U.S. patent application number 14/981036 was filed with the patent office on 2016-04-21 for amphiphilic block copolymers for nucleic acid delivery.
This patent application is currently assigned to BIOCOMPATIBLES UK LIMITED. The applicant listed for this patent is BIOCOMPATIBLES UK LIMITED. Invention is credited to Giuseppe BATTAGLIA, Irene CANTON, Andrew Lennard LEWIS, Peter William STRATFORD.
Application Number | 20160106673 14/981036 |
Document ID | / |
Family ID | 39735310 |
Filed Date | 2016-04-21 |
United States Patent
Application |
20160106673 |
Kind Code |
A1 |
LEWIS; Andrew Lennard ; et
al. |
April 21, 2016 |
AMPHIPHILIC BLOCK COPOLYMERS FOR NUCLEIC ACID DELIVERY
Abstract
The present invention provides a composition comprising vesicles
and encapsulated within the vesicles, nucleic acid comprising less
than 1000 nucleotides, wherein the vesicles comprise an amphiphilic
block copolymer having a hydrophilic and a hydrophobic block.
Methods of forming vesicles and methods of delivering nucleic acid,
in particular, iRNA into cells, are also provided.
Inventors: |
LEWIS; Andrew Lennard;
(Farnham, GB) ; BATTAGLIA; Giuseppe; (Western
Bank, GB) ; CANTON; Irene; (Western Bank, GB)
; STRATFORD; Peter William; (Farnham, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOCOMPATIBLES UK LIMITED |
Farnham |
|
GB |
|
|
Assignee: |
BIOCOMPATIBLES UK LIMITED
Farnham
GB
|
Family ID: |
39735310 |
Appl. No.: |
14/981036 |
Filed: |
December 28, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12991321 |
Jan 28, 2011 |
9254258 |
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PCT/EP2009/055864 |
May 14, 2009 |
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14981036 |
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Current U.S.
Class: |
424/497 ;
435/375; 514/44A |
Current CPC
Class: |
A61K 31/713 20130101;
A61K 48/0041 20130101; C12N 15/111 20130101; C12N 2320/32 20130101;
A61K 9/0019 20130101; A61K 9/1273 20130101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/713 20060101 A61K031/713 |
Foreign Application Data
Date |
Code |
Application Number |
May 15, 2008 |
EP |
08156272.0 |
Claims
1. A composition comprising nanovesicles having an aqueous core and
encapsulated within the aqueous core of the nanovesicles, nucleic
acid comprising less than 1000 nucleotides, wherein the
nanovesicles comprise an amphiphilic block copolymer having a
hydrophilic and a hydrophobic block, wherein the ratio of the
degree of polymerization of the hydrophilic to hydrophobic block is
in the range of 1:2.5 to 1:8, wherein the hydrophobic block has a
degree of polymerization in the range 50 to 250 and the hydrophilic
block has a degree of polymerization of at least 15, the
hydrophilic block comprises ethylenically unsaturated radically
polymerizable monomers comprising a zwitterionic monomer, and
wherein the hydrophobic block comprises (diisopropylamino)ethyl
methacrylate or (diethylamino)ethyl methacrylate monomers.
2. A composition according to claim 1, wherein the nanovesicles
have a diameter in the range 50-1000 nm.
3. A composition according to claim 1 wherein the nucleic acid is
small iRNA and comprises 15 to 25 nucleotide pairs.
4. A composition according to claim 1 in which the zwitterionic
monomer has the general formula Y B X I in which Y is an
ethylenically unsaturated group selected from H.sub.2C.dbd.CR--CO--
A-, H.sub.2C.dbd.CR--C.sub.6H.sub.4-A.sup.1-,
H.sub.2C.dbd.CR--CH.sub.2A.sup.2, R.sup.2O--CO--CR.dbd.CR--CO--O,
RCH.dbd.CH--CO--O--, RCH.dbd.C(COOR.sup.2)CH.sub.2--CO--O,
##STR00009## A is --O-- or NR.sup.1; A.sup.1 is selected from a
bond, (CH.sub.2).sub.lA.sup.2 and (CH.sub.2).sub.lSO.sub.3-- in
which 1 is 1 to 12; A.sup.2 is selected from a bond, --O--,
O--CO--, CO--O, CO--NR.sup.1--, --NR.sup.1--CO, O--CO--NR.sup.1--,
NR.sup.1--CO--O--; R is hydrogen or C.sub.1-4 alkyl; R.sup.1 is
hydrogen, C.sub.1-4-alkyl or BX; R.sup.2 is hydrogen or C.sub.1-4
alkyl; B is a bond, or a straight branched alkanediyl, alkylene
oxaalkylene, or alkylene (oligooxalkylene) group, optionally
containing one or more fluorine substituents; X is a zwitterionic
group.
5. A composition according to claim 4 in which X is a group of the
general formula II ##STR00010## in which moieties A.sup.3 and
A.sup.4, which are the same or different, are --O--, --S--, --NH--
or a valence bond, preferably --O--, and W.sup.+ is a group
comprising an ammonium, phosphonium or sulphonium cationic group
and a group linking the anionic and cationic moieties which is
preferably a C.sub.1-12-alkanediyl group, preferably in which
W.sup.+ is a group of formula --W.sup.1--N.sup.+R.sup.3.sub.3,
--W.sup.1--P.sup.+R.sup.4.sub.3, --W.sup.1--S.sup.+R.sup.4.sub.2 or
--W.sup.1-Het.sup.+ in which: W.sup.1 is alkanediyl of 1 or more,
preferably 2-6 carbon atoms optionally containing one or more
ethylenically unsaturated double or triple bonds,
disubstituted-aryl (arylene), alkylene arylene, arylene alkylene,
or alkylene aryl alkylene, cycloalkanediyl, alkylene cycloalkyl,
cycloalkyl alkylene or alkylene cycloalkyl alkylene, which group
W.sup.1 optionally contains one or more fluorine substituents
and/or one or more functional groups; and either the groups R.sup.3
are the same or different and each is hydrogen or alkyl of 1 to 4
carbon atoms, preferably methyl, or aryl, such as phenyl, or two of
the groups R.sup.3 together with the nitrogen atom to which they
are attached form an aliphatic heterocyclic ring containing from 5
to 7 atoms, or the three groups R.sup.3 together with the nitrogen
atom to which they are attached as heteroaromatic ring having 5 to
7 atoms, either of which rings may be fused with another saturated
or unsaturated ring to form a fused ring structure containing from
5 to 7 atoms in each ring, and optionally one or more of the groups
R.sup.3 is substituted by a hydrophilic functional group, and the
groups R.sup.4 are the same or different and each is R.sup.3 or a
group OR.sup.3, where R.sup.3 is as defined above; or Het is an
aromatic nitrogen-, phosphorus- or sulphur-, preferably nitrogen-,
containing ring, for example pyridine.
6. A composition according to claim 4 in which the zwitterionic
monomer is 2-methacryloyloxyethyl phosphorylcholine.
7. A method for forming a composition according to claim 1, wherein
one of the blocks is pH sensitive, comprising the steps: (i)
dispersing the amphiphilic copolymer is an organic solvent; (ii)
acidifying the pH of the composition formed in step (i); (iii)
adding the nucleic acid to the composition; and (iv) raising the pH
to around neutral to encapsulate the nucleic acid.
8. A method according to claim 7 comprising a preliminary step
before step (i), wherein the amphiphilic copolymer is dissolved in
an organic solvent in a reaction vessel and the solvent is then
evaporated to form a film on the inside of the reaction vessel.
9. An in vitro method of delivering nucleic acid comprising less
than 1000 nucleotides into a cell comprising contacting a
composition according to claim 1 with the cell.
10. A method of treatment by therapy, comprising administering a
composition according to claim 1 to a human or animal body.
11. A method of treatment by therapy according to claim 10, wherein
nucleic acid comprising less than 1000 nucleotides is delivered
into a cell.
12. A composition comprising nanovesicles having an aqueous core
and encapsulated within the aqueous core of the nanovesicles,
nucleic acid comprising less than 1000 nucleotides, wherein the
nanovesicles have a diameter in the range 50-5000 nm and comprise
an amphiphilic block copolymer having a hydrophilic and a
hydrophobic block, wherein the ratio of the degree of
polymerization of the hydrophilic to hydrophobic block is in the
range of 1:2.5 to 1:8, wherein the hydrophobic block has a degree
of polymerization in the range 50 to 250 and the hydrophilic block
has a degree of polymerization of at least 15, the hydrophilic
block is formed from ethylenically unsaturated radically
polymerizable monomers comprising a zwitterionic monomer, and
wherein the hydrophobic block comprises (diisopropylamino)ethyl
methacrylate monomers.
13. A composition according to claim 12, wherein the nanovesicles
have a diameter in the range 50-1000 nm.
14. A composition according to claim 12 wherein the nucleic acid is
small iRNA and comprises 15 to 25 nucleotide pairs.
