RGD-anchored magnetic liposomes for monocytes/neutrophils-mediated brain targeting
Introduction
Blood–brain barrier (BBB) constitutes the major physiological barrier that effectively screens the blood components present in the blood stream before they are allowed to penetrate and reach the brain compartment. Unlike the endothelia of many peripheral tissues, cerebrovascular system has tight intercelluar junctions (Reese and Karnovsky, 1967, Brightman and Reese, 1969). Moreover, because of its highly lipophilic nature, many hydrophilic compounds fail to enter the brain following systemic administration. Hence, management of brain related diseases with presently available therapeutic systems is often a formidable therapeutic attempt. The brain being the major organ of interest for selective delivery of various therapeutically active compounds for effective management of cerebrovascular diseases.
The use of liposomes for drug delivery across the brain capillaries has been documented and examined. Small unilamellar vesicles (SUVs) as well as cationic liposomes coupled with brain drug transport vectors may be transported through the BBB by receptor-mediated or absorptive-mediated transcytosis (Umezawa and Eto, 1988, Wang et al., 1995, Huwyler et al., 1996, Shi and Pardridge, 2000, Shi et al., 2001, Mora et al., 2002, Thole et al., 2002, Zhang et al., 2002). These colloidal carriers are however subjected to conductive opsonization and subsequent opsonic phagocytosis by circulating phagocytes (monocytes and neutrophils) and by macrophages of liver and spleen (Kirsh et al., 1987, Scherphof, 1991). One of the strategies to overwhelm this problem is to magnetize the drug loaded carrier so that it can be retained at or guided to the target site with the help of an external magnetic field of appropriate strength. Retention of magnetic carrier at target site apparently delays reticuloendothelial clearance and neodisposition of the carrier and contained drug. Magnetic liposomes have been investigated for targeted drug carrying potentials (Kiwada et al., 1986, Shinkai et al., 1995, Shinkai et al., 1996, Viroonchatapan et al., 1995, Yanase et al., 1996, Kubo et al., 2000). However, targeting of the drugs to brain using these magnetic liposomes remains an unmet goal because of the stern limitations imposed by impervious capillaries that supply to brain. These capillaries are the major constituents of BBB. Even the liposomes with a diameter as small as 100 nm fail to penetrate via free diffusion through BBB (Sakamoto and Ido, 1993).
It is becoming increasingly apparent that many neurological diseases, such as Alzheimer’s disease, Parkinson’s disease, Prion disease, meningitis, encephalitis and AIDS related dementia, have in common an inflammatory component (Perry et al., 1995). The process of inflammation is characterized by extensive leukocytes (neutrophils and monocytes) recruitment. These cells have unique property of migrating toward inflammation site via the processes known as diapedesis and chemotaxis (Kuby, 1994, Levinson and Jawetz, 1994). Leukocytes are reported to cause BBB breakdown following brain inflammation (Anthony et al., 1997, Anthony et al., 1998, Bolton et al., 1998, Blamire et al., 2000). Studies revealed that leukocytes can cross an intact BBB in healthy condition (Perry et al., 1997).
Phagocytic and exclusive extravasation property of leukocytes make it possible to exploit these cells as carrier system for targeted delivery. Magnetic neutrophils have been prepared in vitro and targeted to lungs under magnetic guidance following intravenous injection (Ranney and Huffaker, 1987). It was therefore envisaged that drug loaded magnetic liposomes can be devised for selective and preferential presentation to blood monocytes/neutrophils that result in both drug and magnetite incorporation into these cells, which subsequently become magnetized cells responding to magnetic field. These magnetic monocytes/neutrophils can then be guided in vivo to the target site, i.e. brain by applying an external magnetic field of appropriate strength. Thus, co-ordinated carrier-biocell strategy, controllable at an external level could be designed with distinctive targeting and therapeutic potentials. Studies revealed that negatively charged liposomes are generally taken up by blood monocytes/neutrophils preferentially and rapidly compared to neutral or positively charged lipid vesicles (Juliano and Stamp, 1975, Poste et al., 1982). Furthermore, these cells (monocytes/neutrophils) express integrin receptors on their surface that selectively bind to small peptide domain Arg-Gly-Asp (RGD) (Aznavoorian et al., 1990, Odekon et al., 1991, Hauzenberger et al., 1993, Saiki et al., 1996). Exquisitely designed studies have exhibited that interaction between RGD domain on integrin molecule and integrin receptor on leukocytes stimulates phagocytosis by polymorphonuclear cells (e.g. neutrophils) (Senior et al., 1992). Furthermore, localized delivery of bioactives to inflammatory sites (rich in integrin molecules or RGD domain) has been achieved using these cells as delivery vehicle (Kao et al., 2002).
In the present study, an anti-inflammatory drug Diclofenac sodium, a model drug was selected for developing drug loaded negatively charged and RGD-coated magnetic liposomes for their preferential presentation to blood phagocytes (monocytes/neutrophils) and evaluating their subsequent targetability to the brain under inflammatory condition.
Section snippets
Materials
l-α-Soya phosphatidyl choline (Soya PC), phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), cholesterol (Chol), RGD peptide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), Sephadex G-50, human recombinant interleukin (IL-1β), concanavallin A and Triton X-100 were purchased from Sigma Chemical Co. (USA). Dextran-Magnetite (DM) was procured from Meitosangyo (Nagoya, Japan). Diclofenac sodium was a kind gift from M/s Promise Pharmaceuticals, India. All other chemicals used were of
Results and discussion
This study attempted to establish the optimized conditions for preparation of magnetic liposomes in order to develop an effective biophysically modulated carrier-cellular co-ordinated targeting strategy. Magnetic liposomes containing diclofenac sodium and dextran magnetite (DM) could be prepared by reverse-phase evaporation method. The prepared system was firstly optimized for the amount of negatively charged lipid (PS). Magnetic liposomes were prepared using different molar ratio of lipids
Conclusion
The negatively charged magnetic liposomes and RGD-coated magnetic liposomes developed in the present study were shown to be selectively taken up by the circulating blood monocytes/neutrophils thus avoiding the uptake by fixed macrophages of the liver. Coating of the liposomes with RGD peptide further increased their uptake via receptor-mediated endocytosis. This uptake in turn imparts magnetic property to these cells and because of their exclusive migration tendency towards inflammatory sites
Acknowledgements
Authors are thankful to M/s Promise Pharmaceuticals, India for the gift sample of diclofenac sodium. Three authors (SJ, VM and PKD) gratefully acknowledge Council of Scientific and Industrial Research (CSIR), New Delhi, India, for providing financial assistance (SRF). The help and facilities provided by the Head, Department of Pharmaceutical Sciences, Dr. Harisingh Gour University, Sagar, M.P., India is duly acknowledged. Authors also extend their heartfelt gratitude to Dr. R.K. Jain, M/s Varni
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