Nanoparticles

Polymeric nanoparticles & Liposomes Nanoparticles are the most extensively investigated drug delivery systems. This includes: Polymeric nanoparticles & Liposomes

Polymeric nanoparticles Nanoparticles are solid colloidal particles ranging in size 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, two different 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 structure with a core containing the drug. Because these systems have very high surface areas, drugs may also be adsorbed on their surface.

MANUFACTURE OF NANOPARTICLES 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.

Nanospheres 1- In situ polymerization of a monomer 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)

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.

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.

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.

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 allowing the polymerization to be followed within the micelles. So, in this case, monomer droplets would essentially act as monomer reservoirs.

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.

Micellar polymerization mechanism.

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.

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

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.

Nanocapsules Interfacial Polymerization Mechanism 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).

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.

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.

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.

Disadvantages of preparation by in situ polymerization of a monomer 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.

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.

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.

2- Dispersion of a Preformed Polymer Nanospheres 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

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.

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.

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).

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. 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.

Preparation of nanospheres by thermal denaturation of albumin

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.

Preparation of nanospheres by desolvation of albumin.

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.

Nanospheres 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.

Emulsification-Based Methods. 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.

Emulsification-solvent evaporation method

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.

Salting-out process

Direct Precipitation-Based Method 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 method

Pharmaceutical Aspects There are some requirements for nanoparticles intended to be used as pharmaceutical dosage forms in humans: to be free of any potentially toxic impurities to be easy to store and to administer to be sterile if for parenteral administration.

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.

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.

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.

Main Methods for the Purification of Nanoparticles on the Laboratory Scale

Freeze-drying. 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

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.

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. 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.

Sterilization Nanoparticles intended to be used parenterally are required to be sterile and apyrogenic. Filtration on 0.22 μm filters is not adequate for nanoparticle suspensions because microorganisms and nanoparticles are generally similar in size (0.25-1 μm). Sterilization may be achieved, either by using aseptic conditions throughout formulation, or by sterilizing treatments such as autoclaving or γ-irradiation.

The choice of the sterilizing treatment depends on the physical susceptibility of the system. Autoclaving (moist heat sterilization) and γ - irradiation May alter the physicochemical properties of the particles in several systems. These modifications occur as a consequence of the 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.

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