TARGETED DRUG DELIVERY SYSTEM.

NANOPARTICLES &

RESEALED ERYTROCYTES.

PRESENTED BY:

K.SAI VANI

256213886014M.PHARM (PHARMACEUTICS)

GUIDED BY:

Dr.Mrs. Yasmin Begum

M.Pharm Ph.D

NANOPARTICLES

TABLE OF CONTENTS: Introduction Concept Ideal characteristics Advantages Disadvantages Polymers used Methods of preparation Novel Nanoparticulate systems

CONTD……

Pharmaceutical aspects of Nanoparticles Characterization of nanoparticles In Vivo fate & Bio distribution of

Nanoparticles Evaluation of nano particles Applications References.

WHAT ARE NANOPARTICLES?

Nano derives from the greek word “nanos” which means Dwarf or Extremely small. It can be used as a prefix for any unit to mean a billionth of that unit.

A nanometer is a billionth of a meter.

DEFINITION:

Nanoparticles are solid colloidal particles ranging from 1to100nm in size.

They consist of micromolecular

materials in which the active ingredients (drug or biologically active material) is dissolved, entrapped, or encapsulated, or absorbed, or attached.

CONCEPT:The basic Concept involved is :

Selective and Effective Localization of pharmacologically active moiety at preselected target(s) in therapeutic concentration,,

Provided restriction of it’s access to non-target normal tissues and cells.

Nanoparticles are mainly taken by :

ReticuloEndothelial System (RES), After the

administration.

Hence are useful to carry drugs to the liver and to cells that are phagocytically active.

By modifying the surface characteristics of the nanoparticles it is possible to enhance the delivery of drugs to spleen relative to the liver.

Distribution of the nanoparticles in the body may be achieved possibly by :

Coating of nanoparticles with certain Serum components, Attachment of antibodies or sulfoxide groups and the use of Magnetic nanoparticles.

IDEAL CHARACTERISTICS: It should be biochemically inert , non

toxic and non-immunogenic.

It should be stable both physically and chemically in Invivo & invitro conditions.

Restrict drug distribution to non-target cells or tissues or organs & should have uniform distribution.

Controllable & Predicate rate of drug release.

Drug release should not effect drug action

Specific Therapeutic amount of drug release must be possessed

Carriers used must be biodegradable or readily eliminated from the body without any problem and no carrier induced modulation in disease state.

The preparation of the delivery system should be easy or reasonable

simple, reproducible & cost effective.  

Based On Method Of Preparation:

Nanocapsules:- Nanocapsules are systems in which the drug is confined to a cavity surrounded by a unique polymer membrane.

Nanospheres:- Nanospheres are matrix systems in which the drug is physically and uniformly dispersed.

NANOPARTICULATE DRUG-DELIVERY SYSTEMS:

CLASSIFICATON OF NANOPARTICLES: Solid Lipid Nanoparticles Polymeric Nanoparticles Ceramic Nanoparticles Hydrogel Nanoparticles Copolymerized Peptide Nanoparticles Nanocrystals and Nanosuspensions Nanotubes And Nanowires Functionalized Nanocarriers Nanospheres Nanocapsules

Solid lipid Nanoparticles: New type of colloidal drug carrier system for i.v. Consists of spherical solid lipid particles in the nm range,

dispersed in water or in aqueous surfactant solution.

Polymeric nanoparticles (PNPs) : They are defined as particulate dispersions or solid

particles with size in the range of 10-1000nm.Composed of synthetic

or semi-synthetic Polymers. Biodegradable polymeric nanoparticles Polylactic acid

(PLA), polyglycolic acid (PGA), Polylactic - glycolic acid (PLGA),

and Polymethyl methacrylate (PMMA) Phospholipids Hydrophobic core

Ceramic Nanoparticles: These are the nanoparticles made up of inorganic

(ceramic) compounds silica, ( Inorganic/metal) titania and alumina.

Exist in size less than 50 nm,which helps them in evading deeper parts of the body.

Hydrogel nanoparticles: Polymeric system involving the self-assembly and

self aggregation of natural polymer amphiphiles cholesteroyl pullulan , cholesteroyl dextran and agarose cholesterol groups provide cross linking points.

Copolymerized Peptide Nanoparticles:

Drug moiety is covalently bound to the carrier instead of

being physically entrapped.

