RESEALED

ERYTHROCYT
ES
-Mohammedali
Sajun
Sem II
Contents :
 Introduction : Biopharmaceuticals and
Erythrocytes
 Resealed Erythrocytes as carrier
 Methods of Preparation
 Characterization
 Application
 Stability
 Case Study
 References
 Biopharmaceuticals
 What are Biopharmaceuticals..?
– All therapeutic, prophylactic and in-vivo diagnostic agents produced
using living organisms or their functional components.

 Biopharmaceuticals or Pharmaceutical Biotechnology

products include :
– DNA based products -- Vaccines

– Antibodies -- Therapeutics

– Cellular carriers of which RESEALED ERYTHROCYTES is of particular

importance.
Erythrocytes
 Erythro= red
 Cytes = cell

 Biconcave discs,
anucleate.
 Filled with hemoglobin
(Hb), a protein that
functions in gas transport
 Contain the plasma
membrane protein
spectrin and other
proteins that:
– Give erythrocytes their
flexibility
– Allow them to change
shape as necessary
 Erythrocytes
 RBC membrane is made up of
phospholipid bilayer with
channels and pores for transfer
of ions and gases respectively.
 Healthy adult male= 4.5
millions/µml
 Healthy adult female= 4.8
millions/µml
 Matured RBCs have no nucleus
Ribosome & Mitochondria .
Therefore all space is available
for drug carrier.
 Reticulocytes- immature
erythrocytes
 Resealed Erythrocytes

Properties as carrier :-
Appropriate size, shape.
Biocompatible & minimum toxic side effects
Minimum leakage before target site is achieved
Should be able to carry broad spectrum of drugs
Appreciable stability during storage period
Should have sufficient space & should carry adequate
amounts of drugs
 What is meant by
Resealed/Loaded Erythrocytes..?
 Advantages of R.E. :-
 A remarkable degree of biocompatibility, particularly when the autologous cells are used for drug loading.
 Complete biodegradability and the lack of toxic product(s) resulting from the carrier biodegradation.
 Avoidance of any undesired immune responses against the encapsulated drug.
 Considerable protection of the organism against the toxic effects of the encapsulated drug, e.g.,
antineoplasms.
 Remarkably longer life-span of the carrier erythrocytes in circulation in comparison to the synthetic
carriers
 In the optimum condition of the loading procedure, the life-span of the resulting carrier cells may be
comparable to that of the normal erythrocytes.
 An easily controllable life-span within a wide range from minutes to months.
 Desirable size range and the considerably uniform size and shape.
 Protection of the loaded compound from inactivation by the endogenous factors.
 Possibility of targeted drug delivery to the RES organs.
 Advantages of R.E. :-
 Relatively inert intracellular environment .
 Availability of knowledge, techniques, and facilities for handling, transfusion, and working with erythrocytes
 Possibility of an ideal zero-order kinetics of drug release.
 Wide variety of compounds with the capability of being entrapped within the erythrocytes.
 Possibility of loading a relatively high amount of drug in a small volume of erythrocytes, which, in turn,
assures the dose sufficiency in clinical as well as animal studies using a limited volume of erythrocyte
samples.
 Modification of the pharmacokinetic and pharmacodynamic parameters of the drug [14,17,20,22];
 Remarkable decrease in concentration fluctuations in steady state in comparison to the conventional methods
of drug administration which is a common advantage for most of the novel drug delivery systems.
 Considerable increase in drug dosing intervals with drug concentration in the safe and effective level for a
relatively long time.
 Possibility of decreasing drug side effects
 Limitations of R.E. :-
 The major problem encountered in the use of biodegradable materials or natural cells as drug carriers is that
they are removed in vivo by the RES as result of modification that occurred during loading procedure in cells.
This, although expands the capability to drug targeting to RES, seriously limits their life-span as long-
circulating drug carriers in circulation and, in some cases, may pose toxicological problems.
 The rapid leakage of certain encapsulated substances from the loaded erythrocytes.
 Several molecules may alter the physiology of the erythrocyte.
 Given that they are carriers of biological origin, encapsulated erythrocytes may present some inherent
variations in their loading and characteristics compared to other carrier systems.
 The storage of the loaded erythrocytes is a further problem provided that there are viable cells and need to
survive in circulation for a long time upon re-entry to the host body. Conditioning carrier cells in isotonic
buffers containing all essential nutrients, as well as in low temperatures, the addition of nucleosides or
chelators, lyophilization with glycerol or gel immobilization have all been exploited to overcome this problem.
 Possible contamination due to the origin of the blood, the equipment used and the loading environment.
Rigorous controls are required accordingly for the collection and handling of the erythrocytes.
 Intravenous Drug Release Mechanisms :
– Passive diffusion into circulation.
– Specialized membrane associated carriers
– Phagocytosis by RES and release into circulation
– Accumulation of drug in RES and slow release from
this system into circulation
– Haemolysis at injection site(if route is other than I.V.)
 Damaged or aged RBCs that lose their
flexibility/integrity are destroyed in the
 Co-encapsulation of
RES. paramagnetic particles or
Macrophages of the RES where RBCs

