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NEW MICROENCAPSULATION METHOD USING AN ULTRASONIC ATOMIZER BASED ON INTERFACIAL SOLVENT EXCHANGE

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Microencapsulation is defined as the process of enclosing micron sized particles of solids or droplets of liquids or gasses in an inert shell, which in turn isolates and protects them from the external environment.

 Particle size below 1µm- nanoparticles, nanocapsules, nanospheres Particles diameter between 3 - 800µm- micro particles or microcapsules or micro

spheres. Particles larger than 1000µm - macro particles.

Micro particles or microcapsules consist of two components: Core material Coating material

Advantages:

to protect the sensitive substances from the external environment. to mask the organoleptic properties like colour, taste, odour of the substance. to obtain controlled release of the drug substance. for safe handling of the toxic materials. to get targeted release of the drug. to avoid adverse effects like gastric irritation of the drug e.g. aspirin is the first drug which is used to avoid gastric irritation.

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Disadvantages:

Incomplete coating.Inadequate stability of sensitive materials.Non-reproducible and unstable release.Economic limitations.

Factors affecting the encapsulation effiency:

Solubility of polymer in organic solventSolubility of organic solvent in waterConcentration of polymer Ratio of dispersed phase to continuous phase (DP/CP ratio) Rate of solvent removalInteraction between the drug and polymerSolubility of drug in continuous phaseMolecular weight of polymer

Techniques of Micro encapsulation:

According to Finch, the main methods of micro encapsulation can be classified as follows:1) Phase separation 2) Interfacial polymerization3) Spray drying, spray congealing4) Solvent evaporation and solvent extraction5) Pan coating

Another new method also introduced so called solvent exchange method using ultra sonic atomizer.

Interfacial solvent exchange method:•Micro encapsulation of protein drugs has been the method of choice for developing long-term delivery formulations.

•In general most proteins undergo inactivation events such as degradation and aggregation within the microparticles during the manufacturing process as well as the release period.

•Protein inactivation in the micro particle system is largely due to extensive exposure of the protein to damaging environments, such as large interfacial area between aqueous and organic phases (w/o), hydrophobic polymers, and their acidic degradation products.

•The hydrophobic interaction between the protein and polymers has been minimized by encapsulating proteins within hydrophilic excipients for increasing the encapsulation efficiency.Eg. Gammahydroxypropylcyclodextrin (g-HPCD) were co-encapsulated with tetanus toxoid in PLGA microparticles.

•In this method, reservoir-type microcapsules were generated using a ultra sonic atomizer system that involves two ink-jet nozzles.

•Series of drops of polymer solution and aqueous drug solution are separately produced using ink-jet nozzles, and then they are induced to collide in the air.

•Following the collision, the two liquid phases are separated as a core.

•Preferred atomizers are ultrasonic ones& it’s configuration is coaxial.

•The solvents -hydrophilic organic solvents - Eg. Acetic acid.

•In this configuration, immediate solvent exchange can occur between water and a hydrophilic organic solvent.

Aims of this method are:

(1)To optimize the microencapsulation process using the solvent exchange technology. (2)To find the best processable PLGA/solvent systems for forming PLGA microcapsules.

(3)To find the optimum physicochemical properties of aqueous micro droplets for formation of stable microcapsules and preservation of protein drugs. (4)To prepare microcapsules loaded with protein drugs and examine the stability of the loaded drugs, their release kinetics, and their long-term stability and

(5)To examine the bioactivity of the protein drugs after release from microcapsules.

Advantages:

It provides a simple and efficient way of making protein-loaded microcapsules.It minimizes the exposure of encapsulated proteins to a large water/organic solvent interfacial area. The method utilizes only a minimal energy for producing microdrops.The contact between the encapsulated drugs and the hydrophobic polymer and their degradation products is minimal in the reservoir-type microcapsules.

Disadvantages:It is time consuming process because solidification doesn't occur readily due to high water solubility of solvents.Encapsulation efficiency decreased upon increase of the ratio (QAq/QPol) of flow rates of aqueous solution (QAq) to polymer solution (QPol).

Materials:1)Poly(lactic-co-glycolic acid) (PLGA; 50/50 lactic acid/glycolic acid). 2)Polyvinyl alcohol (PVA). 3)Sodium dodecyl sulfate (SDS) and ethyl acetate (EA). 4)Coomassie brilliant blue R- 250. 5) DiO (3,3V-dioctadecyloxacarbocyanine perchlorate), fluorescein isothiocyanate labeled

dextran (FITC-dextran), Nile Red, and lysozyme.

