U.S. patent application number 14/144289 was filed with the patent office on 2014-04-24 for methods and systems for generating nanoparticles.
This patent application is currently assigned to Cerulean Pharma, Inc.. The applicant listed for this patent is Cerulean Pharma, Inc.. Invention is credited to David S. Dickey, John Podobinski, J. Michael Ramstack.
Application Number | 20140113137 14/144289 |
Document ID | / |
Family ID | 44657175 |
Filed Date | 2014-04-24 |
United States Patent
Application |
20140113137 |
Kind Code |
A1 |
Podobinski; John ; et
al. |
April 24, 2014 |
Methods And Systems For Generating Nanoparticles
Abstract
In one aspect, the present invention provides a process for
forming polymeric nanoparticles, which comprises using a static
mixer to create a mixed flowing stream of an anti-solvent, e.g., by
introducing a liquid anti-solvent into a static mixer, and
introducing a polymer solution into the mixed flowing anti-solvent
stream such that controlled precipitation of polymeric
nanoparticles occurs. The nanoparticles can then be separated from
the anti-solvent stream.
Inventors: |
Podobinski; John; (Boston,
MA) ; Ramstack; J. Michael; (Lunenburg, MA) ;
Dickey; David S.; (Dayton, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cerulean Pharma, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
Cerulean Pharma, Inc.
Cambridge
MA
|
Family ID: |
44657175 |
Appl. No.: |
14/144289 |
Filed: |
December 30, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13793727 |
Mar 11, 2013 |
8618240 |
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14144289 |
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13479646 |
May 24, 2012 |
8404799 |
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13793727 |
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13023163 |
Feb 8, 2011 |
8207290 |
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13479646 |
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61317783 |
Mar 26, 2010 |
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Current U.S.
Class: |
428/402 |
Current CPC
Class: |
A61K 31/337 20130101;
A61J 1/00 20130101; B82Y 40/00 20130101; A61K 47/593 20170801; A61P
9/00 20180101; C08J 3/14 20130101; Y10S 977/895 20130101; C08L
29/04 20130101; A61K 9/14 20130101; C08L 67/02 20130101; A61P 29/00
20180101; C08F 6/12 20130101; A61P 35/00 20180101; B29B 2009/125
20130101; A61K 47/60 20170801; A61K 9/51 20130101; A61K 47/58
20170801; A61P 31/00 20180101; A61K 9/146 20130101; Y10T 428/2982
20150115 |
Class at
Publication: |
428/402 |
International
Class: |
C08L 67/02 20060101
C08L067/02; C08L 29/04 20060101 C08L029/04 |
Claims
1-136. (canceled)
137. A population of polymeric nanoparticles, comprising: a
plurality of polymeric nanoparticles having an amphiphilic
co-polymer as at least one constituent, wherein said polymeric
nanoparticles exhibit an average particle size equal to or less
than about 100 nm and a polydispersity index in a range of about
0.05 to about 0.1.
Description
RELATED APPLICATION
[0001] The present application claims priority to a provisional
application entitled "Methods and systems for generating
nanoparticles" filed Mar. 26, 2010 and having a Ser. No.
61/317,783, which is herein incorporated by reference in its
entirety.
BACKGROUND
[0002] The present invention relates generally to methods, devices
and systems for fabricating nanoparticles, and more particularly to
such methods, devices and systems that can be employed to generate
polymeric nanoparticles.
[0003] A variety of methods are known for generating nanoparticles.
In one such method, commonly known as nanoprecipitation or flash
precipitation, a polymer solution comprising a polymer dissolved in
a process solvent is brought into contact with another solvent
(also known as anti-solvent) in which the process solvent is
miscible but the polymer is not. As a result, the process solvent
diffuses rapidly into the anti-solvent while the polymer aggregates
into a plurality of nanoparticles.
[0004] The conventional nanoprecipitation processes, however,
suffer from a number of shortcomings. For example, it is difficult
to control predictably the average particle size and the size
distribution of the generated nanoparticles. Further, many
challenges exist in scaling up such processes to generate
nanoparticles on a large scale.
[0005] Accordingly, there is a need for enhanced methods, devices
and systems for generating nanoparticles.
SUMMARY
[0006] In one aspect, the present invention provides a process for
forming polymeric nanoparticles, which comprises introducing an
anti-solvent into a static mixer to create a mixed flowing stream
of the anti-solvent and introducing a polymer-carrying liquid,
e.g., a polymer solution, or a polymer dispersion or a mixed
polymer solution/dispersion, into the mixed flowing stream of the
anti-solvent so as to form polymeric nanoparticles. The polymeric
nanoparticles can be formed via non-reactive or reactive
aggregation of at least one polymer, and in some cases one or more
additives, of the polymer solution, or of the polymer dispersion or
of the mixed polymer solution/dispersion, as well as in some
embodiments a colloid stabilizer of the anti-solvent. For example,
the polymeric nanoparticles can be formed via assembly/growth of at
least one polymer, and in some cases one or more additives, of the
polymer solution, or of the polymer dispersion or of the mixed
polymer solution/dispersion, as well as in some embodiments a
colloid stabilizer of the anti-solvent. An example of reactive
aggregation can include generating the polymeric nanoparticles via
formation of covalent chemical bonds. An example of non-reactive
aggregation can include generating the polymeric nanoparticles via
assembly without formation of covalent chemical bonds.
[0007] For example, the nanoparticles can be formed by
precipitation (e.g., a controlled precipitation through selection
of various parameters, such as the flow rate of the anti-solvent
and/or the flow rate and/or the polymer concentration of the
polymer solution (or of the polymer dispersion or of the mixed
polymer solution/dispersion)). The nanoparticles can then be
separated from the anti-solvent stream. Although in the following
description, various aspects and embodiments of the invention are
primarily described by reference to a polymer solution, the
teachings of the invention can also be practiced with a polymer
dispersion and/or a mixed polymer solution/dispersion.
[0008] The dimensions of the static mixer, e.g., its length and
diameter, can vary over a wide range. By way of example, in some
embodiments the static mixer can have a diameter greater than about
1 cm, or greater than about 2 cm, or greater than about 10 cm, or
larger. For example, the static mixer can have a diameter in a
range of about 1 cm to about 100 cm, or in a range of about 20 cm
to about 80 cm, or in a range of about 30 cm to about 70 cm, or in
a range of about 40 cm to about 60 cm. In some embodiments, the
static mixer can have between about 1 to about 24 mixing elements.
By way of example, the number of the mixing elements can be in a
range of about 12 to about 24. In some embodiments, the number of
mixing elements is in a range of about 1 to about 4. In some
embodiments, the static mixer is configured to cause substantially
isotropic mixing of a fluid over at least about 50%, or at least
about 60%, or at least about 70%, or at least about 80%, or at
least about 90%, or over the entire volume of a portion of a
conduit in which the static mixer is disposed.
[0009] A variety of flow rates, flow velocities and mixing
conditions can be employed. In some embodiments, the anti-solvent
flowing stream is introduced into the static mixer at a flow rate
in a range of about 20 ml/min to about 2000 ml/min, e.g., in a
range of about 20 ml/min to about 1500 ml/min, or in a range of
about 30 ml/min to about 1000 ml/min, or in a range of about 40
ml/min to about 500 ml/min, or in a range of about 20 ml/min to
about 400 ml/min, or in a range of about 20 ml/min to about 300
ml/min, or in a range of about 20 ml/min to about 200 ml/min, or in
a range of about 20 ml/min to about 100 ml/min. In some
embodiments, the anti-solvent flowing stream exhibits an average
axial flow velocity in a range of about 1 cm/sec to about 100
cm/sec (e.g., in a range of about 1.5 cm/sec to about 60 cm/sec).
By way of example, in some embodiments, the anti-solvent flowing
stream can exhibit an average axial flow velocity in a range of
about 1 cm/sec to about 10 cm/sec, or in a range of about 10 cm/sec
to about 20 cm/sec, or in a range of about 20 cm/sec to about 30
cm/sec, or in a range of about 30 cm/sec to about 40 cm/sec, or in
a range of about 40 cm/sec to about 50 cm/sec, or in a range of
about 50 cm/sec to about 60 cm/sec, or in a range of about 60
cm/sec to about 70 cm/sec, or in a range of about 70 cm/sec to
about 80 cm/sec, or in a range of about 80 cm/sec to about 90
cm/sec, or in a range of about 90 cm/sec to about 100 cm/sec. In
many embodiments, the polymer solution is introduced into the mixed
flowing stream of the anti-solvent as a liquid stream.
[0010] A wide range of ratios of the flow rate of the mixed flowing
stream of the anti-solvent relative to that of the polymer solution
stream can be employed. For example, the ratio of the anti-solvent
flow rate relative to the polymer solution flow rate can be in a
range of about 1:1 to about 100:1, e.g., in a range of about 1:1 to
about 10:1, or in a range of about 1:1 to about 20:1, or in a range
of about 1:1 to about 30:1, or in a range of about 1:1 to about
40:1, or in a range of about 1:1 to about 50:1, or in a range of
about 1:1 to about 60:1, or in a range of about 1:1 to about 70:1,
or in a range of about 1:1 to about 80:1, or in a range of about
1:1 to about 90:1. In some embodiments, the flow rate of the
anti-solvent stream is about 10 times greater than the flow rate of
the polymer solution stream. In some embodiments, the polymer
solution is introduced into the mixed flowing stream of the
anti-solvent as a liquid stream at an axial flow velocity in a
range of about 0.5 cm/sec to about 40 cm/sec, for example, in a
range of about 2 cm/sec to about 20 cm/sec.
[0011] The nanoparticles can be formed via precipitation, typically
over a short time period, upon contact of the polymer solution with
the mixed flowing stream of the anti-solvent. For example, the
nanoparticles can be generated via precipitation within a time
period less than about 10 milliseconds (e.g., a time period in a
range of about 1 millisecond to about 10 milliseconds, or in a
range of about 2 milliseconds to about 10 milliseconds), or within
a time period less than about 5 milliseconds (e.g., a time period
in a range of about 1 millisecond to about 5 milliseconds, or a
time period in a range of about 2 milliseconds to about 5
milliseconds) upon exposure of the polymer solution to the mixed
flowing stream of the anti-solvent. For example, in some
embodiments, at least about 50%, or at least about 60%, or at least
about 70%, or at least about 80%, or at least about 90%, or all of
the nanoparticles are formed within a time period less than about
10 milliseconds (e.g., a time period in a range of about 1
millisecond to about 10 milliseconds, or a time period in a range
of about 2 milliseconds to about 10 milliseconds), or within a time
period less than about 5 milliseconds (e.g., a time period in a
range of about 1 millisecond to about 5 milliseconds, or a time
period in a range of about 2 milliseconds to about 5 milliseconds)
upon exposure of the polymer solution to the mixed flowing stream
of the anti-solvent. In an embodiment, the time period over which
the nanoparticles are generated can be adjusted by controlling,
e.g., the flow rate of the anti-solvent flowing stream, the
concentration of the polymer solution, the concentration of the
colloid stabilizer, among others. For example, in an embodiment, as
the flow rate of the anti-solvent flowing stream increases the time
period over which the nanoparticles are generated decreases.
[0012] The polymer solution (and in some embodiments a polymer
dispersion or a mixed polymer solution/dispersion) can be
introduced into the mixed flowing stream of the anti-solvent at a
variety of locations. For example, the static mixer can extend from
a proximal end to a distal end and the polymer solution can be
introduced into the mixed flowing stream of the anti-solvent at an
intermediate location between the proximal and distal ends of the
static mixer. Alternatively, the polymer solution can be introduced
into the mixed flowing stream of the anti-solvent in proximity to
the proximal end of said static mixer. In other embodiments, the
polymer solution can be introduced into the mixed anti-solvent
flowing stream in proximity to the distal end of the static
mixer.
[0013] In a related aspect, the nanoparticles generated by the
above process exhibit a polydispersity index equal to or less than
about 0.25. By way of example, the nanoparticles can exhibit a
polydispersity index in a range of about 0.05 to about 0.1.
[0014] In a related aspect, in the above process for fabricating
nanoparticles, the flow rate of the mixed flowing stream of the
anti-solvent can be changed so as to adjust an average particle
size of the polymeric nanoparticles. By way of example, the flow
rate of the anti-solvent stream can be selected such that the
polymeric nanoparticles exhibit an average particle size equal to
or less than about 200 nm while exhibiting in some cases a particle
size distribution less than about 100 nm. Further, in some
embodiments, the flow rate of the anti-solvent stream can be
selected such that the polymeric nanoparticles will exhibit an
average particle size equal to or less than about 100 nm, e.g., in
a range of about 40 nm to about 100 nm. By way of example, in some
embodiments, the flow rate of the mixed flowing stream of the
anti-solvent can be varied between about 100 ml/min to about 1800
ml/min to adjust the average particle size of the polymeric
nanoparticles in a range of about 100 nm to about 230 nm.
[0015] In a related aspect, the flow rate of the mixed flowing
stream of the anti-solvent can be selected to be in a range in
which an average particle size of the polymeric nanoparticles is
substantially independent of the anti-solvent flow rate.
Alternatively, the flow rate of mixed flowing stream of the
anti-solvent can be selected to be in a range in which an average
particle size of the polymeric nanoparticles is strongly dependent
on the anti-solvent flow rate. For example, in an embodiment, when
the flow rate of the mixed flowing stream of the anti-solvent is
less than about 200 ml/min, e.g., in a range of about 20 ml/min to
about 200 ml/min, or in a range about 20 ml/min to about 100
ml/min, the average particle size of the polymeric nanoparticles is
strongly dependent on the anti-solvent flow rate. For example, in
an embodiment, when the flow rate of the mixed flowing stream of
the anti-solvent is greater than about 200 ml/min, e.g., greater
than about 300 ml/min (e.g., in a range of about 300 ml/min to
about 1000 ml/min, or in a range of about 500 ml/min to about 2000
ml/min), the average particle size of the polymeric nanoparticles
is substantially independent of the anti-solvent flow rate.
[0016] In a related aspect, the average axial flow velocity of the
mixed flowing stream of the anti-solvent or that of the polymer
solution can be selected to be in a range in which an average
particle size of the nanoparticles is substantially independent of
such axial flow velocity. Alternatively, the average axial flow
velocity of the mixed flowing stream of the anti-solvent or that of
the polymer solution can be selected to be in a range in which an
average particle size of the nanoparticles is strongly dependent on
such flow velocity.
[0017] In another aspect, a ratio of a flow rate of the
anti-solvent stream relative to a flow rate of the polymer solution
can be changed so as to adjust an average particle size of the
polymeric nanoparticles.
[0018] In some embodiments, the method for forming polymeric
nanoparticles can include the additional steps of selecting one or
more parameters, e.g., anti-solvent and/or polymer solution flow
rate, polymer concentration in the polymer solution, the average
axial flow velocity of the mixed flowing stream of the anti-solvent
and/or that of the polymer solution, or other parameters discussed
herein, and carrying out the method under such selected conditions.
Optionally, the method can include evaluating a sample of the
nanoparticles produced to determine if the nanoparticles meet one
or more predefined criteria, e.g., average particle size,
polydispersity, drug loading, etc. In some embodiments, if the
sample of the nanoparticles fails to meet the one or more
predefined criteria, one or more of the parameters, such as those
listed above, can be adjusted and the method carried out under the
adjusted conditions. Again, a sample of the nanoparticles produced
can be evaluated to determine if the nanoparticles meet the one or
more predefined criteria. This process can be repeated, if needed,
until a sample of the nanoparticles that meets the one or more
predefined criteria is achieved.
