NANOPARTICLES AS DRUG DELIVERY SYSTEM

FACILITATED BY : PRESENTED BY:PROF. S.J SHANKAR N.ANANTHA LAKSHMIDEPT.OF PHARMACEUTICS IST YEAR M.PHARMPESCP,BANGALORE DEPT. OF PHARMACEUTICS

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Definition :-

• Nano particles are solid colloidal particles ranging in size from 10 nm to 1000nm (1 micron). They consist of macromolecular materials in which the active principle is dissolved, entrapped or encapsulated and/or to which the active principle is adsorbed or attached to and can be used for therapeutic disease management, silicon chip, bio-medical applications etc..

NANO OBSERVED

• Life sciences :- drug delivery, Lab-on- a –chip, drug design• Security :- molecular barcoding, chemical

detection.• Materials :- powder, polymers• Electronics :- LCD, semi-conductors, memory• Energy :- Solar cells, fuel cells, membranes• Nano tools :- STM. AFMs

Some terms in Nano-science include:-

•Nano structure :- 1 to 100 nm•Nano- crystals :- Crystalline solid with grain

sizes 1to 100 nm•Nano coatings :- individual layers or multi-

layer surface coating in the range 1 to 100 nm•Nano powders :- extremely fine powders with

average particles size in range of 1 to 100 nm•Nano fibres :- fibers with a diameter in the

range of 1 to 100 nm

TEM (a, b, and c) images of prepared mesoporous silica nanoparticles with mean outer diameter: (a) 20nm, (b) 45nm, and (c) 80nm. SEM (d) image corresponding to (b). The insets are a high magnification of mesoporous silica particle

TYPES OF NANO PARTICLESThere are two types of nanoparticles depending on the preparation processes:

• Nanospheres• NanocapsulesThe term nanoparticles are a collective name for both nanospheres and nanocapsules.

NANOSPHERES have a monolithic-type structure (matrix) in which drugs are dispersed or adsorbed onto their surfaces or encapsulated within the particles.

NANOCAPSULES are the vesicular system in which the drug is confined to a cavity consisting of an inner liquid core surrounded by a polymeric membrane. In this case the active substance is usually dissolved in the inner core, but may also be adsorbed to the capsule surface

NANO CAPSCULES

Nano capsules are systems in which the drug is confined to a cavity surrounded by a unique polymer membrane.

NANO SPHERES Nanospheres are matrix systems in which the drug is physically and uniformly dispersed.1.PHOSPHOLIPID-chain creates the structure of nanosphere.2.HYDROPHILIC-bioactive/active ingredients.3.LIPOPHILIC-bioactive/active ingredients4.PHOSPOLIPID chain breaking apart5.HYDROPHILIC ingredients slowly releasing.6.LIPOPHILIC ingredients slowly releasing.

PHOTOGRAPHIC VIEW

CLASSIFICATION OF

NANOPARTICLES

SOLID LIPID

POLYMERIC

CERAMIC

HYDROGEL

COPOLYMERIZED

NANOCRYSTALS AND NANOSUSPENSIONS

NANOTUBES AND NANOWIRES

FUNCTIONALIZED NANOCARRIERS

NANOSPHERES

NANOCAPSULES

• SOLID LIPID NANO PARTICLES: New type of colloidal drug carrier system for i.v.Consists of spherical solid lipid particles in the nm

range, dispersed in water or in aqueous surfactant solution.• POLYMERIC NANO PARTICLES:Are defined as particulate dispersions or solid

particles with size in the range of 10-1000nm.Composed of synthetic or semi-synthetic

Polymers. Biodegradable polymeric nanoparticles Polylactic acid (PLA), polyglycolic acid (PGA), Polylactic - glycolic acid (PLGA), and Polymethyl methacrylate (PMMA) Phospholipids Hydrophobic core

•CERAMIC NANO PARTICLES :These are the nanoparticles made up of

inorganic (ceramic) compounds silica, ( Inorganic/metal) titania and alumina. Exist in size less than 50 nm, which helps them in evading deeper parts of the body.

•HYDROGEL NANOPARTICLES: Polymeric system involving the self-assembly

and self aggregation of natural polymer amphiphiles cholesteroyl pullulan , cholesteroyl dextran and agarose cholesterol groups provide cross linking points.

•Copolymerized Peptide Nanoparticles: Drug moiety is covalently bound to the carrier

instead of being physically entrapped.

•Nanocrystals And Nanosuspensions: Pure drug coated with surfactant, Aggregation of

these particles in crystalline form .Drug powder dispersed in aqueous surfactant solution.

• Functionalized Nanocarriers:Biological materials like proteins, enzymes,

peptides etc… are being utilized as a carriers for the drug delivery.

ADVANTAGES :

Particle size and surface characteristics of nanoparticles can

be easily manipulated to achieve both

passive and active drug targeting after

parenteral administration.

They control and sustain release of the drug during the transportation and at

the site of localization, altering organ distribution

of the drug and subsequent clearance of the drug so as to achieve

increase in drug therapeutic efficacy and reduction in side effects.

ADVANTAGES:Controlled release

and particle degradation

characteristics can be readily modulated

by the choice of matrix constituents .

