U.S. patent application number 15/172554 was filed with the patent office on 2016-09-29 for crosslinked polysaccharide beads and their biomedical uses.
The applicant listed for this patent is INSERM (Institut National de la Sante et de la Recherche Medicale), Universite Paris Diderot - Paris 7. Invention is credited to Sidi Mohammed Derkaoui, Catherine Le Visage, Didier Letourneur.
Application Number | 20160279064 15/172554 |
Document ID | / |
Family ID | 42938461 |
Filed Date | 2016-09-29 |
United States Patent
Application |
20160279064 |
Kind Code |
A1 |
Letourneur; Didier ; et
al. |
September 29, 2016 |
Crosslinked Polysaccharide Beads and Their Biomedical Uses
Abstract
The present inventions relates to beads as biocompatible
material adapted for use within the human or animal body. Said
beads are highly useful for tissue engineering, in situ tissue
regeneration, as well as for drug and/or cells delivery. In
addition, said beads may support biotechnological applications such
as cell carriers.
Inventors: |
Letourneur; Didier; (Paris,
FR) ; Le Visage; Catherine; (Paris, FR) ;
Derkaoui; Sidi Mohammed; (Paris, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSERM (Institut National de la Sante et de la Recherche
Medicale)
Universite Paris Diderot - Paris 7 |
Paris
Paris Cedex 13 |
|
FR
FR |
|
|
Family ID: |
42938461 |
Appl. No.: |
15/172554 |
Filed: |
June 3, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13819437 |
Mar 20, 2013 |
9381250 |
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PCT/EP2011/064927 |
Aug 30, 2011 |
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15172554 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 2430/06 20130101;
A61K 9/1075 20130101; A61P 35/00 20180101; A61K 9/0024 20130101;
Y10T 428/2982 20150115; A61L 27/20 20130101; A61L 2430/02 20130101;
C08B 37/0018 20130101; A61K 9/1682 20130101; A61K 47/26 20130101;
A61L 27/20 20130101; C08L 5/00 20130101; A61K 9/1652 20130101; A61L
2430/20 20130101; C08B 37/0021 20130101; A61K 9/0019 20130101 |
International
Class: |
A61K 9/16 20060101
A61K009/16; A61K 9/00 20060101 A61K009/00; A61K 9/107 20060101
A61K009/107 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 31, 2010 |
EP |
10305931.7 |
Claims
1-9. (canceled)
10. A bead obtained by a) preparing an alkaline aqueous solution
comprising at least one polysaccharide and a cross linking agent;
b) dispersing said alkaline aqueous solution into an hydrophobic
phase in order to obtain a water-in-oil (w/o) emulsion; and c)
transforming the w/o emulsion into beads by placing said w/o
emulsion at a temperature from about 4.degree. C. to about
80.degree. C. for a sufficient time to allow the cross-linking of
said at least one polysaccharide; wherein, said polysaccharide is
selected from the group consisting of dextran, pullulan, agar,
alginic acid, starch hyaluronic acid, inulin, heparin, fucoidan,
chitosan and mixtures thereof.
11. The bead according to claim 10, wherein said bead has a size of
from 5 nm to 5 mm.
12-17. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for preparing
polysaccharide beads. The present invention further provides beads
and their uses in various biomedical fields.
BACKGROUND OF THE INVENTION
[0002] Biocompatible materials have received a great interest for
different biomedical applications. The attributes of the ideal
biocompatible material would include the ability to support cell
growth either in vitro or in vivo, the ability to support the
growth of a wide variety of cell types or lineages, the ability to
be endowed with varying degrees of flexibility or rigidity
required, the ability to have varying degrees of biodegradability,
the ability to be introduced into the intended site in vivo without
provoking adverse events, and the ability to serve as a vehicle or
reservoir for delivery of cells, drugs or bioactive substances to
the desired site of action.
[0003] For this purpose, polysaccharides were shown to be the
materials of choice due to their validated biological properties.
Indeed, many studies are currently drawn to materials obtained by
ionotropic gelation (ability of polysaccharides such as pectin,
alginate, carrageenan and gellan to form a gel in the presence of
multivalent ions). However, those techniques are limited since they
are carried out with a little range of polysaccharide type. In
addition, those gels are not suitable for an easy and efficient
administration within a human or animal tissue, independently of
the size and localization of the target tissue. Lee C S et al. have
illustrated the use of calcium-alginate beads also for tissue
engineering of bone (Lee C S et al. Regulating in vivo
calcification of alginate microbeads, Biomaterials 2010, June;
31(18):4926-34). Others have prepared polysaccharide beads from
chitosan based polyelectrolytes for drug delivery systems. Further,
chitosan based polyelectrolyte complexes were proposed as potential
carrier materials in drug delivery systems (Hamman J H et al.,
Chitosan based polyelectrolyte complexes as potential carrier
materials in drug delivery systems, J Biomed Mater Res, 2010).
[0004] However, the above mentioned polysaccharide beads are not
adapted because of the instability of the divalent cations
complexes under physiological conditions, and to the little range
of potential polysaccharides to be used. In addition, the
biocompatible materials currently used can not be easily
administrated within a human or animal tissue, independently of the
size and localization of the target tissue.
[0005] There is thus still a need for a biocompatible material
adapted for an injection within the human or animal body and that
can be used for biological and therapeutic purposes. Particularly,
there is a need for a biocompatible material which would be easily
administrated by injection to the human or animal tissue,
independently of the site of action and of the size of the targeted
region.
SUMMARY OF THE INVENTION
[0006] The inventors filled the foregoing need by providing a
crosslinking method on polysaccharides that allows obtaining beads
without prior chemical modifications of the polysaccharides, and
without the use of organic solvents. Said method is thus easy to
carry out since the polysaccharides are not subjected to any
modification. Thus, the beads present the advantage of being
suitable for therapeutic use, since they are free of any
contamination and organic solvent. The resulting beads in
suspension are stable in physiological fluids. Said beads are
biocompatible and injectable material, useful for tissue
engineering, in situ tissue regeneration, as well as for drug
and/or bioactive substance delivery. In addition, said bead can
support biotechnological applications such as cell carriers in
vitro and in vivo.
[0007] In a first object, the invention relates to a method for
preparing polysaccharide beads comprising the following steps:
[0008] a) preparing an alkaline aqueous solution comprising an
amount of at least one polysaccharide and an amount of a cross
linking agent; [0009] b) dispersing said alkaline aqueous solution
into an hydrophobic phase in order to obtain a w/o emulsion; and
[0010] c) transforming the w/o emulsion into polysaccharide beads
by placing said w/o emulsion at a temperature from about 4.degree.
