Enhancing Flucytosine Anticandidal Activity Using PEGylated Squalene Nanocarrier

Abstract

There is an emerging necessity for improved therapies against Candida‐related infections, with significant implications for global healthcare. Current antifungal agents, limited in number, target specific pathways, but resistance remains a concern. Flucytosine (5FC) exhibits antifungal activity, particularly against Candida. However, monotherapy efficacy is limited, necessitating combination treatments. Herein, we report PEGylated squalene‐based nanocarriers for 5FC loading, aiming to enhance its monotherapy efficacy against Candida strains. The loading of 5FC within micelles was achieved using the ultrasound‐assisted solvent evaporation method. The 5FC‐loaded micelles, together with non‐loaded micelles, were thoroughly characterized and analyzed. STEM and DLS analysis confirmed the core‐shell morphology with nanometric dimensions along with improved colloidal stability. The quantification of drug loading efficiency and drug loading capacity was calculated using the UV‐Vis technique. The in vitro drug‐release studies in simulated physiological conditions showed sustained release within 48 hours. Moreover, the release kinetics calculated using mathematical models showed a Fickian diffusion drug release mechanism in simulated physiological conditions with a slower diffusion rate. The in vitro antifungal activity was tested on Candida albicans, Candida glabrata, and Candida parapsilosis. The results showed improved antifungal activity for the nanotherapeutic and unchanged in vitro toxicity toward normal cells, suggesting promising advancements in 5FC therapy.

Our study focused on improving the effectiveness of the antifungal drug 5‐fluorocytosine (5FC) by loading it in micellar structures formed by PEGylated squalene copolymer. The obtained results for 5FC‐loaded micelles showed in vitro prolonged release under physiological conditions and improved in vitro antifungal activity without cytotoxicity for healthy cells in comparison with the commercial 5FC drug.

1. Introduction

Enhanced therapies for Candida infections are urgently needed due to their significant impact on global healthcare, especially in hospitals. In Europe, the epidemiology of Candida infections has changed notably, with decreases in C. albicans and increases in C. glabrata and C. parapsilosis incidences. ^[1] The emergence of C. auris is a global concern due to its resistance to antifungals and ability to cause severe infections. Fungal infections are challenging to diagnose and treat, requiring complex procedures. Host‐related factors, clinical signs, and mycological evidence are crucial for diagnosis. ^[2]

Fungal infections are a major public health challenge due to their complex diagnostic process and resistance to current antifungal treatments. ^[3] Diagnosing these infections requires labor‐intensive methods like histopathology and culture, along with considering host‐related factors, clinical symptoms, and positive mycological evidence for an accurate diagnosis. ^[4]

In contrast to the various types of antibiotics for bacterial infections, current antifungal drugs are limited, mainly divided into three categories: azoles (e. g., fluconazole, itraconazole, voriconazole, posaconazole), echinocandins (like caspofungin, micafungin, and anidulafungin), and polyenes represented by amphotericin B (AMB). ^[5] These antifungals target different mechanisms, including ergosterol inhibitors (azoles and polyenes), FKS1 inhibitors (echinocandins and ibrexafungerp), and flucytosine, which disrupts RNA and DNA metabolism, often used with polyenes. ^[6]

Flucytosine, also called 5‐fluorocytosine (5FC), was initially explored as a potential cancer treatment but proved ineffective. ^[7] Later, it showed promise against experimental candidiasis and cryptococcosis in mice and was used to treat human infections. ^[8] In 2013, Imperi et al. demonstrated that flucytosine, as a repurposing drug, at clinically relevant dose regimes reduces Pseudomonas aeruginosa, together with its already known properties, pharmacology pathogenicity in a mouse model of lung infection. Also, they demonstrated Flucytosine's in vitro and in vivo action against Pseudomonas aeruginosa, together with its attractive pharmacological properties, prepared the way for clinical studies on anti‐Pseudomonas aeruginosa efficacy. ^[9] Nowadays, it is estimated that its antifungal spectrum is limited, primarily against Candida, Cryptococcus, and certain Cladosporium and Phialophora species. ^[10] Notably, 5FC itself doesn't directly combat fungi; its antimycotic effects arise from its rapid conversion into 5‐fluorouracil (5FU), which inhibits fungal RNA and DNA synthesis.[ ¹¹ , ¹² ]

Using 5FC alone is limited in treating infections from Cryptococcus neoformans, Candida spp., and Torulopsis glabrata due to frequent resistance emergence. Thus, its use as the sole treatment is confined to certain cases like chromoblastomycosis and uncomplicated lower urinary tract and vaginal candidosis. Otherwise, it's combined with other drugs, usually AMB, for systemic mycoses management. ^[13]

5FC causes mild adverse effects in about 6 % of patients, including nausea, vomiting, diarrhea, and occasional abdominal pain. ^[14] However, it can also lead to severe side effects like hepatotoxicity in around 41 % of patients and bone marrow depression.[ ¹⁵ , ¹⁶ ] The exact mechanism of 5FC toxicity is not fully understood but is thought to involve its conversion to 5FU, as detectable levels of 5FU have been found in patients’ urine and serum during 5FC treatment. ^[17]

5FC toxicity is mainly affected by its dosage, requiring careful selection of optimal doses to minimize side effects, especially in patients with renal insufficiency. ^[18] However, thorough monitoring is necessary in all instances. Nanomedicine has been investigated in some studies to reduce 5FC toxicity and improve its administration and pharmacokinetics.[ ¹⁹ , ²⁰ , ²¹ , ²² ]

Recently, there's been notable interest in using nanosystems or nanocarriers to deliver therapeutic drugs due to their ability to efficiently transport drugs to target sites, reduce side effects, and enable controlled release. ^[23] Notably, some nanosystems that were used as efficient treatments for Candida spp. are mentioned in the scientific literature. Among them, the most efficient are: AMB encapsulated into PLGA‐PEG NPs, which proved to be more efficient against C. albicans than free AMB ^[24] ; SLNs and NLCs also loaded with AMB, which proved to be more efficient than free AMB and Fungizone against C. albicans ^[25] ; and SLNs loaded with Fluconazole that were more efficient than free Fluconazole against all the Candida spp. tested.[ ²⁶ , ²⁷ ] All the mentioned nanosystems are based on commercial drugs that are loaded into different nanocarriers. The difference between nanocarriers can result in different behaviors of the nanosystems in biological environments, which translates to improved or diminished efficiency against the studied yeasts. After drug release, nanocarriers must degrade into non‐toxic components cleared by the Reticuloendothelial System (RES).

