INTRODUCTION:

Among the various carriers used for targeting drugs to various body tissues, the cellular carriers meet several criteria desirable in clinical applications, among the most important being biocompatibility of carrier and its degradation products. Leucocytes, platelets, erythrocytes, nanoerythrocytes, hepatocytes, and fibroblasts etc. have been proposed as cellular carrier systems. Among these, the erythrocytes have been the most investigated and have found to possess greater potential in drug delivery. Biopharmaceuticals, therapeutically significant peptides and proteins, nucleic acid-based biological, antigens, anticancer drug and vaccines, are among the recently focused pharmaceuticals for being delivered using carrier erythrocytes. Erythrocytes, also known as red blood cells, and have been extensively studied for their potential carrier capabilities for the delivery of drugs.

The biocompatibility, non-pathogenicity, non-immunogenicity and biodegradability make them unique and useful carriers. Carrier erythrocytes are prepared by collecting blood sample from the organism of interest and separating erythrocytes from plasma. By using various methods the cells are broken and the drug is entrapped into the erythrocytes, finally they are resealed and the resultant carriers are then called "resealed erythrocytes". So many drugs like aspirin, steroid, cancer drug which having many side effects are reduce by resealed erythrocyte. Current review highlights isolation, drug loading methods, Evaluation methods and applications of resealed erythrocytes for drug delivery.¹

Effect of tonicity on Rbc’s

Figure 1. Effect of tonicity on Rbc’s

Basic concept of RBCs

Figure 2. View of Rbc’s

Blood contains different type of cells like erythrocytes (RBC), leucocytes (WBC) and platelets, among them erythrocytes are the most interesting carrier and possess great potential in drug delivery due to their ability to circulate throughout the body, zero order kinetics, reproducibility and ease of preparation1 primary aim for the development of this drug delivery system is to maximize therapeutic performance, reducing undesirable side effects of drug as well as increase patient compliance.²

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 disease tissue or organ.³

Present pharmaceutical scenario is aimed at development of drug delivery systems which maximize the drug targeting along with high therapeutic benefits for safe and effective management of diseases.

Targeting of an active bio molecule from effective drug delivery where pharmacological agent directed specifically to its target site.

Drug targeting can be approaches by either chemical modification or by appropriate carrier.⁴

Erythrocytes:

Red blood cells (also referred to as 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 and circulate for about 100–120 days in the body before their components are recycled by macrophages.

Each circulation takes about 20 seconds. Approximately a quarter of the cells in the human body are red blood cells.

Figure 3. Images of Rbc’s

Anatomy, physiology and composition of RBCs:

RBCs have shapes like biconcave discs with a diameter of 7.8 μm and thickness near 2.2 μm. Mature RBCs have a simple structure. It is also elastic in nature. Their plasma membrane is both strong and flexible, which allows them to deform without rupturing as they squeeze through narrow capillaries. RBCs lack a nucleus and other organelles and can neither reproduce nor carry on extensive metabolic activities. RBCs are highly specialized for their oxygen transport function, because their mature RBCs have no nucleus, all their internal space is available for oxygen transport. Even the shape of RBC facilities it’s function. A biconcave disc has a much greater surface area for the diffusion of gas molecules into and out of the RBC than would; say a sphere or a cube. The red blood cell membrane, a dynamic, semi permeable components of the cell, associated with energy metabolism in the maintenance of the permeability characteristic of the cell of various cations (Na+, K++) and anions (Cl- HCO3 -). Each RBC contains about 280 million hemoglobin molecules. A hemoglobin molecules consists of a protein called globin, composed of four polypeptide chains; a ring like non-protein pigment called a heme, is bound to each of the four chains. At the center of the heme ring combine reversibly with one oxygen molecule, allowing each hemoglobin molecule to bind four oxygen molecules. RBCs include water (63%), lipids (0.5), glucose (0.8%), mineral (0.7%), non-hemoglobin protein (0.9%), meth hemoglobin (0.5%), and hemoglobin (33.67%).