C. to about 80.degree. C. for a sufficient time to allow the
cross-linking of said amount of polysaccharide;
[0011] wherein said polysaccharide is selected from the group
consisting of dextran, pullulan, agar, alginic acid, starch
hyaluronic acid, inulin, heparin, fucoidan, chitosan and mixtures
thereof.
[0012] In one embodiment, the alkaline aqueous solution comprises a
porogen agent. Thus, the invention provides porous polysaccharide
beads.
[0013] In another embodiment, the alkaline aqueous solution
comprises active components, such as for instance hydroxyapatite or
preferably nano-hydroxyapatite.
[0014] In a further embodiment, the alkaline aqueous solution
comprises a drug or a bioactive substance.
[0015] In a second aspect, the invention relates to a
polysaccharide bead obtainable by the method of the invention.
[0016] In a third aspect, the invention relates to a polysaccharide
bead obtainable by the method of the invention for use for in situ
tissue regeneration.
[0017] In a fourth aspect, the invention relates to a
polysaccharide bead comprising hydroxyapatite, preferably
nano-hydroxyapatite, obtainable by the method of the invention for
use for stimulating mineralized bone tissue formation.
[0018] In a fifth aspect, the invention relates to a polysaccharide
bead obtainable by the method of the invention for use as a cell
microcarrier.
[0019] In a sixth aspect, the invention relates to a polysaccharide
bead obtainable by the method of the invention for use for drug,
bioactive substance and/or cell delivery.
[0020] In a seventh aspect, the invention relates to a
polysaccharide bead comprising drug obtainable by the method of the
invention for use for treating cancer.
DETAILED DESCRIPTION OF THE INVENTION
Definition
[0021] As used herein, the term "polysaccharide" refers to a
molecule comprising two or more monosaccharide units.
[0022] As used herein, the term "alkaline solution" refers to a
solution having a pH strictly superior to 7.
[0023] As used herein, the term "aqueous solution" refers to a
solution in which the solvent is water.
[0024] As used herein, the term "porogen agent" refers to any solid
agent which has the ability to form pores within, a solid
structure.
[0025] As used herein, the term "cross-linking" refers to the
linking of one polysaccharide chain to another one with covalent
bonds.
[0026] As used herein, the term "cross-linking agent" encompasses
any agent able to introduce cross-links between the chains of the
polysaccharides of the invention.
[0027] In the context of the invention, the tell is "bead",
"particle", and "sphere" are used in an interchangeable manner and
refer to polysaccharide composition of the invention having a
substantially spherical or ovoid shape.
[0028] As used herein, the term "nanobeads" encompasses bead having
a size of at least 1 nm and inferior to 1000 nm, the term
"microbeads" encompasses beads having a size of at least 1 .mu.m
and inferior to 1000 .mu.m, the term "macrobeads" encompasses beads
having a size of at least to 1 mm.
[0029] As used herein, the term "biodegradable" refers to materials
that degrade in vivo to non-toxic compounds, which can be excreted
or further metabolized.
[0030] As used herein, the term "freeze-drying" refers to the
drying of a deep-frozen material under high vacuum by freezing out
the solvent (ie. water) and then evaporating it in the frozen
state.
[0031] In its broadest meaning, the term "treating", "treatment"
and "therapy" refers to reversing, alleviating, inhibiting the
progress of, or preventing the disorder or condition to which such
term applies, or one or more symptoms of such disorder or
condition.
[0032] As used herein, the expression "bone tissue" refers to
calcified tissues (e.g., calvariae, tibiae, femurs, vertebrae,
teeth), bone trabeculae, the bone marrow cavity, the cortical bone,
which covers the outer peripheries of the bone trabeculae and the
bone marrow cavity, and the like. The expression "hone tissue" also
encompasses hone cells that are generally located within a matrix
of mineralized collagen; blood vessels that provide nutrition for
the bone cells; bone marrow aspirates: joint fluids: bone cells
that are derived from bone tissues; and may include fatty bone
marrow. Finally, bone tissue includes bone products such as whole
bones, sections of whole bone, bone chips, bone powder, bone tissue
biopsy, collagen preparations, or mixtures thereof. For the
purposes of the present invention, the term "bone tissue" is used
to encompass all of the aforementioned bone tissues and products,
whether human or animal, unless stated otherwise.
[0033] As used herein, the expression "bone-related disorders"
includes disorders of bone formation and bone resorption.
Preferably, the expression "bone related disorders" refers to
diseases associated with insufficiency of bone formation or bone
loss. Non-limiting examples of bone related disorders are rickets,
osteoporosis osteomalacia, osteopenia, bone cancer, arthritis,
rickets, bone fracture, bone defects, osteolytic bone disease,
osteomalacia, bone frailty, loss of bone mineral density
achondroplasia, cleidocranial dysostosis, Paget's disease,
osteogenesis imperfecta, osteopetrosis, sclerotic lesions,
pseudoarthrosis, periodontal disease, anti-epileptic drug induced
hone loss, weightlessness induced bone loss, postmenopausal bone
loss, osteoarthritis, infiltrative disorders of bone, metabolic
bone diseases, organ transplant related bone loss, adolescent
idiopathic scoliosis, glucocorticoid-induced bone loss,
heparin-induced bone loss, bone marrow disorders, malnutrition,
calcium deficiency, rheumatoid arthritis, hypogonadism. HIV
associated bone loss, tumor-induced bone loss, cancer-related bone
loss, hormone ablative bone loss, multiple myeloma, drug-induced
bone loss, facial bone loss associated with aging, cranial bone
loss associated with aging, jaw bone loss associated with aging,
skull bone loss associated with aging, and bone loss associated
with space travel. Preferably, the bone related disorders, as used
herein, are bone fracture, large bone defects, rickets,
osteoporosis, osteogenesis imperfecta, osteomalacia, osteopenia,
bone cancer, osteolytic bone disease, bone frailty and/or loss of
bone mineral density.
[0034] As used herein, the expression "cardiac tissue" refers to
the tissues within the heart. This expression encompasses
epicardium, myocardium and endocardium.
[0035] As used herein, the expression "cardiac-related disorders"
refers to pathologies connected with defects of the cardiac tissue.
This expression encompasses the presence of a damages or injured
tissue within the cardiac tissue and the resorption of the cardiac
tissue.
[0036] Non-limiting examples of cardiac-related disorders are
myocardial infarction, stroke, hypertension, coronary heart
disease, congestive heart failure, rheumatic heart disease,
congenital cardiovascular defects, myocarditis, or arrhythmia.
[0037] As used herein, the term `hydroxyapatite`, or "HA" refers to
a naturally occurring mineral form of calcium apatite with the
formula Ca.sub.5(PO.sub.4).sub.3(OH), but is usually written
Ca.sub.10(PO.sub.4).sub.6(OH).sub.2 to denote that the crystal unit
cell comprises two entities. The OH.sup.- ion can be replaced by
fluoride, chloride or carbonate, producing fluorapatite or
chlorapatite. Preferably, for the purpose of the invention, the
OH.sup.- is not replaced. Hydroxyapatite is the major component of
bone and teeth matrix and gives bones and teeth their rigidity.
