Cholesterol anchored arabinogalactan for asialoglycoprotein receptor targeting: synthesis, characterization, and proof of concept of hepatospecific delivery
Graphical abstract
Introduction
Therapeutic efficacy of bioactives apart from their pharmacological effectiveness largely depends upon their concentration at the desired site of action. Systemic metabolism of the bioactives requires administration of higher doses as to compensate the losses. Increased doses lead to adverse drug reactions, which are more common with chemotherapeutic agents due to non-selectivity between host and tumor cells. Target specific drug delivery aims at spatial as well as temporal delivery of the bioactive resulting in negligible and/or reduced exposure to the host cells and therefore reduced side effects. In cancer chemotherapy, the delivery system often aims towards targeting receptors of ligands like transferrin, folic acid, sugars, lectins and growth hormones, which are over-expressed on cell surface etc. Asialoglycoprotein receptor (ASGPR) is a selectin E type receptor present in high density1 (1–5×105 receptors/cell) on the surface of liver hepatocytes and is present on Hepatocellular Carcinoma (HCC) cells.2 ASGPR exhibits receptor mediated endocytosis (RME) and shows selective uptake of moieties with terminal galactose or galactosamine residues.
Use of carbohydrates as ligands for receptor based drug targeting has achieved substantial importance in recent times. Carbohydrate/polysaccharide coated particulate carrier systems have been shown to be selectively taken up by the lungs, liver and spleen depending upon the presence of mannose, galactose or glucose as the terminal residue.3 Apart from delivery of chemotherapeutic agents,4 carbohydrate ligand based particulate carriers have been explored for delivery of genetic materials5 and oral vaccines.6 Liposomes are lipid based biocompatible particulate carrier systems, which have been extensively explored for site specific targeted drug delivery owing to ease of assimilation of cell specific ligands and feasibility of incorporation of hydrophobic and/or hydrophilic agents. Reports on use of carbohydrates/polysaccharides for coating/covalent attachment to liposome for targeting the delivery of bioactive to specific organs such as lungs and liver,3, 7 imparting physical stability8 and increasing the plasma circulation of the carrier systems have appeared in literature. Apart from lactose9 and galactose,10, 11 polysaccharides such as pullulan,12 amylopectin,13 scleroglucan,14 xyloglucan15 etc. have been successfully used for liposome coating. Incorporation of galactose or galactose rich polysaccharide onto the surface of liposome will render the drug delivery system hepatospecific owing to its selective uptake by the ASGPR of hepatocytes.
Early developments were focused towards physical adsorption of carbohydrate solutions on the carrier system.16 Nonetheless, these systems showed poor physical stability and also had substantial chances of ligand being dissociated from the carrier system during or post administration in the biological system. Subsequent studies lead to the development of conjugated systems in which the ligand was covalently attached to a lipid anchor such as long chain fatty acids. Use of polysaccharides, which have numerous terminal monosaccharide units of interest, increases probability of presence of free terminal carbohydrate for receptor ligand binding.
Larch arabinogalactan (AG) is a high molecular weight branched polysaccharide composed of galactose and arabinose units with a trace of uronic acid. The molecular weights of the major fractions of AG in larch are 16,000 and 100,000 Da. Glycoside linkage analysis of AG is consistent with a highly branched structure comprising a backbone of 1,3-linked galactopyranose connected by 1,3-glycosidic linkages, comprised of 3,4,6-,3,6-, and 3,4- as well as 3-linked residues.17 Use of AG as a drug conjugate has been reported for improving the water solubility of an anti-fungal agent.18 AG is reported to have activity against INF-α and also enhancing the activity of natural killer cells.17 However, the high aqueous solubility and poor solubility in organic solvents of AG hampers its successful use in lipid based systems.
The present work aims at design and synthesis of novel polysaccharide lipid conjugate ligand and its incorporation in drug delivery system to render them hepatocyte specificity. We propose hydrophobization of AG by its covalent attachment to a lipid anchor. Cholesterol chloroformate (CCF), a derivative of cholesterol was used as the lipid anchor to conjugate with AG via a bi-functional spacer arm β-alanine (AL).