Nanocrystals And Nanosuspensions:

Pure drug coated with surfactant, Aggregation of these particles in crystalline form .Drug powder dispersed in aqueous surfactant solution.

Functionalized Nanocarriers: Biological materials like proteins, enzymes, peptides

etc… are being utilized as a carriers for the drug delivery.

NANOSPHERES & NANOCAPSULES:

ADVANTAGES: Nano particle can be administered by

parenteral, oral, nasal, occular routes.

By attaching specific ligands on to their surfaces,nano particles can be used for directing the drugs to specific target cells.

Improves stability and therapeutics index and reduce toxic affects.

Both active & passive drug targetting can be achieved by manipulating the particel size and surface characteristics of nano particles

DISADVANTAGES: Small size & large surface area can lead

to particle aggregation .

Physical handling of nano particles is difficult in liquid and dry forms.

Limited drug loading.

Toxic metabolites may form

POLYMERS USED IN PREPARATION:

NATURAL HYDROPHILLI

C

Proteins

Poly-saccharides

SYNTHETIC HYDROPHOB

IC

Pre-polymerized

Polymerized in process

PROTEINS POLYSACCHARIDES

Gelatin Alginate

Albumin Dextran

Lectins Chitosan

Legumin Agarose

Viciline Pullulan

PRE-POLYMERIZED POLYMERIZED IN PROCESS

Poly (E caprolactone)(PECL) Poly Isobutyl cyano acrylates (PICA)

PLA(Poly lactic acid) PBCA(poly butyl cyano acrylates)

Poly lactide co glycolide (PLGA)

PHCA(poly hexyl cyano acrylates)

Polystyrene Poly methyl methacyrlate (PMMA)

PREPARATION TECHNIQUES OF NANOPARTICLES:

Polymerization based methods

Polymer precipitation

methods

Amphiphilic macromolecu

le cross-linking

Amphiphilic macromolecule cross-

linking

Heat Cross-linking

Chemical Cross-linking

Polymerization based Methods

Polymerization of

monomers

Dispersion polymerizatio

n

Interfacial condensation polymerizatio

n

Emulsion polymeriz

ation

Interfacial

complexation

Polymer Precipitation methods

Solvent extraction/ evaporation

Solvent displacement

(nanoprecipitation)

Salting out

AMPHIPHILIC MACROMOLECULAR CROSSLINKING:

Nanoparticles can be prepared from amphiphilic macromolecules, proteins & polysaccharides.

The technique of their preparation involves firstly, the aggregation of amphiphiles followed by further stabilization either by Heat denaturation or Chemical cross linking.

These process may occur in biphasic O/W or W/O type dispersed system.

HEAT CROSS LINKING/CHEMICAL CROSS LINKING:

The cross linking method is exhaustively used for the nano-encapsulation of drug.

The method involves the emulsification of Bovine serum albumin(BSA)/ human serum albumin(HAS) or protein aqueous solution in oil using high pressure homogenization or high frequency sonication.

The high temperature used in the original method restrict the application of method to heat sensitive drugs.

As an alternative to heat stabilization method a chemical cross linking agent, usually glutaraldehyde, is incorporated in to the system.

Though the heat borne drawbacks are obviated, yet a need to remove residual cross-linking agent makes the method cumbersome.

EMULSION CHEMICAL DEHYDRATION:

Chemical dehydration has been reported for producing BSA nanoparticles

Bhargava and Aindo suggested a simple chemical cross-linking method in 1992

Hydroxypropyl cellulose solution in chloroform was used as a continuous phase.

2,2,dimethylpropane(dehydrating agent) was used to translate internal aqueous phase in to a solid particulate dispersion.

This method produce nanoparticles of size (300nm)

POLYMERIZATION BASED METHODS:

Polymers used for nanosphere preparation include:

Poly(methylmethacrylate) Poly(acrylamide) Poly(butyl cyanoacrylate) N-N’metylene-bis-acrylamide etc.

Two different approaches are generally adopted for the preparation of nanospheres using in-situ polymerization technique:

Methods in which the monomer to be polymerized is emulsified in a non-solvent phase(Emulsion polymerization)

(OR)

Methods in which the monomer is dissolved in a solvent that is non solvent for the resulting polymer (Dispersion polymerisation)

EMULSION POLYMERIZATION: The process of emulsion polymerization

can be conventional or inverse, depending upon the nature of the continuous phase in the emulsion

Two different mechanism: Micellar nucleation and polymerization Homogeneous nucleation and

polymerization.

