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2. Use of red cell loader(16):
Novel method for entrapment of non-diffusible drugs into erythrocytes. With 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 2h at room temperature. The process is based on two sequential hypotonic dilutions of washed erythrocytes followed by concentration with a hemo filter and an isotonic resealing of  cells.

3. Hypotonic dilution(17):
Hypotonic dilution was the first method investigated for encapsulation of chemicals into erythrocytes and is the simplest and fastest. 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. The major drawbacks of this method include low entrapment efficiency and a considerable loss of hemoglobin and other cell components.

4. Hypotonic pre-swelling (18):
This method was developed and  modified by Jenner et al. for drug loading. The technique is based upon initial controlled swelling in a hypotonic buffered solution. This mixture is centrifuged at low values. The supernatant is discarded and  cell fraction is brought to  lysis point by L portions of an aqueous solution of  drug to be encapsulated.  The mixture is centrifuged between  drug-addition steps. The lysis point is detected by disappearance of a distinct boundary between  cell fraction and  supernatant upon centrifugation. The tonicity of cell mixture is restored at  lysis point by adding a calculated amount of hypertonic buffer.

Fig.3: Hypotonic presswelling

5. Isotonic osmotic lysis(19):
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  cells because of  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.

6. Hypotonic dialysis(20):
Several methods are based on the principle that semi-permeable dialysis membrane maximizes the intracellular: extracellular volume ratio for macromolecules during lysis and resealing. 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 with isotonic buffer. The drug to be loaded can be added by either dissolving  drug in isotonic cell suspending buffer inside a dialysis bag at the beginning of the experiment or by adding the drug to a dialysis bag after stirring is complete.

Figure 4: Hypotonic dialysis

8. Electro-insertion or electro- encapsulation (21):
The erythrocyte membrane is opened by a dielectric break down. Subsequently, the pores can be resealed byincubation at 37oC in an isotonic medium. The procedure involves suspending erythrocytes in an isotonic buffer in anelectrical discharge chamber. A capacitor in an external circuit is charged to a definite voltage and then dischargedwithin a definite time interval through cell suspension to produce square wave potential.

Figure 5: Electro-insertion or Electro-encapsulation

Characterization of Resealed Erythrocytes (22-24)
* Drug content quantification:
To determine the drug content, packed loaded cells are deproteinized with aceto-nitrile after centrifugation at 3000 rpm for a fixed time interval. The clear supernatant liquid is analysed spectro-photometrically.

* In-vitro drug release and hemoglobin content study:
In-vitro release of drug(s) and hemoglobin are monitored periodically form drug loaded cells. The cells suspension (5% hematocrit in PBS) is stored at 40°C in amber coloured glass containers. Periodically the clear supernatant are withdrawn using a hypodermic syringes equipped with 0. 45 m filter, deproteinized using methanol and are estimated for drug content. The supernatant of each sample after centrifugation is collected and assayed, % hemoglobin release may be calculated using the formula.

* Percent cell recovery and Morphological study:
Percent cell recovery may be determined by counting the no. of intact cells per cubic mm of packed erythrocytes before and after loading the drug. Phase contrast or electron microscope may be used for normal and drug loaded erythrocytes.

* Osmotic fragility and Osmotic shock study:
To study the effect of different tonicities, drug loaded erythrocytes are incubated separately in normal saline solution at 37 ± 2oC for 10 minutes, followed by centrifugation at 2000 rpm for 10 min. For osmotic shock study, dispersing the resealed erythrocyte suspension in distilled water and centrifuged at 300 rpm for 15 min. The supernatant is estimated for percent hemoglobin release spectro-photometrically.

* Turbulence shock study:
It is the measure of simulating destruction of loaded cells during injection. Normal and drug loaded cells are passéd through a 23 gauge hypodermic needle at a flow rate of 10 ml/min which is comparable to the flow rate of blood. It is followed by collection of an aliquot and centrifugation at 2000 rpm for 10 minutes. The hemoglobin in withdrawn sample is estimated. Drug loaded erythrocytes appear to be less resistant to turbulence, probably indicating destruction of cells upon shaking.

