Molecular Pharmaceutics Liposome niosomes Nanoparticle.pdf

By Saurabh Dubey
M.Pharm
Molecular Pharmaceutics (Nano Technology
& Targeted DDS) (MPH 201T)
M. Pharm. Second Semester
Liposomes, Niosomes Resealed Erythrocytes,
Nanoparticles, Solid Lipid Nanoparticles & Dendrimers

Nanoparticles
• Nanoparticles are sub-nanosized colloidal structures composed
of synthetic or semi synthetic polymers.
• Nanoparticles are solid, colloidal particles ranging 10- 1000nm,
consisting of macromolecular materials in which active
ingredient is dissolved/ entrapped/ encapsulated/ absorbed/
attached
• The first reported nanoparticles were based on non-
biodegradable polymeric systems.
❑ polyacrylamide,
❑ polymethylmethacrylate,
❑ polystyrene etc.
• The possibilities of chronic toxicity due to tissue and
immunological response towards these polymers had restricted
their use for systemic administration.
• This problem has been solved by using biodegradable polymers.

Natural Hydrophilic Polymers
Proteins and Polysaccharides have been extensively studied and
characterized.
Proteins
• Gelatin
• Albumin
• Lectins
• Legumin
• Viciline
Disadvantage:
1. Batch to batch variation.
2. Conditional biodegradability.
3. Antigenicity.
Polysaccharides
Alginate
Dextran
Chitosan
Agarsoe
Pullulan

 Solid Lipid Nanoparticles
 Polymeric Nanoparticles
 Ceramic Nanoparticles
 Hydrogel Nanoparticles
 Copolymerized Peptide Nanoparticles
Synthetic Hydrophobic Polymer :
The polymer used are either pre-polymerized or polymerized in process
Pre-polymerized
Poly (ε - caprolactone) (PECL)
Poly (lactic acid) (PLA)
Poly ( lactide -coglycolide) (PLGA)
Polystyrene
Polymerized in process
Poly (isobutylcynoacrylates) (PICA)
Poly (butylcynoacrylates) (PBCA)
Polyhexylcyanoacrylates (PHCA)
Poly (methacrylate) (PMMA)
Classification of Nanoparticles

Preparation Techniques
❑ The appropriate method selection depends on the
physicochemical characteristics of the polymer and the drug to be
loaded.
❑ The preparation technique largely determine the-
• Inner structure
• In vitro release profile
• Biological fate of the systems.

Preparation Techniques of Nanoparticles
1) Amphiphilic macromolecule cross linking via
a) Heat process
b) Chemical process
2) Polymeriazation based method.
a) Polymerization of monomers in situ.
b) Emulsion (micellar) polymerization .
c) Dispersion polymerization.
d) Interfacial condensation polymerization.
d) Interfacial complexation.
3) Polymer precipitation methods
a) Solvent extraction/evaporation
b) Solvent displacement (Nanoprecipitation)
c) Salting out

1. Nanoparticles Preparation by Cross-linking of Amphiphilic
Macromolecules:
• Proteins and polysaccharides are used.
• This technique involves two steps:
a) The aggregation of amphiphile(s)
b) Stabilization either by heat denaturation or chemical cross-
linking.
❑ These processes may occur in a biphasic o/w or w/o type
dispersed systems, which subdivide the amphiphile(s) prior to
aggregative stabilization.
❑ It may also take place in an aqueous amphiphilic solution where
on removal of the solvent by extraction or diffusion, amphiphile(s)
are aggregated as tiny particles and subsequently rigidized via
chemical cross-linking.

Cross-linking in w/o emulsion:

Factors governing the size and shape of the nanoparticles are
mainly,
• Emulsification energy
• Temperature
❑ Most widely used cross-linking agent is glutaraldehyde as 3%
v/v solution.
❑ The problem associated with the use of chemical as a cross-
linking agent is the complete removal of the agent.
Emulsion chemical dehydration:
• Hydroxypropyl cellulose solution in chloroform was used as a
continuous phase.
• 2,2, di-methyl propane (Dehydrating agent) was used to translate
internal aqueous phase in to a solid particulate dispersion.
• produce nanoparticles of size ( 300 nm )

Phase separation in aqueous medium (Desolvation)
• The protein
environment
or polysaccharide
can be desolvated
from an aqueous
by pH change,
temperature or by adding appropriate counter ions .
• Cross linking may be affected simultaneously or
subsequent to desolvation technique .
• This proceeds via three steps Protein dissolution ,
protein aggregation and protein deaggregation
• Here sodium sulphate is used as desolvating agent
• While Alcohol (ethanol and isopropyl alcohol) are added
as deaggregating agent .
• Both lipophilic and hydrophillic drugs could be
entrapped in nanoparticles by this method.

pH Induced Aggregation
• Here protein phase may be separated by change of pH.
• E.g.-Insulin nanoparticles
• Insulin → precipitated → redissolved nanodroplet →
hardened using glutaraldehyde.
• Gelatin & Tween 20 were dissolved in aqueous phase.
pH was adjusted to optimum value.
• Clear solution so obtained was heated to 40 0c &
0C
followed by quenching at 40 for 24 hours &
subsequently left at ambient temperature for 48 hours .
• This lead to gelatin colloidal dispersion .
• Finally colloidal aggregate were cross linked using
glutaraldehyde .

Counter Ion Induced aggregation
• Protein phase is separated due to presence of
counter ions in aqueous phase.
• Aggregation of dispersed phase (polysaccharide)
can be effectively.
• Initiated by adding appropriate counter ions.
• Aggregation can be propagated by adding
secondary specious of counter ions followed by
rigidisation step.
• Eg–Alginate nanoparticles Ca+2 - Gelation
inducing agent.
• Poly ( L lysine )- Propagation of reaction .

2. Nanoparticle-Preparation Using Polymerization Based
Methods:
a. Polymerization of monomers in situ:
Poly acrylate
• derivatives are used as polymers.
• The in situ polymerization process consists of an initiation step
followed by a series of polymerization steps, which results in the
formation of a hybrid between polymer molecules
and nanoparticles.
• Nanoparticles are initially spread out in a liquid monomer or a
precursor of relatively low molecular weight.

(i)Micellar Polymerization Mechanism:
b. Emulsion polymerization

(ii)Homogenous Polymerization Mechanism:

c. Dispersion polymerization
Monomer is dissolved in aqueous medium , which act as a
precipitant ,for subsequently formed polymer.
Polymerization based method involve in situ polymerization method
where drug to be loaded is added to formed polymeric nanoparticles
d. Interfacial Polymerization

3. Polymer precipitation methods:
a. Solvent extraction/evaporation

b. Solvent displacement (nanoprecipitation)

• Liposomes are simple
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cles in which an aqueous
volume is entirely enclosed by a Phospholipids bilayer molecule.
• Spherical vesicles with a phospholipid bilayer
• Liposomes are concentric bilayered vesicles in which an aqueous
volume is entirely enclosed by a membranous lipid bilayer mainly
composed of natural or synthetic phospholipids. OR
• Liposomes (lipid vesicles) are sealed sacs in the micron or
submicron range dispersed in an aqueous environment.
❖ Liposome were first produced in England in 1961 by Alec D.
Bangham
❖The size of a liposome ranges from some 20 nm up to several

How they are formed?
Liposomes are formed by the self-assembly of phospholipid
molecules in an aqueous environment.
ADVANTAGES
1. Biocompatible & Biodegradable
2. Entrap hydrophilic & hydrophobic pharmaceutical agents.
3. Sustained release depot (Propranolol, cyclosporin )
4. Increased efficacy & therapeutic index of drugs ( Actinomycin D)
5. Can be administered through various routes.
6. Lack of antigenic properties
7. Site avoidance effect (avoids non-target tissues).
8. Flexibility to couple with site-specific ligands to achieve active
targeting

DISADVANTAGES
➢ Less stability
➢ Low solubility
➢ Problem of targeting to various tissues due to their large size
➢ Short half life
➢ Leakage of encapsulated drug
➢ High cost of production
➢ Allergic reactions may occur due to liposomal constituents

Basic Components of Liposomal System
There are number of components of liposomes however
Lecithin (mixture of phospholipids) and cholesterol being
main components.
1. Phospholipids (PCs)
• PCs are fatty substances the major structural components
of cell wall & biological membranes.
• Phospholipids are amphipathic moieties with a
hydrophilic head group and two hydrophobic tails.
• Two types of phospholipids exist – phosphodiglycerides
and sphingolipids, together with their corresponding
hydrolysis products.
• Phospholipids have phosphatidyl moiety (tail) with
different head groups (choline, Etahnolamine, Serine)
• The most common phospholipid is phosphatidylcholine
(PC) molecule

Phosphatidylcholine has glycerol bridge links a pair of hydrophobic
acyl hydrocarbon chains having 10-24 carbon atoms with a
hydrophilic polar head group.

