Novel Drug Delivery Carrier Resealed Erythrocytes

    Aklakh Gafar Shaikh*, Sujata Shivaji Waghmare, Zalte Swapnali Nandkumar

Sojar college of pharmacy, Khandvi, Barshi, Solapur, 415408 (MH). India

*Correspondence: aklakshaikh1998@gmail.com

INTRODUCTION

The Drug carrier system including liposomes, nanoparticles, niosomes, resealed erythrocytes etc. act on specific target, promote therapeutic effect of the drug, decrease toxic effect (by increasing drug level and persistence in vicinity of target cells, hence decreasing the drug exposure to non-target cells) and finally increases the dose effectiveness also. Conventional dosage form faces several drawbacks like first pass effect, instability, rapid release of drug, plasma drug fluctuations and require high dose. As a result, the fascinating type of drug targeted novel drug carrier system has attracted a lot. Among the various targeted oriented delivery system, the cellular carrier, micro particulate and vesicular can avoid immune response intravenously by mimic body’s intrinsic component. Erythrocytes, granulocytes, leucocytes and lymphocytes are different cellular carriers. Erythrocytes, the most abundant cells in the human body, have potential carrier capabilities for the delivery of drugs.

Erythrocytes are biocompatible, biodegradable, possess very long circulation half-lives and can be loaded with a variety of chemically and biologically active compounds using various chemical and physical methods. Application of erythrocytes as promising slow drug release or site-targeted delivery systems for a variety of bioactive agents from different fields of therapy has gained. The targeting process of drug can be approaches by either chemical modification or by appropriate carrier. There are many drug delivery carriers has been investigated presently like nanoparticle, microspheres, lipid vesicular carrier, micro emulsion, pharmacosomes, ethosomes, cellular carrier and macromolecule. The targeted or site-specific drug delivery is a very promising goal because it provides one of the most effective ways to improve the therapeutic index (TI) of drug whilst devoicing its potential interaction with non-targeted tissue. There are different carriers has been used for the drug targeting among which cellular carrier offer a greater potential advantages related to its biodegradability, biocompatibility, self-degradability & it also offers high drug loading capacity. [4-10]

Erythrocytes/ Red Blood Cells (RBC):

A healthy female and male has approximately 4.8 million and 5.4 million male RBC/ microliter of blood. RBC are biconcave in shape with thickness of 2.2 micrometre and diameter of 7-8 micro meter. They are necessary for delivery of oxygen in blood organs. Mature RBC have no mitochondria which provide more area for transportation of oxygen. They live only for 120 days. In some species, old RBC’s are recognized depending upon senescent cell antigen and then destroyed by phagocytic cells (in spleen). While in others, RBC are removed from circulation in a random manner.

Characteristics of modified RBC

The pharmacological role of RBC carriage is to boost bioavailability and increase the circulation of drug. But RBC carriage inhibit the circulation of already present longer lasting agents like IgG. e.g.

RBC inhibit the endothelial FcRn mediated immunoglobin recycling mechanism by interacting with them (Sockolosky et al. Moreover, RBC coated with immunoglobin endured phagocytosis through different mechanism such as opsonisation, multivalent involvement of FcRn-gamma and the mechanism is based on degree and nature of modification of RBC. RBC carriage also inhibit the functions of some cargoes. RBC carriage also alter the distribution of drugs in blood circulation in different ways like

• Increase the glomerular filtration and eructation through endothelial intracellular and intercellular pathways.

• Redistribution in blood from marginal plasma layer to main blood stream.

• Increased the intake via spleen

 RBC also changed the excretion of drugs are excreted through urine, bile by shifting to hepatobiliary and reticuloendothelial intake [11-14]

Source and isolation of erythrocyte:

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. To isolate erythrocytes, blood is collected in heparinized tubes by venepuncture. Fresh whole blood is typically used for loading purposes because the encapsulation efficiency of the erythrocytes isolated from fresh blood is higher than that of the aged blood. Fresh whole blood is the blood that is collected and immediately chilled to 4°C and stored for less than two days. The erythrocytes are then harvested and washed by centrifugation. The washed cells are suspended in buffer solutions at various haematocrit values as desired and are often stored in acid–citrate–dextrose buffer at 4 °C for as long as 48 h before use. Jain and Vyas have described a well-established protocol for the isolation of erythrocytes. [15-18]

Requirements for Encapsulation:

