Materials

The VLG was procured from Swapnroop Drugs distributor in Cha. Sambhajinagar (India). Low molecular weight chitosan (50,000–190,000 Da.) was procured from Sigma-Aldrich, USA. Dextran sulfate sodium salt and neutral red provided by SRL Pvt. Ltd. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), α-amylase supplied by HiMedia, India. The L6 cell line was supplied by the National Centre for Cell Science (NCCS), Pune (India). Additional chemicals and reagents were used of analytical grade.

Fabrication of VLG-loaded cross-linked chitosan–dextran nanoparticles (VLG-CDNs)

VLG-encapsulated chitosan–dextran sulfate nanoparticles (VLG-CDNs) were prepared using ionic gelation techniques assisted with microreactor (Amar 2 metal), a microfluidic device supplied by Amar Equipment Pvt. Ltd, India [26, 28, 37,38,39,40]. The nanoparticle fabrication process involves using two inlets to infuse two solutions (solution A and solution B) during the preparation of nanoparticles within the microreactor (Supplementary material S1). In solution A, 50 mg CS was dissolved in the 0.5% acetic acid solution; this solution was stirred for 15–20 min to obtain a clear solution. Tween 80 (0.5% w/v) was introduced to the CS solution as a stabilizer. VLG (25 mg) was dissolved in distilled water and then dropwise added into the CS solution using a syringe. Furthermore, solution B is prepared by dissolving the 0.1% w/v DEX in distilled water. Solutions A and B were constantly stirred at 600 rpm on a magnetic stirrer (REMI, India). Afterward, both solutions were distinctly sonicated for 15 min and filtered using a 0.45 µm filter [37]. The nanoparticle is produced by passing solution A and solution B via a syringe with the help of an infusor (Uni Labs) to uphold a constant flow rate over the specified duration, connecting to the Amar 2 metal microreactor inlets. The resulting nanosuspension from the microreactor outlet was collected in a container under continuous magnetic stirring (600 rpm) at room temperature.

Optimization of nanoparticles using Box–Behnken Design (BBD)

Initially, we conducted preliminary experiments to identify the main factors and their appropriate ranges for optimization. The impact of three key process parameters, namely concentration of polymers and stabilizer and drug content, on particle size, polydispersity index and EE were systematically examined. The BBD is suitable for determining the effects of the independent variable or factors, i.e., drug and excipients, and their allied outcomes on the dependent variables. A BBD comprising 3 factors, 3 levels, and 17 runs was produced for optimization of nanoparticles by using Design-Expert® software version 13.0 (state-Ease Inc., Minneapolis, USA). The independent variables were specified as the concentration of polymers and stabilizer. Moreover, particle size (Y1), PDI (Y2), and EE (Y3) were selected as dependent variables (response). The independent variables are investigated at three distinct levels: low (− 1), medium (0), and high (+ 1), as illustrated in Table 1. The statistical data were displayed, and the quadratic model was fitted to the data depicted in Table 3. Furthermore, the lack of fit F value of the selected model was observed to be significant. To substantiate the selected experimental domain and polynomial equation, two optimal checkpoint formulations (T1 and T2; Table 1) were chosen and a design expert created the 3D response surface plots. The estimated regression coefficients and p value of the factor are detailed in Table 2. Only the model terms deemed statistically significant were taken into consideration.

Table 1 Independent variables and dependent variables: factors and their levels for the Box–Behnken designTable 2 Box–Behnken design independent variables and conforming results for the dependent variablesPhysicochemical characterizationParticle size distribution, polydispersity index, and surface charge or zeta potential

The average particle size and PDI of CDNPs were examined by dynamic light scattering (DLS) using Zetasizer (Nano ZS 90, Malvern Instruments Ltd., UK). The CDNPs were suitable diluted in deionised water at approximately 1:10 ratio, and then, the sample was added to the sample cell and equilibrated at 25 °C for 60 s before measurement [25, 29]. The analysis was executed in triplicate, and the results were expressed as the mean size of particles ± SD. The zeta potential of the CDNPs was assessed through an electrophoretic light scattering technique employing a DLS using a Zetasizer instrument (Nano ZS 90, Malvern Instruments Ltd., UK). A diluted sample in water was allowed to reach stability at 25 °C and was introduced into transparent disposable zeta cells [41]. These experiments were conducted in triplicate.

