One of the most common mucosal infections in females is vaginal candidiasis (VC), which is caused by candida species such as Candida Albicans. It is the second most prevalent mucosal infection after bacterial vaginosis (Johal et al., 2016). Empirical evidence suggests that VC affects a substantial number of women of reproductive age, with estimates ranging from 70%–75%. Additionally, the likelihood of recurrence is anticipated to be in the range of 40%–45%. VC commonly exhibits symptoms such as pruritus, burning, erythema, and edema (Brandolt et al., 2017; Willems et al., 2020). Clotrimazole formulations have been recognized as efficacious pharmacotherapy for this ailment. However, the low biodegradability and limited bioavailability of clotrimazole limit its therapeutic potential. To mitigate these issues, the utilization of nanosystems, such as polymeric micelles, represents a promising strategy to enhance clotrimazole’s solubility and bioavailability. Moreover, incorporating silver nanoparticles into these nanosystems could further potentiate the antifungal properties of clotrimazole, thereby augmenting the overall therapeutic efficacy of this pharmacological intervention.

Clotrimazole, an imidazole derivative with potent antimycotic properties, is primarily utilized topically to manage fungal and dermatophyte infections of the mouth, vagina, and skin. Despite its effectiveness, clotrimazole’s antifungal activity is hindered by its poor water solubility. This challenge has been addressed through the utilization of various formulations, including nano-based structures, to enhance clotrimazole’s bioavailability and efficacy (Santos et al., 2013; Tonglairoum et al., 2017). For the intended purpose, several nano-based systems such as nanocapsules (Santos et al., 2014), nanostructured lipid carriers (NLC) (Nogueira et al., 2022), solid lipid nanoparticles (SLN) (Cassano et al., 2016), liposomes (Abdellatif et al., 2020), nanoemulsions (Soriano-Ruiz et al., 2019), and polymeric micelles (Tonglairoum et al., 2017; Kareem et al., 2019; Catenacci et al., 2020; Albayaty et al., 2021) have been utilized.

Amphiphilic copolymers, comprising both hydrophilic and hydrophobic segments, have gained significant attention as potential nanocarriers. Through self-assembly, these copolymers could form polymer-based amphiphilic nanocarriers with tunable morphology and shape in aqueous solutions. By regulating the balance between the hydrophilic and hydrophobic segments, the synthesis of diverse micelles and vesicles becomes a possibility. Such nanocarriers hold great promise for various applications in drug delivery and biomedicine (Lombardo et al., 2015; Li et al., 2020). Micelle nanocarriers derived from the self-assembly of amphiphilic copolymers at or above critical micelle concentration represent appropriate carriers for encapsulation hydrophobic agents. The core of the micelles can be composed of various polymeric blocks, such as polypropylene glycol, polyesters like PLA (polylactic acid), PCL (polycaprolactone), PLGA (polylactic co glycolic acid), polyamino acids, and lipids (Biswas et al., 2016; Cabral et al., 2018). The hydrophobic core of the micelles serves as the primary site for entrapping lipophilic active ingredients, leading to improved solubility and bioavailability of hydrophobic drugs. The hydrophilic shell of the micelles concurrently creates a stabilizing interface between the two phases, namely, the hydrophobic core and aqueous medium. This interface enhances colloidal stability and effectively prevents undesired interactions with other components (Lombardo et al., 2019). PVP (Polyvinylpyrrolidone), PNIPAm (poly(N-isopropyl acrylamide), PHPMA (Poly(N-(2-hydroxypropyl)methacrylamide), and PEG (polyethylene glycol) are among the nonionic and hydrophilic polymers that are frequently employed in the hydrophilic shell of micelles. PEG is the most commonly used hydrophilic block for the micelle shell due to its high solubility in aqueous media and diverse organic solvents, which provides flexibility in various preparation methods. Moreover, its non-toxicity, low immunogenicity, minimal interaction with other biological components of the body, and ease of biodistribution make it an ideal choice for micelle formulation (Miyata et al., 2011).

