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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
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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]
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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]
17. REFERENCE
1. A. V. Gothoskar, Resealed erythrocyte: A Review pharmaceutical technology march,
2004; 140-158.
2. R. Green and K. J. Widder, Methods in Enzymology (Academic Press, San Diego, 1987),
149.
3. C. Ropars, M. Chassaigne, and C.Nicoulau, Advances in the BioSciences, (Pergamon
Press, Oxford, 1987), 67.
4. D.A. Lewis and H.O. Alpar, “Therapeutic Possibilities of Drugs Encapsulatedin
Erythrocytes,” Int. J. Pharm. 1984; 22: 137-146.
5. A Krantz, Red-cell mediated therapy: opportunities and challenges, Blood Cells Mol.
Diseases. 1997; 23(3): 58-68.
6. Alpar, H.O. and Irwin, W.J. Some Unique Applications of Erythrocytes as Carrier
Systems. Adv. Biosci. (Series). 1987; 67: 1-9.
7. Alvarez, F.J, A. Herra´ez, J.C. Murciano, J.A. Jordan, J.C.Dı´ez, M.C. Tejedor, In vivo
survival and organ uptake of loaded carrier raterythrocytes, J. Biochem.(Tokyo). 1996;
120(2): 286-291.
8. Alvarez, F.J., Jordan, J.A., Calleja, P., Lotero, L.A., Olmos, G., Díez, J.C., Tejedor, M.C.
Cross-Linking Treatment of Loaded Erythrocytes Increases delivery of Encapsulated
Substance to Macrophages Biotechnol Appl. Biochem. 1998; 27(2): 139-143.
9. Alvarez-Guerra, M., Nazaret, C. And Garay, R. P. Erythrocyte act as Bax. B.E, M.D.
Bain, L.D. Fairbanks, A.D. Webster, and R. A. Chalmers, In vitro and in vivo studies
with human carrier erythrocytes loaded with polyethylene glycol-conjugated and native
adenosine deaminase, Br. J. Haematol. 2000; 109(3): 549-554.
10. Bonomo. R. P., A. De Flora, E. Rizzarelli, A.M. Santoro, G.Tabbi, M. Tonetti, Copper
(II) complexes encapsulated in human red blood cells, J. Inorg. Biochem. 1995; 59(4):
773-784.
www.wjpr.net Vol 6, Issue 10, 2017.
170
Warule et al. World Journal of Pharmaceutical Research
11. Morphology 1.Telen MJ, Kaufman ER. The mature erythrocytes. In: Greer JP, Foerster J,
Rodgers GM, Paraskevas F, Glader B, Arber DA, Means RT, editors. Wintrobe’s clinical
hematology. 11th ed. Philadelphia: Lippincott Williams & Wilkins, 2004; 217-247.
12. Guyton AC, Hall JE. Red Blood Cells. 11th ed. Philadelphia: W.B. Saunders, 2000;
382-391.
13. Diem K, Lentner C. DocumentaGeigy, Scientific Tables, Geigy Pharmaceuticals (Ciba-
Geigy Ltd.) 7th ed., 1975; 617-618.
14. Guyton AC, Hall JE. Textbook of Medical Physiology. Philadelphia: W. B. Saunders,
1999; 425-433.
15. Diem K, Lentner C. DocumentaGeigy, Scientific Tables, Geigy Pharmaceuticals
(Ciba-Geigy Ltd.) 7th ed., 1975; 555-561.
16. Spector WS. Handbook of Biological Data. Philadelphia: W. B. Saunders; 1956;
52 & 70.
17. Steinberg MH. Pathobiology of the Human Erythrocyte and Its Hemoglobins, In:
Hoffman R, Benz E, Shattil S, Furie B, Cohen H, editors. Hematology: basic principles
and practice. 4th ed. Philadelphia: Churchill Livingstone, 2005; 442-456.
18. Jia L, Bonaventura C, Bonaventura T, Stamler S. S-nitrosohaemoglobin: a dynamic
activity of blood involved in vascular control. Nature (Lond), 1996; 380: 221-226.
19. Beutler E, Lichtman MA, Coller BS, Kipos TJ. Williams Hematology. New York:
McGraw Hill Inc, 1995; 349–363.
20. Connor J, Schroit AJ. Red blood cell recognition by the reticuloendothelial system.
AdvBiosci (series), 1987; 67: 163-171.
21. Schlegel RA, McEvoy L, Weiser M, Williamson P. Phospholipid organization.
22. Vyas SP and Khar R. K. Resealed Erythrocytesin Targeted and Controlled Drug Delivery:
Novel Carrier Systems, CBS Publishers and Distributors, India, 2002; 87–416.