Resealed Erythrocytes:

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.⁸

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).⁹

Advantages:

1. Biocompatible, particularly when autologous cells are used hence no possibility of triggered immune response.

2. Biodegradability with no generation of toxic products

3. Considerable uniform size and shape of carrier.

4. Relatively inert intracellular environment can be encapsulated in a small volume of cells.

5 Isolation is easy and large amount of drug can be loaded.Prevention of degradation of the loaded drug from inactivation by endogenous chemical.

6 Entrapment of wide variety of chemicals can be possible.

7 Entrapment of drug can be possible without chemical modification of the substance to be entrapped.

8 Possible to maintain steady-state plasma concentration, decrease fluctuation in concentration.

10 Protection of the organism against toxic effect of drug.

11 Targeting to the organ of the RES.

12 Ideal zero-order drug release kinetic.

13 Prolong the systemic activity of drug by residing for a longer time in the body.^11-20

Disadvantages:

1. They have a limited potential as carrier to non-phagocyte target tissue.

^2.Possibility of clumping of cells and dose dumping may be there.^{21, 22}

3. Major problem with this drug carrier is that they are removed in vivo by RES. This seriously limits their useful life as drug carriers and in some cases may pose toxicological problems.

4. Rapid leakage of certain encapsulated material from the loaded erythrocytes.

5. Liable to biological contamination due to the origin of blood.

6. Rigorous and special controls are required for the collectionand handling of erythrocytes.

7. Several molecules may alter the physiology of erythrocytes.

Erythrocytes can be used as carriers in two ways:

1. Targeting particular tissue/organ:

For targeting, only the erythrocyte membrane is used. This is obtained by splitting the cell in hypotonicsolution and after introducing the drug into the cells, allowing them to reseal into spheres. Such erythrocytes are called Red cell ghosts.

2. For continuous or prolonged release of drugs:

Alternatively, erythrocytes can be used as a continuous or prolonged release system, which provide prolonged drug action. There are different methods for encapsulation of drugs within erythrocytes. They remain in the circulation for prolonged periods of time (up to 120 days) and release the entrapped drug at a slow and steady rate.

Isolation of erythrocytes:

Figure 4: Isolation of erythrocytes.

· Blood is collected into heparinized tubes by vein puncture.

· Blood is withdrawn from cardiac/splenic puncture (in small animal) and throughveins (in large animals) in a syringe containing a drop of anti-coagulant.

· The whole blood is centrifuged at 2500 rpm for 5 min. at 4 ±10C in a refrigerated centrifuge.

· The serum and Buffy coats are carefully removed and packed cells washed three times with phosphate buffer saline (pH=7.4).

· The washed erythrocytes are diluted with PBS and stored at 40C for as long as 48 h before use.

· Various types of mammalian erythrocytes have been used for drug delivery, including erythrocytes of mice, cattle, pigs, dogs, sheep, goats, monkeys, chicken, rats, and rabbits.^18,24

Various condition and centrifugal force used for isolation of erythrocytes²⁵:

Sr. no.	Species	Washing Buffer	Centrifugal force (g)
1	Rabbit	10 mmol KH₂PO₄/NaHPO₄	500-1000
2	Dog	15 mmol KH₂PO₄/NaHPO₄	500-1000
3	Human	154 mmol NaCl	<500
4	Mouse	10 mmol KH₂PO₄/NaHPO₄	100-500
5	Cow	10-15 mmol KH₂PO₄/NaHPO₄	1000
6	Horse	2 mmol MgCl2, 10 mmol glucose	1000
7	Sheep	10 mmol KH₂PO₄/NaHPO₄	500-1000
8	Pig	10 mmol KH₂PO₄/NaHPO₄	500-1000

Requirement for encapsulation:

Variety of biologically active substance (5000-60,000dalton) can be entrapped in erythrocytes.

· Non-polar molecule may be entrapped in erythrocytes in salts. Example: tetracycline HCl salt can be appreciably entrapped in bovine RBC.

· Generally, molecule should be Polar & Non polar molecule also been entrapped.

· Hydrophobic molecules can be entrapped in erythrocyte by absorbing over other molecules.