[0019] In some embodiments, at least one attribute of a sample of
nanoparticles produced (e.g., an average particle size,
polydispersity, drug loading, etc), or that of its preparation, can
be compared with a reference value for that attribute. The
reference value can be, e.g., a release parameter or a
manufacturing specification, e.g., one set by a regulatory agency,
e.g., the FDA or EMEA, a compendial authority, or a manufacturer.
In an embodiment, the reference value is a value exhibited by a
preparation previously made by the method. In an embodiment, e.g.,
responsive to whether the attribute meets a reference value for
that attribute a further decision or step is taken, e.g., the
sample is classified, selected, rejected, accepted, or discarded,
released or withheld, processed into a drug product, shipped, moved
to a different location, formulated, labeled, packaged, released
into commerce, exported, imported, or sold or offered for sale,
depending on whether the preselected criterion is met. For example,
based on the result of the evaluation, the batch from which a
sample is taken can be processed, e.g., as just described. For
example, if the criterion is met, the preparation is sold, shipped,
or offered for sale or otherwise released into commerce.
[0020] The polymer solution can comprise a polymer dissolved in a
process solvent, wherein the process solvent is miscible, or at
least partially miscible, with the anti-solvent. In some
embodiments, the concentration of the polymer in the polymer
solution can be changed so as to adjust an average particle size of
the polymeric nanoparticles. A variety of polymers can be employed.
By way of example, the polymer can be any of
poly(lactide-co-glycolide), poly(lactide),
poly(epsilon-caprolactone), poly(isobutylcyanoacrylate),
poly(isohexylcyanoacrylate), poly(n-butylcyanoacrylate),
poly(acrylate), poly(methacrylate), poly(lactide)-poly(ethylene
glycol), poly(lactide-co-glycolide)-poly(ethylene glycol),
poly(epsilon-caprolactone)-poly(ethylene glycol), and
poly(hexadecylcyanoacrylate-co-poly(ethylene
glycol)cyanoacrylate).
[0021] In some embodiments, the polymer solution can include at
least one additive. The additive can be any of a therapeutic agent
or an imaging agent. In some embodiments, such a therapeutic or
imaging agent can be coupled to, associated with, or incorporated
in the polymer. For example, in some embodiments, such a
therapeutic or imaging agent can be conjugated to, or embedded in
the polymer. In some embodiments, multiple different agents can be
coupled to, associated with, or incorporated in the polymer. In
some embodiments, the imaging agent can be coupled to the
therapeutic agent
[0022] By way of example, the therapeutic agent can be, without
limitation, any of an anti-neoplastic agent, an anti-inflammatory
agent, a cardiovascular active agent, or an anti-metabolite.
[0023] In some embodiments, the therapeutic agent can be any of a
taxane, an epothilone, a boronic acid proteasome inhibitor, and an
antibiotic.
[0024] In some embodiments, the imaging agent can be, without
limitation, any of a radioactive or non-radioactive agent, or a
fluorescent agent. Some examples of suitable imaging agents
include, without limitation, Technetium Bicisate, Ioxaglate,
Fluorodeoxyglucose, label-free Raman imaging agents, encapsulate
MRI contrast agent Gd-DTPA, and rhodamine 6G as a fluorescent
agent. In some embodiments, the imaging agent can be radiolabeled
docetaxel (e.g., 3H-radiolabeled docetaxel or 14C-radiolabeled
docetaxel), or radiolabeled paclitaxel.
[0025] The process solvent can include, without limitation, any of
acetone, ether, alcohol, tetrahydrofuran, 2-pyrrolidone,
N-Methyl-2-pyrrolidone (NMP), dimethylformamide (DMF),
dimethylacetamide (DMA), methyl acetate, ethyl formate, methyl
ethyl ketone (MEK), methyl isobutyl ketone (MIBK), methyl propyl
ketone, isopropyl ketone, isopropyl acetate, acetonitrile (MeCN)
and dimethyl sulfoxide (DMSO).
[0026] In some embodiments, the anti-solvent can include an aqueous
solution. By way of example, the aqueous solution can include any
of an alcohol or an ether, and water. In some embodiments, the
anti-solvent can include an organic solvent or a mixture of two or
more organic solvents. For example, the anti-solvent can include,
without limitation, any of methanol, ethanol, n-propanol,
isopropanol, n-butanol, and ethyl ether.
[0027] In some embodiments, the anti-solvent can include a colloid
stabilizer. By way of example, the colloid stabilizer can include,
without limitation, any of poly(vinyl alcohol), Dextran and
pluronic F68, poly(vinyl pyrrolidone), solutol, Tween 80,
poloxamer, carbopol, poly-ethylene glycol, sodium dodecyl sulfate,
poly(.epsilon.-caprolactone), peptides, and carbohydrates.
[0028] In some embodiments, the polymer solution is delivered as a
liquid stream that intersects the anti-solvent stream at a non-zero
angle. The angle can be an acute angle, for example, one in a range
of about 10 degrees to about 90 degrees (e.g., in a range of about
50 degrees to about 90 degrees). In some embodiments, the angle can
be in a range of about 10 degrees to about 170 degrees. In some
other embodiments, the polymer solution is injected into the
flowing stream of the anti-solvent.
[0029] In another aspect, the step of separating the nanoparticles
includes collecting the nanoparticles downstream from the static
mixer as a suspension in a mixture of the anti-solvent and a
process solvent of the polymer solution. At least a portion of the
process solvent can be removed from the suspension in order to
concentrate the suspension. For example, the suspension can be
diafiltered to remove at least a portion of the process
solvent.
[0030] In some embodiments, a lyoprotectant can be added to the
preparation, e.g., the suspension. It can be added prior to or
after the step of concentrating the suspension, to protect the
nanoparticles in a subsequent lyophilization step. By way of
example, the lyoprotectant can be, without limitation, a
derivatized cyclic oligosaccharide, e.g., a derivatized
cyclodextrin, e.g., 2 hydroxy propyl-.beta. cyclodextrin, e.g.,
partially etherified cyclodextrins (e.g., partially etherified
cyclodextrins) disclosed in U.S. Pat. No. 6,407,079, the contents
of which are incorporated herein by this reference.
[0031] In another aspect, a process for forming polymeric
nanoparticles is disclosed, which includes introducing an
anti-solvent into a static mixer so as to generate a mixed flowing
stream of the anti-solvent, and introducing a polymer solution (or
a polymer dispersion or a mixed polymer solution/dispersion) into
the mixed flowing stream of the anti-solvent to generate polymeric
nanoparticles (e.g., via precipitation) such that the polymeric
nanoparticles exhibit a polydispersity index equal to or less than
about 0.25. For example, the polymeric nanoparticles can exhibit a
polydispersity index in a range of about 0.05 to about 0.1.
[0032] In some embodiments, the polymeric nanoparticles can exhibit
an average particle size equal to or less than about 500 nm. For
example, the polymeric nanoparticles can exhibit an average
particle size in a range of about 5 nm to about 500 nm, or in a
range of about 10 nm to about 500 nm, or in a range of about 20 nm
to about 500 nm, or in a range of about 30 nm to about 500 nm, or
in a range of about 40 nm to about 500 nm, or in a range of about
50 nm to about 500 nm.
[0033] In some embodiments, the polymeric nanoparticles can exhibit
an average particle size equal to or less than about 400 nm. For
example, the polymeric nanoparticles can exhibit an average
particle size in a range of about 5 nm to about 400 nm, or in a
range of about 10 nm to about 400 nm, or in a range of about 20 nm
to about 400 nm, or in a range of about 30 nm to about 400 nm, or
in a range of about 40 nm to about 400 nm, in a range of about 50
nm to about 400 nm.
[0034] In some embodiments, the polymeric nanoparticles can exhibit
an average particle size equal to or less than about 300 nm. For
example, the polymeric nanoparticles can exhibit an average
particle size in range of about 5 nm to about 300 nm, or in a range
of about 10 nm to about 300 nm, or in a range of about 20 nm to
about 300 nm, or in a range of about 30 nm to about 300 nm, or in a
range of about 40 nm to about 300 nm, or in a range of about 50 nm
to about 300 nm.
[0035] In some embodiments, the polymeric nanoparticles can exhibit
an average particle size equal to or less than about 200 nm. For
example, the polymeric nanoparticles can exhibit an average
particle size in a range of about 5 nm to about 200 nm, or in a
range of about 10 nm to about 200 nm, or in a range of 20 nm to
about 200 nm, or in a range of about 30 nm to about 200 nm, or in a
range of about 40 nm to about 200 nm, or in a range of about 50 nm
to about 200 nm.
[0036] In some embodiments, the polymeric nanoparticles can exhibit
an average particle size equal to or less than about 100 nm. For
example, the polymeric nanoparticles can exhibit an average
particle size in a range of about of 5 nm to about 100 nm, or in a
range of about 10 nm to about 100 nm, or in a range of about 20 nm
to about 100 nm, or in a range of about 30 nm to about 100 nm, or
in a range of about 40 nm to about 100 nm, or in a range of about
50 nm to about 100 nm.
[0037] In some embodiments, the anti-solvent flow comprises a
stream exhibiting a flow rate in a range of about 20 ml/min to
about 2000 ml/min. In some embodiments, the mixed flowing stream of
anti-solvent exhibits an average axial velocity in a range of about
1 cm/sec to about 100 cm/sec, e.g., in a range of about 1.5 cm/sec
to about 60 cm/sec.
[0038] In the above process for forming polymeric nanoparticles,
the polymer solution can be introduced into the mixed flowing
stream of the anti-solvent at a variety of locations relative to
the static mixer. For example, the polymer solution can be
introduced into the mixed flowing stream of the anti-solvent at an
intermediate location between a proximal end and a distal end of
the static mixer. Alternatively, the polymer solution can be
introduced into the mixed flowing stream of the anti-solvent in
proximity to the proximal end, or the distal end, of the static
mixer.
[0039] In the above process, the polymer solution can be introduced
as a liquid stream into the mixed flowing stream of the
anti-solvent at a variety of flow rates. For example, a flow rate
of the anti-solvent stream relative to a flow rate of said polymer
solution stream can be in a range of about 1:1 to about 100:1,
e.g., in a range of about 1:1 to about 10:1, or in a range of about
1:1 to about 20:1, or in a range of about 1:1 to about 30:1, or in
a range of about 1:1 to about 40:1, or in a range of about 1:1 to
about 50:1, or in a range of about 1:1 to about 60:1, or in a range
of about 1:1 to about 70:1, or in a range of about 1:1 to about
80:1, or in a range of about 1:1 to about 90:1. Further, in some
embodiments, the polymer solution stream is introduced into the
mixed flowing stream of the anti-solvent at a non-zero angle, e.g.,
an acute angle, relative to a flow direction of the anti-solvent
stream. In some embodiments, the polymer solution is injected into
the mixed anti-solvent stream.
[0040] The polymer solution can include a polymer dissolved in a
process solvent, where the process solvent is miscible, or is at
least partially miscible, with the anti-solvent. In some
embodiments, the polymer solution can include at least one
additive, such as a therapeutic agent or an imaging agent. A
variety of therapeutic agents and imaging agents can be employed,
such as those listed above. In some embodiments, one or more of
such agents are coupled to, associated with, or incorporated in the
polymer. In some embodiments, multiple different agents can be
coupled to, associated with, or incorporated in the polymer. In
some embodiments, one or more of such agents are conjugated to, or
embedded in the polymer.
[0041] A variety of polymers, process solvents and anti-solvents
can be employed in the above process. Some examples of such
polymers, process solvents and anti-solvents are provided above. In
some embodiments, the anti-solvent can include a colloid
stabilizer, such as those listed above.
[0042] In another aspect, the invention provides a process for
controlling particle size of nanoparticles formed, e.g., by
precipitation, which comprises introducing an anti-solvent liquid
flow into a static mixer to generate a mixed flowing stream of the
anti-solvent, and introducing a polymer solution into the mixed
flowing stream of the anti-solvent so as to generate a plurality of
polymeric nanoparticles, e.g., by precipitation. The flow rate of
the anti-solvent stream through said static mixer is controlled so
as to adjust an average particle size of the nanoparticles.
[0043] The step of controlling the flow rate of the anti-solvent
stream can include changing the flow rate so as to vary the average
particle size in a range of about 50 nm to about 200 nm.
[0044] In the above process for controlling particle size of
nanoparticles, the polymer solution can comprise a polymer
dissolved in a process solvent that is miscible, or at least
partially miscible, in the anti-solvent. In some embodiments, the
polymer solution can include an additive, such as a therapeutic or
an imaging agent. In some embodiments, one or more of such agents
are embedded in the polymer. In some embodiments, one or more of
such agents are conjugated to the polymer. Some examples of
suitable therapeutic and imaging agents are provided above.
[0045] A variety of polymers, process solvents and anti-solvents
can be employed in the above process. Some examples of such
polymers, process solvents and anti-solvents are provided
above.
[0046] In some embodiments, the anti-solvent can include a colloid
stabilizer. Some examples of suitable colloid stabilizers are
provided above.
[0047] In another aspect, a system for generating polymeric
nanoparticles is disclosed, which comprises a conduit having a
first input port for receiving an anti-solvent, and at least one
static mixer disposed in the conduit to generate a mixed flowing
stream of the anti-solvent, where the static mixer extends from a
proximal end to a distal end. The conduit has a second input port
disposed relative to the static mixer so as to allow introducing a
polymer solution into the mixed flowing stream of the anti-solvent
to generate polymeric nanoparticles, e.g., via precipitation. The
system can further include a device, e.g., a variable pump, adapted
to cause a flow of the anti-solvent from a reservoir to the conduit
and to control a flow rate of the anti-solvent through the static
mixer for adjusting an average particle size of the
nanoparticles.
[0048] In some embodiments, the conduit in which the static mixer
is disposed has an internal diameter of at least about 1 mm, or at
least about 10 mm, or at least about 100 mm, or at least 500
mm.
[0049] In some embodiments, the device for causing the anti-solvent
flow is adapted to control a flow rate of said anti-solvent through
the conduit within a range of about 20 ml/min to about 2000
ml/min.
[0050] In some embodiments, the second input port is located at an
intermediate location between the proximal and distal ends of the
static mixer. In some other embodiments, the second input port is
located in proximity to the proximal end, or the distal end, of the
static mixer. In some embodiments, the second input port is
configured so as to allow introduction of the polymer solution into
the conduit at a non-zero angle, e.g., at an acute angle (e.g.,
wherein the angle between the direction of flow through the conduit
and the direction of flow entering the conduit through the second
input port is in a range of about 50 degrees to about 90 degrees),
relative to a flow direction of the anti-solvent stream.
[0051] In some embodiments, the system includes at least one
injector coupled to the second input port for injecting the polymer
solution into the mixed flowing stream of the anti-solvent.
[0052] In some embodiments, the system can further include a
reservoir for containing a quantity of the polymer solution. A
device adapted to cause a flow of the polymer solution, e.g., a
pump, can cause the polymer solution to flow from the reservoir
through the second input port into the conduit. The device can be
capable of adjusting the flow rate of the polymer solution through
the second port. For example, the device can be adapted to control
the flow rate of the polymer solution through the second input port
in a range of about 4 ml/min to about 200 ml/min, for example, in a
range of about 5 ml/min to about 100 ml/min.
[0053] In the above system, the conduit can comprise an output port
through which the polymeric nanoparticles exit the conduit as a
suspension in a mixture of the anti-solvent and a process solvent
of the polymer solution. A collection vessel coupled to the output
port of the conduit can collect the suspension containing the
nanoparticles. The collection vessel can contain a liquid. In many
embodiments, a stirrer is disposed in the collection vessel for
mixing the liquid.