The current investigation focuses on utilizing PEGylated squalene micellar systems to load 5FC drug, aiming to enhance its efficacy as a monotherapy for treating specific Candida strains. Drawing from our prior research, PEGylated squalene micelles have emerged as promising candidates for drug loading and delivery, offering numerous advantages including cost‐effectiveness, robust colloidal stability, minimal toxicity, high water solubility, efficient drug loading capacity, favourable drug release profiles, among others. ^[28] Initially, the non‐loaded PEGylated squalene micellar nanostructures were synthesized using an ultrasound‐assisted method in ultrapure water or phosphate buffer saline solution (PBS) with a pH of 7.4. Subsequently, 5FC‐loaded micelles were prepared via the ultrasound‐assisted solvent evaporation method. Both non‐loaded and 5FC‐loaded micelles underwent comprehensive characterization to assess particle size, colloidal stability, morphology, loading efficiency, drug loading capacity, in vitro release profiles under simulated physiological conditions, and release kinetics using various mathematical models. The in vitro antifungal efficacy of these systems was evaluated against three yeast strains, namely Candida albicans, Candida glabrata, and Candida parapsilosis, employing a viable cell‐counting method. Additionally, the in vitro cytotoxicity of the developed systems was assessed on Human Gingival Fibroblasts (HGF) cells. The results of our investigation demonstrate that loading the 5FC drug within PEGylated squalene micelles yields significant enhancements in drug administration. Notably, this formulation mitigates the burst‐release effect observed within the initial hours, leading to a sustained release pattern over 48 hours. Furthermore, the antifungal efficacy against certain Candida strains is enhanced. Importantly, the toxicity towards normal HGF cells remains unchanged even at elevated concentrations.

2. Results and Discussion

2.1. Synthesis and Self‐Assembly Behaviour of SQ‐PEG₁₅₀₀‐NH‐Boc Amphiphilic Copolymer

The synthesis and characterization of the SQ‐PEG₁₅₀₀‐NH‐Boc amphiphilic copolymer (4) were extensively described in our previous publication, as illustrated in scheme 1. In summary, the activated ester of squalenic acid (2) underwent a reaction involving nucleophilic attack with the free primary amine from the H₂N‐PEG₁₅₀₀‐NH‐Boc (3), employing TEA in anhydrous DMF as the medium. The methodology for obtaining (3) was also outlined in our prior study. ^[28]

Scheme 1.

The experimental procedure for obtaining SQ‐PEG₁₅₀₀‐NH‐Boc amphiphilic copolymer.

Naturally, the formation of SQ‐PEG₁₅₀₀‐NH‐Boc micelles occurs spontaneously when the copolymer is dissolved in aqueous solutions exceeding the critical micelle concentration (CMC) when the squalene is surrounded by PEG moiety. Conversely, at concentrations below the CMC, the SQ‐PEG₁₅₀₀‐NH‐Boc micelles disaggregate into individual molecular unimers as demonstrated by other scientific studies. ^[29] In a prior investigation, ^[28] the CMC value was determined to be 0.154 mg/mL for SQ‐PEG₁₅₀₀‐NH‐Boc micelles in PBS with pH 7.4, utilizing the pyrene spectrofluorimetric method. ^[30] The SQ‐PEG₁₅₀₀‐NH‐Boc copolymer was employed in its Boc‐protected form to prevent potential undesired physical or chemical interactions with the drugs during experimentation.

2.2. Preparation and Characterization of Non‐Loaded and 5FC‐Loaded SQ‐PEG₁₅₀₀‐NH‐Boc Micelles

a) Preparation of Non‐Loaded SQ‐PEG₁₅₀₀‐NH‐Boc Micelles in PBS 7.4

Achieving a homogeneous dispersion of SQ‐PEG₁₅₀₀‐NH‐Boc micelles in PBS with pH 7.4 involves an initial deconstruction step using ultrasonication, followed by subsequent reformation through magnetic stirring for 30 minutes at room temperature (23 °C). ^[28] Following this procedure, the PBS solution containing SQ‐PEG₁₅₀₀‐NH‐Boc micelles at desired concentrations is stored at 4 °C and analyzed within 48 hours to prevent potential aggregation of the system.

b) Preparation of 5FC‐Loaded SQ‐PEG₁₅₀₀‐NH‐Boc Micelles

The SQ‐PEG₁₅₀₀‐NH‐Boc micelles were loaded with the 5FC‐antifungal drug by employing the ultrasound‐assisted solvent evaporation method (Scheme 2). This process involved dissolving the drug in a THF‐water mixture, followed by injecting the resultant solution into a THF‐water solution containing the SQ‐PEG₁₅₀₀‐NH‐Boc copolymer at concentrations exceeding the CMC value. The mixture of 5FC with the SQ‐PEG₁₅₀₀‐NH‐Boc copolymer was sonicated continuously for a couple of minutes and then subjected to a thermal treatment at 60 °C for 2 hours. This thermal step is essential to achieve equilibrium and optimal homogeneity within the physical mixture of components. Following this, the mixture was allowed to cool to ambient temperature over 24 hours. Subsequently, THF was removed using a rotary evaporator, and the resulting aqueous solution containing both non‐loaded and 5FC‐loaded into SQ‐PEG₁₅₀₀‐NH‐Boc micelles was left for an additional 24 hours in a fume hood under magnetic stirring to eliminate any remaining traces of THF. As depicted in scheme 2, the non‐loaded 5FC drug was eliminated from the aqueous solution using the conventional dialysis method against mQ‐H₂O for 24 hours utilizing an appropriate MWCO membrane. The aqueous solution containing the exclusive 5FC‐loaded micelles was kept at 4 °C until further investigations and analysis.

Scheme 2.

Schematic representation of the experimental procedure for obtaining 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles by ultrasound assisted solvent evaporation method.

c) DLS Analysis in PBS Solutions

The colloidal characteristics of both the non‐loaded and 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles were assessed through dynamic light scattering (DLS) experiments. This evaluation was focused on determining the hydrodynamic diameter (Hd) of particles and their zeta potentials in PBS with a pH of 7.4, at an ambient temperature of 23 °C, and at a concentration of 0.75 mg/mL, which exceeds the CMC value of 0.154 mg/mL. The results are illustrated in figure 1 and summarized in table 1.

