Presented by - Mr. S.SHANMUKHA RAJU

BYS.SHANMUKHA RAJU

PHARMACEUTICS 1ST YEAR II nd semester256811886011

Under the guidance ofMr. AJAY KUMAR.V (M.Pharm)

Dept. of Pharmaceutics KGR INSTITUTE OF TECHNOLOGY

AND MANAGEMENT

Resealed Erythrocyte

As Drug Carrier

Content Introduction Erythrocytes as drug carrier Basic concept of erythrocytesAdvantages & limitationSource & Isolation of ErythrocytesMethod of drug Loading CharacterizationDrug release, Storage Application Conclusion

Introduction:-

Current Research

Idea of drug carrier system

Magic Bullet Concept

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

Erythrocytes Erythro= redCytes = 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

flexibilityAllow them to change

shape as necessary

Erythrocytes

Healthy adult male= 4.5 millions/µmlHealthy adult female= 4.8 millions/µml

Matured RBCs have no nucleus Ribosome & Mitochondria . Therefore all space is available for drug carrier.

Reticulocytes- immature erythrocytes

A. Composition of Erythrocytes

Blood contains about 55% of fluid portion(plasma) 45% of corpuscles or formed elements.

Normal blood cells have extensile, elastic , biconcave and non nucleated configuration with a diameter ranging from 6-9 μ and the thickness is nearly 1-2 μ.

Erythrocytes have a solid content of about 35% most of which is Hb and rest 65% being water.

Lipid content of erythrocytes includes cholesterol, lecithin and cephaelins.

B. Electrolyte composition of erythrocytes:

Although qualitatively similar to that of plasma however, quantitatively it differs from that of plasma.

The concentration of K+ is more in erythrocytes and Na+ in plasma.

The osmotic pressure of the interior of the erythrocytes is equal to that of the plasma and termed as isotonic (0.9% NaCl or normal physiological saline.)

Changes in the osmotic pressure of the medium surrounding the red blood cells changes the morphology of the cells.

If the medium is Hypotonic water diffuses into the cells and they get swelled and eventually loose all their haemoglobin content and may burst.

And if the medium is hypertonic,(i.e. higher osmotic pressure than 0.9% NaCl) they will shrink and become irregular in shape.

Balanced ion solutions like Ringer’s and Tyrode’s soln. which are not only isotonic but also contains ions in proper quantity are used in erythrocyte related experiments.

If blood is placed into a tube and centrifuged, the cells and the plasma will separate. The erythrocytes, which are heavy, will settle down to the bottom of the tube, while the plasma rises up to the top and the leukocytes and platelets will form a thin layer (buffy coat) between the erythrocytes and the plasma. The haematocrit is defined as the percentage of whole blood made up of erythrocytes. Males.......... 40-50% Females....... 38-45%

Advantages of erythrocyte as carrier:-Biodegradable Isolation is easy Non immunogenic

large volume of drug can be encapsulated in small volume of erythrocytesProlong systemic activity of drug

• Protection from premature degration• Prodrug concept (Bioreactor)• Reduce Adverse Effect• Peptide & Enzyme Delivery

Disadvantages :-Possibility of Leakage of cells & dose dumping

Molecule Alter Physiology Of cell

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.

Methods Of Drug Loading In Erythrocytes

Membrane perturbation Electroencapsulation Hypoosmotic lysis Lipid fusion endocytosis

Dilution method Preswell method Osmotic lyses method Dialysis method

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

Isotonic Osmotic Lysis

RBC Isotonically ruptured

RBC

Chemical – urea, polyethylene, polypropylene, and NH4Cl

Physical rupturing

Chemicalrupturing

Drug IsotonicBuffer

Loaded RBC

Resealed RBC

Incubation at 250 C

Preswell Dilutional Haemolysis

RBC0.6%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

Dialysis

RBC

Phosphate buffer

+

80 % Haematocrit value

Placed in dialysis bag with air bubble

Dialysis bag placed in 200ml of lysis buffer with mechanical rotator 2hrs. 4c.