MICELLAR NUCLEATION AND POLYMERIZATION:

It involves the swollen monomers micelles as the site of nucleation and polymerization

The monomers is emulsified in non solvent phase with the help of surfactant molecules.

It leads to formation of monomer-swollen micelles and stabilized monomer droplets.

HOMOGENOUS NUCLEATION AND POLYMERIZATION:

It is applied in the case where the monomer is sufficiently soluble in the continuous outer phase

The nucleation and polymerization stages can directly occur in this phase, leading to the formation of primary chains called oligomer

When the oligomer have reached a certain length they precipitate and form primary particles which are stabilized by the surfactant molecule.

The polymerization rate is dependent on the PH of the medium.

Anionic polymerization takes place in micelle after diffusion of monomer molecules through the water phase and is initiated by negative charged compound

At neutral PH the rate of polymerization is extremely fast.

However at acidic ph i.e,2-4 the reaction rate remains controlled and slow.

Eg:ethyl cynoacrylate- 2hr, hexyl cynoacrylate-10 to 12hr.

DISPERSION POLYMERIZATION: In case of dispersion

polymerization howeverthe monomer instead of emulsified,is dissolved in an aqueous medium which acts as the precipitant for subsequently formed polymer

Nucleation is directly induced in aqueous monomer solution and presence of stabilizer or surfactant is not necessary for the formation of stable nanospheres.

INTERFACIAL POLYMERIZATION: The performed polymer

phase is transformed to an embroynic sheath.

A polymer that eventually become core of nanoparticle and drug molecule to be loaded are dissolved in a volatile solvent.

The solution is poured in to a non-solvent for both polymer and core phase.

The polymer phase is separated as a coacervative phase at O/W interphase.

INTERFACIAL COMPLEXATION: The method is based on the process of

microencapsulation introduced by Lin & Sun 1969.

In case of nanoparticles preparation,aqueous polyelectrolyte solution is carefully dissolved in reverse micelles in an apolar bulk phase with the help of an appropriate surfactant.

Subsequently, competing polyelectrolyte is added to the bulk, which allows a layer of insoluble polyelectrolyte complex to coacervate at the interface.

POLYMER PRECIPITATION METHODS:

Solvent extraction/ evaporation method

Salting out method

Solvent displacement method.

This method involves the formation of a conventional O/W emulsion between a partially water miscible solvent containing the stabilizer.

Ex: PLGA nanospheres

The polymer is solubilized in a solvent (chloroform) and dispersed in gelatin solution by sonication to yield emulsion O/W. the solvent is eliminated by evaporation. For evaporation homogenizer used which breaks the initial coarse emulsion in nanodroplets yielding nanospheres.

DOUBLE EMULSION SOLVENT EVAPORATION METHOD:

NANOPARTICLES PREPARATION USING SALTING OUT TECHNIQUE: The method involves the

incorporation of saturated aqueous solution of (PVA) into an acetone solution of the polymer to form O/W emulsion.

In this technique the miscibility of both phases is prevented by saturation of external aqueous phase.

The precipitation of polymer occurs when sufficient amount of water is added to external phase.

SOLVENT DISPLACEMENT METHOD: This method is based on

the interfacial deposition of a polymer following displacement of a semi-polar solvent miscible with water from a lipophilic solution.

This method involves the use of organic phase completely soluble in aqueous phase inducing immediate polymer precipitation

The solvent is eliminated and the free flowing nanoparticles can be obtained under reduced pressure.

NOVEL NANOPARTICULATE SYSTEM

Solid Lipid Nanoparticles: These are sub-micron colloidal carriers

(50-100nm) which are composed of physiological lipid dispersed in water or in a aqueous surfactant solution.

Micro emulsion technique was used for the production of solid lipid nanoparticles

Homogenization method at higher pressure for either melted or solid lipids has been suggested to obtain SLN.

VERSATILITY:

Parenteral administration Brain delivery Ocular delivery Rectal delivery Oral delivery Topical delivery

ADVANTAGES OF SLN: Small size and relatively narrow size

distribution which provide biological opportunities for site specific drug delivery by SLNs.

Controlled release of active drug over a long period can be achieved

Protection of incorporated drug against chemical degradation.

