This article was downloaded by: [University of California, San Francisco]On: 20 August 2014, At: 08:07Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK

International Journal of Green Nanotechnology: Physicsand ChemistryPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/ugnp20

Preparation of Poly(Lactic Acid) Nanoparticles andOptimization of the Particle SizeTungabidya Maharana a , Bikash Mohanty b & Yuvraj Singh Negi aa Polymer Science and Technology Program, Department of Paper Technology , IndianInstitute of Technology , Roorkee, Saharanpur Campus, Saharanpur, Indiab Department of Chemical Engineering , Indian Institute of Technology , Roorkee, Roorkee,IndiaPublished online: 13 Dec 2010.

To cite this article: Tungabidya Maharana , Bikash Mohanty & Yuvraj Singh Negi (2010) Preparation of Poly(Lactic Acid)Nanoparticles and Optimization of the Particle Size, International Journal of Green Nanotechnology: Physics and Chemistry,2:2, P100-P109, DOI: 10.1080/19430876.2010.532462

To link to this article: http://dx.doi.org/10.1080/19430876.2010.532462

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of theContent. Any opinions and views expressed in this publication are the opinions and views of the authors, andare not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon andshould be independently verified with primary sources of information. Taylor and Francis shall not be liable forany losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoeveror howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use ofthe Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

International Journal of Green Nanotechnology: Physics and Chemistry, 2:P100–P109, 2010Copyright c© Taylor & Francis Group, LLCISSN: 1943-0876 print / 1943-0884 onlineDOI: 10.1080/19430876.2010.532462

Preparation of Poly(Lactic Acid) Nanoparticles andOptimization of the Particle Size

Tungabidya MaharanaBikash Mohanty

Yuvraj Singh Negi

ABSTRACT. This article aims to illustrate an application of the Taguchi method of experimental design(TMED) for the preparation of poly(lactic acid) (PLA) nanoparticles via a nanoprecipitation technique.The effect of four pertinent parameters, such as concentration of PLA, solvent-to–non-solvent (S/NS)volume ratio, molecular weight of PLA, and type of solvent, were studied on the yield and size ofnanoparticles. The first two factors were varied at four levels, whereas the last two factors were variedat two levels and sixteen experiments were carried out. The application of TMED for optimization ofthe essential parameters for nanoprecipitation of PLA nanoparticles has been reported. The optimumcondition based on the reduction of size was found to be at polymer concentration of 10 mg/mL, 0.2 S/NSvolume ratio, low-molecular-weight PLA (PLAL), and dimethyl sulfoxide (DMSO) as solvent. Thesize of PLA nanoparticles obtained was around 100 nm. This case study demonstrates the true powerof a well-planned and designed experiment over the traditional varying one-factor-at-a-time approachto experimentation.

KEYWORDS. polylactic acid, nanoprecipitation, Taguchi method, transmission electron microscopy,zeta potential

INTRODUCTION

Polymer nanoparticles (NPs) have beenproduced for decades for use in a vari-ety of high-performance materials such ashigh-impact-resistant polymers and specialtycoatings long before it was fashionable to

Received 26 September 2010; accepted 9 October 2010.T. Maharana is thankful to the All India Council of Technical Education India for providing a National

Doctoral Fellowship.Tungabidya Maharana and Yuvraj Singh Negi are affiliated with the Polymer Science and Technology

Program, Department of Paper Technology, Indian Institute of Technology, Roorkee, Saharanpur Campus,Saharanpur, India.

Bikash Mohanty is affiliated with the Department of Chemical Engineering, Indian Institute of Technology,Roorkee, Roorkee, India.

Address correspondence to Tungabidya Maharana, Polymer Science and Technology Program, Departmentof Paper Technology, Indian Institute of Technology, Roorkee, 247667, Uttarakhand, Saharanpur Campus,Saharanpur 247001, Uttar Pradesh, India. E-mail: mtungabidya@gmail.com

use the “nano-” label. The extraordinarilylarge surface area on the NPs presents diverseopportunities to place functional groups on thesurface. They can expand/contract with changesin pH or interact with antibodies in special waysto provide rapid ex vivo medical diagnostictests.[1] Important extensions have also been

P100

Dow

nloa

ded

by [

Uni

vers

ity o

f C

alif

orni

a, S

an F

ranc

isco

] at

08:

07 2

0 A

ugus

t 201

4

T. Maharana et al. P101

made in combining inorganic materials withpolymers and in combining different classes ofpolymers together in NP form.[2,3]

