Preparation and characterization of nanoparticles

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Preparation and Characterization of Nanoparticles

Shantanu Tamuly1 and Aman Kumar2
1Department of Veterinary Biochemistry, College of Veterinary Science, Assam Agricultural

University, Khanapara, Guwahati-22, 2Department of Animal Biotechnology, College of Veterinary

Sciences, Lala lajpat Rai University of Veterinary and Animal Sciences, Hisar-125004

Contents

1. Introduction

2. Preparation of nanoparticles

3. Lipid based nanodrug delivery systems

4. Preparation of inorganic nanoparticles

5. Preparation of organic nanoparticles

6. Characterization of nanoparticles

7. Conclusion

8. References

1. Introduction

The term nanotechnology was first used by Professor Noro Taniguchi in 1974. The use of

nanotechnology in the field of medicine has given rise to a new field called nanomedicine. The

medical standing committee of European Science Foundation defined nanomedicine as “the science

and technology of diagnosing, treating and preventing disease and traumatic injury, of relieving pain

and of preserving and improving human health using molecular tools and molecular knowledge of

human body”. The United States National Institute of Health (NIH) considers the nanomedicine as

“an offshoot of nanotechnology that refers to highly specific medical inventions at the molecular scale

for curing disease or repairing damaged tissues such as bone, muscle or nerve”. Professor Peter

Speiser first used nanoparticles as drug delivery system in the year 1973. Many other nanoconstructs

such as drug polymer conjugates were proposed during this period and were subsequently developed

preclinically in 1980s.

2. Preparation of nanoparticles

The description of biodegradable nanoparticles made of poly (methyl cyanoacrylate) and poly (ethyl

cyanoacrylate) was an important milestone during the later part of 1970s. Today more than 25

nanomedicines have been approved for human and veterinary use. Many drugs have low solubility in

water. It is empirically found that when the drugs are converted to nanoparticles (reducing the size of

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drug crystals to nano size, that is less than 1 μm), water solubility of the drug increases that enhances

its efficiency in treating the target malady. The preparation of nanoparticles can be broadly divided

into two categories namely top-down and bottom-up technology

There are different methods of producing nanocrystals in desired shape and size. Fundamentally, three

principles can be used viz. milling, precipitation, and homogenization, and combination of these.

However, the industrially feasible methods utilize the top down technologies that involve reduction of

large-sized macroparticles to nanoparticles. In the bottom up technologies, dissolved molecules are

slowly precipitated in controlled environment. The technology has been used to produce protein and

DNA complex with inorganic nanoparticles such as calcium phosphate nanoparticles; however, it

could not be explored for lipophylic drugs that are poorly soluble in water. He et al. (2000, 2002) for

the first time devised the protocol to synthesize calcium phosphate nanoparticles using bottom up

method under the controlled condition of pH and temperature.

The conversion of macroparticles into nanoparticles leads to an increased surface area to volume ratio

of the particles that again leads to an increased dissolution velocity, thus increase in saturation

solubility increases the gastrointestinal absorption of drugs. So, this increases the bioavailability of a

drug. As far as drug or gene delivery systems are concerned, most emphasis is given to polymer

nanoparticles such as PLG (poly lactide co-glycolide). The most important advantages of use of

polymeric nanoparticles include high drug loading capacity and the feasibility of incorporation of both

polar and non-polar substances that can have the depot effect leading to controlled release. The

important desirable properties that are looked into during selecting a suitable polymer are that the

polymer should be non-toxic, biocompatible and readily processable, non-immunogenic as well as

degraded and eliminated readily from the organism. Finally, the macromolecule selected should

possess suitable functional groups for attaching a particular drug or vaccine antigen.

Coupling of a polymer to a protein antigen imparts several potential advantages. These are: Increased

protection against degradation, easy uptake of polymer-antigen complex by the dendritic cells, and the

depot effect that causes slow release of the vaccine imparting long term immunity.