Drug loading is relatively high and

drugs can be incorporated into the systems without any chemical reaction; this is an important factor for preserving

the drug activity.

Site-specific targeting can be achieved by attaching

targeting ligands to surface of

particles or use of

magnetic guidance.

The system can be used for

various routes of

administration including oral,

nasal, parenteral ,

intra-ocular etc

DISADVANTAGES:

Small size and large

surface area can lead to

particle aggregation.

physical handling of

nanoparticles difficult in liquid and dry forms

In addition, small particles size and large surface area

readily result in limited drug loading and

burst release.

NANOPARTICLES DRUG DELIVERY SYSTEMS

IDEAL CHARACTERISTICS :

It should be biochemical inert , non toxic and non-immunogenic.

It should be stable both physically and chemically in Invivo & invitro conditions.

Restrict drug distribution to non-target cells or tissues or organs & should have uniform distribution.

Controllable & Predicate rate of drug release.

Drug release should not effect drug action

Specific Therapeutic amount of drug release must be possessed

Carriers used must be biodegradable or readily eliminated from the body without any problem and no carrier induced modulation in disease state.

The preparation of the delivery system should be easy or reasonable

simple, reproducible & cost effective.

POLYMERIC NANO PARTICLES• Polymers are macromolecules composed of a large number of

repeating units organized in a chain-like molecular architecture exhibiting a multiplicity of compositions, structures, and properties. It is because of this variety of compositions, structures, and properties that polymers are being used in nanoparticle systems to generate nanoparticles suited for each specific biomedical application. The main use of polymeric nanoparticles is in drug delivery, although they are also used in bioimaging and biosensing assays.

• The use of nanoparticles in drug delivery due to the emerging importance of targeted delivery in medicine, polymeric nanoparticles that are efficient, tissue specific, and most importantly, nontoxic. For the preparation of nanoparticles for drug delivery, there are a variety of methods depending on how the drug will be loaded onto the nanoparticle. The resulting nanoparticle-drug compounds may have the structure of capsules, (polymeric nanoparticles or polymeric nanoconjugates), amphiphilic core/shell (polymeric micelles), or hyperbranched macromolecules of nanometer dimensions (dendrimers)

CRITERIA FOR IDEAL POLYMERIC CARRIER FOR NANOPARTICLES:

Easy to synthesize and characterize

Inexpensive

Biocompatible and Biodegradable

Non-toxic and water soluble

Inert and Non-immunogenic

TYPES OF POLYMERIC CARRIERS :

Natural hydrophilic polymers

Synthetic hydrophobic polymers

The polymers used for the preparation of nano particles are as follows :

NATURAL HYDRO PHILIC POLYMERS • Proteins such as albumin, gelatin, legumin or vicilin as well as

polysaccharides like alginates or agarose have been extensively studied and characterized. • These macromolecules have attracted wide interest as

biomaterials due to intrinsic biodegradability and biocompatibility . • Natural hydrophilic polymers have been classified as proteins

and polysaccharides.PROTEINS POLYSACCHARIDES

GELATIN ALGINATE

ALBUMIN DEXTRAN

LECTIN CHITOSAN

LEGUMIN AGAROSE

NOTE:1. Alginate based drug delivery systems are homo-

compatible and convincingly acceptable for parentral administration of bio actives.

2. In contrast dextran , albumin and gelatin which are though acceptable materials manifest immunogenicity due to use of cross-linking agents which employed during their preparation.

3. Chitosan on the other hand is homo non compatible material and hence it should be restricted to extracorporeal uses only.

DISADVANTAGES:• Batch-batch variation• Conditional biodegradability• Antigenicity. Parentral adminstration of polymeric

nanoparticles get compromised mainly due to antigenicity.

CHITOSANChitosan is a modified natural carbohydrate polymer prepared by the partial N-de acetylation of the crustacean-derived natural biopolymer chitin. There are at least four methods reported for the preparation of chitosan nanoparticles. The four methods are ionotropic gelation, microemulsion, emulsification solvent diffusion and polyelectrolyte complex formation.

GELATINGelatin is extensively used in food and medical products and is a nontoxic alternative. Gelatin NPs are very efficient in delivery and controlled release of the drugs. They are nontoxic, biodegradable, bioactive and inexpensive. Gelatin is a poly ampholyte consisting of both cationic and anionic groups along with a hydrophilic group. It is known that the mechanical properties such as swelling behavior and thermal properties of gelatin NPs depend significantly on the degree of cross-linking between cationic and anionic groups. These properties of gelatin can be manipulated to prepare desired type of NPs from gelatin. Gelatin nanoparticles can be prepared by the desolvation/coacervation or emulsion methods.

SYNTHETIC HYDROPHOBIC POLYMERS

Polymers which are used for nanoparticle preparations are mainly those, which are conceivably employed in the preparation of microspheres.

The polymers used are either pre-polymerized or synthesized before (first group) or during the (second group ) process of nanoparticle preparation.

