Principles and Methods of Bioassay

Bioassay is defined as the estimation of the potency of an active principle in a unit quantity of preparation or detection and  easurement of the concentration of the substance in a preparation using biological methods (i.e. observation of pharmacological effects on living tissues, microorganisms or  immune cells or animal). Hence micro bioassay, radioimmunoassay are also regarded as `bioassay'. Recently `biotechnology' has also been considered for bioassay. Bioassay of the products like erythropoietin, hepatitis-B vaccine etc. is being done through biotechnology. 

 Importance of Bioassay

Bioassays, as compared to other methods of assays (e.g. chemical or physical assay) are less accurate, less elaborate, more laborious, more troublesome and more expensive. However, bioassay is the only method of assay if

(1) Active principle of drug is unknown or cannot be isolated, e.g. insulin, posterior pituitary extract etc. 

(2) Chemical method is either not available or if available, it is  too complex and insensitive or requires higher dose e.g. insulin, acetylcholine.

(3) Chemical composition is not known, e.g. long acting thyroid stimulants. 

(4) Chemical composition of drug differs but have the same pharmacological action and vice-versa, e.g.  cardiac glycosides, catecholamines etc.

Moreover, even if chemical methods are available and the results of bioassay conflict with those of the chemical assay, the bioassay is relied upon and not the chemical assay, since it is the assessment on living organism.

The purpose of bioassay is to ascertain the potency of a drug and hence it serves as the quantitative part of any screening procedure (Research). Other purpose of bioassay is to standardize the preparation so that each contains the uniform specified pharmacological activity. In this way, it serves as a pointer in the Commercial Production of drugs when chemical assays are not available or do not suffice. From the  clinical  point of view, bioassay may help in the diagnosis of  various conditions, e.g. gonadotrophins for pregnancy. 

Principle of Bioassay  

The basic principle of bioassay is to compare the test substance with the International Standard preparation of the same and to find out how much test substance is required to produce the same biological effect, as produced by the standard. The standards are internationally accepted samples of drugs maintained and recommended by the Expert Committee of the Biological Standardization of W.H.O. They represent the fixed units of activity (definite weight of preparation) for drugs. In India, standard drugs are maintained in Government institutions like Central Drug Research Institute, Lucknow, Central Drug Laboratory, Calcutta, etc.

The problem of biological variation must be minimized as far as possible. For that one should keep uniform experimental conditions and assure the reproducibility of the responses. 

Methods of Bioassay for Agonists 

An agonist may produce graded response or quantal response. Graded response means that the response is proportional to the dose and response may lie between no response and the maximum response. By quantal, it is meant that the response is in the form of "all or none", i.e. either no  response or maximum response. The drugs producing quantal effect can be bioassayed by end point method. The drugs producing graded responses can be bioassayed by 

(1) Matching or bracketing method or (2) Graphical method.

1. End Point Method: Here the threshold dose producing a positive effect is measured on each animal and the comparison between the average results of two groups of animals (one receiving standard and other the test) is done. e.g. bioassay of digitalis in cats. Here the cat is anaesthetized with  chloralose and its blood pressure is recorded. The drug is slowly infused into the animal and the moment the heart stops beating and blood pressure falls to zero, the volume of fluid infused is noted down. Two series of such experiments-one using standard digitalis and the other using test preparation of digitalis is done and

then potency is calculated as follows: 
                                      
 Conc. of Unknown = Threshold dose of the Standard X Conc. of Std.                                                   Threshold dose of the Test 

       In case, if it is not possible to measure individual effective dose or if animals are not available, fixed doses are injected into groups of animals and the percentage of mortality at each dose level is determined. The percentage of mortality is taken as the response and then the comparison is done in the same way as done for graded response. 

2. Matching Method: In this method a constant dose of the test is brack doses of standard till the exact match is obtained between test dose and the standard dose.
Initially, two responses of the standard are taken. The doses are adjusted such that one is giving response of approximately 20% and other 70% of the maximum. The response of unknown which lies between two responses of standard dose is taken. The panel is repeated by increasing or decreasing the dose s of standard till all three equal responses are obtained. The dose of test sample is kept constant. At the end, a response of the double dose of the standard and test which match each other are taken. These should give equal responses. Concentration of the test sample can be determined as follows:

                                       Dose of the Standard
Conc. of Unknown =   -------------------------- X Conc. of Std.
                                          Dose of the Test 

This method has following limitations:

1.  It occupies a larger area of the drum as far as tracings are concerned.

2.  The match is purely subjective, so chances of error are there and one cannot determine them.

3.  It does not give any idea of dose-response relationship.
 

However, this method is particularly useful  if the sensitivity of the preparation is not stable. Bioassay of histamine, on guinea pig  ileum is preferably  carried out by this method.