are destroyed include:
photosensitive agentsin
– Peritoneal macrophages erythrocytes along with
– Hepatic kupffer cells drug to be targeted
– Alveolar macrophages of lungs
– Peripheral blood monocytes  Application of Ultrasound
– Vascular endothelial cells waves
 Eg.: Glutaraldehyde (membrane stabilizing
agent) added to loaded RBCs to reduces  Site specific antibody
flexibility of the membrane.
 Also coating RBC with antibody makes
attachment to erythrocyte
them more recognizable by RES membrane.
 Source, Fractionation & Isolation of
Erythrocytes

Source:- mice, cattle, pig, dog, sheep, goat, monkey,

chicken, rat, rabbit & human

Whole blood can be collected by venipuncture or from

orbital sinus in heparinized tube

Red blood cells can be harvested & centrifuged

Different centrifugal force & different buffer composition

for different species is used

Fresh blood is used for loading of drugs.

 Membrane Binding
 Avidin-biotin bridges between drug and
erythrocyte membane.
 Thus also called Avidin-biotin technology.
 Avidin-biotin bridges formed by :
– Attachment to amino group by biotin N-
hydrosuccinimide ester, or
– Oxidation of induced aldehyde groups of the
erythrocyte by biotin hydrazide.
 Methods of Encapsulation
Methods Of Drug Loading In Erythrocytes

Membrane perturbation Lipid fusion endocytosis

Electroencapsulation Hypoosmotic lysis

Dilution Preswell Osmotic lyses Dialysis method

method method method
 Membrane perturbation method

Amphotericin B
Drug
Increased permeability
RBC Resealed RBC
e.g. Chemical agents
of RBC
Resealing Buffer
 Electro-insertion or Electro-encapsulation

3.7 Kv Current for

RBC 2.2 Kv Current for
20 micro sec
Drug 20 micro sec

+ + Loaded RBC
At 250 C Loading suspension Isotonic NaCl
Pulsation
medium Resealing Buffer

Resealed RBC

Fig:- Electro-encapsulation Method

 Entrapment By Endocytosis

RBC Buffer containing ATP,

MgCl2, and CaCl2
+ Loaded RBC Resealed RBC
At 250 C
Drug Resealing Buffer
Suspension

Fig;- Entrapment By Endocytos Method

 Effect of change in tonicity on
RBC

Hypertonic Hypotonic
medium medium

Isotonic
medium
 Dilutional Haemolysis
0.4% NaCl Drug
RBC Membrane ruptured RBC Loaded RBC
Hypotonic Loading buffer

Incubation at 250c Resealing

buffer

Hypotonic med

Resealed Loaded RBC

Isotonic med .
Efficiency  1-8% Enzymes
delivery
Washed
 Isotonic Osmotic Lysis
Physical rupturing

RBC Isotonically ruptured

RBC

Drug Isotonic
Chemical Buffer
rupturing

Loaded RBC

Incubation at
250 C

Resealed RBC

Chemical – urea, polyethylene, polypropylene, and NH4Cl

 Preswell Dilutional Haemolysis

5 min incubation Incubation at 25

0.6%w/v NaCl at 0 0c 0
c
Swelled Loaded
RBC Resealed
RBC Drug + Loading RBC RBC
Resealing Buffer
buffer

Efficiency  72% Fig:- Preswell Method

 Dialysis

80 %
Haematocrit
value

RBC
Placed in dialysis bag Dialysis bag placed in 200ml of lysis buffer
+ with air bubble with mechanical rotator 2hrs. 4c.
Phosphate
buffer
Loading Drug
buffer

Resealed RBC Dialysis bag placed in Resealing buffer with Loaded RBC
mechanical rotator 30 min 37c.

