U.S. patent number 5,460,831 [Application Number 08/147,751] was granted by the patent office on 1995-10-24 for targeted transfection nanoparticles.
This patent grant is currently assigned to The Regents of the University of California. Invention is credited to Andrew Gelman, H. James Hnatyszyn, Nir Kossovsky.
United States Patent |
5,460,831 |
Kossovsky , et al. |
* October 24, 1995 |
**Please see images for:
( Certificate of Correction ) ** |
Targeted transfection nanoparticles
Abstract
A biologically active composition for use in gene therapy and
other transfection procedures. The composition is composed of
nanocrystalline core particles which are coated with a layer that
is designed to allow attachment of transfection agents (DNA/RNA
segments or antisense fragments) without denaturing them. The
composition may further include an exterior targeting membrane
which provides selective targeting of the transfection agents to
specific cell receptors.
Inventors: |
Kossovsky; Nir (Los Angeles,
CA), Hnatyszyn; H. James (Los Angeles, CA), Gelman;
Andrew (Los Angeles, CA) |
Assignee: |
The Regents of the University of
California (Oakland, CA)
|
[*] Notice: |
The portion of the term of this patent
subsequent to January 12, 2010 has been disclaimed. |
Family
ID: |
27356618 |
Appl.
No.: |
08/147,751 |
Filed: |
November 4, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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199 |
Jan 4, 1993 |
5334394 |
|
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690601 |
Apr 24, 1991 |
5178882 |
|
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542255 |
Jun 22, 1990 |
5219577 |
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Current U.S.
Class: |
424/493; 424/490;
424/498; 514/951; 514/970; 424/204.1; 424/494 |
Current CPC
Class: |
A61K
9/0019 (20130101); A61K 9/127 (20130101); A61K
9/5115 (20130101); A61K 9/5161 (20130101); A61K
47/26 (20130101); A61K 48/00 (20130101); A61L
31/10 (20130101); B82Y 5/00 (20130101); C07K
14/005 (20130101); C12N 11/14 (20130101); C12N
15/87 (20130101); A61K 47/6923 (20170801); A61K
47/6929 (20170801); A61K 9/0026 (20130101); Y10S
514/951 (20130101); A61K 2039/53 (20130101); A61K
2039/60 (20130101); C12N 2710/16222 (20130101); C12N
2740/16022 (20130101); A61K 39/00 (20130101); Y10S
514/97 (20130101); A61K 38/00 (20130101) |
Current International
Class: |
A61K
38/42 (20060101); A61K 9/127 (20060101); A61K
39/12 (20060101); A61K 38/43 (20060101); A61K
38/48 (20060101); A61K 9/00 (20060101); A61K
38/28 (20060101); A61K 38/40 (20060101); A61K
38/41 (20060101); A61K 39/21 (20060101); A61K
47/48 (20060101); A61K 48/00 (20060101); A61K
47/26 (20060101); A61K 9/51 (20060101); A61L
31/10 (20060101); A61L 31/08 (20060101); C12N
11/00 (20060101); C12N 11/14 (20060101); C07K
14/05 (20060101); C07K 14/005 (20060101); C12N
15/87 (20060101); C07K 14/16 (20060101); A61K
38/00 (20060101); A61K 009/14 () |
Field of
Search: |
;424/493,494,490,498 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Page; Thurman K.
Assistant Examiner: Spear; James M.
Attorney, Agent or Firm: Poms, Smith, Lande & Rose
Parent Case Text
This is a continuation-in-part of application Ser. No. 08/000,199,
now U.S. Pat. No. 5,334,394 which was filed on Jan. 4, 1993 which
is a continuation-in-part of Ser. No. 07/690,601 which was filed on
Apr. 24, 1991, now U.S. Pat. No. 5,178,882 which is a
continuation-in-part of application Ser. No. 07/542,255 which was
filed on Jun. 22, 1990, now U.S. Pat. No. 5,219,577.
Claims
What is claimed is:
1. A composition of matter comprising:
a biodegradable core particle having a diameter of less than about
1000 nanometers wherein said core particle comprises a
biodegradable ceramic or polymer;
a coating comprising a substance that provides a threshold surface
energy to said core particle which is sufficient to bind
transfection agents without denaturing said agents, said substance
covering at least a part of the surface of said core particle and
wherein said substance is selected from the group consisting of
carbohydrates, citrate, fumarate, succinate, isocitrate,
oxalacetate, realate, pyridoxyl-5-pyrophosphate, thiamine
pyrophosphate, uridine-diphosphate-glucose, glucose-1-phosphate,
adenosine and nicotinamide-adenine-diphosphate;
at least one transfection active agent bound to said coated core
particle wherein said transfection agent bound to said coated core
particle is not denatured.
2. A composition of matter according to claim 1 wherein said core
particle comprises brushite.
3. A composition of matter according to claim 1 which further
includes a target coating covering at least a portion of the
surface of said core particle.
4. A composition of matter according to claim 3 wherein said outer
target coating comprises a phospholipid and targeting ligand.
5. A composition of matter according to claim 1 wherein said
coating comprises a carbohydrate.
6. A composition of matter according to claim 1 wherein said
coating comprise cellobiose, pyridoxal-5-phosphate or citrate.
7. A composition of matter according to claim 1 wherein said
transfection agent is a DNA or RNA segment.
8. A composition of matter according to claim 1 wherein said
transfection agent is an antisense fragment.
9. A composition of matter according to claim 4 wherein said
targeting ligand is a viral envelope.
10. A composition of matter according to claim 3 wherein said core
particle is brushite.
11. A composition of matter according to claim 3 wherein said
coating comprises a carbohydrate.
12. A composition of matter according to claim 3 wherein said
coating comprises cellobiose, pyridoxal-5-phosphate or citrate.
13. A composition of matter according to claim 3 wherein said
transfection agent is a DNA or RNA segment.
14. A composition of matter according to claim 3 wherein said
transfection agent is an antisense fragment.
15. A composition of matter according to claim 1 wherein said
carbohydrate is selected from the group consisting of sucrose,
cellobiose, nystose, triose, dextrose, trehalose, glucose, lactose,
maltose, dextran, nitrocellulose and glycogen.
16. A composition of matter according to claim 3 wherein said
carbohydrate is selected from the group consisting of glucose,
sucrose, cellobiose, nystose, triose, dextrose, trehalose, lactose,
maltose, dextran, nitrocellulose and glycogen.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to synthetic biologically
active compositions which have a microparticulate (nanoparticulate)
core. More particularly, the present invention relates to
biologically active compositions where transfecting DNA or RNA is
attached to a microparticulate core and coated with a targeting
membrane or ligand. These transfection nanoparticles are useful in
delivering the transfecting DNA or RNA to target cells.
