Resealed erythrocyte

Prepared by: Sunil B. Kokate,

Mpharm 2nd sem,

P’ceutics.

GCP A’bad.

CONTENTS

Introduction.

Features of erythrocytes .

Source, fractionation and isolation of erythrocytes.

Methods of drug loading.

In vitro characterization.

Shelf life and storage stability.

Biomedical applications.

References.

Introduction:

Erythrocytes, the most abundant cells with capabilities for the delivery

of drugs, biocompatible, biodegradable, possess long circulation half lives.

Erythrocytes, are studied for their potential carrier capabilities for the

delivery of drugs and drug-loaded microspheres.

Such drug-loaded carrier erythrocytes are prepared simply by collecting blood

samples from the organism of interest, separating erythrocytes from plasma,

entrapping drug in the erythrocytes, and resealing the resultant cellular carriers.

Hence, these carriers are called resealed erythrocytes.

The overall process is based on the response of these cells under osmotic

conditions. Upon reinjection, the drug-loaded erythrocytes serve as slow

circulating depots and target the drugs to a Reticulo Endothelial System (RES).

Figure 2: Hyponotic hemolysis Figure 3: Hyponotic dilution

Figure 1: Composition of blood

Morphology and physiology of erythrocytes

Erythrocytes are the most abundant cells in the human body (5.4 million

cells/mm3 blood in a healthy male and 4.8 million cells/mm3 blood in a

healthy female).

Its crucial role in oxygen delivery to various parts of the body.

These are biconcave discs with an average diameter of 7.8 µm, a thickness

of 2.5 µm in periphery, 1 µm in the center and a volume of 85–91 µm3.

The flexible, biconcave shape enables erythrocytes to squeeze through

narrow capillaries, which may be only 3 µm wide.

Erythrocytes are a highly specialized O2 carrier system in the body. Because

a nucleus is absent, all the intracellular space is available for O2 transport.

Also, mitochondria are absent and energy is generated anaerobically in

erythrocytes.

Erythrocytes live only about 120 days.

Worn-out erythrocytes are removed from circulation and destroyed in the

spleen and liver (RES), and the breakdown products are recycled. The

process of erythrocyte formation within the body is known as erythropoiesis.

In a mature human being, erythrocytes are produced in red bone marrow

under the regulation of a hemopoietic hormone called erythropoietin .

Source and isolation of erythrocytes: Erythrocytes from mammalians like mice, cattle, pigs, dogs, sheep, goats,

monkeys, chicken, rats and rabbits. Fresh whole blood is the blood that is collected heparinized tubes by venipuncture

withdrawn from cardiac/splenic puncture in a syringe containing a drop of anti coagulant, immediately chilled to 4ºC and stored for less than 2 days.

The erythrocytes are then seperated from serum coats by centrifugation at 2500rpm for 5 min at 4 ±1ºC and washed 3 times with phosphate buffer solution (pH= 7.4).

The washed cells are suspended in buffer solutions at various hematocrit values as desired and are often stored in Acid-Citrate-Dextrose(ACD) buffer at 4ºC for as long as 48 h before use.

Properties of resealed erythrocyte of novel drug delivery carriers:

It should be appropriate size, shape and should permit the passage through

capillaries and minimum leakage of drug should take place.

Biocompatible and should have minimum toxic effect.

Ability to carry a broad spectrum of drug.

Specific physicochemical properties by which desired target size could be

recognized.

The degradation product of the carrier system, after release of the drug at the

selected site should be biocompatible. It should be physico-chemically

compatible with drug.

The carrier system should stable during storage.

Haematocrit value:

If blood is placed into a tube and centrifuged, the cells and the plasma will

separate.

The erythrocytes, which are heavy, will settle down to the bottom of the tube,

while the plasma rises up to the top and the leukocytes and platelets will form a

thin layer between the erythrocytes and the plasma.

The haematocrit is defined as the percentage of whole blood made up of

erythrocytes.

Males.......... 40-50% .

Females....... 38-45% .

Sr. no. Advantages of resealed erythrocyte Disadvantages of resealed erythrocyte

1 Biocompatible. A limited potential as carrier to non-phagocyte target tissue.

2 Biodegradable. Clumping of cells .

3 uniform size and shape . Dose dumping .

4 Prevent degradation of loaded drug from endogenous chemicals.

5 Variety of chemicals that can be entrapped.

6 Modification of pharmacokinetic and pharmacodynamic parameters of drug.

7 Non-immunogenic in action.

8 Protect degradation and inactivation of proteins and enzymes .

9 Facilitate incorporation of proteins and nucleic acid in eukaryotic cells by cell infusion with RBC.

Characteristic of Resealed Erythrocytes:

When erythrocytes are suspended in a hypotonic medium they swell to

about one and a half times their normal size, and the membrane rupture in

the formation of pores with diameters of 200 to 500 Å. The pores allow

equilibration of the intracellular and extracellular solution.

