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European Journal of Pharmaceutical Sciences 41 (2010) 78–85
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journa l homepage: www.e lsev ier .com/ locate /e jps
ne-step preparation of pharmaceutical nanocrystals using ultra cryo-millingechnique in liquid nitrogen
oshiyuki Niwaa,∗, Yasuo Nakanishib, Kazumi Danjoa
Department of Industrial Pharmacy, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku, Nagoya 468-8503, Aichi, JapanDevelopment Center, Moriroku Chemicals Co., Ltd., 18F Shin-aoyama-Bldg. East, 1-1-1 Minamiaoyama Minato, Tokyo 107-0062, Japan
r t i c l e i n f o
rticle history:eceived 16 March 2010eceived in revised form 17 May 2010ccepted 30 May 2010vailable online 8 June 2010
eywords:ryo-millingedia milling
a b s t r a c t
A novel micronization technique for pharmaceutical powders has been established using liquid nitrogen(LN2). Different from the conventional dry milling while cooling the milling pot by LN2, the materialswere directly suspended in LN2 together with hard small spherical balls, called beads, and broken downby agitating intensively. The present beads milling in LN2 was named “ultra cryo-milling” in this paper.The operational conditions including the size/amount of beads made of zirconia and agitation speed wereoptimized to obtain the finer particles. It was found that the original crystals were effectively broken downinto submicron particles, and the ultra cryo-milled particles were much finer and more uniform in size andshape than the conventional jet-milled particles. Dried powder was recovered continuously after milling
iquid nitrogenanonizationirconia beadset milling
process because LN2 was spontaneously evaporated at ambient temperature/pressure. Further, it wasshown that this technique is applicable to the drugs with wide range of physicochemical features includingheat-sensitive and water-soluble drugs. However, the resultant fine particles intrinsically tended to formthe agglomerated masses. The crystalline analysis indicated that the both crystal form and crystallinity ofthe original bulk drugs completely remained after ultra cryo-milling process. The results demonstratedthat the ultra cryo-milling would be a fundamental technique to produce the pharmaceutical nanocrystals
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by one step.
. Introduction
Nitrogen is classified as an inert gas due to its poorly reactiveroperty together with noble gases such as helium, argon, and son. Liquefied inert gases, which are obtained by extremely cool-ng or compressing these gases, are attracting attention in manyndustrial fields from the viewpoint of their unique characteris-ics. Especially liquid nitrogen (LN2) exists inexhaustibly in thetmosphere and is considered to be clean resources from the globalnvironmental perspective. It is a cryogenic liquid and widely uti-ized as a coolant or refrigerant in the various industries and thetate-of-the-art scientific research activities such as superconduc-ivity (Hammerl et al., 2000), cryopreservation of cultured cellsMiyamoto et al., 2009), medical sciences (Rossi and Rabin, 2007;raunfelder, 2009), foods industry (Tyler et al., 1988), and so on. In
ddition to cryogenic properties, LN2 has other unique character-stics chemically as well as physically such as poorly reactive andolubilized properties, low viscosity and surface tension, sponta-eous volatility, and wide availability on industrial market.∗ Corresponding author. Tel.: +81 52 839 2662; fax: +81 52 839 2662.E-mail address: [email protected] (T. Niwa).
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928-0987/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.ejps.2010.05.019
© 2010 Elsevier B.V. All rights reserved.