23. Lewis DA and Alpar HO; Therapeutic Possibilities of Drugs Encapsulated in
Erythrocytes, Int. J. Pharm., 1984; 22: 137–146.
24. Zimmermann U; Cellular Drug-Carrier Systems and Their Possible Targeting In Targeted
Drugs, EP Goldberg, Ed., John Wiley & Sons, New York, 1983; 153–200.
25. Jain S and Jain NK; Engineered Erythrocytes as a Drug Delivery System, Indian J.
Pharm. Sci., 1997; 59: 275–281.
26. Telen MJ; The Mature Erythrocytes, in Winthrob’s Clinical Hematology, R. Lee et
al.,Lea & Febiger, Philadelphia, 9th edition, 1993; 101–133.
www.wjpr.net Vol 6, Issue 10, 2017.
171
Warule et al. World Journal of Pharmaceutical Research
27. Guyton AC and Hall JE; Red Blood Cells, Anemia and Polycytemia, Textbook of
Medical Physiology, W.B. Saunders, Philadelphia, 1996; 425–433.
28. Torotra GJ and Grabowski SR; The Cardiovascular System: The Blood, Principles of
Anatomy and Physiology, 7th edition, Harper Collins College Publishers, New York,
1993; 566–590.
29. Jaitely V; Resealed Erythrocytes: Drug Carrier Potentials and Biomedical Applications,
Indian Drugs, 1996; 33: 589–594.
30. Eichler HC; In Vitro Drug Release from Human Carrier Erythrocytes, Adv. Biosci., 1987;
67: 11–15.
31. Summers MP; Recent Advances in Drug Delivery, Pharm. J., 1983; 230: 643–645.
32. Talwar N and Jain NK; Erythrocytes as Carriers of Primaquin Preparation:
Characterization and Evaluation, J. Controlled Release, 1992; 20: 133–142.
33. Lewis DA; Red Blood Cells for Drug Delivery, Pharm. J., 1984; 233: 384–385.
34. Alpar HO and Lewis DA; Therapeutic Efficacy of Asparaginase Encapsulated in Intact
Erythrocytes, Biochem. Pharmacol. 1985; 34: 257–261.
35. K. Adriaenssens et al.; Use of Enzyme-Loaded Erythrocytes in In Vitro Correction
ofArginase Deficient Erythrocytes in Familiar Hyperargininemia, 1976; Clin. Chem., 22:
323–326.
36. Sprandel U; Towards Cellular Drug Targeting and Controlled Release of Drugs by
Magnetic Fields, Adv. Biosci., 1987; 67: 243–250.
37. Eichler HG et al.; In Vivo Clearance of Antibody-Sensitized Human Drug Carrier
Erythrocytes, Clin. Pharmacol. Ther. 1986; 40: 300–303.
38. Baker R; Entry of Ferritin into Human 1Red Cells During Hypotonic Hemolysis, Nature,
1967; 215: 424–425.
39. Vienken J, Jeltsch E and Zimmermann U; Penetration and Entrapment of Large
Particlesin Erythrocytes by Electrical Breakdown Techniques, Cytobiologie, 1978; 17:
182–186.
40. Schlegel RA et al.; Phospholipid Organizationas a Determinant of Red Cell Recognition
by the Reticuloendothelial System, Adv. Biosci., 1987; 67: 173–181.
41. Kinosita K and Tsong TY; Survival of Sucrose-Loaded Erythrocytes in the Circulation,
Nature, 1978; 272: 258–260.
42. Jain S, Jain SK and Dixit VK; Erythrocytes Based Delivery of Isoniazid: Preparation and
In Vitro Characterization, Indian Drugs, 1995; 32: 471–476.
www.wjpr.net Vol 6, Issue 10, 2017.
172
Warule et al. World Journal of Pharmaceutical Research
43. Singh Devendra, Kumar Manish, Singh Talever, Singh L.R., Singh Dashrath. A Review
on Resealed Erythrocytes as aCarrier for Drug Targeting, International Journal of
Pharmaceutical and Biological Archives, 2011; 2(5): 1357-1373.
44. Patel RP, Patel MJ and Patel A. And overview of resealed erythrocytes drug.
45. Deliver, R.P. Patel, journal of pharmacy research, 2009; 2(6): 1008-1012.
46. Gopal V. S., Doijad R.C., and Deshpande P. B. Erythrocytes as a carrier for prednisolone-
in vitro and in vivo evaluation, Pak J. Pharm. Sci, 2010; 23(2): 194-200.
47. Green R and Widder K.J. Methods in Enzymology Academic Press, San Diego,
1987; 149.