15. A composition according to claim 12 in which the zwitterionic
monomer has the general formula Y B X I in which Y is an
ethylenically unsaturated group selected from H.sub.2C.dbd.CR--CO--
A-, H.sub.2C.dbd.CR--C.sub.6H.sub.4-A.sup.1-,
H.sub.2C.dbd.CR--CH.sub.2A.sup.2, R.sup.2O--CO--CR.dbd.CR--CO--O,
RCH.dbd.CH--CO--O--, RCH.dbd.C(COOR.sup.2)CH.sub.2--CO--O,
##STR00011## A is --O-- or NR.sup.1; A.sup.1 is selected from a
bond, (CH.sub.2).sub.lA.sup.2 and (CH.sub.2).sub.lSO.sub.3-- in
which l is 1 to 12; A.sup.2 is selected from a bond, --O--,
O--CO--, CO--O, CO--NR.sup.1--, --NR.sup.1--CO, O--CO--NR.sup.1--,
NR.sup.1--CO--O--; R is hydrogen or C.sub.1-4 alkyl; R.sup.1 is
hydrogen, C.sub.1-4-alkyl or BX; R.sup.2 is hydrogen or C.sub.1-4
alkyl; B is a bond, or a straight branched alkanediyl, alkylene
oxaalkylene, or alkylene (oligooxalkylene) group, optionally
containing one or more fluorine substituents; X is a zwitterionic
group.
16. A composition according to claim 15 in which X is a group of
the general formula II ##STR00012## in which moieties A.sup.3 and
A.sup.4, which are the same or different, are --O--, --S--, --NH--
or a valence bond, preferably --O--, and W.sup.+ is a group
comprising an ammonium, phosphonium or sulphonium cationic group
and a group linking the anionic and cationic moieties which is
preferably a C.sub.1-12-alkanediyl group, preferably in which
W.sup.+ is a group of formula --W.sup.1--N.sup.+R.sup.3.sub.3,
--W.sup.1--P.sup.+R.sup.4.sub.3, --W.sup.1--S.sup.+R.sup.4.sub.2 or
--W.sup.1-Het.sup.+ in which: W.sup.1 is alkanediyl of 1 or more,
preferably 2-6 carbon atoms optionally containing one or more
ethylenically unsaturated double or triple bonds,
disubstituted-aryl (arylene), alkylene arylene, arylene alkylene,
or alkylene aryl alkylene, cycloalkanediyl, alkylene cycloalkyl,
cycloalkyl alkylene or alkylene cycloalkyl alkylene, which group
W.sup.1 optionally contains one or more fluorine substituents
and/or one or more functional groups; and either the groups R.sup.3
are the same or different and each is hydrogen or alkyl of 1 to 4
carbon atoms, preferably methyl, or aryl, such as phenyl, or two of
the groups R.sup.3 together with the nitrogen atom to which they
are attached form an aliphatic heterocyclic ring containing from 5
to 7 atoms, or the three groups R.sup.3 together with the nitrogen
atom to which they are attached as heteroaromatic ring having 5 to
7 atoms, either of which rings may be fused with another saturated
or unsaturated ring to form a fused ring structure containing from
5 to 7 atoms in each ring, and optionally one or more of the groups
R.sup.3 is substituted by a hydrophilic functional group, and the
groups R.sup.4 are the same or different and each is R.sup.3 or a
group OR.sup.3, where R.sup.3 is as defined above; or Het is an
aromatic nitrogen-, phosphorus- or sulphur-, preferably nitrogen-,
containing ring, for example pyridine.
17. A composition according to claim 15 in which the zwitterionic
monomer is 2-methacryloyloxyethyl phosphorylcholine.
18. A method for forming a composition according to claim 12,
wherein one of the blocks is pH sensitive, comprising the steps:
(i) dispersing the amphiphilic copolymer is an organic solvent;
(ii) acidifying the pH of the composition formed in step (i); (iii)
adding the nucleic acid to the composition; and (iv) raising the pH
to around neutral to encapsulate the nucleic acid.
19. A method according to claim 18 comprising a preliminary step
before step (i), wherein the amphiphilic copolymer is dissolved in
an organic solvent in a reaction vessel and the solvent is then
evaporated to form a film on the inside of the reaction vessel.
20. An in vitro method of delivering nucleic acid comprising less
than 1000 nucleotides into a cell comprising contacting a
composition according to claim 1 with the cell.
21. A method of treatment by therapy, comprising administering a
composition according to claim 12 to a human or animal body.
22. A method of treatment by therapy according to claim 21, wherein
nucleic acid comprising less than 1000 nucleotides is delivered
into a cell.
23. A composition comprising nanovesicles and encapsulated within
the aqueous core of the nanovesicles, nucleic acid comprising less
than 1000 nucleotides, wherein the nanovesicles have a diameter in
the range 50-1000 nm and comprise an amphiphilic block copolymer
having a hydrophilic block comprising 2-methacryloyloxy ethyl
phosphorylcholine and a hydrophobic block comprising
2-(diisopropyl)amino ethyl methacrylate, wherein the degree of
polymerisation of the hydrophilic block is 20 to 25 and the degree
of polymerisation of the hydrophobic block is 70 to 75, and wherein
the nucleic acid is siRNA.
24. A composition according to claim 12 wherein the hydrophobic
block consists of (diisopropylamino)ethyl methacrylate
monomers.
25. A composition according to claim 12 wherein the hydrophobic
block consists of 2-(diisopropyl)amino ethyl methacrylate.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. application Ser. No.
12/991,321, filed Nov. 5, 2010; which is a National Stage of
International Application No. PCT/EP2009/055864 filed May 14, 2009,
claiming benefit to European Patent Application No. 08156272.0
filed May 15, 2008, the contents of each of which are incorporated
herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to novel delivery systems for
introducing nucleic acid into cells, more particularly RNA. Methods
of delivering nucleic acid into cells also form part of the
invention.
BACKGROUND OF THE INVENTION
[0003] Recently, researchers have tried to exploit the natural
ability of RNAs to mobilize and transcript genetic information for
therapeutic purposes. Such methods normally involve the
interference with dysfunctional nucleic acids or proteins and/or
the stimulation of the production of therapeutic genes.
Particularly important is a process that uses synthetic
double-stranded RNA, known as RNA interference (RNAi). This
operates via post-transcriptional gene silencing, mediating the
resistance to both endogenous parasitic and exogenous pathogenic
nucleic acids, and therefore regulating the expression of
protein-coding genes. It appears evident that the efficient
cytosolic delivery of RNAs is a vital step in almost all RNAi-based
gene-silencing experiments. Efficient cytosolic delivery of nucleic
acids is, however, a very difficult task. Synthetic siRNAs can be
delivered to cells in culture via electroporation or by using
either cationic lipids or polymers. However, these approaches are
limited by the transient nature of the response and in some cases
by vector-mediated toxicity, (Mittal, V. Improving the Efficiency
of RNA Interference in Mammals. Nature Rev. Genet. 5, 355-365
(2004)).
[0004] WO2002/044321 describes the sequence and structural
requirements for small RNAs mediating RNA interference and
therapeutic uses of interfering RNA. Using a drosophila in vitro
system, it is demonstrated that 19-23 nucleotide short RNA
fragments are the sequence-specific mediators of RNA.
[0005] Alnylam Pharmaceuticals Inc. have developed a variety of
therapeutic compositions comprising siRNAs. Some of these are
disclosed in, for instance, WO2004/030634. Sirna Therapeutics, Inc.
are also active in this area. Their U.S. Pat. No. 7,022,828, for
example, describes siRNA treatment of diseases or conditions
related to levels of IKK-gamma.
[0006] EP1527176 describes novel forms of interfering RNA
molecules. The molecules are double-stranded structures in which
the strands are at least partially complementary to one another,
and wherein the double-stranded structure is blunt ended.
WO2006/069782, by the same Applicant, describes delivery agents for
introducing small nucleic acids, such as those in EP1527176, into
cells. The delivery agents are lipid compositions comprising a
first lipid component, a first helper lipid and a shielding
compound (for instance PEG) which is removable from the lipid
composition under in vivo conditions.
[0007] We have recently reported on a very efficient, non-toxic and
non-inflammatory vector for the delivery of (plasmid) DNA within
human cells, (Lomas, H. et al. Biomimetic pH Sensitive Polymersomes
for Efficient DNA Encapsulation and Delivery. Adv. Mater. Vol 19
(2007), 4238-4243). The combination of this type of polymer with
DNA is described in our patent application WO03/074090. In this
application, 2-(dimethyl)ethyl methacrylate (DMA)-MPC polymers are
used to form DNA-polymer complexes. Depending upon the block
lengths and pendant groups of the respective components of the
copolymer, it is now known that the interaction with DNA can be
tailored to produce DNA condensates (polyplexes) or have the DNA
encapsulated within a vesicle of the material. The latter is based
on the self-assembly of pH sensitive poly(2-methacryloxyethyl
phosphorylcholine)-poly(2-(diisopropylamino)-ethyl methacrylate),
(PMPC-PDPA) block copolymers into nanometer-sized vesicles, also
known as polymersomes, (Du, J., Tang, Y., Lewis, A. L. & Armes,
S. P. pH-Sensitive Vesicles Based on a Biocompatible Zwitterionic
Diblock Copolymer. J. Am. Chem. Soc. 127, 17982-17983 (2005)).
[0008] Tan et al in Biomacromolecules 2007, 8, 448-454 describe
polyethylene oxide-poly(dimethylamino)ethyl methacrylate
(PEO-b-PDMA) and PEO-b-poly(diethylamino)ethyl methacrylate
(PEO-b-PDEA) copolymers as a self-assembling non-viral vector for
plasmid DNA delivery. Similarly, Tan et al in Langmuir vol, 22 No.
8, 2006, 3744-3750 describe complexes of PEO-b-PDEA copolymers with
plasma DNA. Neither of these references discuss the formation of
complexes with smaller strands of nucleic acid.
