Nano Engineered Chitosan-Based Delivery System for Vonoprazan Fumarate for Enhanced Bioavailability: Fabrication and Evaluation

Abstract

Purpose

The main purpose of the present study was to explore chitosan as a polymeric material for the preparation of vonoprazan nanoparticles intended for use as a delivery system for acid-related diseases.

Methods

Vonoprazan-loaded chitosan nanoparticles were formulated using the ionotropic gelation method and characterized in terms of size, zeta potential, polydispersity index, drug entrapment efficiency (EE), FTIR, XRD, Thermal analysis, SEM, in vitro release study at two different pH levels, and drug release kinetics. Acute oral toxicity was assessed to evaluate safety and pharmacokinetic studies were performed to determine bioavailability.

Results

The optimized formulation VCHNP4 demonstrated a mean particle size of 496.5±1.19 nm, a zeta potential of 25.4±3.07 mV, a polydispersity index of 0.474±1.97, and an entrapment efficiency of 78.09±0.41. Surface morphology studies revealed a spherical shape with inclusions of drug-loaded chitosan nanoparticles. Thermal stability was improved, as observed by thermal analysis, and PXRD confirmed the amorphous state of the drug. Saturation solubility testing indicated significantly enhanced solubility of the drug and exhibited pH-dependent drug release, with higher release at pH 1.2 compared to pH 6.8, following the Korsmeyer-Peppas model. Acute oral toxicity studies showed no major differences in the clinical parameters between the control and treatment groups. In vivo pharmacokinetics showed that VCHNPs achieved superior Cmax (307 ± 0.61 ng/mL) compared to VPZ (41 ± 1.01 ng/mL) (p < 0.05).

Conclusions

This study showed that mucoadhesive polymeric nanoparticles of vonoprazan significantly enhanced solubility, pH-dependent release, and improved mucoadhesion, making it a promising approach to improve the oral bioavailability of drugs with poor water solubility.

Plain Language Summary

The ionic gelation method was used to prepare the nanoparticles.

• Vonoprazan Fumarate was successfully loaded into chitosan nanoparticles and showed better stability.

• Prepared nanoparticles exhibited the best release at the gastric region.

• Nanotechnology increases the oral bioavailability of vonoprazan fumarate, and a toxicity study confirmed its safety.

Graphical Abstract

Introduction

Peptic ulcer diseases is a persistent acid-related disorder that usually occurs in the stomach or duodenum, and is a prevalent cause of gastrointestinal hemorrhage. The major factors for peptic ulcer disease are Helicobacter pylori infection and administration of nonsteroidal anti-inflammatory drugs (NSAIDs). In the last decades of the twentieth century, the prevalence of peptic ulcer disease began to decline owing to two significant advancements: the development of effective proton pump inhibitors and the detection of Helicobacter pylori. Although there have been more initiatives to identify and eliminate H. pylori, it still affects as many as 20% of Asian adults.¹ Concurrently, the use of non-steroidal anti-inflammatory drugs (NSAIDs) and aspirin has surged, especially among the elderly, owing to a higher incidence of cardiovascular diseases and rheumatic disorders. Although non-steroidal anti-inflammatory drugs and aspirin are significant risk factors for upper gastrointestinal bleeding, halting these medications poses challenges owing to underlying chronic pain and cardiovascular risks. Therefore, preventive measures must be implemented to reduce the risk of bleeding.²

Vonoprazan has been evaluated for the management of gastric ulcers because of its pharmacodynamic and pharmacokinetic benefits compared with proton pump inhibitors (PPIs). Vonoprazan is an innovative potassium-competitive acid blocker (P-CAB) used to treat erosive esophagitis, gastric and duodenal ulcers, eradicate H. pylori, and prevent ulcer recurrence associated with nonsteroidal anti-inflammatory drugs or low-dose aspirin.³ Vonoprazan has numerous advantages over traditional proton-pump inhibitors (PPIs), such as a rapid onset of action, prolonged acid suppression, reduced inter-individual variability in acid suppression, and no dietary impact on its efficacy. Vonoprazan mitigates various limitations related to traditional proton-pump inhibitors therapy, such as short half-life, susceptibility to degradation in acidic environments, selective inhibition of activated pumps only, the need for multiple administration cycles (3–5) to achieve optimal efficacy, and clinical variability related to CYP2C19 polymorphisms.⁴

In recent years, naturally occurring polymers have attracted considerable attention in the medical field. This is partly due to their biocompatibility with humans, varied chemical structures, and eco-friendly manufacturing procedures. Among these polymers, chitosan has been extensively studied because of its natural abundance, derived from sources such as fungi, crustaceans, and insects, and its proven biocompatibility with minimal adverse effects in humans. Chitosan is a linear polymer composed of β (1 → 4)-d-glucosamine and β (1 → 4)-N-acetylglucosamine units. It is the second most prevalent organic polymer after cellulose and has several applications in medical research, including wound dressings, antibacterial properties, tissue engineering, and drug delivery.⁵

Chitosan, owing to its nontoxic, biocompatible, and biodegradable properties, has significant potential as a drug carrier. The amino and carboxyl groups in chitosan can react with glycoproteins in mucus to establish hydrogen bonds, resulting in a mucoadhesive effect that extends the retention time of drugs and enhances drug bioavailability.⁶ The versatility of this material supports processing into many forms, such as sponges, membranes, gels, scaffolds, microparticles, nanoparticles, nanofibers, and three-dimensional printed constructs.⁷ CHNPs can be synthesized using various techniques, including ionotropic gelation, polyelectrolyte complexation, reverse micellar methods, emulsification, solvent evaporation, and electrospraying.⁸

Ionotropic gelation is the most widely studied method for formulating chitosan nanoparticles because of its simplicity and use of an aqueous medium, which minimizes the risks associated with organic solvents. This method involves crosslinking the cationic amino group of chitosan with a negatively charged polyanionic crosslinker tripolyphosphate (STPP). The resulting complex served as a drug nanocarrier.⁹

However, inadequate water solubility (<1 mg/mL) and limited bioavailability (merely 10%) of Vonoprazan Fumarate (TAK-438) restrict its therapeutic efficacy.¹⁰ Although vonoprazan fumarate effectively suppresses acid, its limited water solubility hinders its oral absorption and dissolution. Conventional methods for treating solubility issues include physical drug modifications like micronization, solid dispersion in carriers, complexation, cryogenic techniques, and supercritical fluid technology; additionally, chemical drug modifications like salt formation, cosolvency, hydrotropy, and prodrug formation have been investigated to improve drug absorption, yet problems including gastric retention and stability still exist. Because of their mucoadhesive properties and ability to enhance solubility and oral bioavailability, chitosan-based nanoparticles are highly beneficial. NP-based delivery systems increase drug solubility, bioavailability, and targeting. Chitosan, a biocompatible and biodegradable polymer, is extensively used in these systems. We hypothesized that encapsulating vonoprazan fumarate into chitosan nanoparticles would improve its solubility, offer sustained drug release, promote mucoadhesion, and hence increase oral bioavailability.

