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MACROPHAGES DEPARTMENT OF PHARMACEUTICS MACROPHAGE Macrophage Definition A type of cell derived from white blood cells ingests (takes in) foreign material. that Macrophages are WBC produced by the differentiation of monocytes in tissues • is a kind of swallowing cell, which means it functions by literally swallowing up other particles or small cells. 3 22/09/2009 Taken from Greek Words (Greek: big eaters, from makros "large" + phagein "eat"; abbr. MΦ). Human macrophages are about 21 micrometres (0.00083 in) in diameter. They move by action of Amoeboid movement. Life time depends on the type of tissue, viability ranges between 6 and 16 days. 22/09/2009 22/09/2009 4 4 Formation of Macrophage Development in bone marrow and passes through the following steps: stem cell committed stem cell monoblast promonocyte monocyte (bone marrow) – monocyte (peripheral blood) macrophage (tissues) Monoblasts(least mature cell of mono nuclear phagocyte system) Differentiate in to monocytes Remain in bone marrow for 24 hours Enter into Peripheral blood From monocytes migrate to Extra vascular tissue( where they differentiate into macrophages) The blood monocytes are young cells (immature macrophage) that already possess migratory, chemotactic, pinocytic and phagocytic activities. Under migration into tissues, monocytes undergo further differentiation (at least one day) to become multifunctional tissue macrophages. MONOCYTE NEUTROPHILL Macrophages are enriched in spleen,lymph,brain,thymus,lungs,liver,conne ctive tissue,bonemarrow. The precursors of macrophages are Monocyte Promonocyte Monoblast all of these cells comes from a common progenitor called the colony forming unit. Function of Macrophage Exhibit 3 main functions in body: 1. Destroy bacteria by phagocytosis 2. Activate other immune function 3. Phagocytose apoptotic cells Function of Macrophage • Once it leaves blood vessel and migrated to tissue, the next job is to EAT the pathogen. This human macrophage, like neutrophil, is a professional "phagocyte" or eating cell (phago = "eating", cyte = "cell"). • Furthermore, the pathogen will be digested by using enzyme from macrophage, in the end resulting antigen and waste material. Steps of phagocytosis: 1.Ingestion through phagocytosis ,a phagosome is formed. 2. the fusion of lysosomes with the phagosome creates a phagolysosome . 3.Pathogen is broken down by enzyme 4.Wastematerial is expelled or assimilated. 1.Pathogens 2.Phagosome 3.Lysosome 4.Wastematerial 5.Cytoplasm 6.Cell membrane 2. Perform Specific Immune Function After digesting a pathogen,present antigen (a molecule, most often a protein found on the surface of the pathogen, used by the immune system for identification)of the pathogen to the corresponding helper T-cell The presentation is done by integrating it into the cell membrane, indicating to other white blood cells that the macrophage is not a pathogen, despite having antigens on its surface. Antigen presentation results in the production of antibodies that attach to the antigens of pathogens, making them easier for macrophages to adhere to with their cell membrane and phagocytose. Secrete hormones cytokines To attract system immune cells to the site and activate cells involved in tissue repair To send signaling path to injury site. Phagocytose apoptotic cells: reduces the potential for an inflammatory response by ensuring that the dying cells are cleared before their intracellular contents are released. Why targeting is required? To activate macrophages Some of the intracellular micro organisms may enter macrophages so in order to kill them, targeting drug to macrophages is necessary. Drug administration in soluble form,fraction of drug entering macrophages is limited so in order to improve amount of drug retained targeting is required. PROBLEMS INVOLVED IN TARGRTING TO MACROPHAGES: It involves in phagocytosis. Enemity towards foreign particles(drug). Carriers for drug delivery to macrophages Liposomes: Liposomes are microscopic vesicles composed of phospholipid bilayers surrounding aqueous compartments. Liposomes have been used extensively as drug carriers. Potential applications in cancer chemotherapy, enzyme therapy, immuno-modulation, antimicrobial therapy, metal detoxification, diagnostics, and topical therapy. Uptake of liposomes by macrophages The complex interaction of liposomes with phagocytic cells is described in different steps: stable adsorption to the cell surface, cellular uptake of intact vesicles by an energy-dependent mechanism. lysosomal degradation of the liposomes and their content. Liposome adsorption to the cell surface seems to be the rate limiting step, since it can be assumed that stably adsorbed vesicles are more