SUMMARY OF THE INVENTION
[0009] In view of the prior art there remains a desire to provide
improved delivery systems for introducing nucleic acid comprising
less than 1000 nucleotides into cells. In accordance with this
desire there is provided in a first aspect of this invention a
composition comprising vesicles and encapsulated within the
vesicles, nucleic acid comprising less than 1000 nucleotides,
wherein the vesicles comprise an amphiphilic block copolymer having
a hydrophilic and a hydrophobic block.
[0010] The second aspect of this invention provides a method for
forming a composition according to the first aspect of the
invention, wherein one of the blocks of the copolymer is pH
sensitive, comprising the steps:
[0011] (i) dispersing the amphiphilic copolymer in an aqueous
media;
[0012] (ii) acidifying the pH of the composition formed in step
(i);
[0013] (iii) adding the nucleic acid to the composition; and
[0014] (iv) raising the pH to around neutral to encapsulate the
nucleic acid.
[0015] The third aspect of this invention provides an in vitro
method of delivering nucleic acid comprising less than 1000
nucleotides into a cell comprising contacting a composition
according to the first aspect of the invention with the cell.
[0016] The fourth aspect of the invention provides a composition
according to the first aspect of the invention for use in a method
of medical treatment by therapy.
[0017] The final aspect of the invention provides a composition
according to the first aspect of the invention for use in a method
of treatment by therapy wherein nucleic acid comprising less than
1000 nucleotides is delivered into a cell.
[0018] The vesicles defined above are biocompatible and do not
undergo any cytotoxic interactions with cells. They have improved
delivery rates of nucleic acid in comparison to the delivery
systems described in the prior art. Although mechanisms for
delivering DNA into cells using block copolymers have already been
disclosed, the delivery of nucleic acid comprising less than 1000
nucleotides in vesicles which preferably have a pH-dependent
dissociation is described herein for the first time. The length of
the nucleic acid has been shown to significantly influence its
interaction with the copolymer. The methods described herein are
suitable for delivery of small nucleic acid molecules.
DETAILED DESCRIPTION OF THE INVENTION
[0019] In the copolymers used in this invention, preferably one of
the blocks comprises pendant groups having a pK.sub.a in the range
3.0 to 6.9. This means that once the vesicles are taken up into
cells, they advantageously dissociate and release nucleic acid
within the cell. Without wishing to be bound by theory the inventor
believes that release of the nucleic acid within the cell can be
explained by the following. Dissociation is promoted by pH
sensitivity of the block copolymer. The mechanism of cell
internalisation (endocytosis) involves engulfment within
phospholipid membranes produced by endocytic organelles such as
trafficking vesicles, phagosomes, or pinosomes (depending on the
precise endocytic pathway). The endocytic organelle detaches from
the cell membrane and takes the vesicles inside the cell for
further processing. Regardless of the endocytic pathway, the
internalised vesicles experience a reduction in local pH from pH
7.4 to pH 5-6 once inside the organelle. This pH drop is sufficient
to trigger the transition from nucleic acid-loaded vesicles to
nucleic acid-copolymer complex. As this transition is confined
within a semi-permeable organelle membrane, the sudden increase in
particle number corresponds to a large increase in osmotic
pressure. This causes lysis of the lipid membrane, releasing the
nucleic acid into the cell cytosol.
[0020] As detailed above, the hydrophobic or the hydrophilic block
of the amphiphilic block copolymer preferably comprises pendant
groups which have a pK.sub.a in the range 3.0 to 6.9. This confers
"pH sensitivity" on the copolymer. By pK.sub.a, is meant the pH
where half of the pendant groups are ionised. pK.sub.a can be
determined by a variety of methods including pH titration, followed
by potentiometric titration, UV spectroscopy and Dynamic Light
Scattering (DLS). An appropriate method should be selected to
measure the pK.sub.a according to the copolymer which is being
analysed and its solubility in the test media.
[0021] DLS is the particularly preferred method for measuring
pK.sub.a. As indicated in the paper by Du et al; J. Am. Chem. Soc
2005, 127, 17982-17983, the DLS signal from
PMPC.sub.25-b-PDPA.sub.120 copolymer in water varies with pH. At a
certain pH the signal rapidly increases as the copolymer undergoes
a transition from being molecularly deassociated to associated. The
pK.sub.a is taken as the pH of the mid-point of this rapid
increase. These experiments are described further in Giacomelli et
al, Biomacromolecules 2006, 7, 817-828. In this reference, the
experiments are performed on micelles of PMPC-b-PDPA block
copolymer, but the techniques may also be used when the phase
transition involves vesicle formation.
[0022] In the specification, the pK.sub.a of a group in a polymer
is determined on the basis of a polymer system (and not assumed to
be the same as the pK.sub.a's of similar moieties in non-polymeric
systems).
[0023] Typically, the hydrophobic block has the pendant groups with
a pK.sub.a in the range 3.0 to 6.9. Preferably, the pK.sub.a of the
pendant groups is in the range 4.0 to 6.9, more preferably 5.5 to
6.9. The vesicles are correspondingly capable of disassociating in
such pH ranges.
[0024] It is preferred that the hydrophobic block comprise pendant
cationisable moieties as pendant groups. Cationisable moieties are,
for instance, primary, secondary or tertiary amines, capable of
being protonated at pH's below a value in the range 3 to 6.9.
Alternatively the group may be a phosphine.
[0025] The nucleic acid may be double or single stranded and may be
DNA or RNA. Typically, the nucleic acid comprises less than 500
nucleotides and preferably has less than 200 nucleotides, more
preferably less than 100 nucleotides. In one preferred embodiment,
the nucleic acid is an oligonucleotide, which is defined herein as
comprising 2-20 nucleotides. The nucleic acid may be a Locked
Nucleic Acid (LNA). This is formed from modified RNA
nucleotides.
[0026] The nucleic acid is preferably an "anti-sense"
oligonucleotide, i.e. preferably has a base sequence which is
complementary to the mRNA, of a gene of interest, which is called
the "sense" sequence. For instance, the nucleic acid may be
anti-sense DNA.
[0027] Anti-sense molecules can be classified as "enzyme-dependent"
or "steric blocking". Enzyme-dependent anti-sense include forms
dependent on RNase H activity to degrade target mRNA, including
ssRNA, RNA and phosphorothioate anti-sense. Steric blocking
anti-sense interferes with gene expression or other mRNA-dependent
cellular processes by binding to a target sequence of mRNA and
sterically hindering gene expression. Steric blocking anti-sense
molecules include peptide nucleic acid, locked nucleic acid and
morpholino anti-sense.
[0028] Preferably, the nucleic acid is an RNA molecule, preferably
an interfering RNA (iRNA) molecule.
[0029] iRNA is typically double stranded and may comprise 200
nucleotides or more. Typically, the iRNA comprises in the range 15
to 100 nucleotide pairs. Interfering RNA acts via a different
mechanism to anti-sense RNA, but both achieve the same effect (gene
silencing). Anti-sense RNA is typically single stranded, whereas
interfering RNA operates via double stranded RNA fragments which
trigger catalytically medicated gene silencing, most typically by
targeting the RNA-induced silencing complex to bind to and degrade
the mRNA.
[0030] Preferably, the iRNA molecule is a small interfering RNA
molecule (siRNA). siRNA typically consist of a double-stranded RNA
structure which comprises between 15 to 25, preferably 18 to 23
nucleotide pairs which are base-pairing to each other, i.e. are
essentially complementary to each other, typically mediated by
Watson-Crick base-pairing. One strand of this double-stranded RNA
molecule is essentially complementary to a target nucleic acid,
preferably a mRNA, whereas the second strand of said
double-stranded RNA molecule is essentially identical to a stretch
of said target nucleic acid.
[0031] The iRNA or siRNA molecule may be flanked on each side and
each stretch, respectively, by a number of additional
oligonucleotides which, however, do not necessarily have to
base-pair to each other.
[0032] The composition of this invention is normally aqueous and
typically therefore the vesicles are in aqueous solution. A typical
pH of the aqueous composition is 7.0 to 7.6, preferably 7.2 to 7.4.
Vesicles are generally substantially spherical and comprise a
bilayered membrane. The bilayer is generally formed from two layers
of amphiphilic molecules, which align to form an enclosed core with
hydrophilic head groups facing the core and the exterior of the
vesicle, and hydrophilic tail groups forming the interior of the
membrane.
[0033] A typical diameter of a substantially spherical vesicle is
in the range 50-5000 nm. More typically, the diameter is in the
range 50-1000 nm. Vesicles having a diameter in this range are
normally termed "nanovesicles". The nanovesicles are preferably
substantially spherical in shape. Typically, the nanovesicles have
a number average diameter of less than 300 nm, preferably less than
250 nm, most preferably less than 200 nm or 150 nm. The thickness
of the bilayer is generally between 2 to 50 nm, more typically
between 5 and 20 nm. These dimensions can be measured by
Transmission Electron Microscopy (T.E.M), and Small Angle X-ray
Scattering (SAXS) (Battaglia et al; JACS 127, 8757 (2005)).
[0034] The nucleic acid is typically associated with the vesicles
via physical or chemical interaction, such as electrostatic or
hydrophobic attraction. Usually the nucleic acid is not covalently
bound to the vesicles and is encapsulated within the vesicles via
physical entrapment alone. Typically, the vesicles are nanovesicles
and the nucleic acid is encapsulated with the aqueous core of the
nanovesicles.
[0035] The composition of this invention is typically an aqueous
composition and has a pH which is substantially neutral. At this pH
the amphiphilic copolymer is uncharged and therefore the nucleic
acid is bound to the structures via physical encapsulation, and not
electrostatic interaction.
