Search Results (214)

Ellagic acid (EA) possesses various biological activities, including anti-inflammatory, whitening, and antioxidant properties. Its practical application is limited by poor aqueous solubility and susceptibility to degradation. To overcome these limitations, this study prepared EA liposomes using the thin-film hydration–ultrasonication method, followed by surface modification with low-molecular-weight chitosan (LM-CS) and medium-molecular-weight chitosan (MM-CS), yielding EA liposomes modified with LM-CS (EA-L-LC) and MM-CS (EA-L-MC), respectively. The formulation and preparation process were optimized using a Box–Behnken design combined with response surface methodology. Under optimal conditions, the mean particle size (MPS), polydispersity index (PDI), Zeta-potential, and encapsulation efficiency (EE) of the different liposomes (unmodified EA-L, EA-L-LC, and EA-L-MC) were determined. Morphological observation and functional group characterization were conducted via transmission electron microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR), respectively. The stability of the various liposomes was compared under different environmental conditions, and their stability and the released amount of EA were evaluated during in vitro digestion. The in vitro antioxidant activity and tyrosinase inhibitory effects of the different liposomes were investigated. After process optimization, the encapsulation efficiency of EA liposomes was effectively enhanced following modification with chitosan of different molecular weights. TEM and FTIR results confirmed that EA was effectively encapsulated, and chitosan was successfully coated onto the outer layer of the liposomes. Compared to unmodified EA liposomes (EA-L), the chitosan-modified liposomes (EA-L-LC and EA-L-MC) exhibited enhanced in vitro antioxidant activity and sustained, slow-release tyrosinase inhibitory effects, along with superior stability across multiple conditions. In vitro digestion experiments demonstrated that EA-L-MC and EA-L-LC achieved slower release rates in simulated gastric fluid compared to EA-L, thereby improving the digestive stability of EA. Full article

Obesity is a major global health burden that is linked to type 2 diabetes, cardiovascular disease, and metabolic syndrome. Chitosan oligosaccharides (COS) are bioactive compounds that are derived from the depolymerization of the chitosan in crustacean shells and are promising candidates for natural anti-adipogenesis effects. However, there is incomplete understanding of the molecular mechanisms by which structurally defined low-molecular-weight COS modulates adipogenic transcription networks and global transcriptional reprogramming. MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry and ¹³C NMR spectroscopy indicated a predominance of dimeric species (DP2) at m/z 344.79, which represents a lower molecular weight fraction and is proposed to improve the membrane permeability and intracellular bioavailability of COS. In a 3T3-L1 preadipocyte model, COS treatment at concentrations of 320–1280 µg/mL dose-dependently reduced intracellular lipid accumulation, triglyceride content, and adipocyte maturation while enhancing lipolysis and insulin-mediated glucose uptake. Western blot analysis indicated dose-dependent downregulation of PPARγ and C/EBPα. Transcriptomic RNA-seq analysis indicated large-scale transcriptional reprogramming with the altered expression of genes involved in PPAR signaling, PI3K-Akt, AMPK, insulin signaling, and fatty acid metabolism pathways among differentially expressed genes. These findings demonstrate that COS suppresses adipogenesis through the coordinated modulation of adipogenic transcription factors and multiple metabolic signaling pathways. The results support its potential as a promising natural compound but warrant preclinical investigation in the context of obesity and metabolic disorders. Full article

Low-molecular-weight chitosan derivatives emerged as promising plant growth biostimulants due to their favorable properties, such as biocompatibility, antibacterial and antifungal activity, enhancement of stress resistance, and yield improvement. In the present study, we evaluated the effect of pea seed priming with two types of chitooligosaccarides (aminochitooligosaccaride and chitooligosaccaride hydrochloride) applied at concentrations of 100 and 500 mg/L under non-stress conditions and 50 mM chronic NaCl stress. We characterized the seed surface topology by atomic force microscopy, the germination process by evaluation of seed germinability and synchrony, root emergence, seed imbibition capacity and ion leakage. Early plant growth and physiological performance were further evaluated in 14-day-old seedlings by measuring leaf water potential, Na⁺ accumulation in roots and leaves, photosystem II activity, leaf pigment content, and membrane stability. The results revealed changes in seed coat topology, i.e., higher surface roughness in 100 and 500 mg/L chitooligosaccaride hydrochloride and 500 mg/L aminochitooligosaccaride primed variants. Concentration-dependent effects of the two chitooligosaccarides under both non-stress and salt stress conditions were evident in 14-day-old seedlings. Under chronic salt stress, seed priming with 100 mg/L chitooligosaccharide hydrochloride and 500 mg/L aminochitooligosaccharide produced the most pronounced improvements in the primary photochemical reactions of photosynthesis, particularly the performance index on an absorption basis and the total performance index. Moreover, the investigated chitooligosaccharide, particularly chitooligosaccaride hydrochloride, preserved membrane integrity and maintained flavonol and anthocyanin levels, indicating a strong protective effect against salt stress. Overall, the data indicate beneficial effects on pea physiological status following seed priming with chitooligosaccarides under chronic salt stress conditions. This highlights the approach as a promising strategy for enhancing plant resilience in challenging environments, and it is worth further investigation and verification at the whole-plant level. Full article

