ORIGINAL ARTICLE

Chitosan-Carboxymethyl-5-Fluorouracil-
Folate Conjugate Particles: Microwave
Modulated Uptake by Skin and
Melanoma Cells
Asif Nawaz1,2 and Tin Wui Wong1,2

5-Fluorouracil delivery profiles in the form of chitosan-folate submicron particles through skin and melanoma
cells in vitro were examined using microwaves as the penetration enhancer. The in vivo pharmacokinetic
profile of 5-fluorouracil was also determined. Chitosan-carboxymethyl-5-fluorouracil-folate conjugate was
synthesized and processed into submicron particles by spray-drying technique. The size, zeta potential,
morphology, drug content, and drug release, as well as skin permeation and retention, pharmacokinetics,
in vitro SKMEL-28 melanoma cell line cytotoxicity, and intracellular trafficking profiles of drug/particles, were
examined as a function of skin/melanoma cell treatment by microwaves at 2,450 MHz for 5 þ 5 minutes. The
level of skin drug/particle retention in vitro and in vivo increased in skin treated by microwaves. This was
facilitated by the drug conjugating to chitosan and microwaves fluidizing both the protein and lipid domains of
epidermis and dermis. The uptake of chitosan-folate particles by melanoma cells was mediated via lipid raft
route. It was promoted by microwaves, which fluidized the lipid and protein regimes of the cell membrane, and
this increased drug cytotoxicity. In vivo pharmacokinetic study indicated skin treatment by microwave-
enhanced drug retention but not permeation. The combination of microwaves and submicron particles syn-
ergized skin drug retention and intracellular drug delivery.
Journal of Investigative Dermatology (2018) 138, 2412e2422; doi:10.1016/j.jid.2018.04.037

INTRODUCTION biological effects as a function of field strength, frequency,

Microwaves are a nonionizing electromagnetic wave char-
acterized by frequencies between 300 MHz and 300 GHz
(Wong, 2014). This is not a form of heat but a form of energy
that interacts with materials in a thermal or nonthermal
fashion. Medically, microwave technology has been adopted
as a part of cancer treatment programs. In recent medical
advances, microwaves are used in hyperthermia ablation for
treatment of renal cancer (Castle et al., 2011), hepatocellular
carcinoma (Liu et al., 2013), intrahepatic primary chol-
angiocarcinoma (Yu et al., 2011), hepatic colorectal metas-
tases (Jones et al., 2011), metastatic liver cancer (Hompes
et al., 2010), benign thyroid nodules (Yue et al., 2013),
dysfunctional uterine bleeding (Bain et al., 2002), and lung
cancer (Vogl et al., 2011). Microwaves can induce different
1
Non-Destructive Biomedical and Pharmaceutical Research Centre,
iPROMISE; and 2Particle Design Research Group, Faculty of Pharmacy,
Universiti Teknologi MARA, Puncak Alam, Selangor, Malaysia
Correspondence: Tin Wui Wong, Non-Destructive Biomedical and
Pharmaceutical Research Centre, iPROMISE, Universiti Teknologi MARA,
Puncak Alam, 42300, Selangor, Malaysia. E-mail: wongtinwui@salam.uitm.
edu.my or wongtinwui@yahoo.com
Abbreviations: C-5-FU-F, chitosan-carboxymethyl-5-fluorouracil-folate; C-F-
FU-F-NP, chitosan-carboxymethyl-5-fluorouracil-folate conjugate submicron
particles; CMFU, carboxymethyl-5-fluorouracil; FTIR, Fourier transform
infrared spectroscopy
Received 31 January 2018; revised 15 April 2018; accepted 16 April 2018;
accepted manuscript published online 30 May 2018; corrected proof
published online 8 August 2018
wave form, and duration of exposure (Rai et al., 1994a,
1994b). These effects are mainly attributed to microwave
heating (Banik et al., 2003). However, recent reports have
shown or suggested that there are nonthermal microwave
effects leading to various types of molecular transformations
and alterations (Herrero et al., 2008).
Microwave radiation has been shown to enhance skin
penetration. It enhances drug permeation transdermally with
no significant temperature rise on skin. The drug permeation
is promoted by microwaves through exerting spacing of lipid
architecture of stratum corneum into structureless domains
(Wong and Anuar, 2013). Melanoma accounts for about 80%
of skin cancer-associated deaths worldwide (Chen et al.,
2010). It originates from melanocytes, which are pigment-
producing cells located in the basal layer of epidermis
(deep skin layer). Most anticancer drugs are hydrophilic and
have low oil/water partition coefficients (Souza et al., 2011).
Their usefulness in the treatment of melanoma is hindered by
inadequate depth of penetration into the dermis (peripheral
skin layers), where tumors are usually located (Taveira &
Lopez, 2011).
Chitosan is a cationic biopolymer with amino and
hydroxyl moieties available in its parent structure for chem-
ical modification. Chitosan and its derivatives have been
reported to exhibit anticancer activities (Huang et al., 2006;
Xia, 2003, 2011), including antimelanoma action (Gibot
et al., 2015). Chitosan interacts with lipids in the skin via
its negatively charged moieties (Taveira et al., 2009). Using

2412 Journal of Investigative Dermatology (2018), Volume 138 ª 2018 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.