* Erythrocyte sedimentation rate (ESR):
It is an estimate of  suspension stability of RBC in plasma and is related to the number and size of  red cells and to relative concentration of plasma protein, especially fibrinogen and α, β globulins. This test is performed by determining the rate of sedimentation of blood cells in a standard tube. Normal blood. ESR is 0 to 15 mm/hr. higher rate is indication of active but obscure disease processes.

* Entrapped magnetite study:
The hydrochloric acid is added to a fixed amount of magnetite bearing erythrocytes and contents are heated at 600°C for 2 hr. Then 20% w/v trichloro-acetic acid is added and supernatant obtained after centrifugation is used to determine magnetite concentration using atomic absorption spectroscopy.

* Self life and Stability and Cross linking of Released Erythrocytes:
Glutaraldehyde (0. 2%) treated erythrocytes in a sintered glass funnel (G-4) are collected by filtration and dried in vacuum (200mm Hg) for 10 hr. Alternatively the erythrocyte suspension is filled into vials and lyophilized at- 400°C to 0. 01 torr using a laboratory lyophilizer. The dried powder is  filled in amber color glass vials and stored at 40°C for month. Improvement in shelf life of  carrier erythrocytes is achieved by storing the cells in powder from, ready for reconstitution at 40°C.


I In Vitro Applications
Carrier RBCs have proved to be useful for a variety of in vitro tests. 1.)  For in vitro phagocytosis cells have been used to facilitate the uptake of enzymes by phago-lysosomes. An inside to this study shows that enzymes content within carrier RBC could be visualized with the help of cytochemical technique. 2.)   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 of fragment of diptheria toxin.(25)

II In Vivo Applications
These  include  the following
1) Slow drug release
Erythrocytes have been used as circulating depot for the sustained delivery of anti-neoplastics, anti-parasitics, veterinary ,antiamoebics, vitamins, steroids, antibiotics, and cardiovascular drugs. (26)

2) Drug targeting
Ideally, drug delivery should be site specific and target oriented to exhibit maximal therapeutic index wit 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  membrane is recognized by macrophages.(27)

3) Targeting reticulo-endothelial system (RES) organs
Surface modified erythrocytes are used to target organs of mononuclear phagocytic system/ reticulo-endothelial system because 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 cross-linking.
• Surface modification with carbohydrates such as sialic acid.(28)

4) Targeting the liver-deficiency/therapy
Many metabolic disorders related to deficient or missing enzymes can be treated by injecting these enzymes. However, the problems of exogenous enzyme therapy include 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 P-glucosidase, P- glucoronidase, and P-galactosidase.The disease caused by an accumulation of glucocerebrosidaes in the liver and spleen can be treated by glucocerebrosidase-loaded erythrocytes.(29)

5) Treatment of parasitic disease
The ability of resealed erythrocytes to selectively accumulate with in RES organs make them useful tool during the delivery of anti parasitic agents. Parasitic diseases that involve harboring parasites in The RES organs can be successfully controlled by this method. Results were favorable in studies involving animal models for erythrocytes loaded with anti malarial, anti leishmanial and anti amoebic drugs.(30, 31, 32)

6) Removal of toxic agents
Cannon et al. reported inhibition of cyanide intoxication with murine carrier erythrocyte containing bovine rhodanase and sodium thiosulphate. Antagonization of organophosphorus intoxication by released erythrocyte containing a recombinate phosphodiestrase also has been reported.(33)

7) Treatment of hepatic tumors
Antineoplastic drugs such as methotrexate (MTX), bleomycin, asparginase and adiramycin have been successfully delivered by erythrocytes. E.g. in a study, the MTX showed a preferential drug targeting to liver followed by lungs, kidney and spleen.(34)

8) Delivery of antiviral agents
Several reports have been cited in the literature about antiviral agents entrapped in resealed erythrocytes for effective delivery and targeting. Because most antiviral drugs are nucleotides or nucleoside analogs, their  entrapment and exit through the membrane needs careful consideration.(35)

9) Enzyme therapy
Many metabolic disorders related to deficient or missing enzymes can be treated by administering these enzymes as resealed erythrocytes. E.g. β-Glucoside, β-glucouronidase, β-galactosidase.(36, 37, 38)

10) 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  RES is very beneficial because the aged erythrocytes are destroyed in RES organs, which results in an accumulation of iron in these organs.(39)

11) 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.(40, 41)



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