presentation on reseald erythrocyte as d drug carrier

1. RESEALED ERYTHROCYTES : AS
DRUG CARRIER
Presented by
Mr. Arjun Bhamare
M.Pharm Second Semister
Dept. of Pharmaceutics.
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2. 2
CONTENT
Introduction
Basic concept of RBC
Drug carrying potential of RBC
Adavantages and Limitations
Source and isolation of RBC
Methods of drug loading
In vitro characterization
Shelf and Storage Stability
Mechanism of Drug release
Applications
Reference

3. • Amongst various carriers vesicular, micro
particulate & cellular carriers meet several
criteria rendering them useful in clinical
applications.
• Erythrocytes have been the most extensively
investigated and found to posses great potential
in novel drug delivery .
• Erythrocytes are loaded with drug/enzymes &
provide target drug delivery system especially in
cancer treatment.
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4.  Erythro= red
 Cytes = cell
 Biconcave discs, anucleate.
 Filled with hemoglobin (Hb),
a protein that functions in
gas transport
 Erythrocyte ghosts: RBC
without hemoglobin
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5. 5
DRUG CARRYING POTENTIAL OF RBC
•The developing RBC has capacity to synthesized hemoglobin however
However, adult RBCs do not have this capacity and serve as carriers for
For hemoglobin
•Cells must be able to release the entrapped drug in a controlled manner
upon reaching the desired target.
•The processing of drug entrapment requires a reversible and transit
permeability change in the membrane, which can be achieved by various
Physical and chemical means.

6. STEPS IN PREPARATION OF RESEALED ERYTHROCYTES
Collection of blood samples (interest)
Separation of erythrocytes from plasma
Entrapping drug in the erythrocytes
Resealing the resultant cellular carriers
Resealed erythrocytes
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7. Biodegradability
with no generation
of toxic products
Wide range of
chemicals can be
entrapped
Ease of circulation
Biocompatibility
probably no changes
of triggered immune
response
Ability to target RES
organs
Advantages
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8. limited potential as
carrier to non-
phagocytic target
tissue
Possibility of Leakage of the
cells and dose dumping may
be there
Disadvantages
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9. Shrinking
Effects of tonicity on RBC’s
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10. SOURCE AND ISOLATION OF RBC’S
 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.
 Fresh whole blood is the blood that is collected and immediately chilled to 4° C
and stored for less than two days.
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11. Drug Loading in Resealed
Erythrocytes
Membrane
Perturbation
Electro
encapsulation
Hypo-Osmotic
Lysis
Entrapment by
Endocytosis
Dilution
method
Dialysis
method
Preswell
method
Osmotic
lysis
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12. Membrane perturbation method
RBC
Amphotericin B
e.g. Chemical agents
Increased
permeability of
RBC
Resealing
Buffer
Drug
Resealed RBC
10
12

13. Electro-insertion or Electro-
encapsulation
RBC 2.2 Kv Current for 20
micro sec
At 250 C
Pulsation
medium
+ +
Drug
Loading suspension
3.7 Kv Current for 20
micro sec
Isotonic NaCl
Loaded RBC
Resealing Buffer
Resealed RBC
Fig:- Electro-encapsulation Method 13

14. DILUTIONAL HAEMOLYSIS
RBC Membrane ruptured RBC Loaded RBC
Resealed Loaded RBC
0.4% NaCl
Hypotonic
Drug
Loading buffer
Resealing
buffer
Incubation at
250c
Efficiency  1-8% Enzymes
delivery
Hypotonic med
Isotonic med.
Washed
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15. PRESWELL DILUTIONAL HAEMOLYSIS
RBC
0.6%w/v NaCl Swelled
RBC Drug + Loading
buffer
5 min incubation at
0 0c
Loaded
RBC
Incubation at 25 0c
Resealing Buffer
Resealed
RBC
Efficiency  72%
Fig:- Preswell Method 15

16. Isotonic Osmotic Lysis
RBC Isotonically ruptured
RBC
Chemical – urea, polyethylene, polypropylene, and NH4Cl
Physical rupturing
Chemical
rupturing
Drug Isotonic
Buffer
Loaded RBC
Resealed RBC
Incubation at 250
C
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17. Entrapment By Endocytosiss
RBC
Drug
Suspension
+
Buffer containing
ATP, MgCl2, and
CaCl2
At 250 C
Loaded RBC
Resealing Buffer
Resealed RBC
Fig;- Entrapment By Endocytosis Method 17

