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Application No.: 11/520,479 1 Docket No.: 638772000109
IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
In re Patent Application of: Neil P. DESAI et al.
Application No.: 11/520,479
Filed: September 12, 2006
For: NOVEL FORMULATIONS OF PHARMACOLOGICAL AGENTS, METHODS FOR
THE PREPARATION THEREOF AND METHODS FOR THE USE THEREOF
Confirmation No.: 8972
Art Unit: 1611
Examiner: T. Love
SUPPLEMENTAL DECLARATION OF NEIL P. DESAI PURSUANT TO 37 C.F.R §
1.132
Commissioner for Patents
P.O. Box 1450
Alexandria, VA 22313-1450
Dear Madam:
I, Neil P. Desai, declare as follows:
1. This declaration is in addition and supplemental to the 37
C.F.R. §1.132 declaration
("the Previous Declaration") previously submitted to the Patent
Office on January 27, 2012.
2. I have reviewed the Office Action dated May 2, 2013. I
understand that claims in the
above-captioned patent application remain rejected as being
obvious over one of Abraxis' earlier
patents, U.S. Pat. No. 5,439,686 ("Desai"), for which I am also
a named inventor, in view of U.S. Pat.
No. 5,407,683 ("Shively"). In this supplemental declaration, I
provide more information about the
data presented in the Previous Declaration as well as the cited
reference Desai.
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Application No.: 11/520,479 2 Docket No.: 638772000109
3. In the Previous Declaration, I presented, in part,
experimental data showing the
advantageous properties of the nanoparticle formulations recited
in the claims of the above-captioned
patent application ("the '479 application"). The experiment
compared the physical stability of two
pharmaceutical formulations (Composition 1 and Composition 2)
containing nanoparticles
comprising a solid core of paclitaxel and an albumin coating at
a paclitaxel concentration of 5 mg/ml.
4. As discussed in the Previous Declaration, upon storage at 40
oc for 24 hours, 1 there was a distinctly visible sediment layer at
the bottom of the vials containing Lot 1 and Lot 2 of
Composition 2 indicating instability of Composition 2. Exhibit
1; See also Exhibit 3 of the Previous
Declaration. Such sedimentation was not observed in the vial
containing Composition 1.
Microscopic observation of the formulations stored at 40 oc for
24 hours at 400x magnification revealed large particles in
Composition 2 indicating particle growth and aggregation, which
were not
observed in Composition 1. Exhibit 2; See also Exhibit 4 of the
Previous Declaration.
5. Further, as discussed in the Previous Declaration, upon
storage at 40°C for 24 hours,
the weight mean diameter of the nanoparticles in Composition 1
remained unchanged. In
Composition 2, by contrast, the weight mean diameter of the
nanoparticles increased significantly
upon storage demonstrating instability of Composition 2. Exhibit
3; See also Table 1 of the Previous
Declaration.
6. The table below summarizes and provides additional particle
size characteristics of the
two different formulations tested in the experiment.2
Formulation Weight Mean 95% Weight 99% Weight Diameter, nm
Distribution (D95), nm Distribution (D99), nm
Composition 1 140 240 282 Composition 2, Lot 1 245 500 633
Composition 2, Lot 2 228 496 638
1 Storage at 40 °C for 24 hours is equivalent to storage at room
temperature for at least three days.
2 Particle size was determined by disc centrifugation method
immediately after reconstitution of the formulations at about 5
mg/ml. Size may differ slightly when using a different measurement
method such as dynamic light scattering.
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Application No.: 11/520,479 3 Docket No.: 638772000109
The 95% and 99% weight distribution in the table above provides
the size in nanometers below which
95% and 99% by weight of the particles lie, respectively. For
example, in Composition 1, 95% of the
particles in the formulation have a particle size below 240 nm.
In Composition 2, 95% of the particles
in the formulation have a particle size below 500 (Lot 1) and
496 nm (Lot 2). In Composition 1, there
was no detectable percentage of nanoparticles that have a size
above 400 nm, with 99% of the
particles lying below 282 nm. In Composition 2, by contrast, at
least 10% of the nanoparticles in the
formulation had a particle size that was above 400 nm, with 99%
of the particles lying below 633 (Lot
1) and 638 nm (Lot 2).
7. The diagram below further illustrates the 99% weight
distribution of the two different
formulations.
Composition 1 Composition 2
s 0 2.82.nm 633nm >1000nm
8. Notably, both Composition 1 and Composition 2 are
albumin-coated paclitaxel
nanoparticle formulations having a particle size below 1000 nm,
yet they behave differently in
stability assays. Composition 1, which contains no detectable
percentage of nanoparticles that have a
size above 400 nm, was shown to be stable at paclitaxel
concentration of 5 mg/ml. By contrast,
Composition 2, which contains nanoparticles slightly greater
than 400 nm, was unstable at the same
paclitaxel concentration under the same conditions. This result
was unexpected.
9. As discussed in the Previous Declaration, physical stability
is a key consideration for
ensuring safety and efficacy of nanoparticle drug products. The
tendency of nanoparticles to
precipitate and/or increase in size (for example by aggregation)
increases as the drug concentration
increases. For example, an increase in drug concentration in a
nanoparticle formulation can result in
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Application No.: 11/520,479 4 Docket No.: 638772000109
an increase in particle concentration, namely, the number of
particles per unit volume. An increase in
particle concentration in tum would increase the frequency of
collision of the particles and thus
increase the tendency of the particles to aggregate and become
unstable. This is demonstrated in
Bums et al., Langmuir 1997, 13, 6413-6420 (Exhibit 4), for
example, which examined particle
aggregation in various formulations having different particle
concentrations. The authors concluded
that "[a]s the particle concentration is increased, the
aggregate growth is more rapid, most likely due
to the increased collision frequency." See also Kallay et al.,
J. Colloid and Interface Science 253,
70-76 (2002) (Exhibit 5) at page 75 ("the aggregation rate is
proportional to the square of the particle
concentratiOn.... . . ")
10. The estimated particle concentration for Composition 1
discussed above, namely, the
albumin-coated solid paclitaxel nanoparticle formulation having
a particle size less than 400 nm at
paclitaxel concentration of 5 mg/ml, is about 8.0 x 1013 /m1.3
The stability of such a formulation at 5
mg/ml or higher was unexpected based on the high particle
concentration.
11. The stability of albumin-coated paclitaxel nanoparticle
formulation having particle
size less than 400 nm is in stark contrast with that of a
different non-albumin based paclitaxel
nanoparticle formulation having particle size less than 400 nm.
In a study conducted to compare the
physicochemical characteristics and stability of two different
commercially-approved nanoparticle
formulations ofpaclitaxel, namely, Abraxane® (an albumin-coated
solid paclitaxel nanoparticle
formulation having particle size less than 400 nm, similar to
Composition 1 described above) and
Genexol-PM ®(a non-albumin polymeric-micelle formulation
ofpaclitaxel having particle size less
than 400 nm), only Abraxane® was shown to be stable at 40 oc
over 24 hours at paclitaxel concentration of5 mg/ml while the
Genexol-PM ®formulation showed excessive precipitation under
these conditions. Ron et al., 99th AACR Annual Meeting Abstract,
No. 5622 (Exhibit 6). This study
further illustrates the difficulty and challenge in obtaining
paclitaxel nanoparticle formulations having
3 The particle concentration is estimated with the assumption
that the average particle size of the particles in the formulation
is about
140 nm and the particle density is about 1165 kg/m3.
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Application No.: 11/520,479 5 Docket No.: 638772000109
particle size less than 400 run that are stable at paclitaxel
concentration of 5 mg/ml or higher, and the
unexpected stability of the claimed albumin-coated solid
nanoparticle formulation.
12. Thus, albumin-coated paclitaxel nanoparticle formulations
having particle size less
than 400 run were stable at 5 mg/ml. This is in stark contrast
with an albumin-coated paclitaxel
nanoparticle formulation which contains particles slightly
greater than 400 run, and a non-albumin
based paclitaxel nanoparticle formulation having particle size
less than 400 run, both shown to be
unstable under the same conditions at paclitaxel concentration
of 5 mg/ml. These results demonstrate
the advantageous and unexpected stability of the albumin-coated
paclitaxel nanoparticle formulation
recited in the claims of the '479 application, especially in
view of the high particle concentration in
such a formulation and the well-known principle that the
aggregation rate of nanoparticles is
proportional to the square of the particle concentration.
13. The Examiner cites Desai as allegedly teaching a stable
albumin-coated nanoparticle
formulation. As discussed in the Previous Declaration, Example 5
of Desai, which the Examiner
relies on as teaching stability of albumin-coated nanoparticle
formulations, refers to the stability of
polymeric shells containing buoyant soybean oil with density
less than water. No drug was present
within the polymeric shell. The stability of the "drugless"
oil-containing polymeric shells discussed
in Example 5 of Desai thus provides no suggestion that a
nanoparticle formulation comprising a solid
core of paclitaxel and an albumin coating would be stable at
paclitaxel concentration of between 5-15
mg/ml. Furthermore, as discussed in the Previous Declaration, an
increase in loading of paclitaxel
within the polymeric shells as taught in Example 4 of Desai
would be expected to increase the particle
size and/or density of the particles, which in tum could
increase the tendency of the particles to
precipitate.
14. Although a separate example in Desai, Example 9, teaches
preparation of polymeric
shells containing a solid core of pharmaceutically active agent
such as paclitaxel, there is no
information about the concentration of the paclitaxel in such
polymeric shell formulation. Nor is
there any indication that the particles in such polymeric shell
formulation are smaller than 400 run.