Based on this hypothesis the present study focused on developing chitosan-based nanoparticles to enhance the delivery of vonoprazan.

Materials and Methods

Vonoprazan Fumarate (99.9% purity) was obtained as a gift from Quaper Pharma Pvt. Ltd ^® (Sargodha, Pakistan), and Chitosan MMW (190–310kDa), acetic acid, Sodium Hydroxide, Hydrochloric acid, sodium tripolyphosphate, and Tween 80 were obtained from Sigma Aldrich (Germany). Monobasic potassium phosphate, disodium hydrogen phosphate, phosphoric acid, and dibasic sodium phosphate were obtained from Sigma-Aldrich (USA). Methanol, ethanol, acetonitrile, and HPLC water were procured from Merck (Darmstadt, Germany). The dialysis membrane used for the dissolution study was acquired from (suppliers), (Lahore, Pakistan). All chemicals used in this study were of analytical grade.

Preparation of Vonoprazan-Loaded Chitosan Nanoparticles

Drug-loaded chitosan NPs were prepared with a slight modification of ionic gelation methods previously reported by Duse L, 2018 et al¹¹ Figure 1 Schematic synthesis of the nanoparticles.

Figure 1.

Schematic diagram for the synthesis of nanoparticles.

Various formulations were prepared containing different concentrations of chitosan as a polymer and sodium tripolyphosphate as a cross-linker. Different ratios of chitosan and SSTPP were used to develop the formulation (Table 1) while maintaining the pH, temperature, and stirring rate. A 0.5% w/v chitosan solution in 2% v/v glacial acetic acid was prepared and the solution was stirred overnight at 500 revolutions per minute (rpm) on a magnetic stirrer (Spectrum Scientific) at 40 °C, filtered, and the pH was adjusted to 4.5. Tween 80 (0.5%) was added and the mixture was stirred for 2 h at 1500 rpm. The drug was added with constant stirring at 700–800 rpm. A 0.1% SSTPP solution was prepared in ultrapure water, filtered through a filter paper, and added at 1000 rpm. After 30 min, the slightly turbid solution was subjected to centrifugation for 15 min at 13,000 rpm, and the supernatant was collected for further characterization. Deionized water was used to wash the nanoparticle pellets and the dried powder was obtained by lyophilization. The formulation was optimized by adjusting the concentrations of chitosan and sodium tripolyphosphate (TPP) while keeping the other variables fixed. The selection criteria included particle size, polydispersity index (PDI), zeta potential, entrapment efficiency, drug loading, and in vitro drug release. The formulation with the most favorable physicochemical properties and release profile was chosen for further characterization and in vivo testing.

Table 1.

Composition of Various Formulation of Nanoparticles

Formulation	VPZ Fumarate(mg)	Chitosan (mg/mL)	SSTPP (mg/mL)	Tween80 (mL)
VCHNP1	10	2	0.5	0.5
VCHNP2	10	3	0.5	0.5
VCHNP3	10	4	0.5	0.5
VCHNP4	10	5	0.5	0.5
VCHNP5	10	6	0.5	0.5
VCHNP6	10	4	0.25	0.5
VCHNP7	10	5	0.25	0.5

Notes: Bold values indicate the concentrations of chitosan and sodium tripolyphosphate (TPP) used as the independent formulation variables in the preparation of chitosan nanoparticles.

Characterization of Nanoparticles

Dynamic Light Scattering Measurements

The hydrodynamic size, polydispersity index (PDI), and zeta potential of the drug-loaded chitosan nanoparticles were determined using a Zetasizer Nano ZS 90 (Malvern Instruments, UK) equipped with a He-Ne laser. The weighed amount of drug-loaded chitosan NPs powder was dispersed in distilled water (used as dispersant), vortexed, and examined at a 90° scattering angle for 30–60 s at 25°C using a disposable sizing cuvette. The applied electrical field facilitated electrophoretic analysis of particle movement. Results are reported as mean ± standard deviation (SD).¹²

Fourier Transform Infrared (FTIR) Analysis

FTIR was employed to assess the interaction and compatibility of the nanoparticle formulation components by identifying their functional groups, which influence the stability of the prepared and optimized formulations. Vibrational frequency peak analysis was carried out employing a Bruker alpha PN 100515/08 spectrometer (Germany) via the KBr pellet method, covering a spectrum range of 4000 to 400 cm⁻¹ and achieving a resolution limit of 2 cm⁻¹. FTIR spectra of vonoprazan, chitosan, SSTPP, chitosan nanoparticles, and drug-loaded chitosan NPs were carried out in pellet form, made from a small sample of potassium bromide, and examined using a Bruker FTIR spectrometer. All data will be obtained in the transmittance mode.¹³

Surface Morphology

SEM analysis of drug-loaded chitosan nanoparticles was performed using a scanning electron microscope (JSM IT 100 Jeol Japan) at an accelerating voltage of 20 kV. The powder samples were then fixed onto aluminum stubs and coated with gold using a sputter coater. Photographic images were obtained at magnifications of 500X and 2000X.¹⁴

Differential Scanning Calorimetry

A DSC study was performed for Vonoprazan, Chitosan, and drug-loaded Chitosan NPs using a Universal V4.1D TA Instruments calorimeter. A 5 mg sample was placed in a sealed aluminum pan. The heating process was performed from to 0–250 °C at a rate of 10 °C/min under a continuous nitrogen supply. A blank sealed pan was used as the reference.¹⁵

Thermogravimetric Analysis

The thermal properties of Vonoprazan, chitosan, and drug-loaded chitosan nanoparticles were studied using a thermogravimetric analyzer (SDT Q600 series, TA Instruments, New Castle, DE, UK) over a temperature range from ambient conditions to 600 °C. Dry samples weighing 5–10 mg were placed in an open platinum pan connected to a microbalance. Heating was performed at a rate of 10 °C/min in a nitrogen environment at a flow rate (fr) of 10 mL/min.

