U.S. patent application number 13/322790 was filed with the patent office on 2012-03-29 for method and apparatus for continuous removal of submicron sized particles in a closed loop liquid flow system.
Invention is credited to Hiroshi Mizukami, Agnes Ostafin.
Application Number | 20120077662 13/322790 |
Document ID | / |
Family ID | 43628342 |
Filed Date | 2012-03-29 |
United States Patent
Application |
20120077662 |
Kind Code |
A1 |
Ostafin; Agnes ; et
al. |
March 29, 2012 |
Method And Apparatus For Continuous Removal Of Submicron Sized
Particles In A Closed Loop Liquid Flow System
Abstract
A method and apparatus for continuous removal of submicron sized
artificial oxygen carriers (rAOC) and other materials such as
cancer cells and bacteria from blood and other liquids. A
centrifuge rotor having a curved shape is offset on a spinning
rotor base and creates contiguous areas of low to high centrifugal
force depending on the distances from the axis of the rotor base.
This creates a density gradient field that separates materials of
different densities input to the centrifuge that exit via different
outputs. A monitor detects any red blood cells (RBC) with the rAOC
before they exit the centrifuge. If there are any RBC detected
logic circuitry changes the speed of rotation of the rotor, and the
flow rate of pumps inputting and removing separated blood and rAOC
to and from the centrifuge until there are no RBC in the rAOC
exiting the centrifuge.
Inventors: |
Ostafin; Agnes; (Layton,
UT) ; Mizukami; Hiroshi; (Pasadena, CA) |
Family ID: |
43628342 |
Appl. No.: |
13/322790 |
Filed: |
August 24, 2010 |
PCT Filed: |
August 24, 2010 |
PCT NO: |
PCT/US2010/046421 |
371 Date: |
November 28, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61236810 |
Aug 25, 2009 |
|
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|
Current U.S.
Class: |
494/1 ; 494/10;
494/60; 494/68 |
Current CPC
Class: |
A61K 31/01 20130101;
A61K 9/5192 20130101; A61K 9/5073 20130101; A61K 9/5169 20130101;
A61K 9/5115 20130101; A61K 9/5123 20130101; B04B 5/0442 20130101;
A61M 1/3687 20130101; B04B 2013/006 20130101; B01D 21/34 20130101;
B04B 2005/045 20130101; A61K 9/0026 20130101; A61K 38/42 20130101;
A61M 1/3696 20140204; A61P 7/00 20180101; B04B 7/08 20130101; A61K
9/5146 20130101; B01D 21/262 20130101; A61P 7/08 20180101; A61M
1/3693 20130101; A61M 1/3679 20130101; B04B 13/00 20130101 |
Class at
Publication: |
494/1 ; 494/68;
494/60; 494/10 |
International
Class: |
B04B 13/00 20060101
B04B013/00; B04B 7/04 20060101 B04B007/04; B04B 15/00 20060101
B04B015/00; B04B 7/12 20060101 B04B007/12 |
Claims
1. A rotor for a centrifuge used to separate components having
different densities from a mixture of the components, the rotor
comprising: a rotor base having a central axis and the rotor base
is rotated about the central axis when the centrifuge is in use; a
first rotor element that is curved and is attached to and has an
orientation extending away from the rotor base, the first rotor
element having a first end and a second end; and a second rotor
element that is curved and is attached to and has an orientation
extending away from the rotor base, the second rotor element having
a first end and a second end, the second end of the first rotor
element being connected to the first end of the second rotor
element to form a composite rotor element; wherein the composite
rotor element is positioned on the rotor base so that the first end
of the first rotor element and the second end of the second end of
the second rotor element are at different distances from the
central axis.
2. The centrifuge rotor of claim 1 further comprising: a centrifuge
housing in which the composite rotor element on the rotor base is
mounted and is rotated; a first output port through the sidewall of
the centrifuge housing for removing a first component of the
mixture of components input to the centrifuge housing; a second
output port through the sidewall of the centrifuge housing for
removing a second component of the mixture of components input to
the centrifuge housing, the spacing between the first and second
output ports being substantially the same spacing as the spacing
between the first end of the first rotor element and the second end
of the second rotor element; an input port through the sidewall of
the centrifuge housing through which the mixture of components is
input to the centrifuge housing, said input port being closer to
the second end of the second rotor element than to the first end of
the second rotor element which is connected to the second end of
the first rotor element to form the composite rotor element.
3. The centrifuge rotor of claim 2 wherein when the rotor base with
composite rotor element mounted thereon is rotated inside the
centrifuge housing the orientation of the composite rotor element
on the rotor base creates a density gradient that separates two
components of the mixture of components that is input to the
centrifuge housing, where the two components have different
densities, and a first of the two components moves in a first
direction inside the centrifuge housing and is removed from the
centrifuge housing at the first output port while a second of the
two components moves in a second, opposite direction inside the
centrifuge housing and is removed from the centrifuge housing at
the second output port.
4. The centrifuge rotor of claim 3 further comprising: a monitor
port through the sidewall of the centrifuge housing, the monitor
port being closer to the second output port at the second end of
the second rotor element than the input port is, the monitor port
being used to extract a sample of the second of the two components
moving toward the second output port, the sample being used to
determine if the first of the two components has been separated
from the second component.
