On the Use of tert -Butanol/Water Cosolvent Systems in Production and Freeze-Drying of Poly-ε-Caprolactone Nanoparticles

RESEARCH ARTICLE – Pharmaceutical Nanotechnology

On the Use of tert-Butanol/Water Cosolvent Systems in Productionand Freeze-Drying of Poly-�-Caprolactone Nanoparticles

TEREZA ZELENKOVA, ANTONELLO A. BARRESI, DAVIDE FISSORE

Dipartimento di Scienza Applicata e Tecnologia, Politecnico di Torino, Torino 10129, Italy

Received 9 July 2014; revised 21 October 2014; accepted 28 October 2014

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24271

ABSTRACT: This work deals with the use of a water/tert-butyl alcohol (TBA) system in the manufacturing process of poly-ε-caprolactone(PCL) nanoparticles, namely in the synthesis stage, using the solvent displacement method in a confined impinging jet mixer (CIJM), andin the following freeze-drying stage. The experimental investigation evidenced that the nanoparticles size is significantly reduced withrespect to the case where acetone is the solvent. Besides, the solvent evaporation step is not required before freeze-drying as TBA is fullycompatible with the freeze-drying process. The effect of initial polymer concentration, flow rate, water to TBA flow rate ratio, and quenchvolumetric ratio on the mean nanoparticles size was investigated, and a simple equation was proposed to relate the mean nanoparticlessize to these operating parameters. Then, freeze-drying of the nanoparticles suspensions was studied. Lyoprotectants (sucrose and mannitol)and steric stabilizers (Cremophor EL and Poloxamer 388) have to be used to avoid nanoparticles aggregation, thus preserving particle sizedistribution and mean nanoparticles size. Their effect, as well as that of the heating shelf temperature, has been investigated by means ofstatistical techniques, with the goal to identify which of these factors, or combination of factors, plays the key role in the nanoparticles sizepreservation at the end of the freeze-drying process. C© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm SciKeywords: nanoparticles; nanotechnology; particle size; stability; freeze-drying; tert-butanol/water cosolvent system; confined impingingjets mixer; solvent displacement; lyoprotectants; steric stabilizers

INTRODUCTION

Polymeric nanoparticles are colloidal systems, submicron-sized, that can be used as a carrier to deliver therapeuticallyactive substances to the target cells or tissues.1 According to themanufacturing procedure, nanospheres or nanocapsules can beobtained. The first ones consist of a dense polymeric matrixwhere the medicament can be dispersed, whereas nanocap-sules are composed of a polymeric shell and an oily or solidnucleus. The effectiveness of the drug mainly depends on thevehicles characteristics, and particularly, on the nanoparticlessize distribution (nano PSD). The nanoparticles size requiredfor parenteral application should range between 100 and 300nm to avoid undesirable interactions with the reticuloendothe-lial system.2–4

Poly-g-caprolactone (PCL) is quite often used in nanoparti-cles production. This biodegradable aliphatic polyester is ob-tained by ring opening polymerization of caprolactone. PCL isa semi-crystalline polymer having a glass transition temper-ature of −60◦C and a melting point of about 62◦C.5 Its bio-compatibility, biodegradability, and nontoxicity, as well as itsslow degradation kinetics make it suitable for pharmaceuticalapplications.1,6

Nanoparticles can be obtained by solvent displacementmethod, also known as nanoprecipitation, based on the mix-ing of two liquid phases (the solvent and the antisolvent).7

The main industrial advantage of this technique is that wa-ter miscible, toxicologically acceptable solvents, as short-chain

Abbreviations used: CIJM, confined impinging jet mixer; DoE, design ofexperiments; PCL, poly-g-caprolactone; PSD, particle size distribution; TBA,tert-butanol; XRD, X-ray diffraction.

Correspondence to: Davide Fissore (Telephone: +39-011-0904693; Fax: +39-011-0904699; E-mail: [email protected])

Journal of Pharmaceutical SciencesC© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

alcohols, can be used.8 After the dissolution of the polymerinto the solvent, the antisolvent is mixed with this system andthis leads to the spontaneous formation of nanoparticles. Themain positive aspect of this method is the great repeatabilityof the nanoparticles size, and the possibility of controlling nanoPSD by adjusting the level of supersaturation, as it affects thesteps of particle nucleation and growth, phase breakdown andnanoparticles agglomeration.8,9 As the formation of nanopar-ticles is almost immediate, the final nanoparticles propertiesare greatly influenced by the mixing of the two individualstreams (the solvent and the antisolvent) and, therefore, spe-cial mixers are recommended to guarantee the best mixingconditions as, for example, the confined impinging jet mixer(CIJM).10–12

The production process of PCL nanoparticles using the sol-vent displacement method in a CIJM, and the removal of theantisolvent using lyophilization, was investigated by Zelenkovaet al.13 to optimize the nanoparticles manufacturing process incase acetone is the solvent used. Before freeze-drying, acetonemust be eliminated from the nanosuspension by evaporationbecause of its low freezing point (−94◦C). The step of solventevaporation in the manufacturing process can be avoided if asolvent compatible with the freeze-drying process is used to dis-solve the PCL. This would allow producing, and subsequentlyfreeze-drying, nanoparticles in sterile conditions, thus resultingin significant benefits for the pharmaceuticals industry.14 A listof solvents in which PCL is soluble (acetyl chloride, aniline, 2-chlorethanol, chloroform, tetrahydrofuran, toluene), partiallysoluble (acetone, acetonitrile, 2-butanone, 2-pentanone, tert-buthyl alcohol, xylenes), and nonsoluble (n-butyl acetate, hex-ane, isopropyl ether, 1-octanol, pentanol, water) was presentedby Bordes et al.6 Tert-butyl alcohol (TBA), carbon tetrachlo-ride, acetic acid, acetonitrile, chlorobutanol, tetrahydrofuran,ethanol, and cyclohexane represent a group of organic solvents

Zelenkova, Barresi, and Fissore, JOURNAL OF PHARMACEUTICAL SCIENCES 1

2 RESEARCH ARTICLE – Pharmaceutical Nanotechnology

tested for freeze-drying of drugs. TBA (in combination withwater) appears to be the organic solvent most frequently usedbecause of its chemical–physical properties as the high vaporpressure (4.1 kPa at 20◦C), the high melting point (24◦C), thelow toxicity, and the miscibility in all proportions with water(although it is quite hydrophobic).15 The quantity of TBA in thesolution has a significant impact on the shape and size of theice crystals and, usually, relatively large needle-shaped crystalsare formed during the freezing stage resulting in a faster dryingif compared to the case when only water is used as solvent.15–18