X-Ray Diffraction

Diffraction analysis of vonoprazan, loaded and unloaded formulations of chitosan-based NPs, was conducted using powder X-ray diffractometry (D-8, Discover, Germany) under operational conditions of 40 mA current and 40 kV voltage, with Cu Kα as the radiation source. The samples were scanned at a rate of 2°/min over a temperature range of 20–65 °C to examine the peak frequencies, shifts, and orientations of vonoprazan and both unloaded and loaded chitosan nanoparticles.¹⁶

Estimation of Percentage Yield and Entrapment Efficiency

The percentage yield of different formulations was calculated using the following formula:¹⁷

(1)

The encapsulation efficiency of the developed nanoparticle formulation was determined using an indirect method. The supernatants of the prepared nanoparticles were examined at 233 nm using a UV spectrophotometer (Shimadzu, UV-1700). The amount of free VPZ in the supernatant was estimated using a standard formula based on the absorbance obtained.¹⁸

(2)

Where VPZ_t is the total concentration of vonoprazan used in the preparation of nanoparticles and VPZ_f is the amount of free drug found in the supernatant.

Determination of Saturated Solubility

This study evaluated the equilibrium solubility of VPZ and chitosan nanoparticles in water and, pH 1.2 hydrochloric acid (HCl) buffer. Excess sample was added to a conical flask with buffer solution, agitated for 24 h, and then centrifuged at 4000 rpm for 15 min. The amount of drug in the supernatant was analyzed using a UV-visible spectrophotometer at 233 nm.¹⁹

Release Studies

Preparation of Standard Stock Solution

The UV-visible spectrophotometry method was used to estimate vonoprazan. The 1000 µg/mL drug solution exhibited maximum absorption (Lambda max) at 233 nm when scanned at 200–300 nm. To prepare standard stock solutions of pure drug, 100 mg vonoprazan was added to a 100 mL volumetric flask, containing 0.1 N HCl, and sonicated to dissolve completely. The working standard solutions were prepared by diluting the stock solution in 0.1 N hydrochloric acid.

In-vitro Release Studies

The in vitro release profile of drug-loaded chitosan nanoparticles was determined using a dialysis membrane (MWCO 12,000 g/mol; Sigma-Aldrich). Drug-loaded chitosan nanoparticles (5 mg) were placed in cellulose dialysis sacs previously soaked in distilled water and tested in dissolution apparatus 2 with 500 mL of media at pH 1.2, 6.8, and maintained at 37 ± 0.5°C. Samples were collected from the medium at various intervals (0, 0.5, 1, 2, 3, 4, 5, 8, 12, 16, and 24 h) and replenished with fresh hydrochloric acid buffer solutions for analysis. Similar dissolution studies were performed for the marketed drug product (Vonozan Tablets) using the same dissolution apparatus while maintaining sink conditions. The samples were filtered and analyzed spectrophotometrically at 233 nm.²⁰

Drug Release Kinetics

The drug release pattern was determined by fitting the results of in vitro release data into many kinetic models, including zero order, first order, Higuchi, Korsmeyer-Peppas, and Hixson-Crowell.²¹

Acute Oral Toxicity

The acute oral toxicity of drug-loaded chitosan nanoparticles was evaluated using the maximal tolerated dose (MTD) method, in accordance with OECD guidelines approved by the ethics committee of Sargodha University, Punjab, Pakistan (Ref. No.SU/ORIC/2077). Healthy adult New Zealand White rabbits (1.5 kg) (approximately 4–6 months old, were acquired from the Animal Laboratory Center at the University of Sargodha. The animals were placed in a clean facility and fed with a standard commercial rabbit pellet diet (approximately 16–18% crude protein, 2–4% crude fat, 12–18% crude fiber; [Safe Rabbit Form Pvt Ltd, Abbotabad, Pakistan] and provided with fresh drinking water ad libitum throughout the study along with tap water.²² Weight variation among the rabbits was maintained within ±20% of the mean weight. Drug-loaded chitosan NPs formulations were administered orally using an intubation cannula. The rabbits received a colloidal dispersion of NPs formulations. Clinical observations (activity, weight, diet, signs of illness, and mortality) were recorded twice daily over two weeks. On the final day of the experiment, the rabbits were fasted overnight for approximately 12 hours (with free access to water) before the induction of anesthesia. After 14 days, gross necropsy and blood biochemistry were conducted, with tissue samples preserved in 10% buffered formaldehyde for 48 h, paraffin-embedded, sectioned, and stained with hematoxylin and eosin for microscopic examination.²³

Pharmacokinetic Study

A pharmacokinetic study was performed to evaluate the bioavailability of pure drug (VPZ) in comparison to drug-loaded chitosan nanoparticles, administered at a dosage of 5 mg/kg, with the approval of the Pharmacy Research Ethics Committee at the University of Sargodha, Pakistan. This study involved six albino rabbits, each weighing 1.5–2 kg, that were fasted for 12 h before dose administration. One group received a pure drug suspension, whereas the second group was administered a drug-loaded chitosan nanoparticle formulation via a silicone rubber gastric intubation tube. Blood samples were collected from the jugular vein at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h intervals. Samples were processed by centrifugation at 4000×g, and the supernatant was preserved at −20°C. Analytical separation was performed using RP-UFLC with a Promosil C18 column (250×4.6 mm, 5µm) and monitored via a UV-visible detector, with chromatograms analyzed through Shimadzu LC Lab Solution version 7.1.1.²⁴

Relative bioavailability (F_rel) of the nanoparticle formulation relative to the pure drug suspension was calculated by using AUC0–∞ values derived by the linear trapezoidal method with extrapolation to infinity; since both formulations were given via the same route and dose, no normalization factor was required, and F_rel > 100% indicated enhanced bioavailability of the nanoparticle formulation.

Statistical Analysis

All tests were performed in triplicate, and the data are presented as mean ± SD. Statistical analysis was performed using Origin Pro 2023. Plasma concentration-time data obtained via RP-UFLC were used to calculate pharmacokinetic parameters for both the pure VPZ suspension and VPZ-loaded chitosan nanoparticle formulation using non-compartmental analysis (PK Solver), with all parameters expressed as mean ± SD (n = 6/group); PK parameters were compared between the two groups using Student’s t-test a p-value < 0.05 was considered statistically significant, and all statistical analyses were performed using GraphPad Prism.²⁴

Accelerated Stability Studies

An accelerated stability study was carried out in accordance with ICH guidelines over a period of six months at 40±2 °C and 75±5% RH to ascertain the formulations’ reliability and efficacy. Drug content and dissolution of drug was established using the assay technique, and changes in physicochemical parameters were evaluated for one, three, and six months.

Results and Discussion

Effect of Test Parameter on Size, Polydispersity, and Zeta Potential of Chitosan NPs

Various factors influence the initial size of nanoparticles during their preparation.²⁵ Table 2 presents the effects of formulation content on the size, polydispersity index, and zeta potential of chitosan NPs. When the concentration of chitosan is low, electrostatic repulsive forces predominate among chitosan molecules owing to the protonation of the amino group in the acidic solution, which leads to smaller nanoparticles. As the concentration of chitosan increases, the molecules come into closer proximity, and greater will be the intermolecular cross-linking increases, resulting in larger nanoparticles. Above the threshold concentration, attractive forces may predominate, causing aggregation and flocculation of nanoparticles.²⁶

Table 2.

