389

Send Orders for Reprints to reprints@benthamscience.net

Pharmaceutical Nanotechnology, 2019, 7, 389-403

RESEARCH ARTICLE
ISSN: 2211-7385
eISSN: 2211-7393

Effect of Acyclovir Solid Lipid Nanoparticles for the Treatment of

Herpes Simplex Virus (HSV) Infection in an Animal Model of
HSV-1 Infection
BENTHAM
SCIENCE

Ritika Kondel1, Nusrat Shafiq1,*, Indu P. Kaur2, Mini P. Singh3, Avaneesh K. Pandey1,
Radha K. Ratho3 and Samir Malhotra1

1
Department of Pharmacology, PGIMER, Chandigarh, 160012, India; 2UIPS, Punjab University,
Chandigarh, 160014, India; 3Department of Virology, PGIMER, Chandigarh, 160012, India

Abstract: Background: Acyclovir use is limited by a high frequency of administra-

tion of five times a day and low bioavailability. This leads to poor patient compliance.
Objectives: To overcome the problem of frequent dosing, we used nanotechnology
platform to evaluate the proof of concept of substituting multiple daily doses of acy-
clovir with a single dose.
Methods: Acyclovir was formulated as solid lipid nanoparticles (SLN). The nanoparti-
Pharmaceutical Nanotechnology

ARTICLE HISTORY
Received: June 25, 2019
Revised: July 17, 2019
Accepted: August 16, 2019
DOI:
10.2174/2211738507666190829161737
cles were characterized for particle size, surface charge and morphology and in vitro
drug release. The pharmacokinetic and pharmacodynamic of SLN acyclovir were
compared with conventional acyclovir in a mouse model.
Results: SLN showed drug loading of 90.22% with 67.44% encapsulation efficiency.
Particle size was found to be of 131 ± 41.41 nm. In vitro drug release showed 100%
release in SIF in 7 days. AUC0-∞ (119.43 ± 28.74 µg/ml h), AUMC0-∞ (14469 ±
4261.16 µg/ml h) and MRT (120.10 ± 9.21 h) were significantly higher for ACV SLN
as compared to ACV AUC0-∞ (12.22 ± 2.47 µg/ml h), AUMC0-∞ (28.78 ± 30.16 µg/ml
h) and MRT (2.07 ± 1.77 h), respectively (p<0.05). In mouse model, a single dose of
ACV SLN was found to be equivalent to ACV administered as 400mg TID for 5 days
in respect to lesion score and time of healing.
Conclusion: The proof of concept of sustained-release acyclovir enabling
administration as a single dose was thus demonstrated.

Keywords: Acyclovir, HSV-1, pharmacodynamic, pharmacokinetic, Solid Lipid Nanoparticles (SLN),

sustained release.

1. INTRODUCTION Acyclovir was the first specific antiviral drug to

Infections due to herpes simplex virus (HSV) are
among the most common viral infections [1, 2].
*Address correspondence to this author at the Department of
Pharmacology, PGIMER, Chandigarh, 160012, India;
Tel: 0172-2755254; E-mail: nusrat_shafiq@hotmail.com
become widely used in the treatment of HSV and
herpes zoster virus (HZV) infections [3, 4]. One of
the major limitations of oral acyclovir is that it
needs to be administered 5 times a day due to its
short half-life and low oral bioavailability [5]. This
is inconvenient and can lead to poor patient com-
pliance.