3. Lipid based nanodrug delivery systems

Lipid based nanodrug delivery systems can be categorised into three main types of drug carriers, viz.

liposomes, lipid nanocapsules, and solid lipid nanoparticles. Liposomes are basically phospholipids

that are amphipathic and they self assemble forming concentric layers. They possess central aqueous

cavity that can accommodate a hydrophilic molecule. The hydrophobic molecules or substances can

be incorporated into the lipid membranes. The important drawback reported in case of liposomes is

their lack of stability. In long term storage, liposomes tend to aggregate and owing to phase state

transition are likely to release the embedded substances. Lipid nanocapsules mimic the physiological

lipoproteins. Their size ranges from 20 to 100 nm. Structurally they can be considered as hybrid of

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polymer nanocapsules and liposomes. These contain an oily core made of medium chain triglycerides

surrounded by a membrane made of mixture of lecithin and pegylated surfactant. Solid lipid

nanoparticles (SLN) are prepared from lipids that are solid at room temperature or at average body

temperature of animals. These solid lipids are stabilized by surfactants. These lipids are triacyl-

glycerols or waxes compared to liposomes and nanocapsules, solid lipid nanoparticles have some

advantages such as better protection of incorporated macromolecules against chemical degradation,

better physical ability and more flexibility in modulating the release of loaded macromolecule.

4. Preparation of inorganic nanoparticles

The preparation of inorganic nanoparticles can be broadly grouped into two categories viz. top down

method and bottom up method.

4.1. Top down method

In this method, the larger macroparticle is broken down into smaller size particles (nanoparticles).

This approach may involve milling or attrition, chemical methods, and volatilization of a solid

followed by condensation of the volatilized components. The most commonly used method for

preparation of nanoparticles of veterinary or medical importance include physical breakage of macro-

or micro- size particles into nanoparticles using high energy waves such as ultrasonic waves. This

approach is rapid but can be used purely for the nanoparticles not having complex with any biological

materials. For example, the calcium phosphate nanoparticle-protein or DNA complex prepared with

this approach would be having significant degradation of protein or DNA component.

4.2. Bottom up method

In this approach, the chemical reactions occur in the solution and the particles are allowed to grow to

size of nanometres. This approach has been followed by many workers in different ways. For instance

the calcium phosphate nanoparticle preparation has been followed by many different methods such

micro-emulsion method (Roy et al. 2003, Bisht et al. 2005) and non-emulsion aqueous method (He et

al. 2000, He et al. 2002). In microemulsion method of preparation of calcium phosphate nanoparticles

includes the preparation of two groups of microemulsions, one containing calcium chloride in hexane

(an organic solvent) and another microemulsion containing sodium phosphate in hexane. Both the

microemulsion are stabilized by surfactants such as bis (2-ethylhexyl) sulphosuccinate). These two

microemulsions are mixed slowly to get calcium phosphate nanoparticles. In this case the

nanoparticles are formed in nano-droplets upon addition of sufficient amount of water. This can be

understood in this way: when the water is added to an organic solvent such as hexane, this leads to

formation of water droplets as water is not miscible in hexane. The micro-droplet formed this way is

highly unstable and tend to aggregate into larger micro-droplet. This aggregation is prevented by the

surfactant that is added to the emulsion. The nanoparticles are formed inside the micro-droplets are

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retrieved by precipitation. The other method for preparation of inorganic nanoparticles, i.e. non-

emulsion aqueous method, was devised by He et al. (2000). This method involves mixing of equi-

molar concentration of calcium chloride and sodium phosphate in buffered medium maintaining a

constant pH with constant stirring for a certain period till the particles attain the desired size (size of a

typical nanoparticle). This technique is cheaper and easier as compared to the top down process.

5. Preparation of organic nanoparticles

The preparation technology of organic nanoparticles can be broadly divided into two categories viz.

Bottom up method (preparative method) and preparation of nanoparticles from preformed polymers

that may be either synthetic or natural.

5.1. Bottom up method (preparative method)

In this method, the monomers of a macromolecule are polymerized in situ. The nanoparticle

preparation by polymerization of monomers can be classified into emulsion polymerization, emulsion

polycondensation and interfacial polymerization. The emulsion polymerization is of two types,

organic and aqueous, depending on the continuous phase.

5.1.1. Emulsion polymerization

This is the fastest method of nanoparticle preparation (Kreuter, 1990). The method can be classified

into two categories based on the use of an organic or aqueous phase. In continuous organic phase

methodology, the monomers or dispersed in an emulsion in which the monomer is not soluble. During

the early stage of polymerization of monomers there is high tendency of polymerization that can be

prevented by addition of surfactants or protective soluble polymers. Normally cyclohexane, n-

pentane, toluene are used as organic phase. In the aqueous continuous phase, the addition of

surfactants or the emulsifiers is not required. The initiation of polymerization occurs when a monomer

molecule dissolved in a continuous phase collides with an initiator molecule that can be an ion or a

free radical. Alternatively, the monomer molecule can be transformed into an initiating radical by

high energy radiation such as gamma radiation or ultraviolet light. The polymerization gets stimulated

when the initiated monomer ions or monomer radical collide with other monomer molecules.

