this presentation contain detail information of resealed erythrocytes from what are the resealed erythrocytes to where they are usesd? basic information like what are RBC and their role, history of resealed erythrocytes, sources and isolation method of erythrocytes,effect of tonicity on RBC's, method of drug loading in erythrocytes, characterization of them, applications

1. RESEALED
ERYTHROCYTES
Semester 2nd graded seminar on:
Presented by :
Jamdhade Vaishali K.
1st Year M. Pharmacy (Pharmaceutics)
Roll No. 5
Guided by :
Dr. Chaudhari Pallavi
Dr. D. Y. Patil college of pharmacy,
Akurdi Pune.
Date: 24/04/2018 1

2. CONTENT
 Introduction
 Advantages and Disadvantages
 Drug carrying potential of erythrocytes
 Sources and isolation
 Effect of tonicity on RBC’s
 Methods of drug loading
 In vitro characterization
 Applications of resealed erythrocytes
 References
2

3. INTRODUCTION
• Blood contains different type of cells like
erythrocytes (RBC), leucocytes (WBC) and platelets,
among them erythrocytes are the most interesting
carrier and posses great potential in drug delivery
due to their ability to circulate throughout the body,
zero order kinetics, reproducibility and ease of
preparation.
• Erythrocytes are loaded with drug/enzymes &
provide target drug delivery system.
3

4. • 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
disease tissue or organ.
4

5. 3
Fig. Composition of blood Fig. RBC
Fig. Composition of erythrocytes
5

6. Erythrocytes are the most abundant cells in the
human body -
5.4 million cells/mm3 blood in a healthy male
and 4.8 million cells/mm3 blood in a healthy
female.
Its crucial role in oxygen delivery to various parts
of the body.
These are biconcave discs with an average diameter
of 7.5 μ, a thickness of 2.5 μ in periphery, 1 μ in the
center and they have life span of about 100-120
day.
6

7. ADVANTAGES
 Biodegradable
 Isolation is easy
 Non immunogenic
 Large volume of drug can be encapsulated in
small volume of erythrocytes
 Prolong systemic activity of drug
 Reduce Adverse Effect
 Peptide & Enzyme Delivery
7

8. DISADVANTAGES
Possibility of Leakage of the cells.
Dose dumping may be there.
Several molecules may alter the physiology of the
erythrocyte.
Liable to biological contamination.
8

9. DRUG CARRYING POTENTIAL OF
ERYTHROCYTES
RBC were discovered in 1658.
The developing RBC has capacity to synthesize
hemoglobin, however, adult RBCs do not have
this capacity and serve as carriers for hemoglobin.
The carrier potentials of these cells was first
realized in early 1970.
The use of cells as drug delivery systems requires
that drug / enzymes which are normally unable to
penetrate the membrane, should be made to
traverse the membrane without causing any
irreversible changes in the membrane structure
and permeability.
9

10. Cells must be able to release the entrapped drug in
a controlled manner upon reaching the desired
target.
The 1st stage in processing of drug entrapment
requires a reversible and transient permeability
change in the membrane, which can be achieved by
various physical and chemical means.
Once loaded these cells can be used either for
carrying the drug to desired site
The erythrocytes recently utilized as carrier for
drugs, enzymes, DNA molecules.
10

11. SOURCES AND ISOLATION
 Sources –
Mouse, Human, Rabbit, Dog, Cow, Goat, Horse, Pig,
Sheep Etc.
 Isolation –
Collection of blood by venipuncture or from orbital sinus
Placed in EDTA, heparin containing tubes. they are used
as anti coagulant
11

12. Effect Of Tonicity On RBC’s12

13. Membrane Perturbation Method
Electro Encapsulation
Endocytosis Method
Hypo-osmotic Lysis
METHOD OF LOADING13

14. Hypo-osmotic
Lysis
Preswell
Method
Dilution
Method
Dialysis
Method
Osmotic-
Lysis
14

15. Dilution Method
Erythrocytes exposed to Hypotonic saline solution
(0.4% NaCl)
Swells until it reaches a critical value of volume or
pressure
Membrane ruptures
15 Hypo-osmotic Lysis

16. Washed erythrocytes (1 volume) treated with loading
material (2-20 volumes) in hypotonic buffer at 0oC for 5
min.
Incubation at 25oC in isotonic solution (0.9% NaCl)
Reseal them with resealing buffer
Membrane ruptures16

17. Dialysis Method
 In this method,
Erythrocyte suspension + Drug solution →
dialysis tube and both ends are tied with
thread.
17

18. This tube is placed in bottle containing 200ml
of swelling solution at stored at 4oC for lysis.
After, the dialysis tube is transferred to 100ml
resealing solution (isotonic PBS, pH 7.4 ) at
room temperature (25o-35oC) for resealing.
These resealed cells are removed and washed
with PBS at 4oC and finally suspended in PBS
solution.
The limitation of dilution method can be
overcome by carrying out lysis and resealing
in the same dialysis tube.
18

19. RBC + Phosphate Buffer
Dialysis bag placed in 200ml of lysis buffer with mechanical
rotator for 2hrs at 40 C
Loaded RBC
Dialysis bag placed in Resealing buffer with mechanical
rotator for 30 min at 25-350C.
Resealed RBC
Placed in dialysis bag with air bubble
19

20. Preswell Method
RBC + Hypotonic solution (0.6% w/v NaCl)
Swelled RBC’s
Drug + Loading buffer incubate at 0oC for 5 min
Loaded RBC
Incubate at 25oC with resealing buffer
Resealed RBC
20