ULTRA SONIC ATOMIZER: An ultrasonic atomizer is a device used to generate such vibrations leading to atomization of a

liquid. The atomizer body consists of three principal sections: 1. Front horn, the atomizing section;2. Rear horn, the rear section, and 3. A section consisting of a pair of disc-shaped piezoelectric transducers.

• Piezoelectric transducers convert electrical energy into high-frequency mechanical motion.

Principles of ultra sonic atomization:

• The direction of vibration is perpendicular to the surface, the liquid film absorbs the vibration energy and creates unique capillary waves.

• When the amplitude of the capillary waves exceeds a critical value, the waves collapse ejecting small drops of the liquid.

• Ultrasonic atomizers operate at low energy levels.

• The velocity of the drops produced from an ultrasonic atomizer is 1-10% that of a air-atomizing nozzle, and this eliminates overspray problems.

Coaxial ultra sonic atomizer

Atomizer configurations:• A coaxial atomizer is preferably used because it efficiently generates a micro drops.• In a coaxial atomizer, two liquids flow under the influence of a single ultrasonic

generator. • One liquid flows through the inner nozzle, and the other flows through the outer one. • Both liquids delivered to the same atomizing surface are broken into microdrops as the

vibration energy is applied on the surface. • Microcapsules can also be produced by submerging the atomizer in a collection bath to

induce the solvent exchange directly by contact between polymer drops and the collection bath.

• Thus formed microparticles are mononuclear microcapsules containing bath materials in the core.

Preparation of microcapsules: Encapsulating Polymer: Polymers are 2 types1. Biodegradable polymers:

Eg.• Homopolymers of poly(lactic acid) (PLA) • Poly(glycolic acid) (PGA)• Poly(lactic acid)-co-(glycolic acid) (PLGA), • Poly(ortho esters), and polyanhydrides.

2.Non-biodegradable polymers:

Eg.• Polyacrylates, polymethacrylates • Polymers of ethylene-vinyl acetates • Non-degradable polyurethanes• Polystyrenes • Polyvinyl chloride, polyvinyl fluoride.

Polymer Solvents:

• The hydrophilic, e.g., organic and water-miscible.

• solvent for the solvent exchange method should have a following requirements:

1. It should possess the right solvency for the polymer (e.g., PLGA).

2. It should be able to spread out easily on the aqueous surface.

3. It should be miscible with water to a certain degree, thus allowing fast phase separation of the polymer.

Process:• PLGA solution (2–5%) in ethyl acetate (PLGA-EA) + Nile Red.

• An aqueous solution + Lysozyme (3%) + Coomassie brilliant blue R-250 + FITCdextran.

• Two solutions were separately into delivered using syringe pumps at controlled flow rates an ultrasonic atomizer through coaxial cables.

• Microcapsules, which were produced upon the onset of atomizer vibration, were collected in a water bath containing 200 ml of 0.5% PVA.

• induce collision between the two groups of liquid microdrops. After 2 h in the PVA bath, the microcapsules were collected using a centrifuge, washed with distilled water.

Confocal Laser Scanning Microscopy (CLSM):

• It is a technique for obtaining high-resolution optical images with depth selectivity.

• Bio- Rad laser scanning confocal imaging system equipped with a krypton/argon laser and a Nikon Diaphot 300 inverted microscope.

• Distribution of the PLGA solution and the encapsulated solution in the microcapsules was examined using a CLSM.

• All confocal fluorescence pictures were taken with a 20 objective lens and excitation at 488 and 568 nm.

Determination of loading and encapsulation Efficiency:

Protein content in the microcapsules was determined using the dimethyl sulfoxide (DMSO) method.

Dimethyl sulfoxide(DMSO) method:

• < 10 mg of microcapsules was precisely weighed and put into a micro centrifuge tube. DMSO (0.2 ml) was added into the tube to dissolve the polymer portion of the microcapsules. Then, 0.8 ml of a 0.05N NaOH solution containing 0.5% SDS was added to the tube and gently mixed.

•Sonication was performed for 90 min at 25oC, samples were centrifuged at 10,000 rpm for 5 min. Supernatants were analyzed using the BCA (bicinchoninic acid) assay method.

In vitro release profile:•Protein release from traditional polymeric microparticles is typically triphasic.

The 3 phases are

(1) an initial burst-release of surface-bound and poorly encapsulated protein,

(2) a second phase consisting of diffusional release

(3) release due to the degradation of the polymer matrix.

•Collected microcapsules were further washed with 10 mM HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.4) containing 0.02% sodium azide and then suspended in 3 ml of fresh HEPES buffer for a release study. •The tubes were then stored in a 37o8C incubator. At selected time intervals, 1 ml of the release buffer was withdrawn and replaced by fresh HEPES buffer.

Polymer degradation profile:

•Weight average molar mass (Mw) of the original PLGA polymers, as well as those in the degrading microcapsules, was determined by gel permeation chromatography.

