U.S. patent application number 12/171275 was filed with the patent office on 2010-07-08 for process for preparing microparticles through phase inversion phenomena.
Invention is credited to Donald E. Chickering, III, Jules S. Jacob, Yong S. Jong, Edith Mathiowitz.
Application Number | 20100172998 12/171275 |
Document ID | / |
Family ID | 26668936 |
Filed Date | 2010-07-08 |
United States Patent
Application |
20100172998 |
Kind Code |
A1 |
Mathiowitz; Edith ; et
al. |
July 8, 2010 |
PROCESS FOR PREPARING MICROPARTICLES THROUGH PHASE INVERSION
PHENOMENA
Abstract
A process for preparing nanoparticles and microparticles is
provided. The process involves forming a mixture of a polymer and a
solvent, wherein the solvent is present in a continuous phase and
introducing the mixture into an effective amount of a nonsolvent to
cause the spontaneous formation of microparticles.
Inventors: |
Mathiowitz; Edith;
(Brookline, MA) ; Chickering, III; Donald E.;
(Pflugerville, TX) ; Jong; Yong S.; (Warwick,
RI) ; Jacob; Jules S.; (Taunton, MA) |
Correspondence
Address: |
Pabst Patent Group LLP
1545 PEACHTREE STREET NE, SUITE 320
ATLANTA
GA
30309
US
|
Family ID: |
26668936 |
Appl. No.: |
12/171275 |
Filed: |
July 10, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10639770 |
Aug 12, 2003 |
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12171275 |
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09853329 |
May 11, 2001 |
6616869 |
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10639770 |
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09442723 |
Nov 18, 1999 |
6235224 |
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09853329 |
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08686928 |
Jul 3, 1996 |
6143211 |
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09442723 |
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60001365 |
Jul 21, 1995 |
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Current U.S.
Class: |
424/490 ;
514/1.1; 514/44R; 514/5.9 |
Current CPC
Class: |
A61K 9/5153 20130101;
A61K 9/1694 20130101; A61K 9/1641 20130101; A61K 9/5138 20130101;
B01J 13/06 20130101; B01J 13/04 20130101; A61K 9/5146 20130101;
A61K 48/00 20130101; A61K 9/5192 20130101; A61K 9/5089
20130101 |
Class at
Publication: |
424/490 ; 514/3;
514/44.R |
International
Class: |
B01J 13/02 20060101
B01J013/02; A61K 9/50 20060101 A61K009/50; A61K 38/28 20060101
A61K038/28; A61K 31/7088 20060101 A61K031/7088 |
Claims
1-40. (canceled)
41. A microencapsulated product prepared by a method comprising:
dissolving a polymer in an effective amount of a solvent,
dissolving or dispersing an agent in said effective amount of said
solvent, wherein said polymer, said agent and said solvent form a
mixture having a continuous phase and wherein the solvent is said
continuous phase, and introducing said mixture into an effective
amount of a nonsolvent to cause the spontaneous formation of a
microencapsulated product, wherein said solvent and said nonsolvent
are miscible and the difference in solubility parameter of
solvent/nonsolvent pairs or the solubility parameter of the
nonsolvent/solvent pairs is less than 6.
42. A microencapsulated product prepared by a method comprising:
dissolving a polymer in an effective amount of a solvent,
dissolving or dispersing an agent in said effective amount of said
solvent, wherein said polymer, said agent and said solvent form a
mixture having a continuous phase and wherein the solvent is said
continuous phase, and introducing said mixture into an effective
amount of a nonsolvent to cause the spontaneous formation of a
microencapsulated product, wherein said step of introducing does
not include emulsification, agitation, and/or stirring, wherein
said solvent and said nonsolvent are a hydrophilic
solvent/nonsolvent pair, wherein the nonsolvent and solvent are
used in effective amounts, wherein the effective amount of
nonsolvent is at least tenfold greater than the amount of
solvent.
43. The microencapsulated product of claim 41, wherein the agent is
dispersed as solid microparticles in the solvent.
44. The microencapsulated product of claim 41, wherein the agent is
contained in microdroplets dispersed in the solvent.
45. The microencapsulated product of claim 41, wherein the agent is
a liquid.
46. The microencapsulated product of claim 41, wherein the agent is
selected from the group consisting of: adhesives, gases,
pesticides, herbicides, fragrances, antifoulants, dies, salts,
oils, inks, cosmetics, catalysts, detergents, curing agents,
flavors, foods, fuels, metals, paints, photographic agents,
biocides, pigments, plasticizers, propellants.
47. The microencapsulated product of claim 41, wherein the agent is
a bioactive agent.
48. The microencapsulated product of claim 47, wherein the
bioactive agent is selected from the group consisting of:
adrenergic agent, adrenocortical steroid, adrenocortical
suppressant, aldosterone antagonist, amino acid, anabolic,
analeptic, analgesic, anesthetic, anorectic, anti-acne agent,
anti-adrenergic, anti-allergic, anti-amebic, anti-anemic,
anti-anginal, anti-arthritic, anti-asthmatic, anti-atherosclerotic,
antibacterial, anticholinergic, anticoagulant, anticonvulsant,
antidepressant, antidiabetic, antidiarrheal, antidiuretic,
anti-emetic, anti-epileptic, antifibrinolytic, antifungal,
antihemorrhagic, antihistamine, antihyperlipidemia,
antihypertensive, antihypotensive, anti-infective,
anti-inflammatory, antimicrobial, antimigraine, antimitotic,
antimycotic, antinauseant, antineoplastic, antineutropenic,
antiparasitic, antiproliferative, antipsychotic, antirheumatic,
antiseborrheic, antisecretory, antispasmodic, antithrombotic,
anti-ulcerative, antiviral, appetite suppressant, blood glucose
regulator, bone resorption inhibitor, bronchodilator,
cardiovascular agent, cholinergic, depressant, diagnostic aid,
diuretic, dopaminergic agent, estrogen receptor agonist,
fibrinolytic, fluorescent agent, free oxygen radical scavenger,
gastrointestinal motility effector, glucocorticoid, hair growth
stimulant, hemostatic, histamine H2 receptor antagonists, hormone,
hypocholesterolemic, hypoglycemic, hypolipidemic, hypotensive,
imaging agent, immunizing agent, immunomodulator, immunoregulator,
immunostimulant, immunosuppressant, keratolytic, LHRH agonist, mood
regulator, mucolytic, mydriatic, nasal decongestant, neuromuscular
blocking agent, neuroprotective, NMDA antagonist; non-hormonal
sterol derivative, plasminogen activator, platelet activating
factor antagonist, platelet aggregation inhibitor, psychotropic,
radioactive agent, scabicide, sclerosing agent, sedative,
sedative-hypnotic, selective adenosine A1 antagonist, serotonin
antagonist, serotonin inhibitor, serotonin receptor antagonist,
steroid, thyroid hormone, thyroid inhibitor, thyromimetic,
tranquilizer, amyotrophic lateral sclerosis agent, cerebral
ischemia agent, Paget's disease agent, unstable angina agent,
vasoconstrictor, vasodilator, wound healing agent, xanthine oxidase
inhibitor.
49. The microencapsulated product of claim 41, wherein the agent is
insulin.
50. The microencapsulated product of claim 41, wherein the agent is
an immunological agent.
51. The microencapsulated product of claim 50, wherein the
immunological agent is an allergen or an antigen selected from the
group consisting of whole inactivated organisms, peptides,
proteins, glycoproteins, carbohydrates, or combinations
thereof.
52. The microencapsulated product of claim 41, wherein the polymer
is a bioadhesive polymer.
53. The microencapsulated product of claim 52, wherein the
bioadhesive polymer is a bioerodible hydrogel.
54. The microencapsulated product of claim 53, wherein the
bioerodible hydrogel is selected from the group consisting of:
polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides,
polyacrylic acid, alginate, chitosan, poly(methyl methacrylates),
poly(ethyl methacrylates), poly butylmethacrylate),
poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly (methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), and poly(octadecl acrylate). Most
preferred is poly(fumaric-co-sebacic)acid.
55. The microencapsulated product of claim 41, wherein the polymer
is selected from the group consisting of: polyamides,
polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene
oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl
ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone,
polyglycolides, polysiloxanes, polyurethanes and copolymers
thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose
ethers, cellulose esters, nitro celluloses, polymers of acrylic and
methacrylic esters, methyl cellulose, ethyl cellulose,
hydroxypropyl cellulose, hydroxy-propyl methyl cellulose,
hydroxybutyl methyl cellulose, cellulose acetate, cellulose
propionate, cellulose acetate butyrate, cellulose acetate
phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose
sulphate sodium salt, poly (methyl methacrylate),
poly(ethylmethacrylate), poly(butylmethacrylate),
poly(isobutylmethacrylate), poly(hexlmethacrylate),
poly(isodecylmethacrylate), poly(lauryl methacrylate), poly (phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl
acetate, poly vinyl chloride polystyrene and
polyvinylpryrrolidone.
56. The microencapsulated product of claim 41, wherein the polymer
is a bioerodible polymer.
57. The microencapsulated product of claim 41, wherein the polymer
is a nonbioerodible polymer.
58. The microencapsulated product of claim 41, wherein the
microencapsulated product consists of particles having an average
particle size of between 10 nanometers and 10 microns.
59. The microencapsulated product of claim 41, wherein the
microencapsulated product consists of particles having an average
particle size of between 100 nanometers and 5 microns.
60. The microencapsulated product of claim 41, wherein the
microencapsulated product consists of particles having an average
particle size of between 100 nanometers and 1 micron.
61. The microencapsulated product of claim 41, wherein the
solvent:nonsolvent volume ratio is greater than 1:40.