· Once encapsulated charged molecule are retained longer than uncharged molecule .The size of molecule entrapped is a significant factor when the molecule is smaller than sucrose and larger than B-galactosidase.^26-31

Methods of drug loading in erythrocytes:

Figure 5. 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:

Figure 6. 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.³³

a) Hypotonic Dialysis method:

Figure 7. Hypotonic Dialysis method

This method was first reported by Klibansky in 1959 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 bag after the stirring is complete.³⁴

b) Hypotonic Pre swelling method:

Figure 8: Hypotonic Pre swelling method

This method was developed by Rechsteiner in 1975 and was modified by Jenner et al. for drug loading.

This method is based on the principle of first swelling 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, cyclophosphamide, methotrexate, insulin, metronidazole, levothyroxine, enalaprilat.

c) Isotonic osmotic lysis method:

Figure 9. Isotonic osmotic lysis method

This method was reported by Schrier et al in 1975. 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. In 1987, 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 37oC.^38-42

Comparison:

Method	% Loading	Advantages	Disadvantages
Dilution	20-40	Fastest and simplest especially for low molecular weight drugs	Entrapment efficiency is less
Presswell	30-45	Better in vivo survival of erythrocytes better structural integrity and membrane Good retention of cytoplasm and good survival in vivo	Time consuming, heterogeneous size distribution of resealed erythrosytes
Isotonic osmotic Lysis	30-90	Better in vivo survival	Impermeable only large molecules, process is time consuming

2) Electro-insertion or Electro encapsulation method:

Figure 10. 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 37°C in an isotonic medium.

The various chemical encapsulated into the erythrocytes are primaquine and related 8- amino quinolone, vinblastine chlorpromazine and related phenothiazine, propranolol, tetracaine and vitamin A.

3) Entrapment by Endocytosis:

Figure 11: Entrapment by Endocytosis

This method was reported by Schrier et al in 1975.

This method involves the addition of one volume of washed packed erythrocytes to nine volume of buffer containing 2.5MM ATP, 2.5MM MgCl₂ and 1MM CaCl₂, followed by incubation for 2 minute at room temperature.

The pores created by this method are resealed by using 154MM of NaCl and incubate at 37⁰C for 2 minute.

Several chemicals are entrapped in erythrocytes by this method are primaquine and related 8- aminoquinoline, vinblastine, chlorpromazine, and related phenothiazine’s, hydrocortisone, tetracaine and vitamin A. ^48-50

4) Chemical Perturbation Of The Membrane:

Figure 12: Chemical perturbation of the membrane

This method is based on the increase in membrane permeability of erythrocytes when the cells are exposed to certain chemicals.

In 1973, Deuticke et al. showed that the permeability of erythrocyte membrane increases upon exposure to polyene antibiotic such as amphotericin B.

In 1980, this method was used successfully by Kitao and Hattori to entrap the antineoplastic drug daunomycin in human and mouse erythrocytes.

However, these methods induce irreversible destructive changes in the cell membrane and hence are not very popular.^51-53

5) Lipid fusion method:

The lipid vesicles containing a drug can be directly fuse to human erythrocytes, which lead to an exchange with a lipid 161 entrapped drug.

The methods are useful for entrapping inositol monophosphate to improve the oxygen carrying capacity of cells and entrapment efficiency of this method is very low (~1%).⁴⁴

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.³⁴

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.³⁵

MECHANISM OF DRUD RELEASE:

The various mechanisms proposed for drug release include:

· Passive diffusion.

· Specialized membrane associated carrier transport.

· Phagocytosis of resealed cells by macrophages of RES, subsequent accumulation of drug into the macrophage interior, followed by slow release.

· Accumulation of erythrocytes in lymph nodes upon subcutaneous administration followed by hemolysis to release the drug.

Shelf and storage stability of resealed RBCs:

· The most common storage media include Hank’s balanced salt solution and acid–citrate–dextrose at 4°C.

· Cells remain viable in terms of their physiologic and carrier characteristics for at least 2 weeks at this temperature.

· The addition of calcium-chelating agents or the purine nucleosides improve circulation survival time of cells upon reinjection.

· Exposure of resealed erythrocytes to membrane stabilizing agents such as dimethyl sulfoxide, dimethyl,3,3-di-thio-bispropionamide, glutaraldehyde, toluene-2-4-diisocyanate followed by lyophilization or sintered glass filtration has been reported to enhance their stability upon storage.

