In silico, in vitro, and in vivo assessment of chitosan-diltiazem nanoparticles against pulmonary fibrosis

ABSTRACT

Aims

Diltiazem (DIL), a calcium channel blocker, has demonstrated potential ininhibiting fibrosis-related processes, including TGF-β activation, collagen production, and epithelial-mesenchymal transition, making it a promising candidate for idiopathic pulmonary fibrosis (IPF). This study evaluates the anti-fibrotic efficacy of DIL-loaded chitosan (DIL-CHT) and trimethyl chitosan (DIL-TMC) nanoparticles through molecular and experimental approaches.

Methods

DIL-CHT and DIL-TMC nanoformulations were developed and analyzed particle size, ζ-potential, entrapment efficiency, and in vitro release. Antifibrotic efficacy in bleomycin (BLM)-induced IPF rat model, was tested at subtherapeutic doses (3 mg/kg/day, i.t.) and DIL alone (10 mg/kg/day, p.o.). DFT (B3LYP/6–31 G**) optimization and molecular docking were conducted to assess electronic properties and interactions among CHT, TMC, and DIL.

Results

DIL-TMC and DIL-CHT nanoparticles were 175.6 nm and 267.8 nm, with entrapment efficiencies of 81.72% and 66.0%, respectively; TMC showed a superior 24-hour sustained release. TMC’s larger HOMO-LUMO gap (ΔE = -0.260 eV vs. −0.253 eV for CHT) suggests greater stability, supporting its enhanced interaction with DIL. TMC nanoparticles significantly reduced BLM-induced IPF symptoms, i.e. BLM induced increased lung index, hydroxyproline accumulation, oxidative stress in lung tissue, and blood pressure.

Conclusions

These findings indicate the strong therapeutic potential of DIL-TMC for IPF with minimal cardiovascular side effects.

1. Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease marked by fibrosis, inflammation, and destruction of lung structure. Damage to alveolar epithelium and abnormal wound repair are key factors in its development [1,2]. IPF can be caused by a multitude of factors, including genetic predispositions, environmental exposures, pharmacological agents, and toxic substances. Histopathological features of IPF include excessive ECM deposition, distorting lung architecture, and irreversible lung function loss [3]. IPF prevalence was determined to be 13.7% based on data from a recent interstitial lung disease (ILD) India registry report, which included 1084 patients [4]. Currently, there are only two drugs available for the treatment of IPF viz., nintedanib and pirfenidone (PFD) but both cause adverse effects and do not as accurately treat disease progression [5].

Drug repurposing, the strategy of finding new uses for existing drugs, offers a faster development timeline, lower costs, higher efficiency, and a reduced failure rate compared to traditional drug discovery [6]. Diltiazem (DIL) is a US FDA-approved drug for hypertension and arrhythmia in 1982 [7]. In the search for a suitable agent for fibrosis treatment, previous literature has illustrated that DIL can be used for evaluation against IPF. Previous studies indicate that diltiazem blocks TGF-β in various fibrotic disorders and, inhibits collagen production and collagen I and II mRNA expression in human PMC cell lines [8]. Another study on pulmonary metastasis demonstrated that DIL inhibited cell epithelial-mesenchymal transition at doses of 1–3 mg/kg, supporting the choice of 1 mg and 3 mg/kg doses in the current research based on these findings [9]. A liver fibrosis study showed DIL could regenerate liver cells at 5 and 15 mg/kg intraperitoneally, guiding the selection of a 10 mg/kg oral dose for lung fibrosis research [10]. Based on the previous studies, it is highlighted that the anti-fibrotic mechanism of DIL could be through the inhibition of matrix metalloproteinases (MMP) enzymes [11], IL-12 [12], and the prevention of oxidative stress [13]. So, these findings kindled us to screen the effectiveness of DIL on IPF. On the other hand, DIL is reported to cause cardiac adverse reactions (bradycardia, AV block, and hypotension) and other side effects like swelling of the ankles, feet, or hands, sudden weight gain, unusual weakness, or fatigue, feeling faint or lightheaded, and unusual weakness or fatigue [14,15]. To overcome these adverse reactions and enhance DIL’s site-specific action, formulated its nanoparticles using long-term retentive, biodegradable, and mucoadhesive polymer chitosan and its water-soluble trimethyl chitosan (TMC).

Chitosan (CHT) is a natural polymer insoluble in water and requires glacial acetic acid for proper solubility, which can cause membrane irritation. To overcome this issue, synthesized a derivative of CHT called TMC, which is freely soluble in water and exhibits greater mucoadhesive properties than CHT [16]. The biodegradability, biocompatibility, mucoadhesiveness, and non-toxicity of TMC make it an ideal biopolymer for various pharmaceutical and biomedical applications, including pulmonary delivery [17].

Nanoparticle formulations for IPF reduce systemic toxicity and dosage frequency, providing efficient, long-acting localized drug delivery [18]. Numerous studies have shown that the local application of medication nanoparticles has a positive impact on pulmonary fibrosis. The size of the nanoparticles has an impact on direct pulmonary delivery. Drug-containing dry powders (DPs) are increasingly administered by respiratory means for local and systemic therapy. To administer medication via the pulmonary route, intratracheal or intranasal administration of medicines is required. Dry powders administered intratracheally tend to localize the drug’s activity and prevent the drug from being metabolized [19,20].

This study hypothesis was based on the DIL effect on IPF via suppression of TGF-β, MMP-1, epithelial-to-mesenchymal transition, and collagen-I expression. Consequently, the current study aimed to investigate the role and effect of DIL against Bleomycin (BLM)-induced IPF in a rat model additionally another objective was to optimize the intratracheal formulation of DIL for effective treatment in IPF with its effective dose and compare the effect of Intratracheal and oral DIL administration in IPF in the rat model. Additionally, this study aimed to compare the effect of both nanoformulations CHTNP’s and TMCNP’s.

2. Methods

2.1. Preformulation study of DIL

Solution of DIL ranging from 1 to 10 ppm in methanol was prepared and analyzed by UV spectrophotometry at 240 nm. A linearity curve was constructed by plotting optical density versus concentration over the DIL range.

