A Review on Resealed Erythrocytes

INTRODUCTION

Blood contains various type of cells like erythrocytes (RBC),WBC and platelets, among them the RBC are the most interesting carrier and posse’s great potential in drug delivery due to their ability to circulate throughout the body, zero order kinetics, reproducibility and ease of preparation. The primary aim for the development of this drug delivery system is to maximize therapeutic performance and reducing undesirable side effects of drug as well as to  increase the  patient compliance.

Erythrocytes

Red blood cells (erythrocytes) are the most common type of blood cells and the vertebrate organism's principal means of delivering oxygen (O₂) to the body tissues via the blood flow through the circulatory system. The cells develop in the bone marrow before their components are recycled by macrophages. Each circulation takes about 20 seconds row and circulate for about 100–120days in the body.

Resealed Erythrocytes

The drug-loaded carrier erythrocytes are prepared 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 as resealed erythrocytes. The overall process is based on the response of these cells under osmotic conditions. Upon re injection, the drug-loaded erythrocytes serve as slow circulating depots and target the drugs to a reticulo endothelial system (RES)

METHODS OF DRUG LOADING IN ERYTHROCYTES:

1) Hypo- osmosis lysis method

In this process, the intracellular and extracellular solute of erythrocytes is exchange by osmotic lysis and resealing .The drug present will be encapsulated within the RBCs by this process.

 a) Hypotonic dilution

In this method, a volume of packed erythrocytes is diluted with 2–20 volumes of aqueous solution of a drug. The solution tonicity is then restored by adding a hypertonic buffer. The resultant mixture is then centrifuged, the supernatant is discarded, and the pellet is washed with isotonic buffer solution.

b) Hypotonic Dialysis method

This method was first reported by Klibansky in1959 and was used in 1977 by Deloach, Ihler and Dale for loading enzymes and lipids. In the process, an isotonic, buffered suspension of erythrocytes with a hematocrit value of 70–80 is prepared and placed in a conventional dialysis tube immersed in 10–20 volumes of a hypotonic buffer. The medium is agitated slowly for 2 h. The tonicity of the dialysis tube is restored by directly adding a calculated amount of a hypertonic buffer to the surrounding medium or by replacing the surrounding medium by isotonic buffer. The drug to be loaded can be added by either dissolving the drug in isotonic cell suspending buffer inside a dialysis bag at the beginning of the experiment or by adding the drug to a Dialysis.

c) Hypotonic Pre swelling method:

This method based on the principle of firsts welling the erythrocytes without lysis by placing them in slightly hypotonic solution. The swollen cells are recovered by centrifugation at low speed. Then, relatively small volumes of aqueous drug solution are added to the point of lysis. The slow swelling of cells results in good retention of the cytoplasmic constituents and hence good survival in vivo. This method is simpler and faster than other methods, causing minimum damage to cells. Drugs encapsulated in erythrocytes using this method include propranolol, asparginase, cyclopohphamide, methotrexate, insulin, metronidazole, levothyroxine, enalaprilat & isoniazid.

d) Isotonic osmotic lysis method

This method was reported by Schrier et al in1975.This method, also known as the osmotic pulse method, involves isotonic hemolysis that is achieved by physical or chemical means. The isotonic solutions may or may not be isotonic. If erythrocytes are incubated in solutions of a substance with high membrane permeability, the solute will diffuse into the cells because of the concentration gradient. This process is followed by an influx of water to maintain osmotic equilibrium. Chemicals such as urea solution, polyethylene glycol, and ammonium chloride have been used for isotonic hemolysis. However, this method also is not immune to changes in membrane structure composition.In1987, Franco et al.developed a method that involved suspending erythrocytes in an isotonic solution of dimethyl sulfoxide (DMSO). The suspension was diluted with an isotonic-buffered drug solution. After the cells were separated, they were sealed at 37 °C.

2) Electro-insertion or Electro encapsulation method

In 1973, Zimmermann tried an electrical pulse method to encapsulate bioactive molecules. Also known as electroporation, the method is based on the observation that electrical shock brings about irreversible changes in an erythrocyte membrane. This method is also called as electroporation. In this method erythrocyte membrane is open by a dielectric breakdown; subsequently the pore of erythrocyte can be resealed by incubation at 37⁰C in an isotonic medium. The various chemical encapsulated into the erythrocytes are primaquin and related 8- amino quinolone, vinblastin chloropromazine and related phenothiazine, propanolol.

6) Loading by electric cell fusion:

This method involves the initial loading of drug molecules into erythrocyte ghosts followed by adhesion of these cells to target cells. The fusion is accentuated by the application of an electric pulse, which causes the release of an entrapped molecule. An example of this method is loading a cell-specific monoclonal antibody into an erythrocyte ghost. An antibody against a specific surface protein of target cells can be chemically cross-linked to drug-loaded cells that would direct these cells to desired cells.

7) Use of red cell loader:

Novel method was developed for entrapment of non diffusible drugs into erythrocytes. They developed a piece of equipment called a “red cell loader”. With as little as 50 ml of a blood sample, different biologically active compounds were entrapped into erythrocytes within a period of 2 h at room temperature under blood banking conditions. The process is based on two sequential hypotonic dilutions of washed erythrocytes followed by concentration with a hem filter and an isotonic resealing of the cells. There was 30% drug loading with 35–50% cell recovery. The processed erythrocytes had normal survival in vivo. The same cells could be used for targeting by improving their recognition by tissue macrophages.

STORAGE:

Store encapsulated preparation without loss of integrity when suspended in hank's balanced salt solution [HBSS] at 40C for two weeks. Use of group 'O' [universal donor] cells and by using the pres well or dialysis technique, batches of blood for transfusion. Standard blood bag may be used for both encapsulation and storage.