3. Graphical method:  This method is based on the assumption of the dose-response relationship. Log-dose-response curve is plotted and the dose of standard producing the same response as produced by the test sample is directly read from the graph. In simpler design, 5-6 responses of the graded doses  of the standard are  taken and then two equiactive responses of the test sample are taken. The height of contraction is measured and plotted against the log-dose. The dose of standard producing the same response as produced by the test is read directly from the graph and the concentration of test sample is determined by the same formula as mentioned before. 

 

The characteristic of log-dose response curve is that it is linear in the middle (20-80%). Thus, the comparison should be done within this range only. In other words, the response of test sample must lie within this range. 

Advantage of this method is that, it is a simple method and chances of errors are less if the sensitivity of the preparation is not changed. Other methods which are based on the dose-response relationship include 3 point, 4 point, 5 point and 6 point methods. In these  methods, the responses are repeated several  times and the mean of each is taken. Thus, chances of error are minimized in these methods. In 3 point assay method 2 doses of the standard and one dose of the test are used. In 4 point method 2 doses of standard and 2 doses of the test are used. In 6 point method 3 doses of standard and 3 doses of the test are used. Similarly one can design 8 point method also. The sequence of responses is followed as per the Latin square method of randomization in order to avoid any bias. 

 The mean responses are calculated and plotted against log-dose and amount of standard producing the same response as produced by the test is determined graphically as well as mathematically:

 

n1 = Lower Standard dose                               
n2 = Higher Standard dose
t    = Test dose
S1 = Response of n1
S2 = Response of n2
T   = Response of test (t)
Cs = Concentration of standard 

Similarly, in 4 point method, amount of standard producing the same response as produced by the test can be determined by graphical method. It is determined mathematically as follows: 

 

t1 = lower dose of test; t2 = higher dose of test; T1 = response of t1; T2 = response of t2. 

Bioassay of Antagonists

Commonly used method for the bioassay of antagonist is simple graphical method. The responses are determined in the form of the percentage inhibition of the fixed dose of  agonist. These are then plotted against  the log dose of the antagonist and the concentration of unknown is determined by finding out the amount of standard producing the same effect as produced by the test.

In this method, two responses of the same dose of agonist  (sub maximal giving approximately 80% of the maximum response) are taken. The minimum dose of standard antagonist is added in the bath and then the response of the same dose of agonist is taken in presence of antagonist. The responses of agonist are repeated every ten min till recovery is obtained. The higher dose of standard antagonist is added and responses are taken as before. Three to four doses of the standard antagonist are used and then one to two doses of test sample of the antagonist is used similarly. The percentage inhibition is calculated, plotted against log dose of antagonist and the concentration of unknown is determined as usual. 

Bioassay of Some Important Drugs 

 Depending upon pharmacological action of various drugs, different preparations may be used. Following chart gives different preparations and the pharmacological activity for which a particular drug is assayed: 

 

Shampoos

Introduction : 

Definition: A shampoo is a preparation of a surfactant (i.e. surface active material) in a suitable form – liquid, solid or powder – which when used under the specified conditions will remove surface grease, dirt, and skin debris from the hair shaft and scalp without adversely affecting the user.

Requirements of a Shampoo:

•  It should effectively and completely remove dust or soil, excessive sebum or other fatty substances and loose corneal cells from the hair.
•  It should produce a good amount of foam to satisfy the psychological requirements of the user
• It should be easily removed on rinsing with water.
•  It should leave the hair non-dry, soft, lustrous with good manageability and minimum fly away.
•   It should impart a pleasant fragnance to the hair.
•  It should not cause any side-effects / irritation to skin or eye. It should not make the hand rough and chapped.

Types of Shampoo : 

• ·         Powder Shampoo
• ·         Liquid Shampoo
• ·         Lotion Shampoo
• ·         Cream Shampoo
• ·         Jelly Shampoo
• ·         Aerosol Shampoo
• ·         Specialized Shampoo
• ·         Conditioning Shampoo
• ·         Anti- dandruff Shampoo
• ·         Baby Shampoo
• ·         Two Layer Shampoo

PRODUCT INGREDIENTS : 

Surfactants are the main component of shampoo. Mainly anionic surfactants are used.