Figure 1.

DLS analysis of non‐loaded and 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles at 0.75 mg/mL in PBS with pH value of 7.4 at ambient temperature of 23 °C. a) Particle hydrodynamic diameter (Hd) distributions; b) Zeta Potentials. All the measurements were done in triplicates and the plots are representing the average values without error bars.

Table 1.

Colloidal characteristics of non‐loaded and 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles at 0.75 mg/mL in PBS with pH value of 7.4 at ambient temperature of 25 °C.

Sample	Average Hd (nm)±SD[a]	Average PDI±SD	Average Zeta Potential (mV)±SD
Non‐loaded micelles	125.33±6.34	0.235±0.091	−6.55±0.40
5FC‐loaded micelles	207.13±8.26	0.335±0.003	−12.36±0.36

[a] Data are presented as mean±SD (n=3).

The plots depicting the particle size measurements of both the non‐loaded and 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles exhibited wide unimodal distributions of Hd (Figure 1a) for each sample. Analysis of the data, represented as mean values with standard deviation (S.D.) (Table 1), revealed an increase in the Hd of the 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles (207.13±8.26 nm) and in the polydispersity index (PDI) (0.335±0.003) compared to the non‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles, which had a Hd of 125.33±6.34 nm and a PDI of 0.235±0.091. This observed difference is likely attributable to the loading of the 5FC drug, primarily on the surface of the SQ‐PEG₁₅₀₀‐NH‐Boc micelles, owing to its hydrophilic properties, as elucidated in our previous study involving another hydrophilic drug. ^[28]

The zeta potential values for both the non‐loaded and 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles are illustrated graphically in figure 1b and summarized as mean values with standard deviation (S.D.) in table 1. These results indicate that loading the 5FC drug in SQ‐PEG₁₅₀₀‐NH‐Boc micelles enhances the colloidal stability of the system, as evidenced by an increase in zeta potential from −6.55±0.40 mV for the non‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles to −12.36±0.36 mV for the 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles.

The DLS analysis reveals that loading the 5FC‐drug into SQ‐PEG₁₅₀₀‐NH‐Boc micelles alters their colloidal properties, leading to an augmentation in the hydrodynamic diameter and an elevation in zeta potential. This indicates that despite the larger size of the micelles, their colloidal stability is enhanced.

d) STEM Analysis

STEM analysis was employed to assess the static morphologies of 5FC‐loaded and non‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles, along with their size distributions (Figure 2). Both samples exhibited core‐shell spherical morphologies with nanoscale dimensions (Figures 2a and 2c). Interestingly, upon closer inspection (red circles), the non‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles (Figure 2a) revealed worm‐like structures on the shell surface, identified as PEG chains enveloping the squalene moieties. However, such formations were absent in the 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles (Figure 2c), likely due to the presence of the 5FC hydrophilic drug on the micelle's surface, interacting with the PEG molecules, corroborating the observations from DLS analysis.

Figure 2.

STEM morphological analysis of the studied samples. a) STEM image of non‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles at 500 nm scale; b) size distribution plot of non‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles (n=120 micelles); c) STEM image of 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles at 1 μm scale; d) size distribution plot of 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles (n=120 micelles).

The size distributions of the examined samples were determined by analysing the sizes of nanostructures in the STEM images using ImageJ software, as depicted in figures 2b and 2d. The non‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles exhibited an average size of 18.82±7.19 nm, with a distribution ranging from 5 to 40 nm (Figure 2b). In contrast, the 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles displayed an increased average size of 62.65±14.37 nm, with a distribution between 30 and 100 nm (Figure 2d). These STEM analysis results align with those from DLS analysis, indicating larger dimensions for the 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles. The convergence of findings from both techniques underscores the potential biomedical applications of the 5FC‐loaded micelles.

2.3. Quantification of Drug Loading Efficiency and Loading Capacity

The loading efficiency (%LE) and drug loading capacity (%DL) of 5FC loaded into SQ‐PEG₁₅₀₀‐NH‐Boc micelles were assessed using the UV‐Vis spectroscopy method via a calibration curve (Figure 3). ^[31] The 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles were prepared in triplicate, and PBS solutions with pH 7.4 were subjected to UV‐Vis analysis to monitor the characteristic absorbance maxima of the drug at 276 nm (Figure 3b). By utilizing the line equations derived from the calibration curve and equations presented in figure 3c, the average values±SD of (%LE) and (%DL) were determined, as shown in table 2.

Figure 3.

UV‐Vis analysis of the studied samples: a) overlapping the absorbance spectra of non‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles, free 5FC and 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles recorded in PBS solutions with pH value of 7.4; b) UV‐Vis spectra of 5FC in PBS with pH 7.4 at concentrations between 0.625 and 30.00 μg/mL; c) linear fitting of the absorbance recorded at 276 nm as function of 5FC concentration (μg/mL) in PBS 7.4.

Table 2.

Loading efficiency (%LE) and drug loading capacity (%DL) of 5FC in SQ‐PEG₁₅₀₀‐NH‐Boc micelles.

Sample[a]	LE (%)	Mean LE (%)±SD	DL (%)	Mean DL(%)±SD
1	29.49	30.20±1.52	2.05	2.07±0.017
2	32.31		2.07
3	28.80		2.09

[a] SQ‐PEG₁₅₀₀‐NH‐Boc/(5FC) replicate number.

The acquired data indicated that the loading efficiency of the 5FC hydrophilic drug within the SQ‐PEG₁₅₀₀‐NH‐Boc micelles was relatively low, with average values of 30.20±1.52 %LE and 2.07±0.017 %DL. These findings align with those observed in our prior investigation involving another hydrophilic drug, suggesting that SQ‐PEG₁₅₀₀‐NH‐Boc micelles are better suited for encapsulating hydrophobic drugs. ^[28]

As outlined in our earlier investigation, SQ‐PEG₁₅₀₀‐NH‐Boc micelles exhibit a core‐shell structure in aqueous environments, with a hydrophobic core surrounded by a hydrophilic shell. ^[32] This configuration facilitates improved encapsulation of hydrophobic drugs within the core, rather than the loading or adsorption of hydrophilic drugs onto the hydrophilic shell. While the %LE and %DL values are not critical parameters for subsequent biomedical applications, they do play a significant role in determining the cost efficiency of the systems.

