RESEALED RESEALED ERYTHROCYTERYTHROCYT

ESES

-Mohammedali SajunMohammedali Sajun

Sem II Sem II

Contents :Contents : Introduction : Biopharmaceuticals and Introduction : Biopharmaceuticals and

ErythrocytesErythrocytes Resealed Erythrocytes as carrierResealed Erythrocytes as carrier Methods of PreparationMethods of Preparation CharacterizationCharacterization ApplicationApplication Stability Stability Case StudyCase Study ReferencesReferences

BiopharmaceuticalsBiopharmaceuticals

What are Biopharmaceuticals..?What are Biopharmaceuticals..?

– All therapeutic, prophylactic and in-vivo diagnostic agents All therapeutic, prophylactic and in-vivo diagnostic agents

produced using living organisms or their functional components.produced using living organisms or their functional components.

Biopharmaceuticals or Pharmaceutical Biotechnology Biopharmaceuticals or Pharmaceutical Biotechnology

products include :products include :

– DNA based productsDNA based products -- Vaccines-- Vaccines

– AntibodiesAntibodies -- Therapeutics-- Therapeutics

– Cellular carriers of which Cellular carriers of which RESEALED ERYTHROCYTESRESEALED ERYTHROCYTES is of is of

particular importance.particular importance.

Erythrocytes Erythrocytes Erythro= redErythro= red Cytes = cellCytes = cell

Biconcave discs, Biconcave discs, anucleate.anucleate.

Filled with hemoglobin Filled with hemoglobin (Hb), a protein that (Hb), a protein that functions in gas functions in gas transporttransport

Contain the plasma Contain the plasma membrane protein membrane protein spectrin and other spectrin and other proteins that:proteins that:– Give erythrocytes their Give erythrocytes their

flexibilityflexibility– Allow them to change Allow them to change

shape as necessaryshape as necessary

Erythrocytes Erythrocytes

RBC membrane is made up of RBC membrane is made up of phospholipid bilayer with phospholipid bilayer with channels and pores for transfer channels and pores for transfer of ions and gases respectively.of ions and gases respectively.

Healthy adult male= 4.5 Healthy adult male= 4.5 millions/µmlmillions/µml

Healthy adult female= 4.8 Healthy adult female= 4.8 millions/µmlmillions/µml

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

ReticulocytesReticulocytes- immature - immature erythrocyteserythrocytes

Resealed ErythrocytesResealed Erythrocytes

Properties as carrier :-Properties as carrier :-

Appropriate size, shape.Appropriate size, shape.

Biocompatible & minimum toxic side effectsBiocompatible & minimum toxic side effects

Minimum leakage before target site is achievedMinimum leakage before target site is achieved

Should be able to carry Should be able to carry broad spectrumbroad spectrum of drugs of drugs

Appreciable Appreciable stabilitystability during storage period during storage period

Should have sufficient space & should carry Should have sufficient space & should carry adequate amounts of drugsadequate amounts of drugs

What is meant by What is meant by Resealed/Loaded Resealed/Loaded Erythrocytes..?Erythrocytes..?

Advantages of R.E. :-Advantages of R.E. :- A remarkable degree of A remarkable degree of biocompatibilitybiocompatibility, particularly when the autologous cells are used for drug , particularly when the autologous cells are used for drug

loading.loading.

Complete Complete biodegradabilitybiodegradability and the lack of toxic product(s) resulting from the carrier and the lack of toxic product(s) resulting from the carrier

biodegradation.biodegradation.

Avoidance of any Avoidance of any undesired immune responsesundesired immune responses against the encapsulated drug. against the encapsulated drug.

Considerable protection of the organism against the toxic effects of the encapsulated drug, e.g., Considerable protection of the organism against the toxic effects of the encapsulated drug, e.g.,

antineoplasms.antineoplasms.

Remarkably Remarkably longer life-spanlonger life-span of the carrier erythrocytes in circulation in comparison to the of the carrier erythrocytes in circulation in comparison to the

synthetic carrierssynthetic carriers

In the optimum condition of the loading procedure, the life-span of the resulting carrier cells may In the optimum condition of the loading procedure, the life-span of the resulting carrier cells may

be comparable to that of the be comparable to that of the normal erythrocytesnormal erythrocytes..

An easily controllable life-span within a wide range from minutes to months.An easily controllable life-span within a wide range from minutes to months.

Desirable Desirable size range size range and the considerably and the considerably uniform size and shapeuniform size and shape..

Protection of the loaded compound from Protection of the loaded compound from inactivationinactivation by the endogenous factors. by the endogenous factors.

Possibility of Possibility of targettargeted drug delivery to the ed drug delivery to the RES organsRES organs..

Advantages of R.E. :-Advantages of R.E. :- Relatively Relatively inert intracellular environmentinert intracellular environment..