The raw materials used in the manufacture of shampoos are:

Principal surfactants: Provide detergency and foam.

Secondary surfactants: Improve detergency, foam and hair condition.

Other additives. CLEANSING ACTION OF SHAMPOO A surfactant consists of two part- one hydrophilic (water loving) while the other is hydrophobic in nature.

Surfactants : 

Surfactants Anionic surfactants are mostly used (good foaming properties). The hydrophilic portion carries a negative charge which results in superior foaming, cleaning and end result attributes. Non-ionic surfactants have good cleansing properties but do not have sufficient foaming power. Cationic surfactants are toxic and are hence not used. However, they may be used in low concentration in hair conditioners. Ampholytics, being expensive, are generally not used. However, they are mainly used as secondary surfactants and good hair conditioners.

ADDITIVES : 

Conditioning agents: Lanolin, mineral oil, herbal extracts, egg derivatives.

Foam builders: Lauroyl monoethanolamide, sarcosinates

Viscosity modifiers : Electrolytes – NH4Cl, NaCl Natural gums – Gum Karaya, tragacanth, alginates Cellulose derivatives – Hydroxy ethyl cellulose, methyl cellulose Carboxy vinyl polymers – Carbopol 934 Others – PVP, phosphate esters.

Sequestering agents: EDTA

Opacifying agents: Alkanolamides of higher fatty acids, propylene glycol, Mg, Ca and Zn salts of stearic acid, spermaceti, etc.

Clarifying agents: Solubilizing alcohols – ethanol, isopropanol Phosphates – Non-ionic solubilizers – polyethoxyated alcohols and esters.

Perfumes : Herbal, fruity or floral fragnances.

Preservatives : Methyl and propyl paraben, formaldehyde (most effective).

Anti-dandruff agents: The shampoos contain small amount of these actives, which are in contact with the scalp for only a short time. In order to be effective the active ingredient must work in the oil-water environment of the scalp and must be readily substantive to the scalp for continuing activity.

Eg: Selenium sulfide, zinc pyrithone, salicylic acid.

Evaluation of Shampoos : 

• ·         Evaluation of Shampoos Performance characteristics
• ·         Foam and foam stability
• ·         Detergency and cleaning action
• ·         Effect of water hardness
• ·         Surface Tension and wetting
• ·         Surfactant content and analysis
• ·         Rinsing Conditioning action Softness Luster Lubricity Body, texture and set retention
• ·         Irritation and toxicity
• ·         Dandruff control
• ·         Microbiological assay
• ·         Eye irritancy test
• ·         Product characteristics
• ·         Fragnance
• ·         Colour
• ·         Consistency
• ·         Package

1.Foam and foam stability: The Ross-Miles foam column test is accepted. 200 ml of surfactant solution is dropped into a glass column containing 50ml of the same solution. The height of the foam generated is measured immediately and again after a specified time interval, and is considered proportional to the volume. Barnett and Powers developed a latherometer to measure the effect of variables such as water hardness, type of soil and quantity of soil on foam speed, volume and stability. Fredell and Read titrated actual standard oiled heads of hair with additive increments of shampoo until a persistent lather end point appeared.

2.Detergency and cleaning action: Cleansing power is evaluated by the method of Barnet and Powers 5gm sample of soiled human hair is placed at 35°c in 200 cc of water containing of 1 gm of shampoo. The flask is shaken 50 times a minute for 4 minutes. Then washed once again with sufficient amount of water, then after filter the hair dried and weighed. The amount of soil is removed under these condition is calculated.

3. Wetting Action: Canvas disk sinking test: A mount veron cotton duck # 6 canvas disk 1 inch in diameter, is floated on the surface of a solution, and the time required for it to sink is measured accurately.

 4. Rinsing: Skilled beauticians are employed to make comparisons on the performance of several shampoos.

5. Conditioning Action: Conditioning action is a difficult property to assess. This is because it is basically dependent on subjective appraisal. No method has been published for measuring conditioning action. The degree of conditioning given to hair is ultimately judged by shampoo user who is making the evaluation on the basis of past experience and present expectations.

