Erythrocyte is biconcave discs , anucleate filled with hemoglobin (Hb), a protein that functions in gas transport.

1. 1
Mr. Sagar Kishor savale
Department of Pharmaceutics
avengersagar16@gmail.com
2015-2016

2. Contents
Introduction
Advantages & disadvantages
Isolation of erythrocytes
Methods of preparation
Characterization
Applications
Novel system & future prospects
Conclusion
References
2

3. Introduction
3

erythro = red and cytes = cell
erythrocyte is red cell.

Erythrocyte is biconcave discs , anucleate filled with hemoglobin (Hb),
a protein that functions in gas transport.

Healthy adult male=4.5millions/µml
Healthy adult female=4.8million/µml

Erythrocytes have a life of around 120 days, whereupon they
degenerate.
 They follow a established life cycle .

4. Advantages
4
 Biodegradability & biocompatibility.
 Circulate throughout the circulatory system.
 Inert environment.
 Prevention of undesired immune response.
 Can be utilized for organ targeting within RES.
 A longer life span as compared to synthetic carriers.
 Decrease in side effect of drugs.
 Increase in drug dosing interval.
 Easy control during life span ranging from minutes to months.
 Large qty. of material can be encapsulated within small volume of cells.

5. Disadvantages
 They are removed in vivo by RES so may cause toxicological problems.
 Rapid leakage of certain encapsulated from the loaded erythrocytes.
 Several molecules may alter the physiology of the erythrocyte.

Lesser standardization in their preparation, compared to other carrier
systems.

The storage of the loaded erythrocytes is a further problem involving
carrier erythrocytes

Liable to biological contamination due to the origin of the blood, the
equipment and the environment
5

6. Erythrocyte Isolation
6
Erythrocytes may be prepared as carriers from
human beings rats , mice , rabbits etc.
Often stored in acid-citrate-dextrose buffer at
4ºC upto 48hrs. prior to use
EDTA/ heparin used as anticoagulant
Blood is centrifuged & refrigerated

7. Various conditions & centrifugal force used for the
isolation of erythrocytes
Sr. no. Species Washing buffer Centrifugal force (g)
1 Rabbit 10mmol KH2PO4/NaHPO4 500-1000
2 Dog 15mmol KH2PO4/NaHPO4 500-1000
3 Human 154mmol NaCl <500
4 Mouse 10mmol KH2PO4/NaHPO4 100-500
5 Cow 10-15mmol KH2PO4/NaHPO4 1000
6 Horse 2mmol MgCl2,10mmol glucose 1000
7 Sheep 10mmol KH2PO4/NaHPO4 500-1000
8 Pig 10mmol KH2PO4/NaHPO4 500-1000
7

8. Drug loading in resealed
erythrocytes
Osmotic lysis
Presswell
method
Dialysis
method
Dilution
method
Membrane
perturbation
Electro-
encapsulation
Hypo-osmotic lysis
Lipid fusion,
Endocytosis
8
Various methods of preparing Resealed Erythrocytes

9. I) Hypo-osmotic lysis method
9
 Erythrocyte have an exceptional capability for reversible shape change

They don’t have internal membrane and no capacity to synthesize
additional plasma membranes, the surface area is inevitably fixed

so increase in volume initially leads to conversion of normal
bioconcave , discocyte(normal erythrocyte) to spherocytes.

Thus the cells becomes spheres as they accommodate additional
volume with a fixed surface area.

The swollen erythrocytes when placed in solution ≤150mOsm/Kg ,the
membrane ruptures, permitting escape of the cellular component.

These ruptured membrane can resealed by raising the salt
concentration to its isotonic levels and upon incubation, the resealed
erythrocytes assemble their normal bioconcave shape and recover
impermeability.

10. (a) Dilutional haemolysis
10
Erythrocytes exposed to hypotonic
medium (0.4% NaCl)
Membrane ruptures & becomes
permeable to macromolecules & ions
One volume of washed erythrocytes
can be treated with2-20volumes of
material to be loaded
Further incubation at 25ºC in an
isotonic medium (0.9% NaCl) &
reseal them

11. Schematic of hypotonic haemolysis
11
Hypotonic dilution is used for loading enzymes such as Bgalactosidase
and B-glucosidase, asparginase, and arginase, as well as bronchodilators
such as salbutamol.

12. (b)Presswell Dilutional Haemolysis
12
Initial controlled swelling in a
hypotonic buffered solution.
Supernatant is discarded
Mixture is centrifuged
Lysis occur
mixture is centrifuged
at low g values.
Addition of 100-150ml aqueous solution of
drug & cell brought to lysis point
Addition of isotonic medium
suspension is incubated at 37oC to
form the resealed erythrocytes

13. Schematic of Presswell Dilutional Haemolysis
13
Drugs encapsulated in erythrocytes using this method include propranolol ,
asparginase , cyclopohphamide, cortisol-21-phosphate ,w1-antitrypsin,
ethotrexate, insulin, metronidazole , levothyroxine , enalaprilate and isoniazide

14. (c) Isotonic osmotic lysis
14

If erythrocytes are incubated in solutions of a substance with
high membrane permeability, the solute will diffuse into the cells
because of the concentration gradient.

This process is followed by an influx of water to maintain osmotic
equilibrium.

Chemicals such as Urea solution , Polyethylene Glycol , and
Ammonium Chloride have been used for isotonic hemolysis.

