Interaction of gelatin with polyenes modulates antifungal activity and biocompatibility of ...

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Open access Full Text article OrIg INal research
Interaction of gelatin with polyenes modulates
antifungal activity and biocompatibility
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m/ rajamani Abstract: Topical application of antifungals does not have predictable or well-controlled
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s.c lakshminarayanan1,2 release characteristics and requires reapplication to achieve therapeutic local concentration
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pre radhakrishnan sridhar3,4 in a reasonable time period. In this article, the efficacy of five different US Food and Drug
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ov Xian Jun loh5 Administration-approved antifungal-loaded (amphotericin B, natamycin, terbinafine, flucon-
ww.donly. Muruganantham azole, and itraconazole) electrospun gelatin fiber mats were compared. Morphological studies
ded from https://wFor personal use NVBMeaaalrnduadhcthhahiaiay1mk,a6unym aKamarl1auitphrai ya3,4 smwcahcoeatomritwvesp i itaetnyhrfh fealeidotbc ,sii tsntit vhctwehoe aer apsi tgg oroararboaicnwstoiesotntrh nva t ezhoodorffe l yfeepoe- olrafiol syfllataeusdmn ceaeoednsnn dramt eozfisauolutsal le mtw-feluedaonns aigt dnopae uolad st s etmfpwnueatonc t-gaisfea goaslal. gd ipanA aiisnntmthcs ootrA genasaeglpsln eestor h.fi genT it elhtfilrweubb soietn e rsas atdtfizr iaoonairlemneg-s laeo.ant aneiHdsrti meofadwuns n.dem gT vtaaehhltreess,
a Jia lin Kwan1
o polyene-loaded mats displayed rapid candidacidal activities as well. Biophysical and rheologi-
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w shou Ping liu1,2
o cal measurements indicate strong interactions between polyene antifungals and gelatin matrix.
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e roger Wilmer Beuerman1,2 As a result, the polyenes stabilized the triple helical conformation of gelatin and the presence
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dici seeram ramakrishna3,4,7 of gelatin decreased the hemolytic activity of polyenes. The polyene-loaded fiber mats were
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m
noncytotoxic to primary human corneal and sclera fibroblasts. The reduction of toxicity with
o 1singapore eye research Institute,
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Na 2signature research Program in complete retention of activity of the polyene antifungal-loaded gelatin fiber mats can provide
of Neuroscience and Behavioral new opportunities in the management of superficial skin infections.
al Disorders, Duke-NUs graduate
urn Medical school, 3Department of Keywords: fungal infections, electrospinning, antifungals, controlled release, drug–matrix
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J Mechanical engineering, National interactions
al University of singapore, 4center for
n
atio Nanofibers and Nanotechnology,
n National University of singapore, Introduction
Inter 5eInngsitniteuetrei nogf, aM*astTeariral s( argeesnecayrc fho ra nd Pathogens from a large group of fungi and yeasts are increasingly impacting the global
science, Technology and research), biota and constitute one of the planet’s biggest killers threatening food security and
3 research link, singapore,
ecosystems.1–3 While superficial fungal infections are the most common form of infec-
6Department of Ophthalmology, Yong
loo lin school of Medicine, National tions, affecting 20%–25% of the world’s population,4 the invasive fungal and yeast
University of singapore, 7NUs
infections associated with excessive use of medical devices and implants and expand-
Nanoscience and Nanotechnology
Initiative, singapore ing numbers of immunocompromised patients are responsible for increased health
care costs, morbidity, and mortality.5–7 In addition, new fungal and yeast pathogens
have emerged that have reduced susceptibility or enhanced resistance to the available
antifungals.8–10 Compared to antibiotics for treating bacterial infections, only limited
correspondence: rajamani categories of antifungal drugs are available for fungal infections: the polyene mac-
lakshminarayanan
rolides, azoles, allylamines, fluoropyrimidines, and echinocandins.11,12
singapore eye research Institute,
11 Third hospital avenue, Due to warm and humid conditions in tropical countries, superficial and subcu-
singapore 168751 taneous fungal infections are relatively common.13 The architecture of the skin pres-
email lakshminarayanan.rajamani@seri.
com.sg ents a formidable barrier for drug penetration, thus impeding the drug efficacy and
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http://dx.doi.org/10.2147/IJN.S58487 permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on
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local bioavailability.14 Therefore, a high concentration of gelatin is used in numerous food and cosmetic applications
the drug at the site of infection is required to achieve clini- as well as in cell-compatible biomaterials.34,35 Using appro-
cal efficacy. A number of strategies such as polymeric and priate solvent composition, electrospinning has been used to
peptide hydrogels, nanoparticles, microemulsion, liposomes, generate gelatin fibers with a wide range of diameters.36 In
niosomes, or ethosomes that can function as drug depots this study, the efficacy of polyene, azole, and allylamine anti-
for delivering the antifungals at the site of infections have fungal drugs (Figure 1) incorporated into gelatin fiber mats
6 been reported.15–20 However, these formulations need to against various yeasts/fungal pathogens were evaluated, their
1
20 be applied frequently to maintain sustained release of the fungicidal kinetics were tested, and their toxicity on primary
v-
No drugs.21 Nonwoven fiber mats produced by electrospinning fibroblast cells was assessed. The interaction between drugs
8-
1 have the potential for a wide range of applications such as and gelatin was investigated by circular dichroism (CD) and
n
o
7 filters, composites, drug carriers, wound dressing, and tissue rheological studies.