Surface morphology

The morphology of optimized VLG-CDNPs was ascertained by employing field emission scanning electron microscopy (FE-SEM) (JSM IT300LV, JEOL, Japan). The nanoparticle samples were dotted on a silica wafer, and air-drying was performed. Afterward, the samples were attached to the aluminum stub anchored on the holder and sputtered with a gold coating for 50 s using a coater (JCE-3000FC, JEOL, Japan). Then, sample holders were placed in the instrument, and photomicrographs were acquired with an accelerating voltage of 10 kV with a secondary electron detector. High-resolution transmission electron microscopy (HR-TEM) was used to analyze the internal structure of nanoparticles with a JEM1400 TEM (Japan). For sample preparation, a diluted nanoparticle suspension was deposited on a carbon-coated copper grid to create a thin film, which was then air-dried at room temperature. The optimized batch of VLG-CDNPs was examined using HR-TEM. The grids were treated with 2% w/v phosphotungstic acid before imaging. Images were captured at various magnifications using a 200 kV electron beam [42, 43].

Encapsulation efficiency (EE)

To determine the entrapment of VLG in the polymer matrix of VLG-CDNPs, the prepared nanosuspension was subjected to centrifugation at 10,000 rpm at 4 °C (Thermo Scientific Sorvall ST 8R). The resulting supernatant was analyzed using a UV–visible spectrophotometer (HITACHI U-2900, Tokyo, Japan) at 205 nm for an unentrapped drug. The EE was determined by using the subsequent equation.

$$}\left(\right)=\frac}}\times 100$$

(1)

Development of spray-dried  formulations

To enhance the long-term stability of VLG-CDNPs, dehydration was executed through the application of a spray drying technology with the addition of matrix formers, yielding a dry powder. Briefly, a VLG-CDNPs nanodispersion and mannitol were meticulously suspended in Milli-Q water in a 1:2 (w/w) ratio. The resulting dispersion underwent continuous magnetic stirring and was subjected to atomization via a 0.5 mm nozzle of a laboratory spray dryer (Spray Mate JISL, Mumbai). The optimization of enmeshed process parameters ensued, with the inlet temperature set at 110 °C, atomization pressure at 1.2 kg/cm2, feed rate at 1 mL/min, and vacuum maintained within the range of 100–110 mm. Ultimately, the prepared spray-dried powders were precisely collected and hermetically stored in a sealed glass in a desiccator. As discernible indicators of the efficacy of the spray drying process, pivotal parameters, including yield, size, and morphology, were examined across varied process conditions. The mean particle size was determined by measuring the projected area diameter of 50 randomly selected particles from SEM micrographs using ImageJ software (Maryland, USA).

Redispersibility

The effectiveness of spray-dried powders to redisperse into nanoparticles was assessed using a previously reported method [44]. The dispersibility of dried powder was measured in terms of redispersibility index (RDI). To investigate the redispersibility of the spray-dried nanoparticle powder, 4 mg of VLG-CDNPs was dispersed in 2 mL of distilled water and stirred for 30 min. Subsequently, the resulting dispersion of VLG-CDNPs nanoparticles subjected for assessment of particle size and zeta potential [42, 44]. The RDI of the nanosuspension was calculated using the subsequent formula.

Solid-state characterizationFourier-transform infrared spectroscopy (FTIR)

FTIR spectra of CS, Dex, VLG, and VLG-CDNPs were obtained using a Bruker Vector 22 FTIR Spectrometer (GmbH, Germany), equipped with crystal sampler attenuated total reflectance (ATR) cell. A small amount of each sample was added to the ATR cell for analyzing spectra. For each sample, 11 scans were recorded between 4000 and 600 cm−1 with a resolution of 2 cm−1 as in the transmission mode [45,46,47,48].

X-ray diffraction study (XRD)

XRD analysis was performed to investigate physical nature of CS, Dex, VLG, and VLG-CDNPs at room temperature. Diffractograms or XRD spectra of powdered samples were acquired using a Bruker AXS (D8Focus XRD) system with Cu Kα radiation (λ = 1.54 Å). The powdered samples were loaded into plastic sample holders, and the surfaces were leveled with a glass slide.