            Variety of bioactive agents (5000-600,000 Daltons in size) can be entrapped in erythrocytes. Once encapsulated, charged molecules are retained longer than uncharged molecules. Both polar and non-polar molecules have been successfully entrapped. Sucrose is used as a marker for encapsulation studies. Hydrophobic molecules may be entrapped by absorbing over other molecules while non-polar molecules may be entrapped in their respective salts. Moreover, if the molecules interact with membrane and cause deleterious effect on membrane structure then that molecule should not be

 DIFFERENT METHODS OF DRUG LOADING:

These methods have been employed for drug loading in erythrocytes:

1. Electron-insertion/Electro encapsulation method

2. Hypo-osmotic lysis method

a) Dialysis method

b) Dilution method

c) Isotonic osmotic lysis method

d) Preswell method

3. Membrane perturbation Method

4. Endocytosis method

5. Lipid fusion method

considered for encapsulation in erythrocytes

1. Electron-insertion/Electro encapsulation method:

It is often referred to as the "electroporation method," which relies on transient electrolysis to build pores that produce the desired membrane permeability for drug loading into erythrocytes. Erythrocytes are suspended in an isotonic buffer within an electrical discharge chamber. It has a capacitor in an external circuit that generates a square-wave potential by being charged to a specific voltage and then discharged through cell suspension in a specific amount of time. It was utilised effectively in 1980 to entrap the anti-cancer medication daunomycin in human and animal erythrocytes. This technique also causes permanent damage to the cell membrane and is hence

2. Hypo-osmotic lysis method

This technique relies on RBCs' capacity to float in isotonic saline solution. Osmotic lysis results from the exchange of intracellular and extracellular RBCs that occurs as a result of resealing. Ultimately, the mediation was enclosed in RBC. not very common. [19-21

a)      Dialysis Technique

This method was used by Klibansky to entrap proteins, then Jarde modified it (Muldoon et al 1987). A proper haematocrit is first created by mixing a medication with a suspension of red blood cells, which is then collected in dialysis tubes.

Air bubbles are used to inflate the inner side volume, which is subsequently shut, leaving just 75% of the inner side volume covered by the suspension of RBC. The function of the air bubble is crucial. The dialysis tubes are put in a bottle with 100ml of swelling solution, which is then shaken at 4O°C, to ensure thorough mixing. The dialysis tube is then placed in a sealing solution (100 ml) at 20 to 30°C, washed with a cold phosphate buffer solution at 40°C, and then resuspended in the phosphate buffer solution once more. Entrapment efficiency for this technique is 30–50%, and recovery efficiency is 70–80%. [22]

b) Dilution Method

It contains RBC that have been placed in 0.4% NaCl (a hypotonic solution), which causes them to rupture and allow the contents of the cells to escape before cell enlargement occurs. 6 times the weight of their initial size). This swelling enables the development of pores with a diameter of 200–500. (Ihler et al. 1987). Treat 1 volume of RBC with 2-20 volume of solution to bring intracellular and extracellular concentrations into balance while letting them to load in hypotonic solution at 0° C for 5 minutes. By combining hypertonic solution, it is possible to restore the solution's tonicity. This technique is used quickly to low molecular weight medicines. Its 1-8% entrapment efficiency is extremely low. The dilution approach can be used with bronchodilators like salbutamol and enzymes like arginase (Adriaenssens et al. 1976), asparaginase (Updike et al. 1983), -galactosidase, and -glucosidase. [23-26]

c) Isotonic osmotic lysis method

This procedure uses polyethylene glycol and is dependent on the transitory permeability of the RBC membrane (Billah et al. 1977). Drugs that are physiologically active diffuse throughout the environment to preserve homeostasis. RBCs are resealed after being cultured in an isotonic solution. This procedure uses polyethylene glycol, ammonium chloride, and urea solution for isotonic osmotic lysi and is suitable for very tiny compounds. [27]

d) Preswell method

This approach was first employed by Rechesteiner (Rechesteiner M. C. 1975), and then Pitt et al. modified it (Pitt et al. 1983). Included in this is the basic enlargement of RBC in a mildly hypotonic solution. Centrifugation was used to recover the swollen cells, and a small amount of the drug's aqueous solution was added until lysis occurred. The best retention of cellular content is achieved when cell swelling occurs gradually. This process is straightforward and creates an RBC carrier. [28,29]

3. Membrane perturbation method:

The permeability of the erythrocytic membrane rises when cells are exposed to polyene antibiotics like ampho- tericin B, which is based on how the membrane permeability of erythrocytes increases when the cells are exposed to particular substances. In 1980, Kitao and Hattori employed this technique to entrap the anti-cancer medi- cation daunomycin in human and animal erythrocytes. They also used halothane for the same purpose. Unfortunately, due to the irrevocable detrimental changes that these approaches have caused in the cell membrane, they are not widely used. [30]