Background: Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is a fatal inflammatory disorder driven by M1 macrophages and the associated inflammatory cascade. Targeted drug delivery to these cells is a promising therapeutic strategy. Methods: L-arginine was conjugated to chitosan of different molecular weights. The resulting curcumin nanocrystals (Arg-CS-Cur) were characterized for conjugation efficiency, zeta potential, stability, and drug release profile. Cellular uptake mechanisms and mitochondrial targeting were investigated in lipopolysaccharide (LPS)-induced M1 macrophages using specific endocytic inhibitors and confocal microscopy. Results: Low-molecular-weight chitosan (MW 50 kDa) showed the highest L-Arg conjugation efficiency (22.31%). The optimized Arg-CS-Cur nanocrystals exhibited high zeta potential (±47.5 mV), excellent stability, and a superior drug release. They were internalized by M1 macrophages more efficiently than unmodified CS-Cur or free curcumin (p < 0.05). Uptake occurred via clathrin-mediated endocytosis (p < 0.001) and was mediated by CAT-2, which was highly expressed in M1 macrophages (p < 0.001). Arg-CS-Cur specifically targeted the mitochondria, reducing ROS and NLRP3 expression, thus inhibiting the NLRP3 inflammasome pathway (p < 0.001). Conclusions: This L-arginine-modified chitosan-based nanodelivery system synergistically exploits CAT-2 and clathrin pathways to deliver curcumin to M1 macrophage mitochondria, inhibiting the NLRP3 inflammasome. This dual-targeted strategy offers a promising approach for treating ALI/ARDS. Full article

The development of sustainable plant protection strategies requires stable and environmentally compatible delivery systems for beneficial microorganisms. In this study, Bacillus subtilis was encapsulated within chitosan-based cast films to evaluate bacterial viability, sustained biological activity, and antifungal efficacy. Films prepared from chitooligosaccharide (COS) and chitosans of low, medium, and high molecular weight (CS-LMW, CS-MMW, CS-HMW) were characterized in terms of morphology, mechanical performance, and pH-dependent swelling behavior. The viscosity of the chitosan solutions increased markedly with molecular weight from 73 cP (COS) to 614 cP (CS-HMW), while film thickness ranged from 34 ± 1.5 to 57 ± 2.3 µm. Mechanical performance improved significantly with increasing molecular weight, with maximum tensile stress exceeding 200 MPa for CS-HMW films, while swelling studies confirmed pronounced pH-dependent behavior consistent with the polyelectrolyte nature of chitosan. Encapsulation effectively preserved bacterial viability and metabolic activity over time. The intrinsic antifungal activity of chitosan synergized with the biocontrol activity of B. subtilis against Fusarium avenaceum and Alternaria solani. The highest antifungal performance was observed for CS-HMW films, which produced inhibition zones up to 84.6 ± 5.0 mm against A. solani. These findings demonstrate that chitosan-based cast films serve as effective carriers for beneficial microorganisms, providing environmental protection and regulated biological activity. The combination of a bioactive polymer matrix with a potent biocontrol agent represents a promising eco-friendly approach to sustainable plant protection. Full article