Availability of knowledge, techniques, and facilities for handling, transfusion, and working with Availability of knowledge, techniques, and facilities for handling, transfusion, and working with

erythrocyteserythrocytes

Possibility of an Possibility of an ideal zero-order kinetics ideal zero-order kinetics of drug release.of drug release.

Wide variety of compounds with the capability of being entrapped within the erythrocytes.Wide variety of compounds with the capability of being entrapped within the erythrocytes.

Possibility of Possibility of loadingloading a relatively a relatively high amounthigh amount of drug in a small volume of erythrocytes, which, in 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 turn, assures the dose sufficiency in clinical as well as animal studies using a limited volume of

erythrocyte samples.erythrocyte samples.

Modification of the pharmacokinetic and pharmacodynamic parameters of the drug [14,17,20,22];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 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 conventional methods of drug administration which is a common advantage for most of the novel

drug delivery systems.drug delivery systems.

Considerable increase Considerable increase in drug dosing intervals in drug dosing intervals with drug concentration in the safe and effective with drug concentration in the safe and effective

level for a relatively long time.level for a relatively long time.

Possibility of Possibility of decreasing drug side effectsdecreasing drug side effects

Limitations of R.E. :-Limitations of R.E. :- The major problem encountered in the use of biodegradable materials or natural cells as drug carriers is The major problem encountered in the use of biodegradable materials or natural cells as drug carriers is

that they are that they are removed in vivo by the RESremoved in vivo by the RES as result of modification that occurred during loading procedure 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 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.long-circulating drug carriers in circulation and, in some cases, may pose toxicological problems.

The The rapid leakage rapid leakage of certain encapsulated substances from the loaded erythrocytes.of certain encapsulated substances from the loaded erythrocytes.

Several molecules may Several molecules may alter the physiology of the erythrocytealter the physiology of the erythrocyte..

Given that they are carriers of biological origin, encapsulated erythrocytes may present some inherent 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.variations in their loading and characteristics compared to other carrier systems.

The The storagestorage of the loaded erythrocytes is a further problem provided that there are viable cells and need 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 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 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 chelators, lyophilization with glycerol or gel immobilization have all been exploited to overcome this

problem.problem.

Possible Possible contaminationcontamination due to the origin of the blood, the equipment used and the loading environment. 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.Rigorous controls are required accordingly for the collection and handling of the erythrocytes.

Intravenous Drug Release Mechanisms :Intravenous Drug Release Mechanisms :– Passive diffusion into circulation.Passive diffusion into circulation.– Specialized membrane associated carriersSpecialized membrane associated carriers– Phagocytosis by RES and release into Phagocytosis by RES and release into

circulationcirculation– Accumulation of drug in RES and slow release Accumulation of drug in RES and slow release

from this system into circulationfrom this system into circulation– Haemolysis at injection site(if route is other Haemolysis at injection site(if route is other

than I.V.)than I.V.)

Damaged or aged RBCs that lose Damaged or aged RBCs that lose their flexibility/integrity are their flexibility/integrity are destroyed in the RES.destroyed in the RES.

Macrophages of the RES where Macrophages of the RES where RBCs are destroyed include:RBCs are destroyed include:– Peritoneal macrophagesPeritoneal macrophages– Hepatic kupffer cellsHepatic kupffer cells– Alveolar macrophages of lungsAlveolar macrophages of lungs– Peripheral blood monocytesPeripheral blood monocytes– Vascular endothelial cellsVascular endothelial cells

Eg.: Glutaraldehyde (membrane Eg.: Glutaraldehyde (membrane stabilizing agent) added to loaded stabilizing agent) added to loaded RBCs to reduces flexibility of the RBCs to reduces flexibility of the membrane.membrane.

Also coating RBC with antibody makes Also coating RBC with antibody makes them more recognizable by RES them more recognizable by RES

Co-encapsulation of Co-encapsulation of paramagnetic particles paramagnetic particles or photosensitive or photosensitive agentsin erythrocytes agentsin erythrocytes along with drug to be along with drug to be targetedtargeted

Application of Application of Ultrasound wavesUltrasound waves

Site specific antibody Site specific antibody attachment to attachment to erythrocyte membrane.erythrocyte membrane.

Source, Fractionation & Source, Fractionation & Isolation of ErythrocytesIsolation of Erythrocytes

Source:- mice, cattle, pig, dog, sheep, goat, Source:- mice, cattle, pig, dog, sheep, goat, monkey, chicken, rat, rabbit & humanmonkey, chicken, rat, rabbit & human

Whole blood can be collected by venipuncture or Whole blood can be collected by venipuncture or from orbital sinus in heparinized tubefrom orbital sinus in heparinized tube

Red blood cells can be harvested & centrifugedRed blood cells can be harvested & centrifuged

Different centrifugal force & different buffer Different centrifugal force & different buffer composition for different species is usedcomposition for different species is used

Fresh blood is used for loading of drugs.Fresh blood is used for loading of drugs.