chitosan as the drug carrier improves the propensity of drug
diffusion into the peripheral skin layers. The effectiveness of
skin drug penetration and retention has been reported to be
aided by the submicron-sized chitosan matrix (Nawaz &
Wong, 2017). Tumor-targeted drug delivery has been
executed using chitosan particles via surface decoration of
the matrix with targeting ligands such as antibodies, hor-
mones, peptides, and small compounds like folic acid and
hyaluronic acid (Jain et al., 2010). Folic acid is well known as
a cancer-targeting ligand that is less expensive, more easily
conjugated to drug carriers, and physicochemically more
stable in applications (Byrne et al., 2008). It has been cova-
lently linked to chitosan particles with the aim of selectively
delivering anticancer drugs to tumor cells (Wu et al., 2006).
Folate receptor exists in malignant tumors, combining with
glycosyl phosphatidylinositol as a membrane glycoprotein
connection (Reddy et al., 2005). Its expression is highly
inhibited in normal tissues and highly expressed or overex-
pressed in several human cancers (Weitman et al., 1992a,
1992b), including melanoma (Skinner et al., 2016;
Sánchez-del-Campo et al., 2009). Melanoma cells have
a-folate receptors (Rodriguez-Lopez et al., 2011; Sánchez-
del-Campo et al., 2009). These folate receptors bind folate
and folate-drug conjugates with very high affinity and shuttle
these bound molecules inside cells via an endocytic mech-
anism (Leamon, 2004).
Thus far, magnetic-based core shell particles, electrospun
mats, liposomes, nanofibers, and microemulsion have been
developed for the purpose of melanoma treatment (Naves
et al., 2017). Using chitosan as the solid submicron particu-
late matrix material aids skin drug delivery and provide ther-
apeutic effects benefiting local disorders such as malignant
melanoma. Melanoma cells have overexpressed folate
receptors (Skinner et al., 2016). Using folic acid as the targeting
ligand, the binding affinity of particles with melanoma cells
can be promoted. The skin and melanoma uptake of chitosan
particles covalently conjugated with folic acid has not been
investigated to the best of our knowledge. Its uptake profiles as
a function of skin/melanoma treatment by microwave have not
been evaluated with respect to the anticancer activity
expression of drug. This study examines the effectiveness of
microwave-assisted drug delivery of chitosan-carboxymethyl-
5-fluorouracil-folate conjugate (C-5-FU-F) submicron parti-
cles (C-5-FU-F-NP) through skin and into the melanoma cells.
The possibility of combining chitosan-folate submicron parti-
cles with microwaves in melanoma cancer treatment is eval-
uated with respect to in vitro drug release, retention, and
permeation, as well as in vivo pharmacokinetics profiles. The
endocytotic uptake profiles of submicron particles are assessed
in vitro against the cellular viability of melanoma cells.
RESULTS AND DISCUSSION
The main chain and amide linkages between the amino
functional group of parent chain and acetyl moiety were
lysed under the influence of microwaves. The formed
chitosan was characterized by molecular weight ¼ 15,370 
544 Da, degree of deacetylation ¼ 92.5  2.7%, solution
viscosity ¼ 3.9  0.0, particle size ¼ 97.3  10.1 nm, and
zeta potential ¼ 71.1  2.1 mV. It was used as the precursor
for conjugate and submicron particle synthesis.
A Nawaz and TW Wong
Microwave Modulated Nanoparticle Delivery
C-5-FU-F conjugate
Modified low molecular weight chitosan was used as the
backbone for 5-fluorouracil and folic acid conjugation. The
C-5-FU-F conjugate was characterized by Fourier transform
infrared spectroscopy (FTIR) and nuclear magnetic resonance
spectroscopy techniques (Figure 1). The conjugate was
characterized by smaller FTIR wavenumbers of the spectral
band at 1,626.8  7.6 cme1 than those of chitosan
(1,645.2  6.1 cme1), carboxymethyl-5-fluorouracil (CMFU)
(1,683.6  2.5 cme1), and folic acid (1,694.8  0.6 cme1).
This was ascribed to the development of amide linkage after
polymer-drug-folate conjugation. The presence of CMFU in
chitosan was reflected by proton signals at 4.3 p.p.m. and
7.7 p.p.m. attributable to CH2 and CH moieties of CMFU and
folic acid by proton signal at 2.7 p.p.m. due to H-22 of folic
acid (Li et al., 2011). The characteristic peaks at 3.7 and
3.1 p.p.m. were ascribed to H-2/H-5/H-6 of chitosan.
Conjugate submicron particles
Near-spherical C-5-FU-F-NP were produced (Figure 2d).
They exhibited a smooth surface morphology. The average
drug content of the submicron particles was 13.4  1.2%,
whereas their encapsulation efficiency was 90.1  8.6%.
The average size and zeta potential of the submicron
particles were 781.7  33.5 nm and 9.0  3.2 mV,
respectively.
Drug release, permeation, and retention
The covalent conjugation of drug onto chitosan reduced
its detachment propensity from the polymer chain and
lowered the in vitro drug dissolution extent of C-5-FU-F-NP
(P ¼ 0.005) (Figure 2a). With reference to in vitro skin
diffusion, the percentages of drug permeation and retention
of C-5-FU-F-NP were nonetheless larger than free CMFU
(Figure 2b and c). The cationic chitosan enabled conjugate
submicron particles to interact with anionic domains of skin
lipids and proteins. This resulted in lipid lamella disorgani-
zation, secondary structural changes of keratin, formation of
larger aqueous pores, and membrane fluidization. The sum-
mative effects increased the intercellular and/or transcellular
penetration extent of drugs (He et al., 2009; Yeh et al., 2011).
In addition, the Tween (Fisher Scientific, Leicestershire, UK)
and Span (Merck, Darmstadt, Germany) contained in the
submicron particles fluidized and extracted skin lipid,
increased the stratum corneum’s water content, and
increased its aqueous pore size (López et al., 2000). The
physicochemical changes reduced the epidermis barrier
function and facilitated transdermal drug transport. Nearly all
drug released from the conjugate submicron particles
permeated across the skin (Figure 2a and b).
Pretreatment of skin by microwaves at 2,450 MHz for 5 þ
5 minutes further elevated the extent of drug permeation to
55.2  15.9% after 24 hours (P ¼ 0.037) (Figure 2b;). This
was due to an increase in submicron particles permeating
across the skin bringing together 5-fluorouracil covalently
linked to chitosan in the matrix, based on FTIR imaging
analysis (Figure 2e). The skin treated by microwaves was
characterized by a higher level of drug retention (P ¼ 0.036)
(Figure 2c). In skin treated with microwaves, a higher par-
ticulate fraction was found in both epidermis (3.5  1.2%)
and dermis (10.9  2.9%) compared with untreated skin
www.jidonline.org 2413
 A Nawaz and TW Wong
Microwave Modulated Nanoparticle Delivery

a Chitosan OH OH

O O
H H
OH H OH OH H O
O O O O O NH
H NH N NH2
H NH OH H
O O N
CH2
H NH NH N
N
O NH
CMFU S C N
O
NH OH Folic acid
F

O HOOO
FITC

HO O O

Chitosan-CMFU-Folic acid Conjugate

FTIR NMR
b

CH2

CH

1H, OH
1H, N-H
2972.3±1.5

1683.6±2.5 13 12 11 10 9 8 7 6 5 4 3 2 1 ppm

c
D2O

H2H5H6
Transmission (%)

2925.1±0.4
1645.2±6.1
9 8 7 6 5 4 3 2 1 ppm

d H-22

1694.8±0.6
13 12 11 10 9 8 7 6 5 4 3 2 1 ppm
e

D2O
CH2

2926.4±2.4 Chitosan Peaks

CH
H2
H2H5H6
1626.8±7.6
80
4000 3500 3000 2500 2000 1500 1000 650 9 8 7 6 5 4 3 2 1 ppm
Wavenumber (cm-1) Chemical shift (ppm)

Figure 1. Chitosan-CMFU-folate conjugation. Profiles of (a) chemical structure of chitosan-CMFU-folate conjugate with FITC labelling, FTIR (left) and
NMR (right) spectra for (b) CMFU, (c) chitosan, (d) folic acid, and (e) chitosan-CMFU-folate conjugate. CMFU, carboxymethyl-5-fluorouracil; FTIR, Fourier
transform infrared spectroscopy; NMR, nuclear magnetic resonance.

