The improvement of diosmin dissolution by PLGA complexes for oral delivery: physicochemical characterization and anti-inflammatory evaluation

Abstract

Diosmin (DSN) is a potent flavonoid used for chronic venous insufficiency, but its therapeutic potential is constrained by poor aqueous solubility and low oral bioavailability (BCS Class IV). To overcome these challenges, a hybrid polymeric nanocarrier system comprising poly(lactic-co-glycolic acid) (PLGA) and (2-hydroxypropyl)-β-cyclodextrin (HPCD), surface-stabilized by chitosan, was successfully engineered via single-emulsion solvent evaporation. Systematic optimization identified a 95 : 5 (w/w) PLGA : HPCD formulation (95P5H-DSN), achieving an encapsulation efficiency of 71.47 ± 1.38% and a drug loading of 12.81 ± 0.25%. The nanoparticles exhibited a uniform spherical morphology with a hydrodynamic diameter of 223 ± 15.72 nm, a polydispersity index of 0.329, and a high positive zeta potential (+70.00 ± 7.35 mV), indicating good colloidal stability. Solid-state analyses (XRD, DSC, and FTIR) verified drug amorphization and successful entrapment within the matrix. In vitro release studies demonstrated pH-dependent behavior that fits the Higuchi diffusion model; the nanocarrier suppressed gastric release at pH 2.0 while providing a controlled and improved dissolution profile at intestinal pH 7.8 (17.73% at 24 h). Furthermore, in LPS-stimulated RAW264.7 macrophages, 95P5H-DSN partially attenuated the intrinsic cytotoxicity of raw DSN and exerted concentration-dependent anti-inflammatory efficacy via nitric oxide suppression. The anti-inflammatory IC₅₀ was 58.95 µg mL⁻¹. These findings establish the PLGA–HPCD nanocomplex as a promising oral drug-delivery platform for enhancing the dissolution and therapeutic efficacy of diosmin.

The synthesis of the PLGA/hydroxypropyl-β-cyclodextrin complex used to deliver hydrophobic diosmin.

Introduction

For patients who are suffering from chronic venous insufficiency (CVI), there is much advice for changing their lifestyle to reduce the impact of potential risks, such as outdoor activities and nutrition, accompanied by using veno-active drugs.¹ Diosmin is a flavone called 3,5,7,-trihydroxy-4-methoxyflavone 7-rutinoside, which is present in the pericarp of citrus, or it can be obtained by dehydrogenation of another flavonoid, hesperidin.² Diosmin (DSN) is beneficial for treating CVI-related conditions, including edema, inflammation, swollen legs, varicose veins, and venous leg ulcers. In addition, diosmin has a strong effect in protecting the blood vessels, increasing the tension and elasticity of the vessel walls, and reducing the permeability of capillary walls.³ Besides the prospects, the main disadvantage of DSN that ruins its widespread acceptance is poor water solubility, leading to a low dissolution rate and impaired uptake from the gastrointestinal tract.⁴ Additionally, after oral administration, diosmin is hydrolyzed to diosmetin, but some reports indicate that plasma diosmetin levels are low, highly variable, and inconsistent across studies. Hence, it is important to reduce the size of diosmin to increase its surface area and ensure better adherence to the mucous layer, thereby enabling it to penetrate and bind to the underlying epithelium. For that, the micronized diosmin form, which is presented in some commercial products such as Daflon 500 mg or µSMIN Plus™, has been shown to ameliorate intestinal absorption to improve its presence in plasma.⁵

Oral administration is an appealing route for delivering the treatment. Nevertheless, the gastrointestinal tract (GIT) has harsh physical, chemical, and biological barriers that influence drug bioavailability. Oral nanocarriers will protect the payload from those barriers while favoring controlled release, as well as targeting specific GIT cellular phenotypes, diffusing through the mucous layer and increasing drug bioavailability.⁶

The oral route remains the most widely used mode of drug administration due to its convenience and high patient acceptance, particularly among pediatric and geriatric populations. Despite these advantages, many biologics and other sensitive therapeutics exhibit poor oral bioavailability because they must overcome multiple physiological barriers within the gastrointestinal tract. Limited aqueous solubility, low epithelial permeability, and instability in acidic or enzymatically active environments all contribute to inefficient absorption. The gastrointestinal tract presents a highly heterogeneous milieu, with pH values ranging from approximately 1–2 in the stomach to 5–7 in the small intestine and 6–7.5 in the colon.⁷ Drug uptake is further restricted by the intestinal epithelium, a tightly regulated barrier composed of enterocytes, goblet cells, Paneth cells, and specialized M cells located in Peyer's patches. Tight junctions between adjacent epithelial cells limit paracellular transport, meaning that orally administered molecules must either traverse the mucus layer and enterocytes via transcellular pathways or be taken up by M cells.⁷ According to the Biopharmaceutics Classification System (BCS), solubility and permeability are the primary determinants of passive absorption. Drugs in BCS classes II and IV often exhibit poor dissolution and limited uptake, whereas class III compounds are hindered mainly by low permeability. These constraints underscore the need for advanced delivery systems that protect drugs during gastrointestinal transit and enhance their interaction with absorptive tissues. Nanoparticles, such as PLGA, solid lipid nanoparticles, hydrogels, polymeric micelles, liposomes, emulsions, nanocrystals, dendrimers, and carbon nanotubes, offer a promising solution, as they can encapsulate therapeutic agents, shield them from enzymatic and chemical degradation, prolong residence time through mucoadhesion, and improve both absorption and bioavailability. Numerous studies have demonstrated that nanoparticle-based oral formulations can significantly enhance systemic exposure compared with conventional dosage forms.⁷ Diosmin is a Biopharmaceutical Classification System IV (BCS IV) compound, meaning it has low solubility and permeability.⁸

Micronization is a useful tool to enhance the bioavailability of poorly water-soluble compounds such as diosmin, hesperidin, and quercetin by producing 1–3 µm particles via mechanical processing (jet milling, ball milling, pin milling). Once again, the smaller the size, the better the dissolution rate as well as the intestinal absorption.⁹ There have been efforts to improve diosmin bioavailability, such as cyclodextrins and chitosan-based nanogels. In 2014, β-cyclodextrin (CD) was first used to create the complex with DSN.¹⁰ The obtained stoichiometry was a 1 : 1 molar ratio between the host and guest components, with an A_L-type phase solubility curve. More specifically, during complex formation, the presence of PEG 6000 significantly increased drug release and antioxidant activity by enhancing the complexing power of β-CD towards DSN.

In 2023, researchers took advantage of nano-chitosan to wrap the diosmin for oral delivery.¹¹ Using the ionic gelation technique, they obtained a particle size of 113.07 ± 12.62 nm, a polydispersity index of 0.266 ± 0.08, a zeta potential of 22.32 ± 0.56 mV, a loading efficiency of 81.56 ± 2.65%, and a loading capacity of 10.25 ± 1.43%, respectively. The formulation was easily eroded at pH 1.2 and 4.5 and swelled at pH 6.8 and 7.4. In 2024, diosmin was encapsulated in lipid chitosan hybrid nanoparticles for acetic acid-induced colitis in rodents.¹² Using a chitosan : lipid weight ratio of 1 : 20 yielded a particle size of 135.53 ± 24.4 nm and a zeta potential of 29.93 ± 0.611 mV, with 88.51% diosmin entrapment. This formulation prevented acetic acid-induced colitis through antioxidant and anti-inflammatory mechanisms, including amelioration of oxidative stress biomarkers and reduction in the expression of NLRP3, caspase-1, and IL-1β.

