INTRODUCTION:

Resealed erythrocytes part of parental control release formulation consisting erythrocyte, also known as red blood cells, have been extensively studied for their potential carrier capabilities for the delivery of drug and drug loaded microspheres. Such drug loaded carrier erythrocyte are prepared simply by collecting blood samples from the organism of interest ,separating erythrocyte from plasma ,entrapping the drug in the erythrocyte and resealing the resultant cellular carriers. Hence these carriers are called resealed erythrocyte. 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⁽¹⁾.

Fig.1: response of erythrocyte under different osmotic conditions.

Erythrocytes:-

Erythrocytes are natural products of the body, biodegradable in nature, isolation of these is easy and large amount of drug can be loaded in small volume of cells, non immunogenic in action and can be targeted to disease tissue or organ, prolong the systemic activity of the drug while residing for a longer time in the body, protect the premature degradation, inactivation and excretion, of proteins and enzymes, act as a carrier for number of drugs, target the drugs within the reticuloendothelial system (RES) as well non RES organs/sites. They have the capacity to carry large amounts of drug; and can behave as a slow-release long-acting system. Potential clinical indications for “RES targeting” include iron over-storage diseases, parasitic diseases, hepatic tumors, cancer and lysosomal storage diseases carriers⁽²⁾.

Fig.2: Erythrocytes

Erythrocytes can be used as carriers in two ways⁽³⁾:-

1. Targeting particular tissue/organ. - For targeting, only the erythrocyte membrane is used. This is obtained by splitting the cell in hypotonic solution 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.

Methods of drug loading⁽⁴⁾:-

Several methods can be used to load drugs or other bioactive compounds in erythrocytes including physical (e.g., electrical pulse method) osmosis-based systems, and chemical methods (e.g., chemical perturbation of the erythrocytes, membrane.

1. Hypotonic hemolytic⁽⁵⁾ :-

This method is based on the ability of erythrocytes to undergo reversible swelling in a hypotonic solution. Erythrocytes have an exceptional capability for reversible shape changes with or without accompanying volume change and for reversible deformation under stress. An increase in volume leads to an initial change in the shape from biconcave to spherical. This change is attributable to the absence of superfluous membrane; hence the surface area of the cell is fixed. The cells assume a spherical shape to accommodate additional volume while keeping the surface area constant. The volume gain is ~25–50%. The cells can maintain their integrity up to a tonicity of ~150 m osm/kg, above which the membrane ruptures, releasing the cellular contents. At this point (just before cell lysis), some transient pores of 200–500 Å are generated on the membrane. After cell lysis, cellular contents are depleted. The remnant is called an erythrocyte ghost.

Fig.3:- Hypotonic haemolytic

2. Use of red cell loader⁽⁶⁾:-

Novel method for entrapment of non-diffusible drugs into erythrocytes. 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 the cells. There was ~30% drug loading with 35–50% cell recovery.

3. Hypotonic dilution⁽⁷⁾:-

Hypotonic dilution was the first method investigated for the 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 preswelling⁽⁸⁾:-

This method was developed and was 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 the cell fraction is brought to the lysis point by L portions of an aqueous solution of the drug to be encapsulated. Adding 100–120. The mixture is centrifuged between the drug-addition steps. The lysis point is detected by the disappearance of a distinct boundary between the cell fraction and the supernatant upon centrifugation. The tonicity of a cell mixture is restored at the lysis point by adding a calculated amount of hypertonic buffer.

Fig.4: Hypotonic presswelling

5. Isotonic osmotic lysis⁽⁹⁾ :-

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.

6. Hypotonic dialysis⁽¹⁰⁾ :-

Several methods are based on the principle that semipermeable 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 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.

Fig.5:- Hypotonic dialysis

7. 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. that the permeability of erythrocytic membrane increases upon exposure to polyene antibiotic such as amphotericin B. In1980, this method was used successfully by Kitao and Hattori to entrap the antineoplastic drug daunomycinin human and mouse erythrocytes. in et al used halothane for the same purpose. However, these methods induce irreversible destructive changes in the cell membrane and hence are not very popular.