[0038] As used herein, the term "nanocristalline hydroxyapatite",
or "nano-hydroxyapatite", or "n-HA", refers to hydroxyapatite
crystal particles having a size comprised between 10 and 100 nm,
preferably between 30 and 60 nm, and most preferably about 50 nm.
n-HA suitable for carrying out the present invention is obtained
for example by precipitation of a solution of phosphoric acid with
a solution of calcium hydroxide.
[0039] As used herein, the terms "non-aqueous phase", "lipophilic
phase", "hydrophobic phase", and "oily phase" may be used in an
interchangeable manner.
[0040] As used herein, "w/o emulsion" or `water-in-oil emulsion",
refers to the dispersion of an aqueous phase into a lipophilic
phase. The term "w/o emulsion" encompasses stable and non-stable
emulsion. In a preferred embodiment, the w/o emulsion is obtained
in the absence of a substantial amount of any surfactant. In a most
preferred embodiment, the w/o emulsion is obtained in the absence
of any surfactant.
[0041] As used herein, "surfactant" or "emulsifier" refers to a
compound that lowers the surface tension of a liquid, the
interfacial tension between two liquids, or that between a liquid
and a solid.
[0042] As used herein, the expression, "in the absence of a
substantial amount of surfactant" refers to the absence of
surfactant or the presence of a quantity of surfactant which does
not interfere with the process of obtaining a w/o emulsion.
Therefore, said expression encompasses the possibility of the
presence of surfactant in a quantity that would not play its role
of lowering the surface tension, therefore not interfering in the
properties or size of the polysaccharide beads obtained by the
method of the invention. As used herein, "in situ tissue
regeneration" refers to the regeneration of a damaged or injured
tissue, possibly leading to the restoration of the function of said
tissue. This term encompasses all the strategies of providing
means, such as the polysaccharide beads of the invention, for
promoting the regeneration of a tissue, especially a defective
tissue.
Crosslinked Polysaccharide Beads and Method for Preparing
Thereof
[0043] In a first object, the invention relates to a method for
preparing polysaccharide beads comprising the following steps:
[0044] a) preparing an alkaline aqueous solution comprising an
amount of at least one polysaccharide and an amount of a cross
linking agent; [0045] b) dispersing said alkaline aqueous solution
into an hydrophobic phase in order to obtain w/o emulsion; and
[0046] c) transforming the w/o emulsion into heads by placing said
w/o emulsion at a temperature from about 4.degree. C. to about
80.degree. C. for a sufficient time to allow the cross-linking of
said amount of polysaccharide,
[0047] wherein said polysaccharide is selected from the group
consisting of dextran, pullulan, agar, alginic acid, hyaluronic
acid, inulin, heparin, fucoidan, chitosan and mixtures thereof.
[0048] Typically, the step b) of dispersing the alkaline aqueous
solution into the hydrophobic phase is performed under mechanical
stirring. Typically, such a dispersing step is performed during 10
min. Alternatively, the emulsification process can be performed
using a high performance disperser, such as Polytron.RTM.
Homogenizer.
[0049] In a preferred embodiment, step b) is performed in the
absence of a substantial amount any surfactant. In a most preferred
embodiment, step b) is performed in the absence of any surfactant.
The polysaccharide beads obtained thereof have a mean diameter of
more than about 10 .mu.m.
[0050] In a specific embodiment, the method of the invention
further comprises the following steps:
[0051] d) submerging said polysaccharide beads into an aqueous
solution; and
[0052] e) washing said polysaccharide beads.
[0053] Typically, the polysaccharide beads are washed in water or
phosphate buffer saline (PBS).
[0054] In another embodiment, the method of the invention further
comprises a step f) of calibrating the polysaccharide beads
according to their size. After performing said step of calibrating,
the person skilled in the art may obtain beads of a size comprised
between about 10 .mu.m to about 5000 .mu.m. Typically, the
polysaccharide beads are calibrated according to their size using
appropriate nylon filter. The person skilled in the art is aware of
the nylon filter adapted for the purpose of the invention.
[0055] In still another embodiment, the method of the invention
further comprises a step g) of freeze-drying said polysaccharide
beads. Freeze-drying may be performed with any apparatus known in
the art. There are essentially three categories of freeze dryers:
rotary evaporators, manifold freeze dryers, and tray freeze dryers.
Such apparatus are well known in the art and are commercially
available such as a freeze-dryer Lyovac (GT2, STERIS Rotary vane
pump, BOC EDWARDS). Basically, the vacuum of the chamber is from
0.1 mBar to about 6.5 mBar. The freeze-drying is performed for a
sufficient time to remove at least 98.5% of the water, preferably
at least 99% of the water, more preferably at least 99.5%.
Typically, the freeze drying step is performed for 24 hours.
[0056] Preferably, the polysaccharide is a mixture of
pullulan/dextran. Typically, the weight ratio of pullulan to
dextran is 75:25 w/w.
[0057] Typically, said cross-linking agent is selected from the
group consisting of trisodium trimetaphosphate (STMP), phosphorus
oxychloride (POCl.sub.3), epichlorohydrin, formaldehydes,
carbodiimides, and glutaraldehydes. Preferably, for the purpose of
the present invention, said cross-linking agent is STMP.
[0058] Typically, the weight ratio of the polysaccharide to the
cross linking agent is in the range from 15:1 to 1:1, preferably
6:1.
[0059] The skilled artisan is aware of the hydrophobic phases
suitable for the purpose of the present invention. Non-limiting
examples of hydrophobic phases are vegetal oils, such as canola
oil, corn oil, cottonseed oil, safflower oil, soybean oil, extra
virgin olive oil, sunflower oil, palm oil, MCT oil, and trioleic
oil. Preferably, for the purpose of the present invention, said
hydrophobic phase is canola oil. Alternatively, said hydrophobic
phase is a silicon fluid. Typically, the quantity of hydrophobic
phase in the w/o emulsion (volume of lipophilic phase/volume of the
water-in-oil emulsion; v/v) represents from about 10% to about 90%
v/v, preferably from about 20% to about 80% v/v, preferably from
about 50% to about 80% v/v and most preferably about 70% v/v of the
w/o emulsion.