Objectives: The identification of novel antimicrobial agents for use in root canal treatment may provide opportunities to improve treatment outcomes. This study aimed to assess the antimicrobial efficacy of different molecular weights of chitosan (CS), and how modification with CS may impact on the antimicrobial, physico-mechanical and biological properties of Biodentine™, a calcium-silicate-based material used in endodontics. Methods: C. albicans biofilms were treated with either 3% sodium hypochlorite (NaOCl) or a 0.05% or 0.1% CS solution for 5 min. The growth medium was replenished, and cells were re-incubated for additional 72 h. Regrowth of biofilms was assessed using a colorimetric XTT assay. Additionally, multispecies biofilms were established and the regrowth of biofilms on Biodentine discs were quantified following the addition of 0.5 wt% and 1 wt% of CS powder using qPCR. The physico-mechanical and biological properties of the new composite of Biodentine and CS were also evaluated. Results: Viability readings revealed significant initial biofilm inhibitory effects of CS solutions, followed by significant regrowth after 72 h. Upon the addition of CS to Biodentine, significant reductions in multispecies biofilm regrowth were determined. Notably, the antibiofilm activity of CS was found to be increased as the molecular weight decreased. The addition of powdered CS of low molecular weight showed a reduction in the mechanical properties of Biodentine, whereas no detrimental effects on the other material properties were noted. Conclusions: Chitosan may not be useful as an alternative irrigant to NaOCl. Addition of CS to Biodentine represents a potential means of augmenting the antimicrobial activity of Biodentine against persistent microorganisms following endodontic therapy. Despite the reductions in mechanical properties of the material, the new composite still represents a viable material option when material strength and hardness are not critical. Full article

The enzymatic production of low-molecular-weight chitosan and chitooligosaccharides (COS), with broad application potential in agriculture, food, medicine, and cosmetics, has emerged as an attractive alternative to chemical chitosan depolymerization owing to its substrate specificity and environmentally benign catalytic action. However, the functional properties of available chitosanases need to be enhanced to meet the demands of industrial COS manufacturing under high temperature and substrate concentrations. In this work, we performed directed evolution on a recombinant Bacillus subtilis chitosanase to increase chitosan hydrolysis performance and thermal resistance. Three rounds of directed evolution screening (~9000 clones) yielded variants MT1, MT2, and MT3 with higher specific activity, achieved through Vmax improvement and increased T_1/2 at 60 °C. HPLC, DLS, and MALDI-TOF results indicate differences in the hydrolysis kinetics and size distribution of COS products over reaction time, suggesting a narrower distribution and a lower average molecular weight. Molecular dynamics simulations and docking studies revealed potential modulation of chitosanase activity via changes in the opening and closing dynamics of the active-site cleft. These results suggest that future efforts targeting the cleft interface could significantly advance both the catalytic performance and the mechanistic understanding of GH46 family chitosanases. Full article

Sustainable agricultural techniques can ensure food security around the world. Hydrogel based soilless culture is an ecological and efficient alternative compared to conventional agriculture. Here, a multi-component hydrogel (pectin, Kelcogel, and chitosan/Se hydrogel, PKCH) was prepared by synthesizing natural biomolecules to cultivate dandelion for stimulate dandelion growth and improve nutritional value. The germination percentage of dandelion on PKCH (88.89%), was significantly higher than that in traditional hydroponics and pure Kelcogel (p < 0.05). Compared with hydroponics, the long-term dandelion cultivation experiments demonstrated that the PKCH cultivation mode enhanced root vitality, further increasing the growth and yield of dandelions (shoot length: 18.36 ± 0.30 cm, root length: 9.28 ± 0.21 cm, main root diameter: 0.94 ± 0.02 cm). The hydrogel substrate was associated with improved nutrient solubilization and sustained release, which may be linked to the accumulation of low-molecular-weight organic acids in the rhizosphere. Exogenous Se was effectively assimilated and transported to the above-ground parts of dandelion, which stimulated the photosynthetic efficiency and nutritional accumulation of dandelion. The polysaccharide content of dandelion reached 69.40 ± 0.13% (expressed as glucose-equivalent total sugars), which demonstrated the potential antioxidant properties and medicinal value. Technical economic analysis revealed the cost-effectiveness of PKCH synthesis and application. This study enriches the application of hydrogels in dandelion cultivation and provides an alternative approach for cultivating dandelion in soilless environments and medicinal crop production techniques. Full article