Besides, the use of organic or organic/water cosolvent systemsmay cause several troubles, for example, difficult handling be-cause of the solvent flammability or explosion potential, to thepresence of residual solvent in the final product and to the highcost.15,16

After nanoparticles synthesis, and before freeze-drying, sev-eral additives (lyoprotectants and steric stabilizers) are gen-erally added to the solution to preserve the PSD, as thefreezing and the drying stages can affect nanoparticles sizestability. A key role in the nanoparticles size preservation dur-ing the processes of lyophilization and storage is played bythe type and concentration of lyoprotectants and steric stabi-lizers used.19–21 Monosaccharides (glucose) and their derivates(mannitol), disaccharides (sucrose, trehalose), polyvinylpyrroli-done (PVP K15), hydroxypropyl-$-cyclodextrin, are the mostfrequently used lyoprotectants. The “vitrification hypothesis”has been proposed to explain the role of lyoprotectants: duringfreezing a glassy system is formed by the saccharides, knownalso as cryo-concentrated phase, where nanoparticles are im-mobilized and remain protected against the ice crystals.22,23

In case mannitol is used as lyoprotectant, its crystallizationcan cause the formation of two separate phases, namely ananoparticles-poor phase and a nanoparticles-rich phase, lead-ing to nanoparticles aggregation. To prevent this event, manni-tol has to remain amorphous during the freezing stage, and thiscan be achieved by adding various excipients, like sodium chlo-ride or steric stabilizers.13,24 Beside lyoprotectants, steric stabi-lizers are added with the goal to provide the best nanoparticlesstability during the lyophilization process. The “water replace-ment theory” is used to explain the mechanism of nanoparti-cles stabilization by means of steric stabilizers: according tothis theory the steric stabilizer is adsorbed onto nanoparticlessurface by means of hydrogen bonds, thus avoiding particle ag-gregation, or even particle fusion.19–21 These substances aregenerally polymers and surfactants, for example, Tween80, Cremophor EL, Poloxamer 388, and polyvinyl alcohol.Beirowski et al.25–27 found that better results can be obtainedusing Poloxamer 388, an amphiphilic nonionic polymer com-posed by hydrophilic ethylene oxide and hydrophobic propyleneoxide28, with respect to Cremophor EL, a heterogeneous non-ionic surfactant, composed generally by oxydated triglyceridesof ricinoleic acid29, even when the lyoprotectants (threhalose,PVP K15, sucrose) are present. Similar conclusions were ob-tained also by Zelenkova et al.13 in a previous work where theproduction of PCL nanoparticles using acetone as solvent wasinvestigated. Anyway, in all studies, the interdependence be-tween the concentration of steric stabilizers and of lyoprotec-tants and the final particle size was highlighted.

After the freezing stage, the frozen solvents are removedfrom the product by sublimation (primary drying). In this stage,it is necessary to maintain the product temperature belowthe collapse temperature of the formulation, although it was

pointed out that in case of product collapse the PCL nanopar-ticles size, in presence of glucose, remain unchanged.24

It has to be remarked that polymeric nanoparticles are usedto deliver therapeutically active substances to the target cells(or tissues) and, therefore, one of the main issues related to theproduction process is the effect of the drug on particle charac-teristics. In this framework, it has to be taken into account that,as shown, among the others, by Jugminder and Mansoor30, theinfluence of the drug can be often neglected when investigatingthe effect of the operating conditions on the final mean parti-cle size in case the drug is entrapped in the polymeric matrixand it does not influences the surface characteristics. In fact,because of the low drug loading generally used, particles aremainly constituted by the polymeric carrier, and the drug doesnot influence significantly the final particle size. Different isthe case of a drug that is mainly adsorbed on the surface of thepolymeric particle, but also in this case, the goal of the synthe-sis process is to get small polymeric particles in such a way thatafter drug adsorption the final size of the particle is still in therange of interest.

This work aims investigating the potential of water/tert-butanol system for the freeze-drying of PCL nanoparticles pro-duced in a CIJM using the solvent displacement method, withthe aim of improving the overall manufacturing process. Thestudy was focused on the effect of the solvent flow rate, of theinitial PCL concentration, of the water-to-tert-butanol flow rateratio and of the quench volumetric ratio on mean size and onthe PSD of PCL nanoparticles. In all cases, nanosuspensionswith a final TBA concentration in water equal to 20%, whichcorresponds to the eutectic composition, were obtained. To en-sure nanoparticles stability during lyophilization, the presenceof additives is required and the effect of sucrose and manni-tol, as lyoprotectants, and of Cremophor EL and Poloxamer388, as steric stabilizers, was studied. In addition, the effectof the heating shelf temperature used in the primary dryingstage on the nanoparticles stability was studied. The influenceof these factors was analyzed using a standard design of exper-iments (DoE) technique aiming to point out the variables, aswell as their reciprocal interactions, playing the key role in thenanoparticles size preservation at the end of the lyophilizationprocess.31

MATERIALS AND METHODS

Production of Nanoparticles

Zelenkova et al.13 and Lince et al.9,12 described in detailthe manufacturing process of PCL nanoparticles. Here, it issummed up in brief. The solvent displacement method wasused to produce nanoparticles. PCL (Mw = 14,000 g mol−1),purchased by Sigma–Aldrich (Steinheim, Germany), was dis-solved in pure tert-butanol (Sigma–Aldrich). Micro-filtratedwater, prepared by Millipore system (Milli-Q RG; MilliporeSystem, Darmstadt, Germany), is used as antisolvent. Forcomparison purposes, in a few experiments acetone (HPLCgrade; Sigma–Aldrich) was used as solvent, in the same con-ditions used for TBA. In case TBA was used, the solution waswarmed up for 30 min to favor PCL dissolution (and then wascooled to 35◦C), whereas in case acetone was used, this step wasnot required. The temperature of the water was about 25◦C inmost experiments; when investigating the effect of TBA, testswere carried out using water at 35◦C. Initial PCL concentration

Zelenkova, Barresi, and Fissore, JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24271

RESEARCH ARTICLE – Pharmaceutical Nanotechnology 3

Figure 1. Simplified scheme of the nanoparticles preparation process with the sketch of the confined impinging jet mixer used for nanoparticlesproduction (din = 1 mm, dout = 2 mm, mixing chamber diameter = 5 mm, total chamber height = 11.2 mm).