1
9. regeneration.22–25 Wound dressings prepared by electrospin-
4
1. Materials and methods
3. ning of biopolymers provide several key attributes, such as
5
y 1 high encapsulation efficiency and sustained delivery of drugs chemicals and reagents
b
m/ for a longer time, that are required for transdermal/topical Gelatin (from porcine skin, high bloom), amphotericin B,
o
s.c delivery of drugs.26 Electrospun fiber mats carrying various natamycin, terbinafine hydrochloride, itraconazole, flucon-
s
e
pr antimicrobial agents, carbon nanotubes, N-halamines, azole, and trifluoroethanol were purchased from Sigma-
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ov vitamin A, and vitamin E have been reported.27–32 However, Aldrich (St Louis, MO, USA). All chemicals were of
ww.donly. the in vitro efficacy of antifungal drugs incorporated into analytical grade and used without further purification.
we
om https://ersonal us eyleetc Ibtnreo etesnpr mduensm ofifob mnesretsrc ahatgaenadii.sn3m3st oyfe aacsttiso/fin,l athmee anvtaoiulasb flue nagnit ihfuans gnaolst egelelacttirno fispbienrn imnga tosf antifungal-loaded
ded frFor p can be grouped into three categories: 1) polyene, azole, and Antifungal drug-loaded gelatin fiber mats were fabricated by
oa allylamine antifungals that target ergosterol or ergosterol the electrospinning method. Gelatin (1 g) was dissolved in
nl
w biosynthetic pathways; 2) echinocandins, which are β-glucan 10 mL of trifluoroethanol at a concentration of 10% weight/
o
d
e synthase inhibitors; and 3) fluoropyrimidines, which target volume (w/v). The drugs were added to the gelatin solution
n
ci
di RNA synthesis. Due to its inherent biodegradability under so that the final antifungal:gelatin ratio was maintained
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m
o physiological environments and nonimmunogenic properties, at 0.25 wt%. The mixture was stirred overnight at room
n
a
N
of
al OH
urn A O OH OH B H O OH
onal Jo HO O OH OH OH OH O OHO O O H OH O OHO
nati O O OH
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Int HO OH O O
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HO OH
NH2
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N
N
N N
N OH
F CI O N
O N N N
O N
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N
F
E H3C CH3
N
CH3
•HCI H3C
Figure 1 chemical structures of antifungals used in this study. (A) amphotericin B; (B) natamycin; (C) fluconazole; (D) itraconazole; (E) terbinafine chloride.
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Dovepress Polyene antifungal-loaded gelatin fiber mats
temperature to achieve a homogenous solution. The over- Kinetics of candidacidal action
night stirred solution was poured into a standard 10 mL To assess the candidacidal properties of the polyene-loaded
syringe that was attached to a steel needle. Electrospinning fiber mats, time-kill kinetics against a Candida albicans
was carried out by introducing a high voltage (direct current DF 2672R strain was carried out. Cultures were grown
high voltage power supply; Gamma High Voltage Research, overnight in Sabouraud dextrose broth and the yeast cell
Inc., Ormond Beach, FL, USA) to the needle as a positive concentration was adjusted to 1.5×105 Colony Forming
6 electrode and a fixed sheet of grounded aluminum foil. The Units (CFU)/mL. Fiber mats and antifungal-loaded fiber
1
v-20 distance between the needle tip and collector was set at 12 mats (0.5×0.5 cm in triplicates) were incubated with 1 mL of
No cm. When the positive electrode reached a critical voltage the cell suspension in a 24-well Nunc® dish (Thermo Fisher
8-
n 1 of 12 kV, the spinning head extruded polymer solution and Scientific, Waltham, MA, USA) and incubated at 37°C with
o
7 the electrospun fibers were deposited on to the aluminum constant shaking. At predetermined time points, 100 µL of
1
49. foil kept at the collector. The emitting rate of the polymer the suspension was withdrawn, serially diluted (102 or 103
1.