48. SHELLY et al. Int J Adv Pharm BiolSci, ISSN 2249-8966, 2013; 3(1): 39.
49. D.M. Brahmankar, sunil B. Jaiswal “controlled release medication” edited Jain M.K.,
Biopharmaceutics and Pharmacokinetics, vallabhprakashan, New Delhi, 2013: 490.
50. Mechanism of drug loading, evaluation and application of erythrocytes as carriers for
drug targeting; Sandip Kumar Singh, Shailesh Kumar Yadav, Ajay Kumar, 1(1): 67-77.
51. Hamidi M and Tajerzadeh H, Carrier Erythrocytes: An Overview, Drug Delivery, 2003;
10: 9–20.
52. Hamidi M, Tajerzadeh H, Dehpour AR, EjtemaeeMehr S, ACE Inhibitionin Rabbits
Upon Administration of Enalaprilat Loaded Intact Erythrocytes, J Pharm Pharmacol,
2001; 53: 1281–1289.
53. Hamidi M, Tajerzadeh H, Dehpour AR, Rouini MR, EjtemaeeMehr S, In vitro
Characterization of Human Intact Erythrocytes Loaded by Enalaprilat, Drug Delivery,
2001; 8: 231–237.
54. Hamidi M, Zarei N, Zarrin AH, Mohammadi Samani S, Preparation and in vitro
characterization of carrier erythrocytes for vaccine delivery, Int. J. Pharm, 2007; 338:
70–78.
55. Jain S, Jain SK, Dixit VK, Erythrocytes Based Delivery of Isoniazid: Preparation and In
Vitro Characterization, Indian Drugs, 1997; 32: 471–476.
56. Jain S, Jain SK, Dixit VK, Magnetically Guided Rat Erythrocytes Bearing Isoniazid:
Preparation, Characterization, and Evaluation, Drug DevInd Pharm, 1997; 23: 999-1006.
57. Jain SK and Vyas SP, Magnetically Responsive Diclofenac Sodium- Loaded
Erythrocytes: Preparation and In- Vitro Characterization, J Microencapsul, 1994; 11(2):
141–151.
58. Jaitely V, Kanaujia P, Venkatesan N, Jain S, Vyas SP, Resealed erythrocytes: carrier
potentials and biomedical application. Indian Drugs, 1996; 33: 549–589.
www.wjpr.net Vol 6, Issue 10, 2017.
173
Warule et al. World Journal of Pharmaceutical Research
59. Jaitely V, Kanaujia P, Vyas SP, Resealed Erythrocytes: Drug Carrier Potentials and
Biomedical Applications, Indian Drugs, 1996; 33: 589–594.
60. Green R and Widder KJ. Methods in Enzymology. Academic Press, San Diego. 1987;
149.
61. Ropars C, Chassaigne M and Nicoulau C. Advances in the Bio Sciences. Pergamon Press,
Oxford, 1987; 67.
62. Lewis DA and. Alpar HO. Therapeutic Possibilities of Drugs Encapsulated in
Erythrocytes. Int J Pharm. 1984; 22: 137–146.
63. Zimmermann U. Cellular Drug-Carrier Systems and Their Possible Targeting In Targeted
Drugs, EP Goldberg, Ed. John Wiley & Sons, New York, 1983; 153-200.
64. Resealed erythrocytes: As a specified tool in novel drug delivery carrier system:
TirupatiRao, Suriya Prabha K., Muthu Prasanna p. October-December, 2004; 496-512.
65. Abhishek Kumar Sah et al. J Chem Pharm Res., 2011; 3(2): 550-555.
66. Sashank shah. I J P B S., 2011(1).
67. Jagadale VL. I J Pharm Edu Res., 2009; 43(4).
68. Magnani M and Rossi L, Erythrocyte Engineering for Drug Deliveryand Targeting,
Biotechnol, Appl Biochem, 1998; 28: 1–13.
69. Magnani M, Bianchi M, Rossi L, Stocchi V, Acetaldehyde Dehydrogenase Loaded
Erythrocytes as Bioreactors for Removal of Blood Acetaldehyde, Alcoholism, Clin Exp
Res., 1989; 13: 849–859.
70. Moorjani M, Lejeune A, Gicquaud C, Lacroix J, Poyet P, Gaudreault RC, Nano
erythrosomes, A New Derivative of Erythrocyte Ghost II: Identification of the
Mechanism of Action, Anticancer Res., 1996; 16(5): 2831-2836.
71. Resealed erythrocytes as a Carrier for drug targeting A review; ashokkumar, mansiverma,
k. k. jha, 2012; 1: 7-15.