[0054] In another aspect, a device for generating nanoparticles is
disclosed, which comprises a conduit having a first input port for
receiving a stream of an anti-solvent and an output port. A static
mixer is disposed in the conduit to cause mixing of the
anti-solvent stream to generate a mixed flowing stream of the
anti-solvent, where the static mixer extends from a proximal end to
a distal end. The conduit has a second input port positioned
relative to the static mixer so as to allow delivery of a polymer
solution into said mixed flowing stream of the anti-solvent for
generating polymeric nanoparticles, e.g., by precipitation.
[0055] In some embodiments, the second input port is positioned at
an intermediate location relative to the proximal and distal ends
of the static mixer. In some alternative embodiments, the second
input port is positioned in proximity to the proximal end, or the
distal end, of the static mixer. In some embodiments, the second
input port is positioned downstream from the static mixer and
sufficiently close to the mixer to allow the delivery of the
polymer solution into the mixed flowing stream of the
anti-solvent.
[0056] In some embodiments, the second input port is configured so
as to introduce the polymer solution into the anti-solvent stream
at a non-zero angle, e.g., at an acute angle, relative to a flow
direction of the mixed flowing stream of the anti-solvent. In some
embodiments, the angle can be in a range of about 50 degrees to
about 90 degrees.
[0057] In some embodiments, the device can further include a
collection tank in fluid communication with the output port for
receiving a suspension containing the polymeric nanoparticles. In
some embodiments, the tank can store a quantity of an aqueous
solution.
[0058] In a related aspect, the above device contains the
anti-solvent and/or the polymer solution discussed above.
[0059] In another aspect, a device for generating nanoparticles is
disclosed, which comprises a conduit having a first input port for
receiving a stream of a liquid anti-solvent and an output port. A
static mixer is disposed in the conduit to cause mixing of the
anti-solvent stream to generate a mixed flowing stream of the
anti-solvent, where the static mixer extends from a proximal end to
a distal end. The device further includes an injector coupled to
the conduit for injecting a polymer solution into the mixed flowing
stream of the anti-solvent.
[0060] In some embodiments, the injector is positioned so as to
inject the polymer solution at an intermediate location between the
proximal and distal ends of the static mixer. Alternatively, the
injector can be positioned to inject the polymer solution in
proximity to the proximal, or the distal, end of the static mixer.
In some embodiments, the injector is configured to inject the
polymer solution into the mixed flowing stream of the anti-solvent
along a direction substantially parallel to the flow direction of
the anti-solvent.
[0061] In a related aspect, the device contains the anti-solvent
and/or the polymer solution discussed above.
[0062] In another aspect, a process for forming polymeric
nanoparticles is disclosed, which comprises using a static mixer to
create a mixed flowing stream of an anti-solvent, and introducing a
polymer solution into the mixed flowing stream of the anti-solvent
such that controlled precipitation of polymeric nanoparticles
occurs. In some embodiments, a flow rate of the mixed flowing
stream of the anti-solvent and/or that of the polymer solution can
be controlled so as to adjust an average particle size of the
nanoparticles.
[0063] In another aspect, a process for monitoring nanoparticles
formed by introducing a polymer solution into a mixed flowing
stream of an anti-solvent is disclosed, which includes selecting
one or more parameters, such as, the flow rate of the anti-solvent
stream, the polymer solution flow rate, the concentration of
polymer in the polymer solution, and the concentration of a colloid
stabilizer in the anti-solvent. The polymer solution is introduced,
under such conditions, into a mixed flowing stream of the
anti-solvent, which is created by introducing the anti-solvent into
a static mixer, so as to form polymeric nanoparticles, e.g., by
precipitation. The nanoparticles produced are then examined to
determine if one or more of their attributes (e.g., their average
particle size or polydispersity index) meet one or more predefined
criteria. If they do not, one or more of the above parameters are
adjusted, and polymer solution is introduced, under the adjusted
conditions, into the mixed flowing stream of the anti-solvent to
generate a new population of nanoparticles. The new population of
the nanoparticles can be examined to determine if one or more of
their attributes meet the predefined criteria. The above steps are
repeated until a population of nanoparticles whose one or more
attributes meet the predefined criteria is achieved.
[0064] In another aspect, a plurality of polymeric nanoparticles
are generated by using the above processes.
[0065] In another aspect, a population of polymeric nanoparticles
having an average particle size in a selected range, e.g., one of
the ranges described above, is generated by using the above
processes.
[0066] In another aspect, a population of polymeric nanoparticles
having a polydispersity index less than about 0.25, e.g., in a
range of about 0.05 to about 0.1, is generated by using the above
processes.
[0067] In a related aspect, a population of polymeric nanoparticles
that includes at least about 10 grams, or at least about 20 grams,
or at least about 30 grams, or at least about 40 grams, or at least
about 50 grams, or at least about 100 grams, or at least about 200
grams, or at least about 300 grams, or at least about 400 grams, or
at least about 500 grams, or at least about 1000 grams of the
nanoparticles is generated by using the above processes.
[0068] In a related aspect, a population of polymeric nanoparticles
having poly(lactic-co-glycolic acid) (PLGA) as at least one
polymeric component is generated by using the above processes. In
some embodiments, the PLGA polymer is attached to a therapeutic
agent. For example, the therapeutic agent can be an anti-neoplastic
agent. In some embodiments, the anti-neoplastic agent is a taxane
(e.g., paclitaxel, docetaxel, larotaxel, or cabazitaxel).
[0069] In another aspect, a pharmaceutically acceptable preparation
of polymeric nanoparticles is generated by using the above
processes. In an embodiment, the pharmaceutically acceptable
preparation includes, e.g., a pharmaceutically acceptable
excipient, e.g., a lyoprotectant. In an embodiment, the
pharmaceutically acceptable preparation is a liquid or a
lyophilized powder.
[0070] In an embodiment, a process described herein further
includes dividing a first pharmaceutically acceptable preparation
made by a process described herein into smaller aliquots and
optionally packaging a plurality of aliquots into gas and/or liquid
tight containers.
[0071] In an embodiment, a process described herein further
includes testing the product (e.g., the preparation of the
nanoparticles) to determine if it meets a preselected reference
value, e.g., a value for concentration, average particle size,
purity, polydispersity index, or other particle properties
described herein.
[0072] Further understanding of the invention can be obtained by
reference to the following detailed description in conjunction with
the associated drawings, which are described briefly below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0073] FIG. 1 is a flow chart depicting various steps in an
exemplary embodiment of a method according to the invention for
generating polymeric nanoparticles,
[0074] FIG. 2 is a flow chart depicting various steps according to
an exemplary embodiment of the invention for performing a
precipitation process for generating polymeric nanoparticles,
[0075] FIG. 3 is a flow chart depicting various steps according to
an exemplary embodiment of the invention for controlling particle
size of nanoparticles formed by nanoprecipitation,
[0076] FIG. 4A schematically depicts a device according to an
exemplary embodiment of the invention for forming
nanoparticles,
[0077] FIG. 4B schematically depicts an alternative implementation
of the device of FIG. 4A,
[0078] FIG. 5A schematically depicts a device according to another
embodiment for generating nanoparticles in accordance with the
teachings of the invention,
[0079] FIG. 5B schematically depicts a device according to another
embodiment for generating nanoparticles in accordance with the
teachings of the invention,
[0080] FIG. 5C schematically depicts a device according to another
embodiment for generating nanoparticles in accordance with the
teachings of the invention,
[0081] FIG. 5D schematically depicts a device according to another
embodiment for generating nanoparticles in accordance with the
teachings of the invention,
[0082] FIG. 5E schematically depicts a device according to another
embodiment for generating nanoparticles in accordance with the
teachings of the invention,
[0083] FIG. 6A schematically depicts a device according to another
embodiment for generating nanoparticles in accordance with the
teachings of the invention in which a plurality of static mixer
units are employed,
[0084] FIG. 6B schematically depicts a device according to an
alternative implementation of the device of FIG. 6A,
[0085] FIG. 6C schematically depicts a device according to another
embodiment of the invention,
[0086] FIG. 7 schematically depicts a system according to an
exemplary embodiment of the invention for generating
nanoparticles,
[0087] FIG. 8A schematically depicts a device according to an
embodiment of the invention for generating nanoparticles, which
employs an injector for injecting a polymer solution into a mixed
flowing stream of an anti-solvent,
[0088] FIG. 8B schematically depicts a device according to another
embodiment of the invention for generating nanoparticles, which
employs an injection system disposed across a conduit through which
a mixed stream of an anti-solvent flows to inject a polymer
solution into the anti-solvent flow,
[0089] FIG. 9 shows a helical static mixer employed in some
prototype devices based on the teachings of the invention for
generating nanoparticles,
[0090] FIGS. 10A and 10B provide two views of a structured-packing
mixer marketed by Sulzer Chemtech USA, Inc. of Oklahoma, U.S.A.
under the trade designation Sulzer SMX, which was employed in some
prototype devices based on the teachings of the invention for
generating nanoparticles,
[0091] FIG. 11A shows a prototype device according to an embodiment
of the invention, which was employed to generate polymeric
nanoparticles,
[0092] FIG. 11B shows a prototype device according to an embodiment
of the invention, which was employed to generate polymeric
nanoparticles,
[0093] FIG. 12 presents data corresponding to Z.sub.ave as a
function of total flow rate for nanoparticles generated in a
prototype device according to the teachings of the invention,
[0094] FIG. 13 presents data corresponding to Z.sub.ave as a
function of anti-solvent flow rate for nanoparticles generated in
two prototype devices according to the teachings of the invention,
and
[0095] FIG. 14 shows particle size distribution exhibited by
polymeric nanoparticles generated by employing a prototype device
according to the teachings of the invention, and
[0096] FIG. 15 shows three graphs corresponding to three
measurements of the particle size distribution exhibited by a
plurality of polymeric nanoparticles generated by employing a
prototype device according to the teachings of the invention as
discussed below in Example 5.
DETAILED DESCRIPTION
[0097] The present invention relates generally to methods, devices
and systems for generating nanoparticles, e.g., polymeric
nanoparticles. In some embodiments, the nanoparticles are formed by
introducing a polymer solution, which comprises one or more
polymer(s) dissolved in a process solvent, into a mixed flowing
stream of an anti-solvent, which is miscible, or at least partially
miscible, with the process solvent but in which the polymer(s)
cannot be dissolved in any appreciable amount, to cause
precipitation of the polymer(s) into a plurality of nanoparticles.
As discussed in more detail below, it has been discovered that
utilizing a static mixer to generate a mixed flowing stream of the
anti-solvent, and introducing the polymer solution into such a
mixed flowing stream to cause precipitation can provide significant
advantages. For example, it allows forming the nanoparticles with a
low polydispersity index over a wide range of anti-solvent (and
polymer solution) flow rates. It has also been discovered that the
anti-solvent flow rate and/or the polymer solution flow rate can be
changed to adjust the average particle size of the fabricated
nanoparticles. The low polydispersity index exhibited by the
nanoparticles can be beneficial in a variety of applications such
as pharmaceutical applications. Moreover, the precipitation process
can be scaled up to form nanoparticles on a large scale.
[0098] The following definitions are provided for a variety of
terms and phrases utilized herein:
Static Mixer:
[0099] The term "static mixer" or "motionless mixer" as used herein
refers to a device that includes one or more substantially
stationary mixing elements, e.g., baffles such as blades, plates,
vanes, that are adapted for placement in the path of a flowing
fluid, e.g., a fluid flowing through a conduit, to produce a
pattern of flow divisions and splits to accomplish mixing, e.g.,
radial mixing via radial circulation or exchange, in the flowing
fluid. Although the stationary mixing elements are typically
immovable within the conduit, some limited movement of the
stationary elements relative to the conduit can occur so long as
such limited movement does not contribute substantially to the
mixing of the flowing fluid. In a static mixer having multiple
stationary elements, these elements are typically arranged in
series and in a staggered orientation relative to one another.
Mixed Flowing Stream:
[0100] The term "mixed flowing stream" as used herein refers to a
flowing stream of a fluid, e.g., a liquid, that exhibits active
motion normal to its direction of flow.
Polymer Solution:
[0101] The term "polymer solution" as used herein refers to a
solution comprising one or more polymers dissolved in a liquid
solvent, which is herein also referred to as process solvent. The
polymer(s) are typically sufficiently soluble in the solvent such
that a concentration of at least about 0.1 percent by weight, and
preferably at least about 0.2 percent by weight, of the polymer(s)
can be dissolved in the solvent at room temperature. In some cases,
the concentration of the polymer(s) that can be dissolved in the
solvent at room temperature can be optionally less than about 10
percent by weight, e.g., less than about 5 percent by weight. The
polymer solution can also include a variety of additives, such as
therapeutic and/or imaging agents or other supplemental additives
useful for the production and/or subsequent use of the
nanoparticles.
[0102] Anti-Solvent:
[0103] The term "anti-solvent" as used herein refers to a liquid,
or a mixture of liquids, which is incapable of dissolving any
appreciable concentration (e.g., a concentration equal to or
greater than about 0.1% at room temperature) of the polymer(s) of
the polymer solution, but is miscible, or at least partially
miscible, with the process solvent. In some embodiments, the
anti-solvent and the process solvent can be mixed in all
proportions to form a homogeneous solution. When combined with the
polymer solution, the anti-solvent causes at least a portion of the
polymer to precipitate.
Average Axial Flow Velocity:
[0104] The phrase "average axial flow velocity" as used herein
refers to a velocity of a fluid, e.g., liquid, along the direction
of flow averaged over a cross-sectional area of the flow, e.g.,
averaged over a cross-sectional area of a conduit through which the
fluid flows. The average axial flow velocity (V.sub.ave) can be
obtained by the following relation:
V ave = Q A Eq . ( 1 ) ##EQU00001##
wherein,
[0105] Q represent the volumetric rate of fluid flow along the
direction of flow (e.g., in units of ml/sec), and
[0106] A represents a cross-sectional area of the flow, e.g., a
cross-sectional area of a conduit through which the fluid
flows.
Nanoparticle:
[0107] The term "nanoparticle" is used herein to refer to a
material structure whose size in any dimension (e.g., x, y, and z
Cartesian dimensions) is less than about 1 micrometer (micron),
e.g., less than about 500 nm or less than about 200 nm or less than
about 100 nm, and greater than about 5 nm. A nanoparticle can have
a variety of geometrical shapes, e.g., spherical, ellipsoidal, etc.
The term "nanoparticles" is used as the plural of the term
"nanoparticle."
Average Particle Size:
[0108] The term "average particle size" is a length dimension which
is designated herein as Z average or Z.sub.ave, and as used herein
refers to the intensity weighted mean hydrodynamic size of an
ensemble collection of particles measured by dynamic light
scattering (DLS). The Z average is derived from a Cumulants
analysis of a measured autocorrelation curve, wherein a single
particle size is assumed and a single exponential fit is applied to
the autocorrelation function. The autocorrelation function
(G)(.tau.)) is defined as follows:
G ( .tau. ) = I ( t ) I ( t + .tau. ) = A [ 1 + B exp ( - 2
.GAMMA..tau. ) ] wherein , Eq . ( 3 ) .GAMMA. = Dq 2 Eq . ( 4 ) q =
2 .pi. n ~ .lamda. 0 sin ( .theta. 2 ) Eq . ( 5 ) D = kT 6 .pi..mu.
