Nanoparticles

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Nanoparticles are the most extensively

investigated drug delivery systems.

This includes:

Polymeric nanoparticles

&

Liposomes

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Nanoparticles are solid colloidal particles ranging insize from 10 to 1,000 nm. They are made of a macromolecular material which can be of synthetic or natural origin.Depending on the process used for their preparation, twodifferent types of nanoparticles can be obtained,nanospheres and nanocapsules. Nanospheres have a matrix-type structure in which a drug is dispersed,whereas nanocapsules exhibit a membrane-wall structurewith a core containing the drug. Because these systems have very high surface areas, drugs may also be adsorbed on their surface.

Polymeric nanoparticles

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Methods of manufacturing nanoparticles

The choice of the manufacturing method depends on the raw material intended to be used and on the solubility characteristics of the active compound to be associated to the particles. The raw material, biocompatibility, the degradation behavior, choice of the administration route, desired release profile of the drug, and the type of biomedical application determine its selection.Thus nanoparticle formulation requires an initial and very precise definition of the needs and objectives to be achieved.

MANUFACTURE OF NANOPARTICLES

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1- In situ polymerization of a monomer

Nanospheres Two different approaches have been

considered for the preparation of

nanospheres by in situ

polymerization, depending on

whether the monomer to be

Polymerized:

is emulsified in a nonsolvent phase

(emulsification polymerization),

or dissolved in a solvent that is a

nonsolvent for the resulting polymer

(dispersion polymerization)

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Emulsification Polymerization.

Depending on the nature of the continuous phase

in the

emulsion, whether, the continuous phase is

aqueous (o/w emulsion), or organic (w/o emulsion).

In both cases the monomer is emulsified in the

nonsolvent phase in presence of surfactant

molecules, leading to the formation of monomer-

swollen micelles and stabilized monomer droplets. 6

The polymerization reaction takes place in the

presence of a chemical or physical initiator.

The energy provided by the initiator creates free

reactive monomers in the continuous phase which

then collide

with the surrounding unreactive monomers and

initiate

the polymerization chain reaction.

The reaction generally stops once full consumption

of monomer or initiator is achieved.

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The mechanism by which the polymeric particles are

formed during emulsification polymerization is by

micellar polymerization, where the swollen-monomer

micelles act as the site of nucleation and

polymerization.

Swollen micelles exhibit sizes in the nanometer range

and thus have a much larger surface area in

comparison with that of the monomer droplets.

Once generated in the continuous phase, free

reactive monomers would more probably initiate the

reaction within the micelles. 8

allowing the polymerization to be followed within

the micelles.

So, in this case, monomer droplets would essentially

act as monomer reservoirs.

As the monomer molecules

are slightly soluble in the

surrounding phase, they

reach the micelles by

diffusion from the monomer

droplets through the

continuous phase, thus

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The drug to be associated to the nanospheres may be

present during polymerization or can be subsequently

added to the preformed nanospheres, so that the

drug can be either incorporated into the matrix or

simply adsorbed at the surface of the nanospheres.

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Micellar polymerization mechanism.11

Dispersion Polymerization

The monomer is no more emulsified but dissolved in

an aqueous medium which acts as a precipitant for

the polymer to be formed.

The nucleation is directly induced in the aqueous

monomer solution.

For Production of Polymethacrylic Nanospheres, water

soluble methyl methacrylate monomers are dissolved

in an aqueous medium and polymerized by y-irradiation or by chemical initiation (ammonium or

potassium peroxodisulfate) combined with heating to

temperatures

above 650C. 12

In the case of chemical initiation, the aqueous medium

must be previously flushed with nitrogen for 1 h in

order to remove its oxygen content, which could

inhibit the polymerization by interfering with the

initiated radicals. Oligomers (primary polymer) are

formed and above a certain molecular weight

precipitate in the form of primary particles. Finally,

nanospheres are obtained by the growth or the fusion

of primary particles in the aqueous phase

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The removal of detergents is very important that

produce very slowly biodegradable and

biocompatible nanoparticles The technique can be

used for vaccination purposes.

where initiation by y-irradiation can be useful for

the production of nanospheres by polymerization in

the presence of antigenic material at room

temperature,

thus preventing its destruction.

