LOADED ERYTHROCYTE: A REVIEW ARTICLE › admin › assets › article_issue › 1504170020.pdf · LOADED ERYTHROCYTE: A REVIEW ARTICLE Snehaprabha Warule*, Jayant Bidkar Shital Bidkar

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Warule et al. World Journal of Pharmaceutical Research

LOADED ERYTHROCYTE: A REVIEW ARTICLE

Snehaprabha Warule*, Jayant Bidkar Shital Bidkar and Ganesh Dama

Sharadchandra Pawar College of Pharmacy, Otur, Pune, Maharashtra.

ABSTRACT

Erythrocytes have been the most interesting carrier and have found to

possess great potential in drug targeting. Resealed erythrocytes are

gaining more popularity because of their ability to circulate throughout

the body, biocompatibility, zero order release kinetics, reproducibility

and ease of preparation. Most of the resealed erythrocytes used as drug

carriers are rapidly taken up from blood by macrophages of

reticuloendothelial system (RES), which is present in liver, lung, and

spleen of the body. The aim of the present review is to focus on the

various features, drug loading technology and biomedical application

of resealed erythrocytes.

KEYWORDS: Resealed erythrocytes, Drug carriers, Macrophages.

1. INTRODUCTION

Erythrocytes, also known as red blood cells (RBC), have been extensively studied for their

potential carrier capabilities for the delivery of drugs and drug-loaded microspheres.[1–3]

Such

drug-loaded carrier erythrocytes are prepared simply by collecting blood samples from the

organism of interest, separating erythrocytes from plasma, entrapping drug in the

erythrocytes, and resealing the resultant cellular carriers. Hence, these carriers are called

resealed erythrocytes. The overall process is based on the response of these cells under

osmotic conditions. Upon reinjection, the drug-loaded erythrocytes serve as slow circulating

depots and target the drugs to a reticuloendothelial system (RES).[4–5]

Blood contains different

type of cells likeerythrocytes (RBC), leucocytes (WBC) andplatelets, among them

erythrocytes are themost interesting carrier and posses great potential in drug delivery due to

their ability tocirculate throughout the body, zero orderkinetics, reproducibility and ease

ofpreparation1 primary aim for the developmentof this drug delivery system is to

maximizetherapeutic performance, reducing undesirableside effects of drug as well as

increase patient compliance.[6-7]

Once in the reticulo-endothelial system, the erythrocyteis

World Journal of Pharmaceutical Research SJIF Impact Factor 7.523

Volume 6, Issue 10, 154-173. Review Article ISSN 2277– 7105

*Corresponding Author

Snehaprabha Warule

Sharadchandra Pawar

College of Pharmacy, Otur,

Pune, Maharashtra.

Article Received on

08 July 2017,

Revised on 27 July 2017,

Accepted on 18 August 2017

DOI: 10.20959/wjpr201710-9125


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attacked by liposomal enzymes that cause the breakage of the cellular membrane and

thedegradation of the haemoglobin by the heme-oxygenase enzyme. Although the greater part

of thedestruction of the old erythrocytes occurs in the reticulo-endothelial system, it is

estimated that upto 10% of the loss of erythrocytes takes place in circulation Erythrocytes

constitute potential biocompatible vectors for different bioactive substances, including drugs,

enzymes and proteins.[8]

2. ERYTHROCYTES MAY BE EMPLOYED FOR TWO MAINPURPOSES

To act as a reservoir for the drug, providing the sustained release of the drug into the body.

To selectively direct the drugs to the reticuloendothelial system of the liver, spleen and

bone marrow, which constitute the usual sites for the destruction of erythrocytes.[9-10]

3. MORPHOLOGY AND PHYSIOLOGY OF ERYTHROCYTES

Erythrocytes, the most abundant cellular constituents of blood (i.e.5, 200,000±300,000and4,

700,000±300,000 cell/mm³ blood in healthy men and women, respectively), represent the

largest cell specific surface among other blood cells (i.e., the highest surface to volume ratio

of 1.9×104 cm/g). The nuclear mature human erythrocyte is one of the most highly

specialized cells. Lacking such cytoplasm organelles as nucleus, mitochondria and

ribosome’s, the red blood cell is unable to synthesize protein, carry out the oxidative reac-

tions associated with mitochondria, or undergo mitosis.[11]

Erythrocytes, produced in bone

marrow by regulatory effect of erythropoietin[12]

, making up more than 99% of the total

cellular space of blood in humans[13]

