U.S. patent application number 14/863539 was filed with the patent office on 2017-06-01 for delivery agent.
This patent application is currently assigned to Thermo Fisher Scientific Baltics UAB. The applicant listed for this patent is Arunas Lagunavicius, Lolita Zaliauskiene. Invention is credited to Arunas Lagunavicius, Lolita Zaliauskiene.
Application Number | 20170152487 14/863539 |
Document ID | / |
Family ID | 55166226 |
Filed Date | 2017-06-01 |
United States Patent
Application |
20170152487 |
Kind Code |
A9 |
Lagunavicius; Arunas ; et
al. |
June 1, 2017 |
DELIVERY AGENT
Abstract
A composition comprising a polycationic agent and a polyanionic
agent, and kits comprising the composition, is provided. In
embodiments, the polyanionic agent is a nucleic acid and the
polycationic agent is a modified polyalkyleneimine polymer.
Inventors: |
Lagunavicius; Arunas;
(Vilnius, LT) ; Zaliauskiene; Lolita; (Vilnius,
LT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lagunavicius; Arunas
Zaliauskiene; Lolita |
Vilnius
Vilnius |
|
LT
LT |
|
|
Assignee: |
Thermo Fisher Scientific Baltics
UAB
Vilnius
LT
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20160024475 A1 |
January 28, 2016 |
|
|
Family ID: |
55166226 |
Appl. No.: |
14/863539 |
Filed: |
September 24, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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|
14147028 |
Jan 3, 2014 |
9481862 |
|
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14863539 |
|
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12901840 |
Oct 11, 2010 |
8951957 |
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14147028 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/59 20170801;
C08G 73/02 20130101; C12N 5/0693 20130101; A61K 47/605
20170801 |
International
Class: |
C12N 5/09 20060101
C12N005/09; C08G 73/02 20060101 C08G073/02 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 12, 2009 |
GB |
0917792.4 |
Claims
1. A composition for delivering a peptide or protein into a cell,
which composition comprises a polycationic agent and a polyanionic
agent, wherein the polyanionic agent is a nucleic acid and the
polycationic agent is a modified polyalkyleneimine polymer having a
repeat unit of formula I or formula V: ##STR00014## where: Z1 and
Z2 each independently represent --H, alkyl or a branching chain; Y1
and Y2 each independently represent a group having formula II or
III: ##STR00015## where: X1 is selected from the group consisting
of --H, -alkyl, --OH, --SH, --CN, --NH.sub.2, --N(alkyl).sub.2,
--NH(alkyl), --CONH.sub.2, ##STR00016## --NHCONH.sub.2; X2 is
selected from the group consisting of --CH.sub.2--, --O--, --S--,
--NH--, or --N(alkyl)-; provided that at least one of X1 and X2 is
a hydrophilic group; m1, m2, n1 and n2 each independently is 0, 1,
2 or 3; and p1 and p2 each independently is 1 or 2.
2-7. (canceled)
8. A kit for delivering a peptide or protein into a cell, which kit
comprises a polycationic agent in a first container and a
polyanionic agent in a second container, wherein the polyanionic
agent is a nucleic acid and the polycationic agent is a modified
polyalkyleneimine polymer having a repeat unit of formula I or
formula V: ##STR00017## where: Z1 and Z2 each independently
represent --H, alkyl or a branching chain; Y1 and Y2 each
independently represent a group having formula II or III:
##STR00018## where: X1 is selected from the group consisting of
--H, -alkyl, --OH, --SH, --CN, --NH.sub.2, --N(alkyl).sub.2,
--NH(alkyl), --CONH.sub.2, ##STR00019## --NHCONH.sub.2; X2 is
selected from the group consisting of --CH.sub.2--, --O--, --S--,
--NH--, or --N(alkyl)-; provided that at least one of X1 and X2 is
a hydrophilic group; m1, m2, n1 and n2 each independently is 0, 1,
2 or 3; and p1 and p2 each independently is 1 or 2.
