Macromolecular Research, Vol. 22, No.

10, pp 1125-1131 (2014)

DOI 10.1007/s13233-014-2162-2

www.springer.com/13233
pISSN 1598-5032 eISSN 2092-7673

Enhanced Therapeutic Efficacy of Lipophilic Amphotericin B Against

Candida albicans with Amphiphilic Poly(N-isopropylacrylamide) Nanogels
Muhammad Qasim, Phornsawat Baipaywad, Nopphadol Udomluck, Dokyun Na*, and Hansoo Park*
School of Integrative Engineering, Chung-Ang University, Seoul 156-070, Korea
Received June 11, 2014; Revised July 15, 2014; Accepted July 20, 2014
Abstract: Candidiasis is a disease with skin rashes caused by the infectious fungus, Candida albicans, and can be
potentially life-threatening, especially in immunodeficient patients. Amphotericin B (AmB) is a potent antifungal
agent with a broad spectrum to treat Candidiasis, but its inherent low solubility and limited skin bioavailability have
prevented its wider clinical use. In this study, we developed poly(N-isopropylacrylamide) (pNIPAM)-based nanogels as a
versatile carrier to enhance AmB solubility and its efficacy. pNIPAM nanogels enhanced the solubility of AmB by 1.6-fold
versus AmB alone. Accordingly, pNIPAM markedly improved the minimal fungicidal concentration (MFC) of AmB
by 8 folds from 3.91 to 0.49 g/mL. We also attached NH2 group to the pNIPAM nanogel to increase surface charges
and investigated its effect on AmB antifungal activity. pNIPAM-NH2 has comparable activity in solid culture to pNIPAM, but 2-fold higher antifungal activity in liquid culture. Our pNIPAM and pNIPAM-NH2 nanogels may be useful
as drug delivery agents for the treatment of local antifungal infections by AmB in solid and liquid environments,
such as on skin and in blood, with high efficacy and sustainability.
Keywords: amphotericin B, solubility, nanogels, poly(N-isopropylacrylamide) (pNIPAM), Candida albicans, Candidiasis.

Introduction

vehicles such as liposomes, colloidal dispersions, micelles, metal

particles, dendrimers, microsphere and nanogels.4,14-16 Of these
diverse delivery agents, nanogels that are a physically and
chemically cross-linked network of polymers have gained great
interest as an efficient drug delivery agent due to advantages
over other agents: easiness of surface modification, high water
content, superior colloidal stability, and large capacity to
encapsulate drugs inside their network.17-21 Nanogels are able
to deliver encapsulated drugs into the cellular membrane and
through the cell membrane as well due to their smooth and
elastic layered surface leading to high permeability.22
N-Isopropylacrylamide (NIPAM) is often utilized as a main
monomer in nanogel fabrication, since poly(N-isopropylacrylamide) (pNIPAM) nanogels are biocompatible and are able to
control release rate of incorporated drugs, and their size can
be easily modified using surfactants such as sodium dodecyl
sulfate (SDS).23 Moreover, they can respond to external stimuli
such as temperature and pH, enabling controlled release of drugs
depending on environmental conditions.24-26 Mono-dispersed
pNIPAM nanogels are fabricated through free-radical crosslinking
polymerization with water soluble radicals:23,27 ammonium
persulfate (APS) and potassium persulfate (KPS) are initiators
of polymerization and di-vinyl groups such as N,N-methylenebisacrylamide (BIS) are cross-linkers.28,29 Morphological and
mechanical properties of pNIPAM nanogels are easily modified
by adjusting the ratio of monomer to cross-linker.25,26,30,31
Moreover, pNIPAM nanogels can copolymerize with other