[0036] Several techniques can be used to demonstrate that nucleic
acid is encapsulated in the vesicles, as opposed to the vesicles
and the nucleic acid simply forming a nucleic acid-copolymer
electrostatically bound complex. Transition electron microscopy
(TEM) and Dynamic Light Scattering (DLS) performed on the
composition of the invention at pH7 allows an observer to view the
presence of discrete vesicles, typically nanovesicles. The form of
the vesicles is not affected by the presence of nucleic acid. Zeta
(Z)-potential measurements also have utility. Such measurements
typically do not demonstrate any charge from the nucleic acid or
copolymer under neutral conditions, which indicates that the
nucleic acid is encapsulated. The use of these methods is described
further in Giacomelli et al; Biomacromolecules 2006, 7, 817-828 and
in Du et al; J. Am. Chem. Soc. 2005, 127, 17982-17983. The presence
of nucleic acid within the vesicles can also be visualised using a
fluorescent probe such as DAPI, which passes through the membrane
of the vesicles and detects nucleic acid in the core.
[0037] Other molecules, such as fluorescent dyes may also be
associated with the vesicles. Preferably, they too are encapsulated
in the core. If the dye bonds to the nucleic acid, the dye may be
used as a reporter molecule to localise the nucleic acid once
encapsulated.
[0038] A suitable label for use in the present invention is any
label which fluoresces when excited with electromagnetic radiation,
and can be associated with the self-assembled structures.
Typically, the fluorescent label is encapsulated within the aqueous
core of vesicles. However, when the fluorescent label is
hydrophobic, more typically it is associated with the hydrophobic
membrane. Fluorescent dyes, such as rhodamine, fluorescein,
BODIPY.RTM. and NBD are particularly suitable.
[0039] In one embodiment of the invention, the hydrophobic block
has a degree of polymerisation of at least 50, more preferably at
least 70. Preferably, the degree of polymerisation of the
hydrophobic block is no more than 250, even more preferably, no
more than 200. Typically, the degree of polymerisation of the
hydrophilic block is at least 15, more preferably at least 20. It
is preferred that the ratio of the degree of polymerisation of the
hydrophilic to hydrophobic block is in the range 1:2.5 to 1:8. All
of these limitations promote vesicle, rather than micelle
formation.
[0040] The hydrophobic and the hydrophilic block should be selected
with regard to the pK.sub.a requirement for the pendant blocks.
[0041] In the invention, although the hydrophilic block may be
based on condensation polymers, such as polyesters, polyamides,
polyanhydrides, polyurethanes, polyethers (including polyalkylene
glycols), polyimines, polypeptides, polyureas, polyacetals or
polysaccharides, preferably the hydrophilic block is based on a
radical polymerised addition polymer of ethylenically unsaturated
monomers. Generally the monomers from which the block is formed
themselves have zwitterionic pendant groups which remain unchanged
in the polymerisation process. It may alternatively be possible to
derivatise a functional pendant group of a monomer to render it
zwitterionic after polymerisation.
[0042] Preferably, the hydrophilic block is formed from
ethylenically-unsaturated zwitterionic monomers. Suitable
ethylenically unsaturated zwitterionic monomers have the general
formula
Y B X I
[0043] In which Y is an ethylenically unsaturated group selected
from H.sub.2C.dbd.CR--CO-A-,
H.sub.2C.dbd.CR--C.sub.6H.sub.4-A.sup.1-,
H.sub.2C.dbd.CR--CH.sub.2A.sup.2, R.sup.2O--CO--CR.dbd.CR--CO--O,
RCH.dbd.CH--CO--O--, RCH.dbd.C(COOR.sup.2)CH.sub.2--CO--O,
##STR00001##
[0044] A is --O-- or NR.sup.1;
[0045] A.sup.1 is selected from a bond, (CH.sub.2).sub.lA.sup.2 and
(CH.sub.2).sub.lSO.sub.3.sup.- in which l is 1 to 12;
[0046] A.sup.2 is selected from a bond, --O--, O--CO--, CO--O,
CO--NR.sup.1--, --NR.sup.1--CO, O--CO--NR.sup.1--,
NR.sup.1--CO--O--;
[0047] R is hydrogen or C.sub.1-4 alkyl;
[0048] R.sup.1 is hydrogen, C.sub.1-4-alkyl or BX;
[0049] R.sup.2 is hydrogen or C.sub.1-4 alkyl;
[0050] B is a bond, or a straight branched alkanediyl, alkylene
oxaalkylene, or alkylene (oligooxalkylene) group, optionally
containing one or more fluorine substituents;
[0051] X is a zwitterionic group.
[0052] Preferably X is an ammonium, phosphonium, or sulphonium
phosphate or phosphonate ester zwitterionic group, more preferably
a group of the general formula II
##STR00002##
[0053] in which the moieties A.sup.3 and A.sup.4, which are the
same or different, are --O--, --S--, --NH-- or a valence bond,
preferably --O--, and W.sup.+ is a group comprising an ammonium,
phosphonium or sulphonium cationic group and a group linking the
anionic and cationic moieties which is preferably a
C.sub.1-12-alkanediyl group,
[0054] preferably in which W.sup.+ is a group of formula
--W.sup.1--N.sup.+R.sup.3.sub.3, --W.sup.1--P.sup.+R.sup.4.sub.3,
--W.sup.1--S.sup.+R.sup.4.sub.2 or --W.sup.1-Het.sup.+ in
which:
[0055] W.sup.1 is alkanediyl of 1 or more, preferably 2-6 carbon
atoms optionally containing one or more ethylenically unsaturated
double or triple bonds, disubstituted-aryl (arylene), alkylene
arylene, arylene alkylene, or alkylene aryl alkylene,
cycloalkanediyl, alkylene cycloalkyl, cycloalkyl alkylene or
alkylene cycloalkyl alkylene, which group W.sup.1 optionally
contains one or more fluorine substituents and/or one or more
functional groups; and
[0056] either the groups R.sup.3 are the same or different and each
is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or
aryl, such as phenyl, or two of the groups R.sup.3 together with
the nitrogen atom to which they are attached form an aliphatic
heterocyclic ring containing from 5 to 7 atoms, or the three groups
R.sup.3 together with the nitrogen atom to which they are attached
as heteroaromatic ring having 5 to 7 atoms, either of which rings
may be fused with another saturated or unsaturated ring to form a
fused ring structure containing from 5 to 7 atoms in each ring, and
optionally one or more of the groups R.sup.3 is substituted by a
hydrophilic functional group, and
[0057] the groups R.sup.4 are the same or different and each is
R.sup.3 or a group OR.sup.3, where R.sup.3 is as defined above;
or
[0058] Het is an aromatic nitrogen-, phosphorus- or sulphur-,
preferably nitrogen-, containing ring, for example pyridine.
[0059] Monomers in which X is of the general formula in which
W.sup.+ is W.sup.1N.sup.+R.sup.3.sub.3 may be made as described in
our earlier specification WO-A-9301221. Phosphonium and sulphonium
analogues are described in WO-A-9520407 and WO-A-9416749.
[0060] Generally a group of the formula II has the preferred
general formula III
##STR00003##
[0061] where the groups R.sup.5 are the same or different and each
is hydrogen or C.sub.1-4 alkyl, and m is from 1 to 4, in which
preferably the groups R.sup.5 are the same preferably methyl.
[0062] In phosphobetaine based groups, X may have the general
formula IV
##STR00004##
in which A.sup.5 is a valence bond, --O--, --S-- or --NH--,
preferably --O--;
[0063] R.sup.6 is a valence bond (together with A.sup.5) or
alkanediyl, --C(O)alkylene- or --C(O)NH alkylene preferably
alkanediyl, and preferably containing from 1 to 6 carbon atoms in
the alkanediyl chain;
[0064] W.sup.2 is S, PR.sup.7 or NR.sup.7;
[0065] the or each group R.sup.7 is hydrogen or alkyl of 1 to 4
carbon atoms or the two groups R.sup.7 together with the heteroatom
to which they are attached form a heterocyclic ring of 5 to 7
atoms;
[0066] R.sup.8 is alkanediyl of 1 to 20, preferably 1 to 10, more
preferably 1 to 6 carbon atoms;
[0067] A.sup.6 is a bond, NH, S or O, preferably O; and
[0068] R.sup.9 is a hydroxyl, C.sub.1-12 alkyl, C.sub.1-12 alkoxy,
C.sub.7-18 aralkyl, C.sub.7-18-aralkoxy, C.sub.6-18 aryl or
C.sub.6-18 aryloxy group.
[0069] Monomers comprising a group of the general formula IV may be
made by methods as described in JP-B-03-031718, in which an amino
substituted monomer is reacted with a phospholane.
[0070] In compounds comprising a group of the general formula IV,
it is preferred that
[0071] A.sup.5 is a bond;
[0072] R.sup.6 is a C.sub.2-6 alkanediyl;
[0073] W.sup.2 is NR.sup.7:
[0074] each R.sup.7 is C.sub.1-4 alkyl;
[0075] R.sup.8 is C.sub.2-6 alkanediyl;
[0076] A.sup.6 is O; and
[0077] R.sup.9 is C.sub.1-4 alkoxy.
[0078] Alternatively X may be a zwitterion in which the anion
comprises a sulphate, sulphonate or carboxylate group.
[0079] One example of such a group is a sulphobetaine group, of the
general formula V
##STR00005##
[0080] where the groups R.sup.10 are the same or different and each
is hydrogen or C.sub.1-4 alkyl and s is from 2 to 4.
