review article nanosuspensions: a promising drug delivery...

827

Review Article

JPP 2004 56 827ndash840

2004 The Authors

Received December 11 2003

Accepted March 30 2004

DOI 1012110022357023691

ISSN 0022-3573

Pharmaceutical Division

University Institute of Chemical

Technology Matunga

Mumbai-400 019 India

V B Patravale

Department of Pharmaceutics

Bombay College of Pharmacy

Kalina Santacruz (E)


Abhijit A Date

Department of Cell Biology

Neurobiology and Anatomy

University of Cincinnati Medical

Center Cincinnati

OH 45267-0521 USA

R M Kulkarni

Correspondence V B Patravale

Pharmaceutical Division

University Institute of Chemical

Technology Matunga


E-mail vbp_muictyahoocoin

Nanosuspensions a promising drug delivery strategy

V B Patravale Abhijit A Date and R M Kulkarni

Abstract

Nanosuspensions have emerged as a promising strategy for the efficient delivery of hydrophobic drugs

because of their versatile features and unique advantages Techniques such as media milling and high-

pressure homogenization have been used commercially for producing nanosuspensions Recently the

engineering of nanosuspensions employing emulsions and microemulsions as templates has been

addressed in the literature The unique features of nanosuspensions have enabled their use in various

dosage forms including specialized delivery systems such asmucoadhesive hydrogels Rapid strides have

been made in the delivery of nanosuspensions by parenteral peroral ocular and pulmonary routes

Currently efforts are being directed to extending their applications in site-specific drug delivery

Introduction

The formulation of poorly water-soluble drugs has always been a challenging problemfaced by pharmaceutical scientists and it is expected to increase because approximately40 or more of the new chemical entities being generated through drug discoveryprogrammes are poorly water-soluble (Lipinski 2002)

The problem is even more intense for drugs such as itraconazole and carbamazepine(belonging to class III as classified by Washington 1996) as they are poorly soluble inboth aqueous and organic media and for drugs having a log P value of 2 (Pouton2000) Such drugs often have an erratic absorption profile and highly variable bio-availability because their performance is dissolution-rate limited and is affected by thefedfasted state of the patient

Traditional strategies such as micronization solubilization using co-solvents the useof permeation enhancers (Aungst 1993 2000) oily solutions (Aungst 1993) and surfact-ant dispersions (Aungst et al 1994) which evolved earlier to tackle the formulationchallenges have limited use Although reasonable success has been achieved in formulat-ing water-insoluble drugs using liposomes (Schwendener amp Schott 1996) emulsions(Floyd 1999 Nakano 2000) microemulsions (Lawrence amp Rees 2000) solid dispersiontechnology (Serajuddin 1999 Leuner ampDressman 2000 Breitenbach 2002) and inclusioncomplexes employing cyclodextrins (Loftsson amp Brewster 1996 Stella amp Rajewski 1997Akers 2002) there is no universal approach applicable to all drugs Hence there is agrowing need for a unique strategy that can tackle the formulation-related problemsassociated with the delivery of hydrophobic drugs in order to improve their clinicalefficacy and optimize their therapy with respect to pharmacoeconomics

Nanosuspensions have revealed their potential to tackle the problems associatedwith the delivery of poorly water-soluble and poorly water- and lipid-soluble drugsand are unique because of their simplicity and the advantages they confer over otherstrategies This review focuses on the various aspects of nanosuspensions and theirpotential as a promising strategy in drug delivery

Nanosuspensions can be defined as colloidal dispersions of nano-sized drug parti-cles that are produced by a suitable method and stabilized by a suitable stabilizer

Methods of production

Media milling (NanoCrystals)This patent-protected technology was developed by Liversidge et al (1992) Formerlythe technology was owned by the company NanoSystems but recently it has beenacquired by Elan Drug Delivery In this method the nanosuspensions are produced

using high-shear media mills or pearl mills The media millconsists of a milling chamber a milling shaft and a recir-culation chamber (Figure 1) The milling chamber ischarged with the milling media water drug and stabilizeras depicted in Figure 1 and the milling media or pearls arethen rotated at a very high shear rate The milling processis performed under controlled temperatures

Principle The high energy and shear forces generated asa result of the impaction of the milling media with thedrug provide the energy input to break the microparticu-late drug into nano-sized particles The milling medium iscomposed of glass zirconium oxide or highly cross-linkedpolystyrene resin The process can be performed in eitherbatch or recirculation mode In batch mode the timerequired to obtain dispersions with unimodal distributionprofiles and mean diameterslt200 nm is 30ndash60min Themedia milling process can successfully process micronizedand non-micronized drug crystals Once the formulationand the process are optimized very little batch-to-batchvariation is observed in the quality of the dispersion

Advantages Drugs that are poorly soluble in both aqueous andorganic media can be easily formulated into nanosus-pensions

Ease of scale-up and little batch-to-batch variation

Narrow size distribution of the final nano-sized pro-duct A comparison of the size of naproxen crystalsbefore and after media milling is given in Figure 2

Flexibility in handling the drug quantity ranging from1 to 400mgmL1 enabling formulation of very diluteas well as highly concentrated nanosuspensions

Disadvantages The major concern is the generation of residues ofmilling media which may be introduced in the finalproduct as a result of erosion (Buchmann et al 1996Muller amp Bohm 1998) This could be problematicwhen nanosuspensions are intended to be administered

CoolantLargedrugcrystals

Chargedwithdrug waterandstabilizer

Re-circubtionchamber

Milling chamber

Screenretainingmilling media in chamber

Millingshaft

MotorMilling media

Nanocrystals

Figure 1 Schematic representation of the media milling process The milling chamber charged with polymeric media is the active component

of the mill The mill can be operated in a batch or recirculation mode A crude slurry consisting of drug water and stabilizer is fed into the

milling chamber and processed into a nanocrystalline dispersion The typical residence time generated for a nanometer-sized dispersion with a

mean diameter of lt200 nm is 30ndash60min Reprinted from Merisko-Liversidge et al 2003 with permission from Elsevier Publications

20

15

10

5

0 1 100

Freq

uen

cy

Nanocrystallinenaproxen aftermedia millingMean 0147 micro mD90 0205 micro m

Naproxen crystalsbefore media millingMean 242 micro mD90 4708 micro m

Particle size (micro m)

Figure 2 The particle size distribution of naproxen crystals before

(~) and after () milling Before milling the drug crystals had a mean

particle size of 242m After being processed for 30min in a media

mill the mean particle size of the nanocrystalline dispersion was

0147m with D90frac14 0205m The particle size measurements were

generated using laser light diffraction in a Horiba LA-910 using

polystyrene nanospheres ranging from 01 to 10m as standards

Reprinted from Merisko-Liversidge et al 2003 with permission from

Elsevier Publications

828 V B Patravale et al

for a chronic therapy The severity of this problem hasbeen reduced to a great extent with the advent of poly-styrene resin-based milling medium For this mediumresidual monomers are typically 50 ppb and the resi-duals generated during the milling processing are notmore than 0005ww of the final product or theresulting solid dosage form

High-pressure homogenizers (Disso Cubes)Disso Cubes technology was developed by R H Muller(Muller et al 1998) The patent rights of Disso Cubes wereinitially owned by DDS (Drug Delivery Services) GmbHbut currently they are owned by SkyePharma plc DissoCubes are engineered using piston-gap-type high-pressurehomogenizers A commonly used homogenizer is the APVMicron LAB 40 (APV Deutschland GmbH LubeckGermany) However other piston-gap homogenizersfrom Avestin (Avestin Inc Ottawa Canada) andStansted (Stansted Fluid Power Ltd Stansted UK) canalso be used A high-pressure homogenizer (Figure 3)consists of a high-pressure plunger pump with a subse-quent relief valve (homogenizing valve) The task of theplunger pump is to provide the energy level required forthe relief The relief valve consists of a fixed valve seat andan adjustable valve These parts form an adjustable radialprecision gap The gap conditions the resistance and thusthe homogenizing pressure vary as a function of the forceacting on the valve An external impact ring forms adefined outlet cross-section and prevents the valve casing

from being damaged due to the flow (Jahnke 1998) Theinstrument is available in discontinuous and continuousversions The continuous version is suitable for optimizingthe various parameters of the homogenization processUse of the discontinuous version is sensible if the drug isvery costly or of limited availability The instrument canbe operated at pressures varying from 100 to 1500 bars Insome instruments a maximum pressure of 2000 bars canbe reached High-pressure homogenizers are availablewith different capacities ranging from 40mL (for labora-tory purposes) to a few thousand litres (for large-scaleproduction) It is advisable to start with the micronizeddrug (particle sizelt25m) for production of nanosus-pensions in order to prevent blocking of the homogeniza-tion gap Hence generally a jet-milled drug is employed asthe starting material for producing Disso Cubes Beforesubjecting the drug to the homogenization process it isessential to form a presuspension of the micronized drugin a surfactant solution using high-speed stirrers Duringthe homogenization process the drug suspension ispressed through the homogenization gap in order toachieve nano-sizing of the drug

Principle During homogenization the fracture of drugparticles is brought about by cavitation high-shear forcesand the collision of the particles against each other Thedrug suspension contained in a cylinder of diameterabout 3mm passes suddenly through a very narrowhomogenization gap of 25m which leads to a highstreaming velocity

