U.S. patent application number 11/409406 was filed with the patent office on 2006-08-24 for materials and methods for drug delivery and uptake.
Invention is credited to Allison Gallup, Laurie B. Gower, Michael Ossenbeck, Vishal M. Patel, Piyush Sheth.
Application Number | 20060188562 11/409406 |
Document ID | / |
Family ID | 31991613 |
Filed Date | 2006-08-24 |
United States Patent
Application |
20060188562 |
Kind Code |
A1 |
Gower; Laurie B. ; et
al. |
August 24, 2006 |
Materials and methods for drug delivery and uptake
Abstract
The subject invention pertains to novel materials and methods
for use in delivering and sequestering substances, such as
pharmacological agents, within a patient. One aspect of the
invention is directed towards core-shell particles having a core
encapsulated within a calcium carbonate shell, with an intermediate
layer composed of an amphiphilic compound surrounding the core.
When the particles of the subject invention are administered to a
patient, they are capable of removing lipophilic drugs by
absorption of the drug through their mineral shell and into their
core. The particles of the subject invention can also be
administered to a patient as controlled release, drug delivery
vehicles. Thus, in another aspect, the subject invention concerns a
method of delivering pharmacological agents by administering the
core-shell particles of the subject invention to a patient in need
of such administration.
Inventors: |
Gower; Laurie B.;
(Gainesville, FL) ; Patel; Vishal M.;
(Gainesville, FL) ; Sheth; Piyush; (Baltimore,
MD) ; Gallup; Allison; (Jacksonville, FL) ;
Ossenbeck; Michael; (Gainesville, FL) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK;A PROFESSIONAL ASSOCIATION
PO BOX 142950
GAINESVILLE
FL
32614-2950
US
|
Family ID: |
31991613 |
Appl. No.: |
11/409406 |
Filed: |
April 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10243340 |
Sep 13, 2002 |
|
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11409406 |
Apr 21, 2006 |
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Current U.S.
Class: |
424/450 |
Current CPC
Class: |
A61K 9/107 20130101;
A61K 9/501 20130101 |
Class at
Publication: |
424/450 |
International
Class: |
A61K 9/127 20060101
A61K009/127 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The subject invention was made with government support under
a research project supported by National Science Foundation Grant
No. EEC-9402989. The government has certain rights in this
invention.
Claims
1. A core-shell particle comprising: (a) a shell, wherein said
shell comprises calcium carbonate; (b) a core; and (c) an
intermediate layer between said shell and said core, wherein said
intermediate layer comprises an amphiphilic compound, and wherein
said core and said intermediate layer are surrounded by said
shell.
2. A method for making a core-shell particle, wherein the method
comprises the steps of: (a) preparing a core; and (b) encapsulating
the core with a calcium carbonate shell.
3. The method according to claim 2, wherein said step (a) comprises
forming an emulsion droplet, and wherein said step (b) comprises
contacting the emulsion droplet with a calcium-containing
solution.
4. The method according to claim 3, wherein the emulsion droplet
comprises an oil phase and an amphiphilic compound, and wherein the
core-shell particle comprises: (a) the calcium carbonate shell; (b)
the core; and (c) an intermediate layer between the calcium
carbonate shell and the core, wherein the intermediate layer
comprises the amphiphilic compound, and wherein the core and the
intermediate layer are surrounded by the shell.
5. The method according to claim 4, wherein the amphiphilic
compound has a partially deprotonated carboxylic acid headgroup
functionality.
6. The method according to claim 4, wherein the amphiphilic
compound is selected from the group consisting of stearic acid and
arachidic acid.
7. The method according to claim 3, wherein said method further
comprises adding a source of Mg ion to the calcium-containing
solution.
8. The method according to claim 3, wherein said method further
comprises adding a short-chained acidic polymer to the
calcium-containing solution.
9. The method according to claim 8, wherein the short-chained
acidic polymer is selected from the group consisting of polyacrylic
acid, polymethacrylate, sulfonated polymer, phosphorylated peptide,
phosphorylated polymer, sulfated glycoprotein, polyaspartic acid,
polyglutamic acid, and copolymers thereof.
10. The method according to claim 9, wherein the short-chained acid
polymer comprises poly-(.alpha.,.beta.)-D,L-aspartic acid.
11. The method according to claim 8, wherein the short-chained acid
polymer is added at a concentration within the range of about 1
.mu.g/ml and about 100 .mu.g/ml.
12. The method according. to claim 3, wherein said forming an
emulsion droplet comprises contacting a hydrophobic compound with
an aqueous solution.
13. The method according to claim 12, wherein said forming an
emulsion droplet further comprises adding an amphiphilic compound
to the aqueous solution.
14. The method according to claim 13, wherein the amphiphilic
compound is selected from the group consisting of stearic acid and
arachidic acid.
15. The method according to claim 3, wherein the calcium-containing
solution comprises CaCl.sub.2.
16. The method according to claim 3, wherein the calcium-containing
solution further comprises Mg.
17. The method according to claim 3, wherein the calcium-containing
solution further comprises MgCl.sub.2.
18. The method according to claim 17, wherein the
calcium-containing solution further comprises CO.sub.3.sup.2-
counterion.
19. The method according to claim 17, wherein said method further
comprises adding CO.sub.3.sup.2- counterion to the
calcium-containing solution.
20. The method according to claim 19, wherein the CO.sub.3.sup.2-
counterion is added to the calcium-containing solution by
peristaltic pumping.
21. A method for sequestering a lipophilic agent within a patient
comprising administering an effective amount of core-shell
particles to the patient, wherein the core-shell particles
comprise: (a) a shell, wherein the shell comprises calcium
carbonate; (b) a core; and (c) an intermediate layer between the
shell and the core, wherein the intermediate layer comprises an
amphiphilic compound, and wherein the core and the intermediate
layer are surrounded by the shell.
22. The method according to claim 21, wherein the core is
hollow.
23. The method according to claim 21, wherein the core comprises an
oil.
24. The method according to claim 23, wherein the core further
comprises an enzyme that degrades the lipophilic agent.
25. The method according to claim 21, wherein the enzyme comprises
a cytochrome P450 enzyme.
26. The method according to claim 24, wherein the lipophilic agent
is absorbed into the core-shell particles, and wherein the enzyme
subsequently degrades the lipophilic agent.
27. The method according to claim 21, wherein the core-shell
particles are administered to the patient intravenously.
28. The method according to claim 21, wherein a toxic amount of the
lipophilic agent is present within the patient prior to said
administration of the core-shell particles.
29. The method according to claim 24, wherein the enzyme is
adsorbed onto the shell of the particles.
30. A method for sequestering a lipophilic agent from the
surrounding environment comprising contacting an effective amount
of core-shell particles with the lipophilic agent, wherein the
core-shell particles comprise: (a) a shell, wherein the shell
comprises calcium carbonate; (b) a core; and (c) an intermediate
layer between the shell and the core, wherein the intermediate
layer comprises an amphiphilic compound, and wherein the core and
the intermediate layer are surrounded by the shell.
31. The method according to claim 30, wherein said contacting is
carried out in vivo.
32. The method according to claim 30, wherein the surrounding
environment is a patient's bloodstream.
33. A method for delivering a biologically active agent to a
patient comprising administering an effective amount of core-shell
particles to the patient, wherein the core-shell particles
comprise: (a) a shell, wherein the shell comprises calcium
carbonate; (b) a core, wherein said core comprises a biologically
active agent; and (c) an intermediate layer between the shell and
said core, wherein said intermediate layer comprises an amphiphilic
compound, and wherein said core and said intermediate layer are
surrounded by said shell.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation of U.S.
application Ser. No. 10/243,340, filed Sep. 13, 2002, which is
hereby incorporated by reference herein in its entirety, including
any figures, tables, nucleic acid sequences, amino acid sequences,
and drawings.
BACKGROUND OF THE INVENTION
[0003] Treatment of drug overdose in humans, whether due to
therapeutic miscalculation, illicit drug use, or suicide attempt,
presents a major problem to the health care industry worldwide. In
the United States alone, over 300,000 patients are admitted to the
emergency rooms because of drug overdose. Treatment of these
patients costs the healthcare industry over ten billion dollars
because of hospital expenses and lost employee productivity. This
does not include the $80 billion associated with alcohol abuse
(Moudgil, B. M., Seventh Year Annual Report. 2001: Engineering
Research Center for Particle Science and Technology, University of
Florida).