2. Description of Related Art
The attachment of biologically active proteins, peptides or
pharmacologic agents to various carrier particles has been an area
of intense investigation. These conjugated biological systems offer
the promise of reduced toxicity, increased efficacy and lowered
cost of biologically active agents. As a result, many different
carrier models are presently available. (Varga, J. M., Asato, N.,
in Goldberg, E. P. (ed.): Polymers in Biology and Medicine. New
York, Wiley, 2, 73-88 (1983). Ranney, D. F., Huffaker, H. H., in
Juliano, R. L. (ed.): Biological Approaches to the Delivery of
Drugs, Ann. New York Acad. Sci., 507, 104-119 (1987).)
Nanocrystalline and micron sized inorganic substrates are the most
common carriers and proteins are the most commonly conjugated
agents. For example, gold/protein (principally immunoglobulin)
conjugates measuring as small as 5 nm have been used in
immunological labeling applications in light, transmission electron
and scanning electron microscopy as well as immunoblotting. (Faulk,
W., Taylor, G., Immunochemistry 8, 1081-1083 (1971). Hainfeld, J.
F., Nature 333, 281-282 (1988).)
Silanized iron oxide protein conjugates (again principally
antibodies) generally measuring between 500 and 1500 nm have proven
useful in various in vitro applications where paramagnetic
properties can be used advantageously. (Research Products Catalog,
Advanced Magnetics, Inc., Cambridge, Mass., 1988-1989.) Ugelstad
and others have produced gamma iron oxides cores coated with a thin
polystyrene shell. (Nustad, K., Johansen, L., Schmid, R., Ugelstad,
J., Ellengsen, T., Berge, A.: Covalent coupling of proteins to
monodisperse particles. Preparation of solid phase second antibody.
Agents Actions 1982; 9:207-212 (id. no. 60).) The resulting 4500 nm
beads demonstrated both the adsorption capabilities of polystyrene
latex beads as well as the relatively novel benefit of
paramagnetism.
Carrier systems designed for in vivo applications have been
fabricated from both inorganic and organic cores. For example,
Davis and Illum developed a 60 nm system comprised of polystyrene
cores with the block copolymer poloxamer, polyoxyethylene and
polyoxypropylene, outer coats that showed a remarkable ability to
bypass rat liver and splenic macrophages. (Davis, S. S., Illum, L.,
Biomaterials 9, 111-115 (1988)). Drug delivery with these particles
has not yet been demonstrated. Ranney and Huffaker described an
iron-oxide/albumin/drug system that yielded 350-1600 nm
paramagnetic drug carriers. (Ranney, D. F., Huffaker, H. H., In,
Juliano, R. L. (ed.): Biological approaches to the delivery of
drugs, Ann. New York Acad. Sci. 507, 104-119 (1987).) Poznasky has
developed an enzyme-albumin conjugate system that appears to
decrease the sensitivity of the product to biodegradation while
masking the apparent antigenicity of the native enzyme. (Poznasky,
M. J.: Targeting enzyme albumin conjugates. Examining the magic
bullet. In, Juliano, R. L. (ed.): Biological approaches to the
delivery of drugs, Annals New York Academy Sciences 1987;
507-211:219.)
Shaw and others have prepared and characterized lipoprotein/drug
complexes. (Shaw, J. M., Shaw, K. V., Yanovich, S., Iwanik, M.,
Futch, W. S., Rosowsky, A., Schook, L. B.: Delivery of lipophilic
drugs using lipoproteins. In, Juliano, R. L.(ed.): Biological
approaches to the delivery of drugs, Annals New York Academy
Sciences 1987; 507:252-271.) Lipophilic drugs are relatively stable
in these carriers and cell interactions do occur although little
detail is known.
In any conjugated biological composition, it is important that the
conformational integrity and biological activity of the adsorbed
proteins or other biological agents be preserved without evoking an
untoward immunological response. Spacial orientation and structural
configuration are known to play a role in determining the
biological activity of many peptides, proteins and pharmacological
agents. Changes in the structural configuration of these compounds
may result in partial or total loss of biological activity. Changes
in configuration may be caused by changing the environment
surrounding the biologically active compound or agent. For example,
pharmacologic agents which exhibit in vitro activity may not
exhibit in vivo activity owing to the loss of the molecular
configuration formerly determined in part by the in vitro
environment. Further, the size and associated ability of the
carrier particle to minimize phagocytic trapping is a primary
concern when the composition is to be used in vivo. All of these
factors must be taken into account when preparing a carrier
particle.
To date, gene therapy in humans has been limited to ex-vivo
protocols in which tissues are transfected in the culture dish and
placed back in the body. In vivo work is still in pre-clinical
development and has been confined to animal models due to a range
of safety and efficacy issues. Such concerns arise primarily from
the use of viral vectors to effect the gene transfer. Retroviral
transfection has generated a lot of interest since it can stably
transfect nearly 100% of targeted cells ex-vivo. Production of
transfecting, replication defective retroviruses, proceeds through
packaging cell lines which in principle are unable to produce wild
type virus. However, low titers of "wild type" (replication
competent) virus have been observed in these systems. In one such
protocol utilizing primates, outbreaks of lymphoma were linked to
the detection of wild type retrovirus from a packaging system.
Besides potential pathogenicity, maintaining useful transfecting
titers of these vectors can be difficult. They are hard to purify
and concentrate since the envelopes (membranes) tend to be
extremely labile. Alternatively, adenoviral vectors have been found
to be considerably more stable. Moreover, these viruses are capable
of transfecting quiescent tissue and producing large amounts of
gene products. Unfortunately, gene expression is often transient
because the viral genome often remains extrachromosomal. Direct
clinical application is also problematic, since replication of the
vector can result in aberrant host protein synthesis leading to
deleterious effects ranging from oncogenesis to cellular
toxicity.
Given the practical concerns of in vivo viral transfection,
nonviral methods are also being developed. At present, most efforts
are centered on receptor mediated transfer because such methods can
provide targeted delivery of DNA (and RNA) in vivo.
Receptor-mediated systems employ ligand DNA (and RNA) complexes
which can be recognized by cell receptors on the cell surface.
Internalization of the complex occurs via the formation of
endocytotic vesicles which allow for transport into the cytoplasm.
