S.Brito Raj , M.

Pharm Sri Venkateswara College of Pharmacy, RVS nagar ,

Targeted drug delivery Magic bullet concept.

Cellular carriers clinical applications

Biocompatibility carrier Erythrocytes : possess greater potential in drug delivery.

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/ l of blood Healthy adult female = 4.8 million RBCs/ l 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.

Normal erythrocyte (Normocyte) - flexible, elastic, biconcave, nucleated structure with a mean diameter of 7.3 m.

Chemical constituents of RBCs Water 63%/Lipids 0.5% Glucose 0.8%

Minerals 0.7%
Non-heamoglobin protein 0.9% Methehemoglobin 0.5% Hemoglobin 33.67%

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.

Hayens solution or toisons solution.

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.

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

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

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

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

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

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

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

A wide variety of biologically active substancea(5000600,000 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

1.

Source: mice, cattle, dog, goat , monkey, rat,rabbit & Human

2.

Fresh Blood is collected into heparinized tubes by

venipuncture+ EDTA or heparin Blood is withdrawn from cardiac/splen (in small animals) and through veins (in large animals) in a syringe containing a drop of anti coagulant. Immediately chilled to 4c & stored for NMT 2 days Centrifuged - 2500 rpm/5 min/4 1c= refrigerated centrifuged Serum & buffy coats are carefully removed Packed cells washed 3 times with PBS (pH = 7.4) Then are diluted with PBS and stored a 41 c

3.

4. 5. 6. 7. 8.

Drug Loading in RE

Membrane perturbation

Electro encapsulation

Hypo-osmotic lysis

Lipid fusion Endocytosis

Dilution method

Dialysis method

Preswell method

Osmotic lysis

Red cells placed in drug solution

Hypotonic < 0.9 % or 0.45 Nacl

Swells & lysed by osmotic & electric shock Spherocyte

+ 1.54 KCl Biconcave discocytes

> 0.9 % Nacl Isotonic, Glutaraldehyde coating

RBC resealed

NOTE: Surface modified with cross linking polymer

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

1.Red cells + drug solution (macro)

3.Swells Spherocyte

4.Pressure/Rupture/ External to internal

2.Hypotonic 0.4 % Nacl

0 c/5 min

Biconcave discocytes

+ 1.54 KCl,25 c

5.0.9 % Nacl Isotonic, Glutaraldehyde coating

RE

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

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

0.6% Nacl Hypotonic 1:5 ratio

H2O ,increased hypotonicity

Swollen cell

RBC

Preswelling

Hypertonic solution

Hypotonic buffer + drug 10 min/oc

R E

Pores recovery

Hemolysis

Advantages

Gentle swelling cells good retention of cytoplasmic

constituents

Good survival- invivo More encapsulation efficiency(72%)

 A. B.

Used to avoid the disadvantages by hypotonic medium Haemolysis by both physical & chemical means Conventional haemolysis in isotonic urea solutions 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

 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

Erythrocyte membrane lysed- Dielectric background

Entrapped = electric pulse of greater than a threshold voltage

of 2kv/cm applied for 20microsecond Resealed by incubation at 37C 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

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 volume

cell volume reach 155%= original

Prevent lysis and balance colloidal osmotic pressure by + macromolecules like BSA Under this osmotic balance ,pores are stay opened at 4c 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) 4c 0.15 ml of erythrocyte suspension

2.high voltage pulsation device Single electric pulse 2.2kV/cm+20sec/25c

osmotic balance + sachraides and proteins 7kV/cm,20sec electric pulse 3:7 (isotonic Nacl & Isoosmotic sucrose)
Electrical perforated erythrocyte cell suspension precooled tube / 4c

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

medium

Advantages

Excellent invivo performance

Normal haemoglobin properties were retained Enclose proteins Prolonged release No mebranolytic or deleterious effect Time consuming Costly

Disadvantages

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.

Erythrocyte

Drug

Drug loaded erythrocyte

ERYTHROCYTE ENDOCYTOSIS PRODUCED BY CATIONS AND TRAPPING OF MOLECULES IN THE INVAGINATION OR INSIDE OUT ENDOCYTOSIS VACUOLES

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 (>0.5

diameter)

3 steps in Endocytosis
1.washed packed erythrocyte+ buffer (ATP,Mgcl2,CaCl2) with drug

incubated for 2 min/ room temperature

2.resealing by + Nacl/

incubation 2 min / 37C,

washed in 5mM imidazole-glycylglycine buffer+154 mM Nacl pH 7.4 30 min/37C 3.entrapment of drug by endocytosis

Physical parameters
Shape & surface morphology Vesicle size & size distribution Drug release Drug content Surface electrical potential Deformability Sterility Pyrogenicity Animal toxicity

Method/instrument used
1.Physical characteristics TEM/SEM/Phase contrast microscopy/optical microscopy TEM/optical microscopy Diffusion cell/dialysis Deproteinization of cell membrane followed by assay of resealed drug/radiolabelling Zeta potential measurement Capillary method 3. Biological characteristics Sterility test Rabbit method, LAL test Toxicity test

2. Cellular characteristics % Hb content Cell volume % Cell recovery Osmotic fragility Deproteinization of cell membrane followed by hemoglobin assay Laser light scatering Neubaurs chamber/haematological analyzer Stepwise incubation with isotonic to hypotonic saline solution and determination of drug and haemoglobin assay

Osmotic shock
Turbulent shock

Dilution with distilled water and estimation of drug and haemoglobin

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 ESR method sedimentation rate

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

Normal & loaded ER (50%)

Incubated 37C in PO4 buffer pH 7.4

Metabolic rotating wheel incubator bath

Sample hypodermic syringe 0.8 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

spectropore membrane filter

Deproteinized (acetonitrile)

% hamoglobin release = A540 of sample A 540 of background

A540 of 100% heam Mean corpuscular Heam = haemoglobin (g/100ml) erythrocyte count (per mm3 x10

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. RBC hypotonic hypertonic swell shrink isotonic to hypotonic (0.1%w/v)
372C/10 min

EVALUATION Drug loaded erythrocyte

Centrifuge 30g/15 min Drug assay/Haemoglobin release

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.

Turbulence shock

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

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.

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,bglucuronidase,

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

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

Reduction in circulatory half life- liver , spleen

With carbohydrates

With sulphydryls

Half life from 27 days to 30 days

125I-CA increase the therapeutic potential of RE

Chemical cross linking

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

Future Prospectus
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

DELIVERY STRATEGIES

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

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.