Biodegradable polymer NPs play a major rolein the controlled release of medicaments in drugdelivery systems. In particular, the use of NP de-vices has received special attention during thepast two decades. The potential of polymer-based NPs as drug delivery systems has beenextensively investigated in recent years.[4–7]

Poly(lactic acid) (PLA) is the most preferredmacromolecule in several biomedical applica-tions because it is one of the most well-knownbioabsorbable polymers. It is also non-toxic andhas good biodegradability.[1]

Numerous methods for the manufacture ofPLA NPs have been investigated.[5,6] Poly-meric nanoparticles are predominantly preparedby wet synthetic routes. Nanoprecipitation isthe most preferred method among the differ-ent methods, such as emulsification, nanopre-cipitation, salting-out, spray drying, and poly-merization, available for preparation of PLAnanoparticles. It is simple, fast, and economic,employs non-toxic solvents, and also has theadvantage of using preformed polymers asstarting materials rather than monomers.[7,8] Itcan also be applied to materials other thansynthetic polymers, including amphiphilic cy-clodextrins, proteins,[9] lipids,[10] and drugs.[5]

For the above reasons, this method is widelyused to prepare NPs for the delivery of activecompounds.

Nanoprecipitation has been extended usingquite complex systems containing three basicingredients (polymer, solvent, and non-solvent)plus a surfactant and, sometimes, even a bi-nary mixture of solvents of the polymer or adrug to be encapsulated.[11–13] The choice ofthe ternary polymer/solvent/non-solvent systemis critical for the success of the method. Thenature of the polymer solvent interactions hasbeen reported to affect the properties of the NPpreparation.[12,14,15] However, apart from the re-quirements that the polymer solvent should bemiscible with the non-solvent of the polymerand that the polymer concentration should below (<2%), no clear guidelines about the in-fluence of each of the three components of thesystem have yet emerged.

Design of experiment (DOE) is a structured,organized method that is used to determine therelationship between the different factors (Xs)affecting a process and the output (Ys) of thatprocess. DOE involves designing a set of ex-periments in which all relevant factors are var-ied systematically.[16] When the results of theseexperiments are analyzed, they help to identifyoptimal conditions, the most influential factorsand those with little influence, as well as theexistence of interactions and synergies betweenfactors.

Therefore, in a nutshell, the present work isdevoted to the preparation and characterizationof PLA NPs to investigate the effect of vari-ous input parameters such as PLA concentration,solvent-to–non-solvent volume ratio, molecularweight of PLA, and solvent on the output pa-rameters such as yield and size of NPs to controlthese in required levels to get an optimum prod-uct. The Taguchi method of DOE was used forthe design and analysis of experiments for theproduction of NPs.

EXPERIMENTAL

Design of Experiment for PLA NPPreparation

For PLA NP preparation by the nanoprecipi-tation method, eight parameters play an impor-tant role. These are the nature of the solvent, thenature of the non-solvent, solvent–non-solventvolume ratio, polymer molecular weight, poly-mer concentration, surfactant type, surfactantvolume, and stirring speed.[1,4–8,17] In addition,time of sonication may play an important role. Itappears that this effect has not been studied byother investigators. However, it was seen practi-cally that sonication for a time period less than20 min does not increase the temperature of thesolution appreciably, whereas longer time peri-ods heat up the solution. Thus, it is not consid-ered as an input variable and was fixed at 20 min.

Among the non-solvents methanol, ethanol,propanol, isopropanol, butanol, and water,methanol was found to be the best. Further,the size of NPs was found to increase in thesequence methanol < ethanol < propanol when

Dow

nloa

ded

by [

Uni

vers

ity o

f C

alif

orni

a, S

an F

ranc

isco

] at

08:

07 2

0 A

ugus

t 201

4

P102 INTERNATIONAL JOURNAL OF GREEN NANOTECHNOLOGY: PHYSICS AND CHEMISTRY

FIGURE 1. Hansen’s two-dimensional graph ofpartial solubility parameters of solvents w.r.t.those of PLA.

10 15 20 25 300

5

10

15

20

25Methanol

Acetone

DMSO

PLA

δ h (J

/cc)

1/2

δv (J/cc)1/2

these were used as non-solvents.[17] Thus,methanol was selected as the non-solvent.Bilati et al.[17] have reported that surfactantswere usually unnecessary for final suspensionstabilization. A review of the cited and relatedliterature shows that contributions of surfactantsare not appreciable[17] and thus surfactants werenot used in the present case.