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 press well or dialysis technique, batches of blood for transfusion. Standard blood bag may be used for both encapsulation and storage.³⁶

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.³⁷

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 mL) with 2.0 mL acetonitrile and centrifugation at 2500 rpm for 10 min. The clear supernatant is analyzed for the drug content by spectrophotometrically.¹¹

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.³⁸

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, hemoglobin and drug released after the procedure are determined. The turbulent fragility of resealed cells is found to be higher.^{46, 49, 50}

5. Erythrocyte sedimentation rate (ESR):

It is an estimate of the suspension stability of RBC in plasma and is related to the number and size of the 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.²⁵

6. Determination of entrapped magnetite:

Atomic absorption spectroscopic method is reported for determination of the concentration of particular metal in the sample. The HCl is added to a fixed amount of magnetite bearing erythrocytes and content are heated at 60⁰C for 2 hours, then 20 %w/v trichloro acetic acid is added and supernatant obtained after centrifugation is used to determine magnetite concentration using atomic absorption spectroscopy.²⁵

7. In vitro stability:

The stability of the loaded erythrocytes is assessed by means of the incubation of the cells in the autologous plasma or in an is osmotic buffer, setting hematocrit between 0.5% and 5% at temperatures of 40C and 370C.⁵¹

8. Hemoglobin release:

The content of hemoglobin of the erythrocytes may be diminished by the alterations in the permeability of the membrane of the red blood cells during the encapsulation procedure. Furthermore, the relationship between the rate of hemoglobin and rate of drug release of the substance encapsulated from the erythrocytes. The hemoglobin leakage is tested using a red cell suspension by recording absorbance of supernatant at 540nm on a spectrophotometer.⁵¹

9. In-vitro drug release and Hb content:

The cell suspensions (5% hematocrit in PBS) are stored at 40C in ambered colour glass container. Periodically clear supernatant are drawn using a hypodermic syringe equipped with 0.45 are filter, deproteined using methanol and were estimated far drug content. The supernatant of each sample after centrifugation collected and assayed, %Hb release may be calculated using formula % Hb release=A540 of sample-A540 of background A540 of 100% Hb.²⁵

10. Osmotic shock:

For osmotic shock study, erythrocytes suspension (1 ml 10% hct) was diluted with distilled water (5 ml) and centrifuge at 300 rpm for 15 minutes. The supernatant was estimated for % hemoglobin release analytically.⁵²

11. Miscellaneous:

Resealed erythrocyte can also be characterized by cell sizes, mean cell volume, energy metabolism, lipid composition, membrane fluidity, rheological properties, and density gradient separation.²⁵

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 cytochemical 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 erythrocyte 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 diphtheria toxin.¹³

In Vivo Applications:

This includes the following

1) Slow drug release:

Erythrocytes have been used as circulating depots for the sustained delivery of antineoplastic, antiparasitics, veterinary antiamoebics, vitamins, steroids, antibiotics, and cardiovascular drugs. ⁶³

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.³⁴

3) Targeting reticuloendothelial system (RES) organs:

Surface modified erythrocytes are used to target organs of mononuclear phagocytic systems/ reticuloendothelial 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 sulfhydryl.

· Surface chemical crosslinking.

· Surface modification with carbohydrates such as sialic acid.³⁵

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 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 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.³³

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.^{11, 18,
46}

6) Removal toxic agents:

Cannon et al. reported inhibition of cyanide intoxication with murine carrier erythrocyte containing bovine rhodanase and sodium thiosulphate. Anatomization of organophosphorus intoxication by released erythrocyte containing a recombinant phosphodiesterase also has been reported.⁴⁶

7) Treatment of hepatic tumors:

Antineoplastic drugs such as methotrexate (MTX), bleomycin, asparginase and Adriamycin 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.⁴⁷

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.⁶⁸

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.^{14,31, 49}

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 the RES is very beneficial because the aged erythrocytes are destroyed in RES organs, which results in an accumulation of iron in these organs.⁴³