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Application No.: 11/520,479 6 Docket No.: 638772000109
Example 9 teaches that "these polymeric shells are examined
under a microscope to reveal opaque
cores .... " The fact that the polymeric shells were viewable
under a microscope to reveal opaque cores
indicates that a substantial portion of the particles in the
formulation taught in Example 9 were larger
than 400 nm. Thus, these formulations taught in Desai differ
from the formulation recited in claims of
the '479 application in at least two aspects: paclitaxel
concentration and particle size.
15. To arrive at the claimed formulation from Desai's
nanoparticle formulations, one
would at least need to: 1) substantially decrease the size of
the particles in the formulation to less than
400 nm; and 2) substantially increase the paclitaxel
concentration to 5-15 mg/ml. Desai provides no
teaching on how to obtain a nanoparticle formulation having a
particle size of less than 400 nm. Nor
would one expect that an albumin-coated solid nanoparticle
formulation having a particle size of less
than 400 nm and paclitaxel concentration of 5-15 mg/ml would
have been stable. Specifically,
according to Example 5 of Desai, the particle concentrations of
the formulations reported therein is
about 7-9 x1010 per ml. See Table 1 at Column 13 of Desai. The
estimated particle concentration of
the albumin-coated paclitaxel nanoparticle formulation having a
particle size less than 400 nm at
paclitaxel concentration of 5 mg/ml, on the other hand, is about
8 x 1013 /ml. This is 1000 fold higher
than those reported in Desai. Since the aggregation rate of
nanoparticles is proportional to the square
of the particle concentration, one would not have expected that
the albumin-coated paclitaxel
nanoparticle formulation having a particle size less than 400 nm
at paclitaxel concentration of 5
mg/ml, whose particle concentration is at least 1000 fold higher
than those reported in Desai, would
be stable.
16. I hereby declare that all statements made herein of my own
knowledge are true and
that all statements made on information and belief are believed
to be true; and further that these
statements were made with the knowledge that willful false
statements and the like so made are
punishable by fine or imprisonment, or both, under Section 1001
of Title 18 of the United States Code,
and that such willful false statements may jeopardize the
validity of the application, any patent issuing
thereon, or any patent to which this verified statement is
directed.
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Applic.atiGn No.: 11:'520,479 7 Docket No.: 638772000109
11/1/2013
Date
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Exhibit 1
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1
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Exhibit 2
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Composition 2, Lot 1, 24 hrs at 40°C, 400x Composition 2, Lot 2,
24 hrs at 40°C, 400x
Composition 1, 24 hrs at 40°C, 400x
2
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Exhibit 3
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Sample Storage Condition Weight Mean Diameter, nm1
0 time 136.9
Composition 1
24 hours at 40°C 135.2
0 time 244.5
Composition 2, Lot 1
24 hours at 40°C 1159.5
0 time 228.0
Composition 2, Lot 2
24 hours at 40°C 561.5
1 Size determined by disc centrifugation method.
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Exhibit 4
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Langmuir 1997, 13, 6413-64
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6414 Langmuir, Vol. 13. No. 24, 1997
F arrest and Wtten. 6 In these experiments metallic oxide
smd-2.2 hema-tite, ~2A kaolinite, 25 bent mite clay, ao and even
carbm black. zr Wthin the published fractal studies nearly all have
coosidered the aggregatim of rolloidal particles in the presence cf
electrolyte, thrugh studying a wide range cf parameters. Sane
include the effect cf varying the electrdyte cmcentratim, a 1o- 1~
I&- 1Q2A2B-31 pH, 8IQ2'ia> tem-perature, a23shear rates,
3233and particle cmcentratim 14 15 m the fractal structure cf the
aggregates.
(6) Fcrrest, S. R.; V\.itten, T. A. J. Phys. A 1979, 12. L 1ffi
(7) Schaefer, D. W; Martin, J. E.; V\.iltzius, P.; Cannell, D. S.
Phys.
Rev. Lett. 1~ 52 2371. (8) Cannell. D. S.; Aubert. C. Phys. Rev.
Lett. 1~ 56 'rn (9) Tang, P.; Cdflesh, D. E.; Chu, B. J. Colloid
Interface Sci. 19'la
126. 3J4. (1G Martin, J. E.; V\.ilcaxm, J.P.; Schaefer, D.;
O:linek, J. Phys. Rev.
A 1ml 41, 4379. (11) Bdle, G.; Cametti, C.; Ccdastefano. P.;
Tartaglia, P. Phys. Rev.
A 1987, 3:i f!Zl. (121 Magazu. S.; Majdino, D.; Mallamace. F.;
Micali, N.; Vasi, C.
Solid State Commun. 1!H}. 7Q 233. (13) Majdino, D.; Mallamace,
F.; Migliardo, P.; Micali, N.; Vasi, C.
Phys. Rev. A 1~ 4Q 4665 (14) Carpineti,M; Ferri, F.; Giglio,M;
Paganini, E.; Perini, U.Phys.
Rev. A 1m) 42. 7?A7. (15) Carpineti, M; Giglio. M; Paganini, E.;
Perini, U. Frog. Colloid
Polym. Sci. 1991, 84. 3J5 (16) lboo, Z; Chu, B. J. Colloid
Interface Sd. 1991. 143, 356 (17) Asnaghi, D.; Carpineti, M;
Giglio. M; Sazzi, MIn Structure
and Dynamics of Strongly Interacting Colloids and Supramolecular
Aggregates in Solution; Chen, S.-H., Huang, J. S., Tartaglia, P.,
Eds.; Kluwer Academic Publishers: Dcrdrecht, The Netherlands, 1002
p 763
(18) Asnaghi, D.; Carpineti, M; Giglio. M; Sazzi, M Phys. Rev. A
199a 45. 1018
(19) Asnaghi, D.; Carpineti, M; Giglio, M; Sazzi, M Frog.
Colloid Polym. Sd. 199a 8Q En
(aJ) V\eitz, D. A.; Huang, J. S.; Lin, MY.; Sung, J. Phys. Rev.
Lett. 1~ 54. 1416
(21) V\.ilcaxm. J.P.; Martin, J. E.; Schaefer. D. W Phys. Rev. A
19'119, $. a>75 (~ Liu, J.; Shih, W Y.; Sarikaya, M; Aksay,l. A.
Phys. Rev. A 1m)
41, 3a:X3. (23) Arnal, R.; Raper, ]. A.; \Mlite, T. D. f Colloid
Interface Sd.
lml 14Q 158 (24) Arnal, R.; Gazeau. D.; \Mlite, T. D. Part.
Part. Syst. Charact.
1994, 11, 315 (25) Herringtm, T. M; Midmcre, B. R. Colloids
Surf. A 1~ 7Q
1ffi (26) Axfcrd, S.D. T.; Herringtm, T. M J. Chem. Soc ..
FaradayTrans.
21994, fa ::m5. (27) Bezcr. P.; Hesse-Bezct, C.; Roosset, B.;
Diraism, C. Colloids
Surf. A 1995, 97, 53 (28) Lin, MY.; Lindsay, H. M; V\eitz, D.
A.; Ball, R. C.; Klein, R.;
lvleakin, P. Nature 19'119, 33Q 3En (29) Lin, MY.; Lindsay, H.
M; V\eitz, D. A; Ball, R. C.; Klein, R.;
Meakin, P. Proc. R. Soc .. London, Ser. A 1!H}. 423 71. (3))
Lin. MY.; Lindsay, H. M; V\eitz. D. A.; Klein, R.; Ball. R. C.;
lvleakin, P. J. Phys.: Condens. Matter 1m) 2. 3::m (31) Lin,
MY.; Lindsay, H. M; V\eitz, D. A.; Ball, R. C.; Klein, R.;
Meakin, P. Phys. Rev. A 19!1), 41, aD5. (~ Tcrres, F. E.;
Russel, W B.; Schowalter, W R. J. Colloid Interface
Sd. 1991, 142. 554. (33) Oles, V. J. Colloid Interface Sci. 199a
154. 351.
Burns et al.
Lin and co-werkers2B-31 have studied the universality cf fractal
rolloid aggregates using beth static and dynamic light scattering.
They investigated the two limiting regimes, diffusim-limited (i.e.,
DLCA), and reactim-limited (i.e., RLCA) colloid aggregatim, fer
three ccm-pletely different rolloids: gdd, silica, and polystyrene.
They frund the aggregatim behavier to be independent of the
detailed chemical nature cfthe rolloid system. The fractal
dimensims of the aggregates were frund to be in goal agreement with
thcse obtained fran calculatioos using ccmputer mcdels. For DLCAall
aggregates had dF = 1.85± QCEand a power-law kinetic growth
behavier, while fer RLCA dF = 2 11 ± Q
-
Light Scattering Study of a Model Colloidal System
measured as a functim of the magnitude of the scattering vecter,
Q. with
(3)
where n 0 is the refractive index of the dispersim medium, (J is
the scattering angle, and A.0 is the in va cu owavelength of the
incident light.
If the individual particles in a fractal aggregate are
mmodisperse and within the Rayleigh-Gans-Debye regime, the
scattered intensity I( OJ fran such an aggregate can be written
as34
I(Qj = kcf>(Qj S(Q) (4)
In the above expressim k0 is a scattering cmstant. P(Qj
isthesingleparticlefcrmfacterandisrelatedtotheshape
cftheprimaryparticle. S(Qj is the interparticle structure facter,
which represents the ccrrelatims between different primary
particles within an aggregate, assuming there are no correlatims
between the aggregates themselves. Thus, it describes the spatial
arrangement of the particles in an aggregate.