Problems arise, however, when the endosomes fuse with lysosomes
which causes the contents to be destroyed. In turn, the
transfection rate for these complexes remain below clinical
efficacy. Some investigators have used fusogenic peptides of
Influenza Hemmoglutin A to disrupt endosome formation which has led
to higher transfection rates. Nonetheless, the data on in vivo
expression suggests that this method may only permit transient
expression of genes.
Besides ligand DNA (and RNA) complexes, lipofection techniques have
also been tried with varying success. Liposomes are specially
susceptible to uptake by the filter organs and in the peripheral
tissues by macrophages which limits their transfection efficiency
and specificity in vivo.
In view of the above, it would be desirable to provide compositions
which can be used to transfect cells with DNA or RNA in both in
vivo or in vitro environments.
SUMMARY OF THE INVENTION
In accordance with the present invention, transfecting DNA or RNA
is attached to a nanocrystalline core particle and coated with a
targeting ligand or membrane to provide a viral transfection system
which may be used in gene therapy. The invention is based in part
on the discovery that the surface of ultrafine particles
(nanocrystalline particles) can be modified with a surface coating
to allow attachment of transfecting DNA or RNA to produce
compositions wherein the naturally occurring structural environment
of the DNA or RNA is mimicked sufficiently so that biological
activity is preserved. The core particle, with the surface coating
and attached transfecting DNA or RNA, is further coated with a
targeting agent, such as ligand or phospholipid membrane complex to
provide targeting of the DNA or RNA to particular cell
receptors.
As a feature of the present invention, the nanocrystalline core may
be composed of brushite. This material is biodegradable,
inexpensive and is found in human beings as a substrate of bone
synthesis. The brushite particles may be synthesized at a nanomeric
size (between approximately 5 nm and 150 nm). This small size
allows the DNA/RNA delivery construct to be small enough to avoid
uptake by the Reticulo-Endothelial System (RES) of the body and
deliver the DNA/RNA to cells in vivo without non-specific toxicity
or loss of drug to macrophages.
As a feature of the present invention, the DNA/RNAparticle
construct is targeted to a specific tissue or cell type. In order
to achieve this targeting, the construct has a targeting ligand or
a primed phospholipid membrane tightly adsorbed to its surface. The
membrane may contain proteins, receptors and carbohydrates which
provide targeting of the vehicle. The membrane also serves to
further maintain the stability of the transfecting DNA or RNA and
the integrity of the construct. This membrane may be derived from
cell membranes, viral envelopes (see U.S. Pat. No. 5,178,882), or
other specifically engineered or synthesized membranes. Due to the
very small size of the biodegradable core particle delivery system,
multiple layers of membranes may be adsorbed to the core particle
to increase the efficiency of targeting. The DNA/RNA transfecting
microparticles in accordance with the present invention have
wide-ranging use depending upon the particular DNA or RNA which is
attached to the biodegradable microparticle core.
The above-discussed and many other features and attendant
advantages of the present invention will become better understood
by reference to the following detailed description.
DETAILED DESCRIPTION OF THE INVENTION
The present invention has wide application to procedures and
methods wherein DNA or RNA are delivered, i.e. transfected, to
cells in vivo or in vitro. These areas of application include gene
therapy. The compositions of the present invention can be used in a
wide variety of other applications where there is a need to target
DNA or RNA to particular cell types in both in vivo and in vitro
environments. The invention may also be used to target other
transfection agents, such as antisense fragments. The term
"transfection agent" as used herein is intended to mean DNA or RNA
segments or antisense fragments which are capable of transfection
into a cell. Exemplary transfection agents include sense DNA, sense
RNA, antisense RNA, and antisense DNA.
The compositions of the present invention include nanocrystalline
core particles (diameters of less than 1000 nm) which are coated
with a surface energy modifying layer that promotes bonding of
proteins, peptides or pharmaceutical agents to the particles. The
coating modifies the surface energy of the nanocrystalline core
particles so that DNA and RNA segments may be attached to the core
particle without significant loss of activity or denaturization.
The result is a biochemically active composition which includes a
biochemically inert core. The end use for the compositions of the
present invention will depend upon the particular transfection
agent which is attached to the coated core particle. For example,
DNA segments, such as human low density lipid receptor, are used in
gene therapy. RNA segments, such as antisense and sense MRNA, are
used in the transfection procedure. Antisense fragments, such as
HIV reverse transcriptase are used in the transfection particles.
Preferred particle sizes are on the order of 10 nm to 150 nm.
The core particles may be made from a variety of inorganic
materials including metals or ceramics. Preferred metals and alloys
include beryllium, silicon, gallium, copper, gold, titanium,
nickel, aluminum, silver, iron, steels, cobalt-chrome alloys, and
titanium alloys. Preferred ceramic materials include brushite,
tricalcium phosphate, alumina, silica, and zirconia. The core
particles may be made from organic materials including carbon
(diamond). Preferred polymers include polystyrene, silicone rubber,
polycarbonate, polyurethanes, polypropylenes,
polymethylmethaacrylate, polyvinyl chloride, polyesters,
polyethers, and polyethylene. Particles made from brushite, are
particularly preferred.
Particles made from the above materials having diameters less than
1000 nanometers are available commercially or they may be produced
from progressive nucleation in solution (colloid reaction), or
various physical and chemical vapor deposition processes, such as
sputter deposition (Hayashi, C., J. Vac. Sci. Technol. A5 (4),
July/August 1987, pgs. 1375-1384; Hayashi, C., Physics Today,
December 1987, pgs. 44-60; MRS Bulletin, January 1990, pgs.
16-47).
Plasma-assisted chemical vapor deposition (PACVD) is one of a
number of techniques that may be used to prepare suitable
microparticles. PACVD functions in relatively high atmospheric
pressures (on the order of one torr and greater) and is useful in
generating particles having diameters of up to 1000 nanometers. For
example, aluminum nitride particles having diameters of less than
1000 nanometer can be synthesized by PACVD using Al
(CH.sub.3).sub.3 and NH.sub.3 as reactants. The PACVD system
typically includes a horizontally mounted quartz tube with
associated pumping and gas feed systems. A susceptor is located at
the center of the quartz tube and heated using a 60 KHz radio
frequency source. The synthesized aluminum nitride particles are
collected on the walls of the quartz tube. Nitrogen gas is used as
the carrier of the Al (CH.sub.3).sup.3. The ratio of Al
(CH.sub.3).sup.3 : NH.sub.3 in the reaction chamber is controlled
by varying the flow rates of the N.sub.2 /Al(CH.sub.3).sub.3 and
NH.sub.3 gas into the chamber. A constant pressure in the reaction
chamber of 10 torr is generally maintained to provide deposition
and formation of the ultrafine nanocrystalline aluminum nitride
particles. PACVD may be used to prepare a variety of other suitable
biodegradable nanocrystalline particles.