3. solution was controlled at 0.6 mL/hour by means of a syringe fold), and poured into a Sabouraud dextrose agar plate. The
5
1
y pump (KD Scientific Inc., Holliston, MA, USA). The as-spun plate was incubated for 48 hours at 37°C for colony counting.
b
m/ fiber mats were exposed for vapor phase crosslinking of The data were expressed in terms of percentage of fungal
o
s.c glutaraldehyde by placing an aqueous solution of glutaral- survival with respect to the positive control (ie, fiber mats
s
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pr dehyde (50%) in a Petri dish kept beneath the fiber mats in without antifungals).
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ov a closed desiccator. After glutaraldehyde vapor treatment,
ww.donly. the sample was vacuum dried for 2 days to remove residual antifungals release properties
om https://wersonal use ghcoolunuttrarsro aall ndgdee hl2ay4tdi hneo muanrasdt sf ,os rore ltsvhpeeen actn.t itvTiefhuleyn. gcarol-slsolaindkedin figb teirm me awtsa asn 3d oTimhfme e ealrnesteicdfut inrngo 1a0sl- pmlouLan doef d fip hbfiobeseprrh mamtea atbstu (sfnfe=r4e)d wsaelirnee w (PeBigSh)e bdu affnedr
ded frFor p (pH 7.0) with constant shaking at 37°C. A 1 mL aliquot of
oa scanning electron microscopy (seM) the above solution was withdrawn at various time intervals,
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w The morphology and diameter of the gelatin crosslinked centrifuged, and the amount of drugs in the supernatant
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e electrospun fibers were observed and determined with the use was determined by spectrophotometry with the calibration
n
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di of an optical microscope (BX51M; Olympus Corporation, method. Each time the mother liquor was reloaded with a
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o Tokyo, Japan) and an SEM (Quanta 200F; FEI, Hillsboro, fresh 1 mL of buffer to maintain the total volume of the
n
Na Oregon, USA). Prior to imaging with the use of SEM, a small release solution at 10 mL. The characteristic absorption
al of section of the fibers on the sample holder was sputter coated bands of each antifungal were used to estimate the amounts
n
ur with gold (JFC-1200 fine coater; JEOL, Tokyo, Japan). of antifungals released. The values were converted into µg
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al SEM was then used to observe the samples at an accelerat- of antifungals released/10 mg of the fiber mat.
n
atio ing voltage of 10–15 kV. The average diameter of the fibers
n Minimum inhibitory concentration
er (n=50) was estimated from four SEM images, each from two
Int independent experiments. (MIc) of antifungals
MIC values for amphotericin B, natamycin, fluconazole, and
radial diffusion assay terbinafine chloride were determined in two independent
Fungus and yeast cell cultures (at a concentration of duplicates by the microdilution method. Overnight cultivated
0.5 McFarland standards) were spread onto the surface of yeast strains were suspended in Sabouraud dextrose broth at a
sterile Sabouraud dextrose agar plates using a cotton swab in starting optical density at 600 nm of ∼0.08 in a flat-bottomed
9 cm diameter Petri dishes. The fiber mats (0.5 cm × 0.5 cm) microtiter plate. A serial dilution of the antifungals in the
and antifungal-loaded gelatin fiber mats were placed on top same broth was mixed with the inoculum to give a final
of the swabbed cultures and incubated at 37°C. Antifungal peptide concentration of 0.1–128 µg/mL. The antifungal
activity of fiber mats was visualized as the diameter of the activity was assessed by monitoring the optical density at
zone of inhibition after incubating plates in the dark for 600 nm in cycles of 30 minutes and an orbital shaking at
48 hours for Candida strains and 72 hours for Fusarium and 100 rpm using an Infinite® M200 microplate reader (Tecan
Aspergillus strains. The assay was performed in two indepen- Group Ltd, Männedorf, Switzerland) for 48 hours at 37°C.
dent duplicates and the average value was reported. Cultures without antifungals were used as positive controls
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and broth alone or with 256 µg/mL of the antifungals served red blood cells to a final concentration of 4% volume/volume
as negative controls. The minimum concentration required (v/v), incubated at 37°C for 1 hour and centrifuged at 3,000
for complete inhibition was assessed by both visible observa- rpm for 10 minutes. To study the effect of gelatin–antifungal
tions as well as by measuring the optical density at 600 nm interactions, the drugs were incubated overnight with an
and taken as the MIC. appropriate amount of gelatin so that the antifungal:gelatin
ratio was 0.25 wt% and added to the rabbit red blood cells.
cD spectropolarimetry
6 The release of hemoglobin in the supernatant was monitored
1
20 Far ultraviolet (UV) CD spectra of gelatin (0.1 mg/mL) was by measuring the hemoglobin absorbance at 576 nm. The
v-
No recorded on a spectropolarimeter (J810; JASCO International readings from cell suspension in PBS (without any additives)
8-
1 Co, Ltd, Tokyo, Japan) in 10 mM PBS (pH 7.0) using a or 1% Triton-X100 were used as 0% or 100% hemolysis,
n
7 o 0.1 cm path length quartz cuvette at 30°C. Spectra were respectively.