In the homogenization gap according to Bernoullirsquosequation the dynamic pressure of the fluid increaseswith the simultaneous decrease in static pressure belowthe boiling point of water at room temperature In con-sequence water starts boiling at room temperature lead-ing to the formation of gas bubbles which implode whenthe suspension leaves the gap (called cavitation) and nor-mal air pressure is reached again The implosion forces aresufficiently high to break down the drug microparticlesinto nanoparticles Additionally the collision of the par-ticles at high speed helps to achieve the nano-sizing of thedrug To improve the efficiency of nano-sizing the addi-tion of viscosity enhancers is advantageous in certain casesas increasing the viscosity increases the powder densitywithin the dispersion zone (homogenization gap)

In order to obtain an optimized formulation the effectof the following process variables should be investigated

Effect of homogenization pressure As the homogenizercan handle varying pressures ranging from 100 to 1500bars the effect of the homogenization pressure on theparticle size should be investigated in each case in order tooptimize the process parameters It is expected that thehigher the homogenization pressure the lower the parti-cle size obtained The studies carried out on RMKP 224-[N-(2-hydroxy-2-methyl-propyl)-ethanolamino]-27-bis(cis-26-dimethylmorpholin-4-yl)-6-phenyl-pteridine rev-ealed that an inverse relationship exists between thehomogenization pressure and the particle size (Mulleramp Bohm 1998 Muller amp Peters 1998 Grau et al 2000)

Nanosuspension

Valve

Valve seat

Macrosuspension

Imp

act

rin

g

Figure 3 Schematic representation of the high-pressure homogeni-

zation process

Nanosuspensions a promising drug delivery strategy 829

Number of homogenization cycles For many drugs it isnot possible to obtain the desired particle size in a singlehomogenization cycle Typically multiple cycles arerequired Hence depending on the hardness of thedrug the desired mean particle size and the requiredhomogeneity of the product homogenization can becarried out in three five or 10 cycles It is anticipatedthat the higher the number of homogenization cyclesthe smaller the particle size obtained The optimumnumber of homogenization cycles can be arrived at byanalysing the particle size and polydispersity index ofthe drug after each cycle


Ease of scale-up and little batch-to-batch variation(Grau et al 2000)

Narrow size distribution of the nanoparticulate drugpresent in the final product (Muller amp Bohm 1998)

Allows aseptic production of nanosuspensions for par-enteral administration

Flexibility in handling the drug quantity ranging from1 to 400mgmL1 thus enabling formulation of verydilute as well as highly concentrated nanosuspensions(Krause amp Muller 2001)

Disadvantages Prerequisite of micronized drug particles Prerequisite of suspension formation using high-speedmixers before subjecting it to homogenization

Emulsions as templatesApart from the use of emulsions as a drug delivery vehiclethey can also be used as templates to produce nanosus-pensions The use of emulsions as templates is applicablefor those drugs that are soluble in either volatile organicsolvent or partially water-miscible solvent Such solventscan be used as the dispersed phase of the emulsion Thereare two ways of fabricating drug nanosuspensions by theemulsification method

In the first method an organic solvent or mixture ofsolvents loaded with the drug is dispersed in the aqueousphase containing suitable surfactants to form an emulsionThe organic phase is then evaporated under reduced pres-sure so that the drug particles precipitate instantaneouslyto form a nanosuspension stabilized by surfactants Sinceone particle is formed in each emulsion droplet it ispossible to control the particle size of the nanosuspensionby controlling the size of the emulsion Optimizing thesurfactant composition increases the intake of organicphase and ultimately the drug loading in the emulsionOriginally organic solvents such as methylene chlorideand chloroform were used (Bodmeier amp McGinity 1998)However environmental hazards and human safety con-cerns about residual solvents have limited their use inroutine manufacturing processes Relatively safer solventssuch as ethyl acetate and ethyl formate can still be con-sidered for use (Sah 1997 2000)

Another method makes use of partially water-misciblesolvents such as butyl lactate benzyl alcohol and triacetinas the dispersed phase instead of hazardous solvents (Trottaet al 2001) The emulsion is formed by the conventionalmethod and the drug nanosuspension is obtained by justdiluting the emulsion Dilution of the emulsion with watercauses complete diffusion of the internal phase into theexternal phase leading to instantaneous formation of ananosuspension The nanosuspension thus formed has tobe made free of the internal phase and surfactants by meansof diultrafiltration in order to make it suitable for adminis-tration However if all the ingredients that are used for theproduction of the nanosuspension are present in a concen-tration acceptable for the desired route of administrationthen simple centrifugation or ultracentrifugation is suffi-cient to separate the nanosuspension

Advantages Use of specialized equipment is not necessary Particle size can easily be controlled by controlling thesize of the emulsion droplet

Ease of scale-up if formulation is optimized properly

Disadvantages Drugs that are poorly soluble in both aqueous andorganic media cannot be formulated by this technique

Safety concerns because of the use of hazardous sol-vents in the process

Need for diultrafiltration for purification of the drugnanosuspension which may render the process costly

High amount of surfactantstabilizer is required as com-pared to the production techniques described earlier

The production of drug nanosuspensions from emulsiontemplates has been successfully applied to the poorlywater-soluble and poorly bioavailable anti-cancer drugmitotane where a significant improvement in the dissolu-tion rate of the drug (five-fold increase) as compared tothe commercial product was observed (Trotta et al 2001)

Microemulsions as templatesMicroemulsions are thermodynamically stable and isotro-pically clear dispersions of two immiscible liquids such asoil and water stabilized by an interfacial film of surfactantand co-surfactant (Eccleston 1992) Their advantagessuch as high drug solubilization long shelf-life and easeof manufacture make them an ideal drug delivery vehicleThere are several research papers available that describethe use of microemulsions as drug delivery vehicles(Constantinides et al 1994 1995 Kim et al 1998 Park ampKim 1999 Kawakami amp Yoshikawa 2002) Recently theuse of microemulsions as templates for the production ofsolid lipid nanoparticles (Gasco 1997) and polymericnanoparticles (Rades et al 2002) has been describedTaking advantage of the microemulsion structure onecan use microemulsions even for the production of nano-suspensions (Trotta et al 2003) Oil-in-water microemul-sions are preferred for this purpose The internal phase ofthese microemulsions could be either a partially miscibleliquid or a suitable organic solvent as described earlier


The drug can be either loaded in the internal phase orpre-formed microemulsions can be saturated with the drugby intimate mixing The suitable dilution of the microemul-sion yields the drug nanosuspension by the mechanismdescribed earlier The influence of the amount and ratio ofsurfactant to co-surfactant on the uptake of internal phaseand on the globule size of the microemulsion should beinvestigated and optimized in order to achieve the desireddrug loading The nanosuspension thus formed has to bemade free of the internal phase and surfactants by means ofdiultrafiltration in order to make it suitable for administra-tion However if all the ingredients that are used for theproduction of the nanosuspension are present in a concen-tration acceptable for the desired route of administrationthen simple centrifugation or ultracentrifugation is suffi-cient to separate the nanosuspension

The advantages and disadvantages are the same as foremulsion templates The only added advantage is the needfor less energy input for the production of nanosuspen-sions by virtue of microemulsions

The production of drug nanosuspensions using micro-emulsions as templates has been successfully applied tothe poorly water-soluble and poorly bioavailable anti-fungal drug griseofulvin where a significant improvementin the dissolution rate of the drug (three-fold increase) ascompared to the commercial product was observed It wasfound that the nature of the co-surfactant affected thedissolution rate of the drug nanosuspension as antici-pated (Trotta et al 2003) However this technique is stillin its infancy and needs more thorough investigation

Formulation considerations

StabilizerStabilizer plays an important role in the formulation ofnanosuspensions In the absence of an appropriate stabil-izer the high surface energy of nano-sized particles caninduce agglomeration or aggregation of the drug crystalsThe main functions of a stabilizer are to wet the drugparticles thoroughly and to prevent Ostwaldrsquos ripening(Rawlins 1982 Muller amp Bohm 1998) and agglomerationof nanosuspensions in order to yield a physically stableformulation by providing steric or ionic barriers The typeand amount of stabilizer has a pronounced effect on thephysical stability and in-vivo behaviour of nanosuspen-sions In some cases a mixture of stabilizers is required toobtain a stable nanosuspension

The drug-to-stabilizer ratio in the formulation mayvary from 120 to 201 and should be investigated for aspecific case Stabilizers that have been explored so farinclude cellulosics poloxamers polysorbates lecithinsand povidones (Liversidge et al 1992) Lecithin is the sta-bilizer of choice if one intends to develop a parenterallyacceptable and autoclavable nanosuspension

Organic solventsOrganic solvents may be required in the formulation ofnanosuspensions if they are to be prepared using anemulsion or microemulsion as a template As these tech-niques are still in their infancy elaborate information on

formulation considerations is not available The accept-ability of the organic solvents in the pharmaceutical arenatheir toxicity potential and the ease of their removal fromthe formulation need to be considered when formulatingnanosuspensions using emulsions or microemulsions astemplates The pharmaceutically acceptable and lesshazardous water-miscible solvents such as ethanol andisopropanol and partially water-miscible solvents suchas ethyl acetate ethyl formate butyl lactate triacetinpropylene carbonate and benzyl alcohol are preferred inthe formulation over the conventional hazardous solventssuch as dichloromethane Additionally partially water-miscible organic solvents can be used as the internalphase of the microemulsion when the nanosuspensionsare to be produced using a microemulsion as a template

Co-surfactantsThe choice of co-surfactant is critical when using micro-emulsions to formulate nanosuspensions Since co-surfact-ants can greatly influence phase behaviour the effect ofco-surfactant on uptake of the internal phase for selectedmicroemulsion composition and on drug loading shouldbe investigated Although the literature describes the useof bile salts and dipotassium glycerrhizinate as co-surfact-ants various solubilizers such as Transcutol glycofurolethanol and isopropanol can be safely used as co-surfact-ants in the formulation of microemulsions