The core particles are coated with a substance that provides a
threshold surface energy to the particle or other surface which is
sufficient to cause binding to occur without that binding being so
tight as to denature biologically relevant sites. Coating is
preferably accomplished by suspending the particles in a solution
containing the dispersed surface modifying agent. It is necessary
that the coating make the surface of the particle more amenable to
protein or peptide attachment.
Suitable coating substances in accordance with the present
invention include carbohydrates, carbohydrate derivatives, and
other macromolecules with carbohydrate-like components
characterized by the abundance of --OH (hydroxyl) side groups. The
coatings may include but are not limited to:
short chain carbohydrates including glucose, sucrose, cellobiose,
nystose, triose, dextrose, trehalose, glucose, lactose, maltose,
etc.
hydroxyl rich weak acids such as citrate, fumarate, succinate,
isocitrate, oxaloacetate, malate, etc.
nucleotide-like molecules with pendant carbohydrate or phosphate
groups such as pyridoxyl-5-pyrophosphate, thiamine pyrophosphate,
uridinediphosphate-glucose, glucose-1-phosphate, adenosine,
nicotinamide-adenine-diphosphate, etc.
derivatives of carbohydrates such as nitrocellulose
complex polymeric carbohydrates and derivatives such as dextran,
glycogen, etc.
Preferred coating materials include cellobiose,
pyridoxyl-5-pyrophosphate and citrate.
In a preferred process for coating, the core particles are
suspended in a coating solution. The coating solution into which
the core particles are suspended contains, for example, from 1 to
30 weight/volume percent of the coating material. The solute is
preferably double distilled water (ddH.sub.2 O). The amount of core
particles suspended within the coating solution will vary depending
upon the type of particle and its size. Typically, suspensions
containing from 0.1 to 10 weight/volume percent are suitable.
Suspensions of approximately 1 weight/volume percent of particles
are preferred.
The core particles are maintained in dispersion in the coating
solution for a sufficient time to provide uniform coating of the
particles. Sonication is the preferred method for maintaining the
dispersion. Dispersion times ranging from 30 minutes to a few hours
at room temperature are usually sufficient to provide a suitable
coating to the particles. The thickness of the coating is
preferably less than 5 nanometers. Thicknesses of the coating may
vary provided that the final core particles include a uniform
coating over substantially all of the particle surface.
The particles are separated from the suspension after coating and
may be stored for future use or redispersed in a solution
containing the DNA or RNA to be attached to the particles.
Alternatively, the coated particles may be left in the suspension
for further treatment involving attachment of the desired DNA or
RNA.
The core particle may be made from a biodegradable ceramic or
polymer. The term "biodegradable" as used herein means any core
particle which decomposes or otherwise disintegrates after
prolonged exposure to a mammalian in vivo environment. To be
biodegradable, the core particle should be substantially
disintegrated within a few weeks after introduction into the body.
Brushite is a preferred biodegradable core particle material.
The DNA or RNA which is applied to the coated particles may be
selected from a wide variety of DNA or RNA segments which are used
to transfect cells during gene therapy. Antisense fragments may
also be used.
Exemplary transfection gene segments include human low density
lipid receptor CDNA, human adenosine deaminase CDNA, human
dystrophan CDNA, and antisense HIV reversed DNA--all subcloned
within appropriate expression vectors. The DNA or RNA transfection
segments may be prepared according to known procedures such as the
procedure described in Manniatis, T., Fritsch, E. F., and Sambrook,
S., Molecular Cloning, Cold Spring Laboratory Press, New York.,
1.0-19.0 (1989). Gene segments are also available commercially from
a number of different suppliers.
The procedure for attaching gene segments or antisense fragments to
the coating on the core particles involves suspending the coated
core particles in an aqueous solution containing the gene segments.
The presence in the solution of materials that may preferentially
attach to the particle surface is often not advantageous. For
example, the dispersion agents present in the solution may create
an undesirable coating on the suspended particles prior to protein
attachment. Water miscible solvents such as methanol or ethanol may
be used. The aqueous solution of coated microparticles can be
agitated sufficiently to provide a uniform suspension of the
particles. Typically, the amount of particles in solution will be
between about 0.5 mg per milliliter of solution and 5 mg per
milliliter of solution. Sonication is a preferred method for
providing a uniform suspension of the coated particles in
solution.
The suspension of coated particles and gene segments must be within
certain parameters for segment attachment and self assembly to
occur. The temperature of the particle solution should be between
1.degree. C. to 45.degree. C. The gene segments or antisense
fragments may be bound to the coated particles in distilled water.
The oxygen tension of the solution is preferably less than 10% in a
solution sparged initially by helium and then gassed with helium,
nitrogen and carbon dioxide. The pH of the solution is preferably
slightly acidic (relative to blood), with a value, preferably, of
between 6.8 to 7.2. An exemplary solution for dispersion of the
coated microparticles and for DNA attachment is an aqueous solution
containing: 0.0360 milligrams MgSo.sub.4 per liter, 0.0609
milligrams MgCl.sub.2.6 H.sub.2 O, 0.0441 milligram CaCl.sub.2.2
H.sub.2 O, 22.823 grams K.sub.2 HPO.sub.4, 13.609 grams KH.sub.2
PO.sub.4, 7.455 grams KCl, and 4.101 gram sodium acetate. The pH of
this solution is adjusted to 6.8.
The coated particle cores with the attached gene segment or
antisense fragment can be separated from the ionic growth medium
and stored for further use. The coated particles may be stored by
any of the conventional methods typically used for storing gene
segment or antisense fragments. For example, the coated particles
may be freeze dried or stored as a suspension in a compatible
solution. When used in gene therapy, the particles coated with a
targeting layer as described below, are injected or otherwise
administered to the individual according to conventional
procedures. Any pharmaceutically acceptable carrier solution or
other compound may be used in administering the DNA/RNA coated
particles to the individual. When used in vitro, the DNA/RNA coated
particles are suspended in solution and used in the same manner as
other gene therapy compounds. The same is true for use of antisense
coated particles. The same protocol and procedures well known for
gene therapy to introduce genes into cells both in vivo and in
vitro may be used wherein the DNA/RNA/antisenseparticle constructs
of the present invention are substituted for other gene therapy
compounds.
