NANOPARTICLES &
RESEALED ERYTROCYTES.
PRESENTED BY:
K.SAI VANI
256213886014
M.PHARM (PHARMACEUTICS)
GUIDED BY:
Dr.M...
 Introduction
 Concept
 Ideal characteristics
 Advantages
 Disadvantages
 Polymers used
 Methods of preparati...
 Pharmaceutical aspects of Nanoparticles
 Characterization of nanoparticles
 In Vivo fate & Bio distribution of
Nano...
 Nano derives from the greek word “nanos”
which means Dwarf or Extremely small. It
can be used as a prefix for any unit...
 Nanoparticles are solid colloidal particles
ranging from 1to100nm in size.
They consist of micromolecular
materials i...
The basic Concept involved is :
 Selective and Effective Localization of
pharmacologically active moiety at preselected...
 Hence are useful to carry drugs to the liver and to
cells that are phagocytically active.
 By modifying the surface c...
 It should be biochemically inert , non toxic
and non-immunogenic.
 It should be stable both physically and
chemicall...
 Drug release should not effect drug action
 Specific Therapeutic amount of drug release
must be possessed
 Carriers...
Based On Method Of Preparation:
 Nanocapsules:-
Nanocapsules are systems in which
the drug is confined to a cavity sur...
 Solid Lipid Nanoparticles
 Polymeric Nanoparticles
 Ceramic Nanoparticles
 Hydrogel Nanoparticles
 Copolymerized...
Solid lipid Nanoparticles:
 New type of colloidal drug carrier system for i.v.
 Consists of spherical solid lipid par...
Ceramic Nanoparticles:
These are the nanoparticles made up of inorganic (ceramic)
compounds silica, ( Inorganic/metal) ...
Copolymerized Peptide Nanoparticles:
Drug moiety is covalently bound to the carrier instead of
being physically entrapp...
 Nano particle can be administered by
parenteral, oral, nasal, occular routes.
 By attaching specific ligands on to th...
 Small size & large surface area can lead to
particle aggregation .
 Physical handling of nano particles is difficult ...
NATURAL
HYDROPHILLIC
Proteins
Poly-saccharides
SYNTHETIC
HYDROPHOBIC
Pre-polymerized
Polymerized in
process
PROTEINS POLYSACCHARIDES
Gelatin Alginate
Albumin Dextran
Lectins Chitosan
Legumin Agarose
Viciline Pullulan
PRE-POL...
Polymerization
based methods
Amphiphilic
macromolecule
cross-linking
Polymer precipitation
methods
 Amphiphilic
macromolecule
cross-linking
Heat Cross-linking
Chemical
Cross-linking
 Polymerization
based
Methods
Polymerization
of monomers
Dispersion
polymerization
Interfacial
condensation
poly...
 Polymer
 Precipitation
methods
Solvent
extraction/
evaporation
Solvent
displacement
(nanoprecipitation)
Saltin...
 Nanoparticles can be prepared from
amphiphilic macromolecules, proteins &
polysaccharides.
 The technique of their p...
 The cross linking method is exhaustively used
for the nano-encapsulation of drug.
 The method involves the emulsifica...
 The high temperature used in the original
method restrict the application of method to
heat sensitive drugs.
 As an ...
 Chemical dehydration has been reported for
producing BSA nanoparticles
 Bhargava and Aindo suggested a simple chemica...
 Polymers used for nanosphere preparation
include:
 Poly(methylmethacrylate)
 Poly(acrylamide)
 Poly(butyl cyanoac...
 Two different approaches are generally
adopted for the preparation of nanospheres
using in-situ polymerization techniq...
 The process of emulsion polymerization can
be conventional or inverse, depending upon
the nature of the continuous pha...
 It involves the swollen
monomers micelles as
the site of nucleation
and polymerization
 The monomers is
emulsified...
 It is applied in the case
where the monomer is
sufficiently soluble in the
continuous outer phase
 The nucleation a...
 The polymerization rate is dependent on the
PH of the medium.
 Anionic polymerization takes place in micelle
after d...