Candida albicans is one of major human fungal pathogens,

which causes mucosal and systemic infections.1 Such infection
is often difficult to eradicate and may emerge as lethal depending on the immune status of patients. Particularly, patients with
immune-compromised diseases such as acquired immune deficiency syndrome (AIDS) are life-threatened by the infection
of C. albicans.2-4 C. albicans often causes vulvovaginal candidiasis in women.5 Clinical studies have shown that pregnant women
are more vulnerable to Candidas in their vaginal flora.5-7 Among
various antifungal drugs Amphotericin B (AmB), possessing
a broad spectrum of antifungal activity,1,8 is used for the treatment
of C. albicans.8-10 AmB is an organic polyene compound that
binds to the membrane sterols of Candidas and forms transmembrane channels, which results in cell leakage and consequently cell death.1,11-13
Despite of the promising antifungal activity of AmB, its
clinical application has been limited due to its poor bioavailability
resulting from its inherent low solubility.1,14 Reported adverse
effects of AmB mainly due to its low solubility include renal
failure, hypokalemia, hypomagnesaemia, and polyuria.12 There
have been many attempts to improve AmB solubility and
thereby reduce its associated cytotoxicity by utilizing efficient
*Corresponding Authors. E-mails: heyshoo@cau.ac.kr or
blisszen@cau.ac.kr
The Polymer Society of Korea

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Scheme I. Delivery of AmB into the fungus cell wall by nanogels.

(a) Fungus cell membrane (b) delivery of AmB to the membrane
via passive diffusion. (c) Delivery mediated by a nanogel system
that binds to membrane.

monomers with different functional groups such as acrylic

acid (AAc) and N-(3-amino propyl) methacrylamide hydrochloride (APMAAHC).30 The cationic charges on the surface
of nanoparticles have been reported to improve drug solubility
and allow strong interaction with biological surfaces with
negative charges such as cell membrane.3,32,33 Easiness in surface modification, biocompatibility, high solubility, and high
permeability make pNIPAM nanogels a promising vehicle
for targeted drug delivery.25
In this study, we fabricated pNIPAM-based nanogels with
different sizes and surface charges to improve the solubility of
AmB and thereby to enhance its antifungal activity (Scheme I).
To investigate the effects of size and surface charge we evaluated
properties of pNIPAM nanogels including solubility, drug release
rate, and antifungal activity in liquid and solid culture.

Experimental
Materials. N-Isopropylacrylamide (NIPAM) was purchased
from Sigma-Aldrich Co LLC Korea and was used in this study
after further purification through recrystallization. Ammonium
persulfate (APS), N,N-methylenebisacrylamide (BIS) and N(3-amino propyl) methacrylamide hydrochloride (APMAAHC)
were purchased from Polysciences, Inc. USA. AmB (Streptomyces sp.) in powder was purchased by Sigma-Aldrich Co
LLC. Methanol at HPLC grade was purchased from Merck
Co Ltd Korea. Muller Hilton (MH) media broth and agar were
purchased form Oxoid Ltd England. C. albicans strain (ATCC
64124) was purchased from ATCC.
1126