The most common natural phospholipid is the
phospatidylcholine (PC ).
Naturally occurring phospholipids used are :
PC: Phosphatidylcholine.
PE: Phosphatidylethanolamine.
PS: Phosphatidylserine
Synthetic phospholipids used are:
DOPC: Dioleoyl phosphatidylcholine
DSPC: Disteroyl phosphatidylcholine
DOPE: Dioleoyl phosphatidylethanolamine
DSPE: Distearoyl phosphatidylethanolamine

Mechanism of formation of liposome
In aqueous media phospholipids as they are not soluble align
themselves closely in planar bilayer sheets or lipid cakes which is
thermodynamically stable.
In which polar head groups face outwards into the aqueous medium,
and the lipidic chains turns inwards to avoid the water phase, giving
rise to double layer or bilayer
•For the liposomes to be formed, upon further hydration, the lipid
cakes(lamella) swells eventually they curve to form a closed vesicles in
the form of spheres
•This spheres are called as liposomes.

PHASE TRANSITION TEMPERATURE (Tc):
• At various temperatures, phospholipid membranes can exist in
different phases.
• The transition from one phase to another can be detected by
technique like micro-calorimetry.
• This is due to the fatty acid chain adopting a new conformation
other than all trans straight chain configuration, such as gauche
configuration state (phenomenon- chain tilt with decrease in
bilayer thickness)

2. Cholesterols
• Incorporation of sterols in liposome bilayer can bring about major
changes in the preparation of these membranes.
• Cholesterol does not by itself form bilayer structure , it acts as fluidity
buffer.
• It inserts into membrane with hydroxyl group oriented towards
aqueous surface & aliphatic chain aligned parallel to acyl chains in the
centre of bilayer.
• It can be incorporated into phospholipid membranes in very high Conc.
upto 1:1 or even 2:1 molar ratios of PC.
• Cholesterol incorporation increases the separation between the choline
head groups and eliminates the normal electrostatic and hydrogen-
bonding interactions.
Role of cholesterol in bilayer formation:
1. Cholesterol act as fluidity buffer.
2. After intercalation with phospholipid
molecules alter the freedom of motion of carbon
molecules in the acyl Chain
3. Restricts the transformations of trans to
gauche conformations.

Cholesterol
• Enhances the stability of the membrane
• Enhances the rigidity of the phospholipid bilayer
• Reduces the permeability of water soluble substance
Cross-section of liposomes

Method of Preparation of Liposomes
A. Passive loading
Involves loading of entrapped
manufacturing procedure.
agents before or during the
B. Active or remote loading
➢Certain types of compounds with ionizable groups and those
with both lipid and water solubility can be introduced into the
liposomes after the formation of the intact vesicle

Preparation methods
• Liposomes can be prepared using a wide range of
combination of lipids with
methods that involve
aqueous media, and somehow affect liposomes
characteristics, such as size, lamellarity and
encapsulation efficiency (EE).
• The recently reported methods can be categorized as
conventional, which mostly involve approaches that are
easy to use at laboratory scale, and novel methods that
appear to be more useful for up-scale production but
require some special equipment

Conventional methods
The most commonly used methods for formulation of liposomes
share the following fundamental stages:
(i) lipids dissolution in organic solvents,
(ii) drying of the resultant solution,
(iii) hydration of dried lipid (using various aqueous media),
(iv) isolation of the liposomal vesicles, and
(v) quality control assays.

Film hydration
• Also known Bangham method, film hydration represents the
simplest and oldest method used in liposome technology.
• In this method, lipids are firstly dissolved in a suitable organic
solvent, and dried down to yield a thin film at the bottom of the
flask.
• The obtained lipid film is hydrated using an appropriate aqueous
medium to produce liposomal dispersion.
• The structural organization of the formed vesicles can be affected
by the hydration conditions.
• A gentle hydration of the lipid film forms giant unilamellar
vesicles (GULV), whereas a hash hydration gives rise to
multilamellar vesicles (MLV) with poor size homogeneity, which
requires an additional downsizing step.
• The most commonly used sizing methods include probe and bath
sonication that afford production of small unilamellar vesicles
(SUV).

• Despite its higher effectiveness, probe sonication is often blamed for potential
contamination (with titanium from the titanium-based nozzle used for mechanical
agitation), and production of local heat that can affect lipids and drugs stability.
• Although the two sonication methods produce liposomes with identical characteristics,
the use of bath sonication remains a better option due to easy control of operational
parameters. Another technique used for liposome sizing includes consecutive extrusion
of the liposomal formulation through polycarbonate filters of defined pore sizes.
• In this method, the number of extrusion cycles is the key parameter to control for
effective homogenization

Reverse phase evaporation
• Reverse phase evaporation is an alternative method to the film
hydration that involves formation of water-in-oil emulsion
between the aqueous phase (containing hydrophilic materials)
and the organic phase (containing lipids and any hydrophobic
materials).
• A brief sonication of this mixture is required for system
homogenization.
• The removal of the organic phase under reduced pressure yields
a milky gel that turns subsequently into liposomal suspension.
• The liposomes can be isolated from the dispersion using
centrifugation, dialysis or sepharose 24 column

Solvent injection
• Solvent injection involves quick injection of the lipid solution (in
ethanol or diethyl ether) into an aqueous medium.
• The experiment is performed either at room or at higher
temperature (e.g., 60°C), depending on whether the organic
solvent is water-miscible or not.
• The liposomes prepared by solvent injection process are mostly
polydispersed and highly contaminated by organic solvents,
especially ethanol due to formation of azeotrope mixture with
water.
Limitations
• Exposure of the therapeutic agents to high temperature and
organic solvent that might affect both the stability and safety of
the liposomal products

Heating method
• Among all the conventional methods, the heating method is
known to be the most attractive method for liposomes
preparation due to its organic solvent free characteristics.
• In the heating method, lipids are hydrated for 1 hour, and heated
for another hour above the transition temperature of the
phospholipids in the presence of a hydrating agent (glycerin or
propylene glycol 3%).
• When cholesterol is part of the formulation, the reaction medium
is heated up to 100°C because of its high melting point.
• Being prepared under heating conditions, the resultant
liposomes can be readily used without any further sterilization
treatments, which minimizes both formulation complexity and
timing.
• In addition, there is no need for further removal of the hydrating
agents employed, since represent physiologically
these
acceptable ingredients that are well-established for
pharmaceutical applications.

• Moreover, the observation that these hydrating agents can prevent
particle coagulation and sedimentation makes them much more
attractive as stabilizer and isotonizing additives.
• The hydroxyl groups of these hydrating agents provide a
cryoprotective effect that makes the heating method an efficient
method for the formulation of inhalable liposomes

Novel preparation methods
Microfluidic channel method
• Microfluidic methods include all the novel techniques that make use of
microscopic channels (in the size range of 5–500 μm).
• In this method, lipids are dissolved in an appropriate organic solvent
(ethanol or isopropanol) and the resultant solution is propelled
perpendicularly or in the opposite direction to the aqueous medium
within the micro-channels.
• The continuous axial mixing of the organic and aqueous solutions leads
to liposomes formation because of local diffusion of phospholipids in
aqueous phase, which encourages the self-assembly process.
• Among many others, the micro hydrodynamic focusing method
represents the most commonly used microfluidic method for liposomes
formulation.
• This method produces small and large unilamellar vesicles, 40–140 nm,
with good size homogeneity (mono dispersed feature).
• The other microfluidic techniques include the microfluidic droplets and
the pulsed jet flow microfluidic methods.