R H , Eq . ( 6 ) ##EQU00002##
wherein,
[0109] I represents the light scattering intensity,
[0110] t represents an initial time,
[0111] .tau. represents the delay time,
[0112] A represents an amplitude (or intercept) of the
autocorrelation function,
[0113] B represents the baseline,
[0114] D represents the diffusion coefficient,
[0115] q represents the scattering vector,
[0116] k represents the Boltzmann constant,
[0117] .lamda..sub.0 represents the vacuum wavelength of a laser
source employed for the light scattering measurements,
[0118] n represents the index of refraction of the medium,
[0119] .theta. represents the scattering angle,
[0120] T represents the absolute temperature (Kelvin),
[0121] .mu. represents the viscosity of the medium, and
[0122] R.sub.H represents the hydrodynamic radius.
[0123] In the Cumulants analysis, the exponential fitting
expression of Eq. (3) is expanded as indicated below as expression
y(.tau.) in Eq. (7) to account for polydispersity, which is defined
in more detail below, or peak broadening,
y ( .tau. ) = 1 2 ln [ G ( .tau. ) - A ] = 1 2 ln [ AB exp ( - 2
.GAMMA..tau. + .mu. 2 .tau. 2 ) ] .apprxeq. 1 2 ln [ AB ] - .GAMMA.
.tau. + .mu. 2 2 .tau. 2 = a 0 - a 1 + a 2 .tau. 2 Eq . ( 7 )
##EQU00003##
[0124] wherein .mu..sub.2 is a fitting parameter and the other
parameters are defined above.
[0125] The dynamic light scattering data can be fit to the above
expression (Eq. (7)) to obtain values of the parameters
.alpha..sub.0, .alpha..sub.1, and .alpha..sub.2. The first Cumulant
moment (.alpha..sub.1) can be utilized to obtain Z.sub.ave as
follows:
Z ave = 1 a 1 kT 3 .pi..mu. [ 4 .pi. n ~ .lamda. 0 sin ( .theta. 2
) ] 2 Eq . ( 8 ) ##EQU00004##
wherein the parameters are defined above.
[0126] The first Cumulant moment (.alpha..sub.1) and the second
Cumulant moment (.alpha..sub.2) can be used to calculate another
parameter known as polydispersity index (PdI), which is discussed
in more detail below, as follows:
PdI = 2 a 2 a 1 2 Eq . ( 9 ) ##EQU00005##
Polydispersity Index:
[0127] The term "polydispersity index" is used herein as a measure
of the size distribution of an ensemble of particles, e.g.,
nanoparticles. The polydispersity index is calculated as indicated
in the above Eq. (9) based on dynamic light scattering
measurements.
[0128] Particle Size Distribution:
[0129] If it is assumed that an ensemble of particles exhibit a
Gaussian size distribution, then the particle size distribution of
such an ensemble is a length dimension that can be defined as the
square root of the standard deviation of the Gaussian distribution
(.sigma..sup.2) as follows:
.sigma..sup.2=PdIZ.sub.ave.sup.2 Eq. (10)
Particle Size Distribution= {square root over (.sigma..sup.2)} Eq.
(11)
wherein Z.sub.ave is defined by Eq. (8) above.
Colloid Stabilizer:
[0130] The term colloid stabilizer as used herein refers to an
additive added to the anti-solvent and/or the polymer solution to
prevent or retard an unwanted alteration of the physical state of
the particles, e.g., a colloid stabilizer can inhibit aggregation
of the nanoparticles. For example, a colloid stabilizer can inhibit
aggregation of the nanoparticles during and/or after their
formation.
Lyoprotectant:
[0131] The term "lyoprotectant," as used herein refers to a
substance present in a lyophilized preparation. Typically it is
present prior to the lyophilization process and persists in the
resulting lyophilized preparation. It can be used to protect
nanoparticles, liposomes, and/or micelles during lyophilization,
for example to reduce or prevent aggregation, particle collapse
and/or other types of damage. In an embodiment the lyoprotectant is
a cryoprotectant.
[0132] In an embodiment the lyoprotectant is a carbohydrate. The
term "carbohydrate," as used herein refers to and encompasses
monosaccharides, disaccharides, oligosaccharides and
polysaccharides.
[0133] In an embodiment, the lyoprotectant is a monosaccharide. The
term "monosaccharide," as used herein refers to a single
carbohydrate unit (e.g., a simple sugar) that can not be hydrolyzed
to simpler carbohydrate units. Exemplary monosaccharide
lyoprotectants include glucose, fructose, galactose, xylose, ribose
and the like.
[0134] In an embodiment, the lyoprotectant is a disaccharide. The
term "disaccharide," as used herein refers to a compound or a
chemical moiety formed by 2 monosaccharide units that are bonded
together through a glycosidic linkage, for example through 1-4
linkages or 1-6 linkages. A disaccharide may be hydrolyzed into two
monosaccharides. Exemplary disaccharide lyoprotectants include
sucrose, trehalose, lactose, maltose and the like.
[0135] In an embodiment, the lyoprotectant is an oligosaccharide.
The term "oligosaccharide," as used herein refers to a compound or
a chemical moiety formed by 3 to about 15, preferably 3 to about 10
monosaccharide units that are bonded together through glycosidic
linkages, for example through 1-4 linkages or 1-6 linkages, to form
a linear, branched or cyclic structure. Exemplary oligosaccharide
lyoprotectants include cyclodextrins, raffinose, melezitose,
maltotriose, stachyose acarbose, and the like. An oligosaccharide
can be oxidized or reduced.
[0136] In an embodiment, the lyoprotectant is a cyclic
oligosaccharide. The term "cyclic oligosaccharide," as used herein
refers to a compound or a chemical moiety formed by 3 to about 15,
preferably 6, 7, 8, 9, or 10 monosaccharide units that are bonded
together through glycosidic linkages, for example through 1-4
linkages or 1-6 linkages, to form a cyclic structure. Exemplary
cyclic oligosaccharide lyoprotectants include cyclic
oligosaccharides that are discrete compounds, such as .alpha.
cyclodextrin, .beta. cyclodextrin, or .gamma. cyclodextrin.
[0137] Other exemplary cyclic oligosaccharide lyoprotectants
include compounds which include a cyclodextrin moiety in a larger
molecular structure, such as a polymer that contains a cyclic
oligosaccharide moiety. A cyclic oligosaccharide can be oxidized or
reduced, for example, oxidized to dicarbonyl forms. The term
"cyclodextrin moiety," as used herein refers to cyclodextrin (e.g.,
an .alpha., .beta., or .gamma. cyclodextrin) radical that is
incorporated into, or a part of, a larger molecular structure, such
as a polymer. A cyclodextrin moiety can be bonded to one or more
other moieties directly, or through an optional linker. A
cyclodextrin moiety can be oxidized or reduced, for example,
oxidized to dicarbonyl forms.
[0138] Carbohydrate lyoprotectants, e.g., cyclic oligosaccharide
lyoprotectants, can be derivatized carbohydrates. For example, in
an embodiment, the lyoprotectant is a derivatized cyclic
oligosaccharide, e.g., a derivatized cyclodextrin, e.g., 2 hydroxy
propyl-.beta. cyclodextrin, e.g., partially etherified
cyclodextrins (e.g., partially etherified .beta. cyclodextrins)
disclosed in U.S. Pat. No. 6,407,079, the contents of which are
incorporated herein by this reference.
[0139] An exemplary lyoprotectant is a polysaccharide. The term
"polysaccharide," as used herein refers to a compound or a chemical
moiety formed by at least 16 monosaccharide units that are bonded
together through glycosidic linkages, for example through 1-4
linkages or 1-6 linkages, to form a linear, branched or cyclic
structure, and includes polymers that comprise polysaccharides as
part of their backbone structure. In backbones, the polysaccharide
can be linear or cyclic. Exemplary polysaccharide lyoprotectants
include glycogen, amylase, cellulose, dextran, maltodextrin and the
like.
Derivatized Carbohydrate:
[0140] The term "derivatized carbohydrate," refers to an entity
which differs from the subject non-derivatized carbohydrate by at
least one atom. For example, instead of the --OH present on a
non-derivatized carbohydrate the derivatized carbohydrate can have
--OX, wherein X is other than H. Derivatives may be obtained
through chemical functionalization and/or substitution or through
de novo synthesis--the term "derivative" implies no process-based
limitation.
Injector:
[0141] The term "injector" as used herein refers to a device that
can force, e.g., propel, a fluid, e.g., a liquid, into a receiving
medium.
[0142] In some of the following embodiments, various methods for
generating nanoparticles are described with reference to steps of
these methods. The order in which the steps of the methods are
discussed is not intended to necessarily indicate the order in
which those steps must be performed.
[0143] With reference to the flow charts of FIGS. 1 and 2, in an
exemplary embodiment of a method according to the teachings of the
invention for forming polymeric nanoparticles, a polymer solution
can be generated by dissolving one or more polymers, such as a
polymer to which a therapeutic or an imaging agent is coupled, or
with which a therapeutic or an imaging agent is associated or in
which a therapeutic or an imaging agent is incorporated (e.g.,
embedded), in a process solvent (step A). Further, an anti-solvent
can be prepared (step B). While the process solvent is miscible, or
at least partially miscible, with the anti-solvent, the polymer is
not soluble in the anti-solvent in any appreciable amount, thereby
allowing a subsequent formation of polymeric nanoparticles via a
precipitation process (step E). For example, the solubility of the
polymer in the anti-solvent at room temperature can be less than
about 0.1% by weight. Further, any additive agent(s) added to the
polymer solution is preferably not soluble in the anti-solvent in
any appreciable amount. In some embodiments, the polymer solution
and the anti-solvent are filter sterilized (steps C and D) prior to
their use in the precipitation process.
[0144] Referring to the flow chart of FIG. 2, in an exemplary
embodiment for performing the precipitation process to generate a
plurality of nanoparticles (step E in the flow chart of FIG. 1), an
anti-solvent is introduced into a static mixer, e.g., a static
mixer disposed in a conduit, to create a mixed flowing stream of
the anti-solvent (step 1). The polymer solution is introduced into
the mixed flowing stream of the anti-solvent such that
precipitation of the polymer into a plurality of polymeric
nanoparticles occurs (step 2). The generated nanoparticles are then
separated from the anti-solvent stream, e.g., in a manner discussed
in more detail below.
[0145] Without being limited to any particular theory, upon contact
of the polymer solution with the anti-solvent stream, precipitation
of the polymer into a plurality of polymeric nanoparticles occurs
as a result of rapid desolvation of the polymer. In particular, the
process solvent diffuses rapidly into the anti-solvent due to its
miscibility with the anti-solvent. The polymer is, however, not
miscible in the anti-solvent and hence aggregates (e.g.,
precipitates) into a plurality of nanoparticles as the process
solvent diffuses into the anti-solvent. The static mixer design,
the choice of the anti-solvent and the process solvent, and in
particular the mixing of the anti-solvent and the process solvent,
can facilitate the mass transfer of solvents, thereby controlling
the size of the nanoparticles formed via precipitation. The
nanoparticles can be formed rapidly, e.g., over a milliseconds time
scale. For example, in some cases the nanoparticles can form in
less than about 10 milliseconds (e.g., in a range of about 1
millisecond to about 10 milliseconds), or less than about 5
milliseconds (e.g., in a range of about 1 millisecond to about 5
milliseconds), subsequent to the contact of the polymer solution
with the mixed flowing stream of the anti-solvent. The rapid
formation of the nanoparticles can be due to interfacial turbulence
at the interface of the solvent and the anti-solvent, which can
result, e.g., from flow, diffusion and surface tension
variations.
[0146] As discussed in more detail below, the introduction of the
polymer solution into a mixed flowing stream of the anti-solvent
results in formation of polymeric nanoparticles with predictable
average particle sizes and a low polydispersity index (PdI) over a
wide range of anti-solvent, and polymer solution, flow rates and
average axial flow velocities.
[0147] In some embodiments, the average particle size (Z.sub.ave)
can be equal to or less than about 500 nm. For example, the
polymeric nanoparticles can exhibit an average particle size in a
range of about 5 nm to about 500 nm, or in a range of about 10 nm
to about 500 nm, or in a range of about 20 nm to about 500 nm, or
in a range of about 30 nm to about 500 nm, or in a range of about
40 nm to about 500 nm, or in a range of about 50 nm to about 500
nm. In some embodiments, the average particle size (Z.sub.ave) can
be equal to or less than about 400 nm. For example, the polymeric
nanoparticles can exhibit an average particle size in a range of
about 5 nm to about 400 nm, or in a range of about 10 nm to about
400 nm, or in a range of about 20 nm to about 400 nm, or in a range
of about 30 nm to about 400 nm, or in a range of about 50 nm to
about 400 nm. In some embodiments, the average particle size
(Z.sub.ave) can be equal to or less than about 300 nm. For example,
the polymeric nanoparticles can exhibit an average particle size in
range of about 5 nm to about 300 nm, or in a range of about 10 nm
to about 300 nm, or in a range of about 20 nm to about 300 nm, or
in a range of about 40 nm to about 300 nm, or in a range of about
50 nm to about 300 nm.
[0148] In some embodiments, the average particle size (Z.sub.ave)
of the nanoparticles can be equal to or less than about 200 nm
(e.g., equal to or less than about 195 nm (and, e.g., equal to or
greater than about 20 nm), equal to or less than about 190 nm (and,
e.g., equal to or greater than about 20 nm), equal to or less than
about 185 nm (and, e.g., equal to or greater than about 20 nm),
equal to or less than about 180 nm (and, e.g., equal to or greater
than about 20 nm), equal to or less than about 175 nm (and, e.g.,
equal to or greater than about 20 nm), equal to or less than about
170 nm (and, e.g., equal to or greater than about 20 nm), equal to
or less than about 165 nm (and, e.g., equal to or greater than
about 20 nm), equal to or less than about 160 nm (and, e.g., equal
to or greater than about 20 nm), equal to or less than about 155 nm
(and, e.g., equal to or greater than about 20 nm), equal to or less
than about 150 nm (and, e.g., equal to or greater than about 20
nm), equal to or less than about 145 nm (and, e.g., equal to or
greater than about 20 nm), equal to or less than about 140 nm (and,
e.g., equal to or greater than about 20 nm), equal to or less than
about 135 nm (and, e.g., equal to or greater than about 20 nm),
equal to or less than about 130 nm (and, e.g., equal to or greater
than about 20 nm), equal to or less than about 125 nm (and, e.g.,
equal to or greater than about 20 nm), equal to or less than about
120 nm (and, e.g., equal to or greater than about 20 nm), equal to
or less than about 115 nm (and, e.g., equal to or greater than
about 20 nm), equal to or less than about 110 nm (and, e.g., equal
to or greater than about 20 nm), equal to or less than about 105 nm
(and, e.g., equal to or greater than about 20 nm), equal to or less
than about 100 nm (and, e.g., equal to or greater than about 20
nm), equal to or less than about 95 nm (and, e.g., equal to or
greater than about 20 nm), equal to or less than about 90 nm (and,
e.g., equal to or greater than about 20 nm), equal to or less than
about 85 nm (and, e.g., equal to or greater than about 20 nm),
equal to or less than about 80 nm (and, e.g., equal to or greater
than about 20 nm), equal to or less than about 75 nm (and, e.g.,
equal to or greater than about 20 nm), equal to or less than about
70 nm (and, e.g., equal to or greater than about 20 nm), equal to
or less than about 65 nm (and, e.g., equal to or greater than about
20 nm), equal to or less than about 60 nm (and, e.g., equal to or
greater than about 20 nm), equal to or less than about 55 nm or 50
nm (and, e.g., equal to or greater than about 20 nm)). For example,
the average particle size can be in a range of about 50 nm to about
200 nm, or in a range of about 100 nm to about 200 nm.