[0060] In one embodiment of the invention, the alkaline aqueous
solution further comprises an amount of a porogen agent. Thus, the
invention also provides porous polysaccharide beads. Non-limiting
examples of porogen agents are sodium chloride, calcium chloride,
ammonium carbonate, ammonium bicarbonate, calcium carbonate, sodium
carbonate, and sodium bicarbonate and mixtures thereof. Preferably,
for the purpose of the invention, said porogen agent is sodium
chloride. Typically, the weight ratio of the polysaccharide to the
porogen agent is in the range from 50:1 to 1:50. In a preferred
embodiment, such weight ratio of the polysaccharide to the porogen
agent is about 12:14.
[0061] Typically, the density of the pores is from about 4% to
about 75%, preferably from about 4% to about 50%. The person
skilled in the art may easily adapt the porosity of the beads
obtained by the method of the invention by adapting the amount of
porogen agents added in the alkaline solution.
[0062] Typically, the size of the pores of the polysaccharide beads
of the invention is comprised between about 1 .mu.m and about 1000
.mu.m. The presence of said pores are highly convenient for
incorporating drugs or cells on the surface of the polysaccharides
heads of the invention.
[0063] In still another embodiment, the alkaline aqueous solution
further comprises hydroxyapatite, preferably nano-hydroxyapatite.
The invention thus provides polysaccharide beads comprising n-HA.
Said polysaccharide beads are founds to be highly appropriate for
use for treating of bone disorders.
[0064] In the context of the present invention, nano-hydroxyapatite
may be a commercial nano-hydroxyapatite, such as those
commercialised by Inframat Corporation or Fluidinova. Preferably,
nanocristalline hydroxyapatite useful in the context of the present
invention is obtained through chemical precipitation at room
temperature of a solution of phosphoric acid, at a concentration
comprised between 0.3 to 1M, preferably 0.6M, with a solution of
calcium hydroxide, at a concentration comprised between 0.5 to
1.5M, preferably 1M.
[0065] Typically, the concentration of nano-hydroxyapatite in the
alkaline aqueous solution (w/v) is comprised between 1 and 10% w/v,
preferably between 1 and 5% w/v, more preferably between 1 and 3%
w/v.
[0066] In a further embodiment, the alkaline solution further
comprises a drug. The invention thus provides polysaccharide beads
comprising a drug, said polysaccharide beads being highly adapted
for administering said drug within a target tissue in the human or
animal body. Typically, said drug is a drug having an acknowledged
therapeutic effect, such as hormones radioactive substance,
fluorescent substance, chemotactic agent, antibiotic, steroidal or
non-steroidal analgesic, immunosuppressant, or anti-cancer drug.
Preferably, said drug is an anti-cancer drug.
[0067] In a further embodiment, the alkaline solution further
comprises a bioactive substance. Typically, said bioactive
substance is a substance known for playing an important role in
various mechanisms such as modification of cellular pathways and
modification of cellular or tissular responses. Said bioactive
substance is chosen among growth factors, cytokines (lymphokines,
interleukins, and chemokines), antioxidant molecules, angiogenic
molecule, anti-angiogenic agents, immunomodulating agents,
proinflammatory cytokines, antiinflammatory cytokines,
plasma-derived bioactive substances, PRP (platelet rich
plasma)-derived substances, soluble adhesion molecules.
[0068] In a further embodiment, the alkaline solution further
comprises iron oxide. The invention thus provides polysaccharide
beads comprising iron oxide particles, highly adapted for use in
cancer treatment based on hyperthermia.
[0069] Typically, the iron oxide particles (Fe.sub.2O.sub.3) may be
obtained by alkaline coprecipitation of iron (III) and iron (II)
salts. Typically, the concentration of iron oxide in the alkaline
aqueous solution is comprised between 0.005 and 0.5 mol/L,
preferably between 0.01 and 0.05 mol/L.
[0070] In one embodiment, gelatin is added to the w/o emulsion
obtained in step b) of the method of the invention. Preferably,
said gelatin is added during step c) of the method of the
invention, i.e. during the cross linking step. Typically, the
concentration of gelatin in the w/o emulsion solution (w/v) is
comprised between 1 and 20% w/v, preferably between 1 and 10% w/v,
more preferably between 5 and 10% w/v. Addition of gelatin has been
shown to enhance adhesion of anchorage-dependent cells onto the
beads during in vitro culture.
[0071] In a further embodiment, the polysaccharide of the invention
is labelled with a fluorescent dye. As used herein, the term
"fluorescent dye" refers to any organic or inorganic molecule that
absorbs electromagnetic radiation at a given wavelength and that
emits electromagnetic radiation of longer wavelength by a
fluorescent mechanism upon irradiation by a source of
electromagnetic radiation, such as a lamp, a photodiode, or a
laser. The invention thus provides polysaccharide beads having a
size of less than 10 .mu.m and comprising a fluorescent agent. Said
beads are highly appropriate for monitoring the development of a
thrombus. Indeed, the inventors have shown that when injected into
a patient likely to be suffering from thrombus, the fluorescent
head may allow assessing and thus preventing a thrombus to occur.
Fluorescent beads are appropriate for monitoring cell infiltration
during in vitro culture.
[0072] A non-limitative list of fluorescent dyes appropriate in the
context of the invention is Fluorescein isothiocyanate (FITC),
Rhodamine, Texas red, fluorescent dyes from the Cy family such as
Cy2, Cy3, Cy5, Cy5.5, Cy7, fluorescent dyes from the Alexa family
such as Alexa 488, Alexa 532, Alexa 546, Alexa 548 Alexa 568, Alexa
594, Alexa 633, Alexa 647 Alexa 660, and indocyanine green.
[0073] Preferably, said polysaccharide is chosen in the group
consisting of dextran labeled with FITC, pullulan labeled with
Rhodamine and their mixtures.
[0074] In one particular embodiment, the method of the invention
may comprise a further step consisting of hydrating the
polysaccharide beads as prepared according to the invention. Said
hydration may be performed by submerging the polysaccharide beads
in a solution, preferably an aqueous solution (e.g., de-ionized
water, water filtered via reverse osmosis, a saline solution, or an
aqueous solution containing a suitable active ingredient) for an
amount of time sufficient to produce a polysaccharide bead having
the desired water content. Typically, when a polysaccharide bead
comprising the maximum water content is desired, the polysaccharide
bead is submerged in the aqueous solution for an amount of time
sufficient to allow the polysaccharide bead to swell to its maximum
size or volume. Typically, the polysaccharide bead is submerged in
the aqueous solution for at least about 1 hour, preferably at least
about 2 hours, and more preferably about 4 hours to about 24 hours.
It is understood that the amount of time necessary to hydrate the
polysaccharide bead to the desired level will depend upon several
factors, such as the composition of the used polysaccharides, the
size (e.g., thickness) of the polysaccharide beads, and the
temperature of the solution, as well as other factors.
[0075] In a second aspect, the invention relates to polysaccharide
beads obtainable by the method of the invention. These
polysaccharide beads are indeed the only ones which have the
remarkable properties provided by the invention.