Except for cyclodextrins and chitosan, poly(lactic-co-glycolic acid) (PLGA) has been intensively studied for many years and has shown enormous potential. The FDA approved PLGA as biocompatible and biodegradable because it can degrade into CO₂ and H₂O, which are easily eliminated from the body, and is widely used for gene transfer, drug delivery, tissue engineering, and molecular imaging.¹³ Back in 2019, DSN was carried by chitosan–PLGA nanoparticles for gastric retention and anti-ulcer activity.⁵ As described, the DSN input, the organic solvent, and the drug : polymer PLGA weight ratio were the key parameters for achieving a high encapsulation rate of about 67.40 ± 2.90%. In 2020, research was done to improve the efficacy of DSN as well as its bioavailability and solubility by using commercial polymeric matrices such as hydroxypropyl starch (HPS) and poly(lactide-co-glycolide) (PLGA)/chitin for atherosclerosis treatment.² They established two methods for carrying out DSN: anti-solvent and acid–base neutralization techniques. Using the anti-solvent method, the nanoparticles contain 84.5% diosmin, have a size of 120 nm, and exhibit a zeta potential of +30 mV. Another attractive study was done in 2019, which mentioned the combination of 2-hydroxypropyl-β-cyclodextrin (HPCD) and PLGA to build nanoparticles loaded with triamcinolone for ocular drug delivery.¹⁴ The HPCD/PLGA nanoparticles obtained were 149.4 ± 3.7 nm in diameter and stable, with a zeta potential of −14.05 ± 0.39 mV, an encapsulation rate of 76.0 ± 4.8%, and a loading capacity of 0.52 ± 0.035%. Meanwhile, in 2021, paclitaxel was entrapped in a type of complexation of HPCD/PLGA nanoparticles.¹⁵ The resulting nanoparticles had a size of 151.03 ± 1.36 nm, a zeta potential of −12.24 ± 1.72 mV, and contained approximately 49.12% of cargo.

So far, the combination of PLGA/hydroxypropyl-β-cyclodextrin (HPCD) or PLGA/lecithin for encapsulating diosmin (DSN) has not been carried out. Hence, this study aims to combine PLGA with HPCD or lecithin to evaluate their ability to carry DSN and to compare their dissolution profiles with those of pure DSN or commercial Daflon 500 mg for oral administration. In addition, the weight ratios of PLGA/HPCD or PLGA/lecithin, the volume of DMSO, and the DSN input were selected for optimization and were physicochemically characterized by DLS, zeta potential, XRD, DSC, FTIR, and FE-SEM.

Experimental

Materials

Diosmin was synthesized at Lab of Organic Synthesis and Polymer Technology (Institute of Chemical Technology, VAST, 99% of purity);¹⁶ poly lactic-co-glycolic acid (PLGA) with LA : GA 50 : 50, M_w 38–54 kDa, acid terminated (eSUNMed Biotechnology, China); chitosan (CS) M_w 50–190 kDa (Sigma Aldrich); poly(vinyl alcohol) (PVA) 87.0–89.0% hydrolyzed, M_w 31–50 kDa (Thermo Scientific); (2-hydroxypropyl)-β-cyclodextrin from Sigma Aldrich; l-α-lecithin from soybean (Thermo Scientific); dichloromethane (DCM); dimethyl sulfoxide (DMSO) (Fisher, USA); ethyl acetate (>99.5%, GHTECH, China) and double distilled water.

Daflon 500 mg is a micronized purified flavonoid formulation (mean particle size = 1.79 µm, with 80% of particles having a size lower than 3.45 µm) composed predominantly of diosmin (90% w/w), with the remaining fraction consisting of flavonoids expressed as hesperidin (10% w/w).¹⁷ Clinically, it is widely used for the management of chronic venous insufficiency and hemorrhoidal disorders due to its well-established vasoprotective and anti-inflammatory properties.¹⁷ Daflon 500 mg was purchased from a local drugstore (manufactured by Les Laboratoires Servier Industries, France); it is taken as 1 tablet twice daily.

Synthesis of PLGA-coated DSN, PLGA/HPCD-coated DSN, and PLGA/lecithin-coated DSN

The synthesis of PLGA nanoparticles containing DSN was done in our previous article,¹⁸ as described in the oil/water single emulsion solvent evaporation technique¹⁹ (or emulsion-solvent evaporation method²). Concisely, 4.5 mg DSN and 60 mg PLGA were dissolved in 5 mL DMSO and 5 mL ethyl acetate, respectively. Subsequently, they were mixed and stirred to form a transparent organic phase. In the meantime, an aqueous solution of PVA at 5% and another solution of chitosan at 0.5% (containing acetic acid at 1%) were prepared. Next, a 20 mL solution containing 0.5% PVA and 0.15% chitosan was stirred at room temperature until homogeneous. Then, the organic phase was added dropwise into the water phase under constant stirring at 600 rpm for 1 min, followed by sonication at 40% amplitude and 100 000 J for 5 min (55 s on and 5 s off), with further stirring overnight to remove the ethyl acetate. The resulting solution was centrifuged to collect the white pellet, then washed 3 times with double-distilled water and freeze-dried for further characterization. The product was coded as 100P-DSN.

To fabricate PLGA/HPCD nanoparticles containing DSN, the PLGA-to-HPCD weight ratio significantly influenced DSN encapsulation and loading. The HPCD was weighed and added to 20 mL of the water phase, followed by the unchanged oil phase and the oil/water emulsification step. In PLGA/lecithin nanoparticles containing DSN, the PLGA-to-lecithin weight ratio also affects DSN encapsulation. Lecithin was weighed and dissolved in 1 mL DCM. Next, this solution was added to the PLGA solution, followed by the DSN solution, with stirring, to obtain a homogeneous oil phase. The preparation of the water phase and the formation of the oil/water emulsion were unchanged. Herein, the weight ratios were designed as shown in Table 1 below (total input of polymers was 60 mg).

Table 1. Formulation design and composition ratios for PLGA/HPCD and PLGA/lecithin nanocomplexes.

Code	PLGA/HPCD weight ratio (%/%)	PLGA (mg)	HPCD (mg)	Lecithin (mg)
100P-DSN	100	60	0	0
95P5H-DSN	95/5	57	3	0
90P10H-DSN	90/10	54	6	0
80P20H-DSN	80/20	48	12	0
95P5L-DSN	95/5	57	0	3
90P10L-DSN	90/10	54	0	6
80P20L-DSN	80/20	48	0	12

Evaluate the effect of DMSO and DSN on the encapsulation ability of 100P-DSN and 95P5H-DSN

In this experiment, 100P-DSN and 95P5H-DSN were chosen for the optimization of carrying DSN. 1.5–6.0 mg of DSN was dissolved in 0.5–7.5 mL of DMSO. The polymers were weighed for the appropriate weight ratios. Meanwhile, the aqueous phase was unchanged for both the as-described 100P-DSN and 95P5H-DSN. Then, the formation and purification of medicated nanoparticles followed the same steps as above.