Examples of antigenic materials used to produce

nanoparticulate were different influenza antigens. 14

Nanocapsules

Nanocapsules is a colloidal carrier with a capsular

structure consisting of a polymeric envelope

surrounding

an oily central cavity containing lipophilic drugs.

Interfacial Polymerization Mechanism

The monomer (isobutyl cyanoacrylate) and a

lipophilic drug (progesterone) are dissolved in an

ethanolic phase containing an oil (Myglyol®,

Lipiodol®) or a non-miscible organic solvent

(benzylic alcohol).

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This mixture is slowly injected through a needle into

a magnetically stirred aqueous phase (pH 4—10)

containing an nonionic surfactant (poloxamer 188).

Upon mixing with the aqueous phase, ethanol

rapidly

diffuses out of the organic phase giving rise to

spontaneous emulsification of the oil/monomer/drug

mixture.

Immediately the monomer molecules polymerize at

the water-oil interface, leading to the formation of

solid wall-structured particles. 16

The mixture immediately becomes milky and

nanocapsules with a mean diameter of 200-300 nm

are formed.

The colloidal suspension can then be concentrated

by evaporation under reduced pressure and filtered.

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For encapsulate hydrophilic compounds such as

doxorubicin and fluorescein, inverse emulsification

polymerization

Technique can be used.

In this procedure, the drug was dissolved in a small

volume of water and emulsified in an organic

external phase

( hexane) containing a surfactant.

An organic solution of cyanoacrylate monomers is

added to the w/o emulsion.

Nanocapsules are formed, resulting from an

interfacial polymerization process around the

nanodroplets.

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1.Most of the carriers produced by polymerization

have inadequate biodegradability properties

preventing their use for regular therapeutic

administration.

Only for vaccination purposes is being suitable

when

achievement of a very prolonged immune

response is desired.

2. The possible inhibition of drug activity due to

interactions with activated monomers present in

polymerization processes.

Disadvantages of preparation by

in situ polymerization of a monomer

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3. It is very difficult to calculate the molecular

weight of the resulting polymerized material due

to the multicomponent nature of the

polymerization media. However, the

determination of molecular weight is very

important as it influences the biodistribution and

release

of the polymeric carrier.

4. The presence of toxic residues due to the

unreacted monomer, initiator, and surfactant

molecules whose elimination requires time-

consuming and not always efficient procedures.

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In order to avoid those limitations and produce

biodegradable, well-characterized, and nontoxic

nanoparticles., already polymerized materials have

been used.

These materials include natural macromolecules

(biopolymers) and synthetic polymers.

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2- Dispersion of a Preformed Polymer2- Dispersion of a Preformed Polymer

Nanospheres Prepared From Natural MacromoleculesNanospheres Prepared From Natural Macromolecules

Due to their biodegradability and biocompatibility,

example of natural macromolecules used for the

manufacture of nanospheres are:

Proteins as albumin, gelatin,

(the most widely used)

Polysaccharides as alginate or agarose

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Two manufacturing techniques are used to produce

nanospheres from natural macromolecules.

1. The first technique is based on the formation

of a w/o emulsion followed by heat denaturation

or

chemical cross-linking of the macromolecule.

2.The second technique is a phase separation

process in an aqueous medium followed by

chemical crosslinking. 23

Emulsification-Based Methods

An aqueous solution of albumin is emulsified at

room temperature in a vegetal oil (cottonseed oil)

and homogenized either by a homogenizer or an

ultrasonication.

Once a high degree of dispersion is achieved, the

emulsion is added drop wise to a large volume of

preheated oil (>120°C) under stirring.

