U.S. patent application number 14/202504 was filed with the patent office on 2014-12-18 for cell surface coating with hyaluronic acid oligomer derivative.
This patent application is currently assigned to Kode Biotech Limited. The applicant listed for this patent is Kode Biotech Limited. Invention is credited to Deborah Adella Blake, Nicolai Bovin, Nicola Lewell Carter, Stephen Michael Henry, Elena Yurievna Korchagina, Alexander Tuzikov, Eleanor Christine Williams.
Application Number | 20140371437 14/202504 |
Document ID | / |
Family ID | 37889086 |
Filed Date | 2014-12-18 |
United States Patent
Application |
20140371437 |
Kind Code |
A1 |
Carter; Nicola Lewell ; et
al. |
December 18, 2014 |
CELL SURFACE COATING WITH HYALURONIC ACID OLIGOMER DERIVATIVE
Abstract
A method of localizing reproduction assisting hyaluronic acid to
reproductive cell surfaces by covalently linking it to lipids is
disclosed.
Inventors: |
Carter; Nicola Lewell;
(Auckland, NZ) ; Blake; Deborah Adella; (Auckland,
NZ) ; Bovin; Nicolai; (Moscow, RU) ; Henry;
Stephen Michael; (Auckland, NZ) ; Korchagina; Elena
Yurievna; (Moscow, RU) ; Williams; Eleanor
Christine; (Auckland, NZ) ; Tuzikov; Alexander;
(Moscow, RU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kode Biotech Limited |
Auckland |
|
NZ |
|
|
Assignee: |
Kode Biotech Limited
Auckland
NZ
|
Family ID: |
37889086 |
Appl. No.: |
14/202504 |
Filed: |
March 10, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13459365 |
Apr 30, 2012 |
8669357 |
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14202504 |
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11992408 |
Nov 3, 2008 |
8183214 |
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PCT/NZ2006/000245 |
Sep 21, 2006 |
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13459365 |
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Current U.S.
Class: |
536/17.1 |
Current CPC
Class: |
C12N 2533/80 20130101;
A61P 15/00 20180101; C08B 37/0072 20130101; C12N 5/0006 20130101;
C07H 17/04 20130101; C12N 5/0604 20130101 |
Class at
Publication: |
536/17.1 |
International
Class: |
C08B 37/08 20060101
C08B037/08 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 21, 2005 |
NZ |
542568 |
Jul 27, 2006 |
NZ |
548784 |
Claims
1-47. (canceled)
48. A method of preparing a water soluble glycosaminoglycan-lipid
construct comprising the steps of reductive amination of the
terminal residue of the glycosaminoglycan prior to condensation
with the N-hydroxysuccinimidyl C.sub.5-7-alkanedioic acid
derivative of phosphatidylethanolamine
49. The method of claim 48 where the glycosaminoglycan is heparan
sulphate or hyaluronic acid.
50. The method of claim 49 where the C5-7-alkanedioic acid is
adipic acid.
51. The method of claim 50 where the phosphatidylethanolamine is
1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or
1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE).
Description
TECHNICAL FIELD
[0001] The invention relates to methods of localising carbohydrates
to the surface of a cell or multi-cellular structure, and
carbohydrate-lipid constructs for use in such methods.
[0002] In particular, the invention relates to carbohydrate-lipid
constructs for localizing hyaluronic acid to the surface of a cell
or multi-cellular structure and their use in methods of in vitro
fertilisation and transplantation of embryos.
BACKGROUND ART
[0003] The development of cells and multi-cellular structures is
influenced by the extracellular matrix (ECM). Hyaluronic acid (HA)
is a major glycosaminoglycan component of the ECM.
[0004] HA is one of the most abundant glycosaminoglycans (GAGS) in
the female reproductive tract (Lee and Ax (1984); Toole (1991)).
Supplementation of both semi-defined and defined culture media with
HA has been shown to improve the development of in vitro matured
and fertilised bovine embryos to the blastocyst stage without
affecting embryo quality and post-freeze survival.
[0005] The inclusion of HA in culture media has been proposed in
order to increase the efficiency of in vitro blastocyst production
from in vitro matured bovine oocytes (Furnus et al. (1998)).
Indeed, in separate studies, the highest rates of implantation and
foetal development after blastocyst transfer were observed when HA
was the macromolecule in the culture media (Gardner et al.
(1999)).
[0006] Several commercial embryo transfer media products
supplemented with HA are available (EmbryoGlue.RTM. Vitrolife,
U.TM. Medicult). Despite the availability of these products, the
basis for the beneficial effects of HA on implantation and foetal
development are not well understood.
[0007] HA may play a biophysical role mediating interactions
between the embryo and the surface of the endometrium. Furnus et
al. (1998) have suggested that HA might benefit embryo development
per se, or regulate the action of factors synthesised by the
embryo, acting in an autocrine manner.
[0008] Gardner et al. (1999) suggested that the highest cell
numbers and hatching rates obtained in their study occurred when
both serum albumin and HA were present in the same medium. It was
proposed by these authors that embryo culture media should contain
both serum albumin and HA, while transfer media need only contain
HA.
[0009] It is an object of the invention to provide
carbohydrate-lipid constructs for use in localising carbohydrate to
the surface of embryos.
[0010] It is a further object of the invention to provide
carbohydrate-lipid constructs for use in influencing the
development of cells and multi-cellular structures.
[0011] It is a yet further object of the invention to provide a
method for improving the likelihood of successful outcomes from
assisted reproductive techniques.
[0012] These objects are to be read disjunctively with the object
to at least provide the public with a useful choice.
DISCLOSURE OF INVENTION
[0013] In a first aspect the invention provides a method of
localising hyaluronic acid to the surface of a cell or
multi-cellular structure including the step of: [0014] Contacting
the cell or multicellular structure with a dispersion of a
carbohydrate-lipid construct of the structure F--S.sub.1-S.sub.2-L
where: [0015] F is an oligomer or polymer of hyaluronic acid
consisting of .beta.1-4 linked disaccharide units of glucuronic
acid .beta.1-3N-acetylglucosamine (GlcUA.beta.1-3GlcNAc); [0016]
S.sub.1-S.sub.2 is a spacer linking F to L; and [0017] L is a lipid
selected from the group consisting of diacyl- and
dialkyl-glycerolipids, including glycerophospholipids.
[0018] Preferably F, S.sub.1, S.sub.2 and L are covalently
linked.
[0019] Preferably F is 15-20 mer.
[0020] Preferably S.sub.1-S.sub.2 is selected to provide a water
soluble construct that stably incorporates into a lipid
bi-layer.
[0021] Preferably L is selected from the group consisting of:
diacylglycerolipids, phosphatidate, phosphatidyl choline,
phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl
inositol, phosphatidyl glycerol, and diphosphatidyl glycerol
derived from one or more of trans-3-hexadecenoic acid,
cis-5-hexadecenoic acid, cis-7-hexadecenoic acid,
cis-9-hexadecenoic acid, cis-6-octadecenoic acid,
cis-9-octadecenoic acid, trans-9-octadecenoic acid,
trans-11-octadecenoic acid, cis-11-octadecenoic acid,
cis-11-eicosenoic acid or cis-13-docsenoic acid. More preferably
the lipid is derived from one or more cis-desaturated fatty acids.
Most preferably L is selected from the group consisting of:
1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE),
1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) and
rac-1,2-dioleoylglycerol (DOG).
[0022] In a first embodiment of the first aspect of the invention L
is a glycerophospholipid and the carbohydrate-lipid construct
includes the substructure:
##STR00001##
where n=3 to 5 and * is other than H. Preferably n is 3.
[0023] In a second embodiment of the first aspect of the invention
L is a glycerolipid and the carbohydrate-lipid construct includes
the substructure:
##STR00002##
where * is other than H, m=3 to 5, and n=9 to 16. Preferably n is
10.
[0024] In the preferred third embodiment of the first aspect of the
invention L is a glycerophospholipid and the carbohydrate-lipid
construct includes the substructure:
##STR00003##
where n=3 to 5 and * is other than H. Preferably n is 3.
[0025] Preferably L is a glycerophospholipid.
[0026] Preferably L-S.sub.1 is an oligomer or polymer of hyaluronic
acid consisting of .beta.1-4 linked disaccharide units of
glucuronic acid .beta.1-3N-acetylglucosamine (GlcUA.beta.1-3GlcNAc)
linked to S.sub.2 via a terminal glycamine residue (gar).
[0027] In a specific embodiment of the first aspect of the
invention the carbohydrate-lipid construct has the structure:
##STR00004##
designated HA-gar-Ad-DOPE (IV).
[0028] M is typically H, but may be replaced by another monovalent
cation such as Na.sup.+, K.sup.+ or NH.sub.4.sup.+.
[0029] In a second aspect the invention consists in a
carbohydrate-lipid construct of the structure F--S.sub.1-S.sub.2-L
where: [0030] F is an oligomer or polymer of hyaluronic acid
consisting of .beta.1-4 linked disaccharide units of glucuronic
acid .beta.1-3N-acetylglucosamine (GlcUA.beta.1-3GlcNAc); [0031]
S.sub.1-S.sub.2 is a spacer linking F to L; and [0032] L is a lipid
selected from the group consisting of diacyl- and
dialkyl-glycerolipids, including glycexophospholipids.