Drug encapsulation efficiency and loading

The calibration curve was built using a DSN 100 ppm solution in 0.2 N NaOH. This solution was diluted to prepare standard solutions at 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 ppm. Then, 2 mg of medicated formulations were dissolved in 8 mL of 0.2 N NaOH with the support of sonication for 15 minutes, followed by 0.22 µm membrane filtration before measuring the absorbance at 268 nm by UV-VIS/NIR JASCO V-77 spectrophotometer, Japan.⁵ The samples were collected in triplicate.

The DSN encapsulation efficiency (EE) and loading (LD) were calculated as follows:^15,20

EE (%) = W_loaded/W_initial × 100 1

LD (%) = W_loaded/W_{nanoformulation} × 100 2

where: W_loaded = weight of DSN loaded in nanoparticles W_initial = initial weight of DSN used.

Size and zeta potential

Particle size distribution of the formulated nanoparticles was characterized using dynamic light scattering (Horiba SZ-100). Before centrifugation, an aliquot of the samples was properly diluted (1 : 100) with DD water and sonicated to achieve a homogeneous suspension of nanoparticles.²¹ The size and PDI were analyzed.

Regarding zeta potential, the post-centrifuged samples were diluted with DD water, sonicated, and measured using a zeta potential meter (Horiba SZ-100).

Stability of the nanoformulation

After centrifugation, the 95P5H nanoformulation was redispersed in DD water, and its stability was evaluated at 4 °C, room temperature (RT), and 40 °C within 3 months.²¹ At the determined time points, the size, PDI, zeta potential, and drug loading capacity. The graphs were generated to provide insights into changes in the nanoformulation's physical properties.

On the other hand, 1 mg of lyophilized 95P5H powder was redispersed in 10 mL of DD water with sonication at 40% amplitude and 100 000 J for 5 min (55 s on and 5 s off). The size, PDI, and zeta potential were measured to evaluate the water redispersion of the 95P5H after 3 days.

XRD

The X-ray diffraction (XRD) patterns of the samples were recorded (X-Ray Diffraction D2 Phaser, Bruker, Germany) with measurements performed over the 2θ angular range from 5–45°.

DSC

Thermal behavior of the samples was analyzed using a differential scanning calorimeter (TA Instruments DSC 25, USA). Approximately 5 mg of each sample was hermetically sealed in aluminum pans and heated from 35 °C to 400 °C at a constant rate of 10 °C min⁻¹ under an argon purge of 20 mL min⁻¹.

FTIR

Fourier transform infrared (FTIR) spectra were obtained using a spectrophotometer (Bruker Tensor 27) in the range of 400–4000 cm⁻¹. The samples were homogeneously blended with potassium bromide (KBr), compressed into discs, and subsequently analyzed.

FE-SEM

The morphological and surface features of diosmin, plain 95P5H nanoparticles, and the medicated formulation were examined using field-emission scanning electron microscopy (FE-SEM, S4800 Hitachi). Samples were affixed to metal stubs with double-sided conductive carbon tape, sputter-coated with a thin gold layer, and imaged at an accelerating voltage of 30 kV.

In vitro dissolution and drug release

This experiment was conducted to evaluate the release of DSN, 100P-DSN, 95P5H-DSN, and commercial Daflon 500 mg at pH 2 and 7.8.^10,22

Phosphate buffer pH 2.0 was prepared by dissolving 0.136 g of potassium dihydrogen phosphate in 800 mL of water, adjusting the pH to 2.0 with hydrochloric acid, and adding sufficient water to bring the final volume to 1000 mL. Phosphate buffer pH 7.8 was prepared by placing 50.0 mL of 0.2 M potassium dihydrogen phosphate in a 200 mL volumetric flask, adding the specified volume of 0.2 M sodium hydroxide/hydrochloric acid, and then adding water to volume 200 mL.

The dialysis bag method was applied to determine the in vitro release for DSN.²³ An accurate amount of each sample (equivalent to 1 mg of DSN) was suspended in 3 mL of the corresponding buffer medium and transferred into a pre-treated dialysis membrane bag (MWCO 10 kDa). The sealed dialysis bag was fully immersed in a receptor vessel containing 20 mL of the dissolution medium and placed in an incubator shaker maintained at 37 ± 0.5 °C and 100 rpm. At predetermined time intervals, 3 mL of the release medium was withdrawn and immediately replenished with an equal volume of fresh, pre-warmed buffer to maintain sink conditions. To quantify the released diosmin, a 1 mL aliquot of the withdrawn sample was mixed with 4 mL of a 10 ppm DSN stock solution (standard addition method), then measured by UV-Vis spectrophotometry at 268 nm. All release assays were performed in triplicate (n = 3), and cumulative drug release percentages were calculated accordingly.

The drug release kinetics were modeled by four models, including zero-order, first-order, Higuchi order, and Korsmeyer–Peppas.^5,24 The zero-order model describes drug release as a direct relationship between elapsed time and the cumulative percentage of drug liberated from the dosage form. This kinetic pattern reflects a system in which the drug is released at a constant rate, allowing the drug concentration in the bloodstream to remain steady during administration. In contrast, the first-order model relates time to the logarithm of the percentage of drug still present in the formulation. It is commonly used to characterize release behavior in which the rate depends on the remaining drug concentration. The Higuchi model expresses drug release as a function of the square root of time and the cumulative percentage released. This approach helps determine whether diffusion is the dominant mechanism governing the release process. The Korsmeyer–Peppas model (C_t/C_∞ = ktⁿ) describes drug release as a power-law relationship between the fractional release (C_t/C_∞) and time. Their equations were respectively shown as follows:

C = k₀t 3

log(100 − C) = log 100 − k_ft/2.303 4

5

log C_t − log C_∞ = log k + n log t 6

Herein, C represents the cumulative percentage of drug released at a given time t, while k₀, k_f, k_H, and k correspond to the rate constants for zero-order, first-order, Higuchi, and Korsmeyer–Peppas release models, respectively. While n is the diffusional exponent that characterizes the release mechanism and is obtained from the slope of the log–log plot. The release mechanism was interpreted using the kinetic model that yielded the highest correlation coefficient (R²), indicating the best fit to the nanoparticles' experimental release profile.

In vitro cytotoxicity

To examine the in vitro cytotoxicity of the test samples, RAW264.7 cells were treated under the same experimental conditions as described above. In brief, cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator containing 5% CO₂ for 48 h. Subsequently, 2.5 × 10⁵ cells per mL were seeded into 96-well microplates. Following treatment, MTT solution (0.5 mg mL⁻¹ in phosphate-buffered saline, PBS) was added to each well and incubated for 4 hours at 37 °C in a humidified atmosphere containing 5% CO₂. The formed formazan crystals were dissolved in DMSO, and absorbances were measured at 540/720 nm using a Tecan Spark spectrophotometer.

Anti-inflammation assay

The anti-inflammatory activity was assessed by measuring the inhibition of nitric oxide (NO) production in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS).^25–27

Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator containing 5% CO₂ for 48 h. Subsequently, 2.5 × 10⁵ cells per mL were seeded into 96-well microplates. Following adherence, they were stimulated with lipopolysaccharide (LPS, 1.0 µg mL⁻¹) for 24 h and treated with test samples at concentrations ranging from 16 to 512 µg mL⁻¹, with cardamonin serving as a positive control. Nitric oxide (NO) production was quantified using the Griess assay, in which cell supernatants were incubated with Griess reagent, and sodium nitrite (NaNO₂) standards were employed to generate a calibration curve. Their absorbances were measured at 570 nm.

