Bursting the Virulence Traits of MDR Strain of Candida albicans Using Sodium Alginate-based Microspheres Containing Nystatin-loaded MgO/CuO Nanocomposites

Introduction

During recent decades, fungal infections have become a serious health issue contributing substantially to human morbidity and mortality.^1,2 These infections range from superficial to invasive or chronic and the effect of these diseases on human health is not generally appreciated. The pathogenic strains of fungi causing life-threatening fungal diseases include Cryptococcus spp., Fusarium spp., Aspergillus spp., and Candida spp. but Candida albicans in particular, is the most ubiquitous pathogenic multidrug resistant strain causing invasive fungal infections.³ To treat these infections, the use of therapeutic agents available presently is limited in terms of their low bioavailability, toxicity, antibiotic resistance and high cost.^4,5 Therefore, there is an increased need to search for new therapeutic options.

Generally, amphotericin B (Amp B) and the azole group of antifungals are the choice of drugs for the treatment of superficial and invasive infections, but excessive use of these drugs has increased the fungal resistance. Nystatin, a polyene macrolide synthesized by Streptomyces noursei, is an antifungal agent and has broader spectrum of antifungal activity than antifungal antibiotics like Amp B and azoles.⁶ Nystatin exerts its therapeutic effect by forming complexes with ergosterol in plasma membrane leading to intracellular content leakage, but its efficacy is restricted to topical infections because of its significant high toxicity when administered intravenously.⁷

Moreover, many inorganic metal oxides have also attracted the attention as antimicrobial agents due to their improved stability and safety which are lacking in organic metals⁸ among the nanoparticles of metal oxides, magnesium oxide NPs have been used to treat various diseases due to their stability under stressful conditions.^9,10 MgO has been used for magnesium supplementation, an important mineral for the human body. MgO shows impressive applications in the medical field such as bone regeneration, heartburn relief and digestive issues, as well as antitumor and antimicrobial agents, and is accepted as a safe biomaterial by the US Food and Drug Administration.¹¹ Raffi et alreported that copper oxide NPs are very stable, strong and have longer shelf life than organic antimicrobials.¹² Cioffi et al studied the antifungal and bacteriostatic properties of CuO NPs.¹³ Studies on antimicrobial activities of both copper oxides and magnesium oxide identified them to be active agents against both fungi and bacteria.¹⁴ But these nanoparticles could be toxic to the human body when administrated directly because of their large and highly reactive surface area.

To overcome the limitations described, special attention has been taken on safe, targeted and modified release, polymer-based delivery systems which provide substantial advantages over the other carrier systems for improved antifungal therapies. Physicochemical properties such as size, surface charge, hydrophilicity, biocompatibility, biodegradability, nontoxicity, high availability and low biopolymer cost have made them a promising alternative.^15,16 Several hydrophilic polymers especially polysaccharides and their derivatives have been used for the synthesis of in-site specific modified release dosage forms.¹⁷ Alginate is an anionic polymer derived from brown seaweed or bacteria, commonly used for decades in several pharmaceutical formulations.¹⁸ It consists of β-d-mannuronic acid (M) and α-L-glucuronic acid (G) repeating units linearly linked by 1.4-glycosidic linkages.¹⁹ Alginate form hydrogels or beads when cross linked with divalent cation such as calcium ion in aqueous solution. This ionotropic gelation is the most universal, simplest and nontoxic method for the synthesis of microspheres and is often employed as supports to deliver bioactive compounds.²⁰ Based on above considerations, the purpose of the present study was to synthesis nystatin-conjugated MgO/CuO composite encapsulated in sodium alginate microspheres. Furthermore, characteristics of the synthesized microspheres in terms of their size, morphology, crystallinity, chemical structure and bioactive compound loading and release was performed. At last the antifungal activity and effect of developed microspheres on virulence traits of Candida was conducted.

Materials and Methods

Materials

Sodium alginate (Gaviscon, Reckitt Benckiser Pakistan Ltd), calcium chloride (Nutriflex Special, B. Braun, Pakistan Pvt), phosphate-buffered saline (PBS, Sigma-Aldrich, USA), fetal bovine serum(FBS, Gibco, Thermo Fisher Scientific, USA), potato dextrose agar (PDA Merck, Germany), sabouraud dextrose agar (SDA, Oxoid Deutschland, Thermo Scientific™, USA), Triton X (Merck, Pvt Ltd, Pakistan), crystal violet (Ricca Chemical, US), nystatin (Wyeth, NJ, USA) were all purchased from commercial vendors. Annexin V binding buffer, propidium iodide and annexin V-FITC kit was obtained from Biolegend. Magnesium oxide and copper oxide nanoparticles were obtained from Nano photocatalysis lab International Islamic University Islamabad.