Other additivesNanosuspensions may contain additives such as bufferssalts polyols osmogent and cryoprotectant depending oneither the route of administration or the properties of thedrug moiety

Post-production processing

Post-production processing of nanosuspensions becomesessential when the drug candidate is highly susceptible tohydrolytic cleavage or chemical degradation Processingmay also be required when the best possible stabilizer isnot able to stabilize the nanosuspension for a longer per-iod of time or there are acceptability restrictions withrespect to the desired route Considering these aspectstechniques such as lyophillization or spray drying maybe employed to produce a dry powder of nano-sizeddrug particles Rational selection has to be made in theseunit operations considering the drug properties and eco-nomic aspects Generally spray drying is more econom-ical and convenient than lyophillization The effect ofpost-production processing on the particle size of thenanosuspension and moisture content of dried nano-sized drug should be given due consideration

Advantages of nanosuspensions

Increase in the dissolution velocity and saturationsolubility of the drugThis is an important advantage that makes nanosuspen-sions amenable to numerous applications The reason


behind the increase in the dissolution velocity and satura-tion solubility of the nanosuspensions can be given asfollows According to the NernstndashBrunner and Levichmodification of the Noyes Whitney dissolution modelequation (Dressman et al 1998 Horter amp Dressman2001) the dissolution velocity of the nanosuspensionincreases due to a dramatic increase in the surface areaof the drug particles from microns to particles of nano-meter size

dXdtfrac14 ((DA)h) (CsXV)

where dXdt is the dissolution velocity D is the diffusioncoefficient A is the surface area of the particle h is thediffusional distance Cs is the saturation solubility of thedrug X is the concentration in the surrounding liquid andV is the volume of the dissolution medium

In addition as described by the Prandtl equation thedecrease in the diffusional distance with increasing curva-ture of ultrafine nano-sized particles contributes to theincrease in the dissolution velocity The Prandtl equation(Mosharraf amp Nystrom 1995) describes the hydrodynamicboundary layer thickness or diffusional distance (hH) forflow passing a flat surface

hHfrac14 k (L12V12)

where L is the length of the surface in the direction offlow k denotes a constant V is the relative velocity of theflowing liquid against a flat surface and hH is the hydro-dynamic boundary layer thickness Corresponding to thePrandtl equation Nystrom and Bisrat (1988) have shownthat for solids dispersed in a liquid medium under agita-tion a decrease in particle size results in a thinner hydro-dynamic layer around particles and an increase of thesurface-specific dissolution rate This phenomenon isespecially pronounced for materials that have mean par-ticle size of less than 2m

The increase in the saturation solubility of the drugwith a decrease in particle size can be explained byOstwaldndashFreundlichrsquos equation

log(CsC)frac14 2V2303RTr

where Cs is the saturation solubility C is the solubility ofthe solid consisting of large particles is the interfacialtension of substance V is the molar volume of the particlematerial R is the gas constant T is the absolute tempera-ture is the density of the solid and r is the radius

Another possible explanation for the increased satura-tion solubility is the creation of high-energy surfaces whendisrupting the more or less ideal drug microcrystals tonanoparticles Lyophobic surfaces from the inside of thecrystal are exposed to the aqueous dispersion mediumduring nanosizing According to OstwaldndashFreundlich Cs

is dependent on the interfacial tension and subsequentlyon the interfacial energy G (Gfrac14 A) Differences in inter-facial energy have a profound effect on the saturationsolubilities of polymorphic forms of the drug the sameexplanation might be valid for the nanosuspension (high-energy form frac14 polymorph II frac14 higher Cs) compared tomicroparticulate suspensions (low-energy form frac14 stable

polymorph I frac14 lower Cs) Dissolution experiments can beperformed to quantify the increase in the saturation solu-bility of the drug when formulated into a nanosuspensionIn a study carried out by Muller and Peters (1998) anincrease in the saturation solubility of RMKP 22 withdecrease in particle size was observed

Improved biological performanceAn increase in the dissolution velocity and saturationsolubility of a drug leads to an improvement in the in-vivo performance of the drug irrespective of the routeused The advantages related to various routes are dis-cussed later in detail

Ease of manufacture and scale-upUnlike nanoparticulate carriers such as polymeric nano-particles which were investigated earlier nanosuspen-sions are easy to manufacture The production processesdescribed earlier are easily scaled up for commercial pro-duction The introduction of nanosuspension productssuch as Rapamune and the NanoCrystal colloidal keto-profen is sufficient to substantiate this

Long-term physical stabilityAnother special feature of nanosuspensions is the absenceof Ostwald ripening which is suggestive of their long-termphysical stability (Peters amp Muller 1996) Ostwald ripening(Rawlins 1982 Muller amp Bohm 1998) has been describedfor ultrafine dispersed systems and is responsible for crystalgrowth and subsequently formation of microparticlesOstwald ripening is caused by the differences in dissolutionpressuresaturation solubility between small and large par-ticles It is in practice an effect based on the higher satura-tion solubility of very small particles as compared to largerones Molecules diffuse from the higher concentrated areaaround small particles (higher saturation solubility) to areasaround larger particles possessing a lower drug concentra-tion This leads to the formation of a supersaturated solu-tion around the large particles and consequently to drugcrystallization and growth of the large particles The diffu-sion process of the drug from the small particles to the largeparticles leaves an area around the small particles that is notsaturated any more consequently leading to dissolution ofthe drug from the small particles and finally complete dis-appearance of the small particles The lack of Ostwaldripening in nanosuspensions is attributed to their uniformparticle size which is created by various manufacturingprocesses The absence of particles with large differencesin their size in nanosuspensions prevents the existence of thedifferent saturation solubilities and concentration gradientsin the vicinity of differently sized particles which in turnprevents the Ostwald ripening effect

VersatilityThe flexibility offered in the modification of surface prop-erties and particle size and ease of post-productionprocessing of nanosuspensions enables them to be incor-porated in various dosage forms such as tablets pelletssuppositories and hydrogels for various routes of admin-istration thus proving their versatility


Characterization of nanosuspensions

The essential characterization parameters for nanosuspen-sions are as follows

Mean particle size and particle size distribution Themean particle size and the width of particle size distri-bution are important characterization parameters asthey govern the saturation solubility dissolution velo-city physical stability and even biological performanceof nanosuspensions It has been indicated by Muller ampPeters (1998) that saturation solubility and dissolutionvelocity show considerable variation with the changingparticle size of the drug

Photon correlation spectroscopy (PCS) (Muller ampMuller 1984) can be used for rapid and accurate deter-mination of the mean particle diameter of nanosuspen-sions Moreover PCS can even be used for determiningthe width of the particle size distribution (polydispersityindex PI) The PI is an important parameter that gov-erns the physical stability of nanosuspensions andshould be as low as possible for the long-term stabilityof nanosuspensions A PI value of 01ndash025 indicatesa fairly narrow size distribution whereas a PI valuegreater than 05 indicates a very broad distributionNo logarithmic normal distribution can definitely beattributed to such a high PI value Although PCS is aversatile technique because of its low measuring range(3 nm to 3m) it becomes difficult to determine thepossibility of contamination of the nanosuspension bymicroparticulate drugs (having particle size greater than3m) Hence in addition to PCS analysis laser diffrac-tometry (LD) analysis of nanosuspensions should becarried out in order to detect as well as quantify thedrug microparticles that might have been generatedduring the production process Laser diffractometryyields a volume size distribution and can be used tomeasure particles ranging from 005ndash80m and incertain instruments particle sizes up to 2000m can bemeasured The typical LD characterization includesdetermination of diameter 50 LD (50) and diameter99 LD (99) values which indicate that either 50 or99 of the particles are below the indicated size TheLD analysis becomes critical for nanosuspensions thatare meant for parenteral and pulmonary delivery Evenif the nanosuspension contains a small number ofparticles greater than 5ndash6m there could be a possibil-ity of capillary blockade or emboli formation as thesize of the smallest blood capillary is 5ndash6m It shouldbe noted that the particle size data of a nanosuspensionobtained by LD and PCS analysis are not identical asLD data are volume based and the PCS mean diameteris the light intensity weighted size The PCS meandiameter and the 50 or 99 diameter from the LDanalysis are likely to differ with LD data generallyexhibiting higher values The nanosuspensions can besuitably diluted with deionized water before carryingout PCS or LD analysis

For nanosuspensions that are intended for intraven-ous administration particle size analysis by the Coultercounter technique is essential in addition to PCS and LD

analysis Since the Coulter counter gives the absolutenumber of particles per volume unit for the differentsize classes it is a more efficient and appropriate techni-que than LD analysis for quantifying the contaminationof nanosuspensions by microparticulate drugs

Crystalline state and particle morphology The assess-ment of the crystalline state and particle morphologytogether helps in understanding the polymorphic ormorphological changes that a drug might undergowhen subjected to nanosizing Additionally when nano-suspensions are prepared drug particles in an amor-phous state are likely to be generated Hence it isessential to investigate the extent of amorphous drugnanoparticles generated during the production of nano-suspensions The changes in the physical state of thedrug particles as well as the extent of the amorphousfraction can be determined by X-ray diffraction analysis(Muller amp Bohm 1998 Muller amp Grau 1998) and can besupplemented by differential scanning calorimetry(Shanthakumar et al 2004) In order to get an actualidea of particle morphology scanning electron micro-scopy is preferred (Muller amp Bohm 1998)

