Clopidogrel loaded nanostructured lipid carrier: Assembly, microstructure and cytotoxicity

Here we designed an optimized NLC for the transport of CLP, an antiplatelet molecule, and further probed its microstructure, cytotoxicity and the stability. NLCs were attained through cavitation technology employing RSM-based factorial design (2³). Amount of lipid (X1), ultrasound power (X2), and sonication time (X3) were independent operational variables while Z-Avg (nm), PDI and ZP (mV) were the studied responses. The designed CLP-NLC was scrutinized for DLS, TEM, FESEM, ATR, PXRD, TGA, rheology, drug release and cytotoxicity. An optimized NLC had Z-Avg (217.5 nm), PDI (0.178), and ZP of -36.4 mV. Morphology investigation showed spherical NLCs. ATR analysis demonstrated H-bonding interactions between CLP and Imwitor ensuring drug solubility and holding in the lipid matrix. PXRD confirmed complete drug amorphization during processing pinpointing the influence of capryol (oil) on the formation of lower ordered crystal lattice of Imwitor. Designed NLCs showed dominance of elastic constituent and shear thinning behavior, an anomalous (n>0.5) type of CLP transport, and an excellent stability over six months. CLP loaded NLC showed significant cellular uptake and marked reduction in cytotoxicity. Thus the ultrasonically designed NLCs can entrap CLP, are biocompatible and safe for the human use, and with the reduced lipid crystallinity modulates the desired drug release.

Highlights

• We successfully devised nanostructured lipid carrier containing an antiplatelet molecule, clopidogrel.
• AFM, TEM & FE-SEM confirmed a spherical morphology of designed NLCs.
• Molecular interactions occurred among the CLP and the excipients during the NLC processing
• CLP amorphization occurred during the processing and confirmed through PXRD.
• Designed NLCs showed an anomalous (non-fickian) drug release, shear thinning nature and an excellent colloidal stability.
• Cytotoxicity study on RAW -264.7 macrophages indicated its biocompatibility and safety for the human use.

Introduction

Clopidogrel bisulfate (CLP) is an antiplatelet molecule beneficial in the treatment of coronary artery disease (CAD), peripheral vascular disease (PVD), and cerebrovascular disease (CVD) (Lemesle et al., 2016, Hussain et al., 2018). However, use of CLP is generally associated with several adverse reactions such as severe neutropenia, various forms of hemorrhage, and pulmonary edema (McCarthy and Kockler, 2003, Erdinler et al., 2007). From biopharmaceutical perspective it belongs to BCS Class-II exhibiting poor aqueous solubility (0.0118 mg/mL, LogP 3.84). Thus, the low aqueous solubility is a foremost factor limiting the oral bioavailability of CLP molecule besides its protonation in stomach, and an extensive P-glycoprotein (Pg-P) efflux (Wessler et al., 2013, Taubert et al., 2006). Therefore, there is a need to develop novel strategies that would allow overcoming the drug solubility issue and thereby maximizing the therapeutic benefit.

As described in earlier research (Kumbhar and Pokharkar, 2013a, Salvi and Pawar, 2019, Daré et al., 2024), LNP possesses a huge potential to carry various drug actives. Nanostructured lipid carriers (NLCs) are the second generation of lipid nanoparticles (LNPs) derived from a blend of solid lipid and oil (Zhu et al., 2020). NLCs are most sought carrier these days due their biocompatibility, feasibility in achieving controlled drug release profile, and the large scale industrial production. Moreover, a high drug pay-load is possible with an alterations in the matrix of NLC. Varghese et al (Varghese et al., 2017). have designed CLP loaded SLNs using cationically modified lauric acid. Despite these attempts, current literature provides minimal information regarding microstructure, cytotoxicity and cellular uptake of CLP confined to NLC. Also to mote that particle size and PDI are the prime factors that influence the endocytosis-dependent (Danaei et al., 2018a) cellular uptake.

Among the various available techniques (Özdemir et al., 2019), hot high pressure homogenization (HPH) is frequently used to produce colloidal lipid carriers. Nevertheless, there remain several issues like availability of the said instrument in the laboratory attributable to its higher cost and temperature induced degradation of active and / or the carrier lipid. Besides an upsurge in particle size can be witnessed with number of cycles and high pressure as a result of augmented kinetic energy resulting in possible destruction of microstructure of the designed system (Muller et al., 2000, Gonzalez-Mira et al., 2010). Also note that HPH produces particles with higher PDI values (Duong et al., 2020) depending on the composition. Lately ultrasound irradiation is emerging as a reliable method to produce diverse nanomaterials for therapeutic applications (Kiss et al., 2019, Hajnorouzi and Afzalzadeh, 2019, Alzate et al., 2020). The mechanism of ultrasound nanosizing is based on bubble implosion and cavitation (Paris et al., 2018), resulting in transient and localized temperature of about 5000 K and pressure surpassing 1000 bar (Sivakumar et al., 2014). Also, with ultrasonic processing it is likely to critically control the operating parameters like, power, and the processing time.

Here, we developed an ultrasonically assisted CLP-NLCs based on response surface methodology (RSM) using 2³ (Three-factor two level) factorial design. The influence of operating variables was thoroughly investigated with respect to assembly, microstructure and subsequent stability of CLP-NLCs. To probe microstructure, the designed NLCs were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), attenuated total reflectance (ATR), powder X-ray diffraction (PXRD), thermo-gravimetric analysis (TGA), and rheology. Besides to establish mechanism of CLP release from NLCs a detailed kinetic study was performed. Furthermore to ascertain biocompatibility of designed CLP-NLCs we performed cytotoxicity examination on RAW-264.7 macrophages using PrestoBlue cell viability assay.

Materials

CLP was obtained from Dr. Reddy’s Laboratory Pvt. Ltd., India. Imwitor 900 K, Dynasan 114, Dynasan 116, Dynasan 118, Miglyol 840 and Miglyol 812 were received as a generous gift from IOI Oleo GmbH, Germany. Compritol 888 pellets, Precirol ATO 5, and Capryol 90 was a kind gift of Gattefossé SAS, Saint-Priest Cedex, France. Span 80, Span 20, Tween 20, Tween 80, stearic acid, lauric acid and oleic acid were purchased from Merck Limited, India. Phosal 53 MCT was a generous gift of Lipoid GmbH.

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Dipak D Kumbhar, Ankita R Wankhede, Poonam P Warade, Suraj S Patil,
Clopidogrel loaded nanostructured lipid carrier: Assembly, microstructure and cytotoxicity,
Chemical Engineering Research and Design,
2025, ISSN 0263-8762,
https://doi.org/10.1016/j.cherd.2025.03.008.