• The microfluidic droplets method involves dissolution of
phospholipids in hexane for preparation of giant liposomes (4–
20 μm).
• In the pulsed jet flow microfluidic method, the conventional film
hydration method has been modified by drying the lipid solution
in microtubes.
• The resultant lipid film is hydrated within the microtubes through
a perfusion process that produces much larger vesicles, 200–
534 μm, with remarkable encapsulation efficiency
• As common advantages, the microfluidic methods offer the
possibility for production of vesicles with desired size, due to the
versatility and flexibility of the methods.
• The disadvantages of these methods include the imperative use of
organic solvent and drastic agitation, as well as difficulty for large
scale production

Supercritical fluidic method
• While being considered as equivalent to the conventional reverse
phase evaporation method, supercritical fluidic technique
represents the most important novel liposome preparation
methods that makes use of a supercritical fluid, such as carbon
dioxide (CO2) maintained under supercritical conditions
(supercritical temperature and pressure).
• In this state, CO2 is an excellent solvent for the lipids. The high-
performance liquid chromatography (HPLC) pump provides a
continuous flow of the aqueous phase into a view cell that
contains the supercritical lipid solution, allowing phase transition
of the dissolved phospholipids.
• Upon sudden decrease in pressure, CO2 gets completely removed
and phospholipids self-assemble into a bilayered vesicular system.
• The supercritical fluidic method affords large unilamellar vesicles
(100–1200 nm) with 5-fold higher encapsulation efficiency than
the equivalent conventional method.

• Apart from being organic solvent-free methods, the supercritical
fluidic method offers many other advantages such as the use of
CO2, as a cheap and environmentally harmless solvent, possibility
for controlling particle size, in situ sterilization and large-scale
production in industrial settings.
• However, the disadvantages of the supercritical fluidic technique,
including particularly its high cost, low yield and use of high
pressures (200–350 bar) which require special infrastructures,
restrict their universal applications for wider developments of
liposomaltechnology

Double Emulsion Method:
1. The organic solution, which already contains water
droplets (W/O) is introduced in to excess aqueous medium
followed by mechanical dispersion causing phase inversion
(O/W).
2. The (W/O/W) multi component vesicle is formed by
double emulsion.
3. Two aqueous compartments being separated from each
other by two phospholipid monolayer.
4. The hydrophobic surfaces of monolayers (tails) face each
other, with a thin film of organic solvent.
5. Removal of organic solvent results in formation of
intermediated sized unilamellar vesicles.

Rapid solvent exchange method:
• Lipid solution in organic solvent is passed through an
orifice of syringe by means of vacuum in to a tube
containing aqueous buffer placed on vortex.
• The organic solvent vaporizes due to vacuum before
contacting aqueous phase.
• The lipid mixture precipitates very quickly in aqueous
buffer forming liposomes.

De-Emulsification method
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Post-preparation treatments
Freeze-thawing
• The freeze-thawing treatment involves freezing the liposomes dispersion in
liquid nitrogen, and subsequently thawing it at the temperature above the
phase transition temperature of the lipids used for formulation.
• Upon freeze-thawing, the liposomal vesicles are subjected to fusion since
the lipid bilayers become fluid and highly permeable, allowing extensive
diffusion of hydrophilic molecules, which leads to important
cryoconcentration.
• These structural modifications encourage encapsulation of hydrophilic
materials that are poorly loaded in liposomes when conventional methods
are used.
• This underlines the reason why freeze-thawing represents an important
treatment in liposome technology.
• Amongst the key parameters to be considered for freeze-thawing
optimization are the number and duration of freeze-thawing cycles.
• These can impact significantly not only the encapsulation efficiency but also
structural characteristics, i.e., liposomes lamellarity and polydispersity.

Freeze-drying
• Commonly known as lyophilization, freeze-drying is a post-preparation
treatment for liposomes that is applied in both laboratory and industrial
settings to preserve the characteristics of liposomal products.
• Freeze-drying involves freezing of the aqueous samples and subsequent
removal of ice by sublimation.
• Freeze-drying represents a very useful treatment for shelf stability of liposomal
suspensions, since water molecules can trigger some chemical reactions and
lead to modification of the cargo or excipients in the formulation.
• Freeze-drying appears to be of great interest when the prepared formulation
contains thermos-sensitive materials such as proteins, nucleic acids, etc., which
might undergo fast degradation when subjected to heat-drying.
• The use of freeze-drying has gained considerable attention in liposome
technology due to improved storage stability of liposomal products.
• Because of potential leakage of liposomes during freeze-drying, addition of
hydrophilic compounds, commonly called cryoprotective agents (such as
carbohydrates), has been established to ensure good stability and quality of the
final product.
• The cryoprotectants commonly used include mannitol, lactose, sucrose and
trehalose. Among these, trehalose is the most reputed cryoprotecting agent
since it preserves liposomes stability during and after freeze-drying treatment

Application of liposomes
❑ Liposomes are used for drug delivery due to their unique properties.
❑ A liposome encapsulates a region on aqueous solution inside a
hydrophobic membrane; dissolved hydrophilic solutes cannot readily
pass through the lipids.
❑ Hydrophobic chemicals can be dissolved into the membrane, and in this
way liposome can carry both hydrophobic molecules and hydrophilic
molecules.
❑ There are three types of liposomes – MLV (multilamillar vesicles) SUV
(Small Unilamellar Vesicles) and LUV (Large Unilamellar Vesicles) used
to deliver different types of drugs.
❑ Liposomes are used as models for artificial cells.
❑ Liposomes can also be designed to deliver drugs in other ways.•
Liposomes that contain low (or high) pH can be constructed such that
dissolved aqueous drugs will be charged in solution.

❑ As the pH naturally neutralizes within the liposome (protons can pass through some
membranes), the drug will also be neutralized, allowing it to freely pass through a
membrane.
❑ These liposomes work to deliver drug by diffusion rather than by direct cell fusion.
❑ Another strategy for liposome drug delivery is to target endocytosis events.
❑ Liposomes can be made in a particular size range that makes them viable targets for
natural macrophage phagocytosis.
❑ These liposomes may be digested while in the macrophage's phagosome, thus releasing
its drug.
❑ Liposomes can also be decorated with opsonins and ligands to activate endocytosis in
other cell types.
❑ The use of liposomes for transformation or transfection of DNA into a host cell is known
as lipofection• Protection against enzyme degradation of drugs
❑ Liposomes are used to protect the entrapped drug against enzymatic degradation whilst
in circulation.
❑ The basis is that the lipids used in their formulation are not susceptible to enzymatic
degradation; the entrapped drug is thus protected while the lipid vesicles are in
circulation in the extracellular fluid.

Drug targeting
❑ The approach for drug targeting via liposomes involves the use of ligands (e.g., antibodies,
sugar residues, apoproteins or hormones), which are tagged on the lipid vesicles.
❑ The ligand recognises specific receptor sites and, thus, causes the lipid vesicles to concentrate
at such target sites.
❑ By this approach the otherwise preferential distribution of liposomes into the
reticuloendeothelial system RES (liver, spleen and bone
❑ marrow) is minimized.
Topical drug delivery
• The application of liposomes on the skin surface has been proven to be effective in drug delivery
into the skin.
• Liposomes increase the permeability of skin for various entrapped drugs and at the same time
diminish the side effect of these drugs.
• Enhanced antimicrobial efficacy
• Antimicrobial agents have been encapsulatedin liposomes for two reasons.
• First, they protect the entrapped drug against enzymatic degradation. For instance, the
penicillins and cephalosporin are sensitive to the degradative action of β-lactamase, which is
produced by certain microorganisms.
• Secondly, the lipid nature of the vesicles promotes enhanced cellular uptake of the antibiotics
into the microorganisms, thus reducing the effective dose and the incidence of toxicity as
exemplified by the liposomal formulation of amphotericin B.

Solid lipid nanoparticles
• The solid lipid nanoparticles(SLN’s) are submicron colloidal
carriers which are composed of physiological lipid, dispersed in
water or in an aqueous surfactant solution.
• They consist of macromolecular materials in which the active
principle is dissolved, entrapped, and or to which the active
principle is adsorbed or attached.
• No potential toxicity problems as organic solvents are not used.
• SLNs are spherical in shape & diameter range from 10-1000nm.
• To overcome the disadvantages associated with the liquid state of
the oil droplets, the liquid lipid was replaced by a solid lipid.
• The reasons for the increasing interest in lipid based system are :
1. Lipids enhance oral bioavailability and reduce plasma profile
variability.
2. Better characterization of lipoid excipients.
3. An improved ability to address the key issues of technology
transfer and manufacture scale-up.