The selected solvent and non-solvent shouldbe non-toxic. Among the solvents such asacetone, chloroform, methyl cyanide (MeCN),dimethyl sulfoxide (DMSO), tetrahydrofu-ran (THF), methyl ethyl ketone (MEK),methylisobutyl ketone (MIBK), methyl propylketone, and isopropyl acetate, DMSO and ace-tone were found to produce smaller NPs whenmethanol was taken as the non-solvent. Thesewere found to produce NPs of almost similarsize.[17] The suitability of the above two sol-vents for the nanoprecipitation of PLA was basedon Hansen’s two-dimensional graph, comparingthe partial solubility parameters of the solventsw.r.t. the partial solubility parameters (δh and δv)of PLA,[5] which is shown in Figure 1. FromHansen’s graph, it is clear that PLA is soluble inacetone and DMSO and insoluble in methanol.A too high polymer concentration in the sol-vent, however, prevents NP formation.[17] Poly-mer concentration should be optimized to ob-

TABLE 1. List of all design parameters alongwith their settings for the experiment

Parameter Range Levels

A: Polymerconcentration

5–20 (mg/mL) 4 (5, 10, 15, 20)

B: S/NS volumeratio

0.05–0.6 4 (0.05, 0.2, 0.35, 0.6)

C: Polymermolecular weight(MW)

PLAL, PLAH 2 (PLAH, PLAL)

D: Solvent (S) Acetone, DMSO 2 (Acetone, DMSO)

tain optimal results. The effect of molar massof PLA has been studied by Legrand et al.,[5]

and they have found that lower molar mass pro-duced smaller NPs and that it is an influentialparameter.

Thus, because the requirement is to producelower dimension NPs with high yield, in thepresent investigation, the four parameters cho-sen for nanoprecipitation are as listed in Table1. Because their effect has not been quantifiedso far, an attempt was made to quantify these ef-fects on the output response like yield (wt%) andsize of NPs using DOE. Two different PLA com-pounds, one having low Mw (PLAL, ca. 98 kDa)and the other having high Mw (PLAH, ca. 178kDa), were selected to study the effect of molec-ular weight of PLA on the size of NP produced.

After deciding the factors and their levels, aTaguchi orthogonal array design (TOAD) [L16(4**2 2**2)] was employed because it wasfound to be the best suited design. The generalfactorial design for the above-described combi-nations of factors and their levels produced 64runs. However, when TOAD was adopted usingMINITAB software (version 13.0, Minitab Inc.,PA, USA), it suggested only 16 runs. The detailsof the values of factors for different experimen-tal runs along with the output parameters (yield[wt%] and NP size) are given in Table 2.

Experimental Procedure for PLANanoparticle Preparation

Materials

The PLA homopolymers synthesized in ourlaboratory and having Mw of 98.470 kDa

Dow

nloa

ded

by [

Uni

vers

ity o

f C

alif

orni

a, S

an F

ranc

isco

] at

08:

07 2

0 A

ugus

t 201

4

T. Maharana et al. P103

TABLE 2. Size and yield of NPs obtained for the 16 experimental runs

Experimental conditions

Yield NP size SNR for SNR for NPRun no. A B C Da (wt%) (nm) PDI yield (db) size (db)

1 5 0.05 PLAL Ac 86.3 134 0.035 38.7222 −42.54212 5 0.20 PLAL Ac 89.9 111 0.072 39.0742 −40.90653 5 0.35 PLAH Di 52.4 220 0.217 34.3916 −46.84854 5 0.60 PLAH Di 46.6 228 0.098 33.3752 −47.15875 10 0.05 PLAL Di 75.9 113 0.128 37.6071 −41.06166 10 0.20 PLAL Di 78.5 109 0.261 37.8919 −40.74857 10 0.35 PLAH Ac 66.7 125 0.228 36.4786 −41.93828 10 0.60 PLAH Ac 52.9 342 0.342 34.4642 −50.68059 15 0.05 PLAH Ac 34.6 221 0.118 30.7941 −46.8878

10 15 0.20 PLAH Ac 39.5 160 0.125 31.9407 −44.082411 15 0.35 PLAL Di 37.8 140 0.109 31.5452 −42.922612 15 0.60 PLAL Di 33.7 270 0.097 30.5655 −48.627313 20 0.05 PLAH Di 27.6 221 0.086 28.8150 −46.887814 20 0.20 PLAH Di 33.6 353 0.13 30.5345 −50.955515 20 0.35 PLAL Ac 28.6 302 0.172 29.1152 −49.600116 20 0.60 PLAL Ac 26.0 335 0.185 28.2928 −50.5009

aAc = acetone; Di = DMSO.