ranged from 5 to 25 mg mL−1. The solution containing the poly-mer and the antisolvent were then inserted in two plastic sy-ringes (the volume of each syringe was equal to 100 mL) andthen fed to the chamber of the CIJM by means of a KDS200 sy-ringe pump (KD Scientific, Holliston, Massachusetts). As soonas the two streams mixed, nanoparticles were formed. A sim-plified scheme of the process and of the CIJM is illustratedin Figure 1. Flow rates (FR) ranged in the interval 5–120 mLmin−1. The water-to-TBA flow rate ratio (W/T) was varied be-tween 1 and 6 reducing the TBA flow rate (FRT) keeping con-stant the flow rate of water (FRW). A set of experiments wherethe FRW was increased keeping constant FRT was also carriedout: in this case W/T ranged between 0.5 and 3. Suspensionscontaining nanoparticles formed in the chamber of the CIJMwere usually quickly diluted (quenched) in a defined volume ofmicro-filtrated water, and kept under gentle stirring. The ratioof the amount of water used for the preparation of nanopar-ticles to that used for the quench stage (“quench volumetricratio”) ranged from 1 to 0.125. To highlight the importanceof the quench stage on the nanoparticles stability, some ex-periments were carried out without quenching the nanoparti-cles. The same operating procedure was used in case acetone isthe solvent. The operating parameters used during the experi-ments are summarized in Table 1.

In this work, the flow fields in the mixer were characterizedby Reynolds number (Rej) of the inlet stream, calculated asshown in Eq. (1):

Rej = Df dinvj

: f(1)

Table 1. Operating Conditions Used for Nanoparticles Synthesis inthe CIJM

Operating Parameters Values

Initial PCL concentration(mg mL−1)

5, 10, 15, 20, 25

Water to tert-butanol flow rateratio (W/T) at constantwaterflow rate (FRW)

1, 2, 3, 6

Water to tert-butanol flow rateratio (W/T) at constanttert-butanol flow rate (FRT)

0.5, 1, 2, 3

Water inlet jet Reynolds number(RejW) (solution flow rate (FR)(mL min−1 at W/T = 1)

124 (5), 248 (10), 496 (20), 744(30), 992 (40), 1488 (60),1985 (80), 2977 (120)

Quench volumetric ratio(percentage of TBA (or acetone)in the final solution)

0.33 (20%), 0.2 (15%), 0.125(10%), no quench (50%)

Table 2. Density and Viscosity (at 25◦C) of the Solvent and of theAntisolvent Used for Nanoparticles Synthesis

Df (kg m−3) : f (kg m−1 s−1)

Acetone 781 3.1 × 10− 4

Tert-butanol 775 3.35 × 10− 3

Water 994 8.5 × 10− 4

where vj is the mean velocity of the stream and din is the inter-nal diameter of the inlet tube (1 mm). The values of the density(Df) and the viscosity (: f) of the solvents and of the antisolventused in this study are shown in Table 2. In this work, the water

DOI 10.1002/jps.24271 Zelenkova, Barresi, and Fissore, JOURNAL OF PHARMACEUTICAL SCIENCES


stream (and the corresponding RejW) has been generally usedas reference.

Nanoparticles suspensions used in the lyophilization runswere prepared as follows: initial PCL concentration was equalto 5 mg mL−1, FR was equal to 80 mL min−1, W/T was equalto 1 and quench volumetric ratio was equal to 0.33 to obtaina final solution at 20% of TBA. Eutectic composition of thelyophilized system was chosen in order to obtain, at the end offreezing stage, a fine and uniform structure of ice–TBA crys-tals, where the nanoparticles can be entrapped, and to preventthe formation of zones with higher or lower concentration ofTBA. Furthermore, it is generally known that numerous com-mercial drugs are freeze-dried in this system (80% water–20%TBA) as Aprostatil (CAVERJECT R© Sterile Powder)32, Amoxi-cillin sodium,33 and Imexon.34

Freeze-Drying of Nanosuspensions

Before lyophilization, lyoprotectants, namely sucrose or manni-tol (Sigma–Aldrich), and steric stabilizers, namely Poloxamer388 (PEG–PPG–PEG Pluronic R© F-108; Sigma–Aldrich) or Cre-mophor EL (Sigma–Aldrich) were added to the nanosuspen-sions. Final concentrations of lyoprotectants was 2.5% (w/w)or 5% (w/w), equal to that of steric stabilizers (thus, the mini-mum concentration of additives was equal to 5% w/w and themaximum was equal to 10%, w/w). In Table 3, all concentra-tions of the additives used in the lyophilization runs presentedin this work are summarized. Nanosuspensions (1.5 mL) werefilled into screw neck DIN 58378-AR10 tubing glass vials withexternal diameter of 18 ± 0.25 mm and wall thickness of 1.2± 0.06 mm. The height of the suspension before freeze-dryingwas equal to height of the dried product after lyophilization(12.2 mm). Laboratory-scale LyoBeta 25 freeze-dryer (Telstar,Terrassa, Spain) was used to carry out the freeze-drying tests.This device is composed of a vacuum-tight chamber (0.2 m3)equipped with four heating shelves (0.5 m2 of total area).The chamber pressure is measured by means of a Baratron626A gauge (MKS Instruments, Andover, Massachusetts). Theproduct temperature measurement was obtained using T-type

Table 3. Composition of Solutions Containing Nanoparticles used inLyophilization Experiments

Test ID Sucrose (%) Cremophor EL (%) Poloxamer 388 (%)

1a 5.0 5.0 0.02a 5.0 2.5 0.03a 2.5 5.0 0.04a 2.5 2.5 0.01b 5.0 0.0 5.02b 5.0 0.0 2.53b 2.5 0.0 5.04b 2.5 0.0 2.5

Test ID Mannitol (%) Cremophor EL (%) Poloxamer 388 (%)

1c 5.0 5.0 0.02c 5.0 2.5 0.03c 2.5 5.0 0.04c 2.5 2.5 0.01d 5.0 0.0 5.02d 5.0 0.0 2.53d 2.5 0.0 5.04d 2.5 0.0 2.5

miniature thermocouples (Tersid, Milano, Italy). Each batchcontained about 50 vials filled with suspensions, and about 200empty vials placed around them for shielding purposes. Shelftemperature (Tshelf) was equal to −50◦C in the freezing stage,and −20◦C in the primary drying at a chamber pressure 5 Pa. Inaddition, to point out how the heating shelf temperature (dur-ing the primary drying stage) affects the nanoparticles size,one set of experiments with Tshelf = −30◦C was carried out. Noshrinkage or collapse of the cake was observed in any case. Laststep, secondary drying, was performed for 6 h at 20◦C and 5 Pa.