5.1.2. Interfacial polymerization

In this method, the organic monomers and the macromolecule (drug or vaccine) are dissolved in the

mixture of an oil and absolute alcohol. This mixture is then added to the aqueous solution containing a

surfactant, the nanoparticles are formed spontaneously by polymerization. The resulting colloidal

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suspension can be concentrated by freeze drying. The most prominent advantage of this method is

high efficiency for drug encapsulation (Couvreur et al. 2002). The main disadvantage of this method

is the possibility of trace remnants of organic solvents that can be removed by a time consuming and a

difficult procedure (Allemann et al. 1993).

5.1.3. Interfacial polycondensation

It involves interfacial polycondensation of lipophyllic monomer and hydrophyllic monomer such as

phtaloyldichloride and diethylenetrianline respectively in the presence or absence of surfactants

(Montasser et al. 2002). These nanoparticles have been reported to be smaller than 500 nm. This

method has been used for preparation of nanoparticle carriers for α-tocopherol (Bouchemal et al.

2004).

5.2. Use of preformed polymers

The preformed polymers can be natural or synthetic. In many cases the polymer suspension prepared

from monomers faces the problem of residual monomers. The residual monomers sometimes show

the toxic reaction. The use of preformed polymers has been proposed to avoid such a problem.

5.2.1. Preparation of nanoparticles from preformed synthetic polymers

5.2.1.1. Emulsification/solvent evaporation (ESE)

This method involves emulsification of polymer macromolecule complex solution into an aqueous

phase. The solvent in which the polymer is suspended is evaporated. The evaporation of solvent

causes the precipitation of the polymer into nanoparticles. The emulsions that are normally used in

this case are oil/water. This method can be efficiently used for liposoluble drugs (Aftabrouchard and

Dorlker 1992). However the limitation of this method is difficulty in large scale production as the

method needs high energy consuming process of homogenization. The frequently used polymers

under this method are poly-lactide-co-glycolic acid (PLGA), ethylcellulose, cellulose acetate

phthalate, poly(ε-caprolactone) etc.

5.2.1.2. Solvent displacement and interfacial deposition

This method involves spontaneous emulsification of the organic internal phase containing dissolved

polymer into the aqueous external phase. The solvent displacement forms nanospheres or

nanocapsules whereas interfacial deposition forms only nanocapsule. The method of solvent

displacement involves precipitation of preformed polymer from an organic solution and diffusion into

and aqueous medium. This leads to precipitation of the polymer deposition in the interphase between

water and the organic solvent caused by fast diffusion of the solvent leading to instantaneous

formation of colloidal suspension (Quintanar-Guerrero et al. 1998). These methods have been used

for PLGA, and poly(methyl vinyl ether-co-meleic anhydride).

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5.2.1.3. Emulsification/solvent diffusion

The polymer that is to be taken for production of nanoparticles is dissolved in partially water soluble

solvent (e.g. polypropylene carbonate) followed by saturation with water. The precipitation of

polymer and eventual formation of nanoparticles is accomplished by diffusion of the organic solvent.

This diffusion is facilitated by emulsification using surfactants. The solvent is finally eliminated by

evaporation. The advantages of this method is high batch to batch reproducibility, ease of large scale

production, easy high encapsulation efficiency (more than 70%) and homogenization is not required.

Disadvantages are possible leakage of water soluble drugs into water and need for elimination of

water. This technique is highly efficient in encapsulating lipophyllic drugs (Quintanar-Guerrero et al.

1998).

5.2.1.4. Salting out with synthetic polymers

This method can be taken as a modification of the emulsification/solvent diffusion method. In this

method, the polymer drug mixture is added to acetone which is subsequently emulsified in an aqueous

medium containing salting agents such as polyvinyl-pyrolidone. This is followed by dilution with

water. This leads to diffusion of acetone into water inducing the formation of aqueous phase. This

method is used to prepare polylactic acid (PLA) and ethylcellulose nanoparticles. This process is

beneficial for encapsulation of protein owing to minimum stress conditions during the preparation

process (Quintanar-Guerrero et al. 1998).