Preparation of Drug-loaded MgO-CuO Conjugate

Stock solution of 20 µL of nystatin (100,000 units/mL) suspension was prepared by diluting it in 980 µL of sterile water. Further, antibiotic-bound biogenic MgO/CuO nanoparticles were prepared by stirring 1 mL of nystatin solution with 1 mL of biosynthesized MgO/CuO nanoparticles for two hours, followed by incubation for 48 h at room temperature. The subsequent functionalized nanoparticles were centrifuged at 6000 rpm for 10 min, and the pellet was resuspended in sterile deionized water, dried in the drying oven, and stored in dark at 4°C in Eppendorf tubes.

Synthesis of Metal Oxide NP-loaded Microspheres

Synthesis of metal oxide NP-loaded microspheres was performed using same method as Kanokpanont et al.²¹ A total of 30 mg of already green synthesized magnesium oxide and copper oxide composite were dissolved in 1 mL of distilled water and stirred separately and 25 mL of sodium alginate solution was prepared by mixing 0.75 g of SA powder in 25 mL of distilled under continuous magnetic stirring for 15 min. Nanoparticles solution was then added to alginate solution and stirred further for two hours. After thorough mixing, the mixture was left for one hour to make it bubble free. This mixture was then passed through a syringe into 50 mL calcium chloride solution. Microspheres formed instantaneously were allowed to harden in the calcium chloride for 15 min and then collected through filtration, washed with distilled water three times and dried for 24 h at room temperature.

Synthesis of Nys-MgO/CuO-loaded Sodium Alginate Microsphere

A 0.75 g of sodium alginate 3% (w/v) was added to 25 mL of distilled water and magnetically stirred for 15 min with gentle heat. Thirty milligrams mg of nystatin-loaded MgO/CuO nanoparticles were added in 1 mL of distilled water in Eppendorf and vortex. This solution was added to sodium alginate solution and stirred up to complete dissolution for two hours. The mixture was allowed to stand for one hour at room temperature to become bubble free. Then 20 mL of sodium alginate solution was pumped slowly into 50 mL of calcium chloride solution (5% w/v) which acted as a cross-linking agent through a syringe with fine stainless-steel needle (0.45 mm in diameter) in a dropwise manner. The distance from the calcium chloride solution to the needle was 6 cm. The microspheres formed immediately and were left for 15 min to harden in the CaCl₂ solution. They were then recovered by filtration, washed with distilled water three times and dried at room temperature for 24 h (Figure 1).

Figure 1 Synthesis of Nys-MgO/CuO nanoparticle-loaded microspheres by ionic gelation method.

Synthesis of Nystatin-loaded Sodium Alginate Beads

Nystatin-loaded microsphere were prepared as a positive control. Briefly, 20 µL of nystatin was dissolved in 2 mL of distilled water and vortex. This solution was added to 0.75 g/25 mL sodium alginate solution and stirred for 30 min. The suspension was elapsed for one hour to remove any air bubbles, introduced into a 5 mL syringe and extruded through a needle into 50 mL of CaCl₂ solution. The formed beads were left for 30 min in a solution of CaCl₂. Drug-loaded microspheres were collected through filtration, washed thrice with distilled water and dried for 24 h at room temperature. Hollow sodium alginate microspheres were prepared as a negative control applying same method described above.

Nanoparticles Entrapment Efficiency

To determine the concentration of Nys-MgO/CuO conjugate entrapped in microspheres,²² the microspheres were weighed and dissolve in 25 mL phosphate buffer (pH 6.8). The suspension was sonicated for 15 mins to lyse microspheres and extracted for 12 h. One milliliter of this solution was then dissolved in 10 mL of PBS and absorbance of Nys-MgO/CuO conjugate in phosphate buffer was determined spectometrically at 285.5 nm. Blank microspheres were also assayed as control. Calibration curve regression equation was calculated for the total sum of conjugate. The efficiency of encapsulation was computed using the following formula:

Release Kinetic Studies

To evaluate the in vitro release of the nystatin-loaded formulation, a vertical Franz diffusion cell system with dialysis membrane (cutoff mol. weight. 12,000) was used. The cellulose acetate membrane was placed carefully on the upper donor chamber separating the receptor chamber. Acetate buffer (pH 5.5) was added to the receptor chamber to maintain sink condition throughout the studies that facilitate the diffusion rate. Accurately weighed quantity of nystatin-loaded microspheres and Nys-MgO/CuO-loaded MS were placed on the upper chambers and allowed to release in a receptor chamber containing acetate buffer media having pH 5.5 at 30±0.2°C while continuous stirring (100 rpm) for approximately 10 h. Samples (1 mL) were withdrawn from the receptor chamber at 0, 1, 2.4, 8, and 12-h time intervals and restored with equal quantity of fresh buffer. Withdrawn samples were analyzed for drug release by UV method at 305 nm.²³ The absorbance of each sample was noted and percent drug release was determined by the following formula.