No toxic metabolites are produced Relatively cheaper and stable. Ease of industrial scale production by hot

dispersion technique.

PREPARATION METHODS OF SLN:Hot Homogenization Technique: Homogenization of melted lipids at

elevated temperature.Cold Homogenization Technique: Homogenization of a suspension of solid

lipid at room temperature.

HOT HOMOGENIZATION TECHNIQUE: Melting of lipid

Dissolution of the drug in the melted lipid

Mixing of the preheated dispersion medium and the drug lipid melt

High pressure homogenization at a temperature above the lipids melting point

O/W-Nano emulsion

Solidification of the nano emulsion by cooling down to room temperature to form SLN.

COLD HOMOGENIZATION TECHNIQUE: Melting of the lipid

Dissolution of the drug in the melted lipid

Solidification of the drug loaded lipid in liquid nitrogen or dry ice

Grinding in a powder mill(50-100 particles)

Dispersion of the lipid in the cold aqueous dispersion medium

Solid lipid nanoparticles.

NANOCRYSTALS & NANOSUSPENSION:

Nanocrystals and Nanosuspensions are two recently introduced aspects to drug delivery research.

The basic theme is to convert micronized drug powders to drug nanoparticles.

PHARMACEUTICAL ASPECTS OF NANOPARTICLES: Should be free from potential toxic

impurities Should be easy to store and administer Should be sterile if parenteral use is

advocated.

Three important process parameters are performed before releasing them for clinical trials:

o Purificationo Freeze dryingo Sterilization.

CROSS-FLOW FILTRATION TECHNIQUE:

This method has been suggested for the purification of nano particles and the method can be scaled up from an industrial stand piont.

In this method the suspension os filtered through membranes with direction of fluid being tangential to surface of membrane.

Depending on the type of membrane used either microfiltration or ultra filtration can be performed.

FREEZE DRYING OF NANOPARTICLESThis technique involves the freezing of the

nanoparticle suspension and subsequent sublimation of its water content under reduced pressure to get freeflowing powder material

ADVANTAGES: Prevention from degradation Prevention from drug leakage, drug desorption. Easy to handle and store and helps in long

term preservation. Readily dispersed in water without

modifications in their physicochemical properties.

STERILIZATION OF NANOPARTICLES: Nanoparticles intended for parenteral use

should be sterilized to be pyrogen free.Sterilization can achieved by: Using aseptic technique throughout their

preparation,processing,and formulation Subsequent sterilization treatments like

autoclaving, irradiation It is deduced from these consideration

that the sterilization of nanoparticles is a critical step that should be systematically investigated during formulation development stage.

INVIVO FATE AND BIODISTRIBUTION OF NANOPARTICLES: Intravenous injection of

colloidal carriers follow their interactions with at least two distinct groups of plasma proteins.

Phagocytosis of particulates by elements of RES is regulated by the presence and balance b/w two groups of serum components.

Opsonins that promote phagocytosis

Dysopsonins that supress the process.

Ex:

Opsonins-immunoglobulins,

fibronectin,tuftsin ,

Dysopsonins- IgA,SecretoryIgA .

SURFACE ENGINEERING OF NANOPARTICLES:

Steric stabilized (stealth) nanoparticles Magnetically guided nanoparticles(Fe3O4) Biomimetic nanoparticles (biomimetic

ligands-salilic acids) Bioadhesive nanoparticles Antibody coated nanoparticles.

EQUIPMENT FOR NANOPARTICLES: Homogenizer Ultra Sonicator Mills Spray Milling Supercritical Fluid Technology Electrospray Ultracentrifugation Nanofiltration

HOMOGENIZER & ULTRA SONICATOR

NANO-MILL MANUFACTURING PLATFORM

EVALUATION OF NANOPARTICLES:

Particle size Density Molecular weight Structure and crystallinity Specific surface area Surface charge & electronic mobility Surface hydrophobicity Invitro release Nanoparticle yield Drug entrapment efficiency

1.PARTICLE SIZE: Photon correlation spectroscopy (PCS) : For

smaller particle. Laser diffractrometry : For larger particle. Electron microscopy (EM) : Required coating of

conductive material such as gold & limited to dry sample.