Targeting of the coated particle and attached gene support or
antisense fragment is accomplished by coating the particles with a
phospholipid membrane complex which includes ligands that are
reactive with receptors on particular cells. Exemplary target
ligands include HIV coat proteins (gp160, 41, 120) corona virus
coat proteins, EBV coat proteins (gp350). Any membrane bound
ligand/receptor may be used. These ligands are attached to the
particle complex in the same manner as attachment of the
transfection agents discussed above.
The lipids used to coat the biodegradable nanocrystalline particle
and bound transfection agent are the same lipids commonly used to
form liposomes. Suitable lipids include phospholipids such as
phosphatidylcholine, cholesterol and phosphatidylserine. The lipid
layer is applied to the nanocrystalline core particle and bound to
a biologically active agent in the same manner as the other
coatings are applied, i.e. by adsorption onto the surface.
The core particle and bound agent do not need to be totally covered
with a lipid layer. Preferably, the amount of lipid used to coat
the particle will be sufficient to coat the entire particle. The
combined layer of lipid and targeting ligand provide for targeting
of the core particle and attached gene segments to the
corresponding cell receptor.
The preferred transfection nanoparticles are selfassembling
complexes of nanometer sized particles, typically on the order 100
nm, that carry an inner layer of transfecting DNA or RNA and an
outer layer of targeting molecules. Functionally, the targeting
molecules, usually referred to as ligands, impart tissue
specificity in the same way a virus finds its host, i.e., the
ligands promote the association of the transfection nanoparticles
to a cell surface by binding to cell surface receptor
molecules.
Construction of the transfection nanoparticles is a simple process
and which occurs spontaneously without apparent covalent
modification. In one exemplary synthesis, nanoparticle dispersions
of tricalcium phosphate (TCP) are prepared from isochoric opposing
streams of 0.750M Calcium Chloride and 0.25M monobasic Sodium
Phosphate. The resulting precipitant is sonicated at 175 Watts at
room temperature for 30 minutes and washed in volumes of 20 mM pH
6.80 phosphate buffer before being mixed with Cesium Chloride
purified transfecting DNA, RNA or antisense. The DNA, RNA or
antisense is then left to adsorb to the particulate surfaces at
room temperature under mild agitation. After the transfection agent
attachment step is completed, membrane specific ligands, typically
prepared from the envelopes of retroviruses, are added to the
dispersion and allowed to adsorb overnight at 4.0.degree. C. in a
stir cell.
The ligand receptor complex can be chosen so as to be unique for
the targeted tissue since tissues can be differentiated by their
component cells, cell surface receptors, and complementary ligands.
Once the interaction takes place, transfection can proceed through
a range of cellular uptake mechanisms, resulting in the dissolution
of the DNA (RNA/antisense) away from the particle complexes,
recombination, and expression in a target cell. Introducing DNA,
RNA or antisense by this method allows for the alteration of the
phenotype of specific cells in a targeted tissue. It will occur
because of the conformational stability of the ligands employed,
the integration locus of the transfecting DNA/RNA/antisense, and
the expression ability of transfecting DNA (RNA/antisense) in the
targeted tissue.
Ligand preparation is as varied as their source. They can be
produced by recombinant means or derived from their natural source.
Preferably, viral envelope ligands are extracted from viruses, such
as Human Immunodeficiency Virus, Epstein-Barr Virus, and murine
ecotropic viral strains. In general, the viral envelopes are
extracted in accordance with known procedures and then combined
with phospholipids in a phosphate buffer. The solution of viral
envelopes and phospholipids are then added to the suspension of
DNA/RNA coated nanoparticles. The viral envelopes and phospholipids
are absorbed onto the nanoparticles to form a targeting
membrane.
Typical preparations of transfection nanoparticles yield in the
neighborhood of a tenth of a microgram of DNA (and RNA) per
microliter of dispersion as gauged by spectrophotometric
determinations over time. If higher concentrations are required,
the DNA (and RNA) is premixed with the substrate solutions and is
allowed to slowly coprecipitate with the core material at a pH of
6.5. The particulate size is controlled by the time wise addition
and surface adsorption of membrane ligands, and by the removal of
substrate by ultrafiltration dialysis. Independent of the synthetic
route chosen, administration of the transfection nanoparticles is
accomplished by both enteral and parenteral routes. The doses are
the same as those used in gene therapy.
The nanoparticle-DNA/RNA constructs of the present invention may be
made without the targeting layer when receptor mediated targeting
is not required. For example, the constructs may be prepared
without the targeting layer when calcium channel uptake of DNA/RNA
or other non-cell receptor uptake is desired. The small size of the
constructs allows them to evade the reticular endothelial system,
thereby increasing the circulation time and transfection
efficiency.
The following non-limiting examples describe certain aspects of the
present invention in greater detail.
Example 1
Preparation of nanocrystalline tin oxide microparticles:
1.5 to 2.0 mg of ultrafine (nanocrystalline) metal powder was
placed in a 1.7 ml screwcap microcentrifuge with 1.5 mls of double
distilled water (ddH.sub.2 O). The ddH.sub.2 O was filtered through
a rinsed 0.45 micron filter-sterilizing unit or acrodisc (Gelman
Scientific). The metal powder was tin oxide with a mean diameter
(by photon correlation spectroscopy) of 140 nm. The mixture was
vortexed for 30 seconds and placed into a water sonicating bath
overnight. The sonication bath temperature was stabilized at
60.degree. C. After a 24-hour sonication, the samples were vortexed
once more for 30 seconds with the resulting dispersion clarified by
microcentrifugation at approximately 16,000 rpm for 15 seconds. The
analysis of particle size was carried out on a Coulter N4MD
sub-micron particle analyzer.
The coating was applied to the tin oxide particles by suspending
the particles in a stock solution of cellobiose. The cellobiose
stock solution was a 292 mM solution made by dissolving 1.000 gram
of cellobiose in 9.00 mls of ddH.sub.2 O. Solution was accomplished
at approximately 70.degree. C. in order to promote quick
dissolution. The resulting cellobiose solution was filter
sterilized through a rinsed 0.45 micron filter with the final
volume being adjusted to 10.00 ml.