5.2.2. Production of nanoparticles from natural polymers

5.2.2.1. Albumin nanoparticles

There are two ways of preparation of albumin nanoparticles:

5.2.2.1.1. Heat treatment: This method is applicable only to drug molecules that are heat resistant.

This method involves emulsification of albumin in cottonseed oil at 25°C (Widder, 1979). The

albumin is denatured by resuspending the emulsion in ether containing formaldehyde. This is

followed by stirring. The particles are retrieved by centrifugation and finally dried by freeze drying.

The main drawback of this method is need for complete elimination of cotton seed oil.

5.2.2.1.2. Chloroform treatment: This method involves emulsification of albumin in chloroform

containing hydroxypropylcellulose and ethylcellulose as stabilizers. The cross-linking of the

macromolecule is performed by addition of glutaraldehyde. Because of the need of chlorinated

solvents, this method did not gain much popularity (Marty et al. 1978, Longo et al. 1982).

5.2.2.2. Gelatin nanoparticles

The emulsion of gelatine is hardened by cooling the emulsion below the gelation point leading to

gelation of the gelatine droplets (nanodroplets). The gelatine nanodroplets are cross-linked with

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formaldehyde. This technique is suitable for heat sensitive drugs. The use of formaldehyde as a cross

linker poses the problem of toxicity (Yoshioka et al. 1981).

5.2.2.3. Alginate nanoparticles

Sodium alginate is a polar polymer that forms gel in the presence of cations such as calcium. The

gelation of diluted sodium alginate occurs with the addition of low concentration of calcium ions

(Kwok et al. 1991). This method is easy to perform and does not need highly sophisticated

equipments. The main disadvantage of this method is elimination of residual oil droplets.

5.2.2.4. Chitosan nanoparticles

These nanoparticles have been used to encapsulate protein such as toxoid, vaccines, anticancer agents,

insulin and nucleic acids. The chitosan spontaneously forms complex with polyanions such as

polyphosphates. There are various methods for producing chitosan nanoparticles have been described

by Agnihotri et al. (2004) and Aktas et al. (2005). The methods for the preparation of nanoparticles

are based on formation of complex between chitosan and polyanions (Calvo et al. 1997). The second

method involves the gelation of a chitosan solution in an emulsion of oil (Tokumitsu et al. 1998). The

chitosan nanoparticle formed by polyanion complex results in chitosan nanoparticle of the diameters

ranging from 200 to 500 nm and that produced by the method of gelation results in nanoparticles of

average diameter of 400 nm. The most important disadvantage of the method of gelation is that it

involves many toxic organic solvents that difficult to remove (Vauthier et al. 2000).

5.2.2.5. Agarose nanoparticles

Agarose nanoparticles are used for administration of therapeutic proteins and peptides. Their

preparation is accomplished by emulsion based technology. Agarose is emulsified in corn oil at 40°C.

The protein or peptides are initially added to agarose solution. The agarose nanodroplets are produced

by homogenization. The gelation of agarose is done by dilution of emulsion with corn oil at 5°C. This

eventually forms protein containing hydrogel nanoparticles (Vauthier and Couvreur, 2000; Wang et

al., 1995; Wang and Wu, 1997)

6. Characterization of nanoparticles

The particle size plays a crucial role in nanoparticle properties and therefore an essential task in

property characterization of nanoparticles is particle sizing. The particle size and size distribution of

nanoparticles can be determined using numerous commercially available instruments. Instruments can

be used for the analysis of dry powders and powders dispersed in suspension. In general, there are two

basic methods of defining particle size. The first method is to inspect the particles and make actual

measurements of their dimensions. Microscopic techniques such as electron microscopy, for example,

measure many dimensional parameters from particle images. The second method utilizes the

relationship between particle behaviour and its size. This often implies an assumption of equivalent

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spherical size developed using a size-dependent property of the particle and relating it to a linear

dimension. Equivalent spherical diameters are the diameters of spheres that have the same or

equivalent length, volume and etc. as irregular particles themselves. An example of this method is

photon correlation spectroscopy (PCS) that the dynamic fluctuation of the scattered light intensity is

the basis for calculation of the average particle size (Staiger et al. 2002). It is not possible to discuss

rationally the size of a particle without considering the three-dimensional characteristics (shape) of the

particle itself. This is because the size of a particle is expressed either in terms of linear dimension

characteristics derived from its shape or in terms of its projected surface or volume. On the other

hand, because the particles being studied are not the exact same size, information is required about the

average particle size and the distribution of sizes about that average. Additionally, by comparing the

results from different instruments with each other, one can obtain extra information about the system.