9-33. (canceled)
34. A method for delivering a peptide or protein into a target
cell, which method comprises contacting the peptide or protein with
a polycationic agent and a polyanionic agent to form a complex and
contacting the complex with the target cell so as to deliver the
peptide or protein thereto, wherein: (1) the polyanionic agent is a
nucleic acid; and (2) the polycationic agent is a modified
polyalkyleneimine polymer having a repeat unit of formula I or
formula V: ##STR00020## where: Z1 and Z2 each independently
represent --H, alkyl or a branching chain; Y1 and Y2 each
independently represent a group having formula II or III:
##STR00021## where: X1 is selected from the group consisting of
--H, -alkyl, --OH, --SH, --CN, --NH.sub.2, --N(alkyl).sub.2,
--NH(alkyl), --CONH.sub.2, ##STR00022## --NHCONH.sub.2; X2 is
selected from the group consisting of --CH.sub.2--, --O--, --S--,
--NH--, or --N(alkyl)-; provided that at least one of X1 and X2 is
a hydrophilic group; m1, m2, n1 and n2 each independently is 0, 1,
2 or 3; and p1 and p2 each independently is 1 or 2.
35-43. (canceled)
44. The method according to claim 34, wherein the peptide or
protein is contacted with the polyanionic agent prior to contacting
the polycationic agent.
45. The method according to claim 34, wherein the peptide or
protein comprises an enzyme, an antibody or an inert protein.
46. The method according to claim 34, wherein the target cell is a
suspension cell, an adherent cell, a primary cell or cultured
cell.
47-48. (canceled)
49. The composition of claim 1, wherein the nucleic acid is
DNA.
50. The kit of claim 8 where the nucleic acid is DNA.
51. The method of claim 34 where the nucleic acid is DNA.
52. A method of making a modified polyalkyleneimine polymer by a
polycondensation reaction, the method comprising reacting at least
a first monomer having two reactive groups and a second monomer
having two reactive groups, the reactive groups selected from --CI,
--Br and --I, tosyl, mesyl --NH.sub.2 and NHR, where R is an alkyl,
in a suitable solvent and at a desired temperature, resulting in
the modified cationic polyalkyleneimine polymer and having a
molecular weight in the range of from 1 to 100 kDa.
Description
RELATED APPLICATIONS
[0001] This application is a Continuation-in-Part of co-pending
U.S. application Ser. No. 14/147,028 filed Jan. 3, 2014; which is a
divisional of U.S. application Ser. No. 12/901,840 filed Oct. 11,
2010 which issued as U.S. Pat. No. 8,951,957; which claims priority
to Great Britain application Serial No. 0917792.4 filed Oct. 12,
2009, each of which is expressly incorporated by reference herein
in its entirety.
FIELD OF INVENTION
[0002] The present invention relates to a composition for
delivering a peptide or protein into a cell, a kit for delivering a
peptide or protein into a cell and a method therefor, as well as
uses of the compositions and kits.
BACKGROUND OF INVENTION
Abbreviations
[0003] P/P--peptide or protein PA--polyanionic agent
pHP--polyhydroxypropyleneimine pWP--sodium phosphotungstate
tribasic hydrate (Na.sub.3O.sub.40PW.sub.12.xH.sub.2O); pW--sodium
polytungstate (Na.sub.6O.sub.39W.sub.12.H.sub.2O); pMoP--sodium
phosphomolybdate hydrate (Mo.sub.12Na.sub.3O.sub.40P.xH.sub.2O);
pMo--ammonium molybdate tetrahydrate
(H.sub.24Mo.sub.7N.sub.6O.sub.24.4H.sub.2O); pP--sodium
polyphosphate (NaPO.sub.3).sub.12-13xNa.sub.2O
[0004] Efficient foreign protein delivery into living cells can
completely bypass transcription-translation processes related to
gene expression, reducing the time required for target protein
synthesis from days to hours.