Statistical analysis

All quantitative experiments, including particle size, polydispersity index (PDI), zeta potential, encapsulation efficiency, drug loading, in vitro drug release, and cell-based assays, were performed in triplicate from three independent preparations. The results are expressed as the mean ± standard deviation (SD). Data visualization was performed using Origin software, and mathematical modeling of in vitro drug-release kinetics was performed in Microsoft Excel.

Results and discussion

PLGA acts as the primary hydrophobic polymer matrix. Its internal cavities specifically encapsulate the lipophilic drug, diosmin.^28,29 HPCD exists within the PLGA core, forming an inclusion complex with the lipophilic diosmin, preventing its recrystallization during solvent evaporation and significantly enhancing the encapsulation efficiency.^30,31 Chitosan coats the outer surface of the PLGA core. Its primary role is to reverse the inherently negative surface charge of PLGA to a highly positive state (as evidenced by our zeta potential results), thereby providing mucoadhesive properties for enhanced intestinal absorption.³² PVA acts as a macromolecular emulsifier in the external aqueous phase during the nanoprecipitation process. It reduces interfacial tension and creates a steric barrier to prevent the nanoparticles from agglomerating during formation and hardening.^33,34 In the emulsification process, an appropriate amount of PLGA was dissolved in an organic solvent together with drugs. In the meantime, the aqueous phase containing anionic polymers (such as PVA and poloxamer) and cationic polymers (chitin and chitosan) was prepared. Afterward, the organic phase was added dropwise into the water phase under high-shear stirring, followed by homogenization to form an oil/water emulsion. The resultant emulsion was stirred overnight to evaporate the organic solvent and harden the oil droplets, resulting in the solid nanocarriers.³⁵

In the synthesis of PLGA-related nanoparticles, chitosan and poly(vinyl alcohol) are two biopolymers that are necessary for the stability and circulation time of the resultant nanocarriers. Poly(vinyl alcohol) was used to lower the surface tension, leading to the non-agglomeration of the formed nanoparticles.³⁶ Chitosan is a cationic, biocompatible, non-toxic, mucoadhesive, and permeable polymer. The coating of the chitosan layer onto the surface of PLGA-related nanoparticles can enhance the residence time of dosage forms, leading to improved bioavailability, lower dose, and minimal side effects.⁵ Additionally, the chitosan layer provides a positive charge that protects the PLGA nanoparticles from size enlargement and enhances the stability of the nanosystems. In addition to the properties of PLGA, such as molecular weight, LA : GA ratio, and acid/ester termination, the concentrations of PVA and chitosan also control both the size and the loading capacity of the nanocarriers. For example, the chitosan concentration should be controlled to below 0.4% to prevent aggregation, as mentioned in previous research.³⁷ Taking PVA into account, the concentration is too high, which can result in bigger PLGA nanoparticles because of a decrease in the net shear stress in the emulsification process.³⁸

Determining the drug-loading efficiency of diosmin is a critical parameter in evaluating the performance of nano-carrier systems, as it directly influences dissolution behavior, bioavailability, and ultimately therapeutic efficacy. Analysis of the formulation parameters (Table 2 and Fig. 1) revealed clear differences in encapsulation efficiency, drug loading, particle size, and size distribution across the PLGA–HPCD (P–H) and PLGA–lecithin (P–L) nanoparticle systems. In the PH series, increasing the PLGA proportion from 80 : 20 to 95 : 5 markedly improved encapsulation efficiency, rising from 42.21 ± 0.32% to 71.47 ± 1.38%, while drug loading also increased to 12.81 ± 0.25%, the highest among all formulations. This composition also produced one of the smallest particle sizes (223.0 ± 15.72 nm), indicating a more compact structure. In contrast, the P–L series exhibited substantially lower encapsulation efficiencies (21.47–36.88%) and larger particle sizes (277.4–352.3 nm), despite lower PDIs. The 100% PLGA formulation demonstrated moderate EE (56.24 ± 5.60%) and LD (10.16 ± 1.00%) with a particle size of 228.4 ± 15.84 nm, confirming that a low proportion of HPCD can enhance drug incorporation. Overall, the 95P5H formulation achieved the most favorable balance of high encapsulation efficiency, high drug loading, and small particle size, making it the most promising candidate for further development despite the polydispersity inherent to the hybrid polymer–cyclodextrin matrix. In this context, HPCD could improve the solubility and stability of diosmin, leading to higher drug entrapment.¹⁴ We hypothesize that HPCD will trap the DSN within its hydrophobic cavity, thereby increasing DSN solubility and retaining it within the nanostructure during self-assembly. Additionally, HPCD captures the hydrophobic segments of PLGA through supramolecular interactions, thereby generating new hydrophilic segments. They are partly or entirely located within the outer hydrophilic nanoparticles, while the other uncoated hydrophobic PLGA segments form an internal lipophilic nanostructure that encapsulates the DSN.¹⁵

Table 2. Physicochemical characteristics (EE%, LD%, particle size, and PDI) of diosmin-loaded nanocomplexes formulated with varying secondary polymer ratios.

Parameter	100P	P–H			P–L
		80 : 20	90 : 10	95 : 5	80 : 20	90 : 10	95 : 5
EE%	56.24 ± 5.60	42.21 ± 0.32	60.22 ± 0.78	71.47 ± 1.38	21.47 ± 1.08	35.97 ± 1.84	36.88 ± 2.26
LD%	10.16 ± 1.00	11.87 ± 0.09	11.78 ± 0.15	12.81 ± 0.25	10.17 ± 0.51	10.79 ± 0.55	13.36 ± 0.50
Size (nm)	228.4 ± 15.84	294.3 ± 59.64	225.0 ± 8.2	223.0 ± 15.72	277.4 ± 7.64	284.4 ± 7.42	352.3 ± 24.93
PDI	0.309 ± 0.098	0.234 ± 0.047	0.604 ± 0.016	0.329 ± 0.030	0.458 ± 0.046	0.297 ± 0.050	0.321 ± 0.066

Fig. 1. Effect of secondary polymer incorporation (HPCD and lecithin) and polymer weight ratios on the encapsulation efficiency (EE%) and loading capacity (LD%) of diosmin-loaded PLGA nanoparticles.

The relatively poor performance of the PLGA–lecithin formulations compared to the PLGA–HPCD nanoparticles primarily stems from the drug (diosmin)'s poor water solubility (non-hydrophilic nature) and the differing stabilization mechanisms of lecithin and HPCD. In the PLGA–lecithin system, lecithin molecules self-assemble at the interface between the oil phase (PLGA) and the aqueous phase. Their hydrophobic tails orient towards the PLGA core, while the hydrophilic heads face the outer aqueous environment. This formed lipid layer acts as a semi-permeable physical barrier, which is highly effective in preventing the reverse diffusion (leakage) of hydrophilic drugs from the core into the continuous phase during the particle hardening process.³⁹ However, because diosmin is a poorly water-soluble, non-hydrophilic compound, lecithin's specific barrier mechanism does not provide a significant advantage in retaining the drug or improving its encapsulation.