(epidermis, 1.3  0.5%; dermis, 4.3  2.1%). Most particles Attenuated total reflectance FTIR analysis indicated that
retained in skin were accumulated in the dermis. This was the skin, especially dermis, was vastly fluidized at O-H and/
deemed helpful for the local treatment of skin malignant or N-H (3,310e3,370 cme1) and asymmetric CH2
melanoma located in the deep skin layers. (2,850e2,950 cme1) domains of ceramide, keratin, and/or

2414 Journal of Investigative Dermatology (2018), Volume 138

 A Nawaz and TW Wong
Microwave Modulated Nanoparticle Delivery

a b
120 CMFU C-5-FU-F-NP 80 CMFU C-5-FU-F-NP

Cumulative Drug Permeation (%)

C-5-FU-F-NP;2450 MHz, 5+5 min
70
Cumulative Drug Release (%)

100
* 60
80 50
*
40
60
30
40 20
10
20
0
0 0 300 600 900 1200 1500
0 300 600 900 1200 1500 Time (min)
Time (min)
c 14 d

12
Average CMFU Retention (%)

*
10

0
CMFU C-5-FU-F-NP C-5-FU-F-NP; 2450 MHz,
5+5 min

e Epidermis Dermis
1.3±0.5% 4.3±2.1%
-498.3 -59.3
-100
-550
-150
-600
-200
Micrometers
Micrometers

-650
(i) -250
-700
-300
-750
-350
-800
-400
-850
-885.7 -446.7
-1703.9 -1600 -1500 -1400 -1300 -1210. 13822.1 13900 14000 14100 14200 14315.
Micrometers Micrometers

-1455.3 3.5±1.2% 10.9±2.9%

419.7
400
-1500
350
-1550
300
Micrometers

Micrometers

-1600
250
(ii) -1650
200
-1700
150
-1750
100
-1800
50
-1842.7 32.3
-1091.9 -1000 -900 -800 -700 -598.1 30716.1 30800 30900 31000 31100 31209.9
Micrometers Micrometers

Figure 2. Drug release, permeation, retention and particle morphology. Profiles of (a) drug release of C-5-FU-F-NP against CMFU (*P < 0.05), (b) skin drug
permeation of C-5-FU-F-NP with untreated skin or skin treated by microwaves at 2,450 MHz for 5 þ 5 minutes against CMFU (*P < 0.05), (c) skin drug retention
of C-5-FU-F-NP with untreated skin or skin treated with microwaves at 2,450 MHz for 5 þ 5 minutes against CMFU (P < 0.05), (d) SEM image of C-5-FU-F-NP
(mean size ¼ 761.8  54.2 nm), and (e) FTIR imaging plots of epidermis and dermis on C-5-FU-F-NP distribution in (i) untreated skin and (ii) skin treated with
microwaves at 2,450 MHz for 5 þ 5 minutes. Population density of C-5-FU-F-NP is represented in the following order: red > pink > green > blue. C-5-FU-F-NP,
chitosan-carboxymethyl-5-fluorouracil-folate conjugate submicron particles; CMFU, carboxymethyl-5-fluorouracil; FTIR, Fourier transform infrared
spectroscopy; min, minute.

www.jidonline.org 2415
 A Nawaz and TW Wong
Microwave Modulated Nanoparticle Delivery

ATR-FTIR
Epidermis Dermis

a
2850.1±0.0
2918.5±0.2 2919.2±0.9

3355.8±5.1 3316.2±2.7

b
Transmission (%)

2850.4±0.3
2919.0±0.9 2926.8±2.8

3362.4±9.5

c 3323.1±5.9

2851.4±0.7
2852.9±1.1
2920.3±0.6 2926.1±2.4

3364.4±9.8 3350.0±7.2
d
2852.3±4.5 2852.9±1.6
2920.4±1.6 2926.7±2.0

3374.1±10.3 3360.6±13.1
80 80
4000 3500 3000 2500 2000 1500 1000 650 3500 3000 2500 2000 1500 1000 650

Wavenumber (cm-1)

Raman
Epidermis Dermis
e 3336.5±6.7 3333.7±2.4

2935.9±1.1 2875.9±0.3
2875.8±0.1 1537.9±0.5 1344.9±1.1 2727.4±5.4
1600-1300

3336.0±2.8
f 3338.9±6.0

2900-2700
2900-2700 1600-1300
1600-1300
Intensity

3349.4±12.8
g 3357.8±3.4

2900-2700 2900-2700
1600-1300 1600-1300

3359.2±4.6 3349.3±10.4
h

2900-2700 2900-2700
1600-1300 1600-1300

3500 3000 2500 2000 1500 1000 500 400 3500 3000 2500 2000 1500 1000 500 400

Raman Shift (cm-1)

Figure 3. ATR-FTIR and Raman spectra of epidermis and dermis. (aed) ATR-FTIR and (eeh) Raman spectra of epidermis (left) and dermis (right) of (a,e)
untreated skin and skin treated with (b,f) C-5-FU-F-NP, (c,g) 2,450-MHz microwaves for 5 þ 5 minutes, and (d,h) 2,450-MHz microwaves for 5 þ 5 minutes and
C-5-FU-F-NP. ATR, attenuated total reflectance; C-5-FU-F-NP, chitosan-carboxymethyl-5-fluorouracil-folate conjugate submicron particles.

lipids when C-5-FU-F-NP was applied (Figure 3a and b).
Similarly, Raman spectroscopy studies were characterized by
reduced band intensity at 3,333e3,337 cme1, 2,700e2,900
cme1, and 1,300e1,600 cme1 corresponding to the respec-
tive O-H and/or N-H, C-H, and N-H/C-N domains of skin
(Figure 3e and f). Further analysis by differential scanning
calorimetry had the skin treated with submicron particles
exhibiting a lower melting temperature at 64.2  0.7  C,
which suggested that the skin lipid was fluidized (Figure 4a
and b). Overall, both skin lipid and protein domains could be
fluidized by C-5-FU-F-NP. This was partly responsible for the
increase in the extent of drug permeation and retention.