5. The centrifuge rotor of claim 4 further comprising: an outwardly
extending end at the first end of the first rotor segment and at
the second end of the second rotor segment, wherein as the rotor
turns inside the centrifuge housing these two ends create a
pressure pushing the first component of the mixture of components
toward the first output port and pushing the second component of
the mixture of components toward the second output port.
6. The centrifuge rotor of claim 5 further comprising: a sensor
connected to the monitor output port to monitor the sample of the
second of the two components moving toward the second output port
and extracted at the monitor port for the presence of any of the
first of the two components, the sensor generating an output signal
if any of the first of the two components is present; and
electronics receiving the output signal from the sensor, the
electronics causing a change in the rate at which the first of the
two components is removed from the centrifuge at the first output
port, and changing the rate at which the second of the two
components is removed from the centrifuge at the second output port
to eliminate the presence of any of the first of the two components
in the sample taken at the monitor output port, thus assuring there
is none of the first of the two components present with the second
of the two components exiting the centrifuge at the second output
port.
7. The centrifuge rotor of claim 6 wherein the electronics also
causes a change in the rate at which the mixture of components is
input to the centrifuge housing to assure there is none of the
first of the two components present with the second of the two
components exiting the centrifuge at the second output port.
8. The centrifuge rotor of claim 2 further comprising: a monitor
port through the sidewall of the centrifuge housing, the monitor
port being closer to the second output port at the second end of
the second rotor element than the input port is, the monitor port
being used to extract a sample of the second of the two components
moving toward the second output port, the sample being used to
determine if the first of the two components has been separated
from the second component.
9. The centrifuge rotor of claim 8 further comprising: an outwardly
extending end at the first end of the first rotor segment and at
the second end of the second rotor segment, wherein as the rotor
turns inside the centrifuge housing these two ends create a
pressure pushing the first component of the mixture of components
toward the first output port and the second component of the
mixture of components toward the second output port.
10. The centrifuge rotor of claim 9 wherein when the rotor base
with composite rotor element mounted thereon is rotated inside the
centrifuge housing the orientation of the composite rotor element
on the rotor base creates a density gradient that separates two
components of the mixture of components that is input to the
centrifuge housing, where the two components have different
densities, and a first of the two components moves in a first
direction inside the centrifuge housing and is removed from the
centrifuge housing at the first output port while a second of the
two components moves in a second, opposite direction inside the
centrifuge housing and is removed from the centrifuge housing at
the second output port.
11. The centrifuge rotor of claim 4 further comprising: a sensor
connected to the monitor output port to monitor the sample of the
second of the two components moving toward the second output port
and extracted at the monitor port for the presence of any of the
first of the two components, the sensor generating an output signal
if any of the first of the two components is present; and
electronics receiving the output signal from the sensor, the
electronics causing a change in the rate at which the first of the
two components is removed from the centrifuge at the first output
port, and changing the rate at which the second of the two
components is removed from the centrifuge at the second output port
to eliminate the presence of any of the first of the two components
in the sample taken at the monitor output port, thus assuring there
is none of the first of the two components present with the second
of the two components exiting the centrifuge at the second output
port.
12. The centrifuge rotor of claim 11 wherein the electronics also
causes a change in the rate at which the mixture of components is
input to the centrifuge housing to assure there is none of the
first of the two components present with the second of the two
components exiting the centrifuge at the second output port.
13. The centrifuge rotor of claim 12 wherein when the rotor base
with composite rotor element mounted thereon is rotated inside the
centrifuge housing the orientation of the composite rotor element
on the rotor base creates a density gradient that separates two
components of the mixture of components that is input to the
centrifuge housing, where the two components have different
densities, and a first of the two components moves in a first
direction inside the centrifuge housing and is removed from the
centrifuge housing at the first output port while a second of the
two components moves in a second, opposite direction inside the
centrifuge housing and is removed from the centrifuge housing at
the second output port.
14. A rotor for a centrifuge used to separate whole blood from
other artificial blood having a density higher than any of the
components of the whole blood, the rotor comprising: a rotor base
having a central axis and the rotor base is rotated about the
central axis when the centrifuge is in use; a first rotor element
that is curved and is attached to and has an orientation extending
away from the rotor base, the first rotor element having a first
end and a second end; and a second rotor element that is curved and
is attached to and has an orientation extending away from the rotor
base, the second rotor element having a first end and a second end,
the second end of the first rotor element being connected to the
first end of the second rotor element to form a composite rotor
element; wherein the composite rotor element is positioned on the
rotor base so that the first end of the first rotor element and the
second end of the second end of the second rotor element are at
different distances from the central axis.
15. The centrifuge rotor of claim 14 further comprising: a
centrifuge housing in which the composite rotor element on the
rotor base is mounted and is rotated; a first output port through
the sidewall of the centrifuge housing for removing the whole blood
from the artificial blood input to the centrifuge housing; a second
output port through the sidewall of the centrifuge housing for
removing the higher density artificial blood input to the
centrifuge housing along with the whole blood, the spacing between
the first and second output ports being substantially the same
spacing as the spacing between the first end of the first rotor
element and the second end of the second rotor element; an input
port through the sidewall of the centrifuge housing through which
the mixture of whole blood and artificial blood is input to the
centrifuge housing, said input port being closer to the second end
of the second rotor element than to the first end of the second
rotor element which is connected to the second end of the first
rotor element to form the composite rotor element.