2.2. Synthesis of TMC

The hydrophilic derivative of CHT i.e. TMC was synthesized according to the previously reported protocol [21] with minor amendments. Reductive methylation of CHT was carried out using methyl iodide (CH₃I) in presence of a base (NaOH) at 60°C to synthesize the TMC. Initially, CHT (2.0 g) and NaI (4.8 g) were dissolved in NMP (80 mL), then NaOH (11 mL, 15%) and CH₃I (11.5 mL) were added. The product was precipitated with ethanol (200 mL), washed, and dried. This mixture was re-dissolved in NMP (80 mL) and heated to 60°C with additional NaI (4.8 g), NaOH (11 mL, 15%), and CH₃I (7 mL). The resultant TMI (N,N,N-trimethyl chitosan iodide) was precipitated, washed, and dissolved in NaCl solution (40 mL, 10% w/v) to exchange iodide with chloride ions, then re-precipitated using ethanol (200 mL), washed with ethanol and acetone repeatedly, and dried to yield the final product (TMC chloride, an off-white water-soluble powder). The TMC so obtained was finally lyophilized [16].

2.3. Confirmation of TMC

2.3.1. Fourier transform infrared spectroscopy–attenuated total reflectance (FTIR – ATR) analysis of TMC

The FTIR-ATR spectra of CHT and TMC were obtained using FTIR spectrophotometer (Shimadzu, FTIR-8300, Tokyo, Japan) in the scanning range of 4000–400 cm⁻¹.

2.3.2. The X-ray diffractograms of TMC

The crystallinity of the CHT and TMC samples was estimated using X-ray diffractometer (Brucker AXS D8 advance®, Karlsruhe, Germany) in a scanning range of 3–80° keeping 0.02° resolution, and the speed of scanning was set at 2.0° min⁻¹. An accelerating voltage of 40 kV was applied while a current intensity of 35 mA was used during the X-ray powder diffraction measurements.

2.3.3. ¹H NMR spectra of TMC

¹H NMR spectra of TMC was acquired on a Bruker Advance II 400 NMR spectrometer (PS751, 400 MHz, Boston, USA), with suppression of the water peak. ¹H NMR spectra was recorded at ambient temperature and the main acquisition parameters were as follows: a pulse of 45º, a recycle delay of 10s, and an acquisition of 128 transients. Time-domain data were apodized with a 0.2 hz exponential function (lb) to improve the signal-to-noise ratio before Fourier transformation [16].

2.4. Preparation of CHT- and TMC-loaded DIL nanoparticles

The resulting nanoparticle suspension underwent probe sonication for 10 cycles to aid dispersion. Subsequently, 3% mannitol was incorporated as a cryoprotectant, and the suspension was lyophilized.

2.5. Preparation of CHT- and TMC-loaded DIL nanoparticles

The ionotropic gelation technique was employed to fabricate the CHT- and TMC-DIL nanoparticles. A 2.5 mg/mL solution of CHT was prepared by dissolving in 0.05% glacial acetic acid and TMC was prepared using distilled water under magnetic stirring at 1000 rpm. An equivalent amount of DIL was added and stirred for 24 h to ensure homogeneity. Then, sodium tripolyphosphate (STPP, 0.25 mg/mL) was incorporated dropwise (0.3 mL/min) under stirring to induce cross-linking, followed by probe sonication for 10 cycles. Finally, the CHT- and TMC nanoparticles were dispersed in 3% mannitol (a cryoprotectant) and lyophilized (VirTis, SP Scientifics, New York, USA) at −70°C and 0.25 mbar to obtain a dry nanoparticle powder [22].

2.6. Formulation parameter

2.6.1. Particle size and ζ-potential

The average particle size, particle size distribution (PDI, polydispersity index), and ζ-potential of the DIL nanoparticles were determined using a Zetasizer (Nano ZS90, Malvern Instruments, UK). Freshly prepared nanoparticle dispersions were appropriately diluted (1:100) with water and analyzed by dynamic light scattering (DLS) at 25°C and at a scattering angle of 90°. The ζ-potential was measured via electrophoretic light scattering after dispersing the nanoparticles in water at an electrophoretic strength of 25 V/m.

2.6.2. Entrapment efficiency

The entrapment efficiency of DIL within CHT and TMC nanoparticles was estimated spectrophotometrically at 240 nm using a UV-visible spectrophotometer (1700, Shimadzu, Tokyo, Japan). The concentration of the drug was then calculated using the established linearity equation derived from the standard curve and calculated using the given formula:

$$\mathrm{\%}\mathit{E}.\mathit{E}.=\frac{\mathit{Total}\mathit{amt}\mathit{ofdrug}\mathit{added}-\mathit{Unentrapped}\mathit{drug}}{\mathit{Total}\mathit{amt}\mathit{of}\mathit{drug}\mathit{added}}\times 100$$

2.6.3. In-vitro DIL release

In vitro drug release study of DIL-loaded CHT and TMC nanoformulation was investigated by USP type I apparatus stirred at a speed of 100 rpm using 900 mL of phosphate buffer solution (pH 7.4) as a dissolution media maintaining 37 ± 0.5°C temperature and 30, 60, 120, 240, 360, 480, 600, 720, and 1440 min as time points for sample withdrawal. The aliquot withdrawn at each time point was analyzed spectrophotometrically for DIL concentration at 240 nm.

2.7. DFT analysis

The structures CHT, TMC, and DIL were optimized using the B3LYP/6-31 G** level of Density Functional Theory (DFT) to gain insights into their electronic and structural properties. The analysis of frontier molecular orbitals (FMOs) highlights the critical role of charge–transfer interactions between the ligand and the binding site of the protein. The highest occupied molecular orbital (HOMO) is the electron-rich, highest-energy orbital, making it energetically favorable for electron removal. In contrast, the lowest unoccupied molecular orbital (LUMO) is the lowest-energy orbital without electrons, making it the easiest orbital to add electrons to [23,24]. These orbitals play a significant role in charge transfer during chemical reactions, along with the energy gap (ΔE) values. The molecular electrostatic potential (MESP) map provides detailed information about the charge distribution within the molecules, helping identify reactive sites for electrophilic and nucleophilic attacks, especially in protein–ligand interactions. Different charge regions are represented by distinct colors, red indicates areas of negative potential (electron-rich regions) that attract protons, blue represents regions of positive potential (electron-deficient) that repel protons, and green indicates neutral regions with zero potential [25,26]. The ionization potential (IP) refers to the energy required to remove an electron from a molecule in the gas phase, while electron affinity (EA) represents the energy gained when an electron is added to a gas-phase molecule [27,28]. Electronegativity (χ) is a measure of an atom’s ability within a molecule to attract electrons. Charge transfer within a molecule is influenced by its chemical softness (σ) and chemical hardness (η). Molecules with high chemical hardness generally exhibit minimal or no charge transfer. The electrophilicity index (ω) measures the ability of a molecule to accept electrons, with higher values indicating stronger electrophilic character [29,30].