6.1. Determination of size of nanoparticles

Transmission electron microscopy, X-ray diffraction, photon size characterization, surface area

analysis (method of Brunauer, Emmett and Teller), and atomic force microscopy are used.

6.1.1. Transmission electron microscopy

This method is used to directly view the nanoparticles and can be used for direct manual measurement

of nanoparticles. Typically, the calculated sizes are expressed as the diameter of a sphere that has the

same projected area as the projected image of the particle. Manual or automatic techniques are used

for particle size analysis. Manual technique is usually based on the use of a marking device moved

along the particle to obtain a linear dimensional measure of the particle added up and divided by the

number of particles to get a mean result (Jillavenkatesa, 2001). TEM images can also be used to judge

whether good dispersion has been achieved or whether agglomeration is present in the system.

Electron microscopy requires elaborate sample preparation and is slow and few particles are

examined. In combination with diffraction studies, microscopy becomes a very valuable aid to the

characterization of nanoparticles (Rawle, 2002).

6.1.2. X-ray diffraction

As a primary characterization tool for obtaining critical features such as crystal structure, crystallite

size, and strain, x-ray diffraction patterns have been widely used in nanoparticle research. Three

methods of Williamson and Hall, Warren and Averbach and Scherrer can be used to calculate

crystallite size, and strain. The simplest and most widely used method for estimating the average

crystallite size is from the full width at half maximum (FWHM) of a diffraction peak using the

Scherrer equation as follows:

×
dXRD=

×

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Where dXRD= Crystallite size; K= constant that is close to unity; λ = diffraction wavelength; β= full-

width half maxima and θ= diffraction angle.

The method of Williamson and Hall is a simplified integral breadth method for deconvoluting size

and strain contributions to line broadening as a function of 2θ. In this method, the y-intercept is used

to calculate crystallite size, while the strain of the particle is calculated from the slope. Warren and

Averbach’s method takes not only the peak width into account but also the shape of the peak. This

method is based on a Fourier deconvolution of the measured peaks and the instrument broadening to

obtain the true diffraction profile. This method is capable of yielding both the crystallite size

distribution and lattice microstrain (Nalwa, 2000; Ungára et al. 2005).

6.1.3. Photon size characterization (Dynamic light scattering analysis)

This method utilizes the relationship between particle behaviour and its size. This method can be used

to study the size distribution of nanoparticles in liquid dispersion. The photon correlation

spectroscopy involves the calculation of average size of particles based on intensity of scattered light.

This method of photon correlation spectroscopy utilizes the dynamic fluctuation of the scattered light

intensity that is shot onto the particle. This fluctuation in the scattered light intensity is the basis for

the calculation of average particle size. This method is suitable for characterizing the particles having

narrow range of size as the larger particles scatter light manifold than the smaller ones. Apart from

that in case of the particles having the property of agglomeration, the characterization using this

method should be compared with other methods such as X-Ray Diffraction, electron microscopy.

6.1.4. Surface area analysis

The method of Brunauer, Emmett and Teller (BET) is commonly used to determine the surface area

of particles. Surface area of particle includes the summation of the area of the exposed surfaces of the

particles per unit mass. The inverse relationship exists between particle size and surface area. The

surface area of particles in powder form is estimated by nitrogen adsorption by the particles. The

amount of gaseous nitrogen adsorbed onto the surface of particles depends on surface area of

particles. The specific surface area of particles that are assumed to be spherical and are in narrow size

distribution, provides the average diameter in nanometres using the following formula.

6000
=

×

Where the dBET= average diameter of particles (nanometres); n = density of particles (g/cm2); S =

specific area of the particles (m2/g)

The particles size measurement made by this method gives a value close to that obtained by electron

microscopy (Staiger 2002, Rawle 2007).