(PLAL) and 178.857 kDa (PLAH) were used forthis purpose. These two PLA samples were syn-thesized via a melt polycondensation method.The polydispersity index (PDI) of PLAL andPLAH are 2.079 and 1.976, respectively. High-performance liquid chromatography (HPLC)-grade methanol was used as non-solvent and wasobtained from Merck (Mumbai, India). HPLC-grade acetone and extra-pure DMSO were alsopurchased from Merck. The purity of all thesesolvents was higher than 99%. Doubly distilleddeionized (DI; 18 M�cm2/cm) water (Milli-Qsystem, Waters Corp., Milford, MA) was usedthroughout the study.

Methods

PLA NPs were prepared by a nanoprecipita-tion technique. The PLA samples were dissolvedin acetone or DMSO to make solutions of con-centrations varying from 5 to 20 mg/mL to formthe diffusing phase. This phase was then addedto the dispersing phase, methanol, by means of a

syringe positioned with the needle directly intomethanol under sonication. The diffusing phaseis not added dropwise but with the needle of thesyringe kept directly in the non-solvent, in or-der to avoid an additional superfluous air–liquidinterface.[8] The freshly formed NPs with sol-vent and non-solvent were then centrifuged for15 min at 15,000 ×g and 4◦C using a HeraeusBiofuge Stratos centrifuge (Thermo Scientific,NC, USA). It was then mixed with 2 mL ofdeionized Millipore water and again centrifugedfor 15 min. This step was repeated twice. Thesamples prepared were subsequently vacuumdried for a period of 24 h to obtain a fine powderand then kept at 4◦C until further use. The yield(wt%) was calculated according to Equation 1.

Yield (wt. %)= wt. of PLA NP obtained

wt. of PLA taken initially×100

(1)The NPs were characterized by different

methods such as transmission electron mi-croscopy (TEM; Tecnai 20 G2 S-TWIN, FEI,

Dow

nloa

ded

by [

Uni

vers

ity o

f C

alif

orni

a, S

an F

ranc

isco

] at

08:

07 2

0 A

ugus

t 201

4

P104 INTERNATIONAL JOURNAL OF GREEN NANOTECHNOLOGY: PHYSICS AND CHEMISTRY

Eindhoren, The Netherlands) and dynamic lightscattering (DLS; Zetasizer 5000, Malvern In-struments Ltd., Worcestershire, UK) for deter-mination of their size, size distribution, and zetapotential.

RESULTS AND DISCUSSION

Taguchi Method of Experimental Designand Its Analysis

The experimental conditions obtained by ap-plying Taguchi method of experimental design(TMED), the orthogonal array design, are givenin Table 2 along with the resulting yield (wt%)and size of NPs. Statistical analysis was carriedout by Minitab (v. 13) software. Because theyield is to be maximized, the larger the bettersignal-to-noise (SNR) ratio is the best approachfor yield and is calculated using Equation 2. Sim-ilarly, because the size of NPs is to be minimized,the smaller the better SNR is the best approachand is calculated using Equation 3 (see Table 2).

S/N = −10[log

(∑(1/Y 2) / n

)](2)

S/N = −10[log

(∑Y 2/n

)](3)

where S/N is the signal-to-noise ratio; Y is (Yi –mean) of the ith response; Yi is the ith response,and n is the number of repetitions in measuringYi .[18]

Analysis of variance (ANOVA) was applied toTable 2 to investigate which factors significantlyaffect the quality characteristics, yield (wt%),and size of NPs. The ANOVAs for yield (wt%)and size of NPs are represented in Tables 3 and4, respectively.

Based on the ANOVA for yield (Table 3),PLA concentration, S/NS volume ratio, and PLAMw were found to be the significant parameters,whereas solvent was found to be insignificant.Again, based on the ANOVA for size of PLANPs (Table 4), PLA concentration was foundto be the most significant parameter followedby S/NS volume ratio and PLA Mw, and sol-vent was found to be the insignificant parameter.However, it is also seen from the main effect

TABLE 3. ANOVA for yield of PLA NPs

Factors DF SS MS F ratio p Value

PLA concentration 3 5286.00 1762.00 70.36 0.000S/NS volume ratio 3 1041.81 347.27 13.87 0.002PLA Mw 1 658.31 658.31 26.29 0.001Solvent 1 91.63 91.63 3.66 0.097Error 7 175.30 25.04Total 15 7253.05

DF = degrees of freedom; SS = sum of squares; MS = meansquares; F = variance ratio; p = probability.

plots (Figures 2 and 3) that type of solvent hasalmost no effect on either yield (wt%) or size ofPLA NPs.