2211-7393/19 $58.00+.00 © 2019 Bentham Science Publishers

390 Pharmaceutical Nanotechnology, 2019, Vol. 7, No. 5
We and others have shown that nanotechnolo-
gy-based drug delivery platforms are able to pro-
vide sustained release plasma profile for various
drugs [6-9]. Acyclovir loaded PLGA microparti-
cles have shown increased efficacy against HSV-1
in cell culture [10]. Majority platforms include
polymer-based systems. However, polymer-based
systems use organic solvent like dichloromethane
(DCM) and acetone which can cause toxicity. Sol-
id lipid nanoparticles (SLN) provide an alternative
drug delivery platform. It comprises of solid lipids
as matrix for drug delivery which has biodegrada-
ble lipids and omits the use of organic solvents
[11]. Camptothecin [12], piribedil [13] and cyclo-
sporine [14] have been encapsulated in SLN and
have shown sustained release profile. Various an-
tivirals including those for HIV have been encap-
sulated in SLN and targeted in vivo [15, 16]. Acy-
clovir SLN has been developed in various studies,
however, they have been developed for dermal and
ocular delivery which is not the desired route for
infections requiring systemic delivery [17-19].
Therefore, with this background, the current
study was planned to prepare SLN based oral sus-
tained release formulation of acyclovir, evaluate
the pharmacokinetics of the formulation and its
efficacy in an animal model of cutaneous herpes
infection.
2. MATERIALS AND METHODS
2.1. Materials
Acyclovir was purchased from Sigma Aldrich,
Inc. St Louis, MO, USA. Soy lecithin (Phospho-
lipon 90H) was received as a gift sample from
Lipoid GmbH, Germany. Compritol 888 ATO®
was a gift sample from Colorcon Asia Pacific pvt.
Ltd., India. Stearic acid and Tween 80 (poly-
sorbate 80) were purchased from Central Drug
House, Mumbai. Chemicals and solvents used
were analytical or HPLC grade. 3-(4,5-Dimethyl-
thiazol-2-yl)-2,5- diphenyl tetrazolium bromide
(MTT) was purchased from Sigma Aldrich, Inc.,
St Louis, MO, USA. Dulbecco’s modified Eagle’s
medium (DMEM), fetal bovine serum (FBS) and
penicillin-streptomycin (50U/ml) hepes buffer 1M,
non-essential amino acids, L-glutamine, phosphate
buffer saline pH 7.2 (PBS) were obtained from
Gibco-BRL, Spain. Crystal violet were purchased
from Hi-Media, India.
Kondel et al.
2.2. Preparation of Acyclovir (ACV) Solid Lipid
Nanoparticles (SLN)
Solid lipid nanoparticles of acyclovir were
prepared by microemulsion method [20]. Briefly,
the lipidic phase (8% lipid) and the aqueous phase
(polysorbate 80, soy lecithin and water) were
heated to 10°C above the lipid melt temperature.
The proportion of surfactant and the volumes of
two phases were so adjusted that a microemulsion
was formed spontaneously upon mixing the two
phases. Hot microemulsion thus formed, was
transferred into the same amount of cold water
(2°C) under constant stirring (Wise Tis HG-15
D, 10,000 rpm) for 20 minutes to obtain SLN. The
prepared SLN was used as such for further studies.
Table 1 describes the composition of acyclovir
solid lipid nanoparticles having both lipid and
aqueous phase.
Table 1. Composition of acyclovir solid lipid nano-
particles.
Aqueous Phase Lipid Phase (0.985 g) (4:1)
Water (4.75 ml) Compritol (0.788 g)
Drug (40% lipid) (0.394g) Stearic acid (0.197 g)
Tween 80 (6.64g) -
Soya lecithin (0.4%) -
2.3. Bioanalytical HPLC Method
Acyclovir was analyzed using HPLC by modi-
fication of the previously described method [21].
(Shimadzu Pump LC-20AD, Prominence liquid
chromatography) with SPD-20A prominence UV-
Vis detector. C-18 column (Hibar 250×4.6 Puro-
spher STAR, RP-18e (5µm) was used. Serial dilu-
tions within the range of 0.2-2.56 μg/ml were
made from stock solution and standard plot was
obtained. The mobile phase consisted of potassium
di-hydrogen phosphate and acetonitrile (KH2PO4:
ACN) (97:3) at pH=2.5 The mobile phase was de-
livered at a flow rate of 1.5 ml/ min and the detec-
tion of acyclovir was carried out at 254 nm. The
injection volume was 20 µl and the analysis was
performed at 30°C (sample 4°C).
Effect of Acyclovir Solid Lipid Nanoparticles for the Treatment
2.4. Solid Lipid Nanoparticles (SLN) Charac-
terization
SLN formulation was characterised for particle
size, PDI and zeta potential using Delsa Nano C,
Beckman Coulter, Inc. Double distilled water
(DDW) was used as a dispersant medium for
diluting the sample to 10-1 concentration. The sizes
of nanoparticles are expressed in nanometer scale.
The polydispersity index (PDI) is the dimension-
less number which indicates the width of the
distribution, having a value between 0 and 1. It is
self-calculated by the software. Zeta potential
measures the surface charge of the particles and it
is also measured by DelsaNano C. It is extremely
important parameter of colloidal stability and
flocculation properties of the colloidal systems.
Transmission electron microscopy (TEM) analy-
sis of the prepared SLNs was carried out to study
sphericity and aggregation. For TEM, samples
were examined by TEM (H 100, Hitachi Ltd.,
Japan). Samples were stained with phosphotun-
gstic acid (PTA, 2%) for 5 minutes and excess
PTA was removed. These were them spread on a
gold grid and examined. Nanoparticles from five
to seven different TEM fields were measured to
determine the mean particles size of nanoparticles.
Fourier Transmission Infrared Spectroscopy (FTIR)
was conducted on samples of free acyclovir, blank
SLN and acyclovir SLN to assess the lipid-drug
interaction obtained using a Perkin Elmer spectro-
meter (Perkin Elmer, Boston, MA). The FT-IR
spectrums were obtained by mixing the samples
with Potassium Bromide and pressed to obtain
pellets which were then scanned in the IR range
from 400 to 4000 cm-1, with a resolution of 8 cm-1.
The concentration of acyclovir in solid lipid
nanoparticles was determined by HPLC as pre-
viously described. The total amount of drug per
unit volume present in the formulation was
determined by suitably disrupting 0.1 ml of the
SLN dispersion in 5 ml chloroform: methanol
(2:1) volumetrically. The total drug content (TDC)
or percent drug loading was determined by using
the equation given below:
TDC = Calculated amount of drug/ml of SLN dispersion ×100
Total amount of drug added/ml of SLN dispersion
Pharmaceutical Nanotechnology, 2019, Vol. 7, No. 5 391
Calculated amount of drug is the quantity of
drug which was determined by HPLC and total
amount of drug is the quantity of the drug which is
added to prepare the SLN.
Encapsulation efficiency (EE) was determined
by analyzing the clear supernatant obtained by
centrifuging the developed SLN dispersions at
8.02 lakh×(g) for 2 h at 4°C using Beckman
Coulter Ultracentrifuge (L100K and 100 Ti rotor).
The EE was calculated as follows:
EE = TDC-Df / TDC ×100
where Df = amount of drug in the clear
supernatant fluid
The drug entrapment, drug loading and the in
vitro drug release were expressed as percentages.
Stability study of ACV SLNs was performed.
ACV SLNs kept for 1 year at 4 – 8°C. After 1
year, particle size, PDI, zeta potential, percent
drug loading and encapsulation efficiency were
measured. Effect of temperature was observed on
the ACV SLNs. As we have to perform the studies
on cell cultures, therefore, autoclaving needs to be
done. ACV SLNs were autoclaved at 121°C, 15
per square inch (psi) above atmospheric pressure
for 15 minutes. After autoclaving, particle size,
PDI, zeta potential, percent drug loading and
encapsulation efficiency were measured.
In vitro release was determined in the following
simulated fluids: simulated gastric fluid (SGF)
pH=1.2; phosphate buffer saline (PBS) pH=6.8;
simulated intestinal fluid (SIF) pH=7.8 by dialysis
bag method using dialysis membrane (molecular
weight cut-off of 12 kDa). The incubation took