Drug Loading capacity is limited
water content
drug degradation
Coexistences of several colloidal species
•
• High
• High pressure induce
•
• Lipid crystallization & drug incorporation - supercooled melts -
gelation phenomenon
• Drug expulsion
Advantages of SLNs:
• Control & target drug release
• Increased drug stability
• High & enhanced drug content
• Feasible for carrying both lipophilic & hydrophilic drug
• Excellent biocompatibility
• Water based technology
• Easy to scale up & sterlize
• Avoid RES
Disadvantages:

Advantages of SLNs over polymeric NPs
Polymeric Nanoparticles Solid Lipid Nanoparticles
• Residual contamination • Avoid residual contamination
• Possible toxicity problems • No toxicity problems
• Expensive production & a lack
of large scale production
method
• Cost effective methods are
available
• Lack of suitable sterilization
method
• Feasible sterilization method
available
• Not stable as compared to
SLNs
• SLNs formulation stable for
even three years have been
developed

SLNs preparation:
• General ingredients include solid lipid, emulsifier & water
• Lipid contains triglycerides, partial glycerides, fatty acids,
steroids, waxes
• Combination of emulsifier might prevent particle agglomeration
Emulsifier include soybean lecithin, egg lecithin, poloxmer etc.
Method of preparation:
• High pressure homogenization
• Hot homogenization
• Cold homogenization
• Ultrasonication
• Solvent emulsification/evaporation
• Micro emulsion
• Using Supercritical Fluid
• By Spray drying

1) High Shear Homogenization:
• It is a reliable and powerful technique, which is used for the
production of SLN.
• High pressure homogenizers push a liquid with high pressure
(100–2000 bar) through a narrow gap (in the range of A few
microns).
• The fluid accelerates on a very short distance to very high
velocity (over 1000 km/H).
• Very high shear stress and cavitations forces disrupt the
particles down to the submicron range.
• Generally 5 -10% lipid content is used but up to 40% lipid
content has also been investigated.
• Two general approaches of HPH are :-
❑ Hot Homogenization.
❑ Cold Homogenization.

Advantages:
1) Easy to handle.
2) High concentration of surfactant and co-surfactant are not
required.
3) Organic solvent free method
Disadvantages:
1) temperature induce drug degradation.
2) partitioning effect.
3) complexity of the crystallization.

Advantages:
1)Low capital cost.
2)Demonstrated at lab scale.
Disadvantages:
1) Larger particle sizes & broader size distribution.
2) does not avoid thermal exposure but minimizes it.

2) Ultrasonication with HPH
• For smaller particle size combination of both ultrasonication and
high speed homogenization is required
Advantages:
1) Equipment used is very common
2) No temperature induced drug degradation
Disadvantages:
1) Potential metal contamination
2) Broader particle size distribution ranging into micrometer range.

Advantages:
1) Avoidance of any thermal stress.
2) Continuous process
Disadvantages:
1) Use of organic solvents.
2) Extremely energy intensive process.

Advantages:
1) Low mechanical energy input.
2) Theoretical stability.
Disadvantages:
1) Extremely sensitive to change.
2) Labor intensive formulation work.
3) Low nanoparticle concentration
5) By Using Supercritical fluid:
Can be prepared by Rapid Expansion of Supercritical Carbon dioxide
solution methods(RESS)
Carbon dioxide with 99.99% is good solvent.
Advantage:-
Solvent less processing.

6) By Using Spray Drying Method:
• Spray drying method is a cheaper method than lyophilization.
• This method causes particle aggregation due to high temperature,
shear forces and partial melting of the particle. The best result by
spray drying method was obtained with SLN concentration of 1%
in solution of trehalose in water or 20% trehalose in ethanol
water mixture(10:90 v/v)
Disadvantage:-
particle aggregation due to high temperature, shear forces & partial
melting of particles.

DENDRIMER
• The word “dendrimer” originated from two Greek words,
dendron :- meaning tree,
meros:- meaning part or segment.
• Dendrimers are a new class of polymeric materials.
• They are highly branched, monodisperse , artificial
macromolecules.
synthetic three-dimensional
• Dendrimers may be defined as
hyper branched, globular
characterized by its highly branched 3D structure
macromolecule, which is
that
provides a high degree of surface functionality.

Dendrimers possess three distinguished components, namely
An initiator core
(i) .
Interior layers
(ii) :- composed of repeating units, radically attached
to the interior core(generations).
Exterior layers
(iii) :- attached to the outermost interior generations
(terminal functionality).

NEED OF DENDRIMERS:-
• Nano-particle drug-delivery systems are most popular one.
• However reticuloendothelial system (RES) uptake, drug leakage,
immunogenicity, haemolytic toxicity, cytotoxicity, restrict the use
of these nanostructures.
• These are overcome by surface engineering the dendrimer such as
Polyester dendrimer, Glyco-dendrimers, PEGylated dendrimers
etc.
• The bioactive agents can be easily encapsulated into the interior
of the dendrimers. or
• Chemically attached i.e. physically adsorbed on to the dendrimer
surface.

METHODS OF SYNTHESIS:-
1) Divergent method.
2) Convergent method.
3) Hypercores & branched monomers method.
4) Double Exponential And Mixed Growth.
5) Other accelerated growth technique

1) Divergent method
Dendrimers starts from the central core and extends toward the
surface i.e. diverging into space.
Two step process:
• Activation of functional surface groups
• Addition of branching monomers units.
❑ Divergent approach is successful for the production of large
quantities of dendrimers.
❑ But it causes some difficulties in the purification of the final
product.

2) Convergent method
• Dendrimer starting
inwards.
from the end groups and progressing
• When the growing wedges are enough large, attached to a
suitable core to give a complete Dendrimer.
• The convergent methodology also suffers from low yields in the
synthesis of large structures.
Advantages:
1.Relatively easy to purify the desired product.

3) Hypercores & Branched Monomers Technique
• Frechet group continued their efforts on research of hypercore &
branched monomers.
• This method involves the pre assembly of oligomeric species,
which can then be linked together to give dendrimer.
• These monomers allow the to design synthetic strategies that are
more convergent in classical synthetic sense of world.

4) ‘Double Exponential’ And ‘Mixed’ Growth
• Double exponential growth, similar to a rapid growth technique
for linear polymers, involves an AB2 monomer with orthogonal
protecting groups for the A and B functionalities.
• This approach allows the preparation of monomers for both
convergent and divergent growth from a single starting
material .
• These two products are reacted together to give an
orthogonally protected trimer, which may be used to repeat the
growth process again

NIOSOMES
• Niosomes basically made of non – ionic surfactant which provide
advantages over the phospholipids because they are more economical
and are chemically more stable as they are not easily hydrolysed or
oxidized during storage.
• The vesicles forming amphiphile is a non-ionic surfactant stabilized by
addition of cholesterol and small amount of anionic surfactant such as
dicetyl phosphate
• Similar to liposomes, in that they are also made up of a bilayer.
• However, the bilayer in the case of Niosomes is made up of non-ionic
• Surface active agents rather than phospholipids.
• Vesicle holds hydrophilic drugs within the space enclosed in the
vesicle, while hydrophobic drugs are embedded within the bilayer
itself.
• Niosomes vesicle would consist of a vesicle forming amphiphile i.e.a
non-ionic surfactant such as Span- 60, which is usually stabilized by the
addition of cholesterol.

Advantages of niosomes:
• They are osmotically active and stable.
• They increase the stability of the entrapped drug.
• The vesicle suspension being water based offers greater patient compliance
over oil based systems
• Since the structure of the niosome offers place toaccommodate hydrophilic,
lipophilic as well as ampiphilic drug moieties, they can be used for a variety of
drugs.
• The vesicles can act as a depot to release the drug slowly andof controlled
release.
• Biodegradable, non-immunogenic and biocompatible.
• Improve the therapeutic performance of the drug molecules by
• Delayed clearance from the circulation
• Protecting the drug from biological environment
• Restricting effects to target cells
• Niosomal dispersion in an aqueous phase can be emulsified in a nonaqueous
phase to
• Regulate the delivery rate of drug
• Administer normal vesicle in external non-aqueous phase.
• Handling and storage of surfactants requires no special conditions.
• Bioavailability of poorly absorbed drugs is increased.
• Targeted to the site of action by oral, parenteral as well as topical routes.