Effect of Various Parameters

The effects of various parameters on yield(wt%) and size of NPs are shown in Figures2 and 3, respectively. Figures 2A and 3A elu-cidate the effect of concentration of PLA onthe yield (wt%) and size of PLA NPs, respec-tively. It can be seen that with the increasein PLA concentration, yield (wt%) decreases,whereas the size of NPs increases because ofthe formation of aggregates, which is due to in-creased intrinsic viscosity with the increase inPLA concentration.[5,14,17,19,20]

Figures 2B and 3B show the effect of S/NSvolume ratio on the yield (wt%) and size of PLANPs, respectively. It can be seen that with theincrease in S/NS volume ratio, yield (wt%) firstincreases and then decreases, whereas the sizeof NPs increases, which is attributed to the in-crease in viscosity with the increase in S/NSvolume ratio.[17] High viscosity hampers the dif-fusion of the solvent toward the non-solvent andaggregates are found to be formed for highly

TABLE 4. ANOVA for size of PLA NPs

Factors DF SS MS F ratio p Value

PLA concentration 3 46,077 15,359 4.41 0.048S/NS volume ratio 3 37,285 12,428 3.57 0.075PLA Mw 1 7921 7921 2.28 0.175Solvent 1 361 361 0.10 0.757Error 7 24,360 3480Total 15 116,004

Dow

nloa

ded

by [

Uni

vers

ity o

f C

alif

orni

a, S

an F

ranc

isco

] at

08:

07 2

0 A

ugus

t 201

4

T. Maharana et al. P105

FIGURE 2. Main effect plots for yield (wt%) of PLA nanoparticles.

PLA conc. (mg/ml) S/NS volume ratio PLA Mw Solvent

5 10 15 20 0.05

0.20

0.35

0.60

PLAL

PLAH

Aceto

ne

DMSO

30

40

50

60

70Y

ield

(w

t. %

)

A B C D

viscous solutions. Thus, removal of the solventalso becomes problematic.

Figures 2C and 3C demonstrate the effect ofmolecular weight of PLA on the yield (wt%)and size of PLA NPs, respectively. It can beseen that with the increase in PLA Mw, yield(wt%) decreases, whereas the size of nanopar-ticles increases. In the case of high-Mw PLA,only a fraction of PLA chains are precipitatedduring nanoprecipitation and the rest leads tothe formation of aggregates, resulting in pooryield (wt%). The smaller size of PLA NPsproduced from low-Mw PLA may be due tothe fact that low-Mw PLA might have certainsurface-active properties that enhanced the for-mation of smaller NPs during nanoprecipita-tion. This hypothesis is supported by the fact

that the hydrophilicity of PLA arises due to po-lar chain ends bearing free carboxylic and hy-droxyl groups. These polar groups present in thehydrophobic backbone of PLA confer an am-phiphilic character to PLA chains. The lower theMw, the higher their hydrophilic/hydrophobicbalance and hence surface-active properties.[5]

Figures 2D and 3D elucidate the effect ofsolvent on the yield (wt%) and size of PLANPs, respectively. It can be seen that larger NPswere formed from acetone with higher yield,whereas smaller NPs with lower yield were ob-tained when DMSO was used as solvent. Thesolubility of PLA in DMSO is higher than inacetone and DMSO has a slightly higher mis-cibility in methanol. This led to faster diffusionof DMSO into methanol, thus creating smaller

FIGURE 3. Main effect plots for size of PLA nanoparticles.

PLA conc. (mg/ml) S/NS volume ratio PLA Mw Solvent

5 10 15 200.

050.

20 0.35 0.6

0PLAL

PLAH

Aceto

ne

DMSO

180

210

240

270

300

NP

siz

e (n

m)

A B C D

Dow

nloa

ded

by [

Uni

vers

ity o

f C

alif

orni

a, S

an F

ranc

isco

] at

08:

07 2

0 A

ugus

t 201

4

P106 INTERNATIONAL JOURNAL OF GREEN NANOTECHNOLOGY: PHYSICS AND CHEMISTRY

TABLE 5. Response table for SNR values foryield

Level PLA conc. S/NS volume ratio PLA Mw Solvent

1 36.3908 33.9846 32.5992 33.61022 36.6104 34.8603 34.1018 33.09083 31.2114 32.88274 29.1894 31.6744Delta 7.4211 3.1859 1.5025 0.5195Rank 1 2 3 4

polymer-containing droplets ready to precipitateas smaller NPs in comparison to acetone. Also,the interaction parameter between the DMSOand methanol pair is smaller than the acetoneand methanol pair, thus leading to smaller NPswith DMSO.[5,21]

Determination of Optimal Conditions

The average SNR of each control factor ateach level for yield (wt%) and size of NPs isgiven in Tables 5 and 6, respectively. The rangeof the SNR, denoted by delta, of each factor isdetermined using Equation 4.