If the ionic strength of the medium then is adjusted to isotonic and the cells

are incubated at 37ºC, the pores will close and cause the erythrocyte to

“Resealed”. Using this technique upto 40% of drug can be entraped in the

resealed erythrocytes from the extracellular solution. So, this systems are

used for targeted delivery via intravenous injection.

Normal aging erythrocytes, slightly damaged erythrocytes and those

coated lightly with antibodies are sequestered in the spleen after

intravenous vein fusion, but heavily damaged or modified erythrocytes are

removed from the circulation by the liver.

Red cells placed in hypotonic

drug solutions

Lysis of cells and entry of

drug

Resealed red cells loaded with drug

Membrane perturbation

Electro encapsulation

Hypo-osmotic lysis

Lipid fusion method

Dialysis method

Preswell method

Dilution method

Method of drug loading in resealed erythrocytes

Osmotic-lysis

Endocytosis method

Normal transport mechanism

Red cell loader used in hypo- somatic lysis.

1) Hypo osmotic lysis method: a) Dialysis method:o In this method, erythrocyte suspension + Drug solution → dialysis tube and both

ends are tied with thread. o This tube is placed in bottle containing 100ml of swelling solution at stored at 4ºC

for lysis.o After, the dialysis tube is transferred to 100ml resealing solution (isotonic PBS,

pH 7.4 ) at room temp. (25º-30ºC) for resealing. o These resealed cells are removed and washed with Phosphate Buffer Solution

(PBS) at 4ºC and finally suspended in PBS solution.o The limitation of dilution method can be overcome by carrying out lysis and

resealing in the same dialysis tube.

b) Dilution method: When these RBC are placed in the hypotonic (0.4% NaOH) they get ruptures

permitting escape of cellular content and equilibrium is achieved within 1min, which results in swelling upto 1.6 time its initial volume.

At 0ºC, opening permits to attain equilibrium for inter- and extra cellular fluids through the pore size of 200-500Å.

Increasing the ionic strength at 37ºC, results in resealing of cell membrane and restoring the osmotic property.

This method is simple and faster, but have very low encapsulation efficiency (1-8%) and lose of cytoplasmic constituents during osmotic lysis.

Low molecular weight drugs like ß-glucosidase and ß-galactosidase can be encapsulated.

Hypotonic medium

Isotonic medium

washed

c) Osmotic lysis:

In this technique, haemolysis in isotonic solution can be achieved by either

chemical or physical means or both.

Erythrocytes are incubated in drug solution with high transerythrocytic

membrane permeability like PEG or NH4Cl or Urea which offers osmotic

diffusion until it reaches equilibrium.

Then the solution was diluted with an isotonic-buffered drug solution.

These cell are separated, washed and resealed at 37ºC.

Eg: Dimethyl sulphoxide (DMSO), Monosaccharides, Sucrose, etc.,

d) Preswell dilutional haemolysis: This technique is based upon initial controlled swelling in hypotonic buffered

solution. The principle involved in this technique is swelling of erythrocytes without lysis

by placing them in slightly hypotonic solution and centrifugation with low speed at 0ºC for 5min.

To this add small volume of drug solution at the point of lysis. This techniques results in retention of cytoplasmic constituents and 72% of drug

entrapment. Eg: Thyroxin, Ibuprofen, etc.,

2) Electro encapsulation:

Used for entrapment of bioactive molecules. It is also known as “Electroporation method”.

The erythrocyte membrane is opened by the dielectric breakdown.

Firstly, erythrocytes are suspended in an isotonic buffered solution in electric discharge

chamber connected to the capacitor and external circuit.

The charge is discharged at definite voltage within definite time interval through cell

suspension to produce a square-wave potential.

The optimum intensity of an electric field is between 1-10 kW/cm and optimum discharge

time is 20-160µs.

The compound which is to be entrapped was added to the medium.

After certain time the cell suspension was transferred to a pre-cooled tubes and kept at 4ºC.

Resealing of electrically perforated erythrocytes membrane is then affected by incubation at

37ºC in an osmotically balanced medium.

Major advantage is uniform distribution of drug loading is achieved and possible to entrap

upto 35% of drug.

Disadvantage is need sophisticated and special instruments and time consuming.