15. (d) Dialysis
15
Washed erythrocytes are mixed with
phosphate buffer saline, pH 7.4
This mixture is placed in dialysis bag
The bag is inflated with air bubble
Sealed dialysis bag is placed in a
bottle containing at least200ml of
lysis buffer (0.1% NaCl, 0ºC)
Placed in a mechanical rotator for
2hrs at 4ºC
Resealing at R.T. for 30 minutes
Mixer
stirrer

16. Comparison of various Hypoosmotic Lysis Methods
16
Method % Loading Advantages Disadvantages
Dilution Method 1-8% Fastest & simplest Entrapment efficiency
is very less
Dialysis 30-45% Better in vivo survival
of erythrocytes, better
structural integrity of
membrane due to
lesser ionic load
Time consuming;
heterogenous size
distribution of
resealed erythrocytes
Presswell Dilution
Method
20-70% Good retention of
cytoplasm constituent
& good survival in vivo
-
Isotonic Osmotic
lysis
- Better in vivo
survillance
Impermeable only to
large molecules,
process is time
consuming

17. II) Chemical perturbation of membrane
17

This method is based on the observation that the permeability of the
erythrocytic membrane is increased when exposed to certain
chemicals
e.g. Daunomycin by Amphotericin B
III) Electro encapsulation

Also known as electroporation, the method is based on the observation
that electrical shock brings about desirable membrane permeability for
drug loading into erythrocytes
e.g. Methotrexate, Isoniazide

18. IV) Entrapment by Endocytosis
18
Addition of one volume of washed packed
erythrocytes to nine volumes of buffer
The pores created by this method are
resealed by using 154 mM of NaCl
The bag is inflated with air bubble
Incubate for 2min at R.T.
Incubate for 2min at 37ºC

19. Characterization
• Physical characterization
• Cellular characterization
• Biological charecterization
19

20. Physical characterization
Parameter Method /instrument used
Shape and surface
morphology
Transmission electron microscopy,
scanning electron microscopy, phase
contrast microscopy, optical microscopy.
Vesicle size and size
distribution
Transmission electron microscopy,
optical microscopy.
Drug release Diffusion cell, dialysis
Drug content Deproteinization of cell membrane followed
by assay of resealed drug, radiolabelling
Surface electrical
potential
Zeta potential measurement
Surface pH pH-sensitive probes
Deformability Capillary method
20

21. Cellular characterization
Parameter Method /instrument used
% Hb content Deproteinization of cell membrane followed
by hemoglobin assay
Cell volume Laser light scattering
% Cell recovery Neubaur’s chamber, hematological analyzer
Osmotic fragility Stepwise incubation with isotonic to
hypotonic saline solutions and determination
of drug and hemoglobin assay
Osmotic shock Dilution with distilled water and estimation
of drug and hemoglobin
Turbulent shock Passage of cell suspension through 30-gauge
hypodermic needle at 10 mL/min flow rate and
estimation of residual drug and hemoglobin, vigorous
shaking followed by hemoglobin estimation
Erythrocyte
sedimentation rate
ESR methods
21

22. Biological characterization
Parameter Method /instrument used
Sterility
Sterility test
Pyrogenicity
Rabbit method, LAL test
Animal toxicity
Toxicity tests
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23. Biomedical application of Resealed Erythrocytes
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1. Erythrocyte as drug/enzyme carrier
Eg. L-asparaginase, actinomycin etc.
2. Erythrocytes as a carrier for proteins & macromolecules
Eg. Insulin, interleukin-2
3. Drug targeting
i. Drug targeting to RES organs
ii. Drug targeting to liver
 Enzyme deficiency/ replacement therapy eg.α-Galactosidase
 Treatment of liver tumours eg. Bleomycin, Carboplatin
 Treatment of parasitic diseases eg. Pentamidine, Primaquine
 Removal of RES Iron overload eg.Desferrioxamine
 Removal of toxic agents eg. Rhodanese, Sodium thiosulphate

24. Contd…
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4. Erythrocyte as circulating bioreactors
 Delivery of antiviral agents eg. Azidothymidine
 Macrophage activation eg. Dexamethsone ,
 Thrombolytic therapy eg. aspirin
 Oxygen deficiency therapies eg. Ionositol hexaphosphate
 Delivery of interleukins

25. Novel systems
• Nanoerythrosomes
• Erythrosomes
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Future perspectives
•This concept further needs optimization. A large
number of work is needed so as to utilize the potentials
as in passive as well as active drug targeting
• Genetic engineering aspects can be coupled to give a
newer dimension to existing concept.

26. Conclusion
• During the past decade, numerous have been
proposed for the use of resealed erythrocytes as
carrier for drugs, enzyme replacement therapy etc.
• In near future, erythrocytes based delivery system
with their ability to provide controlled and site
specific drug delivery will revolutionize disease
management.
26

27. References
1. S.P. Vyas and R.K. Khar, Resealed Erythrocytes in Targeted
and Controlled Drug Delivery: Novel Carrier Systems CBS
Publishers and Distributors, India, 2002, p.387–416.
2. Khar RK and Diwan M (2001). Targeted delivery of drugs. In:
Jain NK (editor). Advances in controlled and novel drug
delivery. CBS Publishers and Distributors, New Delhi,
pp.420-456.
3. Shashank Shah, Novel drug delivery carrier: Resealed
Erythrocytes, International Journal of Pharma and Bio
Sciences, vol-I/ Issue -1/Jan-Mar 2011, p. 394-406
27

28. Contd..
4. Gopal V Shavi, Erythrocytes as carrier for
Prednisolone:In vitro and in vivo evaluation, Pak.
J. Pharm. Sci., Vol.23, No.2, April 2010, pp.194-
200
5. A.V.Gothoskar, Resealed Erythrocytes: A Review,
Pharmaceutical Technology March 2004, p. 140-
158
6. Naseem F., Erythrocyte based new drug delivery
system, IJPS, vol.71, nos.2, March-April 2009,
p.162-163
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