Background: Human Immunodeficiency Virus type 1 (HIV-1) remains a major global health challenge. Despite advances in antiretroviral therapy, new prevention strategies are needed, particularly topical microbicides capable of blocking the earliest steps of viral entry. HIV-1 attachment relies on interactions with heparan sulfate proteoglycans on host cell surfaces; therefore, sulfated heparan-mimetic polymers have been explored as antiviral agents. In this context, sulfated chitosan microparticles are designed to mimic natural glycosaminoglycan receptors, acting as biomimetic decoys that prevent viral attachment and entry. Methods: Low-molecular-weight sulfated chitosan (LMW Chi-S) microparticles were synthesized and characterized (SEM, EDS, DLS, FTIR) following US Patent No. 11,246,839 B2. Their antiviral activity was evaluated by incubating the microparticles with high-viral-load HIV-1-positive plasma (~3.5 × 10⁶ copies/mL) to enable viral binding and removal by pull-down. The performance of the synthesized Chi-S microparticles was compared with established heparinoid controls, including soluble heparin and heparin microparticles. Results: Chi-S microparticles exhibited stronger virus-binding and neutralizing capacity than all heparinoid comparators, achieving up to 70% reduction in viral load relative to untreated HIV-1 plasma. In comparison, soluble heparin and heparin microparticles reduced viral load by approximately 53% and 60%, respectively. Subsequent evaluation across multiple tested concentrations confirmed a consistent antiviral effect, indicating that the synthesized Chi-S microparticles maintain robust virus–particle interactions throughout the concentration range examined. Conclusions: These findings demonstrate that LMW Chi-S microparticles possess potent antiviral properties and outperform classical heparinoid materials, supporting their potential application as topical microbicides targeting early HIV-1 entry mechanisms. Full article

Background/Objectives: Peri-implantitis is a chronic inflammatory condition affecting tissues surrounding dental implants and is characterized by progressive marginal bone loss that can ultimately lead to implant failure. Reduced vascularization and impaired immune clearance in peri-implant tissues contribute to persistent inflammation and limited therapeutic efficacy. MicroRNAs (miRNAs), particularly miR-21, have emerged as key regulators of inflammatory responses and bone remodeling. The objective of this study was to develop a bioactive dental implant coating capable of locally delivering miR-21 to modulate inflammation and promote peri-implant tissue regeneration, thereby preventing peri-implantitis. Methods: Cationic nanoparticles were synthesized using lecithin and low-molecular-weight polyethylenimine (PEI) as a non-viral delivery system for miR-21. Lecithin was employed to enhance biocompatibility, while PEI functionalization provided a positive surface charge to improve miRNA complexation and cellular uptake. The resulting lecithin–PEI nanoparticles (LEC–PEI NPs) were incorporated into a chitosan-based coating and applied to titanium implant surfaces to obtain a sustained miR-21–releasing system (miR21-implant). Transfection efficiency and biological activity were evaluated in human periodontal ligament fibroblasts (hPDLFs) and compared with a commercial transfection reagent (Lipofectamine). Release kinetics and long-term activity of miR-21 from the coating were also assessed. Results: MiR-21-loaded LEC–PEI nanoparticles demonstrated significantly higher transfection efficiency than Lipofectamine and retained marked biological activity in hPDLFs relevant to peri-implantitis prevention. The chitosan-based nanoparticle coating enabled controlled and sustained miR-21 release over time, supporting prolonged modulation of inflammatory and osteogenic signaling pathways involved in peri-implant tissue homeostasis. Conclusions: The miR21-implant system, based on lecithin–PEI nanoparticles incorporated into a chitosan coating, represents a promising therapeutic strategy for peri-implantitis prevention. By enabling sustained local delivery of miR-21, this approach has the potential to preserve peri-implant bone architecture, modulate chronic inflammation, and enhance the osseointegration of titanium dental implants. Full article

