Dharmveer Kohli

Transferrin coupled liposomes as drug delivery carriers for brain targeting of 5-florouracil

Diseases and disorders of the brain are extremely difficult to treat pharmacologically because most drugs are unable to pass across the blood–brain barriers. Complex multi-strand tight junctions between adjacent cerebral endothelial cells and between choroid plexus epithelial cells form a physical barrier and prevent the passage of water soluble drugs from the blood into the brain, whereas the inward passage of lipid soluble drugs is restricted by drug efflux pumps which act as a functional barrier. In the present work, a transferrin-coupled liposomal system for brain delivery of 5-florouracil has been investigated. 5-florouracil and 99mTc-DTPA bearing non-coupled liposomes were prepared by cast film method, which were coupled with the transferrin by incubating these liposomes with transferrin in the presence of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride in saline phosphate buffer (pH 7.4). These liposomal systems were characterized for vesicle size, percent drug entrapment, and in vitro drug release. The size of the liposomes was increased on coupling with transferrin while percent drug entrapment reduced. The results of the in vitro release profile demonstrated that non-coupled liposomal formulation releases a comparatively higher percent (i.e. 74.8±3.21%) of drug than coupled liposomes. Results of in vivo study suggested a selective uptake of the transferrin-coupled liposomes from the brain capillary endothelial cells. In case of coupled liposomes, the level of radioactivity was 17-fold more as compared to the free radioactive agent and 13 times more with the non-coupled liposomes. Therefore, it could be concluded that using transferrin coupled liposomes the brain uptake of the drug could be enhanced.

Transferrin-conjugated liposomal system for improved delivery of 5-fluorouracil to brain

The objective of this study is to achieve the enhanced delivery of 5-fluorouracil to brain through transferrin-coupled liposomes. 5-Fluorouracil-loaded liposomes were prepared by cast film method and characterized for particle size, shape, percent encapsulation efficiency and in vitro drug release. Biodistribution studies were carried out with the help of radiolabelled 5-fluorouracil. 5-Fluorouracil was labelled with 99mTc-DTPA by oxidation–reduction method using stannous chloride and optimized for labelling parameters to get a high labelling efficiency. The in vitro stability was determined to check the efficiency of a system to find out the suitability of the radiolabelled system for in vivo studies. 99mTc-DTPA-labelled 5-fluorouracil bearing non-coupled and coupled liposomes were administered intravenously and biodistribution studies were performed. The distribution of 5-fluorouracil via non-coupled and coupled liposomes was determined in various organs, such as lungs, liver, kidneys, spleen and brain, by measuring the radioactivity using a gamma scintillation unit. The results of in vivo studies confirmed a selective uptake of the transferrin-coupled liposomes from the brain capillary endothelial cells. An average of 10-fold increase in the brain uptake of the drug was observed after the liposomal delivery of 5-fluorouracil, while the transferrin-coupled liposomes caused a 17-fold increase in the brain uptake of 5-fluorouracil. Therefore, it can be concluded that transferrin-coupled liposomes enhance the brain uptake of the drug, like 5-fluorouracil.

Well-defined and potent liposomal hepatitis B vaccines adjuvanted with lipophilic MDP derivatives

UNLABELLED The characterization of immunological cascades of the innate immune system activated by invariant molecular structures termed as pathogen-associated molecular patterns recognized by pattern recognition receptors of macrophages and dendritic cells, have allowed the elucidation of the mechanisms underlying the immunomodulatory properties of adjuvants. Thus, adjuvant-active lipophilic analogues of N-acetyl muramyl dipeptide (MDP) were incorporated in liposomal hepatitis B surface antigen (HBsAg) formulations. The immunoreactivity of the formulations was evaluated by measuring anti-HBs, immunoglobulin G (IgG), and isotype antibody titer and compared with alum-adsorbed HBsAg formulation. The formulations were also evaluated for cell-mediated immune response by HBsAg-specific proliferation of splenocytes and simultaneous estimation of cytokines (interleukin-4 [IL-4], interferon-gamma [IFN-gamma]). Results indicate that the serum IgG and anti-HBs titer obtained after intramuscular administration of liposomal muramyl tripeptide-phosphatidylethanolamine (MTP-PE) and liposomal N-acetylmuramyl-l-alanyl-d-isoglutamine-glycerol dipalmitate (MDP-GDP) antigenic formulations were significantly higher. The incorporation of MTP-PE on the liposomal HBsAg increased the stimulation index (SI) four to five times as compared to plain HBsAg solution, and it also induced significantly higher Th1 cellular immune response with a predominant IFN-gamma level. So it is the novel effective and potentially safe approach in which liposomes act as delivery vehicles for hepatitis B viral antigen to antigen-presenting cells and is ornamented with a biological response modifier that could activate these target cells to enhance the antigen presentation to T lymphocytes. FROM THE CLINICAL EDITOR In this study, adjuvant-active lipophilic analogues on N-acetyl muramyl dipeptide (MDP) were incorporated in liposomal hepatitis B surface antigen (HBsAg) formulations. The immunoreactivity of the formulations was evaluated and found effective, leading to a potentially enhanced immune response against the delivered antigen.