place in rotating vials at 37°C. 10 µl of samples
were withdrawn at pre-set time intervals (0.25 hrs,
0.5 hrs, 1 hrs, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 8 hrs, 12
hrs, 24 hrs, 48 hrs, 60 hrs, 72 hrs, 96 hrs, 120
hrs,144 hrs, 168 hrs) and replaced by an equal
volume of fresh dissolution medium. The aliquots
were then diluted with mobile phase and filtered
through 0.45 µm filters. The samples were
analyzed by HPLC. The cumulative amount of
acyclovir release from the nanoparticles was
392 Pharmaceutical Nanotechnology, 2019, Vol. 7, No. 5
calculated using the following equation:
% Cumulative release= Wt / W0 х 100
Wt is the amount of drug released from the
nanoparticles and present in receiving phase at
time (t) and Wo is the initial amount of drug
present in the nanoparticles. The cumulative
percentage (%) of drug released was plotted
against time. The samples were analysed by HPLC
by the method described above to measure the
amount of acyclovir in the samples.
2.5. Pharmacokinetics Studies
Swiss albino male mice weighing 20-40 g were
used. The protocol was approved by the
Institutional Animal Ethics Committee (IAEC),
PGIMER, Chandigarh (Ref. No. 73/IAEC/444).
Animals were divided into two groups (n=9), ACV
and ACV SLN. They were dosed with 36 mg/kg of
acyclovir, either in a solution form of acyclovir or
as ACV SLN orally using an oral feeding canula.
Blood samples were collected at pre-determined
time points: 0, 15, 30 mins, 1, 2, 4, 8, 12 and 24
hrs after drug administration for ACV and for 0,
15, 30 mins, 1, 2, 4, 8, 12 and 24, 48, 72, 96, 120,
144 and 168 hrs after drug administration for ACV
SLN. Plasma was separated out by centrifuging
the blood samples at 4000 rpm for 10 min at 4°C.
Protein precipitation was carried out by adding
100 µl of 20% perchloric acid in 500 µl of sample,
vortexing it for 1 min and then centrifuging the
samples at 10,000 rpm for 15 min [22]. The super-
natant was collected and then analyzed by stand-
ardized HPLC method. Cmax (Peak plasma
concentration) and Tmax (time to reach peak plasma
concentration) were obtained from actual plasma
concentration versus time data. The area under the
curve (AUC) was calculated using the trapezoidal
rule, from the plasma vs. time plots. AUC0-∞, total
AUC and mean residence time (MRT) were
calculated from the individual AUCs from the
plasma-time plots.
2.6. Cell Culture and Viruses
Vero cells (African green monkey kidney cell
line) were purchased from the National Institute of
Virology, Pune, India. This cell line was grown in
DMEM supplemented with 6% FBS, 1% penicil-
lin-streptomycin, 1% Hepes buffer 1M, 1% non-
Kondel et al.
essential amino acids and 1% l-glutamine. The
maintenance medium for Vero cells was described
above with 2% FBS. Herpes Simplex type 1
(HSV-1) (KOS strain) obtained from National In-
stitute of Virology, Pune, India, was prepared in
aliquots and stored at -80°C until use. Virus titer
was determined by plaque assay in Vero cells and
expressed as plaque-forming units (pfu) per ml.
2.7. Cell Infection
The titer of the virus stocks, determined by
plaque reduction assay, was 3.3 x 106 plaques/ml.
Monolayers of Vero cells grown in 24-well tissue
culture plates, at 80% confluence were infected by
adsorption of HSV-1 (300 µL) supplemented with
2% FBS for 1 hr at 37°C. The inoculum with the
non-adsorbed virus was removed and cells were
washed with the medium. Finally, the infected
cells were treated according to the assay protocol.
2.8. MTT Assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetra-
zolium bromide (MTT) is a yellow water-soluble
tetrazolium dye that is reduced by live cells, but
not by dead ones, to a purple formazan product
that is insoluble in aqueous solutions. Monolayers
of 1x103 Vero cells were placed in 96 well plates.
Treatment was done on these plates with ACV and
ACV SLN over a concentration range of 2.5-250
µg/ml and the plates were incubated for overnight
at 37°C in 5% CO2. MTT (20 µl of 5 mg/ml) was
added to each well plate and incubated for 4 hrs.
Media was discarded and DMSO (200 µl) was
added. Absorbance in each well plate was meas-
ured at 490 nm.
2.9. Plaque Reduction Assay
ACV and ACV SLN were tested for antiviral
activity against HSV-1 by plaque reduction assay
over a concentration range of 0.25µM-8 µM.
1x105 Vero cells were seeded in 24-well plates and
incubated at 37°C and 5% CO2. When the cells
reached 70% confluence, they were infected with
300 µl of 3.3x106 pfu/ml of HSV-1. After
incubation for 1 hr at 37°C to allow viral
adsorption, the plates were washed, and the
medium replaced with maintenance medium
containing different concentrations of ACV and
ACV SLN. After 96 hrs incubation, the medium
Effect of Acyclovir Solid Lipid Nanoparticles for the Treatment
Fig. (1). Calibration curve of standard acyclovir in mice plasma. (A higher resolution / colour version of this figure
is available in the electronic copy of the article).
was removed, and the monolayers were fixed with
10% formaldehyde (200 µl) for 1 hr and then stai-
ned with 300 µl of 1% solution of crystal violet in
30% methanol for 1 hr. Percent plaque reduction
from control was calculated for both the treatment
groups.
2.10. Evaluation of Antiviral Activity in Cuta-
neous Model of HSV-1 Infection
Female BALB/c mice (6 weeks old) were used.
The right mid flank of each mouse was depilated.
One day later, the skin was scratched using a 27-
gauge needle and 40 µL of HSV-1 suspension of
3.3×106 pfu /ml was applied to the scarified area
[23-25]. ACV and ACV SLNs were orally admin-
istered as an oral gavage 24 h after HSV-1 infec-
tion. ACV (400 mg) was given thrice a day for 5
days whereas ACV SLN was a single dose admin-
istration (400mg). Beginning 1 day after infection,
the abraded site was evaluated and scored as fol-
lows: 0, no visible change in the abrasion or surr-
ounding tissue; 0.5, the earliest lesion characteri-
sed by slight swelling near the abrasion and mild
erythema; 1, papule(s) at or around the abrasion; 2,
ulcerated papules at or around the abrasion site with
eschar formation; 3, fusion of ulcerated lesions
into large eschar; 4, large open ulcerated lesion; 5,
death or sacrifice. The animals were evaluated
daily for 11 days beginning 1 day after infection.
2.11. Statistics
PK data were expressed as mean ± standard
deviation (SD). The difference in PK parameters
Pharmaceutical Nanotechnology, 2019, Vol. 7, No. 5 393
and results of plaque reduction assay between the
two groups were analysed by unpaired t-test. For
lesion scoring in animal model, among the control,
ACV and ACV SLN group. Data were analysed by
ANOVA and post-hoc Bonferroni test was used
for between-group comparisons. p value of <0.05
was considered significant.
3. RESULTS
3.1. HPLC Estimation
ACV was standardized for estimation by
HPLC. A standard curve was obtained with 0.2-
25.6 µg/ml concentration range. The correlation
coefficient value of 0.99 was obtained. The stand-
ard curve was found to be eligible for the PK-PD
studies as acyclovir is active against HSV-1 in
concentration range of 0.02-0.9 µg/ ml (Fig. 1).
Recoveries of acyclovir from plasma samples were
found to be 88.11 ± 0.27% which confirms less
sample loss. Inter-day and intra-day variability
were also found to be within range (15%) [26].
3.2. Characterization of ACV Solid Lipid
Nanoparticles
ACV SLN were prepared by microemulsion
technique by optimizing the ratio of the lipid phase
and aqueous phase. ACV SLN had a size of 131 ±
41.41nm with a polydispersity index (PDI) of 0.30
± 0.014. Zeta potential was found out to be -16.00
± 1.90 mV (Table 2).
TEM micrographs showed smooth spherical
surface morphology and non-aggregated nanopar-
394 Pharmaceutical Nanotechnology, 2019, Vol. 7, No. 5 Kondel et al.