[0076] Typically, said polysaccharide beads have a size comprised
from about 5 nm to about 5 mm, preferably about 10 nm to about 1
mm, preferably from about 1 .mu.m to about 100 .mu.m, preferably
from about 1 .mu.m to about 3 .mu.m.
[0077] In one embodiment of the invention, when step b) of the
method of the invention is carried out in absence of surfactant,
the size of the polysaccharide bead obtained thereof is at least
about 10 .mu.m. In another embodiment of the invention, when step
b) of the method of the invention is carried out in the presence of
surfactant, the size of the polysaccharide bead obtained thereof is
less than about 10 .mu.m, and is preferably comprised between about
5 nm and about 10 .mu.m.
[0078] The size of the polysaccharide beads would be chosen with
precaution by the skilled man in regard with the desired use. The
size of the polysaccharide beads of the invention is dependent on
the characteristics and parameters of the method of preparing such
polysaccharide beads. Typically, the size of the polysaccharide
bead of the invention may depend on the nature of the
polysaccharide, the agitation provided during the process and the
distribution of the polysaccharide within the polysaccharide beads.
The person skilled in the art may easily adapt and calibrate the
beads in order to obtain a desired size. Typically, said adaptation
and/or calibration may be performed by the following techniques:
sieving or filtration though nylon filter.
[0079] The method of the invention can further include the step of
sterilizing the polysaccharide beads using any suitable process.
The polysaccharide beads can be sterilized at any suitable point. A
suitable irradiative sterilization technique is for example an
irradiation with Cesium 137, 35 Gray for 10 minutes. Suitable
non-irradiative sterilization techniques include, but are not
limited to, UV-exposure, gas plasma or ethylene oxide methods known
in the art. For example, the polysaccharide beads can be sterilized
using a sterilization system which is available from Abtox, Inc of
Mundelein, Ill. under the trade mark PlazLyte, or in accordance
with the gas plasma sterilization processes disclosed in U.S. Pat.
No. 5,413,760 and U.S. Pat. No. 5,603,895.
[0080] The polysaccharide beads produced by the method of the
invention can be packaged in any suitable packaging material.
Desirably, the packaging material maintains the sterility of the
polysaccharide beads until the packaging material is breached.
[0081] The person skilled in the art may provide desired properties
to the polysaccharide beads according to the invention. Typically,
the person skilled in the art may add a compound chosen in the
group consisting of a biomolecule, an antimicrobial agent, a
surfactant, a differentiation agent, a growth factor and a
fluorescent agent.
[0082] The techniques for incorporating said compounds in the
polysaccharide bead of the invention completely falls within the
ability of the person skilled in the art.
[0083] Typically, because of the presence of pores on the surface
of the polysaccharide beads according to the invention, said
compounds may be incorporated on said head, preferably by sowing.
In this particular embodiment, said compound would be on the
surface of the polysaccharide beads of the invention.
Alternatively, said compounds may be added directly the alkaline
solution of step a) of the method of the invention. In this
particular embodiment, the compound would be within the structure
of the polysaccharide heads of the invention. Alternatively, said
compounds can be incorporated into the heads during a step
consisting of hydrating the polysaccharide beads with a solution of
the compound.
[0084] In one embodiment, the polysaccharide beads of the invention
further comprise one or more biomolecules. Non-limiting example of
biomolecules are drugs, hormones, antibiotics such as gentamicin ou
vancomycin, proteases and anti-proteases, chemotactic agents,
antibiotics, steroidal or non-steroidal analgesics,
immunosuppressants, anti-cancer drugs, short chain peptides,
glycoprotein, lipoprotein, cell attachment mediators, biologically
active ligands, integrin binding sequence, ligands, small molecules
that affect the upregulation of specific growth factors,
tenascin-C, hyaluronic acid, chondroitin sulphate, fibronectin,
decorin, thromboelastin, thrombin-derived peptides, and mixtures
thereof. The use of said biomolecules may enhance treatment
effects, indicate proper orientation, resist infection, promote
healing, increase softness or any other desirable effect. Thus, the
polysaccharide beads of the invention comprising a biomolecule are
highly adapted for use for delivering a drug. The inventors have
included an active drug used as a thrombolytic agent, called
tissue-type plasminogen activator (t-PA).
[0085] In another embodiment, the polysaccharide beads of the
invention further comprise anti-inflammatory agents. Non-limiting
examples of anti-inflammatory agents are indomethacin, salicylic
acid acetate, ibuprofen, sulindac, piroxicam, and naproxen;
thrombogenic agents, such as thrombin, fibrinogen, homocysteine,
and estramustine; and radio-opaque compounds, such as barium
sulfate, gold particles and iron oxide nanoparticles (USPIOs) and
mixtures thereof. In still another embodiment, the polysaccharide
beads of the invention further comprise additives. The amount of
the additive used depends on the particular application and may be
readily determined by one skilled in the art using routine
experimentation.
[0086] In still another embodiment, the polysaccharide beads of the
invention further comprise an antimicrobial agent. Suitable
antimicrobial agents are well known in the art. Non-limiting
examples of suitable antimicrobial agents are alkyl parabens, such
as methylparaben, ethylparaben, propylparaben, and butylparaben;
cresol; chlorocresol; hydroquinone; sodium benzoate; potassium
benzoate; triclosan and chlorhexidine and mixture thereof. Other
examples of antibacterial agents and of anti-infectious agents that
may be used are, in a non-limiting manner, rifampicin, minocycline,
chlorhexidine, silver ion agents and silver-based compositions and
mixtures thereof.
[0087] In still another embodiment, the polysaccharide beads of the
invention further comprise at least one surfactant. Surfactant, as
used herein, refers to a compound that lowers the surface tension
of water. The surfactant may be an ionic surfactant, such as sodium
lauryl sulfate, or a neutral surfactant, such as polyoxyethylene
ethers, polyoxyethylene esters, and polyoxyethylene sorbitan and
mixtures thereof.
[0088] In one embodiment, the polysaccharide beads of the invention
further comprise a differentiation agent. Preferably, such a
differentiation agent is an agent involved in bone formation.
Alternatively, such a differentiation agent is an agent involved in
osteogenesis, angiogenesis or wound healing. Preferably, said
differentiation agent is a growth factor. Non-limiting examples of
growth factor suitable for the purpose of the present invention are
epidermal growth factor (EGF), insulin-like growth factor (IGF-I,
IGF-II), transforming growth factor beta (TGF.beta.), heparin
binding growth factor (HBGF), stromal derived factor (SDF-1);
vascular endothelial growth factors (VEGF), fibroblast growth
factors (FGFs), platelet derived growth factors (PDGF), parathyroid
hormone (PTH), parathyroid hormone related peptide (PTHrP), basic
fibroblast growth factor (bFGF); TGF.beta. superfamily factors;
Bone morphogenetic protein (BMP) preferably BMP2, BMP3, BMP4, BMP5,
BMP7, somatropin, growth differentiation factor (GDF) and mixtures
thereof.