Effect of Chitosan and STPP Concentration on Particle Size, Z.P, PDI, Yield and EE

Formulation	Particle Size (nm)	Zeta Potential mV	PDI	Yield	EE
VCHNP1	265.1±2.13	20.1±2.09	0.384±4.45	68.72±0.53	64.53±1.05
VCHNP2	289.2±3.07	22.2±3.21	0.393±3.92	71.32±0.52	69.12±0.72
VCHNP3	345.9±2.05	24.9±1.90	0.400±2.78	75.24±0.51	71.22±0.59
VCHNP4	496.5±1.19	25.4±3.07	0.474±1.97	86.11±0.33	78.09±0.41
VCHNP5	1163±2.07	*	1.001±4.07	80.30±0.84	70.12±0.14
VCHNP6	693.3±4.13	19.1±3.65	0.468±3.67	72.27±0.16	69.43±0.26
VCHNP7	793.5±3.54	10.9±3.43	0.474±1.87	68.45±0.77	65.21±0.09

Notes: *Zeta potential: Not measurable (or unreliable due to aggregation).

In contrast, decreasing the chitosan/STPP ratio up to a certain threshold limits results in smaller nanoparticles, as chitosan chains cross-link tightly with more available triple phosphate anion molecules. However, a further decrease in this ratio beyond the optimal point can cause the formation of larger particles or aggregates, as depicted by the formulations VCHNP6 (693.3 nm) and VCHNP7 (793.5 nm), which have a lower STPP concentration owing to excessive crosslinking and potential bridging between nanoparticles.²⁷ Similar results were observed in experiments by Bulmer et al²⁸ The polydispersity index (PDI), which reflects uniformity in particle size, is necessary in DDS. A lower PDI value is desirable because it indicates a more consistent particle size distribution, which is crucial for predictable drug release rates. However, achieving low polydispersity is difficult with chitosan-based nanoparticles owing to the different molecular weights of chitosan. The PDI of the nanoparticles tended to increase as the chitosan concentration increased. Increased intermolecular interactions caused by higher chitosan concentrations produced a wider size distribution and, as a result, a larger PDI.²⁹ In our Study, an increase in chitosan concentration from the formulation VCHNP1-VCHNP4, an increase in PDI. In a study conducted by Tsai et al,³⁰ lowering chitosan concentration reduced polydispersity.

The zeta potential refers to the electrical potential difference at the boundaries of a double layer that surrounds a particle in an aqueous solution. The external layer contains free ions, whereas the inner layer binds ions to the surface of the particle, defining its charge. The effects of different CH: STPP mass ratios (2:1 to 5:1) and (4:0.5 to 5:0.5) on the zeta potential of the nanoparticles were investigated. Results: (Table 2) Increasing the CH concentration increased zeta potential. The zeta potential rose from +20.1 for VCHNP1 to +25.4 for VCHNP4 (Figure 2).

Figure 2.

(A) Apparent Zeta potential of VCHNP4 (B) Size distribution of VCHNP4.

Decreasing the sodium tripolyphosphate (SSTPP) ratio in a chitosan-SSTPP system does lead to a decrease in the positive zeta potential of the nanoparticles, as the negatively charged SSTPP interacts with the positively charged chitosan, neutralising the surface charge³¹ as seen in the formulation VCHNP6 and VCHNP7, where the values decrease to 19.1 and 10.9. The zeta potential determines the colloidal system stability, with a solution above ±20 mV considered stable.³²

A high zeta potential is beneficial for controlling drug release rate. Understanding the particle surface charge is crucial for nanoparticle degradation and drug delivery due to its interaction with lysozymes. A high zeta potential can destabilize cell membranes, allowing nanoparticles to escape into cytoplasmic compartments.³³ Positively charged particles permeate better, but a much higher positive zeta potential is toxic to the cells. A zeta potential of +30 is optimal for drug delivery, with reduced cytotoxicity.³⁴

Grenha et al³⁵ showed that zeta potential increased with increasing chitosan concentration. This contradicts previous studies by Bulmer et al and Gan and Wang,³⁶ which suggested that the zeta potential increased with enhanced chitosan/STPP ratios as STPP anions neutralized protonated amino groups.

Percentage Yield and Entrapment Efficiency

The percentage yield of chitosan nanoparticles was greatly controlled by the amount of both chitosan and sodium tripolyphosphate (Table 2). Increasing the concentration of chitosan generally results in increased nanoparticle yield, as observed in the formulation from VCHNP1- VCHNP 4, i.e, 68.72±0.53% to 86.11±0.33%, respectively. This is because more polymer chains are available for crosslinking with STPP.³⁷ However, using too much chitosan may result in larger particles or aggregation, which can reduce overall stability, as in the case of VCHNP5. When the concentration of SSTPP decreased, the yield also decreased, as in the case of formulations VCHNP6 and VCHNP7, with a percentage yield of 72.27± 0.16% to 68.93±0.77%. The SSTPP concentration had a greater and more obvious impact on the yield than chitosan. Optimized STPP levels enhance the crosslinking efficiency and yield. Excess or less STPP can interrupt the optimal nanoparticle fabrication process, lowering yield and causing unstable particles^5,38 Entrapment efficiency of the formulation VCHNP1, VCHNP2, VCHNP3, VCHNP4, VCHNP5, VCHNP6, and VCHNP7 was found to be 64.53±1.05%, 69.12±0.72%, 71.22±0.59%, 78.09±0.41%, 70.12±0.14%, 69.43±0.26% and 65.21±0.09% respectively. The entrapment efficiency exhibited a positive linear correlation with increasing concentration of the CS solution, as demonstrated in Table 2. Increases in the concentration of chitosan (CS) increased the presence of protonated CS (-NH3+) in the system, as illustrated by the increase in zeta potential.

Consequently, the availability of binding sites for the crosslinker increased, leading to a greater entrapment efficiency. These findings were consistent with those of Kalam et al³⁹ However, % EE decreased when the amount of STPP decreased, despite the larger nanoparticle size. This could be attributed to the ability of chitosan to ionically interact with STPP ions, which is related to the presence of fewer STPP anions.⁴⁰

Saturated Solubility Studies

The saturation solubility of pure vonoprazan was evaluated relative to the nanoparticle formulations developed via the ionotropic gelation process, yielding p-values < 0.05. The equilibrium solubility of the drug-loaded chitosan nanoparticles significantly exceeded that of pure VPZ (Figure 3). Similar solubility studies showed a comparable improvement in the solubility of carbamazepine-loaded mesoporous silica particles. The increase in solubility was ascribed to the change in crystalline drugs into amorphous forms.