[0081] Preferably the groups R.sup.10 are the same. It is also
preferable that at least one of the groups R.sup.10 is methyl, and
more preferable that the groups R.sup.36 are both methyl.
[0082] Preferably s is 2 or 3, more preferably 3.
[0083] Another example of a zwitterionic group having a carboxylate
group is an amino acid moiety in which the alpha carbon atom (to
which an amine group and the carboxylic acid group are attached) is
joined through a linker group to the backbone of the biocompatible
polymer. Such groups may be represented by the general formula
VI
##STR00006##
[0084] in which A.sup.7 is a valence bond, --O--, --S-- or --NH--,
preferably --O--,
[0085] R.sup.11 is a valence bond (optionally together with
A.sup.7) or alkanediyl, --C(O)alkylene- or --C(O)NHalkylene,
preferably alkanediyl and preferably containing from 1 to 6 carbon
atoms; and
[0086] the groups R.sup.12 are the same or different and each is
hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or two
or three of the groups R.sup.12, together with the nitrogen to
which they are attached, form a heterocyclic ring of from 5 to 7
atoms, or the three group R.sup.12 together with the nitrogen atom
to which they are attached form a fused ring heterocyclic structure
containing from 5 to 7 atoms in each ring.
[0087] Another example of a zwitterion having a carboxylate group
is a carboxy betaine
--N.sup.+(R.sup.13).sub.2(CH.sub.2).sub.rCOO.sup.- in which the
R.sup.13 groups are the same or different and each is hydrogen or
R.sub.1-4 alkyl and r is 2 to 6, preferably 2 or 3.
[0088] In the zwitterionic monomer of the general formula I it is
preferred that the ethylenic unsaturated group Y is
H.sub.2C.dbd.CR--CO-A-. Such acrylic moieties are preferably
methacrylic, that is in which R is methyl, or acrylic, in which R
is hydrogen. Whilst the compounds may be (meth)acrylamido compounds
(in which A is NR.sup.1), in which case R.sup.1 is preferably
hydrogen, or less preferably, methyl, most preferably the compounds
are esters, that is in which A is O.
[0089] In monomers of the general formula I, especially where Y is
the preferred (alk)acrylic group, B is most preferably an
alkanediyl group. Whilst some of the hydrogen atoms of such group
may be substituted by fluorine atoms, preferably B is an
unsubstituted alkanediyl group, most preferably a straight chain
group having 2 to 6 carbon atoms.
[0090] A particularly preferred zwitterionic monomer is
2-methacryloyloxyethyl-phosphorylcholine (MPC). Mixtures of
zwitterionic monomers each having the above general formula may be
used.
[0091] The hydrophobic block may be formed of condensation
polymers, such as polyethers (including polyalkylene glycols),
polyesters, polyamides, polyanhydrides, polyurethanes, polyimines,
polypeptides, polyureas, polyacetals, or polysiloxanes. One example
of a suitable hydrophobic block is polyalkylene oxide, usually
polypropylene oxide, that is the same type of block as has been
used in the well-studied Pluronic/Poloxamer based systems. One type
of highly hydrophobic block is poly(dimethylsiloxane). In one
preferred embodiment the type of polymer forming the hydrophobic
block is the same as that forming the hydrophilic block. Preferably
the polymer is formed by radical polymerisation of ethylenically
unsaturated monomers.
[0092] Suitable monomers from which the hydrophobic block may be
formed have the general formula VII
Y.sup.1B.sup.1Q VII
[0093] in which Y.sup.1 is selected from
H.sub.2C.dbd.CR.sup.14--CO-A.sup.8-,
H.sub.2C.dbd.CR.sup.14--C.sub.6H.sub.4-A.sup.9-,
H.sub.2C.dbd.CR.sup.14--CH.sub.2A.sup.10,
R.sup.16O--CO--CR.sup.14.dbd.CR.sup.14--CO--O,
R.sup.14--CH.dbd.CH--CO--O--,
R.sup.14CH.dbd.C(COOR.sup.16)CH.sub.2--CO--O,
##STR00007##
A.sup.8 is --O-- or NR.sup.15;
[0094] A.sup.9 is selected from a bond, (CH.sub.2).sub.qA.sup.10
and (CH.sub.2).sub.qSO.sub.3-- in which q is 1 to 12;
[0095] A.sup.10 is selected from a bond, --O--, O--CO--, CO--O--,
CO--NR.sup.15--, --NR.sup.15--CO--, O--CO--NR.sup.15--,
NR.sup.15--CO--O--;
[0096] R.sup.14 is hydrogen or C.sub.1-4 alkyl;
[0097] R.sup.15 is hydrogen, C.sub.1-4-alkyl or B.sup.1Q;
[0098] R.sup.16 is hydrogen or C.sub.1-4 alkyl;
[0099] B.sup.1 is a bond, or a straight branched alkanediyl,
alkylene oxaalkylene, or alkylene (oligooxalkylene) group,
optionally containing one or more fluorine substituents; and
[0100] Q is a cationic or cationisable group of the formula
--NR.sup.17.sub.p, --PR.sup.17.sub.p and SR.sup.17.sub.r, in which
p is 2 or 3, r is 1 or 2, the groups R.sup.43 are the same or
different and each is selected from the group consisting of
hydrogen, C.sub.1-24 alkyl and aryl, or two of the groups R.sup.17
together with the heteroatom to which they are attached from a 5 to
7 membered heterocyclic ring or three R.sup.17 groups together with
the heteroatom to which they are attached form a 5 to 7 membered
heteroaromatic ring, either of which rings may be fused to another
5 to 7 membered saturated or unsaturated ring, and any of the
R.sup.43 groups may be substituted by amino or hydroxyl groups or
halogen atoms, wherein if p is 3, at least one of the groups
R.sup.17 must be halogen.
[0101] Preferably Y.sup.1 is H.sub.2C.dbd.CR.sup.14--CO-A.sup.8-
where R.sup.14 is H or methyl and A.sup.8 is O or NH.
[0102] Preferred groups B.sup.1 are alkanediyl, usually with linear
alkyl chains and preferably having 2 to 12 carbon atoms, such as 2
or 3 carbon atoms.
[0103] Preferably Q is NR.sup.17.sub.2 where R.sup.17 is
C.sub.1-12-alkyl. Preferably both R.sup.17's are the same.
Particularly useful results have been achieved where the groups
R.sup.17 are C.sub.1-4 alkyl, especially ethyl, methyl or
isopropyl.
[0104] Either or both the hydrophobic and hydrophilic blocks may
include comonomers, for instance to provide functionality, control
over hydrophobicity, control over pH sensitivity, pK.sub.aH or
pK.sub.B as the case may be, control over temperature sensitivity
or as general diluents. For instance comonomers providing
functionality may be useful to provide conjugation of pendant
groups following polymerisation and/or vesicle formation, to
targeting moieties, or to provide for conjugation between the
biologically active molecule and the polymer. Alternatively,
functional groups may allow for crosslinking of the polymer
following micelle formation, to confer increased stability on the
micellar structure.
[0105] Examples of suitable comonomers are compounds of the general
formula VIII
##STR00008##
[0106] in which R.sup.18 is selected from hydrogen, halogen,
C.sub.1-4 alkyl and groups COOR.sup.22 in which R.sup.22 is
hydrogen and C.sub.1-4 alkyl;
[0107] R.sup.19 is selected from hydrogen, halogen and C.sub.1-4
alkyl;
[0108] R.sup.20 is selected from hydrogen, halogen, C.sub.1-4 alkyl
and groups COOR.sup.22 provided that R.sup.18 and R.sup.20 are not
both COOR.sup.22; and
[0109] R.sup.21 is a C.sub.1-10 alkyl, a C.sub.1-20 alkoxycarbonyl,
a mono- or di-(C.sub.1-20 alkyl)amino carbonyl, a C.sub.6-20 aryl
(including alkaryl) a C.sub.7-20 aralkyl, a C.sub.6-20
aryloxycarbonyl, a C.sub.1-20-aralkyloxycarbonyl, a C.sub.6-20
arylamino carbonyl, a C.sub.7-20 aralkyl-amino, a hydroxyl or a
C.sub.2-10 acyloxy group, any of which may have one or more
substituents selected from halogen atoms, alkoxy, oligo-alkoxy,
aryloxy, acyloxy, acylamino, amine (including mono and di-alkyl
amino and trialkylammonium in which the alkyl groups may be
substituted), carboxyl, sulphonyl, phosphoryl, phosphino,
(including mono- and di-alkyl phosphine and tri-alkylphosphonium),
zwitterionic, hydroxyl groups, vinyloxycarbonyl and other vinylic
or allylic substituents, and reactive silyl or silyloxy groups,
such as trialkoxysilyl groups;
[0110] or R.sup.21 and R.sup.20 or R.sup.21 and R.sup.19 may
together form --CONR.sup.23CO in which R.sup.23 is a C.sub.1-20
alkyl group.
[0111] It is preferred for at least two of the groups R.sup.18,
R.sup.19, R.sup.20 and R.sup.21 to be halogen or, more preferably,
hydrogen atoms. Preferably R.sup.18 and R.sup.19 are both hydrogen
atoms. It is particularly preferred that compound of general
formula X be a styrene-based or acrylic based compound. In styrene
based compounds R.sup.21 represents an aryl group, especially a
substituted aryl group in which the substituent is an amino alkyl
group, a carboxylate or a sulphonate group. Where the comonomer is
an acrylic type compound, R.sup.21 is an alkoxycarbonyl, an alkyl
amino carbonyl, or an aryloxy carbonyl group. Most preferably in
such compounds R.sup.21 is a C.sub.1-20-alkoxy carbonyl group,
optionally having a hydroxy substituent. Acrylic compounds are
generally methacrylic in which case R.sup.20 is methyl.
