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Antifungal Nanoparticles and Surfaces Cristiana S. O. Paulo,†,‡ Maria Vidal,§ and Lino S. Ferreira*,†,‡ Center of Neurosciences and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal, Biocant, Biotechnology Innovation Center, 3060-197 Cantanhede, Portugal, and Department of Pure and Environmental Sciences, ESAC, Bencanta, 3040-316 Coimbra, Portugal Received August 2, 2010; Revised Manuscript Received August 31, 2010
Nosocomial fungal infections, an increasing healthcare concern worldwide, are often associated with medical devices. We have developed antifungal nanoparticle conjugates that can act in suspension or attach to a surface, efficiently killing fungi. For that purpose, we immobilized covalently amphotericin B (AmB), a potent antifungal agent approved by the FDA, widely used in clinical practice and effective against a large spectrum of fungi, into silica nanoparticles. These antifungal nanoparticle conjugates are fungicidal against several strains of Candida sp., mainly by contact. In addition, they can be reused up to 5 cycles without losing their activity. Our results show that the antifungal nanoparticle conjugates are more fungistatic and fungicidal than 10 nm colloidal silver. The antifungal activity of the antifungal nanoparticle conjugates is maintained when they are immobilized on a surface using a chemical adhesive formed by polydopamine. The antifungal nanocoatings have no hemolytic or cytotoxic effect against red blood cells and blood mononuclear cells, respectively. Surfaces coated with these antifungal nanoparticle conjugates can be very useful to render medical devices with antifungal properties.
1. Introduction Fungi are the fourth most common cause of bloodstream infections in hospitalized patients.1 The design of materials or surfaces that mitigate or prevent fungal colonization or infection with subsequent biofilm formation would, therefore, be highly beneficial for medicine. Several antifungal agents have been proposed in the last years.2 Unfortunately, most of the agents are not currently approved by the Food and Drug Administration (FDA), and their antifungal spectrum and cytotoxicity profile in contact with human cells is practically unknown.3-5 Amphotericin B (AmB) is a potent antifungal agent, approved by the FDA, widely used in clinical practice and effective against a large spectrum of fungi. Few resistant strains of fungi to AmB have been reported so far.6-8 Experimental data indicates that AmB associates with the ergosterol in the fungi membrane, forming pores and, consequently, disrupting the ionic gradient.6 We hypothesize that nanoparticles (NPs) having AmB covalently attached to the surface could either coat surfaces or be directly incorporated in the bulk of materials, to yield medical devices with antifungal properties. Antifungal NPs can produce longer lasting antifungal effectiveness, and can ensure that fungi encountering the antifungal agent are exposed to only high surface concentrations as opposed to low systemic ones created by slow release devices.2 Previously, AmB has been covalently immobilized into carbon nanotubes;9 however, their use to coat surfaceswasnotdemonstrated.Althoughapromisingtechnology,10,11 carbon nanotubes might be expensive for the modification of large surfaces and raise some cytotoxicity concerns in human cells.12 Here we present a novel methodology to immobilize covalently AmB into nanomaterials. Silica nanoparticles (SNP) were chosen due to their noncytotoxicity, low price, high stability and durability, and ease of modification by organosilane * To whom correspondence should be addressed. E-mail:
[email protected]. † University of Coimbra. ‡ Biocant. § ESAC.
chemistry, allowing the incorporation of an array of different functional groups.13 We demonstrate that these NPs have high antifungal activity against several strains of Candida and can be reused without losing their antifungal activity. Importantly, the antifungal activity is not due to the leaching of AmB from the surface of the NP, because media that have been in contact with the antifungal NPs have no significant antifungal activity. We further show that these NPs can be immobilized into flat surfaces forming an antifungal coating.
