U.S. patent application number 11/153935 was filed with the patent office on 2006-01-19 for method of preparing gas-filled polymer matrix microparticles useful for echographic imaging.
This patent application is currently assigned to Point Biomedical Corporation. Invention is credited to Robert E. Short.
Application Number | 20060013771 11/153935 |
Document ID | / |
Family ID | 29419248 |
Filed Date | 2006-01-19 |
United States Patent
Application |
20060013771 |
Kind Code |
A1 |
Short; Robert E. |
January 19, 2006 |
Method of preparing gas-filled polymer matrix microparticles useful
for echographic imaging
Abstract
A method is provided to prepare gas-filled, porous
microparticles having a polymer matrix interior which are useful as
ultrasound echogenic contrast agents. An oil-in-water suspension is
formed, both phases are frozen, then the aqueous and nonaqueous
frozen phases are removed by sublimation. The resulting porous
microparticles can receive a gas and be used as an ultrasound
contrast agent.
Inventors: |
Short; Robert E.; (Los
Gatos, CA) |
Correspondence
Address: |
DECHERT LLP
P.O. BOX 10004
PALO ALTO
CA
94303
US
|
Assignee: |
Point Biomedical
Corporation
San Carlos
CA
|
Family ID: |
29419248 |
Appl. No.: |
11/153935 |
Filed: |
June 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10150449 |
May 17, 2002 |
6919068 |
|
|
11153935 |
Jun 16, 2005 |
|
|
|
Current U.S.
Class: |
424/9.52 |
Current CPC
Class: |
A61K 49/223 20130101;
B01J 13/02 20130101 |
Class at
Publication: |
424/009.52 |
International
Class: |
A61K 49/22 20060101
A61K049/22 |
Claims
1. A microparticle comprising an outer shell enclosing an inner
polymer matrix, wherein the outer shell comprises a biologically
compatible amphiphilic material, the inner polymer matrix is
characterized by a network of interstitial void space surrounded by
a web-like polymer structure, and the outer shell is not covalently
linked to the inner polymer matrix.
Description
1. CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation application of
application Ser. No. 101,150,449, filed May 17, 2002, the
disclosure of which is incorporated herein by reference in its
entirety.
2. FIELD
[0002] This invention pertains to a method of preparing gas-filled
microparticles having a polymer matrix interior. Such
microparticles are useful as a blood pool echogenic contrast agent
for ultrasonic echography.
3. BACKGROUND
[0003] Ultrasound is a modem medical imaging modality using sound
energy to noninvasively visualize the interior structures and
organs of a patient. Pulses of high frequency sound, generally in
the megaHertz (MHz) range, emitted from a hand-held transducer are
propagated into the body where they encounter different surfaces
and interfaces. A portion of the incident sound energy is reflected
back to the transducer that converts the sound waves into
electronic signals which are then presented as a two-dimensional
echographic image on a display monitor.
[0004] One of the advances in ultrasound imaging has been the
development of ultrasonic contrast agents. Use of contrast agents
enables the sonographer to visualize the vascular system which is
otherwise relatively difficult to image. In cardiology for example,
ultrasound contrast injected into the bloodstream permits the
cardiologist to better visualize heart wall motion with the
opacification of the heart chambers. Perhaps more importantly,
contrast can be used to assess perfusion of blood into the
myocardium to determine the location and extent of damage caused by
an infarct. Similarly, visualization of blood flow using ultrasound
contrast in other organs such as the liver and kidneys has found
utility in diagnosing disease states in these organs.
[0005] The first encapsulated contrast agent was developed by
Tickner et al (Final Report NHLB1 Contract No. HR-62917-1A,
National Institute of Health, 1977; U.S. Pat. No. 4,276,885), made
of a gelatin membrane encapsulating a nitrogen bubble. The bubble
diameter, while precise, was not small enough to circulate through
the capillary beds and therefore was not appropriate for systemic
delivery. An agent was later developed to include a lipophilic
material in a saccharide composition to provide a microbubble of
sufficient stability to enable pulmonary capillary transmission
(Circulation 62 (Supp. III): 111-34, 1980).
