Formulation of sesamol loaded mannose surface conjugated smart lipid particles: Assessment of serum liver injury markers
Prashant Gurav, Department of Pharmaceutics, Indira Institute of Pharmacy, India
Manish Kumar Gupta, School of Pharmaceutical Sciences, Jaipur National University, India
Vipul Sansare, Department of Pharmaceutics, Indira Institute of Pharmacy, India
Sesamol is a well-known antioxidant phyto constituent extracted from sesame oil. Numerous landmark investigations have reportedhydroxyl radical scavenging and the hepatoprotective potential of sesamol. Thus present investigation attempted to use mannose surface anchored nanostructured lipid carriers for hepatocytes targeting sesamol with an aim to enhance hepatoprotective potential. The mannose conjugated lipid nanoparticles revealed significantly better reduction of serum liver injury markers compared to non-conjugated one. Mannose conjugation could be a promising strategy for hepatocytes targeting of lipid nanoparticles.
1. Introduction
Liver diseases affect millions of populationworldwide, which are difficult to treat with conventional drug delivery(Bartneck et al., 2014). World health organization has reported 30 to 50% of liver cirrhosisglobally due to alcohol consumption and more than 300 million cases of chronic hepatitisinfections in 2020. Numerous drugs have been investigated for the treatment of diseases associated with the liver.
Majority of conventionally administered drugs undergoes off-target distribution and the efficient therapeutic effect in diseases like hepatocellular carcinoma, hepatitis and liver cirrhosis(Bei et al., 2014). To overcome hurdles associated with conventional drug delivery of drugs the novel nano sized particles like liposomes(Gupta et al., 2021), solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs) (Gupta et al., 2022)and phytosomes(Sansare et al., 2021) were widely utilized by numerous formulation experts(Irache et al., 2008).
The aim of the present work was to design mannose surface anchored sesamol loaded NLCs for hepatocytes targeting. Sesamol loaded NLCs was formulated and surface coated with mannose. The mannose anchored NLCs showed significantly better hepatoprotection with reduction of serum liver injury markers compared to unconjugated NLCs and standards Liv-52.
2. Material and methods
2.1 Material
Sesamol was purchased from Manus Aktteva Biopharma LLP (India). Soya lecithin S-100 was kindly gifted by Lipoid (Germany). Stearic acid wasobtained from Sasol Germany GmbH (Germany). Oleic acid and Tween 20 were purchased from S.D. Fine Chemicals Ltd. (Mumbai, India).
2.2 Design of SM loaded NLCs
SM encapsulated NLCs were formulated by melt homogenization ultrasonication method(Gupta et al., 2022). In practice SM was dissolved in molten stearic acid: oleic acid (8:2) mixture. The soya lecithin S-100 was added in resulting melted medicated lipid phase. An aqueous phase containing 10 ml of distilled water and Tween 20 was injected using a syringe (24 gauge) into molten lipid mass and stirred at 4000 rpm for 10 minutes at 70°C using overhead stirrer (Remi, India).The obtained pre emulsion was subjected to probe sonication (VCX500, Sonics and materials, U.S.A.) at 20% amplitude for 10 minutes and cooled to room temperature. The resulting NLC dispersion was subjected to homogenization using high pressure homogenizer (Stansted, UK) at 10,000 psi for 3 cycles for further particle size reduction.
2.3 Mannose conjugation on SLNs surface
Mannose surface coating on SM loaded NLCs was carried out by ring opening of mannose followed by reaction of its aldehyde group withthe free amine group present over surface of NLCs insodium acetate buffer (pH 4.0) (Fig. 1). Mannosylated NLCs then subjected to dialysis (Molecular weight cut off 12–14 kDa)against distilled water to separateunconjugatedmannose(Sahu et al., 2014).
Fig. 1 Scheme of mannose conjugation on sesamol loaded NLCs
2.4 Characterization of SM loaded NLCs
2.4.1 Assessment of physicochemical properties
Particle size distribution of NLCs were measured using photon correlation spectroscopy (Zetasizer Nano ZS, Malvern instruments, Worcestershire, UK). The entrapment efficiency of SM in the NLCs matrix was determined by the indirect method using Optima Max XP ultracentrifuge (Beckman Coulter, U.S.A.) coupled with UV spectrophotometry.
2.4.2 Assessment of in vivo hepatoprotective potential
The protocol was approved by the Institutional Animals Ethical Committee of Indira Institute of Pharmacy (Approval number: IIP/IAEC/08/2019-20). All animals were purchased from ISO certified, Global bioresearch solution Pvt. Ltd, Shirwal, India.
Rats were randomly divided into five groups, each having 6 rats. Group I was vehicle control (VC) group and received 1 ml/kg BW of olive oil. Group II was positive control (PC) group and received standard hepatotoxic drug CCl4. Group III was standard group and received Liv-52 at the dose of 1 ml/kg BW. Group IV animals was treatment group I and received mannose anchored SM NLCs at the dose of 8 mg/kg BW. Group V was treatment group II and received unconjugated SM NLCs at the dose of 8 mg/kg BW.Hepatic damage to rats was induced by oral administration of 4 ml/kg BW CCl4 (dispersed with an equal volume of olive oil) for 10 days. In VC group 1 ml/kg BW of olive oil was administered for 10 days (no hepatic injury as well as treatment for VC group). All treatments were initiated three days after final dose of CCl4for four weeks(Singh et al., 2015).
