Cytotoxicity and bioavailability assessment from thiamin-phospholipid complexation loaded Ajwain oil based self nanoemulsifying system

Abstract

Poor bioavailability (<5%) of thiamin hydrochloride (TH) primarily relates to its impaired permeability across gut mucosa. To modulate its permeability, a dual system based approach consisting of phospholipid complexation embedded into a self nanoemulsifying system was investigated. Thiamin-Phospholipid Complex (TPC) was developed and its Ajwain oil (AJO)-based self nanoemulsifying formulations (SNFs) were evaluated. Formation of TPC complexation was resulted from molecular interactions held between free bases of thiamin and phospholipid (PL). FTIR characterization reveals that the amino group (-NH₂) in the free base of thiamin was physically interacted to phosphate linkage in PL. Single endothermic event in TPC & TH, reveals complexation between PL & free bases as interpreted from DSC data. Their corresponding melting point was 127 °C and 258 °C, respectively. Heat of fusion (ΔH_f) in TPC was higher than TH or PL. TPC exhibits a poor conductive environment over TH when diluted with ethanol/Tween20 (T2) medium. Lipophilic character of TPC was confirmed from partition coefficient (log P). Exact combination where ternary components produced fully dilutable nanoemulsion system, was identified using ternary diagram drawn between AJO (incorporated with TPC) as oil phase, and Tween20/Polyethylene glycol 600 (PEG600) as Surfactant/co-surfactant (S_mix) at ratios 1:1; 1:2 & 2:1. Seven TPC-loaded self nanoemulsifying formulations (SNFs) were designed which, upon subjected to aqueous phase dilution yielded nanoemulsion with droplet size range 95–190 nm with zeta potential −1.78 ± 0.10 mV. Conductivity and Refractive index (RI) data of SNFs were in the range of 98 ± 7.0 to 231 ± 27 μS/cm and 1.458 to 1.463 units, respectively. In vitro TH release from SNF was determined in hydrochloric (HCl) buffer at pH 1.2, and in phosphate buffer solution (PB) at pH 6.8, data interpretation reveals that TPC delayed the release pattern compared to TH and could be attributed to lipophilic behavior of TPC. Intestinal permeability of drug was assessed from optimized SNF (F1) vs. TH across intestinal gut sac model (Apical to basolateral A→B) resulted in a significant difference in permeability coefficient (1.09 × 10⁻⁵cm/h). Antioxidant potential of SNF was demonstrated in the DPPH method. MTT assay of TPC formulation conducted on MCF-7 and MDA-MB-231 (Breast cancer) cell lines showed the developed system from AJO possessed cytotoxicity effect. Pharmacokinetic assessment showed that optimized SNFs produced more than four-fold enhancement in bioavailability over control (TH solution) in the Wistar rat model. Meanwhile Area under curve (AUC) data obtained from TPC (in coarse emulsion) vs.TH produced a significant difference (p < 0.001). It can be concluded that developed SNFs via phospholipid complexation produced lipophilic transformation of TH and its SNFs modulated permeability as well as bioavailability enhancement of thiamin.

Graphical Abstract

HIGHLIGHTS

• A thiamin-phospholipid complexation (TPC) was developed and its Ajwain oil (AJO) based self-nanoemulsifying formulation (SNF) was investigated for permeability and bioavailability assessment.

• Physical characterization reveals that phospholipid complexation of thiamin was transformed to TPC, owing to its lipophilic nature embedded into an AJO based self nano-emulsifying system.

• TPC modulated the intestinal permeability of thiamin at ex-vivo level however it’s AJO based SNF produced five fold enhancement in bioavailability over TH solution when orally administered at preclinical studies conducted on experimental rats.

• SNF produced cytotoxicity in breast cancer cell lines demonstrated that AJO has antioxidant and cytotoxic activities.

NOVELTY OF WORK

• This study reports the development of phospholipid complexation between free bases of thiamin whereby a highly soluble form of drug i.e. TH was transformed to lipophilic part as TPC.

• Present work describes about the complete dilutability of developed thiamin-phospholipid complex (TPC) into AJO based self nanoemulsifying formulations (SNF).

• Studies conducted which demonstrated modulation of the intestinal permeability of thiamin via the nanoemulsifying system design consisting of a biopharmaceutical classification system (BCS) class III drug.

• A bioavailability enhancement method is reported in this study where the extent of thiamin reached into systemic circulation was 4.5 times higher from complexation approximately.

• This study encompasses a new method of characterization where the lipophilic nature of TPC and its interaction were examined using reported conductometric methods.

• Present work is the first ever reporting of a nanoemulsifying system containing Ajwain oil (AJO) and its physical characterization.

• The underlying work described herein was related to nano emulsification of AJO which further demonstrates in vitro cytotoxicity in breast cancer lines potential.

Keywords:

• Thiamin-phospholipid complex
• phospholipid
• permeability
• bioavailability
• Ajwain oil
• cytotoxicity

Acknowledgement

The authors acknowledge the contribution of Dr. Shweta Dumoga, Department of Pharmaceutical Technology, Meerut Institute of Engineering & Technology, Meerut-250005, India and Dr. Supriya Mehta, Department of Biotechnology, IIT, New Delhi, India, for droplet size and zetasizer studies of samples.

Disclosure statement

No potential conflict of interest was reported by the authors.