•Blank microcapsules were incubated at 37o8C.

•At selected time intervals, take 1ml , freeze-dried, and dissolved in tetrahydrofuran.

•The weight average molar mass was calculated from a calibration curve using a series of polystyrene standards

Lysozyme biological activity assay:

The biological activity of lysozyme in the release medium was determined by measuring turbidity change of a Microcuccus lysodeikticus bacterial cell suspension.

A 0.1 ml aliquot of standard lysozyme solutions with known concentrations and the release samples were added to a cuvette containing 2.9 ml of M. lysodeikticus suspension in HEPES buffer (pH 7.4).

Changes in the turbidity of the cell suspensions were monitored using a Beckman spectrophotometer.

The percentage of retained biological activity (RBA) of lysozyme was calculated by percent RBA=kapparent /ktheoretical.

Here, apparent -the observed rate constant theoretical -the expected value

Examination of encapsulation pattern:

The encapsulation pattern is depend on the flow rate ratio of the two liquids.

A 2% PLGA-EA solution labeled with Nile Red and 0.2% sodium alginate solution containing FITC- dextran were fed into the coaxial ultrasonic atomizer using syringe pumps at controlled flow rates.

The QPol was varied from 0.25 to 1.5 ml/min, while the QAq was fixed at 0.25 ml/min.

Micro drops emerging from the ultrasonic atomizer were captured on a glass plate and observed using a fluorescence microscope.

Fluorescence microscopic images of microdrops

PLGA microdrops labelled &Nile Red (red).aqueous drops labelled & FITC-dextran (green).

Micro encapsulation using an underwater system:

•The coaxial ultrasonic atomizer was operated as submerged under water.

•2% PLGA-EA solution + Nile red flow rate- at 1.5 ml/min -through the outer nozzle .

•An aqueous solution (0.2% sodium alginate or n-decane) flow rate - at 0.25 ml/min - through the inner nozzle.

•Front horn of atomizer immersed in 0.5% PVA bath.

•A part of water bath sample was observed under CLSM.

SUMMARY:

An ultrasonic atomizer generates micro drops of a liquid by vibrating at ultrasonic frequencies.The ultrasonic atomizer have coaxial tubes, through which the two liquids were separately delivered. Reservoir-type microcapsules were obtained are collected in a water bath containing 0.5% PVA using an ultrasonic atomizer.

A-Bright-field microscopic image of the microcapsules B-CLSM cross-sectional image of the microcapsules.

Mechanism of microencapsulation:

Two ink-jet nozzles produced reservoir-type microcapsules when midair collision occurs between the liquid drops of individual liquids.A liquid that is subjected to ultrasonic atomization forms a transitional liquid film on the atomizing surface by absorbing the vibration energy. In a coaxial atomizer, the liquid film might function as an intermediate stage in which the two liquids mix intimately to make a “transient emulsion”, which then breaks up into microcapsules. Once the polymer drops encapsulated the aqueous drops successfully, the polymer layer became phase separated (i.e., precipitated) on the surface of theaqueous drop through the interfacial mass transfer between the two liquids (i.e., solvent exchange).

Drugs:A fine example of an orally administered microencapsulated drug is Cipro XR.

Extended release once-daily tablet to treat urinary tract infections (UTIs).

Cipro -OD: once-daily – high shelf life than cipro XR.

References:

[1] R. Langer, J. Folk man, Polymers for the sustained release of proteins and other macromolecules.[2] S.P. Schwendeman, Recent advances in the stabilization of proteins encapsulated in injectable PLGA delivery systems.[3] C. Perez, I.J. Castellanos, H.R. Costantino, Recent trends in stabilizing protein structure upon encapsulation and release from bioerodible polymers.[4] G. Zhu, S.R. Mallery, S.P. Schwendeman, Stabilization of proteins encapsulated in injectable PLGA.[5] N. Wang, X.S. Wu, A novel approach to stabilization of protein drugs in PLGA micro spheres using agarose hydro gel.[6] H. Okada, T. Heya, Y. Ogawa, T. Shimamoto, One-month release injectable microcapsules of luteinizing hormone.[7] C. Berkland, E. Pollauf, D.W. Pack, K.K. Kim, Uniform double-walled polymer micro spheres of controllable shell thickness.[8] M.D. Blanco, M.J. Alonso, Development and characterization of protein-loaded poly(lactide-co-glycolide) nanospheres.[9] G. Crotts, T.G. Park, Preparation of porous and nonporous biodegradable polymeric hollow micro spheres.[10] Y. Yeo, K. Park, New micro encapsulation technique using an ultrasonic atomizer based on the solvent exchange method.

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