21. 15
Fig. Preswell method
21

22. Osmotic-Lysis Method
RBC
Physical rupturing Chemical rupturing
Isotonically ruptured RBC
Drug + Loading buffer incubate at 0oC for 5 min
22

23. Loaded RBC
Incubate at 25oC with resealing buffer
Resealed RBC
23

24. RBC + Drug Suspension
Use Buffer containing ATP, MgCl2, and CaCl2 At 25o C
Loaded RBC
Incubate at 25oC with resealing buffer
Resealed RBC
Endocytosis24

25. Fig. Endocytosis
25

26. RBC + Drug Suspension
Increased permeability of RBC
Loaded RBC
Incubate at 25oC with resealing buffer
Resealed RBC
Amphotericin B Chemical agent
Membrane Perturbation Method26

27. RBC + Pulsation medium
Drug + Loading suspension
Loaded RBC
Incubate at 25oC with resealing buffer
Resealed RBC
2.2 Kv Current
for 20 micro sec At 25o C
3.7 Kv Current
for 20 micro secIsotonic NaCl
27 Electro-Encapsulation Method

28. Fig. Electro-encapsulation method
28

29. Method
%
Loading
Advantage Disadvantage
Dilution method 1-8
Fastest & simplest
especially for low
molecular weight
drugs
Entrapment efficiency
is very less (1-8%)
Dialysis method 30-45
Better in vitro
survival of
membrane due to
lesser ionic load
Time consuming;
heterogeneous size
distribution of resealed
erythrocytes
Preswell method 20-70
Good retention of
cytoplasm
constituents &
good survival in
vivo
-
Isotonic osmotic
lysis
-
Better in vivo
surveillance
Impermeable to large
molecules , process is
time consuming
COMPARISON OF VARIOUS METHODS
29

30. a) Drug content estimation
b) Percent cell recovery
c) Osmotic fragility
d) Osmotic shock
e) In-vitro drug release and hemoglobin content
f) Erythrocyte sedimentation rate (ESR)
CHARACTERIZATION OF
RESEALED ERYTHROCYTES
30

31. (a) Drug content estimation:
- 0.5ml of loaded cells + 2.0ml of Acetonitrile and
centrifuge at 2500 rpm.
- The supernatant liquid is collected and analyzed for drug
content.
(b) Percent cell recovery:
It is determined by counting no. of intact cells per cubic
mm of packed erythrocytes using laser scattering
(c) Osmotic fragility:
Normal and loaded erythrocytes of drug are incubated
separately in stepwise decreasing % of NaCl solution
(0.9 and 0.1%) at 37oC for 10min and centrifugation at
2000 rpm for 10min. The supernatant examined for drug
and hemoglobin content.
31

32. (d) Osmotic shock:
In this study, 1ml of erythrocyte suspension were diluted
with distilled water of 5ml and centrifuged at 3000rpm for
15min. The supernatent liquid was estimated for hemoglobin
spectrophotometrically.
(e) In-vitro drug release and hemoglobin content:
- Initially collect cell suspension (5% hemotocrit in PBS) and
stored in 4oC in amber colored bottle.
- Periodically the clear supernatant are withdrawn using
hypodermic needle equipped with 0.45μ filter and
deprotinised using methanol and were estimated
for drug content.
% Hemoglobin release =
A540 of sample - A540 of background
A540 of 100% hemoglobin
32
A540 – conventional molar absorbance area coefficient

33. (h) Erythrocyte sedimentation rate (ESR):
- Sedimentation depends upon the size and no. of cells and
relative concentration to the plasma proteins, fibrogen and
α- & s- globulines.
- The test is performed by determination of rate of
sedimentation in the standard tube.
- Normal blood ESR is 0-15mm/hr.
33

34. Drug targeting: these can act as drug carriers and targeting
tool. Used to target organs of mononuclear phagocytic
system/ reticulo endothelial system (RES) because the
changes in the membrane are recognized by macrophages.
Eg: Urease capsules in treatment of kidney failure for
maintenance of serum urea levels.
Targeting RES organs: damaged erythrocytes are rapidly
cleared from circulation of phagocytic Kupffur cells in liver
and spleen.
- Targeting organs like liver and spleen, by modification of
Resealed erythrocyte membrane with various antibiotics,
gluteraldehyde, carbohydrate etc.,
APPLICATION OF RESEALED
ERYTHROCYTES34

35. Treatment of hepatic tumors: Antineoplastic drugs like
Methotrexate, Bleomycin, Asparginase and Adrimycin
delivered by RES
Treatment of parasitic diseases: Antimalarial, antiamoebic
drugs can be delivered.
Removal of RES iron overload: Deferrioxamine, an iron-
chelating drug in erythrocyte ghosts (RBC without Hb) with
a view to promote excretion of iron in patients with excess
body stores.
- Most of the body store of iron is present as intracellular
ferritin and hemosiderin deposits.
35

36. 1. Jain.S., Jain.N.K., resealed erythrocytes as drug
carriers, Edited Jain N.K., Controlled And Novel Drug
Delivery, New Delhi, CBS publishers, New Delhi,
2004, 256-281.
2. Vyas S.P., Khar R.K., Targeted And Controlled Drug
Delivery: Novel Carrier Systems, New Delhi, CBS
publisher, 2004, 387-413.
REFERENCES36

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