Fig.6:- Chemical perturbation of the membrane

8. Electro-insertion or electro encapsulation⁽¹¹⁾ :-

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. In 1977, Tsong and Kinosita suggested the use of transient electrolysis to generate desirable membrane permeability for drug loading. The erythrocyte membrane is opened by a dielectric break down. Subsequently, the pores can be resealed by incubation at 37^oC in an isotonic medium. The procedure involves suspending erythrocytes in an isotonic buffer in an electrical discharge chamber. A capacitor in an external circuit is charged to a definite voltage and then discharged within a definite time interval through cell suspension to produce a square-wave potential. This process can be prevented by adding large molecules (e.g., tetra saccharides tachyose and bovine serum albumin) and ribonucleose.

Fig.7:- Electro-insertion or electro encapsulation

9. Entrapment by endocytosis⁽¹²⁾ :-

This method was reported by Schrier et al. in1975. Endocytosis involves the addition of one volume of washed erythrocytes to nine volumes of buffer containing 2.5 mM ATP, 2.5 mM MgCl2, and 1mM CaCl2, followed by incubation for 2 min at room temperature. The pores created by this method are resealed by using 154 mM of NaCl and incubation at 37^oC for 2 min. The entrapment of material occurs by endocytosis.The vesicle membrane separates endocytosed material from cytoplasm thus protecting it from the erythrocytes and vice-versa.

Fig.8:- Entrapment by endocytosis

10. Loading by electric cell fusion⁽¹³⁾:-

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 chemical lycross-linked to drug-loaded cells that would direct these cells to desired cells.

11. Loading by lipid fusion⁽¹⁴⁾:-

Lipid vesicles containing a drug can be directly fused to human erythrocytes, which lead to an exchange with a lipid-entrapped drug. This technique was used for entrapping inositol monophosphate to improve the oxygen carrying capacity of cells. However, the entrapment efficiency of this method is very low (~1%).

Fig.9:- Loading by lipid fusion

In vitro storage:-

The success of resealed erythrocytes as a drug delivery system depends to a greater extent on their in vitro storage. Preparing drug-loaded erythrocytes on a large scale and maintaining their survival and drug content can be achieved by using suitable storage methods. However, the lack of reliable and practical storage methods has been a limiting factor for the wide-spread clinical use of the carrier erythrocytes.

§ The most common storage media include Hank’s balanced salt solution and acid–citrate–dextrose at 4oC. 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, gluteraldehyde, toluene-2-4-diisocyanate followed by lyophilization or sintered glass filtration has been reported to enhance their stability upon storage.

§ The resultant powder was stable for at least one month without any detectable changes. But the major disadvantage of this method is the presence of appreciable amount of membrane stabilizers in bound form that remarkably reduces circulation survival time.

§ Other reported methods for improving storage stability include encapsulation of a prodrug that undergoes conversion to the parent drug only at body temperature, high glycerol freezing technique, and reversible immobilization in alginate or gelatine gels.

In vivo life span:-

The efficacy of resealed erythrocytes is determined mainly by their survival time in circulation upon reinjection. For the purpose of sustained action, a longer life span is required, although for delivery to target-specific RES organs, rapid phagocytosis and hence a shorter life span is desirable. The life span of resealed erythrocytes depends upon its size, shape, and surface electrical charge as well as the extent of haemoglobin and other cell constituents lost during the loading process.

Cancer:--

Cancer is a term for used diseases in which abnormal cells divide without control and are able to invade other tissues. Cancer cells can spread to other parts of the body through the blood and lymph systems.

Fig.10: Cancer cell

Cancer is a class of diseases characterized by out-of control cell growth. There are over 100 different types of cancer, and each is classified by the type of cell that is initially affected.

Types of Cancer Classified by Body System⁽¹⁵⁾:--

1. Blood Cancer: --The cells in the bone marrow that give rise to red blood cells, white blood cells, and platelets can sometimes become cancerous. These cancers are leukemia or lymphoma.

 Leukemia, acute lymphoblastic leukemia.