Particle charge (zeta potential) The determination ofthe zeta potential of a nanosuspension is essential as itgives an idea about the physical stability of the nano-suspension The zeta potential of a nanosuspension isgoverned by both the stabilizer and the drug itself Inorder to obtain a nanosuspension exhibiting good sta-bility for an electrostatically stabilized nanosuspensiona minimum zeta potential of 30mV is requiredwhereas in the case of a combined electrostatic andsteric stabilization a minimum zeta potential of20mV is desirable (Muller amp Jacobs 2002b)

Saturation solubility and dissolution velocity The deter-mination of the saturation solubility and dissolutionvelocity is very important as these two parameterstogether help to anticipate any change in the in-vivoperformance (blood profiles plasma peaks and bio-availability) of the drug As nanosuspensions areknown to improve the saturation solubility of thedrug the determination of the saturation solubilityrather than an increase in saturation solubility remainsan important investigational parameter The saturationsolubility of the drug in different physiological buffersas well as at different temperatures should be assessedusing methods described in the literature The investiga-tion of the dissolution velocity of nanosuspensionsreflects the advantages that can be achieved over con-ventional formulations especially when designing thesustained-release dosage forms based on nanoparticu-late drugs The dissolution velocity of drug nanosuspen-sions in various physiological buffers should bedetermined according to methods reported in thepharmacopoeia

In-vivo biological performance The establishment of anin-vitroin-vivo correlation and the monitoring of thein-vivo performance of the drug is an essential part ofthe study irrespective of the route and the deliverysystem employed It is of the utmost importance in thecase of intravenously injected nanosuspensions since the


in-vivo behaviour of the drug depends on the organdistribution which in turn depends on its surface prop-erties such as surface hydrophobicity and interactionswith plasma proteins (Blunk et al 1993 1996 Luck et al1997ab) In fact the qualitative and quantitative com-position of the protein absorption pattern observedafter the intravenous injection of nanoparticles is recog-nized as the essential factor for organ distribution(Muller 1989 Blunk et al 1993 1996 Luck et al1997ab) Hence suitable techniques have to be usedin order to evaluate the surface properties and proteininteractions to get an idea of in-vivo behaviourTechniques such as hydrophobic interaction chromato-graphy can be used to determine surface hydrophobicity(Wallis ampMuller 1993) whereas 2-D PAGE (Blunk et al1993) can be employed for the quantitative and quali-tative measurement of protein adsorption after intraven-ous injection of drug nanosuspensions in animals

Applications of nanosuspensions in drug

delivery

Oral drug deliveryThe oral route is the preferred route for drug deliverybecause of its numerous well-known advantages The effi-cacy or performance of the orally administered drug gen-erally depends on its solubility and absorption through thegastrointestinal tract Hence a drug candidate that exhibitspoor aqueous solubility andor dissolution-rate limitedabsorption is believed to possess low andor highly variableoral bioavailability Owing to low oral bioavailability sucha drug candidate would have to be administered in a largerexcess than actually required if it were completely bioavail-able in order to achieve a therapeutically active concentra-tion thus making the therapy costly Orally administeredantibiotics such as atovaquone and bupravaquone reflectthis problem very well Nanosizing of such drugs can lead toa dramatic increase in their oral absorption and subse-quently bioavailability The amelioration in oral bioavail-ability can be attributed to the adhesiveness of the drugnanosuspension increased surface area (due to reductionin particle size by 10ndash50-fold) increased saturation solubil-ity leading to an increased concentration gradient betweenthe gastrointestinal tract lumen and blood and increaseddissolution velocity This enhancement in bioavailabilitywill lead to a subsequent reduction in drug dose renderingthe therapy cost-effective and obliterating any undue drugdumping in the body

Some milestones Atovaquone an antibiotic indicatedfor treating opportunistic Pneumocystis carinii infectionsin HIV patients non-complicated P falciparum malariaand leishmanial infections (Looareesuwan et al 1999)shows poor bioavailability (10ndash15) because of its disso-lution-rate limited absorption and has to be administeredin high doses (750mg twice daily) Administration of ato-vaquone as a nanosuspension resulted in a 25-foldincrease in oral bioavailability as compared to the

commercial product Wellvone which contains the micro-nized drug (Scholer et al 2001)

Danazol a poorly bioavailable gonadotropin inhibitorshowed a drastic improvement in bioavailability whenadministered as a nanosuspension as compared to the com-mercial danazol macrosuspension Danocrine (Liversidge ampCundy 1995) Danazol nanosuspension led to an absolutebioavailability of 823 whereas the marketed danazolsuspension Danocrine was 52 bioavailable In additiondanazol nanosuspension resulted in a reduction in the inter-subject variability and fedfasted ratio of danazol (Figure 4)

Amphotericin B a highly effective polyene antibioticused for systemic mycoses and leishmaniasis lacks oral bio-availability However oral administration of amphotericinB as a nanosuspension produced a substantial improvementin its oral absorption in comparison to orally administeredconventional commercial formulations such as FungizoneAmBisome and micronized amphotericin B (Kayser et al2003) Orally administered amphotericin B nanosuspensionbroughtabout ahighuptakeofnanoparticulatedrug throughthe gastrointestinal tract This is reflected by the considerablereduction it brought about in the number of L donovaniparasites in the liver of infected female Balbc mice as com-pared to other commercial formulations (Figure 5)

Nanosuspensions are also advantageous in achievingquick onset of action for drugs that are completely butslowly absorbed ie those having high tmax values This isillustrated by the study carried out for naproxen a nonster-oidal anti-inflammatory drug A dosage form with fastonset of action would be highly desirable for naproxen Astudy involving a comparison of the pharmacokinetic pro-files of naproxen in the form of nanosuspension suspension(Naprosyn) and tablet (Anaprox) forms revealed that thetime required to achieve Cmax was reduced by approxi-mately 50 for the nanosuspension compared to the sus-pension and tablet (Table 1) Additionally naproxennanosuspension resulted in a 25ndash45-fold increase in the

7

6

5

4

3

2

1

0A B C

Rat

io o

f b

ioav

aila

bili

ty

fed

to

fas

ted

sta

te

Figure 4 Nanocrystalline particles can reduce the absorption varia-

bility resulting from the presence or absence of food The data

compare the performance of various dosage forms of danazol admin-

istered to volunteers in the fed and fasted state at a 200mg dose The

variability observed in the commercial product was significantly

lowered when danazol was formulated using nanoparticles and admin-

istered as a liquid dispersion or a dry filled capsule A frac14 marketed

product B frac14 nanocrystalline capsule C frac14 nanocrystalline disper-

sion Reprinted from Merisko-Liversidge et al 2003 with permission

from Elsevier Publications


AUCs during the first hour of the study (Liversidge ampConzentino 1995 Merisko-Liversidge et al 2003)

Apart from improving oral absorption nanosuspen-sions offer the following advantages

improved dose proportionality

reduced fedfasted state variability (Figure 5)

reduced inter-subject variability

Numerous drug candidates that are poorly water-solubleare required to be taken over a prolonged period of timefor effective medication However many of them cannotbe formulated into sustained-release dosage forms becauseof the risk of dose dumping and poor in-vivo perform-ance Although approaches such as a change in micro-environment and complexation with cyclodextrins haveresulted in the successful incorporation of some poorlywater-soluble drugs in sustained-release dosage forms(Chowdhary et al 2003) these solutions are not applicableto all poorly water-soluble drugs Nanosuspensions onthe other hand enable incorporation of all hydrophobicdrugs in well-established sustained-release technologiesHowever while doing so the effect and the interactionof dosage form excipients with the nanocrystalline drugmust be critically investigated Drug nanosuspensions canalso be incorporated into dosage forms such as tabletscapsules and fast melts by means of standard manufactur-ing techniques Ketoprofen nanosuspension has been suc-cessfully incorporated into pellets to release the drug overa period of 24 h (Remon et al 2001)

In spite of the tremendous potential of nanosuspen-sions in oral delivery formulating compounds as nano-crystalline dispersions is not of value when metabolicandor permeation-related issues affect bioavailabilityHowever in future it should be possible to engineernanosuspensions by using the agents that enhance per-meation (Aungst 2000) andor minimize gut-related meta-bolic issues (Kusuhara et al 1998 Benet et al 1999) Thisamalgamated approach would facilitate delivery of thecompounds belonging to BCS Class IV that exhibit poorwater solubility and poor membrane permeability

Parenteral drug deliveryThe parenteral route is an invasive route Parenteraladministration of drugs is critical and often associatedwith the problems such as the limited number of accepta-ble excipients restrictions on the quantities of excipientsapproved for parenteral use the stringent requirements ofthe aseptic production process safety issues patient non-compliance and biological problems such as allergic reac-tions and thrombophlebitis Despite all these limitationsthe parenteral route still retains its value because of itsspecial advantages such as quick onset of action in case ofemergency reduction in dose of the drug and the ability totarget the drug quickly to the desired site of action espe-cially in the case of severe infections The parenteral routeis often employed as an alternative when the drug is eithernot absorbed through the gastrointestinal tract or under-goes extensive first-pass metabolism

For administration by the parenteral route the drugeither has to be solubilized or have particleglobule sizebelow 5m to avoid the capillary blockade The currentapproaches for parenteral delivery include salt formationsolubilization using co-solvents micellar solutions (Kimet al 2001) complexation with cyclodextrins and recentlyliposomes (Dupont 2002) However there are limitationson the use of these approaches because of limitations ontheir solubilization capacity and parenteral acceptabilityIn this regard liposomes are much more tolerable andversatile in terms of parenteral delivery However theyoften suffer from problems such as physical instabilityhigh manufacturing cost and difficulties in scale-up