Limitations
1. Aggregation: Some charged molecules are added toniosomes to
increase stability of niosomes by electrostatic repulsion which
prevents coalescence. The negatively charged molecules used are
diacetyl phosphate (DCP) and phosphotidic acid. These charged
molecule sare used mainly to prevent aggregation of niosomes)
Fusion(due to repulsive force it can avoided)
2. Leakage and hydrolysis of encapsulated drugs.
3. The aqueous suspension , of niosomes may have to fusion ,
aggregation , limited shelf life due leaking of entrapped drugs , and
hydrolysis of encapsulated drugs.
4. The methods of preparation of multilamellar vesicles such as
extrusion , sonication, are time consuming specliazed equipment for
and may require processing .

TYPES OF NIOSOMES
According to the nature of lamellarity
1. Multilamellar vesicles (MLV) 1-5 μm in size.
2. Large Unilamellar vesicles (LUV) 0.1 – 1μm in size
3. Small Unilamellar vesicles (SUV) 25 – 500 nm in size.
According to the size
1. Small Niosomes (100 nm – 200 nm)
2. Large Niosomes (800 nm – 900 nm)
3. Big Niosomes (2 μm – 4 μm)

Components of niosomes:
• Cholesterol and Non ionic surfactants are the two major components
used for the preparation of niosomes.
1. Cholesterol
• Cholesterol provides rigidity and proper shape.
• Cholesterol provides rigidity and proper shape and reduces leakage of
drug from the Niosome
• Strengthen the non-polar tail of the non-ionic surfactant
• Increase in the entrapment efficiency
2. Surfactants
• The surfactants play a major role in the formation of niosomes.
• The surfactants play a major role in the formation of niosomes.
• Non-ionic surfactants like spans(span 20,40,60,85,80), tweens (tween
20,40,60,80) are generally used for the preparation of Niosomes.
• Few other surfactants that are reported to form niosomes are as follows:
❑ Ether linked surfactant Di-alkyl
❑ chain surfactant Ester linked
❑ Sorbitan Esters
❑ Poly-sorbates

METHODS OF PREPARATION
• Film Method
• Ether Injection Method
• Sonication
• Reverse Phase Evaporation
• Heating Method
• Microfluidization
• Multiple Membrane Extrusion Method
• Transmembrane pH gradient (inside acidic) Drug Uptake Process
(remote Loading)
• The “Bubble” Method
• Formation of Niosomes from Proniosomes

1. Injection method
A) Ether injection method
• Slow injection of an ether solution of niosomal ingredients into an aqueous medium at high
temperature.
• A mixture of surfactant and cholesterol is dissolved in ether (20 ml) and injected into an
aqueous phase (4 ml) using a 14- gauge needle syringe 60°C .
• Niosomes in the form of large unilamellar vesicles (LUV) are formed.
B) Ethanol injection method
• Ethanol injection method offers advantage that it avoids both sonication and high pressure.
• In this technique, surfactant dissolved in ethanol and forcefully injected in aqueous media
using syringe.
• The whole system stirred using magnetic stirrer in order to evaporate ethanol which results in
formation of niosomal vesicles.

2. Thin Film hydration method
• The mixture of surfactant and cholesterol is dissolved in an organic solvent (e.g. diethyl
ether, chloroform, etc.) in a round-bottomed flask
• The organic solvent is removed by low pressure/vacuum at room temperature
• The resultant dry surfactant film is hydrated by agitation at 50-60oC
• Multilamellar vesicles (MLV) are formed.

3. Sonication
• Two techniques of sonication are employed a. Bath sonication and b. Probe sonication
• In probe sonication technique:
• Drug solution in PBS is added into cholesterol/non-ionic surfactant mixtures.
• The whole system is allowed to sonicate for 3 minutes at 60°C through probe sonicator
which results in nano sized vesicles.
• Probe sonication is one of the techniques to reduce the particle size of niosomes.
• Bath sonication is done after film hydration technique for achieving prolonged release
& longer duration of action.
(b)
(a)
FIGURE : PREPARATION OF NIOSOME BY A) BATH SONICATOR B) PROBE SONICATOR

4. Reverse phase evaporation:
• Surface-active agents are dissolved in chlorofom, and 0.25 volume of phosphate saline
buffer (PBS) is emulsified to get w/o emulsion
• The mixture is sonicated and subsequently chloroform is evaporated under reduced
pressure
• The surfactant first forms a gel and then hydrates to form niosomal vesicles
• The vesicles formed are unilamellar and 0.5 μ in diameter.

5. The Bubble method:
• It is novel technique for the one step preparation of liposomes and niosomes without
the use of organic solvents.
• The bubbling unit consists of round-bottomed flask with three necks positioned in
water bath to control the temperature.
• Water-cooled reflux and thermometer are positioned in the first and second neck and
nitrogen supply through the third neck
• Cholesterol and surfactant are dispersed together in the buffer (pH 7.4) at 70°C, the
dispersion mixed for 15 secs with high shear homogenizer and immediately
afterwards “bubbled” at 70°C using nitrogen gas.

6. Micro fluidization
▪ This is a recent technique to prepare smallMLVS.
▪ A microfluidizer is used to pump the fluid at a very high pressure (10,000 psi) through a
screen.
▪ It is then forced along defined micro channels, which direct two streams of fluid to
collide together at right angles, thereby affecting a very efficient transfer of energy.
▪ The lipids/surfactants can be introduced into the fluidizer. The fluid collected can be
recycled until spherical vesicles are obtained.
▪ Uniform and small sized vesicles are obtained.

7. Multiple membrane extrusion method
• In this method, mixture of cholesterol, non-ionic surfactant and dicetyl phosphate is
solubilized in an organic phase and further evaporated to produce transparent film.
• The film is dried in desiccator followed by hydration using aqueous phase containing
drug.
• The whole system is then extruded via series of polycarbonate membranes (Figure 8).
• The resulting suspension contains vesicles of uniform size.

8.Transmembrane pH gradient drug uptake:
• In this method cholesterol and non-ionic surfactants are dissolved in organic solvent like
chloroform.
• The resultant mixture is then vaporized using rotary evaporator to produce watery film
on inner surface of the RBF.
• Hydration of film is done using aqueous 300 mM citric acid solution (pH 4.0) with the
help of cyclo-mixer which gives suspension with multilamellar vesicles.
• Resultant suspension is frozen and thawed 3 times. Further it is sonicated to produce
niosomal suspension which is stirred at high speed on addition of aqueous drug solution.
• pH of the system is increased to 7.0-7.2 with 1M disodium phosphate followed by heating
at 60°C for 10 minutes leads to formation of drug loaded niosomal dispersion.

9. Formation of Niosomes from Proniosomes:
• Proniosome technique includes the coating of a water-soluble carrier such as sorbitol
and mannitol with surfactant.
• The coating process results in the formation of a dry formulation. This preparation is
termed “Proniosomes” which requires to be hydrated before being used.
• The niosomes are formed by the addition of the aqueous phase. This method helps in
reducing physical stability problems such as the aggregation, leaking, and fusion
problem and provides convenience in dosing, distribution, transportation, and storage
showing improved results compared to conventional niosomes.

7. In-vitro methods for niosomes
Franz diffusion cell
The in vitro diffusion studies can be performed by using Franz diffusion cell. Proniosomes
is placed in the donor chamber of a Franz diffusion cell fitted with a cellophane membrane.
The proniosomes is then dialyzed against a suitable dissolution medium at room
temperature; the samples are withdrawn from
the medium at suitable intervals, and analyzed for drug content using suitable method
(U.V spectroscopy, HPLC, etc.) .The maintenance of sink condition is essential.
8. In vivo release study
• In vivo release study was performed using albino rats.
• These albino rats were differentiated into various groups.
• By means of appropriate disposal syringe, a niosomal suspension was injected
intravenously through a tail vein for in vivo study.
9. Stability studies
• To determine the stability of niosomes, the optimized batch was stored in airtight
sealed vials at different temperatures.
• Surface characteristics and percentage drug retained in niosomes and niosomes derived
from proniosomes were selected as parameters for evaluation of the stability, since
instability of the formulation would reflect in drug leakage and a decrease in the
percentage drug retained.
• The niosomes were sampled at regular intervals of time (0,1,2 and 3 months ),observed
for color change, surface characteristics and tested for the percentage drug retained
after being hydrated to form niosomes and analyzed by suitable analytical methods(UV
spectroscopy, HPLC methods etc).