Sufficient cellobiose stock solution was added to 150 microliters
of ultrafine tin oxide dispersion so that the final concentration
of the tin oxide was 1.00 percent (w/v) or 29.2 mM. A typical
volume for preparation was 2.0 mls which was mixed four or five
times by the action of a micro-pipetor. After mixing, the
dispersion was allowed to equilibrate for two hours. Demonstration
of successful coating of the particles was provided by measuring
the mobility of the particles (coated and uncoated) on a Coulter
DELSA 440 doppler energy light scatter analyzer. The coated tin
oxide particles exhibited a relatively low mobility compared to the
non-coated tin oxide particles. Measurements were also taken at
various dilute salt concentrations to ensure that the observations
with respect to mobility were not artifactual. The tests
demonstrate that the particles were coated with the cellobiose.
The coated particles are then used to attach antigenic proteins,
peptides or pharmacological agents to prepare bioreactive
particles.
Example 2
Preparation of nanocrystalline ruthenium oxide particles:
The same procedure was carried out in accordance with Example 1,
except that ruthenium oxide microparticles were substituted for the
tin oxide particles. The ruthenium oxide particles were obtained
from Vacuum Metallurgical Company (Japan).
Example 3
Preparation of the nanocrystalline silicon dioxide and tin oxide
particles:
Nanocrystalline silicon dioxide was acquired commercially from
Advanced Refractory Technologies, Inc. (Buffalo, N.Y.) and tin
oxide was acquired commercially from Vacuum Metallurgical Co.
(Japan). The tin oxide particles were also prepared by reactive
evaporations of tin in an argon-oxygen mixture and collected on
cooled substrates. Nanocrystalline tin oxide was also synthesized
by D.C. reactive Magnetron sputtering (inverted cathode). A 3"
diameter target of high purity tin was sputtered in a high pressure
gas mixture of argon and oxygen. The ultrafine particles formed in
the gas phase were collected on copper tubes cooled to 77.degree.
K. with flowing liquid nitrogen. All materials were characterized
by X-ray diffraction crystallography, transmission electron
microscopy, photon correlation spectroscopy, and Doppler
electrophoretic light scatter analysis. X-ray diffraction samples
were prepared by mounting the powder on a glass slide using
double-sized Scotch tape. CuK.alpha. radiation was used on a
Norelco diffractometer. The spectrum obtained was compared with
ASTM standard data of tin oxide. (Powder Diffraction File, Card
#21-1250. Joint Committee on Power Diffraction Standards, American
Society for Testing and Materials, Philadelphia 1976.) The
specimens for (TEM) were collected on a standard 3 mm diameter
carbon coated copper mesh by dipping into a dispersion of the
(UFP's) in 22-propanol. The samples were examined on a JEOL-STEM
100 CX at an acceleration voltage of 60-80 KV.
To create working dispersions of these metal oxides, 1.5 to 3.0 mg
of metal oxide powder was added to 1.5 ml double distilled H.sub.2
O in a dust-free screw top microcentrifuge tube (Sarsted) and
vortexed for 30 seconds. The mixture was then sonicated for 16 to
24 hours followed by a second 30 seconds vortex. The submicron
fraction was then isolated by pelleting macroparticulates by
microcentrifugation 16,000.times.g for 15 seconds. Approximately
1.3 ml of supernatant was then removed and placed in another
dust-free screw top microcentrifuge tube. A sample was prepared for
photon correlation spectroscopy (Coulter N4MD) and Doppler
electrophoretic light scattering (Coulter delsa 440) analysis by
removing 50 to 100 .mu.l of the dispersion and placing it in a
polystyrene cuvette and diluting it to a final volume of 1.00 ml
with ddH.sub.2 O. The stability of the dispersion was determined by
sequential measurements over a 24-hour period and was found to be
stable. The stability of the dispersion with respect to progressive
salinity of the solvent (increasing conductivity) was similarly
determined. The stability increased with progressive salinity of
the solvent.
1.00 ml of the dispersion was combined and stirred with 8.00 ml of
ddH.sub.2 O and 1.00 ml of 29.2 mM cellobiose stock in a 15.0 ml
capacity ultrafiltration stir cell (Spectra) which has been fitted
with a pre-rinsed 5.times.10.sup.5 molecular weight cutoff type F
membrane (Spectra). The sample was then left to stir for 15
minutes. After stirring, the excess cellobiose was removed by
flushing through the cell chamber 250 ml of ddH.sub.2 O by the
action of a peristaltic pump at a rate that does not exceed 10.0
ml/min. After washing, the filtrate was concentrated by the means
of pressurized N.sub.2 gas to approximately 1.0 ml. Character was
established by the removal of 500 .mu.ul of the treated dispersion
by N4MD analysis. The mean dispersion diameter was re-established
at this step. The stability of the coated dispersion was determined
by sequential measurements over a 24-hour period. The stability of
the coated dispersion with respect to progressive salinity of the
solvent (increasing conductivity) was similarly determined.
The resulting coated nanocrystalline particles are suitable for
attachment of various proteins, peptides and pharmaceutical
agents.
Example 4
Preparation of Tricalcium Phosphate (TCP) Nanocrystalline Particles
Coated With P5P:
1. Using two 60 cc syringes and a T-Luer lock, inject 50 mls of
0.75 m CaCI.sub.2 and 50 mls of 0.25 m Na.sub.2 HPO.sub.4 into a
120 ml pharmaceutical bottle in the cup sonicator. Sonicate for 30
minutes at room temperature to form suspension of TCP
particles.
2. Spin the TCP preparation down in the centrifuge using the bucket
rotor at 3000 rpm for 15 minutes to remove unreacted
components.
3. Resuspend the TCP particles in 50 mls of HPLC grade water and
mix well. Spin down at 3000 rpm for 15 minutes. Repeat step 3,
three times (3.times.).
4. Add 1.0 ml of 100 mg/ml pyridoxal-5-phosphate (P5P) and incubate
for 30 minutes on a rocker arm at room temperature.
5. Lyophilize overnight.
6. Resuspend the P5P-TCP preparation in 50 mls of 0.1 n sodium
hydroxide. Mix well. Spin down at 3000 rpm for 20 minutes. This
removes the excess P5P. (This step may not have to be completed
with all carbohydrates. Centrifugation and subsequent washing steps
may be adequate.)
7. Resuspend in 50 mls of PBS and spin down at 3000 rpm for 15
minutes. Repeat step 8 three times (3.times.). This removes the
sodium hydroxide.
8. Resuspend pellet in 50 mls of HPLC grade water and spin down at
3000 rpm for 15 minutes. Repeat step 9 three times (3.times.). This
removes the PBS.
9. Resuspend the pellet in 4.0 mls of HPLC water and 1.0 ml of 100
mM of sodium citrate to pH 7.2.