At large Qs cr Qr0 » 1, where r0 is the radius c:i the primary
particle, S(Q) is approximately equal to 1. The scattered intensity
is then daninated by the single particle ferm factcr and only the
scattering due to individual particles is seen. At Qs small
canpared to lfo. but large canpared to 1R (i.e .. 1R« Q« Lfo). P(Qj
~ 1andS(QJ reduces to34
(5)
Hence, provided that R is much larger than ro. eq 4 takes the
form of the well-known pavver-law scattering, i.e.,
(~
It is eq 6that was used in estimating dp in this work. In the
remainder cf this paper, any reference to a fractal dimensim will
necessarily imply a mass fractal dimensim.
Potential Energy Calculations. The total pair interactim energy
between particles in a dispersim can be ootained by summatim cfthe
repulsive and attractive interactim energies. Healy et al.
3ti33derivedan expressim fer the repulsive interactim energy
between twoparticles. Assuming low surface potentials, spherical
particles of equal size, electric dwble layers that are thin
canpared to the particle size ([ 1), and small electric dwble-layer
overlap, the repulsive interactim energy, VR. is given by
(7)
where E is the permittivity, 1/Jd is the particle surface
potential, and H is the separatim distance between particles. The
inverse Debye length, K, is related to the cmcentration of a
symmetrical electrolyte, c, by
(8)
where ze is the icn charge, NA is Avcgadro's cmstant, and kT is
the thermal energy. It is custanary to refer to Lk as the thickness
of the diffuse druble layer. It is impcrtant to note that apart
fran fundamental cmstants, K depends mly m the temperature, em
centra tim of electrolyte, and
(34) Teixeira, J. J. Appl. Crystallogr. 19!& 21, 781 (35)
H~. R.; Healy, T. W.; Fuerstenau, D. W. Trans. Faraday Soc.
1900 6Z lim. (33) \1\eise, G. R.; Healy, T. W. Trans. Faraday
Soc. 1970. ffi 49:)
Langmuir, Vol. 13 No. 24. J[£)7 6415
the im charge. Fer a 1-1 type electrolyte, the value of K (in
nm- 1) in water at 25 •c is 32&'12• Clearly, any change in the
imic strength cf a colloidal dispersim will significantly influence
the energy of interactim c:i the particles.
The fcrces cf attracticn between neutral particles are Lmdm-van
der \1\aals dispersim fcroes. Hamaker37
derived the following expressim for the attractive inter-actim
energy, VA. between twospherical particles of radius ro. At a small
interparticle separatim, i.e., H « ro. VA is given by
(9)
where A is the Hamaker cmstant. Hence, the tttal potential
energy cf interactim between two particles in an aquerus dispersim
is ootained by summing the electric dwble layer and van der \1\aals
energies, i.e.,
The repulsive energy is approximately an expmential functim
cfthe distance between the particles with a range cfthe erder of
the thickness cfthe druble layer (l.k). and the van der \1\aals
attractive energy decreases as an inverse power cf the distance cf
the particles. Cmse-quently, van der \1\aals attractim will
predaninate at small and at large distances. At intermediate
distances dwble-layer repulsim usually predaninates. However, this
largely depends m the actual values of the two ferces and hence en
the electrolyte cmcentratim in solutim. 38
Experimental Section Materials. Styrene mmcmer (Aldrich) was
first washed with
lCY/o NaOH (3 x 3J cm3) follo..ved by Millipere water (3 x
2&l cm3) and then vacuum distilled at &l "C. The ammooium
persulfate initiater (Aldrich) and all other reagents emplcy-ed
were cr analytical grade and were used withoot further
purifica-tim. Millipcrewaterwas used in all preparatims.
Afoor-necked, 2L reactim vessel was used and an overhead stirrer
with variable speed cmtrol used to maintain cmstant stirring. The
thermo-stated water bath was able tocmtrol the temperature to
within ±05 ac. and the dialysis tubing was well bdled in Millipcre
water three times befcre use.
Latex Preparation. The polystyrene latices were prepared by the
surfactant-free emulsim pdymerizatim methcd cr Gcxxiwin et al.
~using ammooium persulfate as the initiatcr. A typical
emulsifier-free preparatim was carried rut in the follo.ving way.
The required amoont cr water (aD cm3) was added to the reactim
vessel and the stirrer fitted to me ootlet. Typically, the stirrer
was adjusted to a distance cr aboot 1-2cm from the bottom cr the
flask. A water-rooled reflux cmdenser was added to a secmd ootlet
and the flask immersed in the thermcstated bath. Nitrq;:en was
bubbled throogh the water in the reactioo vessel to remove o.xygen
frcrn the system using a third inlet to the flask. The gas flo..v
was cmtinued throoghoot the reactim, but the flo..v rate was kept
lo..v to minimize evaporation. The stirrer was adjusted to the
required speed (aD rpm) and after stirring for at least 3Jmin with
nitrq;:en passing throogh the system, styrene ( lffi g) was added
via the foorth ootlet. The system was then left to equilibrate fer
another 3J min to allo..v the temperature (8J "C) to attain
equilibrium and to saturate the aqueoos phase with styrene.
The initiatcr (Q 565 g) was dissolved in &l cm3 cf water,
and the solution was preheated to the reactim temperature and then
added to the reactim vessel. A typical reactim time cf 24h was
used. At the end cr the reaction time, the vessel was removed
(37) Hamaker, H. C. Physica 1937, 4, 1058. (38) Shaw, D. J.
Introduction to Colloid and Surface Chemistry, 4th
ed.; Butterwcrth-Heinemann: Oxfcrd, U.K .. I~ Chapter 8 (3=))
Gcxxlwin. J. W.; Hearn, J.; Ho. C. C.; Ottewill, R. H. Colloid
Polym. Sci. 197.ol ~ 461.
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6416 Langmuir, Vol. 13, No. 24. JW7 -••
••••••••• . ••• : ••••• ••• • • • ••• ••• -~-·
•• •
Figure 1. Electrm micrcwaph of the pdystyrene latex particles
used in this study.
frcm the bath and allowed to cool fcr a boot ::0 min. The latex
was then decanted throogh a filter packed with glass wad to remove
any unreacted mmcmer and any a:agulum fcrmed. The
latexwasthendialy.redagainstMillipcrewaterincrdertoremove residual
mencmer, ammcnium persulfate, and any ether icnic species left in
the reactien mixture. The dialysis was ccnsidered ccmplete when the
ccnductance r::f the dialysate became cl05e to that r::f the
Millipcre water after 24 h.
Latex CharacterizatiaL The size and pdydispersity cfthe latex
particles were determined using transmissien electrm micr05ccpy
(JOEL JEM lax> EXII electrm micr05ccpe). Mi-crcwaphs cf a number
r::f representative regicns r::f the sample were taken to obtain
images cf at least 3:X) particles. A ccmmerdal sizing sdtware
package (Optimas 5. la) was then used to calculate the average
particle diameter, and the relative standard deviatien was used
toindicate the pdydispersitycfthe particles. A TEM image eft he
pdystyrene latex spheres is given in Figure 1. clearly indicating
the particle size regularity. The average diameter cfthe pdystyrene
latex particles was foond to be 3:D ± 10 nm.
Electrcphcreticmobilitiescfthelatexparticlesweremeasured using a
Brcx:khaven Zetaplus apparatus. The effect cf pH and
KN(hccncentratien en the particle mobility was examined. The
analysis was perfcrmed at 25 oc. using a particle cencentratien cf
Io-336 wIN. Determinatim cf the stability regien r::f the
pdystyrene latex particles in KN(h was carried oot using an optical
methcxl that relies en the sedimentatim rate cf the aggregated
dispersien. This invdved measurement cfthe percent transmittance
cfthe supernatant fcr latex dispersicns ccntrdled tovarioos salt
ccncentraticns. The transmittance was measured at 540nm using a
Hitachi spectrcphctcmeter after the dispersicns were left
undisturbed fcr 24 h. The results were then plctted against the
ccncentratim cf KN(h and the critical a:agulatien ccncentratien
(ccc) was determined as the inflectim pdnt r:fthe curve.
Fractal Studies. Small-angle static light scattering was used
here to study the structure cfthe latex particle aggregates. The
instrument used was a Malvern Mastersizer S, which has a 633 run
He-Ne laser as the light soorce. The Mastersizer simul-taneoosly
measures scattered intensities at a range cf scattering angles. It
also provides a direct measure cf the average size distributien cf
the material in the scattering cell. Aggr.egatien was achieved by
adding the required amoont cfthe pdystyrene latex particles to a
sdutim r::f KN(h cf known ccncentratien at pH 6and at rcx:m
temperature. Directly after mixing. the sample was gravity fed into
the light scattering cell. The 1~ I(QJ vs 1~ Q plct was then used
to determine the scattering expenent (d. eq ~- Typical experimental
data fran which the scattering expcnents were calculated are
illustrated in Figure 2 The change in thescatteringexpcnents and
the average cfthesize distributien cf the scattering material were
reccrded with time. Fractal dimensien values fcr the aggregates
were determined by taking the absdute value of the limiting
scattering expcnent in the plateau regien (vide infra Figure
8).
...... () ._.
scattering exponent =slope
-4
log1o Q
Burns et al.
2.5
1.5
-3
Figure 2 Typical experimental data frcm which thescatterin~
exponents were estimated. Note that Q and I(QJ are in nm-and
arbitrary units, respectively.
0+-----:=---1------+-----j • • 4 8 t12 -I e • (,) -2 • ~ • .!I!