Methods.
Synthesis of pNIPAM Based Nanogels: pNIPAM nanogels were fabricated through a conventional radical polymerization of NIPAM.23,28 29 Different sizes of pNIPAM nanogels
ranging from 450 to 700 nm were produced depending on the
ratio of cross-linker, BIS. Specifically, 0.95 g of NIPAM with
different ratios of BIS (0.026 g for 50:1, 0.013 g for 100:1,
0.0065 g for 200:1) was dissolved into 195 mL of distilled
water and placed in a three-necked round bottom flask with
constant supply of argon. The solution was purged with argon
for 1 h and heated to 58-65 oC. Then, 5 mL of APS solution
(0.12 g of APS dissolved in 5 mL of water) was injected into
the solution to initiate polymerization and this reaction was
allowed to proceed for 4 h. Argon gas was purged through the
solution until the end of the reaction to avoid any contact of
oxygen, which may intercept radicals and disrupt the polymerization. The resulting dispersion was dialyzed in deionized
water using porous membrane (6-8000Mw, Spectra/Por) and
then freeze-dried. As a result, we obtained three different sizes
of pNIPAM (43027 nm, 56013 nm and 70076 nm). To fabricate nanogels with amine (-NH2) group 0.065 g of APMAAHC
was additionally supplied to the reaction solution (0.95 g of NIPAM
and 0.026 g of BIS) to generate 430 nm of nanogels and obtained
45041 nm of pNIPAM-NH2. The slightly increased size of
pNIPAM-NH2 was due to the addition of co-monomers. The
sizes of nanogels was characterized by dynamic light scattering
(DLS).34
AmB Solubility Assay: The solubility of AmB was measured at 21 oC (room temperature) and pH7 with pNIPAM
nanogel (43027 nm, 56013 nm, and 70076 nm) and with
pNIPAM-NH2 (51140 nm) nanogel. The solubility of AmB
was determined according to the previously reported shaken
flask method with modifications.35 Briefly, an excessive amount
of AmB (1 mg/mL) was added to each vial containing 2 mL of
phosphate buffered saline (PBS), and pNIPAM or pNIPAMNH2 was added into the vial at final concentrations of 0-8 mg/mL.
These mixtures were shaken at 100 rpm for 24 h at room temperature and allowed to stand for additional 24 h for equilibrium. Then, the mixtures were centrifuged at 13,500 rpm for 20
min at room temperature. 200 L of supernatant was taken
from the mixtures and its absorbance was measured at 435 nm
to assess the concentration of dissolved AmB.36 As a reference,
we dissolved AmB in dimethyl sulfoxide (DMSO) at different
concentrations and measured their absorbance at 435 nm.
DMSO dissolves AmB completely and does not interfere with
AmB absorbance.37
AmB Release Assay: The release rate of AmB from nanogels was investigated at room temperature. 2 mg of pNIPAM
or NIPAM-NH2 and 2 mg of AmB were dissolved in 2 mL of
PBS. This mixture was shaken for 24 h at room temperature
and then was centrifuged at 13,500 rpm for 20 min. 1 mL aliquot
was taken from the sample and AmB concentration was measured at 435 nm. Equivalent fresh PBS was added to the sample
to keep its volume constant. Accumulative released concenMacromol. Res., Vol. 22, No. 10, 2014

pNIPAM Nanogels for Enhanced Efficacy of AmB

tration of AmB over 6 days was plotted. Each sample in this

study was in triplicate.
Antifungal Activity Assessment:
Incubation of infectious C. albicans
C. albicans (ATCC 64124) was cultured on MH agar and
MH broth according to the recommended protocol of ATCC.
All culture media were prepared according to the manufacturers instruction. Briefly, freeze-dried pellet was dissolved
with sterile water (5-6 mL) in a test tube at room temperature
for 48 h, since this fungus requires time to escape from a dormant stage. Then, the suspension was spread on solid MH
agar and incubated at 37 oC for 24 h.
Evaluation of Antifungal Activity in Liquid Culture
Antifungal activity was measured according to the broth
micro-dilution method from the Clinical and Laboratory Standards
Institute (CLSI) guidelines.38,39 A single colony was inoculated
into 5 mL of sterile 0.9% NaCl solution. This suspension was
vortexed for 15 seconds and cell density was adjusted to the
density of 0.5 McFarland scale. The initial density of C. albicans
was approximately 1-5106 colony forming units (CFU)/mL,
which was equivalent to 0.5 McFarland scale.35,38 Then the
cells were suspended again in MH media at a final density of
5102 CFU/mL. Individual solutions of pNIPAM or pNIPAMNH2 nanogels with different final concentrations of AmB
(0.03-5 g/mL) were mixed with 1 mL of fungal suspension
(5102 CFU/mL). After 24 h of incubation, 10 L aliquot from
each sample was withdrawn and cultured on MH agar plate at
37 oC for 24 h and minimal fungicidal concentration (MFC)
was determined. MFC is the lowest concentration of AmB to kill
all fungus cells and thereby no colonies appear on an agar plate.
Assessment of Antifungal Activity with Plate Diffusion Method
To investigate the antifungal activity of AmB encapsulated
into nanogels on solid media, C. albicans (1106 cells/mL) was
cultured on MH agar plate and the plate was dried at room
temperature. A well with 8 mm diameter was made in each plate
by gel puncture. Stock solutions of AmB alone and AmB encapsulated into pNIPAM or pNIPAM-NH2 (AmB:nanogel=1:1)
were prepared separately (5 mg/mL) in PBS. 100 L of the
individual stock solution containing 0.5 mg of AmB and 0.5 mg
of nanogel was applied on the plates. The plate was incubated
at 37 oC for 24 h. The antifungal activity was measured as the
average diameter (mm) of zone of inhibition around each well.
Statistical Analysis: All data were statistically analyzed by
one-way analysis of variance (ANOVA) followed by Tukeys
multiple comparisons test. The p-value < 0.05 was considered
as evidence of a significant difference.