16. The centrifuge rotor of claim 15 wherein when the rotor base
with composite rotor element mounted thereon is rotated inside the
centrifuge housing the orientation of the composite rotor element
on the rotor base creates a density gradient that separates the
whole blood from the artificial blood where the components of the
whole blood have a lower density than the artificial blood, and a
first of the whole blood moves inside the centrifuge housing toward
and is removed from the centrifuge housing at the first output port
while the artificial blood moves inside the centrifuge housing
toward and is removed from the centrifuge housing at the second
output port.
17. The centrifuge rotor of claim 16 further comprising: a monitor
port through the sidewall of the centrifuge housing, the monitor
port being closer to the second output port at the second end of
the second rotor element than the input port is, the monitor port
being used to extract a sample of the artificial blood moving
toward the second output port, the sample being used to determine
if the whole blood has been completely separated from the
artificial blood.
18. The centrifuge rotor of claim 17 further comprising: an
outwardly extending end at the first end of the first rotor segment
and at the second end of the second rotor segment, wherein as the
rotor turns inside the centrifuge housing these two ends create a
pressure pushing the whole blood toward the first output port and
the artificial blood toward the second output port.
19. The centrifuge rotor of claim 18 further comprising: a sensor
connected to the monitor output port to monitor the sample of the
artificial blood moving toward the second output port and extracted
at the monitor port to test for the presence of any whole blood
components, the sensor generating an output signal if any of the
first of the two components is present; and electronics receiving
the output signal from the sensor, the electronics causing a change
in the rate at which the first of the two components is removed
from the centrifuge at the first output port, and changing the rate
at which the second of the two components is removed from the
centrifuge at the second output port to eliminate the presence of
any of the first of the two components in the sample taken at the
monitor output port, thus assuring there is none of the first of
the two components present with the second of the two components
exiting the centrifuge at the second output port.
20. The centrifuge rotor of claim 19 wherein the electronics also
causes a change in the rate at which the mixture of whole blood and
artificial blood is input to the centrifuge housing to assure there
is none of the whole blood components present with the artificial
blood exiting the centrifuge at the second output port.
Description
[0001] This application claims priority under 35 U.S.C. 119(e) to
U.S. Provisional Application No. 61/236,810 , filed on Aug. 25,
2009.
Field of the Invention
[0002] The present invention relates to a method and apparatus for
continuous removal of submicron sized particles from blood or other
liquids.
BACKGROUND OF THE INVENTION
[0003] In the prior art there are a range of particulate carriers
intended for the controlled delivery of biologically active
substances within the body. Their sizes range from micron to
submicron, and their compositions range from organic (e.g.
polymers, lipids, surfactants, proteins) to inorganic (calcium
phosphate, silicate, CdSe, CdS, ZnSe, gold and others). Each of
these particulate carriers are designed to carry a chemically or
biochemically reactive substance, and either release it over time,
or at a specific location, or both.
[0004] The size of the individual particulate carriers and their
load capacity is controlled by the amount of material used in the
synthesis, the morphology by which the components assemble, and the
specific composition of the components. The synthesized particulate
carriers have the dual function of being able to solubilize or to
be able to bind to the chemically or biochemically reactive
substances intended for ultimate delivery. The underlying
assumption is that the enclosed reactive substances will ultimately
be released so that they can perform their intended functions. The
particulate carriers themselves usually do not participate in the
release function, except to the extent that they regulate the
timing or location of release of the reactive substances they
carry, and the carrier components must either decompose over time,
or remain as non-active and non-toxic substances that do not cause
any harm.
[0005] In the field of medicine such particulate carriers have been
used to serve as artificial oxygen carriers (AOC) in artificial
blood products. Artificial blood is a product made to act as a
substitute for red blood cells which transport oxygen and carbon
dioxide throughout the body. However, the function of real blood is
complicated, and the development of artificial blood has generally
focussed on meeting only a specific function, gas exchange - oxygen
and carbon dioxide.
[0006] Whole blood serves many different functions that cannot be
duplicated by an AOC. Artificial blood mixable with autologous
blood can support patients during surgery and support transfusion
services in emerging countries with limited healthcare, blood
donations and storage facilities, or high risk of exposure to
disease since screening procedures are too expensive. An AOC is a
blood substitute, which is not dependent upon cross matching and
blood-typing would mean no delay in blood availability, and could
mean the difference between life and death of patients. In prior
art medical applications the residual materials from particulate
carriers are expected to be metabolized and/or excreted over time.
However, the disposal of particulate carriers with natural
metabolism of the patients is extremely difficult.
[0007] Another motivation for developing improved AOC is that
despite significant advances in donated blood screening there are
still concerns over the limited shelf life which is 42 days at
2.degree.-6.degree. C.
[0008] In the era of modern science, several decades of extensive
academic, industry research efforts, clinical trials, and spending
multiple billions of dollars, has led to two major classes of AOCs,
namely emulsified perfluorocarbons (PFC) and polymeric hemoglobins
(Hb). While these two types of AOCs each have some advantages, none
are yet approved for clinical use in the U.S.