D = Delta = SNRmax − SNRmin (4)

Based on the delta statistics, the order of influ-ence is PLA concentration > S/NS volume ratio> PLA Mw > solvent. From Table 5, it can beseen that the greatest variation of SNR is for thelevel 2 of PLA concentration, S/NS volume ratio,and PLA Mw (PLAL) and level 1 of solvent (ace-tone). Similarly, it can be seen from Table 6 thatthe smallest variation of SNR is for the level 2 of

TABLE 6. Response table for SNR values forsize of PLA NPs

Level PLA conc. S/NS volume ratio PLA Mw Solvent

1 −44.3639 −44.3448 −46.9299 −45.89232 −43.6072 −44.1732 −44.6137 −45.65133 −45.6300 −45.32734 −49.4861 −49.2418Delta 5.8789 5.0686 2.3162 0.2410Rank 1 2 3 4

PLA concentration, S/NS volume ratio, and PLAMw (PLAL) and level 2 of solvent (DMSO).Thus, the optimal condition for obtaining higheryield is at 10 mg/mL PLA concentration, 0.2S/NS volume ratio, low-molecular-weight PLA,and acetone, and the optimum levels for size ofNPs are found with polymer concentration of10 mg/mL, 0.2 S/NS volume ratio, PLAL, andDMSO.[22]

Two confirmatory experiments were carriedout to verify the optimal settings of input de-sign parameters. The confirmatory experimentswere quite satisfactory because both of the aboveoptimum conditions produced almost the sameresult. The yield (wt%) and NP size, based onthe optimal settings of the above design param-eters, were found to be 79.3% and 115 nm (PDI:0.089) when acetone was taken as solvent and79.8% and 111 nm (PDI: 0.229) when DMSOwas taken as solvent. The values are almost closeto those obtained from initial experiment (run no.6). These optimum conditions for yield (wt%)and size of NP are acceptable because our mainobjective is to produce smaller size NPs withhigher yield. Thus, based on the delta statisticsfor the size of PLA NPs, the order of influence isPLA concentration > S/NS volume ratio > PLAMw > solvent.

Characterization of Nanoparticles

Particle Size Analysis

Average particle sizes were obtained fromDLS analysis, which measures the hydrody-namic radius, and are given in Table 2. The PDI,a parameter to define the particle size distribu-tion of nanoparticles, was also obtained fromDLS and was found to vary from 0.03 to 0.3,except for run no. 8, as can be seen from Ta-ble 2. Therefore, these preparations could beassumed to be monodisperse. The exceptionalcase for run no. 8 may be due to the high molec-ular weight and high concentration of PLA. PDI< 0.3 is attributed to monodisperse samples,whereas PDI > 0.3 is obtained from the polydis-perse samples.[5] The low values of PDI showedthat the samples had very good dispersity of theparticles and that the samples were uniform.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

alif

orni

a, S

an F

ranc

isco

] at

08:

07 2

0 A

ugus

t 201

4

T. Maharana et al. P107

FIGURE 4. TEM micrographs of PLA nanoparticles: (A) run no. 1; MD: 133.6 nm; (B) run no. 3;MD: 219.6 nm; (C) run no. 11; MD: 139.4 nm; (D) run no. 14; MD: 350.5 nm.

Analysis by TEM

TEM micrographs of PLA NPs obtained fromrun nos. 1, 3, 11, and 14 are shown in Figure 4,among which two micrographs are of PLA NPs

obtained from PLAL samples and the other twoare micrographs obtained from PLAH samples.It can be seen from Figure 4 that spherical andsmooth NPs were produced. The micrographsalso clearly indicate that no hairline cracks

Dow

nloa

ded

by [

Uni

vers

ity o

f C

alif

orni

a, S

an F

ranc

isco

] at

08:

07 2

0 A

ugus

t 201

4

P108 INTERNATIONAL JOURNAL OF GREEN NANOTECHNOLOGY: PHYSICS AND CHEMISTRY

appear on the surface of the NPs. The TEMimages were analyzed using Java-based ImageJ(version 1.41o) image analysis software, devel-oped by National Institutes of Health, to obtainthe particle size diameter data. The diameters ofthe particles were measured and were averagedout to get the mean diameter of the nanoparticles.It was observed that the sizes of PLA NPs ob-tained from TEM are little bit smaller than thoseobtained from DLS measurement, as expected.This is attributed to the fact that the mean sizederived from DLS measurement is the intensity-weighted average hydrodynamic size of the par-ticles being measured, which is influenced byhydration or solvation effects, whereas the TEMsize is a number-weighted average size of a de-hydrated hard sphere. It was observed that thedifference is about 0.5 nm for NPs of size around100 nm and about 3 nm for NPs of size around350 nm.[23] TEM images also illustrated that thesizes of the nanoparticles are not uniformly dis-tributed.