New drug candidates produced in discovery researches haveecome more hydrophobic and less water-soluble as a result of
n vitro highly sensitive affinity assay using the sophisticated high-hroughput screening equipment. Many technologies for particleize reduction of the drug substances have been developed andpplied in the pharmaceutical researches in order to enhance theastrointestinal dissolution and absorption of such poorly water-oluble drug substances. Current micronization technologies cane divided into two categories; one is the build-up processes thatrecipitate the fine particles from dissolved molecules and another
s the break-down processes that start from a large-sized powdero be reduced in size. The mechanical milling techniques in dryondition such as ball milling, hammer milling and jet milling, onef the latter processes, have been traditionally used in pharmaceu-ical manufacturing due to their wide application to the materialsnd simple process. However, it is said that reduction of particleize is limited to 2–3 �m or so, because of the secondary coaggre-ation occurring between particles as a result of Van der Waals’
nteraction and electrostatic force (Jimbo, 1986).In order to overcome the “1-micron limitation”, there has been areak-through in particle size reduction strategies from microniza-ion to nanonization during the last decade. Actually the media
illing and high-pressure homogenization techniques, which were
harmaceutical Sciences 41 (2010) 78–85 79
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T. Niwa et al. / European Journal of P
onducted in the aqueous suspension, have successfully producedhe nanometer-sized drug particles (Jacobs and Müller, 2002; Hecqt al., 2006), hence forming nanosuspension. Especially the high-hear beads milling method in aqueous phase, which is well-knowns “NanoCrystal technology” (Merisko-Liversidge et al., 2003), hasver given four pharmaceutical products currently on marketJunghanns and Müller, 2008). However, the resultant milled sus-ension has to be dried to develop the tablets and capsules, whichre major solid dosage forms. So, such wet milling techniques haveot been popular as a fundamental manufacturing process in phar-aceutical industry so far.In this research, the novel wet milling technique was developed
sing cryogenic fluid, e.g. liquid nitrogen, liquid argon, etc., in ordero overcome the both disadvantages of dry milling and wet millingn aqueous media mentioned above. Actually LN2 was adopted as aispersing media instead of water because few solid materials areissolved in this cryogenic liquid. In addition, LN2 has potentialo become good physical barrier against secondary coaggregationuring milling process because it can penetrate into void spacesnd micropores between and inside the particles due to its inher-nt low surface tension (8.85 mN/m at −196 ◦C, around one-eightho water) and low viscosity (0.158 mPa·s at −196 ◦C, around one-ixth to water) (Vasserman, 1966; Brovik, 1960). Furthermore, itsryogenic property (boiling point: −196 ◦C) is quite attractive toreak the materials because the substances usually become brit-le under such super cold condition. Its spontaneous vaporizations very profitable for this technique because the dry powders areirectly available after milling process. Needless to say, it is advan-ageous that LN2 is industrially available at a reasonable price andnert, nonflammable, and nontoxic. The present beads milling atrozen state was named “ultra cryo-milling” in this paper. There areome cryogenic technologies previously reported in pharmaceuti-al field using LN2 as a dispersing medium as well as a refrigerantspray freezing into liquid (Rogers et al., 2002; Hu et al., 2002);pray freeze drying (Kondo et al., 2009; Niwa et al., 2009); reviewBarot et al., 2006)), but the milling in cryogenic media has not beeneported to develop the pharmaceutical dosages, to our knowledge.he aim of this study is to evaluate the milling efficiency of theresent technique and to establish the standard operational con-ition in order to produce the finer particles. Then, the efficiencynd possibility as a novel milling technique was investigated, whileomparing to conventional jet milling.
. Materials and methods
.1. Chemicals
Phenytoin was purchased from Wako Pure Chemical Co., Ltd.Osaka, Japan). Ibuprofen and salbutamol sulfate were providedy Knoll Co., Ltd. (Germany) and Dolder Co., Ltd. (Switzerland),espectively. Liquid nitrogen (LN2) was manufactured by anuto-supplying equipment for NMR (nuclear magnetic resonance)NS-100, Iwatani Industrial Gases Co., Ltd., Tokyo, Japan). All otherhemicals and solvents were of analytical reagent grade, andeionized-distilled water was used throughout the study.
.2. Manufacturing instruments
The batch-type wet milling machine (RMB-04, Aimex Co., Ltd.,
okyo, Japan) available on the market, which is generally usedn wet milling with beads in aqueous solution, was improved toxecute ultra cryo-milling in LN2. In this study, the 400 mL capac-ty vessel and its equipped rotation shaft and disks were newlyade of zirconia (zirconium oxide) with keeping their original
2
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ig. 1. Schematic diagram of ultra cryo-milling apparatus in LN2. The beads, disks,haft, and inner wall of vessel are made of zirconia.
esign such as shape and size. Zirconia hard small balls, called asbeads” hereafter, with 1 mm, 0.6 mm, 0.3 mm and 0.1 mm in diam-ter (YTZ-1, -0.6, -0.3, -0.1) were purchased from Nikkato Co., Ltd.Osaka, Japan).