2. Materials and Methods 2.1. SNPs Functionalized with AmB. 2.1.1. Preparation and Silanization of SNPs. SNPs of 5 nm in diameter were kindly offered by Eka (Sweden), while SNPs 80 nm were purchased from PlasmaChem GmbH (Germany). Fluorescent-labeled SNP170 were prepared according to the methodology reported by Nyffenegger.14 Silanization of SNP170 and SNP80 was performed in a methanol/ammonia solution (25%; 1:6, v/v), with 2.5% (v/v) of (3-aminopropyl) trimethoxysilane (APTMS, Sigma) and 3-(trihydroxysilyl)propylmethylphosphonate (THPMP, Sigma) in a proportion of 1:4 under magnetic stirring at room temperature for 3 h.15,16 Finally, the SNPs were centrifuged (SNP170: 3200 g for 10 min; SNP80: 20000 g for 40 min) and washed three times with 10 mM MES buffer pH 5.5. SNP5 silanization was carried out in borate/NaOH buffer pH 10.8 (NP concentration of 7.5 mg/mL), under reflux and vigorous magnetic stirring (750 rpm) in the presence of 3-aminopropyldimethyl methoxysilane (APMMS; 87 µL in 1 mL 0.1 M borate buffer, corresponding to 5% of the weight of SNP5 in the suspension) for 3 h. 2.1.2. Preparation and Characterization of DexOxAmB Conjugate. Dextran aldehyde (DexOx) was prepared according to a methodology previously described by us17 with a degree of oxidation of 23% to allow the immobilization of a significant amount of AmB into the backbone of the polymer via imine bond formation, while the remaining aldehyde groups could be used for the attachment of the conjugate into the surface of the NP containing terminal amine groups. A solution of DexOx (10-30 mg/mL, dissolved in 0.1 M borate buffer pH 10) was mixed with an AmB solution (in DMSO or 0.1 M borate buffer pH 10.8) with a predefined concentration to obtain a theoretical degree of derivatization of 4, 17, and 30%. The reaction
10.1021/bm100893r 2010 American Chemical Society Published on Web 09/16/2010
Antifungal Nanoparticles and Surfaces was allowed to proceed for 18 h at room temperature, under magnetic stirring, and shielded from light. Then, a sodium dodecyl sulfate (SDS) aqueous solution (10 mL, 10 mM) was added to the reaction vial to prevent the aggregation of unreacted AmB and the solution transfer to a dialysis membrane of MWCUTOFF of 6-8000 Da. The dialysis was performed in the dark, at 4 °C, for 48 h against 10 mM SDS aqueous solution (changed two times in each day)18 and then against water for 48 h. The solution was then freeze-dried and weighed. The reaction yield was approximately 81%. 2.1.3. Functionalization of SNPs with DexOxAmB. The reaction was initiated by adding a solution of DexOxAmB at variable concentration (3 mL; 1.5, 6, or 12 mg/mL in 0.01 M borate buffer pH 10) to a suspension of silanized SNPs (20 mg in 1 mL 0.01 M borate buffer pH 10) and carried out for approximately 18 h under agitation. To reduce the imine bonds, sodium cyanoborohydride (5 × 10-4 mol, 10 × excess to the imine bonds) was added for 1 h. The whole reaction volume was centrifuged and the pellet resuspended in Milli-Q water. This process was repeated two times before the use of SNPs in subsequent assays. 2.2. SEM and TEM Analysis. SNPs suspended in MES buffer (8 µg/mL) were deposited on 0.5 cm2 glass slides. The solvent was allowed to evaporate and the slides were mounted on a SEM sample stub using conductive carbon cement. The samples were then carbon coated by plasma vapor deposition and analyzed by a Hitachi S4100 SEM. For TEM analysis, the suspension of SNPs (8 µg/mL, in hexane) was spray coated on a TEM 400 mesh grid. The SNPs were then observed by TEM on a JEOL JEM-100 SX microscope at 100 kV. 2.3. Diameter and Zeta Potential of SNPs. The average diameter of SNPs suspended in 2 mL of filtered Milli-Q water was determined by a dynamic light scattering method (DLS) using a Zeta Plus analyzer (Brookhaven) from six independent measurements. The zeta potential of SNPs was determined by a Zeta Plus analyzer from three independent measurements. In this case, an aliquot (20 µL) of SNPs suspended in ethanol (8 mg/mL) was added to 1.5 mL of 0.1 M MES buffer, pH 5.5, and the zeta potential determined. 