RESEALED ERYTHROCYTE

S.Brito Raj , M.PharmSri Venkateswara

College of Pharmacy, RVS nagar , Chittoor

Targeted drug delivery – Magic bullet concept.

Cellular carriers – clinical applications

Biocompatibility carrier

Erythrocytes : possess greater potential in

drug delivery.

INTRODUCTION

ERYTHROCYTE

Erythro – red : cytes-cell

Erythrocytes (RBCs) contain oxygen carrying ptn

hemoglobin, which is a pigment that gives whole blood its

red color.

Healthy adult male = 4.5 million RBCs/ µl of blood

Healthy adult female = 4.8 million RBCs/ µl of blood

Because matured RBCs have no nucleus, all their internal

space is available for oxygen transport.

As they lack mitochondria and generate ATP anaerobically,

they do not use up any oxygen they transport.

Erythrocytes

Normal erythrocyte (Normocyte) - flexible, elastic,

biconcave, nucleated structure with a mean diameter of 7.3

µm.

Chemical constituents of RBC’s

Water – 63%/Lipids – 0.5%

Glucose – 0.8%

Minerals – 0.7%

Non-heamoglobin protein – 0.9%

Methehemoglobin – 0.5%

Hemoglobin – 33.67%

Erythrocytes

Erythrocyte – carrier for oxygen bound to hemoglobin.

Hemoglobin content is about 29

picogram/erythrocyte.

Lack of nucleus , ribosome's & mitochondria

Life span – 120 days.

Enumerated by either visual or electronic procedure.

Visual : cells counted in a hemocytometer.

Hayen’s solution or toison’s solution.

Resealed erythrocytes

Some of hemoglobin is lost & other cellular

constituents are retained , the cells on resealing

lose some of the properties of normal erythrocytes

& are referred as Resealed Erythrocytes

Drug loading in body own erthro when used to

serve as controlled dds

Contain equal amount of protein & lipid.

Outer membrane – negative charge due to

carboxyl groups of salicylic acid.

Encapsulation

Variety of biologically active substances 5000-

600,000 daltons in size .

Molecules should be polar or hydrophillic.

Erythrocyte membrane

Properties

Appropriate size & shape

Possess specific physico-chemical

properties

Biocompatible & minimum toxic side effects

Minimum leakage of drug

Drug should released at the target site in a

controlled manner

Carrier system should have an appreciable

stability during storage

ADVANTAGES

Natural product of the body

Isolation of erythrocytes is easy

Non- immunogenic in action

Targeted to disease organ/tissue/RES

Prolong the systemic activity of drug

Protect premature degradation, inactivation & excretion

of proteins, enzymes & act as carrier for number of drugs

Biodegradable & biocompatible

Circulate in IV for days/Circulatory drug depot

Advantages

Enzymatic inactivation

Erythrocytes have been proposed as carriers for a

wide range of bioactive components including

drugs, enzymes , pesticides, DNA molecules

does not require chemical modification of the

substance to be entrapped. This is in contrast

with other system which involve covalent

coupling of the drug and carrier which may

affect the inherent biological activity of the

parent drug

Advantages

Large amount of drug is encapsulated in small

volume of cell

Serving as circulatory biovectors for enzymes

Versatile carriers in modern pharmaceutical

research ad development

Reduce Adverse effect

Peptide and Enzyme delivery

Non immunogenic

Facilitate incorporation of proteins and

nucleic acid in eukaryotic cells by cell infusion

with RBC

Disadvantages of RE

Small changes in RBC biochemistry – changes the

circulation lifetime RE

Limited potential as carrier to non-phagocytic

target tissue

Possibility of clumping of cells and dose dumping

Permeable to large no. of drugs

Unstable invitro even under the best condition

Problem on long term storage

Limitations

Only bioactives are non-susceptible to

denaturation under hypotonic condition

Molecules alter physiology of cell

Limited potential as carrier to non-phagocytic

target tissue.

Possibility of clumping of cells

Dose dumping

Properties of RE

It should be of appropriate size & shape to permit

the passage through capillaries

Specific physico chemical properties - desired target

site

Biocompatible & physicochemically compatible

Minimum toxic side effect & Minimum leaching of

drug

Target and controlled

Ability to carry a broad spectrum of drugs with

different properties

Appreciable stability during storage

Ease to prepare

Requirement of encapsulation

A wide variety of biologically active

substancea(5000-600,000 daltons)can be

encapsulated

Generally the molecules should be polar or

hydrophilic

The hydrophobic molecules can be entrapped by

absorbing over other molecule

The pore opening size is limited to 400-500A

Non polar molecule in salt form is entrapped

X Molecules which interact with the membrane

and cause deletorious effects on membrane

structure

Isolation of erythrocytes

1. Source: mice, cattle, dog, goat , monkey, rat,rabbit &

Human

2. Fresh Blood is collected into heparinized tubes by

venipuncture+ EDTA or heparin

3. Blood is withdrawn from cardiac/splen (in small animals)

and through veins (in large animals) in a syringe

containing a drop of anti coagulant.