Characterization of Microspheres

X-ray Diffraction (XRD) Analysis

Crystalline structure and purity of bead samples were investigated by X-ray diffraction (XRD) in a wide-angle diffractometer, model D8 Advance Bruker equipped with a LynxEye™ at 60 kV and 60 mA current. The samples were irradiated with monochromatic Cu Kα radiations (1.542 Å).2θ was the diffraction angle used with the values ranging from 10 to 90°, respectively.

FTIR Spectroscopy

Fourier-transform infrared (FTIR) spectra (Thermo Nicolet Nexus FTIR) analyzed the functional groups present in the samples. The sample was crushed to fine powder and mixed with potassium bromide KBr. The mixture was compressed to semi-transparent discs by putting a pressure of 10 tons. The spectrum was measured within 4000–400 cm^–1 range.

FESEM Analysis

To analyze the morphology and particle size of microspheres, scanning electron microscopy (Hitachi SEM SU 3500, Japan) was used with an acceleration voltage of 5 kV and magnification ranging from 25 to 10 k. Randomly selected beads were mounted on the aluminium stub with the help of adhesive double-sided carbon tape, sputter coated with 20 nm gold film and examined in the scanning electron microscope.²⁴ A few beads were cut with sterile medical blade for cross-sectional examinations.

Strains

Two clinical strains of Candida albicans (AH 201) and (AH 267) were used to evaluate the antifungal activity of microspheres. These strains were obtained from the Microbiology Laboratory of Ali Hospital Rawalpindi, Pakistan. The collected strains were incubated for 48 h at 37°C on Sabouraud dextrose agar medium (1% peptone, 1.5% agar, 4% glucose) at pH 5.6. One colony of each isolate was inoculated in 10 mL of Sabouraud dextrose broth and incubated for 24 h at 37°C. Cells were harvested by centrifugation (1500 rpm) for 10 min and washed with PBS. Candida strains were kept as stock cultures in SDA containing 60% glycerol at –80°C for long-term storage.

Antifungal Activity of Synthesized Microspheres

Antifungal susceptibility tests were performed on potato dextrose agar (PDA) plates according to the method of Moeini et alwith slight modifications. Briefly, six to eight beads from two differently synthesized microspheres, MgO/CuO composite loaded microspheres and Nys-MgO/CuO-loaded microspheres were placed on PDA plates swabbed with C. albicans from a parent culture and incubated for 24 h at 37°C.²⁵ Blank SA microspheres were placed as negative control and nystatin-loaded microspheres were placed as positive control. Zone of inhibition was measured the next day.

Effect of Microspheres on Virulence Factors

Germ Tube Development Assay

Germ tube test was performed using the method of Jalal et al with modification to screen the effect of synthesized Nys-MgO/CuO-loaded microspheres on the formation of germ tube.²⁶ Two to three small colonies of Candida were inoculated in a tube containing 0.5 mL of human serum treated with 5 mg of Nys-MgO/CuO-loaded microspheres and incubated for two to three hours at 37°C. After incubation, a drop of culture was transferred to a clean microscope slide to form a smear, a coverslip was placed onto it and examined under a microscope at 40ẋ. Human serum without microspheres was considered as control. For examination of the percentage of germ tubes present, we used light microscope (ECLIPSE TE300, Nikon, Tokyo, Japan); 100 cells were counted each time.

Phenotypic Biofilm Suppression Assay

The effect of Nys-MgO/CuO microspheres on the biofilm production was evaluated by tube method (TM).²⁷ A loopful of microorganism from overnight culture PDA plate was inoculated into tubes containing 10 mL of Sabouraud dextrose broth supplemented with 1% glucose. The tubes were then incubated for 48 h at 35°C. After incubation, the supernatant was decanted, and tubes were washed with PBS (pH 7) and dried tubes were stained with 0.1% w/v crystal violet for 30 min. Excess stain was eliminated by washing the tubes with distilled water three times. Then tubes were inverted to dry and then detected for biofilm formation. Production of biofilm was deemed positive if a clear film lined the test tube wall. Media containing Candida cells without microspheres was considered as control. The inhibition of biofilm formation was analyzed phenotypically by observing color intensity of crystal violet dye and the amount of biofilm formation was scored as 0=absent, 1=weak, 2=moderate, or 3=strong.²⁸ Experiments were performed in triplicate and repeated three times.

