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. 2025 Jul 30:16:1617631.
doi: 10.3389/fmicb.2025.1617631. eCollection 2025.

Synthetic niclosamide-loaded controlled-release nanospheres with high solubility and stability exerting multiple effects against Clostridioides difficile

Yulei Tai  1   2 , Meng Zhang  1 , Yuning Han  1 , Hui Hu  1   2 , Shan Lin  1 , Fangya Zhai  1 , Menglun Tian  1 , Xiaojun Song  3 , Shuangshuang Wan  1   2 , Yu Chen #  1   2 , Dazhi Jin #  1   2   3
Affiliations

Synthetic niclosamide-loaded controlled-release nanospheres with high solubility and stability exerting multiple effects against Clostridioides difficile

Yulei Tai et al. Front Microbiol. .

Abstract

Introduction: Niclosamide (NIC) has significant potential as a clinical therapeutic agent for Clostridioides difficile infection (CDI); however, its strong hydrophobicity hampers its oral bioavailability, and its active effects against C. difficile remain unclear.

Methods: Niclosamide-loaded controlled-release hyaluronic acid-modified poly (lactic-co-glycolic acid) naosphernes (NIC@PLGA-HAs) were synthesized using an oil-in-water emulsion technique and their effects on C. difficile cell growth, spore germination, biofilm formation, and NIC interaction sites with C. difficile toxin B (TcdB) were analyzed.

Results: NIC@PLGA-HAs exhibited enhanced solubility and stability, with a water contact angle on a hydrophilic surface of 65.1° and a zeta potential of 31.57 ± 2.08 mV, and pH-responsive (pH 7.4) controlled-release characteristics compared to free NIC. The NIC@PLGA-HAs killed C. difficile vegetative cells at a minimum inhibitory concentration (MIC) of 4 μg/mL. When C. difficile cells were treated with NIC@PLGA-HAs at the 1/4 MIC, spore germination and biofilm formation were significantly inhibited compared to those in untreated cells (P < 0.01). NIC was found to interact with the receptor-binding domain of TcdB at 24 amino acid sites via an enthalpy-driven reaction (enthalpy change, 36.21 kJ/mol and entropy change, 212.9 J⋅mol/K). In vivo experimental findings in Mongolian gerbils indicated that NIC@PLGA-HAs outperformed free NIC in reducing pathological damage, diarrhea severity, weight loss, and TcdB production and enhanced the survival rate.

Conclusion: These findings presented the therapeutic potential of NIC@PLGA-HAs with high solubility and stability, which simultaneously exerted multiple biological activities against C. difficile.

Keywords: Clostridioides difficile; Niclosamide; biofilm formation; loaded controlled-release nanospheres; multiple effects; spore germination.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

(a) Chemical structures of niclosamide, poly(lactic-co-glycolic acid)-COOH, and hyaluronic acid-NH2. (b) FTIR spectra comparing NIC@PLGA-HA, HA, PLGA, and NIC. (c) TGA graph showing weight loss of NIC, PLGA, HA, and NIC@PLGA-HA with temperature. (d) XRD patterns for NIC, PLGA, HA, and NIC@PLGA-HA. (e) TEM image of NIC@PLGA-HA nanoparticles, scale bar 1 micrometer. (f) Bar graph showing nanoparticle diameter and zeta potential over time, with diameter in blue and zeta in purple.
FIGURE 1
Characterization of NIC@PLGA-HAs. The structural formulas (a) of Niclosamide (NIC), Poly (lactic-co-glycolic acid) (PLGA), and Hyaluronic acid (HA). Fourier Transform infrared spectroscopy (FT-IR) spectra (b), thermogravimetry (TG) curves (c) and X-ray diffraction (XRD) patterns (d) of NIC, PLGA, HA and NIC@PLGA-HAs. TEM image (e) of NIC@PLGA-HAs. The mean particle diameters and zeta potentials (f) of NIC@PLGA-HAs (3.5 mg) synthesized for 5 min under different ultrasonication time.
Three graphs labeled a, b, and c. Graph a shows absorbance versus concentration with a contact angle of 84.5 degrees, featuring a curve fitted to data points and an inset image of a droplet. Graph b displays absorbance versus concentration with a contact angle of 65.1 degrees, also featuring a fitted curve and droplet image. Graph c illustrates cumulative release percentage over time for NIC@PLGA-HA and Free NIC, showing fitted first-order kinetics equations for both.
FIGURE 2
Water-solubility, stability and drug release behavior of NIC@PLGA-HAs. Contact angle images and critical micelle concentrat (CMC) curves of (a) NIC and (b) NIC@PLGA-HAs, and (c) in vitro drug release at pH 7.4 and 37°C. NIC@PLGA-HAs were prepared with the Niclosamide (NIC) (3.5 mg) (mean ± SD, n = 3).
Graph (a) shows CFU concentration over 14 days for untreated, NIC, and NIC@PLGA-HAs, with NIC@PLGA-HAs reducing CFU after day 6. Bar chart (b) compares OD570nm, indicating NIC and NIC@PLGA-HAs significantly reduce OD compared to untreated.
FIGURE 3
NIC@PLGA-HAs inhibited spore germination and biofilm formation of C. difficile. (a) NIC@PLGA-HAs inhibited spore germination at the 1/4 minimal inhibitory concentration (MIC) compared to untreated C. difficile. (b) Biofilm production measured by crystal-violet staining absorbance at 570 nm. The biofilm production was significantly inhibited by NIC@PLGA-HAs in comparison to untreated C. difficile (ns, P > 0.05; **P < 0.01).
Panel a shows microscopy images of cell cultures under different conditions, with red arrows indicating changes. Panel b depicts molecular models of DRBD proteins interacting with NIC, highlighted in red and blue. Panel c features a graph of absorbance versus wavenumber, illustrating spectral changes for different substances. Panel d includes two sets of graphs: one showing enthalpy changes with fitting curves, and another displaying corrected heat rates over time, for two different DRBD conditions interacting with NIC.
FIGURE 4
Interaction between delivery and receptor-binding domain (DRBD) in C. difficile toxin B (TcdB) and Niclosamide (NIC). (a) NIC against TcdB-mediated Caco-2 cell rounding. (b) Views of the interaction between each of TcdB, DRBD, and mutated DRBD and NIC (electrostatic surface). (c) Ultraviolet-visible (UV-vis) monitoring curves when NIC was interacted with DRBD. (d) Isothermal titration calorimetry (ITC) curves when NIC was interacted with DRBD and DRBD mutants, respectively.
Diagram of experimental design and results in a mouse model. (a) Timeline of the experimental setup, including antibiotic treatment and infection. (b) Survival graph showing percentages over seven days for different treatments. (c) Weight loss percentages over six days, comparing various treatments. (d) Bar graph of TcdB concentration in fecal samples for each group, showing significant differences. (e) Histological images of intestinal sections under different treatments: Control, CDI, Vancomycin, NIC, and NIC@PLGA-HA. Scale bar indicates 500 micrometers.
FIGURE 5
The Mongolian gerbils with C. difficile infection (CDI) were treated by NIC@PLGA-HAs. (A) Protocol schematic for the Mongolian gerbils CDI model. (B) Survival of Mongolian gerbils treated and untreated with vancomycin, Niclosamide (NIC) or NIC@PLGA-HAs after C. difficile challenge. (C) Weights of Mongolian gerbil after C. difficile spore challenge. (D) TcdB concentration in fecal samples detected by EILSA (****P < 0.001). (E) HE staining of colon tissues from different groups of Mongolian gerbils. Vancomycin was used as a positive control.
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