[0033] Preferably F, S.sub.1, S.sub.2 and L are covalently
linked.
[0034] Preferably F is 15-20 mer.
[0035] Preferably S.sub.1-S.sub.2 is selected to provide a water
soluble construct that stably incorporates into a lipid
bi-layer.
[0036] Preferably L is selected from the group consisting of:
diacylglycerolipids, phosphatidate, phosphatidyl choline,
phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl
inositol, phosphatidyl glycerol, and diphosphatidyl glycerol
derived from one or more of trans-3-hexadecenoic acid,
cis-5-hexadecenoic acid, cis-7-hexadecenoic acid,
cis-9-hexadecenoic acid, cis-6-octadecenoic acid,
cis-9-octadecenoic acid, trans-9-octadecenoic acid,
trans-11-octadecenoic acid, cis-11-octadecenoic acid,
cis-11-eicosenoic acid or cis-13-docsenoic acid. More preferably
the lipid is derived from one or more cis-desaturated fatty acids.
Most preferably L is selected from the group consisting of:
1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE),
1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) and
rac-1,2-dioleoylglycerol (DOG).
[0037] In a first embodiment of the second aspect of the invention
L is a glycerophospholipid and the carbohydrate-lipid construct
includes the substructure:
##STR00005##
where n=3 to 5 and * is other than H. Preferably n is 3.
[0038] Preferably L is a glycerophospholipid.
[0039] Preferably F--S.sub.1 is an oligomer or polymer of
hyaluronic acid consisting of .beta.1-4 linked disaccharide units
of glucuronic acid .beta.1-3N-acetylglucosamine
(GlcUA.beta.1-3GlcNAc) linked to S.sub.2 via a terminal glycamine
residue (gar).
[0040] In a specific embodiment of the second aspect of the
invention the carbohydrate-lipid construct has the structure:
##STR00006##
designated HA-gar-Ad-DOPE (IV)
[0041] M is typically H, but may be replaced by another monovalent
cation such as Na.sup.+, K.sup.+ or NH.sub.4.sup.+.
[0042] In a third aspect the invention consists in a method of
preparing a carbohydrate-lipid construct of the first embodiment of
the second aspect of the invention of the structure
F--S.sub.1-S.sub.2-L including the steps: [0043] 1. Reacting an
activator (A.sub.1) with a lipid (L) to provide an activated lipid
(A.sub.1-L); [0044] 2. Reductive amination of a carbohydrate (F) to
provide (F--S.sub.1); and [0045] 3. Condensing A.sub.1-L with
F--S.sub.1 to provide the molecule;
[0045] F--S.sub.1-S.sub.2-L
where: [0046] A.sub.1 is an activator selected from the group
including: bis(N-hydroxysuccinimidyl), bis(4-nitrophenyl),
bis(pentafluorophenyl), bis(pentachlorophenyl) esters of
C.sub.5-C.sub.7 carbodioic acids; [0047] L is a lipid selected from
the group consisting of diacyl- and dialkyl-glycerolipids,
including glycerophospholipids; and [0048] F is a carbohydrate.
[0049] Preferably F is an oligomer or polymer of hyaluronic acid
consisting of .beta.1-4 linked disaccharide units of glucuronic
acid .beta.1-3N-acetylglucosamine (GlcUA.beta.1-3GlcNAc).
[0050] Preferably F is 15-20 mer.
[0051] Preferably S.sub.2 is a C.sub.5-C.sub.7 aliphatic diacid.
More preferably S.sub.2 is a C.sub.5-C.sub.7 aliphatic diacid
selected from the group consisting of: --CO(CH.sub.2).sub.3CO--,
--CO(CH.sub.2).sub.4CO-- (adipate), --CO(CH.sub.2).sub.5CO-- and
--CO(CH.sub.2).sub.5NHCO(CH.sub.2).sub.5CO--.
[0052] Preferably L is selected from the group consisting of:
diacylglycerolipids, phosphatidate, phosphatidyl choline,
phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl
inositol, phosphatidyl glycerol, and diphosphatidyl glycerol
derived from one or more of trans-3-hexadecenoic acid,
cis-5-hexadecenoic acid, cis-7-hexadecenoic acid,
cis-9-hexadecenoic acid, cis-6-octadecenoic acid,
cis-9-octadecenoic acid, trans-9-octadecenoic acid,
trans-11-octadecenoic acid, cis-11-octadecenoic acid,
cis-11-eicosenoic acid or cis-13-docsenoic acid. More preferably
the lipid is derived from one or more cis-desaturated fatty acids.
Most preferably L is selected from the group consisting of:
1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE),
1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) and
rac-1,2-dioleoylglycerol (DOG).
[0053] In a preferred embodiment L is a glycerolipid and the
carbohydrate-lipid construct includes the substructure:
##STR00007##
where n=3 to 5 and * is other than H. Preferably n is 3.
[0054] Preferably L is a glycerophospholipid.
[0055] In a specific embodiment the carbohydrate-lipid construct
has the structure:
##STR00008##
designated HA-gar-Ad-DOPE (IV).
[0056] M is typically H, but may be replaced by another monovalent
cation such as Na.sup.+, K.sup.+ or NH.sub.4.sup.+.
[0057] In a fourth aspect the invention consists in a
carbohydrate-lipid construct prepared by a method according to the
third aspect of the invention.
[0058] In a fifth aspect the invention broadly consists in a method
of assisted reproduction including the step of: [0059] contacting
an embryo with a carbohydrate-lipid construct of the second aspect
or fourth aspect of the invention.
[0060] In a sixth aspect the invention broadly consists in a method
of in vitro maturation of an embryo including the step of: [0061]
contacting the embryo with a carbohydrate-lipid construct of the
second aspect or fourth aspect of the invention.
[0062] In a seventh aspect the invention broadly consists in a
method of transferring an embryo to the endometrium with an
improved likelihood of a successful outcome, including the step of:
[0063] contacting the embryo with a carbohydrate-lipid construct of
the second aspect or fourth aspect of the invention.
[0064] In a eighth aspect the invention consists in medium
including a dispersion of carbohydrate-lipid construct according to
the second aspect or fourth aspect of the invention.
[0065] Preferably the medium is an ART or IVM medium.
[0066] In an ninth aspect the invention consists in a medicinal
formulation including a carbohydrate-lipid construct according to
the second aspect or fourth aspect of the invention.
[0067] Preferably the formulation is in a form suitable for
inhalation. More preferably the formulation is in the form of an
aerosol suitable for inhalation.
[0068] In the context of this description the following terms and
phrases have the meanings provided:
[0069] "ART" means assisted reproductive techniques including, but
not limited to, IVF and IVM methods.
[0070] "HA" denotes an oligomer or polymer of HA consisting of
.beta.1-4 linked disaccharide units of glucuronic acid
.beta.1-3N-acetylglucosamine (GlcUA.beta.1-3GlcNAc).
[0071] "Dispersion" means in reference to a carbohydrate-lipid
construct an homogenous suspension or solution of the construct
with or without the inclusion of dispersants or detergents.
[0072] "IVF" means the methods through which male and female
gametes are brought into contact outside the body in order to
accomplish fertilization.
[0073] "IVM" means the methods of in vitro maturation of
embryos.
[0074] "Improved likelihood of a successful outcome" means, in
relation to transferring an embryo to the endometrium, an increased
likelihood of the transplanted embryo implanting and developing to
provide a live birth.
[0075] "Stably incorporates" means that the carbohydrate-lipid
construct incorporates into the lipid bi-layer or membrane for a
time sufficient to effect a change in a biological activity of the
transformed cell or multi-cellular structure.
[0076] "Water soluble" means a stable, single phase system
(including a "dispersion" as defined above) is formed when the
carbohydrate-lipid construct is contacted with water or saline
(such as PBS) in the absence of organic solvents or detergents.
[0077] Exemplary embodiments of the invention will now be described
in detail with reference to the Figures of the accompanying
drawings pages.
BRIEF DESCRIPTION OF DRAWINGS
[0078] FIG. 1--.sup.1H NMR spectrum of HA.sub.10-14 mer (D.sub.2O,
303 K, .delta. ppm) (I).
[0079] FIG. 2--Fluorescence microscopy of red blood cell membranes
following insertion of HA-gar-Ad-DOPE (IV).
[0080] FIG. 3--Fluorescence microscopy of embryos following
insertion of HA-gar-Ad-DOPE (IV): control (top); embryos following
insertion (bottom).
[0081] FIG. 4--Fluorescence microscopy of embryos following
incubation with: Vitrolife Embryo Glue, 20 minutes (top); high
molecular weight HA, 24 hours at 37.degree. C. (bottom).
[0082] FIG. 5--Fluorescence microscopy of embryos following
incubation with: HA-gar-Ad-DOPE (IV), 24 hours at 37.degree. C.
(top); media only, 24 hours at 37.degree. C. (bottom).
[0083] FIG. 6--Fluorescence microscopy of embryos following removal
of the zona pellucida and incubation with: HA-gar-Ad-DOPE (IV), 2
hours at 37.degree. C. (top and middle); media only, 2 hours at
37.degree. C. (bottom).