[0004] Current treatment protocols for overdosed patients vary with
the drug of concern, but are focused on three objectives:
prevention of drug absorption, enhancement of drug excretion, and
administration of pharmacological antidotes. The first two are
accomplished with techniques nonspecific to the ingested drug, such
as emesis, gastric lavage, or use of activated charcoal for the
former objective, and dialysis or hemoperfusion for the latter.
However, since absorption of toxic drugs is very time sensitive,
and since these techniques are applied only once a patient reaches
the emergency room, they are not as effective as would be desired,
with some techniques reported to recover only 30% of the ingested
drug (Rumack, B. H., Poisoning: Prevention of absorption, in
Poisoning and Overdose, M. J. Bayer and B. H. Rumack, Eds., 1983,
p. 13-18). There also currently exist very few specific
pharmacological antidotes to the drugs frequently associated with
life threatening overdose cases (Moudgil, B. M., Seventh Year
Annual Report. 2001: Engineering Research Center for Particle
Science and Technology, University of Florida).
[0005] An important factor influencing drug distribution in the
body is the ability of toxins to bind to blood proteins and
tissues. Certain tissues have strong binding affinities for
specific toxins, causing localized concentration in that tissue.
This is true especially of the kidney and liver, because of their
metabolic and excretory functions. Some toxins bind noncovalently
to albumin, a blood plasma protein, or other proteins. While bound
to protein, the complex becomes pharmacologically inert and is
trapped in the bloodstream due to its large size. Only unbound
drugs are able to cross lipoprotein membranes and exert an effect.
A drug's free molecule concentration is likely to increase during
an overdose, since protein-binding sites are more readily
saturated. Therefore, it is expected that a patient with low levels
of albumin will experience higher toxicity effects than a patient
with normal levels (Lu, F., Basic Toxicology: Fundamentals, Target
Organs, and Risk Assessment. 3rd ed. 1996, Taylor and Francis:
Washington; Fenton, J. J., Toxicology: A Case-Oriented Approach.
2002, CRC Press: Boca Raton; Stine, K. E. and T. M. Brown,
Principles of Toxicology. 1996, CRC Press: Boca Raton).
[0006] Micron-scale and nano-scale core-shell particulate systems,
either hollow or fluid-filled, have become of recent interest.
Core-shell particles find important applications in encapsulation
of a variety of materials for catalysis and controlled release
applications (e.g. drugs, enzymes, pesticides, dyes, etc.); for use
as filler in lightweight composites, pigment, or coating materials;
and in biomedical implant materials (Putlitz, B. Z. et al., Adv.
Mater., 2001, 13:500-+; Walsh, D. and Mann, S., Nature, 1995,
377:320-323; Walsh, D. et al., Adv. Mater., 1999, 11:324-328;
Zhong, Z. et al., Adv. Mater., 2002, 12:206-209; Caruso, F.,
Chem.--Eur. J., 2000, 6:413-419).
[0007] Recently, the use of particulate systems as a treatment for
patients overdosed on lipophilic drugs has been proposed (Moudgil,
B. M., Seventh Year Annual Report. 2001: Engineering Research
Center for Particle Science and Technology, University of Florida).
Several particulate systems, including microemulsions, polymer
microgels, silica nanotubes and nanosponges, and silica core-shell
particles, are currently being investigated for this detoxification
purpose. It has been proposed that, when intravenously administered
to an overdosed patient, such particles will effectively detoxify
the patient's circulatory system of the particular lipophilic toxin
by either: (a) absorption, from the selective partitioning of the
drug molecules from the blood to the hydrophobic core of the
particle; or (b) adsorption of the drug molecules onto surfaces of
surface-functionalized particles. Furthermore, in order to catalyze
the toxin metabolism, and hence its removal from the blood, the
immobilization of toxin-specific catabolic enzymes on or within
particles is being pursued (Moudgil, B. M., Seventh Year Annual
Report. 2001: Engineering Research Center for Particle Science and
Technology, University of Florida).
[0008] Fabrication of hollow sphere particles has been accomplished
using various methods and materials. In general, three fabrication
classes are currently employed: sacrificial cores, nozzle reactor
systems, and emulsion or phase separation techniques (Caruso, F.,
Chem.--Eur. J., 2000, 6:413-419; Wilcox, D. L. and Berg, M., in
Materials Research Society, 1994, Boston: Materials Research
Society). The first involves the coating of a core substrate with a
material of interest, followed by the removal of the core by
thermal or chemical means. In this manner, hollow particles of
yttrium compounds (Kawahashi, N. and Matijevic, E., J. Colloid
Interface Sci., 1991, 143:103-110), TiO.sub.2 and SnO.sub.2 (Zhong,
Z. et al., Adv. Mater., 2002, 12:206-209), and silica (Caruso, F.,
Chem.--Eur. J., 2000, 6:413-419) have been synthesized. Nozzle
reactor systems make use of spray drying and pyrolysis, and their
use has successfully led to the fabrication of hollow glass
(Nogami, M. et al., J. Mater. Sci., 1982, 17:2845-2849), silica
(Bruinsma, P. J. et al., Chem. Mater., 1997, 9:2507-2512), and
TiO.sub.2 (Iida, M. et al., Chem. Mater., 1998, 10:3780) particles.
Emulsion-mediated procedures, or hollow particle synthesis, is a
third common method. This has been used to form latex (Putlitz, B.
Z. et al., Adv. Mater., 2001, 13:500-+), polymeric (Pekarek, K. J.
et al., Nature, 1994, 367:258-260), and silica core-shell particles
(Underhill, R. S. et al., Abstracts ofpapers of the American
Chemical Society, 2001, 221:545).
[0009] Calcium carbonate coated core-shell particles have also been
synthesized. By coating polystyrene beads with calcium carbonate,
followed by removal of the polymer core, hollow particles in the 1
.mu.m to 5 .mu.m size range have been generated (Walsh, D. and
Mann, S., Nature, 1995, 377:320-323; U.S. Pat. No. 5,756,210).
Core-shell particles have also been synthesized using water-in-oil
(Walsh, D. et al., Adv. Mater., 1999, 11:324-328; Enomae, T.,
Proceedings of the 5.sup.th Asian Textile Conference, 1999,
1:464-467), and water-in-oil-in-water (Hirai, T. et al., Langmuir,
1997, 13:6650-6653; Hirai, T. and Komasawa, I., Kagaku Kogaku
Ronbunshu, 2001, 27:303-313) emulsions as templates for calcium
carbonate nucleation. In other processes, Lee et al. (Lee, I. et
al., Adv. Mater., 2001, 13:1617-1620) and Qi et al. (Qi, L. M. et
al., Adv. Mater., 2002, 14:300) respectively use
monolayer-protected gold particles and double-hydrophilic block
copolymer (DHBC)-surfactant complex micelles as templates for
calcium carbonate deposition, resulting in core-shell particles up
to 5 .mu.m in diameter.
[0010] Some of the calcium carbonate core-shell systems discussed
in the scientific literature are generated by using a biomimetic
process (Walsh, D. and Mann, S., Nature, 1995, 377:320-323; Walsh,
D. et al., Adv. Mater., 1999, 11:324-328; Hirai, T. et al.,
Langmuir, 1997, 13:6650-6653; Hirai, T. and Komasawa, I., Kagaku
Kogaku Ronbunshu, 2001, 27:303-313; Qi, L. M. et al., Adv. Mater.,
2002, 14:300). Mineralization in biological systems has been the
focus of intense research because their successful mimicry has
important implications for the synthetic design of superior
materials. Exquisite control of mineral deposition in biosystems is
thought to occur partly due to the presence of an insoluble organic
matrix, along with modulation of the crystal growth process via
soluble macromolecular species, such as acidic proteins and
polysaccharides (Lowenstam, H. A. and Weiner, S., On
Biomineralization, Oxford University Press: New York, 1989).
[0011] As can be understood from the above, there remains a need
for a particulate system that is capable of neutralizing or
eliminating toxic levels of drugs within a patient in a short
period of time, and which can be produced with the high degree of
control associated with biomimetic processes.
BRIEF SUMMARY OF THE INVENTION
[0012] The subject invention pertains to novel materials and
methods for use in delivering and segregating substances, such as
pharmacological agents, within a patient. One aspect of the
invention is directed towards particles having a core encapsulated
by a solid calcium carbonate shell, with an intermediate layer of
amphiphilic molecules surrounding the core. When the particles of
the subject invention are administered to a patient, they are
capable of removing lipophilic drugs by absorption of the drug
through their porous mineral shell and into their core. In one
embodiment, the core of the particles is hollow. In another
embodiment, the core contains a fluid, which is preferably an oil.