6. Microbiological assay: PREPARATION OF PRE-INOCULUM Take the loopful culture of staphylococcus aureus (ATCC6532) aseptically and transfer to sterilized and cooled 100 ml SCDM (broth). Mix well. Incubate the broth at 37oC for 24 hrs. PREPARATION OF MEDIA Soya bean casein digests medium, soya bean casein digest agar and nutrient agar. PREPARATION OF POUR PLATES Sterilized SCD agar (100 ml) is cooled to 40°C and mixed with 5 ml of 24 hrs old pre inoculated culture. This is immediately poured in plates (340 ml each) and allows to set. MAKING THE WELLS ON AGAR PLATES The wells are dig on agar plates with sterilised well digger aseptically. Take 100µml of each sample, add to well aseptically. Incubate the plates at 37oC for 24 hrs to 48 hrs. Observe the effectiveness of sample on culture growing on the agar plate and we can see the effectiveness of sample in the form of zone of inhibition around each well containing different sample.

7. Evaluation of eye irritancy: The test calls for dropping 0.1 ml of liquid shampoo in the conjunctiva sac of one eye of the rabbit , the other eye serving as control. In the case of the first three animals, the treated eye remains unwashed. Since washing the eye may or may not alleviate symptoms of injury. The six remaining animals are divided into two equal groups. In the first of these groups eyes instilled with the substances are washed with 20 ml of lukewarm water two seconds after treatment and in the second group after instillation. Readings are then made at 24, 48 and 72 hr and again four and seven days after treatment. If the lesions have not cleared up in seven days the test material is considered as severe irritant.

8. Viscosity: Viscosity of the liquid shampoo is determined using a Brookefield viscometer 100 mL of the shampoo is taken in a beaker and the spindle is dipped in it for about 5 min and then the reading is taken.

9.Oral toxicity  

1.        Oral toxicity can be given in terms of its lethal dose 50 ( LD/50 ) i.e., number if gms of the material per kg of body weight required to kill half of the test animals used.

2.       Fasting cage animals are taken & dosing is accomplished with the help of stomach tube.

3.       Lower the LD/50 the greater the toxicity

10.Test for pH

1.      Soap based shampoos are more effective in pH of 9.0-10.0.

2.       Synthetic detergent based shampoos are effective pH range of 6.0-9.0.

3.       pH can be measured with the help of pH meter

11.Skin irritation tests

Draize Test in rabbits

·         A set of 6 rabbits is used for testing each material.

·         Shampoos should be tested only for a short duration, that is, not more than 4 hrs as these products come in contact with the skin only for a short duration.

RESEALED ERYTHROCYTES

INTRODUCTION:
Erythrocytes, the most abundant cells in the human body, have potential carrier capabilities for the delivery of drugs. Erythrocytes are bio compatible, 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 a remarkable degree of interest in recent years. Biopharmaceuticals are among the most widely exploited candidates for being delivered to the host body using these cellular carriers. In this review, the potential applications of erythrocytes in drug delivery have been highlighted.

ISOLATION OF ERYTHROCYTES: 

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 venipuncture. 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. To isolate erythrocytes, blood is collected in heparinized tubes by venipuncture.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 4⁰c 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 hematocrit values as desired and are often stored in acid–citrate–dextrose buffer at  4⁰ c as long as 48 h before use. Jain and Vyas have described a well-established protocol for the isolation of erythrocytes. The loading of drugs in erythrocytes was reported separately by Ihler et al. and Zimmermann. In 1979, the term carrier erythrocytes were coined to describe drug-loaded erythrocytes.

Advantages and disadvantages of erythrocytes in drug delivery Advantages

Some of the most important advantages encouraging the use of erythrocytes in

drug delivery include:

• A remarkable degree of biocompatibility, particularly when the autologous cells are used for drug loading.
• Complete biodegradability and the lack of toxic product(s) resulting from the carrier   biodegradation.
• Avoidance of any undesired immune responses against the encapsulated drug.
• Considerable protection of the organism against the toxic effects of the encapsulated drug, e.g. antineoplasms.
• Remarkably longer life-span of the carrier erythrocytes in circulation in comparison to the 
• synthetic carriers. In the optimum condition of the loading procedure, the life-span of the 
• resulting carrier cells may be comparable to that of the normal erythrocytes.
• An easily controllable life-span within a wide range from minutes to months.
•  Desirable size range and the considerably uniform size and shape.
•  Protection of the loaded compound from inactivation by the endogenous factors.
•  Possibility of targeted drug delivery to the RES organs.
•  Relatively inert intracellular environment.
•  Availability of knowledge, techniques, and facilities for handling, transfusion, and working
• with erythrocytes. 
• Possibility of ideal zero-order kinetics of drug release.
•  Wide variety of compounds with the capability of being entrapped within the erythrocytes.
•  Possibility of loading a relatively high amount of drug in a small volume of erythrocytes,
• which, in turn, assures the dose sufficiency in clinical as well as animal studies using a limited
• volume of erythrocyte samples.
• Modification of the pharmacokinetic and pharmacodynamic parameters of the drug.
• Remarkable decrease in concentration fluctuations in steady state in comparison to the
•  conventional methods of drug administration, which is a common advantage for most of the novel drug delivery systems.
• Considerable increase in drug dosing intervals with drug concentration in the safe and effective level for a relatively long time.
• Possibility of decreasing drug side effects.