 Lymphoma

 Multiple Myeloma

2. Bone Cancer:- Bone cancer is a relatively rare type of cancer that can affect both children and adults, but primarily affects children and teens. There are several types of bone cancer, but the most common types are:

 Ewing's Sarcoma

 Osteosarcoma

3. Brain Cancer: - Brain tumors can be malignant (cancerous) or benign (non-cancerous). They affect both children and adults. Malignant brain tumors don't often spread beyond the brain. However, other types of cancer have the ability to spread to the brain. Types of brain cancer include:

 Adult Brain Tumor

 Brain Stem Glioma, Medulloblastoma Childhood

 Cerebellar Astrocytoma , Ependymoma, Childhood

 Visual Pathway and Hypothalamic Glioma,

4. Breast Cancer: -Breast cancer is a common type of cancer that affects women and much less commonly, men.

 Ductal lobular carcinoma in situ

 Inflammatory breast cancer

 Invasive types of breast cancer

5. Digestive/Gastrointestinal Cancers:- This is a broad category of cancer that affects everything from the oesophagus to the anus. Each type is specific and has its own symptoms, causes, and treatments.

 Anal Cancer, Colon Cancer, Pancreatic cancer, Rectal cancer

 Bile Duct Cancer, Extra hepatic cancer

 Carcinoid Tumor, Gastrointestinal cancer

 Esophageal Cancer ,Gallbladder Cancer

 Liver Cancer childhood, Adult Primary Liver Cancer

 Small Intestine Cancer, Stomach Cancer

6. Endocrine Cancers:- The endocrine system is an instrumental part of the body that is responsible for glandular and hormonal activity. Thyroid cancer is the most common of the endocrine cancer types and generally, the least fatal.

 AdrenocorticalCarcinoma, Carcinoid Tumor, Gastrointestinal cancer

 Islet Cell Carcinoma (Endocrine Pancreas) Parathyroid Cancer

 Pheochromocytoma, Pituitary Tumor, Thyroid Cancer

7. Eye Cancer:- Like other organs in the human body, the eyes are vulnerable to cancer as well. Eye cancer can affect both children and adults.

 Melanoma, Intraocular Cancer

 Retinoblastoma

8. Genitourinary Cancers:- These types of cancer affect the male genitalia and urinary tract.

 Bladder Cancer, Kidney Cancer

 Penile Cancer, Prostate Cancer

 Prostate Cancer, Testicular Cancer, Urethral Cancer

 Wilms' Tumor Other Childhood Kidney Tumors

 Renal Pelvis and Ureter Cancer

9. Gynecologic Cancers:- This group of cancer types affect the organs of the female reproductive system..

 Cervical Cancer, Endometrial Cancer, Ovarian Cancer, Uterine Sarcoma.

 Gestational trophoblastic Tumor,

 Vaginal Cancer

10. Head and Neck Cancer:- Most head and neck cancers affect moist mucosal surfaces of the head and neck, like the mouth, throat, and nose. Causes of head and neck cancer vary, but cigarette smoking plays a role. Current research suggests a strong HPV link in the development of some head and neck cancer.

 Hypo pharyngeal Cancer, Laryngeal Cancer, Lip and Oral Cancer

 Metastatic Squamous,

 Neck Cancer, Nasopharyngeal Cancer

 Oropharyngeal Cancer, Paranasal Sinus and Nasal Cavity Cancer

 Parathyroid Cancer.

11.Respiratory Cancers:- Cigarette smoking is the primary cause for cancer affecting the respiratory system. Exposure to asbestos is also a factor.

 Lung Cancer, Small Cell Lung Cancer

 Malignant Mesothelioma

 Thymoma and Thymic Carcinoma

12.Skin Cancers:- Non-melanoma skin cancer is the most common type of cancer among men and women. Exposure to the UV rays of the sun is the primary cause for non-melanoma skin cancer and also melanoma.

 Cutaneous T-Cell Lymphoma

 Kaposi's sarcoma, Merkel Cell Carcinoma

 Melanoma,

 Non-Melanoma Skin Cancer

Advantage of resealed erythrocyte in drug delivery ⁽¹⁶⁾:-

1. The drug loaded erythrocytes serve as slow circulation depots, targets the drug to the reticuloendothelial system (RES), prevents degradation of loaded drug from inactivation by endogenous chemicals, attain steady state concentration of drug and decrease the side-effects of loaded drug.