, occupy a volume of approximately 25 to 30 ml/kg, from

which 71% constitute an aqueous phase.[14]

A total of approximately 760 g of hemoglobin is

contained in the erythrocytes, representing approximately 10% of the total body proteins of

an adult human 10, 12-13. In fact, the major function of the erythrocyte is to encase

hemoglobin and protect it, so it can act as an oxygen transporter for a prolonged period.[17]

Hemoglobin interacts with small diffusible ligands such as O2, CO2 may be involved in the

control of blood pressure.[18]

So, thanks to its hemoglobin content, transport of oxygen from

lung to tissues and the CO2 produced in tissues back to lung is the main role of the

erythrocytes. Erythrocytes draw energy from glucose metabolism via direct glycolysis and

the hexose monophosphate shunt. Erythrocytes are flexible biconcave discs with a cell

diameter of 7 to 9 μm and a thickness of 2 μm.[19]

The biconcave disc shape with the highest

surface to volume ratio is essential for the gas exchange function of erythrocytes. In addition,

this unique shape has a high degree of flexibility required for passage of erythrocytes through


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the capillaries with diameters of 3-4 μm without undergoing extensive remodeling. The

erythrocyte membrane withstands high shear stresses, rapid elongation and folding in the

microcirculation and deformation as the erythrocyte passes through the small fenestrations of

the spleen. The erythrocyte membrane has a specialized structure consisting of a plasma

membrane basic structure including lipids, proteins, and carbohydrates based on the fluid

mosaic model in addition to the cytoskeleton. This structure is necessary for the maintenance

of the integrity of erythrocyte upon exposure to high shear rates in circulation as well as

reticuloendothelial system (RES). Upon decreasing the osmolarity of the surrounding media,

erythrocytes become cup-shaped and finally spherical. This kind of swelling behavior is

necessary for the most methods used for loading the erythrocytes by drugs or other chemi-

cals.

Erythrocytes have a life span of 100 to 120 days in circulation, during which they travel 250

km throughout the cardiovascular system. As a result of the gradual inactivation of the

metabolic pathways of the erythrocyte by ageing, 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 trabecules. The other effective site

for the destruction of the aged or abnormal erythrocytes is the macrophages of the RES

including peritoneal macrophages, hepatic Buffer cells, and alveolar macrophages of the

lung, peripheral blood monocytes, and vascular endothelial cells.[20]

It is well known that

ageing and a series of other factors make the erythrocytes recognizable by the phagocyting

macrophages via changing the chemical composition of the erythrocyte membrane, i.e., the

phospholipids component.[21]

Fig. 1. Schematic representation of Erythrocytes.


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Fig. 2. Schematic representation of Erythrocytes of side view and top view.

4. SOURCES OF ERYTHROCYTES

Various types of mammalian erythrocytes havebeen used for drug delivery, including

erythrocytes ofmice, cattle, pigs, dogs, sheep, goats, monkeys, chicken, rats and rabbits.

5. ISOLATION OF ERYTHROCYTES

To isolate erythrocytes, blood is collected in heparin zed tubes by venipuncture. Fresh whole

bloodis typically used for loading purposes because the encapsulation efficiency of the

erythrocytes isolatedfrom fresh blood is higher than that of the aged blood.

Fresh whole blood is the blood that is collected andimmediately chilled to 4 _C and stored for

less than twodays. 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 at4ºC for as long as 48 h before use.

Advantages of Erythrocytes as Drug Carriers

1. Their biocompatibility, particularly when autologous cells are used, hence no possibilityof

triggered immune response.[22,23,25,29,32]

2. Their biodegradability with no generation of toxic products.[22,23,28,29,32,33]

3. The considerably uniform size and shape of the carrier.[26,27]

4. Relatively inert intracellular environment.[35]

5. Prevention of degradation of the loaded drugfrom inactivation by endogenous

chemicals.[29,31,33,36,37]

6. The wide variety of chemicals that can been trapped.[29,36,38-40]

7. The modification of pharmacokinetic and pharmacodynamics parameters of drug.[25,30,33,36]


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

concentration.[23,25,42,32]

9. Protection of the organism against toxic effects of drugs e.g. antineoplastic.[36]

10. Their ability to circulate throughout the body.[24]

11. The availability of the techniques and facilitiesfor separation, handling, transfusion, and

working with erythrocytes.[28,29]