Drawbacks:

The use of erythrocytes as carrier systems also presents some disadvantages, which can be

summarized as follows:

• 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 as result of modification that occurred during loading procedure in cells. This, although expands the capability to drug targeting to RES, seriously limits their life-span as long-circulating drug carriers in circulation and, in some cases, may pose toxicological problems.
• Te rapid leakage of certain encapsulated substances from the loaded erythrocytes.
• Several molecules may alter the physiology of the erythrocyte.
• Given that they are carriers of biological origin, encapsulated erythrocytes may present some inherent variations in their loading and characteristics compared to other carrier systems.
• The storage of the loaded erythrocytes is a further problem provided that there are viable cells and need to survive in circulation for a long time upon re-entry to the host body.
• Conditioning carrier cells in isotonic buffers containing all essential nutrients, as well as in low temperatures, the addition of nucleosides or chelators, lyophilization with glycerol or gel immobilization have all been exploited to overcome this problem. 
• Possible contamination due to the origin of the blood, the equipment used and the loading environment. Rigorous controls are required accordingly for the collection and handling of the erythrocytes.

METHODS OF DRUG LOADING:

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).the following are types of drug loading: Hypotonic hemolysis, hypotonic dilution, hypotonic preswelling, isotonic osmotic lysis, Chemical perturbation of the membrane. Electro-insertion or electron capsulation, Entrapment by endocytosis, loading by electric cell fusion, loading by lipid fusion.

OSMOTIC SHOCK:

For 0.5 study, erythrocyte suspension (1 ml, 10%) was diluted & centrifuge at 3000 rpm for 15 minute. The supernatant was estimated for % Hb release spectrophotometrically.

TURBULENCE SHOCK:

It is the measure of simulating distribution of loaded cells during injection. In this drug loaded cells are passed through a 23 gauge hypodermic at a flow rate of 10 ml/min which is comparable to the flow rate of blood. It is followed by collecting of an aliquot and centrifugation sample is estimated. Drug loaded erythrocytes appears to be less resistant to turbulence, probably indicating destruction of cells upon shaking.

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.

Use of red cell loader:

Magnani et al. developed a novel method 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 hemofilter 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.

Hypotonic dilution:

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. This reduces the circulation half life of the loaded cells. These cells are readily phagocytosed by RES macrophages and hence can be used for targeting RES organs. Hypotonic dilution is used for loading enzymes such as galactosidase and glucosidase, asparginase and arginase, as well as bronchodilators such as salbutamol.

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 erythrocytic 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. Lin 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.

Entrapment by endocytosis:

This methodwas reported by Schrier et al. in 1975. Endocytosis involves the additionof one volume of washed packed erythrocytesto nine volumes of buffer containing2.5 mM ATP, 2.5 mM MgCl2, and1mM CaCl2, followed by incubation for2 min at room temperature. The porescreated by this method are resealed byusing 154 mM of NaCl and incubationat 37⁰cfor 2 min. The entrapment ofmaterial occurs by endocytosis. The vesiclemembrane separates endocytosed materialfrom cytoplasm thus protecting itfrom the erythrocytes and vice-versa. Thevarious candidates entrapped by thismethod include primaquine and related8–amino–quinolines, vinblastine, chlorpromazineand related phenothiazines,hydrocortisone, propranolol and tetracaine.

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.

 Loading by lipid fusion:

Lipid vesicles containing a drug can be directly fused to human erythrocytes, which lead to an exchange with a lipid-entrapped drug. [35] This technique was used for entrapping inositol mono phosphate to improve the oxygen carrying capacity of cells. However, the entrapment efficiency of this method is very low (1%).