2.4. In Vitro 5FC‐Drug Release from SQ‐PEG₁₅₀₀‐NH‐Boc Micelles

The in vitro release behaviour of 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles was assessed in a 1x PBS solution (pH 7.4) at 37 °C to mimic physiological conditions and compared to free release conditions in mQ‐H₂O at 37 °C. In both studies, control experiments involving free 5FC drug were conducted under similar conditions, and the outcomes are depicted in figure 4.

Figure 4.

In vitro cumulative release of 5FC from free 5FC solution and 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles. a) Cumulative release of 5FC (%) for 96 hours in simulated physiological conditions (PBS with pH of 7.4 and 37 °C); b) Cumulative release of 5FC (%) for 48 hours in simulated physiological conditions (PBS with pH of 7.4 and 37 °C); c) Cumulative release of 5FC (%) for 96 hours in mQ‐H₂O at 37 °C; d) Cumulative release of 5FC (%) for 48 hours in mQ‐H₂O at 37 °C. The experiment was carried out for 96 hours and the results are expressed as means±SD (n=3). *p <0.05, **p <0.01, ***p <0.001 and #p >0.05 (ns) by Student's t‐test.

As presented in figures 4a and 4c, the cumulative release profiles of 5FC from both systems were monitored over 96 hours by sampling the dialysis medium at predetermined time intervals. For the free 5FC release control, the entire drug diffusion across the dialysis membrane was observed within 24 hours, with the release plateau being reached after 4 hours in the case of simulated physiological conditions (Figure 4a) and after 24 hours in the case of free release conditions (Figure 4c), achieving a cumulative release of 94 % and 95 % respectively. Conversely, in simulated physiological conditions, the complete release of the 5FC loaded in the SQ‐PEG₁₅₀₀‐NH‐Boc micelles was achieved after 48 hours, reaching the release plateau at a cumulative release of 78 % (Figure 4a), meanwhile, in free release conditions, the complete release of the 5FC loaded in the SQ‐PEG₁₅₀₀‐NH‐Boc micelles was achieved also after 48 hours, reaching the release plateau at a cumulative release of 52 % (Figure 4c).

For enhanced clarity, figures 4b and 4d illustrates the release profiles for the initial 48 hours of the experiments. In simulated physiological conditions (Figure 4b), the release profile of free 5FC exhibits a burst‐release pattern, with most of the drug being released within the first 4 hours, notably with 82 % released within 1 hour. Similarly, in free release conditions (Figure 4d), the release profile of free 5FC also exhibits a burst‐release pattern, with most of the drug being released within the first 6 hours, notably with 64 % released within 3 hours. In contrast, the release profile of 5FC from the SQ‐PEG₁₅₀₀‐NH‐Boc micelles in simulated physiological conditions reveals a two‐stage kinetic mechanism. Initially, there is a burst‐release of approximately 37 % of the drug within the first hour, followed by a slower release of the drug, culminating in 78 % release within 48 hours. Moreover, the release profile of 5FC from the SQ‐PEG₁₅₀₀‐NH‐Boc micelles in free release conditions reveals also a two‐stage release mechanism with a lighter burst‐release of approximately 17 % of the drug in the first hour and a slower release of the drug with reaching the plateau at 52 % within 48 hours. By comparing the results obtained from the two distinct studies, we can clearly conclude that the cumulative release of 5FC from the SQ‐PEG₁₅₀₀‐NH‐Boc micelles is dependent on the release medium, where in free release conditions the cumulative release of the drug is lower compared to the cumulative release of the drug in simulated physiological conditions.

The outcomes from the in vitro release experiments regarding the 5FC drug demonstrate a distinct improvement in the release profile under physiological conditions when loaded within SQ‐PEG₁₅₀₀‐NH‐Boc micelles compared to free 5FC. Notably, the issue of burst‐release, a significant concern, was effectively avoided. Interestingly, in free release conditions, the release of the drug is limited due to the absence of intermolecular interactions between micelles and medium.

2.5. Drug Release Kinetics Using Different Mathematical Models

To gain deeper insights into the release mechanism of 5FC from SQ‐PEG₁₅₀₀‐NH‐Boc micelles under physiological and free release conditions, and to identify the governing factors, the kinetic data were subjected to fitting with multiple mathematical models, such as Zero‐order, First‐order, Higuchi, Hixson‐Crowell, and Korsmeyer‐Peppas models. This analysis focused on the initial phase of the release curves (Figure 5), encompassing both loaded and free 5FC.

Figure 5.

Linear fitting of the mathematical models applied for the release of loaded 5FC in SQ‐PEG₁₅₀₀‐NH‐Boc micelles and free 5FC in simulated physiological conditions and free release conditions: a) Zero‐order model; b) First‐order model; c) Higuchi model, d) Hixson‐Crowell model; and e) Korsmeyer‐Peppas model.

Through analysis of the correlation coefficients derived from the mathematical models employed, distinct behaviours were discerned among the tested samples (Table 3). Consistent with our prior research, mathematical models exhibiting correlation coefficients exceeding 0.8 were prioritized to determine the most suitable release model for this system. Consequently, for the release data of loaded 5FC in PBS 7.4, all mathematical models proved appropriate, yielding correlation coefficients ranging from 0.88 to 0.99. Notably, the Higuchi (Figure 5c) and Korsmeyer‐Peppas (Figure 5e) models demonstrated superior fitting, boasting the highest correlation coefficients among the tested models.

Table 3.

The data obtained by applying various mathematical models to analyse the release kinetics of free 5FC‐drug and 5FC‐loaded micelles during the initial phase (1–8 hours) of the release process in simulated physiological conditions and free release conditions.

Sample	Zero‐order		First‐order		Higuchi		Hixson‐Crowell		Korsmeyer‐Peppas
	R2	K0	R2	Kt	R2	KH	R2	k	R2	k	n
Loaded 5FC PBS 7.4	0.94	4.49	0.88	−0.09	0.99	17.88	0.97	−0.12	0.99	0.37	0.31
Free 5FC PBS 7.4	0.57	1.26	0.56	−0.01	0.72	5.44	0.64	−0.21	0.86	0.84	0.05
Loaded 5FC mQ‐H2O water	0.83	2.95	0.75	−0.10	0.92	12.04	0.85	−0.06	0.95	0.19	0.40
Free 5FC mQ‐H2O	0.81	7.84	0.72	−0.13	0.91	32.29	0.88	−0.26	0.95	0.34	0.52

Where: R² is the correlation coefficient, K is the proportionality constant, and n is the release exponent.