Mechanistically, the ability of HPCD to form this inclusion complex significantly enhances the apparent solubility of the lipophilic drug and maintains it in a molecularly dispersed state.⁴⁰ More importantly, the presence of HPCD creates a steric barrier that effectively prevents drug molecules from aggregating and recrystallizing on the surface of the nanoparticles during the solvent evaporation phase, thereby improving the physical stability of the particulate system.³⁰ Given the very low aqueous solubility of diosmin, a BCS class IV flavonoid glycoside (log P ≈ 1.36, PubChem CID 5281613), the uncomplexed drug has a strong tendency to precipitate. Therefore, the incorporation of HPCD is essential not only to mitigate this recrystallization but also to enhance the final encapsulation efficiency (EE%) within the polymer matrix.

Next, the 100P nanoparticles and 95P5H samples were investigated to optimize drug entrapment with respect to DMSO volume and diosmin input.

Variation in the amount of DMSO used during nanoparticle preparation produced a distinct influence on both encapsulation efficiency and drug loading in the PH and 100P systems. In Table 3, Fig. 2A and B, at low DMSO volumes (0.5–2.5 mL), EE values remained modest for both formulations, ranging from 40.88–41.71% for P–H and 33.01–43.76% for 100P, with LD values showing minimal fluctuation. Increasing the DMSO volume to 5 mL resulted in a pronounced enhancement in EE, reaching 71.47 ± 1.38% for P–H and 56.24 ± 5.60% for 100P, indicating that this solvent level promotes more efficient solubilization of diosmin and facilitates its incorporation into the polymer matrix. LD values at 5 mL DMSO also remained stable, suggesting that the improvement in EE did not compromise the overall drug-to-polymer ratio. However, further increasing DMSO to 7.5 mL resulted in a sharp decline in EE across both systems. In practice, DMSO increases the encapsulation rate by improving diosmin dissolution, thereby enhancing diffusion of the organic phase into the water phase and strengthening drug-polymer interactions. Meanwhile, excess DMSO can compromise the stability of the emulsion system, leading to reduced drug encapsulation. Collectively, these results demonstrate that 5 mL of DMSO is the optimal solvent volume, yielding the highest encapsulation efficiency for both P–H and 100P formulations while maintaining acceptable drug loading.

Table 3. Effect of organic solvent (DMSO) volume on encapsulation efficiency (EE%) and drug loading (LD%) of 95P5H and 100P formulations.

DMSO (mL)	EE% P–H	LD% P–H	EE% 100P	LD% 100P
0.5	40.88 ± 6.07	12.11 ± 1.80	43.76 ± 4.94	14.27 ± 1.60
2.5	41.71 ± 2.27	12.33 ± 0.67	33.01 ± 3.60	10.04 ± 1.10
5	71.47 ± 1.38	12.81 ± 0.25	56.24 ± 5.60	10.16 ± 1.00
7.5	26.91 ± 1.39	14.68 ± 0.76	41.13 ± 1.91	9.90 ± 0.50

Fig. 2. Optimization of formulation parameters: (A) EE% and (B) LD% as a function of DMSO volume; (C) EE% and (D) LD% as a function of initial diosmin input for 95P5H-DSN and 100P-DSN.

Increasing the initial diosmin amount had a pronounced effect on both encapsulation efficiency and drug loading in the P–H and 100P nanoparticle systems. In Table 4, Fig. 2C and D, at low drug inputs (1.5–3.0 mg), EE values remained modest for both formulations, ranging from 17.86–27.42% in the P–H system and 30.60–33.26% in the 100P system, with correspondingly low LD values. A substantial improvement was observed at 4.5 mg, where EE increased sharply to 71.47 ± 1.38% for P–H and 56.24 ± 5.60% for 100P, accompanied by the highest LD values (12.81 ± 0.25% and 10.16 ± 1.00%, respectively). This indicates that 4.5 mg is the optimal drug input, maximizing diosmin solubilization and incorporation into the polymer matrix. Further increasing the drug amount to 6.0 mg resulted in a marked decline in EE for both systems, suggesting the limit of drug miscibility in the polymer matrix had been reached.⁵ Overall, these results demonstrate that 4.5 mg diosmin provides the most favorable balance of high encapsulation efficiency and drug loading for both P–H and 100P formulations.

Table 4. Influence of initial diosmin input on encapsulation efficiency (EE%) and drug loading (LD%) of 95P5H and 100P formulations.

DSN (mg)	EE% P–H	LD% P–H	EE% P100	LD% P100
1.5	17.86 ± 0.64	5.15 ± 0.19	33.26 ± 7.77	3.28 ± 0.77
3.0	27.42 ± 6.72	7.22 ± 1.77	30.60 ± 2.20	6.24 ± 0.45
4.5	71.47 ± 1.38	12.81 ± 0.25	56.24 ± 5.60	10.16 ± 1.00
6.0	33.70 ± 5.23	7.55 ± 1.17	24.55 ± 4.16	7.79 ± 1.32

Additionally, accurate measurement of particle size and zeta potential is essential in nanocarrier characterization, as these parameters dictate colloidal stability and biodistribution. Herein, Table 5 shows that DSN has micrometer-sized particles with polydispersity and low stability, whereas 100P-DSN has nanoscale dimensions, is moderately monodisperse, and is quite stable, with a zeta potential of 19.1 mV. Meanwhile, 95P5H-DSN had the smallest diameter of 223 ± 15.72 nm with a PDI of 0.329 and high stability, with a zeta potential of 70.00 ± 7.35 mV, owing to the positive chitosan coating through the interaction between the cationic polymer and carboxylic end groups of PLGA.⁴¹

Table 5. Hydrodynamic diameter, polydispersity index (PDI), and zeta potential of free diosmin, 100P-DSN, and the optimized 95P5H-DSN nanoparticles.

Sample	Size (nm)	PDI	Zeta (mV)
DSN	3690 ± 191.7	0.718	−3.35 ± 0.21
100P-DSN	228 ± 15.83	0.309	19.1 ± 7.35
95P5H-DSN	223 ± 15.72	0.329	70.00 ± 7.35

The physicochemical characterization of the formulations demonstrated substantial improvements in nanoparticle properties compared with raw diosmin. Pure DSN exhibited a very large particle size (3690 ± 191.7 nm), high polydispersity (PDI = 0.718), and a slightly negative zeta potential (−3.35 ± 0.21 mV). Incorporation into PLGA nanoparticles (100P-DSN) markedly reduced the particle size to 228 ± 15.83 nm and improved size uniformity (PDI = 0.309), while shifting the zeta potential to a positive value (19.1 ± 7.35 mV), indicating enhanced colloidal stability. Further modification with the PLGA–HPCD hybrid matrix (95P5H-DSN) produced similarly small particles (223 ± 15.72 nm) with a comparable PDI (0.329), but with a substantially higher positive surface charge (70.00 ± 7.35 mV). This pronounced increase in zeta potential suggests stronger electrostatic stabilization and improved dispersion behavior, which are advantageous for biological interactions and nanoparticle stability. Compared with previously reported diosmin nano-systems, the PLGA–HPCD nanoparticles developed in this study demonstrate a favorable balance of physicochemical properties (Table 6). Their particle size (223 nm) is in the range of previously reported polymeric carriers and is accompanied by a strong positive zeta potential (+70.00 mV), which supports good colloidal stability and mucoadhesive potential. Encapsulation efficiency (71.47%) and drug loading (12.81%) are comparable to those of chitosan-coated PLGA (67.4%) and solid lipid nanoparticles (73.46%), although lower than those of poloxamer-based nanocrystals, which prioritize payload over structural stability. While the PDI of the current system is moderately higher than surfactant-rich nanoemulsions, it remains acceptable for hybrid polymer–cyclodextrin matrices. Overall, the current formulation offers a well-balanced combination of stability, encapsulation capacity, and favorable surface characteristics, positioning it as a strong candidate for enhanced oral delivery of diosmin.