Various synthetic polymers used for the preparation of Nanoparticles are :

Pre-polymerised Polymerized in process

Poly(ε-caprolactone) (PECL) Poly (isobutylcyanoacrylates)(PICA)

Poly (Lactic acid) (PLA) Poly(butylcyanoacrylates)(PBCA)

Poly(Lactide-co-glycolide)(PLGA) Polyhexylcyanoacrylates(PHCA)

Polystyrene Polymethyl(methacrylate) (PMMA)

POLY- -CAPROLACTONE Ε(PCL):• poly-ε-caprolactone is degraded by hydrolysis of its ester

linkages under the normal physiological conditions in the human body and has minimal or no toxicity. Therefore, PCL has grabbed the attention of researchers as a candidate of choice for use in drug delivery and long-term implantable devices. PCL's slower rate of degradation compared to polylactides has made it better candidate for making long-term implantable devices. PCL nanoparticles have been prepared mostly by nanoprecipitation, solvent displacement and solvent evaporation.

POLYLACTIC ACID (PLA)• PLA is a biocompatible and biodegradable polymer which is broken

down to monomeric units of lactic acid in the body. Lactic acid is a natural intermediate/by product of anaerobic respiration, which is converted into glucose by the liver during the Cori cycle. Glucose then is used as an energy source in the body. The use of PLA nanoparticles is therefore safe and devoid of any major toxicity. PLA nanoparticles have been mostly prepared by the solvent evaporation, solvent displacement, salting out and solvent diffusion methods. The salting out procedure is based on the separation of a water- miscible solvent from aqueous solution by adding a salting out agent like magnesium chloride or calcium chloride. The main advantage of the salting out procedure is that it minimizes stress to protein encapsulants .

Poly-D-L- lactide-co-glycolide (PLGA)• Poly-D-L- lactide-co-glycolide (PLGA) is one of the most successfully

used biodegradable polymers. It undergoes hydrolysis in the body to produce biodegradable metabolite monomers such as lactic acid and glycolic acid.. Since lactic acid and glycolic acids are normally found in the body and participate in a number of physiological and biochemical pathways, there is very minimal systemic toxicity associated with the use of PLGA for the drug delivery or biomaterial applications. PLGA NPs have been mostly prepared by the emulsification-diffusion, the solvent evaporation and the nano precipitation methods . PLGA nanoparticles have been used to develop protein and peptide based nano medicines, nano-vaccines, and genes containing nanoparticles for in-vivo delivery systems

PREPARATION OF NANO PARTICLES

PREPARATION TECHNIQUES OF NANOPARTICLES :

The selection of the appropriate method for the preparation of nanoparticles depends on the physicochemical characteristics of the polymer and the drug to be loaded.

The preparation techniques largely determine the inner structure, in vitro release profiles and the biological fate of these polymeric delivery systems .

Two types of systems with different inner structures are apparently possible including :

A matrix type system consisting of an entanglement of oligomer or polymer units (Nanoparticles/Nanospheres).

A reservoir type of system comprised of an oily core surrounded by an embryonic polymeric shell

The drug can either be entrapped within the reservoir or the matrix or otherwise be absorbed on the surface of these particulate systems.

The polymers are strictly structured to a nano metric size range particles using appropriate methodologies.

These methodologies are classified as follows :1.AMPIPHILIC MACROMOLECULE CROSS-LINKING a.) Heat cross-linking B.) Chemical cross-linking2.POLYMERIZATION BASED METHODS a.)Polymerization of monomers in situ b.)Emulsion (micellar) polymerization C.)Dispersion Polymerization d.)Interfacial condensation Polymerization e.)Interfacial complexation3.POLYMER PRECIPITATION METHOD a.)solvent extraction/evaporation b.)solvent displacement c.)Salting out

Nano particles preparation by cross-linking of Amphiphilic Macromolecules :• Nano particles can be prepared from amphiphilic macromolecules,

proteins and polysaccharides which have affinity for aqueous and lipid solvents.

• The technique involves firstly , the aggregation of amphiphiles followed by further stabilization either by heat denaturation or chemical cross-linking.

• These processes may occur in biphasic O/W or W/O type dispersed systems.

• It may also take place in an aqueous amphphilic solution where removal , extraction, or diffusion of solvent, amphiphiles are aggregated as tiny particulates and subsequently rigidized via chemical cross-linking.

• The cross-linking generally executed following dispersed phase solvent extraction or depletion.

A.) CROSS-LINKING IN W/O EMULSION• The cross-linking method is exhaustively used for the nano-

encapsulation of drugs,• This method involves the emulsification of bovine serum albumin

(BSA)/ Human serum albumin (HSA) or protein aqueous solution in oil using high pressure homogenization or high frequency sonication.

• The W/O emulsion so formed is then poured into preheated oil ( temperature above 100oc).

• The suspension in the preheated oil maintained above 100oc is held stirred for specified time in order to denature and aggregate the protein contents of aqueous pool completely and to evaporate the water.

• Proteinaceous sub-nanoscopic particles are thus formed where the size of the internal phase globules mainly determines the ultimate size of particulates.

• The particles are finally washed with an organic solvent to remove any adherent or absorbed oil traces and subsequently collected by centrifugation.

• Factors which govern the size and shape of nano partilces are mainly emulsification energy and temperature (used of denaturation and aggregation).

• High temperatures used restrict the application of method to temperature- sensitive drugs. As an alternative to heat stabilzation a chemical cross- linking agent usually glutaraldehyde is incorporated.