• • E -3 • • • • :I. • ._. ~ -4 ••• :c • • • • 0 -5 • • ::e • •
-6~-------------~
pH Figure 3 Effect of pH and icnicstrength en the
electrophcretic mobility of the pdystyrene latex particles. The
ccrresponding electrdyte cencentratims are (•) Io-4 M, (+) 1o-3 M,
and (II) Io- 1 MKNOs.
Results and Discussic:n
The electrophcretic mobility ci the polystyrene latex particles
was measured at three different KN03 cmcen-tratiens and acrcss a
bread range cf pH values. The resultantdataareillustratedinFigure3
Thesemobility curves indicate that as the pH was increased. there
was a significant increase in the ienizatien ci the surface
functienalgroops. AbovepH6 therurvesbegintoflatten rut, suggesting
that the surface ienizatien is canplete in this regien. As
expected, there was a reductien in electrcphcreticmobility of the
particles en increasing the electrolyte ccncentratien.
Generally, the measurement cf sedimentatien rate is a useful
means fer aggregatien studies in colloid science.
-
Light Scattering Study of a Model Colloidal System
100 -r------------------. ...... !!! 80 :::J 0 .c
~ 60
e c 40
i ~ 20 ::.e 0
0
0 0.2 0.4 0.6 0.8 1.2 [KNO:J (M)
Figure 4. Percent transmittance of light (at 540 run) as a
function of the ccncentratim of electrolyte in the pdystyrene latex
dispersion (at pH 6). The onset of aggregation (028 M KNQV and the
critical cmgulation ccncentratim (O 70MKNQV are indicated.
Table 1. Fractal Dimensioos ofPdystyrene Latex Aggregates Fcrmed
in Various Ccncentratioos ofKN03
at Different Initial Particle Ccncentratioosa
[KN031 (M)
040 OtD 06J 070 085 l.CD 1.25 ltD
initial particle cmcentratim (w ;W)
2m 2m 2m 2ill 2ffi 2ffi 217 211 2CE 28 2ffi 2ffi 2ffi l.ffi
l.ffi l.ffi 1.85 1.~ 1~ 1.~ l.ffi 100 1m 1m
• Ncte that since the primary latex particles have a density
cla;e to unity, the weight fractims reptained in this way fer oor
polystyrene latex particles at pH 6is 028M KN03. However, Hunter43
describes the critical coagula-tim a:ncentraticn as the
"electrolyte a:ncentraticn at which slow ccagulaticn gives way to
rapid ccagulaticn". This is determined experimentally as the
ccncentraticn where the settling material leaves behind it an
essentially clear supernatant. On the basis cf this definiticn, the
ccc cf oor latex particles is expected to be closer to Q 7 M KN03.
ltseemsmerelikelythattheccncentraticnof028 M KN0:3 defines the
regicn of stability cf the latex dispersicn; i.e.,
below028Mthedispersicnisstable, while above this ccncentraticn the
dispersicn will be essentially unstable, althoogh the ccagulaticn
kinetics may net be rapid. In ccntrast, 0 7 M KN0:3 ccrrespcnds to
the ccncentration at which rapid ccagulation begins.
Thefractaldimensicnscfthepolystyrenelatexparticle aggregates
fermed in the presence ciKN03 and measured by small-angle static
light scattering are summarized in Table 1. The upper limit c:i the
initial particle ccncentra-ticn was chcsen to avoid the effects
cfmultiple scattering, while the lower limit was selected such that
backgroond
(41) Kotera,A.; Furusawa, K.; Takeda, Y. Kolloid-Z Z Polym.
1970, 2%l 017. ~~otera, A.; Furusawa, K.; Kudo. K. Kolloid-Z Z
Polym. 1973,
(~Hunter, R. J. Foundations of Colloid Science, Oxfcrd Science
Publicatims: Oxfcrd. U.K., 1937; Vd. 1.
Langmuir, Vol. 13 No. 24. HE7 6417
2.50
2.25
• t • • • • • • • 2.00 • • • .... "C • • a • • 1.75
1.50 +-----+-----1-----+------i 0.2 0.6 1.0 1.4 1.8
[KNO:J (M) Figure 5 Dependence of the fractal dimension of
aggregates of polystyrene latex spheres with electrolyte
ccncentratim and initial particle cmcentration: (.&.) Q 0
(JJT(J'!O wIN. noise woold not have a significant effect. The
electrolyte ccncentraticns used in the experiments ranged from 04
to 1. 5M, which allowed beth regimes cf aggregaticn QJLCA and RLCA)
to be c.bserved. This also allowed c.bservation c:i the behavicc cf
the fractal dimensicn at intermediate salt ccncentraticns. The
fractal dimensicns were foond torangefran 1. 78to22Q
whichccrrespcndwell toknawn literature values cf 1.8 and 2 I fer
diffusicn-limited cluster-cluster aggregaticn and reacticn-limited
cluster-cluster aggregaticn, respectively. To illustrate mere
clearly the c.bserved trends with particle a:ncentraticn and icnic
strength, the fractal dimensicns are plctted in Figure 5
Clearly, the structure c:i aggregates fermed frcrn the colloidal
particles differs acccrding to the prevailing a:nditicns within the
aggregating system. At any given particle ccncentraticn, mere
tightly packed ccrnpact structures were fcrmed at low salt
ccncentraticns, while mere q:>en structures were fcrmed at
higher salt a:ncen-traticns. At intermediate salt ccncentraticns,
the fractal dimensicns were foond to be intermediate to the
theereti-cal values predicted by the DLCA and RLCA models. As
explained later in this section, strictly speaking, these
intermediate values shoold be referred to as "effective" fractal
dimensicns.
Acccrding to the DL VO thecry cf call ad stability, the
aggregaticn behavicc of aqueoos colldds is determined by the energy
barrier en the interacticn potential energy curve between two
collddal particles, which in turn is a:ntrolled by the salt
ccncentraticn in the medium. The primary effect cf salt en a
charge-stabilized aqueoos dispersicn is to suppress the extent of
the electricdooble layer (eq 8). This leads to a reducticn in the
repulsive potential energy (eq -n. Since VA is largely unaffected
and is typically attractive, the system becanes mere unstable. The
estimated pctential energy curves fer the polystyrene latex
particles in water at 25 •c are plctted in Figure 6 A value cf 1 x
1o-;;o J was taken fer the Hamaker ccnstant. 43 The surface
pctentials used in the calculations ranged frcrn -:n to -35 mV,
which were estimated frcrn the experimental mobility data (Figure
3).
Observation of these pctential energy diagrams indi-cates two
regimes cibehavier. At salt ccncentrations less than aboot 0 7M,
sane fcrm cf a pctential energy barrier exists, even when the curve
is essentially negative over all particle separaticns. Hence, fcc
salt levels between 0 3 and 0 7 M the pctential energy curves
indicate that
Actavis - IPR2017-01100, Ex. 1024, p. 19 of 33
-
6418 Langmuir, Vol. 13 No. 24, lrE7
80 ....,,.,.,.,..0.1 M
60 f" -o.3M ;~ \ l 1-. -O.SM 40 1\ -0.7M ··········l.OM 20
--l.SM
~ 0 ;; -20 H(nm)
-40
-60
-80
-100
Figure 6. Influence cf the electrolyte concentratim m the total
potential energy of interactim using a Hamaker anstant cf 1 x IQ-aJ
J and surface potentials cf - ::D to -35 m V.
the particles can initially cane to rest in a weak secoodary
minimum fran which deflocculati01 is relatively easy. If the
oolliding particles have suffident thermal energy, they can
surmoont the barrier and enter the deep primary minimum fran which
particle escape is virtually impa;-sible. At greater than aboot Q 7
M salt, no barrier is present and all oollisi01s must result in an
irreversible aggregati01. Such potential energy curves shoold be
canpared with the dJserved transiti01 fran dense toloa:;e
aggregates over a range of coocentrati01s fran 0 7to 1.0 M salt.
Thus, the absence of any barrier to a:Bgulati01 in a potential
energy curve woold indicate the DLCA regime, while the presence of
a significant barrier leads to a higher fractal dimensioo and the
RLCA regime.
The fractal results repated here are coosistent with those by
Zhoo and Chu, 16whoused a canbinati01 c:i static and dynamic light
scattering measurements to study the fractal aggregates c:i
polystyrene spheres induced by the additi01 c:isalt (Q2-1.5MNaCl)
at a coostant pH cf92 Fractal dimensi01s ranging fran 1. 72 (DLCA)
to 2 15 (RLCA) were foond. Intermediate values fer dF were also
cbtained at intermediate salt coocentrati01s. They at-tributed this
to the restructuring cfthe ramified collcidal aggregates, which
they suggest may occur in the presence c:i a finite
interpartideattracti01 energy. Hence, althoogh a different
univalent salt was used at a much higher pH, the trends dJserved
fer the fractal dimensioo c:ithe latex aggregates are similar to
curs.
In cootrast, some c:i the reperted dJservati01s cf the
aggregati01 behavior of pdystyrene latex particles do not agree
with the above findings. Bolle et al. 11 investigated the
aggregati01 c:i pdystyrene particles in a univalent salt (NaCI)
over a wide coocentrati01 range. They foond a value of 1. 75 fer
the fractal dimensioo c:i aggregates in the fast aggregati01 regime
when the salt coocentratioo was high. In low salt cooditi01s a
different kinetic regime was dJserved, hence a different fractal
dimensi01 was expected. However, the growth of the aggregates was
foond to proceed with a fractal dimensi01 (dF = 1. 75) equal to
that fcund fer the DLCA regime. Asnaghi et al. 17- 19 also fcund
for low salt coocentratioos that dF was at first larger than 2 and
therefore dose to the typical RLCA fractal dimensioo but then
gradually reverted to lower values not far fran those typical
cfDLCA They suggested
Burns et al.
that the aggregati01 crossed fran reactim-limited to
diffusi01-limited because as the average duster mass grows,
diffusi01 becanes the rate-limiting process. How-ever, they did
note that decreasing the coocentrati01 c:i salt resulted in higher
fractal dimensi01s that were more stable in time.