[0005] The ideal peptide/protein (P/P) delivery method/agent into
eukaryotic cells should meet three major criteria: i) it should
efficiently bring P/P into the cells; ii) it should protect P/P
against denaturation and degradation; iii) the method itself should
be non-toxic to the host cells.
[0006] In general, P/P delivery or transfection techniques include
physical delivery methods, such as microinjection, electroporation,
and methods based on chemical transfection agents of different
origin that facilitate protein delivery into the cell. A general
characteristic of a protein delivery agent is its ability to form
positively charged complexes with target P/P, which are capable of
attaching themselves to negatively charged cell surfaces, crossing
through the plasma membrane and delivering the captured protein
into the cell. Usually the transfection agent stabilizes the
protein, protects it from degradation and preserves its natural
characteristics during transfection.
[0007] The most popular commercially available current protein
delivery agents are presented in Table 1.
TABLE-US-00001 TABLE 1 Peptide/protein (P/P) delivery into cells.
Company Product Characteristics Method/agent reference Active Motif
Chariot P/P forms non-covalent U.S. Pat. No. 6,841,535 complex with
Chariot peptide GenScript Pep-1 P/P forms non-covalent U.S. Pat.
No. 6,841,535 Corp. complex with Pep-1 peptide Invitrogen Voyager
Voyager products are U.S. Pat. No. 6,184,038 bacterial and
mammalian expression systems for the production of polypeptides
fused with VP22. Krackeler TransVector TransVector is a U.S. Pat.
No. 6,080,724 Scientific bacterial expression Inc./ system for the
production Qbiogene of purified polypeptides fused with Penetratin.
Krackeler Penetratin 1 peptide Penetratin 1 peptide is U.S. Pat.
No. 6,080,724 Scientific activated peptide for Inc./ chemical
conjugation Qbiogene with target P/P. Panomics DeliverX P/P forms
non-covalent U.S. Pat. No. 6,841,535 nanoparticles with virus-
derived amphipathic peptides (MPG peptide technology) Imgenex
ProVectin Encapsulation of P/P in liposomes or association of P/P
with liposomal membrane. Genlantis/ BioPorter P/P forms
non-covalent WO03095641A1 Gene Therapy complexes with Systems
cationic lipids mixture. Pierce Pro-Ject P/P forms non-covalent
complexes with cationic lipids mixture. Stratagene BioTrek P/P
forms non-covalent complexes with lipid formulated reagent.
Synvolux SAINT PhD P/P forms non-covalent EP0755924B1 Therapeutics
complexes with cationic pyridinium amphiphile and a helper lipid.
Targeting ProFect-P1, P/P forms non-covalent Systems ProFect-P2
complexes with: i) lipid reagent (ProFect-P1); ii) with non-lipid
reagent (ProFect-P2). NEB TransPass P P/P forms non-covalent
complexes with non-lipid polimer. Novagen ProteoJuice P/P forms
non-covalent complexes with reagent, which differs substantially
from protein delivery systems based on lipids or protein
transduction domains. Polyplus PULsin P/P forms non-covalent
transfections complexes with cationic amphiphile molecule.
[0008] Based on the origin of material, P/P transfection agents can
be classified into three major groups: i) cationic peptides; ii)
cationic lipids; iii) other cationic reagents/polymers (e.g.
polyamines). As an alternative, transfection agents can be
covalently bound to target peptide/protein: by i) genetic fusion
(e.g. cationic peptides); ii) by chemical conjugation. There are
many known peptide/protein delivery methods that have been
successfully commercialized, however, all of them have certain
limitations in their application or efficacy regarding the type of
target protein, its pI or molecular weight or the types of cells
used for transfection. Therefore, there is a need in the market for
efficient, universal and robust protein delivery agents.