In contrast, regarding the release data for free 5FC in PBS 7.4, only the Korsmeyer‐Peppas model (Figure 5e) demonstrated suitability, exhibiting a correlation coefficient of 0.86. Conversely, the other mathematical models displayed notably inadequate correlation coefficients, falling below 0.80 and ranging from 0.56 to 0.72. In the case of free release conditions, both samples presented similar correlation coefficients between 0.72 and 0.95 with Higuchi and Korsmeyer‐Peppas as the most suitable models exhibiting the highest correlation coefficients. Furthermore, the Korsmeyer‐Peppas model was employed to plot the experimental data as the logarithm of cumulative % drug release against the logarithm of time. This facilitated the determination of the release exponent (n). Notably, three from four samples displayed n values <0.5, suggesting a Fickian diffusion drug release mechanism, meanwhile, the forth (free 5FC in mQ‐H₂O as release medium), presented a n value between 0.5 and 1.0 which is suggesting a non‐Fickian anormal diffusion drug release mechanism. ^[33]

Moreover, the release constant (k) reflects the diffusion constant directly, signifying the rate of drug release (higher values indicate faster release rates, while lower values indicate slower rates). ^[34] In the present investigation (Table 3), the lowest release rates were evident for the study in mQ‐H₂O as release medium with values of 0.34 for free 5FC and 0.19 for the 5FC‐loaded micelles. The highest rates were expectedly observed for the simulated physiological medium study with k values of 0.84 for free 5FC and 0.37 for 5FC‐loaded micelles. This finding implies that despite three from four systems sharing the same release mechanism, the release study of 5FC from SQ‐PEG₁₅₀₀‐NH‐Boc micelles as well as non‐loaded 5FC follows a gradual release pattern in PBS 7.4, consistent with the in vitro drug‐release analyses. Interestingly, as observed in the in vitro drug‐release studies, the 5FC release from the micelles in mQ‐H₂O was slower and limited mainly due to the absence of the intermolecular interactions between micelles and the release medium.

2.6. In Vitro Antifungal Activity of the Studied Nanotherapeutics

The antimicrobial activity screening of the studied samples was performed on three yeast strains represented by C. albicans, C. glabrata, and C. parapsilosis. The diagrams (Figures 6a‐c) show the growth inhibition of the yeast colonies when treated with the samples. There were noticed different antifungal activities against three tested yeast strains. In the case of C. albicans (Figures 6a and 6d) free 5FC was efficient after 2 hours of incubation reducing the fungal colonies to 50 % of viability, the maximum of efficiency being reached after 24 hours (only 20 % cells viability). On the other hand, 5FC‐loaded micelles started to have significant activity after 6 hours of incubation, reducing the yeast colonies to 35 % and reaching 6 % after 24 hours. In the particular case of C. glabrata (as presented in Figures 6b and 6d) free 5FC destroyed all the yeast cells after only 40 minutes of incubation, while 5FC‐loaded micelles reached the same efficiency after 2 hours. Afterwards both samples maintained their efficiency up to 24 hours. C. parapsilosis (Figures 6c‐d) was reduced by 5FC‐loaded micelles to 8 % after 6 hours and totally destroyed after 24 hours, while free 5FC had the same efficiency between 6 and 24 hours of incubation, being able to reduce the yeast cells to about 22–24 %.

Figure 6.

The microbial growth inhibition: a) C. albicans, b) C. glabrata, c) C. parapsilosis expressed as percent of viable yeast colonies vs. incubation time; d) yeast colonies of the three strains after 40 minutes of incubation with free 5FC and 5FC‐loaded micelles.

The presented results are in accordance with the literature regarding the free 5FC antifungal activity on Candida spp., thus confirming the validity of the used protocol for the in vitro antifungal testing's. More than that, our results proved that 5FC‐loaded micelles improved 5FC efficiency in the case of C. albicans, and especially against C. parapsilosis for the tested concentration of 112.5 μg/mL.

2.7. In Vitro Cell Viability of the Studied Nanotherapeutics

The in vitro cytotoxicity of the studied compounds was evaluated by quantitative microscopy using the High Content Screening (HCS) approach. ^[35] HCS is widely used in biological research and drug discovery due to its versatility for analysing the whole cells or components of cells by imaging some fluorescent tags. ^[36] In this study, the in vitro cytotoxicity of free 5FC, non‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles and 5FC‐loaded micelles was tested against Human Gingival Fibroblasts (HGF) cells with an exposure time of 24 hours at three concentrations (112.5, 225, and 450 μg/mL), including the concentration used at the antifungal experiments, and the obtained results are presented in figure 7. The results obtained from the quantitative analysis of dead cells by staining the death cells’ nuclei with red (propidium iodide) (Figure 7a) showed that by loading 5FC‐drug into SQ‐PEG₁₅₀₀‐NH‐Boc micelles, a slight increase in the total number of dead cells could be observed. This increase in cytotoxicity with increasing the concentration could be attributed to a synergic effect between the loaded drug and the nanocarrier, a phenomenon that was observed by others for systems based on metallic nanoparticles and different drugs. ^[37] In the case of free 5FC and non‐loaded micelles, the cytotoxicity is almost non‐existent which confirms the hypothesis of a synergic effect between the two components of the system. Moreover, the quantitative analysis of live cells was possible by staining total cells nuclei (Hoechst) and live cells cytoplasm (Calcein‐AM) and the results are presented in figure 7b. In this case, the differences between the live cells after exposure with studied samples at three concentrations are not very high in comparison with the dead cells analysis.

Figure 7.

In vitro High Content Screening (HCS) analysis of the studied samples after 24 hours of exposure. a) Dead cells quantitative estimation after the treatment with the studied samples; b) live cells quantitative estimation after the treatment with the studied samples; c) cell proliferation and viability under the sample's treatment. Cell nuclei in blue, cytoplasm of living cells in green, dead cells nuclei or remnants of nuclei in red.