The particles of the subject invention can also be administered to
a patient as drug delivery vehicles. Thus, in another aspect, the
subject invention concerns a method of delivering or sequestering
pharmacological agents by administering the calcium
carbonate-encapsulated particles of the subject invention to a
patient in need of such administration.
[0013] The particles of the subject invention can be designed with
various porosities, in order to effectively absorb or release a
selected substance over a period of time.
[0014] In another aspect, the subject invention concerns a method
for making the calcium carbonate core-shell particles of the
subject invention by using a polymer-induced liquid-precursor
(PILP) process.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A-1C show reactants and resulting core-shell
particles of the subject invention. FIG. 1A shows the amphiphilic
nature of stearic acid. FIG. 1B shows reactants utilized to form
core-shell particles of the subject invention. FIG. 1C shows the
structure of a core-shell particle of the subject invention.
[0016] FIGS. 2A and 2B show polarized light micrographs of thin
calcium carbonate films deposited under stearic acid monolayers via
a PILP process. Bar=100 .mu.m.
[0017] FIGS. 3A-3C show cross-polarized light micrographs with
gypsum wave plate of calcium carbonate-coated emulsion droplets.
Bar=200 .mu.m.
[0018] FIGS. 4A-4D show scanning electron micrographs (SEMs) (FIG.
4A-4C) of calcium carbonate-coated core-shell particles and energy
dispersive spectroscopic (EDS) data (FIG. 4D). Particles in FIG. 4B
were crushed by shearing between glass slides, and show the shell
thickness of those particles that were fractured. Bar=10 .mu.m in
FIGS. 4A and 4B; and 2 .mu.m in FIG. 4C.
[0019] FIG. 5 shows in vitro uptake of amitriptyline (AMT) by
CaCO.sub.3 coated core-shell particles from saline solutions.
DETAILED DISCLOSURE OF THE INVENTION
[0020] The subject invention concerns particles having a core
contained within a solid calcium carbonate shell, with an
intermediate layer of amphiphilic molecules surrounding the core.
The subject invention also concerns a method of producing the
calcium carbonate-encapsulated particles by templating a porous
calcium carbonate shell onto the surface of oil-in-water emulsion
droplets using a polymer-induced liquid precursor (PILP) process.
In another aspect, the subject invention pertains to methods for
sequestering lipophilic agents within a patient by administering an
effective amount of the core-shell particles to the patient.
[0021] Briefly, the particles of the present invention can be
produced by blending an oil, such as n-dodecane, with water and an
amphiphile, the latter acting as a surfactant to stabilize the
droplets within water, forming emulsion droplets. The resultant
emulsion droplets can then be introduced into solutions of
CaCl.sub.2, MgCl.sub.2, and a short-chained acidic polymer additive
(such as polyaspartic acid). A CO.sub.3.sup.2- counter ion is then
introduced into the mixture, such as by slow peristaltic pumps,
thereby producing calcium carbonate coated particles that can then
be centrifuged and dried.
[0022] The particles can be constructed in sizes suitable for
particular applications, such as micro-scale or nano-scale
particles. For example, the process of the subject invention can
produce particles, such as microspheres, having a calcium carbonate
shell within the range of about 1 .mu.m to about 200 .mu.m in
diameter. In another embodiment, the shell has a diameter within
the range of about 1 .mu.m to about 50 .mu.m in diameter. In yet
another embodiment, the shell has a diameter within the range of
about 1 .mu.m to about 5 .mu.m in diameter. In order to pass
through the circulatory system of the body, smaller particles can
be constructed having a diameter within the range of about 100 nm
to about 300 nm, for example, using microemulsion templates.
[0023] Advantageously, the method of the subject invention can
produce particles having a shell of uniform thickness. Preferably,
the calcium carbonate shell has a thickness within the range of
about 100 nm to about 1000 nm in thickness. The core-shell particle
of claim 1, wherein said shell has a thickness within the range of
about 200 nm to about 500 nm in thickness.
[0024] The particles of the subject invention are biodegradable and
can be administered to patients for sequestration of a
pharmacological agent (drug uptake) following an overdose, as a
detoxification agent. Detoxification can occur through several
mechanisms. Once administered into a patient (e.g., via the
circulatory system), the particles can absorb a lipophilic drug
into their oily core, or adsorb the drug through dipole/charge
interactions with the mineral shell. Optionally, drug-degrading
enzymes, such as P450 enzymes, can operate within the particles, or
be coated onto or otherwise associated with the surface of the
particles. In another embodiment, the particles can release enzymes
that degrade the drug into harmless catabolites. The calcium
carbonate shell provides stabilization to the emulsion, and
operates as a molecular screen or filter, to avoid saturation of
the particles with proteins and other lipophilic species in the
blood, for example. The particles of the subject invention can also
be administered to a patient as drug delivery vehicles, such as
controlled release drug delivery vehicles, which could occur
through either pores templated into the shell, or via degradation
of the shell.
[0025] Preferably, the particles are of nano-scale dimensions and
non-aggregating, to avoid blockage of blood capillaries (if
administered into the circulatory system), and are biocompatible
(e.g., non-thrombogenic). If the particles are not sufficiently
small to pass through the blood-renal barrier, a biodegradable
material can be included for gradual removal of the particulates
from the blood stream (at a rate slow enough for the body to
tolerate the gradual release of the absorbed toxin). Optionally,
environment-sensitive catabolic enzymes for catalysis of the target
drug are immobilized within the particles; in which case, the
synthesis can be accomplished under benign processing
conditions.
[0026] The subject invention also concerns a method of producing
the calcium carbonate-encapsulated particles of the subject
invention using a polymer-induced liquid-precursor (PILP) process.
Using the novel and facile method of the subject invention, calcium
carbonate "hard" shell--"soft" core particles can be synthesized
under benign conditions. The method of the subject invention
utilizes an oil-in-water emulsion droplet as a template. The
procedure relies on the surface-induced deposition of a calcium
carbonate mineral precursor on to emulsion droplets by a
polymer-induced liquid-precursor (PILP) process, elicited by
including short-chained highly acidic polymers, such as
polyaspartic acid, into crystallizing solutions of calcium
carbonate which are slowly raised in supersaturation. The
deposition of thin films of calcium carbonate onto glass coverslips
using the PILP process has been demonstrated, as described
previously (Gower, L. B. and Odom, D. J., J. Cryst. Growth, 2000,
210:719-734). In those studies, in situ observations revealed that
the acidic polymer transforms the solution crystallization process
into a precursor process by inducing liquid-liquid phase separation
in the crystallizing solution. Droplets of a liquid-phase mineral
precursor can be deposited onto various substrates in the form of a
film or coating, which upon solidification and crystallization,
produces a continuous mineral film that maintains the morphology of
the precursor phase (hence, the name precursor). Using the method
of the subject invention, the PILP process is utilized to coat an
oil droplet in solution, generating a fluid-filled core-shell
particle with a thin uniform shell of calcium carbonate. In some
cases, the precursor phase may not appear to be a liquid, but
instead have solid-like characteristics (e.g. glassy). In either
case, the important aspect is that both are an amorphous precursor
phase, which due to coalescence during the formation of the phase,
lead to a smooth continuous coating of mineral rather than the
traditional solution crystallization of three-dimensional
crystallites. It has also been found that the inhibitory action of
Mg-ion can lead to a similar precursor process, and in the presence
of surfactant, polymer may not be necessary, although optimal
conditions include a combination of Mg-ion and polymer.
[0027] The process of the subject invention can be carried out
under a variety of conditions. For example, in the case of an
aqueous system, the process can be carried out at a temperature of
about 4.degree. C. to about 28.degree. C. For ease of processing,
the process can be carried out at room temperature (about
23.degree. C.). The process is preferably carried out at a pH
within the range of about 7 to about 11 and at 1 atm. More
preferably, the process is carried out at a pH of about 11.
However, the process can be carried out at a pH lower than 7 or
higher than 11 provided a surfactant is utilized that remains
charged at the particular pH. Preferably, the oil:water ratio is
within the range of about 1:8 and about 1:10, by volume. More
preferably, the oil:water ratio is about 1:9, by volume.