.3. Preparation of milled particles
The ultra cryo-milling was carried out by colliding and grindinghe suspended materials with zirconia beads within the vessel insame manner as a conventional wet beads milling or wet mediailling. Liquid nitrogen was used as a dispersing medium instead of
queous solution. The schematic diagram of the preparation appa-atus used for current milling process was illustrated in Fig. 1. Theilling vessel was filled with 550 g of zirconia beads, which packing
olume is equivalent to approximate 150 mL irrespective of beadiameter. Before the operation of milling, the LN2 was graduallyoured into the vessel and the parts of machine (vessel, shaft, disks,nd bead) were pre-cooled to prevent spluttering by intense boil-ng. Then, 15 g of phenytoin, which was mainly used as a modelrug in this study, was suspended in LN2 filling up to 360 mL andhe rotation disks were agitated at a preset speed. At appropriatentervals, LN2 was additionally poured into the vessel to compen-ate for its volatilized volume. After 30 min of agitation, the motoras stopped and the resultant slurry of the drug was separated
rom beads by passing through the sieve with appropriate opening.he dried powder consisted of the milled particles was collectedfter spontaneously evaporating the LN2 at ambient condition. Inrder to avoid adsorbing the moisture from air, the whole operationas performed under atmosphere of nitrogen gas flow.
Apart from the ultra cryo-milling mentioned above, the jetilling was also executed as a reference of milling performance.
0 g of phenytoin particles were size-reduced using jet millachine (A-O, Seishin Enterprise Co., Ltd., Tokyo, Japan) operated
t 0.7 MPa air pressure and a feed rate of 0.4–0.8 g/min. Ibuprofennd salbutamol sulfate were also milled by both methods to con-rm the application range of the current technique. The resultantilled particles were stored in glass vials in a desiccator at room
emperature before the characterization measurement.
.4. Morphology and particle size distribution (PSD)
The morphology and size of the milled particles were observedompared to the original bulk particles under a scanning electron
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0 T. Niwa et al. / European Journal of P
icroscope (SEM, JSM-6060, JEOL Ltd., Tokyo, Japan). The particlesere fixed to the special sample stage and coated using a plat-
num sputtering equipment (JFC-1600, JEOL Ltd.). The particle sizeistribution of the original and milled particles dispersed in dryondition was measured by a laser diffraction scattering methodsing the diffractometer with the dry dispersing unit (LMS-30,eishin Enterprise Co. Ltd., Tokyo, Japan). The particles were dis-ersed into dry air at fixed air pressure of 0.4 MPa. On the otherand, the size distribution dispersed in wet condition was mea-ured by same method using the other diffractometer (SALD-2100,himadzu Corporation, Kyoto, Japan). The particles were dispersednto water with voltexing for 30 s. The diameters at the 10%, 50%,nd 90% of the cumulative volume distribution (D10%, D50%, D90%,espectively) were represented as size distribution. In addition, theumulative weight percentage of the particles less than 1 �m iniameter was defined as “submicron %” to assess the milling effi-iency. The specific surface area of the particles was measured bysurface area analyzer (Nova-1000, Yuasa Ionics Co. Ltd., Osaka,
apan) using argon gas sorption process. The surface area per pow-er unit weight was calculated based on the fitting of the adsorptionata to the BET equation.
.5. Crystalline analysis
X-ray powder diffraction (XRPD) analysis was conducted usingGeiger-Flex difractometer (RAD-2VC, Rigaku Co., Tokyo, Japan)ith CuK�1 radiation and a Ni filter at a voltage of 30 kV and a cur-
ent of 20 mA. Samples were scanned over 2� range of 5–35◦at a ratef 5◦/min. Differential scanning calorimetry (DSC) was performedsing DSC instrument (DSC-60, Shimadzu Co. Ltd., Kyoto, Japan).round 5 mg of each test sample was placed in an aluminum pan.he heating program was carried out using a modulated settingt 10 ◦C/min over 30–310 ◦C under nitrogen gas flow. The heat ofusion was calculated by an endothermic peak area of thermogramt melting point. The crystallinity (Xcr) was calculated according tohe following Eq. (1).
cr = �H
�H0× 100 (1)
here �H0 and �H are the heat of fusion of the original crystalsnd the milled crystals, respectively.