2. Their biocompatibility, particularly when autologous cells are used, hence no possibility of triggered immune response the considerably uniform size and shape of the carrier.

3. Relatively inert intracellular environment. Their biodegradability with no generation of toxic products Prevention of degradation of the loaded drug from inactivation by endogenous chemicals.

4. The wide variety of chemicals that can be entrapped.

5. The modification of pharmacokinetic and pharmacodynamic parameters of drug.

6. Attainment of steady-state plasma concentration decreases fluctuations in concentration.

7. Protection of the organism against toxic effects of drugs (e.g. antineoplastics)

8. Their ability to circulate throughout the body.

9. The availability of the techniques and facilities for separation, handling, transfusion, and working with erythrocytes.

10. The prevention of any undesired immune response against the loaded drug. Their ability to target the organs of the RES.

Disadvantage of resealed erythrocyte in drug delivery⁽¹⁷⁾:-

1. The major problem encountered in the use of biodegradable materials or natural cells as drug carriers is that they are removed in vivo by the RES. This seriously limits their useful life as drug carriers .

2. In some cases may pose toxicological problems.

3. The rapid leakage of certain encapsulated substances from the loaded erythrocytes.

4. Several molecules may alter the physiology of the erythrocyte, Given that they are carriers of biological origin, encapsulated erythrocytes may present greater variability and lesser standardisation in their preparation, compared to other carrier systems,

5. The storage of the loaded erythrocytes is a further problem involving carrier erythrocytes for their possible use in therapeutics.

6. Tests have been performed on their conditioning in suspension in isotonic buffers containing all essential nutrients, as well as in low temperatures, with the addition of nucleosides or chelators, liophylisation, freezing with glycerol or gel immobilization, Liable to biological contamination due to the origin of the blood, the equipment and the environment, such as air.

7. Rigorous controls are required accordingly for the collection and handling of the erythrocytes.

Resealed erythrocyte act on cancer:-

Resealed erythrocyte act on Acute lymphoblastic leukemia:-

Acute lymphoblastic leukemia:- (ALL)⁽¹⁷⁾:-- is cancer of the white blood cells, the cells that normally fight infections. In patients with ALL, the bone marrow produces excess immature white blood cells, called lymphoblasts, which are unable to help the body fight infections.

Fig. 11:- lymphoblastic leukemia cells in bone marrow.

Symptoms:-

Fig.12:-common symptoms acute lymphoblastic leukemia

Initial symptoms are not specific to ALL. The signs and symptoms of ALL are variable but follow from bone marrow replacement and/or organ infiltration.

· Generalized weakness and fatigue ,

· Weight loss and/or loss of appetite

· Frequent or unexplained fever and infections,

· Breathlessness

· Bone pain, joint pains (caused by the spread of "blast" cells to the surface of the bone or into the joint from the marrow cavity)

· Enlarged lymph nodes, liver and/or spleen, Anemia

· Pitting edema (swelling) in the lower limbs and/or abdomen.

L-asparaginase⁽^18-20):-

One of the primary drugs used in the treatment of ALL is L-asparagines (ASNase) from E. coli, which has been in clinical use since 1967. ASNase is an enzyme which hydrolyzes amino acid L-asparagine (ASN) to L-aspartic acid and ammonia. Most human tissues can self-synthesize ASN from L-glutamine by the action of asparagine synthetase (AS). Certain neoplastic tissues, including ALL cells, however, express a significantly lower level of AS, and thus have to rely solely on extracellular source of ASN to maintain protein synthesis. Systemic depletion of ASN by ASNase would therefore impair protein biosynthesis in these cells, leading to their deaths through cellular dysfunction.