2.8. Molecular docking

The optimized structures of CHT, TMC, and DIL were obtained from the DFT calculation. The intermolecular interaction between (A) CHT-DIL and (B) TMC-DIL was executed through the AutoDock vina using the POAP pipeline [31,32]. The maximum grid box was set for CHT-DIL (center x, y, z was −1.4603, −3.0251, −4.3472, and size 25.0 Å) and TMC-DIL (center x, y, z was −0.1319, −0.1412, −0.0756, and size 25.0 Å). The system exhaustiveness was set to 8 and the docking was executed. The interactions formed were analyzed through the LigPlot+ v2.2.8 [33].

2.9. In-vivo experimental study of BLM-induced pulmonary fibrosis in rats

2.9.1. Animals

Sprague Dawley male rats were acquired from the in-house facility of R. C. Patel Institute of Pharmacuetical Education and Research. Approval of the protocol was obtained from the Institutional Animal Ethics Committee (IAEC), approval number (IAEC/2023/II R05). All procedures were performed as per the guidelines provided by the Committee for Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India.

2.9.2. Drugs and polymers

DIL was obtained from carbanion (CAS no.- 33286-22-5, molecular weight-414.519 g/mol), BLM was purchased from neon (brand name-Tumocin) (CAS no.- 9041-93-4, mol. weight-1415.551 g/mol), Pirfenidone was purchased from (CAS no.-53179-13-8, 185.22 g/mol), Low molecular weight CHT (MW 50–190 kDa, degree of deacetylation 75–85%), sodium tripolyphosphate, sodium hydroxide, glacial acetic acid, mannitol, etc. were all analytical and molecular grade.

2.9.3. Induction of blm-induced pulmonary fibrosis

BLM was used to induce pulmonary fibrosis in all groups except the first group. BLM was dissolved in sterile water for injection. Briefly, rats were intratracheally instilled with BLM sulfate (5 mg/kg) using an otoscope.

2.9.4. Experimental design

The rats were randomly divided into six groups (n = 6), and the various treatments were administered for a total of 14 days. On the 15^th day, the animals were sacrificed, and various parameters were evaluated. Group 1 (Control): Normal rats were given saline, p.o. for 14 days. Group 2 (Disease): BLM (5 mg/kg, i.t.) on the day first followed by the vehicle from day 2 to 14. Group 3 (BLM + Standard): BLM at (5 mg/kg, i.t.) dose on day 1 followed by daily pirfenidone (100 mg/kg, p.o.) from day 2–14. Group 4 (BLM + DIL CHT 1 mg/kg/i.t): BLM at (5 mg/kg, i.t.) dose on day 1 followed by daily diltiazem CHT (1 mg/kg, i.t.) from day 2–14. Group 5 (BLM + DIL CHT 3 mg/kg): BLM at (5 mg/kg, i.t.) dose on day 1 followed by daily diltiazem CHT (3 mg/kg, i.t.) from day 2–14. Group 6 (BLM + DIL TMC 3 mg/kg): BLM at (5 mg/kg, i.t.) dose on day 1 followed by daily diltiazem TMC (3 mg/kg, i.t.) from day 2–14. Group 7 (BLM + DIL 10 mg/kg): BLM at (5 mg/kg, i.t.) dose on day 1 followed by daily DIL (10 mg/kg, p.o.) from day 2–14.

2.10. Assessment of physiological parameters

2.10.1. Body weight

Each rat’s body weight (BW) was recorded in grams before BLM administration, and they were subsequently weighed again at the end of the study (on day 14). % change in the BW was determined from day 2 to 14.

$$\%\mathit{Change}\in \mathit{BW}=\frac{\mathit{Final}\mathit{BW}-\mathit{Initial}\mathit{BW}}{\mathit{Initial}\mathit{BW}}\times 100$$

2.10.2. Lung coefficient

Before sacrifice, the rat underwent body weight measurements. Following sacrifice, their lungs were carefully extracted, rinsed with PBS, and then weighed. Lung injury severity was evaluated by calculating the lung coefficient.

$$\mathit{Lung}\mathit{coefficient}=\frac{\mathit{lung}\mathit{weight}\left(\mathit{mg}\right)}{\mathit{body}\mathit{weight}\left(\mathit{g}\right)}$$

2.10.3. Preparation of tissue homogenate for biochemical estimation

A 10% tissue homogenate was prepared using phosphate buffer and KCl.

2.11. Assessment of oxidative stress parameter

2.11.1. Catalase (CAT)

CAT activity in lung tissue homogenates was determined spectrophotometrically by monitoring the decomposition of hydrogen peroxide (H₂O₂) at 240 nm, as described by [34]. Briefly, the reaction mixture contained potassium phosphate buffer (50 mM, pH 7.0) and the tissue homogenate. The reaction was initiated by adding H₂O₂ (30 mM), and the absorbance was recorded for 3 min. One unit of CAT activity was defined as the amount of enzyme required to decompose 1 μmol of H₂O₂ per minute.

2.11.2. Glutathione (GSH)

Reduced glutathione (GSH) levels were estimated in lung tissue homogenates using the Ellman’s reagent (5,5’-dithiobis-(2-nitrobenzoic acid) or DTNB) method. GSH present in the sample reacts with DTNB to form a product that absorbs at 412 nm. The GSH concentration was calculated from a standard curve and expressed as μg/mg of tissue [35].

2.11.3. Superoxide dismutase (SOD)

Superoxide dismutase (SOD) activity was measured spectrophotometrically by monitoring the inhibition of nitro blue tetrazolium (NBT) reduction by superoxide radicals, as per the method of [36]. One unit of SOD activity was defined as the amount of enzyme causing 50% inhibition in the NBT reduction rate.

2.11.4. Lipid peroxidation (LPO)

MDA reacts with thiobarbituric acid to form a colored product that can be quantified spectrophotometrically at 535 nm. MDA was calculated using a standard curve [37].