Eg: Sucrose, Urea, Methotrexate, Glucophorin, etc.,

3) Endocytosis method: In this method, 1 volume of packed erythrocytes + 9 volume of buffer (containing

2.5mM ATP, 2.5mM MgCl2 and 1mM CaCl2) and incubated for 2min at room temperature.

The pores created in this method are resealed by using 154 mM of NaCl and incubate at 37ºC for 2min.

The entrapment of drug was obtained by endocytosis. In this method, the vesicle membrane separates the endocytosed substance from

the cytoplasm containing drug, which are sensitive to inactivation of cytoplasmic enzyme and protect from erythrocyte membrance.

Eg: 8-amino quinolines, Vinblastin, Chlorpromazine, Phenothiazines, Hydrocortison, Propanolol, Tetracine, Vit.A, etc.,

4) Lipid Fusion Method: Lipid vesicles containing a drug can be directly fused to human erythrocytes, which

leads to an exchange with a lipid-entrapped drug. Entrapment efficiency is very low (~1%). Eg: Inositol monophosphate to improve the O2 carrying capacity of RBC.

5) Membrane Perturbation: This method is based on increase in membrane permeability of erythrocytes when

exposed to certain chemicals.

Eg: Amphotericin-B, Daunomycin using Halothane.

6) Normal Transport Mechanism: The biological active components are entrapped in erythrocytes without dispruting the

erythrocyte membrane by incubating in drug solution for varying period of time. 30-35% of drug can be entrapped.

In-vitro storage: The most common storage media is Hank’s Balanced Salt Solution (HBSS) and

Acid-Citrate-Dextrose at 4ºC and can retain their physiological and carrier characteristics for at least 2 weeks.

Addition calcium-chelating agent (or) purine nucleosides improve circulation survival time of cell upon reinjection.

DMSO, dimethyl-3,3-di-thio-bispropionamide, gluteraldehyde, toulene-2,4-diisocynate are used to stabilize the membrane of RSE and enhance stability upon storage.

Disadvantage: At high concentration of membrane stabilizers, reduces circulation survival time.

Sr. No Chemicals Quantity

01. Potassium Phosphate 0.44 mM

02. Potassium Chloride 5.37 mM

03. Sodium Phosphate, Dibasic

0.34 mM

04. Sodium Chloride 136.89 mM

05. D-Glucose 5.55 mM

Hank’s balanced salt solution (HBSS)

Characterization of resealed erythrocytes:

(a) Drug content estimation:

0.5ml of loaded cells + 2.0ml of Acetonitrile and centrifuge at 2500rpm.

The supernatant liquid is collected and analyzed for drug content.

(b) Percent cell recovery:

It is determined by counting number of intact cells per cubic mm of packed

erythrocytes using laser scattering.

(c) In-vitro drug release and hemoglobin content:

Initially collect cell suspension (5% hemotocrit in PBS) and stored in 4ºC in

amber colored bottle.

Periodically the clear supernatant are withdrawn using hypodermic needle

equipped with 0.45µ filter and deprotinised using methanol and were estimated

for drug content.

(d) Osmotic fragility:

Normal and loaded erythrocytes of drug are incubated separately in stepwise

decreasing % of NaCl solution (0.9 and 0.1%) at 37±2ºC for 10min and

centrifugation at 2000 rpm for 10min. The supernatant examined for drug and

hemoglobin content.

(e) Osmotic shock:

In this study, 1ml of erythrocyte suspension were diluted with distilled water of

5ml and centrifuged at 3000rpm for 15min. The supernatent liquid was estimated

for hemoglobin spectrophotometrically.

=A540 of sample - A540 of background

A540 of 100% hemoglobin% Hemoglobin release

(f) Turbulence shock:

It is the measure of simulating destruction of loaded cells during injection.

In this method, the normal and drug loaded cells are passed through a 23 gauge

hypodermic needle at a flow rate of 10ml/min which is comparable to that of blood.

The samples are with drawn and centrifuged for 2000rpm for 10min and estimated

for hemoglobin content.

Drug loaded erythrocytes found to be less resistant to flow and causes distruction.

(g) Determination of entrapped magnitude:

Initially, Hcl is added to a fixed amount of magnitude bearing erythrocytes

and content are heated to 60ºC for 2hrs. to this added 20%w/v Tricholoroacetic acid

is added and supernatent liquid is obtained after centrifugation.

This was analyzed for magnitude concentration by using Atomic

Absorption Spectrocopy.

(h) Erythrocyte sedimentation rate (ESR):

Sedimentation depends upon the size and number of cells and

relative concentration to the plasma proteins, fibrogen and α- & ß-

globulines.

The test is performed by determination of rate of sedimentation in

the standard tube.