Fig.13: Antileukemic bacterial L-asparaginase

Introducing L-asparaginase into red blood cells (RBCs)⁽²¹⁾ :-

The entrapment of E. coli L-asparaginase inside homologous erythrocytes (GRASPA™) is an attractive solution, especially regarding the attenuation of many of the side effects quoted above, as it enables a considerable reduction of the immunological reactions and a protection of the enzyme from plasmatic proteases. The initial cell material (packed RBCs) is selected by the blood bank according to the patient’s (recipient) blood group characteristic. The L-asparaginase encapsulation process can be summarized in six steps

1) RBCs are washed with a saline solution,

2) L-asparaginase is added to the RBCs suspension

3) Following RBC dialysis against a hypotonic solution cells are made permeable. The RBCs swell and pores appear on their membrane allowing the L-asparaginase to enter erythrocytes.

4) To restore isotonicity, a hypertonic solution is added online. RBCs recover their initial shape and the membrane pores reseal. L-aspraginase molecules already within them are definitively entrapped.

5) The RBCs loaed with L-asparaginase are washed to eliminate cell ghosts and extracellular elements

6) A preservative solution (SAG-mannitol) is added to give us the final product: GRASPA™.

Fig.14: Schematic for enzyme loading into intact erythrocytes

Treatment of ALL :-

GRASPA™ opens new perspectives for L-asparaginase use. This helps to improve pharmacodynamic parameters, enzymatic efficacy and also increases general tolerance to the treatment. Such prolonged plasma asparagine depletion, associated with RBCs loaded with L-asparaginase, favours tumour cell elimination. Moreover, the use of L-asparaginase inside erythrocytes highlights the following points:

- Lengthening of the suppression of plasma asparagines.

-Reduction of anaphylactic reactions and the hypersensitivity reduction.

- Modification of the immune response inducing IgG production.

- Pharmacokinetics improvement: 28 days half-life.

- Improvement of the dose/effect relationship since the asparagine depletion is prolonged from 10 to 50 days, although injected.

Resealed erythrocyte act on Multiple myeloma:-

Multiple myeloma⁽²²⁾(from Greek myelo-, bone marrow):-

Also known as plasma cell myeloma or Kahler's disease is a cancer of plasma cells, a type of white blood cell normally responsible for the production of antibodies. Collections of abnormal cells accumulate in bones, where they cause bone lesions, and in the bone marrow where they interfere with the production of normal blood cells.

Fig.15: Myloma cells

Signs and Symptoms: - Patients may not show any signs for quite some time and Myeloma can present itself in many different ways. As the disease progresses, it can cause any of the following problems:

--Bone pain, High calcium levels, Fever

- Anemia, Weight loss, Low platelet count

- Frequent infections ,kidney failure

Treatment⁽²³⁾:-

Multiple Myeloma involves almost all of the bone marrow space in the body. As a result, the disease can only be treated with systemic therapies. Also offered a bone marrow transplantation

. Thalidomide, dexamethasone, melphalan, prednisone, vincristine, adriamycin, decadron are anticancer drugs used in treatment of multiple myloma.

Adriamycin⁽²⁴⁾ :-

It is an anthracycline antibiotic of wide use in Antineoplastic chemotherapy because of its remarkable cytotoxicity toward several solid tumors .Also known as hydroxyl daunorubicin is closely related to the natural product daunomycin and like all, anthracyclines, it works by intercalating DNA. Adriamycin is commonly used in the treatment of a wide range of cancers, including hematological malignancies, many types of carcinoma,multiple myloma and soft tissue sarcomas. Adriamycin's most serious adverse effect is life-threatening heart damage, acute heart arrhythmias. It can also cause neutropenia, alopecia. The drug is administered intravenously, in the form of hydrochloride salt. It the drug was originally isolated in the 1950's from bacteria found in soil samples taken from Castel Del Monte, an Italian castle.

Dialysis Encapsulation of adrimycin in to resesled erythrocyte. ⁽²⁵⁾:-

Adriamycin (doxorubicin) was encapsulated in human erythrocytes by means of a dialysis technique involving transient hypotonic hemolysis followed by isotonic resealing. Up to 1.6 mg of the drug was entrapped per ml of packed erythrocytes, with the efficiency of encapsulation60-80%.It involves following steps;

1. Encapsulation of Adriamycin, ferredoxin,NADP+ reductase and ferredoxin within human erythrocytes was achieved by the hypotonic dialysis-isotonic resealing method .