Figure 3.

Solubility comparison of pure drug and nanoparticle formulation in distilled water and 0.1 N HCl.

Fourier Transformed Infrared Spectroscopy (FTIR)

The chemical structures of the materials used for synthesizing drug-loaded chitosan nanoparticles were analyzed using FTIR spectroscopy. Figure 4 shows the FTIR spectra of CS, SSTPP, VPZ, unloaded chitosan nanoparticles, and drug-loaded nanoparticles, highlighting the specific functional groups of each component. Chitosan exhibited broad peaks at approximately 3375.01 and 3124.64 cm⁻¹, indicative of O–H and N–H stretching vibrations, respectively. Bands at 2866.06, 1647.8, and 1585.96 cm⁻¹ were ascribed to C–H stretching, amide I (C=O stretching), and amide II (N–H bending and C–N stretching), respectively. The signals at 1419.22, 1373.85, and 1322.99 cm⁻¹ correspond to CH₂ bending, CH₃ symmetric deformation, and in-plane bending of primary/secondary O–H groups. Strong peaks at 1059.4 cm⁻¹ (C–N stretching) and 1022.86 cm⁻¹ (C–O skeletal stretching) were identified in CS. These observations closely correspond to those of earlier reports.^41,42

Figure 4.

FTIR analysis of Chitosan, Vonoprazan, STPP, unloaded and vonoprazan loaded NPs.

A Comparison of the FTIR spectra of chitosan, SSTPP, and unloaded CHNPs (Figure 3) showed noticeable peak shifts after nanoparticle formation. The characteristic –NH₂ stretching band of chitosan at 3375.01 cm⁻¹ moved to 3357.18 cm⁻¹ in CHNPs, suggesting the interaction of chitosan with SSTPP. Likewise, the amide I (1647 cm⁻¹) and amide II (1585 cm⁻¹) peaks of chitosan shifted to 1636.25 and 1542.9 cm⁻¹, respectively, indicating participation of chitosan’s amino groups and SSTPP’s phosphate groups in the crosslinking process. Moreover, the intense 1166 cm B-¹ band associated with the –COOH groups of SSTPP disappeared in the CHNPs spectrum, confirming successful and complete crosslinking between chitosan and SSTPP.⁴³ In the case of vonoprazan fumarate, the broad phenolic O–H stretching bands experienced a downward shift of about 60–200 cm⁻¹, overlapping with the C–H stretching region around 3009.5 cm⁻¹. These notable changes suggest that the phenolic O–H groups participated in strong intermolecular hydrogen bonding, resulting in the emergence of new molecular arrangements without the transfer of protons. The peak observed at 3321.7 cm⁻¹ was attributed to N–H stretching. The characteristic pyridine C=N stretching band of VPZ was observed at 1562 cm⁻¹, aligning with its typical range of 1640–1557 cm⁻¹.

The signal observed at 1637.2 cm⁻¹ indicated C=C stretching. Furthermore, the C–N stretching of the aromatic amine was observed at 1333.4 cm⁻¹, which is consistent with the anticipated range of 1340–1260 cm⁻¹. The peaks observed at 1370.5 and 1199.2 cm⁻¹ were assigned to the stretching vibrations of S=N and S=O, respectively.⁴⁴ To determine whether the encapsulation of the drug within the nanoparticles was chemical or physical entrapment, FTIR analysis was employed. Physical entrapment could be anticipated if there were no or very few changes in the IR spectrum compared to the parental compounds, whereas a potential chemical interaction between VPZ and CHNPs would explain a spectral shift.

A comparison of FTIR spectra between unloaded and drug-loaded chitosan NPs (Figure 3) showed no spectral alterations, indicating that VPZ was physically entrapped (encapsulated). While interpreting the FTIR spectra of drug-loaded chitosan NPs, it was noticed that some new peaks appeared that were completely different from the parent compound, and several peaks exhibited shifts towards higher or lower frequencies. Furthermore, a notable increase in the intensity of the C–H stretching bands was observed at 2885.12 and 1378.55. The peak at 1637.2 cm−1 shifted to 1632.55 cm−1, whereas the peak at 1562.0 cm−1 shifted to 1531.92 cm−1. FTIR analysis of the drug-loaded CHNPs formulation showed the disappearance of certain bands associated with vonoprazan, confirming the successful entrapment of the drug within the polymer matrix. These findings are consistent with those reported in a previous study.⁴⁵

Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) was performed on pure chitosan, VPZ, and drug-loaded chitosan nanoparticles, as shown in Figure 5. The DSC profile of chitosan displayed a broad endothermic transition at approximately 94.6°C, extending to an enormous temperature range. This peak signifies the evaporation of physically adsorbed moisture, reflecting the energy necessary to remove water bonded to the polymer.⁴⁶ It also shows an exothermic peak at 300.5, which represents degradation due to depolymerization and dehydration. These results agree with those of Nair and Morris et al⁴⁷

Figure 5.

DSC thermograms of chitosan, vonoprazan, and Vonoprazan loaded NPs.

The DSC thermogram of pure crystalline vonoprazan fumarate shows an endothermic peak at 209.1 °C, indicating its melting point, with no other thermal events up to 200 °C, demonstrating good thermal stability and the absence of hydrated forms. The fumarate salt of VPZ is used commercially because VPZ free base has lower thermal stability, melting at 67.3 °C.⁴⁴ Drug-loaded chitosan nanoparticles retained their original peaks, indicating no component interactions, but exhibited a broader peak and reduced melting point, with an endothermic peak at 126.3°C. This reduction is likely due to the interaction between VPZ and Chitosan, causing broadening and a shift of the chitosan melting peak to lower temperatures.⁴⁶ The decrease in crystallinity observed in encapsulated vonoprazan can be linked to the development of nanoparticles. The objective of DSC analysis was to investigate the anticipated change or decrease in melting point resulting from NP formation, which ultimately signified a shift from a highly crystalline state to a less crystalline state.⁴⁸

Thermogravimetric Analysis

The thermogravimetric analysis (TGA) curve indicated that the sample retained its weight when heated to 100 °C, and the crystal formation lacked planar water and solvent, as illustrated in Figure 6. The first degradation started at 210 °C and 60% weight loss occurred at 380 °C, but vonoprazan showed good thermal stability up to ~200°C, making it suitable for most processing conditions. In the case of the initial polymer degradation at 70 °C due to the loss of moisture, a gradual weight decline between 280–310 °C, where 40% weight loss occurs, and degradation takes place above 350 °C due to the breakdown of the deacylated chain. A 10% weight loss below 100 °C occurred because of the loss of moisture in the VCHNPs formulation, while 50% weight loss occurred at more than 300 °C, indicating efficient encapsulation of the drug as well as good thermal stability within the nanoparticles.