[0112] Preferably the comonomer is a non-ionic comonomer, such as a
C.sub.1-24 alkyl(alk)-acrylate or -acrylamide, mono- or
di-hydroxy-C.sub.1-6-alkyl(alk)-acrylate, or acrylamide,
oligo(C.sub.2-3 alkoxy) C.sub.2-18-alkyl(alk)-acrylate, or
-acrylamide, styrene, vinylacetate or N-vinyllactam.
[0113] For optimum nanovesicle formation, the block copolymers
should have controlled molecular weights. It is preferable for each
of the blocks to have molecular weight controlled within a narrow
band, that is, to have a narrow polydispersity. The polydispersity
of molecular weight should, for instance, be less than 2.0, more
preferably less than 1.5, for instance in the range 1.1 to 1.4.
[0114] In one embodiment of this invention, the monomer from which
the hydrophobic block is formed 2-(diisopropylamino)ethyl
methacrylate (DPA) or 2-(diethylamino)ethyl methacrylate (DEA). The
hydrophobic block is generally not formed from DMA monomers. In
another embodiment, the hydrophilic block is PMPC. Preferably, the
copolymer is a PMPC-b-PDPA block copolymer.
[0115] Preferably, the block copolymer has general formula
PMPC.sub.m-b-PDPA.sub.n, wherein m is in the range 15-30 (for
instance, 25) and n is 50 to 180, preferably 100 to 160, more
preferably 120 to 160.
[0116] The block copolymer may be a simple A-B block copolymer, or
may be an A-B-A or B-A-B block copolymer (where A is the
hydrophilic block and B is the hydrophobic block). It may also be
an A-B-C, A-C-B or B-A-C block copolymer, where C is a different
type of block. C blocks may, for instance, comprise functional,
e.g. cross-linking or ionic groups, to allow for reactions of the
copolymer, for instance in the novel compositions. Crosslinking
reactions especially of A-C-B type copolymers, may confer useful
stability on nanovesicles. Cross-linking may be covalent, or
sometimes, electrostatic in nature. Cross-linking may involve
addition of a separate reagent to link functional groups, such as
using a difunctional alkylating agent to link two amino groups. The
block copolymer may alternatively be a star type molecule with
hydrophilic or hydrophobic core, or may be a comb polymer having a
hydrophilic backbone (block) and hydrophobic pendant blocks or vice
versa. Such polymers may be formed for instance by the random
copolymerisation of monounsaturated macromers and monomers.
[0117] The details of the process for polymerising the monomers
which are used in this invention are to be found on WO 03/074090,
pages 15-24.
[0118] The living radical polymerisation process useful in this
invention has been found to provide polymers of zwitterionic
monomers having a polydispersity (of molecular weight) of less than
1.5, as judged by gel permeation chromatography. Polydispersities
in the range 1.2 to 1.4 for the or each block are preferred.
[0119] An advantage of the present invention is that vesicles may
be loaded with nucleic acid using a pH change system. In such a
process, polymer is dispersed in aqueous liquid in ionised form, in
which it solubilises at relatively high concentrations without
forming vesicles. Subsequently the pH is changed such that some or
all of the ionised groups become deprotonated so that they are in
non-ionic form. At the second pH, the hydrophobicity of the block
increases and vesicles are formed spontaneously.
[0120] The method of forming vesicles with encapsulated nucleic
acid, wherein one of the blocks of the amphiphilic block copolymer
is pH-sensitive, typically involves the following steps:
[0121] (i) dispersing the amphiphilic copolymer in an aqueous
media;
[0122] (ii) acidifying the pH of the composition formed in step
(i);
[0123] (iii) adding the nucleic acid to the composition; and
[0124] (iv) raising the pH to around neutral to encapsulate the
nucleic acid.
[0125] This method preferably comprises a further, preliminary step
wherein the amphiphilic copolymer is dissolved in an organic
solvent in a reaction vessel and the solvent is then evaporated to
form a film on the inside of the reaction vessel.
[0126] By "pH-sensitive", is meant that one of the blocks has a
group which becomes protonated/deprotonated at a particular pH.
This pH is preferably in the range 3.0-6.9. Preferably, one of the
blocks, and typically the hydrophobic block comprises pendant
groups which have a pKa in the range 3.0 to 6.9, for instance, 4.0
to 6.9. Step (ii), of acidifying the composition, typically reduces
the pH to a value below the pK.sub.a of the pendant group.
[0127] In more detail, vesicles of the amphiphilic block copolymer
are typically prepared by dissolving copolymer in an organic
solvent, such as a 2:1 chloroform:methanol mix in a glass
container. Solvent is then typically evaporated under vacuum
leaving a copolymeric film deposited on the walls of the container.
The film is then re-hydrated with an aqueous solution, for instance
using phosphate buffer saline. The pH of the resultant suspension
is decreased to a pH of around 2, to solubilise the film, and then
increased to around 6. When a pH of around 6 is reached, nucleic
acid is added. The pH is then increased to around neutral to
encapsulate the nucleic acid. The dispersion may then be sonicated
and extruded, for instance using a bench top extruder with 200 nm
membranes.
[0128] Further steps in the method may be required in order to
improve the encapsulation efficiency, particularly when the nucleic
acid is RNA. This is because RNA only weakly interacts with the
polymer at pH6. The further steps may include an extrusion step
which breaks down the vesicles, and then allows them to reform in
the presence of the RNA. This may be repeated several, e.g. 2-5
times, in order to increase the encapsulation efficiency.
[0129] An alternative method for forming vesicles with encapsulated
nucleic acid involves forming empty vesicles (by the method
detailed above), or other methods known in the art. Nucleic acid is
then added to an aqueous composition of the vesicles and the
resultant composition is mixed to promote nucleic acid uptake by
the vesicles. Sonication, for instance, may promote nucleic acid
uptake.
[0130] The vesicles used in the invention may be formed from two or
more different block polymers. For instance, they may be formed
from a block copolymer comprising a polyalkylene oxide hydrophilic
block, and from a block copolymer which has a hydrophilic block
formed from a zwitterionic monomer. In this embodiment, in the
method of forming vesicles, a mixture of the two block copolymers
will be used. A suitable mixture would be, for instance, a 75:25
ratio by weight of PMPC-PDPA and PEO-PDPA.
[0131] The introduction of nucleic acid into cells is a desirable
goal clinically. siRNA has the ability to knock down almost any
gene of interest. Disease progression often depends on the activity
of multiple genes, and turning off the activity of a gene with a
siRNA lead to a potential benefit.
[0132] The application file contains at least one drawing executed
in color. Copies of this patent or patent application publication
with color drawing(s) will be provided by the Office upon request
and payment of the necessary fee.
[0133] The invention will now be illustrated by the following
Examples and Figures, wherein;
[0134] FIG. 1 is the .sup.1HNMR spectrum carried out on the diblock
copolymer formed in Example 1;
[0135] FIG. 2 is the Gel Permeation Chromatogram carried out on the
diblock copolymer formed in Example 1;
[0136] FIG. 3 shows (a) DLS particle size distribution of
PMPC.sub.25-PDPA.sub.n unimers at pH 6; (b) TEM micrograph of
PMPC-PDPA unimers (polymer particles in unassembled form) at pH 6;
(c) DLS particle size distribution of PMPC-PDPA polymersomes formed
at pH 7; (d) TEM micrograph of the PMPC-PDPA polymersomes;
[0137] FIG. 4 shows PMPC-PDPA polymersome dispersion correlation
function measured by DLS at different time points;
[0138] FIG. 5 contains Confocal Laser Scanning Micrographs that
show the successful cytosolic polymersome-mediated delivery of
fluorescently labelled (CY3) RNAi to Human Dermal Fibroblast (HDF)
cells;
[0139] FIG. 6 shows Flow Cytometry data showing the successful
cytosolic polymersome-mediated delivery of fluorescently labelled
(CY3) RNAi to HDF cells;
[0140] FIG. 7 shows HDF cells transiently transfected with PEGFP
and treated with EGFP siRNA containing polymeric vesicles;
[0141] FIG. 8 is a histogram showing the values for the green
channel of the HDF cells of FIG. 7;
[0142] FIG. 9 shows fluorescent images of HDFs post immunolabelling
of Lamin A/C after knock-down with polymersomes+anti-Lamin siRNA
(+controls);
[0143] FIG. 10 shows fluorescent images of H1299 cells 48 h post
knock-down with 30 nM polymersomes+anti-EGFP;
[0144] FIG. 11 shows fluorescent images of a pituitary cell line at
1 h with (a) polymersomes alone; (b) polymersomes+Cy3-siRNA; 24 h
with (c) polymersomes alone and (d) polymersome+Cy3-RNA;
[0145] FIG. 12 shows the fluorescent image of the 24 h incubation
of polymersomes+Cy3-siRNA;
[0146] FIG. 13 shows fluorescent images of cell nuclei (A), (D),
(G), siRNA (B), (E), (H) and merged images (C), (F), (I) for naked
siRNA (A-C), polymersomes+siRNA (D-F) and Lipofectamine (G-I);
[0147] FIG. 14 shows real time PCR of relative quantity of ACTH
produced post knock-down with polymersomes+siRNA3 vs
polymersomes+scrambled siRNA with Lipofectamine and PBS
controls;
[0148] FIG. 15 shows Northern Blot results of blood samples from 24
hr-ending mice with samples taken at 1 min, 5 min, 15 min, 30 min
and 24 hours;
[0149] FIG. 16 shows Northern Blot results of organ samples from 30
min and 24 hr ending mice, with organs sampled: Liver (Li), Kidney
(Ki), Spleen (Sp) and Lung (Lu);
[0150] FIG. 17a shows LNA blood clearance results for particle A
from 24 hr-ending mice; and
[0151] FIG. 17b shows LNA blood clearance results for particle B
from 24 hr-ending mice.
