Loaded Erythrocytes

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Loaded erythrocytes:

Loaded erythrocytes Pharmaceutics By Mr. Sachin Shrimant Patil| Guided By Mr. V. T. Deshmukh | Y.T.C. Faculty of Pharmacy (M. Pharm.), Satara

INTRODUCTION:

INTRODUCTION Erythrocytes as a carrier collecting blood samples from the organism of interest, separating erythrocytes from plasma , entrapping drug in the erythrocytes , resealing the resultant cellular carriers. resealed erythrocytes . 2

Basic features of erythrocytes:

Basic features of erythrocytes 3 Erythro :- red Cytes :- cells flexible, elastic, biconcave Diameter :-7.5µm Thickness:-2.0µm

Basic features of erythrocytes:

Basic features of erythrocytes A. Composition of Erythrocytes 4

a. Composition of erythrocytes:

a. Composition of erythrocytes Blood contains- 55 % plasma 45 % corpuscles . Erythrocytes- 35% Hb & 65% water . Lipid content cholestrol , lecithin and cephaelins . 5

b. Electrolyte composition of electrolyte:

b. Electrolyte composition of electrolyte Qualitatively similar but quantitatively differs from plasma . K + is more in erythrocytes Na + is more in plasma The osmotic pressure of erythrocytes is equal to that of the plasma & termed as isotonic ( 0.9 % NaCl or normal physiological saline) Change in the osmotic pressure of the medium - changes the morphology of RBC 6

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If the medium is Hypotonic water, RBC get swelled , loose hemoglobin content and may burst . If the medium is hypertonic, RBC shrinks & beacame irregular in shape Ringer’s & Tyrode’s soln . are used in erythrocyte related experiments i sotonic ions in proper quantity 7

c. Haematocrit value:

c. Haematocrit value Centrifugation Erythrocytes- settle down to the bottom Plasma- top Leukocytes & Platelets- thin layer in between H aematocrit value the percentag e of whole blood made up of erythrocytes . Males.......... 40-50% Females....... 38-45% 8

d. sources & isolation of erythrocytes:

d. sources & isolation of erythrocytes Different mammalian erythrocytes e.g . mice, cattle, pigs, dogs, sheep, goats, monkeys, chicken, rats, and rabbits etc . EDTA or heparin - as anticoagulants 9

Isolation of erythrocytes:

Isolation of erythrocytes Blood is collected into heparinized tubes by venipuncture cardiac/splenic puncture (in small animals) and veins (in large animals) in a syringe containing a drop of anti coagulant. The whole blood is centrifuged at 2500 rpm for 5 min at 4˚C in a refrigerated centrifuge . serum is removed packed cells washed with phosphate buffer saline (pH=7.4 ). The washed erythrocytes are diluted with PBS and stored at 4˚C 10

Advantages of erythrocytes as drug carrier:

Advantages of erythrocytes as drug carrier Autologous cells, biocompatible , no triggered response Uniform size and shape of carrier Relatively inert intracellular environment Biodegradable with no toxic products Prevention of degradation of the loaded drug from inactivation by endogenous chemicals The wide variety of chemicals that can be entrapped The modification of pharmacokinetic & pharmacodynamic parameters of drug attainment of steady-state plasma concentration decreases Drug release at target site in controlled manner Appreciable stability during storage 11

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METHODS OF DRUG LOADING: 12

Hypotonic Haemolysis & Isotonic Resealing Methods :

Hypotonic Haemolysis & Isotonic Resealing Methods Loading of drugs is done by two steps: 2.This step involves restoration of isotonicity to reseal the erythrocytes. 13 1.Hypotonic lysis of cells in a solution containing the drug/enzyme to be entrapped.