Nanosuspensions appear to be a unique approach tosolving the problems mentioned above From the formu-lation perspective nanosuspensions meet almost all therequirements of an ideal drug delivery system for the

Table 1 Human pharmacokinetics of naproxen following oral administration of a nanocrystalline naproxen suspension Naprosyn

suspension and Anaprox tablets in the fed state

Pharmacokinetic parameter Nanocrystalline naproxen Naprosyn Anaprox

tmax (h) 169 105 333 132 32 168

AUC 0ndash1h (mgh1 L1) 3349 912 (273 CV) 1338 81 (611 CV) 741 894 (1207 CV)

Cmax (mgL1) 5089 603 4909 644 5345 716

t12 (h) 1618 442 1616 281 1482 355

Dose 500mgvolunteer nfrac14 23 Reprinted from Merisko-Liversidge et al 2003 with permission from Elsevier Publications

120

100

80

60

40

20

0A B C D E

714

967991985100

Para

site

load

(

)

Figure 5 Percentage reduction of Leishmania donovani parasite

load in livers of infected Balbc mice A frac14 untreated control B frac14Abisome C frac14 fungizone D frac14 amphotericin B (micronized) E frac14amphotericin B (nanosuspension) Reprinted from Kayser et al 2003

with permission from Elsevier Publications


parenteral route Since the drug particles are directlynanosized it becomes easy to process almost all drugsfor parenteral administration Moreover the absence ofany harsh solventsco-solvents andor any potentiallytoxic ingredient in nanosuspensions enables them tobypass the limitations of parenteral administration attrib-uted to conventional formulations strategies Hencenanosuspensions enable significant improvement in theparenterally tolerable dose of the drug leading to a reduc-tion in the cost of the therapy and also improved thera-peutic performance

The maximum tolerable dose of paclitaxel nanosuspen-sion was found to be three times higher than the currentlymarketed Taxol which uses Cremophore EL and ethanolto solubilize the drug In the MV-522 human lung xeno-graft murine tumour model paclitaxel nanosuspensions atdoses of 90 and 100mgkg1 showed no cases of death(nfrac14 9) whereas Taxol at a concentration of 30mgkg1

showed a 22 death rate (Merisko-Liversidge et al 19962003) Similarly the nanosuspensions of other anti-canceragents such as etoposide and camptothecin revealed animprovement in the tolerance level of the drug comparedto the marketed preparations

In addition nanosuspensions have been found toincrease the efficacy of the parenterally administered drug(Merisko-Liversidge et al 2003) A comparison of the effi-cacy of paclitaxel nanosuspension with Taxol using themammary 16-C murine tumour model (nfrac14 5 for eachexperimental group) revealed the superiority of paclitaxelnanosuspension over Taxol in reducing the median tumourburden (Merisko-Liversidge et al 2003) Similarly aphidi-colin a poorly water-soluble new anti-parasitic lead mole-cule when administered as nanosuspension revealed animprovement in EC50 from 200 to 40ngmL1 in compar-ison to DMSO-dissolved drug (Kayser 2000) Recentlyclofazimine a poorly water-soluble anti-leprotic drughas been successfully formulated as a nanosuspensionClofazimine nanosuspension revealed an improvement instability and efficacy over the liposomal clofazimine inM avium-infected female mice (Peters et al 2000)

It is noteworthy that after intravenous administrationof a nanosuspension the drug nanoparticles are seques-tered by mononuclear phagocytic system (MPS) cells andKupffer cells as observed in the case of various othercolloidal drug carriers (Illum et al 1982 Juliano 1988Toster et al 1990 Stolnik et al 1995 Neal et al 1998 Liuet al 2000 Moghimi et al 2001)

The particles are recognized as being foreign bodies andare phagocytosed by the macrophages mainly in the liver(60ndash90) spleen (approximately 1ndash5) and to a very smallextent in the lungs Since this uptake by macrophage is anatural process it is referred to as lsquonatural targetingrsquo in theliterature Natural targeting does not affect the safety pro-file of the drug In fact it helps in increasing the drugtolerance as MPS cells or Kupffer cells can act as a con-trolled-release vehicle for the drug enabling its prolongedaction (Martin et al 1982 Adachi et al 1992 Soma et al2000)

Sequestration by MPS is believed to be the reason forthe increased efficacy of the antibiotics and anti-infectives

in nanosuspension form as they are naturally targeted to themacrophages that are the mainstays of various bacterialand fungal infections Moreover in order to achieve macro-phage targeting in a rapid manner the surface properties ofnanosuspensions could be modulated in a controlled way toalter the plasma protein adsorption pattern A multitude offactors such as the physical properties of the drug particlesthe dose the infusion time the intrinsic solubility of thedrug in the hemodynamic pool of the blood the drugndashplasma protein interaction the plasma protein interactionpattern and the phenomenon of natural targeting influencethe biodistribution and pharmacokinetic profile of thenanoparticulate drug after parenteral administrationNanosuspensions can be administered via different parent-eral routes ranging from intra-articular to intraperitonealto intravenous injection

Currently studies are in progress to identify strategiesfor manipulating the surface properties size and shape ofdrug nanosuspensions in order to eliminate sequestrationby the phagocytic cells of MPS-enriched organs wheneverdesired

Ocular drug deliveryNanosuspensions can prove to be a boon for drugs thatexhibit poor solubility in lachrymal fluids For delivery ofsuch drugs approaches such as suspensions and ointmentshave been recommended Although suspensions offeradvantages such as prolonged residence time in a cul-de-sac (which is desirable for most ocular diseases for effectivetreatment) and avoidance of the high tonicity created bywater-soluble drugs their actual performance depends onthe intrinsic solubility of the drug in lachrymal fluids Thusthe intrinsic dissolution rate of the drug in lachrymal fluidgoverns its release and ocular bioavailability However theintrinsic dissolution rate of the drug will vary because of theconstant inflow and outflow of lachrymal fluids Hencesuspensions may fail to give consistent performanceHowever nanosuspensions by their inherent ability toimprove the saturation solubility of the drug represent anideal approach for ocular delivery of hydrophobic drugsMoreover the nanoparticulate nature of the drug allows itsprolonged residence in the cul-de-sac giving sustainedrelease of the drug To achieve sustained release of thedrug for a stipulated time period nanosuspensions can beincorporated in a suitable hydrogel base or mucoadhesivebase or even in ocular inserts An approach that has recentlybeen investigated to achieve the desired duration of actionof the drug is the formulation of polymeric nanosuspen-sions loaded with the drug The bioerodible as well as water-solublepermeable polymers possessing ocular tolerability(Pignatello et al 2002a) could be used to sustain the releaseof the medication The nanosuspensions can be formulatedusing the quasi-emulsion and solvent diffusion method Thepolymeric nanosuspensions of flurbiprofen and ibuprofenhave been successfully formulated using acrylate polymerssuch as Eudragit RS 100 and Eudragit RL 100 (Bucolo et al2002 Pignatello et al 2002bc) The polymeric nanosuspen-sions have been characterized for drug loading particle sizezeta potential in-vitro drug release ocular tolerability andin-vivo biological performance The designed polymeric


nanosuspensions revealed superior in-vivo performanceover the existing marketed formulations and could sustaindrug release for 24h The scope of this strategy could beextended by using various polymers with ocular tolerability

Pulmonary drug deliveryNanosuspensions may prove to be an ideal approach fordelivering drugs that exhibit poor solubility in pulmonarysecretions Currently such drugs are delivered as suspen-sion aerosols or as dry powders by means of dry powderinhalers The drugs used in suspension aerosols and drypowder inhalers are often jet milled and have particle sizesof microns Because of the microparticulate nature andwide particle size distribution of the drug moiety presentin suspension aerosols and dry powder inhalers the fol-lowing disadvantages are encountered

limited diffusion and dissolution of the drug at the siteof action because of its poor solubility and microparti-culate nature which may affect the bioavailability ofthe drug

rapid clearance of the drug from the lungs because ofciliary movements (Muller amp Jacobs 2002b)

less residence time for the drugs leading to absence ofprolonged effect

unwanted deposition of the drug particles in pharynxand mouth

Nanosuspensions can solve the problems associated withconventional systems because of their versatile nature Thenanoparticulate nature of the drug allows the rapid diffusionand dissolution of the drug at the site of action At the sametime the increased adhesiveness of the drug to mucosalsurfaces (Ponchel et al 1997) offers a prolonged residencetime for the drug at the absorption site This ability of nano-suspensions to offer quick onset of action initially and thencontrolled release of the activemoiety is highly beneficial andis required by most pulmonary diseases Moreover as nano-suspensions generally contain a very low fraction of micro-particulate drug they prevent unwanted deposition ofparticles in the mouth and pharynx leading to decreasedlocal and systemic side-effects of the drug

Additionally because of the nanoparticulate natureand uniform size distribution of nanosuspensions it isvery likely that in each aerosol droplet at least one drugnanoparticle is contained leading to even distribution ofthe drug in the lungs as compared to the microparticulateform of the drug In conventional suspension aerosolsmany droplets are drug free and others are highly loadedwith the drug leading to uneven delivery and distributionof the drug in the lungs Nanosuspensions could be used inall available types of nebulizer However the extent ofinfluence exerted by the nebulizer type as well as thenebulization process on the particle size of nanosuspen-sions should be ascertained