12. The prevention of any undesired immuneresponse against the loaded drug.[35]

13. Their ability to target the organs of the RES.[22,23,29,41]

14. The possibility of ideal zero-order drug-release Kinetics.[37]

15. The lack of occurrence of undesired immuneresponse against encapsulated drug.[22]

16. The large quantity of drug that can beencapsulated within a small volume of cellsensors

dose sufficiency.[22,29,32]

17. A longer life span in circulation as comparedwith other synthetic carriers[24,33,39]

, and

optimum conditions may result in the life span comparable to that of normal

erythrocytes.[40,36]

18. Easy control during life span ranging from minutes to months.[31]

19. A decrease in side effects of drugs.[23,30,32]

20. A considerable increase in drug dosinginterval with drug residing in therapeutic\window

region for longer time periods.[23,30,41,42]

Disadvantages

1. Co-introduction of the erythrocyte membranes, viral envelopes, viral RNA and

residualhemoglobin may haveunpredicted effects onthe cells.

2. A comparatively larger amount of test materialis desired than that for the microcapillary

method.

3. Direct injection into the cell nucleus is notfeasible.

4. They have a limited potential as carrier to no phagocyte target tissue.

5. Possibility of clumping of cells and dose dumping nay is there.[43-47]

6. FACTORS WHICH CONSIDERING RESEALED ERYTHROCYTES AS

CARRIER

1. Appropriate size(s) and shape to permit the passage through the capillaries.

2. It should have minimum side effects and biocompatible.


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3. It should have specific physicochemical properties by which a desired target site could be

recognized.

4. Drug should be released at the target site in a controlled manner.

5. Low leaching/leakage of drug should take place before target site is reached.

6. Physico-chemical compatibility with the drug.

7. It should possess the ability to carry a wide variety of drugs with different properties.

8. It should have sufficient space to carry and eventually to permit the delivery of clinically

adequate amounts of drug.

9. The carrier system should have an appreciable stability during storage.[48]

7. ROUTE OF ADMINISTRATION

Intra peritoneal injection reported that survival of cells in circulation was equivalent to the

cells administered by i.v. injection .They reported that 25% of resealed cell remained in

circulation for 14 days they also proposed this method of injection as a method for extra

vascular targeting of RBCs to peritoneal macrophages. Subcutaneous route for slow release

of entrapped agents. They reported that the loaded cell released encapsulated molecules at the

injection site.[49]

8. MECHANISM OF RELEASE OF LOADED DRUGS

There are mainly three ways for a drug release from the erythrocyte carriers

Phagocytosis: By the process of phagocytosis normally erythrocyte cells removed from

the blood circulation. The degree of cross linking determines whether liver or spleen will

preferentially remove the cells.

Diffusion through the membrane of the cells: Diffusion through the membrane depends

on the drug molecule penetrate through a lipid bilayer i.e. bioactive compound have lipid

solubility.

Using a specific transport system: Most of the drug molecules enter cells by a specific

membrane protein system because the carriers are proteins with many properties analogous to

that of enzymes.[50]

9. 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


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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 4 . 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.[51]

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.[52]

10. CROSSLINKING, STABILITY AND IN-VIVO SURVIVAL OF RESEALED

ERYTHROCYTES

The cells treated with dimethyl sulphoxide (DMSO), toluene 2, 4-di-isocyanate (TD1) and

gluteraldehyde are even resistant to sonication, freezing and thawing. Chemically cross

linking of erythrocytes renders a yield of 55-97% of non-lysed cells. An attempt was made to

get drug loaded cells in lyophilized form. The dried powder was filled in amber color glass

vials and stored at 4ϒC for one month. Improvement in shelf-life of the carrier erythrocytes

was achieved by storing the cells in powder form ready for reconstitution at 4ϒC. This is

important in the large scale manufacturing of drug loaded erythrocytes.[53]

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 hemoglobin and other cell

constituents lost during the loading process.[54]

The various methods used to determine in


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vivo survival time include labeling of cells by 51Cr or fluorescent markers such as

fluorescent isothiocyanate or entrapment of 14C sucrose or gentamicin. The circulation

survival kinetics of resealed erythrocytes show typical bimodal behavior with a rapid loss of

cells during the first 24 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.[55]

11. PHARMACOKINETICS OF DRUGS OR PEPTIDES ADMINISTERED IN

LOADED ERYTHROCYTES

Erythrocytes loaded with drugs and other substances havedifferent release rates. In vitro

studies on release performed witherythrocytes from various animal species loaded with

differentkinds of substances habitually reveal a slow release of the encapsulated substance.[56]