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6.1.5. Atomic force microscopy (AFM)

AFM is the foremost method of measuring and imaging analytes in nanoscale. This method basically

measures the forces that are exerted by particles on the AFM probe. These forces include contact

force Vander waals forces, capillary forces, chemical bonding, electrostatic forces, magnetic forces

and salvation forces. These forces exerted on the AFM probe gives the information of size,

morphology and surface texture of the nanoparticles. AFM consists of cantilever made of silicon

having a probe of radius of the dimension of nanometres. When this tip of the cantilever is brought

near the sample, the deflection produced in the tip is the function of different forces present in the

powdered sample. The deflection is measured by using a laser spot reflected from the top surface of

the cantilever into an array of photodiodes.

6.2. Determination of zeta potential

The nanoparticles in aqueous solution tend to aggregate into large size macroparticles. This

aggregation depends on the charge on the surface of nanoparticles. The zeta potential analysis is a

technique for determining surface charge of nanoparticles. Because of presence of charge of

nanoparticles a double layer of oppositely charged ions form on the surface of nanoparticles. The

electrical potential at the boundary of double layer surrounding the nanoparticle is known as zeta

potential of the nanoparticle. The zeta potential usually has the range of +100 mV to -100 mV. The

nanoparticles that have lower values of zeta potential have greater tendency to aggregate owing to

Van der Waals inter-particle attraction. The zeta potential depends on various factors such as pH of

the buffer in which the nanoparticles are dissolved. For instance, the pH of 7.5 has been found to be

optimum to have maximum zeta potential for calcium phosphate nanoparticle-DNA complex. The

determination of zeta potential is essential for assessing the stability of nanoparticles in aqueous

solution.

7. Conclusion

The introduction of nanotechnology in the field of veterinary science in the form of drug or vaccine

delivery system has immense prospects in improvement of the treatment and prophylaxis. The utility

of nanoparticles is based on altered physicochemical property of particles when they are converted to

the dimension of nanometres. The alteration of physicochemical property of particles is due to

increase in surface to volume ratio. Different methods of preparation of nanoparticles have their own

advantages and disadvantages; selection of the best method depends on the inherent physicochemical

property of the target macromolecule and nanoparticle system (lipid, polymer or inorganic

nanoparticle). For characterization of nanoparticles one method may not yield valid result of

characterization; so, more than one method is utilized for characterization in addition to electron

microscopy.

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8. References

1. Aftabrouchard D and Dorlker E. 1992. Preparation methods for biodegradable microparticles

loaded with water-soluble drugs. STP Pharma Sci. 2: 365-380.

2. Agnihotri SA, Mallikarjuna NN and Aminabhavi TM. 2004. Recent advances on chitosan-based

micro- and nanoparticles in drug delivery. J Control Release 100: 5-28.

3. Aktas Y, Andrieux K, Alonso MJ, Calvo P, Gqrsoy RN and Couvreur P. 2005. Preparation and

in vitro evaluation of chitosan nanoparticles containing a caspase inhibitor. Int J Pharm 298:

378-383.

4. Allemann E, Gurny R and Doekler E. 1993. Drug-loaded nanoparticles preparation methods and

drug targeting issues. Eur J Pharm Biopharm. 39: 173-191.

5. Allen T. 1997. Particle size measurement, Vol. 1& 2, 5th ed. Chapman and Hall.

6. Bisht S, Bhakta G, Mitra S and Maitra A. 2005. pDNA loaded calcium phosphate nanoparticles:

highly efficient non-viral vector for gene delivery. Int J Pharm. 288: 157-168.

7. Bouchemal K, Briancon S, Perrier E, Fessi H, Bonnet I and Zydowicz N. 2004. Synthesis and

characterization of polyurethane and poly(ether urethane) nanocapsules using a new technique

of interfacial polycondensation combined to spontaneous emulsification. Int J Pharm. 269: 89-

100.

8. Calvo P, Remunan-Lopez C, Vila-Jato JL and Alonso MJ. 1997. Chitosan and chitosan/ethylene

oxide-propylene oxide block copolymer nanoparticles as novel carriers for proteins and

vaccines. Pharm Res. 14: 1431-1436.

9. Couvreur P, Barrat G, Fattal E, Legrand P and Vauthier C. 2002. Nanocapsule technology. Crit

Rev Ther Drug Carrier Syst. 19: 99-134.

10. He Q, Mitchell A, Morcol T and Bell SJ. 2002. Calcium phosphate nanoparticles induce

mucosal immunity and protection against herpes simplex virus type 2. Clin Diagn Lab Immunol.

5: 1021-1024.