2416 Journal of Investigative Dermatology (2018), Volume 138


DSC T=66.1±1.5
a T=64.2±0.7
b skin thermogram. C-5-FU-F-NP,
Endotherm
T=63.1±0.8
c T=61.9±1.9
d *
Temperature (°C)
SEM
e f
g h
In the case of microwave treatment, FTIR analysis showed
that the epidermis and dermis of skin treated by microwaves
had larger wavenumbers at 3,310e3,370 cme1, corre-
sponding to O-H and/or N-H bonds of ceramide, keratin,
and/or lipids (Figure 3). The hydrogen bond strength of skin
could possibly be reduced through microwaves interacting
with the keratin and/or polar moieties of ceramide and lipid
materials in stratum corneum. Similarly, the wavenumbers of
FTIR peaks at 2,918.5  0.2 cme1 and 2,850.1  0.0 cme1 of
untreated epidermis and 2,919.2  0.9 cme1 of untreated
dermis increased in skin treated by microwaves (Figure 3).
The transdermal drug diffusion was promoted by the sum-
mative skin fluidization effects. The microwaves irradiating at
2,450 MHz for 5 þ 5 minutes could exert spacing of lipid
architecture of the skin in a random manner with no buildup
of specific structured domains, as suggested by the conver-
sion of skin epidermis into a smoother texture (Figure 4). With
respect to dermis, the treatment of skin by microwaves gave
A Nawaz and TW Wong
Microwave Modulated Nanoparticle Delivery
Figure 4. DSC thermograms and SEM
images. (aed) DSC thermograms and
(eeh) SEM images of (a,e) untreated
skin and skin treated with (b,f) C-5-
FU-F-NP, (c,g) 2,450-MHz
microwaves for 5 þ 5 minutes,
and (d,h) 2,450-MHz microwaves
for 5 þ 5 minutes and C-5-FU-F-NP.
*P < 0.05 with reference to untreated
chitosan-carboxymethyl-5-
fluorouracil-folate conjugate
submicron particles; DSC, differential
scanning calorimetry; SEM, scanning
electron microscopy.
rise to trans-lipid to gauche conformer transformation as
marked by the new FTIR peak at about 2,852 cme1 (Figure 3),
which is responsible for skin fluidization. Both epidermis and
dermis experienced the skin penetration enhancement effect
of microwaves. The effect was particularly marked when
microwave irradiation was used in combination with C-5-FU-
F-NP as the respective physical and chemical penetration
enhancers (Figure 3). The Raman spectroscopy and differen-
tial scanning calorimetry analysis of skin provided similar
outcomes as the FTIR evaluation. The skin treated with mi-
crowaves, especially with applied C-5-FU-F-NP, had a low
endothermic temperature at 61.9  1.9  C, an attribute of
low skin matrix interaction strength (P ¼ 0.006) (Figure 4).
The Raman spectroscopy peaks at 3,333e3,337 cme1,
2,700e2,900 cme1, and 1,300e1,600 cme1 corresponding
to the respective skin O-H and/or N-H, C-H, and N-H/C-N
bonds were characterized by reduced band intensity
(Figure 3). The skin applied with C-5-FU-F-NP and, to a larger
www.jidonline.org 2417
 A Nawaz and TW Wong
Microwave Modulated Nanoparticle Delivery

a C-5-FU-F-NP C-5-FU-F-NP; 2450 MHz, 5+5 min

b
30 10
*

Average Drug Retention (%)

9
25 8
Average Plasma Drug
Concentration (µg/ml)

7
20
6
15 5
4
10 3
2
5
1
0 0
0 5 10 15 20 25 C-5-FU-F-NP C-5-FU-F-NP; 2450 MHz, 5+5 min
Time (h)

c Parameter C-5-FU-F-NP C-5-FU-F-NP; 2450 MHz, 5 + 5 min

Cmax (µg/ml) 14.5 ± 10.5 16.4 ± 10.0
Tmax (h) 8 12

K (h )
-1
0.057 0.036
t1/2 (h) 12.1 18.8
AUC0-t (µg/ml.h) 163.9 ± 22.3 174.4 ± 23.2

Cmax: Peak plasma drug concentration; Tmax : Time at which Cmax was observed
K: Elimination rate constant; t1/2 : Elimination half life
AUC0-t : Area under the plasma concentration-time plot from 0 h to time

Figure 5. Pharmacokinetics. CMFU concentrations in (a) plasma and (b) skin of rats in vivo with reference to rats treated with C-5-FU-F-NP or microwaves at
2450 MHz for 5 þ 5 minutes followed by C-5-FU-F-NP and its (c) pharmacokinetics parameters. *P ¼ 0.133 with reference to C-5-FU-F-NP. C-5-FU-F-NP,
chitosan-carboxymethyl-5-fluorouracil-folate conjugate submicron particles; CMFU, carboxymethyl-5-fluorouracil; h, hour; min, minute.

extent, treated with microwaves had epidermal structure
domain collapse (Figure 4). This then enhanced the trans-
dermal drug delivery propensity.
Pharmacokinetics
Pharmacokinetics analysis in rats indicated that the C-5-FU-
F-NP attained its maximum plasma concentration (i.e., Cmax)
of 14.5 mg/ml 8 hours after administration (Figure 5c). Unlike
the in vitro diffusion study, the in vivo skin drug permeation
rate and extent did not vary significantly as a function of
pretreatment of rats with microwaves at 2,450 MHz for 5 þ 5
minutes, as suggested by insignificant differences between
plasma drug concentration-time profiles of rats administered
with submicron particles (analysis of variance, P > 0.05)
(Figure 5a). The area under the curve0et values were com-
parable in both cases (Figure 5c). Such observations could be
due to physiological differences between living and dead skin
tissue. The epithelial and endothelial barriers of living skin
are provided by tight junctions. The tight junctions are
intercellular locks characterized by extracellular proteins
such as claudins, occludin, tricellulin, and zonula occludin
(Sapra et al., 2012). Microwaves, with relatively long wave-
length, could penetrate the skin volumetrically and are in-
clined to interact with the polar moieties of a skin
constituent. The interaction between microwaves and skin
tight junctions is thought to reduce the width of the para-
cellular pathway. At the endothelium level, such a phenom-
enon could possibly reduce systemic drug availability,
thereby negating the rise in plasma drug level. A reduction in
the permeation tendency of the drug into systemic circulation
was corroborated by a tendency to have increased skin
retention extent of CMFU in rats pretreated with microwaves
(P ¼ 0.133) (Figure 5b). The findings implied that pretreat-
ment of skin with microwaves followed by C-5-FU-F-NP
application was favorable for local skin melanoma treatment.
Subsequent experiments that examined the cytotoxicity and
endocytosis profiles of C-5-FU-F-NP using melanoma cells,
as a function of microwave treatment, were then conducted.
Cytotoxicity
The anticancer property of a therapeutic agent is dependent
on its cytotoxicity. Both free 5-FU and drug-free submicron
particles induced melanoma cell death, with 5-FU being
more potent than the drug-free submicron particles (Figure 6).
The latter were constituted of chitosan, a cationic biopolymer
that had been reported to exhibit anticancer activity (Gibot
et al., 2015). 5-FU inhibits thymidylate synthase enzyme
and is mostly used against basal cell carcinoma and squa-
mous cell carcinoma. Recently, the human melanoma cell
line SKMEL-28 was found to be responsive to 5-FU (Cosco
et al., 2015). With reference to skin delivery, its usefulness
against melanoma treatment is nonetheless hindered by the
inadequate depth of penetration into the deep skin layers
(Neville et al., 2007). In the present investigation, the C-5-
FU-F-NP was found to enhance drug penetration through
skin. Via in vitro cell culture study, C-5-FU-F-NP was asso-
ciated with a higher level of cell death than the free drug and
drug-free submicron particles (Figure 6). This was a resultant
effect of the combination of the cytotoxic drug and submi-
cron particles, as well as possibly an enhancement of intra-
cellular 5-FU delivery through its conjugation to chitosan
submicron particles.
Microwaves negated the viability of SKMEL-28 cell line by
44.7  28.9% when the cell lines were treated with