susceptible to subsequent uptake than vesicles that are only loosely interacting with the cell surface. several conditions to be satisfied for liposomes deliver biologically active agents to mononuclear phagocytes, i.e. macrophages. liposomes must readily bind to and be phagocytosed by free and fixed phagocytes. They must prevent degradation of entrapped drug. They must retain the encapsulated agent for delivery to the intracellular compartment of RES cells. They should have low systemic half-life 8 hrs. Liposomes are the most widely studied carrier in drug targeting to macrophages. Almost all types of drugs involved in macrophage-associated disorders have been studied using liposomes as carrier. However, The extent of liposome binding and subsequent ingestion by macrophages depends on a number of factors of the intended liposomes. These include composition size type surface properties of liposomes Influence of surface charge, liposome size, concentration, and composition on the uptake by macrophages: The potency of a liposome encapsulated drug is affected by liposomes-surface -charge -size Charge: Negatively charged liposomes associate more affectively and deliver their content more efficiently than neutral liposomes Size: small liposomes deliver drugs more effectively than larger liposomes optimal delivery is observed with negatively charged liposomes of 0.05-0.1μm in diameter Composition: systemic evaluation of MLV with different phospholipid composition. Composition: systemic evaluation of MLV with different phospholipid composition reveals that certain classes of phospholipids are recognised preferentially by macrophages. commonly used lipid for formulating liposomesphosphotidyl choline(pc) (neutral charge) Inclusion of negatively charged phospholipids such as phosphotidyl serine phosphotidyl glycol in MLV consisting of pc greatly enhances their binding to macrophages and phagocytosis by macrophages. In contrast neutral MLV’s composed of pc are not efficiently bound by macrophages. The effect of liposome composition, size,concentration and incubation time on the uptake of liposomes by murine bonemarrow macrophages was observed that liposome uptake increased linearly with the incubation time and concentration. Inclusion of increasing amounts of cholesterol and sphingomyelin reduces the targeting to macrophages. Recently developed developed polyethylene glycol- coated liposomes, known as Stealth liposomes, are not readily taken by macrophages in the reticuloendothelial system, however, stay in the circulation for a long period of time. Inclusion of ligands: Fc receptors Mannosyl Galactosyl Fibronectic lipoprotein Complement Many other receptors These macrophage surface receptors determine the control of activities such as Activation Recognition Endocytosis Secretion etc; A usual approach for promoting the uptake of liposomal content by macrophages is to incorporate ligands capable of interacting with macrophage surface receptors. Mannose receptors on the macrophage surface have been exploited by developing neoglycoprotein and mannose residue. If liposomes surface has some mannose residues like manoarachidic acid residue-such liposomes are called mannosylated liposomes these liposomes have more recognising capacity to macrophages 95% of control liposomes without MAE(monoarachidic acid esters)remained in the circulation even at 30 minutes after administration On the contrary,liposomes with MAE were rapidly eliminated from blood. Fc receptors: fragments of immunoglobulins-these fragments used to liposomes-immunogenic liposomes-specificity increases so surface adsorption is improved. Fc receptor-mediated targeting: Both invitro and in vivo uptake of liposomes have been increased extraordinarily by targeting these carriers through Fc surface receptors to macrophages Derksen studied rabbit immunoglobulin and modified mouse monoclonal antibody incorporated liposomes. Coupling of rabbit immunoglobulin with liposomes increases the uptake by rat liver macrophages more than five times compared with control liposomes. In-vivo study demonstrated by that 80–85% of the coupled liposomes were accumulated in the liver within 1-h of the injection this is because of the presence of the Fc moiety in immunoglobulin-coupled liposomes. Fc receptors mediated targeting of liposomes to human monocyte macrophages :the uptake of antibody conjugated liposomes was 4-6 times higher than that of plain liposomes.this increase in uptake was thought to be due to the Fc receptor mediated binding. Grafted liposomes:protein grafted liposomes have more oppourtunity for targeting to liposomes. protein-may be fragments of collagen ,globulin,gelatin,immunoglobulin enhances efficiency. Bovine serum albumin retard the efficiency. Efficiency