10. Sonicate for 15 minutes at room temperature to form suspension
of particles which is ready for attachment of biochemically active
agent.
Example 5
Preparation of TCP Nanocrystalline Particles Coated with
Cellobiose:
The same procedure as described in Example 1 is followed except
that cellobiose is substituted for P5P. The cellobiose coating is
applied to the particles by suspending the particles in a stock
solution of cellobiose. The cellobiose stock solution is a 292 mM
solution made by dissolving 1.000 gram of cellobiose in 9.00 mls of
ddH.sub.2 O. Solution is accomplished at approximately 70.degree.
C. in order to promote quick dissolution. The resulting cellobiose
solution is filter sterilized through a rinsed 0.45 micron filter
with the final volume being adjusted to 10.00 ml.
Sufficient cellobiose stock solution is added to 150 microliters of
the ultrafine biodegradable particle dispersion so that the final
concentration of the particle is 1.00 percent (w/v) or 29.2 mM. A
typical volume for preparation is 2.0 mls which is mixed four or
five times by the action of a micro-pipetor. After mixing, the
dispersion is allowed to equilibrate for two hours. Demonstration
of successful coating of the particles is provided by measuring the
mobility of the particles (coated and uncoated) on a Coulter DELSA
440 doppler energy light scatter analyzer. The coated particles
exhibit a relatively low mobility compared to the non-coated
particles. Measurements are also taken at various dilute salt
concentrations to ensure that the observations with respect to
mobility are not artifactual.
The coated particles are then used to attach antigenic proteins,
peptides or pharmacological agents to prepare bioreactive
particles.
Example 6
Preparation of Nanoparticles with Cellobiose or P5P Coatings:
The tin oxide, ruthenium oxide and silicon dioxide nanoparticles
prepared in Example 1-3 are coated with cellobiose or P5P in the
same manner as TCP.
Example 7
Preparing Meticulously Clean Biodegradable Nanoparticles:
1. Prepare 6 clean sonication tubes with 500 mg of biodegradable
particles per tube.
2. In fume hood, fill tubes with HCl (10N) approx. 8 ml/tube.
3. Sonicate for 30 min. (full power [175 watts]/25.degree. C.);
three tubes per sonication treatment.
3. Centrifuge 30 min. at 2000 rpm.
5. Decant the acidic supernatant (in the fume hood), fill the tubes
with HPLC grade water and then vortex.
6. Sonicate for 30 min [above conditions] and centrifuge for 30
[centrifuging is complete if the supernatant is clear].
7. Decant the supernatant, and fill the tubes with HPLC grade water
and vortex.
8. Repeat steps 7 and 8 two more times.
9. Decant the preparation into a clean glass [pyrex] baking
dish.
10. Anneal at 210.degree. C. overnight.
11. Remove the dried biodegradable crystals by gentle scraping with
a clean unpainted spatula and transfer into 6 clean glass
sonicating tubes.
12. Repeat steps 3 through 8.
13. Prepare a 10 kD (NMWL) 150 ml ultrafiltration cell, empty the
contents only one[no more than 500 mg per filtration run] of the
tubes into the cell, and wash 500 ml of HPLC grade water through
the cell under a N.sub.2 pressure head of 20 psi (regulator
pressure gauge reading).
14. After washing, adjust the preparation volume to 100.0 ml by
using the appropriate volume markings on the side of the cell.
15. Take a concentration measurement by removing 1.0 ml of the
preparation from the cell and lyophilizing it down in a pre-weighed
1.7 ml Eppendorf tube. After lyophilization, take a mass
measurement of the tube with its contents and subtract it away from
the mass of the empty tube. This provides the initial density of
the preparation. Preferably, the concentration or density of the
particles in the solution is about 10 mg/ml. If the initial
density is lower than 10 mg/ml, then the solution should be further
concentrated in the ultrafiltration cell.
Example 8
Coating Meticulously Clean Biodegradable Nanoparticles with a
Molecular Stabilizing Film (Cellobiose):
Incubation/Lyophilization.
1. Sonicate the meticulously clean biodegradable particles (aqueous
dispersion) prepared in Example 7 for 30 minutes at 25.degree. C.
at full power [175 Watts].
2. Then as quickly as possible, exchange suspending medium from
water (stock) to a solution of 500 mM cellobiose using either a
bench top microcentrifuge (30 seconds, full speed of 14,000 RPM)
for small volumes or for larger volumes a floor models centrifuge
(model 21K, in 50 ml centrifuge tubes, 8,000 RPM for a maximum of 2
minutes) Suspend the pelleted particles with 500 mM cellobiose,
sonicate to aid dispersion (approximately 5 minutes at 25.degree.
C. at full power [175 Watts]) and finally set the mixture on a
rocking plate overnight in a cold room [4.degree. C.]
3. The next day portion out the mixture into appropriately sized
vessels for overnight lyophilization.
4. Leave the tubes capped with a layer of parafilm around the cap
and place them in a freezer until the washing step.
5. Reconstitute the particle/cellobiose in a suitable buffer
depending on the application. Suitable buffers are low ionic
strength buffered phosphate (PRB), water, or bicarbonate.
Reconstitution in the buffer is accomplished by vortexing and a 5
minute sonication [175 Watts/25.degree. C.].
6. Wash by repeated centrifugation (using either a bench top
microcentrifuge [30 seconds, full speed of 14,000 RPM] for small
volumes or for large volumes a floor model centrifuge [model 21K,
in 50 ml centrifuge tubes, 8,000 RPM for a maximum of 2 minutes])
and resuspension into the buffer.
7. Take a concentration measurement by removing 1 ml of the
suspension dehydrating it in a lyophilizer in a pre-weighed 1.7 ml
Eppendorf tube, and massing.
8. Calculate the final volume necessary to bring the concentration
to 1 mg/ml. Add enough buffer to bring the concentration of the
particle/cellobiose preparation to 1 mg/ml.
Example 9
Preparing Meticulously Clean Particles of Brushite:
Reagents.
0.75M CaCI.sub.2 : 55.13 g CaCI.sub.2.2H.sub.2 O is dissolved with
HPLC grade water to 0.500 L in a volumetric flask. Filter sterilize
with 0.2 um sterile filtration unit and place in a sterile 500 ml
culture medium flask. Store at room temperature.
0.25M Na.sub.2 HPO.sub.4 : 17.75 g of anhydrous Na.sub.2 HPO.sub.4
is dissolved with HPLC grade water to 0.500 L in a volumetric
flask. Filter sterilize with 0.2 um sterile filtration unit and
place in a sterile 500 ml culture medium flask. Also store at room
temperature.