4. Endocytosis method:

In 1975, Schrier reported it. For endocytosis, nine volumes of buffer containing 2.5 mm ATP, 2.5 mm MgCl₂, and 1 mm CaCl₂ are combined with one volume of washed packed erythrocytes before being incubated for two minutes at room temperature. By adding 154 mm of NaCl and incubating the pores produced by this procedure for 2 minutes at 37°C, the pores are resealed. Endocytosis material is kept apart from the cytoplasm by the vesicle membrane, which also shields it from erythrocytes. This method makes use of a number of medications, including vinblastine, chlorpromazine, phenothiazine, hydrocortisone, tetracaine, and vitamin A5. [31,32]

5. Lipid fusion method:

Direct fusion of drug-containing lipid vesicles with human erythrocytes can result in the exchange of a drug-entrapped in lipid. To increase the ability of cells to carry oxygen, inositol hexaphosphate was entrapped into resealed erythrocytes using this method. The entrapment efficacy of this approach, however, is incredibly low (1%). [33,34]

Applications of resealed erythrocytes:

Applications of resealed erythrocytes 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 [35]

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.

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.

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 either nucleotides or nucleoside analogues, their entrapment and exit through the membrane needs careful consideration. Nucleosides are rapidly transported across the membrane whereas nucleotides are not, and thus exhibiting prolonged release profiles. The release of nucleotides requires conversion of these moieties to purine or pyrimidine bases.

Enzyme therapy:

Enzymes are widely used in clinical practices replacement therapies to treat diseases associated with their deficiency (e.g., Gaucher’s disease, galactosuria), degradation of toxic compounds 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 haemolysis; act as a “circulating bioreactors” in which substrates enter into the cell, interact with enzymes, and generate products; or accumulate enzymes in RES upon haemolysis for future catalysis.

Improvement in oxygen delivery to tissues:

            Haemoglobin is the protein responsible for the oxygen-carrying capacity of erythrocytes. Under normal conditions, 95% of haemoglobin is saturated with oxygen in the lungs, whereas under physiologic conditions in peripheral blood stream only 25% of oxygenated haemoglobin becomes deoxygenated. Thus, the major fraction of oxygen bound to haemoglobin is recirculated with venous blood to the lungs. The use of this bound fraction has been suggested for the treatment of oxygen deficiency. 2, 3-Diphosphoglycerate (2, 3-DPG) is a natural effector of haemoglobin. The binding affinity of haemoglobin for oxygen changes reversibly with changes in intracellular concentration of 2, 3-DPG. [36-41]

In-vitro characterization of Resealed Erythrocytes:

These characterizations are important to ensure them in-vivo performance and therapeutic benefits.

I. Drug Content Determination:

Method: 0.5ml packed loaded erythrocytes are deproteinized with acetonitrile (2 ml) and then centrifuged at 2500 rpm for 10 minutes. Now the clear supernatant liquid is analysed for drug content.

II. In-vitro drug release and Hb content: Both these properties are monitored periodically from drug loaded cells.

Method:

The cell suspension (5% Haematocrit in Phosphate buffer saline) is stored at 40°C in amber coloured glass containers. Periodically the clear supernatant is withdrawn using a hypodermic syringe equipped with 0.45µ filter, deproteinized with methanol and then estimated for drug content. The supernatant of each sample after centrifugation is collected and assayed. Hence, % Hb (Haemoglobin) release is calculated. % Hb release= A540 of sample – A 540 of background A 540=Absorbance at 540 nm, A 540 of 100% Hb

Mean Corpuscular Hb = Hb (g/100 ml) × 10

 Erythrocyte count (millions/cu mm)

III. Percent cell recovery: It is determined by counting the number of intact cells per cubic mm of packed erythrocytes before and after loading the drug.

IV. Morphology: Following types of microscopy are used for the morphological study of normal and drug loaded erythrocytes:

·         Phase contrast microscopy

·         Electron microscopy

V. Osmotic fragility: This method is based on resistance of cells to haemolysis in decreasing concentration of hypotonic saline. It is a reliable parameter for:

·         In-vitro evaluation of carrier erythrocytes with respect to shelf life

·         In-vivo survival of erythrocytes

·         Study of effect of the encapsulated substances

·         For stimulating and mimicking the bioenvironmental conditions that are encountered on in-vivo administration.