[0028] Using the process of the subject invention, the diameter of
the particles can be controlled. For example, the diameter of the
particles can be increased by increasing the size of the emulsion
droplet from which the particles are formed. Vesicular types of
particles (such as unilamellar or multilamellar liposomes) are
feasible as well, which could be used to fabricate core-shell
particles with an aqueous interior surrounded by the mineral shell.
For example, in preparing the particles, a liposome could be
substituted for the emulsion droplet as a template, which would
then be exposed to the amorphous mineral precursor. This could
increase the potential number of applications to include
encapsulated agents that require an aqueous environment, such as
water soluble molecules and macromolecules, biopolymers (e.g.
proteins, DNA) and cells.
[0029] Using the process of the subject invention, the calcium
carbonate shell porosity can be controlled. Because the highly PILP
phase will preferentially deposit on charged or polar regions of
patterned substrates, it is possible to pattern porosity into the
mineral shell by using an organic template with hydrophobic
domains. For example, increased porosity can be obtained by
increasing the quotient of surfactant with uncharged head groups
(such as cholesterol or diolein) in the mixture of surfactants used
to stabilize the emulsion droplet.
[0030] One or more of a variety of short-chained acidic polymers
can be utilized to initiate the amorphous liquid-phase mineral
precursor, including different polymers and biological materials.
As used herein, the term "short-chained acidic polymer" is intended
to mean oligomeric-length scale polymers bearing at least one
acidic functionality on one or more monomers of the polymer chain.
Polyacrylic acid (PAA), polymethacrylates (PMA), sulfonated
polymers, phosphorylated peptides and polymers, sulfated
glycoproteins, polyaspartic acid, polyglutamic acid, and copolymers
of these materials can be utilized to induce the liquid-phase
separation, for example. A range of polymer molecular weights can
be suitable if the other variable of the crystallizing conditions
are appropriately modified to generate the PILP phase.
[0031] Unlike those particles reported previously, using the method
of the subject invention allows one to generate a smooth and
uniform shell of calcium carbonate around the oil droplet, and not
an aggregation of individual crystals, as is common among the
previously published work. Furthermore, in this manner, oil can be
encapsulated within the particle, leading to a "soft" fluidic
core--a feature that is advantageous (although not necessary) for
the effective extraction of lipophilic molecules from aqueous media
by an absorption mechanism.
[0032] Preferably, the shell of the particle of the subject
invention is composed of magnesium-bearing calcium carbonate and is
at least 80% calcium carbonate. More preferably, the shell is
composed of at least 90% calcium carbonate. The Mg-ion is added as
an additional inhibitory agent (to eliminate traditional solution
crystallization), and potentially other ions or molecules could
serve this function, in combination with the polymer.
[0033] The core of the core-shell particle is a void containing a
compound in the oil phase that is incompatible with water.
Preferably, the compound in the oil phase is a hydrophobic
compound, such as an oil. More preferably, the hydrophobic compound
is an organic compound having a solubility to water of not more
than 1 gram per 10 grams of water at 20.degree. C. For example, one
or more of a variety of oils, such as dodecane or hexadecane, can
be incorporated within each particle, occupying its hollow core.
Other organic compounds that can be utilized include, but are not
limited to, cyclohexane, n-hexane, benzene, cottonseed oil,
rapeseed oil, squalane, squalene, waxes, styrene, divinylbenzene,
butyl acrylate, 2-ethylhexyl acrylate, cyclohexyl acryalate, decyl
acrylate, lauryl acrylate, dodecenyl acrylate, myristyl acrylate,
palmityl acrylate, hexadecenyl acrylate, stearyl acrylate,
octadecenyl acrylate, behenyl acrylate, butyl methacrylate,
2-ethylhexyl methacrylate, cyclohexyl methacrylate, decyl
methacrylate, lauryl methacrylate, dodecenyl methacrylate, myristyl
methacrylate, palmityl methacrylate, hexadecenyl methacrylate,
stearyl methacrylate, octadecenyl methacrylate, behenyl
methacrylate, silicone macromonomers, and the like.
[0034] Particles can be loaded with a selected substance or
substances, such as a biologically active agent, by contact with a
solution containing the agent. In one embodiment, the biologically
active agent, such as a detoxifying enzyme, is incorporated within
the emulsion droplet during formation of the core-shell particle.
Loading can be carried out by adding the biologically active agent
to the oil phase prior to emulsification and coating of the
droplet, for example. Because detoxifying enzymes are typically oil
soluble, they can be readily captured into the oil-in-water
emulsion prior to encapsulation with the mineral shell.
[0035] In another aspect, the subject invention pertains to a
method of sequestering a lipophilic agent within a patient by
administering an effective amount of core-shell particles to the
patient, wherein the core-shell particles absorb the lipophilic
agent through their calcium carbonate shell and into their oil
core. The particles can be administered through any of a variety of
routes known in the art, including enteral and parenteral, such as
intravenous. Preferably, the particles are administered into the
circulatory system of the patient, via a blood vessel, such as a
vein or artery. The patient may be suffering from overdose, wherein
a toxic concentration of the lipophilic agent is present within the
patient, such as in the bloodstream. The patient may also be
suffering from harmful drug interaction between the lipophilic
agent and another lipophilic agent or non-lipophilic agent.
[0036] In another aspect, the subject invention pertains to a
method of delivering a biologically active agent to a patient by
administering an effective amount of core-shell particles
containing a selected biologically active agent to the patient,
wherein the core-shell particles can release the biologically
active agent within the patient. The particles can be administered
through any of a variety of routes known in the art, including
enteral, pulmonary, and parenteral, such as intravenous.
Preferably, the particles are administered into the circulatory
system of the patient, such as through a blood vessel.
[0037] The particles of the subject invention can be administered
using any of a variety of means known in the art. For example,
administration of an effective amount of particles can include the
injection of the particles in a blood vessel, such as an
artery.
[0038] Following administration of the particles and drug release
or drug sequestration, the spent particles can, optionally, be
retrieved from the patient using a variety of methods. For example,
if the particles are not sufficiently biodegradable, they can be
filtered from the blood, such as in a dialysis process.
[0039] The term "biodegradable", as used herein, means capable of
being biologically decomposed. A biodegradable material differs
from a non-biodegradable material in that a biodegradable material
can be biologically decomposed into units which may be either
removed from the biological system and/or chemically incorporated
into the biological system.
[0040] The term "biocompatible", as used herein, means that the
material does not elicit a substantial detrimental response in the
patient. It should be appreciated that when a foreign object is
introduced into a living body, that the object may induce an immune
reaction, such as an inflammatory response that can have negative
effects on the patient. As used herein, the term "biocompatible" is
intended to include those materials that cause some inflammation,
provided that these effects do not rise to the level of
pathogenesis.
[0041] The particles of the subject invention can be used as a
vehicle for the delivery of biologically active agents, such as
medical substances in the field of therapeutics. The active agents
may be incorporated in the oil-containing core or chemically bonded
to the calcium carbonate shell, for example.
[0042] As used herein, the terms "incorporated within" or
"otherwise associated with" mean that the particular agent is
contained within the particle of the subject invention or is
directly or indirectly bound to the particle in some fashion. For
example, the biologically active agent can be contained within the
oil core of the particle, or operate as a component of the calcium
carbonate shell or amphiphilic layer. The biologically active agent
can be "free" or bonded to any of the other components of the
particle. The particular agent can be incorporated within, or
otherwise associated with, the particles of the subject invention,
during or subsequent to production of the particles. For example, a
biologically active agent, such as an enzyme, can be attached to
the outer shell through direct adsorption or through a linker
molecule. Alternatively, the agent can be physically entrapped in
the mineral phase, as it is deposited, and subsequently released
upon degradation of the mineral.
[0043] The biologically active agents that can be delivered using
the particles of the subject invention can include, without
limitation, medicaments, vitamins, mineral supplements, substances
used for the treatment, prevention, diagnosis, cure or mitigation
of disease or illness, substances which affect the structure or
function of the body, or drugs. The active agents include, but are
not limited to, antifungal agents, antibacterial agents, anti-viral
agents, anti-parasitic agents, growth factors, angiogenic factors,
anaesthetics, mucopolysaccharides, metals, cells, antibodies,
antibody fragments, and other agents. Because the processing
conditions can be relatively benign, live cells can be incorporated
into the particles during their formation, or subsequently allowed
to infiltrate the particles through tissue engineering
techniques.