B.) EMULSION CHEMICAL DEHYDRATION

• Chemical dehydration has been reported for producing BSA nano particles with a narrow size distribution.• Hydroxypropyl solution in chloroform was used as a

continuous phase while chemical dehydrating agent i.e., 2,2 di-methyl propane , was used to translate internal aqueous phase into a solid particulate suspension.• This method reportedly avoided coalescence of

droplets and could produce nano particles of smaller size (<300nm) .

C.) PHASE SEPARATION IN AQUEOUS MEDIUM ( DESOLVATION )

• The protein or polysaccharide from an aqueous phase can be desolvated by pH change , or change in temperature by adding some appropriate counter ions

• Cross-linking may be affected simultaneously or subsequent to the desolvation step.

• The method essentially proceeds involving three steps :

• Appropriate levels of desolvation and resolvation of the aggregate size are maintained and the aggregated nanoparticulates are cross-linked using glutaraldehyde.

1.PROTEIN DISSOLUTION

2.PROTEINAGGREGATION

3.PROTEIN CROSSDEAGGREGATION

• Solvent competing agent , sodium sulphate is mainly used as a desolving agent while alcohol, i.e., ethanol and isopropanol are carefully added as de solvating or de-aggregating agent.

• This additionally can be optimized turbidometrically using a Nephelometer.

• Only de solvation may give the final product as nanospheres. Desolvation aggregates the protein and turns the suspension colloidal and hence milky in appearance.

• Both lipophilic and hydrophilic drugs could be entrapped in nano particles using this technique.

Example :A two step deslovation method for manufacturing gelatin nano particles. After the first desolvation step , the lower molecular gelatin fractions present in the supernatent were removed by decanting. The high molecular weight fractions present in the sediment were dissolved and then desolvated at pH 2.5 in the second step . Centrifugation and redispersion methods were used to purify particles so obtained

PH INDUCED AGGREGATION• The protein phase may also be separated through PH change. The PH

induced aggregation to prepare nanoparticles has been extensively used and Insulin nanospheres were the successfully prepared first.

• Insulin was precipitated and then redissolved forming nanodroplets , which were hardened using glutaraldehyde . The method yielded nanoparticles of Insulin.

• Similarly in the preparation of Gelatin nanospheres. Gelatin and Tween 20 were dissolved in aqueous phase and the PH of the solution was adjusted to optimum value. The clear solutions so obtained were heated to 40oC followed by its quenching at 4oc for 24 hrs and subsequently left at ambient temperature for 48h. The sequential temperature treatment resulted into a colloidal dispersion of aggregated gelatin the aggregates were finally cross-linked using glutaraldehyde as cross-linking agent. The nanospheres thus resulted were of 200nm average size with uniform dispersity

COUNTER-ION INDUCED AGGREGATION

• Separation of protein phase may also occur by the presence of counter ions in the aqueous medium.

• The aggregation of dispersed phase (polysaccharide) can effectively be initiated by adding some appropriate counter ions

• The aggregation can be propagated by adding secondary species of counter ions followed by rigidization step.

• Alginate nanoparticles have recently prepared using counter ion induced gelation technique, where gelation was induced by ca++ and continued by addition of poly (1-lysine ) . Thus these nanospheres are classically based on polyelectrolyte complex.

• Similarly chitosan nanospheres were prepared by adding triphosphate (highly negatively charged ions) to the medium. The size was found to be critically dependent on the concentration of both i.e., chitosan and tri-phosphate ions.

Nanoparticle preparation using polymerization based methodsTwo different approaches are generally adopted for the preparation of nanospheres using in situ polymerization technique.• Methods in which the monomer to be polymerized is emulsified

in a non-solvent phase (emulsion polymerization) or • Methods in which the monomer is dissolved in a solvent that is

non-solvent for the resulting polymer (dispersion polymerization).In emulsion polymerization method, the monomer is dissolved in an internal phase while in the case of dispersion polymerization , it is taken in the dispersed phase. It is interesting ,that in either of the cases following polymerization , the polymer tends to be insoluble in internal phase or dispersed phase thus results into an ordered suspension of nanospheres.

EMULSION POLYMERIZATION• The process of emulsion polymerization can be conventional or

inverse , depending upon the nature of the continuous phase in emulsion.

• In the former case , the continuous phase is aqueous (O/W emulsion) , whereas in the latter case it is organic (W/O emulsion).

• Two different mechanisms were proposed for the emulsion polymerization process and they include :

Micellar Nucleation and Polymerization

Homogenous Nucleation and Polymerization.

MICELLAR NUCLEATION AND POLYMERIZATION

• The micellar nucleation and polymerization involve the swollen monomer micelles as the site of nucleation and polymerization.

• The monomer is emulsified in the non-solvent phase with the help of surfactant molecules.

• This process leads to the formation of monomer-swollen micelles and stabilized monomer droplets.

• The polymerization reaction proceeds through nucleation and propagation stage 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.

• Being slightly soluble in the surrounding phase , the monomer molecules reach the micelles by diffusion from the monomer droplets through the continuous phase, thus allowing the polymerization to progress within the micelles.

