Resveratrol-Loaded Solid Lipid Nanoparticles Enhance Insulin Sensitivity by Modulating SNARE Protein Expression in Adipose and Muscle Tissues of Type 2 Diabetic Rats

Presentation of the Hypothesis

Encapsulating RES within a lipid core nanoparticle enhances its physicochemical stability and intestinal absorption, leading to greater bioavailability. We hypothesize that SLN‑RES will reduce insulin resistance more effectively than free RES, owing to its antioxidant potency and improved delivery.

Testing the Hypothesis

Spectroscopic analyses were performed to confirm encapsulation efficiency. The second objective was to evaluate SLN‑RES effects on oxidative‑stress biomarkers and its hypoglycemic efficacy in an STZ‑induced type 2 diabetic rat model.

Implications of the Hypothesis

Improved stability and intestinal uptake increase oral RES bioavailability, leading to higher concentrations in target tissues and enhanced hypoglycemic action compared with free RES.

Background

Type 2 diabetes mellitus (T2DM) is characterized by systemic insulin resistance, largely driven by impaired GLUT 2 and GLUT 4 trafficking in muscle and adipose tissues. SNARE proteins—Snap23, Stx4, and Vamp2—anchor GLUT 4 vesicles to the plasma membrane, and their down‑regulation accelerates insulin resistance. Numerous plant‑derived compounds, including resveratrol (RES), have demonstrated metabolic benefits; however, oral RES suffers from poor absorption and rapid degradation. Nanomedicine approaches, particularly solid lipid nanoparticles (SLNs), offer a strategy to enhance RES stability and bioavailability. Prior studies indicate that RES encapsulation in SLNs improves neuroprotective outcomes and suggests potential for metabolic applications. Here, we develop and characterize RES‑loaded SLNs and assess their impact on glycemic control, oxidative stress, and SNARE protein expression in diabetic rats.

Materials and Methods

Materials

High‑purity trans‑resveratrol (>99 %) was sourced from Mega Resveratrol (USA). Streptozotocin (STZ) and nicotinamide (NA) were purchased from Sigma‑Aldrich (Germany). Hydrogenated soybean lecithin (S100) and hydrogenated palm oil (S154) were provided by Lipoid KG and Condea, respectively. TRIzol reagent and cDNA synthesis kit were obtained from Invitrogen (USA). Serum insulin ELISA kits were from Bio‑Equip (China). All other reagents were analytical grade.

Preparation of SLN‑RES

SLN‑RES was prepared via a modified hot homogenization‑ultrasonication method. Briefly, 40 mg S100 and 1 g sorbitol were dissolved in 15 ml distilled water and heated to 70 °C. Simultaneously, 100 mg S154, 70 mg S100, and RES (30–70 mg) were melted at 70 °C; 5 ml chloroform was added to create the organic phase. Rapid injection into the preheated aqueous phase under 1,000 rpm stirring removed chloroform. The suspension was sonicated (2 min) and then injected into 2 °C cold water under continuous stirring (1,000 rpm) for 2 h to solidify the lipid matrix. The resulting powder was washed twice by centrifugation, and free drug was quantified to calculate entrapment efficiency (EE). The effect of RES loading on particle size, PDI, zeta potential, and EE was evaluated (Table 1).

Characterization of SLN‑RES

Dynamic light scattering measured mean diameter, zeta potential, and PDI using a Malvern Zetasizer Nano‑ZS after a 1/30 dilution in water.

Entrapment Efficiency

EE (%) = [(total drug – free drug)/total drug] × 100. Free RES was quantified by UV‑Vis spectroscopy at 310 nm.

In Vitro Drug Release Study

SLN‑RES was dispersed in PBS (pH 7.4) and simulated gastric fluid (pH 1.2). At 0.5, 1, 2, 4, 6, and 8 h, aliquots were withdrawn, filtered (0.24 µm), and free RES measured.

Transmission Electron Microscopy

Particle morphology was examined on a ZEISS TEM grid, revealing spherical shapes and minimal aggregation.

Fourier Transform Infrared Spectroscopy

FTIR spectra (400–4,000 cm⁻¹) were obtained using a PerkinElmer instrument. Samples were mixed with KBr to form pellets.

Animal Study Design

Twenty male Wistar rats (200–250 g) were housed under standard conditions (25 ± 2 °C, 12‑h light/dark). T2DM was induced via a single intraperitoneal injection of STZ (65 mg/kg) and NA (110 mg/kg). Rats with fasting blood glucose >150 mg/dl were considered diabetic. After one week, rats received daily oral gavage of either 10 mg/kg RES or SLN‑RES (10 mg/kg RES content) for 4 weeks. Groups (n = 5) included healthy control (HC), diabetic control (DC), RES, and SLN‑RES. At study end, animals were weighed, anesthetized (ketamine/xylazine), and blood collected by cardiac puncture. Serum and tissues (skeletal muscle, visceral adipose) were harvested and stored at –80 °C for analysis.

Measurement of Fasting Blood Sugar

FBS was measured colorimetrically using a Pars Azmun kit.

Measurement of Serum Insulin

Serum insulin was quantified via a Bio‑Equip ELISA kit. HOMA‑IR was calculated using the standard formula.