4. Immediately chilled to 4˚c & stored for NMT 2 days

5. Centrifuged - 2500 rpm/5 min/4± 1˚c= refrigerated

centrifuged

6. Serum & buffy coats are carefully removed

7. Packed cells washed 3 times with PBS (pH = 7.4)

8. Then are diluted with PBS and stored a 4±1˚ c

Criteria of selection of drug

Water solubility

Resistance against degradation within erythrcyte

Lack of physical and chemical interaction with

erythrocyte membrane

Well defined pharmacokinetic and

pharmacodynamic properties

Methods for entrapmentDrug Loading in

RE

Preswell method

Dialysis method

Dilution method

Lipid fusion

Endocytosis

Hypo-osmotic

lysis

Electro encapsulatio

n

Membrane perturbatio

n

Osmotic lysis

Hypotonic hemolysis & isotonic method

NOTE: Surface modified with cross linking polymer

Red cells placed in drug solution

Swells & lysed by osmotic & electric shock Spherocyte

> 0.9 % Nacl Isotonic, Glutaraldehyde coating

Hypotonic< 0.9 % or 0.45 Nacl

RBC resealed

•incubation at 37 ˚C for 30-40 min•Washed with isotonic buffer 3 times to remove unentrapped components

Biconcave discocytes

+ 1.54 KCl

Dilution method

NOTE: Efficiency-1-8%, low entrapment Simplest & fastest method Low molecular weight Most of the cell content lost by osmotic lysis

1.Red cells + drug solution (macro)

3.Swells Spherocyte

5.0.9 % Nacl Isotonic, Glutaraldehyde coating

2.Hypotonic 0.4 % Nacl

RE

6.Incubation at 37 ˚C for 30-40 minWashed with isotonic buffer 3 times to remove unentrapped components

Biconcave discocytes + 1.54 KCl,25˚ c

4.Pressure/Rupture/External to internal

0˚ c/5 min

Presswell Dilutional Haemolysis(without sudden

lysis)

RBC

0.6% Nacl

Preswelling Swollen cell

H2O ,increased hypotonicity Hypotonic

1:5 ratio

Hypotonic buffer + drug

10 min/o˚c

Hemolysis

Pores recovery

Hypertonicsolution

RE

Presswell Dilutional Haemolysis(without lysis)

Advantages

Gentle swelling – cells good retention of cytoplasmic

constituents

Good survival- invivo

More encapsulation efficiency(72%)

Isotonic osmotic method

Used to avoid the disadvantages by hypotonic medium

Haemolysis by both physical & chemical means

A. Conventional haemolysis in isotonic urea solutions

B. PG induced haemolysis by transient permeability

= resealed with diluting with PG free buffer medium

C. Ammonium chloride induced haemolysis

Adv: Better in vivo surveillance

DA: Impermeable only to large molecules

Process is time consuming

Erythrocyte Dialysis

• Developed due to low entrapment efficiency of dilution method

• Principle: SPM dialysis membrane increase the Intra: extracellular

volume ratio & entrapment of bioactive (30 – 45%)

• High Hematocrit value

• Successful encapsulation of I125 albumin

• Better in vivo survival

• Good structural integrity

• No homogenous size distribution

Dialysis method3 steps for Haemolysis & resealing

Electro insertion or electro encapsulation method

Erythrocyte membrane lysed- Dielectric background

Entrapped = electric pulse of greater than a threshold

voltage of 2kv/cm applied for 20microsecond

Resealed by incubation at 37˚C in osmotic balanced medium

Principle: electrically induced permeability changes at

high membrane potential difference

Electric breakdown is evident when the membrane is

polarized for microsecond using varying voltage

Electromechanical compression of membrane after

breakdown leads to formation of pores

Electro insertion or electro encapsulation method

Extent of pore formation = electric field strength,

pulse duration & ionic strength of the suspending

medium

once the membrane is perforated, regardless of the size of

pores, ions rapidly distributed between the intracellular

and extracellular space to attain donan equilibrium

Colloidal osmotic pressure for Lysis of Haemoglobin (30

osm) this pressure drives water and ion influx as a result

leads to swelling of cells

Membrane rupture cell volume reach 155%= original

volume

Prevent lysis and balance colloidal osmotic pressure

by + macromolecules like BSA

Under this osmotic balance ,pores are stay opened at 4˚c

for 2 days

Drug from drug solution germinate into the cell

3 steps

1.washed erythrocyte (Haematocrit 10-20%) + pulsation

medium

(isotonic saline 150mM,isoosmotic sucrose solution

300mM)