Results and Discussion

Three Different Sizes of pNIPAM and their Effect on
AmB Solubility. AmB is a polyene derivative with a broad
spectrum of antifungal activity.8 Despite of clinical benefits of
AmB to the treatment of candidiasis, its application is limited
due to its inherent low solubility.11 Poor bioavailability of AmB
Macromol. Res., Vol. 22, No. 10, 2014

Figure 1. Average size of fabricated pNIPAM nanogels with different ratios of pNIPAM to BIS. The exact sizes of 200:1, 100:1, and
700:1 are 43027 nm, 56013 nm, and 70076 nm, respectively.

stemming from its low solubility devalues the promising clinical efficacy of AmB.1 Here we develop amphiphilic pNIPAM
nanogels capable of incorporating lipophilic AmB and increasing the solubility of AmB and thereby improving therapeutic
efficacy.
Since the size of pNIPAM nanogels may affect the permeability
of AmB into the cellular membrane25,28,40 and its capacity to
encapsulate AmB, pNIPAM nanogels with different sizes were
prepared by adjusting the ratio of monomer (NIPAM) to crosslinker (BIS) (50:1, 100:1, 200:1) and then encapsulated with
AmB. As the ratio increased, the diameter of pNIPAM nanogel
also increased (Figure 1).
With the three different pNIPAM nanogels, we investigated
the effect of their size on AmB solubility. The pNIPAM nanogels were mixed with AmB and then concentration of dissolved
AmB was measured to calculate solubility. Interestingly, as
shown in Figure 2(a), in most cases the size and concentration
of nanogels did not affect the solubility of AmB significantly.
However, the smallest pNIPAM nanogel (400 nm) at concentrations higher than 2 mg/mL enhanced the solubility of AmB
up to 1.6-fold. This represents that smaller pNIPAM nanogels
have better physical interaction with AmB than do larger pNIPAM nanogels due to higher surface-to-volume ratio.30,35
Release Rate of AmB Encapsulated into pNIPAM Nanogels. AmB loaded into pNIPAM nanogels is continuously released
depending on external conditions.27 In this study, we measured
the release rate of encapsulated AmB in pNIPAM nanogels.
As shown in Figure 2(b), the smallest pNIPAM nanogel (430
nm) releases more AmB compared to larger pNIPAM nanogels (560 and 700 nm). Regardless of size, pNIPAM nanogels
steadily released incorporated AmB into media over several
days. With the concentration of released AmB, we measured
release half-life, meaning the time for releasing half of encapsulated AmB. All the half-lives of the three pNIPAM nanogels
were identical (t1/2=1.0 day): half of AmB was released within
a day. As shown in Figure 2(b), 430 nm pNIPAM nanogel was
able to continuously release AmB over several days.
pNIPAM nanogels enable continuous release of drugs. This
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Figure 2. Solubility, release rate, and antifungal activities of AmB incorporated into pNIPAM nanogels. (a) Relative solubility of AmB
in the presence of pNIPAM nanogels to AmB alone at 21 oC (room temperature). (b) Cumulative AmB release from pNIPAM nanogels
at 21 oC (room temperature). (c) Antifungal activity of AmB on solid media. (d) Antifungal activity of AmB in liquid media. In (a) and
(c), those that show significant difference are marked as * (p-value < 0.05, Tukey test).