62. The microencapsulated product of claim 41, wherein the
solvent:nonsolvent volume ratio is between 1:40 and
1:1,000,000.
63. The microencapsulated product of claim 41, wherein the
solvent:nonsolvent volume ratio is between 1:50 and 1:200.
64. The microencapsulated product of claim 41, wherein the
concentration of the polymer in the solvent is less than 20% weight
per volume.
65. The microencapsulated product of claim 41, wherein the
concentration of the polymer in the solvent is less than 10% weight
per volume.
66. The microencapsulated product of claim 41, wherein the
concentration of the polymer in the solvent is less than 5% weight
per volume.
67. The microencapsulated product of claim 41, wherein the mixture
has a viscosity less than 6 centipoise.
68. The microencapsulated product of claim 41, wherein the mixture
has a viscosity less than 4 centipoise.
69. The microencapsulated product of claim 41, wherein the mixture
has a viscosity less than 3 centipoise.
70. The microencapsulated product of claim 41, wherein the mixture
has a viscosity less than 2 centipoise.
71. The microencapsulated product of claim 41, wherein the solvent
and nonsolvent are hydrophilic pairs.
72. The microencapsulated product of claim 41, wherein the solvent
is a halogenated aliphatic hydrocarbon or a mixture of a
halogenated aliphatic hydrocarbon and another solvent.
73. The microencapsulated product of claim 41, wherein the solvent
is a halogenated aromatic hydrocarbon or a mixture of a halogenated
aromatic hydrocarbon and another solvent.
74. The microencapsulated product of claim 41, wherein the solvent
is an ether or a mixture of an ether and another solvent.
75. The microencapsulated product of claim 41, wherein the solvent
is a cyclic ether or a mixture of a cyclic ether and another
solvent.
76. The microencapsulated product of claim 41, wherein the
microencapsulated product is separated from the nonsolvent.
77. The microencapsulated product of claim 41, wherein a
combination of solvents are used.
78. The microencapsulated product of claim 41, wherein the solvent
is methylene chloride and the nonsolvent is ethanol.
79. The microencapsulated product of claim 41, wherein the
nonsolvent is a mixture of ethanol and water.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/639,770, filed Aug. 12, 2003, now
abandoned, which is a continuation of U.S. patent application Ser.
No. 09/853,329, filed May 11, 2001, now issued as U.S. Pat. No.
6,616,869, which is a divisional of U.S. patent application Ser.
No. 09/442,723, filed Nov. 18, 1999, now issued as U.S. Pat. No.
6,235,224, which is a divisional of U.S. patent application Ser.
No. 08/686,928, filed Jul. 3, 1996, now issued as U.S. Pat. No.
6,143,211 on Nov. 7, 2000, which claims the benefit under 35 USC
section 119 to U.S. Provisional Patent Application Ser. No.
60/001,365 entitled "Process for Preparing Microspheres Through
Phase Inversion Phenomena" filed Jul. 21, 1995 by Edith Mathiowitz,
Donald E. Chickering III, Yong S. Jong and Jules S. Jacob.
BACKGROUND OF THE INVENTION
[0002] Microparticles, microcapsules and microspheres (hereinafter
"microparticles") have important applications in the
pharmaceutical, agricultural, textile and cosmetics industry as
delivery vehicles. In these fields of application, a drug, protein,
hormone, peptide, fertilizer, pesticide, herbicide, dye, fragrance
or other agent is encapsulated in a polymer matrix and delivered to
a site either instantaneously or in a controlled manner in response
to some external impetus (i.e., pH, heat, water, radiation,
pressure, concentration gradients, etc.). Microparticle size can be
an important factor in determining the release rate of the
encapsulated material.
[0003] Many microencapsulation techniques exist which can produce a
variety of particle types and sizes under various conditions.
Methods typically involve solidifying emulsified liquid polymer
droplets by changing temperature, evaporating solvent, or adding
chemical cross-linking agents. Physical and chemical properties of
the encapsulant and the material to be encapsulated can sometimes
dictate the suitable methods of encapsulation, making only certain
methodologies useful in certain circumstances. Factors such as
hydrophobicity, molecular weight, chemical stability, and thermal
stability affect encapsulation. Significant losses are frequently
associated with multiple processing steps. These parameters can be
particularly important in respect of encapsulating bioactive agents
because losses in the bioactivity of the material due to the
processing steps or low yields can be extremely undesirable.
[0004] Common microencapsulation techniques include interfacial
polycondensation, spray drying, hot melt microencapsulation, and
phase separation techniques (solvent removal and solvent
evaporation). Interfacial polycondensation can be used to
microencapsulate a core material in the following manner. One
monomer and the core material are dissolved in a solvent. A second
monomer is dissolved in a second solvent (typically aqueous) which
is immiscible with the first. An emulsion is thinned by suspending
the first solution through stirring in the second solution. Once
the emulsion is stabilized, an initiator is added to the aqueous
phase causing interfacial polymerization at the interface of each
droplet of emulsion.
[0005] Spray drying is typically a process for preparing 1-10
micron sized microspheres in which the core material to be
encapsulated is dispersed or dissolved in a polymer solution
(typically aqueous), the solution or dispersion is pumped through a
micronizing nozzle driven by a flow of compressed gas, and the
resulting aerosol is suspended in a heated cyclone of air, allowing
the solvent to evaporate from the microdroplets. The solidified
particles pass into a second chamber and are trapped in a
collection flask. This process can result in 50-80% loss through
the exhaust vent when laboratory scale spray dryers are used.
[0006] Hot melt microencapsulation is a method in which a core
material is added to molten polymer. This mixture is suspended as
molten droplets in a nonsolvent for the polymer (often oil-based)
which has been heated to above the melting point of the polymer.
The emulsion is maintained through vigorous stirring while the
nonsolvent bath is quickly cooled below the glass transition of the
polymer, causing the molten droplets to solidify and entrap the
core material. Microspheres produced by this technique typically
range in size from 50 microns to 2 mm in diameter. This process
requires the use of polymers with fairly low melting temperatures
(i.e., <150.degree. C.), glass transition temperatures above
room temperature, and core materials which are thermo-stable.
[0007] In solvent evaporation microencapsulation, the polymer is
typically dissolved in a water immiscible organic solvent and the
material to be encapsulated is added to the polymer solution as a
suspension or solution in organic solvent. An emulsion is formed by
adding this suspension or solution to a beaker of vigorously
stirring water (often containing a surface active agent to
stabilize the emulsion). The organic solvent is evaporated while
continuing to stir. Evaporation results in precipitation of the
polymer, forming solid microcapsules containing core material.
[0008] A solvent evaporation process exists which is specifically
designed to entrap a liquid core material in PLA, PLA/PGA
copolymer, or PLA/PCL copolymer microcapsules. The PLA or copolymer
is dissolved in a miscible mixture of solvent and nonsolvent, at a
nonsolvent concentration which is immediately below the
concentration which would produce phase separation (i.e., cloud
point). The liquid core material is added to the solution while
agitating to form an emulsion and disperse the material as
droplets. Solvent and nonsolvent are vaporized, with the solvent
being vaporized at a faster rate, causing the PLA or copolymer to
phase separate and migrate towards the surface of the core material
droplets. This phase separated solution is then transferred into an
agitated volume of nonsolvent, causing any remaining dissolved PLA
or copolymer to precipitate and extracting any residual solvent
from the formed membrane. The result is a microcapsule composed of
PLA or copolymer shell with a core of liquid material.
[0009] In solvent removal microencapsulation, the polymer is
typically dissolved in an oil miscible organic solvent and the
material to be encapsulated is added to the polymer solution as a
suspension or solution in organic solvent. An emulsion is formed by
adding this suspension or solution to a beaker of vigorously
stirring oil, in which the oil is a nonsolvent for the polymer and
the polymer/solvent solution is immiscible in the oil. The organic
solvent is removed by diffusion into the oil phase while continuing
to stir. Solvent removal results in precipitation of the polymer,
forming solid microcapsules containing core material.
[0010] Phase separation microencapsulation is typically performed
by dispersing the material to be encapsulated in a polymer solution
by stirring. While continuing to uniformly suspend the material
through stirring, a nonsolvent for the polymer is slowly added to
the solution to decrease the polymer's solubility. Depending on the
solubility of the polymer in the solvent and nonsolvent, the
polymer either precipitates or phase separates into a polymer rich
and a polymer poor phase. Under proper conditions, the polymer in
the polymer rich phase will migrate to the interface with the
continuous phase, encapsulating the core material in a droplet with
an outer polymer shell.
[0011] A recent patent to Tice (U.S. Pat. No. 5,407,609) involves a
phase separation microencapsulation process which attempts to
proceed more rapidly than the procedure described in the preceding
paragraph. According to Tice, a polymer is dissolved in the
solvent. An agent to be encapsulated then is dissolved or dispersed
in that solvent. The mixture then is combined with an excess of
nonsolvent and is emulsified and stabilized, whereby the polymer
solvent no longer is the continuous phase. Aggressive
emulsification conditions are applied in order to produce
microdroplets of the polymer solvent. After emulsification, the
stable emulsion is introduced into a large volume of nonsolvent to
extract the polymer solvent and form microparticles. The size of
the microparticles is determined by the size of the microdroplets
of polymer solvent. This procedure has the drawback that small
particles can be obtained only with aggressive emulsification
procedures. It also suffers the drawback that multiple processing
steps are required to form the microparticles.