Total Antioxidant Capacity

TAC was determined by the Fe³⁺/Fe²⁺ – TPTZ assay, measuring the blue complex absorbance.

Total Thiol Group

SH groups were measured with DTNB, producing a yellow chromophore.

Lipid Peroxidation Assay

MDA levels were assessed via the thiobarbituric acid reactive substances (TBARS) method using 1,1,3,3‑tetraethoxypropane as standard.

Total Oxidant Status

TOS was measured by the Fe³⁺/xylenol orange complex, calibrated with H₂O₂ and expressed as μM H₂O₂ eq/L.

Quantitative Reverse Transcription Polymerase Chain Reaction

mRNA from frozen tissues was extracted with TRIzol. cDNA synthesis used the RevertAid kit. qRT‑PCR quantified Snap23, Stx4, and Vamp2, normalizing to 18s rRNA. The 2⁻ΔΔCt method calculated relative expression. Experiments were triplicated.

Statistical Analysis

Data were expressed as mean ± SD. One‑way ANOVA (SPSS 16) assessed group differences; p < 0.05 was considered significant.

Results and Discussion

Physicochemical Characterization of SLN‑RES

Increasing the drug‑to‑lipid ratio did not significantly alter particle size or PDI, suggesting multilayer phospholipid formation. The mean size (~250 nm) is optimal for intestinal absorption while avoiding hepatic and splenic clearance. A PDI of 0.4 indicates a moderately heterogeneous population, yet zeta potential of –16.5 mV confers sufficient electrostatic stability. The 79.9 % EE confirms efficient encapsulation, and the SEM/TEM images confirm a smooth, spherical morphology with minimal aggregation.

In Vitro Release Profile

Release studies (Figure 1) showed ~70 % RES release after 6 h at pH 7.4 and after 1 h at pH 1.2, reflecting an initial burst followed by sustained release driven by drug–lipid interactions and diffusion gradients.

In vitro release profiles of RES from SLN‑RES in acidic and physiological conditions.

Morphology Evaluation

TEM (Figure 2) confirmed spherical particles averaging 20–30 nm, with occasional rod‑shaped forms but negligible aggregation, consistent with the high zeta potential and low PDI.

Transmission electron micrograph of SLN‑RES. Bar = 100 nm.

FTIR Validation of RES Loading

FTIR spectra (Figure 3) showed characteristic RES peaks (e.g., O‑H stretch at 3290 cm⁻¹, C=C stretch at 965 cm⁻¹) preserved in SLN‑RES, confirming successful encapsulation. Shifts in lecithin peaks indicated hydrogen‑bond formation and core loading.

FTIR spectrum of SLN, SLN‑RES, S100, and pure RES.

Impact on Body Weight, Glycemia, and Insulin Sensitivity

Diabetic controls (DC) exhibited significant weight loss compared to healthy controls (HC). RES prevented weight loss, whereas SLN‑RES restored body weight to near HC levels (Table 2). Fasting blood glucose and HOMA‑IR improved in both RES and SLN‑RES groups, with SLN‑RES achieving values closer to HC. Serum insulin levels rose only in the SLN‑RES group, indicating enhanced insulin sensitivity.

Oxidative Stress Parameters

Diabetes markedly reduced TAC and SH levels while elevating MDA and TOS. SLN‑RES normalized TAC, MDA, and TOS, whereas free RES only partially restored TAC and failed to reduce MDA, underscoring the importance of improved bioavailability.

SNARE Gene Expression in Adipose Tissue

Both RES and SLN‑RES up‑regulated Snap23, Stx4, and Vamp2 in adipose tissue relative to DC, but the magnitude of change did not differ significantly between formulations, suggesting a plateau in adipose tissue exposure.

The effect of RES and SLN‑RES on Snap23, Stx4, and Vamp2 mRNA in adipose (a–c) and muscle (d–f) tissues. *p < 0.05, +p < 0.01, #p < 0.001.

SNARE Gene Expression in Muscle Tissue

STZ reduced Snap23, Stx4, and Vamp2 expression in skeletal muscle. SLN‑RES restored these genes more effectively than free RES, particularly Vamp2, indicating superior delivery to muscle tissue.

Conclusion

We successfully formulated a stable, bioavailable RES‑loaded SLN suitable for oral administration. SLN‑RES enhanced glycemic control, mitigated oxidative stress, and selectively up‑regulated SNARE proteins in muscle and adipose tissues of diabetic rats, thereby improving insulin sensitivity. Future studies should explore pharmacokinetics and tissue distribution to further validate this therapeutic strategy.

Availability of Data and Materials

All data generated or analyzed during this study are presented in this article.

Abbreviations

RES:

Resveratrol

SLN:

Solid lipid nanoparticle

SLN‑RES:

Resveratrol‑loaded solid lipid nanoparticle

SNAP‑23:

Synaptosomal‑associated protein 23

STX4:

Syntaxin‑4

VAMP‑2:

Vesicle‑associated membrane protein 2

TEM:

Transmission electron microscopy

FTIR:

Fourier‑transform infrared spectroscopy

-SH:

Total thiol group

TAC:

Total antioxidant capacity

MDA:

Malondialdehyde

TOS:

Total oxidant status