Particle Size Measurements

Nanoparticles size was measured before freeze–drying, and atthe end of this process, by means of Dynamic Light Scatteringusing a DLS Zetasizer Nanoseries ZS90 instrument (MalvernInstrument, Malvern, UK). This technique permits to deter-mine the nanoparticles size (z-average) accurately in the inter-val 2 nm to 3 :m. Mie theory, with a product refractive indexequal to 1.570 and product absorption equal to 0.010, was usedto determine the particle size distribution. Before measure-ment, all samples were diluted in water 1:100 (to optimize themeasurement). Each measurement was repeated three times.

X-ray Diffraction

In order to investigate the change of mannitol structure afterthe lyophilization in presence of a steric stabilizer (CremophorEL or Poloxamer 388), the (dried) product was analyzed bymeans of X-ray diffraction (XRD). In addition, the spectra ofpure mannitol (with or without nanoparticles) freeze-dried inan aqueous system were compared with the mannitol spectralyophilized in a water–TBA (20% TBA, w/w) system. A diffrac-tometer X’Pert MRD PRO (Panalytical, Almelo, The Nether-lands) was used, with Cu K" radiation and Ni beta filter, wasused to this purpose. The scan counting time was equal to0.02◦/s, the tension was equal to 40 kV and the current wasequal to 30 mA.

Statistical Analysis of the Results

A 23 factorial DoE was used to evaluate how the percentage oflyoprotectant in the formulation (factor A), the percentage ofsteric stabilizer in the formulation (factor B), and the heatingshelf temperature (during the primary drying stage) (factor C)affect the mean particle size. High (+) and low (−) values ofthese parameters (factor A: 2.5% and 5%, w/w; factor B: 2.5%and 5%, w/w; and factor C: −20◦C and −30◦C) were consid-ered, as it is graphically illustrated in Figure 2: a identifies thecombination of parameter A at the high level (5%, w/w) andparameters B and C at the low levels (2.5%, w/w and −30◦C),ab identifies the combination of parameters A and B at the highlevels (5%, w/w) and parameter C at the low level (−30◦C), abcidentifies the combination of parameters A, B, and C at the highlevels (5%, w/w; 5%, w/w; and −20◦C, respectively), whereas (1)identifies combination of parameters A, B, and C at the lowlevels (2.5%, w/w; 2.5%, w/w; and −30◦C, respectively). For thegeneric j-th combination of the operating parameters, the meanparticle size is evaluated experimentally. The test is repeated ntimes (in our experimental investigation n = 5) and the n valuesof dp obtained in the n tests with the j-th operating conditionsare summed; this values is indicated as dj

p in the followings.At this point, as shown, among the others, by Montgomery,31



Figure 2. Graphical representation of the 23 factorial design used to investigate the effect of lyoprotectant concentration in the formulation(a), of steric stabilizer concentration in the formulation (b), and of heating shelf temperature (c) on the mean particle size after the freeze-dryingprocess.

the single effects of various parameters can be calculated. Theeffect of A results to be equal to:

[da

p − d(1)p

]/n in case the values of B and C are both low;[

dabp − db

p

]/n in case the value of B is high and the value of

C low;[dac

p − dcp

]/n in case the value of C is high and the value of

B low and[dabc

p − dbcp

]/n in case the values of B and C are both high.

By averaging the single effects calculated previously, thetotal effect of A, known also as contrast parameter, on thenanoparticles size is obtained:

E(A) = 14n

[da

p − d(1)p + dab

p − dbp + dac

p − dcp + dabc

p − dbcp

]

= ContrastA

4n(2)

Similarly, the effects of parameters B and C can be calcu-lated. Interactions between two factors can be computed byEq. (3):

E (AB) =[dabc

p − dbcp + dab

p − dbp − dac

p + dcp − da

p + d(1)p

]4n

= ContrastAB

4n(3)

and analogously for BC and AC. The combined effect of thethree parameters is calculated as follows:

E (ABC) =[dabc

p − dbcp − dac

p + dcp − dab

p + dbp + da

p − d(1)p

]4n

= ContrastABC

4n(4)

Values obtained from Eqs. (2)–(4) can be positive or negative:in case the value is positive, it means that when the parameterincreases (from the minimum to the maximum) the observedvariable (the mean particle size) increases, and vice versa whenthe value is negative (the mean size of nanoparticles decreaseswith increasing value of parameter). The analysis of variance“ANOVA”31 was carried out using the Fisher test to verify thesignificance of differences between the arithmetic means of thevarious groups.

RESULTS AND DISCUSSION

Process of nanoparticles production

Lince et al.12 and Zelenkova et al.13 showed that when acetoneis the solvent used to dissolve PCL the mean nanoparticles sizeis greatly influenced by the operating parameters used duringthe manufacturing process, for example, the initial polymerconcentration, the solvent flow rate, the antisolvent to solventflow rate ratio, and the quench volumetric ratio. Equation (6)illustrates the dependence of the nanoparticles mean size (dp)on the polymer concentration in the feed (cp), on the Reynoldsnumber of the water jet (RejW) and on the water to acetone flow



Figure 3. Dependence of nanoparticles mean size on solvent flow ratein case the solvent is acetone (�) or TBA at various temperature of theantisolvent (water) (� = 25◦C) and (� = 35◦C). In the insert corner,the particle size distribution of nanoparticles produced with acetone(dashed line) or TBA (solid line) is shown (FR = 80 mL min−1). Quenchvolumetric ratio = 0.33, cp = 5 mg mL−1, W/T = W/A = 1.

rate ratio (W/A):

dp = A0

(cp

cref

)"

Re$jW(W

/A

)( (5)

where the parameter A0 is strongly influenced by the geometryand type of the reactor used; it can be considered as the meannanoparticles size when RejW = 1, W/A = 1, and cp/cref = 1,where the reference concentration (cref) is equal to 1 mg mL−1.The values of the parameters found in case acetone was usedare: " = 0.29, $ = −0.18, and ( = 0.063 when cp < 1 mg mL−1,and ( = −0.09 when cp > 1 mg mL−1.13 RejW was shown tobe one of the most significant parameters expressing the meansize dependence on the reactor fluid dynamics in case acetoneis used. Anyway, nanoparticles size is also strongly affected bythe initial PCL concentration, whereas W/A does not play akey role.