. Results and discussion
.1. Concept of ultra cryo-milling in liquid nitrogen (LN2)
The ultra cryo-milling is one of the micronization or nanoniza-ion techniques of the particles. The powdery or granular materialsere suspended in liquid phase and pulverized and ground with
o-suspending zirconia beads within the vessel. The preparationrocess is basically same as the well-known wet beads milling tech-ology in aqueous medium, frequently reported in pharmaceuticaleld (Merisko-Liversidge et al., 1996, 2003; Vogt et al., 2008a,b;anaka et al., 2009; Niwa and Hashimoto, 2008). Generally, theaterials are effectively milled to the much finer particles by wetilling methods than dry milling methods such as jet milling
ecause the dispersing liquid penetrated into voids between theroken particles could effectively disturb their re-aggregation assterical barrier. The ultra cryo-milling technique is quite novel
nd differentiated from the conventional wet milling methods in
erms of the dispersing medium. The drug powder is suspendedn LN2, which is a cryogenic liquid around −200 ◦C, and milled byard small beads. When the materials are cooled at such extremelyow temperature, they usually get brittle property. For example,ven the elastic substance like rubber can be fractured as glass.
w0ldd
ceutical Sciences 41 (2010) 78–85
o, the beads milling technique suspended in LN2, named “ultraryo-milling” by the authors, would be advantageous to producehe micronized or nanonized particles. It also should be empha-ized that the present technology is basically discriminated fromry milling one using LN2 as a coolant or a refrigerant, which isommonly known as “cryo-milling or cryo-grinding” (Wu and Ho,006; Chieng et al., 2008; Moribe et al., 2008). LN2 is only used toool the frictional heat generated in such conventional dry millingperation. The zirconia beads were adopted in the current millingrocedure because they have an excellent frictional wear-resistantroperty even at extremely low temperature.
Further, in case of the wet milling in aqueous medium, thereated aqueous suspension should be additionally spray-dried orreeze-dried in order to obtain the dry powder or to circumventhe physical stability issue during storage (Van Eerdenbrugh et al.,007, 2008; Kawabata et al., 2010). In contrast, the drying process
s not required after finishing the milling process because LN2 ispontaneously evaporated at ambient temperature/pressure. Theicronized or nanonized dry particles can be prepared by one oper-
tional step, so it can be said that the ultra cryo-milling is a hybridechnique incorporated both advantages of dry milling and wet
illing in aqueous medium. In addition, the ultra cryo-milling tech-ology can apply even the water-soluble drugs since LN2 does notissolve any substances, which is another advantage different fromet milling in aqueous medium.
.2. Optimization of driving conditions for ultra cryo-millingrocess
The loading of materials into the milling vessel and the drivingonditions on the milling machine were investigated in order toroduce the finer milled particles, ultimately nanometer-sized par-icles. Based on the preliminary manufacturing trials it was foundhe loading of the zirconia beads should not be set over 50% vol-me against the vessel capacity to smoothly circulate the beads
nside the vessel. Further, the evaluation of size distribution of theilled particles indicated that the milling efficiency started to be
eclined under 25% volume of beads loading. Therefore, the loadingf the beads was set to 37.5% volume, that is 150 mL of the packingolume, throughout this study. With respect to the drug loading inhe vessel and agitation time, these two parameters are expected toe closely correlated to successfully perform the milling. In otherords, the larger the amount of drug was loaded, the longer the
gitation time is required. In this report, the drug loading and agi-ation time were fixed to 15 g and 30 min, respectively, irrespectivef the drugs used since the size reduction was reached to a plateaut 30 min.
