Anticandidal potentiality of biosynthesized and decorated nanometals with fucoidan

1 Introduction

Algal polysaccharides (including fucoidan [Fu], laminarin, and alginate from brown algae; agarose, carrageenans, and porphyran from red algae; and ulvan from green algae) significantly represent a precious group of bioactive compounds due to their bioactive physiochemical properties, which encourage their biomedical/therapeutic applications (e.g., antiviral, immunostimulants, anti-inflammatory, anticancer, antioxidant, and antimicrobial agents) [1,2].

Fu had attracted great attention as a valuable polysaccharide with treasurable bioactivities, which is mainly extracted from brown algae species [3]; it is from the main bases of seaweeds’ pharmacological effects. The Fu possessed elevated biosafety, biocompatibility, and numerous bioactivities (including its antioxidant, anticancer, anti-inflammatory, immunomodulatory, antiviral, antiallergic, hypotensive effects, and antimicrobial potentialities) [4,5,6,7,8]. The Fu anticancer potentiality is well documented and was attributed to apoptosis induction in cancer cells, inflammation suppression, and antioxidation properties [6,8,9].

Nanotechnology involved the methods for assembling or modifying the molecules and atoms to become in “nano” scales (with particles’ diameter of 1–100 nm of atoms and up to 1,000 nm for molecules) and the applications of these nanoparticles (NPs) that attained extraordinary and superior physiochemical/biochemical properties [10]. Nanomaterial applications attained great successfulness, particularly in biomedical, pharmaceutical, and healthcare researches. The NPs’ promising efficiency arises from their huge surface area, minute sizes, elevated strength, stability, conduction, and unique biochemical properties [11]. The biomedical applications of NPs included their usage in biosensors and nanomedicine (e.g., for constructing antimicrobial agents, treating diabetic wounds, wound dressing, drug carrying/delivery, and disorder diagnosis) [10,12].

The transformation of materials to nanoforms frequently used different procedures (e.g., biological, physical, and chemical). The physical approaches are highly expensive and energy consuming and require specialized equipment, whereas the chemical protocols frequently generate many toxic and hazardous residues [13,14]. However, the biological NP synthesis (green synthesis) was successfully used to transform various ions/compounds to nanostructures with elevated efficiency, safety, consistency, and bioactivity [15,16]. Many biological systems/derivatives were utilized directly for the biosynthesis of metal NPs, including microorganisms, plant extracts, phyto-constituents, algal extracts, biopolymers, and proteins [16,17,18,19,20,21]. The algal extracts (micro and macro species) and polysaccharides (Fu, carrageenan, alginate, laminaran, etc.) were capable of reducing/stabilizing various metals in nanoforms [12,19,20,22,23,24,25]; higher activities and augmented characteristics were exhibited from the phycosynthesized NPs.

Silver (Ag), the transitional metal with outstanding properties like conductivity and wide antimicrobial spectrum, can be used in numerous biomedical, environmental, and pharmaceutical, wounds and burns healing, food packaging, medical imaging, and hygienic textiles disciplines [26]. Both Ag ions and Ag nanoparticles (Ag-NPs) have forceful wide antimicrobial potentialities. The Ag-NPs are more powerful because of their higher surface area, smaller particle sizes, and great surface energy, which augment the adsorption of Ag-NPs onto microbial cells and interaction with them [12,16,18,27]. The anticandidal activities of Ag-NPs were proved and have attained great attention to fight pathogenic yeasts [13,17,28,29,30].

The biosynthesis of Ag-NPs was successfully achieved; the usage of biopolymers (especially marine polysaccharides) as reducing and stabilizing materials was reported to enhance bioactivity, biosafety, and compatibility of Ag-NPs [12,24,26,28].

The mineral, selenium (Se), is an essential trace element for preserving human health and functions of human body, with a recommended daily intake of 0–300 mg [31]. Selenium nanoparticles (Se-NPs), especially the biosynthesized NPs, were validated for having extraordinary attributes (e.g., antioxidant, anticancer, antibacterial, and antifungal activities) [15,21,32,33]. The biosynthesis of Se-NPs (using plants and algal extracts, biopolymers, and microorganisms) generated more stabilized and compatible NPs with elevated bioactivities [25,31,32,34]. Although numerous investigations reported nanometal biosynthesis using plant derivatives (phytosynthesis), inconsequential studies investigated the direct NP biosynthesis using biopolymers (e.g., Fu), especially with their potential usage as antifungal composites.