[0089] Typically, the growth factor is present at a concentration
comprised from 1 ng to 100 .mu.g per gram of polysaccharide
bead.
[0090] In another embodiment, the polysaccharide beads of the
invention further comprise cells, such as yeast cells, mammalian
cells, insect cells, and plant cells. Preferably, such cell is a
mammalian cell. Non-limiting examples of mammalian cells suitable
for the purpose of the invention are differentiated cells such as
endothelial cells, smooth muscle cells, fibroblasts, chondrocytes,
fibrochondrocytes, osteocytes, osteoblasts, osteoclasts,
synoviocytes, epithelial cells and hepatocytes or stem cells,
embryonic stem cells, human umbilical vein endothelial cells,
induced progenitor stem cells (iPS), mesenchymal stem cells from
different sources, bone marrow, adipose tissue, peripheral blood
progenitor cells, cord blood progenitor cells, genetically
transformed cells and mixtures thereof. Typically, because of the
presence of pores on the surface of the polysaccharide beads
according to the invention, said cells are incorporated on said
beads by well-know techniques such as infiltration through
submerging the polysaccharide beads of the invention in an aqueous
solution comprising said cells. Typically, a suspension of cells is
placed on the lyophilised polysaccharide beads of the invention.
The bead comprising said cells are then incubated in order to
cultivate said cells.
Use of the Crosslinked Polysaccharide Beads According to the
Invention
[0091] In a third aspect, the invention relates to a polysaccharide
bead obtainable by the method of the invention for use for in situ
tissue regeneration. The polysaccharide beads of the invention may
indeed be infiltrated by cells and thus promote tissues
regeneration. Said polysaccharide bead are thus are indeed highly
adapted for injection or deposition within a defective tissue.
Preferably, said defective tissue is cardiac tissue, bone or
cartilage tissue, or muscle tissue. The polysaccharide beads of the
invention are thus highly useful for stimulating heart and muscle
regeneration.
[0092] In one particular embodiment, said polysaccharide beads may
be implanted within the cardiac tissue for treating myocardial
infarction. Therefore, the polysaccharide beads of the invention
are useful for cardiac repair and development.
[0093] In another embodiment, the invention relates to a
polysaccharide bead comprising n-HA obtainable by the method of the
invention for bone regeneration. The inventors have indeed shown
the ability of the porous polysaccharide beads according to the
invention to stimulate the production of an extracellular
mineralized matrix and probably through differentiation of cells
into bone cells. Said polysaccharide beads are thus highly adapted
for use in the treatment of bone related disorders. Indeed, they
are highly effective since their size can be adapted to the size
and localization of the bone to treat or to regenerate.
[0094] For the purpose of the invention, the polysaccharide beads
of the invention may be administrated within a desired location
with the help of any method suitable know by the skilled man.
Typically, the polysaccharide beads may be implanted with the help
of a needle. The needle would be chosen according to the size of
the polysaccharide beads to be administrated. Alternatively, the
polysaccharide beads may be implanted with the help of a catheter.
Such catheter may be porous or may present needle to improve the
administration within the desired location. Macrobeads in bone
defects for instance could also be deposited via a spatula or any
appropriate grip.
[0095] In a fourth aspect, the invention relates to a
polysaccharide bead comprising hydroxyapatite, preferably
nano-hydroxyapatite, obtainable by the method of the invention for
use for stimulating ectopic hone mineralized tissue formation. In
the context of the present invention, the expression "ectopic"
refers to a non osseous tissue. Therefore, the invention also
relates to a polysaccharide bead obtainable according to the method
of the invention for use for inducing mineralized tissue in a
non-osseous site.
[0096] The inventors have shown that administering polysaccharide
beads comprising n-HA according to the invention lead to the
stimulation of a dense collagen network and blood vessel formation
as well as the recruitment of osteoblast-like cells. Such an
administration of polysaccharide beads in subcutaneous site leads
to the formation of a dense mineralized tissue and thus to bone
formation. Preferably, such stimulation of ectopic mineralization
occurs in absence of stem cells and in absence of growth factors
included in the polysaccharide beads prior to injection. Indeed,
the inventors have shown that the polysaccharide beads comprising
n-HA according to the invention have the ability to induce
mineralized tissue in a non-osseous site and in osseous site.
Therefore, the invention provides polysaccharide beads for use for
stimulating mineralized tissue formation in osseous site, as well
as in non-osseous site, in the presence as well as in the absence
of stem cells and/or growth factors.
[0097] In a fifth aspect, the invention relates to a polysaccharide
bead obtainable by the method according to the invention for use as
a cell microcarrier. The polysaccharide beads of the invention have
indeed also been found to be highly adapted for use as a cell
microcarrier, especially for use in bioreactor. Such strategy may
be useful for growing anchorage-dependent cells such as animal
cells, on the surface of the microsphere according to the
invention. Thus, this strategy allows the growth of anchorage
dependent cells in a suspension culture. The use of those
polysaccharide beads is thus an attractive alternative to
conventional monolayer cell culture method.
[0098] In a sixth aspect, the invention relates to a polysaccharide
head obtainable by the method according to the invention for use
for drug and/or cell delivery. In one embodiment, the
polysaccharide beads of the invention may comprise a drug or cells
and are able to deliver said drug or cells within a specific
tissue. Because of its very nature and composition in
polysaccharide, the polysaccharide beads of the invention are
highly inclined to degradation within a specific tissue. Such
disintegration may lead to the delivery of a drug or cells to the
localization of interest. The polysaccharide beads of interest are
porous or not according to the application and type of delivery. In
another embodiment, the polysaccharide beads of the invention are
infiltrated by a drug, a bioactive substance, and/or cells. Said
infiltration is performed by submerging the polysaccharide beads of
the invention in an aqueous solution comprising said drugs and/or
cells. Typically, said solution can be hydrophilic or hydrophobic,
depending on the nature of the drug to be infiltrated within the
polysaccharide beads. Alternatively, the alkaline solution of step
a) of the method of the invention comprises a drug, therefore
providing a polysaccharide bead useful for drug delivery.
[0099] Typically, the suitable drugs are drugs having an
acknowledged therapeutic effect, such as hormones, radioactive
substances, fluorescent substances, chemotactic agents,
antibiotics, steroidal or non-steroidal analgesics,
immunosuppressants, anti-cancer drugs.