[0084] FIG. 7--Fluorescence microscopy of embryos incubated with
HA-gar-Ad-DOPE (IV) (2 hours at 37.degree. C.) after: 24 hours post
incubation (top); 5 hours post incubation (middle); and 2 hours
post incubation (bottom).
[0085] FIG. 8--DIC image of unmodified embryos. Right plate: Same
image but viewed as merged HIS/WIG fluorescent images. Result shows
zero attachment of epithelial cells to the embryo (red).
[0086] FIG. 9--DIC image of VVA modified embryos. Right plate: Same
image but viewed as merged WIB/WIG fluorescent images. Result shows
positive binding of endometrial cells (green) to embryos (red).
Attachment numbers from left to right are 35, 20 and 27.
[0087] FIG. 10--FIG. 10. DIC image of HA.sub.15-20 mer-gar-Ad-DOPE
(IV) modified embryo. Right plate: Same image but viewed as merged
WIB/WIG fluorescent images. Result shows positive binding of 10
endometrial cells (green) to the embryo (red).
[0088] FIG. 11--FIG. 11. DIC image of HA.sub.15-20 mer-gar-Ad-DOPE
(IV) modified embryos. Right plate: Same image but viewed as merged
WIB/WIG fluorescent images. Result shows positive binding of
endometrial cells (green) to embryos (red). Attachment numbers from
left to right are 15, 8 and 9.
[0089] FIG. 12--DIC image of HA.sub.15-20 mer-gar-Ad-DOPE (IV)
modified embryos. Right plate: Same image but viewed as merged
HIS/WIG fluorescent images. Result shows positive binding of
endometrial cells (green) to embryos (red). Attachment numbers from
left to right are 8, 8 and 5.
DETAILED DESCRIPTION
[0090] Hyaluronic acid (HA) is a linear polymer, composed of
repeating disaccharides of glucuronic acid (GlcUA) and
N-acetyl-D-glucosamine (GlcNAc). The polymer can reach a molecular
mass of several million Daltons and is a ubiquitous component of
extracellular matrices, where it is often associated with HA
binding proteoglycans and HA binding proteins.
[0091] CD44 is a broadly distributed cell surface protein thought
to mediate cell attachment to extracellular matrix components or
specific cell surface ligands. CD44 is the principal cell surface
receptor for HA.
[0092] The binding of HA at the cell surface is a complex interplay
of multivalent binding events affected by the size of the
multivalent HA ligand. The minimum receptor binding site is a
hexasaccharide composed of three repeats of .beta.1-4 linked
disaccharide units of glucuronic acid .beta.1-3N-acetylglucosamine
(GlcUA.beta.1-3GlcNAc).
[0093] The overall size of the HA polymer influences binding.
Longer HA polymers result in more receptor-ligand interactions,
thus reducing the probability of dissociation. Monovalent binding
between HA and CD44 is thought to be optimised for a
decasaccharide, although significant binding occurs with the
hexasaccharide. An increase in binding avidity for HA consisting of
between 20 and 24 saccharides indicates the point at which divalent
binding to CD44 occurs.
[0094] HA oligomers and polymers of different sizes are known to
elicit different biological activities. The selection of oligomers
or polymers of different sizes to elicit different biological
responses is contemplated by the inventors.
[0095] Methods for obtaining HA oligosaccharides of uniform size
have been described (Tawada et al. (2002)). Oligosaccharides are
prepared by the digestion of HA polymer with testicular
hyaluronidase enzyme that hydrolyses the .beta.1-4 glycosidic
bond.
[0096] The inventors propose that by localising HA to the surface
of a cell or multi-cellular structure different biological
activities may be elicited. Where the multi-cellular structure is
an embryo improvements may be achieved in one or more of: [0097]
the growth characteristics of the embryo; [0098] the storage
characteristics of the embryo; [0099] the survival of the embryo;
and/or [0100] the likelihood of implantation of the embryo
following transfer to the uterus.
[0101] In respect of this latter improvement oligosaccharides of HA
of sufficient length to enable associative interaction with CD44
expressed on the surface of endometrial cells of the recipient host
is desired. Indeed, longer oligosaccharides are likely to favour
stronger association with these endometrium expressed
receptors.
[0102] Acknowledging the observations of other investigators the
inventors do not discount the possibility of oligosaccharides of HA
localised to the surface of the embryo providing improvements in
other areas, such as embryo growth characteristics. Indeed, the
method of the invention may promote internalisation of surface
localised HA, with consequential effects on intracellular
signalling and embryo development.
[0103] The carbohydrate-lipid constructs may have other medicinal
applications where localization of carbohydrate to the surface of a
cell or multi-cellular structure is advantageous.
[0104] For example, the carbohydrate-lipid construct designated
HA-gar-Ad-DOPE (IV) is particularly suited for use in the
manufacture of medicinal formulations. The construct is soluble in
aqueous media, but readily and stably incorporates into the
membranes of cells (e.g. red blood cells) and multi-cellular
structures (e.g. embryos).
[0105] A proven method of preparing the carbohydrate-lipid
construct designated HA-gar-Ad-DOPE (IV) is provided in Scheme I.
Difficulties have been shown to arise in the preparation of
carbohydrate-lipid constructs comprising HA by other methods as
discussed below. However, the inventors contemplate the feasibility
of preparing a range of carbohydrate-lipid constructs comprising HA
with similar favourable properties.
[0106] These carbohydrate-lipid constructs are distinguished from
those prepared by the methods described in the specifications
accompanying international application no. PCT/NZ02/00214 (WO
03/034074) and PCT/NZ03/00059 (WO 03/087346).
[0107] The methods of localising a carbohydrate to the surface of
the cell or multi-cellular structure described in these
specifications require the use of an endogenously prepared
(biosynthesised) glycolipid or the use of a biotinylated lipid that
is first incorporated into the lipid bi-layer.
[0108] The synthetic carbohydrate-lipid constructs of the present
invention are exogenously prepared and do not comprise
biotin-avidin bridges as a spacer (S.sub.1-S.sub.2) linking the
carbohydrate (F) to the lipid (L). F, S.sub.1, S.sub.2 and L of the
carbohydrate-lipid constructs are covalently linked and can be used
in a one step method of localising the carbohydrate to the surface
of the cell or multi-cellular structure.
##STR00009##
BEST MODE FOR CARRYING OUT THE INVENTION
Preparation of Carbohydrate-Lipid Constructs
Materials and Methods
[0109] Methanol, i-PrOH, CH.sub.2Cl.sub.2, diethyl ether, hexane
and NH.sub.4OAc were from Chimed (Russia). Acetonitrile was from
Cryochrom (Russia). Silica gel 60 RP-18 (40-63 .mu.m) and
NaCNBH.sub.3 were from Merck (Germany). Sephadex G-10 and Sephadex
LH-20 were from Amersham Biosciences AB (Sweden).
[0110] Thin-layer chromatography was performed on silica gel 60
F254 plates (Merck). Compounds were detected by staining with 8%
phosphoric acid in water followed by heating at over 200.degree.
C., or ninhydrine as indicated.
[0111] For activated lipids .sup.1H NMR spectra were acquired on a
Bruker DRX-500 spectrometer. Chemical shifts are given in ppm
(.delta.) relative to CD.sub.3OD.
[0112] For HA-lipid constructs .sup.1H NMR spectra were acquired at
30.degree. C. on a Bruker WM 500 MHz instrument using the signal of
the solvent's residual protons as reference (for
[D.sub.2]H.sub.2O-4.750 ppm).
[0113] Activated lipids were prepared as either the adipate
derivative of glycerophospholipids (Method 1) or the
[p-notrophenoxycarbonylmethylene(polyoxyethylene)]-oxyacetyl
derivative of diacyl glycerolipids (Method 2).
Preparation of Activated Lipids
Method 2 (Preparation of adipate derivative of
1,2-O-distereoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) and
1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE))
[0114] To a solution of bis(N-hydroxysuccinimidyl) adipate (A) (70
mg, 205 .mu.mol) in dry N,N-dimethylformamide (1.5 ml) were added
DOPE or DSPE (L) (40 .mu.mol) in chloroform (1.5 ml) followed by
triethylamine (7 .mu.l). The mixture was kept for 2 h at room
temperature, then neutralized with acetic acid and partially
concentrated in vacuo.
[0115] Column chromatography (Sephadex LH-20, 1:1
chloroform-methanol, 0.2% acetic acid) of the residue yielded the
activated lipid (A-L) (37 mg, 95%) as a colorless syrup; TLC
(chloroform-methanol-water, 6:3:0.5): R.sub.f=0.5 (DOPE-A; III),
R.sub.f=0.55 (DSPE-A).