Differential scanning calorimetry (DSC)

The melting and crystallinity behavior of CS, Dex, VLG, and VLG-CDNPs were analyzed using a differential scanning calorimeter (DSC-7020 Hitachi High-Tech Corporation, Japan). The DSC analysis was performed at a heating range from 30 to 300 °C with a heating rate of 20 °C/min under the stream of nitrogen gas flow (60 ml/min N2). The samples were weighed in the aluminum crucible (~ 5 mg).

Thermogravimetric analysis (TGA)

The thermal stability of VLG and VLG-CDNPs was determined by using a thermogravimetric analyzer (TGA-55, TA Instruments, USA). Each sample was positioned on a platinum HT pan (~ 5 mg), and analysis was guided at a heating rate of 20 °C per/min in the temperature range of 20–600 °C.

In vitro drug release

The in vitro drug release studies were conducted for VLG and VLG-CDNPs in two different media maintained at pH 1.2 and 6.8 using a dialysis bag (cellulose membrane with an average molecular weight cut off 12,000 Da). Briefly, 25 mg of VLG and VLG-CDNPs was introduced in 0.1 M phosphate buffer saline (PBS) maintained at pH 1.2 and 6.8. Nanoformulation and VLG were added in the designated dialysis bag or membranes suspended in a 100 mL beaker containing 0.1 M PBS kept constant stirring at 50 rpm. The samples were extracted and replenished with equivalent volumes of preheated PBS at predefined time intervals (0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h). The chosen time points for the in vitro drug release study of sustained-release nanoparticles are carefully selected to prevent dose dumping, characterize the release profile, and confirm that more than 80% of the drug is released. Initial measurements at 0, 1, 2, 4, and 6 h capture the immediate and early release phases, which is crucial for understanding the initial drug release kinetics. Intermediate measurements at 12 and 24 h assess the nanoparticles’ ability to sustain therapeutic drug levels over a full day, which is essential for once-daily or twice-daily dosing regimens. The levels of VLG in the samples were quantified using a UV–visible spectrophotometer at wavelengths 205 nm (Hitachi U-2900, Japan). These experiments were repeated in triplicate (n = 3).

Stability study of VLG-CDNPs

The VLG-CDNPs powder samples were placed inside glass bottles and subjected to storage for six months. This storage occurred under controlled conditions, specifically at 4 °C and 25 °C, within a stability chamber (Mack Pharmatech, India). Throughout this period, the formulation underwent periodic assessments, including evaluations at the first-, third-, and sixth-month intervals. These evaluations examined their alterations in particle size, PDI, and zeta potential [49].

Cell viability assay

The cytotoxicity of formulation on L6 cells (myoblast cell isolated from the skeletal muscle of a rat) (procured from NCCS Pune) will be examined using a neutral red uptake (NRU) assay. NRU assay is a commonly used test to determine the viability of cells in vitro. The L6 cells (8000 cells/well) were cultured in 96-well microtiter plates for 24 h in Dulbecco’s Modified Eagle’s Medium (DMEM) and complemented with 10% FBS and 1% with antibiotic solution. Moreover, cells are incubated at 37 °C with 5% CO2 to allow the culture to grow and attach to the bottom of the wells. Following the incubation period of 24 h, withdraw the medium and introduce a fresh culture medium into each plate well. Cells were subjected to treatment dilution of VLG and equivalent VLG-CDNPs at various concentrations in wells and then incubated in CO2 incubator (Heal Force-Smart cell CO2 Incubator-Hf-90) for 24 h. After incubation, 100 µL NRU (40 µg/mL in PBS) was introduced to the designated wells and incubated for 1 h. Following this, the medium was extracted, and NRU was dispersed in 100 µL of NRU Destaining solution (50:49:1 ratio of ethanol, water, and acetic acid). Finally, plates were read at 550/660 nm using ELISA plate reader (iMark BioRad-USA) [50].

Nanoparticle uptake cell viability assay

The L6 cell line (1 × 106 cells/well) was cultured on poly-l-lysine-coated glass coverslips in a 6-well microplate. Specific wells were treated with fluorescent CDNPs for 24 h at 37 °C and 5% CO2. To ensure fluorescein isothiocyanate (FITC) absorption onto the nanoparticles, the CDNPs were incubated with a 0.02%w/w FITC solution for 12 h. After incubation, cells were gently rinsed with PBS and fixed with a 4% paraformaldehyde solution. Subsequent steps included permeabilizing cellular membranes with a 0.1% Triton X-100 solution in PBS for 5 min, followed by washes with Milli-Q water. Cells were then stained with a DAPI solution (0.1 mg/ml) for 10 min at room temperature. Micrographs were observed under Cytation 5 (BioTek Instruments, USA) cell imaging multimode reader using a GFP filter cube [51].