The in-vitro release of substances fromloaded erythrocytes responds to a first-order kinetic

process, revealing that the substance permeates the erythrocyte membraneby passive

diffusion. However, human carrier erythrocyte containing enalaprilat have in-vitro, zero-

order release kinetics. The treatment of loaded erythrocytes with glutaraldehyde and other

substances produces a more delayed invitro release of both the encapsulated substance and

the haemoglobin from the loaded erythrocytes. The employment of prodrugs encapsulated in

erythrocytes permits the use of the redcell as a bioreactor that controls the release rate of the

drug.[57]

When the loaded erythrocytes are administered in vivo, circulating cellsused as drug

carriers may alter the pharmacokinetics ofadministered drugs. Encapsulation within

erythrocytes affords thedrug a clearance that depends on the biological half-life of

theerythrocyte, allowing therapeutic levels to be maintained in theblood for long periods of

time, together with the generation of asustained release of the therapeutic agent.[58]

In

vivosurvival of human carrier erythrocytes labeled with 51Crdemonstrates a mean cell life

and cell half-life of 89 to 131 daysand 19 to 29 days, respectively. These changes in

thepharmacokinetics when carrier erythrocytes are used involve theprolonged serum half-life

of the encapsulated substance incomparison to the free substance, an increase in the area

under thecurve of serum concentrations and a greater accumulation of thedrug in the liver and

in the spleen. Enhancedliver targeting in vivo of loaded erythrocytes may be achieved

bymeans of surface treatment with glutaraldehyde and other substance.[59]


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12. ENTRAPMENT METHOD

1. Hypo – osmotic method

Dilution method.

Dialysis method.

Press well method.

Isotonic osmotic lyses.

2. Electrical break down method

3. Endocytosis method

4. Membrane perturbation method

5. Normal transport method

Hypo–osmotic lyses method: In this process, the intracellular and extracellular solutes of

erythrocytes are exchangedby osmotic lyses and resealing. The drugpresent will be

encapsulated within theerythrocytes membrane by this process.

A) Dilution method: The RBC’S are exposed to hypotonicsolution (corresponding to 0.4%

Nacl), theerythrocyte membrane ruptures permittingescape of cellular contents and

equilibrium isachieved with in one minute.The cells up to 1.6 times its originalvolume.The

swelling results in the appearance ofpores of 200 – 500 um in size.The length of time for

which these poresremain open is not fixed. However at 0oC the opening permits long enough

to allow partialresealing of membrane.Increasing the ionic strength to is tonicityand

incubating the cells at 37o C causes thepores to close and restore the osmoticproperties of the

RBC’S. This method was used to entrap b-glucosidase and b– galactosidase. This method is

simplest and fastest yet thecapsulation efficacy is very low i.e. 1 – 8 %.Efficient for

encapsulation of low molecularweight drugs.Most of the cytoplasmic constituents arelost

during the osmotic lyses.[60-63]

B) Dialysis method

Fig. 3. Schematic representation of Dialysis method.


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Erythrocyte Dialyzer

A desired Haematocrit is achieved by mixing washed erythrocyte suspension andphosphate

buffer (pH 7.4) containing drugsolution.This mixture is placed into dialysis bagand then both

ends of the bag are tied withthread. An air bubble of nearly 25 % of theinternal volume is left

in the tube. Duringdialysis bubble serves to blend the content.The tube is placed in a bottle

containing200 ml of lysis buffer solution and placed on amechanical rotator at 4o C for 2

hrs.The dialysis tube is then placed in 200 mlof resealing solution (isotonic PBS pH 7.4)

atroom temperature 25 – 30 o C for resealing.The loaded erythrocytes thus obtained arethen

washed with cold PBC at 4oC. The cellsare finally resuspended in PBC.

Advantages

Good entrapment efficiency is obtainedThe volume of extra cellular solution thatequilibrates

with the intracellular space oferythrocyte during lyses is considerably reduced.

Disadvantages

Time consuming method. The size distribution of loaded ghosts isnot found to be

homogeneous as revealed bystudies with hydro dynamically focusing particle analyzer.[60-63]

C) Press well dilution method

Fig. 4. Schematic representation of Press well dilution method.

Press well Dilution Method

It is based on the principle of first swellingthe erythrocytes without the lyses by placingthem

in slightly hypotonic solution. The swollen cells are recovered bycentrifugation at low speed.

Then, relatively small volumes of aqueousdrug solution are added to the point of lyses i.e.

when there is minimum loss of constituents. The slow swelling of cells results in good

retention of the cytoplasmic constituents and hence good survival in vivo.