[0012] Phase inversion is a term used to describe the physical
phenomena by which a polymer dissolved in a continuous phase
solvent system inverts into a solid macromolecular network in which
the polymer is the continuous phase. This event can be induced
through several means: removal of solvent (e.g., evaporation; also
known as dry process), addition of another species, addition of a
non-solvent or addition to a non-solvent (also known as wet
process). In the wet process, the polymer solution can be poured or
extruded into a non-solvent bath. The process proceeds in the
following manner. The polymer solution undergoes a transition from
a single phase homogeneous solution to an unstable two phase
mixture:polymer rich and polymer poor fractions. Micellar droplets
of nonsolvent in the polymer rich phase serve as nucleation sites
and become coated with polymer. At a critical concentration of
polymer, the droplets precipitate from solution and solidify. Given
favorable surface energy, viscosity and polymer concentrations, the
micelles coalesce and precipitate to form, a continuous polymer
network.
[0013] Phase inversion phenomenon have been applied to produce
macro and microporous polymer membranes and hollow fibers used in
gas separation, ultrafiltration, ion exchange, and reverse osmosis.
Structural integrity and morphological properties of these
membranes are functions of polymer molecular weight, polymer
concentration, solution viscosity, temperature and solubility
parameters (of polymer, solvent and non-solvent). For wet process
phase inversion, polymer viscosities must be greater than
approximately 10,000 centipoise to maintain membrane integrity;
lower viscosity solutions may produce fragmented polymer particles
as opposed to a continuous system. Furthermore, it is known that
the quicker a solution is caused to precipitate, the finer is the
dispersion of the precipitating phase.
[0014] A phase inversion process has been employed to produce
polymer microcapsules. The microcapsules are prepared by dissolving
a polymer in an organic solvent, forming droplets of the solution
by forcing it through a spinneret or syringe needle, (the size of
which droplets determines the size of the final microcapsule), and
contacting the droplets with a nonsolvent for the polymer which is
highly miscible with the polymer solvent, thereby causing rapid
precipitation of the outer layer of the droplet. The microcapsules
must be left in contact with the nonsolvent until substantially all
of the solvent has been replaced with nonsolvent. This process
requires formation of a droplet with dimensions established prior
to contacting the nonsolvent.
[0015] Each of the methods described before require the formation
of an emulsion or droplets prior to precipitation of the final
microparticle. The present invention provides a novel method of
producing microparticles without the requirement of forming an
emulsion prior to precipitation. Under proper conditions, polymer
solutions can be forced to phase invert into fragmented spherical
polymer particles when added to appropriate nonsolvents. We have
utilized this spontaneous microparticle formation phase inversion
process as a rapid, one step microencapsulation technique. The
process is simple to perform, is suitable with a number of
polymeric systems (including many common degradable and
non-degradable polymers typically employed as controlled release
systems), produces extremely small microparticles (10 nm to 10
.mu.m) and results in very high yields.
SUMMARY OF THE INVENTION
[0016] It has been discovered that "phase inversion" of polymer
solutions under certain conditions can bring about the spontaneous
formation of discreet microparticles, including nanospheres. By
using relatively low viscosities and/or relatively low polymer
concentrations, by using solvent and nonsolvent pairs that are
miscible and by using greater than ten fold excess of nonsolvent, a
continuous phase of nonsolvent with dissolved polymer can be
rapidly introduced into the nonsolvent, thereby causing a phase
inversion and the spontaneous formation of discreet
microparticles.
[0017] The process eliminates a step characteristic of the prior
art, that is, creating microdroplets, such as by forming an
emulsion, of the solvent. It likewise eliminates drawbacks
associated with the microdroplet formation step of the prior art.
The microdroplet formation step consumes time, can be disruptive of
the agent to be encapsulated, and can be the limiting factor in
determining the ultimate size of the to formed microparticle. The
process of the invention is simpler and quicker than those prior
art methods because this step is eliminated. The invention has the
advantage that it can be performed very rapidly, the entire process
taking less than five minutes in some cases. The actual phase
inversion and encapsulation can take place in less than 30 seconds.
It also has the advantage of avoiding the agitation and/or shear
forces to which the material to be encapsulated otherwise would be
exposed. Smaller particles are not created by exposing the solvent
to higher and higher agitation and/or shear forces. The
microparticle size is determined instead by nonstress parameters
such as polymer concentration, viscosity, solvent/nonsolvent
miscibility and solvent/nonsolvent volumetric ratios. The invention
also provides micron and even submicron sized polymer particles. It
provides the additional advantage of producing those particles with
minimal losses of the material to be encapsulated. Again,
minimizing losses has important implications on productions
costs.
[0018] It readily will be understood that the process of the
present invention is essentially a single step process, which is
scalable. Automation therefore will be straightforward.
[0019] An additional advantage of the invention is the ability to
produce microparticles characterized by a homogenous size
distribution. Such microparticles will have well defined,
predictable properties.
[0020] According to one aspect of the invention, a method for
microencapsulating an agent to form a microencapsulated product is
provided. A polymer is dissolved in an effective amount of a
solvent. The agent is also dissolved or dispersed in the effective
amount of the solvent. The polymer, the agent and the solvent
together form a mixture having a continuous phase, wherein the
solvent is the continuous phase. The mixture is introduced into an
effective amount of a nonsolvent to cause the spontaneous formation
of the microencapsulated product, wherein the solvent and the
nonsolvent are miscible and 0<|.delta. solvent-.delta.
nonsolvent|<6.
[0021] The microencapsulated product that results can take on a
variety of characteristics, depending upon the agents, polymers,
solvents and nonsolvents employed and the various conditions of the
phase inversion. These parameters may be adjusted so that the
microencapsulated product consists of microparticles having an
average particle size of between 10 nanometers and 10 microns. The
average particle size, of course, may be adjusted within this
range, for example to between 50 nanometers and 5 microns or
between 100 nanometers and 1 micron.
[0022] The particle size is influenced by the solvent:nonsolvent
volume ratio, which preferably is between 1:50 and 1:200. A working
range for the solvent:nonsolvent volume ratio is between 1:40 and
1:1,000,000.
[0023] The polymer concentration in the solvent also can affect the
microparticle size. It is preferred that the polymer concentration
be between 0.1% weight/volume to 5% weight/volume, although higher
polymer concentrations such as 10%, 20% or even higher are possible
depending, inter glia, on the viscosity of the polymer solution,
the molecular weight of the polymer and the miscibility of the
solvent and nonsolvent.
[0024] The viscosity of the polymer/solvent solution also can
affect particle size. It preferably is less than 2 centipoise,
although higher viscosities such as 3, 4, 6 or even higher
centipoise are possible depending upon adjustment of other
parameters.
[0025] The molecular weight of the polymer also can affect particle
size. The preferred range is 2 kDa-50 kDa, although a working range
is 1 kDa-150 kDa. Other polymer sizes are possible depending upon
adjustment of the other parameters. It further is possible to
influence particle size through the selection of characteristics of
the solvent and nonsolvent. For example, hydrophilic
solvent/nonsolvent pairs affect particle size relative to
hydrophobic solvent/nonsolvent pairs.
[0026] The foregoing parameters, alone or in any combination, are
considered important aspects of the invention.
[0027] According to another aspect of the invention, a method for
micro encapsulating an agent to form a microencapsulated product is
provided. A polymer is dissolved in a solvent at a concentration of
between 0.25 and 10% weight per volume. An agent also is dissolved
or is dispersed in the solvent. The polymer, agent and solvent form
a mixture, wherein the viscosity of the mixture is less than 3.5
centipoise. The mixture is introduced into a nonsolvent, wherein
the volume ratio of the solvent:nonsolvent is at least 1:40, to
cause the spontaneous formation of the microencapsulated product,
wherein the solvent and the nonsolvent are miscible and wherein
0<|.delta. solvent-.delta. nonsolvent|<6. Preferably, the
polymer concentration is between 0.5 and 5% weight/volume, the
viscosity is less than 2 centipoise, and the solvent:nonsolvent
ratio is between 1:50 and 1:200.
[0028] According to another aspect of the invention, microparticles
are provided. The microparticles are produced by the processes
described above. It is believed that the processes of the invention
result in products that have different physical characteristics
than microparticles formed according to prior art methods.
[0029] The foregoing aspects of the invention as well as various
objects, features and advantages are discussed in greater detail
below.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The invention involves the discovery that "phase inversion"
of polymer solutions under certain conditions can bring about the
spontaneous formation of discreet microparticles. The process,
dubbed "phase inversion nanoencapsulation" or "PIN", differs from
existing methods of encapsulation in that it is essentially a
1-step process, is nearly instantaneous, and does not require
emulsification of the solvent. Under proper conditions, low
viscosity polymer solutions can be forced to phase invert into
fragmented spherical polymer particles when added to appropriate
nonsolvents.
[0031] Phase inversion phenomenon has been applied to produce macro
and microporous polymer membranes and hollow fibers. The basis for
the formation of such membranes or fibers, as well as the process
of the invention, depends upon the mechanism of microphase
separation. A prevalent theory of microphase separation is based
upon the belief that "primary" particles form of about 50 nm
diameter, as the initial precipitation event resulting from solvent
removal. As the process continues, primary particles are believed
to collide and coalesce forming "secondary" particles with
dimensions of approximately 200 nm, which eventually join with
other particles to form the polymer matrix. An alternative theory,
"nucleation and growth", is based upon the notion that a polymer
precipitates around a core micellar structure (in contrast to
coalescence of primary particles).
[0032] The fact that the present invention results in a very
uniform size distribution of small particles forming at lower
polymer concentrations without coalescing supports the nucleation
and growth theory, while not excluding coalescence at higher
polymer concentrations (e.g., greater than 10% weight per volume)
where larger particles and even aggregates can be formed. (Solvent
would be extracted more slowly from larger particles, so that
random collisions of the partially-solvated spheres would result in
coalescence and, ultimately, formation of fibrous networks.) By
adjusting polymer concentration, polymer molecular weight,
viscosity, miscibility and solvent:nonsolvent volume ratios, the
interfibrillar interconnections characteristic of membranes using
phase inversion are avoided, with the result being that
microparticles are spontaneously formed. As will be seen from the
examples below, as well as the following discussion, the foregoing
parameters are interrelated and the adjustment of one will
influence the absolute value permitted for another.