RELEASE CHARACTERISTICS OF LOADED DRUGS:

There are mainly three ways for a drug to efflux out from the erythrocyte carriers: phagocytosis, diffusion through the membrane of the cells and using a specific transport system. RBCs are normally removed from circulation by the process of phagocytosis. The degree of cross linking determines whether liver or spleen will preferentially remove the cells. Carrier erythrocytes following heat treatment or antibody cross-linking are quickly removed from the circulation by phagocytic cells located mainly in liver and spleen. The rate of diffusion depends upon the rate at which a particular molecule penetrates through a lipid by layer. It is greatest for a molecule with high lipid solubility.

DELIVERY STRATEGIES

As mentioned earlier, there are two major strategies in the delivery of drugs using erythrocytes as carriers which include intravenous slow drug release strategy and target gene delivery.

Intravenous slow drug release strategy:

The normal life-span of an erythrocyte in systemic circulation is about 120 days. As mentioned as an advantage, in the optimum conditions of the loading procedure, the life-span of the resulting carrier cells may be comparable to that of the normal erythrocytes. [36] Erythrocytes have been used as circulating intravenous slow-release carriers for the delivery of antineoplasms, antiparasitics, antiretroviral agents, vitamins, steroids, antibiotics and cardiovascular drugs among others.

A series of mechanisms have been proposed for drug release in circulation from carrier erythrocytes, including passive diffusion out of the loaded cells into circulation, specialized membrane-associated carriers, phagocytosis of the carrier cells by the macrophages of RES and, then, depletion of the drug into circulation, accumulation of the drug in RES upon lysis of the carrier and slow release from this system into circulation, accumulation of the carrier erythrocytes in lymphatic nodes following subcutaneous injection of the cells and drug release upon hemolysis in this sites, and, finally, hemolysis in the injection sites.

Targeted drug delivery:

RES or non-RES ‘targeting’ is another important strategy using erythrocytes as carriers.

RES targeting:

It is a well-known fact that, in physiologic conditions, as a result of the gradual inactivation of the metabolic pathways of the erythrocyte by aging, the cell membrane loses its natural integrity, flexibility and chemical composition. These changes, in turn, finally result in the destruction of these cells upon passage through the spleen. The other effective site for the destruction of the aged or abnormal erythrocytes is the macrophages of the RES including peritoneal macrophages, hepatic Kupffer cells and alveolar macrophages of the lung, peripheral blood monocytes, and vascular endothelial cells. We know that aging and a series of other factors (e.g., stress during non-gentle loading methods) make the erythrocytes recognizable by the phagocyting macrophages via changing the chemical composition of the erythrocyte membrane, i.e., the phospholipids component. Therefore, a considerable fraction of carrier erythrocytes that have undergone some degrees of structural changes during the loading procedure will be trapped by the RES organs, mainly the liver and spleen, within a short time period after re-injection.

A series of approaches have been evaluated to improve RES targeting using carrier erythrocytes. In one of these approaches, the drug-loaded erythrocytes have been exposed to membrane stabilizing agents. This may increase the targeting index of the erythrocytes to RES via decreasing the deformability of these cells.

Non-RES targeting:

Recently, carrier erythrocytes have been used to target organs outside the RES. The various approaches include:

• Co-encapsulation of paramagnetic particles or photosensitive agents in erythrocytes alongwith the drug to be targeted; 
• Application of ultrasound waves; 
• Site-specific antibody attachment to erythrocyte membrane.Chiarantini et al. have reported in vitro targeting of erythrocytes to cytotoxic T-cells by coupling them to Thy-1.2 monoclonalantibody. Price et al. reported the delivery of colloidal particles and erythrocytes to tissuethrough micro vessel ruptures created by targeted micro bubble destruction with ultrasound. 
• In another study, the differential response of photosensitized young and old erythrocytes to photodynamic activation has been studied by Rollan.

 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.

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.

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 byhemolysis to release the drug.

 Routes of administration include intravenous, which is the most common, followed by subcutaneous, intraperitoneal, intranasal, and oral. Studies regarding the improved efficacy of various drugs given in this form in animal models have been reported. Examples include an enhancement in anti-inflammatory effect of corticosteroids in experimentally inflamed rats, increase in half life of isoniazid and levothyroxine.

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.