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.^{43, 50}

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.^{51- 53}

Various applications of resealed erythrosytes

Application	Drug/enzyme/macromolecules
Enzyme deficiency, and Enzyme replacement Therapy	B-galactosidase, B-fructofuronodase, Urease, Glucose – 6- phosphate dehydrogenase, cortical – 2 – phosphate
Thrombolytic activity	Brinase, Aspirin, Heparin
Iron overload chemotherapy	Desferroxamine, Rubomycin, Methotrexate, L - asparginase, Doxorubicin, Daunomycin, Cytosine, Arabinoside
Immuno therapy	Human recombinant interleukin – 2
Circulating carriers	Albumin, Prednisolone, Salbutamol, Tyrosine kinase, Phosphotriesterase
Circulating Bioreacters	Arginase, Uricase, luciferase, acetaldehyde dehydrogenase
Targeting to RES	Pentamidine, Mycotoxin, Imidocarb Dipropionate
Targeting to other than RES	Daunomycin, Methotrexate, Diclofenac sodium

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. These vesicles have been proposed as useful encapsulation systems for macromoleculardrugs.^44-45

Nanoerythrosomes:

These are prepared by extrusion of erythrocyte ghosts to produce small vesicles with an average diameter of 100 nm. Daunorubicin was covalently conjugated to nanoerythrosomes using glutaraldehyde spacer. This complex was more active than free daunorubicin alone.^26-27

CONCLUSION:

During the past decade, numerous applications have been proposed for the use of resealed erythrocytes as carrier for drugs, enzyme replacement therapy etc. The use of resealed erythrocytes looks promising for a safe and sure delivery of various drugs for passive and active targeting.

However, the concept needs further optimization to become a routine drug delivery system. The same concept also can be extended to the delivery of biopharmaceuticals and much remains to be explored regarding the potential of resealed erythrocytes. For the present, it is concluded that erythrocyte carriers are “golden eggs in novel drug delivery systems” considering their tremendous potential. Most of the studies in this area are in the in vitro phase and the ongoing projects worldwide remain to step into preclinical and then, clinical studies to prove the capabilities of this promising delivery system. It is also very effective and safe delivery system for anti-cancer drug with less or without toxicity.

REFERENCES:

1. Singh Devendra, Kumar Manish, Singh Talever, Singh L.R., Singh Dashrath. A Review on Resealed Erythrocytes as a Carrier for Drug Targeting, International Journal of Pharmaceutical and Biological Archives 2011; 2 (5):1357-1373.

2. Patel RP, Patel MJ and Patel A. An overview of resealed erythrocytes drug deliver, R.P. Patel, journal of pharmacy research 2009; 2(6):1008-1012.

3. A. V. Gothoskar. Resealed Erythrocytes: A Review, www. Pharma. Tech. com, pharma. Tech 2004; 142-143.

4. Gopal V. S., Doijad R.C., and Deshpande P. B. Erythrocytes as a carrier for prednisolone- in vitro and in vivoevaluation, Pak J. Pharm. Sci, 2010;(2) 23:194-200.

5. Green R and Widder K.J. Methods in Enzymology Academic Press, San Diego, 1987: 149.

6. G.J. Tortara B. Derrickson. The Cardiovascular System the Blood in Principles of Anatomy and Physiology, New York, NY, 7th ed., 1993:669-672.

7. Guyton AC and Hall JE. Red Blood Cells, Anemia and Polycytemia, in test book of medical physiology, Saunders WB, Philadelphia, PA, 1996: 425-433.

8. Ropars C., Chassaigne M., and Nicoulau C. Advances in the Biosciences, Pergamon Press, Oxford, 1987: 67.

9. Sackmann Erich, Biological Membranes Architecture and Function Handbook of Biological Physics, ed. R. Lipowsky and E. Sackmann Elsevier 1995: 1.

10. Rajendra Jangde. An Overview of Resealed Erythrocyte for Cancer Therapy, Asian J. Res. Pharm. Sci. 2011;1(4):83-92.

11. V. Jaitely et al. Resealed Erythrocytes: Drug Carrier Potentials and Biomedical Applications, Indian Drugs, 1996; 33:589–594.

12. H.O. Alpar and D.A. Lewis. Therapeutic Efficacy of Asparaginase Encapsulated in Intact Erythrocytes, Biochem. Pharmacol. 1985; 34:257–261.