The effect of agitation speed of the rotation shaft and disksn particle size was also investigated under the fixed conditionentioned above. The particle size distribution was measured
n such two dispersion ways as the recovered dry powder wasispersed into (1) compressed air and (2) water to simulate theispersion behaviors (1) in the consecutive powder processing,.g. mixing or fluidized-bed granulation (dry condition), and (2) inhe gastrointestinal fluid after oral administration (wet condition),espectively. The cumulative distribution curves of the particlesrepared at slow and fast agitation are shown in Fig. 2, in whichhe left and right figures are results in the dry and wet disper-ion conditions, respectively. As expected, the distribution curvesere found to be shifted to smaller size when the agitation speed
as increased from 550 to 1600 rpm with both bead sizes of 1 and.3 mm. It was generally known that the beads are vertically circu-ated passing through the holes of the disks and the gap betweenisks and vessel wall during the agitation as illustrated in Fig. 1. Therug particles are pulverized and ground by the collision impact
T. Niwa et al. / European Journal of Pharmaceutical Sciences 41 (2010) 78–85 81
F enyto1 d (�)w
gbgmddtttswt
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tlaetf
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tmmTtfgtaparticles and the original bulk particles were observed by SEM as
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ig. 2. Effect of agitation speed on particle size distribution of ultra cryo-milled ph600 rpm and (©) 550 rpm using 1-mm-sized zirconia beads, and at (�) 1600 rpm anhile dispersing the milled particles in dry air (left) and water (right).
enerated when the particles are powerfully sandwiched betweeneads with high speed. Therefore, the more powerful collision andrinding between beads generated by faster agitation at 1600 rpmight result in successful milling in both bead sizes. Comparing
ispersion ways there is little difference in both the dry and wetistributions. The much higher speed over 1600 rpm was foundo be not good way to reduce the milled size in the current sys-em because the particles and beads tended to move together athe same speed. In addition, such intense agitation is also likely toplash and overflow LN2. Therefore, the further increase of speedas avoided from the performance and researcher’s safety perspec-
ives.On the other hand, it was found that the milling performance
as not remarkably affected by the diameter of zirconia beads inhe range of 0.1–1 mm at 1600 rpm of agitation as shown in Fig. 3.he increase of size of hard beads might give the higher collisionower per one sphere, whereas the decrease of beads size wouldnhance the collision frequency in the system. Therefore, theseesults suggested that the magnitude of milling derived from colli-ion energy could be dependent on the number of collision as wells the weight of a bead as also reported by Takatsuka et al. (2009).
n the conventional wet milling in aqueous medium, the smallereads with 0.05 or 0.1 mm in diameter are generally used in ordero acquire high performance (Vogt et al., 2008a,b; Tanaka et al.,009), but such small beads are not easy to handle in the prepara-stut
ig. 3. Effect of beads diameter on particle size distribution of ultra cryo-milled pheny600 rpm using (�) 1 mm, (�) 0.6 mm, (�) 0.3 mm, and (×) 0.1 mm-sized zirconia beads.ir (left) and water (right).
in particles. The phenytoin particles were ultra cryo-milled under agitating at (�)550 rpm using 0.3 mm-sized zirconia beads. The size measurement was performed
ion process. The effective fracture was achieved even using mucharger beads with 1 mm in diameter, which is one of the feature anddvantage in this cryo-technology. The beads with 1 mm in diam-ter were selected as a standard condition in this research becausehe milled products suspended in LN2 could be easily separatedrom beads by passing the suspension through the sieve.
.3. Ultra cryo-milling compared to jet milling with respect toanonization and crystalline property
In order to evaluate the performance of the ultra cryo-milling,he phenytoin powders were also jet-milled. The reason why the jet
illing was adopted as a control is as follows: (1) Dry powder of theilled particles is directly available in both milling processes. (2)
he ultra cryo-milling is regarded as an alternative size-reducingechnique to the traditional and conventional milling one, which isollowed by the consecutive manufacturing process, i.e. blending,ranulating or tableting. (3) Jet milling is known as the most effec-ive way among the conventional dry milling, e.g. hammer millingnd ball milling. The morphological appearances of the both milled
hown in Fig. 4. The original crystals of phenytoin (A) were effec-ively broken into the submicron to 2–3 �m particles in size byltra cryo-milling (Fig. 4, B-1 and B-2). On the other hand, the par-icles larger than 5 �m in size were frequently observed together
toin particles. The phenytoin particles were ultra cryo-milled under agitating atThe size measurement was performed while dispersing the milled particles in dry
82 T. Niwa et al. / European Journal of Pharmaceutical Sciences 41 (2010) 78–85
Fig. 4. Scanning electron microphotographs of phenytoin particles. Key: (A) original particles; (B-1 and B-2) ultra cryo-milled particles in LN2; (C-1 and C-2) jet-milledparticles.