[0044] The terms "pharmaceutically active agent", "biologically
active compound", "biologically active agent", "active agent",
"active compound" and "drug" are used herein interchangeably and
include pharmacologically active substances that produce a local or
systemic effect in a human or non-human animal. The terms thus mean
any substance intended for use in the diagnosis, cure, mitigation,
treatment or prevention of disease or in the enhancement of
desirable physical or mental development and conditions in a human
or non-human animal.
[0045] Examples of antimicrobial agents that can be delivered using
the particles of the present invention include, but are not limited
to, isoniazid, ethambutol, pyrazinamide, streptomycin, clofazimine,
rifabutin, fluoroquinolones, ofloxacin, sparfloxacin, rifampin,
azithromycin, clarithromycin, dapsone, tetracycline, erythromycin,
cikprofloxacin, doxycycline, ampicillin, amphotericine B,
ketoconazole, fluconazole, pyrimethamine, sulfadiazine,
clindamycin, lincomycin, pentamidine, atovaquone, paromomycin,
diclarazaril, acyclovir, trifluorouridine, foscarnet, penicillin,
gentamicin, ganciclovir, iatroconazole, miconazole, Zn-pyrithione,
and silver salts, such as chloride, bromide, iodide, and
periodate.
[0046] Growth factors that can be incorporated into or otherwise
associated with the particles of the present invention include, but
are not limited to, basic fibroblast growth factor (bFGF), acidic
fibroblast growth factor (aFGF), nerve growth factor (NGF),
epidermal growth factor (EGF), insulin-like growth factors 1 and 2
(IGF-1 and IGF-2), platelet-derived growth factor (PDGF), tumor
angiogenesis factor (TAF), vascular endothelial growth factor
(VEGF), corticotropin releasing factor (CRF), transforming growth
factors alpha and beta (TGF-.alpha. and TGF-.beta.), interleukin-8
(IL-8), granulocyte-macrophage colony stimulating factor (GM-CSF),
bone morphogenic protein (BMP), the interleukins, and the
interferons.
[0047] Other agents that can be incorporated into or otherwise
associated with the particles of the subject invention include acid
mucopolysaccharides including, but not limited to, heparin, heparin
sulfate, heparinoids, dermatan sulfate, pentosan polysulfate,
chondroitin sulfate, hyaluronic acid, cellulose, agarose, chitin,
dextran, carrageenin, linoleic acid, and allantoin.
[0048] Proteins that can be incorporated into or otherwise
associated with the particles of the subject invention include, but
are not limited to, collagen (including cross-linked collagen),
fibronectin, laminin, elastin (including cross-linked elastin),
osteonectin, bone sialoproteins (Bsp), alpha-2HS-glycoproteins,
bone Gla-protein (Bgp), matrix Gla-protein, bone
phosphoglycoprotein, bone phosphoprotein, bone proteoglycan,
protolipids, bone morphogenetic protein, cartilage induction
factor, platelet derived growth factor and skeletal growth factor,
or combinations and fragments thereof.
[0049] Other biologically active agents that can be incorporated
into or otherwise associated with the particles of the subject
invention include genetically-modified or non-genetically modified
cells. Thus, the particles of the subject invention can contain
such cells within their core and be administered to a patient. The
cells can be non-stem cells (mature and/or specialized cells, or
their precursors or progenitors) or stem cells. Thus, the cells can
range in plasticity from totipotent or pluripotent stem cells
(e.g., adult or embryonic), precursor or progenitor cells, to
highly specialized or mature cells, such as those of the pancreas.
In one embodiment, the cells are genetically modified to produce a
biologically active agent, such as a detoxifying enzyme.
[0050] Stem cells can be obtained from a variety of sources,
including fetal tissue, adult tissue, cord cell blood, peripheral
blood, bone marrow, and brain, for example. Stem cells and non-stem
cells (e.g., specialized or mature cells, and precursor or
progenitor cells) can be differentiated and/or genetically
modified. Methods and markers commonly used to identify stem cells
and to characterize differentiated cell types are described in the
scientific literature (e.g., Stem Cells: Scientific Progress and
Future Research Directions, Appendix E1-E5, report prepared by the
National Institutes of Health, June, 2001). The list of adult
tissues reported to contain stem cells is growing and includes bone
marrow, peripheral blood, brain, spinal cord, dental pulp, blood
vessels, skeletal muscle, epithelia of the skin and digestive
system, cornea, retina, liver, and pancreas.
[0051] The active agents incorporated within, or otherwise
associated with, the particles of the subject invention can exhibit
modified release characteristics. Release of the active agent can
be controlled using a variety of methods. For example, biologically
decomposable conjugates can be utilized. Alternatively, release of
the active agent can be controlled by inserting the active agent in
various components of the particle that have a different
biodegradability. For example, if used in medical or agricultural
applications, it may be desired to be able to control the release
dosage and release rate of active agents. In one embodiment, the
particles exhibit a decreasing (decaying) rate of release
(first-order release kinetics). In another embodiment, the
particles exhibit a constant rate of release (zero-order release
kinetics). In another embodiment, the particles exhibit one or more
sudden releases, or bursts, after a certain delay time.
[0052] The particles of the subject invention can be utilized to
administer hormones, for example. An important field of application
is the development of therapeutic systems for the controlled
release of an anti-diabetic agent, such as insulin, in the
treatment of pancreatic diabetes. The particles of the subject
invention can also be utilized to administer anti-tumor compounds,
such as cytotoxic agents, for the treatment of cancer.
[0053] Larger micro-scale particles of the invention can contain
cells. According to the methods of the invention, such particles
can be utilized to deliver the cells, and/or active agents produced
by the cells, in vivo. Examples of cells that can be incorporated
within, or otherwise associated with, the particles of the subject
invention include, but are not limited to, stem cells, precursor or
progenitor cells, chondrocytes, pancreatic cells, hepatocytes, and
neural cells. Such cells can be released from the particles upon
degradation of the shell in vivo.
[0054] The surface of the particles can be modified using surface
modification methods known to those of ordinary skill in the art.
For example, the amphiphilic layer composition can be varied to
vary the uncharged head group domain size.
[0055] As used herein, the term "lipophilic" is intended to mean
oil soluble. Examples of lipophilic drugs include amitriptyline,
bupivicaine, and amiodarone.
[0056] As used herein, the term "oil" is intended to mean any
nonpolar, water-insoluble compound.
[0057] As used herein, the terms "amphiphile" "amphiphilic
compound", and "surfactant" are used herein interchangeably and
intended to mean a compound having at least one hydrophilic (polar)
portion and at least one hydrophobic (nonpolar) portion, such as
stearic acid and arachidic acid. Typically, amphiphiles exhibit
amphiphilic behavior in which their molecules become concentrated
at the interface between a polar solvent and a nonpolar solvent.
Preferably, the amphiphilic compounds used in the subject invention
have molecules with at least one hydrophilic head group and at
least one hydrophobic tail. More preferably, the amphiphilic
compound has a partially deprotonated carboxylic acid headgroup
functionality.
[0058] The particles of the subject invention can be formulated in
any of a variety of forms or shapes in the micro- or nano-scale
size range (e.g., microparticles or nanoparticles). The particles
of the present invention can be, for example, capsules (e.g.,
microcapsules or nanocapsules), or spheres (e.g., microspheres or
nanospheres).
[0059] The core-shell particles of the subject invention can be
formulated and administered as a pharmaceutical composition,
containing a pharmaceutically acceptable carrier or diluent. The
pharmaceutical compositions of the subject invention can be
formulated according to known methods for preparing
pharmaceutically useful compositions. Formulations are described in
a number of sources which are well known and readily available to
those skilled in the art. For example, Remington's Pharmaceutical
Science (Martin E W [1995] Easton Pa., Mack Publishing Company,
19.sup.th ed.) describes formulations which can be used in
connection with the subject invention. Formulations suitable for
parenteral administration include, for example, aqueous sterile
injection solutions, which may contain antioxidants, buffers,
bacteriostats, and solutes which render the formulation isotonic
with the blood of the intended recipient; and aqueous and
nonaqueous sterile suspensions which may include suspending agents
and thickening agents. The formulations may be presented in
unit-dose or multi-dose containers, for example sealed ampoules,
vials, and disposable syringes made of glass or plastic, and may be
stored in a freeze dried (lyophilized) condition requiring only the
condition of the sterile liquid carrier, for example, water for
injections, prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powder, granules, tablets,
etc. It should be understood that, in addition to the ingredients
particularly mentioned above, the formulations of the subject
invention can include other agents conventional in the art having
regard to the type of formulation in question. The pharmaceutical
compositions can be included in a container, pack, or dispenser,
together with instructions for administration.