[0116] .sup.1H NMR (CDCl.sub.3/CD.sub.3OD, 2:1), .delta.:
[0117] DSPE-A--5.39 (m, 1H, --OCH.sub.2--CHO--CH.sub.2O--), 4.53
(dd, 1H, J=3.42, J=11.98, --CCOOHCH--CHO--CH.sub.2O--), 4.33 (dd,
1H, J=6.87, J=11.98, --CCOOHCH--CHO--CH.sub.2O--), 4.23 (m, 2H,
PO--CH.sub.2--CH.sub.2--NH.sub.2), 4.15 (m, 2H, --CH.sub.2--OP),
3.61 (m, 2H, PO--CH.sub.2--CH.sub.2--NH.sub.2), 3.00 (s, 4H,
ONSuc), 2.81 (m, 2H, --CH.sub.2--CO (Ad), 2.48 (m, 4H, 2x
(--CH.sub.2--CO), 2.42 (m, 2H, --CH.sub.2--CO (Ad), 1.93 (m, 4H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 1.78 (m, 4H, 2x
(COCH.sub.2CH.sub.2--), 1,43, 1.47 (2 bs, 40H, 2OCH.sub.2), 1.04
(m, 6H, 2CH.sub.2).
[0118] DOPE-A (III)--5.5 (m, 4H, 2x (--CH.dbd.CH--), 5.39 (m, 1H,
--OCH.sub.2--CHO--CH.sub.2O--), 4.58 (dd, 1H, J=3.67, J=11.98,
--CCOOHCH--CHO--CH2O--), 4.34 (dd, 1H, J=6.61, J=11.98,
--CCOOHCH--CHO--CH.sub.2O--), 4.26 (m, 2H,
PO--CH.sub.2--CH.sub.2--NH.sub.2), 4.18 (m, 2H, --CH.sub.2--OP),
3.62 (m, 2H, PO--CH.sub.2--CH.sub.2--NH.sub.2), 3.00 (s, 4H,
ONSuc), 2.8 (m, 2H, --CH.sub.2--CO (Ad)/2.50 (m, 4H, 2x
(--CH.sub.2--CO), 2.42 (m, 2H, --CH.sub.2--CO (Ad), 2.17 (m, 8H, 2x
(--CH.sub.2--CH.dbd.CH--CH.sub.2--), 1.93 (m, 4H,
COCH.sub.2CH.sub.2CH.sub.2CH.sub.2CO), 1.78 (m, 4H, 2x
(COCH.sub.2CH.sub.2--), 1,43, 1.47 (2 bs, 40H, 2OCH.sub.2), 1.04
(m, 6H, 2CH.sub.3).
Method 2 (Preparation of
rac-1,2-Dioleoyl-3-[p-nitrophenoxycarbonylmethylene(polyoxyethylene)]-oxy-
acetylglycerol)
[0119] Biscarboxymethyl ether of polyethylene glycol
(polymerization degree of 9-16) is dried by co-evaporation with
benzene and subsequent stored under vacuum. Thionyl chloride (0.86
ml, 12 mmol) is added dropwise to a stirred mixture of diacid (0.6
g, .about.1 mmol) and heated Na.sub.2CO.sub.3 (0.42 g, 4 mmol).
[0120] The reaction mixture is stirred at room temperature until
gas evolution ceased (.about.4 h), evaporated, and twice
resuspended in ethyl ether and evaporated for the removal of
residual thionyl chloride. The residue is resuspended in ethyl
ether and filtered through Kieselgur.
[0121] The upper phase of the filtrate with separated phases is
evaporated. The residue is dissolved in dry dioxane, evaporated,
and dried in a vacuum over NaOH to give diacid dichloride of
carboxymethyl[poly(oxyethylene)]oxyacetic acid (D).
[0122] A solution of rac-1,2-dioleoylglycerol (DOG; L) (90 mg, 0.14
mmol) and triethylamine (0.1 ml, 0.72 mmol) in dry chloroform (3
ml) is added with stirring to diacid dichloride (D) (0.59 g,
.about.0.89 mmol). The reaction is carried out with stirring at
room temperature for 24-36 h (TLC monitoring in systems B and C)
with an occasional addition of triethylamine (10 .mu.l portions,
0.2 ml in total).
[0123] The reaction mixture is diluted with chloroform (.about.10
ml) and washed with 0.1 N HCl (10 ml) (phases separated by
centrifugation). The aqueous phase is twice extracted with
chloroform. The combined organic extracts are washed with water and
evaporated. The residue is dried by co-evaporation with benzene and
applied onto a gel filtration column (1.times.100 cm) equilibrated
with solvent system A to give a mixture of mono- and di-acylation
products.
[0124] The mixture is separated by chromatography on a silica gel
column in a gradient of 10:1 methanol-acetic acid solution in
chloroform (from 2 to 10%) to give (D-L) as a colorless oil;
Rf.about.0.6 (B, elongated spot); MS, m/z: 1149.9 m/z: 1149.9
[M+H.sub.2O-1].sup.+ (36), 1194.1 [M+H.sub.2O-1].sup.+ (69), 1238.1
[M+H.sub.2O-1]+(100), 1282.0 [M+H.sub.2O-1].sup.+ (92), 1326.0
[M+H.sub.2O-1].sup.+ (85), 1370.3 [M+H.sub.2O-1].sup.+ (75), 1414.2
[M+H.sub.2O-1].sup.+ (60), and 1458.1 [M+H.sub.2O-1].sup.+ (42);
.sup.1H NMR (CD.sub.2OD): 1.09 (6H, t, CH.sub.2), 1.50 (40H, m
CH.sub.2), 1.80 (4H, br. quintet, CH.sub.2CH.sub.2COO), 2.23 (8H m,
CH.sub.2CH.dbd.CH), 2.52 (4H, t, CH.sub.2COO), 3.83 (52H m,
OCH.sub.2CH.sub.2O), 4.31 (2H, s, OCH.sub.2COO-diglyceride), 4.38
(2H, s, OCH.sub.2COOH), 4.48 and 4.59 (4H, a set of multiplets, C1'
and C3'), 5.48 (1H, m, C2'), 5.54 (4H, m, CH.dbd.CH).
[0125] A solution of p-nitrophenyl trifluoroacetate (52 mg, 0.22
mmol) in dry pyridine (0.2 ml) is added to acid (D-L) (85 mg,
.about.0.07 mmol) and stirred at room temperature for 7 h. The
reaction mixture is separated on a Sephadex LH-20 column
(0.8.times.50 cm) equilibrated with solvent system A supplemented
with 1% CH.sub.3COOH to yield (A.sub.2-D-L) as a light yellow
chromatographically homogeneous oil; Rf 0.75 (D, a, b, c).
[0126] .sup.1H NMR:
[0127] DOG-D-A.sub.2--0.88 (6H, t, CH.sub.3), 1.29 (40H, m,
CH.sub.2), 1.61 (4H, m, CH.sub.2CH.sub.2COO), 2.01 (8H, m,
CH.sub.2CH.dbd.CH), 2.31 (4H, t, CH.sub.2COO), 3.65 (52H, m,
OCH.sub.2CH.sub.2O), 4.16 (2H, s, OCH.sub.2COO-diglyceride), 4.17
(2H, s, t OCH.sub.2COONp), 4.22, 4.29, and 4.38 (4H, a set of dd,
C1' and C3'), 5.27 (1H, br. quintet, C2'), 5.35 (4H, m, CH.dbd.CH),
7.34 (2H, d, J2'3'=J5'6'=9.15, H2', H6'), 8.29 (2H, d,
J3'2'=J5'6'=9.15, H3', H5')
[0128] The activated lipids prepared by Method 1 or Method 2 may be
condensed with the primary amine of a carbohydrate derivative to
provide a carbohydrate-lipid construct.
Condensing Activated Lipid with Aminopropyl-HA
[0129] Condensing activated lipid with the aminopropyl derivative
of hyaluronic acid according to either of the following prophetic
methods was found to provide low to no yield of carbohydrate-lipid
construct.
Method 3
[0130] To a solution of activated DOPE (A.sub.1-L) (33 .mu.mol) in
N,N-dimethylformamide (1 ml) 30 .mu.mol of 3-aminopropyl glycoside
of HA and 5 .mu.l of triethylamine is added. The mixture is stirred
for 2 h at room temperature. Column chromatography (Sephadex LH-20
in 1:1 chloroform-methanol is followed by silica gel in ethyl
acetate-isopropanol-water, 4:3:1 (v/v/v).
Method 4
[0131] To a solution of the ester (A.sub.2-D-L) (.about.6.25
.mu.mol) in dry DMSO (0.2 ml) and TEA (3 .mu.l, 20 .mu.mol) is
added to 3-aminopropyl glycoside of HA (6.48 .mu.mol). The reaction
mixture is stirred at room temperature for 24 h, mixed with a drop
of water, and separated on a gel filtration column (0.6.times.35
cm) equilibrated with solvent system A.
[0132] The failure of these methods of preparation was attributed
to the instability of the 3-aminopropyl glycoside of HA in
solution. An alternative method of preparing a primary amine of the
carbohydrate hyaluronic acid was therefore developed (Scheme
I).
[0133] The reductive amination of HA provided a carbohydrate with a
terminal glycamine residue (gar) that could then be condensed with
an activated lipid such as
rac-1,2-dioleoyl-3[p-nitrophenoxycarbonylmethylene(polyoxyethylene)]-oxya-
cetylglycerol (DOG) to provide a range of carbohydrate-lipid
constructs.