2. Briefly, washed erythrocytes were placed in a dialysis bag, at a final hematocrit of 70% together with either Adriamycin or ferredoxin.

3. NADP+ reductase and ferredoxin or both, dissolved in 0.9% NaCl. Transient hemolysis was achieved by placing the dialysis bag in 10 vol of 10 mM sodium phosphate and 10 mM sodium bicarbonate (pH 7.4) containing 20 mM glucose, and gently mixing by rotation for20 min at 4°C

4. Ten milliliters of the lysed erythrocytes were left for 10 min at 39°C then mixed with 1.0 ml of a resealing isotonic solution (5 mM adenine/100 mM inosine/100 mM sodium pyruvate/100 mM sodium phosphate/100 mM glucose/12% NaCl.

5. Incubated for 30 min at 39°C.

6. The Resealed erythrocytes were washed three times in 0.9% NaCl and once more in autologous plasma to remove extracellular bound material before being used in the treatment.

Treatment of Multiple myeloma by adriamycin encapsulated erythrocyte⁽²⁵⁾ :-

Adriamycin encapsulated in erythrocyte are very effective in treatment o multiple myloma. It is parentrally given to the patients. They are capable of causing breaks in DNA strand by activating Topoisomerase-ll. The encapsulation potential of adriamycin in human erythrocytes was found to be remarkably good. Thus, the hypotonic Encapsulation of Adriamycin in autologous human erythrocytes may represent a therapeutic strategy for the slow release in circulation of this antineoplastic drug in order to reduce or prevent its adverse effects and especially the delayed cardiotoxicity that limits its use in patients with neoplastic disease.

Applications of resealed erythrocytes⁽²⁶⁾:-

Resealed erythrocytes have several possible applications in various fields of human and veterinary medicine. Such cells could be used as circulating carriers to disseminate a drug within a prolonged period of time in circulation or in target-specific organs, including the liver, spleen, and lymph nodes. A majority of the drug delivery studies using drug-loaded erythrocytes are in the preclinical phase. In a few clinical studies, successful results were obtained.

1. Slow drug release:-

Erythrocytes have been used as circulating depots for the sustained delivery of antineoplastics, antiparasitics, veterinary antiamoebics, 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/ reticuloendothelial system because the changes in the membrane are recognized by macrophages. However, resealed erythrocytes also can be used to target organs other than those of RES.

3. Targeting RES organs: –

Damaged erythrocytes are rapidly cleared from circulation by phagocyte Kupffer cells in liver and spleen. Resealed erythrocytes, by modifying their membranes, can therefore be used to target the liver and spleen. The various approaches to modify the surface characteristics of erythrocytes include Surface modification with antibodies, gluteraldehyde, sialic acid, sulphydryl and Surface chemical cross-linking e.g. delivery of 125 I-labeled carbonic anhydrase loaded in erythrocytes cross-linked with sulfo succinmidyl propionate.

4.Targeting the liver- Enzyme deficiency/replacement 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.

5. Treatment of hepatic tumors:-

Hepatic tumors are one of the most prevalent types of cancer. Antineoplastic drugs such as methotrexate, bleomycin, asparginase and Adriamycin have been successfully delivered by erythrocytes. Agents such as daunorubicin diffuse rapidly from the cells upon loading and hence pose a problem.

6. Treatment of parasitic diseases:-

The ability of resealed erythrocytes to selectively accumulate within 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.

7. Removal of RES iron overload;-

Desferrioxamine-loaded erythrocytes have been used to treat excess iron accumulated because of multiple transfusions to thalassemic patient’s. 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.

8. Removal of toxic agents:-

Cannon et al. reported inhibition of cyanide intoxication with murine carrier erythrocytes containing bovine rhodanase and sodium thiosulfate. Antagonization of organ phosphorus in toxication by resealed erythrocytes containing a recombinant phospho diestrase also has been reported.

9. Targeting organs other than those of RES:-

Recently, resealed erythrocytes have been used to target organs outside the RES. The various approaches include Entrapment of paramagnetic particles, photosensitive material along with the drug and Antibody attachment to erythrocyte membrane to get specificity of action .Zimmermann proposed that the entrapment of small paramagnetic particles into erythrocytes might allow their localization to a particular location under the influence of an external magnetic field.