HOMOGENOUS NUCLEATION AND POLYMERIZATION• Homogenous nucleation and polymerization applies in

cases where the monomer is sufficiently soluble in the continuous outer phase.• The nucleation and polymerization stages occur in this

phase leading to the formation of primary chains called Oligomers.• Both micelles and droplets play the role of monomer

reservoirs throughout the polymer chain length.• When the oligomers have reached a certain length ,

they precipitate and form primary particles, which are stabilized by the surfactant molecules provided by the micelles and the droplets.

• Poly(alkylcyanoacrylate) (PACA) , a biodegradable polymer are essentially prepared by emulsion polymerization.

• Water insoluble monomer is emulsified in an external acid aqueous phase that contains a stabilizer.

• Monomer polymerized rapidly following anion polymerization mechanism.

• The polymerization rate is dependent on the PH of the medium.

• Anionic polymerization takes place in micelles after diffusion of monomer molecules through the water phase and is initiated by negatively charged compound.

• Thus at neutral PH the rate of polymerization being extremely fast leads to formation of aggregates.

• However , at acidic PH ,i.e., PH 2-4 , the reaction rate remains well controlled and slow, thus producing uniform sized nanospheres of relatively high molecular weight.

• During polymerization the medium is stirred in order to maintain size and dispersibility of phase undergoing polymerization.

• The polymerization is continued for carried time depending upon the monomer, where alkyl chain length is the determinant , i.e., longer the chain length, longer is the time of polymerization needed.

• For instance, with ethyl cyanoacrylate it is 2 h and for hexyl cyanoacrylate the polymerization is conducted for 10-12 h.

• The colloidal suspension on completion of polymerization is finally neutralized and lyophilized in the presence of some cryo protectants, i.e., glucose.

• Water-soluble drugs may be associated with PACA nanoparticles either by dissolving the drug in the aqueous polymerization medium or by incubating the blank nanospheres with an aqueous solution of drug.

• The drug molecules are entrapped within the polymer matix and are also adsorbed onto the surface of the nanoparticles. The drug molecules are physically adsorbed only on the surface.

• Polymer based nano capscule preparation and incorporation/loading of lipophilic drug.

• The monomer and drug are dissolved in a mixture of polar (methanol/acetone) solvents, and oil (benzyl benzoate, coconut oil) using a lipophilic surfactant , i.e., lecithin.

• The oil phase is then added to an aqueous phase containing a hydrophilic surfactant. Polaxmer 188 is often used.

• After oil phase is dispersed into an aqueous phase, critically two processes occur at the oil-water interface.

• Diffusion of polar solvents from oil phase to the bulk aqueous phase and monomer polymerization at the O/W interface.

• The polymerization process is catalyzed by OH- ions.• Thus on completion of polymerization a nanocapscular system

results where lipophilic drug dissolved in oil is incorporated in a polymeric sac.

• Conversely, a dispersion system, W/O type, where dispersed aqueous phase contains the drug and stabilizer , which organic phase is typically consisted of an organic solvent ( chloroform/n-hexane) and a monomer , can be used to prepare nano capscules based upon inverse emulsification polymerization mechanism.

• The interfacial OH- ions initiated polymerization occurs at W/O interface enveloping aqueous phase with its drug contents in the form of nano capscules.

• The inverse polymerization process was rapidly adopted for the preparation of poly(alkyl cyanoacrylate) or PACA nanospheres.

• In these studies , the drug was dissolved in a small amount od water or hydrophilic solvent (methanol) and emulsified in an organic phase (i.e., isooctane, cyclohexane-chloroform, hexane) in the presence of large amount of surfactants.

• Alkylcyanoacrylate monomers were then added directly or dissolved in an organic solvent to the preformed W/O emulsion under stirring.

• Hydrophilic compounds such as doxorubicin, fluorescein, and methylene blue as well as lipophilic compounds such as triamcinolone acetonide were sufficiently incorporated into PACA nanoparticles prepared using this technique.

DISPERSION POLYMERIZATION• In dispersion polymerization the monomer instead of being

emulsified , is dissolved in a aqueous medium which acts as a precipitant for subsequently formed polymer.

• Polymerization based methods essentially involve in situ controlled polymerization of appropriate monomers where drug may be added to monomeric phase or may be added to monomeric phase or may be added to the formed polymeric nano particulates dispersion for adsorpive loading

• The monomer is introduced into the dispersion medium (phase) of an emulsionor an inversed emulsion into non-solvent based polymeric solution.

• The polymerization is initiate by adding a catalyst and proceeds with nucleation phase followed by a growth phase (propagation).

• On the other hand,in the case of dispersion polymerization,the nucleation is directly inducedin the aqueous monomer solution and the presence of stabilizer or surfactants is not absolutely necessary for the formation of stable nanospheres.

• The method is used to prepare biodegradable polyacrylamide and poly methyl-methacrylate(PMMA) nanoparticles.

• The acrylamide or methyl methacrylate monomer is dissolved in aqueous phase and polymerized by γ-irradiation or by chemical initiation (ammonium or potassium peroxdisulphate) combined with heating to temperature above 65oc.

• The polymerization is initiated by γ-irradiation from a 60C source at elevated temperature.