F left) a(
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ig. 5. Particle size distribution profiles of phenytoin particles dispersed in dry air (�) jet-milled particles.
ith 2–3 �m particles on jet milling (Fig. 4, C-1 and C-2). Thesehotos proved that the ultra cryo-milled particles were much finernd more uniform in size and shape than the jet-milled parti-les although the kinetic energy, namely collision energy, in theet milling seems to be much higher than that in the ultra cryo-
illing. The difference in size and shape would be attributed tohe frequency of mechanical impact on the materials derived fromhe both methods. The collision and grinding with hard beads was
epeated during running in the batch-type process of the ultra cryo-illing method, assuming to lead to production of the smaller andore uniform particles. In addition, the transformation of the crys-als to fragile characteristics under cryogenic condition is likely toe another reason to promote nanonization effectively. The visual
Acpwn
able 1epresentative particle sizes of the original and milled particles of phenytoin measured a
Analysis Samples D10% (�m
Dry dispersion Original particles 4.26Ultra cryo-milled particles 0.79Jet-milled particles 1.14
Wet dispersion Original particles 6.84Ultra cryo-milled particles 0.62Jet-milled particles 1.11
a D10%, D50% and D90% are diameters at the 10%, 50% and 90% of the population distributb Submicron (%) is the weight ratio of the particles smaller than 1 �m in size in the cum
nd water (right). Key: (�) original particles; (�) ultra cryo-milled particles in LN2;
bservation revealed that the ultra cryo-milling was more effectiveltrafine-processing technique than jet milling. Thus, the milling toubmicron-sized drug particles, named nano-milling, was realizedy the ultra cryo-milling.
The particle size distribution profiles and the representativearticle sizes of each particle are summarized in Fig. 5 and Table 1,espectively. The original phenytoin powder, available as an ana-ytical reagent grade, had sharp particle distribution with 5–20 �m.
s also indicated in SEM photographs, the ultra cryo-milled parti-les were found to have smaller distribution compared to jet-milledarticles by both dry and wet dispersion. However, the size rangeas a bit broadened from around 0.3 toward 8 �m, which wasot consistent with visual observation. The size distribution largerfter dry and wet dispersion.
)a D50% (�m)a D90% (�m)a Submicron (%)b
8.51 15.3 0.002.02 5.23 21.53.24 6.82 9.07
12.7 21.1 1.011.84 5.27 27.54.29 7.95 9.06
ion.ulative distribution curve.
T. Niwa et al. / European Journal of Pharmaceutical Sciences 41 (2010) 78–85 83
F inal ap
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ig. 6. X-ray powder diffraction patterns (left) and DSC profiles (right) of the origarticles in LN2; (C) jet-milled particles.
han 2–3 �m detected by laser diffraction scattering method wasssumed to be caused by the agglomerated particles because a partf the milled particles might not be fully mono-dispersed as anndividual particle in the air as well as water. In spite of insuf-cient dispersion of the milled particles, the submicron % value,hich was an index expressing the extent of nano-milling, was
ignificantly increased to 22–28% compared to original (0%) andet-milled particles (9%). However, the size distribution of ultraryo-milled particles was not smaller than that of the aqueousedia milled particles, which shows 100–200 nm or less in average
iameter if the appropriate dispersing agent is applied (Merisko-iversidge et al., 2003).