18. APPLICATIONS OF RESEALED ERYTHROCYTES
Slow drug release :Mechanisms proposed for drug release include
 Passive diffusion
 Specialized membrane associated carrier transport
 Phagocytosis
 Accumulation of erythrocytes in lymph nodes
Drug targeting
Targeting RES organs
Targeting the liver: Enzyme deficiency/replacement therapy
Treatment of hepatic tumors
Treatment of parasitic diseases
Removal of RES iron overload
Removal of toxic agents
Targeting organs other than those of RES include
Entrapment of paramagnetic particles along with the drug
Entrapment of photosensitive material
The use of ultrasound waves
Antibody attachment to erythrocyte membrane to get specificity of
action
Delivery of antiviral agent
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19. Design and Characterization of Anticancer Resealed
Erythrocytes
Drug : Methotrexate (MTX)
Category : Folate Antagonist
Problem : A short distribution half-life (1.5–3.5 h)
Short tumor exposure time.
Implication : Acute lymphoblastic leukaemia (ALL)
Choriocarcinoma
Materials & Methods:
Pure Drug: Methotrexate (MTX)
Cross-linking Agents: Glutaraldehyde
Buffer: Phosphate Buffer Solution.
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20. Preparation of erythrocyte suspensions
Human peripheral blood in Hanks PBS solution is centrifuged
(800×g, 10 min., 4° C)
plasma & buffy coat is subsequently removed
RBCs were washed three times in isotonic Hanks-PBS
Last wash
the cell suspension is centrifuged to obtain packed RBCs
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21. ENCAPSULATION OF METHOTREXATE IN ERYTHROCYTES BY
HYPOTONIC HEMOLYSIS
Erythrocytes (Immediately after isolation)
Kept into Hypotonic Solution
Swelling Of erythrocytes
Drug Encapsulated Within erythrocytes
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22. Kept into isotonic solution
Resealed erythrocytes collected by centrifugation
Washing(Hanks-PBS Solution)
Lyophilization
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23. IN VITRO CHARACTERIZATION
1)Scanning Electron Microscopy (SEM)
Gluteraldehyde treated
(biconcave But a slight
Change in their shape)
Unloaded
(biconcave)
DMSO treated
(uniconcave)
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24.  Drug Content
 Osmotic Fragility
 Morphology and Percent Cellular Recovery
 Physical Characterization
 Biological Characterization
- Sterility
- Pyrogenecity
- Animaltoxicity
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Self and Storage Stability of Resealed RBC
The most common storage media is Balance salt
solution
The cell remain viable of their carrier
characteristic for at least 2 weeks
It should store at temperature less than 4 C

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Mechanism of Drug Release
 Passive diffusion.
Specialized membrane associated carrier transport.
Accumulation of Erythrocytes in lymph node subcutaneous
Administration followed by hemolysis to release the drug

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Application of Resealed Erythrocytes
•Erythrocytes as carrier for enzymes
•Erythrocytes as carrier for Drug
•Erythrocytes for Drug targeting
- Drug targeting to reticuloendothelial system
- Drug targeting to liver
-Treatment of liver tumour
- Treatment of parasitic disease
- Treatment of Iron reload

28. References
2. Doijad R.C et al “Design and characterisation of anticancer engineered
resealed
erythrocytes” International journal of pharmaceutical science and
nanotechnology Volume 1 Issue 3 October-December 2008 pp.243-250.
3. Gupta A. et al “Cell based drug delivery system through resealed
erythrocytes – A
review” International journal of pharmaceutical science and drug research
2010
Volume 2 Issue 1 pp.23-30.
4.Gothoskar A. V “Resealed erythrocytes : a Review” Pharmaceutical
technology
March 2004 pp.140-158.
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6. Rathod S.S et al “Design and evaluation of drug loaded erythrocytes”:
International
Journal of PHARMTECH RESEARCH Volume 2 Issue 2 Apr-Jun 2010 pp.1550-
1557.
7. Shah S. “Novel drug delivery carrier: Resealed erythrocytes” International
journal
Pharma and Bio sciences Vol. 2 Issue 1 Jan-Mar 2011 pp.394-406.
8. ss G.I. et al “Asian Journal of Biochemistry Application and Safety of
Erytrocytes
as a Novel Drug Delivery System “ Volume 4 2011 pp.309-321.

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