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Healthy adult male = 4.5 million RBCs/ l of blood Healthy adult female = 4.8 million RBCs/ l of blood Because matured RBCs have no nucleus, all their internal space is available for oxygen transport.
As they lack mitochondria and generate ATP anaerobically, they do not use up any oxygen they transport.
Normal erythrocyte (Normocyte) - flexible, elastic, biconcave, nucleated structure with a mean diameter of 7.3 m.
Minerals 0.7%
Non-heamoglobin protein 0.9% Methehemoglobin 0.5% Hemoglobin 33.67%
Erythrocyte carrier for oxygen bound to hemoglobin. Hemoglobin content is about 29 picogram/erythrocyte. Lack of nucleus , ribosome's & mitochondria
Some
of
hemoglobin
is
lost
&
other
cellular
constituents are retained , the cells on resealing lose some of the properties of normal erythrocytes & are
as controlled dds
Contain equal amount of protein & lipid. Outer membrane negative charge due to carboxyl groups of salicylic acid.
Encapsulation
Appropriate size & shape Possess specific physico-chemical properties Biocompatible & minimum toxic side effects
Enzymatic inactivation
does not require chemical modification of the substance to be entrapped. This is in contrast with other system
parent drug
of cell
Serving as circulatory biovectors for enzymes Versatile carriers in modern pharmaceutical research
ad development
Reduce Adverse effect Peptide and Enzyme delivery Non immunogenic Facilitate incorporation of proteins and nucleic acid in eukaryotic cells by cell infusion with RBC
circulation lifetime RE
Limited potential as carrier to non-phagocytic target tissue Possibility of clumping of cells and dose dumping Permeable to large no. of drugs
Only bioactives are non-susceptible to denaturation under hypotonic condition Molecules alter physiology of cell
A wide variety of biologically active substancea(5000600,000 daltons)can be encapsulated Generally the molecules should be polar or hydrophilic
1.
2.
3.
4. 5. 6. 7. 8.
Drug Loading in RE
Membrane perturbation
Electro encapsulation
Hypo-osmotic lysis
Dilution method
Dialysis method
Preswell method
Osmotic lysis
incubation at 37 C for 30-40 min Washed with isotonic buffer 3 times to remove unentrapped components
3.Swells Spherocyte
Biconcave discocytes
+ 1.54 KCl,25 c
NOTE: Efficiency-1-8%, low entrapment Simplest & fastest method Low molecular weight Most of the cell content lost by osmotic lysis
6.Incubation at 37 C for 30-40 min Washed with isotonic buffer 3 times to remove unentrapped components
RBC
Preswelling
Hypertonic solution
R E
Pores recovery
Hemolysis
Advantages
A. B.
Used to avoid the disadvantages by hypotonic medium Haemolysis by both physical & chemical means Conventional haemolysis in isotonic urea solutions PG induced haemolysis by transient permeability = resealed with diluting with PG free buffer medium
C.
Ammonium chloride induced haemolysis Adv: Better in vivo surveillance DA: Impermeable only to large molecules Process is time consuming
Electric breakdown is evident when the membrane is polarized for microsecond using varying voltage
Extent of pore formation = electric field strength, pulse duration & ionic strength of the suspending medium
Prevent lysis and balance colloidal osmotic pressure by + macromolecules like BSA Under this osmotic balance ,pores are stay opened at 4c for 2 days Drug from drug solution germinate into the cell
3 steps
osmotic balance + sachraides and proteins 7kV/cm,20sec electric pulse 3:7 (isotonic Nacl & Isoosmotic sucrose)
Electrical perforated erythrocyte cell suspension precooled tube / 4c
medium
Advantages
Disadvantages
Amphotericin B interacts with the cholesterol of plasma membrane of eukaryotic cells causing change in permeability of the membrane.
