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Interaction of gelatin with polyenes modulates antifungal activity and biocompatibility of electrospun fiber mats.

Lakshminarayanan R, Sridhar R, Loh XJ, Nandhakumar M, Barathi VA, Kalaipriya M, Kwan JL, Liu SP, Beuerman RW, Ramakrishna S - Int J Nanomedicine (2014)

Bottom Line: Terbinafine-loaded mats were effective against three filamentous fungal species.Among the two azole antifungals compared, the itraconazole-loaded mat was potent against Aspergillus strains.As a result, the polyenes stabilized the triple helical conformation of gelatin and the presence of gelatin decreased the hemolytic activity of polyenes.

View Article: PubMed Central - PubMed

Affiliation: Singapore Eye Research Institute, Singapore ; Signature Research Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore.

ABSTRACT
Topical application of antifungals does not have predictable or well-controlled release characteristics and requires reapplication to achieve therapeutic local concentration in a reasonable time period. In this article, the efficacy of five different US Food and Drug Administration-approved antifungal-loaded (amphotericin B, natamycin, terbinafine, fluconazole, and itraconazole) electrospun gelatin fiber mats were compared. Morphological studies show that incorporation of polyenes resulted in a two-fold increase in fiber diameter and the mats inhibit the growth of yeasts and filamentous fungal pathogens. Terbinafine-loaded mats were effective against three filamentous fungal species. Among the two azole antifungals compared, the itraconazole-loaded mat was potent against Aspergillus strains. However, activity loss was observed for fluconazole-loaded mats against all of the test organisms. The polyene-loaded mats displayed rapid candidacidal activities as well. Biophysical and rheological measurements indicate strong interactions between polyene antifungals and gelatin matrix. As a result, the polyenes stabilized the triple helical conformation of gelatin and the presence of gelatin decreased the hemolytic activity of polyenes. The polyene-loaded fiber mats were noncytotoxic to primary human corneal and sclera fibroblasts. The reduction of toxicity with complete retention of activity of the polyene antifungal-loaded gelatin fiber mats can provide new opportunities in the management of superficial skin infections.

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(A) Ultraviolet absorption spectra of amphotericin B in three different media. The four peaks observed in the monomeric amphotericin B are labelled. The concentration of amphotericin used was 50 μg/mL. The Y-axis for the amphotericin B in gelatin is represented separately. (B) The relative aggregation state of amphotericin B as measured by the intensity ratio of peak I and peak IV of the absorption spectra in three different media. (C) Circular dichroism spectrum of amphotericin B in three different media. Note the bisignate nature of amphotericin B in phosphate buffered saline indicating a complex structure. (D) Hemolytic activity of amphotericin B and natamycin in the presence/absence of gelatin. For all these studies, the antifungal:gelatin ratio was 0.25 wt%. (E) Ultraviolet absorption spectra of amphotericin B released from the gelatin matrix. For a comparison, the absorption spectra of free amphotericin B (10 μg/mL) in buffer is also shown.Note: ***P<0.001.Abbreviations: AmpB, amphotericin B; DMSO, dimethyl sulfate; PBS, phosphate buffered saline; AU, arbitrary unit.
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f9-ijn-9-2439: (A) Ultraviolet absorption spectra of amphotericin B in three different media. The four peaks observed in the monomeric amphotericin B are labelled. The concentration of amphotericin used was 50 μg/mL. The Y-axis for the amphotericin B in gelatin is represented separately. (B) The relative aggregation state of amphotericin B as measured by the intensity ratio of peak I and peak IV of the absorption spectra in three different media. (C) Circular dichroism spectrum of amphotericin B in three different media. Note the bisignate nature of amphotericin B in phosphate buffered saline indicating a complex structure. (D) Hemolytic activity of amphotericin B and natamycin in the presence/absence of gelatin. For all these studies, the antifungal:gelatin ratio was 0.25 wt%. (E) Ultraviolet absorption spectra of amphotericin B released from the gelatin matrix. For a comparison, the absorption spectra of free amphotericin B (10 μg/mL) in buffer is also shown.Note: ***P<0.001.Abbreviations: AmpB, amphotericin B; DMSO, dimethyl sulfate; PBS, phosphate buffered saline; AU, arbitrary unit.