[0033] In the preferred processing method, a mixture is formed of
the agent to be encapsulated, a polymer and a solvent for the
polymer. The agent to be encapsulated may be in liquid or solid
form. It may be dissolved in the solvent or dispersed in the
solvent. The agent thus may be contained in microdroplets dispersed
in the solvent or may be dispersed as solid microparticles in the
solvent. The phase inversion process thus can be used to
encapsulate a wide variety of agents by including them in either
micronized solid form or else emulsified liquid form in the polymer
solution.
[0034] The loading range for the agent within the microparticles is
between 0.01-80% (agent weight/polymer weight). When working with
nanospheres, an optimal range is 0.1-5% (weight/weight).
[0035] In general, the agent includes, but is not limited to,
adhesives, gases, pesticides, herbicides, fragrances, antifoulants,
dies, salts, oils, inks, cosmetics, catalysts, detergents, curing
agents, flavors, foods, fuels, metals, paints, photographic agents,
biocides, pigments, plasticizers, propellants and the like. The
agent also may be a bioactive agent. The bioactive agent can be,
but is not limited to: adrenergic agent; adrenocortical steroid;
adrenocortical suppressant; aldosterone antagonist; amino acid;
anabolic; analeptic; analgesic; anesthetic; anorectic; anti-acne
agent; anti-adrenergic; anti-allergic; anti-amebic; anti-anemic;
anti-anginal; anti-arthritic; anti-asthmatic; anti-atherosclerotic;
antibacterial; anticholinergic; anticoagulant; anticonvulsant;
antidepressant; antidiabetic; antidiarrheal; antidiuretic;
anti-emetic; anti-epileptic; antifibrinolytic; antifungal;
antihemorrhagic; antihistamine; antihyperlipidemia;
antihypertensive; antihypotensive; anti-infective;
anti-inflammatory; antimicrobial; antimigraine; antimitotic;
antimycotic, antinauseant, antineoplastic, antineutropenic,
antiparasitic; antiproliferative; antipsychotic; antirheumatic;
antiseborrheic; antisecretory; antispasmodic; antithrombotic;
anti-ulcerative; antiviral; appetite suppressant; blood glucose
regulator; bone resorption inhibitor; bronchodilator;
cardiovascular agent; cholinergic; depressant; diagnostic aid;
diuretic; dopaminergic agent; estrogen receptor agonist;
fibrinolytic; fluorescent agent; free oxygen radical scavenger;
gastrointestinal motility effector; glucocorticoid; hair growth
stimulant; hemostatic; histamine H2 receptor antagonists; hormone;
hypocholesterolemic; hypoglycemic; hypolipidemic; hypotensive;
imaging agent; immunizing agent; immunomodulator; immunoregulator;
immunostimulant; immunosuppressant; keratolytic; LHRH agonist; mood
regulator; mucolytic; mydriatic; nasal decongestant; neuromuscular
blocking agent; neuroprotective; NMDA antagonist; non-hormonal
sterol derivative; plasminogen activator; platelet activating
factor antagonist; platelet aggregation inhibitor; psychotropic;
radioactive agent; scabicide; sclerosing agent; sedative;
sedative-hypnotic; selective adenosine A1 antagonist; serotonin
antagonist; serotonin inhibitor; serotonin receptor antagonist;
steroid; thyroid hormone; thyroid inhibitor; thyromimetic;
tranquilizer; amyotrophic lateral sclerosis agent; cerebral
ischemia agent; Paget's disease agent; unstable angina agent;
vasoconstrictor; vasodilator; wound healing agent; xanthine oxidase
inhibitor.
[0036] Bioactive agents include immunological agents such as
allergens (e.g., cat dander, birch pollen, house dust, mite, grass
pollen, etc.) and antigens from pathogens such as viruses,
bacteria, fungi and parasites. These antigens may be in the form of
whole inactivated organisms, peptides, proteins, glycoproteins,
carbohydrates or combinations thereof. Specific examples of
pharmacological or immunological agents that fall within the
above-mentioned categories and that have been approved for human
use may be found in the published literature.
[0037] The agent is added to the polymer solvent, preferably after
the polymer is dissolved in the solvent. The solvent is any
suitable solvent for dissolving the polymer. Typically the solvent
will be a common organic solvent such as a halogenated aliphatic
hydrocarbon such as methylene chloride, chloroform and the like; an
alcohol; an aromatic hydrocarbon such as toluene; a halogenated
aromatic hydrocarbon; an ether such as methyl t-butyl; a cyclic
ether such as tetrahydrofuran; ethyl acetate; diethylcarbonate;
acetone; or cyclohexane. The solvents may be used alone or in
combination. The solvent chosen must be capable of dissolving the
polymer, and it is desirable that the solvent be inert with respect
to the agent being encapsulated and with respect to the
polymer.
[0038] The polymer may be any suitable microencapsulation material
including, but not limited to, nonbioerodable and bioerodable
polymers. Such polymers have been described in great detail in the
prior art. They include, but are not limited to: polyamides,
polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene
oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl
ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone,
polyglycolides, polysiloxanes, polyurethanes and copolymers
thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose
ethers, cellulose esters, nitro celluloses, polymers of acrylic and
methacrylic esters, methyl cellulose, ethyl cellulose,
hydroxypropyl cellulose, hydroxy-propyl methyl cellulose,
hydroxybutyl methyl cellulose, cellulose acetate, cellulose
propionate, cellulose acetate butyrate, cellulose acetate
phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose
sulphate sodium salt, poly (methyl methacrylate),
poly(ethylmethacrylate), poly(butylmethacrylate),
poly(isobutylmethacrylate), poly(hexlmethacrylate),
poly(isodecylmethacrylate), poly(lauryl methacrylate), poly (phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl
acetate, poly vinyl chloride polystyrene and
polyvinylpryrrolidone.
[0039] Examples of preferred non-biodegradable polymers include
ethylene vinyl acetate, poly(meth) acrylic acid, polyamides,
copolymers and mixtures thereof.
[0040] Examples of preferred biodegradable polymers include
synthetic polymers such as polymers of lactic acid and glycolic
acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic
acid), poly(valeric acid), poly(caprolactone),
poly(hydroxybutyrate), poly(lactide-co-glycolide) and
poly(lactide-co-caprolactone), and natural polymers such as
algninate and other polysaccharides including dextran and
cellulose, collagen, chemical derivatives thereof (substitutions,
additions of chemical groups, for example, alkyl, alkylene,
hydroxylations, oxidations, and other modifications routinely made
by those skilled in the art), albumin and other hydrophilic
proteins, zein and other prolamines and hydrophobic proteins,
copolymers and mixtures thereof. In general, these materials
degrade either by enzymatic hydrolysis or exposure to water in
vivo, by surface or bulk erosion. The foregoing materials may be
used alone, as physical mixtures (blends), or as co-polymers. The
most preferred polymers are polyesters, polyanhydrides,
polystyrenes and blends thereof.
[0041] Particularly preferred are bioadhesive polymers. A
bioadhesive polymer is one that binds to mucosal epithelium under
normal physiological conditions. Bioadhesion in the
gastrointestinal tract proceeds in two stages: (1) viscoelastic
deformation at the point of contact of the synthetic material into
the mucus substrate, and (2) formation of bonds between the
adhesive synthetic material and the mucus or the epithelial cells.
In general, adhesion of polymers to tissues may be achieved by (i)
physical or mechanical bonds, (ii) primary or covalent chemical
bonds, and/or (iii) secondary chemical bonds (i.e., ionic).
Physical or mechanical bonds can result from deposition and
inclusion of the adhesive material in the crevices of the mucus or
the folds of the mucosa. Secondary chemical bonds, contributing to
bioadhesive properties, consist of dispersive interactions (i.e.,
Van der Waals interactions) and stronger specific interactions,
which include hydrogen bonds. The hydrophilic functional groups
primarily responsible for forming hydrogen bonds are the hydroxyl
and the carboxylic groups. Numerous bioadhesive polymers are
discussed in that application. Representative bioadhesive polymers
of particular interest include bioerodible hydrogels described by
H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules,
1993, 26:581-587, the teachings of which are incorporated herein,
polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides,
polyacrylic acid, alginate, chitosan, poly(methyl methacrylates),
poly(ethyl methacrylates), poly butylmethacrylate),
poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly (methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), and poly(octadecl acrylate). Most
preferred is poly(fumaric-co-sebacic)acid.
[0042] Polymers with enhanced bioadhesive properties can be
provided wherein anhydride monomers or oligomers are incorporated
into the polymer. The oligomer excipients can be blended or
incorporated into a wide range of hydrophilic and hydrophobic
polymers including proteins, polysaccharides and synthetic
biocompatible polymers. Anhydride oligomers may be combined with
metal oxide particles to improve bioadhesion even more than with
the organic additives alone. Organic dyes because of their
electronic charge and hydrophobicity/hydrophilicity can either
increase or decrease the bioadhesive properties of polymers when
incorporated into the polymers. The incorporation of oligomer
compounds into a wide range of different polymers which are not
normally bioadhesive dramatically increases their adherence to
tissue surfaces such as mucosal membranes.
[0043] As used herein, the term "anhydride oligomer" refers to a
diacid or polydiacids linked by anhydride bonds, and having carboxy
end groups linked to a monoacid such as acetic acid by anhydride
bonds. The anhydride oligomers have a molecular weight less than
about 5000, typically between about 100 and 5000 daltons, or are
defined as including between one to about 20 diacid units linked by
anhydride bonds. In one embodiment, the diacids are those normally
found in the Krebs glycolysis cycle. The anhydride oligomer
compounds have high chemical reactivity.