Table 2. Results of parameters used for characterization of nanoparticles PDI, particle size, zeta potential
results are of 3 sets of experiments.

Percent Drug Percent Encapsula-

Particle Size (nm) Zeta Potential (mV) PDI
Loading tion Efficiency
(Mean ± S.D) (Mean ± S.D) (Mean ± S.D)
(Mean ± S.D) (Mean ± S.D)

131 ± 41.44 -16 ± 1.90 0.30 ± 0.014 90.22 ± 1.18% 67.44 ± 6.21%

103.7 nm

Focused view

drug slnzd.tif
Print Mag: 347000x @ 7.0 in 100 nm
2:35:50 p 04/24/15 HV = 80.0kV
Direct Mag: 200000x
AMT Camera System

Fig. (2). Transmission Electron Microscopy (TEM) micrograph of Acyclovir SLN showing spherical nanoparticles.
(A higher resolution / colour version of this figure is available in the electronic copy of the article).

ticles (Fig. 2). The drug loading and encapsulation
efficiency were found out to be 90.22 ± 1.18% and
67.44 ± 6.21% respectively. In ACV SLNs, FT-IR
spectra peak shifted from 3521 cm-1 to 3404 cm-1
in -NH stretch, and shift in 1485 cm-1 to 1472 cm-1
due to C-OH group was also noted (Fig. 3) (Table 3).
In vitro drug release study demonstrated a biphasic
release pattern where it was observed that there
was an initial burst release of drug followed by a
sustained release. An initial burst release of a drug
was 51%, 45% and 52% in SIF, PBS and SGF
respectively in the first 24 hours. 87% and 95% of
entrapped drug were released until 7 days in PBS
and SGF. 100% drug release was observed in SIF
in 7 days (Fig. 4).
3.3. Stability of ACV SLN
Stability of ACV SLN was assessed for drug
loading, particle size, polydispersity index and
zeta potential. After storage for 1 year, percent
drug loading was 85%. Particle size, PDI and zeta
potential changed to 152 nm, -31.11 and 0.318,
Effect of Acyclovir Solid Lipid Nanoparticles for the Treatment Pharmaceutical Nanotechnology, 2019, Vol. 7, No. 5 395

1960.63
784.57
8090.53 73.56 755.56
1538.54 1417.42 719.53630.52
3185.46 1601.45 885.4 679.52 532.48
259.4 561.55
3309.40 1001.40 545.44
3430.16 1629.34 1387.37
3404.37 1472.30 1350.29 959.31
1463.29 1297.53
1736.22 1250.29

2870.1 1966.72 719.71 526.71

2850.5 1643.67 847.66
%T 2916.5 1111.6
1298.60 950.58
3437.56 1463.57 1350.57 1249.58
1736.53

2850.39
107.35
2917.33 2102.16 416.18
650.13 506.13
1262.11 867.11 575.9
3521.5 2928.4 1309.6 1217.6 1012.6 901.5 779.6 652.6
3309.1 2856.4 1611.1 1455.2 1539.2 1183.3 1005.2 753.6 627.6
3471.1 3184.1 2790.4 1715.0 1577.1 1345.4 1105.1
3441.1 2716.3 1632.0 1541.3 1049.3

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0
cm-1
Ritika Kondel-1-sp- 1/11/2016 - ACV
Ritika Kondel-2-sp- 1/11/2016 - BSNS
Ritika Kondel-3-sp- 1/11/2016 - ACV SCNS

Fig. (3). FT-IR spectra of acyclovir SLNs, Blank SLNs and free acyclovir. (A higher resolution / colour version of
this figure is available in the electronic copy of the article).

Fig. (4). In vitro release of Acyclovir SLN in three simulated fluids; SIF (pH=7.8), SGF (pH=1.2), PBS (pH= 6.8).
(A higher resolution / colour version of this figure is available in the electronic copy of the article).

Table 3. Major bands of FT-IR spectra of acyclovir, blank SLNs and acyclovir SLNs.

Band Acyclovir Blank SLNs Acyclovir SLNs Functional Groups (Frequency Range)

1 3521.3 3437.56 3404.37, 3436. 36 -NH stretch (3100-3500 cm-1

2 1485.2 - 1472. 30 -C-OH group (1500-1065 cm-1)

3 1715.0 1736.53 1736.53 -C=O group (1700-1800)

4 3309.1 - 3309.40 -OH stretch (3200-3500 cm-1)

396 Pharmaceutical Nanotechnology, 2019, Vol. 7, No. 5 Kondel et al.

Table 4. Characterisation of formulation before and after 1 year of storage at 4-8°C and effect of sterilisation
on formulation.

Parameters Initial Final After Sterilisation

Particle Size (nm) 131.37 ± 41.44 151.9 ± 3.31 246.6 ± 16.2*

PDI 0.30 ± 0.014 0.315 ± 0.023 0.229 ± .079
*
Zeta potential -16.03 ± 1.63 -30.6 ± 1.89 -12.0 ± 3.57
% Drug loading 90.3 ± 1.18 85 ± 2.44 85 ± 2.18
*
Values are expressed as mean ± S.D; p<0.05.

Fig. (5). Plasma concentration- time profile of orally administered free acyclovir (36 mg/kg) and acyclovir solid
lipid nanoparticles (equivalent 36 mg/kg of acyclovir in SLNs) in mice dosed orally. (n=3) mice were taken at each
time point from 0 hour to 168 hours. Time points were shown as logarithmic scale. (A higher resolution / colour
version of this figure is available in the electronic copy of the article).

respectively (Table 4). Sterilisation did not have
any effect on percent drug loading, though it did
increase the particle size of SLN from 13 nm to
256.6 nm.
3.4. Comparative Assessment of Pharmacoki-
netics of Free and Nanoformulation of Acyclo-
vir in Mice
ACV SLN demonstrated a prolonged release of
drug for up to 7 days in comparison to ACV (Ta-
ble 5). The drug levels observed in both the groups
corresponds to a concentration which is needed for
antiviral activity (0.02-0.9 µg/ ml) [5]. Plasma
concentrations were maintained for up to 7 days in
ACV SLN as compared to 2.5 hrs in the ACV
group. The Cmax was significantly decreased (p<0.05)
for ACV SLNs as compared to ACV (3.59 ± 3.00
vs. 10.54 ± 5.46 µg/ ml). The AUC0-∞ (119.43 ±
28.74 µg/ ml h), AUMC 0-∞ (14469 ± 4261.16 µg/
ml h) and MRT (120.10 ± 9.21 hrs) were signifi-
cantly higher for ACV SLN as compared to ACV
AUC0-∞ (12.22 ± 2.47 µg/ ml hrs), AUMC0-∞
(28.78 ± 30.16 µg/ ml hrs) and MRT (2.07 ± 1.77
hrs), respectively (p<0.05, Table 5) (Fig. 5).
3.5. Pharmacokinetic/Pharmacodynamic Index
of Acyclovir Solid Lipid Nanoparticles
EC50 against HSV-1 isolates ranged from (0.02
to 13.5 µg/ mL) [5, 27, 28]. Time above EC50 was
found to be significantly higher for ACV SLN
equivalent to (36 mg/kg) of acyclovir as compared
to ACV (168 hours v/s 4 hours) (Table 6).
Effect of Acyclovir Solid Lipid Nanoparticles for the Treatment Pharmaceutical Nanotechnology, 2019, Vol. 7, No. 5 397

Table 5. Pharmacokinetic parameters of acyclovir and acyclovir solid lipid nanoparticles (Cmax, Tmax, AUC,
AUMC, MRT; n=3).