EXAMPLE 1a
Procedure for the ATRP Synthesis of the PMPC.sub.25-PDPA.sub.70
Diblock Copolymer
[0152] In a typical ATRP procedure, a Schlenk flask with a magnetic
stir bar and a rubber septum was charged with Cu(I)Br (25.6 mg,
0.178 mmol) and MPC (1.32 g, 4.46 mmol). ME-Br initiator (50.0 mg,
0.178 mmol) and bpy ligand (55.8 mg, 0.358 mmol) were dissolved in
methanol (2 mL), and this solution was deoxygenated by bubbling
N.sub.2 for 30 minutes before being injected into the flask using a
syringe. The [MPC]:[ME-Br]:[CuBr]:[bpy] relative molar ratios were
25:1:1:2 and the reaction was carried out under a nitrogen
atmosphere at 20.degree. C. After 65 minutes, a deoxygenated
mixture of DPA (2.67 g, 12.5 mmol, 70 eq.) and methanol (3 mL) were
injected into the flask. After 48 h, the reaction solution was
diluted by addition of isopropanol (about 200 mL) and then passed
through a silica column to remove the catalyst. Typically, the
yield would be 50-75% as some of the material is not easily
recovered from the silica column.
Characterisation:
[0153] .sup.1NMR was carried out at pH2 in D.sub.2O (see FIG. 1).
GPC analysis of this diblock copolymer was conducted in a 3:1
chloroform/methanol mixture in the presence of 2.5 mM LiBr using
poly(methyl methacrylate) standards (see FIG. 2). Typical results
are presented in Table 1.
TABLE-US-00001 TABLE 1 Properties of the PMPC-PDPA diblock
copolymer Copolymer composition by TEM entry .sup.1H NMR in
D.sub.2O/DCl at pH 2 M.sub.n,GPC M.sub.w/M.sub.n Morphologies 1
PMPC.sub.25-PDPA.sub.70 32,900 1.16 vesicles + micelles
EXAMPLE 1b
Synthesis of PEO-PDPA Copolymer
[0154] The procedure is based on that followed by Vamvakaki et al
in Macromolecules; 1999; 32(6) pp 2088-2090.
[0155] The monohydroxy-capped poly(ethylene oxide) (PEO) was
donated by Inspec U.K. GPC analyses gave Mw/Mn's of 1.10 for PEO;
degrees of polymerization were either 22 or 45 for PEO. In a
typical synthesis, PEO (5.0 g) dissolved in 100 mL of dry THF was
added to a round-bottomed flask under dry nitrogen. Potassium
naphthalene (2.50 mmol) in THF was added via a double-tipped
needle, and the reaction solution was stirred at 30.degree. C. for
1-2 h to form the alcoholate macro-initiator. Freshly distilled
tertiary amine methacrylate (5-15 mL) was added, and the
polymerization was allowed to proceed for 4 h prior to quenching
with methanol. In some cases the polymerizations were conducted at
35 or 50.degree. C. Solvent was removed under vacuum, the copolymer
was redissolved in dilute HCl, and the water-insoluble naphthalene
was removed by filtration. PEG.sub.113-PDPA.sub.71 and
PEG.sub.10-PDPA.sub.30 were obtained in high yields (95-100%) with
good control over copolymer molecular weight.
EXAMPLE 2
Generic Preparation of PC Polymersomes
[0156] 20 mg of polymer described in Example 1
(PMPC.sub.25-PDPA.sub.70) was dissolved in 7.5 mL of a 2:1
chloroform:methanol mixture and the vial left open in a fumehood to
evaporate completely and leave a thin film of polymer around the
bottom of the vial. The polymer was dissolved by addition of 2 mL
of PBS at pH2 to give a clear solution. The solution pH was raised
to pH6 at which point some turbidity resulted; this is the stage
when the species to be encapsulated was added to the polymersome
solution. The pH was then raised to pH7.3 to induce polymersome
closure and encapsulation of the species. The maximum concentration
of polymer that should be used is around 10 mg/mL. Otherwise the
structures that form interact with one another and discrete
polymersomes do not form.
[0157] At this stage, 1 mL polymersomes corresponds to about 8 mg
polymer. The polymersomes have a size in the order of 400 nm and
the encapsulation efficiency around 5%. To improve on this, the
solution was sonicated to induce break down and reformation of the
polymersomes. The solution was put into a syringe and passed
through a 200 nm extruder mesh which has been previously thoroughly
cleaned in ethanol, into a receiver syringe. The solution was
passed back and forth a minimum of 25 times. This improves
encapsulation efficiency to .about.20% and polymersome size is
reduced to about 200 nm. The extruded material was passed again
through the Sepharose column to remove the unencapsulated material.
The eluant was collected in fractions every 100 .mu.L and fraction
8/9 contained the polymersomes, as evident from the turbidity. The
final volume collected increased from 1.2 to 1.4 mL. The
polymersomes were then placed in the fridge and stored before
use.
Characterisation:
[0158] Transmission Electron Microscopy.
[0159] Samples were mounted on pre-coated carbon-coated copper
grids. These grids were submerged for 20 seconds into the copolymer
solution and then in uranyl formate water solution (2% w/w).
Imaging was performed on a Philips CM100 instrument operating at
100 kV equipped with a Gatan 1 k CCD Camera.
[0160] Dynamic Light Scattering (DLS).
[0161] Dynamic light scattering measurements were performed on
Brookhaven Instruments 200SM laser light scattering goniometer
using a He--Ne 125 mW 633 nm laser. PMPC.sub.25-PDPA.sub.70
polymersome dispersions were diluted, if necessary, with filtered
PBS to a concentration of 1 mg/ml and placed into glass vials.
Single scans of ten minutes exposure were performed and particle
sizes were estimated using the CONTIN multiple pass method of data
analysis at angles of 30.degree., 90.degree. and 120.degree.. For
analysis of the colloidal stability of the polymersomes with time,
correlation between the average count rate histories and
correlation functions at each angle were analyzed. FIG. 3 shows the
results from TEM and DLS analysis on the polymer solution at
different pHs.
EXAMPLE 3
Polymersome Stability on Storage
[0162] It was investigated using DLS whether storing the vesicles
under different conditions affected their long term colloidal
stability. The vesicles were stored at 5 mg/ml and 1 mg/ml, and
stored at both room temperature and 5.degree. C. for each of the
concentrations. Measurements were taken on Day 1 (within 2 hours of
the preparation of the vesicles), Day 2 (within 24 hrs of the
preparation of the vesicles), Day 3 (within 48 hrs of the
preparation of the vesicles), Day 4, Day 9 and Day 90. The graph in
FIG. 4 shows that the correlation functions measured by DLS at
different time points are almost identical indicating that the
polymersomes' particle size distribution has not changed with time.
They are therefore stable over an extended period, even at room
temperature.
EXAMPLE 4
Encapsulation of siRNA into Polymersomes
[0163] The block copolymer described in Example 1 self-assembles
into vesicles at neutral pH, and dissolves completely as unimers at
endocytic pH as described in Example 2. This transition can also be
exploited for achieving efficient encapsulation of siRNA. Indeed,
as the pH of the solution is still acidic the copolymers are
dissolved molecularly. At this stage RNA can added to the solution
and the pH is quickly raised. At neutral pH the vesicles start to
form and therefore encapsulate the RNA within their aqueous core.
Successful encapsulation can be demonstrated by a fluorescence
assay based on the ability of diamidino-2-phenylindole (DAPI) to
bind to nucleic acids. After encapsulation and GPC separation from
the original solution, the RNA loaded polymersomes were added to
primary human dermal fibroblast (HDF) cells. After 2 hours
incubation the cells were analysed by confocal laser scanning
microscopy (FIG. 5) which shows the presence of fluorescent siRNA
distributed throughout the cytoplasm of the cell. Fluorescence flow
cytometry (FIG. 6) demonstrates that the siRNA has been delivered
to a large proportion of the cell population. Both techniques
therefore confirm the efficient cytosolic delivery of Cy3 labelled
RNA.
[0164] These data suggest that PMPC-PDPA polymersomes are able to
efficiently encapsulate and deliver RNA within cell cytosols
without affecting their viability. PMPC-PDPA polymersomes are
therefore a valid alternative to lipid formulations.
EXAMPLE 5
siRNA Delivery with Polymeric Vesicles
[0165] Primary human dermal fibroblasts were transiently
transfected (using the CaPO.sub.4 method) with Plasmid Enhanced
Green Fluorescent Protein (PEGFP) (from amaxa). Transfection
efficiency was proven to be above 80% after 24 hrs. siRNA (from
amaxa) was encapsulated in polymeric vesicles (37.5 micrograms/ml
of vesicles) and added to transfected fibroblasts (1:10 dilution).
Fluorescence micrographs were taken at .lamda..sub.ex=495
nm/.lamda..sub.em=515 nm for detection of EGFP expression at 48 h
and 72 h. Optical micrographs of the same fields of views were
taken to identify the total amount of cells per field of view. An
N=1 experiment was performed with 6 samples per treatment.