Efficiency  30-45%
Characterization
Characterization
 Applications
 R.E. used as carrier for :-
– Drugs
– Enzymes
– Peptides and protiens

 Erythrocytes for drug targeting

– Drug targeting to reticuloendothelial system
– Drug targeting to liver
-Treatment of liver tumors
-Treatment of parasitic diseases
-Removal of RES iron overload
-Removal of toxic agents
 Applications
Delivery of antiviral agents
Oxygen deficiency therapy
Microinjection of macromolecules

Novel systems
Nanoerythrosomes
Erythrosomes
Applications
Applications
Applications
Applications
 Stability
 Membrane stabilizing agents like dimethyl
sulfoxide may be added during preparation.
 However they reduce the circulation time of such
erythrocytes as seen earlier.
 Other approaches for increasing stability include :
– encapsulation of prodrug that undergoes conversion
into parent drug only at body temperature,
– high glycerol freezing technique and
– reversible immobilization in alginate or gelatin gels.
 Marketed Products
 Majority of the R.E. products mentioned in this persentation are in the
clinical stage.

 A couple of examples of marketed formulations are :-

– An antiviral drug : AZEDOTHYMIDINE.
– AN anti-myobacterial drug : ETHAMBUTOL.

 These two drugs are used in mycobacterium avium infection and some
bacterial infections in AIDS patients.

 AZTpEMB , AZTpEMBpAZT , AZTp2EMB are some of the compounds,

synthesized which contain anti-retrovial and antimicrobial activity are
used in the treatment of M.AVIUM infection and in bacterial infections
of AIDS.
 Carmen Lizano, Silvia Sanz, Jose Luque,
– In-vitro and In-vivo studies of glutamate dehydrogenase
encapsulated into mouse erythrocytes by a hypotonic dialysis
method
 Life Science, Vol. 65, No.26, pp.2781-2789, 1999.

 Outcome : Loaded GDH erythrocytescan be used as a potential

carrier system for the in-vivo removal of high levels of ammonium
from blood.

 Glutamate Dehydrogenase  3.8 % (low)

(GDH) entrapment yield :

 Carrier Cell Recovery yield :  56 %

 Mean Cell Haemoglobin  Constant

Content (MCHC) :
 4 different RBC suspensions were
prepared :
– Native control RBCs
– Native control RBCs in the presence of
enzyme (non-specific GDH binding)
– Dialyzed/resealed RBCs (control, unloaded)
– Dialyzed/resealed enzyme loaded RBCs
(loaded)
 Ammonia content determined using
commercial kit from Sigma Diagnostics.
 Method based on reductive amination of 2-
oxoglutarate using GDH and reduced
NADPH
 2-Oxoglutarate + NH4+ + NADPH + H+
GDH

L-Glutamate + NADP+ + H2O

 References
 Mehrdad Hamidi, Adbolhossein Zarrin, Mahshid Foroozesh,
– Applications o carrier erythrocytes in delivery of biopharmaceuticals
 Journal of Controlled Release, 118(2007) 145-160, June 2006.

 Carmen G. Millan, Maria L. Marinero, Jose M. Lanao,

– Drug, enzyme and peptide delivery using erythrocytes as carriers
 Journal of Controlled Release, 95(2004) 27-49, December 2003.

 R. P. Patel, M. J. Patel, N. A. Patel,

– An Overview of Resealed Erythrocyte Drug Delivery
 Journal of Pharmacy Research, 2009, 2(6),1008-1012 .

 Carmen Lizano, Silvia Sanz, Jose Luque,

– In-vitro and In-vivo studies of glutamate dehydrogenase encapsulated into mouse
erythrocytes by a hypotonic dialysis method
 Life Science, Vol. 65, No.26, pp.2781-2789, 1999.
 References
 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.

 Vyas S.P., Khar R.K.,

– Resealed Erythrocytes,
 Targeted And Controlled Drug Delivery: Novel Carrier Systems , New Delhi, CBS
publisher, 2004, 387-413.

 A.V. Gothoskar,
– ‘Resealed Erythrocytes - A Review’,
 Pharmaceutical Technology, March 2004.

 Indian Journal of Pharmaceutical Education & Research Vol. 43(4), Oct-Dec, 2009 , 375-
386

 Journal of Controlled Release 95 (2004) 27– 49

THANK YOU !