Normal blood ESR is 0-15mm/hr. Higher rate indicates active but

obscure disease processes.

Applications of resealed erythrocytes:

Drug targeting: These can act as drug carriers for target organs of mononuclear

phagocytic system/ reticuloendothelial system (RES) because the changes in the

membrane are recognized by macrophages.

Eg: Urease capsules in treatment of kidney failure for maintenance of serum urea

levels.

Targeting RES organs: Damaged erythrocytes are rapidly cleared from circulation

of phagocytic Kupffur cells in liver and spleen.

Targeting organs like liver and spleen, by modification of Resealed erythrocyte

membrane with various antibiotics, gluteraldehyde, carbohydrate, sulphydryl, etc.,

Treatment of hepatic tumors: Antineoplastic drugs like Methotrexate, Bleomycin,

Asparginase and Adrimycin delivered by RES.

Treatment of parasitic diseases: Antimalarial, antileishmanial and antiamoebic drugs

can be delivered.

Eg: Pentamidine loaded immunoglobin-G coated erythrocytes against macrophage-

cantained leishmania.

Removal of RES iron overload: Desferrioxamine, an iron-chelating drug in

erythrocyte ghosts with a view to promote excretion of iron in patients with excess

body stores.

• Most of the body store of iron is present as intracellular ferritin and hemosiderin

deposits.

In vivo survival & immunological consequences :

A rapid loss of cells during first 24 hrs followed by much slower loss

afterwards.

The first phase represents the cells that are severally damaged during the drug

loading procedures.

The second phase has a half life of the orders of weeks for different mammalian

erythrocytes.

Resealed erythrocytes prepared from RBCs of chicken, rats and rabbits

exhibited relatively poor circulation profile as compared against unloaded

normal erythrocytes.

Release characteristics of loaded drugs:

There are mainly three ways for a drug to efflux out from the erythrocyte carriers:

1) Phagocytosis

2) Diffusion through the membrane of the cells.

3) Using a specific transport system.

RBCs are normally removed from circulation by the process of phagocytosis.

The degree of cross-linking determines whether liver or spleen will preferentially

remove the cells .

Carrier erythrocytes following heat treatment or antibody cross-linking are quickly

removed from the circulation by phagocytic cells located mainly in liver and spleen.

Many substances enter cells by a specific membrane protein system because the

carriers are proteins with many properties analogous to that of enzymes, including

specificity.

Circulating carrier• ß-galactosidase, ß-fructo-furonodase• urease

Enzyme deficiency , replacement therapy

• Albumin, Salbutamol, Prednisolone• Tyrosine kinase

Iron overloaded • Brinase, Aspirin• Heparin

Thrombolytic activity • Human recombinant interlukin -2

Immunotherapy • Desferoxaine

Application • Drug/enzyme/macromolecule

Application • Drug/enzyme/macromolecule

Chemotherapy• Rubomucin• Methotrexate• Daunorubicin• Doxorubicin

Circulating bioreactor• Acetaldehyde• Arginase

Novel Systems

a) Nanoerythrosomes : An erythrocytes based new drug carrier, named nanoerythrosome has

been developed which is prepared by extrusion of erythrocyte ghosts to

produce small vesicles having an average diameter of 100 nm.

Daunorubicin (DNR) was covalently conjugated to the nEryt (nEryt-

DNR) using glutaraldehyde as homobifunctional linking arm. This led to

a complex that is more active than free DNR both in vitro and in vivo.

Daunorubicin (DNR) conjugated to these nanoerythrosomes has a higher

antineoplastic index than the free drug.

b) Erythrosomes:

Erythrosomes are specially engineered vesicular systems in which

chemically cross-linked human erythrocyte cytoskeletons are used as a

support upon which a lipid bilayer (phosphatidyl choline)is coated.

These vesicles have been proposed as useful encapsulation systems for

macromolecular drugs.

References

1) Vyas SP and Khar R.K.: Resealed erythrocytes in targeted and controlled drug

delivery systems. CBS publishers and distributors India. 3rd edition. 2002; 387‐

416.

2) Jain S and Jain N.K.: Engineered erythrocytes as a drug delivery system. Indian J.

Pharm. Sci. 1997; 59, 275–281.

3) R. P. Patel et al. L:An overview of resealed erythrocyte drug delivery.. Journal of

pharmacy research. 2009; 1008-1012.

4) 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. 1991; 28,

219.

5) Torotra G.J. and Grabowski S.R. :The cardiovascular system, The blood in

principles of anatomy and physiology. Harper collins college publishers, New

York, 7th edition. 1993; 566–590.

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