Flow Cytometry Analysis

C. albicans cells were cultured in Sabouraud broth at 37°C until late log phase. The cells were centrifuged to remove supernatant, suspended in PBS supplemented with 2% glucose and counted in a Neubauer chamber. Suspensions containing 10⁶ cells/mL were incubated with 10 mg of Nys-MgO/CuO microspheres agent for eight and 24 h. After antifungal treatment, yeast cells were centrifuged, washed and resuspended in PBS. Cells were then incubated with 1% FBS and Annexin V binding buffer for 20 min and then stained with 5 μL propidium iodide and 5 μL Annexin V-FITC. These stained cells were kept in the dark for 30 min at room temperature. Suspension of untreated cells was also stained with PI and Annexin V-FTIC and used as control. After staining, cells were analyzed with FACScan flow cytometer (Beckman Coulter Cytomics FC500).

Efflux of Cytoplasmic Content

Damage to the cell membrane causes cytoplasmic substances such as DNA and RNA to be released from the cell, which can be detected by UV spectrophotometers below 260 nm.²⁹ Four aliquots of yeast suspensions first containing only Sabouraud broth as negative control, second with 5 mg blank MS as positive control and third and fourth with 5 mg MgO/CuO and Nys-MgO/CuO MS were incubated at 30°C overnight. The mixture was then filtered using 0.22 μm drainage pin filter to remove yeast and microspheres. OD values were monitored under 260 nm UV.

Hemolytic Activity Against Human Erythrocytes

Hemolytic activity of Nys-MgO/CuO MS was evaluated against human erythrocytes. Sterile blood (5 mL) was withdrawn from a healthy human after obtaining consent of the participant by briefing her about the study and that the provided blood will be solely used for research purposes. The blood was added directly to 50 mL of sterile blood agar base which was cooled to 45 to 50°C. Flask was swirled immediately to avoid the formation of bubbles and froth on the surface. The mixture was poured immediately into the petri dishes and allowed to solidify. Wells of 6 mm diameter were cut aseptically into the agar and different concentrations of microspheres were added to the wells; two beads (6.6 mg), four beads (13.3 mg), six beads (21 mg) and 0.1% Triton X was used as positive control. Plates were incubated for 24 h at 37°C and then hemolytic reaction (zone of hemolysis around well in millimeters were noted if produced) on plates were observed by holding up the plate to a light source.³⁰ Formation of a clear visible enlightened area indicate hemolysis of RBCs.

Statistical Analysis

SPSS version 20 (IBM) was used for statistical analysis. Results were expressed as mean ±SD unless otherwise stated. One way ANOVA was applied to the data following Tukey–Kramer test to determine the statistical significance. P-value ˂0.05 was chosen as level of significance.

Results and Discussion

Synthesis of Microspheres

Sodium alginate and drug-composite encapsulated alginate beads were prepared by simple ionic gelation technique by extruding the sodium alginate mixture into aqueous calcium chloride used as cross-linker through a syringe of 24-gauge needle. Morphological images of wet and dry microspheres are presented in Figure 2. Microspheres obtained were spherical and rough in shape. Air dried microspheres were larger in size than oven dried because of inadequate dehydration owing to air drying process.^31,32 Blank sodium alginate microspheres were transparent crystal-like in appearance while nystatin-loaded microspheres were pale yellow. Similarly, microspheres containing only MgO/CuO composite were dark brownish-black while Nys-MgO/CuO-loaded microspheres were light brownish-black in color.

Figure 2 (A) Morphology of wet microspheres; sodium alginate beads (i), Mg/CuO composite-loaded SA beads (ii), nystatin loaded SA beads (iii), Nys-MgO/CuO loaded SA beads (iv). (B) Morphology of dry microspheres; (a) air dried sodium alginate beads, (b) oven dried sodium alginate beads.

Nanoparticle Entrapment Efficiency

The encapsulation efficacy of Nys-MgO/CuO NPs in sodium alginate was found to be 80%. A possible reason for the high encapsulation efficiency of Nys-MgO/CuO NPs in sodium alginate could be the good solubility of nanoparticles in the solvent, and the close contact between the components.

Release Kinetic Studies

Figure 3 depicts the release profile of nystatin from sodium alginate microspheres and from MgO/CuO SA matrix. Results showed that there is comparatively high release of drug from sodium alginate microspheres encapsulating only drug than from sodium alginate microsphere encapsulating Nys-MgO-CuO NPs. There were two phases in the release profile. The first phase indicates the quick release of the drug, followed by slow release.³³ Our result confirmed that drugs conjugated with MgO/CuO nanoparticles and encapsulated in sodium alginate have increased stability in acid medium and have sustained and prolonged release of drug. This stability and controlled release are attributed by sodium alginate.

Figure 3 In vitro drug release profile of nystatin from sodium alginate microsphere and from Nys-MgO/CuO-loaded sodium alginate beads.