[0149] In general, a wide range of anti-solvent and polymer
solution flow rates can be employed. By way of example, the flow
rate of the anti-solvent stream can be in a range of about 20
ml/min to about 2000 ml/min, and the flow rate of the polymer
solution can be in a range of about 4 ml/min to about 200 ml/min,
for example, in a range of about 5 ml/min to about 100 ml/min. In
some embodiments, the flow rate of the anti-solvent stream is
substantially greater than the flow rate of the polymer solution
into the anti-solvent stream. For example, the flow rate of the
anti-solvent stream can be at least about 2 times, or at least
about 3 times, at least about 5 times, or at least about 10 times,
greater than the flow rate of the polymer solution. In some other
embodiments, the anti-solvent flow rate and the polymer solution
flow rate can have a 1:1 ratio.
[0150] In some embodiments, the concentration of the polymer
solution and/or the concentration of the anti-solvent can be
changed so as to adjust the average particle size of the
nanoparticles.
[0151] In many embodiments, the static mixer generates a mixed
flowing stream of the anti-solvent that presents a substantially
isotropic mixed anti-solvent environment to the incoming polymer
solution, thus ensuring that the formed nanoparticles will exhibit
a low polydispersity index. For example, the static mixer can
create sufficient radial and/or tangential motion of the
anti-solvent to rapidly create a substantially uniform mixed
environment, thus facilitating formation of nanoparticles with a
low polydispersity index. For example, as indicated above, the
polydispersity index can be equal to or less than about 0.25, e.g.,
in a range of about 0.05 to about 0.1. By way of example, in some
embodiments, the static mixer generates a mixed anti-solvent
environment that is substantially isotropic over at least about
50%, or at least about 60%, or at least about 70%, or at least
about 90%, or 100% of the volume of the conduit in which the static
mixer is disposed.
[0152] Further, a mixed flowing stream of the anti-solvent can
allow the introduction of the polymer solution at a variety of
velocities (and corresponding momentum values) into the
anti-solvent stream. Even at a low momentum, the polymer solution
will encounter a highly mixed anti-solvent environment, which will
lead to formation of nanoparticles with predictable average
particle size and polydispersity index.
[0153] In some embodiments, the polymer solution can be introduced
into the mixed flowing stream of the anti-solvent at an
intermediate location of the static mixer. Alternatively, the
polymer solution can be introduced into the anti-solvent flowing
stream in proximity to a proximal or a distal end of the mixer. In
some embodiments, the polymer solution is introduced into the mixed
flowing stream of the anti-solvent at a non-zero angle, e.g., an
acute angle, relative to the anti-solvent flow direction. For
example, the polymer solution stream can intersect the anti-solvent
flowing stream at an angle in a range of about 50 degrees to about
90 degrees. In some embodiments, the polymer solution is injected
into the mixed flowing stream of the anti-solvent either at an
angle relative to the direction of the anti-solvent flow or
substantially parallel to the direction of the anti-solvent
flow.
[0154] In some embodiments, rather that utilizing a single static
mixer unit, multiple static mixer units can be employed to provide
a mixed flowing stream of an anti-solvent within a selected portion
of a conduit. In some such embodiments, the static mixer units are
oriented in a staggered configuration relative to one another so as
to enhance the mixing of the anti-solvent.
[0155] A variety of polymers, process solvents, and anti-solvents
can be employed in the precipitation process to form nanoparticles.
By way of example, the polymers can include the following monomers
(or sub-units): acrylates, acrylonitriles such as methacrylnitrile,
vinyls, aminoalkyls, styrenes, and lactic acids. Some examples of
acrylates include, without limitation, methyl acrylate, ethyl
acrylate, propyl acrylate, n-butyl acrylate, isobutyl acrylate,
2-ethyl acrylate, and t-butyl acrylate. Some examples of vinyls
include, without limitation, vinyl acetate, vinylversatate,
vinylpropionate, vinylformamide, vinylacetamide, vinylpyridines and
vinylimidazole. Some examples of aminoalkyls include, without
limitation, aminoalkylacrylates, aminoalkylmethacrylates and
aminoalkyl(meth)acrylamides.
[0156] In some embodiments, the polymer can be an amphiphilic
copolymer that is formed of monomers exhibiting different
hydrophilic and hydrophobic properties. For example, in some
embodiments, the polymer has a hydrophilic portion and a
hydrophobic portion. In some embodiments, the polymer is a block
copolymer. In some embodiments, the amphiphilic copolymer is formed
of blocks (groups) of monomers or sub-units, where some blocks are
substantially hydrophobic while other blocks are substantially
hydrophilic. For example, in diblock copolymers the blocks are
arranged as a series of two blocks having similar hydrophobic or
hydrophilic properties while in triblock copolymers, the blocks are
arranged as a series of three blocks having similar hydrophobic or
hydrophilic properties. In some embodiments, the amphiphilic
polymer comprises two regions, one of which is hydrophilic and the
other hydrophobic, where the two regions together comprise at least
about 70% by weight of the polymer (e.g., at least about 80%, at
least about 90%, at least about 95%).
[0157] In some embodiments, the hydrophobic portion of the polymer
is a biodegradable polymer (e.g., PLA, PGA, PLGA, PCL, PDO,
polyanhydrides, polyorthoesters, or chitosan). In some embodiments,
the hydrophobic portion of the polymer is PLA. In some embodiments,
the hydrophobic portion of the polymer is PGA. In some embodiments,
the hydrophobic portion of the polymer is a copolymer of lactic and
glycolic acid (e.g., PLGA).
[0158] In some embodiments, the hydrophilic portion of the polymer
is polyethylene glycol (PEG). In some embodiments, the hydrophilic
portion of the polymer has a molecular weight of from about 1 kDa
to about 20 kDa (e.g., from about 1 kDa to about 15 kDa, from about
2 kDa to about 12 kDa, from about 6 kDa to about 20 kDa, from about
5 kDa to about 10 kDa, from about kDa to about 10 kDa, from about 5
kDa to about 7 kDa, from about 6 kDa to about 8 kDa, about 6 kDa,
about 7 kDa, about 8 kDa, or about 9 kDa). In some embodiments, the
ratio of molecular weight of the hydrophilic to hydrophobic
portions of the polymer is from about 1:20 to about 1:1 (e.g.,
about 1:10 to about 1:1, about 1:2 to about 1:1, or about 1:6 to
about 1:3).
[0159] In some embodiments, the hydrophilic portion of the polymer
terminates in a hydroxyl moiety prior to conjugation to an agent.
In some embodiments, the hydrophilic portion of the polymer
terminates in an alkoxy moiety. In some embodiments, the
hydrophilic portion of the polymer is a methoxy PEG (e.g., a
terminal methoxy PEG).
[0160] In some embodiments, the hydrophilic portion of the polymer
is attached to the hydrophobic portion through a covalent bond. In
some embodiments, the hydrophilic polymer is attached to the
hydrophobic polymer through an amide, ester, ether, amino,
carbamate, or carbonate bond (e.g., an ester or an amide).
[0161] In some embodiments, the polymer is a biodegradable polymer
(e.g., polylactic acid (PLA), polyglycolic acid (PGA),
poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),
polydioxanone (PDO), polyanhydrides, polyorthoesters, or chitosan).
In some embodiments, the polymer is a hydrophobic polymer. In some
embodiments, the polymer is PLA. In some embodiments, the polymer
is PGA.
[0162] In some embodiments, the polymer is a copolymer of lactic
and glycolic acid (poly(lactic-co-glycolic acid) (PLGA)). In some
embodiments, the polymer is a PLGA-ester. In some embodiments, the
polymer is a PLGA-lauryl ester. In some embodiments, the polymer
comprises a terminal free acid prior to conjugation to an agent. In
some embodiments, the polymer comprises a terminal acyl group
(e.g., an acetyl group). In some embodiments, the ratio of lactic
acid monomers to glycolic acid monomers is from about 0.1:99.9 to
about 99.9:0.1. In some embodiments, the ratio of lactic acid
monomers to glycolic acid monomers is from about 75:25 to about
25:75 (e.g., about 50:50 or about 75:25).
[0163] In some embodiments, the average molecular weight of the
polymer is from about 1 kDa to about 20 kDa (e.g., from about 1 kDa
to about 15 kDa, from about 2 kDa to about 12 kDa, from about 6 kDa
to about 20 kDa, from about 5 kDa to about 10 kDa, from about 7 kDa
to about 10 kDa, from about 5 kDa to about 7 kDa, from about 6 kDa
to about 8 kDa, about 6 kDa, about 7 kDa, about 8 kDa, or about 9
kDa). In some embodiments, the polymer has a glass transition
temperature of about 20.degree. C. to about 60.degree. C. In some
embodiments, the polymer has a polymer polydispersity index equal
to or less than about 2.5 (e.g., less than or equal to about 2.2,
or less than or equal to about 2.0).
[0164] By way of further illustration, some examples of suitable
polymers include poly(lactide-co-glycolide), poly(lactide),
poly(epsilon-caprolactone), poly(isobutylcyanoacrylate),
poly(isohexylcyanoacrylate), poly(n-butylcyanoacrylate),
poly(acrylate), poly(methacrylate), poly(lactide)-poly(ethylene
glycol), poly(lactide-co-glycolide)-poly(ethylene glycol),
poly(epsilon-caprolactone)-poly(ethylene glycol), and
poly(hexadecylcyanoacrylate-co-poly(ethylene
glycol)cyanoacrylate).
[0165] In some embodiments, the polymer can include one or more
grafted moieties, e.g., alkyl chains of 4 to 18 carbons, such as a
grafted butyl group. In some embodiments, such grafted moieties can
enhance the salvation of the polymer in the process solvent and/or
the stability of the polymeric nanoparticles formed in the
subsequent steps.
[0166] In some embodiments, a single agent is attached to a single
polymer, e.g., to a terminal end of the polymer. In some
embodiments, a plurality of agents are attached to a single polymer
(e.g., 2, 3, 4, 5, 6, or more). In some embodiments, the agents are
the same agent. In some embodiments, the agents are different
agents. In some embodiments, the agent is a therapeutic agent or an
imaging agent.
[0167] In an embodiment, the agent is poorly soluble in water,
e.g., it has a solubility of less than about 1 mg/liter, or about
0.9 mg/liter, or about 0.8 mg/liter, or about 0.7 mg/liter, or
about 0.6 mg/liter, or about 0.5 mg/liter in unbuffered water (pH
of 7). In an embodiment, the agent has a molecular weight of
between about 200 to 1500, 400 to 1500, 200 to 1000, 400 to 1000,
200 to 800, or 400 to 800 Daltons.
[0168] In some embodiments, the therapeutic agent is an
anti-neoplastic agent. In some embodiments, the anti-ncoplastic
agent is an alkylating agent, a vascular disrupting agent, a
microtubule targeting agent, a mitotic inhibitor, a topoisomerase
inhibitor, an anti-angiogenic agent or an anti-metabolite. In some
embodiments, the anti-neoplastic agent is a taxane (e.g.,
paclitaxel, docetaxel, larotaxel or cabazitaxel). In some
embodiments, the anti-neoplastic agent is an anthracycline (e.g.,
doxorubicin). In some embodiments, the anti-neoplastic agent is an
epothilone (e.g., ixabepilone, epothilone B, epothilone D,
BMS310705, dehydelone or ZK-epothilone). In some embodiments, the
anti-neoplastic agent is a platinum-based agent (e.g., cisplatin).
In some embodiments, the anti-neoplastic agent is a pyrimidine
analog (e.g., gemcitabine, premetrexed, floxuridine, fluorouracil
(5-FU)).
[0169] In some embodiments, the anti-neoplastic agent is
paclitaxel, attached to the polymer through the 2' or 7 carbon
position, or both the 2' and 7 carbon positions. In some
embodiments, the agent is linked to the polymer through the 7
position and has an acyl group at the 2' position (e.g., wherein
the agent is a taxane such as paclitaxel, docetaxel, larotaxel or
cabazitaxel).
[0170] In some embodiments, the anti-neoplastic agent is docetaxel.
In some embodiments, the anti-neoplastic agent is
docetaxel-succinate. In some embodiments, the anti-neoplastic agent
is doxorubicin. In some embodiments, the anti-neoplastic agent is
larotaxel. In some embodiments, the anti-neoplastic agent is
cabazitaxel.
[0171] In some embodiments, the therapeutic agent is an agent for
the treatment or prevention of cardiovascular disease. In some
embodiments, the therapeutic agent is an agent for the treatment or
prevention of an inflammatory or autoimmune disease.
[0172] In some embodiments, the agent is attached directly to the
polymer, e.g., through a covalent bond. In some embodiments, the
agent is attached to a terminal end of the polymer via an amide,
ester, ether, amino, carbamate or carbonate bond. In some
embodiments, the agent is attached to a terminal end of the
polymer. In some embodiments, the polymer comprises one or more
side chains and the agent is directly attached to the polymer
through one or more of the side chains.
[0173] In some embodiments, a single agent is attached to a
polymer. In some embodiments, multiple agents are attached to a
polymer (e.g., 2, 3, 4 or more agents). In some embodiments, the
agents are the same agent. In some embodiments, the agents are
different agents.
[0174] In some embodiments, the agent is doxorubicin, and is
covalently attached to the polymer through, e.g., an amide
bond.
[0175] In some embodiments, the polymer-agent conjugate is:
##STR00001##
wherein about 40% to about 60% of R substituents are hydrogen
(e.g., about 50%) and about 40% to about 60% are methyl (e.g.,
about 50%); and wherein n is an integer from about 90 to about 170
(e.g., n is an integer such that the molecular weight of the
polymer-agent conjugate is from about 6 kDa to about 11 kDa).
[0176] In some embodiments, the agent is paclitaxel, and is
covalently attached to the polymer through, e.g., an ester
bond.
[0177] In some embodiments, the polymer-agent conjugate is:
##STR00002##
wherein about 40% to about 60% of R substituents are hydrogen
(e.g., about 50%) and about 40% to about 60% are methyl (e.g.,
about 50%); and wherein n is an integer from about 90 to about 170
(e.g., n is an integer such that the molecular weight of the
polymer-agent conjugate is from about 6 kDa to about 11 kDa).
[0178] In some embodiments, the polymer-agent conjugate is:
##STR00003##
wherein about 40% to about 60% of R substituents are hydrogen
(e.g., about 50%) and about 40% to about 60% are methyl (e.g.,
about 50%); and wherein n is an integer from about 90 to about 170
(e.g., n is an integer such that the molecular weight of the
polymer-agent conjugate is from about 6 kDa to about 11 kDa).
[0179] In some embodiments, the paclitaxel is attached through both
the 2' and the 7 carbons. In some embodiments, the polymer-agent is
provided as a mixture containing one or more or all of,
drug-polymer species coupled through the 2' carbon, drug-polymer
species coupled through the 7 carbon, and drug-polymer species
coupled through both the 2' and the 7 carbons.
[0180] In some embodiments, the agent is paclitaxel, and is
covalently attached to the polymer via a carbonate bond.
[0181] In some embodiments, the agent is docetaxel, and is
covalently attached to the polymer through, e.g., an ester
bond.
[0182] In some embodiments, the polymer-agent conjugate is:
##STR00004##
wherein about 40% to about 60% of R substituents are hydrogen
(e.g., about 50%) and about 40% to about 60% are methyl (e.g.,
about 50%); and wherein n is an integer from about 90 to about 170
(e.g., n is an integer such that the molecular weight of the
polymer-agent conjugate is from about 6 kDa to about 11 kDa).
[0183] In some embodiments, the polymer-agent conjugate is:
##STR00005##
wherein about 40% to about 60% of R substituents are hydrogen
(e.g., about 50%) and about 40% to about 60% are methyl (e.g.,
about 50%); and wherein n is an integer from about 90 to about 170
(e.g., n is an integer such that the molecular weight of the
polymer-agent conjugate is from about 6 kDa to about 11 kDa).