The Candida sp. (including ∼150 species) could have threatening health concerns because they possess endosymbiosis for humans and cause numerous infections, especially in immunosuppressed hosts. About 80% of Candida infections (candidiasis) are caused by Candida albicans, albeit the infections from non-Candida albicans (including Candida glabrata and Candida parapsilosis, Candida tropicalis, Candida krusei, and Candida auris) became more frequent [35]. The Candida sp. infections, either superficial (e.g., vaginal, oral, or mucocutaneous) or profound (e.g., septicemia or myocarditis), are currently serious challenges to humans; many Candida species could acquire multiple resistances to accustomed antifungal agents [36]. Many positive attempts were conducted for achieving effectual anticandidal agents from NP formulations/composites [13,32,37]; the main challenge for that is the cytotoxicity of metal NPs toward mammalian tissues [30], which could be minimized via conjugation/capping of these NPs within polymeric molecules [12,28,34].

Accordingly, our goals in this study were to extract Fu from the macro-brown algae, Cystoseira barbata, and to use the extracted Fu for innovative biosynthesis of Ag-NPs and Se-NPs. The anticandidal activities and action of Fu-synthesized NPs were also investigated toward many pathogenic Candida sp. strains.

2 Materials and methods

2.3 Fu extraction

The Fu was extracted from C. barbata powder with hot water using slightly modified protocols [4,38]. Dried algae powder (10 g) was immersed in 100 mL of methanol–chloroform–DW (4:2:1) mixture and kept at 25 ± 2°C (room temperature; RT) overnight under occasional stirring to eliminate most of algal pigments, proteins, and lipids. After attaining the treated algae biomass by filtration and washing with DW, it was soaked in 500 mL of 0.1 M HCl at 85 ± 2°C for 125 min, then the supernatant was decanted, and the defatted biomass was washed by acetone and dried overnight. Subsequently, biomass was put in 500 mL of CaCl₂ solution (1%, pH = 7) with mild stirring for 30 min, then at a constant state for 12 h at RT to precipitate alginate. The precipitated fraction was separated via centrifugation (Sigma 2–16 KL centrifuge; Sigma Lab. GmbH, Germany) at 8,500×g for 38 min at RT. The Fu in supernatant was precipitated by adding three volumes of ethanol (95%) and keeping at 4 ± 1°C overnight. Precipitated Fu was recovered via centrifugation (9,450×g) for 35 min, then Fu pellets were washed with ethanol and DW, frozen at −20°C, and lyophilized. A flowchart is illustrated (Figure A1 in Appendix) to display the step-by-step protocol used for Fu extraction.

Figure 1

Visual appearance and UV-Vis spectra of Fu-synthesized Ag-NPs and Se-NPs. Photographs indicated NP solutions’ color after 0, 5, 10, 20, and 30 min of reaction for A, B, C, D, and E, respectively.

3 Results

3.3 Characteristics of Fu-synthesized metal NPs

3.3.1 Optical analysis

The optical investigation of Fu-mediated NPs (Fu/Ag-NPs and Fu/Se-NPs), via direct observation and UV-Vis spectrophotometric examination, confirms NP formation after Fu interactions (Figure 1). The direct observation of Fu/metal solutions indicated gradual transformation of color from clear appearance to deep blackish brown (for Fu/Ag-NPs) and reddish orange (for Fu/Se-NPs) within 30 min of reaction; no further color changes were observed after that. The highest NP-absorption peaks (λ _max), via UV-vis analysis, were recorded at 260 and 426 nm for Fu/Se-NPs and Fu/Ag-NPs, respectively (Figure 1).

Figure 2

FTIR spectra of extracted Fu and polymer-decorated NPs of silver (Fu/Ag-NPs) and selenium (Fu/Se-NPs). The main influencing bands in Ag-NPs and Se-NPs synthesis are indicated by black and orange lines, respectively.

3.3.2 FTIR spectroscopy

The FTIR analysis of produced molecules (i.e., Fu and its composites with Ag-NPs and Se-NPs) was conducted to elucidate the molecular biochemical bonding and the interactions between composited agents and the potential Fu groups responsible for NP synthesis. The extracted Fu from C. barbata had the key characteristic features of accustomed Fu biopolymers, as proved from its detected biochemical bonding via FTIR analysis (Figure 2). The FTIR spectrum of Fu (Fu in Figure 2) designated the presence of functional sulfated groups/ponds in the Fu structure and their detectable levels.