In this study, aggregate structures were also fcund to becane
less canpact as the latex particle coocentrati01 was increased (cf.
FigureS). Verylittlehasbeenrepated 01 the effects of particle
coocentrati01 01 the aggregate structure of collcidal systems.
Carpineti et al. 14. 15 have studied the aggregati01 of polystyrene
spheres at a fixed salt coocentrati01 (3)mM MgC12) in the fast
aggregati01 regime and varying particle coocentrati01 fran 1 x 10'1
to 5 x 1010partides.6n3• The data showed that at larger
coocentratiOOS they COOSistently foond dF = 1.61 ± 0(£, but as the
coocentratioo was decreased, dF grew to 1.83 ± 002. In cootrast,
Martin et al. 10 have provided experimental evidence fer an
increase in the fractal dimensi01 with an increase in the
coocentratioo of aggregating silica particles, in the rapid
aggregati01 regime.
In cur study, three particle coocentrati01s were examined:
l.C:Ox 10'1, 185x 10'1, and 3 70x 10lpartides/ an3, with each being
investigated over a wide range of salt c01centratioos. Our results
exhibited trends similar to those c:i Carpineti et al. 01
increasing the particle coocentratioo at a fixed level of
electrolyte; that is, the fractal dimensi01 was dJserved to
increase with a decrease
inthenumberc:iinitialpartidesintheaggregatingsystem. However, the
particle coocentratioo had its most pro-ncunced effect oo the
aggregate structure at low i01ic strengths, i.e., in the slow
aggregatioo regime.
Our werk reveals that there is a noticeably larger variatioo in
the fractal dimensi01 with particle coocen-tration at lower salt
coocentrati01s. At high levels cfsalt, where there is nopctential
energy barrier toaggregati01, the fractal dimensioo does net vary
significantly with changes in the particle coocentrati01. This can
be attributed to the fact that all duster (cr particle) collisi01s
wculd result in permanent coo tact; hence recoofigurati01 c:i
dusters wculd be effectively prevented. In cootrast, under slow
growth cooditi01s, dusters (cr particles) cwld undergo a number of
collisi01s befere they became permanently locked intopositi01,
since they may initially 01ly be in a weak secoodary minimum. Thus,
particles attached to the edge of an aggregate will have the
ability to "roll aroond" as the structure develcps as they attempt
to find their minimum energy state. The ability c:i such a particle
to sample a number of possible coofcrmati01s within an aggregate
will depend oo the prcbability of ancther particle colliding with
it in a given time, which may effectively lock it in positi01. This
will, c:i ccurse, be influenced by the frequency c:i particle
cdlisioos within the aggregating system. This suggests that if the
primary particle coocentratioo is reduced, the frequency of
cdli-sioos is also reduced and hence there will be mere time fer
the particles to restructure and form a denser ag-gregate.
C01sequently, the fractal dimensioo will be higher at lower
particle coocentratioos, as cur results indicate.
The rate at which a dispersien will a:Bgulate depends en the
frequency with which particles encwnter me ancther and the
prcbability that the energy barrier to a:Bgulatioo can be overcane
when collisiens take place. Figure 7illustrates cur findings 01 the
dependence of the relative rate of aggregate growth en the
electrdyte coocentratioo and hence the pctential energy barrier to
aggregati01. In excess electrdyte, the aggregate grows rapidly and
to a canparatively large size. This growth
Actavis - IPR2017-01100, Ex. 1024, p. 20 of 33
-
Light Scattering Study of a Model Colloidal System
14~--------------------------·
12 (a)
10
8
6
4
2
0
14 ... ! 12 (b) E 10 ca :g
.;'E 8 m::::&. 6 e-m m 4 ca c
2 : :E
0
14
12 (c)
10
8
6
4
2
0 0 so 100 150 200
Time (min)
Figure 7. Variatirn of the mean ag®ate size with time at
different particle crnrentratirns: (a) a aJ70'!6; (b) a CXB'J'J6;
(c) QCXBJ'J6 wIN. The ccrrespcnding crncentratirns ci electrdyte
are (0) a4M, ¢:1) a5M, () a6M, (LI.) a7M, (e) a85M, •> 1.0 M,
(+) 1.25 M. and (•) 1.5 M KNOs. Fer the particle crncentratirn ci Q
CXBJ'A. wIN, a 4 M KNOs. has been excluded fer clarity.
shows a power-law behavier and is, therefere, assodated with
diffusirn-limited cluster-cluster aggregatim. Fur-thermere, since
every particle callisirn results in a permanent em tact, the number
ci single particles present becanes rapidly reduced. This, in
canbinatim with the decreasing probability of cluster-cluster
aggregatirn due to the reduced mcbility of these clusters, results
in an eventual plateau of the aggregate size. The intrcx:luctim
cfarepulsiveenergybarrierbyredudngthecrncentratirn of electrolyte
reduces the size of the aggregates and causes the aggregatirn
kinetics to be slowed. The reductirn in the sticking probability of
the particles also prcx:luces a linear growth in the size of the
aggregates over the time scale examined, where singlets are still
readily available to attach to the existing aggregates. The canmm
exprnential growth behavier ci reactirn-limited cluster-cluster
aggregatim cannot readily be identified in this
Langmuir, Vol. 13 No. 24, JW7 6419
study due to the relatively shert time scale ci the
cbservatirn.
An interestingsituatirn arises when the latex dispersirn is
aggregated at intermediate electrolyte crncentratirns. The
aggregate size appears to grow in mere of a stepwise fashirn, which
suggests a transitim fran slaw aggregatirn to rapid aggregatirn at
this level ci electroJ.yte. This stepwise growth, althoogh not
clearly visible at Q CXBJ'J6 wIN particles, appears to begin aroond
Q 7 M KNO:> Therefere, at the intermediate salt levels oor data
indicate that there was a crcssover behavier fran RLCA to DLCA as
the aggregatim progressed. Acccrding to the scale-invariant
definitirn of fractal aggregates, the previoosly reperted fractal
dimensirns at these intermediate salt crncentratims shoold, in a
strict sense, be termed "effective" fractal dimensirns, as already
suggested in the literature. 18
In all, these results indicate that due to the law sticking
prcbability of the particles at law levels of electrolyte the
growth cfthe aggregates is slaw but results in crnsistently higher
fractal dimensirns. In crntrast, fast aggregatirn ch>erved under
high salt crnditirns gives larger aggregate sizes but less dense
structures, ch>ervatims crnsistent with a high prcbability of
particle sticking. Furthermere, the growth of the aggregates is
dependent m the number of particles present in the aggregating
system. As the particle crncentratirn is increased, the aggregate
growth is more rapid, mcst likely due to the increased col.lisim
frequency. The fractal dimensirn ci the aggregates is ch>erved
to decrease under such crnditirns, with this effect being mere
prmoonced at the law salt cmcentratirns.
In this study the change in the slcpe ci the scattering curves
(i.e., the scattering exprnent) was alsommitered with time and the
results are illustrated in Figure 8 The results indicate that in
excess electrdyte the scattering expment is cmstant over time,
while at law electrolyte levels a growth in the scattering expment
was ch>erved. The absolute value of the scattering exprnent will
represent the mass fractal dimensim of the aggregated particles,
provided that the aggregate size is much larger than that of the
primary particles. In these experiments it was noted that the
limiting scattering exprnents ccrresprnded to sufficiently large
aggregates, and thus, a fractal dimensirn coold be assigned at this
pdnt. As a result, the scattering exprnents cbtained in the initial
stages of the aggregatim, with particular cmcem to the law salt
crnditirns, do net ccrresprnd to a fractal dimen-sirn fer the
aggregating system. Nevertheless, the change in the scattering
exprnent with time does provide a measure ci the relative rate ci
aggregate growth and reactim kinetics fer varioos aggregating
cmditirns. It was foond that the lower the salt cmcentratirn used
in the aggregatim, the lmger the time required to achieve a
constant scattering expment, hence slower reactirn kinetics.
Similarly, lmger times were required fer the aggregates to becane
scale invariant when the particle crncentratim was lowered.