The goal of this work was to investigate if the mean particlesize dependence on cp, RejW, W/T is similar to that expressedby Eq. (5) in presence of a different solvent, namely TBA, and todetermine the parameters ", $, and (. Firstly, the dependenceof the particle diameter on the solvent flow rate using acetoneor TBA was compared, and results are given in Figure 3; for afixed geometry and mixer size, RejW is simply proportional toFRW. The initial PCL concentration was equal to 5 mg mL−1,the antisolvent to solvent flow rate ratio was equal to 1, andthe quench volumetric ratio was equal to 0.33. It appears thatwhen increasing the flow rate, the mean nanoparticles size de-creases in case acetone was used, whereas with TBA, the flowrate has a poor effect on the nanoparticles size, and smallernanoparticles (around 205 nm, with maximum deviation lowerthan 10%) are produced if compared with the case when acetonewas used. The negligible effect of mixing on the nanoparticlesmean size can be motivated by the low diffusivity of PCL in thewater–TBA solution and the high viscosity of TBA (the viscos-ity of TBA is 10 times higher with respect to acetone, and thediffusivity of PCL in acetone is more than seven times higherwith respect to TBA). Thus, it may be assumed that the processof nanoparticles growth is suppressed in favor of nanoparticlesnucleation.

Figure 4. a) Comparison between mean nanoparticles size depen-dence on RejW obtained with different quench volumetric ratios: � =no quench (TBA 50%), • = 1 (TBA 33%), � = 0.5 (TBA 25%), � = 0 .33(TBA 20%), � = 0.2 (TBA 15%), o = 0.125 (TBA 10%). (b) Comparisonbetween the particle size distribution of nanoparticles prepared withvarious quench volumetric ratios: no quench (solid line), 1 (dash-dottedline), 0.33 (dashed line). cp = 5 mg mL−1, W/T = 1, FR = 80 mL min−1.

In order to verify whether the initial temperature of theantisolvent influences the mean nanoparticles size, in one ex-periment, the temperature of the antisolvent (water) beforethe mixing was set equal to that of the TBA stream (35◦C).It appears from Figure 3 that the temperature of the antisol-vent poorly affects the mean nanoparticles size. From the PSDcurves it appears that the nanoparticles size prepared usingTBA are not only smaller, but also less disperse, then in case ofacetone.

The effect of the quench volumetric ratio (the ratio betweenthe amount of water employed for producing nanoparticles andthe amount of water employed for quenching them) on the meannanoparticles size was studied because it was expected that theamount of water employed for the quench stage can play a keyrole in the stabilization of the nanoparticles. Figure 4 shows themean particles size versus RejW when varying the quench volu-metric ratio in the range from 1 to 0.125 at constant cp = 5 mgmL−1 and W/T = 1 (results are also shown in case quench is notused). From Figures 4a and 4b, the step of quench appears to befundamental for the stabilization of nanoparticles, as withoutthe quench nanoparticles at least two times bigger are pro-duced as aggregation (and possibly ripening) of nanoparticlesoccurred as pointed out by the curve of particle size distribution,whereas when the quench was used, nanoparticles appear to bestable and smaller. A quench volumetric ratio equal or lowerto 0.5 has a negligible impact on the mean nanoparticles size,



Figure 5. Comparison between mean nanoparticles size dependenceon RejW [ (a) and on W/T (b) and (c)] for different initial concentrationsof polymer (� 5 mg mL−1; � = 10 mg mL−1, • = 15 mg mL−1, � = 20mg mL−1, and o = 25 mg mL−1). In (a), W/T = 1, in (b) FRW = 120 mLmin−1(FRT ranges from 20 to 120 mL min−1) and in (c) FRT = 40 mLmin−1 (FRW ranges from 20 to 120 mL min−1). Dashed lines identifythe values calculated with Eq. (8).

as it fluctuates around 205 nm and the maximum deviation isnever higher than 10%. Even thought slightly larger nanopar-ticles were obtained for the quench volumetric ratio equal to1, nanoparticles appear to be stable and the PSD curves aremonomodal also in this case. These findings are consistent withthe results obtained by Zelenkova et al.13 in case acetone wasused as solvent. As the desired final composition of the water–TBA system, that has to undergo the lyophilization process, is20% of TBA, following experiments related to the production ofnanoparticles were carried out using a quench volumetric ratioequal to 0.33.

The polymer concentration in the feed is another parameterthat can strongly influence the mean nanoparticles size. Figure5a shows the nanoparticles size versus RejW varying the initialPCL concentrations at W/T equal to 1 (the quench volumetricratio was 0.33). From these results, the parameters A0, ", and$ can be determined by best fitting, thus obtaining:

dp = 119.75(

cp

cref

)0.29

Re0.0084jW (6)

Good agreement between the values measured and thosecalculated using Eq. (6) was obtained, as shown by dashed linesin Figure 5a. With higher initial PCL concentration (from 5 to20 mg mL−1), an increase in the mean nanoparticles size was

observed. It was again evidenced that the feed flow rate (and,thus, the Re) plays a negligible role on the dimension of thenanoparticles.

Subsequently, the influence of W/T for various values ofthe initial PCL concentrations (5–25 mg mL−1) on the meannanoparticles size was studied and results are summarized inFigures 5b and 5c. The effect of W/T on dp can be expressedroughly by a simple power law, as in Eq. (5), and parameter (can be calculated by means of best fitting. In fact, from Eq. (5)we get that:

(W/T)( = dp

A0

(cp

cref

)"Re$jW

(7)

and the exponent ( may be easily calculated using the valuesof parameters A0, ", and $ previously obtained by looking forthe best fit of the various data set obtained when W/T is equalto 1. It was found that ( is equal to −0.23 and, thus, the finalform of equation can be expressed as follows:

dp = 119.75(

cp

cref

)0.29

Re0.0084jW (W/T)−0.23 ,

(cp

cref

)< 20 (8)

In Figures 5b and 5c, the experimentally measured and thecalculated values of the mean particle diameters are compared.It appears from Figure 5b, where W/T varied from 1 to 6 atconstant FRW, equal to 120 mL min−1 (FRT ranges from 20 to120 mL min−1), that when rising the initial polymer concen-tration also dp increases, and this is in agreement with theresults illustrated in Figure 5a. The same trend was observedalso when FRT was maintained constant at 40 mL min−1, vary-ing FRW in the interval from 20 to 120 mL min−1 (Fig. 5c) forcp ranging from 5 to 15 mg mL−1. For cp higher or equal to 20mg mL−1, the size does not increase following the same rule(that would correspond to the dotted lines) but it decreases. Acomplex dependence of the size on polymer concentration whenvarying the water to solvent flow rate ratio had been observedalso in previous works by Di Pasquale et al.35 and Zelenkova etal.13 in case acetone is used for producing nanoparticles.