2.11.5. Nitric oxide (NO)

NO levels were quantified using the Griess reagent assay. Equal volumes of sample supernatant and Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 2.5% phosphoric acid) were incubated for 10 minutes at room temperature. The absorbance was measured at 548 nm, and nitrite concentration was determined using a standard curve prepared with sodium nitrite solutions [38].

2.12. Assessment of fibrotic markers in lung tissues

2.12.1. Hydroxyproline content

Hydroxyproline is formed as a modification to the peptide chain after translation. Hydroxyproline concentration is determined by the reaction of oxidized hydroxyproline with 4-(Dimethylamine) benzaldehyde (DMAB), which results in a colorimetric (560 nm) product, proportional to the hydroxyproline present [39].

2.13. Histopathological examination

In H&E staining, hematoxylin colors cell nuclei with blue, and eosin colors cytoplasm/extracellular matrix with pink [40]. The whole procedure was followed according to a given reference [41]. Masson’s trichrome staining highlights collagen in blue, muscle and erythrocytes in red, and nuclei in black [42]. The whole procedure was followed according to a given reference [43].

2.14. Hemodynamic parameter

For the assessment of hemodynamic parameters, rats were anesthetized using Ketamine 80 mg/kg, the right carotid artery of each rat was cannulated using a polyethylene cannula (PE 30) filled with heparinized saline (100 IU/mL) and connected to a physiological pressure transducer (MLT844) and the signal amplified by bio-amplifier, systolic pressure, diastolic pressure, and mean pressure was recorded using the power lab data acquisition (DAQ system, AD Instrument, Australia with LabChart-5 pro software.

3. Results

3.1. Synthesis of TMC

The highly cationic derivative TMC was synthesized using reductive methylation [16] (Supplementary Figure S1). The TMC so obtained was preliminarily found to be off-white colored powder and freely soluble in water. The yield of the reaction was satisfactorily obtained to be 55.73%.

3.2. Confirmation of TMC

Supplementary Figures S2A and 2B represent the FTIR-ATR spectra of CHT and TMC, respectively. A band centered at 1487 cm⁻¹ in the FTIR spectra of TMC demonstrating angular deformation due to asymmetric stretching of the C−H bonds of methyl groups (CH₃ antisym deformation) is the primary evidence of successful trimethylation on the CHT backbone. The absence of bands in the range 1500–1600 cm⁻¹ on the TMC spectrum due to the N−H bending demonstrated that the methylation of amino groups on CHT has effectively occurred.

Two major diffraction peaks at 2θ = 9.93° and 19.61° were observed in the X-ray diffractogram of CHT, which is attributed to its crystalline structure. Whereas the X-ray diffractogram of TMC revealed several well-precise peaks at 27.71°, 32.06°, 45.85°, 56.46°, 56.84°, and 66.82° owing to the formation of crystalline regions at a periodic distance. The methylation of CHT disrupts hydrogen bonds among amino groups, leading to the disappearance of diffraction peaks seen in bulk CHT shown in Supplementary Figures S2C and 2D.

Supplementary Figure S3 displays the ¹H NMR spectrum of TMC, with its chemical structure and respective peak assignments. The appearance of a sharp peak at 3.4 ppm in the spectrum attributed to the protons of trimethyl groups confirms the success of the reductive methylation reaction and the formation of quaternized amino groups. In addition, the absence of a peak in the range of 2.8–3.1 ppm indicates that the N-dimethylation has been prevented appropriately.

3.3. Formulation parameter

3.3.1. Physicochemical characteristics

The particle size and ζ-potential profiles of DIL-loaded CHT and TMC nanoparticles are presented in Supplementary Figure S4. The average particle size of DIL-loaded CHT- and TMC nanoparticles was found to be 267.8 (Supplementary Figure S4A) and 175.6 nm (Supplementary Figure S4B), respectively. The particle size data indicated that the CHT- and TMC-based carriers are nanometric in size, which is essential for rapid absorption across the lung mucosal membrane following pulmonary administration. The polydispersity indices were found to be 0.480 (Supplementary Figure S4A) and 0.391 (Supplementary Figure S4B) for CHT- and TMC-based nanoparticles indicating the homogeneous distribution, which is essential for colloidal stability of the prepared nanoparticulate formulations.

The ζ-potential values of DIL-CHT and DIL-TMC nanoparticles were found to be 5.10 (Supplementary Figure S4C) & 12.2 mV (Supplementary Figure S4D), respectively. The high cationicity of TMC nanoparticles over CHT nanoparticles indicates the possibility of higher mucosal retention of the former over later. The high cationicity of TMC is attributed to the incorporation of three methyl groups on the nitrogen atoms of CHT, making it a quaternized derivative.

The entrapment efficiency of DIL-CHT and DIL-TMC nanoparticles were found to be, 66.0 and 81.72%, respectively. The high entrapment of DIL in TMC nanoparticles compared to CHT nanoparticles is attributed to the more inter-chain space provided by the quaternized TMC, accommodating high amounts of DIL and thereby ensuring that the adequate amount of DIL is available at the target site to elicit the therapeutic response.

3.4. In-vitro drug release study of dil nanoformulations

The in vitro release reports of DIL-loaded CHT- and TMC nanoformulations in phosphate buffer (pH 6.8) are shown in Supplementary Figures S5A and 5B. The DIL was released in a sustained manner in both the formulations while the pattern of DIL release was also comparable in both the prepared formulations. The cumulative percent of DIL released from the formulations during 24 h was found to be ~97% (CHT nanoparticles) and ~89% (TMC nanoparticles). Among the prepared nanoparticulate formulations, the TMC-based nanoformulation exhibited a better-sustained release profile of DIL over a period of 24 h.

The findings of the in vitro release of DIL from DIL-CHT, and DIL-TMC nanoparticles elucidated the congruity of the drug release from almost assembled with first-order release kinetics, implying a release based on its availability of initial concentration shown in Supplementary Figure S5C and 5D.

3.5. DFT analysis

The energy gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) is a key indicator of a molecule’s chemical stability and is represented in Figure 1(a). A larger HOMO-LUMO gap corresponds to a harder, more stable, and less reactive molecule, as it requires more energy for electron transfer or excitation. In this study, CHT (ΔE = −0.253488 eV), DIL (ΔE = −0.17365 eV), and TMC (ΔE = −0.260003 eV) exhibit relatively small energy gaps, which suggest that these compounds are highly reactive and possess significant electron transfer capacity. Among them, DIL shows the smallest energy gap, indicating the highest reactivity, while TMC has the largest energy gap, making it the most stable compound in the series.