At the end of treatment schedule, rats were anesthetized using ether and blood was removed through retro-orbital plexus. Serum from blood samples was separated by centrifuging and stored at −20°C for quantification of serum liver injury markers. Serum liver injury markers like ALT, AST were estimated using diagnostic kits (Reckon diagnostic, India). The manufacturer protocol was used to perform the assays(Singh et al., 2015).
3.1 Results and discussion
3.1 Design and characterization of mannose anchored SM loaded NLCs
The melt homogenization ultrasonication technique was successfully utilized for the design of SM loaded NLCs. The mannose surface coating on preformed drug loaded NLCs was carried out by ring opening andreaction between aldehyde group of mannose and free amino grouppresent on the surface of NLCs in acetate buffer (pH 4.0) (Fig. 1). This reaction results in the formation of Schiff’s base (–N–CH–) and bond between mannose and NLCs surface(Sahu et al., 2014).
Both mannose conjugated and unconjugated NLCs were characterized for particle size and entrapment efficiency. The unconjugated and mannose anchored SM NLCs revealed particle size of 142.8 and 145.1 nm respectively. The numerous scientific experts have proved that nano sized particles with size less than 200 nm can efficiently entrapped in hepatic cells(Mao et al., 2005)(Liang et al., 2005). Both NLCs showed particle size less than 200 nm which proved its suitability for hepatocytes targeting. Entrapment efficiency of SM in unconjugated and mannose anchoredSM NLCs was found to be 71.23 and 69.62 %.
3.2 Assessment of in vivo hepatoprotective potential
The hepatic cells damage results in release of AST and ALT in blood stream. The increase in AST and ALT levels due to administration of CCl4 in rats confirmed hepatic damage. CCl4administration in rats resulted in 2368.06% elevation in ALT level compared to group I (VC). Four weeks treatment with unconjugated SM NLCs and mannose anchored SM NLCs resulted significant reduction in increased ALT levels by 51.48 ± 3.61% and 60.41 ± 3.46% respectively as represented in Table 1. Liv-52 administration in rats resulted 45.75 ± 2.78% reduction in ALT level. Thusdesigned mannose anchored SM NLCs were successful in significant reduction (p < 0.05) of elevated ALT level in rats compared to unconjugatedSM NLCs. This could be attributed due to hepatocytes targeting of SM loaded NLCs.
The AST level was elevated by 980.73% due to administration of CCl4compared to group I (VC). The unconjugated SM NLCs reduced increased AST level by 74.62 ± 3.73%. However mannose anchored SM NLCs showed significantly better (p < 0.05) reduction of increased AST compared to conventional SM NLCs.
Table 1. % inhibition in ALT and AST levels in treatment groups with respect to CCl4 group (n=6).
| Group | % Inhibition of ALT versus CCl4 group | % Inhibition of AST versus CCl4 group |
| Group III | 45.75 ± 2.78 | 73.45 ± 3.51 |
| Group IV | 60.41 ± 3.46 | 79.82 ± 2.81 |
| Group V | 51.48 ± 3.61 | 74.62 ± 3.73 |
4. Conclusion
In current investigation, mannose anchored SM NLCs were designed for hepatocytes targeting in order to enhance hepatoprotective potential of SM. Melt homogenization ultrasonication technique was found to be effective method for design of SM loaded NLCs. The selected method was found to be suitable for production of NLCs with better particle size and entrapment efficiency. The particle diameter of NLCs less than 200 nm confirmed its suitability for hepatocyte targeted drug delivery. In vivo hepatoprotective potential confirmed hepatocytes targeting potential of mannose anchored NLCs by effectively reducing serum liver injury markers. Thus mannose surface conjugation could be viable alternative for hepatocyte targeting of drug loaded nano sized particles.
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Prashant B. Gurav (M.Pharm-Pharmaceutics). He as 14 years of experience in teaching and currently associated with Indira Institute of Pharmacy, Sadavali as an HOD of Pharmaceutics. He has also guided 35 postgraduate students. He has authored many research & review papers in indexed journals and presented papers in international conferences.
Manish Kumar Gupta (PhD- Pharmaceutical Sciences). Currently he is associated with Jaipur National University, Jaipur as Professor (Pharmaceutics) and Coordinator. He has several research papers published in Journals of repute and presented at national/ international conferences. He has guided 10 postgraduate students. Presently, 6 students are pursuing Ph.D. under his guidance.
Vipul Ajit Sansare (M.Pharm-Pharmaceutics). His area of specialization is Novel Drug Delivery Systems. Currently he is associated with Indira Institute of Pharmacy, Sadavali as an assistant professor of Pharmaceutics. He has authored 24 research papers in indexed journals, published 6 books, presented 9 papers in international conferences and received three awards in international conferences.