EVALUATION OF RESEALED ERYTHROCYTES:

After loading of therapeutic agent on erythrocytes, the carrier cells are exposed to physical, cellular as well as biological evaluations.

1. Shape and Surface Morphology

The morphology of erythrocytes decides their life span after administration. The morphological characterization of erythrocytes

is undertaken by comparison with untreated erythrocytes using either transmission (TEM) or Scanning electron microscopy (SEM). Other methods like phase contrast microscopy can also be used.

2. Drug Content

Drug content of the cells determines the entrapment efficiency of the method used. The process involves deproteinization of packed, loaded cells (0.5 m L) with 2.0 m L acetonitrile and centrifugation at 2500 rpm for 10 min. The clear supernatant is analyzed for the drug by spectrophotometrically.

3. Cell Counting and Cell Recovery

This involves counting the number of red blood cells per unit volume of whole blood, usually by using automated machine it is determined by counting the no. of intact cells per cubic mm of packed erythrocytes before and after loading the drug.

4. Turbulence Fragility

It is determined by the passage of cell suspension through needles with smaller internal diameter (e.g., 30 gauges) or vigorously shaking the cell suspension. In both cases, haemoglobin and drug released after the procedure are determined. The turbulent fragility of resealed cells is found to be higher.

APPLICATIONS OF RESEALED ERYTHROCYTES

In Vitro Applications

Carrier RBCs have proved to be useful for a variety of in vitro tests. For in vitro phagocytosis cells have been used to facilitate the uptake of enzymes by phagolysosomes. An inside to this study showed that enzymes content within carrier RBC could be visualized with the help of cyto chemical technique. The most frequent in vitro application of RBC mediated microinjection. A protein or nucleic acid to be injected into eukaryotic cells by fusion process. Similarly, when antibody molecules are introduced using erythrocytic carrier system, they immediately diffuse throughout the cytoplasm. Antibody RBC auto injected into living cells have been used to confirm the site of action .

In Vivo Applications

This includes the following

1) Slow drug release

Erythrocytes have been used as circulating depots for the sustained delivery of anti-neoplastics, anti-parasitics, veterinary ant-iamoebics, vitamins, steroids, antibiotics, and cardiovascular drugs.

2) Drug targeting

Ideally, drug delivery should be site specific and target oriented to exhibit maximal therapeutic index with minimum adverse effects. Resealed erythrocytes can act as drug carriers and targeting tools as well. Surface modified erythrocytes are used to target organs of mononuclear phagocytic system/ RES because the change in the membrane is recognized by macrophages.

3) Targeting reticulo endothelial system (RES) organs

Surface modified erythrocytes are used to target organs of mononuclear phagocytic systems/ reticulo endothelial system because it maximal the changes in membrane are recognized by macrophages. The various approaches used include:

• Surface modification with antibodies (coating of loaded erythrocytes by anti‐Rh or other types of antibodies)

• Surface modification with glutaraldehyde.

• Surface modification with sulphydryl.

• Surface chemical crosslinking.

• Surface modification with carbohydrates such as sialic acid.

4) Targeting the liver-deficiency/therapy

Many metabolic disorders related to deficientor missing enzymes can be treated by injecting these enzymes. However, the problems of exogenous enzyme therapy include a shorter circulation half life of enzymes, allergic reactions, and toxic manifestations .these problems can be successfully overcome by administering the enzymes as resealed erythrocytes. The enzymes used include Pglucosidase, P- glucoronidase, and Pgalactosidase.The disease caused by an accumulation of glucocerebrosidaes in the liver and spleen can be treated by glucocerebrosidase-loaded

Enzyme therapy

Many metabolic disorders related to deficientor missing enzymes can be treated byadministering these enzymes as resealed erythrocytes. E.g. β‐ Gl coside, β‐glucouronidase, β‐galactosidase.

Removal of RES iron overloads

Desferrioxamine-loaded erythrocytes have been used to treat excess iron accumulated because of multiple transfusions to thalassemic patients. Targeting this drug to the RES is very beneficial because the aged erythrocytes are destroyed in RES organs, which results in an accumulation of iron in these organs.

Targeting Non RES

Erythrocytes loaded with drugs have also been used to target organs outside the RES The various approaches for targeting non‐RES  organs include:

·        Entrapment of paramagnetic particles along with the drug.

·        Entrapment of photosensitive material.

·        Use of ultrasound waves.

·        Antibody attachment to erythrocytes membrane to get specificity of action.

·        Other approaches include fusion with

·        liposome, lectin pre‐treatment of resealed cells etc.

ROUTE OF ADMINISTRATION

Intra peritoneal injection reported that survival of cells in circulation was equivalent to the cells administered by i.v. injection .They reported that 25% of resealed cell remained in circulation for 14 days they also proposed this method of injection as a method for extra vascular targeting of RBCs to peritoneal macrophages. Subcutaneous route for slow release of entrapped agents. They reported that the loaded cell released encapsulated molecules at the injection site.

NOVEL APPROACHES

Erythrosomes:

These are specially engineered vesicular systems that are chemically cross-linked to human erythrocytes’ support upon which a lipid bilayer is coated.This process is achieved by modifying a reverse-phase evaporation technique.

Nano erythrosomes:

These are prepared by extrusion of erythrocyte ghosts to produces small vesicles with an average diameter of 100nm. Daunorubicin was covalently conjugated to nano erythrosomes using gluteraldehyde spacer. This complex was more active than free daunorubicin alone.