Characterization

X-ray Diffraction (XRD)

To investigate the entrapment and physical state of nystatin, MgO/CuO composite and Nys-MgO/CuO conjugate in sodium alginate beads, X-ray diffraction data have been attained. The XRD of blank sodium alginate MS and nystatin-loaded alginate MS are given in Figure 4A. These samples show no distinctive XRD peaks suggesting that the drug was not present in a crystalline state but rather in an amorphous shape in bead matrix.³⁴ This clearly indicates that a shift in the crystalline state of the drug was caused by ionotropic gelation process during the preparation of beads. Two diffraction peaks at 2θ values 13.5° and 29.8° were observed for sodium alginate due to the reflection of their amorphous (110) plane from glucuronate unit and (200) plane from mannuronate unit. Figure 4B shows XRD pattern for MgO/CuO-loaded alginate MS and Nys-MgO/CuO-loaded alginate MS in semicrystalline structural form. The amorphous structure in Figure 4B ensures the presence of sodium alginate while crystalline structure ensures the presence of MgO/CuO and Nys-MgO/CuO. Diffraction peaks for MgO/CuO nanocomposite observed at 39.6°, 44.2°, 64.7°, 76.2°, and 78.61° represents the crystalline planes at (111), (200), (220), (311), and (222), respectively. An additional peak for CuO was observed at 34.26° representing crystal plane at (−111). The observed diffraction reflections of MgO and CuO are in good agreement with JCPDS card No. 77–2364 and JCPDS card No. 89–2531.^35,36 In XRD patterns of Nys-MgO/CuO-loaded formulation a substantial reduction in peak intensities was noticed. The peak depends on the size of the crystal, but the characteristic nystatin peaks have been integrated with the polymer blend matrix in the present study for Nys-MgO/CuO MS, thus inducing amorphous phase nature. However, in the case of drug-loaded beads, the crystallinity of the drug at the crystal size detection limit is hard to investigate. XRD results suggest nystatin dispersion at molecular level in the polymer blend matrix.³⁷ The crystalline size of MgO/CuO loaded alginate MS and Nys-MgO/CuO-loaded alginate MS samples is measured via Scherrer’s equation centered at 44.2° (200) of MgO is found 24.0 and 30.0 nm, respectively.

Figure 4 XRD pattern of (A) (a) blank sodium alginate MS, (b) Nys-MS, (B) (c) MgO/CuO-loaded alginate MS, (d) Nys-MgO/CuO-loaded alginate MS.

FTIR Spectroscopy

The FTIR spectrum bands for bare sodium alginate were observed at 3422.5, 2921.8, 1629.5 cm^–1 (Figure 5a).³⁸ All samples exhibited broad and wide spectrum at wave number 3600 cm⁻¹–3200 cm⁻¹ which is attributed to free hydroxyl group O–H stretching and bending vibrations of water molecules. In the FTIR spectrum of sodium alginate the peak at 1629 cm⁻¹ was assigned to –COO^– symmetric stretching. Peak obtained at 2921 cm⁻¹ was assigned to –CH₂ stretching.³⁹ The cross-linking of sodium alginate with CaCl₂ showed a shift of C=O peak from 1629 cm⁻¹ to 1639 cm⁻¹ indicating an ionic bonding between calcium ion and carboxyl groups of sodium alginate.⁴⁰ The FTIR spectrum of nystatin drug had absorption peaks at wave numbers 3412.2, 2921.7, 1627.3, 1442.2, 1031, 543.1 cm⁻¹. (Figure 5b).⁴¹ The absorption peak at wave number 618 cm⁻¹ was assigned to the stretching vibration of Cu-O bonding in monoclinic crystal structure of CuO (Figure 5c and d).^42,43 The characteristic bands at wavenumber 3600–3400, 1639, ~600–800 cm⁻¹ were assigned to functional groups of MgO structure.⁴⁴ The stretching vibration mode ~600–800 cm^–1 indicated Mg-O-Mg bonds.⁴⁵ Some peaks of nystatin, MgO and CuO overlap somewhat with one another and with the intense peak of sodium alginate. Balamurugan et al investigated the MgO NPs and found the vibration mode in the range of 487–677 cm⁻¹ wave number, demonstrating the bonds of Mg-O-Mg.⁴⁶

Figure 5 FTIR spectra of (a) sodium alginate MS (b) nystatin-loaded alginate MS, (c) MgO/CuO-loaded alginate MS, (d) Nys-MgO/CuO-loaded alginate MS.