13. D.A. Lewis. Red Blood Cells for Drug Delivery, Pharm. J., 1984; 32: 384–385.

14. R. Baker. Entry of Ferritin into Human Red Cells during Hypotonic Haemolysis, Nature, 1967; 215: 424–425.

15. U. Sprandel. Towards Cellular Drug Targeting and Controlled Release of Drugs by Magnetic Fields, Adv. Biosci. (Series), 1987; 67: 243–250.

16. K. Kinosita and T.Y. Tsong. Survival of Sucrose-Loaded Erythrocytes in the Circulation, Nature, 1978; 272, 258–260.

17. H.G. Eichler et al. In Vivo Clearance of Antibody-Sensitized Human Drug Carrier Erythrocytes, Clin. Pharmacol. Ther, 1986; 40: 300– 303.

18. M.P. Summer. Recent Advances in Drug Delivery, Pharm. J., 1983; 230, 643–645.

19. H. C. Eichler et.al. In Vitro Drug Release from Human Carrier Erythrocyte AS Carrier System, Advance in Bioscience, 1987; 67:11-15.

20. J. R. DeLoach. Methods in Enzymology, Academic Press, New York, 1987; 235.

21. Carmen Gutierrez Millan, Maria Luisa Sayalero Marinero, Aranzazu Zarzuelo Castaneda and Jose M. Lanao. Drug, enzyme and peptide delivery using erythrocytes as carriers, Journal of Controlled Release, 2004; 95(1): 27-49.

22. Mehrdad Hamidi, Adbolhossein Zarrina, Mahshid Foroozesha and Soliman Mohammadi-Samania. Applications of carrier erythrocytes in delivery of biopharmaceuticals, Journal of Controlled Release, 2007; 118(2):145-160.

23. G.M. Ihler. Erythrocyte Carriers, Pharmacol. Ther.1989; 20:151– 169

24. Torotra G.J. and Grabowski S.R. The Cardiovascular System: The Blood, in Principles of Anatomy and Physiology, Harper Collins College Publishers, New York, NY, 7th ed., 1993; 566–590.

25. Shashank shah. Novel Drug Delivery Carrier: Resealed Erythrocytes, International Journal of Pharma and Bio Sciences, 2011; 2(1): 394‐406.

26. D. A. Tyrrell, B.E. Ryman. Encapsulation of PEG Urease/PEG-AlaDH within Sheep Erythrocytes and Determination of the System’s Activity in Lowering Blood Levels of Urea in Animal Models, biochemistry soc.trans, 1976; 4:677.

27. J.R. DeLoach, G. M. Ihler, Carrier Erythrocyte, Biochem. Biophys. Acta. 1979. 496-136.

28. R. Goldman, T. Facchinetti, D. Bach, A. Raz, M. Shintizky. Activation of Phospholipase A2 by Adriamycin in vitro. Role of Drug-Lipid Interactions, Biochem. Biophys. Acta,1979. 512.

29. M. Hamidi and H.tajerzadeh. Carrier Erythrocyte an Overview, Drug Delivery, 2003; 10:9-20.

30. R. L. Lopez-Marques, L. R. Poulsen, S. Hanisch, K. Meffert, M. J. Buch-Pedersen, M. K. Jakobsen, T. G. Pomorski, and M. G. Palmgren. Intracellular Targeting Signals and Lipid Specificity Determinants of the ALA/ALIS P4-ATPase Complex Reside in the Catalytic ALA {alpha}- Subunit. Mol. Biol. Cell 2010: 21, 791-801

31. X. Zhou and T. R. Graham. Reconstitution of phospholipid translocase activity with purified Drs2p, a type-IV P-type ATPase from budding yeast Proc. Natl. Acad. Sci. USA 2009; 106: 16586-16591

32. J.R. Deloach, R.L. Harris, and G.M. Ihler. An Erythrocyte Encapsu- lator Dialyzer

33. Used in Preparing Large Quantities of Erythrocyte Ghosts and Encapsulation of a Pesticide in Erythrocyte Ghosts, Anal. Biochem.1980; 102, 220–227.

34. J.R. Deloach and G.M. Ihler, A Dialysis Procedure for Loading of Erythrocytes with Enzymes and Lipids, Biochim. Biophys. Acta. 1977; 496, 136–145.