[0134] In the following exemplary method HA is condensed with the
N-oxysuccinimide ester DOPE-Ad-Nos via a terminal glycamine residue
(gar).
Preparation of HA-glycamine (II)
[0135] HA (HA.sub.15-20 mer)(I)(36 mg) was dissolved in 5M
NH.sub.4OAc (3.6 mL). The solution was kept for 21 h at 40.degree.
C. After addition of aqueous 2M NaCNBH.sub.3 in five sequential
portions (40 .mu.L of 2M NaCNBH.sub.3--for 3 h; 80 .mu.L-18 h; 160
.mu.L-8 h; 160 .mu.L-21 h; 160 .mu.L-21 h) the mixture was kept at
40.degree. C.
[0136] Desalting of the reaction mixture by gel-permeation
chromatography on a Sephadex G-10 column (1.8.times.40 cm,
eluent-aqueous 0.1M Py.AcOH) and freeze-drying gave 32.8 mg of
HA-glycamine (II) in admixture with I.
[0137] TLC (eluent i-PrOH/MeOH/MeCN/water 4:3:6:4): HA-glycamine
(II) (ninhydrine-positive) R.sub.f=0.2; HA oligomer (I)
R.sub.f=0.31.
Preparation of HA-gar-Ad-DOPE (IV)
[0138] HA-glycamine (II) (32.8 mg) was dissolved in the mixture of
i-PrOH (1.5 mL) and water (0.75 mL). To the rapidly stirred
solution of II a solution of N-oxysuccinimide ester DOPE-Ad-Nos
(III) (34 mg, 35 .mu.M) in CH.sub.2Cl.sub.2 (0.2 mL) was added
followed by aqueous 1M Na.sub.2CO.sub.3 in two portions (85 and 45
.mu.L) with 45 min interval. The mixture was stirred for 45 min and
then acidified with AcOH (30 .mu.L).
[0139] Gel-permeation chromatography of the reaction mixture on
Sephadex LH-20 column (1.8.times.35 cm, eluent MeCN/water 2:1,
0.03M Py.AcOH) gave 42.5 mg of HA-gar-Ad-DOPE (IV) in admixture
with I.
Separation of HA Oligomer (I) and HA-gar-Ad-DOPE (IV)
[0140] A solution of the crude product in water was slowly put on a
C.sub.18 reverse phase column (1.2.times.7 cm, water). Elution with
water and water/MeOH 10:1 provided I (12.3 mg). Elution with
water/MeOH 1:3 and then with water/MeOH/CHCl3 5:15:1 gave IV.
[0141] This fraction was evaporated and the residue (thin film on
the flask walls) was extracted with hexane (2.times.2 mL) and ether
(2.times.2 mL) then dissolved in water (1.5 mL) and
freeze-dried.
[0142] Yield of IV was 20.6 mg (.about.50%).
[0143] TLC: R.sub.f=0.33, eluent i-PrOH/MeOH/MeCN/water
4:3:6:4.
[0144] .sup.1H NMR of IV, Na-salt (500 MHz, D.sub.2O, 2 mM
NaHCO.sub.3, 30.degree. C.): .delta.=5.473 (m, 2-CH.dbd.CH-- of
DOPE), 5.328 (m, OCH.sub.2CHCH.sub.2O of DOPE), 4.574 and 4.473 (m,
HA: H-1 of GlcNAc, H-1 of GlcA; CO--OCHCHCH.sub.2 of DOPE), 4.246
(dd, J=12.3 Hz, J=6.8 Hz, CO--OCHCHCH.sub.2 of DOPE), 4.027 (t,
J=5.7 Hz, POCH.sub.2CH.sub.2N of DOPE), 3.95-3.34 (HA: H-2/H-6 of
GlcNAc, H-2/H-5 of GlcA; POCH.sub.2CHCH.sub.2 of DOPE), 2.413 (m, 2
CH.sub.2C00 of DOPE), 2.302 (m, 2 CH.sub.2CON), 2.049 (m, 2
CH.sub.2CH.dbd.CHCH.sub.2 of DOPE), 2.039 (m, NCOCH.sub.3 of
GlcNAc), 1.630 (m, 2 CH.sub.2CH.sub.2CON and 2 CH.sub.2CH.sub.2C00
of DOPE), 1.306 (m, --CH.sub.2-- of DOPE), 0.892 (.about.t, 2
CH.sub.3 of DOPE) ppm.
Approximation of Size of HA Oligomer Used in the Synthesis of
HA-gar-Ad-DOPE (IV)
[0145] The average "n" value of the HA oligomer of the
HA-gar-Ad-DOPE construct (IV) was considered to be the same as that
of starting HA oligomer (I).
[0146] For an HA.sub.n1-n2 mer the average "n" value may be
estimated by .sup.1H-NMR. Assuming the GlcNAc reducing end ratio
.alpha./.beta. to be 60/40 (the normal ratio for free GlcNAc) the
average "n" value for HA.sub.10-14 mer was calculated as
.about.12.2 (FIG. 1). For HA.sub.15-20 mer (I) this approach
provided an average "n" value of .about.13.
Insertion of HA-Gar-Ad-DOPE (IV) into Red Blood Cell Membranes.
[0147] Packed Group O cells were washed three times with PBS by
adding 7.times. the amount of PBS to RBC in a test tube. A Pasteur
pipette washed used to gently mix the contents of the test tube.
The test tube was centrifuged for 1 minute on at low speed in order
to get the RBC's to stack at the bottom of the tube.
[0148] Using a plastic pipette the supernatant was gently removed
and the cells resuspended in another 7 volumes of PBS. Washing was
repeated two times until the supernatant was clear. After the last
wash the supernatant was removed.
[0149] A 10 mg/ml solution of IV was diluted to the desired
concentration with PBS, e.g. for 5 mg/mL solution, 5 .mu.L+5 .mu.L
PBS.
[0150] For insertion of IV, RBCs were resuspended in the diluted
(where necessary) solution at a ratio of 1 part solution to 3 part
stacked cells. For each sample or control to be tested, 5 of sample
or control solution was added to 15 .mu.l resuspended cells in a
1.5 mL eppendorf.
[0151] The mixes were incubated: [0152] 3 hours at 21.degree. C.,
mixing every hour, then for 18 hours at 4.degree. C.; 2 hours at
37.degree. C.; [0153] 4 hours at 37.degree. C.; [0154] Overnight
(O/N) at 4.degree. C.; or [0155] Overnight (O/N) at 37.degree.
C.
[0156] Transformed RBCs were washed 3 times, by adding 1 mL PBS and
centrifuging for 1 min at a low speed. Washing of the RBCs was then
performed as described above. A 3% cell suspension was prepared by
adding 0.3 .mu.L of washed transformed RBCs to 97 .mu.L PBS in a
Kimble glass test tube.
[0157] In Kimble glass test tubes 30 .mu.L of the 3% cell
suspension+30 .mu.L 1% BSA/PBS diluted anti-HA antibody
(Biogenesis, cat. no. 5029-9990) were mixed for test samples, or 30
.mu.L 3% cell suspension+30 .mu.L 1% BSA/PBS alone for controls.
The mixtures were incubated at 37.degree. C. for 30 min. The
incubated mixtures were then centrifuged at high speed for 10 s and
assessed for agglutination.
[0158] The samples were washed three times with PBS by repeated
centrifugation at high speed and resuspension. 30 .mu.L 1% BSA/PBS
diluted donkey anti-sheep antibody (Invitrogen, cat. no. A-11015)
was then added. The samples were then assessed for
agglutination.
[0159] The results are presented in Table 1 and FIG. 2.
TABLE-US-00001 TABLE 1 Insertion of HA-gar-Ad-DOPE (IV) into red
blood cell membranes Insertion conditions Anti- O/N O/N 4 hr 2 hr
O/N bodies 3 hr 21.degree. C., 18 hr 4.degree. C. 2 hr 37.degree.
C. 4.degree. C. RT 37.degree. C. 37.degree. C. RT Sample (.alpha.)
5 mg/mL 1 mg/mL 1 mg/mL 1 mg/mL 5 mg/mL KODE15-20 .alpha.-HA 1:10
1:50 1:100 1:10 1:10 1:50 1:100 1:10 1:10 1:10 1:10 1:10 +++ + -
+++ +++ +++ ++ + .alpha.- 1:20 1:20 1:20 1:20 1:200 1:200 1:200
1:200 1:200 1:200 1:200 1:20 sheep ++ ++ - ++++ ++ (+) +(-) +++ ++
++ + + Fluores- cence KODE10-14 .alpha.-HA 1:10 1:50 1:100 1:10
1:10 1:50 1:100 1:10 1:10 1:10 1:10 +++ +/- - ++ ++ +++ ++ ++
.alpha.- 1:20 1:20 1:20 1:20 1:200 1:200 1:200 1:200 1:200 1:200
1:200 sheep ++++ - - +++ + - - ++ ++ + + Code KBL64 KBL64 KBL64
KBL640 KBL64 KBL64 KBL64 KBL64 KBL64 KBL64 KBL64 KBL640 07E34 07E35
07E35 7E34 07E71 07E71 07E71 07E86 07E86 07E86 07E86 7E111
Insertion of HA-gar-Ad-DOPE (IV) into embryos
Preparation of .alpha.-HA Microdrops
[0160] 25 .mu.L .mu.-HA (1:20dil) solution was pipetted into the
middle of well of a 4-well plate. The drop was covered with 0.8 mL
mineral oil. A further 25 .mu.L of .mu.-HA (1:20dil) solution was
pipetted through the mineral oil onto the microdrop. Microdrops
were incubated at 37.degree. C.