SUMMARY OF THE INVENTION
[0009] In a first aspect, the present invention provides a
composition for delivering a peptide or protein into a cell, which
composition comprises a polycationic agent and a polyanionic agent,
wherein the polyanionic agent comprises an inorganic polyphosphate
or a polyoxometalate.
[0010] There is further provided a kit for delivering a peptide or
protein into a cell, which kit comprises a polycationic agent in a
first container and a polyanionic agent in a second container,
wherein the polyanionic agent comprises an inorganic polyphosphate
or a polyoxometalate.
[0011] In a further aspect, the present invention provides use of a
composition for delivering a peptide or protein into a target cell,
which composition comprises a polycationic agent and a polyanionic
agent, wherein: [0012] (1) the polyanionic agent comprises a
polyphosphate or a polyoxometalate; and/or [0013] (2) the
polycationic agent comprises a polyalkyleneimine.
[0014] There is further provided use of a kit for delivering a
peptide or protein into a target cell, which kit comprises a
polycationic agent in a first container and a polyanionic agent in
a second container, wherein; [0015] (1) the polyanionic agent
comprises a polyphosphate or a polyoxometalate; and/or [0016] (2)
the polycationic agent comprises a polyalkyleneimine.
[0017] It has surprisingly been found that a composition comprising
a polycationic agent and a polyanionic agent in which the
polyanionic agent comprises a polyphosphate or a polyoxometalate
has improved properties for delivering a peptide or protein into a
cell. Peptide or protein delivery according to the invention is
characterised by a more efficient, uniform and robust performance
over a wide range of peptides and proteins of interest, including
those of different pI and those of different molecular mass. When
compared with prior art methods, it was found that transfection
efficiency according to the invention was increased.
[0018] Without wishing to be bound by theory, it is thought that
positively charged cationic agents alone are capable of interacting
efficiently with negatively charged peptides or proteins. They are
thought to be condensed into nano-sized complexes termed polyplexes
which deliver encased peptide or protein material into the cell.
Whilst such transfection reagents as in the prior art may be
capable of delivering negatively charged biomolecules such as
nucleic acids and proteins, different proteins have neutral,
negative or positive charges and therefore are not thought to
interact with the cationic agents with the same efficiency.
According to the invention, by adding a polyanionic agent, its
interaction with the peptide or protein of interest may assist in
overcoming this problem and ensure that the polyanionic agent
coated protein will have a negative charge. This enables it to
interact efficiently with the polycationic agent and subsequently
be transported through the cell membrane. This is thought to give
rise to delivery agents which are more efficient, and which offer a
more uniform and robust performance over a wide range of peptides
or proteins of interest rather than just some.
[0019] Typically, the polyanionic and polycationic agents of the
invention interact with each other ionically. They may also
interact ionically with the peptide or protein of interest. This is
an advantageous property because there is no need, for example, for
the polyanionic agent to have a functional group for covalent
coupling to the peptide or protein. Likewise, there is no need for
the polycationic agent to have such a functional group.
Accordingly, the composition does not need to incorporate an agent
for covalent coupling to the peptide or protein. Advantageously, no
agent for covalent coupling is present in the composition.
[0020] The polycationic agent of the invention may be lipidic, for
example comprising a plurality of cationic lipids which may be
organised in a supramolecular structure such as a liposome. A
polycationic agent which comprises a cationic polymer is preferred.
Such cationic polymers may be polylysine or polyarginine,
Particularly suitable cationic polymers include polyalkyleneimine
polymers such as polyethyleneimines (such as described in U.S. Pat.
No. 6,013,240) or polyhydroxyalkyleneimines. Cationic
polyalkyleneimine polymers having a repeat unit comprising a
hydrophilic group are described herein in which the hydrophilic
group is in the polymer backbone or pendant from the polymer
backbone and wherein the hydrophilic group preferably comprises a
heteroatom. Polyhydroxyalkyleneimine polymers are preferred, such
as poly(2-hydroxy)propyleneimine.