Determination of Zeta Potential

Zeta (ζ )-potential of PLA NPs was deter-mined in aqueous medium, at different pHs.ζ -potential as a function of pH is presented inFigure 5. The surface of PLA NPs has a nega-tive ζ -potential and thus it is most likely to favorthe electrostatic adsorption of polycations.[1,18]

The positively charged adsorbed drug particleson the PLA NP surface can interact with the neg-atively charged body cells. The negative surface

FIGURE 5. Variation of zeta potential with pH.

3 .5 4 .0 4 .5 5 .0 5 .5 6 .0 6 .5 7 .0 7 .5

−3 5

−3 0

−2 5

−2 0

−1 5

−1 0

Zet

a po

tent

ial (

mV

)

pH

charge of PLA NPs originates from free car-boxylic acid groups at the chain ends of the PLApolymer. Nanoparticles with a ζ -potential above(+/−) 30 mV have been shown to be stable insuspension, because the surface charge preventsaggregation of the particles and hence favors thedrug release from the drug-loaded PLA NPs.

ζ−potential value (−33.9 mV) suggested astable system at pH 7.4, which is the normal pHof blood. The pH of the stomach is 3. Normalurine pH averages about 6.0. Saliva has a pHbetween 6.0 and 7.4. PLA nanoparticles are sta-ble at higher pH. As the pH was decreased, themagnitude of the ζ -potential values decreaseddue to protonation of the carboxylic acid chainsof the PLA particle surface at low pH values.[18]

It seems obvious from Figure 5 that the particleaggregates are formed at low pH values, leadingto lower potential. Alkaline pH, biochemicallyspeaking, is slow and cool. In the present case,at alkaline pH the ζ -potential is higher. Thus, itcan be concluded that drugs encapsulated intothe PLA NPs can help slow the release of drugsinto the body because the pH of extracellularfluid is 7.4.

CONCLUSIONS

Smaller PLA NPs were prepared by a nano-precipitation method using acetone and DMSOas solvents. Systematic investigation of thesynthesis parameters showed that it is possi-ble to prepare NPs of smaller size and withrelatively narrow PDI. TMED and its analysissuggested that PLA concentration and solvent-to–non-solvent volume ratio are the most influ-ential parameters and lower-molecular-weightPLA produced smaller NPs. Acetone and DMSOwere found to be equally good solvents. The op-timum yield of the PLA NPs was obtained with10 mg/mL PLA concentration, 0.2 S/NS volumeratio, low-molecular-weight PLA, and acetone,and the optimum levels for size of nanoparti-cles were found with polymer concentration of10 mg/mL, 0.2 S/NS volume ratio, PLAL, andDMSO. At the optimum conditions the yield andsize were found to be around 79% and 115 nm,respectively.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

alif

orni

a, S

an F

ranc

isco

] at

08:

07 2

0 A

ugus

t 201

4

T. Maharana et al. P109

REFERENCES

1. Sussman, E. M.; Clarke, M. B., Jr.; Shastri, V. P.Single-step process to produce surface-functionalized poly-meric nanoparticles.Langmuir 2007, 23, 12275–12279.

2. Nakayama, N.; Hayashi, T. Preparation and char-acterization of poly(L-lactic acid)/TiO2 nanoparticlenanocomposite films with high transparency and effi-cient photodegradability. Polymer Degrad. Stabil. 2007, 92,1255–1264.

3. Konno, T.; Kurita, K.; Iwasaki, Y.; Nakabayashi,N.; Ishihara, K. Preparation of nanoparticles com-posed with bioinspired 2-methacryloyloxyethyl phos-phorylcholine polymer. Biomaterials 2001, 22, 1883–1889.

4. Lassalle, V.; Ferreira, M. L. PLA nano- and mi-croparticles for drug delivery: An overview of the methodsof preparation. Macromol. Biosci. 2007, 7, 767–783.