Parity diagrams in Figure 6 demonstrate the good agreementbetween the experimentally measured data of nanoparticlesmean size and the values calculated with Eq. (8) for variousvalues of initial PCL concentrations ranging from 5 to 15 mgmL−1. The value of W/T in Figure 6a was equal to 1, in Figure6b it ranged from 1 to 6 at constant FRW (120 mL min−1),varying FRT, and in Figures 6c and 6d, W/T was ranging from0.5 to 3, FRT being constant at 40 mL min−1 with variable FRW.From Figure 6c, it appears that the parameters values of Eq. (8)are not valid when the PCL concentration in the feed is equalor higher than 20 mg mL−1. For this reason, new values forthe parameters were determined using the same procedure asbefore:

dp = 962(

cp

cref

)−0.48

Re0.0085jW (W/T)−0.23,

(cp

cref

)≥ 20 (9)

and good agreement between the experimentally measureddata and values calculated by the Eq. (9) was found, as it isillustrated in Figure 6d.

In case acetone is used, the solvent must be taken awayby evaporation before the lyophilization process because of



Figure 6. Comparison between experimentally measured values ofmean nanoparticle diameter obtained from five different initial con-centrations of polymer (� = 5 mg mL−1; � 10 mg mL−1; • = 15 mgmL−1; � = 20 mg mL−1, and o = 25 mg mL−1) and values calculatedwith Eq. (8) (a), (b), and (c) and with Eq. (9) (d). In (a), W/T = 1 andFRW ranges from 5 to 120 mL min−1. In (b), W/T = 1, 2, 3, 6; FRW =120 mL min−1 and FRT ranges from 20 to 120 mL min−1. In (c) and(d), W/T = 0.5, 1, 2, 3; FRT = 40 mL min−1 and FRW ranges from 20 to120 mL min−1.

low compatibility with the freeze-drying process (low freezingpoint). In case TBA was used, the step of evaporation is notrequired, and the solution can be lyophilized after the addi-tion of the excipients, namely the steric stabilizers and thelyoprotectants. By this way, the nanoparticles can be preparedunder sterile conditions, as mentioned before. To ensure thebest stability and uniformity of nanoparticles prepared for thelyophilization, nanoparticles were produced under the givenconditions: cp = 5 mL min−1, FR = 80 mL min−1, W/T = 1.Quench volumetric ratio was equal to 0.33 with the objectiveto obtain the eutectic concentration of the binary mixture, thatis, 80% (w/w) water–20% (w/w) TBA, as explained in the Intro-duction.

Lyophilization of formulations containing nanoparticles

The inadequate long term stability in water and the difficulthandling during storage can seriously impair the application of

Figure 7. (a) Dependence of the mean size of nanoparticles on timefor various storage temperatures (� = 25◦C, � = 8◦C). (b) PSD ofnanoparticles stored at 8◦C for various times: t = 0 day (dashed line),t = 23 days (dotted line), t = 87 days (solid line). Quench volumetricratio = 0.33, W/T = 1, cp = 5 mg mL−1, FR = 80 mL min−1.

nanoparticles.19 In Figure 7a, the mean sizes of nanoparticlesstored under different temperatures (8◦C, 25◦C) for 3 monthsare compared. In Figure 7b, the PSD immediately after theirsynthesis and that after 1 and after 3 months of storage at8◦C is illustrated (similar results were obtained for nanopar-ticles stored at 25◦C). The mean size of nanoparticles appearsto be well preserved for almost 1 month in both cases, but af-ter 3 months, nanoparticles stability is no longer preserved, asnanoparticles become bigger and tend to aggregate. The samevalues of PCL concentration (5 mg mL−1), FR (80 mL min−1),W/T (1), and quench volumetric ratio (0.33) were used in allthe runs.

Freeze-drying can allow longer term stability, but steric sta-bilizers and lyoprotectants are necessary to guarantee the high-est size stability of nanoparticles during the freeze-drying pro-cess. In Figure 8, the mean nanoparticles size before and afterthe freeze-drying process are compared. The primary dryingstep was carried out at various temperatures, namely at −20◦Cand −30◦C, in order to identify its influence on the mean sizeof nanoparticles. The final concentrations of the additives (ly-oprotectants and steric stabilizers) in the formulation contain-ing nanoparticles ranged from 5% to 10%. It appears at a firstsight that the mean size of nanoparticles increases after thelyophilization process in all cases. However, the increase in thenanoparticles mean size was smaller when the primary dryingwas carried out at lower temperature (−30◦C), in comparisonwith the case when the higher temperature (−20◦C) was used.



Figure 8. Comparison of the size of the nanoparticles obtained from sixteen different suspensions (the detailed compositions are listed inTable 3; left side sucrose, right side mannitol, upper line with Cremophor EL, lower line with Poloxamer 388) before freeze-drying (�) and afterfreeze-drying carried out at different heating shelf temperatures ( = −20◦C and � = −30◦C). cp = 5 mg mL−1, FR = 80 mL min−1, W/T = 1,quench volumetric ratio = 0.33, Pchamber = 5 Pa.