Hypotonic haemolysis:

Hypotonic haemolysis RBC Membrane ruptured RBC Loaded RBC Resealed Loaded RBC 0.4% NaCl Hypotonic Drug Loading buffer Resealing buffer Incubation at 25 0 c Efficiency  1-8% Enzymes delivery Hypotonic med Isotonic med . Washed 14

Isotonic Osmotic Lysis:

Isotonic Osmotic Lysis RBC Isotonically ruptured RBC Chemical – urea, polyethylene, polypropylene, and NH 4 Cl Physical rupturing Chemical rupturing Drug Isotonic Buffer Loaded RBC Resealed RBC Incubation at 25 0 C 15

Hypotonic pre-swelling:

Hypotonic pre-swelling RBC 0.6%w/v NaCl Swelled RBC Drug + Loading buffer 5 min incubation at 0 0 c Loaded RBC Incubation at 25 0 c Resealing Buffer Resealed RBC Efficiency  72% Fig:- Preswell Method 16

Loading by red cell loader:

Loading by red cell loader Magnani and coworkers , 1998 developed a novel method for the entrapment of non-diffusible drugs into human erythrocytes . 50ml of blood The method is based on two sequential and controlled hypotonic dilutions of washed red blood cells . Subsequent isotonic resealing of erythrocytes allow a 35-50 % cell recovery 30 % entrapment of drug 17

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18

Hypotonic dilution:

Hypotonic dilution 1st method, simplest & fastest Procedure: Packed erythrocytes + 20 vol. aq. Solution of drug Hypertonic buffer to restore tonicity Centrifugation Supernatant discarded & pellet is washed 19

Hypotonic dilution:

Hypotonic dilution Drawbacks: Low entrapment efficiency Loss of Hb & other cell content Reduces circulation half life of loaded cell . Use- targeting RES organs e.g.- Enzymes- galactosidase , glucosidase , asparginase , arginase bronchodilators- salbutamol 20

Entrapment By Endocytosis :

Entrapment By Endocytosis RBC Drug Suspension + Buffer containing ATP, MgCl 2 , and CaCl 2 At 25 0 C Loaded RBC Resealing Buffer Resealed RBC Fig;- Entrapment By Endocytos Method 21 Examples: propranolol, chlorpromazine

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Chemical perturbation of Membrane RBC e.g. Amphotericin B Chemical agents Increased permeability of RBC Resealing Buffer Drug Resealed RBC 22 Drwabacks Destructive changes in cell membrane Examples: Antineoplastics like daunomycin, halothane

Lipid fusion:

Lipid fusion Lipid vesicles containing drug can be directly fused with human erythrocytes leading to exchange of lipid entrapped drug. T his technique is used for loading of inositol mono phosphate into resealed erythrocytes for increased oxygen carrying capacity. The encapsulation efficiency by this method is however very low i.e. (1 %). 23

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Method % Loading Advantages Disadvantages Dilution 1-8% Fastest and simplest for low mol. Wt. Less Entrapment efficiency Pre-swell Dilution 20-70% Good retention of cytoplasm constituents and good survival in-vivo --- Isotonic Lysis --- Better in vivo surveillance Time consuming, Impermeable to large molecules 24

characterization:

25 1)Drug conent determination centrifuge at 3000 rpm Loaded cells clear supernatent Assay of drug content characterization

characterization:

26 characterization 2)Percent cell recovery:- Counting the intact cell numbers in per cubic mm after & before loading the drug. 3)Morphology:- E lectron microscope can be used.

characterization:

27 4 )Osmotic shock:- determination of % Hb spectrophotometrically 5 ) Turbulence shock:- distribution of loaded cell during injection by comparing flow rate of blood 6 ) Erythrocyte Sedimentation Rate:- Suspension stability of RBC plasma Number & size of cells & protein characterization

Shelf life & storage stability:

Shelf life & storage stability Hank’s balanced salt solution ( HBSS ) at 4ºC for two weeks Suspending cells in oxygenated HBSS with 1 % soft gelatin . Cells recovered after liquefying the gel by placing tubes in water bath at 37ºC followed by centrifugation. Cryopreservation of Erythrocytes in liquid nitrogen 28