2418 Journal of Investigative Dermatology (2018), Volume 138

 A Nawaz and TW Wong
Microwave Modulated Nanoparticle Delivery

Death Percentage

90
a
80

Average Cell Death (%)

70
60
50
40
30
20
10
0
5-FU Blank C-5-FU-F-NP 2450 MHz, 5+5 min C-5-FU-F-NP;
nanoparticles 2450MHz, 5+5 min

ATR-FTIR
b

2933.3±0.0
1078.1±0.5
2963.2±2.4
1403.0±0.7
Transmission (%)

1547.2±1.1

3292.6±3.4 1651.7±2.3
c

1078.8±0.1
2971.5±1.9 1404.3±0.6
1550.5±0.6

1653.8±0.3

3370.5±15.3
80
4000 3500 3000 2500 2000 1500 1000 650
Wavenumber (cm-1)

Total Cellular Fluorescence

d e
50000 Endocytic Inhibitors
45000
Total Cell Fluorescence

45000 2450 MHz 5+5 min; Endocytic Inhibitors

Total Cell Fluorescence

40000
40000
(A.U)

35000
(A.U)

35000

30000 30000

25000 25000

20000 20000
C-5-FU-F*-NP C-5-FU-F*-NP; Control Chlorpromazine Genistein Wortmannin Nystatin
2450 MHz, 5+5 min

Figure 6. In vitro cell culture studies. (a) Death percentage of melanoma cells after treatment with free drug, drug-free submicron particles, C-5-FU-F-NP, 2450-
MHz microwaves for 5 þ 5 minutes, and a combination of C-5-FU-F-NP and 2450-MHz microwaves for 5 þ 5 minutes. ATR-FTIR spectra of (b) melanoma cells
and (c) melanoma cells treated with microwave at 2450 MHz for 5 þ 5 minutes. Total cellular fluorescence of melanoma cells incubated with C-5-FU-F*-NP (d)
without and with pretreatment of cells with microwaves at 2450 MHz for 5 þ 5 minutes and (e) with pre-incubation of cells with different endocytic inhibitors
and with pretreatment of cells with microwaves at 2450 MHz for 5 þ 5 minutes followed by endocytic inhibitors. Chlorpromazine: clathrin pathway; genistein:
caveolae pathway; wortmannin: macropinocytosis; nystatin: lipid raft pathway. 5-FU, 5-fluorouracil; ATR, attenuated total reflectance; AU, arbitrary unit; C-5-
FU-F-NP, chitosan-carboxymethyl-5-fluorouracil-folate conjugate submicron particles; FTIR, Fourier transform infrared spectroscopy.

microwaves at the frequency of 2,450 MHz for 5 þ 5 mi- outcomes of attenuated total reflectance FTIR studies. The
nutes. The cytotoxic effect of microwaves was apparently FTIR peaks at 3,292.6  3.4 cme1 corresponding to an O-H
greater than that of the free drug and was comparable to that and/or N-H moiety and 2,963.2  2.4 cme1 corresponding to
of C-5-FU-F-NP. The cytotoxicity of microwaves could be an a CH2 moiety of both lipids and/or proteins of the untreated
attribute of its interaction with the cell membrane/nuclei of cells (Srisayam et al., 2014) were shifted to higher wave-
the melanoma. The microwaves could fluidize the lipid/ numbers at 3,370.5  15.3 and 2,971.5  1.9 cme1
protein regimes of melanoma cells, as reflected by the (Figure 6) when the cells were treated with microwaves. The