Applications
1. Anti-neoplastic Treatment
Doxorubicin, the anthracyclic antibiotic with broad spectrum anti-tumor activity, is
formulated in niosomal preparation for targeted delivery.
Niosomal delivery of this drug to mice bearing S-180 tumor increased their life span and
decreased the rate of proliferation of sarcoma.
Niosomal entrapment increased the half-life of the drug, prolonged its circulation and
altered its metabolism.
Intravenous administration of methotrexate entrapped in niosomes to S-180 tumor
bearing mice resulted in total regression of tumor and also higher plasma level and slower
elimination.
2. Leishmaniasis Treatment
Leishmaniasis is a disease caused by parasite genus Leishmania which invades the cells of
the liver and spleen.
Most Commonly prescribed drugs for the treatment are the derivatives of antimony – which,
in higher concentrations – can cause liver, cardiac and kidney damage.
Use of niosomes as a drug carrier showed that it is possible to administer the drug at high
levels without the triggering the side effects, and thus showed greater efficacy in treatment.

3. Studying immune response
• Due to their immunological selectivity, low toxicity and greater stability; niosomes are
being used to study the nature of the immune response provoked by antigens.
• Non-ionic surfactant vesicles have clearly demonstrated their ability to function as
adjuvants following parenteral administration with a number of different antigens and
peptides.
4. Transdermal Drug Delivery System
• Niosomes have application in topical and transdermal products both containing
hydrophobic and hydrophilic drugs.
• Drugs encapsulated for topical and transdermal delivery are lidocaine, estradiol,
cyclosporin, erythromycin, alpha-interferon etc.
• Slow penetration of drug through skin is the major drawback of transdermal route of
delivery. An increase in the penetration rate has been achieved by transdermal delivery
of drug incorporated in niosomes.
5. Niosomes as Drug Carriers
• Niosomes have also been used as carriers for iobitridol, a diagnostic agent used for X-
ray imaging.
• Topical niosomes may serve as solubilization matrix, as a local depot for sustained
release of dermally active compounds, as penetration enhancers, or as rate-limiting
membrane barrier for the modulation of systemic absorption of drugs.

6. Ophthalmic drug delivery
• From ocular dosage form like ophthalmic solution, suspension and ointment it is
difficult to achieve
• excellent bioavailability of drug due to the tear production, impermeability of corneal
epithelium,non productive absorption and transient residence time.
• Niosomal and liposomal delivery systems can be used to achieve good bioavailability of
drug.
• Bioadhesive-coated niosomal formulation of acetazolamide prepared from span 60,
cholesterol stearylamine or dicetyl phosphate exhibits more tendencies for reduction of
intraocular pressure as compared to marketed formulation (Dorzolamide).
7. Drug Targeting
• Niosomes possess beneficial ability of targeting site of action.
• Targeting of drugs to reticulo-endothelial system (RES) is successfully done using
niosomes.
• The RES holds up niosome vesicles and this uptake of niosomes is influenced by
opsonins (circulating serum factors). Opsonins render the niosome for clearance.
• This process of localization of active pharmaceutical ingredient can be useful in the
treatment of cancer cells, different parasitic infections and can also be applicable to
target particular organ other than RES40.

Resealed erythrocytes carriers
• A large number of drug carriers are available today but an ideal carrier is
still lacking.
• Among the various carriers used for targeting of drugs to various body
tissues, the erythrocytes meet several criteria desirable in clinical
applications, among the
most important being biocompatibility, nom-immunogenicity of carrier and
its degradation products.
• Leukocytes, platelets and erythrocytes have been proposed as cellular
carrier systems.
• Among these, the erythrocytes have been the most investigated and have
found to possess great potential in drug delivery.
Rationale: Damaged erythrocytes are removed from circulation by
phagocytic reticulo-endothelial cells.

Properties of resealed erythrocyte as novel drug carriers
1. The drug should be released at target site in a controlled manner.
2. It should be appropriate size, shape and should permit the passage through capillaries
and minimum leakage of drug should take place.
3. It should be biocompatible and should have minimum toxic effect.
4. It should possess the ability to carry a broad spectrum of drug.
5. It should possess specific physicochemical properties by which desired target site could
be recognized.
6. The degradation product of the carriers system, after release of the drug at the selected
site should be biocompatible.
7. It should be physico-chemically compatible with drug.
8. The carrier system should have an appreciable stability during storage.
Advantages:
1. They are the natural product of the body which are biodegradable in nature.
2. Isolation of erythrocyte is easy and larger amount of drug can be encapsulated in a small
volume of cells.
3. The entrapment of drugs does not require any chemical modification of the substance to
be entrapped.
4. They are non-immunogenic , non-toxic in action and can be targeted to disease tissue/
organ.
5. They prolong the systemic activity of drug while residing for a longer time in the body
6. They protect the premature degradation, inactivation and excretion of proteins and
enzymes
7. They can target the drugs within reticuloendothelialsystem.

8. They facilitate incorporation of proteins and nucleic acids in eukaryotic cells by cell
infusion with RBC.
9. A longer life span in circulation as compared to other synthetic carrier.
10. Isolation of RBC is easy, collection and storage techniques are well established.
Disadvantages:
1. They have a limited potential as carrier to non-phagocytic target tissue & compounds
extensively metabolized in liver.
2. Possibility of clumping of cells and dose dumping.
3. Fragility of RBC membrane, their permeability to various drugs and leakiness.
4. These are not suitable for highly polar and non-diffusible drugs (heparin, gentamycin).
Isolation of RBC
1. Blood is collected into heparinized tubes by vein puncture.
2. Blood is withdrawn from cardiac/splenic puncture( in small animals) and through veins
(in large animals) in a syringe containing a drop of anti coagulant.
3. The whole blood is centrifuged at 2500 rpm for 5 min at 4±10 C in a refrigerated
centrifuge.
4. The serum and buffy coats are carefully removed and packed cells washed three times
with phosphate buffer saline (pH=7.4).
5. The washed erythrocytes are diluted with PBS and stored at 4oC until used.
• Erythrocytes from mammalians like mice, cattle, pigs, dogs, sheep, goats, monkeys,
chicken, rats and rabbits.
• EDTA or heparin can be used as anticoagulants agents.

Drug Entrapment methods
• The following methods have been employed for drug entrapment in erythrocytes:
1. Hypo- osmotic lysis method
a. Dilution method
b. Dialysis method
c. Pre-swell method
d. Isotonic osmosis lysis method
2. Electrical breakdown method
3. Endocytosis method
4. Membrane perturbation method
5. Normal transport method
6. Lipid fusion method
7. Osmotic pulse method/ Incubation method
1. Hypo- osmotic lysis method:
In this process, the intracellular and extracellular solutes are exchanged by osmotic lysis and
resealing.
The drug present will be encapsulated with in the erythrocytes membrane .
a. Dilution method

1. Erythrocytes have little capacity to resist increase in volume.
2. When these RBC are placed in the hypotonic (0.4% NaCl), erythrocyte volume increases
by 50-75% they get ruptures permitting escape of cellular content and equilibrium is
achieved
within 1min, which results in swelling up to 1.6 time its initial volume.
3. At 0oC, opening permits to attain equilibrium for inter- and extra cellular fluids through
the pore size of 200-500A0.
4. Increasing the ionic strength at 37oC, results in resealing of cell membrane and restoring
the osmotic property.
5. This method is simple and faster, but have very low encapsulation efficiency (1-8%) and
lose of cytoplasmic constituents during osmotic lysis.
6. Low mol. wt. drugs like s-glucosidase and s-galactosidase can be encapsulated.
7. These cells loose most of cell constituents and are removed by RE cells, thus viability in-
vivo is reduced.
b. Dialysis method:
1. In this method, erythrocyte suspension + Drug solution, loaded in dialysis tube with 25%
air bubble and both ends are tied with thread.
2. This tube is placed in bottle containing 100ml of swelling solution, stored at 4oC for lysis.
3. After, the dialysis tube is transferred to 100ml resealing solution (isotonic PBS, pH 7.4) at
room temp. (25-30oC) for resealing.
4. These resealed cells are removed and washed with PBS at 4oC and finally suspended in
PBS solution.
5. High entrapment efficiency (30-40%) is attained by carrying out lysis and resealing in the

Large molecular weight substances are entrapped by using low hematocrit erythrocyte
suspension.
Disadvantages:
1. Time consuming method.
2. Drug concentration in individual ghosts may vary.