also influenced by concentration of cross linking agent like glutaraldehyde or gelatin. NANOPARTICLES OR MICROSPHERES: Nanoparticles and microspheres are under extensive study for delivering drugs to macrophages. They may be polymeric—biodegradable or nonbiodegradable and proteinaceous in nature. The size range covered by microparticles is between 1 and 1000 mm. Microspheres are monolithic or matrix-type microparticles, whereas microcapsules are of reservoir type. In contrast to microspheres, nanoparticles are in the size ranging between 10 and 1000 nm. Uptake of microspheres/nanoparticles by macrophages: As that of liposomes, size, surface property,composition, concentration, and hydrophilicity or lipophilicity of microspheres and nanoparticles play a significant role in the uptake by macrophages. For phagocytosis to occur particles are first contacted by pseudopods of macrophages and engulfed into the cytoplasm by lamellipods . Hydrophobic and relatively large microspheres are more susceptible to phagocytosis than their hydrophilic counterparts. Likewise, nanoparticles with lipophilic coating are better phagocytosed than their hydrophilic counterparts. The extent of phagocytosis can be improved by coating the particle surface with opsonic materials and activating macrophages with various activating factors. Incubation time and dose of the vehicles can also control the process of phagocytosis. Because of the scarcity of experimental data. Influence of microsphere size, composition,concentration and surface property. The influence of surface charge and size of microspheres on their phagocytosis by mouse peritoneal macrophages were studied by using polystyrene and phenylated polyacrolein microspheres of different diameter as well as modified cellulose microsphere with different surface charge. Size:maximum phagocytosis took place when their size was in the range of 1-2 μm and the uptake was maximum with in a one hour period of incubation. charge: for both negatively and positively charged particles ,the extent phagocytosis was increased.with increasing zetapotentials and was lowest when zetapotential was zero(some charge is required) Surface hydrophobicity: Hydrophobic microspheres prepared from benzoyl cellulose were most susceptible to phagocytosis and the non-ionic hydrophilic spheres with opsonic materials and amphiphiles can one was the least. Similarly nanoparticles made from Polyalkylcyanoacrylate polymethylmethacrylate, and human serum albumin microspheres have been used to study the influence of various parameters on the uptake by human macrophages. The more lipophilic polymethylmethacrylate were phagocytosed better than polyalkylcyanoacrylate nanoparticles of similar size. Polybutylcyanoacrylate nanoparticles coated with lipophilic Pluronic F68—a biocompatible poloxamer—increased phagocytosis by nearly 50%, shows more uptake; while Pluronic F108 had no influence or no change. Coating of microspheres with opsonic materials and amphiphiles, and activation of macrophages: coating of microspheres with opsonic materials and amphiphiles can modify significantly the extent of phagocytosis by macrophages depending on the state of macrophage activation. A study about the influence of several proteins on the uptake of macrophages revealed that Gamma-globulin Human fibronectin, Bovine tuftsin and Gelatin - enhance the phagocytosis, while bovine serum albumin reduces the phagocytosis of cellulose microspheres. It was also demonstrated that precoating or surface immobilization with gelatin was the most effective method to enhance the phagocytosis among all other opsonic proteins. Phagocytic uptake tests carried out at 37(degree centigrade) showed that microspheres coated with any surfactants cause a decrement in the phagocytosis in both condition— either in the presence or absence of serum. Microspheres from some biodegradable substances such as copolymers of polylactic acid and polyglycolic acid,cross-linked potato starch, hydroxyethyl starch and cross linked starch, dextran, lichenan and mannan are found to besuccessfully phagocytosed by macrophages. drugs used to target macrophages Cephalosporins Pencillins Amino glycoside antibiotics Immunomodulators interferons interleukins muramyl dipeptide muramyl tripeptide Anticancer drugs doxorubicin daunorubicin under clinical trials Marketed formulations for macrophages ambisome – with amphotericinB Used in the treatment of leishmaniasis- parasite disorder. References M.J. Auger, J.A. Ross, in: C.E. Lewis, J.O’D. McGee (Eds.),the natural Immune System: The Macrophage, Oxford University Press, New York, 1992, pp. 2–74. I.J. Fidler, Targeting of immunomodulators to mononuclear phagocytes for therapy of cancer, Adv. Drug Deliv. Rev.2(1988) 69–106. THANK YOU