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Advantages: Simple and quicker than dialysis method. Under optimum conditions

resealederythrocytes can survive in – vivo as long and native RBC’S.

D) Isotonic osmotic lyses technique: If erythrocytes are incubated in solution of asubstance

with high transerythrocyticmembrane permeability the solute will diffuseinto the cells due to

inwardly direct chemicalpotential gradient. This will be followed bywater uptake until

osmotic equilibrium isrestored. A transient permeability in erythrocyte wallcould be

produced using propylene glycolwhich also allows the drugs/ agents to diffusein.The lysed

erythrocytes are resealed underisotonic condition by dilution glycol free medium.[60-63]

E) Electrical breakdown method

Fig. 5. Schematic representation of Electrical breakdown method.

Electrical breakdown of a cell membraneis observed when the membrane is polarized very

rapidly (in nano to micro seconds) usingvoltage of about 2kV/ cm for 20μ sec whichlead to

the formation of pores and entrapment of drugs. Electrical breakdown probably takes placein

the lipoid regions or at the lipid proteinjunction in the membrane. Pores formed are stable and

it is possibleto control pore size. Subsequently the pores can be resealedby incubation at 37º

C in osmotically balanced medium.

Disadvantage-of this method is that it is very expensive.

F) Endocytosis method

Fig. 6. Schematic representation of Endocytosis method.


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Intracellular vesicles could be inducing inerythrocytes containing small molecules drugsor

virus from external medium. This method is efficient for loading largerparticles such as virus

(up to 1000 nmdiameter), enzymes and small molecules. In this method the vesicle

membraneseparates the endocytoced substance fromthe cytoplasm containing the drug which

aresensitive to inactivation by cytoplasmic enzymes and also protect the

erythrocytemembrane. The contents of vesicles, however mayrelease into erythrocytes

cytoplasm dependingupon the nature of material.

Ex. Entrapment of glucose, insulin and b –glucouronidase by a chlorpromazine induced

endocytosis has been reported.

G) Membrane perturbation method

Antibiotics such as Amphotericin – Bdamage micro–organisms by increasing thepermeability

of their membrane to metabolite and ions. This property could be exploited forloading of

drugs in to erythrocytes.Amphotericin – B was used to loaded erythrocytes with antileukamic

drugs. Amphotericin – B interacts with thecholesterol of the plasma membrane ofeukaryotic

cells causing change inpermeability of the membrane.

Normal transport mechanism

It is possible to load erythrocytes withdrugs without disrupting the erythrocytic membrane in

any way by incubating the drugand erythrocytes for varying period of time. After infusion the

drug would in general, exit from the cell following the kineticscomparable to those observed

for entry.

Lipid fusion method

Lipid vesicles containing ionsitolhexaphosphate with human erythrocytes the incorporated

ionositol hexaphosphate in erythrocytes provided a significant lowering ofthe O2 affinity for

haemoglobin in intact erythrocyte.[60-63, 64]

13. CHARACTERIZATION OF RESEALED ERYTHROCYTES

1. Drug content determination: After centrifugation at 3000 rpm for a fixed time interval

drug loaded erythrocyte cells are deproteinized with acetonitrile. The clear supernatant liquid

is analyzed for drug content.[65]

2. In-vitro drug release and Hb content: The erythrocyte cell suspensions which have

hemocrit value 5% are stored at 40C in amber colored glass container. Clear supernatant are


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drawn using a hypodermic syringe equipped with 0.45 filter for fixed time intervals and

deproteined using methanol and were estimated for drug content. After centrifugation

supernatant of each sample is collected and assayed, %Hb release calculated by using

formula. % Hb release=A540 of sample-A540 of background A540 of 100% Hb. Laser light

scattering technique may also be used to evaluate haemoglobin content of individual resealed

erythrocytes.

3. Percentage cell recovery: Percentage cell recovery can be determined by counting the

number of intact cells per cubic mm of packed erythrocytes before and after loading of the

bioactive compound with the help of haemocytometer.