www.jidonline.org 2419
 A Nawaz and TW Wong
Microwave Modulated Nanoparticle Delivery
amide I and II of protein regimes at 1,651.72.3 and 1,547.2
 1.1 cm-1 similarly had their wavenumbers increased
(Figure 6). The summative observations suggest that micro-
waves interacted with the cells via O-H/N-H, CH2, and C-N
moieties, fluidizing the membrane and/or other cellular
components. The level of cytotoxicity is ultimately dependent
on the degree of membrane disruption (Weber et al., 2008).
The combination of C-5-FU-F-NP and microwaves brought
about an average cell death rate of 67.4  16.7% (Figure 6).
The interaction of microwaves and lipid/protein contents of
the cells could induce cell death and synergistically increase
the membrane permeability of cells toward submicron par-
ticles/drug, thereby enhancing the cytotoxicity of C-5-FU-F-
NP. The latter was evidenced by an enhanced intracellular
trafficking of fluorescence-labelled C-5-FU-F*-NP by mela-
noma cells treated with microwaves (Figure 6).
Endocytosis
Physicochemical properties such as particle size, shape,
molecular weight, surface charge, and composition play a
key role in the cellular uptake of polymeric nanoparticles
(Chavanpatil & Panyam, 2006). The submicron particles may
enter cells via different pathways as a function of their
physicochemical attributes. The C-5-FU-F*-NP was
endocytosed by the melanoma cells via a combination of
receptor-mediated pathways in the progress of lipid rafts >
macropinocytosis > clathrin z caveolae (Figure 6e). As in
previous studies (Huang et al., 2002), chitosan submicron
particles tended to be endocytosed via the clathrin-mediated
pathway at the respiratory epithelium (A549 cells) and in-
testinal epithelium (Caco-2 cells) regardless of their sizes. It
was suggested that chemical composition can be more
important than size in defining the said pathway of endocy-
tosis (Huang et al., 2002). The self-assembled cationic
nanoparticles of hydrophobically modified chitosan, on the
other hand, use multiple pathways for cellular entry
including clathrin, caveolae, and macropinocytosis routes
(Nam et al., 2009). In this study, conjugation of chitosan
submicron particles with folate and 5-FU likewise rendered
them with the ability to use multiple pathways for intracel-
lular trafficking by melanoma cells. The pretreatment of
melanoma cells by microwaves before their incubation with
C-5-FU-F*-NP promoted the submicron particles being
endocytosed via the lipid raft-mediated pathways and
appeared to reduce the endocytosis tendency of particles by
means of clathrin, caveolae, and macropinocytosis pathways
when compared with untreated melanoma cells (Figure 6e).
This might be associated with microwaves enabling the
fluidization of lipid domains of the cellular membrane, as
inferred from the present studies of changes in skin epidermis
and dermis in response to microwave irradiation.
CONCLUSION
The extent of skin drug penetration and retention of 5-FUe
and folate-conjugated chitosan submicron particles was
facilitated through treating the skin with microwaves (2,450
MHz, 5 þ 5 minutes), as a result of fluidization of epidermal
and dermal protein/lipid domains, which in turn increased
the diffusion of particles/drug into the skin tissue. The extent
of cellular uptake of submicron particles by melanoma and
2420 Journal of Investigative Dermatology (2018), Volume 138
their cytotoxicity were significantly enhanced by pretreating
these cells with microwaves. The intracellular trafficking of
submicron particles by melanoma cells was mediated via
clathrin, caveolae, macropinocytosis, and lipid raft endocy-
totic pathways. It was promoted by pretreatment of cells with
microwaves through facilitating the endocytosis of submicron
particles via lipid raft pathways. The combination of micro-
waves and submicron particle technology synergistically
promoted drug/particle uptake by skin tissue and melanoma
cells, as well as cytotoxicity, instead of being a mere sub-
micron particulate system.
MATERIALS AND METHODS
Materials
Chitosan (Zhejiang Aoxing Co. Ltd., Zhejiang, China) was selected as
the matrix polymer, with 5-FU AoBo Bio-Pharmaceutical Technology
Co. Ltd., Shanghai, China) as the anticancer drug and folic acid (Acros
Organics, Morris, New Jersey) as the cancer-targeting ligand. Other
materials are listed in the Supplementary Materials online.
Preparation of low molecular weight chitosan
Low molecular weight chitosan was prepared by subjecting 2%
weight/weight solution of chitosan dissolved in 2% volume/volume
acetic acid (Merck, Germany) solution to microwave irradiation at
800 W for 9 minutes in the presence of 0.04% weight/weight of
sodium chloride and characterized as reported by Nawaz and Wong
(2016) (see Supplementary Materials).
Preparation of C-5-FU-F-NP. For preparation of carbox-
ymethyl-5-FU, we use the method reported by Tada (1975) with
slight modifications as described by Nawaz and Wong (2017). The
C-5-FU-F conjugate was synthesized using the modified method of
Li et al. (2011) (see Supplementary Materials). FTIR spectroscopy
(Spectrum RX1 system, PerkinElmer, Waltham, MA) and Ultra-
shieldPlus 500 nuclear magnetic resonance (Bruker, Bremen,
Germany) techniques were used to evaluate the conjugation status of
C-5-FU-F. High-performance liquid chromatography was used to
determine the carboxymethyl-5-FU content of the conjugate. At least
three replicates were conducted for each experiment, with the re-
sults averaged. The conjugate submicron particles were prepared by
nanospray-drying (TwinNanoSpray; UiTM, Puncak Alam, Malaysia)
50 mg conjugate with 0.6% weight/weight Tween 20 and 0.4%
weight/weight Span 20 in 99.75 g of 1% volume/volume acetic acid
solution (see Supplementary Materials).
Physicochemical characterization
The particle size and zeta potential of C-5-FU-F-NP were determined
by photon correlation spectroscopy technique (Malvern Zetasizer
Nano ZS 90, Malvern Instruments Ltd., UK). Scanning electron mi-
croscopy (Quanta FEG 450; FEI, Eindhoven, The Netherlands) was
used to evaluate the surface morphology of C-5-FU-F-NP (Al-Azi
et al., 2014). The C-5-FU-F-NP was subjected to acid hydrolysis and
had its carboxymethyl-5-FU content determined by high-performance
liquid chromatography analysis (Zhang et al., 2010). The drug release
profile of C-5-FU-F-NP was assayed by Franz diffusion cell (Hanson
MicroettePlus, Chatsworth, CA). Both drug content and encapsulation
efficiency of the submicron particles were determined (Nawaz &
Wong, 2017). At least three replicates were conducted for each
sample, and the results were averaged (see Supplementary Materials).