Figure 3

TEM imaging of Fu-synthesized Ag-NPs and Se-NPs.

For the Fu-synthesized Ag-NP spectrum (Fu/Ag-NPs in Figure 2), the composite had the main distinctive peaks of Fu. The varied peaks from the Fu spectrum could indicate the potential responsible bonds/groups for Ag-NP synthesis. Comparing to the Fu FTIR spectrum, the band at 1425.44 cm⁻¹ was shifted to 1385.41 cm⁻¹, which indicates the potential transformation of C–C stretching of aromatics to C–H alkenes stretching after compositing with Ag-NPs.

The FTIR spectrum of Fu-synthesized Se-NPs (Fu/Se-NPs in Figure 2) also had the main characteristic peaks of the Fu spectrum; the novel altered bands in the Fu spectrum due to Se-NPs synthesis/interaction were the new band at 3748.21 cm⁻¹ that may designate the Se-NPs binding to –OH groups and other function groups of Fu.

3.3.3 Size and charge of NPs

The DLS of produced NPs (Fu/Ag-NPs and Fu/Se-NPs) indicated that Fu could effectually reduce both metal ions to NPs form, with minute Ps ranges and means (Table 3). The Ps means of synthesized NPs were 12.86 and 16.18 nm for Ag-NPs and Se-NPs, respectively. The Fu-synthesized NPs exhibited persuasive negative ζ potentialities of −35.8 and −34.7 mV for Ag-NPs and Se-NPs, respectively.

Table 3

Particle size (Ps) distribution and ζ potential of Fu-synthesized Ag-NPs and Se-NPs

Nanoparticles	Ps range (nm)	Mean diameter (nm)	Median (nm)	Zeta potential (mV)
Fu/Ag-NPs	3.82–36.44	12.86	12.04	−35.8
Fu/Se-NPs	5.27–43.54	16.18	15.84	−34.7

3.3.4 TEM imaging

The TEM imaging of Fu/Ag-NPs and Fu/Se-NPs was used for inspecting the shapes, sizes, and surface morphologies of the composited nanocomposites. The TEM images (Figure 3) indicated that both Fu synthesized and decorated nanometals had mostly spherical shapes and good distribution within the biopolymer matrix. Many NPs were embedded in Fu as combined clusters; the estimated Ps average diameters were ∼12.13 nm for Ag-NPs and ∼15.54 nm for Se-NPs. These results matched the acquired data by DLS measurements of corresponding Ps distributions (Table 3).

Figure 4

Consequences of exposing Candida sp. (C. albicans and C. parapsilosis) to Fu/Se-NPs (S) and Fu/Ag-NPs (A) on morphological attributes of cells after exposure for 0, 5, and 10 h.

4 Discussion

The Fu was extracted from C. barbata extract; the extract analysis results match a preceding investigation that recommended the usage of hot water extraction to attain the highest percentages from l-fucose and uronic acid, during Fu extraction, compared to salt extraction procedure [3]. The elevated contents of sulfated monosaccharide (e.g., fucose) in isolated Fu from brown algae species were documented, which are chiefly responsible for Fu bioactivities and interactions [5,8,49,50]. Moreover, the obtained percentages here of Fu monosaccharides and yield are comparable with extracted Fu from other algal species, including Ecklonia maxima, Laminaria pallida, Splachnidium rugosum, Padina tetrastromatica, Fucus vesiculosus, numerous Fucus sp., and Sargassum sp. [2,6,51].

The color changes of NP-containing solutions are principally correlated with the surface plasmon resonance (SPR) excitation of synthesized NPs, the deepness of solution color is indicative of size of the formed NPs [14,23]. The obtained λ _max values for Fu/Se-NPs and Fu/Ag-NPs are in accordance with those of phycosynthesized Ag-NPs and Se-NPs with algal derivatives and the incorporated NPs with biopolymers and algal polysaccharides [16,23,24,25,31].