11. He Q, Mitchell AR, Johnson SL, Bartak CW, Morcol T and Bell SJ. 2000. Calcium phosphate

nanoparticle adjuvant. Clin Diagn Lab Immunol. 7: 899-903.

12. Jillavenkatesa A, Dapkunas SJ and Lum, Lin-Sien H. 2001. Particle size characterization, NIST

Recommended Practical Guide special publication 960-1, Technology Administration U.S

Department of Commerce.

13. Kreuter J. 1990. Large-scale production problems and manufacturing of nanoparticles. In: Tyle

P (ed) Specialized drug delivery system. Marcel Dekker, New York. p. 257-266.

14. Kwok KK, Groves MJ and Burgess DJ. 1991. Production of 5-15 Am diameter alginate-

polylysine microcapsules by an air atomization technique. Pharm Res. 8: 341-344.

15. Longo WE, Iwata H, Lindheimer TA and Goldberg EP. 1982. Preparation of hydrophilic

albumin microspheres using polymeric dispersing agents. J Pharm Sci. 71: 1323-1328.

11

16. Marty JJ, Oppenheim RC and Speiser P. 1978. Nanoparticles – a new colloidal drug system

delivery system. Pharm Acta Helv. 53: 17-23.

17. Montasser I, Fessi H and Coleman AW. 2002. Atomic force microscopy imaging of novel type

of polymeric colloidal nanostructures. Eur J Pharm Biopharm. 54: 281-284.

18. Nalwa HS. 2000. Handbook of nanostructrured materials and nanotechnology. Vol 2:

Spectroscopy and theory. Academic Press. p. 155-167.

19. Quintanar-Guerrero D, Allemann E, Fessi H and Doelker E. 1998. Preparation techniques and

mechanism of formation of biodegradable nanoparticles from preformed polymers. Drug Dev

Ind Pharm. 24:1113-1128.

20. Rawle A. 2002. The importance of particle sizing to the coatings industry. Part 1: Particle size

measurement. Advances in color science and technology. 5, 1, 1.

21. Rawle AF. 2007. Micron sized nano-materials, powder technology. Advances in color science

and technology. 174, 6.

22. Roy I, Mitra S, Maitra A and Mozumdar S. 2003. Calcium phosphate nanoparticles as novel

non-viral vectors for targeted gene delivery. Int J Pharm 250: 25-33.

23. Staiger M, Bowen P, Ketterer J and Bohonek J. 2002. Particle size distribution measurement and

assessment of agglomeration of commercial nanosized ceramic particles. J Disper Sci Tech 23,

5, 619.

24. Tokumitsu H, Ichikawa H, Fukumori Y, Hiratsuka J, Sakurai Y and Kobayashi T. 1998.

Preparation of gadopentetate-loaded chitosan nanoparticles for gadolinium neutron capture

therapy of cancer using a novel emulsion droplet coalescence technique. Proc 2nd World

Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology, APGI/APV,

Paris. p. 641-642.

25. Ungára T, Tichy G, Gubicza J and Hellmiga RJ. 2005. Correlation between subgrains and

coherently scattering domains. Powder Diffraction. 20(04): 366-375.

26. Vauthier C and Couvreur P. 2000. Development of polysaccharide nanoparticles as novel drug

carrier systems. In: Wise DL (ed) Handbook of pharmaceutical controlled release technology.

Marcel Dekker, New York 7. p. 413-429.

27. Vauthier C, Couvreur P. Development of polysaccharide nanoparticles as novel drug carrier

systems. In: Wise DL, editor. Handbook of pharmaceutical controlled release technology. New

York7 Marcel Dekker; 2000. p. 413- 29.

28. Wang N, Wu XS, Mesiha M. 1995. A new method for preparation of protein-loaded agarose

nanoparticles. Pharm Res. 12:S257.

29. Wang N, Wu XS. 1997. Preparation and characterization of agarose hydrogel nanoparticles for

protein and peptide drug delivery. Pharm Dev Technol. 2:135- 42.

30. Widder KJ, Flouret G and Senye A. 1979. Magnetic microspheres: synthesis of a novel

parenteral drug carrier. J Pharm Sci. 68: 79-82.

12

31. Yoshioka T, Hashida M, Muranishi S and Sezaki H. 1981. Specific delivery of mitomycin C to

the liver, spleen, and lung: nano- and microspherical carriers of gelatin. Int J Pharm. 8: 131-

141.

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