Biological characterization
Skin C-5-FU-F-NP and 5-FU permeation and retention were exam-
ined in vitro using skin harvested from healthy male Sprague Dawley
rats (Anuar & Wong, 2013; Nawaz & Wong, 2017). Skin C-5-FU-F-
NP content and distribution were characterized with a PerkinElmer
Spotlight 400 imaging system (Nawaz & Wong, 2016). At least three
replicates were conducted for each sample, and the results were
averaged (see Supplementary Materials).
Microwave treatment of skin
Microwaves were used to modify the physicochemical properties of
skin. They were applied to the top epidermal surfaces using a vector
network analyzer system (R&S ZVA, Rohde & Schwarz, Germany),
where 1 mW of microwaves (0.14 mW/cm2) was delivered at 2,450
MHz for 5 þ 5 minutes nonthermally, with no observable skin
damage and in the contact mode to avoid electromagnetic loss (Jain
et al., 2010). The permeation/retention profiles of drug/submicron
particles were characterized against untreated skin. The physico-
chemical changes of skin and their relations to skin drug/submicron
particle transport were assessed by subjecting skin to attenuated total
reflectance FTIR, Raman spectroscopy, differential scanning calo-
rimetry, and scanning electron microscopy analysis (Nawaz &
Wong, 2017).
Pharmacokinetics analysis
Two groups of male Sprague Dawley rats (200e250 g each, n ¼ 6/
group; LAFAM, Malaysia) were anesthetized and had the dorsal
region shaved. Group A was placed with 15 mg of solid submicron
particles. Group B was first treated by microwaves at 2,450 MHz
for 5 þ 5 minutes from the direction of epidermis using a contact
mode vector network analyzer system followed by 15 mg of solid
submicron particles. The blood sample was collected periodically
and the plasma drug concentration was examined by high-
performance liquid chromatography. From the same experiment,
the treated skin area was removed from the killed rats and skin
drug retention was analyzed using the high-performance liquid
chromatography. All experiments were approved by the university
ethics committee based on international guidelines (Organisation
for Economic Co-operation and Development Environment,
Health, and Safety) for animal experimentation (see Supplementary
Materials).
Cell culture study
The cytotoxicity of C-5-FU-F-NP as a function of microwaves was
evaluated in vitro on SKMEL-28 melanoma cells (ATCC, Manassas,
VA) using MTT assay. Free 5-FU (150 mg/200 ml of phosphate buffer
saline, pH 7.4) and/or submicron particles (1,110 mg/200 ml) were
introduced with cells pretreated with microwaves at 2,450 MHz for
5 þ 5 minutes using a non-contact mode vector network analyzer
system when required. The biological alterations of melanoma cells
after their treatment with microwaves were examined by subjecting
the freeze-dried cell line to attenuated total reflectance FTIR spec-
troscopy analysis. The cellular uptake of submicron particles was
evaluated by first covalently linking FITC (Acros Organics) to C-5-
FU-F conjugate to form C-5-FU-F*, followed by nanospray-drying
as previously described and cellular internalization profiling of C-
5-FU-F*-NP by a laser scanning confocal microscope (TCS SPE
Leica; Leica, Wetzlar, Germany). In the same study, the endocytic
pathways of submicron particles were examined through intro-
ducing and incubating melanoma cells with pharmacological
membrane entry inhibitors: 7 mg/ml chlorpromazine (Calbiochem,
A Nawaz and TW Wong
Microwave Modulated Nanoparticle Delivery
San Diego, CA) (Chiu et al., 2010) were used to inhibit the formation
of clathrin vesicles, 200 mmol/L genistein (Calbiochem) (Perumal
et al., 2008) to inhibit caveolae pinching, 500 nmol/L Wortmannin
(Calbiochem) (Chiu et al., 2010) to inhibit phosphatidylinositol
3-kinase (macropinocytosis), and 25 mg/ml nystatin (Calbiochem)
(Ivanov, 2008) to interact with cholesterol (lipid rafts). At least three
replicates were conducted, and results were averaged (see
Supplementary Materials).
Statistical analysis
Results were expressed as a mean of at least three experiments with
the corresponding standard deviation. Statistical data analysis was
carried out using SPSS software, version 18.0 (IBM, Armonk, NY)
and a statistically significant difference was denoted by P less than
0.05. Student t test and analysis of variance/post hoc analysis by
Tukey honest significance test were used when applicable.
CONFLICT OF INTEREST
The authors state no conflict of interest.
ACKNOWLEDGMENTS
The authors wish to thank MOSTI, MOHE, and UiTM (0141903; Bestari
Perdana) for facility and fund support and Gomal University Pakistan for PhD
scholarship support.
SUPPLEMENTARY MATERIAL
Supplementary material is linked to the online version of the paper at www.
jidonline.org, and at https://doi.org/10.1016/j.jid.2018.04.037.
REFERENCES
Al-Azi SOSM, Tan YTF, Wong TW. Transforming large molecular weight
pectin and chitosan into oral protein drug nanoparticulate carrier. React
Funct Polym 2014;84:45e52.
Anuar NK, Wong TW. Microwave: effects and implications in transdermal
drug delivery. Prog Electromagn Res 2013;141:619e43.
Bain C, Cooper KG, Parkin DE. Microwave endometrial ablation versus
endometrial resection: a randomized controlled trial. Obstet Gynecol
2002;99:983e7.
Banik S, Bandyopadhyay S, Ganguly S. Bioeffects of microwaveeea brief
review. Bioresour Technol 2003;87:155e9.
Byrne JD, Betancourt T, Brannon-Peppas L. Active targeting schemes for
nanoparticle systems in cancer therapeutics. Adv Drug Deliv Rev 2008;60:
1615e26.
Castle SM, Salas N, Leveillee RJ. Initial experience using microwave ablation
therapy for renal tumor treatment: 18-month follow-up. Urology 2011;77:
792e7.
Chavanpatil AKMD, Panyam J. Nanoparticles for cellular drug delivery:
mechanisms and factors influencing delivery. J Nanosci Nanotechnol
2006;6:2651e63.
Chen Y, Bathula SR, Yang Q, Huang L. Targeted nanoparticles deliver siRNA
to melanoma. J Invest Dermatol 2010;130:2790e8.
Chiu YL, Ho YC, Chen YM, Peng SF, Ke CJ, Chen KJ, et al. The characteristics,
cellular uptake and intracellular trafficking of nanoparticles made of
hydrophobically-modified chitosan. J Control Release 2010;146:152e9.
Cosco D, Paolino D, Maiuolo J, Di Marzio L, Carafa M, Ventura CA, et al.
Ultradeformable liposomes as multidrug carrier of resveratrol and
5-fluorouracil for their topical delivery. Int J Pharm 2015;489:1e10.
Gibot L, Chabaud S, Bouhout S, Bolduc S, Auger FA, Moulin VJ. Anticancer
properties of chitosan on human melanoma are cell line dependent. Int J
Biol Macromol 2015;72:370e9.
He W, Guo X, Xiao L, Feng M. Study on the mechanisms of chitosan and its
derivatives used as transdermal penetration enhancers. Int J Pharm
2009;382:234e43.
Herrero MA, Kremsner JM, Kappe CO. Nonthermal microwave effects
revisited: on the importance of internal temperature monitoring and
agitation in microwave chemistry. J Org Chem 2008;73:36e47.
www.jidonline.org 2421
 A Nawaz and TW Wong
Microwave Modulated Nanoparticle Delivery