Table 6. Comparative physicochemical properties of 95P5H-DSN with previously reported diosmin-loaded nanosystems.

No.	Composition	Size and PDI	Zeta potential	EE and LD	Ref.
1	Chitosan–poloxamer 188 nanocrystals	Nanorod	+40.43 ± 0.15 mV	EE%	21
		368.93 ± 0.47 nm		93.29 ± 0.58
		PDI = 0.23 ± 0.01
2	Compritol ATO 888 (140–450 mg) as solid lipid and soy lecithin (25–85 mg) as co-surfactant	37.48 nm	None	EE% 73.46%	42
		PDI = 0.29		LD% 9.075%
3	Lauroglycol 90 (LG90®) as oil, Tween-80 as surfactant, and transcutol-HP (THP) as co-surfactant	Nanospheres	−23.4 mV	EE%	43
		41 ± 0.07 nm		87 ± 0.81%
		PDI = 0.073 ± 0.008
4	Chitosan-coated PLGA	Nanospheres	+27.40 ± 2.90 mV	EE%	5
		337.6 ± 41 nm		67.4 ± 2.9%
		PDI 0.3 ± 0.09
5	Phytosome used:	316 ± 2.45 nm	−27.1 ± 0.12 mV	LD%	44
	Lipoid® S100, Lipoid® S75, and Lipoid® S PC-3	PDI 0.50 ± 0.01		28.40 ± 0.01%
6	PLGA–HPCD	Amorphous nanoparticles	70.00 ± 7.35 mV	EE%	Current study
		223 ± 15.72 nm		71.47 ± 1.38%
		PDI 0.329		LD%
				12.81 ± 0.25%

The stability of formulation 95P5H was investigated to inform future use. The variations in size, PDI, zeta potential, and LD% were recorded at 3 conditions, including 4 °C, room temperature (RT), and 40 °C within three months, and are exhibited in the following tables and figures.

In Table 7 and Fig. 3A–C, at 4 °C, the formulation remained relatively stable, with particle size increasing moderately from 223 ± 15.7 nm to 258.9 ± 46.1 nm after 3 months, while PDI values fluctuated without indicating severe aggregation. The zeta potential decreased from 70.00 ± 7.35 mV to 55.75 mV, yet remained sufficiently positive to maintain colloidal stability. Storage at room temperature induced more pronounced changes, with particle size expanding to 305.1 ± 48.3 nm and zeta potential declining to 35.95 mV by month 3, suggesting gradual destabilization and partial aggregation. The most significant instability occurred at 40 °C, where particle size increased sharply to 335.57 ± 108.5 nm, and the zeta potential dropped from strongly positive values to near-neutral (−0.65 mV), indicating substantial loss of electrostatic stabilization and a higher tendency for particle agglomeration due to the system's high kinetic energy.⁴²

Table 7. Colloidal stability metrics (hydrodynamic size, PDI, and zeta potential) of 95P5H-DSN stored across different temperatures (4 °C, RT, and 40 °C) over 3 months.

No.	Month	Size (nm)	PDI	Zeta (mV)
4 °C	0	223 ± 15.7	0.329 ± 0.03	70.00 ± 7.35
	1.5	262.0 ± 36.0	0.761 ± 0.10	59.85 ± 2.76
	3	258.9 ± 46.1	0.242 ± 0.13	55.75 ± 2.33
RT	0	223 ± 15.7	0.329 ± 0.03	70.00 ± 7.35
	1.5	251.4 ± 57.3	0.253 ± 0.08	47.9 ± 8.49
	3	305.1 ± 48.3	0.425 ± 0.07	35.95 ± 1.34
40 °C	0	223 ± 15.7	0.329 ± 0.03	70.00 ± 7.35
	1.5	289 ± 50.3	0.552 ± 0.17	26.70 ± 4.67
	3	335.57 ± 108.5	0.497 ± 0.17	−0.65 ± 0.35

Fig. 3. Long-term physicochemical stability profiles of 95P5H-DSN nanoparticles stored at 4 °C, room temperature (RT), and 40 °C over 3 months: (A) hydrodynamic diameter, (B) polydispersity index, (C) zeta potential, and (D) drug loading capacity.

The drug loading of 95P5H-DSN nanoparticles (Table 8 and Fig. 3D) showed gradual changes during storage, with the extent of variation strongly dependent on temperature. At 4 °C, LD decreased slightly from 12.81 ± 0.25% to 11.61 ± 2.06% at 1.5 months and 10.97 ± 3.13% after 3 months, indicating minimal drug loss and good retention of formulation. Storage at room temperature showed a similar trend, with LD values declining to 9.91 ± 1.42% at 1.5 months and stabilizing at 10.73 ± 1.30% by month 3, indicating moderate but acceptable stability. In contrast, storage at 40 °C resulted in a more pronounced reduction in LD, dropping to 8.67 ± 1.06% at 1.5 months and 8.56 ± 0.56% at 3 months, consistent with accelerated drug diffusion or matrix relaxation at elevated temperatures.

Table 8. Drug loading retention (LD%) of 95P5H-DSN during 3 months of storage at 4 °C, RT, and 40 °C.

Month	4 °C	RT	40 °C
0	12.81 ± 0.25	12.81 ± 0.25	12.81 ± 0.25
1.5	11.61 ± 2.06	9.91 ± 1.42	8.67 ± 1.06
3	10.97 ± 3.13	10.73 ± 1.3	8.56 ± 0.56

The results in Table 9 show that lyophilized 95P5H becomes unstable after three days. Particle size increases from 223 ± 15.7 nm to 572.03 ± 55.94 nm, indicating strong aggregation. The rise in PDI from 0.329 ± 0.03 to 0.455 ± 0.15 confirms a more heterogeneous particle population. Most notably, the zeta potential drops sharply from +70 ± 7.35 mV to +21.4 ± 1.61 mV, greatly reducing electrostatic repulsion and allowing particles to cluster due to inadequate protection during lyophilization or storage.

Table 9. Reconstitution stability (hydrodynamic diameter, PDI, and zeta potential) of lyophilized 95P5H-DSN after 3 days in aqueous media.