Different forms of drug loaded rbc::

Different forms of drug loaded rbc : more than 80% erythrocyte ghosts loaded with drug or enzyme appear as biconcave under electron microscope. Less than 20% cells show abnormal morphology, and the rest appear as spherocytes or other destroyed forms. “Erythrocytes ghosts”- RBC- haemolysis - washing with large volumes of hypotonic medium - looses all Hb - on resealing the resultant cells appear as pale or transparent 29

Release characteristics of the loaded drugs:

Release characteristics of the loaded drugs There are mainly three ways: ◦Phagocytosis ◦Diffusion through the membrane of the cells ◦Using a specific transport system. liver or spleen 30

Application of resealed erythrocytes:

Application of resealed erythrocytes Erythrocytes as drug/ enzyme carriers: Erythrocytes as carriers for enzymes. Erythrocytes as carriers for drugs. Erythrocytes as carriers for proteins and macromolecules. Drug targeting: Drug targeting to RES organs Surface modification with antibodies Surface modification involving sulphydryls , glutaraldehyde , antibodies. 31

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Drug targeting to Liver Enzyme deficiency/replacemen t therapy Treatment of liver tumors Treatment of parasitic diseases Removal of RES Iron Overload Targeting to sites other than RES- rich organs Magnet-responsive Erythrocyte Ghosts. Photosensitized Erythrocytes. Antibody Anchored Erythrocytes ( Immunoerythrocytes ). Erythrocytes as Circulating Bioreactors 32

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Application Drug/ Enzyme/ Molecule Enzyme deficiency, replacement therapy ß- galactosidase , ß- fglucosidase , urease Thrombolytic activity Aspirin, Heparin Immuno -therapy Human recombinant Interleukin-2 Circulating carriers Albumin, Salbutamol, Tyrosine kinase, Prednisolone Targeting to RES Pentamidine , Mycotoxine 33

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Application Drug/ Enzyme/ Molecule Iron overload Desferroxamine Chemotherapy Rubomucin , Methotrexate, Doxorubicin, Daunomycin , Cytosine, Adriamycin Circulating Bioreactor Arginase , Acetaldehyde dehydrogenase Targeting to sites other than RES Daunomycin , Diclofenac sodium, Methotrexate 34

Novel systems:

Novel systems Nanoerythrosomes : Extrusion of erythrocyte ghosts to produce small vesicles with an average diameter of 100nm . Example: Daunorubicin conjugated to nanoerythrosomes using gluteraldehyde spacer. This complex is more active. 35

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B. Erythrosomes : specially engineered vesicular systems in which, erythrocytes are used as a support upon which a lipid bilayer is coated. These vesicles have been proposed as useful encapsulation systems for macromolecular drugs . 36

Future prespective:

Future prespective A large amount of valuable work is needed so as to utilize the potentials of erythrocytes in passive as well as active targeting of drugs. Diseases like cancer could surely find its cure. Genetic engineering aspects can be coupled to give a newer dimension to the existing cellular drug carrier concept. 37

References:

References S.P. Vyas and R.K. Khar , Resealed Erythrocytes in Targeted and Controlled Drug Delivery, Novel Carrier System, 387-416. S . Jain and N.K. Jain, Resealed Erythrocytes as drug Carriers in Controlled and Novel Drug Delivery, 256-291. An Overview of Resealed Erythrocyte Drug DeliveryR . P. Patel et al.Journal of Pharmacy Research 2009, 2(6),1008-1012. P.C . Mangal and A. Kaur , Electroporation of Red Blood Cell Membrane and its Use as a Drug Carrier System, Ind. Journal of Biochemistry Biophysiology . 28, 219, 1991. www.pharmatech.com www.pharmatutor.org www.nature.com www.medline.com 38

Thank you!:

Thank you! 39

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