Figure 6.

TGA thermograms of chitosan, vonoprazan, and VCHNPs.

Powder X-Ray Diffraction

The X-ray powder diffractions that it is represented with the 2 θ ± 0.2 ° angles of diffraction, are at 7.64, 11.48, 12.7,13.2, 15.96, 17.76, 18.04, 19.56, 21.68, 22.32, 24.36, and 25.96 due to its crystal structure. The studies reported in the literature support this finding. The magnitudes of the XRD peaks were associated with the crystalline form of the material. All the drug peaks were visible in the chitosan-loaded nanoparticles, but the intensity of the peaks was noticeably reduced, and there was a dramatic reduction in d-spacing, similar to that reported by Sing and Pathak et al⁴⁹ When compared to the diffractograms of loaded and unloaded nanoparticles, a broad peak appeared at 2θ = 11.42 °in unloaded chitosan NPs, whereas the drug-loaded chitosan NPs showed peaks at 7.68,11.3, and 13.0°, confirming the entrapment of vonoprazan into chitosan nanoparticles with reduced intensities. This demonstrated that, in contrast to the pure drug, the drug in the loaded nanoparticle was amorphous. The reduction in crystallinity is associated with crosslinking between the reactive groups of chitosan and sodium tripolyphosphate, as well as additional hydrogen-bonding interactions with the drug. These alterations indicate that intermolecular interactions disturb the initial crystalline structure and demonstrate the miscibility of the drug within the nanoparticle matrix.⁵⁰

A more amorphous state dissolved more rapidly than the crystalline form, which increased the dissolution rate of the drug. It has a higher internal energy and thermodynamic properties.⁵¹ Figure 7 shows the X-ray diffractograms of the pure drug, loaded, and unloaded nanoparticles.

Figure 7.

PXRD diffractograms of Vonoprazan, vonoprazan-loaded and unloaded NPs.

Scanning Electron Microscopy

The SEM study of drug-loaded chitosan nanoparticles demonstrated predominantly spherical particles with noticeable inclusion, indicating effective drug incorporation (Figure 8). The particles exhibited a uniform size distribution and were distributed uniformly within the sample. The nanoparticles were well dispersed with minimal signs of clumping, indicating a stable colloidal formulation. Similar results were reported by Ramachandran T, 2024 et al⁵²

Figure 8.

SEM analysis of VCHNPs at different magnifications.

In-vitro Release Study

Nanoparticle (NP) formulations demonstrated that sustained release of Vonoprazan (VPZ) was achieved with NPs, which was attributed to the cross-linking between the amino groups of chitosan and the phosphate ions of tripolyphosphate (STPP) and the mucoadhesive nature of chitosan. At 5 hours, 88.51 ± 0.12% of Vonopraan was released from the NPs. The mucoadhesive properties of chitosan allow for better retention of the drug in the absorption window, enabling controlled and prolonged release of VPZ in the gastric mucosa. This suggests that NPs could enhance the bioavailability of the drug significantly by ensuring prolonged availability for absorption, which is anticipated to improve its therapeutic efficacy, especially near its site of action, that is, the esophagus. These findings coincide with the studies previously performed by Modi J, Joshi G, Sawant K, 2013 et al.⁵³

Higher drug release was observed due to the pH-sensitive properties of chitosan. At a lower pH (pH 1.2), the amino groups of chitosan are ionized or “swollen” when they absorb hydrogen ions, causing an increase in solubilization of the polymer, thereby increasing the diffusion and delivery rate of drugs. Conversely, at a higher pH (pH 6.8) chitosan has less swelling and becomes a tighter solid matrix that restricts diffusion and drug delivery.

The release characteristics observed will be useful since these nano-particles are being developed as a gastro-retentive mucoadhesive system. Chitosan’s mucoadhesive properties provide an extended duration for the particles to remain within the gastric lumen prior to transit into the intestine. Given that vonoprazan is stable at low pH and rapidly absorbed upon ingestion, the increased amount of drug released during its time in the stomach would improve the bioavailability of the drug.⁵⁴ Protonation of the amino groups of chitosan, which increases solubility and polymer swelling, is responsible for the increased drug release at acidic pH (Figure 9). Deprotonation leads to decreased solubility and the emergence of a dense matrix at a higher pH (6.8) (Figure 10), which restricts drug diffusion and drug release.⁵⁵

Figure 9.

In vitro drug release profiles of VCHNPs formulations at (pH 1.2). Data are presented as mean ± standard deviation (SD), n = 6.

Figure 10.

In vitro drug release profiles of VCHNPs formulations at (pH 6.8). Data are presented as mean ± standard deviation (SD), n = 6.

Therefore, to elucidate the precise mechanism of release, the data obtained from in vitro drug release studies were fitted into different kinetic models, as shown in Table 3, following the Korsmeyer–Peppas equation. The regression coefficient was 0.9924, indicating an excellent model fit.⁵⁶ Chitosan, a hydrophilic swellable polymer, is recommended for spherical systems, such as nanoparticles. “n values less than 0.5 indicate Fickian diffusion, while values between 0.5 and 1.0 indicate anomalous transport”.⁵⁷

Table 3.

Correlation Coefficient (R²) and Release Exponent (n) of Different Kinetic Models

Sr No	Drug release kinetic		VCHNP1	VCHNP2	VCHNP3	VCHNP4	VCHNP6	VCHNP7
(A) Simulated Gastric fluid
1	Zero order Model	R2	0.9011	0.9828	0.9826	0.9637	0.9394	0.8973
2	First order Model	R2	0.9748	0.9423	0.9206	0.8811	0.8358	0.7813
3	Higuchi Model	R2	0.9001	0.8364	0.7963	0.7366	0.6850	0.6284
4	Korsmeyer- peppas	R2	0.9647	0.9843	0.9873	0.9840	0.9909	0.9924
		n	0.533	0.479	0.469	0.373	0.413	0.496
5	Hixson-crowell	R2	0.7222	0.5594	0.4806	0.0167	0.2436	0.5948
(B) Simulated Intestinal fluid
1	Zero-order Model	R2	0.6779	0.4960	0.4416	0.1315	0.1518	0.5453
2	First-order Model	R2	0.7427	0.5893	0.5111	0.3860	0. 2876	0.6183
3	Higuchi Model	R2	0.9922	0.9728	0.9042	0.8704	0.8770	0.9525
4	Korsmeyer- peppas	R2	0.9949	0.9741	0.9071	0.9475	0.9052	0.9525
		n	0.511	0.666	0.717	0.786	0.938	0.908
5	Hixson-crowell	R2	0.9363	0.9580	0.9511	0.9313	0.9079	0.8738

Clinical and General Signs of Toxicity

In accordance with OECD guideline 423, the acute oral toxicity of the drug-loaded chitosan nanoparticle formulation (VCHNP4) was evaluated in male rabbits over a 14-day observation period at a dose of 2000 mg/kg. Throughout the trial, none of the animal died. Daily monitoring of body weight, food intake, and water intake revealed no discernible differences between the treatment and control groups (Table 4). No anomalies were found in the physical or behavioral observations; the fur, skin, eyes, and activity patterns were all normal. No obvious signs of organ abnormalities, vomiting, or discharge from the nasal cavity.⁵⁸ Overall, the absence of pathological or clinical alterations suggests that the VCHNP4 formulation was safe at the tested dosage and did not cause acute toxicity in rabbits.