In vitro glucose uptake assay

L6 cells (8 × 104 cells/well) were cultured in 96-well plates and permitted to grow in glucose-free DMEM for 24 h. After an incubation period, the cell culture media was carefully extracted, and the cells were subsequently rinsed once with Krebs–Ringer-phosphate-HEPES (KRPH) buffer. The cells were subjected to treatment with control, test sample, insulin, and metformin control for 1 h. After 1 h, cells are washed twice with KRPH buffer and cultured in glucose-free DMEM. The cell was incubated with or without 2-deoxy-D-glucose (2-DG) (10 mM) in KRPH buffer containing 2% of BSA for 40 min. Subsequently, the cells underwent three consecutive washes with PBS to eliminate any residual 2-deoxy-d-glucose (2-DG). Following this, cell lysis was performed using an extraction buffer, followed by a single freeze–thaw cycle. Subsequently, the samples were subjected to heat treatment at 85 °C for a duration of 40 min to destroy endogenous nicotinamide adenine dinucleotide phosphate (NADP), and the resulting lysates were centrifuged at 500 rpm for 2 min. The supernatant was examined for 2-deoxyglucose-6-phosphate (2-DG6P) content utilizing the Enzyme Assay Kit (GOD-POD), and the absorbance was measured at 505 nm using a microplate reader. The lysates from cells that were not subjected to 2-DG were employed to realize the blank value. Data were quantified in nanomoles of 2-DG by comparison with a 2-DG6P standard. Furthermore, for experimental control, insulin (0.1 U/mL) was used as the positive control, and metformin (1 mM) was utilized as the negative control [52].

Assessment of enzyme inhibitory activityα-Amylase inhibition activity

The assessment of α-amylase inhibitory activity was done following the previously described procedure [53, 54]. Briefly, different concentrations of VLG (0–50 μg/mL) and equivalent VLG-CDNP samples were prepared in a phosphate buffer. 10 μL of an α-amylase solution (20 mg/mL) and test samples were dispensed in each well of a 96-well plate, and the resulting mixture was incubated for 10 min. The enzymatic reaction was initiated by introducing 50 μL of 0.1% soluble starch, followed by an additional 15-min incubation. After this, 100 μL of GOD-POD reagent was added to the mixture, and the plate was permitted to undergo incubation at room temperature for 10 min. The absorbance was quantified at 490 nm using a microplate reader (iMark, BioRad).

α-Glucosidase inhibition activity

The inhibition of the α-glucosidase enzyme was carried out as earlier described [52, 54]. Briefly, 10 µL of α-glucosidase enzyme was added to 50 µL phosphate buffer (100 mM, pH 6.8), and aliquots of 20 µL test samples (Acarbose, VLG, and VLG-CDNPs) were reacted inside the 96-well plate and incubated for 37 °C for 15 min. After that, 20 µL aliquot of 5 mM 4-Nitrophenyl-β-d-glucopyranoside (PNPG) was added in each well to quickly initiate an enzymatic reaction at 37 °C for 20 min. Subsequently, 50 µL of a 0.1 M sodium carbonate solution was introduced into each well to halt the enzymatic reaction. The variations in absorbance due to the release of p-nitrophenol were measured at 405 nm using an ELISA microplate reader (iMark BioRad). The obtained outcomes will be presented as percentage inhibition, and this value will be determined using the following formula.

$$\% \text=(1- _/_) \times 100$$

(3)

where A1 is the absorbance with the test substance, and A0 is the absorbance without the test substance (control).

Statistical analysis

Statistical analysis was conducted using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Differences between the two groups were assessed using Student’s t test. For comparisons among multiple groups, one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test was employed with a level of significance set at probability *p < 0.05, **p < 0.01, and ***p < 0.001. A p value of < 0.05 was considered statistically significant. All experimental findings are presented as the mean ± standard deviation (SD) derived from a minimum of three measurements for each trial.