• The redox catalyst may be used as a chemical initiator , i.e., potassium peroxidisulphate.

• The oligomers formed subsequently aggregate and above a certain molecular weight precipitate in the form of primary particles and propagate as nanosphere which may or may not be stabilized by surfactant molecules.

• The monomer concentration has linear effect on the size of nanospheres where size increases with increasing monomer concentration , decreasing initiator concentration and decreasing temperature.

• Being very slowly biodegradable and biocompatible , PMMA nanoparticles have been considered as optimal polymeric systems for vaccination purpose.

• For this application, initiation by γ-irradiation is useful for the production of nano particles by polymerization in the presence of antigenic material ,because it operates at temperatures suitable for preservation of heat sensitive antigenic materials , as their structure and nature remain unaffected.

• The antigenic materials entrapped in PMMA nanoparticles using this technique include influenza virion , influenza sub unit antigen ,bovine serum albumin , HIV-1 and HIV-2 antigens.

• Besides PMMA nano particles , co polymeric methacrylic acid based nano particles are also prepared by dispersion polymerization process using blends of methyl methacrylate with one or several other acrylic acid derivatives ( eg. Hydroxyethyl methacrylate, methacrylic acid , ethylene glycol dimethyl acrylate, sulphopropylmethacrylate).

• These copolymer nanoparticles were developed within the intention of modifying the surface properties of the nanoparticles, namely the hydrophilicity and charge, which are important parameters governing the in vivo distribution of the particles.

INTERFACIAL POLYMERIZATION

• The preformed polymer phase is finally transformed to an embryonic sheath.• A polymer that eventually becomes core of nano particles

and drug molecules to be loaded are dissolved in a volatile solvent.• The solution is then poured into a non-solvent for both

polymer and core phase.• The polymer phase is separated as a coacervate phase at

O/W interface.• The resultant mixture instantaneously turns milky owing to

the formation of nano capscules .• Interfacial polymerization for the encapsulation of proteins,

enzymes , antibodies and cells .

INTERFACIAL COMPLEXATION

• The method is based on the process of microencapsulation .In the case of nano particle preparation, aqueous poly electrolyte solution is carefully dissolved in reverse micelles in an apolar bulk phase with the help of an appropriate surface active agent. Subsequently competing poly electrolyte is added to the bulk, which allows a layer of insoluble poly electrolyte complex to coacervate at the interface.

NANO PARTICLE PREPARATION USING POLYMER PRECIPITATION METHOD• SOLVENT EXTRACTION :In this method , the hydrophobic polymer ( except dextran,

mainly polyester) and /or a hydrophobic drug is dissolved in a particular organic solvent followed by its dispersion in a continuous phase , in which polymer is insoluble.

External phase also contains a stabilizer.Depending upon solubility miscibility technique they are

designated as :

1.Solvent extraction

2. Solvent evaporation

Using an organic solvent that is completely soluble in continuous aqueous phase (i.e., acetone ) – nano precipitation.

By evaporation of the organic solvent at room temperature or at accelerated temperatures or using vaccum.

By incorporating additional amount of water into the ultraemulsion ( to extract or diffuse the solvent).

Increasing the solubility of the organic solvent in the external medium by adding an alcohol (i.e., isopropanol).

The polymer precipitation occurs as a consequences of the solvent extraction or evaporation , which can be brought up by :

SOLVENT EXTRACTION METHOD

• This method involves the formation of a conventional O/W emulsion between a partially water miscible solvent containing the polymer and the drug and an aqueous phase containing the stabilizer.

• The subsequent removal of solvent or the additions of water to the systems so as to affect the fusion of the solvent to the external phase are two variants of the solvent extraction method.

• Ex: in a classical procedure plga nanospheres, the polymer solubilised in a solvent and dispersed in gelatin solution by sonicationto yield emulsion(o/w), then the solvent is eliminated by evaporation.

• For the evaporation purpose apart from sonication,high speed or pressure homogenization methods are widely employed.

• The homogenizer breaks the initial coarse emulsion in nanodroplets , yielding nanosphere with narrow size distribution.

DOUBLE EMULSION SOLVENT EVAPORATION METHOD

• Emulsion solvent evaporation technique has been further modified and a double emulsion ( or multiple emulsion ) of water in oil in water type has been used.

• PLGA nanoparticles were prepared loaded with bovine serum albumin using double emulsion solvent evaporation method , Owing to the high solubility of the protein in water , the double emulsion technique has been chosen as one of the appropriate methods.

• BSA and PLGA are dissolved separately in aqueous and organic phases respectively (containing the stabilizer) and subjected to ultra sonication to yield a W/O emulsion . The W/O emulsion further added to a PVA aqueous solution to yield the water-in-oil-in-water (W/O/W) double emulsion. The organic solvent is allowed to evaporate while being stirred first at atmosphere pressure for 16 h and then gradually at reduced pressure (from 100mm Hg to 30mm Hg) to yield nanoparticles .

Schematic representation of EC microparticles preparation using double-emulsification the W/O1/O2 method.

SOLVENT DISPLACEMENT OR NANOPRECIPITATION• This method is based on the interfacial deposition of a polymer

following displacement of a semi – polar solvent miscible with water from a lipophilic solution .