4. Morphology: Phase contrast optical microscopy, transmission electron microscopy and

scanning electron microscopy are the microscopic methods used to evaluate the shape, size

and surface features of loaded erythrocytes.[66]

5. Osmotic shock: 1ml of 10%erythrocyte suspension was diluted with 5ml of water and

centrifuged the above mixture at 3000rpm for 15minutes. The supernatant was estimated for

% Hb release Spectrophotometrically.[67]

6. Turbulence shock: This parameter indicates the effects of shear force and pressure by

which resealed erythrocyte formulation are injected on the integrity of the loaded cells. 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.[65]

7. Determination of entrapped magnetite: Determination of the concentration of a

particular metal element present in a sample can be determined by Atomic Absorption

spectroscopy. To a fixed amount of magnetite bearing erythrocyte add the HCl and these

contents are heated at 600C for 2 hours and then 20%w/v trichloroacetic acid is added. These

contents were centrifuged and collect supernatant. From this supernatant determine magnetite

concentration using absorption spectroscopy.[65]

8. Erythrocyte sedimentation rate (ESR): It is an estimate of stability of suspension of

erythrocyte cells in plasma and is related to the number and size of the red cells and to

relative concentration of plasma proteins like fibrinogen and α, β globulins. This test is


167


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.[66]

9. Miscellaneous: Resealed erythrocyte can also be characterized by cell sizes, mean cell

volume, energy metabolism, lipid composition, membrane fluidity, rheological properties,

and density gradient separation.[66]

14. APPLICATION

In Vivo Applications-This includes the following.

1) Slow drug release: Erythrocytes have been used as circulatingdepots for the sustained

delivery oantineoplastics, antiparasitics, veterinaryantiamoebics, vitamins, steroids,

antibiotics, and cardiovascular drugs.[54]

2) Drug targeting: Ideally, drug delivery should be site specificand target oriented to exhibit

maximaltherapeutic index with minimum adverseeffects. Resealed erythrocytes can act

asdrug carriers and targeting tools as well.Surface modified erythrocytes are used totarget

organs of mononuclear phagocyticsystem/ RES because the change in themembrane is

recognized by macrophages.[25]

3) Targeting reticuloendothelial system (RES) organs

Surface modified erythrocytes are used totarget organs of mononuclear phagocytic

systems/reticuloendothelial system because the changes in membrane are recognized by

macrophages.

The various approaches used include.

• Surface modification with antibodies (coating of loaded erythrocytes by anti‐Rhor other

types of antibodies).

• Surface modification with glutaraldehyde.

• Surface modification with sulphahydryl.

• Surface chemical cross linking.

• Surface modification with carbohydrates such as silica acid.[55]


168


4) Targeting the liver-deficiency/therapy

Many metabolic disorders related todeficient or missing enzymes can be treatedby injecting

these enzymes. However, theproblems of exogenous enzyme therapy include a shorter

circulation half life ofenzymes, allergic reactions, and tomanifestations .these problems can

besuccessfully overcome by administering the Enzymes as resealed erythrocytes. The

enzymes used include Pglucosidase, International glucoronidase, and Pgalactosidase. The

disease caused by an accumulation of glucocerebrosidaes in the liver and spleencan is treated

by glucocerebrosidase-loaded erythrocytes.[68]

15. NOVEL APPROACHES

Erythrosomes

These are speciallyengineered vesicular systems that arechemically cross-linked to

humanerythrocytes’ support upon which a lipidbilayer is coated. This process is achievedby

modifying a reverse-phase evaporationtechnique. These vesicles have been, proposed as

useful encapsulation systems for macromolecular drugs.[69]

Nanoerythrosomes

These are prepared byextrusion of erythrocyte ghosts to producesmall vesicles with an

average diameter of100 nm. Daunorubicin was covalently conjugated to nanoerythrosomes

using gluteraldehyde spacer. This complex was more active than free daunorubicin alone.[70]

Other

Significant advances have been made withthe use of erythrocyte for specific targetingto cells

of the immune system. Antiviraldrugs can be pretreated to deliver drugdirectly to

macrophages. Several laboratorytechniques have developed for the encapsulation of allosteric

effector of hemoglobin, inositol hexa phosphate, which is effective at oxygen delivery, much

more effective than normal erythrocytes.[54,55,70]

Future Perspective

A large amount of valuable work is neededso as to utilize the potentials of erythrocytesin

passive as well as active targeting of drugs.

Diseases like cancer could surely find it scure.

Genetic engineering aspects can becoupled to give a newer dimension to theexisting

cellular drug carrier concept.[71]


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16. CONCLUSION

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 the delivery of

biopharmaceuticals and much remains to be explored regarding the potential of resealed

erythrocytes.[71]

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LOADED ERYTHROCYTE: A REVIEW ARTICLE › admin › assets › article_issue › 1504170020.pdf · LOADED ERYTHROCYTE: A REVIEW ARTICLE Snehaprabha Warule*, Jayant Bidkar Shital Bidkar