RESEALED RESEALED ERYTHROCYTESERYTHROCYTES

By: D. Vinay Kumar

Introduction:Erythrocytes, the most abundant cells in the human body, have potential carrier

capabilities for the delivery of drugs. Erythrocytes are biocompatible, biodegradable, possess long circulation half lives and can be loaded with a variety of biologically active compounds using various chemical and physical methods.

Erythrocytes, also known as red blood cells, have been extensively 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 reticuloendothelial system (RES).

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withdrawn from cardiac/splenic puncture in a syringe containing a drop of anti coagulant, immediately chilled to 4ºC and stored for less than 2 days.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 buffer at 4ºC for as long as 48 h before use.

Properties of resealed erythrocyte of novel drug delivery carriers:The drug should be released at target site in a controlled manner.It should be appropriate size, shape and should permit the passage through capillaries and Minimum leakage of drug should take place.It should be biocompatible and should have minimum toxic effect.It should possess the ability to carry a broad spectrum of drug.It should possess specific physicochemical properties by which desired target size could be recognized.The degradation product of the carriers 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 have an appreciable stability during storage.

Advantages: Their biocompatibility, particularly when autologous cells are used, hence no

possibility of triggered immune response. Their biodegradability with no generation of toxic products. The considerably uniform size and shape of the carrier. Relatively inert intracellular environment . 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 and pharmacodynamic parameters of drug

attainment of steady-state plasma concentration decreases. They are non-immunogenic in action and can be targeted to disease tissue/organ. They prolong the systemic activity of drug while residing for a longer time in the

body. They protect the premature degradation, inactivation and excretion of proteins and

enzymes and act as a carrier for number of drugs. They facilitate incorporation of proteins and nucleic acid in eukaryotic cells by

cell infusion with RBC. Disadvantage: They have a limited potential as carrier to non-phagocyte target tissue. Possibility of clumping of cells and dose dumping may be there.

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, 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

METHODS OF DRUG LOADING IN RESEALED ERYTHROCYTES

Osmotic-lysis

Endocytosis method

Normal transport mechanism

HYPO OSMOTIC LYSIS METHOD Dialysis method:- In this method, erythrocyte suspension + Drug solution → dialysis tube and both

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

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

pH 7.4 ) at room temp. (25º-30ºC) for resealing. - These resealed cells are removed and washed with PBS at 4ºC and finally

suspended in PBS solution.- The limitation of dilution method can be overcome by carrying out lysis and

resealing in the same dialysis tube.

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 mol. wt. drugs like ß-glucosidase and ß-galactosidase can be encapsulated. 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.,

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.,

ELECTRIC BREAKDOWN TECHNIQUE:ELECTRIC BREAKDOWN TECHNIQUE:- Used for entrapment of bioactive molecules. It is also known as “Electroporation Used for entrapment of bioactive molecules. It is also known as “Electroporation

method”..method”..- The erythrocyte membrane is opened by the dielectric breakdown. The erythrocyte membrane is opened by the dielectric breakdown. - Firstly, erythrocytes are suspended in an isotonic buffered solution in electric Firstly, erythrocytes are suspended in an isotonic buffered solution in electric

discharge chamber connected to the capacitor and external circuit.discharge chamber connected to the capacitor and external circuit.- The charge is discharged at definite voltage within definite time interval through The charge is discharged at definite voltage within definite time interval through

cell suspension to produce a square-wave potential.cell suspension to produce a square-wave potential.- The optimum intensity of an electric field is between 1-10 kW/cm and optimum The optimum intensity of an electric field is between 1-10 kW/cm and optimum

discharge time is 20-160discharge time is 20-160µs.µs.- The compound which is to be entrapped was added to the medium.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 After certain time the cell suspension was transferred to a pre-cooled tubes and kept

at 4at 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.,

ENDOCYTOSIS METHOD:- In this method, 1vol. of packed erythrocytes + 9 vol. 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 154mM 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, VitA, etc.,

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.

MEMBRANE PERTURBATION:- This method is based on increase in membrane permeability of erythrocytes when

exposed to certain chemicals.

Eg: Amphotericin-B, Daunomycin using Halothane.

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 and acid-citrate-dextrose at 4ºC and can retain their physiological and carrier characteristics for at least 2weeks.

- 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.

Disadv.:- At high conc. of membrane stabilizers, reduces circulation survival time.