The valorization of shrimp shell waste is crucial for promoting sustainability and a circular economy. This study aimed to extract chitin from the exoskeletal residues of deep-water rose shrimp (Parapenaeus longirostris) sourced from the Marmara Sea and synthesize low-molecular-weight chitosan (LMWC) via conventional, microwave-, and autoclave-assisted deacetylation pathways. The shell biomass was subjected to sequential demineralization (1 M HCl) and deproteinization (1 M NaOH), yielding 14.42% chitin. The extracted chitin was then converted to LMWC using the three methods, and the products were characterized using FT-IR spectroscopy, titration, viscometry, SEM, and TGA. The results demonstrated that the autoclave-assisted method achieved the highest degree of deacetylation (DD) at 95%, significantly outperforming the conventional method (81%) and the microwave-assisted method (67%). The autoclave-synthesized chitosan also exhibited the lowest viscosity (33 cP), confirming its low molecular weight. Morphological analysis showed that chitin exhibited a well-defined fibrous structure. After deacetylation, this structure transformed into a rough and porous surface morphology. Thermal analysis further demonstrated that the laboratory-synthesized chitosan exhibited higher thermal stability than the commercial chitosan sample. In conclusion, the autoclave-assisted method proved to be highly efficient for producing low-molecular-weight chitosan with a high degree of deacetylation. However, the conventional method remains the most practical option for scalable industrial production due to its simplicity and well-established infrastructure. Moreover, the laboratory-synthesized chitosan exhibited higher thermal stability, increased porosity, and a higher degree of deacetylation compared to commercially available chitosan, which may offer functional advantages in applications requiring enhanced reactivity, solubility, or thermal resistance. Overall, the findings provide valuable insights into selecting appropriate deacetylation strategies for producing low-molecular-weight chitosan with tailored properties, thereby bridging the gap between laboratory-scale synthesis and potential industrial applications. Full article

In this study, biopolymer chitosan–glucan from fruiting bodies of Agaricus bisporus (Cs-Agrif) was extracted and characterized as a sustainable alternative to commercial low molecular weight (LMW) chitosan obtained from crab shells (Cs-1). Cs-Agrif was prepared through an alkaline treatment process that included deproteination and deacetylation in the same step. The obtained sample was evaluated for its molecular weight, rheological behavior, degree of deacetylation (DD), crystallinity, and β-glucan and phenolic contents. Furthermore, the antioxidant properties of the prepared chitosan were determined under in vitro conditions using four spectrophotometric methods. Finally, its antimicrobial activity was tested against two pathogenic bacteria, one yeast, and mycotoxigenic fungi. Cs-Agrif had low molecular weight, of 45.70 ± 5.20 kDa, with pseudoplastic flow behavior. The degree of deacetylation was 92.7%. FT-IR and XRD analyses confirmed a chitosan-like structure and lower crystallinity in Cs-Agrif compared to pure commercial chitosan. The mushroom-derived chitosan contained β-glucans and phenols, indicating a chitosan–glucan complex. Antimicrobial assays showed low Cs-Agrif microbicidal concentrations (≤2.5 mg mL⁻¹) for Escherichia coli, Enterococcus faecalis, and Candida albicans. The growth of Aspergillus flavus was significantly reduced after five days of incubation. The laboratory-prepared Cs-Agrif exhibited strong antioxidant activity at 5 mg mL⁻¹, comparing to standards. Mushroom-derived chitosan–glucan biopolymer displays excellent physicochemical, antimicrobial, and antioxidant properties, confirming its potential use in biomedicine, food, and the pharmaceutical and cosmetic industries, among many others. Full article

High-performance spun bionanocomposite fibers, composed of high-molecular-weight chitosan (HMW), low-molecular-weight chitosan “oligomers” (LMW), and cellulose nanofibers (CNFs), were successfully fabricated via gel spinning of viscous aqueous chitosan (CHI) based formulations into a NaOH coagulation bath. The X-ray diffraction (XRD) analysis revealed that the incorporation of cellulose nanofibers contributed to enhance crystallinity of chitosan in spun fibers. The spinning process, which comprised sequential acidic solubilization, basic neutralization, stretching, and drying steps, produced chitosan/CNF composite fibers with high crystallinity, further enhanced by the incorporation of low molecular weight chitosan. The cellulose nanofibers seem to promote CHI crystallization, by acting as nucleation sites for the nucleation and growth of chitosan crystals, with those latter of LMW further enhancing crystallization and orientation due to higher mobility of shorter polymer chains. Two-dimensional XRD patterns demonstrated the preferential alignment of both CNFs and chitosan crystals along the fiber axis. Increasing the proportion of short-chain chitosan led to a reduction of the viscosity of collodion, facilitating the spinning of solutions with higher polymer concentrations. The X-ray diffraction (XRD) analysis revealed that the addition of low-molecular-weight chitosan (LMW), with an intermediate molecular weight Mw of ~4.4 × 10⁴ g/mol, produced the most significant improvements in the crystallinity index (CrI) and orientation. This structural enhancement corresponded to superior mechanical properties like Young’s modulus, yield stress σ_y, and stress-at-break σ_b of the processed composite fibers. By incorporating that intermediate molecular weight chitosan, a Young’s modulus as high as 20 GPa was achieved for the spun composite fibers, which was twice higher than the modulus of around 10 GPa obtained by adding the lowest molecular weight chitosan of Mw ~ 2.9 × 10⁴ g/mol in the composite, and largely above the modulus of around 5 GPa obtained for fiber just spun with chitosan without incorporation of cellulose nanofibers. Full article