 In case of dispersion
polymerization
howeverthe monomer
instead of emulsified,is
dissolved in an aqueous
medium whi...
 The performed polymer
phase is transformed to
an embroynic sheath.
 A polymer that
eventually become core
of nanop...
 The method is based on the process of
microencapsulation introduced by Lin & Sun
1969.
 In case of nanoparticles pre...
 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 contai...
 The method involves the
incorporation of saturated
aqueous solution of (PVA)
into an acetone solution of
the polymer...
 This method is based on the
interfacial deposition of a
polymer following
displacement of a semi-polar
solvent misci...
Solid Lipid Nanoparticles:
These are sub-micron colloidal carriers (50-
100nm) which are composed of physiological
lipi...
 Parenteral administration
 Brain delivery
 Ocular delivery
 Rectal delivery
 Oral delivery
 Topical delivery
 Small size and relatively narrow size
distribution which provide biological
opportunities for site specific drug deliv...
Hot Homogenization Technique:
 Homogenization of melted lipids at elevated
temperature.
Cold Homogenization Technique:...
Melting of lipid
Dissolution of the drug in the melted lipid
Mixing of the preheated dispersion medium
and the drug lip...
High pressure homogenization at a
temperature above the lipids melting point
O/W-Nano emulsion
Solidification of the na...
Melting of the lipid
Dissolution of the drug in the melted lipid
Solidification of the drug loaded lipid in liquid
nitr...
Grinding in a powder mill(50-100 particles)
Dispersion of the lipid in the cold aqueous
dispersion medium
Solid lipid n...
 Nanocrystals and Nanosuspensions are two
recently introduced aspects to drug delivery
research.
 The basic theme is ...
 Should be free from potential toxic impurities
 Should be easy to store and administer
 Should be sterile if parente...
 This method has been
suggested for the
purification of nano
particles and the
method can be scaled
up from an indus...
This technique involves the freezing of the
nanoparticle suspension and subsequent
sublimation of its water content unde...
 Nanoparticles intended for parenteral use
should be sterilized to be pyrogen free.
Sterilization can achieved by:
 U...
 Intravenous injection of
colloidal carriers follow their
interactions with at least two
distinct groups of plasma
pr...
 Steric stabilized (stealth) nanoparticles
 Magnetically guided nanoparticles(Fe3O4)
 Biomimetic nanoparticles (biomi...
 Homogenizer
 Ultra Sonicator
 Mills
 Spray Milling
 Supercritical Fluid Technology
 Electrospray
 Ultracentr...
 Particle size
 Density
 Molecular weight
 Structure and crystallinity
 Specific surface area
 Surface charge &...
1.PARTICLE SIZE:
 Photon correlation spectroscopy (PCS) : For
smaller particle.
 Laser diffractrometry : For larger p...
2.Density :
 Helium or air using a gas pycnometer
 Density gradiant centrifugation
3. Molecular weight :
 Gel perme...
5. Surface charge & electronic mobility :
 Surface charge of particle can be determined by
measuring particle velocity ...
6.Surface Hydrophobicity :
 Important influence on intraction of nanoparticles with
biological environment.
 Several ...
8.Nano particle yield:
9.Drug entrapment efficiency:
Company Technology API Route of
administration
Novavax, USA Micellar
nanoparticles
Testosterone S.c.
BioAUiance,
Fra...
Rapamune
(Merck & Co. Inc) (Wyeth-Ayerst Laboratories)
OLAY MOISTURIZERS
ABRAXANE
(American Biosciences, Inc.)
(Proct...
Nanoparticles are one of the
novel drug delivery systems, which can be of
potential use in controlling and targeting dru...
 Vyas S.P. , Khar R.K. Targeted & Controlled Drug
Delivery, Novel Carrier Systems, CBS Publication
,2002 ,Page No.249-2...
1
 Introduction
 Source of erythrocytes
 Isolation of erythrocytes
 Drug loading in erythrocytes
 Factors effecting...
 Present pharmaceutical scenario is aimed at
development of drug delivery systems which maximize
the drug targeting alo...
 Erythrocytes also known as red
blood cells and have extensively
studied for their potential carrier
capabilities for ...