Transmission electron microscopy (TEM) : Easier method & Permits differntiation among nanocapsule & nanoparticle

Atomic force microscope Laser force microscope Highresolution

Scanning electron microscope microscope

2.Density : Helium or air using a gas pycnometer Density gradiant centrifugation

3. Molecular weight : Gel permeation chromatography using

refractive index detector.

4. Structure & Crystallinity : X-ray diffraction Thermoanalytical method such as, 1) Differential scanning calorimetry 2) Differential thermal analysis 3) Thermogravimetry

5. Surface charge & electronic mobility : Surface charge of particle can be determined by

measuring particle velocity in electrical field.

Laser Doppler Anemometry tech. for determination of Nanoparticles velocities.

Surface charge is also measured as electrical mobility.

Charged composition critically decides bio-distribution of nanoparticle .

Zeta potential can also be obtain by measuring by the electronic mobility.

6.Surface Hydrophobicity : Important influence on intraction of nanoparticles with

biological environment. Several methods have been used,

1. Hydrophobic interaction chromatography.

2. Two phase partition.

3. contact angle measurement.

7. Invitro release : Diffusion cell Recently introduce modified Ultra-filtration tech. Media used : phosphate buffer

8.Nano particle yield:

9.Drug entrapment efficiency:

APPLICATIONS:

PRODUCTS:Company Technology API Route of

administration

Novavax, USA Micellar nanoparticles

Testosterone S.c.

BioAUiance, France

Polydsohexylcyanoacrylate)nanoparticles

Doxorubicin i.v.

AmericanBioscience, USA

Albumin-Drugnanoparticles

Paclitaxel i.v.

Wyeth Pharmaceutical, USA

Drug Nanoparticles

Rapamycin Oral

BioSante, USA Calcium phospahtenanoparticles

Insulin Oral

EMEND Rapamune

OLAY MOISTURIZERS

(Merck & Co. Inc) (Wyeth-Ayerst Laboratories)

(American Biosciences, Inc.) ABRAXANE

(Proctor and Gamble)

CONCLUSION: Nanoparticles are one of

the novel drug delivery systems, which can be of potential use in controlling and targeting drug delivery as well as in cosmetics textiles and paints. Judging by the current interest and previous successes, nanoparticulate drug delivery systems seems to be a viable and promising strategy for the biopharmaceutical industry.

REFERENCE: Vyas S.P. , Khar R.K. Targeted & Controlled

Drug Delivery, Novel Carrier Systems, CBS Publication ,2002 ,Page No.249-277,331-387.

www.pharmainfo.net/reviews/nanoparticles-and-its-applications-field-pharmacy

Nanoparticles –A Review by VJ Mohanraj & Chen Y, Tropical Journal of Pharmaceutical Research 2006; 5(1): 561-573

Google.com(images) Jain N. K., Controlled and novel Drug

Delivery, 1st edition 2001, CBS Publication; 292 - 301.

1

CONTENTS: Introduction Source of erythrocytes Isolation of erythrocytes Drug loading in erythrocytes Factors effecting resealed erythrocytes Advantages of erythrocytes Method of drug loading In vitro characterization of erythrocytes Applications

INTRODUCTION: Present pharmaceutical scenario is aimed at

development of drug delivery systems which maximize the drug targeting along with high therapeutic benefits for safe and effective management of diseases.

Target drug delivery system indeed a very attractive goal because in this system targeting of an active bio molecule from effective drug delivery where pharmacological agents directed specifically to its target.

Various cellular carriers has been used for drug targeting among which cellular carriers(leukocytes, platelets and erythrocytes) offer a great potential.

Erythrocytes also known as red blood cells and have extensively studied for their potential carrier capabilities for delivery of drugs .

Diameter - 7-10µ

Life span - 120days

No. of cells/L of blood - 5X1012,4.5X1012

Shape,  nucleus type - biconcave disc like, anucleate

Cytoplasm - pink(Hb),halo in centre

Functions - transports Hb that binds to O2 &CO2

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 hemoglobin content and may burst.

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.

HAEMATOCRIT VALUE: 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%

SOURCE OF ERYTHROCYTES: Different mammalian erythrocytes have been

used for drug loading, resealing and subsequent use in drug and enzyme delivery.

E.g. mice, cattle, pigs, dogs, sheep, goats, monkeys, chicken, rats, and rabbits etc.