Scanning Electron Microscopy (SEM)

Surface morphology of beads were examined by scanning electron microscopy (SEM). Optical micrographs of beads in Figure 6 illustrate that the beads have a rough spherical shape of average size 8–10 μm and a highly porous surface, which easily allows the diffusion of drug conjugated nanoparticles from microspheres when come into contact with an aqueous environment (Figure 6C, D, G and H). The porous surface observed through SEM also proved the sustained in vitro release of drug from microspheres. It was observed that the size of dried bare sodium alginate microspheres was smaller than other combinations of microsphere and wet bare microspheres were larger in size than other microsphere formulations (Figure 6A). It is evident that nystatin-loaded MS and Nys-MgO/CuO MS (Figure 6B and F), have rougher surface than the other alginate bead formulations. Due to the incorporation of drug and nanoparticles, their surfaces have attained roughness, and increased concentration has made the surface more porous. The morphological features MgO/CuO composite-loaded microspheres shown in Figure 6E suggest rough structures with many cracks ascribed to rapid process of drying.³⁷ The cross-sectional images of Nys-MgO/CuO-loaded MS (Figure 7) showed that the drug conjugated nanoparticles were found in the inner surface and were dispersed well in the polymer matrix. It also confirmed that the surface of microspheres become permeable upon swelling in aqueous medium.⁴⁷

Figure 6 SEM images of blank sodium alginate MS (A and C), nystatin-loaded alginate MS (B and D), MgO/CuO-loaded alginate MS (E and G), Nys-MgO/CuO-loaded alginate MS (F and H).

Figure 7 SEM images of cross-section of Nys-MgO/CuO microsphere. Arrows indicates the dispersed nanoparticles.

Antimicrobial Activity

The means of zones of inhibition of C. albicans by microspheres at 24 h were measured. Table 1 shows the antifungal activity of synthesized microspheres against C. albicans. Nys-MgO/CuO alginate microspheres represented an excellent inhibitory effect against both strains of C. albicans than MgO/CuO alginate microspheres in in-vitro. The synergistic activity of Nys-MgO/CuO-loaded sodium alginate microspheres showed zone of inhibition of 24.5±1.7 mm while zone of inhibition of Mg/CuO-loaded sodium alginate microspheres was computed as 19.2±1.6 mm for C. albicans strain (AH201). For C. albicans strain (AH267) zone of inhibition measured for Nys-MgO/CuO and MgO/CuO microspheres was 14.3±1.2 mm and 1.3±0.61 mm respectively. Nystatin-loaded microspheres showed 0.41±0.23 mm and 0.5±0.21 mm. Sodium alginate microsphere (control) did not demonstrate any antifungal activity in both strains (Figure 8).

Table 1 Zone of Inhibition Showed by Synthesized Microspheres Against Candida albicans

Figure 8 Antimicrobial activity of synthesized microspheres on Candida albicans (a) blank SA beads (control), (b) nystatin-loaded SA beads, (c) Nys-MgO/CuO-loaded SA beads, (d) MgO/CuO-loaded SA beads.

CuO NPs in the concentration of 50 µg/mL were used by Khan et al showed higher antimicrobial activity against growth and biofilm formation.⁴⁸ A study conducted by Kong et al demonstrated that MgO NPs can efficiently inhibit the growth and two-phase morphological transformation of C. albicans.⁴⁹ Nystatin antifungal activity on Candida was studied previously. For one of them, the bioactivity of 30 µg/mL nystatin was 18–24 mm in agar well diffusion.⁵⁰ In another study, nystatin has shown antifungal effect against C. albicans at MIC₅₀ equal to 25 µg/mL. MIC₅₀ standard of nystatin for susceptible Candida species is 4–7 µg/mL; therefore, the strains of Candida in this study showed considerable resistance to nystatin. However, nystatin has been reported to be successful compared to azole and amphotericin B against resistant strains of Candida in clinical trials.⁵¹ Results of our study showed combined antifungal effect of nystatin-loaded MgO/CuO NPs.

Effect on Virulence Factors

Germ Tube Development Assay

The most important virulent factors in C. albicans are the morphological transformations between yeast and filamentous forms. Development of germ tube or hyphae initiates adherence and biofilm formation in C. albicans which play key role in colonization and hence pathogenesis.Infections could be reduced by blocking the transformation of yeast buds into hyphae. For instance, hyphal-inducing conditions include stimuli such as increased temperature, pH, serum, nutrient hunger, and cell density.⁵² In present study, the effect of biosynthesized sodium alginate microsphere encapsulating Nys-MgO/CuO on development of germ tube in clinical isolates of C. albicans has been investigated. Germ tube formation is expressed as a percentage of that for control cells incubated in absence of microspheres. Our experiments confirmed that Nys-MgO/CuO-loaded SA microspheres significantly inhibit the serum induced mycelia formation by 75% after 2.5 h of incubation and 92% after three hours of incubation at concentration of 5 mg/mL (Figures 9 and 10).^52,53 Jalal et al published similar findings that silver NPs inhibited germ tube formation at 0.25 mg/mL by 97.1%.⁵⁴ In another study, complete inhibition of germ tube formation in Candida spp. by CuO NPs at 300 mg/L was reported.⁵⁵ However, the exact inhibition mechanism is not clear.

Figure 9 A (stained), B (unstained); formation of germ tube in untreated cells after 1 h (I), after 2.5 h (II). Inhibition of germ tube formation in cells treated with Nys-MgO/CuO MS for 2 h (III), for 3 h (IV). The arrows indicate the formation of germ tubes (I, II) and inhibition of growth of germ tubes (III, IV).