Figure 1.

(a) DFT-Based frontier molecular orbital energies calculations of HOMO, LUMO, and maximum electrostatic potential (MESP). (b) Molecular docking models of (a) CHT-DIL complex and (b) TMC-Diltiazem DIL complex.

The molecular electrostatic potential (MESP) plots further illustrate the chemical reactivity of these compounds. The color distribution on the MESP plots, primarily ranging from green to blue, reflects areas susceptible to nucleophilic and electrophilic attacks. The darker blue regions, particularly on the pyranose ring of CHT and TMC, suggest zones where nucleophilic attack is likely. In contrast, slight yellow regions observed between the central pyranose ring and the hydroxyl and methoxy groups of both CHT and diltiazem indicate areas prone to nucleophilic interactions, supporting the compounds’ overall reactivity.

Electronegativity values of the compounds were found to be lower than their ionization potentials (IP), indicating a propensity to release electrons, which aligns with their nucleophilic character. TMC has lower electronegativity compared to CHT implying that it has lower electron withdrawing propensity, and stability over CHT. The chemical softness values for CHT, diltiazem, and TMC are notably higher than their chemical hardness values, reinforcing the idea that these molecules can undergo various chemical reactions, as supported by their relatively small HOMO-LUMO gaps. The negative values of electronegativity, or electronic chemical potential (μ), reflect the tendency of electrons to depart from the system in equilibrium, implying that these compounds are stable yet reactive. Among the three, diltiazem exhibits a slightly more negative chemical potential, which suggests it is more reactive toward electron donors and can readily participate in chemical reactions.

Lastly, the global electrophilicity index (ω), which measures a molecule’s ability to act as an electrophile, further distinguishes diltiazem as the strongest electrophile with the highest index value. This indicates its superior capacity to accept electrons during chemical reactions. In comparison, CHT and TMC show moderate electrophilicity index values, making them relatively weaker electrophiles. Molecular orbitals (HOMO, LUMO), and various reactivity indices like electronegativity, chemical hardness, and electrophilicity properties of CHT, TMC, and DIL were represented in Table 1.

Table 1.

DFT-Based frontier molecular orbital (FMO) energy calculations of the CHT, TMC, and DIL.

Property	Energy (E)	HOMO	LUMO	Energy Gap (ΔE)	Ionization Potential (IP)	Electron Affinity (EA)	Electronegativity (χ)	Chemical Hardness (η)	Chemical Softness (σ)	Chemical Potential (μ)	Electrophilicity Index (ω)
Chitosan	−1813.310049	−0.224254	0.029234	−0.253488	0.224254	−0.029234	0.09751	0.126744	3.94495	−0.09751	0.063371
Diltiazem	−1662.682422	−0.208512	−0.034862	−0.17365	0.208512	0.034862	0.121687	0.17365	2.87935	−0.121687	0.08682
Trimethylchitosan	−1967.152314	−0.210808	0.049195	−0.260003	0.210808	−0.049195	0.0808065	0.13000	3.8461	−0.080806	0.065

a. Energy gap(ΔE) = E_HOMO − E_LUMO. b. Ionization potential (IP) = −E_HOMO. c. Electron affinity (EA) = −E_LUMO. d. Electronegativity (χ) = (IP + EA)/2. e. Chemical hardness (η) = (IP − EA)/2. f. Chemical softness (s) = 1/2η. g. Chemical potential (μ) = −(IP + EA)/2. h. Electrophilic index (ω) = μ²/2η

3.6. Molecular docking

The binding energy (BE) of the CHT-DIL complex is calculated to be −3.0 kcal/mol, indicating the stability of the interaction. CHT forms four hydrogen bonds (represented by green dashed lines) with DIL, with the following positions and distances: O1 - N2 (2.77 Å), O3 - O1 (3.11 Å), O2 - O2 (2.90 Å), and O5 - N3 (3.27 Å), respectively. In comparison, the TMC-DIL complex shows a BE of −3.3 kcal/mol, suggesting a slightly stronger interaction than the CHT-DIL complex. The hydrogen bonds formed between TMC and DIL also include four bonds, with the positions and distances: O3 - O8 (3.12 Å), O4 - O3 (3.10 Å), O5 - C26 (3.70 Å), and O1 - N7 (3.93 Å). These results suggest that the methylation of CHT (as seen in TMC) enhances the binding affinity with DIL, leading to a slightly more stable complex (Figure 1(b)). The TMC-DIL complex shows a lower binding energy (−3.3 kcal/mol) compared to the CHT-DIL complex (−3.0 kcal/mol), which suggests that TMC forms a more stable interaction with DIL. Both complexes display hydrogen bonding as the key interaction, but the TMC-DIL complex involves additional methylation, which may contribute to more varied interactions and possibly more steric compatibility, resulting in stronger binding. The modification of CHT to TMC enhances the binding affinity with DIL by introducing different interaction sites through the methylated groups, thereby making TMC a more favorable carrier for DIL. This concept is useful in illustrating how slight chemical modifications, such as methylation, can affect drug-carrier interactions, potentially influencing the efficacy of drug delivery systems.

3.7. In-vivo parameters

3.7.1. Effect of DIL nanoformulation on % change in body weight and lung index

Intratracheal administration of BLM (5 mg/kg) significantly reduced body weight by days 13 (p < 0.05) and 14 (p < 0.01) compared to the control group. Treatment with PFD, DIL 1 mg/kg, DIL 3 mg/kg (CHT), DIL 3 mg/kg TMC, and 10 mg/kg oral resulted in an observable increase in body weight compared to the BLM-treated groups (Figure 2(a)). Additionally, an increase in the lung coefficient (LC) was observed in the BLM group compared to the normal group. The standard therapy PFD group exhibited a normal lung index similar to the control group. Treatment with both DIL 1 mg/kg and DIL 3 mg/kg TMC resulted in a lung index approaching normal levels compared to the BLM group, whereas DIL 3 mg/kg (CHT) did not mitigate the increased lung index (Figure 2(b)).

Figure 2.