4˚c 0.15 ml of

erythrocyte suspension

2.high voltage pulsation device

Single electric pulse 2.2kV/cm+20µsec/25˚c

osmotic balance + sachraides and proteins 7kV/cm,20µsec electric pulse 3:7 (isotonic Nacl

& Isoosmotic sucrose)

Electrical perforated erythrocyte

cell suspension precooled tube / 4˚c

3.Resealing by incubation at 37˚c in an osmotically

balanced medium

Advantages

Excellent invivo performance

Normal haemoglobin properties were retained

Enclose proteins

Prolonged release

No mebranolytic or deleterious effect

Disadvantages

Time consuming

Costly

MEMBRANE PERTURBATION METHOD

permeability by chemical agents (halothane)

Low molecular weight substance entrapment

Observation = permeability of the erythrocytic membrane is

increased, when it is exposed to some chemical agents.

Antibiotics such as amphotericin B damage microorganisms by

increasing the permeability of their membrane to metabolites and

ions. This property could be exploited for loading of drug into

erythrocytes.

Amphotericin B interacts with the cholesterol of plasma

membrane of eukaryotic cells causing change in permeability of

the membrane.

Endocytosis method

Erythrocyte Drug Drug loaded erythrocyte

ERYTHROCYTE ENDOCYTOSIS PRODUCED BY CATIONS

AND TRAPPING OF MOLECULES IN THE INVAGINATION

OR INSIDE OUT ENDOCYTOSIS VACUOLES

Endocytosis method

principle: drug entrapment in erythrocyte ghost by

endocytosis

the vesicle membrane seperates the endocytosed

substance from the cytoplasm, which may shelter drug

prone to inactivation in erythrocyte or alternatively

protects the erythrocyte from the drug

the swollen ghost with endocytic vacuole (>0.5µ

diameter)

1.washed packed erythrocyte+ buffer (ATP,Mgcl2,CaCl2) with drugincubated for 2 min/

room temperature

2.resealing by + Nacl/

incubation 2 min / 37˚C,

washed in 5mM imidazole-glycylglycine buffer+154 mM Nacl pH

7.4

30 min/37˚C

3.entrapment of drug by endocytosis

 

3 steps in Endocytosis

Characterization parameters - REPhysical parameters

Method/instrument used

1.Physical characteristics

Shape & surface morphology

TEM/SEM/Phase contrast microscopy/optical microscopy

Vesicle size & size distribution

TEM/optical microscopy

Drug release Diffusion cell/dialysis

Drug content Deproteinization of cell membrane followed by assay of resealed drug/radiolabelling

Surface electrical potential

Zeta potential measurement

Deformability Capillary method

3. Biological characteristics

Sterility Sterility test

Pyrogenicity Rabbit method, LAL test

Animal toxicity Toxicity test

2. Cellular characteristics

% Hb content Deproteinization of cell membrane followed by hemoglobin assay

Cell volume Laser light scatering

% Cell recovery Neubaur’s chamber/haematological analyzer

Osmotic fragility

Stepwise incubation with isotonic to hypotonic saline solution and determination of drug and haemoglobin assay

Osmotic shock Dilution with distilled water and estimation of drug and haemoglobin

Turbulent shock Passage of cell suspension through 30-gauge hypodermic needle at 10 mL/min flow rate and estimation of residual drug and haemoglobin, vigorous shaking followed by haemoglobin estimation

Erythrocyte sedimentation rate

ESR method

In vitro characteristicsDrug content

Packed loaded ER (0.5 ml)

Deproteinized + acetonitrile (2ml)

Centrifugation

2500 rpm for 10 min

Clear supernatant (drug) /cell settled

Analyzed for the drug content using

spectrophotometrically

In vitro haemoglobin content

Normal & loaded ER (50%) Incubated 37˚C in PO4 buffer pH 7.4

Metabolic rotating wheel incubator

bath

Sample hypodermic syringe 0.8µ spectropore membrane

filter

Deproteinized (acetonitrile)

Estimate the amount of drug release

% haemoglobin at 540 nm spectrophotometrically

% haemolysis – comparing the absorbance of supernatant &

absorbance of complete hydrolysis of same number of cells in

distilled water

% hamoglobin release = A540 of sample – A 540 of background

A540 of 100% heam

Mean corpuscular Heam = haemoglobin (g/100ml) x10

erythrocyte count (per mm3

Osmotic fragility

simulates and mimics the bio-environment conditions that are encountered on invivo administration

in vitro handling and the effect of loaded contents on

the survival rates of the erythrocytes. RBChypotonic hypertonic

swell shrinkEVALUATIONDrug loaded erythrocyte isotonic to hypotonic

(0.1%w/v) 37±2ºC/10 min

Centrifuge 30g/15 min

Drug assay/Haemoglobin release

Osmotic shock

It is a sudden exposure of drug loaded erythrocyte to the environment , which is far from isotonic to evaluate the ability of RE to withstand the stress and maintain the integrity as well as appearance.