Figure 10 Inhibition of germ tubes in C. albicans incubated with Nys-MgO/CuO MS at 2.5 h and 3 h. The data is presented as the mean ±SD and analyzed using ANOVA. *P<0.01 and **P<0.001 refers to statistical significance of both groups from the control.

Phenotypic Biofilm Suppression Assay

Biofilm formation is another important virulence trait in C. albicans. It plays a key role in pathogenesis. C. albicans form a biofilm on medical devices such as urinary catheter, cardiac valves, artificial vascular bypass device and pacemakers. The sessile cells within biofilm act as a barrier for drug diffusion, are highly resistant to drugs, and are difficult to eliminate. Antibiofilm agent for handling such serious infections is therefore urgently required. To some extent, Candida’s loss of ability to form biofilm may indicate less virulence when directly invading and colonizing the host tissue (Figure 11). Qualitative suppression of biofilm formation in the presence of Nys-MgO/CuO alginate beads was investigated in the present study by test tube method and biofilm inhibition was examined phenotypically (Figure 12).⁵⁶ It was demonstrated that Nys-MgO/CuO alginate microspheres inhibit biofilm formation on tubes when stained with crystal violet (Figure 12B and C) while control untreated normal cells remained bound together within extracellular matrix and formed strong biofilm (Figure 12A). A study by Jalal et al⁵⁴ showed complete inhibition of the formation of biofilm in C. albicans by Ag NPs. Recently Eshed et al reported that low concentrations of CuO NPs prevent biofilm formation in Streptococcus mutans.⁵⁷ The antibiofilm effect is primarily due to the interruption of cell wall and enhanced penetration of drug and nanoparticles into the biofilm structure that disturbs cell membrane lipidomes. Inhibition of blastospores and hyphae forms also suppresses the development of biofilms in C. albicans.⁵⁸

Figure 11 Stages of biofilm formation and effects of Nys-MgO/CuO microspheres on biofilm formation.

Figure 12 Control untreated cells incubated for 24 h (A), cells treated with Nys-MgO/CuO for 24 h (B), for 48 h (C). Key: score for biofilm formation (0=absent, 1=weak, 2=moderate, 3=strong).

Flowcytometry

Flow cytometry enables the rapid detection of drug conjugated nanoparticles internalization in live yeast.⁵⁹ C. albicans exhibited increased damage to cell membrane and leakage of cell inclusion when treated with Nys-MgO/CuO microspheres, as determined by flow cytometry analysis. Phosphatidylserine is a phospholipid enriched in the inner leaflet of the plasma membrane that is exposed to calcium-dependent stimuli in the outer leaflet during early apoptosis.PI, an intact impermeable dye will only move through the cells when they are compromised or dead. In contrast Annexin V-FITC has strong attraction for outer leaflet phosphatidylserine. Consequently, cells can be marked with Annexin V-FITC only for the duration of early apoptosis while late apoptotic cells or necrotic cells can be marked with both Annexin V-FITC and PI.⁶⁰ In combination with PI, we acquired fast and precise assessment of cellular structural damage based on flow cytometric analysis. Figure 13A revealed 30.28% dead cells (left upper quadrant) and 29.99% late apoptotic cells (right upper quadrant) with incubation time of eight hours and Figure 13B demonstrates 94.16% late apoptotic cells. Our results showed that the cell population in the right upper quadrant in Figure 13B which indicates late apoptotic cells increased to 94.16% with increase in incubation time with Nys-MgO/CuO microspheres (24 h) in comparison to Figure 13A (29.99%) which was incubated for eight hours. We confirmed that the cell death caused by Nys-MgO/CuO MS exhibits late apoptotic features (Table 2). Collectively, the data presents that the Nys-MgO/CuO MS elevated the membrane permeability of the C. albicans cells, possibly resulting in cell injury or death.

Figure 13 Flow cytometry images of C. albicans after incubation with Nys-MgO/CuO for (A) 8 h, (B) 24 h.

Table 2 Flowcytometry Results of C. albicans Cells Treated with Nys-MgO/CuO

Efflux of Cytoplasmic Content

Drug-conjugated antimicrobial nanoparticles exhibit antibiotic activity on the target cell membrane through the formation of pores that cause leaks of important metabolites leading to cell lysis. Disruption of the cell membrane structure is the primary destroying mechanism for antifungal agents. We examined whether Nys-MgO/CuO MS would cause leakage of cellular components. The leakage of cytosolic components was evaluated using cell suspension supernatant preincubated with nystatin loaded MS, MgO/CuO composite loaded MS and Nys-MgO/CuO loaded MS with concentration of (10 mg/10 mL). The absorbance recorded at 260 nm was 0.141 nm, 0.055 nm and 0.096 nm respectively which revealed a significant release of intracellular constituents (DNA or proteins) from severely destroyed membranes of C. albicans. This data give evidence for flow cytometry analysis (Figure 13) that the disruption of cell membranes occurred, and cells stained with both annexin V and PI (PI binds with released fragmented DNA).