[0044] The oligomers can be formed in a reflux reaction of the
diacid with excess acetic anhydride. The excess acetic anhydride is
evaporated under vacuum, and the resulting oligomer, which is a
mixture of species which include between about one to twenty diacid
units linked by anhydride bonds, is purified by recrystallizing,
for example from toluene or other organic solvents. The oligomer is
collected by filtration, and washed, for example, in ethers. The
reaction produces anhydride oligomers of mono and poly acids with
terminal carboxylic acid groups linked to each other by anhydride
linkages.
[0045] The anhydride oligomer is hydrolytically labile. As analyzed
by gel permeation chromatography, the molecular weight may be, for
example, on the order of 200-400 for fumaric acid oligomer (FAPP)
and 2000-4000 for sebacic acid oligomer (SAPP). The anhydride bonds
can be detected by Fourier transform infrared spectroscopy by the
characteristic double peak at 1750 cm.sup.-1 and 1820 cm.sup.-1,
with a corresponding disappearance of the carboxylic acid peak
normally at 1700 cm.sup.-1.
[0046] In one embodiment, the oligomers may be made from diacids
described for example in U.S. Pat. No. 4,757,128 to Domb et al.,
U.S. Pat. No. 4,997,904 to Domb, and U.S. Pat. No. 5,175,235 to
Domb et al., the disclosures of which are incorporated herein by
reference. For example, monomers such as sebacic acid,
bis(p-carboxy-phenoxy)propane, isophathalic acid, fumaric acid,
maleic acid, adipic acid or dodecanedioic acid may be used.
[0047] Organic dyes, because of their electronic charge and
hydrophilicity/hydrophobicity, may alter the bioadhesive properties
of a variety of polymers when incorporated into the polymer matrix
or bound to the surface of the polymer. A partial listing of dyes
that affect bioadhesive properties include, but are not limited to:
acid fuchsin, alcian blue, alizarin red s, auramine o, azure a and
b, Bismarck brown y, brilliant cresyl blue aid, brilliant green,
carmine, cibacron blue 3GA, conga red, cresyl violet acetate,
crystal violet, eosin b, eosin y, erythrosin b, fast green fcf,
giemsa, hematoylin, indigo carmine, Janus green b, Jenner's stain,
malachite green oxalate, methyl blue, methylene blue, methyl green,
methyl violet 2b, neutral red, Nile blue a, orange II, orange G,
orcein, paraosaniline chloride, phloxine b, pyronin b and y,
reactive blue 4 and 72, reactive brown 10, reactive green 5 and 19,
reactive red 120, reactive yellow 2,3, 13 and 86, rose bengal,
safranin o, Sudan III and IV, Sudan black B and toluidine blue.
[0048] The working molecular weight range for the polymer is on the
order of 1 kDa-150,000 kDa, although the optimal range is
21(13a-501(1 h. The working range of polymer concentration is
0.01-50% (weight/volume), depending primarily upon the molecular
weight of the polymer and the resulting viscosity of the polymer
solution. In general, the low molecular weight polymers permit
usage of a higher concentration of polymer. The preferred
concentration range according to the invention will be on the order
of 0.1%-10% (weight/volume), while the optimal polymer
concentration typically will be below 5%. It has been found that
polymer concentrations on the order of 1-5% are particularly useful
according to the methods of the invention.
[0049] The viscosity of the polymer solution preferably is less
than 3.5 centipoise and more preferably less than 2 centipoise,
although higher viscosities such as 4 or even 6 centipoise are
possible depending upon adjustment of other parameters such as
molecular weight. It will be appreciated by those of ordinary skill
in the art that polymer concentration, polymer molecular weight and
viscosity are interrelated, and that varying one will likely affect
the others.
[0050] The nonsolvent, or extraction medium, is selected based upon
its miscibility in the solvent. Thus, the solvent and nonsolvent
are thought of as "pairs". We have determined that the solubility
parameter (.delta.(cal/cm.sup.3).sup.1/2) is a useful indicator of
the suitability of the solvent/nonsolvent pairs. The solubility
parameter is an effective protector of the miscibility of two
solvents and, generally, higher values indicate a more hydrophilic
liquid while lower values represent a more hydrophobic liquid
(e.g., .delta..sub.i water=23.4 (cal/cm.sup.3).sup.1/2 whereas
.delta..sub.i hexane=7.3 (cal/cm.sup.3).sup.1/2). We have
determined that solvent/nonsolvent pairs are useful where
0<|.delta. solvent-.delta. nonsolvent|<6
(cal/cm.sup.3).sup.1/2. Although not wishing to be bound by any
theory, an interpretation of this finding is that miscibility of
the solvent and the nonsolvent is important for formation of
precipitation nuclei which ultimately serve as foci for particle
growth. If the polymer solution is totally immiscibile in the
nonsolvent, then solvent extraction does not occur and
nanoparticles are not formed. An intermediate case would involve a
solvent/nonsolvent pair with slight miscibility, in which the rate
of solvent removal would not be quick enough to form discreet
microparticles, resulting in aggregation of coalescence of the
particles.
[0051] It, surprisingly, was discovered that nanoparticles
generated using "hydrophilic" solvent/nonsolvent pairs (e.g., a
polymer dissolved in methylene chloride with ethanol as the
nonsolvent) yielded approximately 100% smaller particles than when
"hydrophobic" solvent/nonsolvent pairs were used (e.g., the same
polymer dissolved in methylene chloride with hexane as the
nonsolvent).
[0052] Similarly, it was discovered, surprisingly, that the
solvent:nonsolvent volume ratio was important in determining
whether microparticles would be formed without particle aggregation
or coalescence. A suitable working range for solvent:nonsolvent
volume ratio is believed to be 1:40-1:1,000,000. An optimal working
range for the volume ratios for solvent:nonsolvent is believed to
be 1:50-1:200 (volume per volume). Ratios of less than
approximately 1:40 resulted in particle coalescence, presumably due
to incomplete solvent extraction or else a slower rate of solvent
diffusion into the bulk nonsolvent phase.
[0053] It will be understood by those of ordinary skill in the art
that the ranges given above are not absolute, but instead are
interrelated. For example, although it is believed that the
solvent:nonsolvent minimum volume ratio is on the order of 1:40, it
is possible that microparticles still might be formed at lower
ratios such as 1:30 if the polymer concentration is extremely low,
the viscosity of the polymer solution is extremely low and the
miscibility of the solvent and nonsolvent is high. Thus, as used in
connection with the claims, the polymer is dissolved in an
effective amount of solvent, and the mixture of agent, polymer and
polymer solvent is introduced into an effective amount of a
nonsolvent, so as to produce polymer concentrations, viscosities
and solvent:nonsolvent volume ratios that cause the spontaneous and
virtually instantaneous formation of microparticles.
[0054] As will be seen from the examples below, a variety of
polymers have been tested in the methods of the invention,
including polyesters such as poly(lactic acid),
poly(lactide-co-glycolide) in molar ratios of 50:50 and 75:25;
polycaprolactone; polyanhydrides such as poly(fumaric-co-sabacic)
acid or P(FA:SA) in molar ratios of 20:80 and 50:50;
poly(carboxyphenoxypropane-co-sebacic) acid or P(CPP:SA) in molar
ratio of 20:80; and polystyrenes or PS.
[0055] Nanospheres and microspheres in the range of 10 nm to 10
.mu.m have been produced according to the methods of the invention.
Using initial polymer concentrations in the range of 1-2%
(weight/volume) and solution viscosities of 1-2 centipoise, with a
"good" solvent such as methylene chloride and a strong non-solvent
such as petroleum ether or hexane, in an optimal 1:100 volume
ratio, generates particles with sizes ranging from 100-500 nm Under
similar conditions, initial polymer concentrations of 2-5%
(weight/volume) and solution viscosities of 2-3 centipoise
typically produce particles with sizes of 500-3,000 nm. Using very
low molecular weight polymers (less than 5 kDa), the viscosity of
the initial solution may be low enough to enable the use of higher
than 10% (weight/volume) initial polymer concentrations which
generally result in microspheres with sizes ranging from 1-10
.mu.m. In general, it is likely that concentrations of 15%
(weight/volume) and solution viscosities greater than about 3.5
centipoise discreet microspheres will not form but, instead, will
irreversibly coalesce into intricate, interconnecting fibular
networks with micron thickness dimensions.
[0056] It is noted that only a limited number of microencapsulation
techniques can produce particles smaller than 10 microns, and those
techniques are associated with significant losses of polymer, the
material to be encapsulated, or both. This is particularly
problematic where the active agent is an expensive entity such as
certain medical agents. The present invention provides a method to
produce nano to micro-sized particles with minimal losses. The
described methods can result in product yields greater than 80% and
encapsulation efficiencies as high as 100%.
[0057] The methods of the invention also can produce microparticles
characterized by a homogeneous size distribution. Typical
microencapsulation techniques produce heterogeneous size
distributions ranging from 10 .mu.m to mm sizes. Prior art
methodologies attempt to control particle size by parameters such
as stirring rate, temperature, polymer/suspension bath ratio, etc.
Such parameters, however, have not resulted in a significant
narrowing of size distribution. The present invention can produce,
for example, nanometer sized particles which are relatively
monodisperse in size. By producing a microparticle that has a well
defined and less variable size, the properties of the microparticle
such as when used for release of a bioactive agent can be better
controlled. Thus, the invention permits improvements in the
preparation of sustained release formulations for administration to
subjects.