This leads to the immediate vaporization of the

water contained in the droplets and to the

irreversible denaturation of the albumin which

coagulates in the form of solid nanospheres. 24

The suspension is then cooled

at room temperature or in an

ice bath.

For complete removal of the

oil, wash the particles using

large amounts of organic

solvent (e.g., ether, ethanol,

acetone).

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Large amounts of organic solvents are required

to obtain nanospheres free of any oil or

residues.

It is very difficult to produce small nanospheres

(<500 nm) with narrow-size distributions, due to

the instability of the emulsion prior to hardening

by heat or crosslinking.

Disadvantages of this technique:

The purification step may cause particle wastes.

The hardening step by heat denaturation may be

harmful to heat-sensitive drugs. This can be

avoided by the use of a crosslinking agent.

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Preparation of nanospheres by thermal denaturation of Preparation of nanospheres by thermal denaturation of albuminalbumin

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Phase Separation-Based Methods in an Aqueous Medium

The particles are formed in an aqueous medium by a

phase

separation process and are stabilized by cross linking

with

glutaraldehye.

Gelatin and albumin nanospheres can be produced

by the slow addition of a desolvating agent (neutral

salt or alcohol) to the protein solution to the form

protein aggregates

The nanospheres are obtained by crosslinking of

these aggregates with glutaraldehyde.

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Preparation of nanospheres by desolvation of Preparation of nanospheres by desolvation of albumin.albumin. 29

The major disadvantage of this

technique is the necessity for

using hardening agents

(glutaraldehyde) that may react

with the drug and may cause

toxicity to the nanoparticle

formulations.

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Nanospheres Prepared From Synthetic PolymersNanospheres Prepared From Synthetic Polymers

Examples of synthetic polymers used for the

preparation

of nanospheres:

Polylactic acid (PLA), poly(glycolic acid) (PGA),

polylactic-co-glycolic acid) (PLGA), poly(e-

caprolactone)

(PCL), and poly(Polyhydroxybutyrate) (PHB).

These polyesters polymers exhibit biodegradability

and biocompatibility.

Under physiological conditions, they are degraded

into safe products as glycolic acid and lactic acid.

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Polyesters nanoparticles can be produced using two

different methods.

The method is based on the emulsification of an

organic solution of the polymer (chloroform,

methylene chloride, ethyl acetate), in an aqueous

phase (o/w emulsion) containing surfactants (e.g.,

polysorbate, poloxamer, sodium dodecyl sulfate).

The extraction of the solvent from the nanodroplets

is achieved by evaporation of the organic solvent at

room temperature under stirring.

Emulsification-Based Methods.

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Emulsification-solvent evaporation method 33

A second method is based on the direct

precipitation of the solubilized polymer by salting

out process

Two different salting-out agents, magnesium

chloride and magnesium acetate, were used,

providing an acidic or a basic aqueous phase,

respectively.

Although the salting-out process has proved

suitable for

the production of large quantities of highly drug-

loaded

nanospheres, the use of large amounts of salts

may raise a problem concerning compatibility with

active compounds.

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Salting-out process 35

This method allows nanospheres to be obtained

without prior emulsification.

This technique involves the use of an organic solvent

that is completely miscible with the aqueous phase,

typically (acetone, ethanol or methanol).

In this case, the polymer precipitation is directly

induced in an aqueous medium (non solvent), by

progressive

addition under stirring of the polymer solution.

This method is limited to drugs that are highly

soluble

in polar solvents, but only slightly soluble in water

(e.g.,

indomethacin).

Direct Precipitation-Based MethodDirect Precipitation-Based Method

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Direct precipitation methodDirect precipitation method 37

There are some requirements for nanoparticles

intended to be used as pharmaceutical dosage forms

in humans:

(1)to be free of any potentially toxic impurities

(2)to be easy to store and to administer

(3)to be sterile if for parenteral administration.

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Purification.