1
9. recorded from 260 nm to 190 nm (for gelatin) and 450 nm to
4
3.1. 300 nm (for amphotericin B) in 0.1 nm steps at a scan rate of cytotoxicity of antifungal-
5
y 1 50 nm/minute. The final spectrum is the average of four scans. loaded fiber mats
b
m/ For the variable temperature CD experiments, ellipticity at Both the antifungal-loaded and bare fiber mats were adhered
o
s.c 224 nm was monitored over a temperature of 10°C–60°C to microscope cover slips (n=4) and plated onto 24-well Nunc
s
pre at a heating rate of 5°C/minute. To study the influence of dishes. Human corneal and sclera fibroblasts were grown in
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ov antifungals on the secondary structure, gelatin was incubated HyClone® Dulbecco’s Modified Eagle’s Medium (Thermo
ww.donly. with 0.25 wt% antifungals and the CD measurements were Fisher) with 10% fetal bovine serum antimycotic solution con-
m https://wsonal use rRechoerdoeldo ags ibceafol rce.haracterization tsauilnfianteg, 5a0n0d u2n.i2t5s/ mµgL/ mpeLn iacmillpinh oGte, r0ic.1in m Bg /(mSLig mstrae-pAtoldmriycchi)n.
oer The cells were seeded at a concentration of ∼4×104 cells/well
ded frFor p of antifungal–gelatin interactions and incubated with the fiber mats at 37°C for 24 hours. After
oa Rheological experiments were performed at room tem- equilibrating the plates to room temperature, an equal volume
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ow perature using a controlled strain rheometer (ARES-G2; of CellTiter-Glo® reagent was added (Promega Corporation,
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ne TA Instruments, New Castle, DE, USA). The rheometer is Madison, WI, USA). The adenosine triphosphate content was
ci
di equipped with two sensitive force transducers for torque rang- determined by recording the luminescence on a Tecan Infinity
e
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o ing from 0.05 mN/m to 200 mN/m. The gel was placed onto M200 plate reader as per the manufacturer’s instruction.
n
Na parallel-plate geometry (25 mm in diameter). The dynamic
al of storage modulus (G′) and loss modulus (G″) of the gel were Uniaxial tensile tests
n
ur examined as a function of frequency from 0.1–100 rad/ second. Mechanical properties of electrospun fibrous membranes
o
J
al The measurements were carried out at strain amplitude (γ 0) were determined with a tabletop uniaxial testing machine
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atio of 5% to ensure the linearity of viscoelasticity. (Model 3345; Instron, Norwood, MA, USA) with the use of
n
er a 10 N load cell under a crosshead speed of 5 mm/minute
Int UV spectroscopy of amphotericin B at ambient conditions (25°C and 70% relative humidity).
UV spectra of amphotericin B were recorded on a UV1800 All samples were prepared in the form of rectangular shape
double beam spectrophotometer (Shimadzu, Kyoto, KYT, with dimensions of 20×10 mm from the electrospun fibrous
Japan) at room temperature. To determine aggregation status membranes. The thicknesses of samples were measured
of amphotericin B, the spectra were recorded in dimethyl with a digital micrometer. At least five samples were tested
sulfoxide (DMSO), 10 mM PBS buffer (pH 7.0), and in the for each type of electrospun fiber mats and the average was
presence of gelatin. reported.
hemolytic activity of polyenes statistical analysis
in the presence of gelatin For statistical analysis, the data were analyzed by using
Hemolytic activity of polyenes in the presence of gelatin Prism® 6.0 software (GraphPad Software, Inc., La Jolla, CA,
was determined against rabbit red blood cells.37 Briefly, USA). All the experimental data were expressed as mean ±
two independent triplicates of 32 µg/mL and 64 µg/mL of standard deviation. Two-tailed Student’s t-test and one-way
amphotericin B or natamycin in PBS was mixed with rabbit analysis of variance with Newman–Keuls post hoc analysis
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Dovepress Polyene antifungal-loaded gelatin fiber mats
were used for determining the significance of differences.
A B
P,0.05 was considered significant.
Results and discussion
Morphology of antifungal-
loaded fiber mats
6
1 Gelatin and antifungal-loaded gelatin fiber mats were
0
2
v- electrospun from trifluoroethanol solution and their anti-
No C D
8- fungal activities were evaluated. After an initial optimiza-
1
on tion, 10% w/v of gelatin was used and the final antifungal
7
1 concentration was maintained at 0.25% weight/weight (w/w)
9.
4
1. in order to minimize bead formation. Figure 2A–F show
3.