Erythrocyte
Drug
ERYTHROCYTE ENDOCYTOSIS PRODUCED BY CATIONS AND TRAPPING OF MOLECULES IN THE INVAGINATION OR INSIDE OUT ENDOCYTOSIS VACUOLES
endocytosis
the vesicle membrane seperates the endocytosed substance from the cytoplasm, which may shelter drug prone to inactivation in erythrocyte or alternatively protects the erythrocyte from the drug
3 steps in Endocytosis
1.washed packed erythrocyte+ buffer (ATP,Mgcl2,CaCl2) with drug
Physical parameters
Shape & surface morphology Vesicle size & size distribution Drug release Drug content Surface electrical potential Deformability Sterility Pyrogenicity Animal toxicity
Method/instrument used
1.Physical characteristics TEM/SEM/Phase contrast microscopy/optical microscopy TEM/optical microscopy Diffusion cell/dialysis Deproteinization of cell membrane followed by assay of resealed drug/radiolabelling Zeta potential measurement Capillary method 3. Biological characteristics Sterility test Rabbit method, LAL test Toxicity test
2. Cellular characteristics % Hb content Cell volume % Cell recovery Osmotic fragility Deproteinization of cell membrane followed by hemoglobin assay Laser light scatering Neubaurs chamber/haematological analyzer Stepwise incubation with isotonic to hypotonic saline solution and determination of drug and haemoglobin assay
Osmotic shock
Turbulent shock
Deproteinized
+ acetonitrile (2ml)
Centrifugation 2500 rpm for 10 min Clear supernatant (drug) /cell settled
Deproteinized (acetonitrile)
simulates and mimics the bio-environment conditions that are encountered on invivo administration in vitro handling and the effect of loaded contents on the survival rates of the erythrocytes. RBC hypotonic hypertonic swell shrink isotonic to hypotonic (0.1%w/v)
372C/10 min
It is a sudden exposure of drug loaded erythrocyte to the environment , which is far from isotonic to evaluate the ability of RE to withstand the stress and maintain the integrity as well as appearance. RE + distilled water 3 min Centrifugation 3000 rpm / 15 min Release of hemoglobin to varying degrees estimated spectrophotometriccally.
Turbulence shock
The effect of shear force and pressure by which RE formulation are injected , on the integrity of the loaded cells. Loaded erythrocyte passes through 23 guage hypodermic needle
Phagocytosis
Diffusion through the membrane of cells Using of specific transport system Carrier erythrocyte following the heat treatment or antibody crosslinking are removed from the circulation by phagocytic cell located in the liver and spleen.
It is greatest for the molecule with lipid high solubility zero order release kinetics
Targeting
Drug targeting surface modification target the organs of mononuclear phagocytic system /REs because the changes in the
Target in liver ,lymph node and spleen by modifying membranes Treat hepatic tumour, Parasitic disease Antiviral carrier / Enzyme therapy Improvement in oxygen delivery to tissue Microinjection of macromolecules
b-fructouronidase,b-galactosidase
DRUG TARGETING
With antibodies
Circulatory half life is shortened-by coating ER with antibodies Which target spleen macrophages Heavily modified cells-liver macrophage
With glutaraldehyde
With carbohydrates
With sulphydryls
Magnet responsive erythrocyte Ferrofluids-colloidal suspension magnetite+Fe3o4). External magnetic field Improve the stability Targeting/ Reduce cytotoxicity Especially inflammatory drugs
Photosensitized erythrocyte (haematoprophyrin derivative) Antibody anchored erythrocytes(immunoerythrocytes) Antviral agent Thrombolytic therapy Oxygen defieciency therapy
Future Prospectus
Erythrosome:
Nanoerythrosome:
100 nm erythrocyted
DELIVERY STRATEGIES
Mechanisms passive diffusion, specialized membrane-associated carriers, phagocytosis of the carrier cells by the macrophages of RES: depletion of the drug in circulation, accumulation of the drug
in RES, upon lysis of the carrier and slow release from this system
into circulation, accumulation of the carrier erythrocytes in lymphatic nodes following subcutaneous injection of the cells and
Erythrosomes : Erythrosomes are specially engineered vesicular systems in which chemically cross-linked human erythrocyte cytoskeletons are used as a support upon a lipid bilayer is coated.