The hydrophobic and semi-crystalline polymer, poly(ɛ-caprolactone) (PCL), consists of repeating units of five non-polar methylene groups and a relatively polar ester group. The polymer is synthesized through the ring-opening polymerization method using ɛ-caprolactone monomers. Due to its high degree of crystallinity and hydrophobicity, modification of PCL with polyethylene glycol (PEG) to form PCL-PEG copolymers has been proposed (Wei et al., 2009). Several studies have explored the use of PCL-PEG-PCL (LGL) amphiphilic copolymers in the synthesis of micelle nanocarriers containing lipophilic drugs (Gou et al., 2009; Grossen et al., 2017).

Silver nanoparticles (AgNPs) are a type of inorganic nanocarrier that exhibit antibacterial effects against various types of bacteria, including Gram-positive and Gram-negative bacteria. Moreover, AgNPs have demonstrated antifungal properties against Candida species. In a study conducted by Panasek et al., it was found that AgNPs at concentrations of approximately 1 mg/L exhibit noteworthy antifungal activities against Candida species. Furthermore, the study revealed that these nanoparticles do not induce any cytotoxic effects on human fibroblast cells. These findings suggest that AgNPs could be a potential therapeutic agent for controlling Candida infections without causing harm to human cells (Panáček et al., 2009).

Several studies have demonstrated the synergistic antibacterial effects resulting from the combination of silver nanoparticles with antibiotics such as vancomycin, amoxicillin, penicillin G, ampicillin, kanamycin, and erythromycin (Lee and Jun, 2019; Mikhailova, 2020). Moreover, incorporating silver nanoparticles into support materials like polymeric micelles has been instrumental in minimizing aggregation, leading to enhanced antibacterial and antifungal properties (Huang et al., 2017).

The combined strategy of loading nanocarriers into hydrogels has garnered significant attention across various research disciplines. Notably, thermosensitive hydrogels have emerged as a promising candidate for vaginal drug delivery owing to their sol-gel phase transition properties. These hydrogels exhibit a unique property of transitioning from a free-flowing sol state at low temperatures to a gel-like state at 37°C, providing sustained drug release with adjustable kinetics while offering ease of use and prolonged drug residence time. Moreover, the incorporation of nanocarriers in these hydrogels offers reduced leakage and enhanced efficacy, thereby reducing the frequency of administration (Taurin et al., 2018). PEG-PCL-PEG (GLG) is a hydrogel that possesses biodegradability, amphiphilicity, and thermosensitivity, rendering it an attractive material for use in diverse biomedical applications (Gong et al., 2007). In a study conducted by Frank, it was indicated that the incorporation of polymeric nanocapsules into mucoadhesive semi-solid formulations can effectively augment adhesion and drug diffusion through the vaginal mucosa. This innovative approach holds immense promise for the development of novel therapeutic strategies that can effectively combat a wide range of gynecological disorders (Frank et al., 2017).