Potential of transferrin and transferrin conjugates of liposomes in drug delivery and targeting

Targeted drug delivery has gained recognition in modern therapeutics and attempts are being made to explore the potential of cell biology-related bioevents in the development of specific and target-oriented systems. In connection to modern therapeutic systems, most of the emphasis has been laid upon the bioconjugated drug delivery systems. Bioconjugates involve the linking of two or more molecules to form a novel complex having the combined properties of its individual components. The nature of the linking agent between the pharmacologic agent and the delivery-augmenting moiety dictates the degree of successful delivery and its outcome. The component for the bioconjugated drug delivery includes receptors and ligands, where the receptors act as molecular targets or portals whereas ligands with receptors provide selective and specific trafficking towards the targeting site. Recently, a number of bioconjugated systems have been discovered for the site-specific presentation and delivery of various bioactive substances using biorelevant ligands, including antibodies, glycoprotein, viral proteins, and molecules of endogenous origin. In this review, the potential of transferrin (Tf) and Tf conjugates of liposomes in site-specific drug delivery systems are discussed. Tf is an abundant component of serum with the capacity to bind and transport iron, while Tf receptor (TfR), a dimeric transmembrane glycoprotein, is present on the surface of the most proliferating, higher eukaryotic cells. Tf expression is also found in nonproliferating tissues, such as hepatocytes, tissue macrophages, pituitary cells, pancreatic islet cells, and the endothelium of brain capillaries. Tumor cells frequently carry elevated numbers of TfRs compared with corresponding normal cells, and reduced serum levels of Tf are often observed in patients with tumors. In the past, various strategies have been developed, which include coupling of the liposomal surface with Tf by using various linking agents. Low-molecular weight drugs and proteins as well as liposomes can be linked with Tf. The Tf-coupled vesicular system is physicochemically stable in the bioenvironment and is site-specific. The aim of coupling liposomes with Tf is to improve the physical and biochemical stability of liposomes and make them appropriate for targeting specific organs and cells. Tf may be widely applied either as a carrier or targeting ligand in the active targeting of anticancer agents, proteins, and genes to primarily proliferating malignant cells that overexpress TfRs. Tf has been used as a molecular conjugate to deliver DNA to erythroleukemic, lung, and liver cell lines. Tf can also be modified with the positive charge N-acylurea groups to make them suitable for electrostatic binding of DNA, in order to achieve a well defined DNA-binding ligand for receptor-mediated gene transfer. Association of Tf with lipoplexes, in particular the negatively charged ternary complexes, significantly overcomes the inhibitory effect of serum and facilitates efficient transfection in many cell lines, including HeLa, K-562 cells, and lung carcinoma cells Calu-3 and H-292 cells. Tf-lipoplex has demonstrated high efficiency in tumor-targeted gene delivery and long-term therapeutic accuracy in systemic p53 gene therapy for both human head and neck cancer and prostate cancer. Tf and Tf-coupled liposomal drug delivery systems may prove particularly valuable to enable the use of a drug that seems to be ineffective or toxic if delivered systematically. The delivery of drugs to the brain has been particularly challenging because of the presence of the blood-brain barrier, which restricts the passage of most therapeutic agents into the brain. Therefore, active targeting of the brain is crucial for effective treatment of brain diseases. The anti-TfR antibody, such as OX26, when coupled with therapeutic agents, has shown potential in drug and gene delivery to the brain.

Humoral and cell-mediated immune-responses after administration of a single-shot recombinant hepatitis B surface antigen vaccine formulated with cationic poly(l-lactide) microspheres

The present investigations were aimed to compare the humoral and cell-mediated immune responses between recombinant hepatitis B surface antigens (HBsAg) adsorbed L-PLA microspheres (Ms) vaccine (single-shot) and marketed alum-HBsAg vaccine (two-doses). The blank cationic (cetyltrimethyammoniumbromide) microspheres were prepared by the double emulsion (w/o/w) solvent evaporation technique. The HBsAg was adsorbed onto the surface of blank cationic microspheres. These microspheres were characterized in vitro for their size, shape, adsorption-efficiency, in-process stability, and HBsAg release studies. Specific humoral immune responses (IgM and IgG) and cell-mediated immune responses (cellular-proliferation) assay including release of interferon-gamma (IFN-γ), interleukin-2 (IL-2), and nitric oxide (NO) from host’s cells stimulated with HBsAg or lipopolysaccharide (LPS)/ concanavalin A (con A) in-vitro were determined. Based on these findings, it was concluded that the single injection (using subcutaneous-route) of the polymeric microspheres produced better immune response (both humoral and cell-mediated) than two injections of a conventional alum-HBsAg vaccine. These data demonstrate high potential of polymeric microspheres for their use as a carrier adjuvant for hepatitis B vaccine.