Embryo Glue.TM. Treatment
[0161] Embryos were transferred to microdrops containing Embryo
Glue.TM. and incubated at 37.degree. C. (+5% CM for 20 min.
Preparation of Pronase Microdrops
[0162] 25 .mu.L 0.5% Pronase solution was pipetted into the middle
of a well of a 4-well plate. The drop was covered with 0.8 ml,
mineral oil. A further 25 .mu.L of 0.5% pronase solution was
pipetted through the mineral oil onto the microdrop. Microdrops
were incubated at 37.degree. C. (no CO.sub.2).
Removal of Zona Pellucida
[0163] Washed embryos were transferred from KH media to the pronase
microdrops and incubated on a 37.degree. C. (no CO.sub.2) thermal
plate until all the zona pellucidas were removed (c. 5 min.). The
embryos were then washed 4.times. in KH media.
Reaction with Primary Antibody
[0164] Embryos from experiments were washed 4.times. in KH media
taking care to rinse the micro handling pipette between each
washing step. Embryos were then placed in microdrops with
.alpha.-HA antibody and incubated in a 37.degree. C. incubator (no
CO.sub.2) for 45 to 50 minutes.
[0165] Embryos were recovered and washed 4.times. in MV Wash.
Preparation of Alexa Fluor (AF) .alpha.-Sheep Microdrops
[0166] 25 .mu.L AF .alpha.-sheep (1:100dil) solution was pipetted
into the middle of well of 4-well plate. The drop was covered with
0.8 mL mineral oil. A further 25 .mu.L of AF .alpha.-sheep
(1:100dil) solution was pipetted through the mineral oil onto the
microdrop. Microdrops are incubated at RT in the dark.
[0167] Washed embryos were placed in microdrops with AF
.alpha.-sheep antibody. The four-well plates were then placed in
the dark and incubated at room temperature (RT) for 30 minutes.
[0168] Embryos were recovered and washed 4.times. in MV Wash.
Imaging
[0169] Embryos were placed on a glass slide and covered with
mineral oil. The slide was then stored in dark before imaging on a
Olympus BX51 Fluorescent microscope.
[0170] Experiment 1
[0171] Embryos were placed in pre-warmed, de-gassed microdrops
containing: [0172] HA.sub.15-20 mer-gar-Ad-DOPE (IV) [0173]
HA.sub.15-20 mer-gar-Ad-DOPE (IV)+HA.sub.15-20 mer [0174]
HA.sub.15-20 mer [0175] Control (media alone) [0176] KC media
alone
[0177] Embryos in microdrops were then incubated at 37.degree. C.
(+5% CO.sub.2) for 24 hours.
[0178] The results are presented in FIG. 3.
[0179] Experiment 2
[0180] Embryos were placed in pre-warmed, de-gassed microdrops
containing: [0181] HA.sub.15-20 mer-gar-Ad-DOPE (IV) (zona
pellucida removed); [0182] High molecular weight (HMW) HA; [0183]
HA.sub.15-20 mer-gar-Ad-DOPE (IV); [0184] KC Media with Embryo
Glue.TM.; [0185] KC media alone (control).
[0186] Embryos in microdrops were then incubated at 37.degree. C.
(+5% CO.sub.2) overnight.
[0187] The results are presented in Table 2 and FIGS. 4 and 5.
TABLE-US-00002 TABLE 2 Insertion of HA-gar-Ad-DOPE (IV) into
embryos AF Number of Treatment .alpha.-HA .alpha.-sheep embryos
Fluorescence IV 1:20 1:100 14 2+ (zona pellucida removed) HMW HA
1:20 1:100 10 -- IV 1:20 1:100 12 2+ Embryo Glue .TM. 1:20 1:100 8
-- Control 1:20 1:100 12 --
[0188] Experiment 3
[0189] Embryos were placed in pre-warmed, de-gassed microdrops
containing: [0190] HA.sub.15-20 mer-gar-Ad-DOPE (IV) (zona
pellucida removed) [0191] KC media alone (zona pellucida removed)
(control)
[0192] Embryos in microdrops were then incubated at 37.degree. C.
(+5% CO.sub.2) for 2 hours. Retention of HA.sub.15-20
mer-gar-Ad-DOPE (IV) was observed after 2, 5 and 24 hours.
[0193] The results are presented in FIGS. 6 and 7.
HA.sub.15-20 mer-gar-Ad-DOPE (IV) In Vitro Murine Embryo Toxicity
Studies
[0194] Studies were performed to assess the effects of overnight
insertion of HA.sub.15-20 mer-gar-Ad-DOPE (IV) on murine embryo
morphology and development.
Preparation of Mouse Embryos
[0195] Pre-pubescent C57/CBA F1 generation mice (21-30 days old)
were superovulated by intrapertoneal injection of 5 IU of FSH
(Folligon, Intravet, NZ) between 15:30 and 17:30, and 48 hours
later with 5 IU of human chorionic gonadotrophin (Chorulon,
Intravet, NZ).
[0196] Each donor mouse was placed with a CBA male stud of proven
fertility and checked for a seminal plug the following morning (day
0.5 post-coitus).
[0197] On day 1.5 post-coitus donor mice were sacrificed by
cervical dislocation. Uterine horns including the oviduct were
excised from the abdomen and placed on a sterile Petri dish.
[0198] Two-cell embryos were flushed from the oviduct using
in-house handling media (HM), collected and cultured in human
embryo culture media (HECM) in 5% CO.sub.2 at 37.degree. C. until
time of the experiment.
Preparing Experimental and Control Microdrops
[0199] Both sets of microdrops were prepared and equilibrated in 5%
CO.sub.2 at 37.degree. C. for at least 2 hours before use.
[0200] Experimental microdrops were 2 mg/mL HA.sub.15-20
mer-gar-Ad-DOPE (IV). 30 .mu.L of HECM was placed centrally in a
well of a 4-well culture dish. The drop was then overlayed with 0.9
mL sterile mineral oil and 20 .mu.L of stock HA.sub.15-20
mer-gar-Ad-DOPE (IV) at 5 mg/mL in HECM was added (mixed gently by
pipetting).
[0201] Control drops were made in a similar fashion, but 20 .mu.L
of HECM was added after the sterile mineral oil was overlaid (whole
drop contained HECM).
Preparation of Embryos
[0202] On day 2.5 post coitus, 20 healthy randomly selected embryos
were placed into each group and incubated in 5% CO.sub.2 at
37.degree. C. overnight in either HA.sub.15-20 mer-gar-Ad-DOPE
(IV)-containing or control media.
[0203] All embryos (now day 3.5) were washed in 37.degree. C. HM
and placed into 1 mL of HM kept on a 37.degree. C. heat plate under
aluminium foil (keeping groups separate).
[0204] Embryos were transferred in HECM microdrops and incubated in
5% CO.sub.2 at 37.degree. C. and assessed for embryo morphology or
development following further culture
Grading of Murine Blastocysts
[0205] Blastocysts were graded on Day 3.5, Day 4.5 and Day 5.5 post
coitus. Blastocysts were graded on two separate scales: [0206] i)
the rate of embryo development, predicted by the size of the fluid
filled blastocoel; and [0207] ii) the health of the embryos,
related to the amount of degradation/fragmentation visualised.
[0208] The grading system used to describe the rate of embryo
development was as follows:
TABLE-US-00003 Blastocyst score Visual factors 5 No blastocoel 4
Blastocoel less than half the volume of the embryo 3 Blastocoel
greater than half the volume of the embryo 2 Blastocoel completely
fills the embryo 1 Blastocoel volume is larger than that of an
early blastocyst Hatching Hatching blastocyst Hatched Fully hatched
from zona pellucida
[0209] The alphabetical grading used to predict the health of the
embryo was as follows:
TABLE-US-00004 Blastocyst degradation Visual factors C Dark
degenerative material, irregular sizing of blastomere cells, highly
distorted shape B Minimal degenerative material Minor cytoplasmic
fragmentation, minimal distortion of blastomere size and blastocyst
shape A No degenerative material, uniform blastomere sizes, uniform
blastocyst shape
[0210] Combining these two scales together gave 15 blastocyst
grades from the poorest 5C to the healthiest 1A.
[0211] In practice the 1A and 2A were often grouped together as the
marginal visual difference between these two and discrepancy noted
between observers meant that statistically it was more credible to
group these together.