• Solvent displacement method involves the use of an organic phase ,which is completely soluble in the external aqueous phase.

• The organic solvent diffuses instantaneously to the external aqueous phase, inducing immediate polymer precipitation because of the complete miscibility of both the phases.

• Consequently neither separation nor extraction of the solvent is required for the polymer precipitation .

• After nanoparticle preparation, the solvent is eliminated an the free flowing nanoparticles can be obtained under reduced pressure. This method is particularly useful for drugs that are slightly soluble in water.

• If the drug is highly hydrophilic , it diffuses out into the external aqueous phase whereas if the drug is highly hydrophobic , it may precipitate in the aqueous phase as nano crystals , which further grow during storage.

• In case of hydrophilic polymer , an aqueous solution of polymer is dispersed or emulsified in oil phase.

• The precipitation of polymer proceeds on addition of acetone. Using this technique ovalbumin loaded dextran nano spheres of ~1μm size were prepared. The nano spheres were fairly stable and uniform in size.

• However, the loading efficiency of lipophilic drugs , such as indomethacin , metipranol , betaxolol in nano particles of PLA, PLGA and PECL has been increased using a modified solvent displacement method.

• In this method the drug is dissolved in a small volume of an appropriate oil and then diluted in the polar organic solvent ( actone/ethanol/methanol). When the organic solution is dispersed in the aqueous phase , the polymer precipitates around the nanodroplets forming a reservoir system

SALTING OUT • Salting out process is one of the most commonly used methods

used to prepare nano particles.• This method involves the incorporation of a saturated aqueous

solution of polyvinyl alcohol (PVA) into an acetone solution of the polymer under magnetic stirring to form O/W emulsion.

• However, the process differs from nano precipitation technique as in the latter the polymeric solution (in acetone ) is completely miscible with the external aqueous medium.

• But in the salting out technique the miscibility of both the phases is prevented by the saturation of the external phase with PVA.

• The precipitation of the polymer occurs when a sufficient amount of water is added to the external phase to allow complete diffusion of the acetone from the internal phase into the aqueous phase. This technique is suitable for drugs and polymers that are soluble in polar solvents such as acetone or ethanol.

SUPERCRITICAL FLUID TECHNOLOGY

• The need to develop environmentally safer methods for the production of PNP has motivated research on the utility of supercritical fluids as more environmental friendly solvents, with the potential to produce PNPs with high purity and without any trace of organic solvent. Supercritical fluid and dense gas technology are expected to offer an interesting and effective technique of particle production, avoiding most of the drawbacks of the traditional methods.

• Two principles have been developed for the production of nanoparticles using supercritical fluids:

1. Rapid expansion of supercritical solution (RESS) 2. Rapid expansion of supercritical solution into liquid solvent (RESOLV).

Rapid expansion of supercritical solution • the solute is dissolved in a supercritical fluid to form a

solution, followed by the rapid expansion of the solution across an orifice or a capillary nozzle into ambient air.

• The high degree of super saturation, accompanied by the rapid pressure reduction in the expansion, results in homogenous nucleation and thereby, the formation of well-dispersed particles.

• The RESS experimental apparatus consists of three major units: a high-pressure stainless steel mixing cell, a syringe pump, and a pre-expansion unit.

• The concentration and degree of saturation of the polymer have a considerable effect on the particle size and morphology of the particles

Experimental set-up for preparation of polymer nanoparticles by rapid expansion of supercritical fluid solution

Rapid expansion of supercritical solution into liquid solvent • A simple, but significant modification to RESS involves

expansion of the supercritical solution into a liquid solvent instead of ambient air,

• Poly (heptadecafluorodecyl acrylate) nanoparticles having an average size of less than 50 nm. Even though in RESS technique no organic solvents used for the formation of PNPs, the prime products obtained using this technique are micro scaled rather than nanoscaled, which is the main drawback of RESS.

• In order to overcome this drawback a new supercritical fluid technology known as RESOLV has been developed. In RESOLV the liquid solvent apparently suppresses the particle growth in the expansion jet, thus making it possible to obtain primarily nano sized particles.

Experimental set-up for the rapid expansion of supercritical fluid solution into liquid solvent process

EVALUATION OF NANOPARTICLES Particle size Density Molecular weight Structure and crystallinity Specific surface area Surface charge & electronic mobility Surface hydrophobicity Invitro release Nanoparticle yield Drug entrapment efficiency

PARTICLE SIZE• Particle size and size distribution are the most important

characteristics of nanoparticle systems.• They determine the in vivo distribution, biological fate, toxicity

and the targetting ability of nano particle systems• In addition they can also influence the drug loading, drug release

and stability of nano particles Photon correlation spectroscopy (PCS) : For smaller particle. Laser diffractrometry : For larger particle. Electron microscopy (EM) : Required coating of conductive

material such as gold & limited to dry sample. Transmission electron microscopy (TEM) : Easier method &

Permits differntiation among nanocapsule & nanoparticle. Atomic force microscope Laser force microscope Highresolution

Scanning electron microscope microscope

2.Density : Helium or air using a gas pycnometer Density gradiant centrifugation3. Molecular weight : Gel permeation chromatography using refractive

index detector.4. Structure & Crystallinity : X-ray diffraction Thermoanalytical method such as, 1) Differential scanning calorimetry 2) Differential thermal analysis 3) Thermogravimetry

5. Specific surface area :Sorptometer

specific surface area A = 6 Density * diameter of particle6. Surface charge & electronic mobility :Surface charge of particle can be determined by measuring particle

velocity in electrical field.Laser Doppler Anemometry tech. for determination of Nanoparticles

velocities.Surface charge is also measured as electrical mobility.Charged composition critically decides bio-distribution of nanoparticle .