4. SUMMARY
[0006] This invention pertains to a novel method of preparing
gas-filled polymer matrix microparticles suitable for use as an
ultrasound contrast agent. The method of preparation comprises the
steps of
[0007] 1. dissolving a polymer in a substantially water-immiscible
solvent;
[0008] 2. emulsifying the polymer solution in an aqueous medium,
optionally containing suitable surfactants and bulking agents;
[0009] 3. reducing the temperature of the emulsion wherein both
aqueous and water-immiscible phases become frozen;
[0010] 4. removing water from the aqueous and solvent from
water-immiscible phase by means of sublimation, resulting in the
formation of polymer matrix microparticles;
[0011] 5. introducing a gas into the polymer matrix
microparticles.
[0012] Step 2 may be modified such that the aqueous medium also
contains a biologically compatible amphiphilic material which
encapsulates the emulsion droplets. Upon crosslinking, the
amphiphilic material becomes a contiguous outer layer of the
microparticle.
[0013] The method may also include, after step 2, the step of
replacing the aqueous medium with a second aqueous medium. This
additional step is useful when the components of an aqueous medium
optimized for emulsion of the polymer solution are different from
the components of an aqueous medium optimized for lyophilization.
The additional step may be achieved by centrifugation or by
diafiltration.
[0014] Also provided is a method of ultrasound imaging of organs or
tissue of a patient comprising the steps of:
[0015] 1. injecting into a patient a suspension of gas-filled
polymer matrix microparticles in a physiologically acceptable
aqueous liquid carrier, where the microparticles have a mean size
of about 1 to 10 microns and are made from a biodegradable
synthetic polymer, and
[0016] 2. echographically imaging the patient at an organ or tissue
of interest.
5. DESCRIPTION OF DRAWINGS
[0017] In the accompanying drawings:
[0018] FIG. 1 is a plot of frame number vs. acoustic densitometry
backscatter taken on an ultrasonic scanner as described in Example
3 for a test of the microparticles made in accordance with Example
1.
[0019] FIG. 2 is a plot of sound intensity in MI.sup.2 vs. peak
backscatter as described in Example 3.
[0020] FIG. 3 is a plot of sound intensity in MI.sup.2 vs.
fragility slope as described in Example 3.
6. DETAILED DESCRIPTION
[0021] The present invention provides a method of manufacturing
gas-filled porous microparticles having a polymer matrix interior.
Such microparticles are useful as an ultrasonic contrast agent.
These microparticles, being porous, rely on the hydrophobicity of
the polymer to retain the gas within. The microparticles may be
produced to also include an outer layer of a biologically
compatible amphiphilic material, thus providing a surface for
chemical modification to serve various purposes.
[0022] The process of manufacture utilizes a different emulsion
solvent removal technique from those typically used to produce
solid polymer microspheres. Typically, the solvent undergoing phase
change is evaporated. According to the process of the present
invention, solvent removal is effected by sublimation by
lyophilization. In an evaporation process, mobile polymer molecules
in the liquid phase will cohere to form a solid microsphere when
solvent is removed. However, the initial freezing in lyophilization
immobilizes the polymer molecules so that when solvent is removed
under vacuum, a network of interstitial void spaces surrounded by a
web-like polymer structure remains. This porous structure can then
be filled with a gas.
[0023] The fabrication of the matrix microparticles starts with the
preparation of the polymer and water-immiscible solvent solution.
Preferred polymers are biodegradable synthetic polymers such as
polylactide, polycaprolactone, polyhydroxyvalerate,
polyhydroxybutyrate, polyglycolide and copolymers of two or more
thereof. The requirements for the polymer solvent are that it is
substantially water-immiscible and practicably lyophilizable. By
practicably lyophilizable it is meant that the solvent freezes at a
temperature well above the temperature of a typical lyophilizer
minimum condensing capability and that the solvent will sublimate
at reasonable rate in vacuo. Suitable solvents include p-xylene,
cyclooctane, benzene, decane, undecane, cyclohexane and the like.