Enhancement of T-helper type I immune responses against hepatitis B surface antigen by LPS derivatives adjuvanted liposomes delivery system

Currently, there is a clinical need for more effective vaccine for hepatitis B that induces robust cell-mediated immune response capable of viral clearance in chronic hepatitis B infection. In the present study, hepatitis B vaccines formulations were designed by loading the hepatitis B surface antigen into liposomes adjuvanted with rough lipopolysaccharide (Re-LPS) and lpxL1 LPS using conventional rotatory evaporation method and were characterized for various parameters, such as vesicle shape and surface morphology, size and size distribution, entrapment efficiency, turbidity, and in vitro release pattern. The immunoreactivity in mice was evaluated by measuring anti-HBs IgG titer and compared with alum-adsorbed HBsAg solution, plain HBsAg, and liposomal HBsAg formulations. The formulations were also evaluated for cell-mediated immune response by HBsAg specific proliferation of spleenocytes after secondary immunization and re-stimulation in vitro with the same antigen. Simultaneous estimation of cytokines (IL-4, IFN-γ) was also carried out. Ex vivo cellular uptake study was performed by fluorescence microscopy. Results indicate that the serum IgG titer obtained after i.m administration of Re-LPS- and lpxL1 LPS-adjuvanted liposomal HBsAg formulation was equivalent to alum-adsorbed HBsAg formulation but was more responsive, sustained, and significantly higher than the corresponding liposomal HBsAg and plain HBsAg formulations. Incorporation of lpxL1 LPS into the liposomal HBsAg increased the stimulation index (SI) 6–10 times as compared with plain HBsAg. Re-LPS- and lpxL1 LPS-adjuvanted liposomal HBsAg formulations induced stronger cellular immune response with a predominant Interferon-gamma (IFN-γ) level than those induced by free HBsAg alone, alum-adsorbed HBsAg, and non-adjuvanted liposomal HBsAg. Probably, the possible mechanism for the enhancement of cellular immunity in addition to humoral immunity by LPS-adjuvanted liposomal HBsAg formulation is due to marked enhancement of immunological presentation and recruitment of antigen via macrophage and antigen-presenting cells (APCs).

Polymeric lamellar substrate particles as carrier adjuvant for recombinant hepatitis B surface antigen vaccine.

Blank polymeric lamellar substrate particles (PLSP) of poly (l-lactide) were prepared and recombinant hepatitis B surface antigen (rHBsAg) was adsorbed onto these particles. The physical characteristics of blank PLSPs or PLSP-rHBsAg in vitro and its immunological responses in Balb/c mice were investigated. The average size of the particles was less than 10microm. Antigen adsorption efficiency was found to be 62.66+/-1.26%. Immunization with PLSP-rHBsAg resulted in upregulation of specific cellular (lymphoproliferation, IFN-gamma and NO release) as well as IgG response in animals. These responses were higher than those produced by two-dose schedule of alum-adsorbed antigen (alum-rHBsAg). Thus in conclusion, in terms of convenience and efficacy PLSP-rHBsAg is superior to alum-rHBsAg.

Development and characterization of lectin-functionalized vesicular constructs bearing amphotericin B for bio-film targeting

The long-term goal of this work will be to develop a topical formulation for oropharyngeal candidosis. Liposomes were prepared by the vesicle extrusion technique from mixtures of dipalmitoylphosphatidylcholine, cholesterol, and stearylamine incorporating a reactive phospholipid, the m-maleimidobenzoyl-N-hydroxysuccinimide ester derivative of dipalmitoylphosphatidylethanolamine, which was conjugated with the N-succinimidyl-S-acetylthioacetate derivatives of succinyl concanavalin A. Morphology of liposomes was studied by transmission electron microscopy and bio-film architecture using fluorescence microscopy. Lectinized vesicles were put into contact with bovine submaxillary mucin, to determine the in vitro activity and specificity. Targeting study was performed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The liposomes were found to have a diameter in the range of 360–450 and 390–510 nm for uncoated and coated formulations, respectively. The MTT assay showed a strong association of lectin-bearing liposomes with candidal bio-film. The results suggest that surface-modified liposomes can effectively target candidal bio-film in vitro.