[0100] Typically the bioactive substance is a chosen among growth
factors, cytokines (lymphokines, interleukins, and chemokines),
antioxidant molecules, angiogenic molecule, anti-angiogenic agents,
immunomodulating agents, proinflammatory cytokines,
antiinflammatory cytokines, plasma-derived bioactive substances,
PRP (platelet rich plasma)-derived substances, soluble adhesion
molecules.
[0101] Typically, the polysaccharide beads of the invention loaded
with a drug and/or a bioactive substance are administrated within
the human or animal body by injection usually subcutaneous,
intramuscular or intraosseous. The loaded polysaccharide beads are
then able to release the drug and/or bioactive substance in a
consistent way over a long period of time.
[0102] Preferably, said drug is an anti-cancer drug. Thus, the
invention provides polysaccharide bead comprising an anti-cancer
drug, highly useful for treating cancer. The polysaccharide beads
of the invention loaded with anticancer drugs are appropriate for
use for treating solid tumors, for instance, sarcomas, carcinomas,
and lymphomas. Treatment of solid tumors varies based on the type,
location and stage of tumor. Once at the target site, the drug is
released from the polysaccharide beads creating a high local
concentration in the tumor tissue.
[0103] The polysaccharide beads of the invention may also be used
as drug depot implant. Said way of administering a drug is
advantageous since it allows a slow release of an effective
therapeutic amount of said drug into a desired location over a
prolonged period of time. The drug is thus delivered in optimal
conditions. Said polysaccharide bead loaded with a drug, used as
drug depot implant may be administered according to suitable
techniques, known by the skilled person in the art. For example,
said polysaccharide beads may be administered by injection by
syringe and/or catheter, or forceful injection by gun. Drug depot
implant strategy may be performed with various type of drug, such
as decanoate salts or esters. Typically, said polysaccharide bead
used as a drug depot implant are advantageous for administrating
antibiotics and/or anti-cancer drugs within a defective bone. For
example, said polysaccharide bead may comprise gentamicin and/or
vancomycin and be applied via an intraosseous administration.
[0104] In a seventh aspect, the invention relates to a
polysaccharide bead comprising iron oxide obtainable by the method
according to the invention for use for treating cancer. The
polysaccharide beads of the invention comprising iron oxide are
adapted for use for local heat generation (hyperthermia) in cancer
treatment. When exposed to a high frequency magnetic field, the
polysaccharide bead comprising iron oxide generates heat through
oscillation. Direct injection of said polysaccharide beads into
solid tumors, followed by exposure to an alternating magnetic
field, is capable of inducing tumor regression. Polysaccharide
beads loaded with both magnetic nanoparticles and anticancer drugs
such as doxorubicin or paclitaxel allow to combine hyperthermia and
drug delivery strategies for treating cancer.
[0105] In an eighth aspect, the invention relates to a
polysaccharide bead comprising a fluorescent dye obtainable by the
method according to the invention for use for real-time assessment
of cardiac perfusion, and acute intravascular thrombi. Preferably,
said bead have a size of less than 10 .mu.m. Preferably, the
invention relates to a polysaccharide head comprising a fluorescent
dye obtainable by the method according to the invention for use for
assessing thrombus in a subject likely to develop one. Indeed, the
polysaccharides beads of the invention comprising a fluorescent dye
are highly appropriate for intravenous injection. Cardiac perfusion
can then easily be imaged in real time.
FIGURE LEGEND
[0106] FIG. 1: Macroscopic view of beads as macro/microcarriers.
The top panel evidences the beads after crosslinking (left), after
freeze-drying (middle), and after hydratation (right). The bottom
panel is an example of beads observed on confocal microscopy
incubated with vascular cells that attached to the bead surfaces
for further use in vitro in a bioreactor, or in vivo for cell
delivery. FITC-dextran was used in the composition of the beads and
cells were previously labeled with TRITC-phalloidin for
identification on the bead surface.
[0107] FIG. 2: The morphology of freeze-dried porous heads was
analyzed by scanning electron microscopy (left, scale bar: 50
.mu.m). After rehydradation in PBS, hydrated beads were observed
with Environmental Scanning Electron Microscopy (ESEM Philips XL
30, Netherlands) (right, scale bar: 200 .mu.m).
[0108] FIG. 3: Fluorescent porous beads prepared with FITC-dextran
were observed with a fluorescent microscope before size calibration
(left, size ranging from 20 .mu.m to 1 mm) and after calibration
(middle, diameter: 620 .mu.m). Cell infiltration inside porous
beads was assessed using confocal microscopy (right). Cells were
identified by labeling with TRITC-phalloidin. Cells infiltrated the
beads and were observed within the pores (mean diameter of beads:
660 .mu.m)
[0109] FIG. 4: Macroscopic views and confocal images of injectable
porous microbeads. On the top left panel, beads were labeled with
alcian blue to evidence the beads coming out of the needle.
Different beads were prepared that varied in their size: 22.+-.2
.mu.m, 97.+-.7 .mu.m and 164.+-.23 .mu.m.
[0110] FIG. 5: Fluorescent beads were injected subcutaneously
without leakage into a female C57 black mouse using a G25 gauge
needle (left). Subcutaneous tissue was excised 24 hours later for
histopathology analysis (right). Beads remained at the site of
implantation and were visually assessed in the subcutaneous
sample.
[0111] FIG. 6: Fluorescent beads (arrows) were observed on 8 .mu.m
sections of subcutaneous tissue 24 hours after injection (left,
fluorescent microscopy). Beads (arrows) were also observed on
alcian blue/nuclear red stained sections (right, light microscopy).
Scale bars: 100 .mu.m.
[0112] FIG. 7: Polysaccharide bead according to the invention
covered with Fibroblast 3T3 after 2 days of culture.
EXAMPLES
Preparation of Macro/Micro Beads
[0113] A water-in-oil (w/o) emulsification process was performed to
obtain polysaccharide micro/macro beads.
[0114] Beads were prepared using a blend of pullulan/dextran 75:25
(pullulan, MW 200,000, Hayashibara Inc; Dextran MW 500,000,
Pharmacia), prepared by dissolving 9 g of pullulan and 3 g of
dextran into 40 mL of distilled water.
[0115] Chemical cross-linking was carried out using trisodium
trimetaphosphate STMP (Sigma) under alkaline condition. 100 .mu.L
of 10M sodium hydroxide was added to 1 g of the polysaccharide
blend, followed by the addition of 100 .mu.L of water containing 30
mg of STMP. This polysaccharide/NaOH/STMP mixture was then
dispersed into 100 mL of canola oil under mechanical stirring for
10 min.
[0116] The w/o emulsion was then cross-linked at 50.degree. C. for
20 min. Resulting heads were collected by centrifugation (2000 rpm,
for 3 min), washed extensively with PBS (10.times.) then 0.025%
NaCl solution, calibrated according to their size using nylon
filters and freeze-dried for 24 h until complete removal of water
(FIG. 1, top).