For a better understanding of the harmless effects of the studied compounds for the HGF cells, in figure 7c is presented a picture with overlays of the three channels for all the tested compounds at three concentrations, together with control images. In this picture, no morphological changes can be observed and moreover, a very good distribution of the living cells was observed for the studied samples at concentrations of 112.5 and 225 μg/mL in comparison with the control cells at the same concentrations. Interestingly, in the case of non‐loaded micelles and non‐loaded 5FC, a slight proliferation of the HGF cells can be observed, phenomenon which was not observed in the case of 5FC‐loaded micelles. This phenomenon can be attributed to a better compatibility of the above‐mentioned compounds which are stimulating the division of the cells. However, at the concentration of 450 μg/mL, the living cells seems to be fewer in comparison with the other two concentrations, the numerical distribution being comparable with the control sample, meaning that the cytotoxicity of the free 5FC, non‐loaded micelles and 5FC‐loaded micelles at this concentration is insignificant. It can be assumed that all the compounds that were tested did not exhibit cytotoxic effects up to 450 μg/mL for the HGF normal cells.

3. Conclusions

This study aimed to improve the effectiveness of the antifungal drug 5‐fluorocytosine (5FC) by loading it in micellar structures formed by PEGylated squalene copolymer. Using the ultrasound‐assisted solvent evaporation method, satisfactory loading efficiency (30.20±1.52 %) and drug loading capacity (2.07±0.017 %) were achieved. Both non‐loaded and 5FC‐loaded micelles had spherical “core‐shell” morphologies with nanometric dimensions (measured with STEM) of around 20 and 65 nm, respectively. Hydrodynamic diameters, measured by DLS, of non‐loaded and loaded micelles were 125.33±6.34 nm and 207.13±8.26 nm, respectively. Zeta potential analysis indicated improved colloidal behavior for 5FC‐loaded micelles (−12.36±0.36 mV) compared to non‐loaded micelles (−6.55±0.40 mV). The STEM analysis results align with those from DLS analysis, both indicating larger dimensions for the 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles. In contrast to the dialysis bag's sudden release of free 5FC, loaded 5FC produced a steady two‐stage in vitro release over a 48‐hour period in both studied release media. Mathematical modeling favored the Higuchi and Korsmeyer‐Peppas models for three from four studied systems, while only the Korsmeyer‐Peppas model was suitable for non‐loaded 5FC in PBS 7.4 as release medium. Also, three of four samples exhibited a Fickian diffusion drug release mechanism, while the non‐loaded 5FC in free release conditions presented a non‐Fickian anormal diffusion drug release mechanism. Slower diffusion rates were obtained in the case of 5FC‐loaded micelles in free release conditions study mainly due to the lack of intermolecular interactions between micelles and release medium. In vitro antifungal studies showed enhanced efficacy against C. albicans, particularly in combating C. parapsilosis at 112.5 μg/mL concentration, while maintaining biocompatibility with Human Gingival Fibroblasts even at higher concentrations up to 450 μg/mL. These findings suggest the potential of 5FC‐loaded PEGylated micelles as effective delivery systems for future in vivo antifungal studies.

Experimental Section

Materials: Boc‐protected PEGylated Squalene (SQ‐PEG₁₅₀₀‐NH‐Boc) was obtained and characterized as in our previous study; ^[28] Flucytosine (5FC) Pharmaceutical Secondary Standard C.R.M. (Supelco, USA); tetrahydrofuran (THF) anhydrous ≥99.9 % (Sigma‐Aldrich, Germany); N,N‐dimethylformamide (DMF) anhydrous 99.8 % (Sigma‐Aldrich, Germany); methylene chloride (DCM) puriss ≥99.9 % (Honeywell, Riedel‐de Haen, Germany); n‐hexane 99 % (J.T.Baker, Germany); triethylamine (TEA) >99 % (TCI, Germany); sodium chloride (NaCl) ACS >99 % (Honeywell | Fluka, USA); Potassium chloride (KCl) ReagentPlus ≥98 % (Honeywell | Fluka, USA); anhydrous magnesium sulfate (MgSO₄) ≥99.5 % (Sigma‐Aldrich, Japan); sodium phosphate dibasic dihydrate (Na₂HPO₄ x 2H₂O) puriss 98.5–101 % and sodium phosphate monobasic monohydrate (NaH₂PO₄ x H₂O) ACS ≥98 % were purchased from Honeywell | Fluka, Germany; double‐distilled water (dd‐H₂O) obtained with an FiSTREEM Cyclon WSC044.MH3.4 system, ultra‐pure water (mQ‐H₂O) obtained with an TKA‐GenPure 08.2204 system. All chemicals were utilized in their original purchased state without undergoing any purification process.

Ultraviolet‐Visible Spectroscopy (UV‐Vis): Absorbance spectra were acquired using the Lambda 35 UV‐Vis Spectrophotometer (Perkin Elmer). Solutions containing the compounds at predetermined concentrations were examined within quartz cuvettes possessing a 10 mm optical path. UV‐Vis spectra were captured in double beam mode, with the solvent serving as a reference. The analysis of the absorption spectra was conducted using Origin Pro v2020b software developed by OriginLab.

Particle size analysis and zeta potential measurements: The colloidal characteristics of both the PEGylated squalene non‐loaded micelles (SQ‐PEG₁₅₀₀‐NH‐Boc) and 5FC‐loaded micelles (SQ‐PEG₁₅₀₀‐NH‐Boc/(5FC)) were assessed in phosphate‐buffered saline (PBS) solutions with a pH of 7.4 at 23 °C. The analysis was conducted using a Malvern ProRED Particle Size Analyzer (Malvern Panalytical). Particle size measurements were performed utilizing DTS0012 disposable 10x10 plastic cells (Malvern Panalytical), while zeta potential assessments were carried out using DTS1070 disposable folded capillary cells (Malvern Panalytical). The acquired data were processed using ZS XPLORER Software Version 3.3.0.42 from Malvern Panalytical.

Scanning Transmission Electron Microscopy (STEM): STEM analysis was performed using a Verios G4 UC Scanning Electron Microscope (Thermo Scientific, Czech Republic) operating in STEM Mode at 30 kV, fitted with a STEM 3+ detector. Both samples (non‐loaded and 5FC‐loaded micelles) were prepared in the same conditions by applying the diluted aqueous samples, after removing the THF, onto 400 Mesh carbon‐coated copper grids (TED PELLA) and air‐drying them for 24 hours under dust‐free conditions at room temperature (23 °C). The acquired images were analyzed using ImageJ 1.48r software.