would allow local therapy at the site of infection with a single

one-time application and allow a high concentration of drugs,
which is difficult to achieve safely via systemic therapy with a
single dose. Local therapy with pNIPAM is particularly advantageous for pregnant women, where there are concerns about
birth defects with adverse antifungal agents.41
Antifungal Activity of AmB with pNIPAM Nanogels. Efficient treatment of skin infection requires proper dosage of
drugs to maintain high therapeutic index, and thus drug bioavailability must be sufficient to inhibit the growth of infectious pathogens. Here we investigated the antifungal activity
of AmB incorporated into various pNIPAM nanogels. First
we investigated the antifungal activity of pNIPAM nanogels
on solid agar plate, which is similar to human skin; zone of
inhibition was measured to evaluate antifungal activity on solid
media. A small hole was made at the center of MH agar plate
where cells were grown like a lawn, pNIPAM nanogels containing AmB, were loaded into the hole, and then a cleared zone
(diameter) was measured one day after application. Consistent
with the earlier experiment results on solubility, the 430 nm
pNIPAM nanogel showed the largest zone of inhibition, while
larger pNIPAM nanogels showed no significant antifungal activ1128

ity compared with AmB alone (Figure 2(c)). The average zone
of inhibition of 430 nm, 560 nm, and 700 nm pNIPAM nanogels were 56.51 mm, 251 mm, and 21 mm1 for 700 nm,
respectively.
We also measured antifungal activity in liquid media that
could be similar to our blood environment. Measured MFCs
in liquid media are shown in Figure 2(d). All pNIPAM nanogels
regardless of their size remarkably improved the antifungal
activity of AmB at least 4 folds compared with AmB in the
absence of pNIPAM. As shown in Figure 2(d), the smallest
(430 nm) pNIPAM nanogel had the lowest MFC, meaning
highest antifungal activity. As mentioned earlier (Figure 2(b)(c)), the smallest pNIPAM nanogel had higher solubility for
AmB and higher release rate than other pNIPAM nanogels,
so that it could increase the local concentration of AmB and
thereby kill infectious C. albicans more effectively. In addition,
higher antifungal activity of 430 nm pNIPAM nanogel could
stem from its relatively higher permeability into the fungus
cell membrane.42
Consequently, the high solubility and capacity of 430 nm
pNIPAM nanogel for AmB allowed high antifungal activity of
AmB on both liquid and solid media. This demonstrates that 430 nm
Macromol. Res., Vol. 22, No. 10, 2014

pNIPAM Nanogels for Enhanced Efficacy of AmB

pNIPAM nanogel is an efficient delivery nano-agent for AmB

and effective method to treat the infection of C. albicans.
Comparison of pNIPAM and pNIPAM-NH2 Nanogels
on AmB Solubility and Antifungal Activity. Upon superficial fungal infections, pathogens harbor epidermal layers of
skin and thus treatment of such infections requires drugs to
penetrate the stratum corneum to reach to a target site.43 Naturally
skin has negative charges.35 Cationic nanogels are expected to
be an efficient drug delivery agent compared with other polymeric delivery agents, e.g. dendrimers, carbon nano-tubes,

Figure 3. Average size of fabricated pNIPAM nanogels and pNIPAM-NH2 nanogels (200:1). The exact sizes are 45723 nm and
51140 nm, respectively.