[0058] The invention also provides further methods for controlling
the size of the four microspheres. This is particularly useful
where the material to be encapsulated must first be dispersed in
the solvent and where it would be undesirable to sonicate the
material to be encapsulated. The mixture of the material to be
encapsulated and the solvent (with dissolved polymer) can be frozen
in liquid nitrogen and then lyophilized to disperse the material to
be encapsulated in the polymer. The resulting mixture then can be
redissolved in the solvent, and then dispersed by adding the
mixture to the nonsolvent. This methodology was employed in
connection with dispersing DNA, shown in the examples below.
[0059] As mentioned above, the methods of the invention can be, in
many cases, carried out in less than five minutes in the entirety.
It is typical that preparation time may take anywhere from one
minute to several hours, depending on the solubility of the polymer
and the chosen solvent, whether the agent will be dissolved or
dispersed in the solvent and so on. Nonetheless, the actual
encapsulation time typically is less than thirty seconds.
[0060] After formation of the microcapsules, they are collected by
centrifugation, filtration, and the like. Filtering and drying may
take several minutes to an hour depending on the quantity of
material encapsulated and the methods used for drying the
nonsolvent. The process in its entirety may be discontinuous or a
continuous process.
[0061] Because the process does not require forming the solvent
into an emulsion, it generally speaking may be regarded as a more
gentle process than those that require emulsification. As a result,
materials such as whole plasmids including genes under the control
of promoters can be encapsulated without destruction of the DNA as
a result of the emulsification process. Thus the invention
particularly contemplates encapsulating materials such as plasmids,
vectors, external guide sequences for RNAase P, ribozymes and other
sensitive oligonucleotides, the structure and function of which
could be adversely affected by aggressive emulsification conditions
and other parameters typical of certain of the prior art
processes.
[0062] Included below are several examples of the present invention
and the novel products produced thereby. Most of these examples
product microparticles ranging in size from 100 nanometers to 10
microns. Although illustrative of the advance in the art achieved
by the present invention, it is expected that those skilled in
polymer science and microencapsulation processes will, on the basis
of these examples, be able to select appropriate polymers,
solvents, nonsolvents, solution modifiers, excipients, diluents,
encapsulants and so on to spontaneously form microparticles
exhibiting desirable properties, including properties desirable for
medical applications such as sustained release of bioactive
compounds or oral delivery of drug compounds.
[0063] The following non-limiting examples describe the preparation
of microspheres by the phase inversion method in which a polymer
dissolved in a continuous phase solvent system coalesces into a
solid macromolecular network in which the polymer is the continuous
phase (Kestling, et. al., Materials Science of Synthetic Membranes,
p. 132-164 (1985)). This event can be induced through several
means: removal of solvent (e.g. by evaporation), addition of
another species, addition of a non-solvent or addition to a
non-solvent (wet process). In the latter, the polymer solution can
be poured or extruded into a non-solvent bath. The method and
materials of the present invention will be further understood by
reference to these non-limiting Examples.
EXAMPLES
Example 1
Preparation of Microspheres by Phase Inversion
Nanoencapsulation
Methods:
[0064] A variety of polymers have been used to fabricate "PIN"
nanospheres including: polyesters, such as poly (lactic acid) or
PLA, poly(lactic-co-glycolide) or PLGA in molar ratios of 50:50 and
75:25, polycaprolactone or PLC; polyanhydrides, such as
poly(fumaric-co-sebacic) acid or P(FA:SA) in molar ratios of 20:80
and 50:50 poly(carboxyphenoxypropane-co-sebacic) acid of P(CPP:SA)
in molar ratio of 20:80; and polystyrenes or PS. Polymers with
molecular weights ranging from 1-112,000 kDa have been successfully
used to fabricate nanospheres (see Table 1 below). Unless otherwise
indicated all reagents used were obtained from Sigma Chemical
Company of St. Louis, Mo. or Aldrich Chemicals of Milwaukee,
Wis.
Results:
[0065] 1. Preparation of a Drug Free Nanosphere:
[0066] 5 ml of 1% polyvinylphenol (w/v) (PVP, Polysciences, Inc.)
in methylene chloride was rapidly added to 200 ml of petroleum
ether without stirring. The mixture was immediately filtered and
the resulting nanospheres were air dried on the filter paper.
[0067] The dried nanospheres were examined by scanning electron
microscopy (SEM) (data not shown). The micrographs revealed a
monodisperse preparation of distinct nanospheres ranging in size
from 10 to 100 nm. The low size range of the nanospheres is
characteristic of nanospheres formed using low concentrations of
polymer (1-5% w/v).
[0068] 2. Preparation of Microspheres (and Nanospheres) Including a
Microencapsulated Fluorescent Low Molecular Weight, Hydrophilic
Dye:
[0069] 5 ml of 5% polylactic acid-2 KDa (PLA) (Boehringer
Ingleheim, Inc.) in methylene chloride (w/v) containing 0.1% (w/v)
rhodamine 6 G (2.0% w/w) was added quickly to 200 ml of petroleum
ether without stirring. The mixture was immediately filtered and
the resulting microspheres were air dried on the filter paper.
[0070] A large batch of the same microspheres was formed by rapidly
adding 100 ml of 5% PLA (w/v) in methylene chloride containing 0.1%
(w/v) rhodamine 6 G to 4 liters of petroleum ether without
stirring. This mixture was immediately filtered and the resulting
microspheres were air dried on the filter paper.
[0071] Both sets of microspheres were examined by SEM and were
found to consist of a monodisperse preparation of distinct
microspheres. Both preparations of microspheres ranged in size from
0.5 to 5.mu.. The fluorescent dye was entrapped within the
microspheres. Analysis of the polymer content of the microspheres,
revealed that 4.9 .mu.m of the original 5.0 .mu.m of polymer was
recovered, providing an overall yield recovery of 98%.
[0072] 3. Preparation of Microspheres (and Nanospheres) with
Microencapsulated Sodium Chloride Crystals:
[0073] 0.3 g of spray-dried NaCl, having an average particle size
of 0.1-10.mu., cubic morphology, was dispersed by probe sonication
and stirred into 10 ml of 5% PLA (w/v) in methylene chloride. The
salt loading was 37.5% w/w. This mixture was rapidly added to 400
ml of petroleum ether and immediately filtered. The resulting
microspheres were air dried on the filter paper. In some
experiments the resulting microspheres were incubated for 1.5 hours
in 0.9% NaCl (w/v), washed with distilled water and air dried.
[0074] The untreated sodium chloride microspheres consisted of a
monodispersed preparation of distinct microspheres ranging in size
from 0.5 to 5.mu., as determined by SEM. The salt crystals were
entirely entrapped by the microspheres. No free cubic crystals of
salt were observed in the preparation. SEM of the saline treated
microspheres revealed that in some instances these microspheres had
a sponge-like morphology, which may be useful for an ultrasound
imaging agent.
[0075] 4. Preparation of Microspheres Having a Diameter Greater
than 10.mu., Using the Phase Inversion Method:
[0076] 5 ml of 10% PVP 9-1 KDa (Polysciences Inc) (w/v) in
methylene chloride was rapidly added to 200 ml of petroleum ether
without stirring. The mixture was immediately filtered and the
resulting microspheres were air dried on filter paper.
[0077] Examination of the dried microspheres by SEM revealed that
the microspheres consisted of discrete spherical particles in the
size range of 2 to 20.mu.. The results suggest that microspheres
prepared from low molecular weight polymers (less than 50 KDa)
having concentrations between 5 and 10% (w/v) were larger in size
(up to 20.mu.). Therefore, the resulting microsphere size can be
controlled by manipulating the polymer concentration.
[0078] 5. Preparation of Hydrophobic Protein Microspheres Coated
with Bioadhesive Polymers by Phase Inversion:
[0079] A hydrophobic protein, such as zein F 4000 (prolamine),
derived from corn, was dissolved with sodium salicylate in 70%
ethanol (EtOH), such that the concentration of zein and sodium
salicylate was 7% w/v to yield a 1:1 weight ratio. The solution was
spray dried to produce microspheres in the range of 1 to 20.mu.,
having an average diameter of 5 to 7.mu.. 200 mg of the zein
microspheres were vortexed and briefly bath-sonicated in 2.5 ml of
10% poly(fumaric-co-sebacic acid) 20:80 6 KDa, (P(FA:SA)
(synthesized according to the procedure of Domb and Langer, Journal
of Polymer Science, v. 25, p 3373-3386 (1987)) (w/v) in methylene
chloride and rapidly added to 400 ml of petroleum ether without
stirring. The mixture was immediately filtered and air dried on the
filter paper.
[0080] The average diameter of the uncoated zein microspheres was
determined to be 5 to 7.mu. by SEM and the average diameter of the
coated microspheres was found to be greater than 30.mu..
[0081] 6. Microspheres were Coated with Polymer to Produce Coated
Microspheres Having a Diameter Greater than 20 .mu.m Using Phase
Inversion:
[0082] 0.5 g of glass beads were vortexed and bath sonicated for 1
minute in 2 ml of 20% polycaprolactone 76 Kda (PCL) (Aldrich)
(w/v). This mixture was drained and added to petroleum ether with
vigorous shaking. The petroleum ether was drained and the beads
were air dried.
[0083] SEM of the resultant air dried product indicated that the
beads were uniformly coated with polymer. The surface texture of
the coating was rough. Examination at a higher magnification
revealed that the roughness was attributable to polymer
spherulites, measuring 10-20.mu. in length.
[0084] 7, The Use of Polymers Having Low Glass Transition
Temperatures Produces Globular Aggregates Rather than
Microspheres.