C. Pre-swell dilution technique
This technique is based upon initial controlled swelling of RBC with out lysis in hypotonic
bufferedsolution.
1. Erythrocyte suspension (2ml, 50% hematocrit), centrifuged at 1000 rpm for 10 min at
4oC to obtain packed RBC.
2. Remove supernatant. Collect packed RBC + 4 ml of 0.65% NaCl (hypotonic pre-swelling
solu.) and centrifuge 600 rpm for 5 min to collect swollen RBC cells.
3. To swollen RBC, add small volumes of drug solution until they reach the point of lysis.
4. Point of lysis is detected by appearance of thin layer of white ghosts on centrifugation.
5. After 10 min hypertonic saline
solu. Is added and suspension is
incubated at 37oC for 10 min to
restore isotonicity and resealing.
6. Cells are washed thrice with
washing buffer to remove
hemoglobin and unentraped
drug. Cells are finally suspended
in PBS buffer.
This techniques results in good
retention of cytoplasmic
constituents, high drug
entrapment (72%) and good in-
vivo survival.
Eg: Thyroxin, Ibuprofen, etc.,

d. Isotonic osmotic lysis technique:
In this technique, haemolysis in isotonic solution can be achieved by either chemical or
physical means or both.
1. Propylene glycol increases transient permeability and drug diffusion through
erythrocyte wall without disturbing isotonicity.
2. The lysed erythrocytes are resealed under isotonic condition by dilution with a glycol
free medium.Eg: DMSO, monosaccharides, sucrose.

1. ELECTRIC BREAKDOWN TECHNIQUE
Principle: Electrical break down of cell membrane is observed when the membrane is
polarized very rapidly (nano-microseconds) using voltage of 1 volt.
• This is due to electromechanical compression of membrane which leads to formation of
pores.
• The break down at lipid region / lipid-protein junction in membrane.
• At this potential difference, increase in membrane conductance is observed.
• The potential difference across the membrane can be built up either directly (inter/ intra
cellular electrodes) or indirectly by applying electric field pulse to cell suspension.
• Mechanical rupture of membrane occurs when polarized with 50-350 mv for the period
exceeding 10 µsec resulting in 10 pores/cm with pore size 3 nm.
Method:
1. Firstly, erythrocytes are suspended in an isotonic buffered solution in electric discharge
chamber connected to the capacitor and external circuit.
2. The charge is discharged at definite voltage within definite time interval through cell
suspension to produce a square-wave potential.
3. The optimum intensity of an electric field is between 1-10 kW/cm and optimum
discharge time is 20-160μs.
4. The compound which is to be entrapped was added to the medium.
5. After certain time the cell suspension was transferred to a pre-cooled tubes and kept at
4oC.
6. Resealing of electrically perforated erythrocytes membrane is then done by incubation
at 37oC in an osmotically balanced medium.

• Major advantage is uniform distribution of drug loading is achieved and possible to entrap
up to 35% of drug.
• Disadvantage is need sophisticated and special instruments and time consuming.
• This is inferior to dialysis and pre-swell methods.
Eg: Sucrose, Urease, Methotrexate, Isoniazid, Glucophorin.

3. ENDOCYTOSIS METHOD:
• Intra cellular vesicles with small molecules, drugs, enzymes, viruses (100 nm) can be
induced in erythrocytes.
• The vesicle membrane separates the endocytosed substance from the cytoplasm, thus
drug which are sensitive to inactivation of cytoplasmic enzyme are protected.
1. In this method, 1vol. of packed erythrocytes + 9 vol. of buffer (containing 2.5mM ATP,
2.5mM MgCl2 and 1mM CaCl2) and incubated for 2min at room temperature.
2. The pores created in this method are resealed by using 154mM of NaCl and incubate at
37oC for 2min.
3. The entrapment of drug was obtained by endocytosis.
• Endocytosis can be induced by exposure to certain membrane activated drugs as given
below.
Eg: 8-amino quinolines, Vinblastin, Chlorpromazine, Phenothiazines, hydrocortisone,
propanolol, tetracine, Vit-A, etc.,

4. MEMBRANE PERTURBATION:
• Antibiotics such as amphotericin-B damage micro-organisms by increasing the
permeability of their membrane to metabolites and ions.
• This property could be exploited for loading of drug into erythrocytes.
• Amphotericin-B was used to load erythrocytes with anti-leukaemic drug daunomycin.
• Amphotericin-B interacts with the cholesterol of the plasma membrane of eukaryotic
cells causing change in permeability of the membrane.

5. NORMAL TRANSPORT MECHANISM:
• The biological active components are entrapped in erythrocytes without dispruting the
erythrocyte membrane by incubating in drug solution for varying period of time.
• After infusion the drug would, exit from the cell following kinetics compared to those
observed for entry.
• Drug of 30-35% can be entrapped.
6. Lipid fusion method:
• Lipid vesicles containing drug can be directly fused with human erythrocytes leading to
exchange of lipid entrapped drug.
• This technique was used for loading inositol hexaphosphate into resealed erythrocytes.
• Cell fusion takes place by cell swelling, followed by cell adhesion.
• This method gives very low encapsulation efficiency (1%).
• Fusion can be induced by carboxylic acids, their esters, retinol, tocopherol.
7. Osmotic pulse method/ Incubation method:
This method has 4 steps.
Step-1: DMSO incubation:
The RBC suspension can be incubated with different concentration of DMSO with variable
hematocrit.
Step-2: Isotonic dilution with the material to be encapsulated:
The drug can be added with constant mixing.
Step-3: Post dilution incubation with cellular swelling:
The RBC swell and drug is encapsulated through pores.
Step-4: Return to original shape:
Drug loaded resealed erythrocytes are obtained.

CHARACTERIZATION OF RESEALED ERYTHROCYTES
1. Drug content estimation,
2. In-vitro drug release and hemoglobin content,
3. Percent cell recovery,
4. Morphology,
5. Osmotic fragility,
6. Osmotic shock,
7. Turbulence shock,
8. Determination of entrapped magnetite,
9. Erythrocyte sedimentation rate (ESR),
10. Zeta sedimentation ratio
11. Hematological parameters
12. Biochemical parameters
13. Lipid content
14. Membrane fluidity
15. Rheological parameters.

1. Drug content:
• Packed loaded erythrocytes (0.5ml) are first deproteinized with acetonitrile (2ml) and
subjected to centrifugation at 2500 rpm for 10 min.
• The clear supernatant is analyzed for the drug content using specified estimation
methodology for entrapped drug.
2. In-vitro drug release and hemoglobin content:
• Initially collect cell suspension (5% hemotocrit in PBS) and stored in 4oC in amber
colored bottle.
• Periodically the clear supernatant are withdrawn using hypodermic needle equipped
with 0.45μ filter and deprotenised using methanol and were estimatedfor drug
content.
• Hemoglobin Conc. is measured spectrophotometrically after conversion of
hemoglobin, oxyhemoglobin, cyanamethamoglobin to hematin.
• This conversion is done by adding strong base (NaOH) to pH 10 / alkaline cyanide
potassium ferricyanide reagent.
• Results are expressed in “g hemoglobin/ 100 ml of blood”

3. Percent cell recovery:
It is determined by counting no. of intact cells per cubic mm of packed erythrocytes before and
after drug loading. The counting is performed in a haemocytometer.
4. Morphology:
Phase contrast & electron microscopy for normal and drug loaded RBC.
• For electron microscopy sample is washed with cacodylate buffer and stained negatively with
0.01% uranyl acetate.
• SEM/ TEM – monitoring and evaluation of all stages of carrier cell preparation.
5. Osmotic fragility:
This indicates resistance of cell to haemolysis in decreasing conc. of hypotonic saline.
This is reliable parameter for in-vitro shelf life, in-vivo survival and effect of encapsulated
substances.
Normal and loaded erythrocytes of drug are incubated separately in stepwise decreasing % of
NaCl solution (0.9 and 0.1%) at 37oC for 10min and centrifugation at 2000 rpm for 10min.
The supernatant examined for drug and hemoglobin content.
Increased osmotic fragility indicates acquired hemolytic anemia, increased resistance is
observed in thalassemia, sickle cell anemia, hypochromic anemia.
6. Osmotic shock:
• Drug loaded erythrocytes are suddenly exposed to an environment, which is far from isotonic
and the ability of resealed erythrocytes to withstand the stress, maintain their integrity as
well as appearance is evaluated.
• In this study, 1ml of erythrocyte suspension were diluted with distilled water of 5ml and
centrifuged at 3000rpm for 15min.
•The supernatant liquid was estimated for hemoglobin spectrophotometrically.