[0184] In some embodiments, the docetaxel is attached through both
the 2' and the 7 carbons. In some embodiments, the polymer-agent is
provided as a mixture containing one or more or all of,
drug-polymer species coupled through the 2' carbon, drug-polymer
species coupled through the 7 carbon, and drug-polymer species
coupled through both the 2' and the 7 carbons.
[0185] In some embodiments, the agent is docetaxel, and is
covalently attached to the polymer through a carbonate bond.
[0186] In some embodiments, the agent is attached to the polymer
through a linker. In some embodiments, the linker is an alkanoate
linker. In some embodiments, the linker is a PEG-based linker. In
some embodiments, the linker comprises a disulfide bond. In some
embodiments, the linker is a self-immolative linker. In some
embodiments, the linker is an amino acid or a peptide (e.g.,
glutamic acid, branched glutamic acid or polyglutamic acid).
[0187] In some embodiments the linker is a multifunctional linker.
In some embodiments, the multifunctional linker has 2, 3, 4 or more
reactive moieties that may be functionalized with an agent. In some
embodiments, all reactive moieties are functionalized with an
agent. In some embodiments, not all of the reactive moieties are
functionalized with an agent (e.g., the multifunctional linker has
four reactive moieties, and only one, two or three react with an
agent.)
[0188] In some embodiments, the polymer-agent conjugate is:
##STR00006##
wherein about 40% to about 60% of R substituents are hydrogen
(e.g., about 50%) and about 40% to about 60% are methyl (e.g.,
about 50%); and wherein n is an integer from about 90 to about 170
(e.g., n is an integer such that the molecular weight of the
polymer-agent conjugate is from about 6 kDa to about 11 kDa).
[0189] In some embodiments, two agents are attached to a polymer
via a multifunctional linker. In some embodiments, the two agents
are the same agent. In some embodiments, the two agents are
different agents. In some embodiments, the agent is docetaxel, and
is covalently attached to the polymer via a glutamate linker.
[0190] In some embodiments, the polymer-agent conjugate is:
##STR00007##
wherein about 40% to about 60% of R substituents are hydrogen
(e.g., about 50%) and about 40% to about 60% are methyl (e.g.,
about 50%); and wherein n is an integer from about 90 to about 170
(e.g., n is an integer such that the molecular weight of the
polymer-agent conjugate is from about 6 kDa to about 11 kDa).
[0191] In some embodiments, four agents are attached to a polymer
via a multifunctional linker. In some embodiments, the four agents
are the same agent. In some embodiments, the four agents are
different agents. In some embodiments, the agent is docetaxel, and
is covalently attached to the polymer via a bis(glutamate)
linker.
[0192] In some embodiments, the polymer-agent conjugate is:
##STR00008##
wherein 2'-docetaxel is:
##STR00009##
wherein about 40% to about 60% of R substituents are hydrogen
(e.g., about 50%) and about 40% to about 60% are methyl (e.g.,
about 50%); and wherein n is an integer from about 90 to about 170
(e.g., n is an integer such that the molecular weight of the
polymer-agent conjugate is from about 6 kDa to about 11 kDa).
[0193] In some embodiments, the polymer, e.g., the hydrophilic
portion of an amphiphilic copolymer, comprises a terminal
conjugate. In some embodiments, the terminal conjugate is a
targeting agent or a dye. In some embodiments, the terminal
conjugate is a folate or a rhodamine. In some embodiments, the
terminal conjugate is a targeting peptide (e.g., an RGD peptide).
By way of example, the targeting agent can be covalently linked to
the polymer. In some embodiments, the targeting agent can be
capable of binding to, or otherwise associating with, a target
biological entity, e.g., a membrane component, a cell surface
receptor, a prostate specific membrane antigen, or the like. In
some embodiments, the targeting agent can cause the nanoparticles
administered to a subject to become localized to a tumor, a disease
site, a tissue, an organ, a type of cell, e.g., a cancer cell. In
some embodiments, the targeting agent can be selected from the
group of nucleic acid aptamers, growth factors, hormones,
cytokines, interleukins, antibodies, integrins, fibronectin
receptors, p-glycoprotein receptors, peptides and cell binding
sequences.
[0194] In some embodiments, a radiopharmaceutical agent e.g., a
radiotherapeutic agent, a radioimaging agent, or other radioisotope
can be coupled to, associated with or incorporated in the polymer,
e.g., embedded in the polymer.
[0195] In some embodiments, the process solvent is an organic
solvent (or a mixture of two or more organic solvents). In some
embodiments, the process solvent is capable of dissolving at least
about 0.1%, or at least about 0.2%, by weight of the polymer at
room temperature.
[0196] Some examples of suitable process solvents include, without
limitation, acetone, ether, alcohol, tetrahydrofuran,
2-pyrrolidone, N-Methyl-2-pyrrolidone (NMP), dimethylformamide
dimethylacetamide (DMA), methyl acetate, ethyl formate, methyl
ethyl ketone (MEK), methyl isobutyl ketone (MIBK), methyl propyl
ketone, isopropyl ketone, isopropyl acetate, acetonitrile (MeCN)
and dimethyl sulfoxide (DMSO).
[0197] In some embodiments, the anti-solvent can be an aqueous
(water-based) solution, another solvent, a combination of a solvent
and an aqueous solution, or a combination of one or more organic
solvents. In some embodiments, the anti-solvent can be purified
water. Some other examples of suitable anti-solvents include,
without limitation, methanol, ethanol, n-propanol, isopropanol,
n-butanol, ethyl ether, and water:ethanol (e.g., 50:50). In some
cases, the anti-solvent can be a liquefied gas, such as carbon
dioxide under adequate pressure.
[0198] In some embodiments, the anti-solvent can include a colloid
stabilizer, e.g., to inhibit aggregation of the formed
nanoparticles. Some examples of suitable colloid stabilizers
include, without limitation, poly(vinyl alcohol) (PVA), Dextran and
pluronic F68, poly(vinyl pyrrolidone), solutol, Tween 80,
poloxamer, carbopol, poly-ethylene glycol (PEG), sodium dodecyl
sulfate, poly(.epsilon.-caprolactone), peptides, and carbohydrates.
Another example of a colloid stabilizers includes, without
limitation, a PEG-lipid (e.g., PEG-ceramide, d-alpha-tocopheryl
polyethylene glycol 1000 succinate,
1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] or
lecithin). In some embodiments, the PVA is from about 5 to about 45
kDa, for example, the PVA is from about 5 to about 30 kDa (e.g.,
the PVA is from about 5 to about 20 kDa), and up to about 98%
hydrolyzed (e.g., about 85% hydrolyzed). In some embodiments, the
viscosity of the PVA (4% PVA in water), measured by utilizing the
falling ball method, is in a range of about 2.5 to about 6.5 mPasec
(e.g., in a range of about 2.5 to about 3.5 mPasec at a temperature
of about 20.degree. C.). In some embodiments, the viscosity of the
PVA (4% PVA in water), measured by utilizing the falling ball
method, is in a range of about 3.4 to about 4.6 mPasec.
[0199] In some embodiments, the polymer solution can have one or
more additive molecules. As discussed above, in some embodiments,
the additive molecules are embedded in the polymer prior to
formation of the polymer solution. In other embodiments, the
additive molecules can become embedded in the polymeric
nanoparticles during the precipitation process. For example, in
some embodiments, the additive molecule can be conjugated to the
polymer, e.g., via covalent bonding, and the conjugated polymer can
be dissolved in a process solvent to form the polymer solution. In
other cases, the additive molecules can be present in the polymer
solution without being conjugated to the polymer and become
subsequently trapped in polymeric nanoparticles during the
precipitation process.
[0200] By way of example, the additive molecules can be a
therapeutic, or an imaging agent or a combination of therapeutic
and imaging agents. Some examples of suitable therapeutic agents
include, without limitation, anti-neoplastic agents,
anti-inflammatory agents, cardiovascular active agents, and
anti-metabolites.
[0201] In some embodiments, the imaging agent can be coupled, e.g.,
conjugated to the polymer, for incorporation in the nanoparticles.
In other embodiments, the imaging agent can be coupled, e.g., via a
chelating agent, to a therapeutic agent, which is in turn coupled,
e.g., conjugated, to the polymer. The imaging agents can include,
e.g., radioactive, non-radioactive, or fluorescent labels. Some
examples of imaging agents include, without limitation,
radiopharmaceuticals such as Technetium Bicisate, Ioxaglate, and
Fluorodeoxyglucose, label-free Raman imaging agents, encapsulate
MRI contrast agent Gd-DTPA, and rhodamine 6G as a fluorescent
agent. In some embodiments, the imaging agent can be radiolabeled
docetaxel (e.g., 3H-radiolabeled or 14C-radiolabeled docetaxel), or
radiolabeled paclitaxel.
[0202] Referring again to the flow chart of FIG. 1, the
nanoparticles formed in the precipitation process can be collected,
e.g., as a suspension (step F). For example, the formed
nanoparticles entrained in a mixture of anti-solvent and process
solvent (in many cases, mostly anti-solvent) flowing downstream
from the static mixer can be introduced into a tank, e.g., a tank
containing a liquid, e.g., deionized water. A suspension of the
nanoparticles can then be collected from the tank.
[0203] The collected nanoparticles can be subjected to a variety of
processes, which may be conducted aseptically, to yield aqueous or
non-aqueous solutions, dispersions or powders. For example, as
discussed in more detail below, a concentrated suspension of the
nanoparticles can be lyophilized to yield a powder containing the
nanoparticles, e.g., a sterile powder. In some applications, such a
sterile powder of the nanoparticles can be subsequently
reconstituted into sterile injectible solutions or dispersions.
[0204] For example, with continued reference to the flow chart of
FIG. 1, the collected suspension containing the nanoparticles can
be diafiltered and concentrated (step G). By way of example, the
suspension containing the nanoparticles can be diafiltered, e.g.,
to remove at least a portion of the process solvent, the colloid
stabilizer or other additives added to the anti-solvent. In some
embodiments, the diafiltration (also known in the art as crossflow
filtration) can be performed in multiple steps. In some such
embodiments, the nanoparticles can be washed, e.g., by using
deionized water, between successive diafilteration steps. Further,
in some embodiments the diafilteration is conducted preferably in a
continuous fashion, i.e., by adding wash solution during the
diafiltration process.
[0205] In some embodiments in which the polymeric nanoparticles
include therapeutic and/or imaging agents, the concentration of
these agents can be monitored (step H) while the suspension
containing the nanoparticles is being concentrated. While in some
cases such monitoring is performed continuously, in other cases it
can be performed at multiple discrete times. By way of example,
high pressure liquid chromotagraphy (HPLC) can be employed to assay
the suspension as the volume of the anti-solvent and process
solvent mixture is reduced.
[0206] With continued reference to the flow chart of FIG. 1, in
this exemplary embodiment, a lyoprotectant is added to the
concentrated suspension of the nanoparticles to protect the
nanoparticles from damage and/or to retard permanent aggregation of
the nanoparticles when subsequently subjected to lyophilization.
The lyoprotectant can also facilitate the resuspension of the
nanoparticles. Some examples of suitable lyoprotectants include,
without limitation, a derivatized cyclic oligosaccharide, e.g., a
derivatized cyclodextrin, e.g., 2 hydroxy propyl-.beta.
cyclodextrin, e.g., partially etherified cyclodextrins (e.g.,
partially etherified 8 cyclodextrins) disclosed in U.S. Pat. No.
6,407,079, the contents of which are incorporated herein by this
reference.
[0207] In step (J), the concentrated suspension containing the
nanoparticles and the lyoprotectant can then be stored in one or
more suitable vessels, e.g., vials, and lyophilized in a manner
known in the art (step K). The vials can then be sealed to protect
the nanoparticles from spoilage. By way of example, the
lyophilization can be achieved by initially freezing the
concentrated suspension followed by a primary drying phase in which
the ambient pressure to which the concentrated suspension is
subjected is lowered (e.g. to a few millibars) while supplying
enough heat to cause sublimation of frozen liquid, mostly frozen
water in many implementations at this stage. In a secondary drying
phase, unfrozen liquid (e.g., water molecules), if any, can be
removed by raising the temperature above that in the primary. In
some embodiments, upon completion of the freeze-drying process, an
inert gas, such as nitrogen, can be introduced into the vessel
containing the lyophilized nanoparticles prior to sealing the
vessel.
[0208] As discussed above, the use of a static mixer in generating
a mixed flowing stream of an anti-solvent into which a polymer
solution is introduced for generating nanoparticles advantageously
allows operating the precipitation process at a variety of
anti-solvent, and polymer solution, flow rates. It has been
discovered that the anti-solvent flow rate and/or the polymer
solution flow rate can be adjusted to control the average particle
size of the formed nanoparticles in a predictable manner. Hence, in
another aspect, the invention provides a method for controlling
particle size of nanoparticles formed by precipitation.
[0209] For example, with reference to the flow chart of FIG. 3, in
such a method, an anti-solvent flow is introduced into a static
mixer to generate a mixed flowing stream of the anti-solvent (step
1). A polymer solution is then introduced into the mixed flowing
stream of the anti-solvent so as to provide a plurality of
nanoparticles by precipitation (step 2). The flow rate of the
anti-solvent through the static mixer is controlled (step 3) so as
to adjust an average particle size of the nanoparticles.
[0210] By way of example, the flow rate of the anti-solvent through
the static mixer can be changed in a range of about 20 ml/min to
about 2000 ml/min so as to vary the average particle size in a
range of about 50 nm to about 200 nm.
[0211] In some embodiments the flow rate of the anti-solvent is
changed, while maintaining the flow rate of the polymer solution
substantially constant, so as to adjust the average particle size
of the nanoparticles. For example, in some embodiments that utilize
high ratios of the flow rate of the anti-solvent relative to that
of the polymer solution, e.g., ratios of 10:1 or higher, the
average particle size can be controlled by adjusting only the
anti-solvent flow rate. In other embodiments, the polymer solution
flow rate is changed, while maintaining the flow rate of the
anti-solvent substantially constant, no as to adjust the average
particle size of the nanoparticles. Alternatively, both the
anti-solvent flow rate and that of the polymer solution can be
concurrently changed so as to adjust the average particle size of
the nanoparticles.
[0212] In some embodiments, the concentration of the polymer in the
polymer solution and/or the concentration of the colloid stabilizer
added to the anti-solvent can be changed so as to adjust the
average particle size of the nanoparticles.
[0213] The above processes for generating nanoparticles with
controlled average particle sizes and a low polydispersity index
find a variety of applications, such as nanopharmaceutical
therapeutic applications. For example, they can be employed to
fabricate nano-scale drug delivery systems for selectively
targeting disease tissue. For example, polymeric nanoparticles
embedded with therapeutic agents can be formed for selectively
targeting diseases and disorders such as oncological diseases,
autoimmune diseases as well as cardiovascular diseases.
[0214] The ability to predictably control the average particle size
and particle size distribution afforded by the methods of the
invention allows optimizing selective delivery of such therapeutic
nanoparticles. For example, the average particle size of the
nanoparticles can be selected to allow their preferential
accumulation in cancerous tumors via their passage through leaky
blood vessels of such tumors. Further, a narrow size distribution
of the nanoparticles can be employed to ensure that the therapeutic
nanoparticles can effectively target cancerous tumors.