Budesonide a poorly water-soluble corticosteroid hasbeen successfully formulated as a nanosuspension for pul-monary delivery (Muller amp Jacobs 2002b) A good rela-tionship was obtained between increasing the drugconcentration in the formulation and the number ofmicrograms of drug delivered per 2 s actuation

Targeted drug deliveryThe need to target drugs to specific sites is increasing dayby day as a result of therapeutic and economic factorsNanoparticulate systems have shown tremendous poten-tial in targeted drug delivery especially to the brain(Schroeder et al 1998 Kreuter 2001) Successful targetingof the peptide dalargin to the brain by employing surface-modified polyisobutyl cyanoacrylate nanoparticles hasbeen a major achievement in targeted delivery (Kreuteret al 1997)

Likewise nanosuspensions can be used for targeteddelivery as their surface properties and in-vivo behaviourcan easily be altered by changing either the stabilizer orthe milieu Their versatility and ease of scale-up and com-mercial production enables the development of commer-cially viable nanosuspensions for targeted deliveryNatural targeting of MPS by nanosuspensions has alreadybeen described However the natural targeting processcould pose an obstacle when macrophages are not thedesired targets Hence in order to bypass the phagocyticuptake of the drug its surface properties need to bealtered as in the case of stealth liposomes (Gregoriades1995 Allen 1997 Lasic et al 1997 Papisov 1998 Woodle1998 Vyas et al 2000) The engineering of stealth nano-suspensions (analogous to stealth liposomes) by usingvarious surface coatings for active or passive targeting ofthe desired site is the future of targeted drug deliverysystems

Another good example is targeting of Cryptosporidiumparvum the organism responsible for cryptosporidiosisby using surface-modified mucoadhesive nanosuspensionsof bupravaquone (Kayser 2001 Muller amp Jacobs 2002a)A considerable difference has been observed in the efficacyof bupravaquone nanosuspensions when delivered withand without mucoadhesive polymers (Kayser 2001Muller amp Jacobs 2002a) Mucoadhesive bupravaquonenanosuspensions because of their prolonged residence atthe infection site revealed a 10-fold reduction in the infec-tivity score of Cryptosporidium parvum as compared to thebupravaquone nanosuspensions without mucoadhesivepolymers (Table 2) Similarly conditions such as pulmon-ary aspergillosis can easily be targeted by using suitable

Table 2 Anticryptosporidial activity of bupravaquone and its

formulation as a nanosuspension in neonatal mice

Compound formulation Infectivity score

Ileum Caecum

Bupravaquone in DMSO 156 018 046 07

Bupravaquone nanosuspension 117 015 067 012

Bupravaquone mucoadhesive

nanosuspension

017 012 017 014

PBS (control) 20 02 20 02

Reprinted from Kayser 2001 with permission from Elsevier

Publications


drug candidates such as amphotericin B in the form ofpulmonary nanosuspensions instead of using stealth lipo-somes (Kohno et al 1997) With the advent of polymericnanosuspensions loaded with drug (as described in thesection on ocular delivery) it should be possible to targetsites such as the colon or bacteria such as H pylori bysuitable modifications in the formulation strategy Overallnanosuspensions have indicated a good potential in tar-geted drug delivery but this has yet to be fulfilled

Conclusion

Nanosuspensions appear to be a unique and yet commer-cially viable approach to combating problems such aspoor bioavailability that are associated with the deliveryof hydrophobic drugs including those that are poorlysoluble in aqueous as well as organic media Productiontechniques such as media milling and high-pressure homo-genization have been successfully employed for large-scaleproduction of nanosuspensions The advances in produc-tion methodologies using emulsions or microemulsions astemplates have provided still simpler approaches for pro-duction but with limitations Further investigation in thisregard is still essential Attractive features such asincreased dissolution velocity increased saturation solu-bility improved bioadhesivity versatility in surface modi-fication and ease of post-production processing havewidened the applications of nanosuspensions for variousroutes The applications of nanosuspensions in parenteraland oral routes have been very well investigated andapplications in pulmonary and ocular delivery have beenrealized However their applications in buccal nasal andtopical delivery are still awaiting exploration The devel-opment of stealth nanosuspensions laced with functiona-lized surface coatings capable of eliciting passive or activetargeting as per the requirement can be regarded as thefuture step in the nanosuspension research

References

Adachi Y Arii S Funaki N Higashitsuji H Fijita SFurutani M Mise M Zhang W Tobe T (1992)Tumoricidal activity of Kupffer cells augmented by anticancerdrugs Life Sci 51 177ndash183

Akers M J (2002) Excipientndashdrug interactions in parenteralformulations J Pharm Sci 91 2283ndash2300

Allen T M (1997) Liposomes opportunities in drug deliveryDrugs 54 8ndash14

Aungst B J (1993) Novel formulation strategies for improvingoral bioavailability of drugs with poor membrane permeationor presystemic metabolism J Pharm Sci 82 979ndash986

Aungst B (2000) Intestinal permeation enhancers J PharmSci 89 429ndash442

Aungst B J Nguyen N Rogers N J Rowe S Hussain MShum L White S (1994) Improved oral bioavailability of anHIV protease inhibitor using Gelucire 4414 and Labrasolvehicles BT Gattefosse 87 49ndash54

Benet L Z Izumi T Zhang Y Silverman J A Wacher V J(1999) Intestinal MDR transport proteins and P-450 enzymes asbarriers to oral drug delivery J Control Release 62 25ndash31

Blunk T Hochstrasser D F Sanchez J C Muller B W(1993) Colloidal carriers for intravenous drug targetingPlasma protein adsorption patterns on surface-modified latexparticles evaluated by two-dimensional polyacrylamide gelelectrophoresis Electrophoresis 14 1382ndash1387

Blunk T Hochstrasser D F Luck M A Calvor A MullerB W Muller R H (1996) Kinetics of plasma protein adsorp-tion on model particles for controlled drug delivery and drugtargeting Eur J Pharm Biopharm 42 262ndash268

Bodmeier R McGinity J M (1998) Solvent selection in thepreparation of poly (DL-lactide) microspheres prepared bysolvent evaporation method Int J Pharm 43 179ndash186

Breitenbach J (2002) Melt extrusion from process to drugdelivery technology Eur J Pharm Biopharm 54 107ndash117

Buchmann S Fischli W Thiel F P Alex R (1996) Aqueoussuspension an alternative intravenous formulation for animalstudies Eur J Pharm Biopharm 42 S10

Bucolo C Maltese A Puglisi G Pignatello R (2002)Enhanced ocular anti-inflammatory activity of ibuprofen car-ried by an Eudragit RS 1001 nanoparticle suspensionOphthalmic Res 34 319ndash323

Chowdhary K P Reddy G K Rao S (2003) A novelapproach for controlled release of nifedipine through cyclo-dextrin complexation Proceedings of the InternationalSymposium on Innovations in Pharmaceutical Sciences andTechnology Vol 5 Controlled Release Society ndash IndianChapter pp 133 (abstr 55)

Constantinides P P Scarlart J P Smith P L (1994)Formulation and intestinal absorption enhancement evalua-tion of water in oil microemulsions incorporating medium-chain triglycerides Pharm Res 11 1385ndash1390

Constantinides P P Lancaster C Marcello J Chiossone DOrner D Hidalgo I Smith P L Sarkahian A B Yiv S HOwen A J (1995) Enhanced intestinal absorption of a RGDpeptide from wo microemulsion of different composition andparticle size J Control Release 34 109ndash116

Dressman J B Amidon G L Reppas C Shah V P (1998)Dissolution testing as a prognostic tool for oral drug adsorp-tion immediate release dosage forms Pharm Res 15 11ndash22

Dupont B (2002) Overview of the lipid formulations of ampho-tericin B J Antimicrob Chemother S1 31ndash36

Eccleston G M (1992) Microemulsions In Swarbrick SBoylan J C (eds) Encyclopedia of pharmaceutical tech-nology Vol 9 Marcel Dekker New York pp 375ndash421

Floyd A G (1999) Top ten considerations in the developmentof parenteral emulsions Pharm Sci Technol 4 134ndash143

Gasco M R (1997) Solid lipid nanospheres form warm micro-emulsions Pharm Technol Eur 9 32ndash42

Grau M J Kayser O Muller R H (2000) Nanosuspensionsof poorly soluble drugs ndash reproducibility of small-scale pro-duction Int J Pharm 196 155ndash157

Gregoriades G (1995) Engineering liposomes for drug deliveryprogress and problems TIBTECH 13 527ndash537

Horter D Dressman J B (2001) Influence of physicochemicalproperties on dissolution of drugs in the gastrointestinal tractAdv Drug Deliv Rev 46 75ndash87

Illum L Davis S S Wilson C G Thomas N W Frier MHardy J G (1982) Blood clearance and organ disposition ofintravenously administered colloidal particles Effect of parti-cle size nature and shape Int J Pharm 2 135ndash136

Jahnke S (1998) The theory of high-pressure homogenizationIn Muller R H Benita S Bohm B H L (eds) Emulsionsand nanosuspensions for the formulation of poorlysoluble drugs Medpharm Scientific Publishers Stuttgartpp 177ndash200


Juliano R L (1988) Factors affecting the clearance kinetics andtissue distribution of liposomes microspheres and emulsionsAdv Drug Deliv Rev 2 31ndash54

Kawakami K Yoshikawa T (2002) Microemulsion formula-tion for enhanced absorption of poorly soluble drugs IPrescription design J Control Release 81 65ndash74