EDTA or heparin can be used as anticoagulants agents

ISOLATION OF ERYTROCYTES: Blood is collected into heparinized tubes by venipuncture

Blood is withdrawn from cardiac/splenic puncture(in small animal) and through veins (in large animals) in a syringe containing a drop of anti coagulant.

The whole blood is centrifuged at 2500 rpm for 5 min at

4 ±1.0o C in a refrigerated centrifuge.

The serum coats are carefully removed and packed cells

washed three times with phosphate buffer saline (pH=7.4).

The washed erythrocytes are diluted with PBS and stored

at 40oC until used.

METHODS OF DRUG LOADING: Hypotonic hemolysis. Hypotonic dilution. Hypotonic preswelling. Hypotonic dialysis. Use of red cell loader. Isotonic osmotic lysis. Chemical perturbation of the membrane. Electro-insertion or electroencapsulation. Entrapment by endocytosis. Loading by lipid fusion.

HYPOTONIC HAEMOLYSIS Erythrocytes to undergo

reversible swelling in a hypotonic solution.

An increase in volume leads to an initial change in the shape from biconcave to spherical.

The cells can maintain their integrity up to a tonicity of 150mosm/kg above which the membrane ruptures, releasing the cellular contents.

Membrane ruptured RBC Loaded RBC Resealed Loaded RBC

0.4% NaCl

Hypotonic

Drug

Loading buffer

Resealing buffer

Incubation at 250c

HYPOTONIC HEMOLYSIS:

RBC

Chemicals – Urea, Polyethylene, Polypropylene, and NH4Cl

HYPOTONIC HEMOLYSIS:

These ruptured erythrocytes as drug carriers is based on the fact that the ruptured membranes can be resealed by restoring isotonic conditions.

Upon incubation at 25oc the cells resume their original biconcave shape and recover original impermeability.

HYPOTONIC DILUTION: In this method, a volume of packed

erythrocytes is diluted with 2–20 volumes of aqueous solution of a drug.

The solution tonicity is then restored by adding a hypertonic buffer.

The resultant mixture is then centrifuged, the supernatant is discarded, and the pellet is washed with isotonic buffer solution.

HYPOTONIC DILUTION:

Compounds that can be encapsulated are enzymes such as -galactosidase and -glucosidase , asparginase,and arginase, salbutamol.

RBC

Hypotonic / Drug solution

Membrane ruptured RBC

Isotonic solution

Resealed erythrocytes

HYPOTONIC PRESWELLING:

The technique is based upon initial controlled swelling in a hypotonic buffered solution. This mixture is centrifuged at low g values.

The supernatant is discarded and the cell fraction is brought to the lysis point by adding 100–120 L portions of an aqueous solution of the drug to be encapsulated.

The mixture is centrifuged between the drug-addition steps.

The lysis point is detected by the disappearance of a distinct boundary between the cell fraction and the supernatant upon centrifugation.

The tonicity of a cell mixture is restored at the lysis point by adding a calculated amount of hypotonic buffer.

Then, the cell suspension is incubated at 37 C to reanneal the resealed erythrocytes

Loaded RBC Resealed Loaded RBC

Loading bufferResealing

buffer

Incubation at 250c

HYPOTONIC PRESWELLING

0.6%w/v NaCl

5 min incubation at 0 0c

Swelled

RBC RBC

Drugs: Propranolol, Asparginase, Methotrexate, Insulin , Metronidazole , Levothyroxine, Isoniazid.

ISOTONIC OSMOTIC LYSIS: This method, also known as the osmotic pulse

method, involves isotonic hemolysis that is achieved by physical or chemical means.

The isotonic solutions may or may not be isoionic.

If erythrocytes are incubated in solutions of a substance with high membrane permeability, the solute will diffuse into the cells because of the concentration gradient.

This process is followed by an influx of water to maintain osmotic equilibrium.

ISOTONIC OSMOTICS LYSIS

RBC

Physical/chemical rupturing

Isotonic buffer

Drug

Isotonically ruptured RBC

Resealed RBC

loaded RBC

Incubation at 25oc

Compounds encapsulated are – Urea, polyethylene, polypropylene, and NH4Cl

ENTRAPMENT BY ENDOCYTOSIS:-

RBC

Drug Suspension

+

Buffer containing ATP, MgCl2, and CaCl2

At 250 C

Loaded RBC

Resealing Buffer

Resealed RBC

Drugs: primaquine ,quinolines, vinblastine, chlorpromazine, phenothiazines, propranolol, vitamin A.