[0060] The particles of the subject invention can be applied as a
film or coating on a substrate. The substrate can be composed of
any material, such as metal, polymer, and/or ceramic materials.
[0061] The term "patient", as used herein, refers to any vertebrate
species. Preferably, the patient is of a mammalian species.
Mammalian species which benefit from the disclosed methods of drug
delivery and/or detoxification include, and are not limited to,
apes, chimpanzees, orangutans, humans, monkeys; domesticated
animals (e.g., pets) such as dogs, cats, guinea pigs, hamsters,
Vietnamese pot-bellied pigs, rabbits, and ferrets; domesticated
farm animals such as cows, buffalo, bison, horses, donkey, swine,
sheep, and goats; exotic animals typically found in zoos, such as
bear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,
giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie
dogs, koala bears, kangaroo, opossums, raccoons, pandas, hyena,
seals, sea lions, elephant seals, otters, porpoises, dolphins, and
whales.
[0062] The particles of the subject invention can be used in novel
therapeutic systems in which ferrous components are associated with
the particles so as to impart magnetic properties to the particles.
The magnetic properties of the particles can induce and control the
release of the active agent via a "magnetic switch" that may be
operated from outside the body. In some therapeutic approaches,
systems of particles and active agents can be selectively
accumulated in their target area using external magnetic fields.
For treating very special problems, small magnets can be implanted
within the patient for local control in the target area, e.g., a
tumor area.
[0063] The particles of the subject invention are useful in
diagnostic applications, as well. For example, the particles of the
subject invention can incorporate, or otherwise be associated with,
visualization markers, and are applicable for many special
indications such as magnetic resonance (MR) lymphography after
intravenous or local interstitial administration, tumor
visualization, visualization of functions or malfunctions, of
plaque (atherosclerosis imaging), thrombi and vascular occlusions,
MR angiography, perfusion imaging, infarct visualization,
visualization of endothelial damages, receptor imaging,
visualization of blood-brain barrier integrity, etc., as well as
for differential diagnosis, in particular, for distinguishing
tumors/metastases from hyperplastic tissue.
[0064] The particles are also useful for industrial applications,
such as use as light-weight pigment/filler particles or as
platelets for high contrast print gloss.
[0065] The terms "comprising", "consisting of", and "consisting
essentially of" are defined according to their standard meaning and
may be substituted for one another throughout the instant
application in order to attach the specific meaning associated with
each term.
MATERIAL AND METHODS
[0066] Emulsion Substrate Svnthesis. Oil-in-water emulsion droplets
were synthesized by blending in a household kitchen blender,
n-dodecane oil (SIGMA-ALDRICH) and distilled water in 1:9 volume
ratio, stabilized with 1% w/v stearic acid (SIGMA-ALDRICH) (per oil
phase volume). The distilled water was adjusted to the desired pH
using 0.1M NaOH (FISHER SCIENTIFIC) prior to emulsification.
[0067] Particle Synthesis. Immediately after preparing the
emulsion, as indicated above, 1 mL of the emulsion was pippetted
into 35 mm FALCON polystyrene petri dishes, followed by 1 mL of an
80 mM/400 mM CaCl.sub.2/MgCl.sub.2 solution (SIGMA-ALDRICH)
(freshly prepared using distilled water, and filtered by 0.2 .mu.m
ACRODISC syringe filters). Next, 36 .mu.L of a freshly prepared and
filtered 1 mg/mL solution of poly-(.alpha.,.beta.)-D,L-aspartic
acid (MW 8600) (ICN/SIGMA-ALDRICH) was transferred to each petri
dish by micropipet. The petri dishes were then covered by parafilm,
which was punched with a small hole, into which the outflow end of
the tubing from an ultra-low flow peristaltic pump (FISHER
SCIENTIFIC) was inserted. At a rate of approximately 0.025 mL/min,
2 mL of a freshly prepared and filtered solution of 300 mM
(NH.sub.4).sub.2CO.sub.3 (SIGMA-ALDRICH) was pumped into each petri
dish (taking about 80 minutes to complete). The resulting product
was collected and centrifuged at 8000 rpm for 10 minutes, rinsed
with saturated CaCO.sub.3 (SIGMA-ALDRICH), then re-centrifuged
under the same conditions. After a rinsing with ultrapure ethanol
(FISHER SCIENTIFIC), the product was re-centrifuged a final time
under the same conditions, and then left to dry in air
overnight.
[0068] Determination of Particle Morphology and Composition. The
dried particles were examined by an OLYMPUS BX60 polarized light
microscope, using a gypsum wave-plate in order to observe both
amorphous and crystalline phases. For scanning electron microscopy
(SEM) observations, particle samples were spread onto aluminum
studs, and then gold-coated and examined with a JEOL 6400 SEM.
Energy Dispersive Spectroscopy (EDS) was used for elemental
composition analysis of the particle shell. For diffraction
studies, dried particles were adhered to double-sided tape, and
analyzed in a PHILIPS APD 3720 X-ray instrument.
EXAMPLE 1
Formation of Free-Standing Films of Calcium Carbonate Under
Langmuir Monolayers
[0069] As a preliminary step to core-shell particle fabrication,
and to better understand the deposition of calcium carbonate films
on surfactant templates, the formation of freestanding films of the
mineral under Langmuir monolayers spread at the air-liquid
interface was investigated. FIGS. 2A and 2B show polarized light
micrographs of mineral films deposited under stearic acid
monolayers. The micrographs were taken using a gypsum wave plate,
which renders amorphous material to appear as the same magenta
color as the background. As seen by the lack of birefringence in
FIG. 2A, the initial film is amorphous and optically isotropic
(iso). Interestingly, the film cracked like a brittle glass when
scooped onto a coverslip, which is not typical for an amorphous
calcium carbonate (ACC) phase (granular ACC precipitates are
produced from highly supersaturated solutions). If the films are
removed from solution and let to dry in air, they crystallize in
either spherulitic (sph) or single-crystalline (sc) patches (FIG.
2B). Similar results were obtained under arachidic acid
monolayers.
[0070] Repeating this experiment using cholesterol or diolein
surfactants, in contrast, did not yield the uniform mineral film
under the monolayer. Both stearic acid and arachidic acid
surfactants have partially deprotonated carboxylic acid headgroup
functionalities, while cholesterol and diolein surfactants, which
bear alcohol moieties, remain polar but uncharged. Therefore, the
surface charge on the monolayer is thought to play an important
role in attracting mineral species and the ion-binding polymer to
the surface, serving to increase ion saturation, and induce the
deposition of the mineral precursor.
EXAMPLE 2
Surface-Induced Deposition of a Mineral Shell onto a Charged
Emulsion Droplet
[0071] Using stearic acid as a surfactant, n-dodecane oil was
dispersed in water to form an oil-in-water emulsion. To coat these
emulsion droplets, they were first combined with Ca.sup.2+
dissolved in aqueous solution, along with polyaspartic acid to
induce the PILP process. Mg.sup.2+ ions were also added to enhance
the inhibitory action of the polymer, which helps to inhibit
traditional crystal growth from solution (as opposed to from the
precursor phase). The CO.sub.3.sup.2- counterion was subsequently
pumped into the above mixture using ultra-low-flow peristaltic
pumps. To monitor its effect on mineral deposition, the surface
charge on the surfactant layer was varied by adjusting the pH of
the aqueous solutions between 7 and 11 (pK.sub.a of stearic acid is
10.15).
[0072] FIG. 3A shows freshly coated particles synthesized in this
manner at pH 7. As detected from the lack of birefringence under
cross-polarized light, the particles, as expected, initially had an
amorphous CaCO.sub.3 shell. After rinsing the particles with
saturated CaCO.sub.3 and ethanol, the particles were allowed to dry
in air. FIG. 3B shows particles synthesized at pH 8 that were
allowed to age in air for 1 week. The presence of birefringence in
some of the spherical shells can now be detected, indicating an
amorphous to crystalline phase transformation had taken place in
the mineral shell, as was observed in the thin flat films.
