min, accumulation of SLN in the lipid epider-
mis meant that more aconitine was released than was
Figure 7. Ten-hour time course of aconitine concentration transferred to the skin microcirculation, and the Cmax was
after application of drug-containing solid lipid nanoparticles reached. Subsequent epidermal saturation with SLN led
(SLN7) to the abdomen, scapularis, and chest regions of
Delivered by Publishing Technology to the decrease
to: xingin liwei
drug concentration as it was trans-
Sprague-Dawley rats in vivo. (n = 5).
IP: 166.111.120.71 On: Tue,ferred 20 Jan 2015
to the 16:48:29
microcirculation faster than it was delivered.
Copyright: American Scientific Publishers
In contrast, the drug concentration after tincture applica-
the SLN lipid core promoted particle size stability, and the tion was relatively moderate at Tmax (time to peak con-
high lipophilicity of aconitine may have contributed to the centration, 60 min). The aconitine in tincture displayed
stability of its encapsulation.17
18 diffusion behavior during percutaneous absorption, con-
forming to Fick’s first law and producing a more simple
In Vivo Dermal Microdialysis Studies skin drug concentration profile over time.
Aconitine absorption after topical application of SLN7 or The regional comparisons of skin aconitine concentra-
tincture was investigated in vivo using skin microdialy- tions over time are presented in Figure 7 and Table IV.
sis. There was no significant difference between RR from The concentration of aconitine from SLN applied to the
dialysis and from retrodialysis (drug delivery) in vitro abdomen and chest initially increased more rapidly than
(p > 005), satisfying the prerequisite for retrodialysis use in the scapular region. However, the scapular drug con-
in the in vivo RR studies.39 The in vitro and in vivo centration continued to rise and achieved the highest Cmax
probe recoveries were consistently above 30% (Table II). of the compared skin regions within the test period at
In the in vitro and in vivo recovery validation studies 510 min, whilst Cmax and AUC0−t were lowest in the chest.
using STD as perfusates, drug concentration in the per- These regional permeability differences may relate to skin
fusate showed a linear correlation with drug loss from structure.40
41 The current study found that rat epidermal

Table IV. Pharmacokinetic parameters of aconitine loaded in SLN via microdialysis after applied on different regions of rat skin
in vivo. (n = 5).

Parameter unit Abdomen Chest Scapular region

Tmax min 300.00 ± 00.00 420.00 ± 00.00† 510.00 ± 00.00†∗

Cmax g/mL 1.21 ± 0.11 0.73 ± 0.06† 1.45 ± 0.32†∗
AUC0−t min/g/mL 346.31 ± 25.21 291.91 ± 15.63 397.64 ± 25.38∗
MRT0−t min 314.92 ± 16.35 337.45 ± 28.70 405.99 ± 18.59†∗

Note: Compared with the pharmacokinetic parameters from the abdomen, † p < 0
05; compared with the pharmacokinetic parameters from the chest, ∗ p < 0
05;
Abbreviations: SLN, solid lipid nanoparticle; Tmax , time to peak concentration; Cmax , peak concentration; AUC, area under the concentration-time curve; MRT,
mean residence time.

358 J. Biomed. Nanotechnol. 11, 351–361, 2015

Yong-Tai et al. Solid Lipid Nanoparticles Formulated for Transdermal Aconitine Administration and Evaluated In Vitro and In Vivo

(a)

Figure 9. In vivo skin blood flow of various rat skin regions

over 1 h. (n = 5).

(b)

Figure 10. Writhing times of various mice groups over 15 min

after being induced by 0.6% acetic acid aqueous solution (v/v)
intraperitoneally. Abbreviations: NSG, normal saline group
(20 L per mouse); PCG, positive medicine control group
Delivered by Publishing Technology to: xing liwei
(20 mg of Votalin [Diclofenac Diethylamine Emulgel] per
IP: 166.111.120.71 On: Tue, 20 Jan 2015 16:48:29
mouse); BBG, blank based group (20 L SLN suspension
Copyright: American Scientific Publishers
without aconitine per mouse); TTG, aconitine-loaded tincture
group (aconitine content 0.25‰ [w/v], 20 L per mouse); SLN-
LDG, aconitine-loaded solid lipid nanoparticles (SLN7, aconi-
(c) tine content 0.25‰ [w/v]) groups administered with low dosage
(10 L per mouse); SLN-MDG, middle dosage (15 L per
mouse); SLN-HDG, high dosage (20 L per mouse). Compared
with NSG, † p < 0
05; compared with TTG, ∗ p < 0
05. (n = 10).