AUC AUMC
Cmax(µg/ml) Tmax(h) AUC (µg/ml*h)(0-24) MRT (h)
Group (µg/ml*h) (0-∞) (µg/ml* h)(0-∞)
(Mean ± S.D) (Mean ± S.D) (Mean ± S.D) (Mean ± S.D)
(Mean ± S.D) (Mean ± S.D)

ACV 10.54 ± 5.46 0.33 ± 0.144 15.11 ± 3.21 10.56 ± 1.30 28.78 ± 30.16 2.07 ± 1.77
ACV SLN 3.59 ± 3.00 1±0 10.14 ± 1.21 119.43 ± 28.74 14469.73 ± 4261 120.10 ± 9.21

Fig. (6). Effect of Acyclovir and Acyclovir SLN on non-infected Vero cells viability at different drug concentration
demonstrating cell toxicity. Each point represents the mean ± S.D (n=3). (A higher resolution / colour version of
this figure is available in the electronic copy of the article).

Table 6. Time above EC50 for free acyclovir and
acyclovir solid lipid nanoparticles (Acyclo-
vir SLNs) at an EC50 (0.02-13.5 µg/ml).
Drugs Time>EC50 (Hours)
Free acyclovir (36 mg/kg) 4 icity was observed at concentration above ≤100
Acyclovir NPs
168
(equivalent to 36 mg/kg)
3.6. Comparative Antiviral Efficacy of ACV
and ACV SLN
3.6.1. Cellular Toxicity
To investigate cytotoxicity, uninfected Vero
cells were treated with ACV and ACV SLN and
then subjected to cell viability assay. The amount
of surviving cells after incubation with an ACV
and ACV SLN during a 24 hours incubation was
estimated by MTT assay. The viability was ex-
pressed in percent by comparison with non-treated
control. At higher concentration of ACV tested,
250 µg/ml, cell viability was 19.1% (Fig. 6). How-
ever, at 100 µg/ml, cell viability observed was
60.5% (Fig. 6). Similar results were obtained for
ACV SLN and no significant difference was ob-
served between the two treatment groups. Cytotox-
µg/ ml for both the treatment groups.
3.6.2. Plaque Reduction Assay
The antiviral efficacy of ACV SLN against
HSV-1 was assessed by plaque reduction assay in
a monolayer of Vero cells. ACV showed a concen-
tration-dependent response on plaque formation.
As shown in Fig. (4), ACV decreased the percent
(%) plaque count from 61% at the lowest (0.25
µM) to 2% at the highest concentration (8 µM).
On the other hand, ACV SLN showed better
antiviral efficacy at lowest concentration (0.25
µM) (Fig. 7) as no plaque formation occurred in
wells having ACV SLN in concentration range of
(0.5 µM - 8 µM) (p<0.05). The effective concen-
398 Pharmaceutical Nanotechnology, 2019, Vol. 7, No. 5 Kondel et al.

Fig. (7). Plaque reduction assay at different drug concentration of Acyclovir and Acyclovir SLN. Percentage plaque
formation reduction from the control was shown as percentage HSV-1 plaques formed in both the treatment groups.
(A higher resolution / colour version of this figure is available in the electronic copy of the article).

Fig. (8). Comparative pharmacodynamic activity of Acyclovir and Acyclovir SLN was shown. Treatment of HSV-1
infected mice was given after 24h infection. Control mice were given saline. ACV was given 3 times a day treat-
ment for consecutive 5 days. ACV SLNs were given once administration during five days (n=6). (A higher resolu-
tion / colour version of this figure is available in the electronic copy of the article).