[0117] Human endothelial cells cultured with beads can be observed
on their surface (FIG. 1, bottom). For confocal analysis,
FITC-dextran was used and cells were labeled with
TRITC-phalloidin.
[0118] In another experiment, (w/o) emulsification process was
conducted using a high performance disperser (Polytron.RTM.
Homogenizer) in order to obtain smaller beads (<50 .mu.m).
Preparation of Porous Macro/Micro Beads
[0119] In another experiment, porous microbeads could be prepared.
In this process, porogen agent such as NaCl (14 g) was added into
the polysaccharide solution before cross-linking.
[0120] The resulting macro/micro beads were further characterized
by confocal analysis and electronic microscopies. Beads prepared
with NaCl as a porogen agent were porous (FIG. 2). The morphology
of freeze-dried porous beads was evidenced by scanning electron
microscopy (FIG. 2, left). After rehydradation in PBS, hydrated
porous beads were observed with Environnemental Scanning Electron
Microscopy (FIG. 2, right)
[0121] Interestingly, using a process of crosslinking, porous micro
or macrobeads were obtained (FIG. 3, left). By changing the
experimental conditions, micro/macro beads of different sizes could
be obtained from 1 .mu.m to a size higher than 1 mm. Polytron
equipment provided beads with a size comprised between 1 and 30
.mu.m, while using mechanical stirring provides beads with a size
comprised between 10 .mu.m and 1 mm (FIG. 4).
[0122] Through calibration, the inventors have met the burden to
obtain a large scope of beads with different size. More precisely,
the inventors have obtained polysaccharides heads according to the
invention of a size of: [0123] 100 to 200 .mu.m; [0124] 200 to 300
.mu.m; [0125] 300 to 500 .mu.m; [0126] 500 to 700 .mu.m; and [0127]
700 to 1000 .mu.m.
Cell Infiltration
[0128] The Inventors assessed cell infiltration inside porous
microbeads using confocoal microscopy. After incubation of beads
with human endothelial cells, the cell infiltration inside the
FITC-labeled porous beads was thus observed (FIG. 3, middle and
right panels).
Injection of the Beads
[0129] The microbeads of the invention have a size highly
appropriate for injection through a needle (FIG. 4), and allow a
depot of a bead suspension. Fluorescent beads (100-200 .mu.m) were
injected subcutaneously into female C57 black mice using a G25
gauge needle, with no immediate nor late leakage observed (FIG. 5).
No reaction was observed at the injection site 24 hours later.
After sacrifice, subcutaneous tissue was excised and analyzed with
histopathology techniques.
[0130] Beads were observed on 8 .mu.m sections, either using a
fluorescence microscope (FITC-labeled green beads and red
autofluorescence background) or a light microscope following alcian
blue/nuclear red staining protocols (FIG. 6).
Incorporation of Nano-Hydroxyapatite
[0131] Nano-hydroxyapatite (n-HA) was prepared by wet chemical
precipitation using a 0.6M solution of Phosphoric acid (H3PO4
Rectapur, Prolabo.RTM., France) and a 1M solution of calcium
hydroxide (CaOH2 Alfa Aesar, Germany). The suspension of n-HA was
included in the alkaline solution of polysaccharides in the
starting solution at a 6% w/w. The resulting polysaccharide
macrobeads contained n-HA dispersed in the 3D structure of the
beads. The inventors have then discovered bone formation after
implantation of the said beads in a condyle defect in rats.
Preparation of Polysaccharide Beads According to the Invention with
a Thrombolytic Agent:
[0132] Beads according to the invention were prepared in the range
of 1 to 10 microns containing tPA (American Diagnostica, tPA
single-chain recombinant tissue plasminogen activator (IPA)).
9 g of pullulan, dextran 3 g, 1.2 g of fucoidan and 14 g NaCl in
were mixed in 40 ml of water. 300 mg of the solution were isolated
and 30 .mu.L of 10 M NaOH were added before mixing. 30 .mu.L of
STMP (300 mg dissolved in 1 mL of STMP water) were then added. The
mixture is injected into the oil (30 ml of canola oil, 0.35 mg of
Span 80+Tween 80 0.12 mg) and the mixture was stirred with polytron
(small propeller) at full speed for 2 minutes. The breaker is then
put in an oven at 50.degree. C. for 20 minutes. After removing the
beaker from the oven, the mixture is balanced in 10.times.PBS under
magnetic stirring for 30 min. The supernatant was removed and two
rinses in 0.01% SDS, 3 rinses (1 h) in saline 0.025% NaCl were
performed. Finally, a lyophilisation step is performed.
[0133] The inventors obtained different types of beads with the
following composition of polysaccharide: [0134] 75% pullulan+25%
dextran [0135] 25% DEAE pullulan, 75% neutral pullulan [0136] 50%
pullulan+(25% dextran, 25% neutral dextran) [0137] 25% DEAE
pullulan, 75% neutral pullulan, [0138] 50% DEAE dextran, 50%
neutral dextran.
[0139] The activity was then assessed with 5 mg of beads in
Eppendorf tubes. For this purpose, 50 .mu.L of t-PA (200 UI/mL) or
beads according to the invention containing t-Pa were incubated 1
hour, and rinsed 3 times with 500 L of buffer solution (PBS-0.1%
HSA-0.01% Tween 20).
[0140] Colorimetric measurement after adding 50 .mu.L of 2 mM
substrate in S444 demonstrated that more than 30% of native t-PA
activity was maintained in the beads.
Infiltration of Cells on Polysaccharide Heads According to the
Invention
[0141] The seeding of the cells is performed in sterile 1.5 ml
eppendorf tubes. In each of those tubes, 4 mg of lyophilized beads
are placed within each tube. A cell suspension is prepared,
containing from 2 to 3.times.10.sup.6 cells in 15 to 20 .mu.L of a
culture medium for each of the eppendorf tubes. The cell suspension
is placed within each eppendorf tube, which are then incubated for
30 min. 500 .mu.L of culture medium is then added in each tubes.
The tubes are then incubated for 2 h at 37.degree. C. in order to
optimize the adhesion of the cells on the beads. The beads are then
transferred in culture wells. 500 .mu.L of culture medium is added
to each wells. The cells are thus infiltrated in the beads of the
invention and cultivated the appropriate time.
[0142] The inventors performed said techniques for having heads
infiltrated with: fibroblasts 3T3 (see FIG. 7), human umbilical
vein endothelial cells, human mesenchymal stem cells, or lewis rat
mesenchymal stem cells.
[0143] The inventors thus obtained polysaccharide bead comprising
cultivated cells.
* * * * *