Critical Micelle Concentration (CMC): In accordance with our prior publication, the critical micelle concentration (CMC) of the SQ‐PEG₁₅₀₀‐NH‐Boc copolymer was determined in PBS aqueous solution with a pH of 7.4. ^[28] The CMC value was determined using the pyrene method with a FluoroMax‐4 fluorescence spectrophotometer (HORIBA Scientific). ^[32] Various concentrations of SQ‐PEG₁₅₀₀‐NH‐Boc solutions were prepared within the range of 3.24x10⁻⁸ M to 13x10⁻³ M by combining PBS aqueous solution with a pH of 7.4 (1 mL) with pyrene (1.0x10⁻⁷ M in dd‐H₂O) (1 mL). The samples were magnetically stirred at 60 °C for 90 minutes and equilibrated overnight at room temperature (23 °C) under continuous stirring. The fluorescence spectra of pyrene were measured, with excitation at 334 nm, and the emission spectra were recorded within the range of 349–600 nm (averaging 3 scans). The acquired spectra were processed using baseline subtraction and normalization with Origin Pro v2020b software from OriginLab.

Quantification of loading efficiency and loading capacity: The loading efficiencies (%LE) and drug loading capacities (%DL) of the 5FC drug within SQ‐PEG₁₅₀₀‐NH‐Boc micelles were assessed using a UV‐vis spectrophotometer at 276 nm, referencing a standard calibration curve derived from 5FC in PBS with a pH value of 7.4. The %LE and %DL were determined following Equations (1) and (2) as outlined below:

$${\displaystyle \%LE=\frac{Weightofencapsulateddrug\left(mg\right)}{Weightofinitialdrug\left(mg\right)}\times 100}$$ (1)

$${\displaystyle \%DL=\frac{Weightofencapsulateddrug\left(mg\right)}{Weightofmicelles\left(mg\right)}\times 100}$$ (2)

Phosphate Buffer Saline (PBS) solution preparation: The PBS solution with a pH value of 7.4 was prepared using the technique reported elsewhere.[ ³⁸ , ³⁹ ]

In vitro drug release studies: To evaluate the in vitro release behaviour of 5FC from 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles, the common dialysis technique was employed in PBS medium with a pH of 7.4 at 37 °C.[ ⁴⁰ , ⁴¹ ] For demonstrative purposes to define the differences between the controlled release and free release conditions, similar studies were carried out in ultrapure water. In brief, 3 mL of 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles were enclosed within dialysis bags (MWCO 10 kDa), sealed at both ends with Teflon threads, and fully immersed in 40 mL of release medium. The release systems were positioned on a heated multi‐post magnetic stirrer and agitated at 100 rpm at 37 °C. At predetermined intervals (0, 1, 2, 3, 4, 5, 8, 24, 48, 72, and 96 hours), 2 mL of the release medium was withdrawn for analysis, and an equivalent volume of fresh medium was replenished to maintain a constant volume. The drug release experiments spanned 96 hours and were conducted in triplicate. UV‐vis absorption measurements at 276 nm were utilized to quantify the released amount of 5FC from the tested systems. The cumulative release profile of the 5FC drug was determined using an equation outlined elsewhere. ^[42] For comparison, an equivalent amount of free 5FC, as found in 5FC‐loaded micelles, was subjected to similar release assessments.

Drug release kinetics: The in vitro release kinetics of free 5FC and 5FC‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles were thoroughly examined by subjecting experimental data to rigorous analysis through various mathematical models for both studied release conditions (PBS 7.4 and mQ‐H₂O as release medium). These models, including Zero‐order, First‐order, Higuchi, Hixson‐Crowell, and Korsmeyer‐Peppas, were carefully selected based on their established efficacy and relevance in similar studies outlined in the existing literature.[ ⁴² , ⁴³ ]

In vitro antimicrobial activity: The antimicrobial activity screening of the studied samples was performed by using viable cell‐counting method. ^[44] Three yeast strains were used, represented by Candida albicans ATCC10231, Candida glabrata ATCC20019, and Candida parapsilosis ATCC22019 that were refreshed on Sabouraud dextrose agar (SDA) at 37 °C. Microbial suspensions were prepared with these cultures in sterile Sabouraud dextrose broth medium to obtain optical turbidity comparable to that of 0.5 McFarland standards. 500 μL of the samples (112.5 μg/mL as concentration of 5FC) were placed into solutions which contained 0.5 mL of the yeast suspensions and 4.5 mL 1X PBS and incubated up to 24 hours in a shaker at 37 °C. A control experiment was also conducted. 1 μL of the control samples and of the treated samples were removed at determined periods of incubation time (10 minutes, 20 minutes, 40 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 24 hours) and spread on Plate Count Agar (PCA) plates. The number of colonies was counted after 24 hours of incubation at 37 °C. All tests were carried out in triplicate to verify the results. After incubation, the plates were analysed with SCAN1200®, version 8.6.10.0 (Interscience), and the number of colonies was expressed as the mean±standard deviation (SD) performed with GraphPad Prism software version 7.00 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com). The ratio of inhibited the growth of bacteria (IRG) was calculated using the Equation 3:

$${\displaystyle IRG\left(\%\right)=\frac{\left(Na-Nb\right)}{Na}x100\%}$$ (3)

where: Na and Nb are the average values of colonies of the control group and the experimental groups, respectively.

Cell cultures for the in vitro cytotoxicity experiments: Human Gingival Fibroblasts (HGF) cells were cultivated using Minimum Essential Medium‐Alpha Eagle containing 10 % fetal bovine serum (Gibco) and 1 % antibiotic/antimycotic (1 % penicillin‐streptomycin‐amphotericin B, 10 K/10 K/25 μg in 100 mL) from Lonza (Verviers, Belgium) at 37 °C under a humidified atmosphere with 5 % CO₂. The medium was changed every 3–4 days, and the cells were maintained in culture dishes until they reached subconfluency. For cell passages, detachment was carried out by washing with PBS and treatment with Tryple detachment agent (Thermofisher).