RE + distilled water 3 min

Centrifugation 3000 rpm / 15 min

Release of hemoglobin to varying degrees

estimated spectrophotometriccally.

The effect of shear force and pressure by which RE formulation are injected , on the integrity of the loaded cells.

Loaded erythrocyte passes through

23 – guage hypodermic needle

flow rate of 10 ml /min .

aliquot of the suspension

withdrawn

Centrifuge -2000 rpm for 10 min

hemoglobin content was measured

Turbulence shock

Released characteristics of RE; drug to efflux out from the cell

Phagocytosis Diffusion through the membrane of cells Using of specific transport system Carrier erythrocyte following the heat treatment or

antibody crosslinking are removed from the circulation by

phagocytic cell located in the liver and spleen. The rate of diffusion depends upon the rate at which a

particular molecules penetrate through the lipid Bilayer It is greatest for the molecule with lipid high solubility zero order release kinetics Group of ER means the drug is released by constant

fraction of cell is removed from the group each day.

Application of RE

Enhanced drug delivery/ prolonged drug release

Targeting

Drug targeting – surface modification target the organs of

mononuclear phagocytic system /REs because the changes

in the membrane are recogonized by macrophages

Target in liver ,lymph node and spleen by modifying

membranes

Treat hepatic tumour, Parasitic disease

Antiviral carrier / Enzyme therapy

Improvement in oxygen delivery to tissue

Microinjection of macromolecules

RE as Drug / Enzyme carriers (disseminate bioactive agent over

prolonged period of time in circulation)

Enzyme –catalase, urease, arginase, asparginase,b-glucuronidase,

b-fructouronidase,b-galactosidase

Alcohol dehydrogenase – metabolism of ethanol

RE as an drug carriers (antineoplastic

agents,antibiotics,vit,steroids)

Prolong half-life

RE – carriers for proteins and macromolecules(insulin)

DRUG TARGETING

1. Surface modification (liver & spleen)

2. Chemical modification (organs- mononuclear phagocyte system)

Surface modification

With antibodies

Circulatory half life is shortened-by coating ER with antibodies

Which target spleen macrophages

Heavily modified cells-liver macrophage

With glutaraldehyde

Localized in the liver kupffer cells

With carbohydrates

Reduction in circulatory half life- liver , spleen

With sulphydryls

Half life from 27 days to 30 days

Chemical cross linking

125I-CA increase the therapeutic potential of RE

Magnet responsive erythrocyte

Ferrofluids-colloidal suspension magnetite+Fe3o4).

External magnetic field

Improve the stability

Targeting/ Reduce cytotoxicity

Especially inflammatory drugs

Photosensitized erythrocyte (haematoprophyrin derivative)

Antibody anchored erythrocytes(immunoerythrocytes)

Antviral agent

Thrombolytic therapy

Oxygen defieciency therapy

Erythrosome:

specially engineered vesicular system

crosslinked to human erythrocytes support

upon which a Bilayer is coated.

process is achieved by modifying a reverse

phase evaporation technique

encapsulate macromolecular drug.

Nanoerythrosome:

100 nm erythrocyted

Future Prospectus

Intravenous slow drug release strategy: delivery of

antineoplasms, antiparasitics, antiretroviral agents,

vitamins, steroids, antibiotics and cardiovascular drugs .

Mechanisms passive diffusion, specialized membrane-

associated carriers, phagocytosis of the carrier cells by the

macrophages of RES: depletion of the drug in circulation,

accumulation of the drug in RES, upon lysis of the carrier

and slow release from this system into circulation,

accumulation of the carrier erythrocytes in lymphatic

nodes following subcutaneous injection of the cells and

finally, hemolysis at the injection sites.

DELIVERY STRATEGIES

Recent developments novel systems

Nanoerythrosomes : An erythrocyte based new drug

carrier, named nanoerythrosome has been developed

which is prepared by extrusion of erythrocyte ghosts to

produce small vesicles having an average diameter of

100 nm.

Eg.,Daunorubicin was covalently conjugated to

nanoerythrosomes using gluteraldehyde spacer.

Erythrosomes : Erythrosomes are specially engineered

vesicular systems in which chemically cross-linked

human erythrocyte cytoskeletons are used as a support

upon a lipid bilayer is coated.