[0009] Chemically and biologically inert, emulsified, sterilized
perfluorocarbons (PFCs) are stable in storage at low temperatures
2-5.degree. C. for over a year. Further, PFCs are relatively
inexpensive to produce and can be made devoid of any biological
materials eliminating the possibility of spreading an infectious
disease via a blood transfusion. Because they are not soluble in
water they must be combined with emulsifiers able to suspend tiny
droplets of PFC in the blood. In vivo the perfluorocarbon is
ultimately expelled via the lungs after digestion of the emulsifier
by the macrophage/monocyte system. In addition, PFCs are
biologically inert materials that can dissolve about fifty times
more oxygen than blood plasma but less oxygen than red blood cells.
For instance, a mixture consisting of 70% blood and 30%
perfluorocarbon by volume can provide the needed 5 ml of oxygen per
100 ml of blood if the partial pressure of oxygen in the lungs can
be increased to 120 mm Hg by having the patient breath air with an
oxygen partial pressure of approximately 180 mm Hg.
[0010] Perfluorocarbons (PFC) dissolve more oxygen than water, but
still less than normal blood. To supply the needed amount of oxygen
in circulation, patients may require supplemental oxygen. Highly
hydrophobic PFC requires emulsifiers to stabilize the droplet in
blood. These emulsifiers interact with proteins and emulsifiers
found in blood leading to instability. As a result, large
quantities of PFC in circulation in the blood cannot be tolerated.
Small amounts of PFC escape from the blood into the lungs where it
is vaporized and breathed out. Large amounts of PFC and emulsifier
can have a negative effect on lung function.
[0011] Crosslinked, polymerized or encapsulated hemoglobin (pHb)
based artificial oxygen carriers (AOC) are late-comers compared
with perfluorocarbon based AOCs described in previous paragraphs,
and are attracting increasing attention because their oxygen
delivery characteristics are similar to that of the red blood cells
(hereinafter referred to as RBC).
[0012] Polymeric hemoglobins (pHb) bind O.sub.2 and CO.sub.2, with
a binding mechanism much like that of red blood cells (RBC), but
even a small quantity of unpolymerized Hb left in the circulation
can become very toxic. As an artificial oxygen carrier (AOC), a
large amount of pHb needs to be injected into a person. Premature
breakdown can increase the risk of toxicity, and such a large
amount can overtax the body's natural removal processes.
Polymerized Hb remains costly. Animal sources of Hb run the risk of
transferring, among other things prion-based diseases. Recombinant
Hb is a promising approach. It requires high quality separation and
purification procedures, that add to the cost.
[0013] While both polymeric hemoglobins (Hb) and perfluorocarbons
(PFC) based AOC products deliver oxygen in significant quantities
to cells and tissue, their side effects, such as nitric oxide
related vasoconstriction, stroke, cardiac arrest, flu-like symptoms
and long term chemical toxicity, have forced the termination of all
the clinical trials in the U.S. An all out effort to reduce the
toxicity of relatively large quantity of AOC injected into a body
by metabolic decompositions has failed.
[0014] In view of the many problems experienced with artificial
blood products and particulate carriers intended for the controlled
delivery of biologically active substances within the body,
particulate artificial oxygen carriers (AOC) have been developed
that minimize the above described problems in the prior art with
non-particulate AOCs. The particulate AOCs are designed to be
continually circulated in a closed loop fluid circulation system,
are less subject to turbulent breakup, chemical decomposition, or
accumulation of debris, and are capable of exchange of small ions
and gases.
[0015] However, while particulate AOC artificial oxygen carriers
minimize the problems of earlier AOCs that are described above,
they break down in time in the blood so there is a need in the art
for a way to remove them from the body after they have served their
purpose as an artificial oxygen carrier.
SUMMARY OF THE INVENTION
[0016] The need in the prior art described in the previous
paragraph is satisfied by the present invention. To satisfy the
above listed need in the prior art the present invention is a
specialized centrifugal rotor that utilizes density gradient
separation to efficiently remove particulate artificial oxygen
carriers (hereinafter referred to as retrievable AOCs or rAOC) from
blood or other biofluids. In addition, the rAOC is retrieved from a
patients system as soon as its medical purpose is accomplished in
order to alleviate the physiological stress on already compromised
patients.
[0017] With the present invention the particulate rAOCs can be
retrieved at any desired time using continuous flow separation
employing density-gradient centrifugation, which may be
supplemented with magnetic fields, affinity filtration or other
methods, without suffering damage, or inflicting damage on other
materials that may already be present in the flowing fluid.
[0018] Other applications for the present invention include removal
and concentration of metastatic cancer cells from circulating
blood, retrieval of low copy mammalian, bacterial or virus cells,
and tissue and organ imaging. Depending on the application, the
specific design requirement of these materials in terms of their
size and composition may vary, but common to all of them are the
properties summarized earlier, and the tailored ability for
continuous retrieval from circulating fluids using the methods
listed in the previous paragraph.
[0019] To remove the carrier particles from the blood one or more
of the following continuous flow separation methods may be used:
(a) centrifugation, (b) magnetic fields, and/or (c) affinity
filtration without suffering damage or inflicting damage on other
materials that may already be present in the flowing fluid. It is
contemplated that particulate rAOCs be removed from the bloodstream
as soon as possible after they have performed their function, but
prior to degradation of the particulate rAOCs, and subsequent
development of detrimental side effects.