On the basis of the results given in Figure 8 it appears that
the transitim fran slaw to fast aggregatim can be readily
ch>erved. If rapid aggregatirn is assumed to be the regim in
which the scattering exprnent is cmstant over time, then the
critical coagula tim cmcentraticn can be readily determined. It
appears that this change occurs between Q70and Q85MKNO:> This is
cmsistent with the results in Figure 7, where the above
crncentraticns ci electrolyte ccrrespcnd to a stepwise growth in
aggregate size. These results also canpare faverably with
experi-mental data cbtained fran turbidity measurements (Figure 4)
using the definitim provided by Hunter. 43 Hence, fractal
measurements appear to also provide a
Actavis - IPR2017-01100, Ex. 1024, p. 21 of 33
-
642D Langmuir, Vol. 13, No. 24, lfE7
2.20 -..----------------..,
2.05
1.90
(a)
1.75
2.20
-c CD c 2.05 0 C1. )( CD m c
"i: 1.90 ! cu ()
(b) (/)
1.75
2.20
2.05
1.90
(c)
1.75 ......... .___--+-------+------1 0 50 100 150 200
Time (min) Figure 8 Variatirn of the scattering exponent with
time at different particlecrncentratirns: (a) OCXJ7CY;6; (b)
OCXJ3EP;6; (c) OCXBY;6 w ;W. The ccrresponding ccncentrations of
electrdyte are (O) 04M, ¢:1) 05M, (0) 06M, (e.) 07M, (e) 085M,
•> 1.0 M, (+) 1.25 M. and (•) 1.5 M KN0:3. Fcc the particle
ccncentraticn of 0 CXB:J';6 w ;W, 0 4 M KNOs has been excluded fcc
clarity.
sensitive methcx:l fcr the estimatirn of the critical
coogu-latirn ancentratioo. Based oo these observatioos, the
"effective" fractal dimensirn e
-
Exhibit 5
Actavis - IPR2017-01100, Ex. 1024, p. 23 of 33
-
Journal of Colloid and Interface Science 253, 70-76 (2002) doi:
10.1 006/jcis.2002.8476
Stability of Nanodispersions: A Model for Kinetics of
Aggregation of Nanoparticles
Nikola Kallay1 and Suzana Zalac2
Depanment of Chemistry, Faculty of Science, University of
Zagreb, Marulicev trg 19, P. 0. Box 163, HR I 0000 Zagreb,
Croatia
Received October 29, 2001; accepted May 9, 2002
In the course of aggregation of very small colloid particles
(nanoparticles) the overlap of the diffuse layers is practically
com-plete, so that one cannot apply the common DLVO theory. Since
nanopoarticles are small compared to the extent ofthe diffuse
layer, the process is considered in the same way as for two
interacting ions. Therefore, the Br0nsted concept based on the
Transition State The-ory was applied. The charge of interacting
nanoparticles was calcu-lated by means of the Surface Complexation
Model and decrease of effective charge of particles was also taken
into account. Numerical simulations were performed using the
parameters for hematite and rutile colloid systems. The effect of
pH and electrolyte concentra-tion on the stability coefficient of
nanosystems was found to be more pronounced but similar to that for
regular colloidal systems. The effect markedly depends on the
nature of the solid which is char-acterized by equilibrium
constants of surface reactions responsible for surface charge,
i.e., by the point of zero charge, while the speci-ficity of
counterions is described by their association affinity, i.e., by
surface association equilibrium constants. The most pronounced is
the particle size effect. It was shown that extremely small
particles cannot be stabilized by an electrostatic repulsion
barrier. Addition-ally, at the same mass concentration,
nanoparticles aggregate more rapidly than ordinary colloidal
particles due to thier higher number COncentration. © 2002 Elsevier
Science (USA)
Key Words: stability of nanodispersions.
INTRODUCTION
It is commonly accepted that the stability of colloidal systems
is, in most cases, the result of an extremely slow aggregation
pro-cess. The main reason for such a slow aggregation process is a
high electrostatic energy barrier, and in some cases a protective
layer of adsorbed chains. The theory of Colloid Stability
consid-ers collision frequency and efficiency (1, 2). Collision
frequency was theoretically solved by Smoluchowski (3), while the
basis for evaluation of the collision efficiency was given by Fuchs
( 4 ). In order to use the Fuchs theory one should know the
interaction energy as a function of the distance between
interacting parti-
1 To whom correspondence should be addressed. Fax:
+385-1-4829958. E-mail: nkallay@ prelog.chem.pmf.hr.
2 Present address: PLIVA d. d., R&D-Research, Prilaz baruna
Filipovica 25, HR 10000 Zagreb, Croatia.
0021-9797/02$35.00 © 2002 Elsevier Science (USA) Alt" rights
reserved.
70
cles. The effect of dispersion forces was solved by Hamaker (5),
Bradly (6), and de Boer (7), while electrostatic repulsion could be
evaluated on the basis of the Derjaguin, Landau, Vervey, Overbeek
(DLVO) theory (1, 8). Recently, more sophisticated models were
elaborated (9-14). In most of the cases the theory of Colloid
Stability explains the experimental data, especially if the correct
values of the electrostatic surface potentials, as obtained from
the Surface Complexation model (15-19), are used (20-22). However,
small particles, with sizes below 10 nm (called nanoparticles),
generally do not show electrostatic sta-bilization. According to de
Gennes (23), the reason for the in-stability of nanocolloidal
systems might be in their low charge (surface charge density times
surface area). In some cases sta-ble systems of nanoparticles could
be prepared (24, 25) but no kinetic measurements were
published.
In this paper we analyze the theoretical aspect of the kinetics
of aggregation of nanoparticles based on the Brj1insted theory (26,
27), which was developed for the salt effect on the kinetics of
ionic reactions (primary salt effect). The reason for such a choice
lies in the fact that the classical DLVO approach cannot be used
for nanoparticles: nanoparticles are small with respect to the
thickness of the electrical diffuse layer, so that in the course of
the collision of two nanoparticles a complete overlap of two
diffuse layers takes place. Let us consider extension of the
diffuse layer. According to the Gouy-Chapman theory, depending on
the ionic strength and surface potential, the latter is reduced to
10% of its original value at a distance of 2 to 2.5 reciprocal K
values. This means that at the ionic strength of w-z mol dm-3 the
diffuse layer is extended up to 6 nm from the surface. As shown on
Fig. 1, in such a case overlap of diffuse layers of two
nanoparticles is practically complete. In the case of ordinary
colloid particles the overlap is partial so that the DLVO theory is
applicable.
A nanoparticle surrounded by a diffuse layer is similar to an
ion situated in the center of an ionic cloud. In the course of
colli-sion two nanoparticles in contact have a common diffuse layer
or "ionic cloud." Therefore, interaction of nanoparticles could be
considered in a manner similar to that for two interacting ions,
and consequently described by the Br!11nsted theory. This theory
considers the "transition state" or "activated complex" which is a
pair of two interacting ions with a common ionic cloud. The
Actavis - IPR2017-01100, Ex. 1024, p. 24 of 33
-
STABILITY OF NANODISPERSIONS 71
FIG. 1. Overlap of electrical interfacial layers for two
ordinary colloid par-ticles (r = 30 nm) and for two nanoparticles
(r = 3 nm).
equilibration of the transition state is fast, while the
transforma-tion of the transition state into product(s) is slow,
and thus the rate determining step.
THEORY
Introduction of the Brf!lnsted Concept to Kinetics of
Aggregation of Nanoparticles
The quantitative interpretation of kinetics of aggregation of
nanoparticles will follow the Bnzmsted concept (26, 27). It will be
based on the Transition State theory using the activity
coef-ficients as given by the Debye-Hiickellimiting law.
Aggregation of two charged nanoparticles A ZA and Bzs could be
represented by
[1]
where z denotes the charge number. The rate of aggregation v is
proportional to the product of concentrations of interacting
particles (AZA](BZB]
[2]
where k is the rate constant (coefficient) of aggregation.
According to the Brjlinsted concept, in the course of aggrega-
tion two charged nanoparticles undergo reversible formation of
the transition state with charge number being equal to the sum of
the charges of interacting species. The transition state ABZA+zs
undergoes the next step (binding) which is slow and is therefore
the rate determining step
[3]
Note that equilibration of the interface may result in a change
of the total charge of the doublet. In such a case ZA + ZB f=. ZAB.
Since the equilibration of the first step is fast, and the second
process is slow, the overall rate of reaction (v) is proportional
to
the concentration of the transition state
[4]
where k' is the rate constant (coefficient) of the second
process. Equilibration of the first step is fast so that one
calculates the concentration of the transition state [ABzA+zs] from
the rele-vant equilibrium constant K=~' taking into account the
activity coefficients y of reactants and of the transition
state
[5]
The equilibrium constant K =ft is defined in terms of
activities, and consequently its value does not depend on the ionic
strength; i.e., it corresponds to infinite dilution. Equations [4]
and [5] result in
[6]
According to the above equation, the overall rate constant, as
defined by Eq. [2], is given by
k = k' K=~' _y_(A_z_A )_y"7'(B_z_s) y(ABZA+Zs) [7]
It is clear that the overall rate constant k depends on the
ionic strength of the medium through activity coefficients.
Activity coefficients could be obtained from the Debye-Hiickel
equation derived for ionic solutions. The same equation is assumed
to be applicable for extremely small particles i of charge number
z;
2 1/2 Z; AnHic
logy;=- 1/2. 1 +able
[8]
The ionic strength Ic for 1 : 1 electrolytes is equalto their
concen-tration. The Debye-Hiickel constant AnH depends on the
electric permittivity of the medium e( =eoer)
[9]
where L is .the Avogadro constant and R, T, and F have their
usual meaning. (For aqueous solutions at 25°C: AnH = 0.509 mol-112
dm312 .) Coefficient bin Eq. [8] is equal to
b= (2F2)1/2 eRT '
[10]
while parameter a is the distance of closest approach of the
interacting charges, which is in the case of nanoparticles
related
Actavis - IPR2017-01100, Ex. 1024, p. 25 of 33
-
72 KALLAY AND ZALAC
to their radius. By introducing Eq. [8] into Eq. [7] one obtains
electrical interfacial layer
1/2 , # 2ZAZBAoHic
logk = logk +log K + 112
. 1 +able
[11]
Equation [11] suggests that the plot of the experimental log k
value as the function of J}/2 j(l + ab/}12 ) should be linear with
the slope of 2ZAZBAoH' which is true if charges of interacting
species do not depend on the ionic strength. However, as it will be
shown later, the charge of a colloidal particle decreases with
ionic strength due to association of counterions with surface
charged groups.
