Branched amphotericin functional poly(N- iso propyl acrylamide): an antifungal polymer

Abstract

Branched poly(N-isopropylacrylamide) was functionalized with Amphotericin B (AmB) at the chain ends to produce an antifungal material. The polymer showed antifungal properties against AmB-sensitive strains of Candida albicans, Fusarium keratoplasticum and Aspergillus flavus (minimal inhibitory concentration ranged from 5 to 500 µg ml^-1) but was not effective against an AmB resistant strain of C. albicans nor against Candida tropicalis. The polymer end groups bound to the AmB target, ergosterol, and the fluorescence spectrum of a dye used as a solvatochromic probe, Nile red, was blue shifted indicating that segments of the polymer became desolvated on binding. The polymer was less toxic to corneal and renal epithelial cells and explanted corneal tissue than the free drug. Also, the polymer did not induce reactive oxygen species release from peripheral blood mononuclear cells, nor did it cause a substantial release of the proinflammatory cytokines, tumour necrosis factor-α and interleukin-1β (at 0.5 mg ml^-1).

Keywords: branched polymer; fungi; smart polymers.

Figure 1.

Synthesis of HB-PNIPAM-AmB. (a) Schematic diagram showing a generalized structure of HB-PNIPAM with chain end functionality (blue) and a chain end segment with an amphotericin end group. SEC data: (b) RI and UV data for HB-PNIPAM-AmB and AmB. (c) Molar mass distributions of as prepared polymer (HB-PNIPAM-Py) and HB-PNIPAM-AmB. Asterisk denotes molar masses were obtained by size exclusion chromatography in methanol. Hash indicates functionality (F) expressed as molar percentage of chain ends carrying an AmB moiety determined from ¹H nuclear magnetic resonance (NMR). Circled times symbol represents amount of AmB per unit mass of polymer derived from UV absorbance at 405 nm (extinction coefficient = 9 × 10¹¹ mol⁻¹ cm⁻¹).

Figure 2.

(a) Peak wavelength and (b) emission intensity of Nile red mixed with HB-PNIPAM-AmB alone (open circle) or with equivalent molar amounts of ergosterol (filled circle) or with HB-PNIPAM-Py alone (open red circle) or with equivalent molar amounts of ergosterol (filled red circle). Polymer concentrations = 1 mg ml⁻¹ in aqueous solution. Asterisks indicate significance: asterisks, between comparisons of HB-PNIPAM-AmB; asterisks, between comparisons of HB-PNIPAM-Py; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

Cell viability, relative to untreated controls: (a) AmB and (b) HB-PNIPAM-AmB with rabbit limbal epithelial cells. (c) Human cells in explanted human cornea, (d) AmB and (e) HB-PNIPAM-AmB with human renal epithelial cells (MTT). Data obtained after exposure to HB-PNIPAM-AmB (filled square) or AmB (open circle) for 48 h. Error bars are standard error of the mean (n = 3). All concentrations of HB-PNIPAM-AmB are the weight of total polymer per unit volume. The data show that AmB is more toxic to epithelial cells and tissue than the polymer. Significance: (a,b,e,d) indicated between treated and untreated cells; (c) between HB-PNIPAM-AmB and AmB treated; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

Relative metabolic activity (MTT, optical density of sample/optical density of untreated control) of PBMCs in contact with HB-PNIPAM-AmB. PBMCs derived from four anonymous donors. The data are relative to cell only controls and compared to H₂O₂ positive controls. Asterisks indicate significance. The black asterisks indicate that all of the HB-PNIPAM-AmB concentrations were significantly different to the H₂O₂ control. The brown asterisks, red asterisks and blue asterisks indicate that individual concentrations of HB-PNIPAM-AmB were significantly different to the H₂O₂. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5.

Release of reactive oxygen species in contact with HB-PNIPAM-AmB. Asterisks indicate significance relative to positive control (TBHP): *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 6.

Release of TNF-α following treatment with HB-PNIPAM-AmB concentrations of cytokines in supernatants (cells seeded at 20 000 cells per well) up to 24 h after treatment with HB-PNIPAM-AmB or LPS. Asterisks and plus signs indicate significance. The black asterisks indicate that all of the HB-PNIPAM-AmB concentrations were significantly different to the LPS control at the stated time point. The brown asterisks, red asterisks and blue asterisks indicate significant differences between individual concentrations of HB-PNIPAM-AmB with LPS. *p < 0.05, **p < 0.01, ***p < 0.001. The black plus signs indicate that all of the HB-PNIPAM-AmB were significantly different to the untreated cells at the stated time points. The brown plus, red plus and blue plus signs indicate significant differences between individual concentrations of HB-PNIPAM-AmB and LPS. ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001.

Figure 7.

Release of IL-1β following treatment with HB-PNIPAM-AmB concentrations of IL-1β in supernatants (cells seeded at 20 000 cells per well) up to 24 h after treatment with HB-PNIPAM-AmB or LPS. There is no significant increase in the release of IL-1β following treatment with 0.5 mg ml⁻¹ of HB-PNIPAM-AmB but concentrations of over 5 mg ml⁻¹ stimulated release of increased amounts of this cytokine. Asterisks and plus signs indicate significance. The black asterisks indicate that all of the HB-PNIPAM-AmB concentrations were significantly different to the LPS control at the stated time point. The brown asterisks, red asterisks and blue asterisks indicate significant differences between individual concentrations of HB-PNIPAM-AmB with LPS. *p < 0.05, **p < 0.01, ***p < 0.001. The black plus signs indicate that all of the HB-PNIPAM-AmB were significantly different to the untreated cells at the stated time points. The brown plus, red plus and blue plus signs indicate significant differences between individual concentrations of HB-PNIPAM-AmB and LPS. ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001.