The effect of milling operation on the crystalline property ofhenytoin was investigated by X-ray powder diffraction (XRPD)nd DCS. XRPD patterns and DSC curves of the original and theilled particles shown in Fig. 6 indicated that the crystal form of
he original bulk particles remained after both milling processesince the positions of the diffraction peaks and endothermic peakerived from the crystal of the drug were not changed at all. Therystal habit of three samples was also identical although several
rystal habits of phenytoin were previously reported (Nokhodchit al., 2003). However, the jet-milled product shows a peak widen-ng and the resolution of the peaks is somewhat becoming worsen whole XRPD chart (C). As reported by Steckel et al. (2003) andhikhalia et al. (2006), milling is a high-energy process causings(lat
ig. 7. Scanning electron microphotographs of (A) ibuprofen and (B) salbutamol sulfatet-milled particles.
nd milled particles of phenytoin. Key: (A) original particles; (B) ultra cryo-milled
he crystal lattice to be disordered on the surface of the newlyormed particles. On the other hand, the intensity of the peaksn XRPD profile of the ultra cryo-milled product appears to benchanged even after milling treatment. In order to evaluate themorphous/crystalline property quantitatively, the relative ratio ofeat of fusion against the original bulk, which was defined as crys-allinity (Xcr), was calculated from the endothermic peak area onSC thermogram. The crystallinity of the jet-milled particles was
lightly decreased (Xcr = 81%), but the ultra cryo-milled particlesere kept crystallinity of the original one (Xcr = 101%). The acti-
ated energy level was assumed to be immediately relaxed underuper cold liquid. These results suggest that the ultra cryo-millingn LN2 is novel milling technique producing submicron-sized crys-als maintaining same crystalline state as the original one. So, theltra cryo-milled product was named nanocrystals in this paper.
.4. Expansion of application for ultra cryo-milling
In order to make sure that the present technique is applicableo the drugs with various characteristics, ibuprofen and salbutamol
ulfate were also applied as model drugs with low melting point75–77 ◦C) and water-soluble property, respectively. The morpho-ogical appearances of each milled particles were observed by SEMs exhibited in Fig. 7. As shown in the milled particles of pheny-oin (see Fig. 4), the uniform particles in size and shape were alsoe particles. Key: (1) original particles; (2) ultra cryo-milled particles in LN2; (3)
84 T. Niwa et al. / European Journal of Pharmaceutical Sciences 41 (2010) 78–85
Table 2Mean size and percentage of submicron particles in the original and milled particles of ibuprofen and salbutamol sulfate measured after dry and wet dispersion.
Materials Samples D50% (�m)a, submicron (%)b
Dry dispersion Wet dispersion
Ibuprofen Original particles 50.6 0.00 28.5 0.00Ultra cryo-milled particles 2.47 9.20 9.34 1.57Jet-milled particles 6.61 3.05 7.00 0.00
Salbutamol sulfate Original particles 67.2 0.00 NMc NMUltra cryo-milled particles 4.80 19.4 NM NMJet-milled particles 5.73 7.83 NM NM
a D50% is diameter at the 50% of the population distribution.b Submicron (%) is the weight ratio of the particles smaller than 1 �m in size in the cumc NM: not measured because the samples were dissolved in water.
F(
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ig. 8. Specific surface area of the original and milled particles of each drug. Key:black) phenytoin, (gray) ibuprofen, (white) salbutamol sulfate.