In the previous work,13 mannitol was found less suitable aslyoprotectant than sucrose, probably because of its crystallinecharacter. In this case study, the difference between the twolyoprotectants (when the same type of steric stabilizer at thesame concentration is present) is less evident. However, takinginto account not only the mean value of the particle diame-ter, but also the experimentally measured variability aroundthis mean value (shown in Fig. 8), it appears that in case ofhigh mannitol concentration (5%), processed at high tempera-ture (−20◦C), particle diameter increases, in particular whenPoloxamer 388 is used as steric stabilizer (tests 1d/2d vs. 1c/2c).Particle diameter increase is less evident when using manni-tol in case Cremophor EL is the steric stabilizer (tests 1c/2c vs.1a/2a). In case mannitol concentration is lower (2.5%), the effectof the steric stabilizer is roughly the same observed in presenceof sucrose (at the same concentration). From the graphs, it ap-pears that Cremophor EL is better than Poloxamer 388 as asteric stabilizer comparing the cases when the same lyoprotec-tant was used. However, it is quite difficult to determine fromthe obtained data which factor plays the key role in preserv-ing the nanoparticles size. For this reason, experimental datawere analyzed with the objective to evaluate which factor orcombination of factors, namely the percentage of lyoprotectantin the formulation (A), the percentage of steric stabilizer inthe formulation (B), the heating shelf temperature in the pri-mary drying stage (C), has the greatest influence on the meansize of nanoparticles at the end of the freeze-drying process.Estimated single effects and their combination for given solu-tions are illustrated in Figure 9. As illustrated in the Materi-als and Methods section, the effect of the parameter A on themean particle diameter represents how much the mean particle

diameter changes when parameter A is modified. Taking intoaccount that there are also other two parameters, namely Band C, actually four cases have to be considered, thus resultingin Eq. (2). Similarly, the effects of the other parameters (andof their combinations) can be calculated using the equationsgiven by Montgomery,31 thus evidencing their relative impor-tance. It appears that the temperature in the primary dryingstage affects significantly nanoparticles stability for all the for-mulations. It is also clear that the concentration of the stericstabilizers, particularly Cremophor EL, plays a considerablerole in the nanoparticles stabilization when using mannitol (assteric stabilizer). This phenomenon can be explained by thepoor lyoprotective capacity of mannitol caused by its crystal-lization in the freezing stage. This statement is supported alsoby the fact that the mean nanoparticles size is almost indepen-dent of the amount of steric stabilizer in presence of sucrose.

Although the amount of steric stabilizer present in themedium does not greatly influence the mean nanoparticles sizeduring the freeze-drying process, its presence is indispensablefor preserving the PSD of the nanoparticles, as it is shown inFigure 10. In Figure 10a, the percentage of particle with a di-ameter respectively smaller than 300, 500, and 1000 nm beforeand after the freeze-drying process is shown and in Figure 10b,the curves of PSD are illustrated. It appears that the stericstabilizer is indispensable during the lyophilization process forthe preservation of the PSD. It was again confirmed that Cre-mophor EL is superior to Poloxamer 388.

It has been previously shown by Zelenkova et al.13 that man-nitol crystallization in the freezing stage in aqueous systemcan be slightly suppressed by the addition of various stericstabilizers, as Cremophor EL and Poloxamer 388, and, thus,



Figure 9. Effect of single factors [lyoprotectant (a), steric stabilizer (b), and heating shelf temperature (c)] and of their combination on the sizeof nanoparticles after freeze-drying process in case formulations are stabilized with Cremophor EL and sucrose (a), with Poloxamer 388 andsucrose (b), with Cremophor EL and mannitol (c), and with Poloxamer 388 and mannitol (d).

the PSD and the mean nanoparticles size is better preservedby the amorphous mannitol. In this work, it was necessaryalso to determine whether and how the presence of TBA (20%,w/w) affects the crystalline structure of the mannitol at theend of the lyophilization process. For this purpose, the freeze-dried samples were analyzed using XRD. Total concentration ofthe excipients (mannitol and steric stabilizers) in the nanosus-pensions before lyophilization varied between 5% (w/w) and7.5% (w/w) and all samples were prepared and analyzed in thesame operating conditions. In Figure 11, the XRD spectra ofthe freeze-dried formulations are shown. Figure 11a shows themannitol spectra in case the steric stabilizers are not present,and with (last two specimens) or without (first two specimens)the nanoparticles lyophilized in aqueous systems (first andthird sample) and in water–TBA (20%, w/w TBA) systems (sec-ond and fourth sample). The first two spectra of Figures 11band 11c refer to mannitol with Cremophor EL or Poloxamer388, at various concentrations, and with nanoparticles, and thethird one was prepared without nanoparticles. All samples werelyophilized in water–TBA (20% TBA, w/w) systems. It appearsthat not only the steric stabilizers, like the presence of nanopar-ticles, as found in previous work, but also the presence of TBA insolution, strongly affect the mannitol behavior, forcing the for-mation of mannitol *-phase (reference spectrum is illustratedin the Fig. 11d), whereas with pure mannitol in water, repre-sented in the first spectrum in Figure 11a, the orthorhombic

polymorphs of " and $ phases prevail. Thus, the presence ofTBA (20%, w/w) in the formulation containing pure mannitolwithout the nanoparticles appears to promote the formationof mannitol *-phase, and this effect is similar to that of thenanoparticles or of the steric stabilizer (see the second spectrumin Fig. 11a). Nevertheless, when the nanoparticles are presentin the solution, the final phase distribution does not differ fromthe spectrum of the sample lyophilized in aqueous medium, asevidenced by last two spectra in Figure 11a. Although the pres-ence of nanoparticles in pure mannitol causes the formation ofsome amorphous mannitol (as it appears in both cases in Fig.11a), the aggregation of nanoparticles occurred as illustratedin Figure 10. The steric stabilizer favors the formation of theamorphous phase (see Figs. 11b and 11c). It was observed thatthe amount of the amorphous fraction slightly increases whenincreasing the quantity of the steric stabilizer, but not propor-tionally, and it is less than expected. It should be noted thatthe same prevailing mannitol phase was formed regardless ofthe nanoparticles presence, as shown in the lowest spectra inFigures 11b and 11c by the specimens containing the highestconcentration of steric stabilizers (a similar phenomenon wasobserved also at lower concentrations).