Mentions: It has been shown that the toxicity of amphotericin B for mammalian cells depends on the level of aggregation.45,46 In aqueous solutions, amphotericin B exists in an aggregated form containing about 2,000 molecules, whereas in DMSO the monomeric form is predominant below 5 mM.47 The characteristic absorption bands in UV and CD spectroscopic methods can differentiate between the different states of aggregation. Therefore, the UV absorption spectra of amphotericin B under various conditions were recorded. In DMSO, amphotericin B displayed four clear characteristic absorption bands around 350, 368, 388, and 412 nm (Figure 9A). In buffer, the intensity of well-defined absorption bands decreased and a broad band around 340 nm was observed. However, the presence of gelatin lowers the extent of aggregation of amphotericin B and all four major peaks were visible (Figure 9A). The band around 412 nm is a measure of monomeric form of amphotericin B, whereas the band at 346 nm is assigned to an aggregated form.48 The ratio of two peak intensities at 346 nm and 412 nm determines the extent of aggregation of amphotericin B. In DMSO, a peak ratio of 0.5 was observed as the concentration of amphotericin B was varied from 1–50 μg/mL, indicating the presence of monomeric forms (Figure 9B). In buffer, the peak ratios were 1.5 at lower and 3.1 at higher concentrations of amphotericin B. However, in the presence of gelatin, the peak ratio was 0.47 below 20 μg/mL and increased to 0.73 at 50 μg/mL of amphotericin B, thus confirming that gelatin prevents the aggregation of amphotericin B in aqueous solution (Figure 9B).


Interaction of gelatin with polyenes modulates antifungal activity and biocompatibility of electrospun fiber mats.

Lakshminarayanan R, Sridhar R, Loh XJ, Nandhakumar M, Barathi VA, Kalaipriya M, Kwan JL, Liu SP, Beuerman RW, Ramakrishna S - Int J Nanomedicine (2014)

(A) Ultraviolet absorption spectra of amphotericin B in three different media. The four peaks observed in the monomeric amphotericin B are labelled. The concentration of amphotericin used was 50 μg/mL. The Y-axis for the amphotericin B in gelatin is represented separately. (B) The relative aggregation state of amphotericin B as measured by the intensity ratio of peak I and peak IV of the absorption spectra in three different media. (C) Circular dichroism spectrum of amphotericin B in three different media. Note the bisignate nature of amphotericin B in phosphate buffered saline indicating a complex structure. (D) Hemolytic activity of amphotericin B and natamycin in the presence/absence of gelatin. For all these studies, the antifungal:gelatin ratio was 0.25 wt%. (E) Ultraviolet absorption spectra of amphotericin B released from the gelatin matrix. For a comparison, the absorption spectra of free amphotericin B (10 μg/mL) in buffer is also shown.Note: ***P<0.001.Abbreviations: AmpB, amphotericin B; DMSO, dimethyl sulfate; PBS, phosphate buffered saline; AU, arbitrary unit.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043707&req=5