Membrane BindingMembrane Binding

Avidin-biotin bridges between drug Avidin-biotin bridges between drug and erythrocyte membane.and erythrocyte membane.

Thus also called Avidin-biotin Thus also called Avidin-biotin technology.technology.

Avidin-biotin bridges formed by :Avidin-biotin bridges formed by :– Attachment to amino group by biotin N-Attachment to amino group by biotin N-

hydrosuccinimide ester, orhydrosuccinimide ester, or– Oxidation of induced aldehyde groups of Oxidation of induced aldehyde groups of

the erythrocyte by biotin hydrazide. the erythrocyte by biotin hydrazide.

Methods of Methods of EncapsulationEncapsulation

Methods Of Drug Loading In ErythrocytesMethods Of Drug Loading In Erythrocytes

Membrane perturbation

Electroencapsulation

Hypoosmotic lysis

Lipid fusion endocytosis

Dilution method

Preswell method

Osmotic lyses

method

Dialysis method

Membrane perturbation methodMembrane perturbation method

RBC

Amphotericin B

e.g. Chemical agents

Increased permeability of

RBC Resealing Buffer

Drug

Resealed RBC

Electro-insertion or Electro-Electro-insertion or Electro-encapsulationencapsulation

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 EndocytosisEntrapment By Endocytosis

RBC

Drug Suspension

+

Buffer containing ATP, MgCl2, and

CaCl2At 250 C

Loaded RBC

Resealing Buffer

Resealed RBC

Fig;- Entrapment By Endocytos Method

Effect of change in tonicity Effect of change in tonicity on RBCon RBC

Hypertonic medium

Isotonic medium

Hypotonic medium

Dilutional HaemolysisDilutional 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 LysisIsotonic 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 Preswell Dilutional HaemolysisHaemolysis

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

DialysisDialysis

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%

CharacterizationCharacterization

CharacterizationCharacterization

ApplicationsApplications

R.E. used as carrier for :-R.E. used as carrier for :-– DrugsDrugs– EnzymesEnzymes– Peptides and protiensPeptides and protiens

Erythrocytes for drug targetingErythrocytes for drug targeting– Drug targeting to reticuloendothelial systemDrug targeting to reticuloendothelial system– Drug targeting to liverDrug targeting to liver

-Treatment of liver tumors-Treatment of liver tumors-Treatment of parasitic diseases-Treatment of parasitic diseases-Removal of RES iron overload-Removal of RES iron overload-Removal of toxic agents-Removal of toxic agents

ApplicationsApplications

Delivery of antiviral agentsDelivery of antiviral agents

Oxygen deficiency therapyOxygen deficiency therapy

Microinjection of macromoleculesMicroinjection of macromolecules

Novel systemsNovel systems

NanoerythrosomesNanoerythrosomes

ErythrosomesErythrosomes

ApplicationsApplications

ApplicationsApplications

ApplicationsApplications

ApplicationsApplications

StabilityStability

Membrane stabilizing agents like dimethyl Membrane stabilizing agents like dimethyl sulfoxide may be added during preparation.sulfoxide may be added during preparation.

However they reduce the circulation time of However they reduce the circulation time of such erythrocytes as seen earlier.such erythrocytes as seen earlier.

Other approaches for increasing stability Other approaches for increasing stability include :include :– encapsulation of prodrug that undergoes conversion encapsulation of prodrug that undergoes conversion

into parent drug only at body temperature, into parent drug only at body temperature, – high glycerol freezing technique andhigh glycerol freezing technique and– reversible immobilization in alginate or gelatin gels.reversible immobilization in alginate or gelatin gels.

Marketed ProductsMarketed Products

Majority of the R.E. products mentioned in this persentation Majority of the R.E. products mentioned in this persentation are in the clinical stage.are in the clinical stage.

A couple of examples of marketed formulations are :-A couple of examples of marketed formulations are :-– An antiviral drug : AZEDOTHYMIDINE.An antiviral drug : AZEDOTHYMIDINE.– AN anti-myobacterial drug : ETHAMBUTOL.AN anti-myobacterial drug : ETHAMBUTOL.

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

AZTpEMB , AZTpEMBpAZT , AZTp2EMB are some of the AZTpEMB , AZTpEMBpAZT , AZTp2EMB are some of the compounds, synthesized which contain anti-retrovial and compounds, synthesized which contain anti-retrovial and antimicrobial activity are used in the treatment of M.AVIUM antimicrobial activity are used in the treatment of M.AVIUM infection and in bacterial infections of AIDS.infection and in bacterial infections of AIDS.