Furthermore, the Maltese cross pattern in the birefringence (see
FIG. 3C, which is a magnification of FIG. 3B, lower right)
indicates a spherulitic crystalline structure of the shell. The
polycrystalline nature of spherulites suggests that the shell is
naturally porous (without requiring patterning of the deposition
process), although it is at a very fine scale since the particles
appear smooth at relatively high magnification.
[0073] Under scanning electron microscopy (SEM), the morphology and
uniformity of the particles were better judged. From these
observations, particles synthesized at pH 11 yielded the best
results--fairly monodisperse, uniformly spherical particles of
diameter ranging between 1-5 .mu.m (see FIG. 4A). Since the
formation of a shell around the emulsion droplets was most enhanced
at this pH setting, the increased surface charge at pH 11, compared
to lower pHs, was deemed to be instrumental in the deposition of
the PILP precursor. A sample of these particles was sheared between
glass slides, and SEM of the resulting product is pictured in FIG.
4B. The presence of spherical shell fragments and hollow cores
confirms the core-shell structure of the fabricated particles. The
shell thickness, based on SEM images such as those shown in FIGS.
4A-4C, was observed to be between 200 and 500 nm in thickness. No
specific correlation between particle diameter and shell thickness
was noticed, although it is thought that by controlling the
reaction time, this property might be tailorable. The shell is a
smooth uniform coating (FIG. 4C), and although it appears to be
spherulitic, it is not composed of an aggregation of individual
crystals that nucleated from solution on the template, but rather
it transformed from an amorphous precursor phase. Using Energy
Dispersive Spectroscopy (FIG. 4D), the presence of Ca, Mg, and O in
the particle shell was confirmed, suggesting that the mineral is a
Mg-bearing CaCO.sub.3 phase (which is also seen for PILP films
deposited onto solid substrates).
[0074] In summary, the synthesis of core-shell particles was
carried out using an oil-in-water emulsion as a substrate. The
reaction chemistry was conducted at a consistent final Ca.sup.2+
and CO.sub.3.sup.2- concentration of 20 mM and 150 mM respectively,
with a polymer level varied between 0 and 300 .mu.g/ml, and a Mg
level varied between 0 and 100 mM. Peristaltic pumping was employed
to introduce the CO.sub.3.sup.2- counterion into the reaction
container. This pumping technique was utilized to synthesize
core-shell particles under the following conditions: Ca/Mg=20/100
mM; CO.sub.3.sup.2-=150 mM; polymer=10 .mu.g/ml; and a pH 11
environment. Since these conditions seemed to yield the best
particles, further testing was conducted on particles fabricated
under these conditions.
EXAMPLE 3
Deposition of Mineral Shell in Absence of PILP-Enhancing
Polymer
[0075] When a PILP-enhancing polymer was not included in the
reacting solution, core-shell particles could, under certain
conditions, still be synthesized successfully. In some tests, the
particles made without polymer were indistinguishable from those
made with polymer under optical microscopy. Under SEM, however, the
particles formed without polymer did not always match the quality
of those made with polymer--a portion of the product was not
uniformly spherical but of some modified amorphous shape. The
reason these particles formed even in the absence of polymer is
most likely due to the relatively high amount of Mg used (at a Ca
to Mg ratio of 1 to 5). Mg is a potent crystal growth inhibitor and
may have elicited a PILP-like mechanism in the formation of the
shell.
[0076] A series of tests were therefore conducted, varying both the
polymer and Mg levels in the synthesis procedure, to determine
their effects. In the absence of Mg, no particles formed at all.
Instead, polymer-modified crystals were abundant at all levels of
polymer (tested between 10 and 300 .mu.g/ml). In a second set of
experiments, polymer concentration was maintained at either 10
.mu.g/ml or 100 .mu.g/ml, and the final concentration of Mg in the
reacting solution was varied between 20 and 100 mM. In this case,
core-shell particles were successfully synthesized at Mg levels as
low as 20 mM, and at polymer concentrations of 10 .mu.g/ml.
[0077] Apparently, a small amount of Mg (at 1:1 ratio of Ca/Mg) is
necessary to promote the formation of a core-shell particle.
However, increased polymer level significantly perturbed the
process. Particles formed at Mg=80 mM and at a polymer
concentration of 10 and 100 .mu.g/ml, respectively, were compared.
At 100 .mu.g/ml, the particles formed poorly--without uniformity in
shape or sizes, while the lower polymer samples formed normally.
This trend held true at every Mg level tested (20, 40, 60, and 80
mM)--the particles with higher polymer doses did not form as well
as with lower polymer doses. Since more polymer is likely to better
inhibit mineral nucleation, the higher doses may not have allowed a
shell to deposit or solidify very well on the emulsion droplet. In
addition, because of the charge associated with the acidic polymer
(especially at the pH 11 condition of the experiment), the high
polymer level may have compromised the stability or function of the
emulsion droplet.
EXAMPLE 4
Degradability of Particles Svnthesized using PILP and
PILP-Enhancing Polymer
[0078] Because the shells of the core-shell particles are generated
via PILP, they are in a metastable amorphous state. This suggests
that the particle shells are susceptible to biodegradation once
reintroduced into the blood. This is considered an important
advantage of a CaCO.sub.3 core-shell particle for use in drug
detoxification, as it facilitates the removal of particle
components from the blood stream. To determine whether these
particles are indeed degradable, samples of dried particles were
dispersed in buffered saline solutions, and monitored for several
weeks. The particles were continually stirred while in solution to
simulate the constant agitation expected if they were flowing
within the circulatory system.
[0079] Particles dispersed in phosphate buffered saline solutions
(PBS) (pH .about.7.4) at a concentration of approximately 4 mg/mL
of and 16 mg/mL lost their spherical shape due to dissolution as
early as a week after dispersion, and the remaining material
eventually recrystallized into several crystal morphologies.
"Concentration" in this case is defined as mg of particles per mL
of solution (saline, blood, etc.), and the tested concentrations
are within the range that is proposed for detoxification of an
overdosed patient. While the particle shell is degradable, the
component materials remained as insoluble precipitates if not
sufficiently diluted. In the blood stream, the reprecipitation is
less likely since the larger volume will dilute the ionic species
created during particle degradation.
EXAMPLE 5
Drug-Uptake Efficiency of Particles Synthesized using PILP and
PILP-Enhancing Polymer
[0080] To determine whether these particles were capable of
uptaking lipophilic drugs, High Performance Liquid Chromatography
(HPLC) was employed. The test drug used for these studies was
amitriptyline (AMT). AMT is the most widely prescribed tricyclic
anti-depressant (TCA) in the United States. The drug is a
significant cause for hospitalizations due to toxicity and has been
reported as the most common cause of drug related deaths and
suicide. Other drugs in this class include clomipramine,
desipramine, imipramine, norclomipramine, nortriptyline, and
trimipramine, but AMT is more typically prescribed. AMT is a highly
lipophilic drug and is thought to effectively treat depression by
blocking the physiological inactivation of biogenic amines.
[0081] Particles were introduced into saline solutions isotonic to
blood at concentrations of 0.01%, 0.025%, and 0.05% (1%=1 mg
particles/10 mL solution). AMT was then added and concentrated to 1
mM in the mixture. That mixture was sonicated for 5 minutes and
then filtered by centrifugation for 30 minutes, during which time
the particles were presumably absorbing the AMT. The amount of AMT
remaining in the resulting filtrate was then assessed by HPLC.
[0082] Three different samples were tested for comparison purposes.
The first sample was dried core-shell particles containing the oily
core. The second sample was core-shell particles that were calcined
at 240.degree. C. for 1.5 hours while simultaneously vacuum dried
to evaporate any n-dodecane oil in or on the particle (b.p. of
n-dodecane=216.degree. C.). From optical microscopy observations,
the structure of these treated core-shell particles remained
unaffected. The third sample was commercial CaCO.sub.3 obtained
from SIGMA-ALDRICH (mostly calcite crystals). The hypothesis was
that particles with oil would absorb significantly larger amounts
of AMT from solution than both particles without oil and the
commercial calcite samples, since the advantage of partitioning the
lipophilic molecules into the oily core was not available to the
latter two.