In vitro quantitative microscopy: At the beginning of the experiment, 1500 cells/well were seeded in 384‐well plates (Cellvis Mountain View, CA, USA). The next day, cells were subjected to treatment with sample solutions at concentrations of 450, 225 and 112.5 μg/mL (calculated as concentration of 5FC). For control, wells with cells that were not treated were used. The treated plates were incubated for 24 hours at 37 °C, 5 % CO₂ and humidified atmosphere. At the end of the incubation, the cells were stained for 15 minutes with Hoechst, Calcein‐AM and propidium iodide to distinguish the cell nuclei, the cytoplasm of live cells, respectively the nuclei of dead cells. After the staining step, fluorescence microscopy images were acquired using the high content screening tool ImageXpress® Confocal HT.ai (Molecular Devices, San Jose, CA, USA) and the MetaXpress program, using the DAPI channels for Hoechst, FITC for Calcein‐AM and Texas Red for propidium iodide. The data analysis was done with the same software, through the “cell scoring” module. Live cells were counted considering the double positive elements on the DAPI and FITC channels, and dead cells were counted by correlating the double positive elements on the DAPI and Texas Red channels.

Statistical Analysis: Statistical analysis was conducted using GraphPad Prism 7.00 for Windows (GraphPad Software, La Jolla California, CA, USA). A Student's t‐test was utilized to compare the studied nanotherapeutics, with each group comprising three replicates. Results are expressed as means±standard deviation (S.D.), with statistical significance determined when calculated probability (p) values were less than 0.05.

Synthesis of SQ‐PEG₁₅₀₀‐NH‐Boc: the SQ‐PEG₁₅₀₀‐NH‐Boc amphiphilic compound was synthesized and fully characterized as detailed in our previously published paper. ^[28] In summary, the activated ester of squalenic acid (SQ‐COONHS) (0.1 g, 0.21 mmol) was dissolved in anhydrous DMF (3.33 mL), followed by the addition of TEA (0.032 g, 0.31 mmol) to the solution. Subsequently, H₂N‐PEG₁₅₀₀‐NH‐Boc (0.498 g, 0.31 mmol) was dissolved in anhydrous DMF (6.67 mL) and added dropwise to the aforementioned solution. The reaction mixture was stirred for 24 hours at room temperature (23 °C) under nitrogen atmosphere. The following day, the mixture was diluted with dd‐H₂O and subjected to extraction using a DCM:n‐hexane mixture in a 1 : 1 volume ratio. The combined organic phases were washed with dd‐H₂O and brine, dried over anhydrous MgSO₄, and concentrated under reduced pressure using a rotary evaporator (10 mbar, 40 °C) to yield an amorphous solid residue. This residue was then dissolved in mQ‐H₂O (20 mL) and subjected to sonication three times for four minutes at 120 W (sonication pulse was set at 2 s with 4 s between pulses) as described in the literature. ^[45] Following sonication, the solution underwent filtration using an Amicon Ultra‐15 filter unit (Merck, Germany) with a molecular weight cutoff (MWCO) of 3000 Da at 5000xg for 60 minutes. The residue obtained was washed twice with mQ‐H₂O and subjected to filtration once more. Subsequently, the resulting solution was diluted with mQ‐H₂O and dialyzed against mQ‐H₂O for 48 hours using a 3500 Da MWCO membrane (SnakeSkin from ThermoScientific, USA). The solution containing SQ‐PEG₁₅₀₀‐NH‐Boc was recovered from the dialysis tube and subjected to freeze‐drying under reduced pressure to yield a white fluffy solid, amounting to 0.419 g (99 %), which was stored for further analysis.

Preparation of SQ‐PEG₁₅₀₀‐NH‐Boc non‐loaded micelles: the preparation of non‐loaded SQ‐PEG₁₅₀₀‐NH‐Boc micelles followed the procedure outlined in our prior research, employing the ultrasound‐assisted method. ^[28] Initially, the SQ‐PEG₁₅₀₀‐NH‐Boc copolymer was dissolved in mQ‐H₂O or PBS solution at a concentration exceeding the critical micelle concentration (CMC) and stirred for 24 hours at room temperature (23 °C) to facilitate the self‐assembly of SQ‐PEG₁₅₀₀‐NH‐Boc molecules into micellar structures within the aqueous medium. Subsequently, to disintegrate the micelles, which tended to form larger aggregates, a high‐power UP200St−G Ultrasonic System equipped with a UP200St‐TD Probe‐Type transducer (HIELSCHER Ultrasonics) was employed, subjecting the solution to ultrasonication at 120 W for 10 minutes. Following this, the micelles were allowed to reassemble by maintaining the solution containing SQ‐PEG₁₅₀₀‐NH‐Boc molecules under stirring at room temperature (23 °C) for 30 minutes. The resulting aqueous solution containing SQ‐PEG₁₅₀₀‐NH‐Boc micelles was stored at 2–4 °C for later analyses.

Preparation of 5FC‐loaded micelles: 5FC‐loaded micelles were prepared via the ultrasound‐assisted solvent evaporation method. ^[28] Initially, the SQ‐PEG₁₅₀₀‐NH‐Boc copolymer (approximately 0.03 g) was dissolved in a mixture of mQ‐H₂O (6 mL) and THF (6 mL) with continuous sonication. Concurrently, the 5FC commercial drug (approximately 0.003 g) was dissolved in a mixture of mQ‐H₂O (2 mL) and THF (2 mL) with continuous sonication. The 5FC solution was gradually added to the SQ‐PEG₁₅₀₀‐NH‐Boc solution under continuous sonication, followed by an additional 3‐minute sonication period after the injection completion. Subsequently, the mixture underwent thermal treatment at 60 °C for 2 hours under magnetic stirring and was left to cool to room temperature (23 °C) over a 24‐hour period. The following day, THF evaporation was performed, and the remaining aqueous solution was stirred in a fume hood for another 24 hours to ensure complete evaporation of residual THF. Finally, to eliminate non‐loaded 5FC, the resulting aqueous solution was dialyzed against mQ‐H₂O for 24 hours using a 3500 Da MWCO membrane. The 5FC‐loaded micelles recovered from the dialysis bag were stored at 4 °C until further experimentation.

Conflict of Interests

The authors declare no conflict of interest.

Craciun B.-F., Rosca I., Peptanariu D., Pinteala M., ChemMedChem 2025, 20, e202400432. 10.1002/cmdc.202400432

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.