tration to achieve 50% protection against virus
replication (EC50) when using ACV SLN and
ACV was 0.003 µM and 0.296 µM, respectively.
3.6.3. Cutaneous Mouse Model of HSV-1 Infec-
tion
For this purpose, infected mice were divided
into three groups and were treated with saline,
ACV and ACV SLNs after 24 hours of infection.
Lesion development in mice was assessed for 11
days. It was observed that as compared to the
saline-treated mice, treatment with ACV and ACV
SLNs both significantly reduced the lesion score in
infected mice and also reduced the time of healing
of lesions (p<0.005). At day 1, there was no
significant difference observed in lesion scores
between all the treatment groups. On the subse-
quent days, significant difference was observed in
both the treatment groups when compared to the
saline-treated group (p<0.005) (Fig. 8). Time of
healing was also found to be significantly dec-
reased in treatment groups as compared to control
group (9 days vs. 4 days). When ACV and ACV
SLNs groups were compared, no significant differ-
ence was observed in lesion scoring and time of
healing (Fig. 9).
4. DISCUSSION
In the current study, we developed solid lipid
nanoparticles (SLNs) of acyclovir containing
Effect of Acyclovir Solid Lipid Nanoparticles for the Treatment
Fig. (9). Representative images depicting HSV lesions in BalB/C mice at day 3. A) Control mice given saline; B)
Free acyclovir group mice given 3 times a day administration for 5 days; C) Acyclovir SLNs group mice given
once for five days; D,E,F focused images of above images above groups. (A higher resolution / colour version of
this figure is available in the electronic copy of the article).
suitable lipids and surfactants which are biodeg-
radable and biocompatible.
4.1. Preparation and Characterisation of Acy-
clovir Solid Lipid Nanoparticles
We were able to achieve a drug loading of
67.44%. Previously, Seyfoddin A 2013, have pre-
pared acyclovir SLNs the microemulsion tech-
nique and have used Tween 80 as a surfactant and
have reported encapsulation efficiency of 13.32%
[29]. They have tried with different lipids and
different drug lipid ratio to get the best encap-
sulation efficiency. We have used compritol 888
ATO as a lipid and Tween 80 as a surfactant to
prepare microemulsion. The high encapsulation
efficiency of 67.44% demonstrates that the lipid
combination used have synergistic interaction with
the drug molecule. By combining the two lipids, it
resulted in better incorporation of acyclovir into
the lipid mix melt into the empty spaces of the
imperfect lipid matrix. Most of the other studies
which have developed acyclovir nanoparticles
have developed using different lipids and most of
them have not prepared nanoparticles for oral
delivery [30-33].
Acyclovir solid lipid nanoparticles had an
average size of 131 ± 41.41 nm with total drug
loading (TDC) of 90.22%. Particle size less than
Pharmaceutical Nanotechnology, 2019, Vol. 7, No. 5 399
200 nm is desirable for sustained release action as
they remain inaccessible to the reticuloendothelial
system (RES) and can show prolonged action [34].
A high total drug capacity holds promise in the
drug development process as scaling up the for-
mulation may not be a wasteful excess [34]. The
suspension was also found to be stable over one-
year showing non-significant change in particle
size and total drug loading. The nanoparticles
exhibited low negative zeta potential of -16 ± 1.90
mV which could be due to terminal carboxylic
group of stearic acid. Under TEM, nanoparticles
had smooth surface and were found to be spherical
in shape. The particle size observed with TEM
micrographs corresponds well with number distri-
bution pattern. FTIR spectra of acyclovir showed
an -NH stretch at 3521.3 cm-1, -C=O stretch at
1715.0 cm-1 and -C-OH stretch at 1485 cm-1. The
shift in the stretch of these groups indicates
interaction with the lipids. In ACV SLNs groups a
higher shift was observed at all these groups which
suggest an interaction of acyclovir with compritol
888 ATO and stearic acid both which resulted in
good encapsulation efficiency of 67.44%. In vitro
drug release of ACV SLN showed an initial phase
of burst release of drug in first 24 hours due to the
presence of unentrapped drug onto the nanopar-
ticles. Afterwards, it showed a sustained release of
drug by diffusion from the lipid core, wherein the
400 Pharmaceutical Nanotechnology, 2019, Vol. 7, No. 5
drug was homogeneously distributed. A slow
release of drug is may be accounted by the high
lipophilicity of the compritol 888 ATO. Higher
carbon chain length of compritol AT0 888
contribute to higher sustained release [35].
Further, amorphous nature of formulation leads to
high solubility [35].
4.2. Pharmacokinetics of Acyclovir Solid Lipid
Nanoparticles
Oral administration of acyclovir solid lipid
nanoparticles led to a prolonged drug release with
levels above EC50 for 7 days in comparison to 4
hours in the free drug group. Several reasons may
be given for improved oral bioavailability of
acyclovir after administration of acyclovir solid
lipid nanoparticles. Firstly, smaller particle size
increases the surface area of particles itself which
leads to increased absorption from the gastro-
intestinal tract [36]. Bioadhesion of SLN to the
mucosa of the gastrointestinal tract due to lipid
moieties may increase the residence time at the gut
wall. This close contact with the epithelial mem-
branes may enhance the drug absorption thus
leading to increased oral bioavailability [36].
Secondly, absorption can also be facilitated by
presence of fatty acids and mono or diacylgly-
cerols being converted by triglycerides present in
the nanoformulation by gastrointestinal lipase
[37]. Thirdly, lipids present in SLN increase lipo-
protein formation which stimulates lymphatic lipid
flux. This increased lymphatic delivery of drug
leading to enhanced bioavailability. Lastly, nano-
particles are preferentially taken by the M-cells
present in the Peyer's patches of the gut-associated
lymphoid tissue (GALT) followed by shuttling
into the systemic circulation and release of drug in
the blood. This specialised uptake mechanism can
also improve absorption of drug [38]. Similar
studies have shown increased bioavailability of
drug encapsulated in solid lipid nanoparticles [37,
39, 40]. A significant increase in the MRT of acy-
clovir in case of acyclovir solid lipid nanoparticles
as compared to free acyclovir was due to change in
the acyclovir absorption and elimination after
encapsulating into solid lipid nanoparticles. This
may be also because of slow release of drug from
the nanoparticles due to higher biodegradation