15 the morphology of gelatin and various antifungal-loaded
y
m/ b gelatin fibers imaged after glutaraldehyde crosslinking. The
o images show smooth surface and absence of any beading,
c
s. E F
es pores, or physical defects in various antifungal loaded fiber
pr
e mats. The average diameters of individual fibers in various
v
o
ww.donly. antifungal-loaded mats are shown in Figure 2G. Fiber mats
we without antifungals had a mean diameter of 0.8±0.4 µm.
m https://sonal us Tpohley eanvee raangtei fduinagmael-tleoras doefd thmea itns dhivaidd uaa lla firgbeerr sa nind bbortoha dtheer
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nl age diameters of the fibers remained higher for natamycin
w m) ***
cine do (e1le.8c±tr1o.s0p µumn )g ceolamtipna, raend tion carmeapsheo tienr idciianm Be (t1er.4 w±0a.s9 o µbmse)r. vFeodr meter (µ *** ***
medi with an increasing concentration of gelatin or solvent com- e dia ***
ano position.36 In the absence of these parameters, the above Fibr
N
of results indicate significant interactions between the poly- 0
nal enes and gelatin matrix. Among the two azoles, addition of Control Fluc Itra Terb Amph Nata
ur
Jo fluconazole did not affect the diameter distribution of the Figure 2 scanning electron micrographs showing the electrospun gelatin
al fibers loaded with various antifungals. (A) No antifungals; (B) amphotericin B;
on fibers (0.8±0.4 µm), whereas incorporation of itraconazole (C) natamycin; (D) fluconazole; (E) itraconazole; (F) terbinafine. (G) histogram
nati increased the dia meter moderately (1.2±0.6 µm). The aver- showing the effects of various antifungals on the average fiber diameter.
er Notes: ***P,0.001; scale bar =50 µm.
nt age diameter of terbinafine-loaded fibers (1.4±0.6 µm) was
I
larger compared to the control fiber mats.
Type Culture Collection (Manassas, VA, USA) and the
clinical isolates of C. albicans (Figure 3A and B). The other
antifungal properties
antifungal-loaded fiber mats displayed significant variability
of electrospun fiber mats in anti-Candida activity. Fluconazole- and terbinafine-loaded
The antifungal properties of drug-loaded fiber mats were fiber mats were the least effective as they showed clear
evaluated by radial disc diffusion assay against a panel zone of inhibition against only one strain of C. albicans
of C. albicans, and the zone of inhibition was measured (Figure 3C and D). Itraconazole-loaded fiber mats, on the
(Figures S1–S7). Figure 3 shows the activity of antifungal- other hand, inhibited the growth of two American Type Culture
loaded fiber mats (expressed as the zone of inhibition) against Collection strains of C. albicans and a weaker inhibitory
various C. albicans strains. The results suggest that the anti- activity against the other three strains (Figure 3E).
fungal activity depends on the type of antifungals loaded. The in vitro antifungal activities of the fiber mats were
Polyene-loaded fiber mats displayed excellent antifungal determined against four filamentous pathogens as well. As
properties, suppressing the growth of both the American was observed before, both the polyene antifungals inhibited
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lakshminarayanan et al Dovepress
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ati Figure 3 Efficacy of drug-loaded gelatin fiber mats against yeast strains. The antifungal activity against Candida albicans strains is expressed as the zone of inhibition measured
n by radial diffusion assay. Fiber mats loaded with (A) amphotericin B (*P,0.05 compared to C. albicans aTcc 10231 strains; **P,0.01 compared to C. albicans aTcc 10231
nter strains; @@P,0.01 compared to C. albicans aTcc 2091 strains; @@@P,0.001 compared to C. albicans aTcc 2091 strains; #P,0.05 compared to C. albicans aTcc 24433);
I (B) natamycin (*P,0.05 compared to C. albicans aTcc 10231 strains); (C) terbinafine (***P,0.001 compared to all the groups); (D) itraconazole (***P,0.01 compared to all
the groups except C. albicans aTcc 24433 strains; @@@P,0.001 compared to all the groups except C. albicans aTcc 2091 strains); and (E) fluconazole. ***P,0.001 compared
to all the groups. (F) Representative photographs showing the zone of inhibition of gelatin (upper panel) and amphotericin B-loaded gelatin (lower panels) fiber mats.
Note: The absence of bars in the graph indicates no inhibition against the particular strain.
Abbreviation: aTcc, american Tissue culture collection.
the growth of all four filamentous fungi (Figure 4A and B). chloride-loaded fiber mats were effective against filamentous
Itraconazole-loaded fiber mats showed strong inhibition for fungi. Among the two azoles, itraconazole-loaded fiber mats
two Aspergillus strains, whereas fluconazole-loaded fiber were superior compared to fluconazole-loaded fiber mats.
mats had no activity against all the filamentous pathogens
In vitro release of antifungals from gelatin
tested (Figure 4D and E). Of all the antifungals, terbinafine
chloride-loaded fiber mats displayed the strongest inhibitory fiber mats and MIC of antifungals
activity against filamentous fungal species (Figure 4C). These The cumulative release profile for various antifungals was
results suggest that polyene-loaded fiber mats retained the determined by UV spectrophotometry (Figure 5). The amount
broad-spectrum antifungal properties, whereas terbinafine of antifungals released is expressed as µg/mg of fiber mats.