[0020] To meet the criteria for retrievability of the above
described particulate rAOC particles of the present invention from
blood during their use, the particulate material must be submicron
sized (50 nm-700 nm) hollow particles filled with a high density
perfluorocarbon (PFC) and/or a poly hemoglobin (pHb) liquid. The
hollow particles have one or two rigid reinforcing shells. The
exterior surface of these particulate shells are coated with
molecules containing exposed functional groups (COOH, NH.sub.2, SH
etc.) convenient for the crosslinking of either more than one
particle, or proteins like antibodies, cell receptor targets,
polyhemoglobin, hemoglobin etc.
[0021] The single shell coated emulsion particles (rAOC) of the
present invention have a higher density than other components of
blood such as red blood cells, white blood cells and plasma.
Accordingly, centrifugal forces may be utilized to separate the
particles from other blood components, but density gradient is used
rather than a sedimentation velocity method as in the prior art. In
the prior art red blood cells are the furthest moving particles in
a centrifugal field, but with the present invention the novel AOC
is the furthest moving particles in the centrifugal field. With the
AOC being the furthest moving particles in a centrifugal field they
may be separated from all other blood components.
[0022] rAOCs in the blood have a higher density than the blood and
are separated therefrom by continuous flow density gradient
centrifugation that utilizes the higher density of the rAOC
particles to accomplish their separation. Affinity filtration may
also be used to separate the rAOC nano or sub-nano size particles
from the blood.
[0023] In addition, paramagnetic materials may be added to the
higher density PFC in each nanoparticle, and the magnetic
susceptibility is used for the retrieval of the polymerized
hemoglobin. The flowing liquid containing paramagnetic and
diamagnetic materials (the natural blood component) must be exposed
to a magnetic field during the centrifugal separation so that they
will deviate in the direction of the flow of particles with
paramagnetic materials away from the diamagnetic particles, thus
making it possible to separate and collect both types of
particles.
Description of the Drawing
[0024] The invention will be better understood upon reading the
following Detailed description in conjunction with the drawings in
which:
[0025] FIG. 1 is a perspective view of the novel centrifuge that
utilizes density gradient separation to efficiently remove
particulate artificial oxygen carriers from blood or other
biofluids;
[0026] FIG. 2 is a top view of the novel centrifuge that better
shows the novel rotor used in the centrifuge;
[0027] FIG. 3 is a linear graphical representation of the novel
rotor of the centrifuge;
[0028] FIG. 4 is a block diagram of the circuits required for
operation of the novel centrifuge that utilizes density gradient
separation to efficiently remove particulate artificial oxygen
carriers from blood or other biofluids;
[0029] FIGS. 5A and 5B are transmission electron microscope images
of submicron sized blood substitutes optimized for use with the
described invention;
[0030] FIG. 6 is a cross sectional diagram showing how a single
shelled rAOC is constructed; and
[0031] FIG. 7 is a cross sectional representation of a double
shelled, dual core oxygen carrier (DCOC) that wraps a PFC emulsion
core wrapped with a first shell on the outside of which is PolyHB
that is wrapped with a second shell; and
DETAILED DESCRIPTION
[0032] Prior art coated particulate carriers intended for the
controlled delivery of biologically active or medicinal substances
within the body, or to serve as artificial oxygen carriers (AOC),
break down in time in the blood so there is a need in the art for a
way to remove them from the body after they have served their
purpose. Hereinafter, only AOC are specifically mentioned but the
teaching also applies to particulate carriers intended for the
controlled delivery of biologically active or medicinal substances
within the body.
[0033] To meet the criteria for coated/particulate artificial
oxygen carriers that can be temporarily substituted for blood, and
for the retrievability of such coated AOCs (hereinafter referred to
only as retrievable rAOC) from blood using the present invention,
the rAOCs described herein are particulates having shells 12 (see
FIGS. 5A and 5B) that must be submicron sized (50-1000 nm) hollow
particles around a high density perfluorocarbon (PFC) emulsified
nanoparticle. The reinforcing shell 12 is rigid and consists of a
combination of lipids and inorganic materials like calcium
phosphate, silicate, or biocompatible organic polymers such as, but
not exclusively: polycaprolactone, polylactic acid, polyglycolic
acid, polyethylene oxide, chitosan or chondroitin. The rAOCs
nanoemulsion core particles 11 are denser than blood and the higher
density is used to retrieve them from blood using a special
centrifuge. Such shelled rAOCs are shown in and described very
briefly with reference to FIGS. 5, 6, and 7.
[0034] Simply, the novel means of the present invention for
removing such rAOCs from blood comprises having a novel centrifuge
rotor 24 that creates a density gradient that separates the rAOCS
from the blood. In the prior art separation of mixed components is
based sedimentation velocity. This is possible because the density
of rAOC is 1.98 g/ml, while the density of most of the blood
components are only slightly over 1.0 g/ml. A mixture of blood and
rAOCs withdrawn from the body are input to a specific point in the
centrifuge where the rotation of the centrifuge rotor 24 causes the
blood to flow in one direction and the rAOCs to flow in the
opposite direction, and they are both removed from the centrifuge.
Before the separated rAOCs are retrieved a sample of the rAOC flow
is removed from the centrifuge and input to a red blood cell (RBC)
sensor which looks for any red blood cells. If any red blood cells
are detected electronics of the system adjusts the speed of the
pumps inputting and removing the RBC and rAOC from the centrifuge
until no RBC are detected in the rAOCs to be removed from the
centrifuge. In addition, the rotational speed of the novel rotor
inside the centrifuge may also be adjusted. This is shown in and
described hereinafter in greater detail with reference to FIG.