Fig:- Entrapment By Endocytosis Method

HYPOTONIC DIALYSIS: In the process, an isotonic, buffered suspension of erythrocytes

with a hematocrit value of 70–80 is prepared and placed in a conventional dialysis tube immersed in 10–20 volumes of a hypotonic buffer.

The medium is agitated slowly for 2 h.

The tonicity of the dialysis tube is restored by directly adding a calculated amount of a hypotonic buffer to the surrounding medium or by replacing the surrounding medium by isotonic buffer.

The drug to be loaded can be added by dissolving the drug in isotonic cell suspending buffer inside a dialysis bag at the beginning of the experiment.

HYPOTONIC DIALYSIS

Phosphate buffer

+Placed in dialysis

bag with air bubbleDialysis bag placed in 200ml of lysis

buffer with mechanical rotator 2hrs. 4c.

DrugLoading buffer

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

Resealed RBC

DRUGS: gentamicin, pentamidine, interlukin-2 , desferroxamine and recombinant erythropoietin.

Hypotonic Dialysis

ELECTRO-INSERTION OR ELECTRO-ENCAPSULATION

This method is based on the observation that electrical shock brings about irreversible changes in an erythrocyte membrane.

The use of transient electrolysis to generate desirable membrane permeability for drug loading.

The erythrocyte membrane is opened by a dielectric breakdown.

Subsequently, the pores can be resealed by incubation at 37OC in an isotonic medium.

ELECTRO-INSERTION OR ELECTRO-ENCAPSULATION

RBC

2.2 Kv Current for 20 micro sec

At 250 C

Pulsation medium

++

Drug

Loading suspension

3.7 Kv Current for 20 micro sec

Isotonic NaCl

Loaded RBC

Resealing Buffer

Resealed RBC

DRUGS: Urease , Methotrexate ,isoniazid , human glycophorin ,DNA fragments, and latex particles of diameter 0.2 m.

Electro-insertion or Electro-encapsulation

Fig:- Electro-encapsulation Method

ENTRAPMENT BY RED CELL LOADER:

It is a novel method for entrapment of nondiffusible drugs into erythrocytes.

They developed a piece of equipment called a “red cell loader”.

With as little as 50 mL of a blood sample, different biologically active compounds were entrapped into erythrocytes within a period of 2 h at room temperature under blood banking conditions.

The process is based on two sequential hypotonic dilutions of washed erythrocytes followed by concentration with a hemofilter and an isotonic resealing of the cells.

There was 30% drug loading with 35–50% cell recovery. The processed erythrocytes had normal survival in vivo. The same cells could be used for targeting by improving

their recognition by tissue macrophages.

RED CELL LOADER

CHEMICAL PERTURBATION METHOD:

“THIS METHOD IS BASED ON THE FACT THAT THE PERMEABILITY OF THE ERYTHROCYTES INCREASES ON EXPOSURE TO CERTAIN

CHEMICAL AGENTS .”

However, these methods induce irreversible destructive changes in the cell membrane and hence are not very popular.

RBC

Amphotericin B

Drug

RBC with increased permeability

Resealing Buffer

Resealed RBC

ROUTES OF ADMINISTRATION

Routes of administration include:

i. Intravenous (most common).ii. subcutaneous. iii.Intraperitoneal.iv.Intranasal.

MECHANISM OF DRUG RELEASE There are mainly three ways for a drug to efflux out

from erythrocyte carriers.

Phagocytosis. Diffusion through the membrane of the cell. Using a specific transport system. 

INVITRO CHARACTERIZATION:Resealed erythrocytes after loading are characterized for following

parameters.

1. Drug Content: Packed loaded erythrocytes are 1st deproteinized with acetonitrile and

subjected to centrifugation at 2500rpm for 10min. The clear supernatant is analyzed for the drug content.

2. IN VITRO DRUG AND HAEMOGLOBIN RELEASE:

Normal and loaded erythrocytes are incubated at 37+ 20c in phosphate buffer saline (pH-7.4) at 50% haematocrit in a metabolic rotating wheel incubator bath.

Periodically,the samples are with drawn with the help of a hypodermic syringe fitted with a 0.8 µ spectrophore membrane filter.

Percent haemoglobin can similarly calculated at various time intervals at 540run spectrophotometrically.