[0166] FIG. 7 shows HDF cells transiently transfected with PEGFP
and treated with EGFP siRNA containing polymeric vesicles. The
siRNA treatment was performed for 24 hours and images were taken 48
and 72 h after treatment. The percentage of cells expressing EGFP
was calculated with respect to the total number of cells per field
of view in siRNA treated and untreated (EGFP control) groups. The
degree of EGFP knockdown with the siRNA containing vesicles was
calculated and normalised to the control (untreated group). EGFP
production was considerably reduced with the treatment of vesicles
containing EGFP siRNA (FIG. 8). The maximum of EGFP expression
silencing was observed after 48 h treatment. A decrease in the
expression was observed after 72 h, but this was less efficient
than at 48 h. The vesicles containing the siRNA were stable (and
effective) at 4 degrees Celsius for up to 5 days. Treatment with
the vesicles did not alter the proliferative ability of the cells
over the 72 h experimental time, as measured by nuclear counter
labelling.
EXAMPLE 6
Knock-Down of Lamin A/C Using Polymersomes
[0167] 1. siRNA Encapsulation
[0168] Lamins are intermediate filament-type proteins which form
major components of the cellular nuclear lamina. The nuclear lamina
is a matrix of protein located next to the inner nuclear membrane.
In general terms, lamin proteins are involved in important cellular
tasks such as nuclear stability, chromatin structure and gene
expression. Mammals have two main type lamins, A and B. A type
lamin A/C was used in this experiment.
[0169] Two 20 mg PMPC.sub.20-PDPA.sub.75 films were rehydrated with
4 ml PBS at pH2; filter sterilised and taken up to pH6. At this
point the following samples were made: [0170] 1) Control
polymersomes (1 mL of PMPC.sub.20-PDPA.sub.75 polymersome solution)
[0171] 2) siRNA neg control (Ambion, cat #: AM4636) 5 .mu.M (final
volume: 1 mL of PMPC.sub.20-PDPA.sub.75 polymersome solution)
[0172] 3) siRNA anti Lamin A/C (Ambion, cat #: AM 4619) 5 .mu.M
(final volume: 1 mL of PMPC.sub.20-PDPA.sub.75 polymersome
solution)
[0173] Samples were taken up to pH 7, sonicated for 15 mins at
4.degree. C. and passed through a GPC (Sepharose 4B) column under
sterile conditions.
[0174] After the column, a picogreen curve was vesicleed using the
control polymersome sample as diluent. The final concentration of
siRNA encapsulated inside was calculated from a standard curve. The
final concentration was approximately 500 nM, with an efficiency of
10%.
[0175] 2. Knock-Down Experiments
[0176] HDF (human dermal fibroblast) cells were seeded on 96 well
plates. After 24 hours, medium was replaced and cells were treated
as follows: [0177] siRNA-polymersomes (either negative control or
anti-lamin A/C): siRNA final concentrations on cells of 40, 20, 10,
5 and 1 nM [0178] Polymersomes empty (at concentrations of polymer
equivalent to those ones used above) [0179] PBS a (volumes
equivalent to the ones used above) [0180] Naked siRNA (at a
concentration of 40 nM) [0181] Lipofectamine control (negative
control and anti-lamin siRNA, at a concentration of 20 nM,
following manufacturer's instructions).
[0182] Experiments were performed in triplicate wells. Cells were
incubated with the treatments for 24 h and afterwards, the
transfectant medium was removed and cells were re-fed with new
medium. After 48 h, the medium was removed, the monolayer washed
with PBS and cells were fixed with 10% formalin for 1 h prior to
immunolabelling.
[0183] 3. Immunolabelling
[0184] Cell monolayers were washed a further three times in PBS,
prior to cell permeabilisation with 0.1% Triton X100 for 20
minutes. Subsequently, cells were washed three times with PBS.
Unreactive binding sites were blocked by adding a 5% dried milk
powder solution to each well for 1 hour. A further three washes in
PBS were performed prior to incubation with the first antibody
solution. The first antibody (mouse monoclonal Jol 3 to Lamin
A+C-Nuclear envelope marker, Abcam) was added at a concentration of
1:100 in 1% PBS-dried milk powder. Cells were incubated in the
antibody solution at 4.degree. C. for 18 hours. The cells were then
washed three more times in PBS. Afterwards, cells were incubated
for 1 hour at room temperature with rabbit polyclonal to mouse
IgG-FITC (Abcam) in 1:1000 1% PBS-milk powder. Three further washes
in PBS were performed. After this time, the fluorescent staining
was visualised using an AXON image express system (Axon
Instruments/Molecular Devices, Union City, Calif.). Briefly,
fluorescence micrographs of immunolabelled samples were taken using
epifluorescent illumination at .gamma..sub.ex 495 nm .gamma..sub.em
515 nm (for FITC visualisation).
[0185] Significant reductions in fluorescence were seen with 1-20
nM anti-Lamin polymersomes. Significant cell death occurred with
lipofectamine with both neg siRNA and anti-Lamin siRNA (FIG.
9a-i).
EXAMPLE 7
Knock-Down of EGFP Using Polymersomes
[0186] H1299 cells with stable expression of Enhanced Green
Fluorescent Protein (EGFP) plated at 25,000 or 50,000 cells/well in
12 well plates were exposed to a 30 nM or 60 nM concentration of
polymersomes containing anti-EGFP siRNA, with mismatch RNA and PBS
as controls. Some significant off-target effects were observed as a
consequence of delivering high levels of RNA and either causing
cell toxicity or hyperproliferation and over-expression. Off-target
effects are the result of the SiRNA interfering with the expression
or function of genes or proteins other than the target. Some
off-target effects can be eliminated using lower concentrations of
SiNRA. At 30 nM or 60 nM with 50,000 cells at 48 hrs, a 50% knock
down of EGFP could be observed (FIG. 10)
EXAMPLE 8
Knock-Down of POMC Gene in Pituitary Cells Using Polymersomes
[0187] In a model of Cushing's Disease, the Propiomelanocortin
hormone (POMC) gene was knocked-down using polymersomes containing
siRNA3, reducing the expression of Adenocorticotropic hormone
(ACTH) (both in pituitary cell lines and in tumour cells extracted
from patients). The pituitary cells are very sensitive to
transfection agents, lipofectamine causing >30% cell death in
cell lines and 100% cell death in cells taken from patients. In
FIG. 11, Cy3-labelled siRNA was encapsulated and delivered to the
pituitary cell lines. The fluorescent images clearly show siRNA
delivery into the cells (FIG. 11), with a very homogeneous
distribution of the labelled RNA within the cytosol (FIG. 12).
Naked siRNA is not delivered without a vector, and lipofectamine is
toxic to the cells (FIG. 13). The siRNA was shown to remain stable
for at least 8 days post encapsulation.
[0188] A knock-down experiment was performed on the pituitary cell
line with real time PCR used to determine the relative quantity of
ACTH being produced. Some off target effect was seen with
Lipofectamine with increased expression due to toxicity.
Polymersome with scrambled siRNA control produced some unexplained
knock-down but the most effective knock-down was seen using
Polymersomes+siRNA3 at 5 nM.
EXAMPLE 9
In Vivo Study of Polymersomes Containing LNA
[0189] Non-fluorescent polymersomes and fluorescent polymersomes
(covalently labelled with rhodamine) encapsulating Locked Nucleic
Acid (LNA) were evaluated separately and labelled as particles A
& B. Polymersomes were prepared as described in Example 2. The
maximum injection volume possible for administration per mouse was
200 .mu.L. Two groups of 10 mice were divided for evaluation of
particle A and particle B.
[0190] Particle A: Non-fluorescent polymersomes with LNA. 5 mice
per group. Amount of LNA per mouse 5 .mu.g/200 .mu.l.
[0191] Particle B: Rhodamine-fluorescent polymersomes. 5 mice per
group. Amount of LNA per mouse 11.2 .mu.g/160 .mu.l.
[0192] Mice 1-5 were sacrificed 30 mins after injection on the tail
vein. Mice 6-10 were sacrificed after 24 hours. Blood samples were
collected with a Pasteur pipette by retro-orbital blood collection
(no anaesthetized mice) from the orbital sinus behind the eyeballs.
Sampling of blood was performed after 1 min, 5 mins, 15 mins, 30
mins and 24 hours.
[0193] Blood samples were placed into EDTA containing eppendorf
tubes to stop coagulation. The early time points were added to 1
.mu.l 0.1M EDTA containing tubes. The end points at 30 mins and 24
hrs were collected into 2 .mu.l 0.25M EDTA containing tubes.
[0194] Organ samples (at 30 min time point and 24 h) were taken
from random distal areas (approx 1/4 of the kidney and lung, 1/5
spleen and about 1/10 of liver). Organs were kept in eppendorf
tubes containing RNAlater.TM. (Ambion) to stop RNA from degrading
(lung and liver with 600 .mu.l RNAlater.TM., spleen and kidney with
400 .mu.l RNAlater.TM.). Organ samples were kept overnight at room
temperature to permeabilise the RNAlater.TM. and then stored at
2-4.degree. C.
[0195] LNA deposition within blood and organs was analysed by
Northern blot analysis. For blood clearance analysis the intensity
of the LNA signals was measured by Quantity ONE programme
system.
[0196] The results from Northern blots showed both particles were
present in the blood stream for at least 24 hours, hence
demonstrating a long circulation time (FIGS. 16 and 17a+b).
Northern blots of the organ samples revealed a relatively
consistent delivery of RNA to liver, kidney, spleen and lungs for
both particles in all animals (FIG. 16).
* * * * *