10. Delivery of antiviral agents:-

Several reports have been cited in the literature about antiviral agents entrapped in resealed erythrocytes for effective delivery and targeting.. Resealed erythrocytes have been used to deliver deoxycytidine derivatives, recombinant herpes simplex virus type 1 (HSV-1) glycoprotein B, azidothymidine derivatives, azathioprene, acyclovir, and fludarabin ephosphate.

11. Enzyme therapy:-

Enzymes are widely used in clinical practice as replacement therapies to treat diseases associated with their deficiency (e.g., Gaucher’s disease,galactosuria), degradation of toxic compound secondary to some kind of poisoning (cyanide,organophosphorus), and as drugs. The problems involved in the direct injection of enzymes into the body have been cited. One method to overcome these problems is the use of enzyme-loaded erythrocytes. These cells then release enzymes into circulation upon hemolytic act as a “circulating bioreactors” in which substrates enter into the cell, interact with enzymes, and generate products or accumulate enzymes in RES upon hemolytic for future catalysis. The first report of successful clinical trials of the resealed erythrocytes loaded with enzymes for replacement therapy is that of ß -glucoserebrosidase for the treatment of Gaucher’s disease. The disease is characterized by in born deficiency of lysosomal ß-glucoserebrosidase in cells of RES thereby leading to accumulation of ß-glucoserebrosides in macrophages of the RES.

Novel approaches:-

Erythrosomes⁽^27-29) :-

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 macromolecular drugs.

Nanoerythrosomes⁽^30-31):-

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 gluteraldehyde spacer. This complex was more active than free daunorubicin alone, both in vitro and in vivo.

Future perspectives:-

The concept of employing erythrocytes as drug or bio active carrier still needs further optimization a large amount of valuable work is needed so us to utilize the potential of erythrocyte in passive and as well as active targeting of drugs. Disease like cancer would surely find it cure. Genetic engineering aspects can be coupled to give a newer dimension to the existing cellular drug concept.

CONCLUSION:-

The use of resealed erythrocytes looks promising for a safe and effective 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. It is very effective and safe delivery system for anti cancer drug with or without less toxicity.

REFERENCES:

1. A.V.Gothoskar Resealed Erythrocytes A Review www. Pharma. tech. com, pharma. tech march (2004),142-143

2. G.J. Torotra and S.R. Grabowski, “The Cardiovascular System: The Blood,” in Principles of Anatomy and Physiology , Publishers, New York, NY, 7th ed., (1993), pp. 566–590.

3. G.M. Ihler,“Erythrocyte Carriers,”Pharmacol. Ther.(1989).20, 151–169

4. M. Hamidi and H. Tajerzadeh, “Carrier Erythrocytes: An Overview,” Drug delivery(2003)10, 9–20 .

5. J.R. Deloach, R.L. Harris, and G.M. Ihler, “An Erythrocyte Encapsu- lator Dialyzer Used in Preparing Large Quantities of Erythrocyte Ghosts and Encapsulation of a Pesticide in Erythrocyte Ghosts,”Anal. Biochem. (1980)102, 220–227

6. E. Pitt et al., “Encapsulation of Drugs in Intact Erythrocytes: An Intravenous Delivery System,” Biochem. Pharmacol. 22, (1983) 3359–3368 .

7. 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

8. D.A. Lewis and H.O. Alpar, “Therapeutic Possibilities of Drugs Encapsulated in Erythrocytes,” Int. J. Pharm. 22, (1984)137–146

9. H. Davson and J.F. Danielli Dannen Conn.(Hanfer Publishing Co Germany, 1970), p. 80.

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

11. K. Kinosita and T.Y. Tsong , “Hemolysis of Human Erythrocytes by a Transient Electric Field,” Proc. Natl. Acad. Sci. USA 74, (1977)1923–1927.