 Although qualitatively similar to that of plasma
.however, quantitatively it differs from that of plasma.
 The concen...
 If the medium is Hypotonic, water diffuses into
the cells and they get swelled and eventually
loose all their hemoglob...
 If blood is placed into a tube and
centrifuged, the cells and the plasma
will separate.
 The erythrocytes, which are...
 Different mammalian erythrocytes have been
used for drug loading, resealing and
subsequent use in drug and enzyme deli...
 Blood is collected into heparinized tubes by venipuncture
 Blood is withdrawn from cardiac/splenic puncture(in small
...
 Hypotonic hemolysis.
 Hypotonic dilution.
 Hypotonic preswelling.
 Hypotonic dialysis.
 Use of red cell loader. ...
 Erythrocytes to undergo
reversible swelling in a
hypotonic solution.
 An increase in volume leads to
an initial cha...
Membrane ruptured RBC Loaded RBC Resealed Loaded RBC
0.4% NaCl
Hypotonic
Drug
Loading buffer
Resealing
buffer
Incub...
 These ruptured erythrocytes as drug carriers is based on the
fact that the ruptured membranes can be resealed by restor...
 In this method, a volume of packed
erythrocytes is diluted with 2–20 volumes of
aqueous solution of a drug.
 The sol...
RBC
Hypotonic /
Drug solution
Isotonic solution
Membrane ruptured RBC
Compounds that can be encapsulated are enzymes ...
 The technique is based upon initial controlled swelling
in a hypotonic buffered solution. This mixture is
centrifuged ...
Loaded RBC Resealed Loaded RBC
Loading buffer
Resealing
buffer
Incubation at
250c
HYPOTONIC PRESWELLING
0.6%w/v NaC...
 This method, also known as the osmotic pulse
method, involves isotonic hemolysis that is
achieved by physical or chemi...
RBC
Physical/chemical
rupturing Isotonic buffer
Drug
Isotonically ruptured RBC
loaded RBC
Incubation at
25oc
Resea...
RBC
+
Drug
Suspension
Buffer containing ATP,
MgCl2, and CaCl2
At 250 C
Loaded RBC
Resealing Buffer
Resealed RBC
...
Fig:- Entrapment By Endocytosis Method
 In the process, an isotonic, buffered suspension of
erythrocytes with a hematocrit value of 70–80 is prepared
and plac...
+
Phosphate
buffer
Placed in dialysis
bag with air bubble
Dialysis bag placed in 200ml of lysis
buffer with mechanic...
Hypotonic Dialysis
 This method is based on the observation that electrical
shock brings about irreversible changes in an
erythrocyte memb...
RBC
2.2 Kv Current for
20 micro sec
At 250 C
+
Pulsation
medium
Drug
+
Loading
suspension
3.7 Kv Current for
2...
Electro-insertion or Electro-encapsulation
Fig:- Electro-encapsulation Method
 It is a novel method for entrapment of nondiffusible drugs
into erythrocytes.
 They developed a piece of equipment ca...
“THIS METHOD IS BASED ON THE FACT THAT THE PERMEABILITY
OF THE ERYTHROCYTES INCREASES ON EXPOSURE TO CERTAIN
CHEMICAL AG...
Routes of administration include:
i. Intravenous (most common).
ii. subcutaneous.
iii.Intraperitoneal.
iv.Intranasal.
 There are mainly three ways for a drug to efflux out
from erythrocyte carriers.
 Phagocytosis.
 Diffusion through t...
Resealed erythrocytes after loading are characterized for following
parameters.
1. Drug Content:
Packed loaded erythroc...
 Percent haemoglobin can similarly calculated at various time
intervals at 540run spectrophotometrically.
 Laser light...
 To evaluate the effects of varying tonicities,drug loaded erythrocytes are
incubated with saline solutions of different...
5.TURBULENCE SHOCK:
 This parameter indicates the effect of shear force and pressure by which
resealed erytrocytes form...
Erythrocytes as drug/ enzyme carriers:
 Erythrocytes as carriers for enzymes.
