Hybrid Verapamil‑Dextran Nanostructured Lipid Carriers: Statistically Optimized Formulation for Enhanced Cellular Uptake and Sustained Release

Abstract

Verapamil, a calcium‑channel blocker used for hypertension, angina, and arrhythmias, suffers from low oral bioavailability (20–35%) due to extensive first‑pass metabolism. This study presents the development of hybrid verapamil‑dextran nanostructured lipid carriers (HVD‑NLCs) aimed at boosting cellular uptake and improving bioavailability. Formulations were produced by high‑shear homogenization and optimized through a 2⁴ full factorial design. The lead preparation (VER‑9) achieved a particle size of 192.3 ± 3.0 nm, a polydispersity index of 0.553 ± 0.075, and an entrapment efficiency of 93.3 ± 2.7 %. Dextran sulfate inclusion prolonged release (~85 % after 48 h) in both simulated gastric (pH 1.2) and intestinal (pH 6.8) media. Differential scanning calorimetry confirmed no chemical interaction between drug and excipients, while wide‑angle X‑ray scattering showed the drug existed in an amorphous state within the NLC matrix. Transmission and scanning electron microscopy revealed uniformly spherical nanoparticles. In Caco‑2 cells, VER‑9 markedly outperformed free verapamil solution and a verapamil‑dextran complex, delivering 10.9‑ and 135‑fold higher intracellular concentrations, respectively. Refrigerated storage (5 ± 3 °C) maintained stability over 6 months. Collectively, HVD‑NLCs represent a promising carrier system that significantly enhances verapamil cellular uptake and provides sustained release, potentially translating into improved therapeutic efficacy.

Background

Verapamil, an L‑type calcium‑channel blocker and α‑adrenergic antagonist, is a cornerstone therapy for hypertension, supraventricular tachyarrhythmia, angina, and cluster headache. Classified as BCS Class I, it is well absorbed (≤90 %) yet only 20–35 % of an oral dose reaches systemic circulation because of pronounced first‑pass hepatic metabolism. Enhancing its intestinal uptake is therefore a critical formulation challenge.

Nanostructured lipid carriers (NLCs) have emerged as superior lipid‑based nanocarriers compared with solid lipid nanoparticles, offering higher drug loading, improved physical stability, and controlled release. Their lipid matrix promotes chylomicron formation, potentially elevating oral bioavailability. However, encapsulating hydrophilic drugs such as verapamil in NLCs is difficult. We therefore engineered hybrid NLCs by complexing verapamil with the anionic polymer dextran sulfate, forming a counter‑ion complex that can be entrapped in the lipid matrix, and studied the impact of formulation variables on particle attributes using a factorial design.

Methods

Materials

Verapamil HCl, Compritol 888 ATO®, oleic acid, Tween 80®, Poloxamer 188, trehalose, and other reagents were sourced from commercial suppliers. Caco‑2 cells were obtained from ATCC.

Preparation of HVD‑NLCs

Lipid phase (Compritol + oleic acid) was melted at 85 °C. Verapamil dissolved in a 1:1 w/w mixture of Tween 80® and Poloxamer 188 was heated to 85 °C and added dropwise to the lipid phase under high‑speed homogenization (24 000 rpm). Dextran sulfate solution (1:1 w/w to verapamil) was incorporated during homogenization. The mixture was cooled to room temperature and then lyophilized using trehalose as cryoprotectant.

Statistical Optimization

A 2⁴ full factorial design varied homogenization time (A), solid lipid concentration (B), liquid lipid concentration (C), and surfactant concentration (D). Dependent responses were particle size (PS), polydispersity index (PDI), zeta potential (ZP), and entrapment efficiency (%EE). Design‑Expert® software generated polynomial models and desirability functions; formulations with desirability >0.85 were selected.

Characterization

PS, PDI, and ZP were measured by dynamic light scattering. Entrapment efficiency was determined after separation of free drug by Sephadex G‑25 column and HPLC quantification. Lyophilization trials employed trehalose (1:1 and 1:2 lipid:cryoprotectant ratios). In vitro release was evaluated in SGF (pH 1.2) and SIF (pH 6.8) using a dialysis bag (12 000 Da cutoff). DSC and WAXS assessed drug–excipient compatibility and crystalline state. TEM and SEM examined morphology. Caco‑2 cellular uptake was quantified after 6 h incubation and passive lysis, followed by HPLC analysis.

Stability

Lyophilized VER‑9 was stored at 5 ± 3 °C, 25 ± 2 °C/60 % RH, and 40 ± 2 °C/75 % RH for 6 months; PS, PDI, ZP, and %EE were monitored.

Results and Discussion

Factorial Design Outcomes

All four factors significantly influenced PS, PDI, ZP, and %EE (p < 0.05). Increasing homogenization time, liquid lipid, and surfactant concentrations reduced particle size, while higher solid lipid and surfactant levels increased zeta potential. %EE decreased with prolonged homogenization but increased with surfactant concentration. Interaction terms (AC, BD, CD) further modulated these effects.

Optimized Formulation (VER‑9)

VER‑9 exhibited 192.3 ± 3.0 nm size, 0.553 ± 0.075 PDI, −46.8 ± 1.2 mV ZP, and 93.3 ± 2.7 % EE. Lyophilization with trehalose (1:1 ratio) preserved these attributes upon reconstitution.

In Vitro Release

VER‑9 released ~85 % verapamil over 48 h in both SGF and SIF, whereas free drug dissolved >95 % within 4 h. The sustained release is attributed to the electrostatic complex with dextran sulfate and the lipid matrix.

DSC and WAXS

DSC curves showed loss of verapamil’s melting peak in VER‑9, confirming its amorphous state. WAXS patterns displayed disappearance of crystalline verapamil peaks, supporting the amorphous dispersion within NLCs.

Morphology

TEM images revealed spherical particles <200 nm. SEM images of lyophilized VER‑9 showed intact morphology when trehalose was present, confirming its protective role.

Cellular Uptake

VER‑9 delivered 10.9‑fold higher intracellular verapamil than free solution and 135‑fold higher than the verapamil‑dextran complex (p < 0.01). The improved uptake is likely due to enhanced chylomicron-mediated transport and the lipid carrier’s ability to fuse with cell membranes.

Stability

At 5 ± 3 °C, VER‑9 remained stable for 6 months with negligible changes in PS, PDI, ZP, and EE. Samples stored at 25 °C/60 % RH or 40 °C/75 % RH aggregated within 1 month and could not be quantified.

Conclusion

The hybrid verapamil‑dextran NLCs (VER‑9) represent a robust, lyophilizable platform that delivers sustained release, high entrapment, and markedly enhanced cellular uptake of verapamil. Its stability at refrigerated temperatures suggests suitability for clinical development, potentially translating into improved oral bioavailability and therapeutic outcomes.

Abbreviations

%EE

Percentage of entrapment efficiency

DMEM

Dulbecco’s modified Eagle’s medium

DSC

Differential scanning calorimetry

FBS

Fetal bovine serum

HVD‑NLCs

Hybrid verapamil‑dextran nanostructured lipid carriers

ICH

International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use

IDL

Intermediate‑density lipoprotein

LDL

Low‑density lipoprotein

NLCs

Nanostructured lipid carriers

PBS

Phosphate‑buffered saline

PDI

Polydispersity index

PS

Particle size

RH

Relative humidity

SEM

Scanning electron microscope

TEM

Transmission electron microscope

VLDL

Very low‑density lipoprotein

WAXS

Wide‑angle X‑ray scattering

ZP

Zeta potential