The main distinctive peaks in FTIR spectrum of Fu were detected at 501.89 cm⁻¹ (sulfated C–O–S at C-4 of fucopyranose); 1088.97 cm⁻¹ (O═S═O symmetric vibration in sulfate esters); 1240.42 cm⁻¹ (CH₃ in fucose and O-acetyls); 1425.44 and 1413.31 cm⁻¹ (CH₂ in galactose and xylose); 1632.42 cm⁻¹ (stretched C–O vibration of O acetyl); and 3428.11 cm⁻¹ (indicates stretched O–H group) [9,52,53,54]. The intense signal at ∼1,089 cm⁻¹ indicated the strong stretched vibrations of sulfoxide (S═O), which evidences the significant amount of sulfate groups in the extracted Fu [55,56]. In the Fu-synthesized Ag-NP spectrum (Fu/Ag-NPs in Figure 2), the sulfate peak (at 816.23 cm⁻¹) was also shifted to 873.15 cm⁻¹ in the Fu/Ag-NPs, which is assumingly attributed to the sulfate group involvement in the Ag-NP synthesis [5,6]. Additionally, the differed bands from the Fu spectrum included the band at 501.89 cm⁻¹ (C–O–S) that became less intense and shifted to 494.31 cm⁻¹ and the band at 1413.31 cm⁻¹ (CH₂) that became sharper and has increased intensity. These changes are mostly due to Ag ion reduction into Ag-NPs and interactions with the formed NPs [24]. For the FTIR spectrum of Fu-synthesized Se-NPs (Fu/Se-NPs in Figure 2), the emerged/altered band from the Fu spectrum indicated the responsible groups/bonds for Se-NP synthesis. The Fu band at 1240.42 cm⁻¹ (CH₃) was disappeared and the band at 3673.21 cm⁻¹ was vanished in Fu/Se-NPs spectrum, indicating Se-NPs interactions with Fu biochemical groups. A novel band at 771.48 cm⁻¹ was also emerged in the Fu/Se-NPs spectrum, which is assumingly attributed to Se-NP conjugation with Se–O and/or –OH groups of Fu [57,58].

Compared to Ps of synthesized Ag-NPs using Fus of F. evanescens and Saccharina cichorioides (64 and 53 nm, respectively) [24], and from Spatoglossum asperum (20–46 nm) [22], the used Fu here from C. barbata exhibited a stronger reducing activity, which enabled the generation of lesser size Ag-NPs. Furthermore, the achieved miniature Ps of Fu-mediated Se-NPs, as innovative results, validated the high potentiality of C. barbata for reducing/decorating metal NPs [23].

The ζ potential can precisely indicate the surface charge of individual molecules and additionally can measure the formed electric dual layer by adjoining ions in solution; regularly, NPs with ζ values of ≥+30 mV or ≤−30 mV possess high stability due to electrostatic inter-particle repulsion [59]. The elevated ζ potentials of Fu-synthesized NPs indicate the capability of Fu biopolymer for stabilizing metal NPs. Harmonized results for ζ potential of Fu-synthesized Ag-NPs were reported with negativity up to −36 mV [26] and in −27 to −30 mV range [24].

The acquired results from TEM and DLS analysis of Fu-synthesized NPs validated the potentiality of Fu in reducing/stabilizing NPs. Recent studies indicated matched shapes and combinations of Fu-synthesized Ag-NPs [24]; the NPs had oval/spherical and good distribution attributes. As a main goal of this study was to achieve effectual nanometals with reduced toxicity to human cells, the decoration/embedding of synthesized NPs within the Fu matrix could assist to reach this target. The amalgamation of nanometals into biopolymers composites was significantly appointed to augment their stabilization, generate additional bioactivity via conjugation with biomolecules, enhance metal NPs biocompatibility, and accordingly diminish their humanoid cytotoxicity [16,23,60,61,62]. Accordingly, the synthesized/capped NPs with Fu are assumed to possess elevated biosafety and nontoxicity. Since no cytotoxicity assays were conducted in the present study, further biotoxicity examinations are recommended to confirm the expected biosafety of Fu-synthesized NPs.