Glutamate Dehydrogenase Glutamate Dehydrogenase (GDH) entrapment yield :(GDH) entrapment yield :

Carrier Cell Recovery yield :Carrier Cell Recovery yield :

Mean Cell Haemoglobin Mean Cell Haemoglobin Content (MCHC) :Content (MCHC) :

3.8 % (low)3.8 % (low)

56 %56 %

ConstantConstant

Carmen Lizano, Silvia Sanz, Jose LuqueCarmen Lizano, Silvia Sanz, Jose Luque,,– In-vitro and In-vivo studies of glutamate In-vitro and In-vivo studies of glutamate

dehydrogenase encapsulated into mouse erythrocytes dehydrogenase encapsulated into mouse erythrocytes by a hypotonic dialysis methodby a hypotonic dialysis method

Life Science, Vol. 65, No.26, pp.2781-2789, 1999.Life Science, Vol. 65, No.26, pp.2781-2789, 1999.

Outcome : Loaded GDH erythrocytescan be used as a Outcome : Loaded GDH erythrocytescan be used as a potential carrier system for the in-vivo removal of high potential carrier system for the in-vivo removal of high levels of ammonium from blood.levels of ammonium from blood.

4 different RBC suspensions were 4 different RBC suspensions were prepared :prepared :– Native control RBCsNative control RBCs– Native control RBCs in the presence of Native control RBCs in the presence of

enzyme (non-specific GDH binding)enzyme (non-specific GDH binding)– Dialyzed/resealed RBCs (control, Dialyzed/resealed RBCs (control,

unloaded)unloaded)– Dialyzed/resealed enzyme loaded RBCs Dialyzed/resealed enzyme loaded RBCs

(loaded)(loaded)

Ammonia content determined using Ammonia content determined using commercial kit from Sigma Diagnostics.commercial kit from Sigma Diagnostics.

Method based on reductive amination Method based on reductive amination of 2-oxoglutarate using GDH and of 2-oxoglutarate using GDH and reduced NADPHreduced NADPH 2-Oxoglutarate + NH2-Oxoglutarate + NH44

+ + + NADPH + H+ NADPH + H+ +

GDHGDH

L-Glutamate + NADPL-Glutamate + NADP++ + H + H22OO

ReferencesReferences Mehrdad Hamidi, Adbolhossein Zarrin, Mahshid Foroozesh,Mehrdad Hamidi, Adbolhossein Zarrin, Mahshid Foroozesh,

– Applications o carrier erythrocytes in delivery of biopharmaceuticalsApplications o carrier erythrocytes in delivery of biopharmaceuticals Journal of Controlled Release, 118(2007) 145-160, June 2006.Journal of Controlled Release, 118(2007) 145-160, June 2006.

Carmen G. Millan, Maria L. Marinero, Jose M. Lanao,Carmen G. Millan, Maria L. Marinero, Jose M. Lanao,– Drug, enzyme and peptide delivery using erythrocytes as carriersDrug, enzyme and peptide delivery using erythrocytes as carriers

Journal of Controlled Release, 95(2004) 27-49, December 2003.Journal of Controlled Release, 95(2004) 27-49, December 2003.

R. P. Patel, M. J. Patel, N. A. Patel,R. P. Patel, M. J. Patel, N. A. Patel,– An Overview of Resealed Erythrocyte Drug DeliveryAn Overview of Resealed Erythrocyte Drug Delivery

Journal of Pharmacy Research, 2009, 2(6),1008-1012Journal of Pharmacy Research, 2009, 2(6),1008-1012 ..

Carmen Lizano, Silvia Sanz, Jose Luque,Carmen Lizano, Silvia Sanz, Jose Luque,– In-vitro and In-vivo studies of glutamate dehydrogenase encapsulated In-vitro and In-vivo studies of glutamate dehydrogenase encapsulated

into mouse erythrocytes by a hypotonic dialysis methodinto mouse erythrocytes by a hypotonic dialysis method Life Science, Vol. 65, No.26, pp.2781-2789, 1999.Life Science, Vol. 65, No.26, pp.2781-2789, 1999.

ReferencesReferences Jain.S., Jain.N.K., Jain.S., Jain.N.K.,

– Resealed Erythrocytes as drug carriers, Resealed Erythrocytes as drug carriers, Edited Jain N.K., Edited Jain N.K., Controlled And Novel Drug DeliveryControlled And Novel Drug Delivery, New Delhi, , New Delhi,

CBS publishers, New Delhi, 2004, 256-281.CBS publishers, New Delhi, 2004, 256-281.

Vyas S.P., Khar R.K., Vyas S.P., Khar R.K., – Resealed Erythrocytes,Resealed Erythrocytes,

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

A.V. Gothoskar, A.V. Gothoskar, – ‘‘Resealed Erythrocytes - A Review’,Resealed Erythrocytes - A Review’,

Pharmaceutical Technology, March 2004.Pharmaceutical Technology, March 2004.

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

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

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