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om https://wersonal use E 100 ATFC. sCo l3a6n3i6 ATFC. Cso 2la6n6i71 AAT. CfuCm 9ig0a9tu0s6 AA.T bCrCas 1lie6n4s0i4s F 0 ATFC. sCo l3a6n3i6 ATFC. Cso 2la6n6i71 AAT. CfuCm 9ig0a9tu0s6 AA.T bCrCas 1lie6n4s0i4s
ded frFor p m)
a m
nlo n (
w o
do biti
e hi
n n
dici of i
me ne
o o
n Z
a
N
of 0
al F. solani F. solani A. fumigatus A. braseliensis
n
ur ATCC 3636 ATCC 26671 ATCC 90906 ATCC 16404
o
J
nal Figure 4 Efficacy of drug-loaded gelatin fiber mats against filamentous fungi. The antifungal activity against Aspergillus and Fusarium strains is expressed as the zone of inhibition
natio cmoemaspuarreedd btoy Fruasdaiarilu mdi ffsuoslaionni aaTsscacy. 3F6ib3e6r smtraatins sl;o @adP,ed0 .w05it hc o(mAp) aarmedp htoo tAe.r ibcrians iBlie n(*si*s* aP,Tc0.c00 116 c4o0m4 pstarraeidn st)o; ( CAs)p teergrbilliunsa fibnraes i(l*ie*n*sPis, a0.T0c01c c o16m4p0a4r)e; d( Bto) Fn.a stoalmanyic ainT (c*cP, 306.0356
er strains; @@@P,0.001 compared to A. brasiliensis aTcc 16404); (D) itraconazole (***P,0.001 compared to F. solani aTcc 3636 strains; @@@P,0.001 compared to F. solani
Int aTcc 26671 strains; #P,0.05 compared to A. fumigatus aTcc 90906 strains); and (E) fluconazole. (F) Representative photographs showing the zone of inhibition of gelatin
(upper panel) and amphotericin B-loaded gelatin (lower panels) fiber mats.
Note: The absence of bars in the graph indicates no inhibition against the particular strain.
Abbreviation: aTcc, american Tissue culture collection.
The release profile was similar for all the antifungals, the MIC of all the antifungals alone against five different
although the amount of released drugs was different. The C. albicans strains was determined. Table 1 compares the
sustained release of polyene antifungals from fiber mats determined MIC of various antifungal agents. Amphotericin
indicated significant drug–gelatin interactions.38 At the end of B displayed the lowest MIC followed by natamycin against
3 days, the amount of amphotericin B and natamycin released all the C. albicans strains examined. Interestingly, four
in the medium was 9.5 µg/mL and 8.5 mg/mL, respectively. C. albicans strains showed resistance to fluconazole as the
The amount of terbinafine released was 7.3 µg/mL. Of all MIC was .64 µg/mL. A good correlation was observed
the antifungals, fluconazole was released in maximum between the amounts of antifungals released and MIC values
amounts (14 µg/mL). To understand the loss of activity for of free antifungals against particular strains of C. albicans.
other antifungals when incorporated into the gelatin matrix, Thus, poor antifungal properties and release characteristics
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16 The changes in morphology of C. albicans incubated
with polyene-loaded fiber mats were investigated by SEM.
)
dat C. albicans incubated with gelatin alone had smooth and intact
em
s surfaces (Figure 7A). However, cells treated with the polyene
ar
ee
elb antifungals had extensive surface deformation and leakage of
s rf fi some intracellular components (Figure 7B and C). The damage
6 galg o Amphotericin B caused by antifungals was more noticeable in the amphotericin
8-Nov-201 Antifung/10 mµ TNeartbaimnayfcinine Bre-sluolatds ecdo nfifibremr m thaatst tthhaen p ionl yneantaem-lyocaidne-dlo fiabdeerd mfibaetsr rmetaatisn. Tedh ethsee
n 1 ( Fluconazole antifungal activity even in the presence of gelatin.