Laser light scattering may also be used to evaluate haemoglobin content of individual resealed erythrocytes.

3.OSMOTIC FRAGILITY: It is reliable parameter for invitro evaluation of carrier erytrocytes

with respect to shelf life, invivo survival & effect of encapsulated substances.

When RBC are exposed to solution of varying tonicities, this shape changes due to osmotic balance.

To evaluate the effects of varying tonicities,drug loaded erythrocytes are incubated with saline solutions of different tonicities at 37+20c for 10min.

The suspension after centrifugation for 15min, 2000rpm is assayed for drug or haemoglobin release.

4.OSMOTIC SHOCK: Osmotic shock describes a sudden exposure of drug loaded erytrocytes

to an environment, which is far from isotonic to evaluate the ability of resealed erythrocytesto withstand the stress and maintain their integrity as well as appearance.

Incubating the resealed erythrocytes with distilled water for 15min followed by centrifugation at 3000rpm for 15min, may cause the release of haemoglobin to varying degrees which could be estimated spctrophotometrically.

5.TURBULENCE SHOCK: This parameter indicates the effect of shear force and pressure by which

resealed erytrocytes formulations are injected, on integrity of the loaded cell.

Loaded erythrocytes are passed through a 23-gaug hypodermic needle at a flow rate of 10min. After every pass,aliquot of the suspension is withdrawn and centrifuged at 300g for 15 min, and haemoglobin content, leached out is estimated spectrophotometrically.

6.MORPHOLOGY&PERCENT CELLULAR RECOVERY:

Phase contrast optical microscopy, transmission electron microscopy & scanning electron microscopy are the microscopic methods used to evaluate the shape, size & surface features of loaded erythrocytes.

Percent cell recovery can be determined by assessing the number of intact erytrocytes remaining per cubic mm with the help of haemocytometer.

APPLICATIONS:Erythrocytes as drug/ enzyme carriers: Erythrocytes as carriers for enzymes. Erythrocytes as carriers for drugs. Erythrocytes as carriers for proteins and macromolecules.

Drug targeting: Drug targeting to RES organs Surface modification with antibodies. Surface modification with Glutaraldehyde. Surface modification involving sulphydryls.

Drug targeting to Liver: Enzyme deficiency/replacement therapy Treatment of liver tumors Treatment of parasitic diseases Removal of RES Iron Overload

NOVEL SYSTEMS Nanoerythrosomes :

An erythrocytes 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.

Daunorubicin (DNR) was covalently conjugated to the

nEryt (nEryt-DNR) using glutaraldehyde as homobifunctional linking arm. This led to a complex that is more active than free DNR both in vitro and in vivo.

Daunorubicin (DNR) conjugated to these nanoerythrosomes has a higher antineoplastic index than the free drug.

ERYTHROSOMES. These are specially engineered vesicular systems

that are chemically cross-linked to human erythrocytes’ support upon which a lipid bilayer is coated.

This process is achieved by modifying a reverse-phase evaporation technique.

These vesicles have been proposed as useful encapsulation systems form macromolecular drugs.

CONCLUSION The use of resealed erythrocytes looks promising for a

safe and sure delivery of various drugs for passive and active targeting.

However, the concept needs further optimization to become a routine drug delivery system.

The same concept also can be extended to the delivery of biopharmaceuticals and much remains to be explored regarding the potential of resealed erythrocytes.

REFERENCE:1. R. Green and K.J.Widder, Methods in Enzymology

(Academic Press, San Diego, 1987), p. 149.2. C. Ropars, M. Chassaigne, and C.Nicoulau, Advances in

the BioSciences, (Pergamon Press, Oxford, 1987), p. 67.3. D.A. Lewis and H.O. Alpar, “Therapeutic Possibilities of

Drugs Encapsulated in Erythrocytes,” Int. J. Pharm. 22, 137–146 (1984).

4. U. Zimmermann, Cellular Drug-Carrier Systems and Their Possible

5. Targeting In Targeted Drugs, EP Goldberg, Ed. (John Wiley & Sons, New York, 1983), pp. 153–200.

6. S. Jain and N.K. Jain, “Engineered Erythrocytes as a Drug Delivery

7. S.P. Vyas and V.K. Dixit, Pharmaceutical Biotechnology 1 (CBS Publishers & Distributors, New Delhi, 655.(1999).

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