Table 4.

Influence on Body Weight, Food Consumption, and Water Intake of Rabbits Group A and Group B (VCHNPs)

Observation	Group A (Control)	Group B(VCHNPs)
Sign of illness	No	No
Body weight (Kg)
Day-1	1.87±0.08	1.89±0.15
Day-3	1.89±0.71	1.90±0.19
Day-7	1.91±0.13	1.94±0.29
Day-14	1.93±0.12	1.96±0.14
Food Consumption (g)
Day-1	73.63±0.05	77.30±1.27
Day-3	74.81±0.23	75.16±0.29
Day-7	77.32±0.08	76.04±0.22
Day-14	75.20±0.04	75.35±0.14
Water intake (mL)
Day-1	198.5±0.05	201.5±0.13
Day-3	201.6±0.12	206.5±0.82
Day-7	200.4±0.15	203.7±1.39
Day-14	203.5±1.13	198.6±1.15

Note: All values are expressed as mean ±SD.

Biochemical Blood Analysis

Blood serves as a critical indicator of chemical toxicity, leading to various physiological and pathological conditions.¹⁴ The blood chemistry, liver function test, renal function test, and lipid profiles of the placebo (control) and treatment groups are shown in Table 5. Haematological, hepatic, renal, and lipid profiles of rabbits treated with drug-loaded chitosan-based nanoparticles showed no significant changes compared to the controls, with all values remaining within normal reference ranges. The platelet distribution width (PDW) and mean platelet volume (MPV) were slightly increased because of the cationic and immunomodulatory properties of chitosan, which can cause mild platelet activation and immune interaction. These variations were minimal and within the physiological limits. Overall, these findings indicate that the VCHNP4 formulation is well tolerated and has no adverse hematological or biochemical effects, suggesting its safety for future pharmacological studies.

Table 5.

Biochemical Blood Analysis of Group A and Group B (Chitosan Nanoparticles)

Parameter	Group A(Control)	Group B (VCHNPs)
Hb (g/dL)	12.10±0.14	11.9±1.12
WBCs (103/μL)	6.90±1.19	5.33±0.16
RBCs (106/μL)	4.06±1.12	3.42±1.06
Platelet (103/μL)	354±1.13	297±1.52
Hematocrit (%)	35.8±1.89	34.1±0.19
Monocytes (%)	03±1.12	02±1.12
Neutrophils (%)	58±1.88	59.8±0.68
Lymphocytes (%)	38.7±1.76	69.3±1.14
Basophils (%)	0.1±1.55	0.0±0.00
MCV (fl)	73.2±0.05	70.4±1.34
MCH (pg)	22.7±0.03	20.4±1.41
MCHC (g/dL)	30.4±0.15	28.9±1.21
MPV (fl)	8.3±0.06	10.5±1.45
PDW (fl)	16.4±0.01	17.2±1.19
ALT (U/L)	49±1.74	45±1.21
Alkaline Phosphate (U/L)	148±1.98	138±1.92
Total bilirubin (mg/dL)	0.5±0.19	0.6±0.12
Creatinine (mg/dL)	0.8±1.12	1.0±1.98
Urea (mg/dL)	29±0.23	30±0.1.90
Cholesterol (mg/dL)	52±0.14	58±1.84
Triglyceride (mg/dL)	106±0.26	109±1.54

Note: All values are expressed as mean ±SD.

Histopathological Examination

No histopathological lesions were observed in the liver, heart, kidney, intestine, or stomach of the VCHNP-treated group (Figure 11). Organ weights within the normal ranges are illustrated in Table 6. The livers of rabbits exhibited a normal morphology of hepatic cells radiating from a central vein, also sinusoids, and Kupffer cells.⁵⁹ The tubules in the kidneys maintained a normal epithelial lining arrangement and lumen thickness. Oval-to-round enteroendocrine cells were dispersed within the glandular epithelium. The glands were encased in collagenous and smooth muscle fibers. The folded mucosa of the fundic part comprises the epithelium and lamina propria, which carry fundic glands surrounded by submucosa containing diffuse smooth muscle bundles.⁶⁰ Histological examination of the small intestine revealed intact intestinal villi with well-preserved epithelium and lamina propria.⁶¹ The cardiac muscle showed numerous branching cells that contained one or two central nuclei interconnected by intercalated discs. These are manifested as excellent lines between the cardiomyocytes.⁶² Thus, the vital organs were found to be devoid of major pathologies.

Figure 11.

(A) Hepatic histology of VCHNPs rabbits showing normal hepatocytes and portal veins. (B) Renal histology showing normal renal tubules and glomeruli. (C) Stomach showing well-diffused smooth muscle bundles; (D) Small intestine showing normal villi; (E) Heart showing normal myocardial integrity.

Table 6.

Effect of Oral Administration of Vonoprazan-Loaded Chitosan Nanoparticles on the Organ Weight (g) of Rabbits

Treatments Groups	Control	Treatment
Liver	28.6±1.71	37.1±0.18
Spleen	0.4±1.03	0.3±1.13
Stomach	16.0±1.11	16.6±1.20
Intestine	4.12±1.40	4.5±0.2.11
Kidney	3.5±1.04	3.6±1.12
Heart	3.6±1.07	3.5±1.18

Note: All values are expressed as mean ±SD.

Pharmacokinetic Study

The Peak plasma concentrations of the oral VPZ solution and the optimized VCHNP4 nanoparticle formulation were compared using pharmacokinetic studies in two groups of healthy rabbits (Figure 12). The evaluation included several key pharmacokinetic parameters, as shown in Table 7. Plasma concentration data were analyzed with a non-compartmental model using Kinetica 4.4, revealing significant pharmacokinetic differences between the drug suspension and drug-loaded chitosan nanoparticles, highlighting the benefits of the VCHNP4 formulation. The formulation exhibited a higher Lambda-z (0.186 ± 1.23 h⁻¹) in comparison to the VPZ suspension (0.077 ± 0.34 h⁻¹), indicating a more rapid elimination rate.