4.
[0035] FIG. 1 is a perspective view of the novel centrifuge rotor
24 that utilizes density gradient separation to efficiently remove
particulate artificial oxygen carriers (rAOC) from blood (RBC) or
other biofluids. The case of the centrifuge and input and output
ports therethrough are not shown in FIG. 1 to make the drawing
simpler so the invention can be better understood. Rotor 24
comprises a circular rotor base 25 that is mounted on an axis 27 to
a motor driven shaft (not shown). As shown in FIG. 1 rotor base 25
is rotated in a counter clockwise direction for the rotor 24
configuration shown and described herein. This direction is
important, based on the arrangement of rotor elements 26a and 26b
and their position on rotor base 25, to create a density based
gradient that separates RBC (output at port 29) from the rAOC
(output at port 28) from a mixture of RBC and rAOC that is input to
the centrifuge at port 31. Distances d3, d4 and dr are shown in all
of FIGS. 1, 2 and 3 to better understand how the Figures relate to
each other. The thickness of rotor 26a,26b is 0.5 cm, the width is
2 cm, and the length is 15 cm, and the volume of the rotor will be
only 15 ml.
[0036] Rotor 24 is made up of two curved elements 26a and 26b that
are joined together to form a curved element 26a,26b that is
oriented perpendicular to rotor base 25. The curvature of element
26b is slightly larger than the curvature of element 26a, and
curved composite element 26a,26b is offset on rotor base 25 as may
be seen in FIG. 1, but is better seen in the top view of FIG. 2. In
FIG. 1 the far left end and the far right end of curved element
26a,26b curve outward a small amount to direct the flow of
separated whole blood to output port 29 and to direct the
separated/retrieved rAOC to output port 28 where they exit the
centrifuge via their respective ports 28, 29 (not shown) through
the case wall (not shown) of the centrifuge. The different
curvatures of elements 26a and 26b and the position of the
composite curved element 26a,26b on rotor base 25 create differing
distances d3, d4 and dr in FIG. 1 where d4>dr>d3. These
distances are shown in FIGS. 1, 2 and 3 to help understand rotor 24
in all the Figures. As shown in FIGS. 1, 2 and 3 a mixture of whole
blood (RBC) and AOCs is typically extracted from a body (not shown)
and is input to the centrifuge at input port 31. As mentioned above
the length of rotor 26a,26b is 15 cm but the separation capacity
per unit time could be increased by enlarging the width of the
rotor 26a,26b to greater than 2 cm. In an alternative embodiment of
the invention the curvatures of rotor segments 26a and 26b may be
the same.
[0037] FIG. 2 is a top view of the novel rotor 24 used in a
centrifuge. As previously mentioned the different curvatures of
rotor elements 26a and 26b and the offset of composite rotor
element 26a,26b on rotor base 25 are best seen in FIG. 2. More
particularly, rotor 26a,26b being belt shaped in the general shape
of an ellipsoid with overlapping ends. With rotor 26a,26b being off
centered on base 25 regions of high, medium and low centrifugal
force are created depending on the distances from the axis of
rotation 27. As previously mentioned the far left end and the far
right end of curved composite element 26a,26b curve outward a small
amount to direct the flow of separated whole blood (RBC) to output
port 29 and to direct the separated/retrieved rAOC to output port
28 where they exit the centrifuge via their respective ports 28, 29
(not shown) through the case wall (not shown) of the centrifuge.
The curvature of composite rotor element 26a,26b and its position
on rotor base 25 is best seen in this Figure. Input 31 where the
composite mixture of RBC and rAOC is input to the centrifuge is
offset from the junction of rotor elements 26a and 28b and is
closer to rAOC output port 28 by a circumferential distance "x" as
shown. The reason for this is described further in this Detailed
Description. The other input and output ports have been previously
described with reference to FIG. 1 so the description is not
repeated here. While two rotor segments are shown in FIGS. 1 and 2,
in alternative embodiments of the invention there may be more than
two rotor segments.
[0038] FIG. 3 is a linear graphical representation of the novel
rotor 24 of the centrifuge. This Figure shows how the distance
between the face of composite rotor elements 26a,26b and the axis
of rotation 27 of rotor 24 changes. Thus, the magnitude of
centrifugal force at different regions of rotor 24 are depicted by
the distance from the axis of rotation 27, which is stretched and
shown as the dotted line at the top of FIG. 2. The distances d3, d4
and dr are shown in all of FIGS. 1, 2 and 3 to better understand
how the Figures relate to each other. The rate of change in
distance is basically linear except where rotor element 26a meets
rotor element 26b. This is due to the fact the curvature of element
26a is different than the curvature of element 26b. In alternative
embodiments of the invention the rate of change in distance may be
uniform, and in another alternative embodiment the rate of change
may be non-linear. Distances d3, d4 and dr between the face of
rotor element 26a,26b and axis 27 are shown to link FIG. 3 with
FIGS. 1 and 2. The input port 31 and output ports 28, 29 and 30 and
their relative position with respect to the linear depiction of
rotor 24 is shown.