A growing scientific interest in bioactive compounds from sea cucumbers is contributing to a broader recognition even in regions where their consumption is not common. This study evaluated the biological potential of a Holothuria forskali extract obtained through different extraction methods, including water extraction, ethanol–water extraction, and enzymatic hydrolysis. The hydrolysate (H), rich in low-molecular-weight peptides, yielded the highest antioxidant (30.6 ± 0.6 mg VitC Eq/g sample for ABTS and 10.7 ± 0.1 mg GAEs/g sample for Folin-reactive substances) and ACE-inhibitory (82.6%) activities. Based on these results, the hydrolysate was selected for encapsulation in two nanostructured delivery systems for comparative purposes: chitosan nanoparticles (NPs) and rapeseed lecithin liposomes (LPs). Both nanostructures were characterized in terms of size, ζ-potential, and polydispersity and subjected to simulated in vitro gastrointestinal digestion (GIDv) to assess their stability and mucoadhesive properties. After digestion, antioxidant activity increased in both systems, particularly in liposomes. Although encapsulation initially reduced ACE-inhibitory activity, gastrointestinal digestion restored or enhanced it, especially in liposomal formulations (≈37% inhibition). The mucoadhesive potential of the nanostructures after DGIv, focusing on their interactions with mucin, was assessed. Liposomal digests significantly increased viscosity in the presence of mucin, while chitosan nanoparticles decreased it, suggesting the formation of soluble complexes with reduced hydrodynamic volume. Electrostatic and hydrogen bonding interactions between chitosan and mucin were particularly evident in the NPH formulation. The rheological synergism parameter (Δη) revealed more negative values for NPs and NPHs, indicating stronger mucoadhesive interactions compared to controls and suggesting their suitability for mucosal delivery. These findings support the use of H. forskali hydrolysates as a source of functional bioactive compounds and highlight the potential of chitosan-based nanocarriers for enhancing their stability, bioaccessibility, and mucoadhesive properties in functional food or nutraceutical applications. Full article

Background/Objectives: Glaucoma is a multifactorial eye disease that can cause optic nerve damage and irreversible blindness. It is considered a significant public health problem worldwide. Topical intraocular pressure (IOP)-lowering eye preparations are used to prevent or slow further damage. Previously, we demonstrated that nimodipine (NMD), a calcium channel blocker, significantly reduced IOP after a single drop of NMD/HPMC suspension. The current study was designed to develop NMD chitosan nanoparticles (NMD-CS NPs) to improve the NMD IOP-lowering efficacy. Methods: NMD-CS NPs were prepared using the spontaneous-emulsification solvent diffusion method. Three different types of chitosan, carboxymethyl CS (CMCS), low molecular weight CS (LCS), and medium molecular weight CS (MCS), were used. Different concentrations of polymers, various stabilizers, and two pHs were used for formulation optimization. NMD-CS NPs were characterized regarding their particle size, polydispersity index (PDI), zeta potential, DSC, FTIR, and encapsulation efficiency. NMD-CS NPs were incorporated into eye drops and characterized in terms of their in vitro release, cytotoxicity, transcorneal permeability, and in vivo efficacy. Results: The optimized NMD-CS NPs demonstrate a small particle size with a narrow size distribution and acceptable zeta potential values. DSC and FTIR results confirmed the complete entrapment of NMD inside the NPs. NMD-CS NP eye drops successfully sustained NMD release without any burst effect. These NPs demonstrated a Higuchi non-Fickian diffusion mechanism and 79.41% improved corneal permeability. Cytotoxicity studies revealed that NMD formulations are nontoxic. After a single topical ocular application, NMD-MCS NP eye drops induced a significantly superior effect to Timolol maleate eye drops with regard to the %IOP reduction and duration of action. Conclusions: Evaluation results of NMD-CS NP eye drops show their positive effect in a preclinical animal model as a promising glaucoma therapy. Full article