 Erythrocytes as carriers for drugs.
 ...
Nanoerythrosomes :
 An erythrocytes based new drug carrier, named
nanoerythrosome has been developed which is prepared ...
These are specially engineered vesicular systems
that are chemically cross-linked to human
erythrocytes’ support upon w...
 The use of resealed erythrocytes looks promising for a
safe and sure delivery of various drugs for passive and
active ...
1. R. Green and K.J.Widder, Methods in Enzymology (Academic
Press, San Diego, 1987), p. 149.
2. C. Ropars, M. Chassaigne...
ANY QUERIES???
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
Nano particles and released erythrocytes
of 125

Nano particles and released erythrocytes

Published on: Mar 3, 2016
Source: www.slideshare.net


Transcripts - Nano particles and released erythrocytes

  • 1. NANOPARTICLES & RESEALED ERYTROCYTES. PRESENTED BY: K.SAI VANI 256213886014 M.PHARM (PHARMACEUTICS) GUIDED BY: Dr.Mrs. Yasmin Begum M.Pharm Ph.D
  • 2.  Introduction  Concept  Ideal characteristics  Advantages  Disadvantages  Polymers used  Methods of preparation  Novel Nanoparticulate systems
  • 3.  Pharmaceutical aspects of Nanoparticles  Characterization of nanoparticles  In Vivo fate & Bio distribution of Nanoparticles  Evaluation of nano particles  Applications  References.
  • 4.  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.
  • 5.  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.
  • 6. 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.
  • 7.  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.
  • 8.  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.
  • 9.  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.
  • 10. 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.
  • 11.  Solid Lipid Nanoparticles  Polymeric Nanoparticles  Ceramic Nanoparticles  Hydrogel Nanoparticles  Copolymerized Peptide Nanoparticles  Nanocrystals and Nanosuspensions  Nanotubes And Nanowires  Functionalized Nanocarriers  Nanospheres  Nanocapsules
  • 12. 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
  • 13. 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.
  • 14. 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.
  • 15.  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
  • 16.  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
  • 17. NATURAL HYDROPHILLIC Proteins Poly-saccharides SYNTHETIC HYDROPHOBIC Pre-polymerized Polymerized in process
  • 18. 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)
  • 19. Polymerization based methods Amphiphilic macromolecule cross-linking Polymer precipitation methods
  • 20.  Amphiphilic macromolecule cross-linking Heat Cross-linking Chemical Cross-linking
  • 21.  Polymerization based Methods Polymerization of monomers Dispersion polymerization Interfacial condensation polymerization Emulsion polymeriza tion Interfacial complexat ion
  • 22.  Polymer  Precipitation methods Solvent extraction/ evaporation Solvent displacement (nanoprecipitation) Salting out
  • 23.  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.
  • 24.  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.
  • 25.  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.
  • 26.  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)
  • 27.  Polymers used for nanosphere preparation include:  Poly(methylmethacrylate)  Poly(acrylamide)  Poly(butyl cyanoacrylate)  N-N’metylene-bis-acrylamide etc.
  • 28.  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)
  • 29.  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.
  • 30.  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.
  • 31.  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.
  • 32.  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.
  • 33.  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.
  • 34.  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.
  • 35.  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.
  • 36.  Solvent extraction/ evaporation method  Salting out method  Solvent displacement method.
  • 37.  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.
  • 38.  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.
  • 39.  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.
  • 40. 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.
  • 41.  Parenteral administration  Brain delivery  Ocular delivery  Rectal delivery  Oral delivery  Topical delivery
  • 42.  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.
  • 43. Hot Homogenization Technique:  Homogenization of melted lipids at elevated temperature. Cold Homogenization Technique:  Homogenization of a suspension of solid lipid at room temperature.
  • 44. Melting of lipid Dissolution of the drug in the melted lipid Mixing of the preheated dispersion medium and the drug lipid melt
  • 45. 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.
  • 46. Melting of the lipid Dissolution of the drug in the melted lipid Solidification of the drug loaded lipid in liquid nitrogen or dry ice
  • 47. Grinding in a powder mill(50-100 particles) Dispersion of the lipid in the cold aqueous dispersion medium Solid lipid nanoparticles.