Results
TABLE-US-00005 [0212] TABLE 3 Summarising embryo grade on days 3.5,
4.5 and 5.5 post coitus after an overnight incubation in 2 mg/mL
HA.sub.15-20 mer-gar-Ad-DOPE (IV) Embryo Control HA.sub.15-20
mer-gar-Ad-DOPE (IV) Grade D 3.5 D 4.5 D 5.5 D 3.5 D 4.5 D 5.5 5A
149 0 0 155 2 1 4A 29 0 0 24 0 0 3A 5 0 0 9 0 0 2A 5 6 1 3 3 0 1A 3
11 0 3 6 2 HgB 8 115 116 5 111 100 HdB 0 6 16 0 5 19 All B Grade 7
11 6 6 12 2 All C Grade 0 2 1 0 5 3 Total 206 151 140 205 144
127
Summary
[0213] The results showed that HA.sub.15-20 mer-gar-Ad-DOPE (IV)
modified murine embryos which were in vitro cultured to day 5.5
post coitus showed no untoward consequences in morphology or
development.
HA.sub.15-20 mer-gar-Ad-DOPE (IV) In Vivo Toxicity Studies Using
Murine Embryo Transfers
[0214] Studies were performed to assess the effects of modifying
murine embryos with HA.sub.15-20 mer-gar-Ad-DOPE (IV) on
implantation and viability when transferred into pseudo-pregnant
mice.
Methodology
[0215] Mouse embryos experimental and control microdrops, and
modified embryos were prepared as per the methods described above
(HA.sub.15-20 mer-gar-Ad-DOPE (IV) in vitro murine embryo toxicity
studies).
[0216] On day 3.5 post coitus 5 embryos were selected from the
HA.sub.15-20 mer-gar-Ad-DOPE (IV) modified embryo and 5 from the
unmodified embryo groups.
[0217] The selection process used a computer generated randomised
list (specifying the embryos, order in which groups were
transferred and the side groups were transferred into).
[0218] Embryos were placed into 1 mL of HM kept on a 37.degree. C.
heat plate under aluminium foil until transfer. Groups were kept in
separate wells.
Preparation of Pseudo-Pregnant Recipient Mice
[0219] In order to obtain a receptive endometrium within a
recipient mouse, the recipient must be mated with a vasectomised
male mouse of proven sterility. The act of coitus maintains the
corpus luteum of ovulated follicles and appropriate levels of
progesterone for implantation.
[0220] On day 0.5 post coitus of the donor mice, approximately 8
CBA/C57 F1 generation females between 60 and 100 days old and in
estrus were selected from a large pool and mated with vasectomised
CBA male mice.
[0221] Recipient mice were checked for seminal plugs the following
morning, denoting pseudo-pregnancy (day 0.5 post coitus). The
specified programming meant recipient mice were asynchronous by
minus 1 day compared to the embryos. The rationale was that the
embryos would "wait" for a receptive endometrium, but a receptive
endometrium would not "wait" for embryo.
[0222] Mice were kept in separate cages until the day of
transfer.
Embryo Transfer in a Dual Horn Fertile Model
[0223] Day 3.5 post coitus embryos were transferred into day 2.5
post coitus recipients. As described above the order and side of
transfer for each group was determined by computer generated
randomised lists.
[0224] Recipient mice were anaesthetised with 0.8 mL of Avertin
(made in-house) and an incision was made on the side of the
abdomen, just above the hip. The ovary was secured by grasping the
fat pad above it with a serrafin clamp and withdrawing outside of
the body.
[0225] All surgery was preformed on a 37.degree. C. heat plate.
[0226] A 29 g needle was used to create a hole through the uterine
horn. Five embryos were loaded into a fire-pulled and polished
capillary pipette (approx. 150-170 .mu.m in diameter) with mineral
oil and air gaps for stabilization.
[0227] The pipette was inserted into the uterine horn through the
pre-prepared hole and expelled until an air-gap was visible. The
uterine horn and ovary were replaced into the abdominal cavity and
the body wall and skin were sutured closed.
[0228] Mice were identified through ear marks and observed until
conscious.
Assessment of Fetal Outcomes
[0229] Recipients were euthanized on day 15 post-embryo transfer.
An incision was made in the abdomen exposing the entire uterus.
Viability of each fetus was checked by gently pinching with
forceps, followed by excision of the uterus.
[0230] Each fetus was excised out of the uterine horns and
separated from placenta. Each fetus and its corresponding placenta
were weighed.
Results
[0231] A summary of the results is provided in Table 4.
TABLE-US-00006 TABLE 4 Summary of 14 experiments transferring 2
mg/ml overnight HA15-20mer modified embryos (exp) and unmodified
embryos (ctrl) into the left or right horns of a recipient mouse.
Fetus HA.sub.15-20 mer-gar- Variable Ad-DOPE (IV) Ctrl embryos
introduced 70 70 Mean weight (g) 0.96 0.98 Resorptions 8.00 10.00
Losses 8.00 7.00 Implant sites 62 63 Viable fetuses 54 53 Fetus
resorption 8 10 embryo loss 8 7 Mean fetus weight (g) 0.96 0.98 SD
(fetus weight) 0.099 0.095 Mean placenta weight (g) 0.11 0.11
Summary
[0232] When comparing the implantation rates of experimental and
control groups through binary logistic regression, a p-value of
0.530 resulted. Thus there was no significant difference between
the two groups.
[0233] When comparing the fetal weights of experimental and control
groups through general linear modelling, a p-value of 0.140
resulted. Thus, there was no significant difference between the two
groups.
[0234] These results show that in a fertile animal there are no
untoward consequences of HA.sub.15-20 mer-gar-Ad-DOPE (IV)
modification on pregnancy or in utero development.
Rosetting of HA.sub.15-20 mer-gar-Ad-DOPE (IV) Inserted Murine
Embryos with Single Cell HEC-1A Human Epithelial Cells
[0235] Studies were performed to determine the level of adherence
of single cell human epithelial cells from endometrial cell line
HEC-1A (Human Endometrial Carcinoma sub-adherent cell line, ATCC
HTB-112) to murine embryos inserted with HA.sub.15-20
mer-gar-Ad-DOPE (IV) in vitro.
[0236] Embryos at the hatching to hatched stage had their zona
pellucidae removed and were incubated with:
A) HA.sub.15-20 mer-gar-Ad-DOPE (IV) (experimental); B) media alone
(blank--HECM, human embryo culture media); or C) lectin VVA (Vicia
villosa, Milton Adams BA 4601-2).
[0237] Embryos were then incubated with epithelial cells and
visualized for attachment using fluorescence microscopy.
[0238] Treatment Group C served as a positive control of maximal
rosetting.
[0239] Embryos were pre-stained with a fluorescent dye SNARF (red
label, 5-(and -6)-chloromethyl SNARF.RTM.-1, acetate, Molecular
Probes #C6826) and endometrial epithelial cells were stained with
fluorescent dye CMFDA (green label--CellTracker.TM. Green CMFDA,
5-chloromethylfluorescein diacetate, Molecular Probes # C7025) in
order to easily visualize attachment of epithelial cells to
embryos.
TABLE-US-00007 Group Treatment A. Experimental HA.sub.15-20
mer-gar-Ad-DOPE (IV) (2 mg/mL) in HECM B. Blank (Negative control)
HECM C. Rosette Positive control Lectin VVA (0.2 mg/mL) in HECM
Preparation of Murine Embryos and Microdrops
[0240] Murine embryos and microdrops were prepared as per the
methods described above (HA.sub.15-20 mer-gar-Ad-DOPE (IV) in vitro
murine embryo toxicity studies) but with HECM media (with or
without VVA)
Removal of Zona Pellucida
[0241] Embryos were transferred to 0.5% pronase (Sigma #P8811) 50
.mu.L microdrops and placed in a 37.degree. C. incubator for 6
minutes or until the zona pellucidae (ZP) were removed (checked
every 2 minutes).
[0242] Embryos were washed 3.times. in 50 .mu.L in-house handling
media (HM) drops and transferred to HM holding well (1 mL).
Staining Embryos with SNARF
[0243] The following steps were performed protected from light.
[0244] Embryos were transferred to 2 .mu.M SNARF 50 .mu.L
microdrops and incubated for 40 minutes in a 37.degree. C.
incubator, in an aluminium foil covered container.
[0245] The embryos were then washed 2.times. in HM media.
[0246] Embryos were transferred to HM media microdrops and
incubated a further 40 minutes in the 37.degree. C. incubator.
Modification of Embryos
[0247] The following steps were performed protected from light.
[0248] Embryos transferred to Group A microdrops were incubated at
37.degree. C.+5% CO.sub.2 for 2 hr.
[0249] Embryos transferred to Group B microdrops were incubated at
37.degree. C.+5% CO.sub.2 for 2 hr.
[0250] Embryos transferred to Group C microdrops were incubated at
37.degree. C.+5% CO.sub.2 for 40 min.
[0251] Treated embryos were washed 1.times. HM media (1 mL),
transferred to HECM microdrop, and incubated at 37.degree. C. 5%
CO.sub.2 for a further hour and 10 min.
Staining HEC-1A Epithelial Cells
[0252] The following steps were performed protected from light.
[0253] Cells were centrifuged 200 g for 10 min and the supernatant
removed.
[0254] 500 .mu.L 3 .mu.M CMFDA in 3% PVP (polyvinyl pyrrolidone,
Medicult AB, #10890001)/1.times. PBS was added to cells and the
cell gently resuspended.