[0215] Further, in some embodiments, polymeric nanoparticles can be
employed for sustained drug delivery. For example, in some
embodiments, a therapeutic agent can be entrapped within a
nanoparticle formed of a biodegradable polymer. As the polymer
coating is degraded, the entrapped agent can be released into a
subject to whom the nanoparticles have been delivered.
[0216] In addition, the nanoparticles can be formed so as to be
masked from a host's immune system, thereby exhibiting reduced
immunogenicity and antigenecity. By way of example, the polymer
nanoparticles can be PEGylated to extend their circulation time in
a host. PEGylated nanoparticles can evade a subject's immune
system, and PEGylated chemotherapeutic nanoparticles can lower the
toxicity of the therapeutic agent and reduce unwanted side
effects.
[0217] In some embodiments, the above processes for generating
polymeric nanoparticles can allow controlling the amount of a
therapeutic or an imaging agent that can be loaded onto a
nanoparticle (e.g., coupled to, associated with or incorporated
into the nanoparticles), and controlling the release rate of such
an agent upon introduction of the nanoparticle into a subject. For
example, the amount of a therapeutic agent that can be loaded onto
a nanoparticle can vary based on the degree of branching exhibited
by a linker attached to the polymer from which the nanoparticle is
formed and to which the agent can be coupled.
[0218] The applications of nanoparticles, and particularly
polymeric nanoparticles, formed in accordance with the teachings of
the invention are not limited to therapeutic applications. For
example, the nanoparticles can also be employed in imaging
applications.
[0219] The polymeric nanoparticles can be delivered to a subject in
a variety of ways. For example, the nanoparticles can be combined
with suitable supplemental additives, such as water, ethanol,
propyleneglycol, polyethyleneglycol, glycerol, vegetable oils, and
ethyloleate, for parenteral injection into a subject. In some
embodiments, the nanoparticles can be administered orally, e.g.,
via encapsulation of lyophilized nanoparticles using known
excipients. In some embodiments, the nanoparticles can be
administered by inhalation, e.g., via nebulization, propellant or a
dry powder device. In some embodiments, the nanoparticles can be
administered mucosally (e.g., via vaginal or rectal mucosa). In
some embodiments, the nanoparticles can be applied in a topical
form to a subject's tissue. In some embodiments, the nanoparticles
can be administered ophthalmically.
[0220] FIG. 4A schematically depicts a device 40 according to an
embodiment of the invention for generating polymeric nanoparticles.
The device 40 includes a conduit 42, e.g., a hollow tube, that
extends axially from a first input (inlet) port 44, through which a
fluid, e.g., an anti-solvent, can be introduced into the conduit,
and an output (outlet) port 46. A static mixer 48 is disposed in
the conduit to receive the fluid entering the conduit through the
input port 44. The exemplary static mixer 48 extends from a
proximal end (PE) to a distal end (DE), and includes a plurality of
stationary baffles 48a that cause mixing of the fluid as it flows
through the mixer. Different types of static mixers can be employed
in the device 40. By way of example, some suitable static mixers
are disclosed below in the Examples section. By way of further
examples, static mixers disclosed in U.S. Pat. Nos. 3,286,992 and
4,511,258 entitled, respectively, "Mixing Device," and "Static
Material Mixing Apparatus," which are herein incorporated by
reference in their entirety, can be employed. By way of another
example, in some embodiments, static mixers marketed by Chemineer,
Inc. of Ohio, U.S.A. under the trade designation Kenics static
mixers can be employed.
[0221] The device 40 further includes a second input port 50
through which a second fluid (e.g., another fluid such as a polymer
solution as discussed below) can be introduced into the fluid
flowing axially along the conduit 42 through the static mixer 48.
In this embodiment, the second input port 50 is disposed at an
intermediate location between the proximal end (PE) and the distal
end (DE) of the static mixer 48.
[0222] While in this embodiment the second port is configured to
introduce a stream of fluid, e.g., a polymer solution, into the
fluid, e.g., anti-solvent, flowing axially through the conduit at
an angle of about 90 degrees relative to the axial flow direction,
in other embodiments the second port can be configured such that
the direction of the flow of the second fluid would intersect the
axial flow direction at an angle other than 90 degrees.
[0223] By way of example, FIG. 4B schematically depicts another
embodiment of a device in which the second input poll 50 is
configured to introduce a fluid into the conduit along a direction
that forms a non-orthogonal angle with the direction of axial
flow.
[0224] In use, an anti-solvent flowing stream is established
through the conduit 42 via the input port 44, e.g., causing the
anti-solvent to flow from a reservoir (not shown) into the conduit,
for example, via pumping, as discussed in more detail below. The
static mixer causes mixing of the flowing anti-solvent so as to
provide a mixed flowing stream of the anti-solvent before the
stream reaches the second input port 50. Once a mixed flowing
stream of the anti-solvent has been established, a polymer solution
can be introduced into the anti-solvent stream via the second input
port 50 (e.g., by causing the polymer solution to flow from a
reservoir (not shown) into the conduit, for example, via pumping,
as discussed in more detail below).
[0225] As discussed above, the contact of the polymer solution with
the anti-solvent results in precipitation of the polymer into a
plurality of polymeric nanoparticles that are carried by the stream
of the anti-solvent away from the static mixer. The formed
nanoparticles can then be collected as a suspension in a mixture of
anti-solvent and the process solvent. As discussed above, in many
embodiments, the rate of flow of the anti-solvent through the
conduit 42 is substantially greater than the flow rate of the
polymer solution into the conduit, e.g., by a factor of about 10 or
more. Hence, in such embodiments, the nanoparticles are surrounded
primarily by the anti-solvent--including any additive(s) such as a
colloid stabilizer added to the anti-solvent--as they move down the
conduit to a collection device--though the collection device
receives typically the process solvent and, in some cases additives
added to the process solvent, as well. Further, in many
embodiments, the flow rate of the anti-solvent is sufficiently fast
to ensure that the polymer solution entering the conduit would
interact with a fresh batch of anti-solvent that is substantially
free of process solvent and polymeric material that had previously
entered the conduit.
[0226] While in the above device 40 the second input port is
positioned at an intermediate location relative to proximal and
distal ends of the static mixer, in other embodiments the second
input port can be positioned in proximity to the proximal end or
the distal end of the static mixer. By way of example, FIG. 5A
schematically depicts a device 52 according to an alternative
implementation of the above device 40 for generating nanoparticles
in which the second input port 50 is positioned in proximity to the
distal end (DE) of the static mixer. For example, the input port 50
can be offset from the distal end of the mixer by one or two mixing
elements.
[0227] By way of another example, FIG. 5B schematically depicts a
device 54 according to another alternative implementation of the
above device 40 in which the second input port 50 is positioned in
proximity to the proximal end (PE) of the static mixer, e.g.,
offset by one or two mixing elements from the proximal end. In some
embodiments, the static mixer is preferably selected to generate a
mixed flowing stream of the anti-solvent over a short length of the
mixer. In some embodiments the static mixer can be chosen such that
its mixing effect on the anti-solvent flow would even be present
slightly upstream from the proximal end of the mixer. In such
embodiments, as shown schematically in FIG. 5C, the second input
port can be positioned slightly upstream from the static mixer but
sufficiently close to its proximal end such that the incoming
polymer solution would interact with a mixed flowing stream of the
anti-solvent.
[0228] The static mixer can also be chosen such that its mixing
effect on the anti-solvent flow is present downstream from the
distal end of the mixer. In such embodiments, as shown
schematically in FIG. 5D, the second input port 50 of a device 58'
can be positioned slightly downstream from the static mixer 48 but
sufficiently close to its distal end DE (e.g., within approximately
1-2 mixing element lengths) such that the incoming polymer solution
would interact with a mixed flowing stream of the anti-solvent.
[0229] In some embodiments, a plurality of input ports for
introducing the polymer solution into a mixed flowing stream of
anti-solvent can be provided. For example, as shown in FIG. 5E, a
device 58'' can include a plurality of polymer solution input ports
50a, 50b on opposite sides of the conduit 42. In other embodiments,
the plurality of polymer solution input ports can be located on the
same side of the conduit and/or can be spaced at various intervals
from each other along the length of the conduit 42.
[0230] In other embodiments, rather than utilizing a single static
mixer unit, a plurality of static mixer units can be employed. For
example, FIG. 6A schematically depicts a device 60 according to an
embodiment of the invention that, similar to the above device 40,
includes a conduit 42 for receiving a flowing stream of an
anti-solvent through a first input port 44 as well as a second
input port 50 for introducing a stream of a polymer solution into
the mixed flowing stream of the anti-solvent. In device 60, a
plurality of static mixer units 61a, 61b, 61c are disposed within
the conduit 42 to cause mixing of the flowing anti-solvent. The
static mixer units are disposed in series, preferably in staggered
orientation relative to one another. While FIG. 6A illustrates the
input port 50 as being positioned roughly in the center of one of
the static mixer units 61b, in other embodiments the input port 50
can be positioned differently. For example, in some embodiments, as
illustrated in FIG. 6B, a device 62 can include a second input port
50 that is positioned between adjacent static mixer units 61a, 61b
so as to deliver the polymer solution to into a gap between those
units.
[0231] FIG. 6C schematically illustrates a device 62' according to
another embodiment of the invention that includes a conduit 42 in
which static mixer units 61a, 61b are disposed. The device 62'
includes two input ports 50a, 50b, which are positioned on opposite
sides of the conduit 42, for the introduction of a polymer solution
into the conduit. The static mixer units are separated from one
another so as to provide a gap in the vicinity of the input ports
50a,50b such that a polymer solution can be introduced through the
gap into the conduit.
[0232] FIG. 7 schematically depicts a system 70 according to an
embodiment of the invention for generating polymeric nanoparticles
in which any of the above devices 40, 52, 54, 56, 58, 58', 58'',
60, 62 in any of their various implementations can be incorporated.
The system 70 includes a reservoir 72 for storing a polymer
solution and another reservoir 74 for storing an anti-solvent, such
as a mixture of deionized water and a colloid stabilizer. A device
78, e.g., a gear pump, is fluidly connected at its input to the
reservoir 74 and is fluidly coupled at its output to first input
port 44 of the device 40 to cause a flow of the anti-solvent from
the reservoir into the conduit 42. A static mixer 48 is disposed
within the conduit 42. As discussed above, the flow of the
anti-solvent stream through the static mixer generates a mixed
flowing stream of the anti-solvent.
[0233] Another device 76, e.g., another gear pump, is fluidly
connected at its input to the reservoir 72 and at is output to the
second input port 50 of the device 40 to cause a flow of the
polymer solution from the reservoir 72 via the second port into the
mixed flowing stream of the anti-solvent to cause precipitation of
the polymer into a plurality of polymeric nanoparticles. As
discussed above, although in many embodiments the flow rate of the
anti-solvent is substantially greater than that of the polymer
solution, e.g. by a factor of 10, a variety of anti-solvent and
polymer solution flow rates can be employed.
[0234] In this embodiment, both of the devices 76 and 78 arc
variable pumps that can adjust the flow rate of the anti-solvent
and the polymer solution, respectively, for introduction into the
device 40. By way of example, in this implementation, the device 78
can adjust the flow rate of the anti-solvent in a range of about 20
ml/min to about 2000 ml/min, and the device 76 can adjust the flow
rate of the polymer solution in a range of about 4 ml/min to about
200 ml/min, or, in some embodiments, in a range of about 5 ml/min
to about 100 ml/min. As noted above, in some embodiments, the
devices 76 and 78 are gear pumps. An example of suitable gear pump
is a pump marketed by Cole-Parmer Instrument Company of Illinois,
U.S.A. under the trade designation Ismatec.
[0235] The output port 46 of the device 40 is in fluid
communication with a collection vessel 84. The formed nanoparticles
are entrained in a fluid stream comprising a mixture of the
anti-solvent and the process solvent (in many cases the
anti-solvent is the major component of the fluid stream) that
carries the nanoparticles via the output port 46 into the
collection vessel 84, which may contain a liquid, e.g., deionized
water. In some embodiments, the collection vessel is not pre-filled
with a liquid. A suspension containing the nanoparticles can be
collected from the collection vessel to be concentrated and in some
embodiments lyophilized, e.g., in a manner discussed above.
[0236] As discussed above, it has been discovered that the use of a
static mixer to cause mixing of the anti-solvent stream allows
generating nanoparticles with a low polydispersity index over a
wide range of flow rates, e.g., a polydispersity index equal to or
less than about 0.25 (e.g., in a range of about 0.05 to about 0.1).
In addition, the flow rate can be adjusted to obtain a desired
average particle size. In this embodiment, the variable pump 78
allows changing the flow rate of the anti-solvent to "dial" the
average particle size of the nanoparticles generated via
nanoprecipitation.
[0237] In some embodiments, one or more injectors can be employed
to introduce the polymer solution into the mixed flowing stream of
the anti-solvent. By way of example, FIG. 8A schematically shows a
device 81 according to such an embodiment of the invention for
generating nanoparticles, which includes a conduit 83 in which two
static mixer units 85a and 85b are disposed. Similar to the
previous embodiments, the conduit 83 includes an input port 87
through which a fluid, e.g., anti-solvent, can be introduced into
the conduit and an output port 89 through which the fluid exits the
conduit. In this embodiment, an injector 91 is coupled to the
conduit at an intermediate location between the static mixer units.
The injector 91 includes an inlet port 93 for receiving a fluid,
e.g., a polymer solution, and an output nozzle 95 that is
positioned within the conduit and is configured to inject the fluid
into the mixed flowing stream of the anti-solvent. Although the
illustrated nozzle faces downward (it is aimed substantially
perpendicular to the axial direction of the anti-solvent flow), the
nozzle can also be aimed at other angles with respect the direction
of the anti-solvent flow. For example, the nozzle can include a 90
degree bend such that it is aimed parallel to the direction of the
anti-solvent flow towards the mixer's distal end.
[0238] FIG. 8B schematically depicts another device 97 according to
the teachings of the invention that employs an injection system for
injecting a polymer solution into a mixed flowing stream of the
anti-solvent. The device 97 includes a conduit 99 in which a two
static mixer 101a and 101b are disposed. An injection system 103
extends across the conduit within a gap between the two static
mixer units. The injection system includes a plurality of injection
nozzles 105 that are configured to inject a polymer solution in a
downstream direction and with sufficient velocity such that the
polymer solution would be introduced into a mixed flowing stream of
an anti-solvent through the mixer from mixer's proximal end to its
distal end.
EXAMPLES
[0239] The following examples are provided for further elucidation
of various aspects of the invention. The examples are intended only
for illustrative purposes and do not necessarily represent optimal
ways of practicing the invention and/or optimal results that can be
obtained.
[0240] A prototype system based on the system shown in FIG. 7 above
was assembled to generate polymeric nanoparticles in accordance
with the above teachings, as discussed in the following examples.
In some of the following examples, a helical static mixer similar
to that shown in FIG. 9 marketed by Cole-Parmer Instrument Company
of Illinois, U.S.A. was employed. The helical static mixer includes
alternating left and right-hand twists that cause a fluid flowing
through the mixer to move from the wall of a conduit in which the
mixer is disposed to the center of the mixer and from the center to
the wall in an alternating fashion. In some other examples, a
static mixer known as a "structured-packing mixer" marketed by
Sulzer Chemtech USA, Inc. of Oklahoma, U.S.A. under the trade
designation Sulzer SMX, shown in FIGS. 10A and 10B, was employed.
This mixer includes a lattice of mixing elements that is oriented
at 45 degrees relative to the direction of the flow.
[0241] At least two prototype devices were constructed:
[0242] I. A 5 mm internal diameter (ID) helical mixer device was
constructed by inserting a 5 mm OD polyacetal helical mixer
(Cole-Parmer) into a 5 mm ID polypropylene tube fitted with a
barbed polypropylene "Y" fitting on one end. (as shown in FIG.