  • 48.  Nanocrystals and Nanosuspensions are two recently introduced aspects to drug delivery research.  The basic theme is to convert micronized drug powders to drug nanoparticles.
  • 49.  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 Purification o Freeze drying o Sterilization.
  • 50.  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.
  • 51. This 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.
  • 52.  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.
  • 53.  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 .
  • 54.  Steric stabilized (stealth) nanoparticles  Magnetically guided nanoparticles(Fe3O4)  Biomimetic nanoparticles (biomimetic ligands-salilic acids)  Bioadhesive nanoparticles  Antibody coated nanoparticles.
  • 55.  Homogenizer  Ultra Sonicator  Mills  Spray Milling  Supercritical Fluid Technology  Electrospray  Ultracentrifugation  Nanofiltration
  • 56.  Particle size  Density  Molecular weight  Structure and crystallinity  Specific surface area  Surface charge & electronic mobility  Surface hydrophobicity  Invitro release  Nanoparticle yield  Drug entrapment efficiency
  • 57. 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
  • 58. 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
  • 59. 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.
  • 60. 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
  • 61. 8.Nano particle yield: 9.Drug entrapment efficiency:
  • 62. Company Technology API Route of administration Novavax, USA Micellar nanoparticles Testosterone S.c. BioAUiance, France Polydsohexyl cyanoacrylate) nanoparticles Doxorubicin i.v. American Bioscience, USA Albumin-Drug nanoparticles Paclitaxel i.v. Wyeth Pharmaceutical, USA Drug Nanoparticles Rapamycin Oral BioSante, USA Calcium phospahte nanoparticles Insulin Oral
  • 63. Rapamune (Merck & Co. Inc) (Wyeth-Ayerst Laboratories) OLAY MOISTURIZERS ABRAXANE (American Biosciences, Inc.) (Proctor and Gamble)
  • 64. 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.
  • 65.  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.
  • 66. 1
  • 67.  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
  • 68.  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.
  • 69.  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
  • 70.  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.
  • 71.  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.
  • 72.  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%
  • 73.  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
  • 74.  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.
  • 75.  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.
  • 76.  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.
  • 77. 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
  • 78.  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.
  • 79.  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.
  • 80. RBC Hypotonic / Drug solution Isotonic solution Membrane ruptured RBC Compounds that can be encapsulated are enzymes such as - galactosidase and -glucosidase , asparginase,and arginase, salbutamol. Resealed erythrocytes
  • 81.  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
  • 82. Loaded RBC Resealed Loaded RBC Loading buffer Resealing 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.
  • 83.  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.
  • 84. RBC Physical/chemical rupturing Isotonic buffer Drug Isotonically ruptured RBC loaded RBC Incubation at 25oc Resealed RBC Compounds encapsulated are – Urea, polyethylene, polypropylene, and NH4Cl
  • 85. 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.
  • 86. Fig:- Entrapment By Endocytosis Method
  • 87.  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.
  • 88. + Phosphate buffer Placed in dialysis bag with air bubble Dialysis bag placed in 200ml of lysis buffer with mechanical rotator 2hrs. 4c. Loading Drug buffer Dialysis bag placed in Resealing buffer with mechanical rotator 30 min 37c. Resealed RBC DRUGS: gentamicin, pentamidine, interlukin-2 , desferroxamine and recombinant erythropoietin.
  • 89. Hypotonic Dialysis
  • 90.  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.
  • 91. 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.
  • 92. Electro-insertion or Electro-encapsulation Fig:- Electro-encapsulation Method
  • 93.  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.
  • 94. “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
  • 95. Routes of administration include: i. Intravenous (most common). ii. subcutaneous. iii.Intraperitoneal. iv.Intranasal.
  • 96.  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.
  • 97. 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.
  • 98.  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.
  • 99.  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.
  • 100. 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.
  • 101. 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
  • 102. 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.
  • 103. 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.
  • 104.  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.
  • 105. 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).
  • 106. ANY QUERIES???

Related Documents