[0255] Cells were incubated in a 37.degree. C. shaking water bath
for 45 min (resuspended occasionally).
[0256] Cells were centrifuged 200 g for 10 min and the supernatant
removed.
[0257] Cells were gently resuspended in 500 .mu.L Calcium and
Magnesium Free Hanks Balanced Salt Solution (CMF-HBSS,
Gibco-Invitrogen, #14170112) plus 2% Fetal Bovine Serum (FBS,
Gibco-Invitrogen, #10091-130).
[0258] Cells were incubated in a 37.degree. C. shaking water bath
for a further 30 min.
[0259] The epithelial cells were then washed 2.times. by
resuspending cells in CMF-HBSS-2% FBS, centrifuging 200 g for 10
min, and removing supernatant. Washing was repeated.
Preparation of Terasaki Plates
[0260] A row number and column letter was allocate to each
experimental and control group, marking clearly.
[0261] 8 .mu.L of HM media was aliquotted into each well identified
for use.
Incubation of Embryos and HEC-1A Epithelial Cells
[0262] The following steps were performed in the dark as much as
possible.
[0263] The epithelial cells were used at 20-25.times.10.sup.6 cells
per mL.
[0264] Embryos from each group were placed in their respective
wells on the Terasaki tray, ensuring that there were no more than 3
embryos per well and the embryos were separated.
[0265] Using a wide-bore handling pipette, the re-suspended
epithelial cells were gently aspirated (gently mixing prior to use
to remove clumps) over the embryos ensuring that epithelial cells
surrounded the embryos and covered the base of the well.
[0266] The lid was placed firmly on Terasaki tray and the cells
were incubated at room temperature for 30 minutes covered in
aluminium foil.
[0267] Wells were checked after 10 min and every 5 minutes
thereafter to ensure cells did not dry out.
Visualisation of "Rosetted" Embryos
[0268] The following steps were performed in the dark as much as
possible.
[0269] Using a wide-bore handling pipette embryo and epithelial
cells were gently transferred to 1 mL HM media (pre-warmed).
[0270] A 5 .mu.L drop of HM media was placed onto a microscope
slide.
[0271] Embryos were transferred into the HM media drop on the
microscope slide (being careful not to transfer free epithelial
cells) and covered with mineral oil (<10 .mu.L) by placing
around the circumference of the media and then gently covering the
top of the media.
[0272] Each microscope slide was viewed under an Olympus BX51
fluorescent microscope under 100.times. magnification, taking three
pictures of each embryo; one DIC (Differential Interference
Contrast microscopy), and two fluorescent (WIB and WIG filters, 550
nm and 620 nm respectively).
[0273] The two fluorescent images were merged using Olysia
BioReport. (FIGS. 8 to 12)
Scoring Adherence
[0274] The number of endometrial cells attached to each embryo at
the centre plane of focus was recorded for every embryo. Results
are presented in Tables 5 to 7.
TABLE-US-00008 TABLE 5 Epithelial Rosett cells Rosette Assay (n)
Controle attached Unmodified HA15-20mer (VVA) 0 11 4 0 1 2 3 0 2-3
3 4 0 4-5 2 4 0 6-10 0 8 1 11-20 0 1 5 20-40 0 0 10 Mean* 1.06 4.73
23.5
TABLE-US-00009 TABLE 6 Epithelial Rosette cells Rosette Assay (%)
Control attached Unmodified HA15-20mer (VVA) 0 61% 17% 0% 1 11% 13%
0% 2-3 17% 17% 0% 4-5 11% 17% 0% 6-10 0% 33% 6% 11-20 0% 4% 31%
20-40 0% 0% 63%
TABLE-US-00010 TABLE 7 Epithelial Rosette cells Rosette Assay
(mean) Control attached Unmodified HA15-20mer (VVA) Mean 1.06 4.73
23.5 % of Max 4.5% 20.1% 100% (VVA)
[0275] The rosette assay demonstrated at least a four-fold increase
in attachment of epithelial cells to HA.sub.15-20 mer-gar-Ad-DOPE
(IV) modified embryos.
HA.sub.15-20 mer-gar-Ad-DOPE (IV) Transformed Red Blood Cells
Incubated with HMW HA, Bovine Serum Albumin Solutions or Human
Serum
[0276] Studies were performed to test whether HA.sub.15-20
mer-gar-Ad-DOPE (IV) transformed red blood cells (RBCs) associated
with high molecular weight HA (HMW HA), albumin or compatible serum
which would cause them to agglutinate/aggregate.
Insertion of HA.sub.15-20 mer-gar-Ad-DOPE (IV) into RBCs
[0277] HA.sub.15-20 mer-gar-Ad-DOPE (IV) @ 10 mg/mL to 5 mg/mL was
diluted in Celpresol (CSL #063321301).
[0278] 15 .mu.L of washed group O RBC, then 5 .mu.L of HA.sub.15-20
mar-gar-Ad-DOPE (IV) or Celpresol (untreated) was added to an
eppendorf tube and mixed.
[0279] Tubes were incubated at room temperature for 3 hr, then
4.degree. C. overnight with occasional mixing.
[0280] Transformed RBCs were washed two times with PBS and then
suspended as 5% in Celpresol.
High Molecular Weight HA (HMW HA), Bovine Serum Albumin (BSA) and
Serum Solutions.
[0281] HMW HA was hyaluronic acid sodium salt from Streptococcus
equi from Fluka BioChemika Cat #53747 (MW 1.5-1.8.times.106 Da).
Solutions of 0.5 and 2.5 mg/ml were prepared in Celpresol.
[0282] BSA was bovine serum albumin Gibco Cat#30063-572. Solutions
of 2%, 4%, 6%, 8% and 10% (w/v) were prepared in PBS.
[0283] Serum was human serum not containing antibodies directed
against the group O cells.
Incubating Transformed RBCs with HMW HA Solutions
[0284] 5% suspensions were made from washed transformed group O
RBCs.
[0285] 30 .mu.L HA.sub.15-20 mer-gar-Ad-DOPE (IV) RBC or untreated
RBC was added to 60 .mu.L HMW HA, bovine serum albumin solution or
human serum, in duplicate.
Samples were Either: centrifuged for 10 s in an immufuge then
assessed for agglutination; or incubated 30 mins at 37.degree. C.,
centrifuged and then assessed for agglutination.
[0286] The results showed that in the presence of high molecular
weight protein (albumin) or high molecular weight HA cells coated
with HA as HA.sub.15-20 mer-gar-Ad-DOPE (IV) are able to interact
as visualised by agglutination via non antibody mediated
interactions.
[0287] Although the invention has been described by way of
exemplary embodiments it should be appreciated that variations and
modifications may be made without departing from the scope of the
invention. Furthermore where known equivalents exist to specific
features, such equivalents are incorporated as if specifically
referred to in this specification.
INDUSTRIAL APPLICABILITY
[0288] The invention has application in the preparation of media
and medicinal formulations.
TABLE-US-00011 TABLE 8 Agglutination scores - HA.sub.15-20
mer-gar-Ad-DOPE (IV) RBC Immediate Centrifugation HA.sub.15-20
mer-gar-Ad-DOPE (IV) RBC Untreated RBC HMW HA RT RT mg/mL 1 2 3 4 0
-- -- -- -- 0.5 -- -- -- -- 2.5 2+ 2+ -- --
TABLE-US-00012 TABLE 9 Agglutination scores - HA.sub.15-20
mer-gar-Ad-DOPE (IV) RBC 37.degree. C. incubation then
centrifugation HA.sub.15-20 mer-gar-Ad-DOPE (IV) RBC Untreated RBC
HMW HA 37.degree. C. 37.degree. C. mg/mL 1 2 3 4 0 -- -- -- -- 0.5
-- -- -- -- 2.5 1+ 3+ -- --
TABLE-US-00013 TABLE 10 Agglutination scores - BSA Immediate
Centrifugation HA.sub.15-20 mer-gar-Ad-DOPE (IV) RBC Untreated RBC
HMW HA RT RT mg/mL 1 2 3 4 2% -- -- -- -- 4% vw vw -- -- 6% 2+ 2+
-- -- 8% 2+ 2+ -- -- 10% 3+ 3+ -- --
TABLE-US-00014 TABLE 11 Agglutination scores - BSA 37.degree. C.
incubation then Centrifugation HA.sub.15-20 mer-gar-Ad-DOPE (IV)
RBC Untreated RBC HMW HA 37.degree. C. 37.degree. C. mg/mL 1 2 3 4
2% -- -- -- -- 4% 1+ 1+ -- -- 6% 2+ 2+ -- -- 8% 3+ 3+ -- -- 10% 3+
3+ -- --
TABLE-US-00015 TABLE 12 Agglutination scores - Serum Immediate
Centrifugation HA.sub.15-20 mer-gar-Ad-DOPE (IV) RBC Untreated RBC
Serum RT RT Sample 1 2 3 4 1 3+ 2+ -- -- 2 4+ 3+ -- -- 3 4+ 4+ --
-- 4 4+ 4+ -- -- 5 4+ 4+ -- -- 6 4+ 4+ -- -- 7 4+ 4+ -- -- 8 4+ 4+
-- --
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