Effect of lung-targeted nanoformulations of DIL on physiological parameters on BLM-induced pulmonary fibrosis. (a) % change in body weight (b) lung index. Data represented as mean ± SEM (n = 6). Statistical analysis by two-way ANOVA and One-way ANOVA followed by Bonferroni multiple comparison posttest. p < 0.0001, DFn = 6, DFd = 35, F (6, 35) = 3.3. ****p < 0.0001, **p < 0.01, *p < 0.05 as compared to the BLM group; ##p < 0.01 as compared to the control group.

3.7.2. Effect of DIL nanoformulation on oxidative stress parameters

As shown in Figure 3A, BLM (5 mg/kg) administration by intratracheally significantly decreases CAT activity compared to the control (p < 0.01), indicating compromised antioxidant defense. Both DIL nanoformulations (CHT and TMC) show improvement (p < 0.05), with DIL 3 mg/kg TMC and DIL 10 mg/kg oral significantly increasing CAT activity beyond control levels (p < 0.0001). Similar trends are observed with GSH levels, which are crucial for the antioxidant defense system. BLM significantly depletes GSH (p < 0.01); however, DIL formulations, particularly DIL 1 mg/kg CHT and DIL 10 mg/kg oral, restore or even elevate GSH levels (p < 0.01) as shown in Figure 3(b). Intratracheal administration of BLM causes a significant decrease in SOD enzymatic activity (p < 0.01). Although all DIL formulations show a trend toward improvement, DIL 3 mg/kg TMC significantly increases SOD activity compared to the BLM group (p < 0.05) represented in Figure 3(c).

Figure 3.

Effect of lung-targeted nanoformulations of DIL on oxidative stress parameter against BLM-induced pulmonary fibrosis. (a) level of CAT, (b) level of GSH, (c) level of SOD activity, (d) level of NO, (e) level of LPO, and (f) hydroxyproline content. Data represented as mean ± SEM (n = 6). Statistical analysis by one-way ANOVA followed by Bonferroni multiple comparison posttest. p < 0.0001, DFn = 6, DFd = 35, F (6, 35) = 16. ****p < 0.0001, **p < 0.01, *p < 0.05 as compared to the BLM group; ##p < 0.01 as compared to the control group.

The fourth graph shows that BLM significantly increases NO levels (p < 0.01), a marker of inflammation and oxidative stress. All DIL formulations significantly reduce NO levels: DIL 1 mg/kg CHT (p < 0.05), DIL 3 mg/kg CHT (p < 0.01), DIL 3 mg//kg TMC (p < 0.05), and DIL 10 mg/kg oral (p < 0.05) shown in Figure 3(d). Similarly, BLM significantly increases MDA levels (p < 0.01), indicating LPO. DIL 1 mg/kg CHT (p < 0.05), and DIL 3 mg/kg (p < 0.05) show a decrease in MDA levels (Figure 3(e)).

As shown in Figure 3(f) intratracheal BLM (5 mg/kg) significantly increased lung tissue hydroxyproline content (**p < 0.01), indicating elevated collagen levels. Conversely, treatment with DIL (3 mg/kg) in both nanoformulation and oral administration notably decreased hydroxyproline content, suggesting reduced collagen. DIL at 3 mg/kg via TMC nanoparticles exhibited effects comparable to the control group.

3.8. Histopathological evaluation

3.8.1. Effect of DIL nanoformulation on MT staining and H&E staining

Representative images of histochemical features of the lung tissue in the MT-stained sections showed increased collagen deposition in the BLM group, indicating fibrosis generation. The collagen deposition was reduced by various treatments with DIL nanoformulation, aligning with the reduction in hydroxyproline content (Figure 3(f)). Further, the lung tissue histology (as shown in Figure 4(a)) of the control group shows moderate fibrosis, lower cellularity, and larger alveolar sizes, indicating healthy lung tissue. BLM 5 mg/kg exhibits the highest fibrosis by increased cellularity, and reduced alveolar size, confirming its role in inducing fibrosis for this experimental model. PFD 100 mg/kg shows reduced fibrosis and moderate cellularity compared to the BLM group, suggesting a protective effect. DIL CHT 1 mg/kg exhibits high fibrosis but lower cellularity and larger alveolar sizes, indicating less severe inflammation but still significant fibrosis. DIL CHT 3 mg/kg has the lowest fibrosis percentage, moderate cellularity, and smaller alveolar sizes, indicating a significant protective effect against fibrosis. DIL TMC 3 mg/kg shows minor fibrosis, the highest cellularity, and smaller alveolar sizes, suggesting minor inflammation but some preservation of alveolar structures. DIL 10 mg/kg, p.o. exhibits moderate fibrosis, the highest cellularity, and smaller alveolar sizes. The control group alveolar architecture was found to be intact, with normal thin alveolar walls and minimal cellular infiltrates, representing healthy lung tissue. Whereas BLM 5 mg/kg shows a significant increase in cellularity, thickening of the alveolar septa, and infiltration of inflammatory cells, indicative of lung injury and fibrosis. PFD 100 mg/kg group lung structure appears relatively preserved with less thickening of the alveolar walls and fewer infiltrating cells compared to the BLM group, suggesting a protective effect. DIL CHT 1 mg/kg showed moderate cellular infiltration and thickening of the alveolar walls, although less severe than the BLM group. This indicates a partial protective effect at this dose. DIL CHT 3 mg/kg: Reduced cellular infiltration and alveolar wall thickening compared to the BLM group, suggesting an improved protective effect. Whereas DIL TMC 3 mg/kg shows similar to the DIL CHT 3 mg/kg group, showing reduced cellularity and thickening of alveolar walls. This suggests that the TMC formulation also has a protective effect. DIL 10 mg/kg, p.o. showed a significant reduction in cellular infiltration and alveolar wall thickening, indicating a protective effect (Figure 4(b)).

Figure 4.

(a) histopathological alterations in lung tissue (MT staining and visualization at 10X magnification). (b) effect of lung-targeted nanoformulations of DIL on histopathological alterations in lung tissue against BLM-induced pulmonary fibrosis (H&E staining and visualization at 10X).

3.9. Effect of DIL nanoformulation on hemodynamic parameters

The effects of 3 mg/kg DIL nanoformulation (i.t.) and plain drug (i.v. and p.o.) on rat blood pressure (systolic arterial pressure; SAP), diastolic arterial pressure (DAP), and mean arterial pressure (MAP) were investigated in order to evaluate the safety of DIL, which is approved for the treatment of hypertension, angina, and arrhythmia. 3 mg/kg DIL nanoformulation showed SAP, DAP, and MAP toward normal compared to DIL i.v and p.o (Figure 5).