Extensive research has been conducted on the use of the vaginal route for pharmacological applications. Compared to the oral route, the vaginal route offers several advantages such as the elimination of hepatic first-pass metabolism, the absence of drug interactions, and the prevention of drug destruction in the gastrointestinal tract. As a result, vaginal drug delivery allows for the possibility of loading lower doses of drugs, which can reduce the occurrence of side effects. Additionally, for drugs targeting the female reproductive system, the use of the vaginal route can lead to higher drug concentrations and improved effectiveness (Ensign et al., 2014; Das Neves et al., 2015). Facchinatto et al. designed chitosan nanoparticles (Clt-NPs) loaded with clotrimazole as a topical treatment for vulvovaginal candidiasis. The Clt-NPs were found to have proper selectivity index values for clinical isolates of Candida spp. Similarly, Takahashi and his colleagues synthesized a silver-decorated PVCL-PVA-PEG graft copolymer that demonstrated high antibacterial activity as a drug delivery system effective against biofilm infections (Facchinatto et al., 2021; Takahashi et al., 2021). Considering all of these findings, there remains a gap in the development of a robust drug delivery system capable of loading temperature-sensitive hydrogel with polymeric micelles containing silver nanoparticles, and evaluating its therapeutic efficacy. The main objective of this study focuses on countering Candida albicans fungal infection using a PCL-PEG-PCL triblock copolymer composed of an inner PCL core and an outer PEG shell. By encapsulating clotrimazole within the hydrophobic core and decorating the shell with silver nanoparticles, an effective antifungal approach is achieved. Initially, the PCL-PEG-PCL triblock copolymer is synthesized via ring-opening polymerization. Subsequently, polymeric micelles (PMs) are formed using the nano-precipitation method, enabling the simultaneous loading of clotrimazole into the core. The micelles are chemically modified to incorporate silver nanoparticles onto their surface. Silver-beared clotrimazole-loaded micelles are then loaded into the PEG-PCL-PEG hydrogel. This drug delivery system enhances biocompatibility, solubility, and clotrimazole efficacy. Furthermore, the presence of silver nanoparticles contributes to the augmented antifungal properties. The adhesive properties of the PEG-PCL-PEG hydrogel facilitate increased drug diffusion and sustained drug release, thereby prolonging the drug’s residence time. This system could prove to be an ideal choice for releasing clotrimazole and silver nanoparticles topically.

The FTIR spectrum of LGL reveals four absorption bands that provide important information about the chemical groups present in the sample. An absorption band appearing at a wavelength of approximately 1723 cm⁻¹ corresponds to the ester carbonyl group (C=O) of ɛ-caprolactone, while the absorption band observed at 1,105 cm⁻¹ corresponds to the PEG -OCH₂CH₂ repeating units. Additionally, two absorption bands appear at wavelengths of around 1,240 and 3,620 cm⁻¹, which belong to the -COO- bands and terminal hydroxyl groups of PCL, respectively.

The FTIR spectrum of GLG displays an absence of absorption within the 2,270–2,250 cm⁻¹ range. This lack of absorption establishes the complete disappearance of hexamethylene diisocyanate -NCO groups. Such disappearance is attributed to a coupling reaction between the -NCO and -OH groups. Moreover, the absorption band observed at 1,527 cm⁻¹ can be attributed to the N-H group which demonstrates the formation of tri-block copolymers. Thus, the accuracy of tri-block copolymer synthesis is confirmed by the FTIR spectrum. Additionally, the copolymer formation and the number-average molecular weight determination of copolymers were further confirmed through the use of HNMR spectrum. The LGL HNMR spectrum shows several peaks corresponding to different methylene protons in the copolymer. Specifically, the peak at 1.4 ppm and 1.65 ppm are related to -(CH₂)₃- methylene protons, while the peak at 2.32 ppm is associated with -OCCH₂- methylene protons. Additionally, the peak at 4.06 ppm corresponds to -CH₂OOC- methylene protons in PCL units, and the higher intensity peak at 3.65 ppm is attributed to -CH₂CH₂O- methylene protons in PEG units. Moreover, short peaks at 3.82 and 4.23 ppm refer to -O-CH₂-CH₂- methylene protons in the PCL blocks’ PEG terminal units. In contrast, the GLG HNMR spectrum displays peaks at 3.6 and 3.38 ppm referring to -CH₂CH₂O- and terminal groups -OCH₃ in the PEG blocks, respectively. Similarly, the peaks at 1.35 and 1.62 ppm correspond to -(CH₂)₃- methylene protons, and the peak at 2.3 ppm correlates with -OCCH₂- methylene protons. Furthermore, the peak at 4.06 ppm corresponds to -CH₂OOC- methylene protons of PCL units, and the short peaks at 4.20 and 3.82 ppm relate to O-CH₂-CH₂- methylene protons of PEG terminal unit attached to the PCL blocks. Finally, GPC analysis was utilized to determine the M_n, M_w, and polydispersity of copolymers. Table 2 presents a comprehensive summary of all the results obtained. The PCL/PEG ratio of copolymers was calculated using the HNMR spectrum, which revealed a ratio of 1.54:1 and 2.32:1 for LGL and GLG copolymers respectively. The values obtained through experimentation exhibit a degree of similarity to the corresponding feeding ratios presented in Table 1. However, they are not entirely identical due to the derivation of the data in Table 1 being based on theoretical assumptions. On the other hand, the values calculated above are derived from actual experimental results. Consequently, we can draw the conclusion that the experimental data aligns with the anticipated values, thus confirming the accuracy of the conducted analysis.