35. U. Benatti et al. Comparative Tissue Distribution and Metabolism of Free Versus Erythrocyte Encapsulated Adriamycin in the Mouse, Adv. Biosci. (Series) 1987. 67, 129– 136.

36. Rechsteiner M.C. Uptake of Protein by Red Cells, Exp. Cell Res. 1975; 43: 487–492.

37. Field W.N., Gamble M.D., and Lewis D.A. A Comparison of Treatment of Thyroidectomized Rats with Free Thyroxin and Thyroxin Encapsulated in Erythrocytes, Int.J. Pharm.1989;51,175–178.

38. Bird J., Best R., and Lewis D.A. The Encapsulation of Insulin in Erythrocytes, J. Pharm. Pharmacol.1983; 35, 246–247.

39. A. Zanella et al. Desferrioxamine Loading of Red Cells for Transfusion, Adv. Biosci. (Series) 1987; 67, 17–27.

40. G. Fiorelli et al. Transfusion of Thalasemic Patients with Desferrioxamine Loaded Standard Red Blood Cell Units, Adv. Biosci. (Series) 1987; 67, 47–54.

41. M.C. Villareal et al. Approach to Optimization of Inositol Hexaphosphate Entrapment into Human Red Blood Cells, Adv. Biosci. (Series) 1987; 67, 55–62.

42. C. Hurel et al. Optimization of Desferrioxamine Loading in Red Blood Cells, Adv. Biosci. (Series) 1987; 67, 37–46.

43. M.C. Villareal et al. Modification of Cardiac Parameters in Piglets after Infusion of IHPLoaded Red Blood Cells, Adv. Biosci. (Series) 1987; 67: 81–88.

44. Gopal VS, Kumar AR, Usha NA, Karthik A, Udupa N. Effective drug targeting by erythrocytes as carrier systems. Curr. Trends Biotechnology. Pharm. 2007. 1,18‐33.

45. Zimmermann U., Riemann F., and Pilwat G. Enzyme Loading of Electrically Homogenous Human Red Blood Cell Ghosts Prepared by Dielectric Breakdown, Biochim. Biophys. Acta.1976; 436, 460– 474.

46. Tson g T.Y. and Kinosita K. Use of Voltage Pulses for the Pore Opening and Drug Loading, and the Subsequent Resealing of Red Blood Cells, Bibl Haematol.1985; 51: 108 – 114.

47. Zimmermann U., Pilwat G., and Riemann F. Preparation of Erythrocyte Ghosts by Dielectric Breakdown of the Cell Membrane, Biochim Biophys Acta.1975; 375 (2):209–219.

48. Jagadale VL, Aloorkar NH, Dohe GH, Gehlot MV. Resealed erythrocytes: an effective tool in drug targeting, Indian J. Pharm. Educ. Res. 2009; 43(4):375‐383.

49. Schrier SL. Shape Changes and Deformability in Human Erythrocyte Membrane, J. Lab. Clin. Med. 1987; 110 (6): 791-797.

50. Kinosita K and Tsong TY. Hemolysis of Human Erythrocytes by a Transient Electric Field, Proc, Natl, Acad. sci. USA, 1977; 74: 1923-1927.

51. Deloach JR, “Encapsulation of Exogenous Agents in Erythrocytes and the Circulating Survival of Carrier Erythrocytes,” J, Appl. Biochem. 1983; 5(3): 149-157.

52. B. Deuticke, M. Kim, and C. Zolinev. The Influence of Amphotericin-B on the Permeability of Mammalian Erythrocytes to Nonelectrolytes, anions and Cations, Biochim. Biophys. Acta. 1973; 318: 345–359.

53. T. Kitao, K. Hattori, and M. Takeshita. Agglutination of Leukemic Cells and Daunomycin Entrapped Erythrocytes with Lectin in Vitro and In Vivo, Experimentia 1978; 341: 94–95.

54. W. Lin et al. Nuclear Magnetic Resonance and Oxygen Affinity Study of Cesium Binding in Human Erythrocytes, Arch Biochem Biophys.1999; 369, (1): 78–88.

Received on 28.12.2018 Modified on 20.01.2019

Research J. Pharm. and Tech. 2019; 12(5):2603-2611.

DOI: 10.5958/0974-360X.2019.00437.2