Figure 5.

Effect of DIL nanoformulation on rat arterial blood pressure (SAP, DAP, and MAP).

4. Discussion

The limited therapeutic options in treating IPF underscore an abrupt need for repositioned drugs till novel and specific antifibrotic therapies are invented [1,2]. The existing molecules, nintedanib, and pirfenidone, have adverse effects and tend to slow down the progression of the disease instead of providing a complete cure [5]. DIL, a calcium channel blocker used to treat high blood pressure, has shown the ability to hinder important processes associated with fibrosis, including the activation of TGF-β, the generation of collagen, and the transition of epithelial cells to mesenchymal cells, and this study developed two DIL nanoformulations using CHT and TMC via ionotropic gelation method for the management of IPF [21]. CHT is a natural polymer obtained from chitin and is not soluble in water; hence, Glacial acetic acid is used to solubilize it. However, the use of acetic acid may result in membrane irritation and to address this challenge, developed a modified form of CHT called TMC, that dissolves readily in water and has a superior mucoadhesive property [16]. The biodegradability, biocompatibility, muco-adhesivity, and non-toxicity of TMC render it an excellent biopolymer for a range of pharmaceutical and biomedical applications, including pulmonary delivery [17]. In this study, TMC was prepared and characterized using FTIR-ATR, XRD, and ¹H NMR, confirming that TMC exhibited distinct physicochemical characteristics compared to CHT. Notably, TMC demonstrated a similar drug release pattern to CHT and showed an improved delayed-release profile.

The optimized batch of DIL nanoformulations prepared had a particle size in the range of 200–500 nm and nanoparticles of this size range reach the lung alveoli to a greater extent as proved by Dhand et al. [44]. The encapsulation efficiency of both CHT and TMC formulations is over 65% and this can be considered satisfactory [45]. Similar encapsulation rates have been reported for nifedipine (NIF) dry powder formulation reported for the treatment of IPF [45]. The drug release study of DIL nanoparticle formation prepared revealed a first-order kinetics up to 24 hours indicating an increased bioavailability and lower excretion of DIL in the system. The DFT and molecular docking analysis highlight the superiority of TMC over CHT in nanoparticle preparation. TMC exhibits a larger HOMO-LUMO energy gap (ΔE = −0.260 eV) compared to CHT (ΔE = −0.253 eV), indicating greater stability and lower reactivity. The molecular electrostatic potential (MESP) plots further support TMC’s enhanced reactivity by showing more favorable regions for nucleophilic attacks, particularly on the pyranose ring. Additionally, TMC’s lower electronegativity suggests less electron-withdrawing propensity and higher chemical stability compared to CHT. Docking studies reinforce this finding, as TMC forms a more stable complex with DIL, resulting in better interactions and binding energy. Overall, TMC’s superior electronic properties and molecular interactions make it a more effective carrier for DIL, positioning it as the preferred choice for nanoparticle formulations.

Further, prepared DIL nanoformulations were tested for anti-fibrotic activity in the BLM-induced IPF rat model. BLM-induced fibrosis is a well-established preclinical model in rodents and in the present study, a dose of 5 mg/kg was chosen to produce pulmonary fibrosis [46]. The induction of fibrosis by BLM is time-, day-, and dose-dependent. Based on the literature and a pilot study, a single intratracheal administration of 5 mg/kg using a digital otoscope-assisted method was employed, with a study duration of 15 days to induce IPF in rats. Starting on the second day, the drugs were administered by oral gavage to the PFD group (100 mg/kg) and the DIL group (10 mg/kg). The DIL CHT (1 and 3 mg/kg) and TMC (3 mg/kg) formulations were delivered via the intratracheal route under light isoflurane anesthesia every 24 hours up to the 14th day. A digital otoscope-assisted device, fabricated in our lab, was used to administer the powdered formulation. On the 15th day, the rats were sacrificed to evaluate their physical parameters (body weight and lung index) and oxidative stress markers, including SOD, GSH, CAT, LPO, NO, and HYP. Additionally, performed the histopathological analysis hematoxylin & eosin and Masson trichome stained lung tissue sections under 10× microscopy and graded the extent of fibrosis using the Ashcroft scale [47].

Every day, the rats’ body weight was noted and recorded the percentage changes in each group. The BLM-induced body weight loss for prevented by the DIL CHT (1 and 3 mg/kg), TMC (3 mg/kg), and oral DIL (10 mg/kg) treatments. In previous findings, it has been reported that BLM-induced IPF reduces the body weight of rats [48]. These treatments also decreased the lung index which may be due to reduced accumulation of fibrous connective tissues in the lungs of rats receiving DIL treatments. IPF is characterized by an excessive accumulation of collagen and other proteins in the lung tissue. The observed decrease in the lung index may be attributed to the reduced inflammatory processes that often precede fibrosis.

An association has been made between oxidative stress and the production of superoxide anions, which ultimately leads to a chain of inflammatory events [49]. Based on this, it may be concluded that inhibiting oxidative stress is an important strategy for preventing fibrosis and the consequences that are associated with it. Previously conducted research has shown that the development of fibrosis is a consequence of an imbalance of oxidants [48]. CAT plays a significant role in fibrosis by mitigating oxidative stress, thereby potentially reducing tissue damage and inflammation, which are key processes in the progression of fibrosis [50]. In the present study after administration of BLM, observed a reduction in CAT level. However, animal treatment with DIL nanoformulations had increased CAT levels comparable to the control group. Another important antioxidant, GSH, plays a crucial role in mitigating fibrosis by regulating oxidative stress and inflammation [51]. DIL nanoformulations including the DIL CHT 1 (mg/kg) inhibited the BLM-induced reduction in GSH. A similar effect of retaining the SOD activity was observed in the rats receiving DIL nanoformulations indicating their efficacy in preserving the SOD activity. A reduced SOD activity is implicated progression of fibrosis [52]. These findings align with the previous available study where the level of CAT, GSH, and SOD were reduced in the fibrotic lung tissues and protected by antifibrotic agents [53].