The anticandidal activities of each NPs (Ag-NPs and Se-NPs) were formerly indicated from many investigations [13,28,30,33]. The anticandidal potentiality of Se-NPs was illustrated to correlate with Ps of NPs; the lesser sizes provided higher activity [32,37]. The Se-NPs (at 70–300 nm size range) that conjugated with biopolymers (e.g., chitosan and BSA) exhibited potent antimycotic activity against C. albicans, with an minimum inhibitory concentration (MIC) of 25 µg·mL⁻¹, which exceeded the anticandidal activity of plain Se-NPs that had an MIC of 400 µg·mL⁻¹ [33]. Regarding Ag-NP anticandidal activity, the investigations indicated their powerful action to destroy yeast cells and hinder their metabolic activities, mainly due to reactive oxygen species (ROS) generation, which influences the microbial cell activity [10,30].

The incorporation of NPs (Ag-NPs and Se-NPs) into biopolymer matrix/composite was reported to stimulate their anticandidal activities [28,33,34,61]; this was attributed to NPs stabilization and good distribution within the polymer matrix and the ability of a loaded polymer to adhere to yeast cells, which facilitates NP interaction with the cell membranes. Additionally, the reduced/stabilized Ag-NPs using Fu from F. vesiculosus, in microwave-assisted procedure, had strong antibacterial actions (mainly due to leakage of intracellular proteins and ROS oxidative stress), which were attributed to Fu/Ag-NP binding and interaction with microbial cells [12]. The antimicrobial activities (antibacterial and antimycotic) of Fu were also reported against oral pathogens [7], where they suggested that Fu antimicrobial properties are based on its structures, for example, sugar chains’ branching and sulfate groups number, the extraction procedure, and the capability of such sulfated polysaccharides to bind with proteins and further cellular molecules.

Accordingly, the conjugation/decoration of synthesized NPs within Fu matrix is strongly suggested to augment their combined anticandidal actions, as the Fu can effectually bind to surface of the cells and enable attachment/interaction of NPs with cell membranes and interior vital components.

The exposed Candida sp. (i.e., C. albicans and C. parapsilosis) were chosen for SEM analysis because they exhibited the most resistant and sensitive patterns, respectively, toward NPs in previous experiments (antimycotic assays). Thus, it was assumed from their analysis to provide potential mechanisms of NP anticandidal actions. The proposed antimicrobial actions of metal NPs were suggested to depend on their potentiality for ROS generation, their interactions with cells barriers (leading to deformation and disruption of cell walls), affecting the permeability of cell membranes, inhibition of DNA and protein synthesis, and suppression of metabolic regulation genes [10,11,63]. The generated ROS from metal NPs could additionally interfere/inhibit amino acid construction and DNA replication and could seriously lead to cell membrane damage [10]. The integration of Se-NPs with biopolymer matrix (e.g., chitosan) provided more forceful anticandidal action toward C. albicans, which triggered the formation of destructive bores in yeast membranes [28,34]. This action surpassed the plain Se-NPs anticandidal action [32], which supposed that NPs incorporation/stabilization with biopolymers (Fu in current study) can augment the bioactivity and action of the composite. The Ag-NPs were also reported to possess potent anticandidal actions via interference with Candida sp. membranes and interior components, and pore formation in such membranes, which led to lysis and death of mycotic cells [13].

As the forcefulness of NP antimicrobial potentiality is simultaneous and involves multiple mechanisms, even antimicrobial-resistant microbes could not acquire the resistances to such action mechanisms, which is considered as main advantages of NPs as microbicidal agents, contrary to customary antibiotics [33]. However, the conjugation of NPs with biopolymers, for example, Fu, could elevate their biosafety for practical applications in human practices, for example, pharmaceutical drugs, skin care, antiseptic wash, or topical formulations [62].

5 Conclusion

The Fu was extracted from C. barbata algae and was innovatively and effectually used for the green synthesis of metal NPs (Ag-NPs and Se-NPs) via the facile protocol. The synthesized Fu/Ag-NPs and Fu/Se-NPs had favorable characteristics, for example, mean Ps of 12.86 and 16.18 nm, respectively, elevated ζ potential negativity, and good distribution within the Fu matrix. The anticandidal actions of Fu-synthesized NPs were proved against many Candida sp. via qualitative and quantitative assays and by ultrastructure imaging. The potentialities of Fu/Ag-NPs and Fu/Se-NPs as powerful anticandidal agents with minimized toxicity for human tissues could enable their future applications in practical formulations for controlling pathogenic Candida sp. Further investigations can be suggested for confirming the biosafety of generated nanocomposites and their activities against other pathogenic microbes.