Treatment of hepatic tumors:

Hepatic tumors are one of the most prevalent types of cancer. Antineoplastic drugs such as methotrexate, bleomycin has been successfully delivered by erythrocytes. Agents such as daunorubicin diffuse rapidly from the cells upon loading and hence pose a problem. This problem can be overcome by covalently linking daunorubicin to the erythrocytic membrane using gluteraldehyde as a spacer. The resealed erythrocytes loaded with carboplatin show localization in liver.

Treatment of parasitic diseases:

The ability of resealed erythrocytes to selectively accumulate within RES organs make them useful tool during the delivery of antiparasitic 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 antimalarial, antileishmanial and antiamoebic drugs.

Removal of RES iron overload

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.

Removal of toxic agents:

Cannon et al. reported inhibition of cyanide intoxication with murine carrier erythrocytes containing bovine rhodanase and sodium thiosulfate. Antagonization of organophosphorus intoxication by resealed erythrocytes containing a recombinant phosphodiestrase also has been reported.

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. 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. Resealed erythrocytes have been used to deliver deoxycytidine derivatives, recombinant herpes simplex virus type 1 (HSV-1) glycoprotein B, azidothymidine derivatives, azathioprene, acyclovir, and fludarabine phosphate.

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 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 hemolysis act as a “circulating bioreactors” in which substrates enter into the cell, interact with enzymes, and generate products or accumulate enzymes in RES upon hemolysis for future catalysis.

The most important application of resealed erythrocytes in enzyme therapy is that of asparginase loading for the treatment of pediatric neoplasm. This enzyme degrades aspargine, an amino acid vital for cells. This treatment prevents remission of pediatric acute lymphocytic leukemia. There are reports of improved intensity and duration of action in animal models as well as humans. Other enzymes used for loading resealed erythrocytes include urease, galactose-1-phosphate uridyl transferase, uricase, and acetaldehyde dehydrogenase.

Improvement in oxygen delivery to tissues:

Hemoglobin is the protein responsible for the oxygen-carrying capacity of erythrocytes. Under normal conditions, 95% of hemoglobin is saturated with oxygen in the lungs, whereas under physiologic conditions in peripheral blood stream only 25% of oxygenated hemoglobin becomes deoxygenated. Thus, the major fraction of oxygen bound to hemoglobin 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 hemoglobin. The binding affinity of hemoglobin for oxygen changes reversibly with changes in intracellular concentration of 2, 3-DPG. This compensates for changes in the oxygen pressure outside of the body, as the affinity of 2, 3-DPG to oxygen is much higher than that of hemoglobin.

Microinjection of macromolecules:

Biological functions of macromolecules such as DNA, RNA, and proteins are exploited for various cell biological applications. Hence, various methods are used to entrap these macromolecules into cultured cells (e.g., microinjection). A relatively simple structure and a lack of complex cellular components (e.g., nucleus) in erythrocytes make them good candidates for the entrapment of macromolecules. In microinjection, erythrocytes are used as microsyringes for injection to the host cells. The microinjection process involves culturing host eukaryotic cells in vitro. The cells are coated with fusogenic agent and then suspended with erythrocytes loaded with the compound of interest in an isotonic medium. Sendai virus (hemagglutinating virus of Japan, HVJ) or its glycoproteins or polyethylene glycol have been used as fusogenic agents. The fusogen causes fusion of co-suspended erythrocytes and eukaryotic cells. Thus, the contents of resealed erythrocytes and the compound of interest are transferred to host cell. This procedure has been used to microinject DNA fragments, proteins, nucleic acids to various eukaryotic cells.

Advantages of this method include quantitative injection of materials into cells, simultaneous introduction of several materials into a large number of cells, minimal damage to the cell, avoidance of degradation effects of lysosomal enzymes, and simplicity of the technique. Disadvantages include a need for a larger size of fused cells, thus making them amenable to RES clearance, adverse effects of fusogens, and unpredictable effects on cell resulting from the co- introduction of various components. Hence, this method is limited to mainly cell biological applications rather than drug delivery.

Other applications of resealed erythrocytes include

•  surface modification with antibodies 
• surface modification with gluteraldehyde 
• surface modification with carbohydrates such as sialic acid 
• entrapment of paramagnetic particles along with the drug 
• Entrapment of photosensitive material 
• antibody attachment to erythrocyte membrane to get specificity of action

 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 macromolecular drugs.

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 gluteraldehyde spacer. This complex was more active than free daunorubicin alone.

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.