Method:

Normal and drug - loaded erythrocytes are incubated separately in stepwise decreasing % of NaCl solution (0.9%) at 37oC±2oC for 10 minutes, followed by centrifugation at 2000 rpm for 10 minutes. Then the supernatant liquid is examined for drug and haemoglobin content.

VI. Osmotic shock:

This is used to describe a sudden exposure of drug loaded erythrocytes to an environment, which is far from isotonic so as to evaluate the ability of resealed erythrocytes to withstand the stress and maintain their integrity as well as appearance.

Method: Erythrocyte suspension (10% haematocrit,1 ml) was diluted with distilled water (5 ml) and centrifuged at 300 rpm for 15 minutes. Supernatant was estimated for % Hb release spectrophotometrically.

VII. Turbulence shock: This parameter indicates the effects of shear and pressure, by which resealed erythrocyte formulations are injected, on the integrity of the loaded cells. Drug loaded erythrocytes appear to be less resistant to turbulence because resealing of erythrocytes makes them sensitive towards turbulence/ Mechanical agitation and hence estimation of turbulence shock study provides their expected performance in-vivo.

Method: Loaded erythrocytes (10% haematocrit,5 ml) are passed through 23-gauge hypodermic needle at a flow rate of 10 ml/minute (which is comparable to the flow rate of blood). After every pass, aliquot of suspension is withdrawn and then centrifuge at 2000 rpm for 10-15 minutes. Now the Hb content is estimated spectrophotometrically.

VIII. Determination of entrapped Magnetite: Resealed erythrocytes are entrapped with magnetite to make them Magnoresponsive.

Method:

Magnetite bearing erythrocytes and Hydrochloric acid are heated at 60ºC for 2 hours. Now 20% w/v trichloroacetic acid is added. Centrifugation is done and supernatant is examined for Magnetite concentration using atomic absorption spectroscopy.

IX. Erythrocyte Sedimentation Rate (ESR): ESR is the estimation of suspension stability of RBC in plasma and is related to:

·         Number and size of red cells.

·         The relative concentration of plasma proteins (especially fibrogen, alpha and beta globulins) This test is performed by determining the ESR of blood cells in a standard tube of ESR apparatus. Higher rate of ESR is indication of active but obscure disease processes. The normal blood ESR is found to be 0 to 15 mm/hour.

X. The Zeta Sedimentation Ratio: It is based on a measure of the closeness with which RBC’s will approach each other after standardized cycles of dispersion and compaction.

XI. Miscellaneous: Lipid composition, Membrane fluidity, rheological properties, density gradient separation, energy metabolism, Biological characterization (sterility test using aerobic and anaerobic cultures, Pyrogenicity using rabbit fever response or LAL test, animal toxicity study

In vitro storage

The success of resealed erythrocytes as a drug delivery system depends to a greater extent on them 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 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 dime- thyl sulfoxide, dimethyl,3,3-di-thio-bispropionamide, glutaraldehyde, toluene-2-4-diisocyanate followed by lyophilisation 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 gelatin 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.

The various methods used to determine in vivo survival time include labelling of cells by 51Cr or fluorescent markers such as fluorescein is thiocyanate or entrapment of 14C sucrose or gentamicin. The circulation survival kinetics of resealed erythrocytes show typical bimodal behaviour with a rapid loss of cells during the first24 h after injection, followed by a slow decline phase with a half-life on the order of days or weeks. The early loss accounts for 15–65% loss of total injected cells. The erythrocytic carriers constructed of red blood cells of mice, cattle, pigs, dogs, sheep, goats, and monkeys exhibit a comparable circulation profile with that of normal unloaded erythrocytes. On the other hand, resealed erythrocytes prepared from red blood cells of rabbits, chickens, and rats exhibit relatively poor circulation profile. [42-52]

CONCLUSION:

In this paper, various 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. The Resealed erythrocytes had also been employed for effective delivery of numerous drugs as depicted by many researchers for the treatment of cancer, tumour, and arthritis and also for effective treatment of the poisoning. However, in near future, erythrocytes based delivery system with their ability to provide controlled and site specific drug delivery will revolutionize in effective treatment of various disease. For the present, it is concluded that erythrocyte carriers are “Nano Device in field of Nanotechnology” considering their tremendous potential and prospective.

Future perspectives:

Following are some future perspectives of Resealed Erythrocytes:

·         A large amount of valuable work is needed so as to utilize the potentials of erythrocytes in passive as well as active targeting of drugs.

·         Diseases like cancer could surely find its cure.

·         Genetic engineering aspects can be coupled to give a newer dimension to the existing cellular drug carrier concept.

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