2.4. Quantification of Amine Groups in Silanized SNPs. Amine groups were quantified by the ninhydrin assay.19 In a scintillation vial, 1 mL of SNP suspension (20 mg/mL in water) was added to 1 mL of ninhydrin reagent, mixed and then immersed in boiling water. After 15 min, the vials were removed from the water bath and 15 mL of an ethanol/water mixture (1:1, v/v) was added to the reaction, which was then allowed to cool at room temperature for 15 min, in the absence of light. The particle samples were centrifuged before reading to avoid the interference of suspension turbidity in the absorbance reading. The absorbance at 570 nm was converted into concentration by using a calibration curve with solutions of glycine (10-45 mM). 2.5. Quantification of DexOxAmB Immobilized into SNPs. DexOxAmB immobilized into SNPs was determined by the anthrone colorimetric assay.20 This assay quantifies the amount of dextran immobilized into the SNPs. SNPs coated with DexOx and suspended in water were centrifuged, washed with distilled water, centrifuged, and finally resuspended in water. The anthrone assay was performed on the supernatant after each washing step. For supernatants with negligible absorbance at 620 nm (8 (>3.7)
0.03 0.2 (0.09)
0.08 1 (0.5)
1000 (10)
1000-2000 (10-20)
300 (3)
100 (1)
a
The values in parentheses refer to the amount of AmB (µg) in the conjugates or nanoparticles. b Data corresponds to the average of three independent measurements.
4. Discussion
Figure 4. Antifungal activity of silver NPs. (A) The activity was measured against 1 × 105 cells of C. albicans for 8 h. (B) Yeast growth kinetics in the presence of silver NPs. A suspension of NPs was incubated with 1 × 105 cells/mL of C. albicans in YPD medium and the absorbance of the medium measured at 600 nm overtime. Silver NPs above 0.01 mg/mL interfere with absorbance measurements and were not included in the graph. The results are expressed as average ( SD (n ) 3).
were detected by a MTT assay after 24 h exposure to NP-coated surfaces when compared to a glass surface.
This work reports a methodology to create nonleaching antifungal surfaces. The method comprises three steps: (i) immobilization of AmB to silica nanoparticles, (ii) the treatment of the substrate surface with an adhesive layer, and (iii) the coating of the substrate with the silica nanoparticles functionalized with AmB. This method offers the opportunity to create durable antifungal nanocoatings in any material surface, regardless of the nature of the bulk material. Several approaches have been described in the last years to render medical devices with antifungal properties.8,28,29 Typically, they are based on incorporation of a leachable antiseptic or antibiotic in the surface of the device. However, these approaches have limited life spans, with the majority of antimicrobial agent being released within a matter of days. In addition, large concentrations of the active agent might be required for encapsulation in these formulations, making them unsuitable for application to certain materials and devices, which may be structurally impaired by the high drug loading. Furthermore, these approaches might be permissive to the
Figure 5. Antifungal activity and biocompatibility of a substrate coated with SNP5 functionalized with DexOxAmB. (A) Antifungal activity of SNP5- DexOxAmB immobilized at glass coverslips on planktonic C. albicans cells. (A.1) CFU counts at the medium. Counts are normalized relatively to the control (SNP5-DexOx) and expressed as average ( SD (n ) 4). (A.2) Photographs of coverslips coated with SNP5-DexOxAmB (left) and SNP5-DexOx (right) plated on YPD agar after incubation in medium with C. albicans. (B) Hemocompatibility of the coated substrates. Release of hemoglobin was measured after 24 h of contact of red blood cells (2 × 108 cells per mL, in PBS) with the coverslips. Results are expressed as average ( SD (n ) 3). PBS with 20 mM SDS was used as positive control. (C) Cytotoxicity of the coated substrates. The metabolism of blood mononuclear cells (5 × 105 cells/mL, in EGM-2 medium) in contact with the coated substrates was evaluated by a MTT assay after 24 h of contact. Results are expressed as average ( SD (n ) 3).