5. Legrand, P.; Lesieur, S.; Bochot, A.; Gref, R.; Raat-jes, W.; Barratt, G.; Vauthier, C. Influence of polymer be-haviour in organic solution on the production of polylactidenanoparticles by nanoprecipitation. Int. J. Pharm. 2007,344, 33–43.

6. Wei, Q.; Wei, W.; Lai, B.; Wang, L. Y.; Wang, Y.X.; Su, Z. G.; Ma, G. H. Uniform-sized PLA nanoparti-cles: Preparation by premix membrane emulsification. Int.J. Pharm. 2008, 359, 294–297.

7. Jain, R. A. The manufacturing techniques of vari-ous drug loaded biodegradable poly(lactide-co-glycolide)(PLGA) devices. Biomaterials 2000, 21, 2475–2490.

8. Fessi, H.; Puisieux, F.; Devissaguet, J. P.; Am-moury, N.; Benita, S. Nanocapsule formation by interfacialpolymer deposition following solvent displacement. Int. J.Pharm. 1989, 55, R1–R4.

9. Duclairoir, C.; Nakache, E.; Marchais, H.; Orec-chioni, A. M. Formation of gliadin nanoparticles: Influenceof the solubility parameter of the protein solvent. ColloidPolymer Sci. 1998, 276, 321–327.

10. Trotta, M.; Debernardi, F.; Caputo, O. Preparationof solid lipid nanoparticles by a solvent emulsification-diffusion technique. Int. J. Pharm. 2003, 257,153–160.

11. Niwa, T.; Takeuchi, T.; Hino, T.; Kunou, N.;Kawashima, Y. Preparations of biodegradable nanospheresof water-soluble and insoluble drugs with D,L-

lactide/glycolide copolymer by a novel spontaneous emul-sification solvent diffusion method, and the drug releasebehavior. J. Contr. Release 1993, 25, 89–98.

12. Murakami, H.; Kobayashi, M.; Takeuchi, H.;Kawashima, Y. Further application of a modified spon-taneous emulsification solvent diffusion method to vari-ous types of PLGA and PLA polymers for preparation ofnanoparticles. Powder Tech. 2000, 107, 137–143.

13. Chorny, M.; Fishbein, I.; Danenberg, H. D.; Golomb,G. Study of the drug release mechanism from tyrphostinAG-1295-loaded nanospheres by in situ and external sinkmethods. J. Contr. Release 2002, 83, 389–400.

14. Thioune, O.; Fessi, H.; Devissaguet, J. P.; Puisieux,F. Preparation of pseudolatex by nanoprecipitation: Influ-ence of the solvent nature on intrinsic viscosity and inter-action constant. Int. J. Pharm. 1997, 146, 233–238.

15. Galindo-Rodriguez, S. A.; Puel, F.; Briancon, S.;Allemann, E.; Doelker, E.; Fessi, H. Comparative scale-upof three methods for producing ibuprofen-loaded nanopar-ticles. Eur. J. Pharm. Sci. 2005, 25, 357–367.

16. Montgomery, D. C. Design and Analysis of Experi-ments, 5th ed.; John Wiley & Sons: New York, 2004.

17. Bilati, U.; Allemann, E.; Doelker, E. Developmentof a nanoprecipitation method intended for the entrapmentof hydrophilic drugs into nanoparticles. Eur. J. Pharm. Sci.2005, 24, 67–75.

18. Roy, R. K. A Primer on the Taguchi Method; VanNostrand Reinhold: New York, 1990.

19. Stainmesse, S.; Orecchioni, A. M.; Nakache, E.;Puisieux, F.; Fessi, H. Formation and stabilization of abiodegradable polymeric colloidal suspension of nanopar-ticles. Colloid Polymer Sci. 1995, 273, 505–511.

20. Stainmesse, S.; Fessi, H.; Devissaguet, J. P.;Puisieux, F.; Thies, C. Process for the preparation of dis-persible colloidal systems of a substance in the form ofnanoparticles; US005133908A, 1992.

21. Hirsjarvi, S.; Peltonen, L.; Hirvonen, J. Surface pres-sure measurements in particle interaction and stability stud-ies of poly(lactic acid) nanoparticles. Int. J. Pharm. 2008,348, 153–160.

22. Jahanshahi, M.; Sanati, M. H.; Hajizadeh, S.; Babaei,Z. Gelatin nanoparticle fabrication and optimization ofthe particle size. Phys. Status Solidi 2008, 205, 2898–2902.

23. http://www.malvern.com/FAQ

Dow

nloa

ded

by [

Uni

vers

ity o

f C

alif

orni

a, S

an F

ranc

isco

] at

08:

07 2

0 A

ugus

t 201

4