[0083] Results of this uptake study are shown in FIG. 5. The
particles with oil were shown to extract 83% of AMT at the lower
particle concentrations and upwards of 97% at 0.025% and 0.05% w/v
levels. Surprisingly, the particles without oil were able to
extract just as much drug as the oil-filled particles at the 0.025%
and 0.05% levels, and only 10% less at 0.01%. While the commercial
CaCO.sub.3 did absorb the least amount of AMT, the quantity of drug
that was extracted from solution was also unexpectedly high (as
high as 50%). The commercial crystals used in this last sample
measured as large as 40.mu.m, and therefore the sample's surface
area did not compare well to particle samples tested. Therefore, a
fourth sample was tested for drug uptake--that of commercial
CaCO.sub.3 crystals that were mill ground for 18 hours to reduce
the crystal size, and therefore increase the surface area, to a
range comparable to the particles samples. In this case, the sample
was able to uptake up to 85% of the AMT drug, as shown in FIG. 5.
In light of this, it is feasible that nano-scale CaCO.sub.3
crystals could uptake over 90% of AMT from saline solutions. This
is an important finding as it suggests that the partitioning the
drug into the oily core of the particle may not be the most
prevalent mechanism of drug uptake as initially thought. The drug
may also be adsorbing significantly to the particle or crystal
surfaces.
EXAMPLE 6
Drug Detoxification using Calcium Carbonate Core-Shell
Particles
[0084] The core-shell particles of the subject invention are
particularly useful for the detoxification of lipophilic drugs
within a patient in need of such detoxification. In one embodiment,
an effective amount of core-shell particles are administered to the
patient, such as through intravenous injection, wherein the unbound
lipophilic drug (e.g., unbound to blood protein) is simply absorbed
through the calcium carbonate shell of the particles, and into
their core, effectively partitioning the lipophilic drug from the
patient's bloodstream, for example. The particles can then be
allowed to degrade, releasing the lipophilic drug over a period of
time that is not harmful to the patient. Alternatively, the
particles can be retrieved from the patient using known methods of
particulate retrieval. In another embodiment, one or more
drug-detoxifying enzymes (also referred to herein as a
drug-detoxifying system) are incorporated within, or otherwise
associated with, the particles of the subject invention. In another
embodiment, compounds which act as inducers of endogenous drug
detoxifying enzymes can be incorporated within, or otherwise
associated with, the particles of the subject invention.
[0085] Drug biotransformation usually involves two phases, phase I
and phase II. Phase I reactions are classified typically as
oxidations, reductions, or hydrolysis of the parent drug. Following
phase I reactions, the metabolites are typically more polar
(hydrophilic), which increases the likelihood of their excretion by
the kidney. Phase I metabolic products may be further metabolized.
Phase II reactions often use phase I metabolites that can catalyze
the addition of other groups, e.g., acetate, glucuronate, sulfate,
or glycine to the polar groups present on the intermediate.
Following phase II reactions, the resultant metabolite is typically
more readily excreted. The drug detoxifying enzymes utilized in the
subject invention can catalyze phase I reactions, phase II
reactions, or both phase I and phase II reactions, for example.
[0086] Most phase I reactions are catalyzed by the cytochrome P450
(CYP) enzyme system, which is a superfamily consisting of
heme-containing isozymes (van der Weide and, J. and Steijns, L.,
"Cytochrome P450 Enzyme System: Genetic Polymorphisms and Impact on
Clinical Pharmacology", Ann. Clin. Biochem., 36:722-729, 1999). At
least 74 CYP gene families, of which 14 are ubiquitous in all
mammals, have been described thus far (Nelson, D. R. et al., "P450
Superfamily: Update on New Sequences, Gene Mapping, Accession
Numbers, and Nomenclature", Pharmacogenetics, 6:1-42, 1996). The
enzymes belonging to the families CYP1, CYP2, and CYP3 catalyze the
oxidative biotransformation of exogenous compounds, including many
drugs, (pro)carcinogens, (pro)-mutagens, and alcohols. Other CYP
families are involved in the metabolism of endogenous substances,
such as fatty acids, prostaglandins, and steroid and thyroid
hormones. Specific catalytic activities that have been observed
with regard to some P450 isoforms in in vitro assays include
testosterone 6-hydroxylase activity of CYP3A4, dextromethorphan
O-deethyolase activity of CYP2D6, tolbutamide 4-hydroxylase
activity of CYP2C9, phenacitin O-deethylase activity of CYP1A2,
(S)-Mephenytoin 4'-hydroxylase activity of CYP2C19, chorozoxazone
6-hydroxylase activity of CYP2E1, coumarin 7-hydroxylase activity
of CYP2A6, lauric acid 12-hydroxylase activity of CYP4A11, and
paclitaxel 6-hydroxylase activity of CYP2C8. As indicated above,
one or more P450 enzymes can be incorporated within, or otherwise
associated with the particles of the subject invention.
Alternatively, inducers of endogenous P450 enzyme activity can be
incorporated within, or otherwise associated with, the particles of
the present invention. For example, there are over twenty different
CYP enzymes within the human body, with at least six of the enzymes
(CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A) accounting for
the metabolism of nearly all clinically useful medications.
Examples of P450 enzymes and their corresponding substrate
specificities are listed in Table 1. TABLE-US-00001 TABLE 1
Examples of Cytochrome P450 (CYP) Enzymes and Corresponding
Substrates Enzyme Substrates CYP1A2 caffeine, clozapine,
fluvoxamine, haloperidol, paracetamol, theophylline, acetominophen,
amitriptyline, antipyrine, clomipramine, enoxacin, imipramine,
olanzapine, ondansetron, phenacetin, propranolol, tacrine,
R(-)warfarin, verapamil CYP2A6 coumarin CYP286 cyclophosphamide
CYP2C8 arachidinic acid, paclitaxel, retinoic acid, warfarin CYP2C9
cyclophoshamide, diclofenac, hexobarbital, ibuprofen, mefanamic
acid, naproxen, phenytoin, piroxicam, tenoxicam, thiotepa,
tolbutamide, TCAs, torsemide, S(-)warfarin CYP2C19 amitriptyline,
clomipramine, diazepam, heoxbarbital, imipramine, lansoprozole,
mephenytoin, mephobarbital, mclobemide, omeprazole, proguarnil,
-propranolol CYP2D6 antiarrhythmics such as encainide, flecainide,
mexiletine, propafenone; antipsychotics such as clozapine
haloperidol, perphenazine, reduced haloperidol, risperidone,
thioridazine; beta-blockers such as bufuralol, metoprolol,
propranolol, timolol; opiates such as codeine, dextromethorphan,
hydromorphone, methadone, oxycodone, tramadol; TCAs such as
amitriptyline, clomipramine, desipramine, imipramine,
norclomipramine, nortriptyline, trimipramine; SSRIs such as
fluoxetine, paroxetine; miscellaneous antidepressants such as
maprotiline, nefazodone, venlafaxine; antihypertensives;
debrisoquin; methlenedioxymethamphetamine (Ecstasy); ondansetron;
phenformin; sparteine; tacrine; terfenadine; tropisetron; verapamil
CYP2E1 acetominophen, chlorzoxazone, ethanol, enflurane, halothane,
acetone, paracetamol CYP3A4 antiarrhythmics such as amiodarone,
lidocaine, propafenone, quinidine; antidepressants such as
bupropion, sertraline, TCAs (amitriptyline, clomipramine,
desipramine, imipramine, norclomipramine, nortriptyline,
trimipramine), venlafaxine; benzodiazepines such as alprazolam,
diazepam, midazolam, triazolam; calcium channel blockers such as
diltiazem, felodipine, nifedipine, nimodipine, nisoldipine,
verapamil; nonsedating antihistamines such as astemizole,
terfenadine; acetaminophen; alfentanil; amiodarone; codeine;
cyclosporin A/G; carbamazepine; cyclophosphomide; cortisol;
dapsone; dexamethasone; dextromethorphan; doxorubicin; erythromycin
(N--CH3); ethinylestradial etoposide; fentanyl; felodipine;
ifosfamide; lansoprozole; lidocaine; lomustine; lavastatin;
omeprazole; ondansetron; progesterone; tamoxifen; taxol;
testosterone; triacetyloleandomycin (TAO); vincristine;
vinblastine; vinorebine; warfarin; methadone Note: SSRIs =
selective serotonin reuptake inhibitors; TCAs = tricyclic
antidepressants
[0087] The drug-detoxifying enzyme can be contained within the core
of the core-shell particles or otherwise associated with the
particles. For example, the drug-detoxifying enzyme can be adsorbed
onto the calcium carbonate shell of the particles.
[0088] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0089] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application.
* * * * *