Section snippets
Results and discussion
It is reported that ASGPR present on hepatocytes specifically bind to terminal galactose residues of carbohydrate/polysaccharides. Use of hydrophobized carbohydrates is reported for incorporation into liposomal systems intended for organ targeting. Most of these conjugations are esterification and amidation reactions involving hydroxyl/amino group of carbohydrates and carboxylic acid group of the lipid anchor.8, 19 Poor specificity of carbohydrate chemistry does not allow selective
Conclusion
Novel hydrophobized AG was successfully synthesized and incorporated into liposomes. Incorporation of CHOL-AL-AG in liposome did not affect the bilayer structure of liposome. Liposomes exhibited particle size of 200 nm required for targeting tumor and infectious cells. Presence of galactose residues on the surface of liposome was confirmed by in vitro RCA binding assay, essential for binding with ASGPR. SML exhibited enhanced uptake by ASGPR expressing HepG2 cells owing to enhanced binding of
Materials and methods
Arabinogalactan (AG) from larch wood, cholesterol chloroformate (CCF), Ricinus communis agglutinin (RCA) were purchased from Sigma–Aldrich, India; β-alanine (AL), cholesterol (CHOL), tetrahydrofuran (THF) HPLC grade, dimethyl sulfoxide (DMSO) AR grade, chloroform (CHCl3) AR grade and Carbonyl diimidazole (CDI) were purchased from Fischer-Qualigens, India, N-pyrolidone (Soluphor-P) was a gift sample from BASF, India, Dimethyl amino pyridine (DMAP), sodium hydroxide pellets (NaOH) and absolute
Acknowledgments
Authors are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi, India (01(2159)/07/EMR-II) for funding the project and fellowship to Pathak Pankaj. Authors are thankful to Lipoid, Germany for providing gift samples of phospholipids, Khandelwal Labs, India for gift sample of doxorubicin. Authors would also like to thank Amrutlal Mody Research Fund and Lady Tata Memorial Trust for providing fellowship to Mr. Sameer Padhye and Mr. Vivek Dhawan respectively.
References (22)
- et al.
J Biol Chem
(2002) - et al.
Biochim Biophys Acta
(2000) - et al.
Int J Pharm
(2006) - et al.
Glycosylated cationic liposomes for carbohydrate receptor–mediated gene transfer
- et al.
Int J Pharm
(2014) - et al.
Int J Pharm
(1997) - et al.
Eur J Pharm Biopharm
(2007) - et al.
Carbohydr Res
(2013) - et al.
J Pharm Sci
(1991) - et al.
Int J Pharm
(2006)
Carbohydr Res
Cited by (32)
Mitochondria targeted liposomes of metformin for improved anticancer activity: Preparation and evaluation
2022, Journal of Drug Delivery Science and TechnologyPolysaccharide and monosaccharide guided liver delivery of Sorafenib Tosylate – A nano-strategic approach and comparative assessment of hepatospecificity
2022, International Journal of PharmaceuticsCitation Excerpt :Nagarsenker and co-workers have investigated delivery of doxorubicin incorporated in a liposomal system modified using palmitoylated arabinogalactan (Shah et al., 2014) as the ligand. Arabinogalactan modified with cholesterol incorporated in liposomes encapsulating doxorubicin was also explored and elicited a greater therapeutic effect in a tumorigenic model in rats (Pathak et al., 2015, 2016). Previously, we have reported design and synthesis of biocompatible targeting ligand based on agarose (Dhawan et al., 2021).
Polysaccharide conjugates surpass monosaccharide ligands in hepatospecific targeting – Synthesis and comparative in silico and in vitro assessment
2021, Carbohydrate ResearchCitation Excerpt :Daunorubicin hydrochloride was converted to base daunorubicin and was loaded in liposomes with and without targeting ligands. The liposomes were prepared as per procedure described previously [34]. After 4 h, the cells were washed with DMEM (Dulbecco's Modified Eagle's Medium) and imaged using a confocal microscope (Ziess LSM 780, Carl Ziess Germany).
New properties of chia seed mucilage (Salvia hispanica L.) and potential application in cosmetic and pharmaceutical products
2021, Industrial Crops and ProductsCitation Excerpt :Studies on the biological activities of chia seed have shown important pharmacological activities, such as antioxidant, antimicrobial, and inhibitory activity against acetylcholinesterase and butyrylcholinesterase (Kobus-Cisowska et al., 2019; Hrnčič et al., 2020). These physicochemical and biological properties of chia seeds make this natural raw material a strong candidate for the development of health products (Pathak et al., 2015; Timilsena et al., 2016; Sansone et al., 2019). Extensive studies concerning chia mucilage biopolymer brought attention to its potential as a renewable raw material to produce environmentally friendly products (Fernandes et al., 2020).
Functionally engineered ‘hepato-liposomes’: Combating liver-stage malaria in a single prophylactic dose
2020, International Journal of PharmaceuticsCancer nanotechnology: Enhancing tumor cell response to chemotherapy for hepatocellular carcinoma therapy
2019, Asian Journal of Pharmaceutical SciencesCitation Excerpt :Meng and colleagues successfully demonstrated the galactose-installed photo-crosslinked pH-sensitive degradable micelles (Gal-CLMs) for active targeting chemotherapy have a great potential for hepatocellular carcinoma chemotherapy (Fig. 3) [126]. In another study, cholesterol arabinogalactan anchored liposomes (CHOL-Al-AG) carrying DOX targeting ASGPR receptors on mammalian hepatocytes in HCC were studied where improved delivery of DOX was reported in terms of stability, loading efficiency, tumor regression both in vitro and in vivo compared to unmodified liposomes [127]. Integrins are transmembrane receptors which are responsible for cell–cell and cell-extracellular matrix interactions [128].