time of acyclovir solid lipid nanoparticles [40].
Kondel et al.
4.3. Pharmacodynamics of Acyclovir Solid Lipid
Nanoparticles
Efficacy studies were carried out both in cell
culture by plaque reduction assay and in the
animal model of cutaneous HSV-1 infection. 50%
inhibition observed with the treatment groups
corresponds to the value which is above the
reported EC50 value (0.02-13.5 µg/ml ≈ 0.08-60
µM). Approximately 100 times improvement in
the efficacy was observed in the ACV SLNs group
when compared to ACV alone. This improvement
in antiviral efficacy could be due to the longer
duration of contact of the drug-loaded SLNs with
the infected cells [10]. Jalon et al., 2003 have also
shown a significant decrease in IC50 value (0.15 ±
0.03 µM), in cell plates treated with ACV
microparticles as compared to drug solution alone
(0.27 ± 0.02 µM). The difference in EC50 value of
the reported study, when compared to our study,
maybe due to the reason that we are using
nanoparticles unlike microparticles used in their
study. Similar studies also showed increase
efficacy of drugs incorporated in the SLN through
increased cellular uptake in the cells [41-44].
Efficacy study done in an animal model of
cutaneous herpes infection showed that acyclovir
given three times a day for consecutive five days
was comparable with a single dose of acyclovir
solid lipid nanoparticles. The lesions observed in
control animals are scored up to 3 which corres-
ponds to large lesions fused into large eschar,
whereas, for the treatment groups maximum lesion
score of 2 was observed. The score of 2 corres-
ponds to small ulcerated papules leading to eschar
formation. Time of healing was also found to be
decreased in treatment groups as compared to
control group (9 days vs. 4 days). However, no
significant difference was observed in lesion sco-
ring and time of healing (p<1.000). The pharma-
cokinetic study observed significant increase in
MRT in the ACV SLN group when compared to
ACV alone (120.10 h vs. 2.07 h). The efficacy
results observed are in accordance with the phar-
macokinetics results which demonstrate that the
drug levels were maintained above EC50 for up to
7 days in ACV SLN group as compared to 4 hours
in ACV alone group. This could be a valid reason
for the comparable effect shown when free was
given acyclovir given 3 times a day for five days
Effect of Acyclovir Solid Lipid Nanoparticles for the Treatment
and a single dose of ACV SLN. The animal model
of cutaneous herpes used in our study is a well-
validated model and has been used to evaluate ef-
ficacy in HSV-1 and HSV-2 infections models in
various studies [45, 46].
There are limitations to our current study. We
were not able to show the antiviral efficacy of our
nanoparticles through a genotypic assessment like
polymerase chain reaction (PCR) and western
blotting. We have shown the efficacy in one ani-
mal model of herpes infections. Screening needs to
be done on different models of herpes infections.
CONCLUSION
In conclusion, our study showed that the
developed single dose acyclovir solid lipid
nanoparticles have shown comparative efficacy to
the multiple-dose regimen of conventional
acyclovir. They found to be potential carriers for
oral therapy in the treatment of HSV-1 infection.
In future, we will plan for the ACV SLN toxicity
studies and its efficacy studies in HSV-2 infection.
ETHICS APPROVAL AND CONSENT TO
PARTICIPATE
Ethical approval from the Institutional Animal
Ethics Committee, PGIMER, Chandigarh, India
(Ref. No. 73/IAEC/444).
HUMAN AND ANIMAL RIGHTS
No humans were used. We have followed
OECD guidelines for the PK-PD experiments and
the protocol was approved by the Institute Animal
Ethics committee.
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERIALS
The raw data that support the finding of the
results of this study are available from the
corresponding author, [Dr. NS], and the first au-
thor [Dr. RK] upon request.
FUNDING
The work was funded by Life Science Research
Board (LSRB), DRDO, New Delhi, India (No.
DLS/81/4822/LSRB-257/SH&DD/2012).
Pharmaceutical Nanotechnology, 2019, Vol. 7, No. 5 401
CONFLICT OF INTEREST
The authors confirm that this article content has
no conflict of interest.
ACKNOWLEDGEMENTS
Declared none.
REFERENCES
[1] Arvin A, Campadelli-Fiume G, Mocarski E, et al.
Human herpesviruses: biology, therapy, and im-
munoprophylaxis. Cambridge University Press: Cam-
bridge 2007.
[2] Hay CM, Reichman RC. Antiviral drugs. In: Gold-
smith LA, Katz SI, Gilchrest BA, Paller AS, Leffell
DJ, Wolff K, Eds. Fitzpatrick’s Dermatology in Gen-
eral Medicine, 7th ed. McGraw-Hill: New York 2008;
pp. 2203-11.
[3] Chang YC, Madkan VK, Sra K, Carrasco DA, Tyring
SK. Systemic antiviral agents In: Wolff K, Ed. Com-
prehensive Dermatologic Drug Therapy. WB Saun-
ders: Philadelphia 2007; pp. 101-24.
[4] Dollery C. Therapeutic drugs. 2nd ed. Churchill Liv-
ingstone: Edinburgh 1999; pp. A39-44.
[5] Goodman L, Gilman A, Brunton L, Lazo J, Parker K.
Goodman & Gilman’s the pharmacological basis of
therapeutics, 13th ed. McGraw-Hill: New York 2006;
pp. 1106-7.
[6] Arora A, Shafiq N, Jain S, Khuller GK, Sharma S,
Malhotra S. Development of sustained release
“NANOFDC (fixed dose combination)” for hyperten-
sion - an experimental study. PLoS One 2015; 10(6):
e0128208.
[7] Arora A, Shafiq N, Jain S, et al. Development of sus-
tained release “Nanopolypill” of ischemic heart dis-
ease drugs - an experimental study. Curr Nanosci
2014; 10(6): 816-26.
[8] Kumar G, Malhotra S, Shafiq N, Pandhi P, Khuller
GK, Sharma S. In vitro physicochemical characteriza-
tion and short term in vivo tolerability study of ethi-
onamide loaded PLGA nanoparticles: potentially ef-
fective agent for multidrug resistant tuberculosis. J
Microencapsul 2011; 28(8): 717-28.
[9] Kumar G, Sharma S, Shafiq N, Khuller GK, Malhotra
S. Optimization, in vitro-in vivo evaluation, and short-
term tolerability of novel levofloxacin-loaded PLGA
nanoparticle formulation. J Pharm Sci 2012; 101(6):
2165-76.
[10] de Jalón EG, Blanco-Príeto MJ, Ygartua P, Santoyo
S. Increased efficacy of acyclovir-loaded microparti-
cles against herpes simplex virus type 1 in cell cul-
ture. Eur J Pharm Biopharm 2003; 56(2): 183-7.
[11] Müller RH, Mäder K, Gohla S. Solid lipid nanoparti-
cles (SLN) for controlled drug delivery - a review of
the state of the art. Eur J Pharm Biopharm 2000;
50(1): 161-77.
402 Pharmaceutical Nanotechnology, 2019, Vol. 7, No. 5
[12] Yang S, Zhu J, Lu Y, Liang B, Yang C. Body distri-
bution of camptothecin solid lipid nanoparticles after
oral administration. Pharm Res 1999; 16(5): 751-7.
[13] Demirel M, Yazan Y, Müller RH, Kiliç F, Bozan B.