f9-ijn-9-2439: (A) Ultraviolet absorption spectra of amphotericin B in three different media. The four peaks observed in the monomeric amphotericin B are labelled. The concentration of amphotericin used was 50 μg/mL. The Y-axis for the amphotericin B in gelatin is represented separately. (B) The relative aggregation state of amphotericin B as measured by the intensity ratio of peak I and peak IV of the absorption spectra in three different media. (C) Circular dichroism spectrum of amphotericin B in three different media. Note the bisignate nature of amphotericin B in phosphate buffered saline indicating a complex structure. (D) Hemolytic activity of amphotericin B and natamycin in the presence/absence of gelatin. For all these studies, the antifungal:gelatin ratio was 0.25 wt%. (E) Ultraviolet absorption spectra of amphotericin B released from the gelatin matrix. For a comparison, the absorption spectra of free amphotericin B (10 μg/mL) in buffer is also shown.Note: ***P<0.001.Abbreviations: AmpB, amphotericin B; DMSO, dimethyl sulfate; PBS, phosphate buffered saline; AU, arbitrary unit.
Mentions: It has been shown that the toxicity of amphotericin B for mammalian cells depends on the level of aggregation.45,46 In aqueous solutions, amphotericin B exists in an aggregated form containing about 2,000 molecules, whereas in DMSO the monomeric form is predominant below 5 mM.47 The characteristic absorption bands in UV and CD spectroscopic methods can differentiate between the different states of aggregation. Therefore, the UV absorption spectra of amphotericin B under various conditions were recorded. In DMSO, amphotericin B displayed four clear characteristic absorption bands around 350, 368, 388, and 412 nm (Figure 9A). In buffer, the intensity of well-defined absorption bands decreased and a broad band around 340 nm was observed. However, the presence of gelatin lowers the extent of aggregation of amphotericin B and all four major peaks were visible (Figure 9A). The band around 412 nm is a measure of monomeric form of amphotericin B, whereas the band at 346 nm is assigned to an aggregated form.48 The ratio of two peak intensities at 346 nm and 412 nm determines the extent of aggregation of amphotericin B. In DMSO, a peak ratio of 0.5 was observed as the concentration of amphotericin B was varied from 1–50 μg/mL, indicating the presence of monomeric forms (Figure 9B). In buffer, the peak ratios were 1.5 at lower and 3.1 at higher concentrations of amphotericin B. However, in the presence of gelatin, the peak ratio was 0.47 below 20 μg/mL and increased to 0.73 at 50 μg/mL of amphotericin B, thus confirming that gelatin prevents the aggregation of amphotericin B in aqueous solution (Figure 9B).

Bottom Line: Terbinafine-loaded mats were effective against three filamentous fungal species.Among the two azole antifungals compared, the itraconazole-loaded mat was potent against Aspergillus strains.As a result, the polyenes stabilized the triple helical conformation of gelatin and the presence of gelatin decreased the hemolytic activity of polyenes.

View Article: PubMed Central - PubMed

Affiliation: Singapore Eye Research Institute, Singapore ; Signature Research Program in Neuroscience and Behavioral Disorders, Duke-NUS Graduate Medical School, Singapore.

ABSTRACT
Topical application of antifungals does not have predictable or well-controlled release characteristics and requires reapplication to achieve therapeutic local concentration in a reasonable time period. In this article, the efficacy of five different US Food and Drug Administration-approved antifungal-loaded (amphotericin B, natamycin, terbinafine, fluconazole, and itraconazole) electrospun gelatin fiber mats were compared. Morphological studies show that incorporation of polyenes resulted in a two-fold increase in fiber diameter and the mats inhibit the growth of yeasts and filamentous fungal pathogens. Terbinafine-loaded mats were effective against three filamentous fungal species. Among the two azole antifungals compared, the itraconazole-loaded mat was potent against Aspergillus strains. However, activity loss was observed for fluconazole-loaded mats against all of the test organisms. The polyene-loaded mats displayed rapid candidacidal activities as well. Biophysical and rheological measurements indicate strong interactions between polyene antifungals and gelatin matrix. As a result, the polyenes stabilized the triple helical conformation of gelatin and the presence of gelatin decreased the hemolytic activity of polyenes. The polyene-loaded fiber mats were noncytotoxic to primary human corneal and sclera fibroblasts. The reduction of toxicity with complete retention of activity of the polyene antifungal-loaded gelatin fiber mats can provide new opportunities in the management of superficial skin infections.

Show MeSH
Related in: MedlinePlus