DrugLoading buffer

Loaded RBCDialysis bag placed in Resealing buffer with mechanical rotator 30 min 37c.

Resealed RBC

Efficiency 30-45%

Electro-insertion or Electro-encapsulation

RBC 2.2 Kv Current for 20 micro sec

At 250 CPulsation medium

+ +

Drug

Loading suspension

3.7 Kv Current for 20 micro sec

Isotonic NaCl

Loaded RBC

Resealing Buffer

Resealed RBC

Fig:- Electro-encapsulation Method

Entrapment By Endocytosis:-

RBC

Drug Suspension

+

Buffer containing ATP, MgCl2, and CaCl2

At 250 C

Loaded RBC

Resealing Buffer

Resealed RBC

Fig;- Entrapment By Endocytos Method

2)Endocytosis Method

Membrane perturbation method

RBC

Amphotericin B

e.g. Chemical agents

Increased

permeability of RBCResealing Buffer

Drug

Resealed RBC

4)Lipid Fusion Method1

In this method,RBC’s are fused with lipid vessicle containing inositol hexaphosphate.

This provides significant lowering of oxygen affinity for Hb in intact erythrocytes.

Disadv:-Low encapsulation efficiency.Ex:-Tyrosine kinase.

5)Electropration2

Erythrocytes membrane strong external electrical field

Pores induced on membrane

Ex: Enzyme Alcohole aldehyde dehydrogenase,

Diclofenac sodium

In vitro characterization

In vitro characterization

Mechanism of release of resealed erythrocytes

Shelf & storage stability of resealed erythrocytes

In vivo survival & immunological consequences

Some Important Aspects Of Resealed Erythrocytes

Hank’s balance salt solution (HBBS) at 4c for 2 weeks

Oxygenated HBBS containing 1% soft bloom gelatin

Standard blood bag for both encapsulation and storage

Cryopreservation of erythrocytes at liquid nitrogen temp

Storage

Resent drugs which we can encasulateA. Antineoplastic drugs:- 1. Actinomycine DResent trend:-If encapsulation of Actinomycine D-DNA

is perform,complex is retained in cell for longer time. 2. Methotrexate 3. Bleomycine 4. DoxorubicincineResent trend:-RBCs loaded with Doxorubicin HCL

increase half life and bioailability. 5. Etoposide 6. Carboplatin

B. ACE Inhibitors:- Ex:- Enaprilat C. Anti-Infective agents:- 1. Gentamycine 2. Primaquine 3. MetronidazoleD. Systemic Corticosteroids:- Ex:- DexamethasoneE. As a Enzyme carriers:- 1. Alcohole dehydrogenase 2. Aldehyde dehydrogenase 3. Alcohole oxidase 4. L asparginase 5. Uricase 6. Uriase 7. Urokinase

Applications of resealed erythrocytes

Erythrocytes as carrier for enzymes

Erythrocytes as carrier for drugs

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 of resealed erythrocytes

Delivery of antiviral agentsOxygen deficiency therapyMicroinjection of macromolecules

Novel systemsNanoerythrosomesErythrosomes

Recent Developments

Nanoerythrosomes:- These are small vesicles having size as that of

liposome's. Prepared by extrusion of RBC ghosts, having average diameter 100nm.Spheroidal,appear to be stable, and maintain both cytotoxic and antineoplastic activity.

ConclusionResealed Erythrocytes is found to be most promising targeted

drug deliery system to Reticuloendothelial system.Due to non antigenic and non immunogenic nature,it is well

compatable with human immune system.It is found to be good carrier for various biomolecules like

Enzymes,Peptides etc.Though use of Resealed Erythrocytes is promising,much area

is found to be unexploerd and technology to develop this dosage form is not economical which increase the cost of dosage form.

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

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

Journal of Controlled Release 95 (2004) 27– 49

Any Questions or Suggestions?

Any Questions or Suggestions?