Estimation of the Equilibrium Constant of the Transition State
Formation
To analyze the effect of repulsion between two charged
par-ticles on the equilibrium constant Ki' we shall split the Gibbs
energy of the transition state formation !), i' G0 into
electrostatic term, !), i' G~1 , and the rest, which we shall call
the chemical term, !), i' G~h. The latter includes van der Waals
dispersion attraction
-RTlnKi' = -RTln(K;K~)=!),.i'G0
= !),i'G~h + !),i'G~I•
where !),.i'G~h = -RT InK; and !),i'G~1 = -RT InK~.
[12]
As noted before, the equilibrium constant Ki' is based on the
activities of the interacting species and its definition (Eq. [5])
considers the corrections for the nonideality. It corresponds to
the zero-ionic strength so that the value of K~ could be ob-tained
considering simple Coluombic interactions between two
nanoparticles. Accordingly, the (molar) electrostatic energy
be-tween particles A ZA and Bzs of radii r A and rB in the medium
of the permittivity e is
[13]
where rA + rB is the center to center distance between
inter-acting particles in close contact. In the case of two
identical particles (r A= rB = r and ZA = ZB = z)
[14]
This approach, based on the Coulomb law, could be tested by the
Hogg-Healy-Fuerstenau (HHF) theory (9). For two equal spheres of
the same surface potential cp, separated by surface to surface
distance x, the electrostatic interaction energy, expressed on the
molar scale, is equal to
where K is the Debye-Hiickel reciprocal thickness of the
K = (2IcF2) 1/2 eRT
[16]
At the zero-ionic strength Uc ---+ 0) the surface potential cp
of a sphere of radius r and the charge number z is
ze cp=--.
4xer [17]
(Note that cp potential is in fact the electrostatic potential
at the onset of diffuse layer.) Under such a condition the diffuse
layer extends to infinity (K ---+ 0), so that for zero separation
(x ---+ 0) Eq. [15] reads
[18]
The comparison ofEq. [18] with Eq. [14] shows that HHF theory
results in "-'30% lower value of energy than the Coulomb law. This
discrepancy is not essential for the purpose of this study, so that
in further analysis we shall use the Coulomb expression.
By introducing Eqs. [12] and [13] into Eq. [11] for the rate
constant of aggregation of nanoparticles A ZA and B'8 , one
ob-tains
2 2 A 1112 lo k= lo k' + lo Ki' _ ZAZBF + ZAZB DH c g g g ch
4xeL(rA+rB) 1+ab!}12
[19]
or in another form
( B !}
12 ) log k = log ko - ZAZB - 2AoH l/2 , [20] rA+rB 1+ablc
where
and
F 2 ln 10 B=---
4xeLRT
ko=k'K~.
[21]
[22]
At high ionic strength the counterion association is so
pro-nounced that the effective charge number of nanoparticles
ap-proaches to zero. In such a case the electrostatic repulsion
dimin-ishes and the aggregation is controlled by the diffusion (k =
kdiff), as described by the Smoluchowski theory. Accordingly,
ko = kdiff· [23]
Actavis - IPR2017-01100, Ex. 1024, p. 26 of 33
-
STABILITY OF NANODISPERSIONS 73
The stability coefficient (reciprocal of the collision
efficiency), commonly defined as W = kctiff/ k, is then equal
to
k ( B 1IJ2 ) log W = log ko = ZAZB - 2AnH e l/2 . rA +rB 1
+able
[24]
In the case of aggregation of identical nanoparticles the above
equation is reduced to
ko 2 (B 1112 ) log W = log -k = 2z - - AnH 112
• r 1 +able
[25]
Evaluation of the Charge Number
For a given electrolyte concentration, the stability coefficient
of the nanodispersion could be obtained by Eq. [24] (or by Eq.
[25], in the case of uniform particles), once the charge number of
particles is known. The surface potential (as used in the theory of
Colloid Stability) and charge number are deter-mined by the ionic
equilibrium at the solid/liquid interface which will be considered
here for metal oxide particles dispersed in aqueous electrolyte
solutions. The Surface Complexation model (2-pK concept) considers
(15-22) amphotheric surface =MOH groups, developed by the hydration
of metal oxide surfaces, that could be protonated (p) or
deprotonated (d)
=MOH+H+--+ =MOHi;
r(MOHi) Kp = exp(F¢0 / RT) a(H+)r(MOH) [26]
=MOH --* =MO- + H+;
r(MO-)a(H+) Kct= exp(-F¢0 /RT) . [27] r(MOH)
Kp and Kct are equilibrium constants of protonation and
depro-tonation, respectively, ¢0 is the potential of the 0-plane
affecting the state of charged surface groups MOHi and Mo-, r is
the surface concentration (amount per surface area), and a is
activity in the bulk of solution.
Charged surface groups bind counterions, anions A- (surface
equilibrium constant Ka), and cations c+ (surface equilibrium
constant Kc)
From the d-plane (onset of diffuse layer, potential ¢ct), ions
are distributed according to the Gouy-Chapman theory.
The total concentration of surface sites rtot is equal to
rtot = r(MOH) + r(MOHi) + r(MO-)
+ r(MO- · c+) + r(MOHi · A-).
Surface charge densities in the 0- and f3-planes are
ao = F(r(MOHi) + r(MOHi ·A-)
- r(Mo-)- r(Mo- . c+))
ap = F(r(MO- · c+)- r(MOHi ·A-)).
[30]
[31]
[32]
The net surface charge density a 8 corresponding to the charge
fixed to the surface is opposite in sign to that in the diffuse
layer act
The relations between surface potentials, within the fixed part
of electrical interfacial layer (ElL), are based on the constant
capacitance concept
[34]
where C 1 and C2 are capacities of the so-called inner and outer
layer, respectively. The general model of ElL could be simplified
(19) by introducing rfJtJ =¢ct. which corresponds to C2 --* oo. The
equilibrium in the diffuse layer is described by the Gouy-Chapman
theory. For planar surfaces (relatively large particles)
2RTeK . as= -ad= --F- smh(-F¢ct/2RT) [35]
and for small spherical particles (nanoparticles)
[36]
Once the system is characterized, the Surface Complexation
=MOHi+A---* =MOHi -A-;
r(MOHi ·A-) K - exp(-FA."fRT)---'---"---.--a- 'f',_,
a(A-)r(MOHi)
=Mo- + c+ --* =Mo- . c+;
r(Mo-. c+)
[28] model enables calculation of the colloid particle charge
num-ber under given conditions. This means that one should know
equilibrium constants of surface reactions, capacitances of in-ner
and outer layers, and total density of surface sites. By an
Kc = exp(F¢fl/ RT) a(C+)r(MO ) , [29] iteration procedure one
obtains the net surface charge density as (defined by Eq. [33])
from which the particle charge number is
where ¢tJ is the potential of f3-plane affecting the state of
asso-ciated counterions. [37]
Actavis - IPR2017-01100, Ex. 1024, p. 27 of 33
-
74 KALLAY AND ZALAC
Numerical Simulation and Discussion 1 o
...-----r--r---------------,
The above theory, developed for kinetics of aggregation of
nanoparticles (nanocoagulation), will be demonstrated on a few
examples. Two systems (hematite and rutile) under different
con-ditions will be examined. The values of equilibrium parameters,
used in calculation of the particle charge number, were obtained by
interpretation of adsorption and electrokinetic data for ordi-nary
colloid particles (21, 22). It was assumed that these param-eters
approximately describe the properties of corresponding nanosystems.
In the evaluation the Gouy-Chapmen equation for spherical
interfacial layer, Eq. [36], was used. Once the charge number was
obtained, the stability coefficient was calculated via Eq.
[25].
Figure 2 demonstrates the effect of electrolyte concentration on
the stability of hematite nanodispersions containing parti-cles of
r = 3 nm. It is obvious that the stability of the system decreases
rapidly with electrolyte addition. At pH 4, particles are
positively charged so that association of anions with the surface
charged groups takes place. Nitrate ions were found to aggregate
the system more effectively with respect to the chlo-ride ions,
which is due to lower values of the surface associa-tion
equilibrium constant of the latter counterions. The effect of
electrolyte concentration is explicitly included in Eq. [25]
through ionic strength. However, particle charge number also
depends on the electrolyte concentration due to counterion
as-sociation so that both effects result in a decrease of stability
at
5
KCI
HEMATITE pH=4 r=3nm
oL---~--~---~----~~--~----~
·3 ·2 -1
lg(lJmol dm-3)
0
FIG. 2. Effect of electrolytes on the stability of hematite
aqueous nanodispersion (r = 3 nm) at T = 298 K and pH 4, as
obtained by Eq. [25]. The charge number was calculated by the
Surface Com-plexation model (Eqs. [26]-[39]) using parameters
obtained (21) with hematite colloid dispersion (r =60 nm): f 101 =
1.5 x to-5 mol m-2; Kp = 5xl04; Kd=l.5xl0- 11 ; pHpzc=7.6;
K(N03)=1410; K(Cl-)=525; C1(N03") = 1.88 Fm-2; C1(Cl-) = 1.81 F
m-2; C2 = oo; Er =sf so =78.54.