Time	Size (nm)	PDI	Zeta potential (mV)
0	223 ± 15.72	0.329 ± 0.03	70.00 ± 7.35
3 days	572.03 ± 55.94	0.455 ± 0.15	21.4 ± 1.61

X-ray diffraction (XRD) was used to examine how the interaction between DSN and the carrier affects the physical state of DSN in the 95P5H formulation. As shown in Fig. 4A, the diffraction spectra are compared across free diosmin, plain 95P5H nanoparticles, the physical mixture of DSN with plain 95P5H, and the encapsulated 95P5H-DSN complex. Regarding pure DSN, it had strong peaks at 2θ from 12.5 to 27.5°, especially a sharp peak at 15.63° (Fig. S1), which reflects the crystalline structure of DSN.¹² The plain nanocarrier 95P5H, it has an amorphous structure with a broad peak at 2θ = 20.2° (Fig. S2). The physical mixture of DSN and plain 95P5H nanoparticles still exhibited the corresponding DSN peaks, indicating that there was no incorporation of diosmin into the inner structure of the plain 95P5H nanocarriers (Fig. S3). Nevertheless, the characteristic peaks of DSN were absent in the optimized 95P5H-DSN sample, indicating that DSN was present within the nanocarriers and resulting in an amorphous state (Fig. S4).

Fig. 4. Solid-state characterization of raw DSN, plain 95P5H, physical mixture, and 95P5H-DSN nanoparticles by (A) X-ray diffraction (XRD) patterns, (B) differential scanning calorimetry (DSC) thermograms, and (C) FTIR spectra.

Secondly, DSC measurements were conducted to assess changes in thermal behavior. As shown in Fig. 4B, the curves correspond to DSN alone, plain 95P5H nanoparticles, their physical mixture, and DSN-loaded 95P5H nanoparticles (Fig. S5). Diosmin expressed a broad endothermic peak at 138 °C corresponding to dehydration and a sharp endothermic melting peak at 286 °C (Fig. S6).¹² The specific peaks of DSN also appeared in the thermogram of the physical mixture (Fig. S7) and disappeared in the medication form (Fig. S8), indicating that the diosmin crystallites were embedded within the nanoparticles, forming an amorphous state. These data are consistent with the XRD measurements.

In addition, FTIR measurements were conducted to investigate potential chemical interactions and confirm encapsulation. Herein, from the illustration in Fig. 4C, the FTIR characteristic peaks of diosmin were specified at 1661 and 1610 cm⁻¹ owing to the aromatic ketonic carbonyl stretching C O vibration. Meanwhile, the peak at 1502 cm⁻¹ corresponded to the C C bond in the aromatic ring (Fig. S9).^5,12 Plain 95P5H nanoparticles express the peak at 2952 cm⁻¹ due to the linear CH₂ stretching and at 1759 cm⁻¹ due to the ester bond (Fig. S10).⁵ The physical mixture between DSN and 95P5H still displayed the distinctive peaks of DSN (Fig. S11), which was proof of physical interaction, whilst the medicated 95P5H didn't have any corresponding signals of the drug (Fig. S12); thereby, the nanoparticles were formed to encapsulate the DSN, which left the IR signals of the medicated formulation lower than those of the plain nanoparticles.

Field emission scanning electron microscopy (FE-SEM) plays a crucial role in elucidating the surface morphology and structural integrity of nanocarriers. By providing high-resolution images, FE-SEM enables direct visualization of particle shape, size uniformity, and surface characteristics, which are essential parameters for assessing formulation stability and drug encapsulation efficiency. In Fig. 5, DSN introduces a rod-shaped structure with microscale width and length. It has a crystallite structure, as confirmed by the XRD diffractogram (Fig. S13). Meanwhile, the plain 95P5H nanoparticles exhibited smooth, spherical morphologies with diameters of approximately 100–150 nm (Fig. S14). However, we observed some adherence of chitosan-coated nanoparticles due to residual surfactant and chitosan on their surfaces.³⁸ Furthermore, lyophilization also induces particle aggregation.²¹ To prevent the aggregation of the formed nanoparticles, the use of cryoprotectants such as trehalose, sucrose, lactose, and glucose, etc. can be considered.⁴⁵ Finally, the medicated dosage of 95P5H-DSN consisted of uniform amorphous nanoparticles (Fig. S15), as evidenced by XRD measurements. Studies have noted that dynamic light scattering reports the hydrodynamic size, which reflects not only the nanoparticle core but also the surrounding polymeric components and the associated hydration shell. As a result, the measured diameters appear larger than the actual dimensions of the solid particles.²¹

Fig. 5. Field-emission scanning electron microscopy (FE-SEM) micrographs showing the surface morphology and shape of free diosmin, plain 95P5H nanocarriers, and drug-loaded 95P5H-DSN nanoparticles.

The in vitro release profiles of free diosmin (DSN), the commercial formulation (Daflon), PLGA nanoparticles (100P), and PLGA–HPCD nanoparticles (95P5H) were evaluated at pH 2.0 (gastric condition) and pH 7.8 (intestinal condition) over 24 h (Fig. 6). In gastric conditions (pH 2.0), all formulations exhibited restricted drug release due to the low intrinsic aqueous solubility of diosmin under acidic conditions. However, the polymeric nanocarriers (100P and 95P5H) exhibited reduced cumulative release (∼6.94% and 5.38% at 24 h) compared with free DSN (∼10.39%) and Daflon (∼7.46%). The hydrophobic PLGA matrix effectively encapsulates the flavonoid core, serving as a physical diffusion barrier that prevents premature leakage and acid-induced degradation in the stomach. Conversely, in simulated intestinal fluid (pH 7.8), a pronounced enhancement in release rate and extent was observed at 100P and 95P5H, driven by the weak ionization and improved solubility of diosmin at slightly alkaline pH. Notably, the nanoformulations containing hydroxypropyl-β-cyclodextrin (95P5H) demonstrated the highest cumulative release (∼17.73% at 24 h), compared with 100P (∼15.02%), commercial Daflon (∼12.15%), and free DSN (∼10.32%). The superior release was due to host–guest complexation facilitated by HPCD, which preserves the lipophilic agent in an amorphous dispersion and promotes faster diffusion into the surrounding medium.⁴⁰ These findings highlight the potential of the HPCD-tailored PLGA nanocarrier as a robust oral delivery system that protects vulnerable lipophilic drugs during gastric transit while enhancing their release in the intestinal tract.

Fig. 6. In vitro cumulative drug release profiles of free DSN, commercial Daflon 500 mg, 100P, and 95P5H nanoparticles over 24 h in gastric fluid (pH 2.0) and intestinal fluid (pH 7.8).

At pH 7.8, the 95P5H release profile (Table 10) fits the Higuchi model (R² = 0.9190), indicating diffusion-driven release.²¹ However, the Korsmeyer–Peppas exponent (n = 0.9725) reveals Super Case-II transport. This can demonstrate a synergistic mechanism where sustained diosmin release is governed by both Fickian diffusion and the relaxation, swelling, and erosion of the polymer matrix.

Table 10. In vitro release kinetic parameters and mathematical model fitting for 95P5H nanoparticles under gastric (pH 2.0) and intestinal (pH 7.8) conditions.