PCL has a negative surface charge that can be mitigated by the addition of neutral PEG blocks, leading to less negative values of total zeta potential (Cu et al., 2011; Suk et al., 2016). Figure 3 demonstrates that the zeta potential results of the PM solution at pH 7.3 revealed a negative surface charge of the micelles, indicating that the adsorption of silver ions on the micelles was caused by the electrostatic interaction between positively charged ions and negatively charged micelles. When silver nitrate solution was added to the PM solution, Ag⁺ ions were adsorbed to the negative surface of the PM. Subsequently, the introduction of sodium borohydride to the solution resulted in the reduction of the silver ions, ultimately leading to the development of silver nanoparticles on the polymeric micelles. The yellowish color illustrated in Figure 1 confirms the accuracy of the reduction of silver ions. The presented FTIR spectrum in Figure 4A pertaining to clotrimazole drug indicates several absorption bands, each occurring at specific wavelengths. In particular, the absorption bands found at the wavelengths 753.07, 822.49, 904.45, 1208.18, and 1273.75 cm⁻¹ signify the bending vibration of C-H bonds, while those at 1040.41 and 1079.94 cm⁻¹ correspond to the stretching vibration of C-N bonds in the aromatic ring of imidazole. Similarly, the absorption band at the wavelength of 1438.64 cm⁻¹ denotes the stretching vibration of the C=C bond of the aromatic ring of imidazole, whereas the absorption band occurring at 1592.91 cm⁻¹ signifies the stretching vibration of the C=N bond in the aromatic ring of imidazole. The absorption band that appears at the wavelength of 3055.66 cm⁻¹ corresponds to the stretching vibration of the C-H bond in the aromatic ring. In Figures 4B, a comparison between the FTIR spectra of clotrimazole, blank micelle, and clotrimazole-loaded micelle is presented. Notably, the absorption bands of clotrimazole and polymer present in the clotrimazole-loaded micelle demonstrate that the Clt has indeed been loaded in the polymeric micelle. The average size of PMs, as determined by ImageJ software and depicted in Figure 5, closely aligns with the findings from dynamic light scattering (DLS). Moreover, the accurate representation of the micelles’ morphological structure was observed.

The EDX spectroscopy and elemental mapping analysis depicted in Figures 6A–E reveals the presence of oxygen, carbon, and silver elements in the structure of PM-Ag. PEG-PCL polymeric micelles are primarily composed of carbon and oxygen, which is confirmed by the higher peak intensity of these two elements. Additionally, the existence of silver in the PM-Ag structure is supported by peak observation at an energy of about 3 kV. Comparing the UV-vis spectroscopy of the PM and PM-Ag (Figure 6F), it is evident that the special peak at 410 nm related to the surface plasmon resonance of silver nanoparticles confirms the presence of silver in the PM-Ag solution.