Kayser O (2000) Nanosuspensions for the formulation of aphi-dicolin to improve drug targeting effects against Leishmaniainfected macrophages Int J Pharm 196 253ndash256

Kayser O (2001) A new approach for targeting to Crypto-sporidium parvum using mucoadhesive nanosuspensionsresearch and applications Int J Pharm 214 83ndash85

Kayser O Olbrich C Yardley V Kiderlen A F Croft S L(2003) Formulation of amphotericin B as nanosuspension fororal administration Int J Pharm 254 73ndash75

Kim C K Gao Gao Z Choia H G Shin H J Park K MLim S J Hwang K J (1998) Physicochemical characteriza-tion and evaluation of a microemulsion system for oral deliv-ery of cyclosporin A Int J Pharm 161 75ndash86

Kim C K Hwang Y Y Chang J Y Choi H G Lim S JLee M K (2001) Development of a novel dosage form forintramuscular injection of titrated extract of Centella asiaticain a mixed micellar system Int J Pharm 220 141ndash147

Kohno S Otsubo T Tanaka E Maruyama K Hara K(1997) Amphotericin B encapsulated in polyethylene glycol-immunoliposomes for infectious diseases Adv Drug Del Rev24 325ndash329

Krause K Muller R H (2001) Production and characterisa-tion of highly concentrated nanosuspensions by high pressurehomogenization Int J Pharm 214 21ndash24

Kreuter J (2001) Nanoparticulate systems for brain delivery ofdrugs Adv Drug Del Rev 47 65ndash81

Kreuter J Petrov V E Kharkevich D A Alyautdin R N(1997) Influence of the type of surfactant on the analgesiceffects induced by the peptide dalargin after 1st delivery acrossthe bloodndashbrain barrier using surfactant coated nanoparticlesJ Control Release 49 81ndash87

Kusuhara H Suzuki H Sugiyama Y (1998) The role ofp-glycoprotein and canalicular multispecific organic aniontransporter in the hepato-biliary excretion of drugs J PharmSci 87 1025ndash1040

Lasic D Ceh B Winterhalter M Frederik P M Vallner J J(1997) Stealth liposomes from theory to product Adv DrugDel Rev 24 165ndash177

Lawrence M J Rees G D (2000) Microemulsion-based mediaas novel drug delivery systems Adv Drug Del Rev 45 89ndash121

Leuner C Dressman J (2000) Improving drug solubility fororal delivery using solid dispersions Eur J Pharm Biopharm50 47ndash60

Lipinski C (2002) Poor aqueous solubility ndash an industry wideproblem in drug discovery Am Pharm Rev 5 82ndash85

Liu Y Bacon E R Ballinger K Black C D V Illig KMcIntire G LWang P P OrsquoNeil N Kinter L Desai V C(2000) Pharmacokinetic and hepatic disposition of Bis [1-ethoxycarbonyl)propyl)5-acetylamino-236-triiodoisophthalatein rats and isolated perfused rat livers Drug Metab Dispos 28731ndash736

Liversidge G G Conzentino P (1995) Drug particle sizereduction for decreasing gastric irritancy and enhancingabsorption of naproxen in rats Int J Pharm 125309ndash313

Liversidge G G Cundy K C (1995) Particle size reduction forimprovement of oral bioavailability of hydrophobic drugsI Absolute oral bioavailability of nanocrystalline danazole inbeagle dogs Int J Pharm 127 91ndash97

Liversidge G G Cundy K C Bishop J F Czekai D A(1992) Surface modified drug nanoparticles US Patent5145684

Loftsson T Brewster M E (1996) Pharmaceutical applicationsof cyclodextrins J Pharm Sci 85 1017ndash1025

Looareesuwan S Chulay J D Canfield C J HutchinsonD B (1999) Atovaquone and proguanil hydrochloridefollowed by primaquine for treatment of Plasmodium vivaxmalaria in Thailand Trans R Soc Trop Med Hyg 93637ndash640

Luck M Schroder W Harnisch S Thode K Blunk TPaulke B-R Kresse M Muller R H (1997a)Identification of plasma proteins facilitated by enrichment onparticulate surfaces Analysis by two-dimensional electrophor-esis and N-terminal microsequencing Electrophoresis 182961ndash2967

Luck M Paulke B R Schroder W Blunk T Muller R H(1997b) Analysis of plasma protein adsorption on polymericnanoparticles with different surface characteristics J BiomedMater Res 1 478ndash485

Martin F Caignard A Olsson O Jeannin J F Leclerc A(1982) Tumoricidal effect of macrophages exposed to adria-mycin in vivo or in vitro Cancer Res 42 3851ndash3857

Merisko-Liversidge E Sarpotdar P Bruno J Hajj S Wei LPeltier N Rake J Shaw J M Pugh L Polin L Jones JCorbett T Cooper E Liversidge G G (1996) Formulationand anti-tumor activity evaluation of nanocrystalline suspen-sions of poorly soluble anti-cancer drugs Pharm Res 13272ndash278

Merisko-Liversidge E Liversidge G G Cooper E R (2003)Nanosizing a formulation approach for poorly-water-solublecompounds Eur J Pharm Sci 18 113ndash120

Moghimi S M Hunter A C Murray J C (2001) Long-circulating and target-specific nanoparticles theory to prac-tice Pharmacol Rev 53 283ndash318

Mosharraf M Nystrom C (1995) The effect of particle size andshape on the surface specific dissolution rate of micronizedpractically insoluble drugs Int J Pharm 122 35ndash47

Muller B W Muller R H (1984) Particle size analysis of latexsuspensions and microemulsions by photon correlation spec-troscopy J Pharm Sci 73 915ndash918

Muller R H (1989) Differential opsonization A new approachfor the targeting of colloidal drug carriers Arch Pharm 322700

Muller R H Bohm B H L (1998) Nanosuspensions InMuller R H Benita S Bohm B H L (eds) Emulsionsand nanosuspensions for the formulation of poorly solubledrugs Medpharm Scientific Publishers Stuttgart pp 149ndash174

Muller R H Grau M J (1998) Increase of dissolution velocityand solubility of poorly water soluble drugs as nanosuspen-sion Proceedings World Meeting APGIAPV Paris Vol 2pp 623ndash624

Muller R H Peters K (1998) Nanosuspensions for the for-mulation of poorly soluble drugs I Preparation by a size-reduction technique Int J Pharm 160 229ndash237

Muller R H Jacobs C (2002a) Buparvaquone mucoadhesivenanosuspension preparation optimisation and long-term sta-bility Int J Pharm 237 151ndash161

Muller R H Jacobs C (2002b) Production and characteriza-tion of a budesonide nanosuspension for pulmonary adminis-tration Pharm Res 19 189ndash194

Muller R H Becker R Kruss B Peters K (1998)Pharmaceutical nanosuspensions for medicament administra-tion as system of increased saturation solubility and rate ofsolution US Patent No 5858410


Nakano M (2000) Places of emulsions in drug delivery AdvDrug Del Rev 45 1ndash4

Neal J C Stolnik S Garnett M C Davis S S Illum L(1998) Modification of copolymers poloxamer 407 and polox-amine 908 can affect the physical and biological properties ofsurface modified nanospheres Pharm Res 15 318ndash324

Nystrom C Bisrat M (1988) Physiochemical aspects of drugrelease VIII The relation between particle size and surfacespecific dissolution rate in agitated suspensions Int J Pharm47 223ndash231

Papisov M (1998) Theoretical considerations of RES-avoidingliposomes molecular mechanics and chemistry of liposomeinteractions Adv Drug Del Rev 32 119ndash138

Park K Kim C K (1999) Preparation and evaluation offlurbiprofen-loaded microemulsion for parenteral deliveryInt J Pharm 181 173ndash179

Peters K Muller R H (1996) Nanosuspensions for the oralapplication of poorly soluble drugs In Proceedings EuropeanSymposium on Formulation of Poorly-available Drugs for OralAdministration APGI Paris pp 330ndash333

Peters K Muller R H Borner K Hahn H Leitz K SDiederichs J E Ehlers S (2000) Preparation of a clofaziminenanosuspension for intravenous use and evaluation of its ther-apeutic efficacy in murine Mycobacterium avium infectionJ Antimicrob Chemother 45 77ndash83

Pignatello R Bucolo C Spedalieri G Maltese A Puglisi G(2002a) Flurbiprofen-loaded acrylate polymer nanosuspen-sions for ophthalmic application Biomaterials 23 3247ndash3255

Pignatello R Bucolo C Ferrara P Maltese A Puleo APuglisi G (2002b) Eudragit RS100 nanosuspensions for theophthalmic controlled delivery of ibuprofen Eur J PharmSci 16 53ndash61

Pignatello R Bucolo C Puglisi G (2002c) Ocular tolerabilityof Eudragit RS 100 and RL 100 nanosuspensions as carrier forophthalmic controlled delivery J Pharm Sci 91 2636ndash2641

Ponchel M Montisci J Dembri A Durrer C Duchene D(1997) Mucoadhesion of colloidal particulate systems in thegastrointestinal tract Eur J Pharm Biopharm 4 25ndash31

Pouton C W (2000) Lipid formulations for oral administrationof drugs non-emulsifying self-emulsifying and lsquoself-micro-emulsifyingrsquo drug delivery systems Eur J Pharm Sci 11S93ndashS98

Rades T Davies N Watnasirichaikul S Tucker I (2002)Effects of formulation variables on characteristics of poly(ethylcyanoacrylates) nanocapsules prepared from wo micro-emulsions Int J Pharm 235 237ndash246