12. S.L. Schrier “Energized Endocytosis in Human Erythrocyte Ghosts,” J. Clin. Invest. 56 (1), (1975) 8–22.

13. L.H. Li “Electrofusion Between Heterogeneous-Sized MammalianCells in a Pellet: Potential Applications in Drug Delivery and Hybridoma Formation,” Biophys J. 71 (1), (1996). 479–486 .

14. C. Nicolau and K. Gersonde, “Incorporation of Inositol Hexaphosphate into Intact Red Blood Cells, I: Fusion of Effector-Containing Lipid Vesicles with Erythrocytes,”Naturwissenschaften 66 (11), (1979)563–566

15. Cancer Facts and Figures 2010 at the American Cancer Society.

16. S. Jain and N.K. Jain, “Engineered Erythrocytes as a Drug Delivery System,” Indian J. Pharm. Sci. 59, (1997)275–281.

17. Malignancies of Lymphoid Cells. Clinical Features, Treatment, and Prognosis of Specific Lymphoid Malignancies. 16th Chapter 97.

18. Broome JD. L-asparaginase: discovery and development as tumor-inhibitory agent. Cancer treat. Rep. (1981);65(suppl.4):111-4.

19. Rizzari C, Zucchetti M, Sparano P, Lo Nigro L, Arico M, Milani M, D’Incalci M. L-asparagine depletion and L-asparaginase activity in children with acute lymphoblastic leukemia receiving i.m. or i.v. Erwinia C. or E. coli Lasparaginase as first exposure. Ann Oncol. (2000);11(2):189-93.

20. Billet AL, Carls A, Gelber RD et al. Allergic reactions to Erwinia asparaginase in children with acute lymphoblastic leukaemia who had previous allergic reactions to Escherichia Coli asparaginase. Cancer. (1992);70:201-6.

21. Dr Yann GODFRIN, PhD, Prof Yves BERTRAND, MD, L-asparaginase Introduced into Erythrocytes for the Treatment of Leukaemia (ALL) Volume 1 Issue 1 • June/July 2006 ,10-13. a b c d Raab MS, Podar K, Breitkreutz I, Richardson PG, Anderson KC (July 2009). "Multiple myeloma". Lancet 374 (9686): 324–39.

22. ^ a b c International Myeloma Working Group "Criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma Working Group". Br. J. Haematol. 121 (5): (2003). 749–57.

23. Arcamone, F., Penco, S., Vigevano, A. and Redaelli, S. Cancer Chemotherapy. Rep. 6, (1975): 123-129

24. Bonadonna, G., Monfardini, S., De Lena, M. and Fossati Bellani, F. Brit. Med. J. 3, (1969) : 503-506.

25. Proc. Nati. Acad. Sci. USA, Encapsulation of Adriamycin in human erythrocytes Medical Sciences Vol. 83, pp.7029-7033

26. H.O. Alpar and W.J. Irwin, “Some Unique Applications of Erythrocytes as Carrier Systems,” Adv. Biosci. (series) (1987) 67, 1–9 .

27. Legha, S. S., Benjamin, R. S., Mackay, B., Ewer, M., Wallace, S., Valdivieso, M., Rasmussen, S. L., Blumenschein, G. R. and Freireich, E. J. Cuppoletti J. et al., “Erythrosomes: Large Proteo liposomes Derived from Cross-Linked Human Erythrocyte Cytoskeletons and Exogenous Lipid,” Proc. Natl. Acad. Sci. USA 78 (5) ,(1981) 2786–2790 .

28. .C.Y. Jung, Methods in Enzymology (Academic Press, New York, NY, 1987), pp. 149–217.

29. S.P. Vyas and V.K. Dixit, Pharmaceutical Biotechnology 1 (CBS Publishersand Distributors, New Delhi, (1999), pp. 655.

30. .M. Moorjani et al., “Nanoerythrosomes, A New Derivative of Erythrocyte Ghost II: Identification of the Mechanism of Action,”Anticancer Res. 16 (5A), (1996).2831–2836.

31. A. Lejeune et al., “Nanoerythrosomes, A New Derivative of Erythrocyte Ghost: III. Is Phagocytosis Involved in the Mechanism of Action?,” Anticancer Res. (1997)17, 5A .

Received on 19.09.2011 Accepted on 30.09.2011

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