[0039] Whole blood including rAOCs obtained from a person who is
connected in a closed loop system with a density gradient
centrifuge is input to the centrifuge at input port 31. The whole
blood is separated from the rAOC because the density of the rAOCs
is greater than the density of the whole blood and any of its
individual components. The whole blood is output at output port 29
and is returned to the person from whom the blood and rAOCs was
withdrawn. The rAOCs are output at port 28 and stored for future
use or disposal. In addition, at a particular location near where
the rAOCs exit the centrifuge via rAOC output port 28, a small
sample is removed from the density gradient centrifuge and exits
the centrifuge at monitor output port 30. The sample is input to a
red blood cell sensor 32 of a control circuit 38 to be checked for
the presence of any remaining red blood cells (RBC) with the rAOCs
about to exit the centrifuge. This is better shown in and described
with reference to FIG. 4. If any RBC are detected control circuit
38 adjusts the speed of the blood and rAOC pumps 36 and 37 that are
part of circuit 38 to permit the centrifuge to fully separate any
remaining RBC from the rAOC before the rAOC reaches monitor output
port 30. This feedback operation assures that only rAOCs exit rAOC
output port 28.
[0040] The centrifugal field generated in the density gradient
centrifuge as novel rotor 24 turns about its axis 27 (FIGS. 1 and
2) creates a density gradient field that changes between output
ports 28 and 29. Depending on the shape of rotor elements 26a and
26b, how they are joined, and how they are positioned on rotor base
25 this density field may change uniformly or it may non-linearly.
The result is that the lower density whole blood fraction is
separated from the higher density rAOC fraction. In an alternative
embodiment another output port may be added somewhere between
output ports 28 and 29 to separate intermediate density fractions
of blood. The separated whole blood and rAOC are withdrawn through
their respective output ports as previously described. The whole
blood collected may be subjected to further fractionation. For
example, further fractionation may be used to separate platelets
and white blood cells from the whole blood in a manner known in the
art.
[0041] More particularly as novel rotor 24 turns the density
gradient field it creates causes the less dense, faster moving
fractions of whole blood to move toward whole blood output port 29
and the more dense rAOC, however, migrate toward an area of the
chamber having the greatest centrifugal force. By selecting the
proper fluid in flow and out flow rates through the centrifuge, the
physical dimensions of the rotor, and the speed of rotation of the
rotor in the centrifuge, faster moving cells and slower moving
cells may be separately extracted from the separation chamber and
subsequently collected. In this manner, white blood cells and
platelets may be separated and subsequently collected in separate
collect reservoirs. Therefore, the combination of density
centrifugation and centrifugal elutriation provides methods of
separating blood components based on both density and sedimentation
velocity properties.
[0042] The basic design of the centrifuge rotor 26a,26b is a belt
shaped semicircular rotor placed slightly off-centered from the
axis of rotation as shown in FIGS. 1 and 2. FIG. 1 is a three
dimensional view of the rotor 26a,26b on the spinning rotor base
25, and FIG. 2 is a top view of rotor 26a,26b on the spinning rotor
base 25. In FIG. 3 the rotor 26a,26b is shown stretched out in a
linear configuration to help show the location of the rotor on
rotor base 25 with respect to axis of rotation 27.
[0043] The semicircular rotor 26a,26b consists of two curved
segments 26a and 26b, one segment (26b) slightly more distanced
from the axis of rotation 27 than the other segment (26a) and
therefore experiencing higher centrifugal force, while the other
segment (26a) is closer to the axis of rotation and therefore
experiences less centrifugal force than segment (26b). A mixture of
the blood and high-density particles (rAOC) enter the outer wall of
the higher centrifugal force segment 26b as indicated as "Whole
blood and rAOC input 31) in FIGS. 1, 2 and 3.
[0044] With reference to FIG. 3, as the centrifugation begins the
rAOC of the input mixture 31 remain at the wall of the furthest out
rotor segment 26b, as it is the most dense material and moves
towards the higher centrifugal field. This is to the right in FIG.
3 and the output is indicated as "Flow of rAOC F.sub.r". In FIGS. 1
and 2 this is clockwise and the output is indicated as "rAOC output
28". All the blood components move toward the left in FIG. 3 toward
closer rotor segment 26a because their densities are smaller and
they essentially float on top of the rAOC. In FIGS. 1 and 2 this is
counterclockwise and the blood components output is indicated as
"Whole blood output 29".
[0045] More particularly, as the blood and rAOC continue to be
injected into rotor 26a, 26b at input 31 (shown in FIGS. 1-3), the
blood components move towards the lower centrifugal field while the
rAOC move to the higher centrifugal field. The thickness of belt
shaped rotor 24 is only 5 mm. The separation of the rAOC and blood
is carried out very quickly and form layers based are density of
the particles. With separation being accomplished quickly it is
possible maintain the rate of rAOC and blood inflow sufficiently
fast to make the process "continuous-flow density separation". As
mentioned above the rAOC leave the rotor at output 28 at the end of
highest centrifugal force, while the blood components move leave
the rotor at output 29 at the end of lowest centrifugal force. The
semicircular rotor has a small offset, bend and protrusion near the
junction of segments 26a and 26b to make the separation of rAOC
from the blood complete. In FIGS. 1, 2 and 3 this indicated by the
number 40, but offset 40 is best seen in FIGS. 2 and 3. More
specifically, it is possible to enhance the change of centrifugal
force by creating a protrusion at the site where distinctive
separation of two layers is made, since their sedimentation
coefficients are predominantly a function of (1-.rho./.delta.), the
particulates will be positioned close to the outer wall of the
rotor when the density equilibrium is established.