Polylactide in xylene is a preferred solution.
[0024] The concentration of polymer in solution will dictate the
void volume of the end product which, in turn, will impact acoustic
performance. A high concentration provides lower void volume and a
more durable microparticle. Acoustic modalities particularly
relying on bubble breakage will exhibit performance variation
depending on polymer concentration. Polymer molecular weight also
has an effect. A low molecular weight polymer produces a more
fragile particle. Optionally, additives may be used in the polymer
organic phase. Plasticizers to modify elasticity of the polymer or
other agents to affect hydrophobicity of the microparticle can be
added to modify the mechanical, and thus acoustic, characteristics
of the microparticle. Such plasticizers include the phthalates or
ethyl citrates. Agents to modify hydrophobicity include fatty acids
and waxes.
[0025] The polymer solution is then emulsified in an aqueous phase.
The aqueous phase may contain a surface active component to enhance
microdroplet formation and provide emulsion stability for the
duration of the fabrication process. Surface active components
include the poloxamers, tweens, brijs. Also suitable are soluble
proteins such as gelatin, casein, albumin, or synthetic polymers
such as polyvinyl alcohol.
[0026] Addition of viscosity enhancers may also be beneficial as an
aid in stabilizing the emulsion. Useful viscosity enhancers include
carboxymethyl cellulose, dextran, methyl cellulose, hydroxyethyl
cellulose, polyvinyl pyrrolidone, and various natural gums such as
gum arabic, carrageenan, and guar gum.
[0027] The range of ratios of the organic phase to the aqueous
phase is typically between 2:1 and 1:20 with a 2:1 to 1:3 ratio
range preferred.
[0028] If the aqueous phase is also to serve as the suspending
medium during the lyophilization step, other components which may
be included in the aqueous phase are ingredients suitable as
bulking agents such as polyethylene glycol, polyvinyl pyrrolidone,
sugars such as glucose, sucrose, lactose, and mannitol. Salts such
as sodium phosphate, sodium chloride or potassium chloride may also
be included to accommodate tonicity and pH requirements.
[0029] A variety of equipment may be used to perform the
emulsification step including colloid mills, rotor-stator
homogenizers, ultrasonic homogenizers, high pressure homogenizers,
microporous membrane homogenizers, with microporous membrane
homogenization preferred because the more uniform shearing provides
for a more monodisperse population of emulsion droplets.
[0030] Size of the droplets formed should be in a range that is
consistent with the application. For example, if the microparticles
are to be injected intravenously, then they should have diameters
of less than 10 microns in order to pass unimpeded through the
capillary network. The size control can be empirically determined
by calibration on the emulsification equipment.
[0031] If it is desired to provide an optional outer layer of a
biologically compatible material, the material is first solubilized
in the aqueous phase. This outer layer material will typically be
amphiphilic, that is, have both hydrophobic and hydrophilic
characteristics. Such materials have surfactant properties and thus
tend to be deposited and adhere to interfaces such as the outer
surface of the emulsion droplets. Preferred materials are proteins
such as collagen, gelatin, casein, serum albumin, or globulins.
Human serum albumin is particularly preferred for its blood
compatibility. Synthetic polymers may also be used such as
polyvinyl alcohol.
[0032] The deposited layer of amphiphilic material can be further
stabilized by chemical crosslinking. If proteinaceous, suitable
crosslinkers include the aldehydes like formaldehyde and
glutaraldehyde or the carbodiimides such as dimethylaminopropyl
ethylcarbodiimide hydrochloride. To crosslink polyvinyl alcohol,
sodium tetraborate may be used.
[0033] Provision for the outer layer is preferably achieved by
diluting the prepared emulsion into an aqueous bath containing the
dissolved crosslinker. This outer crosslinked layer also has the
advantage of increasing the stability of the emulsion droplets
during the later processing steps.