Model	Constant	95P5H
		pH 2	pH 7.8
Zero order	k 0	0.2239	0.7298
	R 2	0.7952	0.8076
First order	k f	0.0023	0.0080
	R 2	0.8003	0.8256
Higuchi	k H	1.2757	4.1524
	R 2	0.9073	0.919
Korsmeyer–Peppas	n	0.0614	0.9725
	k	1.06	0.074
	R 2	0.8826	0.9007

DSN is recognized for its therapeutic effects in many applications, including antioxidant, vascular health, anti-diabetes, antimicrobial, and protective effects on the liver, cardiovascular, and retinal systems.⁴⁶ Also, DSN exhibited potential anti-inflammatory effects by suppressing cytokines, including IL-6, IL-12, and TNF-α, as discussed in a previously reported article using LPS-stimulated RAW264.7 macrophages via NO inhibition.⁴⁷ Accordingly, the ability of both DSN and 95P5H-DSN to inhibit NO production was assessed by the same model, as depicted in Table 11. As a negative control, DMSO did not induce cytotoxicity in RAW264.7 cells. Conversely, cardamonin was utilized as a positive control; at concentrations of 0.3 µM and 3.0 µM, it reduced cell viability to 86.77% and 80.84%, while inhibiting NO production by 46.95% and 87.98%, respectively. Plain DSN significantly reduced cell viability.⁴⁷ In our dataset, free diosmin (DSN) exhibited cytotoxicity at elevated concentrations, with viability decreasing to 10.54 ± 0.26% at 256 µg mL⁻¹ and 50.62 ± 0.89% at 128 µg mL⁻¹ relative to the LPS control. At these levels, NO inhibition could not be reliably assessed because the marked reduction in viable RAW264.7 cells would inherently suppress NO production independent of anti-inflammatory activity. Only at concentrations where DSN maintained viability-72.02 ± 0.69% at 64 µg mL⁻¹ and 81.25 ± 0.87% at 32 µg mL⁻¹-was NO inhibition interpretable, with measurable suppression observed only at 32 µg mL⁻¹ (19.54 ± 1.23%). Blank 95P5H nanoparticles also exhibited dose-dependent cytotoxicity, with viability ranging from 25.57 ± 1.58% at 2000 µg mL⁻¹ to 80.69 ± 1.69% at 250 µg mL⁻¹. NO inhibition was detectable only at the lowest concentration (20.32 ± 0.98%), corresponding to the range where cell viability remained sufficiently high to support meaningful interpretation. In contrast, the diosmin-loaded nanoparticles demonstrated improved biocompatibility, most evident at 256 µg mL⁻¹, and viability (>80%) at ≤64 µg mL⁻¹. Viability remained above 80% at 64 µg mL⁻¹ (80.67 ± 1.09%), 32 µg mL⁻¹ (85.64 ± 0.65%), and 16 µg mL⁻¹ (90.32 ± 1.54%), enabling reliable assessment of NO inhibition. At 64 µg mL⁻¹, 95P5H-DSN produced a robust NO suppression (55.56 ± 0.58%), exceeding the inhibition observed with free DSN at comparable viability levels. Lower concentrations also showed measurable anti-inflammatory activity (22.23 ± 1.32%, at 32 µg mL⁻¹; 11.14 ± 0.98%, at 16 µg mL⁻¹). The IC₅₀ was reached at 58.95 µg mL⁻¹. These results demonstrate that encapsulation not only mitigates diosmin-induced cytotoxicity but also enhances its anti-inflammatory efficacy, providing a clear, concentration-dependent comparison between free DSN and 95P5H-DSN.

Table 11. Cytocompatibility and anti-inflammatory activity (NO inhibition) in LPS-stimulated RAW264.7 macrophages following treatment with free diosmin and 95P5H-DSN.

Sample	Concentration	Cell viability (%)	NO inhibition (%)
DMSO	1%	103.98 ± 0.84	—
Cardamonin	0.3 µM	86.77 ± 0.54	46.95 ± 1.23
	3.0 µM	80.84 ± 0.63	87.98 ± 0.94
LPS	1.0 µg mL−1	100.0 ± 0.13	0.0 ± 0.9
DSN	256 µg mL−1	10.54 ± 0.26	—
	128 µg mL−1	50.62 ± 0.89	—
	64 µg mL−1	72.02 ± 0.69	—
	32 µg mL−1	81.25 ± 0.87	19.54 ± 1.23
Blank 95P5H nanoparticles	2000 µg mL−1	25.57 ± 1.58	—
	1000 µg mL−1	45.25 ± 0.78	—
	500 µg mL−1	70.52 ± 1.78	—
	250 µg mL−1	80.69 ± 1.69	20.32 ± 0.98
Nano diosmin (95P5H-DSN)	256 µg mL−1	32.56 ± 1.05	—
	128 µg mL−1	52.89 ± 0.84	—
	64 µg mL−1	80.67 ± 1.09	55.56 ± 0.58
	32 µg mL−1	85.64 ± 0.65	22.23 ± 1.32
	16 µg mL−1	90.32 ± 1.54	11.14 ± 0.98

Emerging advances in stimuli-responsive nanocarriers and multifunctional therapeutic platforms provide valuable guidance for the future development of diosmin delivery systems. Technologies such as pH-triggered PLGA–MSN carriers⁴⁸ and NIR-activated CO-releasing hydrogels⁴⁹ demonstrate how smart materials can be engineered to respond to specific pathological microenvironments, enhance drug stability, and improve therapeutic precision. Building on these concepts, future diosmin formulations could incorporate microenvironment-responsive release mechanisms, mucoadhesive or targeting ligands, or hybrid polymer-inorganic matrices to further improve oral absorption and biological performance. Additionally, integrating multi-agent strategies, such as combining diosmin with anti-inflammatory or antimicrobial components, may broaden its therapeutic applications beyond vascular disorders. Our ongoing project is a dual combination of diosmin and hesperidin with promising prospects. These innovations highlight a clear pathway for evolving the current PLGA–HPCD system into a more adaptive and multifunctional platform capable of addressing complex gastrointestinal and systemic barriers with greater efficacy.

Conclusions

In this study, a hybrid PLGA–HPCD nanoparticle delivery system coated with chitosan was systematically formulated and evaluated to enhance the dissolution and bioactivity of diosmin. The optimized formulation (95P5H-DSN) demonstrated promising physicochemical attributes, including a nanoscale hydrodynamic diameter (223 ± 15.72 nm), a robust positive surface charge (+70.00 ± 7.35 mV), and a high encapsulation efficiency (71.47 ± 1.38%). Solid-state characterization (XRD, DSC, and FTIR) confirmed the successful encapsulation and transition of crystalline diosmin into an amorphous dispersion within the host–guest hybrid matrix. In dissolution assays, the 95P5H-DSN nanoparticles reduced premature release under gastric conditions (pH 2.0) and provided a slow, diffusion-controlled release in intestinal fluid (pH 7.8), resulting in a higher cumulative release compared to both unformulated DSN and commercial Daflon 500 mg. Biologically, the nanoformulation improved cell viability in RAW264.7 macrophages compared to the free drug, while exerting concentration-dependent anti-inflammatory efficacy through nitric oxide suppression. Collectively, the engineered PLGA–HPCD nanoplatform offers a promising strategy for improving the oral delivery, dissolution behavior, and therapeutic potential of poorly soluble flavonoids.

Author contributions

Anh-Khoa Ton and Truc Thi-Anh Phan: conceptualization, investigation, data curation, formal analysis, writing – original draft, writing – review and editing. Mi Thi-Tra Tran: investigation, data curation, formal analysis Thi-Kim-Chi Huynh: validation, supervision. Hoang-Phuc Nguyen: investigation. Thi-Cam-Thu Nguyen: writing – original draft. Thi-Kim-Dung Hoang: conceptualization, funding acquisition, writing – review and editing, project administration.

Conflicts of interest

There are no conflicts to declare.

Data availability

All required data supporting the findings are available in the manuscript. The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra06567h.