Rawlins E A (1982) Solutions In Rawlins E A (ed)Bentleyrsquos textbook of pharmaceutics 8th edn BailliereTindall London p 6

Remon J P Vergote G J Vervaet C Driessche I Hoste SSmedt S Demeester J Jain R A Ruddy S (2001) An oralcontrolled release matrix pellet formulation containing nano-crystalline ketoprofen Int J Pharm 219 81ndash87

Sah H (1997) Microencapsulation technique using ethyl acetateas a dispersed solvent effects on its extraction rate on the

characteristics of PLGA microspheres J Control Release47 233ndash245

Sah H (2000) Ethyl formate ndash alternative dispersed solvent usefulin preparing PLGA microspheres Int J Pharm 195 103ndash113

Scholer N Krause K Kayser O Muller R H Borner KHahn H Liesenfeld O (2001) Atovaquone nanosuspensionsshow excellent therapeutic effect in a new murine model ofreactivated toxoplasmosis Antimicrob Agents Chemother 451771ndash1779

Schroeder U Sommerfeld P Ulrich S Sabel B (1998)Nanoparticulate technology for delivery of drugs across thebloodndashbrain barrier J Pharm Sci 87 1305ndash1307

Schwendener R A Schott H (1996) Lipophilic 1-beta-D-arabi-nofuranosyl cytosine derivatives in liposomal formulations fororal and parenteral antileukemic therapy in the murine L1210leukemia model J Cancer Res Clin Oncol 122 723ndash726

Serajuddin A T M (1999) Solid dispersion of poorly water-soluble drugs early promises subsequent problems and recentbreakthroughs J Pharm Sci 88 1058ndash1066

Shanthakumar T R Prakash S Basavraj R M Ramesh MKant R Venkatesh P Rao K Singh S Srinivas N R(2004) Comparative pharmacokinetic data of DRF-4367 usingnanosuspension and HP--CD formulation Proceedings of theInternational Symposium on Advances in Technology andBusiness Potential of New Drug Delivery Systems MumbaiVol 5 B V Patel Educational Trust and B V Patel PERDCentre p 75 (abstr 55)

Soma C E Dubernet C Barratt G Benita S Couvreur P(2000) Investigation of the role of macrophages on the cyto-toxicity of doxorubicin and doxorubicin-loaded nanoparticleson M5076 cells in vitro J Control Release 68 283ndash289

Stella V J Rajewski R A (1997) Cyclodextrins their future indrug formulation and delivery Pharm Res 14 556ndash567

Stolnik S Illum L Davis S S (1995) Long circulating micro-particulate drug carriers Adv Drug Del Rev 16 195ndash214

Toster S D Muller R Kreuter J (1990) Modification of thebody distribution of poly (methylmethacrylate) nanoparticlesin rats by coating with surfactants Int J Pharm 61 85ndash100

Trotta M Gallarate M Pattarino F Morel S (2001)Emulsions containing partially water miscible solvents forthe preparation of drug nanosuspensions J Control Release76 119ndash128

Trotta M Gallarate M Carlotti M E Morel S (2003)Preparation of griseofulvin nanoparticles from water-dilutablemicroemulsions Int J Pharm 254 235ndash242

Vyas S P Katare Y K Mishra V Sihorkar V (2000)Ligand directed macrophage targeting of amphotericin Bloaded liposomes Int J Pharm 210 1ndash14

Wallis K H Muller R H (1993) Determination of the surfacehydrophobicity of colloidal dispersions by mini-hydrophobicinteraction chromatography Pharm Ind 55 1124ndash1128

Washington C (1996) Stability of lipid emulsions for drugdelivery Adv Drug Del Rev 20 131ndash145

Woodle M C (1998) Controlling liposome blood clearance bysurface-grafted polymers Adv Drug Del Rev 32 139ndash152


using high-shear media mills or pearl mills The media millconsists of a milling chamber a milling shaft and a recir-culation chamber (Figure 1) The milling chamber ischarged with the milling media water drug and stabilizeras depicted in Figure 1 and the milling media or pearls arethen rotated at a very high shear rate The milling processis performed under controlled temperatures

Principle The high energy and shear forces generated asa result of the impaction of the milling media with thedrug provide the energy input to break the microparticu-late drug into nano-sized particles The milling medium iscomposed of glass zirconium oxide or highly cross-linkedpolystyrene resin The process can be performed in eitherbatch or recirculation mode In batch mode the timerequired to obtain dispersions with unimodal distributionprofiles and mean diameterslt200 nm is 30ndash60min Themedia milling process can successfully process micronizedand non-micronized drug crystals Once the formulationand the process are optimized very little batch-to-batchvariation is observed in the quality of the dispersion


Ease of scale-up and little batch-to-batch variation

Narrow size distribution of the final nano-sized pro-duct A comparison of the size of naproxen crystalsbefore and after media milling is given in Figure 2

Flexibility in handling the drug quantity ranging from1 to 400mgmL1 enabling formulation of very diluteas well as highly concentrated nanosuspensions

Disadvantages The major concern is the generation of residues ofmilling media which may be introduced in the finalproduct as a result of erosion (Buchmann et al 1996Muller amp Bohm 1998) This could be problematicwhen nanosuspensions are intended to be administered

CoolantLargedrugcrystals

Chargedwithdrug waterandstabilizer

Re-circubtionchamber

Milling chamber

Screenretainingmilling media in chamber

Millingshaft

MotorMilling media

Nanocrystals

Figure 1 Schematic representation of the media milling process The milling chamber charged with polymeric media is the active component

of the mill The mill can be operated in a batch or recirculation mode A crude slurry consisting of drug water and stabilizer is fed into the

milling chamber and processed into a nanocrystalline dispersion The typical residence time generated for a nanometer-sized dispersion with a

mean diameter of lt200 nm is 30ndash60min Reprinted from Merisko-Liversidge et al 2003 with permission from Elsevier Publications

20

15

10

5

0 1 100

Freq

uen

cy

Nanocrystallinenaproxen aftermedia millingMean 0147 micro mD90 0205 micro m

Naproxen crystalsbefore media millingMean 242 micro mD90 4708 micro m

Particle size (micro m)

Figure 2 The particle size distribution of naproxen crystals before

(~) and after () milling Before milling the drug crystals had a mean

particle size of 242m After being processed for 30min in a media

mill the mean particle size of the nanocrystalline dispersion was

0147m with D90frac14 0205m The particle size measurements were

generated using laser light diffraction in a Horiba LA-910 using

polystyrene nanospheres ranging from 01 to 10m as standards

Reprinted from Merisko-Liversidge et al 2003 with permission from

Elsevier Publications









Nanosuspension

Valve

Valve seat

Macrosuspension

Imp

act

rin

g


zation process








































hHfrac14 k (L12V12)


























delivery







7

6

5

4

3

2

1

0A B C

Rat

io o

f b

ioav

aila

bili

ty

fed

to

fas

ted

sta

te

























tmax (h) 169 105 333 132 32 168


Cmax (mgL1) 5089 603 4909 644 5345 716

t12 (h) 1618 442 1616 281 1482 355


120

100

80

60

40

20

0A B C D E

714

967991985100

Para

site

load

(

)































Ileum Caecum




nanosuspension

017 012 017 014



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Conclusion


References











































































































Nanosuspension

Valve

Valve seat

Macrosuspension

Imp

act

rin

g


zation process








































hHfrac14 k (L12V12)


























delivery







7

6

5

4

3

2

1

0A B C

Rat

io o

f b

ioav

aila

bili

ty

fed

to

fas

ted

sta

te

























tmax (h) 169 105 333 132 32 168


Cmax (mgL1) 5089 603 4909 644 5345 716

t12 (h) 1618 442 1616 281 1482 355


120

100

80

60

40

20

0A B C D E

714

967991985100

Para

site

load

(

)































Ileum Caecum




nanosuspension

017 012 017 014



Publications



Conclusion


References










































































































































hHfrac14 k (L12V12)


























delivery







7

6

5

4

3

2

1

0A B C

Rat

io o

f b

ioav

aila

bili

ty

fed

to

fas

ted

sta

te

























tmax (h) 169 105 333 132 32 168


Cmax (mgL1) 5089 603 4909 644 5345 716

t12 (h) 1618 442 1616 281 1482 355


120

100

80

60

40

20

0A B C D E

714

967991985100

Para

site

load

(

)































Ileum Caecum




nanosuspension

017 012 017 014



Publications



Conclusion


References

















































































































delivery







7

6

5

4

3

2

1

0A B C

Rat

io o

f b

ioav

aila

bili

ty

fed

to

fas

ted

sta

te

























tmax (h) 169 105 333 132 32 168


Cmax (mgL1) 5089 603 4909 644 5345 716

t12 (h) 1618 442 1616 281 1482 355


120

100

80

60

40

20

0A B C D E

714

967991985100

Para

site

load

(

)































Ileum Caecum




nanosuspension

017 012 017 014



Publications



Conclusion


References

















































































































tmax (h) 169 105 333 132 32 168


Cmax (mgL1) 5089 603 4909 644 5345 716

t12 (h) 1618 442 1616 281 1482 355


120

100

80

60

40

20

0A B C D E

714

967991985100

Para

site

load

(

)































Ileum Caecum




nanosuspension

017 012 017 014



Publications



Conclusion


References






























































































































Ileum Caecum




nanosuspension

017 012 017 014



Publications



Conclusion


References





































































































Conclusion


References




































































































review article nanosuspensions: a promising drug delivery...

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