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development of fungi resistance when the concentration of the released antifungal agent is below MIC. In contrast, our approach involves a covalent surface functionalization with an antifungal agent that substantially does not leach into solution. This technology has several advantages: first, it will produce longer lasting antifungal effectiveness; second, it will avoid possible sensitization and systemic toxicity effects of antifungal agents; and third, permanent tethering will ensure that fungi encountering the antifungal agents are exposed to only high surface concentrations as opposed to low systemic ones created by slow release devices. Recently, we have developed a gel formed by a polysaccharide-based hydrogel impregnated with AmB that kills Candida albicans essentially by contact and remains biologically active for at least 53 days.30 Also, others have demonstrated the effective immobilization of AmB in a polysaccharide-based gel to control the release of the antifungal agent.31 However, in both cases, the low mechanical properties of the gels make them less appropriate as coatings for devices where extensive manipulation is needed. In this work we developed a platform to overcome this limitation based on the coating of a substrate with a thin layer of antifungal nanoparticles. We demonstrate that AmB can be immobilized to SNPs by a polysaccharide linker (DexOx) to form antifungal nanoparticle conjugates. The coupling of AmB to a polysaccharide is advantageous as it can increase the solubility of AmB in aqueous solution, thereby facilitating its immobilization into the nanoparticles. Furthermore, part of the aldehyde groups in DexOx can be used for attachment to the nanoparticle surface. Previously, AmB has been conjugated to several polysaccharides, including arabinogalactan, dextran, and gum Arabic.31-33 We selected dextran due to its intrinsic ability to limit protein and cell adhesion.34 Under the conditions tested, we could immobilize up to 50 µg of AmB per mg of SNP5. We found that SNPs with low diameter (∼22 nm; Table 1) have lower MIC than SNPs with higher diameter (∼337 nm; Table 1). However, the most fungicidal formulation of SNP functionalized with AmB presents a higher MIC than the one observed for the unconjugated AmB. Several reasons might explain this phenomenon, including (i) SNPs aggregation (see Table 1 and section 3.4), (ii) the inappropriate orientation of AmB immobilized at the surface of the NP, and (iii) the excessive number of attachment points of DexOxAmB into the surface of SNP reducing the overall mobility of AmB. These hypotheses should be explored in future work to improve the overall system. Our results indicate that the fungicidal activity of the nanoparticle formulation is mainly mediated by contact. AmB belongs to the group of polyenes and its mode of action involves the disruption of fungal membrane.6 Several mechanisms have been proposed, including AmB-induced formation of pores in the presence of ergosterol,35 AmB-induced membrane defects,36 and AmB-induced oxidative events.37 Importantly, we demonstrate that SNPs functionalized with AmB and immobilized on top of a substrate by a chemical adhesive maintains their antifungal activity without leaching. This is in line with our previous results showing that AmB physically entrapped in dextran-based hydrogels can kill fungi by contact.30 Previous reports show that polysaccharide-based hydrogel with physical- or chemical-AmB was biocompatible, did not cause hemolysis in human blood, and was effective in killing Candida albicans when incubated with microorganisms and implanted in mice.30,31 Our preliminary results show that the antifungal nanocoatings have no measurable hemolytic or
Paulo et al.
cytotoxic effect against red blood cells and blood mononuclear cells, respectively, and therefore should be further evaluated in vitro and in vivo in more stringent tests.
5. Conclusions We have prepared and characterized SNPs functionalized with AmB that present high antifungal activity after several cycles of use. These nanomaterials can be potentially attached to any substrate, regardless of its chemical nature, by a chemical adhesive formed by polydopamine. Either in suspension or as coatings, these nanomaterials offer an alternative to current technologies involving the use of silver NPs, because they present high activity against several strains of Candida and other formulations based on AmB. Acknowledgment. We thank Rui de Carvalho (University of Coimbra) for NMR analysis, Andrade Ramos (Coimbra Institute of Engineering) for helpful discussions, and the financial support of Fundac¸a˜o para a Cieˆncia e a Tecnologia (SFRH/BD/35270/2007; fellowship to C.P.), Biocant Ventures, QREN (Project No. 5402), and the MIT-Portugal Program (focus in Bioengineering). Supporting Information Available. Preparation of SNP170, preparation and characterization of DexOx and DexOxAmB (1H NMR and UV-vis spectra), and stability of SNP-DexOxAmB in YPD medium. This material is available free of charge via the Internet at http://pubs.acs.org.
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BM100893R