Hompes R, Fieuws S, Aerts R, Thijs M, Penninckx F, Topal B. Results of single-
probe microwave ablation of metastatic liver cancer. Eur J Surg Oncol
2010;36:725e30.
Huang M, Ma Z, Khor E, Lim LY. Uptake of FITC-chitosan nanoparticles by
A549 cells. Pharm Res 2002;19:1488e94.
Huang R, Mendis E, Rajapakse N, Kim SK. Strong electronic charge as an
important factor for anticancer activity of chitooligosaccharides (COS). Life
Sci 2006;78:2399e408.
Ivanov AI. Pharmacological inhibition of endocytic pathways: is it specific
enough to be useful? Methods Mol Biol 2008;440:15e33.
Jain A, Jain SK, Ganesh N, Barve J, Beg AM. Design and
development of ligand-appended polysaccharidic nanoparticles for
the delivery of oxaliplatin in colorectal cancer. Nanomedicine
2010;6:179e90.
Jones C, Badger SA, Ellis G. The role of microwave ablation in the manage-
ment of hepatic colorectal metastases. Surgeon 2011;9:33e7.
Leamon CP, Reddy JA. Folate-targeted chemotherapy. Adv Drug Deliv Rev
2004;56:1127e41.
Li P, Wanga Y, Zeng F, Peng Z, Kong LX. Synthesis and characterization of
folate conjugated chitosan and cellular uptake of its nanoparticles in HT-29
cells. Carbohydr Res 2011;346:801e6.
Liu Y, Zheng Y, Li S, Li B, Zhang B, Yuan Y. Percutaneous microwave ablation
of larger hepatocellular carcinoma. Clin Radiol 2013;68:21e6.
López AFCM, Llinares F, Cortell C, Herraez M. Comparative enhancer effects
of Span 20 with Tween 20 and Azone on the in vitro percutaneous
penetration of compounds with different lipophilicities. Int J Pharm
2000;202:133e40.
Nam HY, Kwon SM, Chung H, Lee SY, Kwon SH, Jeon H, et al. Cellular
uptake mechanism and intracellular fate of hydrophobically modified
glycol chitosan nanoparticles. J Control Release 2009;135:259e67.
Naves LB, Dhand C, Venugopal JR, Rajamani L, Ramakrishna S, Almeida L.
Nanotechnology for the treatment of melanoma skin cancer. Prog Biomater
2017;6(1e2):13e26.
Nawaz A, Wong TW. Microwave as skin permeation enhancer for trans-
dermal drug delivery of chitosan-5-fluorouracil nanoparticles. Carbohydr
Polym 2017;157:906e19.
Nawaz A, Wong TW. Quantitative characterization of chitosan in the skin by
Fourier-transform infrared spectroscopic imaging and ninhydrin assay:
application in transdermal sciences. J Microsc 2016;263:34e42.
Neville JA, Welch E, Leffell DJ. Management of nonmelanoma skin cancer in
2007. Nat Clin Pract Oncol 2007;4:462e9.
Perumal OP, Inapagolla R, Kannan S, Kannan RM. The effect of surface
functionality on cellular trafficking of dendrimers. Biomaterials 2008;29:
3469e76.
Rai S, Singh UP, Mishra GD, Singh SP. Samarketu. Additional evidence of
stable EMF-induced changes in water revealed by fungal spore germina-
tion. Electromagn Biol Med 1994a;13:253e9.
Rai S, Singh UP, Mishra GD, Singh SP. Samarketu. Effect of water’s microwave
power density memory on fungal spore germination. Electromagn Biol Med
1994b;13:247e52.
Reddy JA, Allagadda VM, Leamon CP. Targeting therapeutic and imaging
agents to folate receptor positive tumors. Curr Pharm Biotechnol 2005;6:
131e50.
Rodriguez-Lopez JN, Cabezas-Herrera J, Sanchez L, Montenegro MF, Cabezas-
Herrera J. Novel antifolates as produgs for the treatment of melanoma. In:
Murph M, editor. Research on melanoma—a glimpse into current directions
and future trends. INTECH Open Publisher; 2011. p. 101e24.
Sánchez-del-Campo L, Montenegro MF, Cabezas-Herrera J, Rodrı́guez-
López JN. The critical role of alpha-folate receptor in the resistance of
2422 Journal of Investigative Dermatology (2018), Volume 138
melanoma to methotrexate. Pigment Cell Melanoma Res 2009;22:
588e600.
Sapra MB, Jindal AK, Tiwary. Tight junctions in skin: new perspectives. Ther
Deliv 2012;3:1297e327.
Skinner CC, McMichael EL, Jaime-Ramirez AC, Abrams ZB, Lee RJ,
Carson WE III. Folate-conjugated immunoglobulin targets melanoma
tumor cells for NK cell effector functions. Melanoma Res 2016;26:
329e37.
Souza JG, Gelfuso GM, Simão PS, Borges AC, Lopez RF. Iontophoretic
transport of zinc phthalocyaninetetrasulfonic acid as a tool to improve drug
topical delivery. Anticancer Drugs 2011;22:783e93.
Srisayam M, Weerapreeyakul N, Barusrux S, Tanthanuch W, Thumanu K.
Application of FTIR microspectroscopy for characterization of biomole-
cular changes in human melanoma cells treated by sesamol and kojic acid.
J Dermatol Sci 2014;73:241e50.
Tada M. Antineoplastic agents. The preparation of 5-fluorouracil-1-acetic acid
derivatives. Bull Chem Soc Jpn 1975;48:3427e8.
Taveira SF, Lopez RFV. Topical administration of anticancer drugs for skin
cancer treatment. In: Porta C, editor. Skin cancers—risk factors, prevention
and therapy. INTECH Open Publisher; 2011. p. 247e72.
Taveira SF, Nomizo A, Lopez RF. Effect of the iontophoresis of a chitosan gel
on doxorubicin skin penetration and cytotoxicity. J Control Release
2009;134:35e40.
Vogl TJ, Naguib NN, Lehnert T, Nour-Eldin NE. Radiofrequency, microwave
and laser ablation of pulmonary neoplasms: clinical studies and technical
considerations—review article. Eur J Radiol 2011;77:346e57.
Weber N, Ortega P, Clemente MI, Shcharbin D, Bryszewska M, de la
Mata FJ, et al. Characterization of carbosilane dendrimers as effective
carriers of siRNA to HIV-infected lymphocytes. J Control Release
2008;132:55e64.
Weitman SD, Lark RH, Coney LR, Fort DW, Frasca V, Zurawski VR, et al.
Distribution of the folate receptor GP38 in normal and malignant cell lines
and tissues. Cancer Res 1992a;52:3396e401.
Weitman SD, Weinberg AG, Coney LR, Zurawski VR, Jennings DS,
Kamen BA. Cellular localization of the folate receptor: potential role in
drug toxicity and folate homeostasis. Cancer Res 1992b;52:6708e11.
Wong TW. Electrical, magnetic, photomechanical and cavitational waves to
overcome skin barrier for transdermal drug delivery. J Control Release
2014;193:257e69.
Wong TW, Anuar NK. Physicochemical modulation of skin barrier by
microwave for transdermal drug delivery. Pharm Res 2013;30:90e103.
Wu J, Liu Q, Lee RJ. A folate receptor-targeted liposomal formulation for
paclitaxel. Int J Pharm 2006;316:148e53.
Xia W, Liu P, Zhang J, Chen J. Biological activities of chitosan and chitooli-
gosaccharides. Food Hydrocoll 2011;25:170e9.
Xia WS. Physiological activities of chitosan and its application in functional
foods. J Chin Institute Food Sci Technol 2003;3:77e81.
Yeh TH, Hsu LW, Tseng MT, Lee PL, Sonjae K, Hoe YC, Sung HW. Mechanism
and consequence of chitosan-mediated reversible epithelial tight junction
opening. Biomaterials 2011;32:6164e73.
Yu MA, Liang P, Yu XL, Cheng ZG, Han ZY, Liu FY, et al. Sonography-guided
percutaneous microwave ablation of intrahepatic primary chol-
angiocarcinoma. Eur J Radiol 2011;80:548e52.
Yue W, Wang S, Wang B, Xu Q, Yu S, Yonglin Z, et al. Ultrasound guided
percutaneous microwave ablation of benign thyroid nodules: safety and
imaging follow-up in 222 patients. Eur J Radiol 2013;82:11e6.
Zhang Z, Zhang Q, Wang J, Shi X, Zhang J, Song H. Synthesis and drug
release in vitro of porphyran carrying 5-fluorouracil. Carbohydr Polym
2010;79:628e32.