S.NoS.No Chemicals Chemicals Quantity Quantity

01.01. Potassium Phosphate Potassium Phosphate 0.44 mM 0.44 mM

02.02. Potassium Chloride Potassium Chloride 5.37 mM 5.37 mM

03.03. Sodium Phosphate, Dibasic Sodium Phosphate, Dibasic 0.34 mM 0.34 mM

04.04. Sodium Chloride Sodium Chloride 136.89 mM 136.89 mM

05.05. D-Glucose D-Glucose 5.55 mM5.55 mM

Hank’s balanced salt solution (HBSS)

CHARACTERIZATION OF RESEALED ERYTHROCYTES

(a) Drug content estimation,

(b) In-vitro drug release and hemoglobin content,

(c) Percent cell recovery,

(d) Osmotic fragility,

(e) Osmotic shock,

(f) Turbulence shock,

(g) Erythrocyte sedimentation rate (ESR),

(h) Determination of entrapped magnetite,

(a)(a) Drug content estimation:Drug content estimation:

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

- The supernatant liquid is collected and analyzed for drug content.- The supernatant liquid is collected and analyzed for drug content.

(b)(b) Percent cell recovery:Percent cell recovery:

It is determined by counting no. of intact cells per cubic mm of packed It is determined by counting no. of intact cells per cubic mm of packed erythrocytes using laser scattering.erythrocytes using laser scattering.

(c)(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)(d) Osmotic fragility:Osmotic fragility:

Normal and loaded erythrocytes of drug are incubated separately in stepwise Normal and loaded erythrocytes of drug are incubated separately in stepwise decreasing % of NaCl solution (0.9 and 0.1%) at 37decreasing % of NaCl solution (0.9 and 0.1%) at 37±2ºC for 10min and ±2ºC for 10min and centrifugation at 2000 rpm for 10min. The supernatant examined for drug and centrifugation at 2000 rpm for 10min. The supernatant examined for drug and hemoglobin content.hemoglobin content.

(e)(e) Osmotic shock:Osmotic shock:

In this study, 1ml of erythrocyte suspension were diluted with distilled water of 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 5ml and centrifuged at 3000rpm for 15min. The supernatent liquid was estimated for hemoglobin spectrophotometrically.for hemoglobin spectrophotometrically.

=A540 of sample - A540 of background

A540 of 100% hemoglobin% Hemoglobin release

(f)(f) Turbulence shock:Turbulence shock:- It is the measure of simulating destruction of loaded cells during injection.- 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 - 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 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 blood. The samples are with drawn and centrifuged for 2000rpm for 10min and estimated for hemoglobin content.estimated for hemoglobin content.- Drug loaded erythrocytes found to be less resistant to flow and causes - Drug loaded erythrocytes found to be less resistant to flow and causes distruction.distruction.

(g)(g) Determination of entrapped magnitude:Determination of entrapped magnitude:- Initially, Hcl is added to a fixed amount of magnitude bearing erythrocytes and - Initially, Hcl is added to a fixed amount of magnitude bearing erythrocytes and content are heated to 60content are heated to 60ºC for 2hrs. ºC for 2hrs. - to this added 20%w/v Tricholoroacetic acid is added and supernatent liquid is - to this added 20%w/v Tricholoroacetic acid is added and supernatent liquid is obtained after centrifugation.obtained after centrifugation.- This was analyzed for magnitude concentration by using AAS.- This was analyzed for magnitude concentration by using AAS.

(h)(h) Erythrocyte sedimentation rate (ESR):Erythrocyte sedimentation rate (ESR):- Sedimentation depends upon the size and no. of cells and relative concentration - Sedimentation depends upon the size and no. of cells and relative concentration to the plasma proteins, fibrogen and to the plasma proteins, fibrogen and αα- & ß- globulines. - & ß- globulines. - The test is performed by determination of rate of sedimentation in the standard - The test is performed by determination of rate of sedimentation in the standard tube. tube. - Normal blood ESR is 0-15mm/hr. Higher rate indicates active but obscure - Normal blood ESR is 0-15mm/hr. Higher rate indicates active but obscure disease processes.disease processes.

Applications of resealed erythrocytes: Drug targeting: these can act as drug carriers and targeting tool. Used to target

organs of mononuclear phagocytic system/ reticuloendothelial system 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.

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