roduced after ultra cryo-milling treatment in both ibuprofen (A-) and salbutamol sulfate (B-2) although the original sizes of theseompounds (A-1, B-1) were much larger than that of phenytoin aslso indicated in Table 2. These photos revealed that the ultra cryo-illed particles size seemed to be somewhat larger than that in
ase of phenytoin, but the fine particles from submicron to 2–3 �mn size were mainly produced in both drugs. The water-solublerugs, which are not applicable to wet milling in aqueous medium,ere also successfully micronized. In contrast, the jet-milled parti-
les of ibuprofen had round and edgeless structure (A-3) probablyue to the partial melt around the surface of crystals during thereatment. This surface fusion might decrease the brittleness of
aterials, resulting in larger particles than those of ultra cryo-illing. Further, the stick-shaped or plate-shaped coarse crystals
aving larger major/minor axis ratio were still remained in the jet-illed particles of salbutamol sulfate (B-3). As a result, about 20%eight of ultra cryo-milled salbutamol product could be dispersed
s submicron-sized particles in the dry condition in spite of noth-ng in original particles as shown in Table 2. The nano-dispersionf ibuprofen-milled particles was not good in dry and wet condi-ions probably due to strong tendency of agglomeration. Especiallyt was found that the dispersed ibuprofen particles were immedi-tely re-assembled to form the agglomerates in water, resulting innconsistent submicron % in wet condition with those in dry condi-
ion. The spectroscopic analysis in HPLC demonstrated that all threerugs were not chemically decomposed at all during treatmentnder cryogenic condition.Finally, the specific surface area was evaluated to estimate theharmaceutical performance of each particle (Fig. 8). The ultra
(
ulative distribution curve.
ryo-milled particles had two times higher specific surface areaalues than the jet-milled particles in any drugs as anticipatedrom morphological viewpoint. Their areas were enlarged 3.5, 3.7,nd 7.0 times wider than those of original particles in case ofhenytoin, ibuprofen, and salbutamol sulfate, respectively. Sup-ose all particles supplied to the measurement are composed ofame sized-spheres (diameter = d, cm) with smooth surface and noores, and the particle density is 1.3 g/cm3, the surface area per uniteight is expressed as 4.6/d [cm2/g]. When the specific surface area
alues are 1, 3, and 7 m2/g, the diameters of spherical particles areathematically calculated to 4.6, 1.5, and 0.66 �m, respectively.
he ultra cryo-milled particles of phenytoin having 2.7 m2/g of spe-ific surface area value are considered to be an assembly of sameized-spheres with 1.7 �m in diameter. Although the actual parti-les have irregular shape, rough surface and pores, the theoreticalalue calculated from specific surface area is not assumed to beide of the mark. Accordingly, the ultra cryo-milled particles are
uperior to jet-milled particles in enlargement of surface area. It istrongly expected that this technology would improve the dissolu-ion behavior for poorly water-soluble drugs.
. Conclusion
In conclusion, the ultra cryo-milling technique was developeds a fundamental micronization processing of drug substances.he standard operational conditions were optimized to producehe submicron-sized crystalline particles, named nanocrystals. Theenefits of the novel ultra cryo-milling technique established in theresent experiment are summarized as follows:
1) Microparticulation: With this technique it is feasible to breakthe drug crystals into submicron-sized particles, which arenot usually produced by conventional dry milling methodsincluding the most powerful jet milling. Liquid nitrogen playskey roles of a dispersion medium disturbing coaggregationbetween particles as well as a coolant relaxing mechanical heat.
2) Quasi-drying process: This technique is strongly orientedtoward the formulation design of solid dosage forms, e.g. tabletsand capsules because dry powder is directly available aftertreatment. The spontaneous vaporization of LN2 is highly dif-ferentiated from wet milling in aqueous medium.
3) Mild process for crystalline structure: The crystal form of thedrug is kept unchanged even after breaking down. The pro-cess without causing amorphism has no concern about physicalstability during storage for developing the pharmaceutical
products.4) Wide application: This technique is applicable to the materi-als with wide range of physicochemical properties. There is noalternative milling technique for the heat-sensitive, oxidizableand water-soluble materials in particular. The treatment of the
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biomaterials or biomedicines such as phospholipids, proteins,enzymes, and antibodies would have potential to develop thenovel drug delivery particles such as spontaneously formingvesicles in biological fluids, e.g. liposome, micelle, complex.
Finally, the milled particles intrinsically tend to agglomer-te each other after evaporating LN2. In order to enhance theiredispersion property in dry and wet conditions, the additionalormulation design would be required. Some excipients improv-ng the aqueous wettability would be indispensable for especiallyoorly water-soluble drugs. The authors’ consecutive research ono-milling with anti-adhesion agents or aqueous dispersing agentsill be reported in our following paper.
cknowledgement
The authors are grateful to Ms. Tomomi Yamamoto for her excel-ent technical assistance throughout this work.
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