An analogous trend can be pointed out for both steric sta-bilizers, even thought the final composition of ", $, and *-mannitol phases is different in the examined samples. WhenCremophor EL was added to the formulations, the mannitol



Figure 10. (a) Percentage of nanoparticles with diameter smallerthan 300, 500, and 1000 nm before freeze-drying (�) and after freeze-drying of three different suspensions: � = sucrose 5% (w/w), � =sucrose 2.5% (w/w) with Poloxamer 388 2.5% (w/w) and • = sucrose2.5% (w/w) with Cremophor EL 2.5% (w/w). (b) Comparison of PSD ofnanoparticles before lyophilization (solid line) and after freeze-drying(dash-dotted line = sucrose 5%, w/w, dashed line = sucrose 2.5%, w/wwith Poloxamer 388 2.5%, w/w, and dotted line = sucrose 2.5%, w/wwith Cremophor EL 2.5%, w/w). cp = 5 mg mL−1, FR = 80 mL min−1,W/T = 1, quench volumetric ratio = 0.33, Pchamber = 5 Pa, Tshelf =−20◦C.

orthorhombic $-phase was almost absent. Instead, after theaddition of Poloxamer 388 a higher amount of $-phase wasobserved and the intensity of the peaks that identify the $-mannitol phase (peaks are marked with an asterisk) increasedproportionally with the amount of Poloxamer 388 in the sam-ple. Similar results were obtained also by Zelenkova et al.13

using an aqueous system for the lyophilization.Finally, it can be noted that steric stabilizers are required for

preserving the nanoparticles size (and the PSD) with mannitol,and their amount is generally related with nanoparticles sta-bility (this is especially true for the Cremophor EL) as pointedout in Figure 8, where it appears that the Cremophor EL issuperior to Poloxamer 388 in the nanoparticles size preserva-tion (nanoparticles were smaller). These finding can be con-firmed by XRD analysis: in presence of Cremophor EL moreamorphous phase (around 12%) is formed with respect to whatoccurs when Poloxamer 388 is used. To conclude, nanoparticlessize and PSD is preserved in presence of steric stabilizers notonly because of the formation of an amorphous phase, that im-mobilizes the nanoparticles, but also thanks to the hydrogenbonds formed between polar group localized on the superfi-cies of PCL nanoparticles and the steric stabilizers, as alreadyexplained.

Figure 11. Comparison of XRD spectra of freeze-dried water–TBA(20%, w/w TBA) formulations containing nanoparticles and mannitol–steric stabilizer in diverse concentrations: (a) from top to bottom: 5%(w/w) mannitol without nanoparticles (water solution), 5% (w/w) man-nitol without nanoparticles (20%, w/w TBA in water) 5% (w/w) mannitolwith nanoparticles (water solution), 5% (w/w) mannitol with nanopar-ticles (20%, w/w TBA in water). (b) From top to bottom: 2.5% (w/w)mannitol and 2.5% (w/w) Cremophor EL, 2.5% (w/w) mannitol and 5%(w/w) Cremophor EL with and without nanoparticles (last spectrum)(20%, w/w TBA in water). (c) From top to bottom: 2.5% (w/w) manni-tol and 2.5% (w/w) Poloxamer 388, 2.5% (w/w) mannitol and 5% (w/w)Poloxamer 388 with and without nanoparticles (last spectrum) (20%,w/w TBA in water). (d) Reference spectrum of mannitol *–phase (JPDFreference number 00–022–1794).

CONCLUSIONS

The use of a water/tert-butanol cosolvent system (20%, w/wTBA) in the production and lyophilization of PCL nanoparticleswas presented in this paper. Solvent displacement method wasused to get nanoparticles in a CIJM. The influence of the poly-mer concentration in the feed, of the feed flow rate, of the waterto tert-butanol flow rate ratio (W/T), and of the quench volumet-ric ratio on the nanoparticles size and on the PSD was studied.It was pointed out that the feed flow rate has a poor effect onthe nanoparticles size, in contrast to the polymer concentra-tion in the feed, which significantly affects the nanoparticlessize. Based on the experimental results, a simple equation wasdetermined, looking for best fit with experimentally measuredvalues, to describe the dependence of the nanoparticles mean



size on the polymer concentration in the feed, on the RejW andon the water to tert-butanol flow rate ratio.

Before the lyophilization process it is not required to elimi-nate the solvent, and this brings many benefits. The influenceof the chosen lyoprotectants (namely sucrose and mannitol), to-gether with that of the steric stabilizers (namely Poloxamer 388and Cremophor EL), in various concentrations at two differentheating shelf temperatures (−20◦C and −30◦C) on the meannanoparticles size at the end of the process of lyophilizationwas studied. Finally, the experimentally measured data werestatistically analyzed and it was determined that the heatingshelf temperature is a factor which has an important impacton the mean nanoparticles size. It was also found that sucroseconcentration strongly affects the mean size of the nanoparti-cles and it is almost independent of the amount of the stericstabilizer present in the medium. In case mannitol was used,the steric stabilizer concentration, especially of Cremophor EL,has a stronger effect on the nanoparticles mean size. It was ob-served that Cremophor EL has better properties for the preser-vation nanoparticles mean size in comparison with Poloxamer388, especially with mannitol. These findings were confirmedby XRD analysis.

LIST OF SYMBOLS

A Percentage of lyoprotectant in the formulation con-taining nanoparticles

A0 Parameter used in Eq. (5) (corresponding to meanparticle size when W/A = 1, RejW = 1, cp/cref = 1)(nm)

B Percentage of steric stabilizer in the formulationcontaining nanoparticles

C Heating shelf temperaturecp Polymer concentration (mg mL−1)cref Reference concentration (mg mL−1)din Inlet jet diameter (m)dout Outlet jet diameter (m)dp Mean nanoparticle diameter (z-average) (nm)dpCAL Calculated value of nanoparticle diameter (m)dpEXP Experimental value of nanoparticle diameter (m)dj

p Sum of the values of mean nanoparticle diameter ob-tained in the n tests carried out in the j-th operatingconditions (m)

E(i) Effect of the i-th operating parameter on the meannanoparticles size (m)

FR Flow rate (mL min−1)FRT tert-Butanol flow rate (mL min−1)FRW Water flow rate (mL min−1)Mw Molecular weight (g mol−1)n Number of repetitions of the testPchamber Chamber pressure (Pa)Rej Reynolds number of the inlet streamRejW Water inlet jet Reynolds numberTshelf Heating shelf temperature (◦C)t Time (s)vj Inlet jet velocity (m s−1)W/A Water flow rate to acetone flow rate ratioW/T Water flow rate to tert-butanol flow rate ratio

Greeks" Parameter used to calculate dp in Eqs. (5) and (6)$ Parameter used to calculate dp in Eqs. (5) and (6)

( Parameter used to calculate dp in Eqs. (5) and (6): f Fluid viscosity (kg m−1 s−1)Df Fluid density (kg m−3)

Acknowledgment

The contribution of Viviana Negro to the experimental investi-gation is gratefully acknowledged.

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Documents

On the Use of tert -Butanol/Water Cosolvent Systems in Production and Freeze-Drying of Poly-ε-Caprolactone Nanoparticles