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PRESENTED BY V.SUHASINI M.PHARM (PHARMACEUTICS)

RESEALED ERYTHROCYTES

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INTRODUCTION Erythrocytes (RBCs) have potential carrier

capability for the delivery of drug 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

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Carrier erythrocytes are prepared by collecting blood sample and separating erythrocytes from plasma. By using various methods

1st the cells are broken and the drug is entrapped into the erythrocytes, finally they are resealed carriers are then called "resealed erythrocytes".

So many drugs like aspirin, steroid, cancer drug which having many side effects are reduce by resealed erythrocyte.

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Erythro = red and cytes = cell Erythrocytes (RBCs) contain oxygen carrying

protein hemoglobin, which is a pigment that gives whole blood its red color

A healthy adult male has about 4.5 million RBCs/μl of blood , and a healthy adult female has about 4.8 million

Immature RBC are called “RETICULOCYTES. So matured (RBCs) called erythrocyte have no

nucleus all their internal space is available for oxygen transport

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biconcave

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Isolation of erythrocytes The cellular content is about 40-50% of the blood

volume and contains erythrocytes(red blood cells,RBC),Leukocytes(while blood cells, WBC) and thrombocytes (platelets).the primarily water (90 to 92%) and protein(7%)

Blood is collected into heparinized tubes by venipuncture method withdrawn from cardiac case of small animal) and

through veins (in case large animals) into a syringe

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The whole blood is centrifuged at 2500 rpm for 5 min at 40C in refrigerated centrifuge

The serum and buffy coats are carefully removed and packed cells washed for three times with phosphate buffer saline pH=7.4

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Various animal erythrocytes used for isolationSR.NO

SPCIES WASHING BUFFER

CENTRIFUGAL FORCE(G)

1 Rabbit 10mmol KH2PO4/NaHPO4 500-1000

2 Dog 15mmol KH2PO4/NaHPO4 500-1000

3 Human 154mmol NaCl <500

4 Mouse 10mmol KH2PO4/NaHPO4 100-500

5 Cow 10-15mmol KH2PO4/NaHPO4 1000

6 Horse 2mmol MgCl2,10mmol glucose

1000

7 Sheep 10mmol KH2PO4/NaHPO4, 500-1000

8 Pig 10mmol KH2PO4/NaHPO4 500-1000

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ELECTROLYTE COMPOSITION OF ERYTHROCYTES: Electrolyte are qualitatively similar to that of

plasma .however, quantitatively it differs from that of plasma.

The concentration of Na+ and K+ is more in erythrocytes in plasma.

The osmotic pressure of the interior of the erythrocytes is equal to that of the plasma and termed as isotonic (0.9% NaCl or normal physiological saline.)

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If the medium is Hypotonic, water diffuses into the cells and they get swelled and eventually loose all their hemoglobin content and may burst.

if the medium is Hypertonic,(i.e. higher osmotic pressure than 0.9% NaCl) they will shrink and become irregular in shape.

Balanced ion solutions like Ringer’s and Tyrode’s soln. which are not only isotonic but also contains ions in proper quantity are used in erythrocyte related experiments.

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Drug loading in erythrocytesWhen erythrocytes are suspended in a hypotonic

medium they swell, and the membrane rupture in the formation of pores with diameters of 200 to 500 Å.

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.

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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

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ADVANTAGES OF ERYTHROCYTES AS CARRIER

1. A remarkable degree of biocompatibility 2. Complete biodegradability and the lack of toxic

product resulting from the carrier biodegradation 3. Avoidance of any undesired immune responses 4. Considerable protection of the organism against toxic

effect of the encapsulation drug (e.g) antineoplastic agent

5. Remarkably longer life span of the carrier erythrocytes in circulation in comparison to the synthetic carrier

The entrapment of drug also does not require the chemical modification of the substance to be entrapped

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DISADVANTAGES The rapid leakage of certain encapsulated

substances from the loaded erythrocytes Several molecules may alter the physiology of

the erythrocyte The storage of the loaded erythrocytes is a

further problem Possible contamination due to the origin of the

blood , the equipment used and the loading environment

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VERIES METHOD OF DRUG LOADING

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MEMBRANE PERTURBATION METHOD This method is based upon the increase in

membrane permeability of erythrocytes when the cells are exposed to certain chemicals.

EX: It showed that the permeability of erythrocytic

membrane increases upon exposure to polyene antibiotic such as amphotericin B.

this method was used successfully to entrap the antineoplastic drug daunomycin in human and mouse erythrocytes .

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ELECTRO-INSERTION OR ELECTROENCAPSULATION

This method is also known as electroporation, method

Electrical breakdown is achieved by membrane polarization for microseconds using varied voltage of 2kv/cm is applied for 20 µsec.

Electrolysis used to generate desirable membrane permeability for drug loading .

The extent of pore formation depends upon the electric field strength, pulse duration and ionic strength of suspending medium.

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- 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.

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- The various drugs entrapped by this method include primaquine and related 8–amino–quinolines,vinblastine, chlorpromazine and related phenothiazines, hydrocortisone, propranolol, tetracaine, and vitamin A .

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HYPO OSMOTIC LYSIS METHOD Dilution method:

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Examples: hypotonic dilution is used for loading enzymes such as galactosidase and glucosidase, asparginase , as well as bronchodilators such as sulbutamol

Limitations 1. Low entrapment efficiency and a

considerable loss of hemoglobin and other cell components

2. Reduction in the circulation half life of the loaded cells

3. Only for low molecular weight drug

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HYPO OSMOTIC LYSIS METHODDialysis 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.

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HYPO OSMOTIC LYSIS METHODOsmotic 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

trans erythrocytic 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.,

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HYPO OSMOTIC LYSIS METHOD 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.,

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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.- the vesicle membrane separates endocytosed substance

from the cytoplasm and protect the erythrocyte membrance.Eg: 8-amino quinolines, Vinblastin, Chlorpromazine,

Phenothiazines, hydrocortison, propanolol, tetracine, VitA, etc.,

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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.

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In vitro characterization

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In vitro characterization

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Shelf Life And Storage Stability

Encapsulated product and carrier both show satisfactory self-stability when stored in Hank’s balanced salt solution(HBSS) example Potassium Phosphate at 4o C for two weeks.

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APPLICATIONS OF RELEASED ERYTHROCYTES

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Treatment of hepatic tumors: Antineoplastic drugs like Methotrexate, Bleomycin, Asparginase and Adrimycin delivered by RES.

Treatment of parasitic diseases: Antimalarial, and antiamoebic

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

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Conclusion Numerous applications have been proposed

for the use of resealed erythrocytes as carrier for drug, enzymes replacement therapy etc

Resealed erythrocytes technology will remain an active area for further research

Erythrocytes based delivery system with their ability to provide controlled and site specific drug delivery

For the present it is concluded that erythrocyte carriers are “GOLDEN EGGS IN NOVEL DRUG DELIVERY SYSTEM”

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REFERENCES Targeted & controlled drug delivery , novel carrier

systems by S.P.VYAS & R.K.KHAR. Resealed Erythrocytes: A Novel Carrier for Drug

Targeting Abhishek Kumar Sah, Ashish Rambhade, Alpna Ram and

Sunil K. Jain. J. Chem. Pharm. Res., 2011, 3(2):550-565 http://pharmawiki.in/ppt-resealed-erythrocytes

/ http://www.ijrpc.com/files/04-355.pdf http://www.ijppsjournal.com/Vol4Issue3/4374.p

df http://ijpbs.net/volume2/issue1/pharma/_38.pd

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