Zeta potential can also be obtain by measuring the electronic mobility.7. Surface Hydrophobicity :Important influence on intraction of nanoparticles with biological

environment.Several methods have been used,

1. Hydrophobic interaction chromatography.

2. Two phase partition.

3. contact angle measurement.

8. Invitro release :Diffusion cellRecently introduce modified Ultra-filtration tech.Media used : phosphate buffer

9. Nanoparticle yield :% yield = Actual weight of product

Drug entrapment % = Mass of drug in Nanoparticles *100 Mass of drug used in formulation

Total weight of excipient & Drug

10. Drug entrapment efficiency :

CHARACTERIZATION OF NANOPARTICLES

EQUIPMENT USEDHomogenizerUltra SonicatorMillsSpray MillingSupercritical Fluid TechnologyElectrosprayUltracentrifugationNanofiltration

HOMOGENISER SONICATOR SPRAY MILLING

SUPER CRITICAL FLUID TECHNOLOGY

NANO MILL- MANUFACTURING PLATFORM

NANO FILTRATION

NANO CRYSTALS

NANO CRYSTALS AND NANOSUSPENSIONS

Drug nanocrystals are pure solid drug particles with amean diameter below 1000 nm. A nanosuspensionconsists of drug nanocrystals, stabilizing agents such assurfactants and/or polymeric stabilizers, and a liquiddispersion medium. The dispersion media can be water,aqueous solutions, or nonaqueous media. The term .drugnanocrystals. implies a crystalline state of the discreteparticles, but depending on the production method theycan also be partially or completely amorphous. Drugnanocrystals have to be distinguished from polymericnano particles, which consist of a polymeric matrix andan incorporated drug. Drug nanocrystals do not consist ofany matrix material.

PROPERTIES OF NANO CRYSTALS

PRODUCTION OF NANOCRYSTALS

• There are three possible methods involved in the production of nano crystals and they are :

1. PRECIPITATION METHOD: The drug is dissolved in a solvent and subsequently added to a nonsolvent, leading to the precipitation of finely dispersed drug nanocrystals. This method is used to prepare ointments containing finely dispersed, precipitated drugs.

2. MILLING METHOD: Bead or a pearl mill to achieve particle size diminution. Milling media, dispersion medium (generally water), stabilizer and the drug are charged into the milling chamber. Shear forces of impact, generated by the movement of the milling media, lead to particle size reduction. There are two basic milling principles. Either the milling medium is moved by an agitator, or the complete container is moved in a complex movement leading consequently to a movement of the milling media.

3. HOMOGENIZATION METHOD• there are three important technologies namely: Microfluidizer

technology (IDD-P™™ technology), Piston gap homogenization in water (Dissocubes® technology) and in water mixtures or in nonaqueous media (Nanopure® technology).

PRODUCTS IN MARKET

NANOSUSPENSION• Nanosuspensions are colloidal dispersions of nanosized drug

particles stabilized by surfactants. They can also be defined as a biphasic system consisting of pure drug particles dispersed in an aqueous vehicle in which the diameter of the suspended particle is less than 1μm in diameter.

ADVANTAGES:Enhance the solubility and bioavailabilty of drugsSuitable for hydrophilic drugs.Higher drug loading can be achievedDose reduction is possibleEnhance the physical and cheical stability of drugs.Provides a passive drug targeting .

PREPARATION OF NANO SUSPENSIONS

PREPARATIVE TECHNIQUES FOR NANOSUSPENSION WITH MERITS AND DEMERITS

• EVALUATION OF NANO SUSPENSIONS :IN-VITRO EVALUATION :- Particle size and size distribution Partice charge (zeta potential) Crystalline state and morphology Saturation solubility and dissolution velocityIN-VIVO EVALUATIONEVALUATION FOR SURFACE – MODIFIED

NANOSUPSENSIONS:- Surface hydrophilicity Adhesion properties Interaction with body proteins

APPLICATIONSBIOAVAILABILITY ENHANCEMENT: Bioavailability enhances

drug with poor solubility , poor permeability or poor solubility in GI tract that leads to poor oral bioavailability.

OCULAR ADMINSTRATION: Delivery of poorly soluble drug in cul-de-sac suspensions and ointments.

PULMONARY ADMINSTRATION: The nanoparticulate delivery of the drug allows the rapid diffusion and dissolution of the drug at the site of action .Nano suspension offers rapid onset of action and then control release of the active moiety is highly beneficial.

TARGETED DRUG DELIVERY: Nano suspensions can be designed for targeted delivery of antimycrobial, fungal or leishmanial drugs to macrophages.

APPLICATIONS