7. Turbulence shock:
• It is the measure of simulating destruction of loaded cells during injection.
• In this method, the normal and drug loaded cells are passed through a 23 gauge
hypodermic needle at a flow rate of 10ml/min which is comparable to that of blood.
• The samples are with drawn and centrifuged for 2000rpm for 10min and estimated for
hemoglobin content.
• Drug loaded erythrocytes found to be less resistant to turbulence indicating destruction
on shaking.
8. Determination of entrapped magnetite:
• Magnetically responsive drug loaded erythrocytes are developed by loading iron
compound (magnetite) in RBC.
• Initially, Hcl is added to a fixed amount of magnetite bearing erythrocytes and contents are
heated to 60oC for 2hrs.
• To this added 20%w/v Tricholoroacetic acid is added and supernatent liquid is obtained
after centrifugation.
• This was analyzed for magnetite concentration by using AAS (Atomic Absorption
Spectroscopy).

9. Erythrocyte sedimentation rate (ESR):
• Sedimentation depends upon the size and no. of cells and relative concentration to the
plasma proteins.
• ESR increases with rise in O2, cholesterol, fibrinogen, α-globulin. ESR decreases with rise
in Co2, albumin, nucleoprotein, lecithin.
• The test is performed in a standard tube placed vertically.
• Height of supernatant plasma in mm separated out at the top of the vertical column of
blood after 1 hr is noted.
• Normal blood ESR is 0-15mm/hr. Higher rate indicates active but obscure disease
progress.
10. Zeta sediment ratio:
This measures the closeness with which RBC approach each other after dispersion.
Velocity of RBC aggregation can be measured in a rheoscope.
11. Hematological parameters:
This predicts effect of loaded drug on integrity of RBC.Parameters include protein content,
hemoglobin content, mean corpuscular volume (MCV), mean corpuscular hemoglobin
(MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width
(DW).
12. Biochemical parameters:
This is helpful in detection of presence of disease.Parameters include cell debris, content
measurement of thoil, glutathione, oxidized glutathione, creatinine, initial hemolysisand
K+ leakage rate, etc.,

13. Lipid content:
Lipid content and the spatial arrangement of lipids on the external surface of resealed
erythrocytes affects their in-vivo half life.
The longer half-life of loaded RBC indicated it maintains cell surface similar to normal RBC
which prevents its removal by RE cells.
14. Membrane fluidity:
This is measured by a hydrophobic fluorescent probe and the values are related to lipid
composition.
15. Rheological parameters:
Viscosity and relative density are measured by brook field viscometer and relative density
bottle respectively

Applications of resealed erythrocytes
These have been proposed for a variety of applications in human and veterinary medicines.
1. Applications In-Vitro
2. Applications In-Vivo
a) Targeting bioactive agents to RE system,
b)Erythrocytes as circulating carriers,
c) Erythrocytes as circulating bioreactors,
d)Erythrocytes in enzyme delivery,
e) Prevention of thromboembolism,
f) Treatment of poisoning,
g) Prodrug,
h)Drug targeting other than RES.

1.Applications In-Vitro
❑ Carrier RBC is used in in-vitro tests.
❑ For in-vitro phagocytosis, cells have been used to facilitate the uptake of enzymes by
phagolysosomes.
❑ The enzyme content with in carrier RBC can be visualized with cytochemical technique.
❑ Biochemical defects like glucose-6-phosphate dehydrogenase deficiency can be treated
with resealed erythrocytes.
❑ RBC are most frequently used in microinjections.
❑ Ribosome inactivation proteins and nucleic acids are injected in to RBC by fusion
process.
❑ Anti-body injected RBC are useful to confirm the site of action of diphtheria toxin.
2. Applications In-Vivo
a) Targeting bioactive agents to RE system
• Resealed erythrocytes with modified characteristics are removed form circulation by
phagocytic cells located in liver and spleen.
• This means bioactive agents can be targeted to these organs using erythrocytes.
• The drug entrapped erythrocytes are used for RES
• Targeting in treatment of following diseases.
(i). Treatment of lysosomal storage disease:
Resealed erythrocytes have been proposed to deliver lysosomal enzymes to lysosomes of
the erythrophagocyticcells, thus resulting in replacement of the missing enzyme.
Ex: β- glucoronidase, β-galactoronidase and β-glucosiade.

(ii) Treatment of Gaucher’s disease:
Gaucher’s disease is due to accumulation of glucocerebroside from catabolised
erythrocytes and leukocytes in spleen, liver and bone marrow macrophages. This disease
was treated by encapsulating glucocerebrosidase in erythrocyte.
(iii) Treatment of liver tumors:
Anticancer agents like bleomycin, adriamycin, Lasparaginase, doxorobucin and
methotrexate are encapsulated in erythrocyte to treat hepatic carcinomas.
(iv) Treatment of parasitic diseases.
The ability of resealed erythrocytes to selectively accumulate within RES organs make
them useful tool during the delivery of anti-parasitic agents.
Eg: Pentamidine loaded immunoglobin-G coated erythrocytes against macrophage-
containing leishmania.Liver targeting– Glutaraldehyde treated erythrocytes (Primaquine
phosphate {anti-malarial} + Metronidazole{anti-amoebic})
(v) Removal of RES iron overload:
Most of the body store of iron is present as intracellular ferritin and hemosiderin deposits.
Desferrioxamine, an iron-chelating drug in erythrocyte ghosts with a view to promote
excretion of iron in patients with excess body stores.
(vi) Targeting of mycotoxins to RES:
Erythrocytes can target large doses of T-2 toxin (tricothecene mycotoxin) to macrophages
of liver and spleen for treatment of Schistosomiasis and prolong the retention time of it.

b) Erythrocytes as circulating carriers:
Various bioactive agents are encapsulated in erythrocytes for their slow release in
circulation for treatment of parasitic diseases in cattle.
Ex: anti cancers, vitamins, steroides, antibiotics, Homodiumbromide, Imidicocrab
dipropionate, tetracycline.
c) Erythrocytes as citculating bioreactors:
To diminish the level of circulating metabolites(arginine, uric acid) enzyme (arginase,
urease) loaded erythrocytes are used.
d) Erythrocytes in enzyme delivery:
Enzyme loaded erythrocytes are used to replace missing enzyme in metabolic disorders/
to degrade toxic compounds in blood.
Diseases ---Gauchers disease, kidney failure, hyperuricemia.
Enzymes --- L-Aspariginase (Leukemia), araginase, alcohol oxidase, acetaldehyde
dehydrogenase, hexokinase, etc.,
Adv:-This eliminate or minimize the problems related toimmunologic responses and
toxicity.
e)Prevention of thromboembolism:
Encapsulated heparin is liberated from circulating erythrocytes at the site of thrombus
formation thus reducing the risk of further thrombus growth.
Ex: Aspirin + ferromagnetic colloid loaded →Thrombosis.

f) Treatment of poisoning:
Rhondanase + Sodium thosulphate loaded erythrocytes are used for treatment of cyanide
poisoning by converting cyanide to less toxic thiocyanate.
g) Prodrug:
2,3-dideoxycytidine (ddCyd) is potent antiviral for killing HIV.
2,3-dideoxycytidine-5-phosphate (ddCMP) is prodrug loaded in erythrocytes for treatment
of HIV.
Adv: Toxicity is reduced, slow delivery with long plasma Conc.
h) Drug targeting other than RES:
Urease loaded erythrocytes are used in treatment of kidney failure.

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