Hemolytic Activity

Hemolytic activity was assessed by using petri dishes containing nutrient agar supplemented with 5 mL RBCs. However, it allows the evaluation of hemolysis that appears on the agar as an easily visible light region. Results represented in Figure 14A and B showed that Nys-MgO/CuO microspheres exhibited no hemolytic activity, but at a relatively high concentration (Figure 14C). There was absence of enlightenment around the well containing 6 mg and 14 mg Nys-MgO/CuO MS while Triton X (control) showed 33 mm hemolytic halo and a clearly enlightened area. Our result confirmed that at dosage range of 5–14 mg/mL, these microspheres do not exercise any hemolytic effect on human erythrocytes (Table 3). A research carried out by Amiriet al found that CuONPs with a concentration of 100–5000 μg/mL had no toxic impact on human cells.⁶¹ Similarly, a study by Patel et al proved that MgO NPs do not show any significant cytotoxicity at the range 150–250 µg/mL.⁶² Boros-Majewska et al also reported that nystatin at low concentrations do not cause cytotoxicity.⁶³

Figure 14 Hemolytic activity of Nys-MgO/CuO MS against human blood at different concentrations key: control showed formation of 33 mm halo due to lysis of RBCs (A) 6.6 mg (two beads), (B) 14 mg (four beads), (C) beads showed hemolysis at conc. of 21 mg (six beads).

Table 3 Hemolytic Activity of Nys-MgO/CuO Microspheres on Human RBCs (Erythrocytes)

Mechanism of Action of Synthesized Microspheres on Candida albicans

Nys-MgO/CuO loaded microspheres exhibited positive effects against yeast. Penetration of drug and nanoparticles released from microspheres within fungal cell wall causes denaturation of proteins, inactivation of enzymes, creation of ROS and DNA damage.⁶⁴ The release of drug as well as composite might have interacted with the cell membrane permeability by binding with ergosterol, a major component of the cell membrane and forming a polyene-microsphere-ergosterol complex. This complex causes changes in the efflux pump and forms pores in the cell membrane.⁶⁵ Leakage of K⁺ ion makes the extracellular environment acidic and impairs the metabolism that leads to cell death. Suppression of lanosterol conversion into ergosterol may be caused by inhibition of enzyme 1–4 α demethylase encoded by gene ERG II which is involved in ergosterol biosynthesis. High accumulation of lanosterol causes toxicity and changes in the membrane fluidity and chitin synthase activity. Microspheres may also cause deletion/mutation in gene FKS1 that will inhibit the activity of 1, 3β glucan synthase leading to disruption in cell glucan synthesis which results in cell wall damage (Figure 11).⁶⁶ Moreover, the electrostatic interaction between the cell wall and antifungal material allows the antifungal material to invade the cell and produce ROS which in turns denatures enzymes and proteins and interact with DNA and RNA leading to cytotoxicity and cell apoptosis (Figure 15).⁶⁷ Moreover, inhibition of Candida morphogenesis by Nys-MgO/CuO nanoparticles invariably inhibit biofilm formation. Cell wall damage and existence of yeast and hyphae forms also inhibit biofilm formation.⁶⁸

Figure 15 Hypothetical mechanism of action of Nys-MgO/CuO microspheres against C. albicans.

Conclusion

This study illustrates the successful development of novel drug-composite loaded microspheres by ionic gelation method for the treatment of C. albicans infections. The method of preparation is simple, fast, and inexpensive, and can be performed at room temperature under mild conditions in aqueous surroundings. SEM revealed the size and surface structure of microspheres, and XRD confirmed the purity and crystallinity. FTIR analyzed the functional groups and entrapment of Nys-MgO/CuO nanoparticles. The synergistic activity of drug-conjugated nanoparticles encapsulated in alginate beads exhibited strong anticandidal activity than drug or nanocomposite alone. Flow cytometry revealed cell membrane damage leading to the release of cytoplasmic content and cell death. Toxicity imposed by nanoparticles upon direct contact with the human body was reduced by using a nontoxic, biocompatible, and biodegradable alginate-polymer based antifungal delivery system providing a safe and modified release. The nystatin composite-loaded microsphere systems could be effectively adapted in applications where a sustained release of this antifungal is beneficial to prevent or minimize infections. The obtained microspheres have promising potential in antifungal agent delivery application as anticandidal material. Further, in vivo study must be carried out to explore the biocompatibility and undesired adverse impact of Nys-MgO/CuO MS for their potential therapeutic application.