Depending on the preparation method, various

toxic impurities can be found in the

nanoparticulate suspensions including:

organic solvents, residual monomers,

polymerization initiators, electrolytes, surfactants,

stabilizers, and large polymer aggregates.

The necessity for and degree of purification are

dependent on the final purpose of the formulation

developed.

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For example, the stabilizer PVA,

frequently used to prepare

polyester nanoparticles, is not

acceptable for parenteral

administration, whereas it is not so

critical for oral and ocular

administration. Polymer aggregates can be easily removed by

simple filtration.

The removal of other impurities requires more

complicated procedures as gel filtration, dialysis,

and ultracentrifugation. 40

However, these methods are incapable of

eliminating molecules with high molecular weight.

Using cross-flow filtration technique, the

nanoparticle suspension is filtered through

membranes, with the direction of the fluid being

tangential to the surface of the membranes to

avoid the clogging of the filters.

It was shown that by using a microfiltration

membrane (porosity of 100 nm), nanoparticles

produced by the salting out process could be

purified of the salts.41

Main Methods for the Purification of Nanoparticles on the Laboratory Scale

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Freeze-drying (lyophilization) represents one of the

most useful methodologies to ensure the long-term

conservation of polymeric nanoparticles

This technique involves the freezing of the

suspension and the elimination of its water content

by sublimation under reduced pressure, where

nanoparticles are obtained in the form of a dry

powder that is easy to handle and to store. Freeze-

dried nanoparticles are usually readily redispersible

in water without modification of their

physicochemical properties

Freeze-drying.

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Nanocapsules composed of an oily core surrounded

by a tiny polymeric wall tend to aggregate during

the freeze-drying process.

This problem can be solved by desiccating these

systems in the presence of an appropriate

lyoprotective and cryoprotection agent such as

mono- or disaccharides

(e.g., lactose, sucrose, glucose).

The mechanisms by which sugars protect

nanoparticles

during freeze-drying is that during freezedrying

sugars may interact with the solute of interest (e.g.,

liposome, protein) through hydrogen-bonding. 44

It has to be kept in mind that the addition of sugar

may affect the isotonicity of the final

nanoparticulate suspension, and that a subsequent

step of tonicity adjustment may be required prior to

any parenteral or ocular administration.

As a result, the solute

might be maintained in a

"pseudo-hydrated“ state

during the dehydrating

step of freeze-drying, and

would therefore be

protected from damage

during dehydration and

subsequent rehydration.

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Nanoparticles intended to be used parenterally Nanoparticles intended to be used parenterally

are required to be sterile and apyrogenic.are required to be sterile and apyrogenic.

Filtration on 0.22 Filtration on 0.22 μμm m filters is not adequate for filters is not adequate for

nanoparticle suspensions because microorganisms nanoparticle suspensions because microorganisms

and nanoparticles are generally similar in size and nanoparticles are generally similar in size

(0.25-1 (0.25-1 μμm).m).

Sterilization may be achieved, either by using Sterilization may be achieved, either by using

aseptic conditions throughout formulation, or by aseptic conditions throughout formulation, or by

sterilizing treatments such as autoclaving or sterilizing treatments such as autoclaving or γγ--

irradiation.irradiation.

Sterilization

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The choice of the sterilizing treatment depends on The choice of the sterilizing treatment depends on

the physical susceptibility of the system. the physical susceptibility of the system.

Autoclaving (moist heat sterilization) and Autoclaving (moist heat sterilization) and γγ -

irradiationirradiation

May alter the physicochemical properties of the May alter the physicochemical properties of the

particles in several systems. particles in several systems.

These modifications occur as a consequence of the These modifications occur as a consequence of the

cleavage or cross-linking of the polymeric chains.cleavage or cross-linking of the polymeric chains.

The final formulation would therefore result from a

rational balance between conditions maintaining

the formulation integrity upon sterilization and the

final purpose of the formulation.47

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