[0046] Near at the exit port 28 of the rAOC, there is a monitor
output port 30, from which small samples are taken of the rAOC
flowing toward its output 28 to test the purity of the rAOC. The
testing of the rAOC is shown in and described with reference to
FIG. 4. The purity of the rAOC might change slowly over time during
centrifugal retrieval of the rAOC so the relative flow rates of
pumps 36 and 37 must be adjusted to maintain the purity of the rAOC
output at its port 28. The addition of all out-flows of the rAOC
and blood should equal to the inflow of the blood and rAOC, i.e.
Fbr=Fr+Fm+Fb.
[0047] In FIG. 4 is a block diagram of circuits required for
successful operation of the novel centrifuge that utilizes density
gradient separation to efficiently remove particulate artificial
oxygen carriers (rAOC) from blood or other biofluids. The circuits
first comprise a red blood cell (RBC) sensor 32 that receives the
previously mentioned sample output from the centrifuge at monitor
output 30. The concentration of any contaminating low density RBC
in the sample taken at output 30 is detected
spectrophotometrically. The output from RBC sensor 32 is amplified
by amplifier 33 and is then input to two logic circuits 34 and 35.
Circuits 34 and 35 are programmed to respond to any output from
sensor 32 to provide output signals that will change the operation
of pumps 36 and 37 which thereby can change either or both of the
flow rate of lower density blood flowing out at blood output 29 and
higher density rAOC flowing out at blood output 28. In addition,
there can be a programmed logic circuit 38 that responds to the
output from sensor 32 and, in cooperation with logic circuits 34
and 35, provides and output at 39 to the motor that rotates rotor
24 to change its rotational speed.
[0048] FIGS. 5 A&B shows typical electron microscope pictures
of the shelled rAOC particles 11. The shells 12 of these novel rAOC
particles 11 are coated with molecules containing exposed
functional groups (COOH, NH.sub.2, SH etc.) convenient for the
crosslinking of either more than one particle, or proteins like
antibodies, cell receptor targets, polyhemoglobin, hemoglobin etc.
Outer ring or shell 12 is a gas permeant calcium phosphate or
polymer coating, while the interior is an oxygen carrying center
containing a hemoglobin (HB) 13 nanoparticle and/or a
perfluorocarbon (PFC) 14 nanoparticle.
[0049] Very briefly, single shell rAOCs 11 are made as follows.
Nanoemulsion particles 13 are made from a mixture of
perfluorooctylbromide (PFOB) 21, 1,2-dioleoyl-sn-glycero-phosphate
(DOPA)22 and water, preferably by a stirring process, but other
methods known in the art may be utilized.
The outer surface of the perfluorooctylbromide (PFOB) nanoparticles
11 has a surface of 1,2-dioleoyl-sn-glycero-phosphate (DOPA) 22
surrounding a nanomulsion particle 21. The uncoated
(non-mineralized) nanoemulsion particles 13 have a negatively
charged surface of PO.sub.3.sup.- created by using phosphatidic
acid to stabilize the nanoemulsion particles. Since the synthesis
of nanoemulsion particles takes place under basic conditions, the
surface charge density of the nanoemulsion is quite high with zeta
potentials nearing -50 mV.
[0050] To coat the negatively charged nanoemulsions particles 13
they may be mixed with 2:00 .mu.l of 0.1 M phosphoric acid
solution. Next, a CaCl.sub.2 solution is added followed by a CEPA
solution to coat the nanoemulsion particles and arrest further
calcium phosphate deposition. In this process positively charged
calcium ions from the phosphoric acid are attracted to the
negatively charged PO.sub.3.sup.- on the surface of the
nanoemulsion particles 13 (DOPA) as shown in FIG. 6. The
accumulation of calcium ions at the periphery of the nanoemulsion
particles increases the local concentration past the stability
point for calcium phosphate precipitation resulting in
precipitation of calcium phosphate onto the nanoemulsion particles
to form a shell. The finished shelled, particles function well as
oxygen carriers in blood.
[0051] A second shell and second oxygen carrier may be added as
shown in FIG. 7. First, Polylysine/Hb is deposited layer by layer
onto the negatively charged carboxylated surface of the first shell
made as described above. Then a mixture of perfluorocarbon (PFC)
and Polyhemoglobin (PolyHB) is coated over the first shell and the
same previously described method is used to place a second shell
over the PFC and PolyHB. The second shell makes the rAOC particles
tougher and even better able to withstand being retrieved from
circulating blood using the continuous flow density gradient
separation technique described above. The finished shelled,
particles function well as oxygen carriers in blood.
[0052] The novel density gradient separation technique taught and
claimed herein may be used to separate other mixtures of substances
having different densities. It may be used to separate and remove
metastatic cancer cells from circulating blood. It may also be used
for retrieval of low copy mammalian, bacterial or virus cells from
blood. It may also be used to remove materials added to blood to
enhance tissue and organ imaging. Depending on the application, the
specific design requirement of these materials in terms of their
size and composition may vary, but common to all of them are the
properties summarized earlier, and the tailored ability for
continuous retrieval from circulating fluids.
[0053] While what has been described herein is the preferred
embodiment of the invention it will be understood by those skilled
in the art that numerous changes may be made without departing from
the spirit and scope of the invention.
* * * * *