o
7 0
1
49. 0 12 24 36 48 60 72 Interaction of gelatin with antifungals
1. Time, hours
3. Since the fiber diameter of gelatin mats was doubled in
5
1
y Figure 5 Cumulative release profile of antifungals form gelatin fiber mats. the presence of polyene-loaded antifungals, the interac-
b
m/ tion between antifungals and gelatin was investigated
o
s.c are responsible for apparent loss of activity of azoles and using spectroscopy and rheometry. First, the changes in
s
e
pr terbinafine upon incorporation into the gelatin matrix. secondary structure of gelatin were monitored by CD
e
v
o spectropolarimetry. Figure 8A depicts the changes in the
ww.donly. Kinetics of candidacidal action secondary structure of the gelatin solution and gelatin upon
m https://wsonal use oSifn cpe obolythe tnhee p-ololyaendee-ldoa dfiebd efibre rm maattss displayed generally iTnhceu bCaDti osnp ewctirtha o0f.2 g5e lwatti%n sahmowpheodt ear sictrionn Bg naengda ntiavtea mtroyucignh.
oer good inhibitory activity, the remaining experiments were around 197 nm and no broad positive band in the n-π* region
ded frFor p performed with amphotericin B- and natamycin-loaded fiber (Figure 8A). However, gelatin incubated with natamycin
a
o mats. After incubation for 52 hours, both the polyene-loaded and amphotericin B displayed a strong maximum in the
nl
ow fiber mats resulted in considerable loss of viability ($2 log n-π* region (Figure 8A). The positive maximum was more
d
e reduction in viability) of C. albicans (Figure 6A), suggest- pronounced for natamycin compared to amphotericin B. The
n
ci
di ing potent candidacidal action. To gain further insights, the presence of a positive band around 225 nm is a hallmark for
e
m
o kinetics of candidacidal action after exposing C. albicans triple helical structure associated with collagen.39,40 Using
n
a
N (1.5×105 CFU/mL) to amphotericin B- or natamycin-loaded the equation reported by Fujitsu et al, the relative helical
of
al fiber mats was also investigated (Figure 6B). At various content ([C] ) of gelatin in the presence of polyenes was
n rel
ur time intervals, the supernatant was diluted (102 or 103 fold), estimated.41 Positive or negative (C) indicates increased or
Jo rel
al plated on Sabouraud dextrose agar, and the number of yeast decreased triple helical content, respectively. (C) at 10°C for
n rel
o
ati colonies was counted after 48 hours. For both the polyene amphotericin B- and natamycin-loaded gelatin was 0.8 and
n
er antifungal-loaded fiber mats, a steep decrease in the viability 2.4, respectively, indicating greater stability of triple helix
nt
I of C. albicans was observed during early incubation time. structure in the presence of natamycin.
After 24 hours of incubation, amphotericin B-loaded fiber To further confirm these observations, the variable
mats caused 1.6 log CFU/mL decrease (97.3% killing), temperature CD was recorded by monitoring the change in
10
whereas natamycin caused 1.2 log log CFU/mL decrease ellipticity at 225 nm. Thermal transition has been used to
10
(93% killing) in the viability of C. albicans. investigate the helix–coil transition in collagen or collagen
Table 1 Minimum inhibitory concentration of antifungals against various strains of Candida albicans
Antifungals Minimum inhibitory concentration in µg/mL against
C. albicans C. albicans C. albicans C. albicans C. albicans
ATCC 10231 ATCC 2091 ATCC 24433 DF 2672R DF 1976R
ampB 1.25 1.25 1.25 1.25 1.25
Nata 10 5 10 10 10
Terb 16 64 .64 8 16
Fluco .64 .64 16 .128 .128
Abbreviations: ampB, amphotericin B; aTcc, american Type culture collection; C. albicans, Candida albicans; Fluco, fluconazole; Nata, natamycin; Terb, terbinafine chloride.
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Dovepress Polyene antifungal-loaded gelatin fiber mats
A
B
150 ** 1000,000
** 100,000
y
a viabilit U/mL 10,000
d F
di C 1,000
an Gelatin
6 c
01 % 100 AmpB fibre mat
v-2 Natamycin fibre mat
o
N 10
8- 0 0 4 8 12 16 20 24
1
n No AmpB Natamycin Time, h
o antifungal
7
1
9. Figure 6 (A) Candidacidal properties of the fiber mats loaded with amphotericin B and natamycin. (B) Kinetics of candidacidal action of amphotericin B and natamycin-
4
1. loaded fiber mats.