[0021] A cationic polyalkyleneimine polymer having a repeat unit
comprising a hydrophilic group is provided. In one embodiment, the
hydrophilic group is in the polymer backbone. In one embodiment,
the hydrophilic group is pendant from the polymer backbone. In one
embodiment, preferably, there are as many imine groups as
hydrophilic groups. (i.e. a 1:1 ratio). In another embodiment,
preferably the ratio of imine groups:hydrophilic groups is 2:1.
Typically, the hydrophilic group, referred to elsewhere herein as
X1 or X2, comprises a heteroatom. The cationic polyalkyleneimine
polymer according to the invention may be linear or branched.
According to the present invention, the term "branched" means
branching of the polyalkyleneimine polymer per se. "Branched
polyalkyleneimine polymer" does not mean a polyalkyleneimine
polymer having a second, different polymer grafted thereto.
[0022] Preferably, in the polymer having a repeat unit comprising a
hydrophilic group, said repeat unit has a structure as shown in
formula V, more preferably formula I:
##STR00001##
where: Y1 and Y2 each independently represent a group having
formula II or III; Z1 and Z2 each independently represent --H,
alkyl or a branching chain; X1 represents --H, -alkyl, or a
hydrophilic group, X2 represents --CH2-, --CH(alkyl), --C(alkyl)2-
or hydrophilic group, provided that at least one of X1 and X2 is a
hydrophilic group; m1, m2, n1 and n2 each independently is 0, 1, 2
or 3; and p1 and p2 each independently is 1 or 2.
[0023] When Z1 and/or Z2 are branching chains it will be understood
that Z1 and/or Z2 each represent another cationic alkyleneimine
polymer chain having a repeat unit comprising a hydrophilic group.
In this way, the cationic alkyleneimine polymer will be branched.
Preferred alkyl Z1 and Z2 groups include
--CH3, and --C2H5. Other repeat units may be present in the
polymer.
[0024] When Z1=Z2 and Y1=Y2, the cationic alkyleneimine polymer can
be a homopolymer of a repeat unit having a structure as shown in
formula IV:
##STR00002##
where Z=Z1=Z2 and Y=Y1=Y2. The polymer could be further defined as
a homopolymer of AA (formula VII) type or BB (formula VIII)
type.
[0025] When Z1.noteq.Z2 and/or Y1.noteq.Y2, the cationic
alkyleneimine polymer can be a copolymer of a first repeat unit
having a structure as shown in formula V and a second, different
repeat unit having a structure as shown in formula VI:
##STR00003##
[0026] Such polymers could be defined as a copolymer of AA*
(formula IX) and BB* (formula X) types. The cationic alkyleneimine
polymer may be a regular alternating AB copolymer (formula XI),
though a random AB copolymer may be considered also in one
embodiment.
[0027] Preferred cationic alkyleneimine polymers according to the
invention have a repeat unit selected from one of formulae VII to
XI:
##STR00004##
where Z1=Z2 and, with reference to general formula I, Y1=Y2=a group
having formula II.
##STR00005##
where Z1=Z2 and, with reference to general formula I, Y1=Y2=a group
having formula III.
##STR00006##
where Z1 is the same as or different from Z2. In other words, with
reference to general formula I, Y1.noteq.Y2 and Y1 and Y2 each
independently represent a group having general formula II.
##STR00007##
where Z1 is the same as or different from Z2. In other words, with
reference to general formula I, Y1.noteq.Y2 and Y1 and Y2 each
independently represent a group having general formula III.
##STR00008##
where Z1 is the same as or different from Z2. In other words, with
reference to general formula I, Y1.noteq.Y2 and Y1 represents a
group having formula II and Y2 represents a group having formula
III.
[0028] Throughout this specification, the use of "*" means that X1*
can be different from X1, X2* can be different from X2, m1* can be
different from m1, n1* can be different from n1, m2* can be
different from m2, and n2* can be different from n2.