[0034] Provision of a separate outer layer also allows for charge
and chemical modification of the surface of the microparticles
without being limited by the chemical or physical properties of
material present inside the microparticles. Surface charge can be
selected, for example, by providing an outer layer of a type A
gelatin having an isoelectric point above physiological pH or by
using a type B gelatin having an isoelectric point below
physiological pH. The outer surface may also be chemically modified
to enhance biocompatibility, such as by pegylation, succinylation,
or amidation, as well as being chemically binding to the surface
targeting moiety for binding to selected tissues. The targeting
moieties may be antibodies, cell receptors, lectins, selecting,
integrins, or chemical structures or analogues of the receptor
targets of such materials.
[0035] Optionally prior to lyophilization the outer aqueous phase
may be replaced by a second aqueous phase. This would allow the
first aqueous phase to be optimized for emulsification, then
replaced by a second aqueous phase optimized for lyophilization.
Replacement may be achieved by means of diafiltration or by
centrifugation.
[0036] The emulsion is then lyophilized. This involves first
freezing both the water immiscible organic phase in the emulsion
droplets and the suspending aqueous phase, then removing both
phases by sublimation in vacuo. The process produces a dry cake
containing porous polymer matrix microparticles.
[0037] The microparticles are porous and thus can receive a gas.
Introducing a selected gas into the lyophilization chamber after
the drying step will fill the interstitial voids within the
microparticle matrix interior. Alternatively, a selected gas can be
exchanged for the gas used to repressurize the lyophilization
chamber.
[0038] Any gas may be used, but biologically inert gases such as
air, nitrogen, helium, oxygen, xenon, argon, helium, carbon
dioxide, and halogenated hydrocarbons such as perfluorobutane,
perfluoropropane or sulfur halides such as sulphur hexafluoride are
preferred. Depending upon the application, one may select the gas
based on its solubility in blood. For example, perfluorocarbons
have low solubility while carbon dioxide has very high solubility.
Such differences in solubility will influence the acoustic
performance of the microparticle.
[0039] The dry cake can be reconstituted in an aqueous medium to
form a suspension of gas-filled microparticles. The microparticle
suspension when injected into the bloodstream, tissue, or cavity
serves as a contrast agent for ultrasound. Typical applications for
which the present invention can be used include visualization of
myocardial perfusion, quantification of renal function, and
delineation of tumor vascularization.
[0040] The following examples are provided by way of illustration
and are not intended to limit the invention in any way.
EXAMPLE 1
Fabrication of Gas-Filled Microparticle Having a Polymer Matrix
Interior
[0041] An aqueous solution of 1% poly (vinyl alcohol) and 2.8%
mannitol was prepared. Separately, a polymer solution containing 6%
poly(DL-lactide) in p-xylene was prepared. To 40.0 g of the polymer
solution was combined 50.0 g of the aqueous solution in a jacketed
beaker maintained at 30.degree. C. The mixture was then emulsified
to create an oil-in-water emulsion using a circulating system
consisting of a peristaltic pump and a sintered metal filter having
a nominal pore size of 7 microns. After circulating for 6 minutes,
35.0 g of the resultant emulsion was diluted with 177.9 g of a 2.8%
mannitol solution also maintained at 30.degree. C. After 15 minutes
of continuous stirring, a portion of the diluted emulsion was
aliquoted into 10 ml vials and then lyophilized. After the drying
cycle was complete, nitrogen gas was introduced into the
lyophilization chamber to a pressure slightly below atmospheric and
the vials stoppered.
[0042] Microscopic inspection of the reconstituted product revealed
discret gas-filled microparticles.
EXAMPLE 2
Fabrication of Gas-Filled Microparticle Having a Polymer Matrix
Interior and Comprising an Outer Layer
[0043] A solution of 5.4% human serum albumin (hsa) was prepared by
dilution of a 25% solution and the pH adjusted to 4 with HCl.