Figure 12.

Plasma concentration-time profile of VPZ in rabbits after equivalent single oral dose administration of VPZ and VCHNPs.

Table 7.

Comparative Pharmacokinetic (PK) Profile of VPZ Drug Suspension and VCHNPs

Sr.No	Parameters	VPZ Suspension (Mean ± SD)	VCHNPs (Mean ± SD)	p-value
1	Lambda_z	0.077± 0.34	0.186± 1.23	<0.0001
2	t1/2 (h)	3.76± 1.67	8.96± 1.20
3	Tmax (h)	2 ± 1.53	4 ± 1.84
4	Cmax (ng/mL)	41 ± 1.01	307 ± 1.61
5	Clast_obs/Cmax	0.365± 1.14	0.169± 1.89
6	AUC 0-t (ng/mL/h)	133.33± 2.09	3459.87± 1.15
7	AUC 0-∞ (ng/mL/h)	214.85± 1.13	4132.61± 1.18
8	AUMC 0-∞ (ng/mL/h)	1331.67 ± 1.04	60,069.51± 1.24
9	MRT	6.19± 1.62	14.53± 1.20

The half-life (t_1/2) of VCHNP4 was extended (8.96±1.20 h), in contrast to (3.76 ±1.67 h) for the drug suspension, reflecting prolonged systemic retention. The Tmax was higher (4±1.84 h for VCHNP4 than for 2±1.53 h, which showed a controlled and sustained mechanism of drug release. The increased values of Cmax for VCHNP4 307 ±1.61 ng/mL compared to the drug, i.e, 41±1.01 ng/mL, indicate improved solubility and absorption.

The area under the concentration–time curve for VCHNP4 was much higher than VPZ suspension (3459.87±1.15 ng·h/mL compared to 133.33±2.09 ng·h/mL), showing enhanced bioavailability and a more efficient systemic drug delivery profile. The reduced Clast_obs/Cmax ratio for VCHNP4 (0.169±1.89 compared with 0.365±1.14) facilitated steady plasma concentrations over time. The area under the moment curve (AUMC) was 60069.51 ±1.24 compared to 1331.33 ±1.04, and the mean residence time (MRT) was 14.53 ±1.20 versus 6.19 ±1.61, indicating that the elimination of the drug will be slower and reside in the blood and targeted tissue for a prolonged time as compared to the free drug, which will be cleared abruptly.⁶³ The results highlight the improved pharmacokinetic profile of the VCHNP4 formulation achieved via its nanoparticle-based formulation. The pharmacokinetic advantages of the chitosan-based NPs formulation were highlighted by the Student’s t-test findings, which showed significant differences (p ≤ 0.05) between the pure drug and the VCHNPs formulations. The relative bioavailability of the VPZ-loaded chitosan nanoparticle formulation was found to be approximately 1923.8% (≈19.2-fold) higher than that of the pure drug suspension, calculated as (AUC0–∞ nanoparticle/AUC0–∞ suspension) × 100, indicating a marked improvement in oral bioavailability attributable to the nanoparticle delivery system. Pharmacokinetic studies highlight the efficacy of mucoadhesive chitosan nanoparticles as a drug delivery system that can enhance bioavailability, sustain release, and improve the therapeutic performance of poorly absorbed or unstable drugs.⁶⁴

Accelerated Stability Studies

The accelerated stability study assessed the chitosan nanoparticle formulation for 1, 3 and 6 months at 40 ± 2°C and 75 ± 5% RH, following ICH recommendations. Table 8 depicted drug content showed little deterioration and stayed within normal limits, according to the results. Dissolution profiles showed no appreciable variations from the original formulation, indicating that the nanoparticles maintained their release characteristics. Overall, the analysis indicates that the formulation has good stability and a respectable shelf-life potential.

Table 8.

Accelerated Stability Studies of Optimized Formulation (VCHNP4)

Feature	1 Month	3 Month	6 Month
	VCHNP4	VCHNP4	VCHNP4
Assay	93.76 ± 2.3	91.20 ± 2.6	90.56 ± 3.0
% DD	78.96 ± 1.7	74.89 ± 1.8	70.96 ± 1.9

Conclusion

Vonoprazan-loaded Chitosan nanoparticles were synthesized via an ionotropic gelation process using sodium tripolyphosphate as the cross-linking agent. The concentrations of chitosan and STPP, as well as the CS/STPP weight ratio, were optimized to achieve the optimal particle size and highest drug entrapment efficiency. Chitosan has numerous benefits for nanoparticle development, such as biocompatibility, biodegradability, and low immunogenicity. A higher positive charge density imparts mucoadhesive characteristics, rendering it an ideal option for drug administration to mucosal regions. The nanoparticles exhibited an effective entrapment efficiency, moderate particle size, and high positive zeta potential. FTIR analysis revealed a strong interaction between VPZ and chitosan. DSC, TGA, and PXRD analyses revealed no incompatibilities between the drug and polymer, indicating that the drug was incorporated into the polymer matrix in an amorphous form and was thermally stable In vitro study observed sustained drug release over 5 h, adhering to the Fickian transport mechanisms from nanoparticles. This is expected to increase the absorption, bioavailability, and therapeutic efficacy of vonoprazan. Acute oral toxicological studies and in vivo pharmacokinetic data have confirmed that VCHNPs are non-toxic, biocompatible, safe, and have improved oral bioavailability. Consequently, Vonoprazan delivered via chitosan nanoparticles could be a suitable alternative to the traditional dosage form for the treatment of acid-related diseases. However, additional long-term stability studies, comprehensive toxicity assessment, and detailed preclinical investigations are needed before clinical translation.

Limitation of the Study

The present study has some limitations that should be acknowledged. The pharmacokinetic study was conducted with a relatively small sample size, and the safety evaluation was limited to acute oral toxicity following OECD Guideline 423. Histopathological assessment was qualitative, without quantitative scoring. Although accelerated stability studies demonstrated acceptable stability of the optimized formulation, long-term stability under recommended storage conditions was not evaluated. Furthermore, no biodistribution or active targeting studies were performed, as the formulation was developed as a mucoadhesive oral nanoparticulate delivery system to enhance the oral bioavailability of vonoprazan. Future studies should focus on long-term stability, extended toxicity evaluation, biodistribution, and clinical translation to further establish the therapeutic potential of the developed formulation.

Data Sharing Statement

The authors confirm that data supporting the findings of this study are available within the article.

Ethical Approval Statement

This study was approved by the Research Ethics Committee of the Faculty of Pharmacy, University of Sargodha, Sargodha, Punjab, Pakistan (Ref. No.SU/ORIC/2077). All experimental procedures were conducted according to institutional ethical guidelines.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that they have no conflicts of interest.