magnetic particles, etc.,3,16,35,44,45 since the positive charges of

nanogels facilitate the interaction with skin membrane and
enhance the penetration of drugs to a target site.
To investigate the effect of pNIPAM nanogels surface charge
on the therapeutic properties of AmB, we additionally manufactured
small-sized pNIPAM-NH2 following the protocol for fabricating
430 nm pNIPAM nanogel (Figure 3). The size of pNIPAMNH2 was 51140 nm, which was slightly larger than pNIPAM
nanogel due to additional co-monomer (APMAAHC). With
this pNIPAM-NH2 nanogel, we evaluated its solubility, release
rate, and antifungal activity of encapsulated AmB and compared with those of AmB incorporated into 430 nm pNIPAM
nanogel (Figure 4). Unexpectedly, we could not find any significant improvement in solubility, release rate, and antifungal
activity of AmB on solid media. Rather, the addition of NH2
to pNIPAM nanogel reduced solubility significantly. However,
we found 2-fold improvement in the antifungal activity in liquid media.
In detail, surface charge of nanogels affects its swelling ratio
that could contribute to difference in AmB solubility.46 On the
contrary to pNIPAM nanogels, the addition of NH2 group negatively affected the AmB solubility (Figure 4(a)). The additional
amine groups (NH2) on the surface of nanogels could increase
steric hindrance enough to interfere with AmB incorporation.47
Despite the reduced solubility, AmB release rate from pNIPAM-

Figure 4. Solubility, release rate, and antifungal activities of AmB encapsulated in pNIPAM-NH2 nanogel. (a) Relative solubility of
AmB in the presence of various concentrations of pNIPAM and PNIPAM-NH2 nanogels at 21 oC (room temperature). (b) Cumulative
drug release profile of AmB loaded into pNIPAM and pNIPAM-NH2 nanogels at 21 oC (room temperature). (c) Antifungal activity of
AmB on solid media. No significant difference in zone of inhibition between pNIPAM and pNIPAM-NH2 (p-value > 0.05) (d) Antifungal
activity measured as MFC in liquid media. In (a) and (c) those that show significant difference are marked as * (p-value < 0.05, Tukey test).
Macromol. Res., Vol. 22, No. 10, 2014

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NH2 was similar to that of 430 nm pNIPAM (Figure 4(b)). These

results demonstrate that solubility of AmB is dependent on the
surface charge of nanogels, but the release rate of AmB is
independent.
To investigate the effect of surface charge on antifungal
activity, we evaluated the antifungal activity of AmB encapsulated
into pNIPAM-NH2 in liquid and solid media. pNIPAM-NH2
nanogel showed similar antifungal activity on solid media to
pNIPAM nanogel (Figure 4(c)), but showed two-fold higher
activity in liquid media (Figure 4(d)). This increase in antifungal
activity in liquid media by pNIPAM-NH2 nanogel is consistent
with the previously reported studies on the effect of NH2charged nanoparticles on antifungal activity.3,30,35 The higher
activity of pNIPAM-NH2 in liquid culture would be due to
increased interaction between the positively charged NH2 in
pNIPAM-NH2 and the negative charges on the fungus cell
membrane (Scheme I).8,11 In addition, in liquid media nanogels
can move freely to the cell membrane with higher chance of
interaction, but in case of solid media the interaction of pNIPAM
and pNIPAM-NH2 nanogels with fungus cell membrane is
limited to the rate of diffusion.
Consequently, for the treatment of infection on skin that is
similar to solid media 430 nm pNIPAM nanogel is a promising agent for the delivery of AmB for the effective treatment
of C. albicans. For the treatment of infection in blood that is
similar to liquid media pNIPAM-NH2 nanogel would be a
more effective delivery agent.

Conclusions
Here we fabricated pNIPAM nanogels and a modified version of pNIPAM with NH2 as a drug delivery agent for AmB
to effectively treat the infection of C. albicans. The encapsulation of AmB into our nanogels showed increased solubility
and higher cell permeability, leading to higher antifungal activity. The successful application of our pNIPAM nanogels to
lipophilic AmB represents that our nanogels can be used as a
drug delivery agent for other water insoluble drugs and can
be applied to the treatment that requires continuous and prolonged presence of drugs at a target site, e.g. skin.
Acknowledgments. This work was supported by a grant from
the Basic Science Research Program (2011-0013819, NRF2013R1A1A1076136), and Mid-career Researcher Program
(2011-0028796) through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education, Science
and Technology.

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