[0085] 5 ml of 1% ethylene vinyl acetate 55 KDa (EVA) (Du Pont,
Inc.) (w/v) in methylene chloride containing 0.1% (w/v) of
rhodamine 6 G (10.0% w/w, encapsulant) was rapidly added to 200 ml
of petroleum ether without stirring. The mixture was immediately
filtered and the resulting composition was air dried on the filter
paper. The dried composition was examined by SEM and found to be in
the form of globular aggregates. The fluorescent dye was entrapped
by the globular aggregates. The results indicate that polymers
having low glass transition temperatures (i.e., below ambient) tend
to coalesce during phase inversion.
Example 2
Drug Release Profile from Microspheres Created by Phase Inversion
Nanoencapsulation
[0086] 1. Release of Dicumarol from Dicumarol Containing
Polyanhydride(Fa:Sa) (P(FA:SA)) Microspheres:
[0087] Dicumarol containing microspheres were formed by adding 0.1
g spray dried dicumarol (40% w/w) to 5 ml of 5% polyanhydride
(FA:SA) 20:80 (w/v) in methylene chloride. The mixture was rapidly
added to 100 ml of petroleum ether without stirring and immediately
filtered. The resulting microspheres were washed with petroleum
ether to remove loosely adherent drug on the surface of the
microspheres and then air dried on the filter paper.
[0088] Aliquots of dicumarol containing microspheres, containing
approximately 5 mg of dicumarol, were used in studies to examine
the release of drug from the microsphere. 5 mg of spray dried
dicumarol was used as a control. The dicumarol containing
microspheres or the spray dried dicumarol were separately incubated
in 10 ml of phosphate-buffered saline, pH 7.2 (PBS) at room
temperature for 10 hours. Periodically, 100 .mu.l samples of the
incubation fluid were withdrawn and analyzed for dicumarol
concentration using a UV spectrophotometric assay. The release of
dicumarol from the encapsulated microspheres was at least ten-fold
less than the control, spray dried drug after three hours.
[0089] 2. Release of Small Highly Water Soluble Drug can be
Optimized by Producing Microcapsules by the Phase Inversion
Method:
[0090] Salicylic acid was encapsulated in PVP (1-7 KDa
Polysciences) by spray drying a 1:1 ratio of 10% (w/v) solution of
each component in acetone at 65.degree. C. The particles were mixed
with a 5% P(FA:SA) 20:80 solution (w/v) in methylene chloride so
that the final loading of the drug was 16% (w/w) with respect to
the P(FA:SA). 10 ml of this mixture was poured into 200 ml of
petroleum ether. The resulting microspheres were collected by
filtration and air dried.
[0091] Aliquots of PVP or P(FA:SA)-encapsulated-PVP microspheres
containing approximately 40 mg of salicylic acid were incubated in
10 ml of phosphate-buffered saline, pH 7.2 (PBS) at room
temperature for 10 hours. As a control, 40 mg of salicylic acid
alone was subjected to the same conditions. Periodically, 100 .mu.l
samples of the incubation fluid were collected and analyzed for
dicumarol concentration using a visible spectrophotometric assay.
Although the release of salicylic acid from PVP-microspheres was
not significantly different from the dissolution of stock salicylic
acid, the release of salicylic acid from P(FA:SA) coated
microspheres was observed to be markedly decreased. An improved
linearity of drug release was also observed. SEM of the coated
microspheres indicated that the beads were uniformly coated with
polymer, and had a particle size of 10.mu.. These results indicate
that phase inversion encapsulation can produce controlled release
of a small highly water soluble drug and also that multiple polymer
systems can be used to optimize delivery of drugs by this
method.
[0092] 3. Emulsions of Proteins can be Released from Microspheres
Produced by Phase Inversion Encapsulation:
[0093] 0.5 ml of 20 mg FITC-BSA/ml (Sigma Chemical Co.) of
phosphate-buffered saline (PBS) was re-suspended in 10 ml of 1% PLA
2 KDa (w/v) in methylene chloride to yield a protein loading of
9.1% (w/w). The mixture was probe-sonicated for three cycles of 10
seconds duration and quickly poured into 400 ml of petroleum ether.
The resulting microspheres were filtered and air dried.
[0094] 11.0 mg of the microspheres were incubated in 5 ml of PBS pH
7.2 at 37.degree. C. Periodically, 50 .mu.l samples of the
incubation fluid were collected and analyzed for FITC-BSA using a
visible spectrophotometric assay The results of the assay indicated
that the entire loading of the encapsulant was released into the
incubation fluid within 30 minutes. These results suggest that the
phase inversion encapsulation process may be used to entrap
proteins, and that these emulsions of proteins in microspheres are
rapidly released.
[0095] 4. Release of Insulin from Nanospheres Composed of PLA and
Poly (Fumaric Acid):
[0096] Micronized zinc insulin was incorporated into a 5% (w/v)
polymer solution of a 4:1 blend of PLA 24 KDa and poly (fumaric
acid) in methylene chloride at a loading of 4.4+/-0.7% (w/w). This
mixture was dispersed into petroleum ether (1:100
solvent/nonsolvent volume ratio) and the resulting nanospheres were
collected by filtration and air dried.
[0097] Insulin release from the nanospheres was studied over a 22
hour time period. After 1 hour, approximately 24% of the total
insulin was released and at the end of 5 hours, nearly 45% of the
drug had released form the nanospheres. The rate of release of
insulin slowed down between 5 and 22 hours. At the end of the
experiment 53% of the initial loading remained encapsulated in the
nanospheres.
Example 3
Microspheres Produced by Phase Inversion Encapsulation Exhibit
Enhanced Bioavailability of Encapsulated Drugs In Vivo
[0098] 1. Oral Delivery of Microparticles:
[0099] Studies were conducted to determine the fate of orally
administered P(FA:SA)20:80 microparticles. The microparticles
contained rhodamine and had a particle size range of between 0.1
and 1.0 micrometers. Rats were fed a single dose of 30 mg of such
microparticles. As early as one hour posted-feeding of a single
dose, microparticles were observed to traverse the mucosal
epithelium by passing between absorptive cells (paracellular
route). In addition, microparticles were seen crossing through
follicle associated epithelium (FAB) and into the Peyer's patches.
After three and six hours, an even greater number of microparticles
were seen between epithelial cells and in the Peyer's patches.
Focal areas demonstrated massive amounts of nonselective uptake, by
both absorptive cells and Peyer's patches. Liver samples showed
large numbers of nanospheres with apparently normal looking
hepatocytes. Spleen sections also showed nanospheres, but fewer
than in the liver. At twelve hours, large numbers spheres were
still observed in between villous epithelial cells and in the
Peyer's patches. Similar sections were observed even at twenty-four
hours post-feeding.
[0100] This experiment showed extensive uptake of microparticles
extending over at least twenty-four hours, following a single oral
dose. Microparticles apparently crossed the epithelial boundary by
passing in between cells. The observed uptake did not seem to be
limited to the FAE overlying the Peyer's patches; uptake occurred
diffusely by absorptive epithelium as well as FAE.
[0101] Transmission electron microscopy experiments using
electron-opaque tracers such as micronized ferric oxide or 5 nm
colloidal gold that had been microencapsulated with bioadhesive
P(FASA) 20:80 were also conducted. The findings demonstrated that
nanospheres in great number were indeed being taken up by
absorptive epithelial cells lining the small intestine. In a
typical thin section of an absorptive cell, up to one hundred
nanospheres could be counted. While the results of light microscopy
indicated a paracellular means of entry, these electron micrographs
showed many microparticles within cells. The mechanism of entry is
not known although several particles were occasionally observed in
clear "endocytotic" vesicles located directly beneath the terminal
web region in proximity to the apical microvillous border. The
range of particle sizes observed in the cytoplasm of cells was
40-120 nm, well below the resolution of normal light optics and
therefore undetectable by light microscopy. Nanoparticles were
visualized in the cytoplasm, inside membranous profiles of the
endoplasmic reticulum and Golgi apparatus and generally in the
supranuclear (apical) portion of the absorptive cell. Occasionally,
nanoparticles were seen near the basal aspects of the cell. Spheres
were often found near the lateral borders of the cell, in the
intracellular spaces and in close apposition to the tight
junctions. These findings suggest that translocation of nanospheres
via the transcellular route occurred in addition to paracellular
movement.
[0102] 2. Oral Delivery of Insulin:
[0103] Insulin was encapsulated in P(FA)-PLGA(50:50) polymer blends
using the phase inversion Nanoencapsulation methods. After
measuring fasting blood glucose levels, fasted rats were injected
subcutaneously with an initial glucose load and then fed either a
suspension of nanospheres containing 20 IU zinc-insulin (micronized
FeO was included an electron dense tracer) in saline or else sham
fed saline only. Blood glucose levels (BGL) were assayed at
intervals after feeding.
[0104] The controls showed the expected response to the glucose
load. BGL rose by 40 mg/dL after three hours and then slowly
started to return towards baseline. In contrast, animals fed the
encapsulated insulin formulation had consistently lower blood
glucose levels than the control animals at three of the four time
points that were sampled. After 1.5 hours, the BGL was 20 mg/dL
below baseline compared to 30 mg/dL above baseline for control
animals. At three hours the BGL of the nanoparticle treated animals
rose to 20 mg/dL above baseline compared to a 40 mg/dL rise for the
control animals (not statically different). At four hours, the BGL
of the nanoparticle-fed animals was nearly 30 mg/dL below baseline,
compared to a BGL of 20 mg/dL above base line for the control
animals. After five hours, the glucose levels of the test group
were lower than at four hours, while the levels of the control
animals were still 35 mg/dL above baseline. Because the animals fed
the encapsulated insulin preparation were better able to regulate
the glucose load, it is clear that the insulin was not harmed by
the encapsulation method, that the insulin survived the environment
of the stomach, the insulin crossed the intestinal barrier and the
insulin was released from the nanoparticles in a bioactive form. A
widespread distribution of insulin-loaded nanospheres also was
observed. The spheres were observed in great numbers, traversing
the mucosal epithelium in the small intestine, in the Peyer's
patches, in the lamina propria, in the lacteals and in the blood
vessels of the gut wall. Nanoparticles also were observed in spleen
and other tissue samples. Thus, systemic delivery of both insulin
and nanoparticles was demonstrated.