5
HEMATITE
KN03 r=3nm
oL-----~--~----~--~~----~----~
-3 -2 -1 0
lg(IJmol dm-3)
FIG. 3. Effect of pH on the stability of hematite aqueous
nanodispersion (r = 3 nm) in the presence of potassium nitrate at T
= 298 K. The parameters used in calculations are the same as in
Fig. 2.
higher electrolyte concentrations. Figure 3 demonstrates the
ef-fect of the activity of potential determining H+ ions. At lower
pH values particles are more positively charged, the system is more
stable, and higher electrolyte concentration is necessary for
aggregation. The effect of particle size on the stability of the
system is dramatic. As shown in Fig. 4, systems with smaller
10
HEMATITE KN03 pH=4
~
~
5 r=3nm
oL---~-----L---=~==~~----~--~ ·3 -2 0
FIG. 4. Effect particle size on the stability of hematite
aqueous nanodisper-sions in the presence of potassium nitrate at pH
4 and T = 298 K. The parameters used in calculations are the same
as in Fig. 2.
Actavis - IPR2017-01100, Ex. 1024, p. 28 of 33
-
STABILITY OF NANODISPERSIONS 75
10~------~------------------------.
5 RUTILE pH= 9.5 LiN03
r=3nm
oL---~----~~==~~L---~--~ -3 -2 -1 0
lg(lJmol dm-3)
FIG. S. Effect of kind of material on the stability of aqueous
nanodisper-sion (r = 3 nm) at T = 298 K. For hematite particles,
the parameters used in calculations are the same as in Fig. 2. For
rutile rods (length is I 00--240 nm and width is 45 nm) the
parameters are (22): r 1o1 = w-5 mol m-2 ; Kp = 6 x 107 ; Kd =6 x
w-5; pHpzc =6.0; K(Li+) =380; C1(Li+)= 1.58 F m-2 ; Cz =oo.
particles are markedly less stable. In fact, such a system is
un-stable even at low electrolyte concentrations. It is interesting
to analyze the particle size effect on the basis of Eq. [25],
assum-ing that surface charge density does not depend significantly
on the particle size. In such a case the particle charge number is
proportional to r 2 so that first term associated with B is
pro-portional to r 3• Accordingly, one would expect an increase of
stability coefficient with particle size. The second term,
associ-ated with ionic strength, acts in the opposite direction.
However, this term is not significant at low ionic strength so that
the first one prevails. At high ionic strength the second term
becomes influential, but also the charge density and charge number
are reduced so much that log W approaches zero for any particle
size. A similar conclusion would be obtained if one assumes that
surface potential does not depend significantly on parti-cle size.
However, according to Eq. [36], the size effect on the stability
will be less pronounced. The reality is between the above-noted
extremes, but the use of the Surface Complexa-tion model enabled us
to avoid such speculations. Generaly, one may conclude that smaller
particles are less stable. Indeed, as shown for zirconia (24) the
nanodispersion coud be stable at very high surface potentials which
were achieved 6 pH units below the point of zero charge. The
specificity of the material comprising nanoparticles could be seen
through the values of the equilibrium parameters. Figure 5 shows
the difference between the predicted behavior of hematite and
rutile nanosystems. The pH values were chosen to be approximately
equally far from the point of zero charge, but in the opposite
direction. At pH 9.5 rutile is negatively while at pH 4 hematite is
positively charged.
The absolute values of surface potentials are approximately the
same. However, due to the different values of the equilibrium
parameters, rutile was found to be significantly less stable than
hematite.
It may be concluded that application of the Br¢nsted concept to
the stability of nanodispersions shows that the presence of
electrolytes may completely reduce the stability, and that the
electrolytes with counterions exhibiting higher affinity are more
effective for association at the interface. Lower surface
poten-tial (pH closer to the point of zero charge) slightly reduces
the stability. In the above analysis the parameters used for
calcula-tions may differ from the reality since they were obtained
from measurements with relatively large colloid particles; however,
the general behavior of nanosystems may be still explained. The
comparison between hematite and rutile showed how sensitive the
stability of nanosystems is on the inherent characteristics of the
solid. Different equilibrium parameters result in a very pronounced
difference in the stability. However, the major char-acteristics of
the system are the particle size; smaller particles are
significantly less stable. The interpretation based on the Br1
-
76 KALLAY AND ZALAC
8. Deijaguin, B. V., and Landau, L., Acta Physicochim., USSR 14,
552 (1941).
9. Hogg, R., Healy, T. W., and Fuerstenau, D. W., Trans. Faraday
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10. Bhattachaijee, S., Elimelech, M., and Borkovec, M., Croat.
Chem. Acta 71, 883 (1998).
11. Semmler, M., Ricka, J., and Borkovec, M., Colloids Surf.
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(2000). 14. Sun, N., and Walz, Y., J. Colloid Interface Sci. 234,
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Soc., Faraday Trans. I
70, 1807 (1974). 16. Davis, J. A., James, R. 0., and Leckie, J.
0., J. Colloid Interface Sci. 63,
480 (1978). 17. Westall, J., and Hohl, H., Adv. Colloid
Interface Sci. 12, 265
(1980).
18. Dzombak, D. A., and Morel, F. M. M., "Surface Complexation
Modelling." Wiley Interscience, New York, 1990.
19. Kallay, N., Kova~evic, D., and Cop, A., in "Interfacial
Dynamics" (N. Kallay, Ed.), Surfactant Science Series, Vol. 8.
Dekker, New York, 1999.
20. Kallay, N., and Zalac, S., J. Colloid Interface Sci. 230, 1
(2000). 21. Colic, M., Fuerstenau, D. W., Kallay, N., and
Matijevic, E., Colloid Surf.
59, 169 (1991). 22. Kallay, N., Colic, M., Fuerstenau, D. W.,
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Colloid Polym. Sci. 272,554 (1994). 23. de Gennes, P.-G., Croat.
Chem. Acta 71, 833 (1998). 24. Peyre, V., Spalla, 0., Belloni, L.,
and Nabavi, M., J. Colloid Interface Sci.
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Actavis - IPR2017-01100, Ex. 1024, p. 30 of 33
-
Exhibit 6
Actavis - IPR2017-01100, Ex. 1024, p. 31 of 33
-
Comparison of physicochemical characteristics and stability
onhree novel tonnu1at1ons ot paclitaxeJ: ADraxane, Nanoxei, ana
uenexo1 rM -... rage 1 v• ~
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99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA
Drug Delivery and Targeting: Poster Presentations -Proffered
Abstracts
Abstract #5622
Comparison of physicochemical characteristics and stability of
three novel formulations of paclitaxel: Abraxane, Nanoxel, and
Genexol PM
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Niles Ron, Jon Cordia, Andrew Yang, Sherry Ci, Phithi Nguyen,
Melissa Hughs and Neil Desai
Abraxis Bioscience, Inc., Los Angeles, CA
Background: Abraxane (Abraxis BioScience, Inc., Los Angeles, CA,
approved in USA and Canada), Nanoxel (Dabur Pharma, H.P., India,
approved in India), and Genexol PM (Samyang Pharmaceuticals, Seoul,
Korea, approved in Korea) are 3 commercially approved, novel
formulations of paclitaxel. Abraxane consists of albumin-bound
injectable nanoparticles of paclitaxel, while Genexol and Nanoxel
(utilizing cosolvents) are polymeric-micelle formulations. Abraxane
and Genexol are lyophilized products approved for 25°C ± 2°C
storage, while Nanoxel is a liquid formulation approved for 2-8°C
storage. This study investigated the physicochemical
characteristics and short-term stability of the 3 products under
recommended clinical use conditions and under accelerated
conditions. Methods: The drugs were reconstituted and prepared per
the instructions provided in the respective package inserts.
Abraxane and Genexol were reconstituted using the recommended
saline diluent, while Nanoxel was mixed and diluted in 10%
dextrose. Each drug was reconstituted to 0.7 mg/mL and 5 mg/mL.
Physical stability was monitored both visually and microscopically;
particle size was measured and monitored over time at room
temperature (RT, measured to be 23°C) and 40°C using photon
correlation spectroscopy (PCS) (Zetasizer 3000, Malvern, UK).
Chemical purity was measured by reduced reversed-phase HPLC
(Shimadzu Scientific Instruments, MD). Results: Following
reconstitution, Abraxane was determined to be stable both
physically and chemically
Actavis - IPR2017-01100, Ex. 1024, p. 32 of 33
-
Comparison of physicochemical characteristics and· stability of
three novel fonnulations ofpaclitaxel: Abraxane, Nanoxel, and
Genexol PM-... Page 2 of2
at RT and 40°C over 24 hrs, with no evidence ofnanoparticle size
growth at either 0.7 mg/mL or 5 mg/mL. While reconstituted Genexol
was stable at RT over 24 hrs, micelle instability resulting in
precipitation of paclitaxel in the form of large needle-like
crystals for both 0. 7 mg/mL and 5 mg/mL formulations was seen
between 2 to 4 hrs at 40°C. These observations were confirmed using
orthogonal techniques, visual assessment from photomicrographs, and
PCS particle size measurement. For Nanoxel at 0.7 mg/mL, a minor,
but consistent, increase in particle size was observed at RTover 24
hrs. However, significant aggregation, particle-size growth, and
crystallization were seen within 4 hrs at 40° C. HPLC data
comparing pre- and post-filtration confirmed that the crystal
formation for both Nanoxel and Genexol resulted from paclitaxel
precipitation and aggregation. In addition, analytical results
showed that Nanoxel had slightly lower paclitaxel purity as
compared to either Abraxane or Genexol. Conclusions: Nanoparticle
albumin-bound paclitaxel, Abraxane, showed excellent
physicochemical stability as compared to the micellar formulations,
Nanoxel and Genexol. Particle size growth and crystal formation
were readily apparent in Nanoxel and Genexol, especially in the
short term under accelerated conditions.
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Actavis - IPR2017-01100, Ex. 1024, p. 33 of 33