LPO measured in terms of MDA, indicates the extent of oxidative stress-induced lipid peroxidation and damage to the cell membrane. BLM-induced IPF resulted in a rise in the LPO levels and the DIL treatment including the CHT (1 mg/kg) formulation reduced the LPO. These findings are also supported by a previous study of DIL where it decreased the level of MDA [13]. NO plays a significant role in fibrosis by regulating inflammation and fibroblast activity, influencing tissue remodeling and scar formation [54]. In this study, observed the BLM instillation led to an increase in NO levels in lung tissue and treatment with the DIL nanoformulation reduced NO levels. This effect was more prominent at the DIL (3 mg/kg) dose.

Previously, DIL demonstrated potential in preventing peritoneal fibrosis by inhibiting collagen synthesis and TGF-β1 production in human peritoneal mesothelial cells (PMCs). It suppressed IL-1β-induced expression of collagen I and III mRNA and reduced TGF-β1 production at both mRNA and protein levels. The inhibitory effects were mediated through the suppression of stress-activated JNK and p38 MAPK pathways [8]. DIL, at doses of 5 mg/kg and 15 mg/kg, enhances hepatic regeneration by inhibiting TGF-β1 following partial liver resection in rats [10] and also demonstrated TGF-β1 inhibition in liver cell cultures [55]. In an experimental study involving rats, DIL was administered at a dose of 25 mg/kg/day following isoproterenol-induced myocardial injury. The results indicated that DIL improved myocardial structure by reducing fibrosis and enhancing the expression of connexin 43 (Cx43), a protein critical for cardiac cell communication. This suggests that diltiazem may play a significant role in preventing structural remodeling of the heart, which is crucial for maintaining cardiac function [56]. These studies support DIL as a promising anti-fibrotic candidate due to its ability to inhibit collagen synthesis, TGF-β1 production, and myocardial remodeling without causing cardiovascular side effects.

Importantly, safety is a major concern over the drug efficacy, so this study assessed the effect of DIL nanoformulations on the cardiovascular safety measures in rats and the results were compared with the intravenous administration of DIL. The results showed that DIL (3 mg/kg) nanoformulations intratracheally did not affect normal rat BP, whereas the intravenous 3 mg/kg and oral 10 mg/kg at the same dose induced a decrease in the BP.

Hydroxyproline is an amino acid found in mammalian collagen and is elevated in fibrosis conditions [57]. Elevation of Hydroxyproline by BLM treatment reduced by our treatment drug DIL (3 mg/kg). The TMC group showed a more noteworthy effect. MT staining visually confirmed this, showing reduced collagen deposition in DIL-treated groups. Furthermore, H&E staining revealed that DIL nanoformulations mitigated BLM-induced lung tissue destruction and morphological alterations.

The superiority of the TMC nanoformulation over CHT is a key finding. This can be attributed to TMC’s enhanced water solubility, which eliminates the need for acidic solvents that can irritate, and its greater mucoadhesive properties, which may prolong pulmonary retention and action. The in vitro drug release studies support this, showing that the TMC formulation provided a more sustained release profile over 24 h compared to CHT.

A notable strength of the study is the comparative analysis of different DIL formulations and routes. The intratracheal DIL nanoformulations, particularly at 3 mg/kg, demonstrated efficacy comparable or superior to both the standard oral treatment (pirfenidone) and oral DIL (10 mg/kg). This suggests that the nanoformulation approach not only mitigates systemic side effects but also enhances therapeutic efficacy, likely due to targeted pulmonary delivery and sustained release. The study also addresses safety concerns. Given DIL’s primary use in cardiovascular conditions, the researchers prudently assessed its impact on blood pressure in normal rats. The finding that intratracheal DIL nanoformulations did not significantly alter blood pressure, unlike intravenous administration, further supports the safety and specificity of this delivery method.

5. Conclusion

Idiopathic Pulmonary Fibrosis (IPF) is a severe lung disease marked by the excessive deposition of extracellular matrix and scar tissue formation, leading to reduced lung function and significant morbidity. Current treatments, pirfenidone and nintedanib, are limited in efficacy and come with substantial side effects, underscoring the need for new therapeutic options. DIL, a drug used for hypertension and arrhythmia, has found promise in inhibiting fibrotic pathways, particularly through the TGF-β pathway. In the present study, the prepared DIL nanoformulations demonstrated significant antifibrotic effects in BLM-induced fibrosis in rats via reducing oxidative stress markers and improving physical and histopathological parameters. Despite some cardiovascular risks associated with DIL, the intratracheal delivery of DIL nanoparticles showed promising safety profiles and anti-fibrotic efficacy. These findings suggest that DIL, especially in nano-formulated forms (mainly water-soluble polymer TMC), could be a new treatment option for IPF, warranting further clinical investigation. Alongside, DFT and molecular docking studies demonstrate that TMC offers superior stability, reactivity, and binding efficiency compared to CHT, making it a more promising candidate for developing DIL-loaded nanoparticles for antifibrotic therapy; hence further research is needed to assess its safety and understanding of its molecular mechanisms.

Funding Statement

This paper was not funded.

Article highlights

• DIL-TMC nanoparticles, with sizes 175.6 nm, show high entrapment efficiencies (81.72%), with more sustained release compared to DIL-CHT.

• DFT and molecular docking revealed TMC’s superior stability and enhanced interaction with DIL for better therapeutic effects.

• DIL-TMC nanoparticles significantly reduced fibrosis symptoms in a bleomycin-induced IPF mouse model, improving lung health and oxidative stress.

• DIL, a promising candidate for idiopathic pulmonary fibrosis.

Authors contribution

Nandeeni Punase: Methodology, Resources, Investigation, Writing – original draft. Ganesh V. Jamdar, Ghanshyam Mapare and Niharika Patil: Methodology, Investigation. Vishal S. Patil and Formal analysis, Investigation, and Writing – original draft, Revision, and Drafting. Narendra Nagpure: Formal analysis, Investigation. Chandrakantsing V. Pardeshi: Formal analysis, Software, Resources, Writing – review & editing. Chandragouda R. Patil: Investigation, Resources, Software, Writing – review & editing, Validation, Project administration, Methodology, Funding acquisition, Conceptualization.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Ethical declaration

The study was reviewed and approved by Institutional Animal Ethics Committee of R.C. Patel Institute of Pharmaceutical Education and Research (Approval No. IAEC/CPCSEA/RCPIPER/2023–24/02).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/20415990.2025.2478803.