[0105] 3. Encapsulation and Oral Delivery of Dicumarol:
[0106] Dicumarol containing microspheres were produced as described
above in Example 2, subsection 1. Equal doses of dicumarol, spray
dried dicumarol and polyanhydride (FA:SA) 20:80 encapsulated
dicumarol (25 mg drug/kg body weight) suspended in 1.5 ml maple
syrup were fed to catheterized rats (250-350 g). Blood samples were
taken at regular intervals and serum was assayed for dicumarol
concentrations using a UV spectrophotometric method.
[0107] The results of the in vivo studies indicate that the
polyanhydride (FA:SA) microcapsule formulation had significantly
increased bioavailability compared to the unencapsulated
formulations, including the micronized drug. At 12 hours
post-feeding, the serum concentrations for the polyanhydride
(FA:SA) formulations were significantly higher than for the
controls. At 48 hours post-feeding, the serum levels of dicumarol
the controls had returned to baseline, while those animals fed the
bioadhesive polyanhydride formulation had detectable drug
concentrations for at least 72 hours.
TABLE-US-00001 TABLE 1 ORAL BIOAVAILABILITY OF DICUMAROL STOCK
SPRAY P(FA:SA) 20:80 DICUMAROL DICUMAROL "PIN"ENCAPSULATED CONTROL
CONTROL DICUMAROL C MAX (ug/ml 11.53 .+-. 1.10* 17.94 .+-. 1.22
18.63 .+-. 1.76* T MAX (hrs) 9.87 .+-. 1.76 9.42 .+-. 1.36 10.61
.+-. 0.02 t1/2 (half life) (hrs) 18.25 .+-. 3.30 16.21 .+-. 0.87
17.92 .+-. 0.41 AUC (area under curve) 171.48 .+-. 33.16 232.10
.+-. 19.20.noteq. 363.59 .+-. 70.95.noteq. (ug/ml-hrs) *=
Significantly different at p < .03 .noteq.= Significantly
different at p < .005 (means .+-. std error)
[0108] These results indicate that phase inversion encapsulation of
drugs in bioadhesive formulations, such as the polyanhydride
(FA:SA) can increase bioavailability.
[0109] 4. Incorporation of DNA into Polymeric Nanospheres by Phase
Inversion
[0110] This example provides a description of the incorporation of
plasmid DNA into poly(fumaric acid:sebacic acid) 20:80 (P(FA:SA))
using a phase inversion technique.
[0111] Materials. P(FA:SA) 20:80 (synthesized by a method of A.
Domb & R. Langer, Journal of Polymer Science, 1987, v. 25, p.
3373-3386), a reporter plasmid pCMV/.beta.gal (Clonetech),
methylene chloride (Fisher) and petroleum ether (Fisher) were used
to construct the nanospheres.
[0112] Methods. 200 mg of P(FA:SA) in methylene chloride (1%
wt/vol) is vortexed (30 sec) with 2 mg of pCMV/.beta.gal in
distilled water (1 mg/ml), frozen in liquid nitrogen and
lyophilized overnight to disperse the DNA in the polymer. The
purpose of this step was to reduce the particulate size and prevent
aggregation of the DNA. DNA present in the disperse phase of the
emulsion would not be able to aggregate due to the physical
separation induced by the continuous polymer phase. The resulting
mixture was redissolved in 2 ml of methylene chloride, poured into
200 ml of petroleum ether and filtered to recover microspheres
encapsulating the DNA.
[0113] Results. Polymer nanoparticles produced using this technique
were analyzed to determine whether DNA was encapsulated within the
nanoparticles. Plasmid DNA was extracted from the nanoparticles and
subjected to agarose gel electrophoresis. The results indicate that
DNA was encapsulated without degradation. Thus, the phase inversion
technique can be used to incorporate very large intact molecular
weight plasmid DNA (7.2.times.10.sup.6 Daltons) in biodegradable
nanoparticles.
Example 4
Processing Parameters
[0114] A variety of polymers, solvents, viscosities, non-solvents,
drugs, and concentrations were tested in phase inversion
experiments. Table 3 summarizes the results of many of these
tests.
TABLE-US-00002 TABLE 3 Concen- Non- Concen- Polymer MW tration
Viscosity Solvent Solvent Drug tration Product polystyrene 2K 5%
MeCl.sub.2 pet ether rhodamine 0.1% polystyrene 2K 10% MeCl.sub.2
pet ether rhodamine 0.1% polystyrene 50K 1% MeCl.sub.2 pet ether
none -- polystyrene 50K 1% MeCl.sub.2 pet ether rhodamine 0.1% 1-5
.mu.m polystyrene 50K 3% MeCl.sub.2 pet ether rhodamine 0.1%
polystyrene 50K 5% MeCl.sub.2 pet ether rhodamine 0.1% 500 nm-2
.mu.m polystyrene 50K 10% MeCl.sub.2 pet ether rhodamine 0.1% 1-4
.mu.m polystyrene 50K 15% MeCl.sub.2 pet ether rhodamine 0.1% 1-10
.mu.m & aggr polystyrene 50K 20% MeCl.sub.2 pet ether rhodamine
0.1% large aggregate polystyrene 50K 1% MeCl.sub.2 ethanol
rhodamine 0.1% polystyrene 50K 5% MeCl.sub.2 ethanol rhodamine 0.1%
<100 nm polystyrene 50K 10% MeCl.sub.2 ethanol rhodamine 0.1%
<100 nm-3 .mu.m polycapro- 72K 1% 3.188 MeCl.sub.2 pet ether
rhodamine 0.1% 1-3 .mu.m lactone polycapro- 72K 5% 7.634 MeCl.sub.2
pet ether rhodamine 0.1% 1-3 .mu.m lactone large aggr polycapro-
112K 1% 4.344 MeCl.sub.2 pet ether rhodamine 0.1% 500 nm-5 .mu.m
lactone polycapro- 112K 5% MeCl.sub.2 ethanol rhodamine 0.1% Large
lactone aggregate polyvinyl- 1.5-7K 1% acetone pet ether none --
250 nm-1 .mu.m phenol polyvinyl- 1.5-7K 5% acetone pet ether none
-- phenol polyvinyl- 1.5-7K 10% " acetone pet ether none -- phenol
polyvinyl- 9-11K 1% acetone pet ether none -- 100 nm-2 .mu.m phenol
polyvinyl- 9-11K 5% acetone pet ether none -- 250 nm-2.5 .mu.m
phenol polyvinyl- 9-11K 10% acetone pet ether none -- 500 nm-10
.mu.m phenol polylactic 2K 1% 0.876 MeCl.sub.2 pet ether rhodamine
0.1% 100 nm acid polylactic 2K 5% 1.143 MeCl.sub.2 pet ether
rhodamine 0.1% 500 nm-2 .mu.m acid polylactic 2K 10% 2.299
MeCl.sub.2 pet ether rhodamine 0.1% 1-10 .mu.m acid brittle
polylactic 24K 1% 1.765 MeCl.sub.2 pet ether rhodamine 0.1% 100 nm
acid polylactic 24K 5% 2.654 MeCl.sub.2 pet ether rhodamine 0.1%
500 nm-1 .mu.m acid polylactic 24K 10% 3.722 MeCl.sub.2 pet ether
rhodamine 0.1% 10 .mu.m aggr acid polylactic 40-100K 1% 2.299
MeCl.sub.2 pet ether rhodamine 0.1% acid polylactic 40-100K 5%
2.832 MeCl.sub.2 pet ether rhodamine 0.1% acid polylactic 40-100K
10% 6.122 MeCl.sub.2 pet ether rhodamine 0.1% acid polylactic 100K
1% 2.566 MeCl.sub.2 pet ether rhodamine 0.1% 100 nm acid polylactic
100K 5% 4.433 MeCl.sub.2 pet ether rhodamine 0.1% 500 nm-2 .mu.m
aggr acid polylactic 100K 10% 8.256 MeCl.sub.2 pet ether rhodamine
0.1% film/aggr acid ethylene- 55K 1% MeCl.sub.2 pet ether rhodamine
0.1% Globular vinyl acetate strands ethylene- 55K 5% MeCl.sub.2 pet
ether rhodamine 0.1% coalesced vinyl acetate strands ethylene- 55K
10% MeCl.sub.2 pet ether rhodamine 0.1% continuous vinyl acetate
sheet PAN/PVC 1% 2.566 acetone pet ether none -- coarse 1-20 .mu.m
PAN/PVC 5% 15.903 acetone pet ether none -- 100 .mu.m aggr
[0115] Each of the foregoing patents, patent applications and
references is herein incorporated by reference in its entirety.
Having described the presently preferred embodiments, in accordance
with the present invention, it is believed that other
modifications, variations and changes will be suggested to those
skilled in the art in view of the teachings set forth herein. It
is, therefore, to be understood that all such variations,
modifications, and changes are believed to fall within the scope of
the present invention as defined by the appended claims.
[0116] Each of the foregoing patents, patent applications and
references is herein to incorporated by reference in its entirety.
Having described the presently preferred embodiments, in accordance
with the present invention, it is believed that other
modifications, variations and changes will be suggested to those
skilled in the art in view of the teachings set forth herein. It
is, therefore, to be understood that all such variations,
modifications, and changes are believed to fall within the scope of
the present invention as defined by the appended claims.
[0117] Those of ordinary skill in the art will readily ascertain
numerous equivalents of the foregoing examples. Such equivalents
are intended to be embraced by the following claims.
* * * * *