Formulation and in vitro-in vivo evaluation of
piribedil solid lipid micro- and nanoparticles. J Mi-
croencapsul 2001; 18(3): 359-71.
[14] Penkler L, MuÈller RH, Runge SA. Pharmaceutical
cyclosporin formulation with improved biopharma-
ceutical properties, improved physical quality and
greater stability, and method for producing said for-
mulation United States Patent US 6551619B1, 1999.
[15] Abd-Rabou AA, Bharali DJ, Mousa SA. Taribavirin
and 5-fluorouracil-loaded pegylated-lipid nanoparticle
synthesis, p38 docking, and antiproliferative effects
on MCF-7 breast cancer. Pharm Res 2018; 35(4): 76.
[16] Jain AK, Jai SK. Galactosylated poly (d, l-lactic-co-
glycolic acid) nanoparticles for liver targeted delivery
of acyclovir. J Biomed Pharm Res 2013; 2: 7-14.
[17] Bhalekar MR, Upadhaya PG, Madgulkar AR, Kshir-
sagar SJ, Dube A, Bartakke US. In vivo bioavailabil-
ity and lymphatic uptake evaluation of lipid nanopar-
ticulates of darunavir. Drug Deliv 2016; 23(7): 2581-
6.
[18] Cavalli R, Donalisio M, Bisazza A, et al. Enhanced
antiviral activity of acyclovir loaded into nanoparti-
cles. Methods Enzymol 2012; 509: 1-19.
[19] Seyfoddin A, Al-Kassas R. Development of solid
lipid nanoparticles and nanostructured lipid carriers
for improving ocular delivery of acyclovir. Drug Dev
Ind Pharm 2013; 39(4): 508-19.
[20] Bhandari R, Kaur IP. Pharmacokinetics, tissue distri-
bution and relative bioavailability of isoniazid-solid
lipid nanoparticles. Int J Pharm 2013; 441(1-2): 202-
12.
[21] Emami J, Bazargan N, Ajami A. HPLC determination
of acyclovir in human serum and its application in bi-
oavailability studies. Res Pharm Sci 2009; 4: 47-54.
[22] Brown SD, White CA, Chu CK, Bartlett MG. Deter-
mination of acyclovir in maternal plasma, amniotic
fluid, fetal and placental tissues by high-performance
liquid chromatography. J Chromatogr B Analyt
Technol Biomed Life Sci 2002; 772(2): 327-34.
[23] Lipipun V, Kurokawa M, Suttisri R, et al. Efficacy of
Thai medicinal plant extracts against herpes simplex
virus type 1 infection in vitro and in vivo. Antiviral
Res 2003; 60(3): 175-80.
[24] Docherty JJ, Smith JS, Fu MM, Stoner T, Booth T.
Effect of topically applied resveratrol on cutaneous
herpes simplex virus infections in hairless mice. An-
tiviral Res 2004; 61(1): 19-26.
[25] Kurokawa M, Ochiai H, Nagasaka K, et al. Antiviral
traditional medicines against herpes simplex virus
(HSV-1), poliovirus, and measles virus in vitro and
their therapeutic efficacies for HSV-1 infection in
mice. Antiviral Res 1993; 22(2-3): 175-88.
[26] Guidelines for the validation of analytical methods
used in residue depletion studies www.ema.europa.
eu/docs/en_GB/document...guideline/2010/.../WC500
040499.pdf2010.
Kondel et al.
[27] Food Drug Administration. Zovirax®. Available at:
https://www.accessdata.fda.gov/drugsatfda_docs/label
/2001/018604s018lbl.pdf
[28] O’Brien JJ, Campoli-Richards DM. Acyclovir. An
updated review of its antiviral activity, pharmacoki-
netic properties and therapeutic efficacy. Drugs 1989;
37(3): 233-309.
[29] Seyfoddin A, Al-Kassas R. Development of solid
lipid nanoparticles and nanostructured lipid carriers
for improving ocular delivery of acyclovir. Drug Dev
Ind Pharm 2013; 39(4): 508-19.
[30] Hasanovic A, Zehl M, Reznicek G, Valenta C. Chi-
tosan-tripolyphosphate nanoparticles as a possible
skin drug delivery system for aciclovir with enhanced
stability. J Pharm Pharmacol 2009; 61(12): 1609-16.
[31] Cortesi R, Ravani L, Menegatti E, Drechsler M, Es-
posito E. Colloidal dispersions for the delivery of
acyclovir: a comparative study. Indian J Pharm Sci
2011; 73(6): 687-93.
[32] Bhosale U, Kusum DV, Jain N. Formulation and op-
timization of mucoadhesive nanodrug delivery system
of acyclovir. J Young Pharm 2011; 3(4): 275-83.
[33] AK, Jain, J. S. Galactosylated poly (d, l-lactic-co-
glycolic acid) nanoparticles for liver targeted delivery
of acyclovir. J Biomed Pharm Res 2013; 2: 7-1.
[34] Rohit B, Pal KI. A method to prepare solid lipid na-
noparticles with improved entrapment efficiency of
hydrophilic drugs. Curr Nanosci 2013; 9: 29-33.
[35] Aburahma MH, Badr-Eldin SM. Compritol 888 ATO:
a multifunctional lipid excipient in drug delivery sys-
tems and nanopharmaceuticals. Expert Opin Drug De-
liv 2014; 11(12): 1865-83.
[36] Qi J, Lu Y, Wu W. Absorption, disposition and
pharmacokinetics of solid lipid nanoparticles. Curr
Drug Metab 2012; 13(4): 418-28.
[37] Hu L, Tang X, Cui F. Solid lipid nanoparticles
(SLNs) to improve oral bioavailability of poorly sol-
uble drugs. J Pharm Pharmacol 2004; 56(12): 1527-
35.
[38] Italia JL, Yahya MM, Singh D, Ravi Kumar MNV.
Biodegradable nanoparticles improve oral bioavaila-
bility of amphotericin B and show reduced ne-
phrotoxicity compared to intravenous fungizone.
Pharm Res 2009; 26(6): 1324-31.
[39] Zhang Z, Lv H, Zhou J. Novel solid lipid nanoparti-
cles as carriers for oral administration of insulin.
Pharmazie 2009; 64(9): 574-8.
[40] Müller RH, Runge S, Ravelli V, Mehnert W, Thüne-
mann AF, Souto EB. Oral bioavailability of cyclo-
sporine: solid lipid nanoparticles (SLN) versus drug
nanocrystals. Int J Pharm 2006; 317(1): 82-9.
[41] Zhuang CY, Li N, Wang M, et al. Preparation and
characterization of vinpocetine loaded nanostructured
lipid carriers (NLC) for improved oral bioavailability.
Int J Pharm 2010; 394(1-2): 179-85.
[42] Hariharan S, Bhardwaj V, Bala I, Sitterberg J,
Bakowsky U, Ravi Kumar MNV. Design of estradiol
loaded PLGA nanoparticulate formulations: a poten-
tial oral delivery system for hormone therapy. Pharm
Res 2006; 23(1): 184-95.
Effect of Acyclovir Solid Lipid Nanoparticles for the Treatment
[43] Sharma CP, Kalarikkal N, Sandeep K. Thomas S,
Pothen LA. Evaluation of in vitro cytotoxicity and
cellular uptake efficiency of zidovudine-loaded solid
lipid nanoparticles modified with aloe vera in glioma
cells. Mater Sci Eng C 2016; 66: 40-50.
[44] Baek JS, Cho CW. Surface modification of solid lipid
nanoparticles for oral delivery of curcumin: im-
provement of bioavailability through enhanced cellu-
lar uptake, and lymphatic uptake. Eur J Pharm Bio-
pharm 2017; 117: 132-40.
Pharmaceutical Nanotechnology, 2019, Vol. 7, No. 5 403
[45] Mosallaei N, Jaafari MR, Hanafi-Bojd MY, Golmo-
hammadzadeh S, Malaekeh-Nikouei B. Docetaxel-
loaded solid lipid nanoparticles: preparation, charac-
terization, in vitro, and in vivo evaluations. J Pharm
Sci 2013; 102(6): 1994-2004.
[46] Ahmadnia S, Moazeni M, Mohammadi-Samani S,
Oryan A. In vivo evaluation of the efficacy of albend-
azole sulfoxide and albendazole sulfoxide loaded sol-
id lipid nanoparticles against hydatid cyst. Exp Para-
sitol 2013; 135(2): 314-9.