11A). The mixer was extended through one of the arms of the "Y"
fitting. The aqueous phase (i.e., anti-solvent) was directed via
1/4 inch tubing through the mixer-containing arm. The organic phase
(i.e., polymer solution) was directed via 1/8 inch tubing through
the other (empty) arm. A 1/4 inch to 1/8inch reducer was connected
to the bottom port to provide a slight back pressure.
[0243] II. An 8 mm ID modified SMX mixer device was constructed by
modifying a standard 1/4 inch Sulzer (Sulzer Chemtech USA, Tulsa,
Okla.) SMX mixer with an 1/8 inch ID side entry port midway along
the static mixer length. The mixer was fabricated from 316L
stainless steel mixer (FIG. 11B). It was 3 inches long with an 8 mm
bored ID and contained 8 SMX type elements. The mixer was
configured such that the aqueous phase flowed through the mixer
main body while the organic phase entered the side port.
Example 1
[0244] The following process was used to characterize the effect of
organic and aqueous flowrates on average particle size and
polydispersity using the prototype helical mixing device depicted
in FIG. 11A.
[0245] 2 grams of 5050 PLGA (5050DLG 1AP, Mw=6.4 kD, Mn=2.9 kD,
Lakeshore Biomaterials, Birmingham, Ala.) were dissolved in 158
grams of acetone and sonicated for 30 seconds at room temperature.
Separately, 10 grams of poly vinyl alcohol (PVA) (80% hydrolyzed,
Mw 9-10 kD, Aldrich, St Louis, Mo.) was dissolved in 2 kilograms of
water at room temperature. Each solution was translucent and
visually free of undissolved material.
[0246] The organic and aqueous phases were transferred to glass
reservoirs, the bottom outlet of each was connected to
pre-calibrated magnetically driven gear pumps (Ismatec,
Cole-Parmer) via flexible tubing to the respective ports of the
helical mixer device. A particle size sample was collected by first
initiating the aqueous flow, then the organic flow to predetermined
flow rates. After a few moments of flushing, a small suspension
sample was collected at the tubing outlet and then pumps were
turned off in reverse order. The pump settings were then readjusted
and a subsequent particle size sample taken. At the conclusion of
the experiment, each sample particle size was measured via Malvern
Zetasizer Model Nano S (Malvern Instruments, Southborough, Mass.).
In most cases each particle size measurement was conducted in
duplicate and the results averaged.
[0247] Total flow rates in the range of about 25 to 500 ml/min with
the organic phase relative to the aqueous phase flow rate (O:W)
ratios of 1:5 and 1:10 were employed and the results shown
below:
TABLE-US-00001 TABLE 1 O:W Qo Qw Qt Z-Avg D(v)50 D(v)90 D(v)10
Ratio (ml/min) (ml/min) (ml/min) (nm) PdI (nm) (nm) (nm) 1:5 73.0
365.0 438.0 84 0.089 68 109 47 60.0 301.0 361.0 94 0.052 82 122 59
47.0 237.0 284.0 102 0.073 90 138 63 34.3 172.0 206.3 111 0.093 97
156 67 21.5 107.4 128.9 123 0.059 112 175 77 12.9 64.4 77.3 140
0.072 131 211 86 4.3 21.5 25.8 180 0.065 183 268 126 1:10 46.4
463.5 509.9 87 0.082 71 114 48 38.6 386.5 425.1 92 0.071 78 121 54
30.1 300.0 330.1 98 0.091 81 134 55 21.5 214.0 235.5 110 0.076 92
157 62 12.9 128.0 140.9 121 0.066 110 173 75 4.3 42.9 47.2 152
0.061 147 232 97
[0248] In the above table and the tables that follow, Q.sub.o and
Q.sub.w refer to flow rates of the organic and the aqueous phases,
respectively. Q.sub.t refers to the total flow rate. Dv50 is
defined as the particle size below which the sizes of 50% of the
particles lie, Dv90 is defined as the particle size below which the
sizes of 90% of the particles lie, and Dv10 is defined as the
particle size below which the sizes of 10% of the particles
lie.
[0249] The polydispersity index remains less than about 0.1 over
the entire tested flow rate range.
[0250] FIG. 12 presents data corresponding to Z.sub.ave as a
function of the total flow rate, indicating that the average
particle size decreases as the flow rate increases from about 25
ml/min to about 500 ml/min. At flow rates less than about 200-300
ml/min, average particle size decreases at a much faster rate than
flow rates greater than 200-300 ml/min. In other words, two flow
rate regimes can be discerned from the data: one in which the
average particle size is strongly flow dependent and another in
which the average particle size can be considered as substantially
independent of the flow rate. FIG. 12 also shows that average
particle sizes appear to be substantially independent of the O:W
ratio within the range of 1:5 and 1:10.
[0251] These data show that the average particle size can be tuned
("dialed") by changing the flow rates (principally the anti-solvent
rate) through the mixer while ensuring that the polydispersity
index remains low. In other words, for a given desired average
particle size, the flow rates can be selected to achieve the target
particle average size.
Example 2
[0252] The process described above in Example 1 was again conducted
but the polymer concentration in the polymer solution was increased
to 2%. Data was collected at both 1:5 and 1:10 O:W ratios.
[0253] The results are shown in the table below:
TABLE-US-00002 TABLE 2 O:W Qo Qw Qt Z-Avg D(v)50 D(v)90 D(v)10
ratio (ml/min) (ml/min) (ml/min) (nm) PdI (nm) (nm) (nm) 1:5 73.0
365.0 438.0 110 0.058 97 151 68 60.0 301.0 361.0 121 0.057 109 172
75 47.0 237.0 284.0 129 0.012 121 177 87 34.3 172.0 206.3 137 0.059
129 200 88 21.5 107.4 128.9 157 0.042 155 233 105 12.9 64.4 77.3
176 0.05 178 264 120 4.3 21.5 25.8 207 0.061 214 307 150 1:10 46.4
463.5 509.9 119 0.058 108 166 75 38.6 386.5 425.1 126 0.055 115 182
79 30.1 300.0 330.1 132 0.012 124 183 88 21.5 214.0 235.5 140 0.059
133 203 93 12.9 128.0 140.9 155 0.053 152 232 101 4.3 42.9 47.2 191
0.026 194 283 134
[0254] As in Example 1, PdI was maintained at less than 0.1 for all
flow conditions.
[0255] The Z.sub.ave versus flow rate is plotted in FIG. 12
together with the data in Example 1. The data shows that increasing
the polymer concentration from 1% to 2% results in an increase in
the average particle size in a controlled fashion and with a
similar dependency on the flow rate as that observed for the 1%
polymer concentration.
Example 3
[0256] The process discussed above in Example 1 was again conducted
by utilizing the modified SMX mixer (shown in FIG. 11B) to test the
effect of flow rate on average particle size. Using freshly
prepared 1 wt % PLGA and 0.5 wt % PVA solutions and maintaining the
W:O ratio at 10:1, a series of experiments detailed below were
conducted. [0257] a) In the first experiment, the SMX mixer was
utilized with a set of pumps with a total flow rate range of 100 to
500 ml/min. [0258] b) In the second experiment, the same mixer was
used but with another set of pumps with a larger flow rate in a
range of 500 to 2000 ml/min. [0259] c) In the third experiment, the
helical mixer was used with the pumps used in a) but at a single
flow rate combination. [0260] d) In the final experiment, the SMX
mixer was used with pumps used in b) but at 3 flow rate
combinations.
[0261] The results are shown in the Table 3 below:
TABLE-US-00003 TABLE 3 Qo Qw Qt Z-Ave D10(v) D50(v) D90(v)
Experiment (ml/min) (ml/min) (ml/min) (nm) PdI (nm) (nm) (nm) a 10
100 110 232 0.115 151 250 425 15 150 165 215 0.082 147 225 349 20
200 220 191 0.036 130 194 287 25 250 275 185 0.068 123 188 290 30
300 330 191 0.100 111 197 338 35 350 385 166 0.053 110 166 249 45
450 495 149 0.074 94 143 227 b 45 450 495 168 0.057 112 168 252 60
660 660 152 0.062 97 148 231 75 750 825 140 0.064 86 131 212 90 900
990 132 0.069 81 121 196 105 1050 1155 127 0.076 75 114 190 120
1200 1320 123 0.053 77 112 174 135 1350 1485 115 0.077 67 100 165
150 1500 1650 121 0.074 69 105 179 180 1800 1980 113 0.084 65 97
164 C 45 450 495 121 0.151 59 97 199 45 450 495 128 0.025 84 119
178 D 105 1050 1155 128 0.049 84 120 181 150 1500 1650 111 0.078 68
98 156 180 1800 1980 107 0.062 63 91 147
[0262] The data shows that in most cases the PO was maintained at
less than 0.1. A plot of Z.sub.ave versus anti-solvent flow rate is
shown in FIG. 13. From this Figure, it can be seen that the data
from the three experiments using the SMX mixer are comparable and
may be represented by a single curve. The data obtained by
utilizing the helical mixer indicates that smaller sized particles
were generated compared to the particles generated by utilizing the
SMX mixer for the same flow rate. This can be due to a greater
mixing intensity achieved in the helical mixer device, which has a
smaller diameter.
Example 4
[0263] Utilizing the helical mixer shown in FIG. 11A, the following
process was employed to fabricate a 3-gram batch of docetaxel
PEGylated nanoparticles.
[0264] 2.52 grams of docetaxel custom conjugated PLGA (Mw=6.6 kD,
Mn=3.0 kD, drug loading: 8%, AMRI Albany, N.Y.), and 0.480 grams of
5050DL-PLGA mPEG 2 kD (Mw: 10.6 kD, mPEG Mw: 2.0 kD, Lakeshore
Biomaterials, Birmingham, Ala.) were dissolved in 237 grams of
acetone at room temperature. Separately, a total of 15 grams of
poly vinyl alcohol (PVA) (80% hydrolyzed, Mw 9-10 kD, Aldrich, St.
Louis, Mo.) was dissolved in 3 kilograms of water at room
temperature. Each solution was translucent and visually free of
undissolved material.
[0265] Referring to FIG. 7, the flow of the aqueous phase
(anti-solvent) was initiated at a flow rate of 220 mL/min via the
aqueous inlet through the conduit of the device in which the static
mixer was disposed. Once the flow of the aqueous phase was
established, the flow of the polymer solution was initiated at 22
mL/min from an organic solution vessel (not shown) through the
organic inlet into the flowing stream of the aqueous phase. Once
the organic solution vessel had emptied, the organic pump was
turned off and the aqueous pump remained on for several moments to
flush out the mixer. The recovered suspension collected in a
collection vessel was milky white and no large particles were
distinguishable by the naked eye.
[0266] The nanoparticle suspension was diafiltered and concentrated
in the following two-step process. The suspension was initially
diafiltered to remove dissolved PVA and acetone by recirculation
through a 300 kD MWCO filter cassette (Pall Omega Centramate Medium
Screen cassette, 0.093 m.sup.2), at a recirculation flow rate of
200 ml/min and a transmembrane pressure (TMP) of 1.5 bar using 44 L
of deionized water. Upon completion, the nanoparticles suspension
was drained from the cassette and the cassette was rinsed twice by
recirculating fresh deionized water through the cassette and
respective tubing (2.times.250 ml). The rinse volumes were
collected and combined with the initially recovered nanoparticles
suspension.
[0267] The washed nanoparticles suspension was subsequently
concentrated using three smaller 300 kD MWCO filters (Millipore
Pellicon XL, 50 cm.sup.2 with Biomax membrane) plumbed in parallel
at a recirculation rate of about 25 ml/min and a TMP of 1 bar. Drug
concentration was continuously monitored by HPLC assay as the
suspension volume was reduced. A portion of the suspension was
collected when the drug content level reached 1.7 mg/ml and the
remainder was collected at a drug content level of 3.2 mg/ml. Each
suspension was stored at 4.degree. C. The yield based on docetaxel
recovery was about 85.5%.
[0268] FIG. 14 shows the particle size distribution obtained by the
Zetasizer. The average particle size (Z.sub.ave) was measured to be
about 97.5 nm, Dv90 (defined as the particle size below which the
sizes of 90% of the particles lies) was determined to be about
130.3 nm, and Dv50 (defined as the particle size below which the
sizes of 50% of the particle lie) was determined to be about 85.2
nm. Further, the polydispersity index (PdI) was measured to be
about 0.066.
Example 5
[0269] Utilizing the modified SMX mixer shown in FIG. 11B, the
following process was employed to fabricate a 10-gram batch of
docetaxel PEGylated nanoparticles. 6.044 grams of docetaxel custom
conjugated PLGA (Mw=9.8 kD, Mn=5.7 kD, drug loading: 7.6%, AMRI
Albany, N.Y.), and 4.003 grams of 5050DL-PLGA mPEG 2 kD (Mw: 13.0
kD, mPEG Mw: 2.0 kD, Lakeshore Biomaterials, Birmingham, Ala.) were
dissolved in 791 grams of acetone at room temperature. Separately,
11 L of 0.5% solution of PVA was prepared by combining 1.1 L of
previously prepared stock solution of 5% PVA with 9.9 L of RODI
water. (The stock solution was prepared by dissolving 110 gm of PVA
(80% hydrolyzed, Mw: 9-10 kD, Sigma-Aldrich, St. Louis, Mo.) into
2200 ml of RODI water and heating the solution at 80.degree. C. for
3 hr. The solution was cooled to room temperature, filtered and
stored at 4.degree. C.
[0270] Referring to FIG. 7, the flow of the aqueous phase
(anti-solvent) was initiated at a flow rate of 608 ml/min via the
aqueous inlet through the conduit of the device in which the static
mixer was disposed. Once the flow of the aqueous phase was
established, the flow of the polymer solution was initiated at 60.8
ml/min from an organic solution vessel (not shown) through the
organic inlet into the flowing stream of the aqueous phase. Once
the organic solution vessel was empty, the organic pump was turned
off and the aqueous pump remained on for several moments to flush
out the mixer. The recovered suspension, approximately 11 liters,
was collected in 20 liter polypropylene carboy.
[0271] The nanoparticle suspension was diafiltered and concentrated
using a tangential flow filter (TFF) (GE Healthcare hollow fiber
cartridge, polysulfone membrane, 500 kD NMWC, 0.14 m.sup.2) in a
three-step process. Using a recirculation rate of 1160 ml/min and a
transmembrane pressure (TMP) of less than 20 psi, the suspension
was initially concentrated to approximately 1 liter, diafiltered
with 12 liters of RODI water, and concentrated a final time to 206
ml. The suspension was recovered and measured by HPLC for docetaxel
content and stored at 4.degree. C. The yield based on docetaxel
recovery was about 96%.
[0272] FIG. 15 shows three graphs corresponding to three
measurements, obtained by Malvern Zetasizer Model Nano S (Malvern
Instruments, Southborough, Mass.), of the particle size
distribution of the nanoparticles. The average particle size
(Z.sub.ave) was measured to be about 80.35 nm, Dv90 (defined as the
particle size below which the sizes of 90% of the particles lies)
was determined to be about 103 nm, and Dv50 (defined as the
particle size below which the sizes of 50% of the particle lie) was
determined to be about 69.2 nm. Further, the polydispersity index
(PdI) was measured to be 0.052.
[0273] All publications referred to herein, including patents,
published patent applications, articles, among others, are hereby
incorporated by reference in their entirety.
[0274] Those having ordinary skill in the art will appreciate that
various changes can be made to the above embodiments without
departing from the scope of the invention.
* * * * *