Separately, 0.99 gm poly(D-L lactide) was dissolved in 29.0 gm
p-xylene. In a jacketed beaker maintained at 40.degree. C., the
resulting polylactide solution was combined with 30 gms of the
previously prepared hsa solution and a coarse emulsion was formed
using magnetic stirring. A peristaltic pump was used to pump the
coarse emulsion through a porous sintered metal filter element with
a 2 .mu.m nominal pore size. The emulsion was recirculated through
the element for approximately 15 minutes until the average droplet
size was less than 10 microns. The emulsion was diluted into 350 ml
of a 40.degree. C. aqueous bath containing 1.0 ml of a 25%
glutaraldehyde solution and 1.4 ml of 1N NaOH. After 15 minutes,
0.75 gm of poloxamer 188 surfactant was dissolved into the aqueous
bath. The emulsion microdroplets were retrieved by centrifugation
at 2000 rpm for 10 minutes, formulated into an aqueous solution
containing polyethylene glycol, glycine, and poloxamer 188,
aliquoted into 10 ml vials, and then lyophilized. After the drying
cycle was completed, nitrogen gas was introduced into the
lyophilization chamber to a pressure slightly less than atmospheric
and the vials were stoppered.
[0044] Microscopic inspection of the reconstituted product revealed
discrete gas-filled microparticles.
EXAMPLE 3
Contrast Efficacy of Gas-Filled Microparticles Having a Polymer
Matrix Interior
[0045] An Agilent 5500 ultrasonic scanner was used for this study
to measure the acoustic backscatter and fragility from a suspended
matrix particle. This scanner has the capability of measuring the
acoustic density (AD) as a function of time within a region of
interest (ROI) displayed on the video monitor. The scanner was set
to the 2D harmonic mode with send frequency of 1.8 MHz and receive
frequency of 3.6 MHz. The test cell was a 2 cm diameter tube
running the length of a Doppler flow phantom manufactured by ATL
Laboratories of Bridgeport, Conn. Microparticle agent made in
accordance with Example 1 was first reconstituted with deionized
water. The resulting suspension was diluted into a 1 liter beaker
containing water and then circulated through flow phantom using a
peristaltic pump (Masterflex L/S manufactured by Cole-Parmer). To
insure that the agent remained uniformly suspended in the beaker,
mixing using a VWR Dylastir magnetic stirrer in conjunction with a
2 cm coated plastic stir bar was maintained throughout the duration
of the testing. When data was to be collected, the pump was turned
off resulting in no flow within the phantom.
[0046] The scanner transducer (s4 probe) was placed directly over
the flow phantom within a water-well designed into the phantom. It
was oriented 90 degrees to the flow axis such that the image of the
flow tube on the monitor was circular. The ROI (21.times.21) was
positioned by the operator within the image of the tubing lumen to
be at the top center about 1 mm away from the top wall and free of
any bright echoes caused by the wall. The scanner was set to the
acoustic densitometry (AD) mode. This mode permits the scanner to
read the mean densitometry within the ROI as a function of time
using a triggered mode. The triggering interval was selected to be
200 milliseconds. For each test run, suspended agent was circulated
into the flow tube and then flow was discontinued. Using a
triggering interval of 200 milliseconds, the sample was then
insonated and the acoustic densitometry within the ROI was measured
at each frame. The tests were repeated at several scanner power
levels.
[0047] From the AD decay curve at each power setting, a linear
regression curve was fit through the first three or four data
points. The zero time intercept provides the peak backscatter
produced by the agent as seen in FIG. 1. The slope of that curve is
identified as the fragility slope and is a measure of the fragility
of the agent. These two measurements are plotted respectively
against intensity in FIGS. 2 and 3. Note that the backscatter of
the matrix microparticle increases linearly with ultrasound
intensity (FIG. 2) and this is considered typical behavior. The
plot of fragility slope (FIG. 3) provides some additional
information regarding the agent. First, it indicates that the agent
is breaking. Secondly, its intercept with the x-axis in FIG. 3
identifies the point where it begins to break. Thus for this agent,
at fragiligy slope -17.4 the agent begins to fail at an MI.sup.2
value of intensity of 0.0868, which is a value of MI of 0.295. Thus
for values of MI less than 0.295, the agent will not fail and
therefore if drug were encapsulated within it would not be
released.
* * * * *