The micelles exhibited an encapsulation efficiency of 64.53% and a drug loading of 14.6%. Figure 7 shows that the drug release profile from H-PM-Clt is similar to that of the PM-Clt solution, with a lower total percentage of drug release observed in the hydrogel. Specifically, PM-Clt had a total percentage of drug release of 53%, while H-PM-Clt had a total percentage of 44.8%. The similarity in the slopes of the two graphs indicates that the drug release rates for both samples were almost identical.

Polymeric micelles have a unique characteristic of penetrating fungal cells through endocytosis and acting as a delivery vehicle for drugs into the cells which enhances their pharmacological activity (Nelemans and Gurevich, 2020). As shown in Figure 8, the viability of cells remains high, exceeding 80%, when micelle concentration is at or below 125 μg/mL. Nevertheless, as the concentration of micelles rises beyond this threshold, there is an observable rise in cytotoxicity levels. In the control group, the cell viability was 100%. In a similar study, the toxicity effect of Ag-decorated MPEG-g-PU vesicles on HUVEC cells was performed and the results displayed that by concentration rising of silver nanoparticles, the cell viability decreases which at 20 μg/mL of silver concentration the cell viability declined to 72% while this concentration is much higher than the MIC and MBC of designated micelles. In Huang et al.’s study, silver-decorated curcumin-loaded Poly(aspartic acid)-PCL polymeric micelles showed very low toxicity to mammalian cells across all investigated concentrations (Huang et al., 2017; Su et al., 2017).

In Figure 9, it is demonstrated that pure PM is not effective against fungi at any concentration considered. However, the viability of fungal cells decreases as the concentration of PM-Ag and PM-Clt increases. The growth inhibition of PM-Ag is more pronounced than PM-Clt, but PM-Clt-Ag displays a synergistic effect in inhibiting the growth of Candida albicans cells. The antifungal activity of H-PM-Clt-Ag is less than PM-Clt-Ag but higher than PM-Clt and PM-Ag. According to Section 3.3, the comparison chart of PM-Clt and H-PM-Clt indicates that loading polymeric micelles into hydrogels leads to a significant decrease in drug release rate. Therefore, H-PM-Clt-Ag exhibits lower antifungal activity when compared to the PM-Clt-Ag sample. As shown in Figures 8, 9, at 125 μg/mL concentration the cell viability and fungal viability of H-PM-Clt-Ag were approximately 95% and 30% respectively. At 250 μg/mL concentration, the cell viability was reduced to 50% and fungal viability reached 15%. Depending on the severity of the disease, the physician determines that losing several healthy cells to provide more effective treatment will be the preferable way of healing. The antibacterial potential of Ag-decorated MPEG-g-PU vesicles against E. coli and S. aureus bacteria examined by Su et al. revealed that concentrations of 5.24 and 10.48 μg/mL of Ag produced the greatest inhibition of growth. However, when compared to PM-Ag, PM-Clt-Ag, and H-PM-Clt-Ag, which exhibited 28.5, 26.4, and 22.9 μg/mL of silver content at 125 μg/mL concentration of micelle measured by ICP, it becomes apparent that higher levels of silver are necessary to achieve the desired antifungal activity. Huang’s study demonstrated that the bacterial viability of silver-decorated curcumin-loaded Poly(aspartic acid)-PCL polymeric micelles at 125 μg/mL concentration of the micelle was approximately 45% against S. aureus and 40% against P. aeruginosa. However, the current research study provides evidence for a higher level of effectiveness in terms of antifungal activity (Huang et al., 2017; Su et al., 2017).

To summarize this study, a core/shell polymeric micelle containing clotrimazole and silver nanoparticles was developed and demonstrated significant antifungal activity against Candida albicans. The combination of clotrimazole and silver nanoparticles had a synergistic effect, enhancing the antifungal properties of the drug delivery system. Additionally, the silver-beared polymeric micelles were found to be cytocompatible with HEK293T cells at certain concentrations. These results suggest that a thermosensitive hydrogel system loaded with silver-beared polymeric micelles with desirable properties may be suitable for treating vaginal candidiasis.