Brushite synthesis.
About a half hour before synthesis, prepare the sonicator by
cooling down the cup horn. This is accomplished by adjusting the
low temperature thermostat on the water condenser to 4.degree. C.
and dialing a setting of "4" on the peristatic circulator. Once the
4.degree. C. mark is reached, prepare 50.0 ml of 0.75M CaCI.sub.2
and 50.0 ml of 0.25M Na.sub.2 H.sub.2 PO.sub.4 and load into 50 ml
syringes. The syringes are then to be connected to a 3-way luer
lock connector so that they are set in diametric
opposition--allowing the remaining luer port to be free to dispel
product. Once the mixing apparatus is set up, place a sterile 120
ml sonicating flask in the cup horn and slowly power up the
sonicator to 100% power. Position the mixing apparatus so that the
free luer port is over the sonicating flask. Expel syringe contents
into the flask as rapidly and evenly as possible so as to empty
each syringe roughly at the same time. Then quickly secure a
polypropylene liner over the sonicating flask and let sonicate for
an additional 15 minutes.
Brushite washing.
Roughly divide the preparation into two 50 ml blue top
polypropylene tubes and pellet at 2000 rpm for 10 minutes (room
temperature). Reconstitute by vortexing each pellet with sterile
HPLC grade water to 50 ml (or tube capacity) and pellet at 2000 rpm
for 10 minutes. Repeat this wash 3 more times and reconstitute the
last pellets to 50.0 ml. Transfer the dispersion to a sterile 120
ml sonicating flask with polypropylene liner. Place the flask in a
previously cooled sonicator cup horn at 1.degree. C. Sonicate at
100% power for 60 minutes.
Example 10
Coating Meticulously Clean Particles of Brushite with a Molecular
Stabilizing Film of Pyridoxyl-5-Pyrophosphate:
Brushite/Pyroxidal 5 phosphate (vitamine B6).
Pellet 100 ml of the dispersion prepared in Example 10 so that the
entire contents can be transferred to a 50 ml conical tube. Adjust
the tube volume to 40.0 ml. Then transfer the contents in 10 ml
aliquots to four 15 ml conical tubes. Dissolve 1000 mg of
Pyroxidal-5-phosphate with 800 .mu.l of 10N NaOH and adjust with
water to 10 mls. Filter sterilize this clear yellow solution with a
0.2 .mu.m acrodisc and add 2.5 ml aliquots to each of the
previously prepared 4 brushite tubes. Vortex each tube a few
seconds to make certain that the contents are well dispersed.
Lyophilize overnight [approx. 16 hrs] at the low drying rate
setting. The next morning resuspend in 50 ml aliquots of sterile
HPLC grade water five more times. Pellet once more and transfer the
pellets to four 15 ml conical tubes and adjust the final
preparation volume with water to 40.0 ml.
Example 11
Coating Meticulously Clean Particles of Brushite With a Molecular
Stabilizing Film of Citrate:
Brushite/citrate.
Pellet the 100 ml of the dispersion prepared in Example 13 so that
entire contents can be transferred to a 50 ml conical tube. Adjust
the tube volume to 40.0 ml. Then transfer the contents in 10 ml
aliquots to four 15 ml conical tubes. Add 10 ml of 100 mM citrate
to each of the 15 ml conicals and nutate for 30 minutes at room
temperature. Lyophilize overnight [approx. 16 hrs] at the low
drying rate setting. The next morning resuspend in 50 ml aliquots
of sterile HPLC grade water five more times. Pellet once more and
transfer the pellets to four 15 ml conical tubes and adjust the
final preparation volume with water to 40.0 ml.
Example 12
Attachment of Transfection Agents:
The following procedure is used to attach DNA or RNA segments to
any of the coated particles described in the preceding
Examples:
A dispersion of 40 mg/ml of coated nanoparticles in 20 mM pH 6.80
phosphate buffer is prepared. To this dispersion, 1.00 ug of cesium
chloride purified DNA or RNA fragments are added and left to absorb
at room temperature under mild agitation for approximately 16
hours. Specific DNA fragment used in this example is the human
deaminase gene.
Example 13
Attachment of Targeting Ligands and Phospholip Membrane:
The procedure described in the example for isolating and attaching
the targeting ligands and phospholip membrane may be used for all
of the particle/coating/DNA or RNA combinations set forth in the
preceding examples.
10.sup.6 transforming units of virus is incubated with Triton
extraction buffer (1.0% of Triton.times.100/0.25 mM DTT 10 mM Tris
pH 7.4 1.0 mM MgCl). Extract is then ultracentrifuged at 100K*g for
2.0 hrs (35 rpm SW50.1 Beckman rotor) at 4.0.degree. C. to remove
nucleocapsid. Removal of triton and envelope protein enrichment is
accomplished by incubation with a 300 ul slurry of polystyrene
micro beads (Spectra Gel D2) and subsequent 100 kD ultrafiltration
into phosphate buffer 5 mg/ml of phosphotidyl choline and 5 mg/ml
phosphotidyl serine. An alternative method of viral extraction is
as follows. A mixture of phosphotidyl choline (5 mg/ml) and
phosphotidyl serine (5 mg/ml) in 20 mM pH 7.4 phosphate buffer is
sonicated for 30 minutes at 4.0.degree. C. 10.sup.6 units per ml of
virus is then added to 1.0 ml of the phospholipid mixture and left
to sonicate at 5 sec cycles per minute for 30 minutes at
4.0.degree. C. Nucleocapsid is removed by ultracentrifugation at 35
krpm at 4.0.degree. C. Intervening layers of carbohydrate may be
first adsorbed to the TCP-DNA/RNA complex prior to the addition of
the ligand/membrane components.
Example 14
A construct is composed of a brushite core with an adsorbed first
layer consisting of human deaminase gene in an expression cassette
with a albumin enhancer and limited terminal repeats for genomic
integration. The construct includes an adsorbed second layer of
cellobiose and an adsorbed third layer of membrane proteins from
human low density lipids (LDL receptor ligands).
The entire contents of all references cited hereinabove are hereby
incorporated by reference.
Having thus described exemplary embodiments of the present
invention, it should be noted by those skilled in the art that the
within disclosures are exemplary only and that various other
alternatives, adaptations and modifications may be made within the
scope of the present invention. Accordingly, the present invention
is not limited to the specific embodiments as illustrated herein,
but is only limited by the following claims.
* * * * *