53. Notes: There was a significant decrease in the viability of Candida albicans exposed to antifungal-loaded fiber mats; **P,0.01.
y 1 Abbreviation: ampB, amphotericin B.
b
m/
o
c
s. mimetic peptides.42,43 For gelatin without antifungals, a linear in ellipticity, indicating clear helix–coil transition (Figure 8B
s
e
pr change in ellipticity ([θ] ) with temperature was observed and C). As was observed before, natamycin promoted the
ve 225
ww.doonly. shuegligxe ssttriuncgtu dree n(Faitguurered 8cBo)n. fHoormweavtieorn, g aenladt inla icnkc uobfa tae dt rwipitlhe hSeulgicaarsl caonndf opromlyaotilos nc amno prero stiegcnti fithcea nntaltyi vthea sntr aumctpuhroe taenridc ipnr Bo-.
we
m https://sonal us natamycin and amphotericin B displayed a nonlinear change mote helix–coil transition of gelatin by crosslinking through
oer
ded frFor p
a
o
nl
w
o
d
e
n
ci
di
e
m
o
n
a
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al
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ur
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al
n
o
ati
n
er
nt
I
Figure 7 Morphology of Candida albicans grown on polyene-loaded fiber mats. Scanning electron micrographs of C. albicans treated with (A) gelatin fiber mats without
antifungals; (B) amphotericin B-loaded fiber mats; and (C) natamycin-loaded fiber mats.
Note: Inset scale bar =10 µm.
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A B
−2,000 4,500
−1ol 195 205 215 225 235 245 255 265 −1ol
m m
d d
2m 2m
g c g c
de de
ov-2016 [], θMRW GGGeeelllaaatttiiinnn++AnamtapmB ycin [], θ225 Gelatin
N Gelatin+AmpB
8- −25,000 0
n 1 Wavelength, nm 10 20 30 40 50 60
9.17 o C 8,000 D1,000 Wavelength, nm
3.1.4 −1ol
5 m
m/ by 1 2cm d Pa)
s.co deg G' (
ovepres [], θ225 Gelatin AGmelpaBtin+gelatin
m https://www.dsonal use only. 0 10 Gela2t0in+naWtaam3vy0eclinengt4h0, nm 50 60 000.1 Fr1equencyN,a rtaamd1y/0sceinc+gelatin 100
oer Figure 8 Interaction between polyene antifungals and gelatin probed by circular dichroism spectropolarimetry and rheological studies. (A) Far ultraviolet circular dichroism
ded frFor p sTpheec tcroonpcoelnatrrimateiotrny o off agnetliafutinng aanlsd wgealsa t0i.n2 5in cwutb%a twedit hw ritehs ppeoclyt etnoe g aenlatitfiunn. g(aDls) aFtr 3e0q°ucen. cTyh eswrmeeapl d oefn sattourraagtieo nm oofd guelulast i(ng i′n) tohfe g perlaetsinen acned o afn (tBif)u anmgapl-hlooatedreidc inge Bla atinnd. (C) natamycin.
a
o Abbreviation: ampB, amphotericin B.
nl
w
o
d
e
cin hydrogen bonding.41,44 The results suggest that the greater was increased to 851±7 Pa and 848±9 Pa in the presence of
di
e stabilization of gelatin by the two polyenes is attributed to a amphotericin B and natamycin, respectively. Combined with
m
o
n significantly higher number of hydroxyl groups present (ten CD studies, it is likely that the increased association between
a
N
of in amphotericin B and five in natamycin). However, at this the gelatin chains brought about by the supramolecular inter-
nal stage, it is difficult to conceive the increased triple helical actions between the polyenes and the protein chains through
ur
o propensity in the presence of natamycin over amphotericin B, hydrogen bonding results in increased storage modulus and
J
al
n though the latter has a higher number of −OH groups. In the increased diameter of the fibers. This interesting phenomenon
o
ati presence of terbinafine, a small increase in helical propensity certainly warrants further investigation and suggests that
n
er
nt was observed, whereas fluconazole had no significant influ- rheology, along with spectrometry methods, could provide
I
ence on the secondary structure of gelatin (Figure S8). The valuable information on drug–matrix interactions.
estimated (C) was 0.78 in the presence of terbinafine and
rel
Gelatin stabilizes the monomeric
0.5 for fluconazole-loaded gelatin, thus confirming a weak
interaction between fluconazole and gelatin. forms of amphotericin B
Rheological studies of the drug-loaded gelatin solutions It has been shown that the toxicity of amphotericin B for
were carried out to determine if the interaction of antifungals mammalian cells depends on the level of aggregation.45,46 In
modified the physical properties of the gels. Macroscopically, aqueous solutions, amphotericin B exists in an aggregated
a nonflowable gel was obtained with or without drug. This form containing about 2,000 molecules, whereas in DMSO the
material did not flow even when the sample vial was inverted. monomeric form is predominant below 5 mM.47 The character-
A solid-like behavior was observed with G′ greater than G″ istic absorption bands in UV and CD spectroscopic methods
under these conditions, and both the moduli were almost inde- can differentiate between the different states of aggregation.
pendent of frequency at higher frequency ranges (Figures 8D Therefore, the UV absorption spectra of amphotericin B under
and S9). The frequency-independent value of G′ for gelatin various conditions were recorded. In DMSO, amphotericin B
solution without any antifungals was 767±27 Pa. The G′ value displayed four clear characteristic absorption bands around
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