Figure 11. Pain thresholds of mice groups exposed to hot

Figure 8. Microscopy images of rat skin sections from vari-
ous regions using paraffin microtomy and hematoxylin eosin
(HE) staining. Arrows indicate the thickness of the epidermis
((a) abdomen; (b) chest; (c) scapular region; magnification
×400).
thickness was about 70 m on the abdomen, 40 m on
the chest and 110 m on the scapular region (Fig. 8). Skin
blood flow was highest in the chest region, and lowest in
the scapularis (Fig. 9). These regional data are consistent
water-induced pain (HWPM). Abbreviations: NSG, normal
saline group (20 L per mouse); PCG, positive medicine con-
trol group (20 mg of Votalin [Diclofenac Diethylamine Emulgel]
per mouse); BBG, blank based group (20 L SLN suspension
without aconitine per mouse); TTG, aconitine-loaded tincture
group (aconitine content 0.25‰ [w/v], 20 L per mouse); SLN-
LDG, aconitine-loaded solid lipid nanoparticles (SLN7, aconi-
tine content 0.25‰ [w/v]) groups administered with low dosage
(10 L per mouse); SLN-MDG, middle dosage (15 L per
mouse); SLN-HDG, high dosage (20 L per mouse). Compared
with NSG, † p < 0
05; compared with TTG, ∗ p < 0
05. (n = 10).

J. Biomed. Nanotechnol. 11, 351–361, 2015 359

Solid Lipid Nanoparticles Formulated for Transdermal Aconitine Administration and Evaluated In Vitro and In Vivo
Figure 12. Latent period (latent phase) and times of lick-
ing paws (I phase and II phase) of mice groups exposed to
formaldehyde-induced pain (FIPM). Abbreviations: NSG, nor-
mal saline group (20 L per mouse); PCG, positive medicine
control group (20 mg of Votalin [Diclofenac Diethylamine
Emulgel] per mouse); BBG, blank based group (20 L SLN
suspension without aconitine per mouse); TTG, aconitine-
loaded tincture group (aconitine content 0.25‰ [w/v], 20 L
per mouse); SLN-LDG, aconitine-loaded solid lipid nanopar-
ticles (SLN7, aconitine content 0.25‰ [w/v]) groups adminis-
tered with low dosage (10 L per mouse); SLN-MDG, middle
dosage (15 L per mouse); SLN-HDG, high dosage (20 L per
mouse). Compared with NSG, † p < 0
05; compared with TTG,
∗
p < 0
05. (n = 10).
with the idea that SLN-controlled release of aconitine lev-
els depend on the balance between drug accumulation in
Delivered by Publishing
the epidermis and its removal by microcirculation, indicat- Technology
IP: 166.111.120.71 On:
Tue, 20
ing that higher sustained levels of drug release in dermis
Copyright: American Scientific
could be achieved in regions with thicker skin and lower
blood flow. This can be observed clearly after 390 min in
Figure 7.
Figure 13. Weight increment of right auricles of various mice
groups with dimethylbenzene-induced pain (DIPM). The left
ears acted as controls. Abbreviations: NSG, normal saline
group (20 L per mouse); PCG, positive medicine control
group (20 mg of Votalin [Diclofenac Diethylamine Emulgel]
per mouse); BBG, blank based group (20 L SLN suspen-
sion without aconitine per mouse); TTG, aconitine-loaded tinc-
ture group (aconitine content 0.25‰ [w/v], 20 L per mouse);
SLN-LDG, aconitine-loaded solid lipid nanoparticles (SLN7,
aconitine content 0.25‰ [w/v]) groups administered with low
dosage (10 L per mouse); SLN-MDG, middle dosage (15 L
per mouse); SLN-HDG, high dosage (20 L per mouse). Com-
pared with NSG, † p < 0
05; compared with TTG, ∗ p < 0
05.
(n = 10).
360
Yong-Tai et al.
In Vivo Anti-Inflammatory and Analgesic Effects
In the present studies, the aconitine-loaded SLNs (SLN7)
showed the satisfactory pharmaceutical effects on the
inflammatory and physical models of pain (Figs. 10–13).42
The delivery vehicle (BBG) had no significant influence
(p > 005) on the results (Figs. 10–13). No significant
dose-dependent effects (p > 005) were present in the
experimental groups exposed to SLN. However, aconitine
administered using SLN showed more efficacy in the four
pain models tested than the same amount (0.25‰ w/v)
administered in the tincture (p < 005). This probably
reflected the improved transdermal permeability of aconi-
tine delivered by SLN.
CONCLUSION
The in vitro and in vivo study data showed that SLN pro-
vided effective topical delivery of aconitine. This novel
aconitine-loaded nanocarrier produced superior transder-
mal permeability and deposition in epidermis, as demon-
strated by the in vitro skin permeation and in vivo
microdialysis studies. SLN-delivered aconitine produced
improved anti-inflammatory and analgesic effects, com-
pared to aconitine tincture.
Acknowledgments: This work was financially sup-
ported by the National Natural Science Fund of
to: xing liwei
China
(81303234),
Jan 2015 Youth Scientific Research Fund
16:48:29
(20134q098) from Shanghai Health Bureau, and Students
Publishers
Research Fund (2012) from Shanghai Municipal Education
Committee.
Declaration of Interest
The authors report no declarations of interest.
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