[0029] X1 and X1* independently are selected from --H, alkyl, or a
hydrophilic group, provided that in formula IX at least one of X1
and X1* represents a hydrophilic group.
[0030] Preferably, X1 and X1* independently are selected from the
group consisting of --H, alkyl, --OH, --SH, --NH.sub.2,
--N(alkyl).sub.2, --NH(alkyl), --CONH.sub.2, --NHCONH.sub.2,
--CN
##STR00009##
provided that in formula IX at least one of X1 and X1* represents a
hydrophilic group (i.e. not H or alkyl in the list above).
[0031] X2 and X2* independently are selected from the group
consisting of --CH.sub.2--, --CH(alkyl)-, C(alkyl).sub.2- and a
hydrophilic group provided that in formula X at least one of X2 and
X2* represents a hydrophilic group.
[0032] Preferably, X2 and X2* independently are selected from the
group consisting of --CH.sub.2--, --O--, --S--, --NH--,
--N(alkyl)-, provided that in formula X at least one of X2 and X2*
represents a hydrophilic group (i.e. not --CH.sub.2-- in the list
above).
[0033] A preferred polymer according to the invention is poly
(2-hydroxypropyleneimine) (pHP), i.e. a homopolymer having an AA a
repeat unit of formula XII:
##STR00010##
[0034] Other preferred polymers according to the invention include
poly (2-hydroxypropyleneimine ethyleneimine) (pHPE) and poly
(2-hydroxypropyleneimine propyleneimine) (pHPP), i.e. AB copolymers
having an AB repeating unit of formula XIII or XIV:
##STR00011##
[0035] The degree of polymerisation (d) of polymers having a repeat
unit as shown in any one of general formulae I, or VII to XIV
preferably is >1, more preferably in the range from 1 to 1000,
more preferably from 30 to 500. Accordingly, a polymer according to
the invention may have the formula:
repeat unit of general formula I .sub.d
[0036] The molecular weight of the cationic polyalkyleneimine
polymer according to the first aspect preferably is in the range of
from 1 to 100 kDa, more preferably 5 to 30 kDa. Molecular weight
can be measured by size exclusion chromatography (SEC). For a
linear polymer, the molecular weight will provide a direct
indication of chain length i.e. the degree of polymerisation.
However, SEC provides scarce information on polymer branching.
[0037] A cationic polyalkyleneimine polymer of the invention may be
obtained by any suitable method.
[0038] A second aspect of the present invention provides a method
of making a modified polyalkyleneimine polymer as defined by a
polycondensation reaction.
[0039] A preferred polycondensation reaction is between first
monomers each having two reactive groups and second monomers each
having two reactive groups, said reactive groups being selected
from --Cl, --Br and --I, tosyl, mesyl --NH.sub.2 and NHR. A
preferred polycondensation reaction is between first monomers each
having two reactive groups selected from --NH.sub.2 or NH (alkyl)
and second monomers each having two halide reactive groups,
selected from --Cl, --Br, --I, tosyl and mesyl.
[0040] A -tosyl or mesyl group may be used in place of a halide
reactive group.
[0041] Random copolymers can be obtained by using more than two
different monomers in the polymer feed. For example, using three
monomers Hal-A-Hal, NH.sub.2-A-NH.sub.2, and NH.sub.2--B--NH.sub.2
in the polymer feed for the polycondensation reaction will produce
random AB type copolymers. Higher order polymers two also may be
made. For example, using 3 monomers Hal-A-Hal,
NH.sub.2--B--NH.sub.2, Hal-C-Hal in the polymer feed for the
polycondensation reaction will produce random ABC type
copolymers.
[0042] It will be appreciated that repeat units as illustrated
throughout this application may be derived from a single monomer
carrying suitable reactive groups. Similarly, repeat units as
illustrated throughout this application may be derived from two or
more monomers, each carrying suitable reactive groups.
[0043] The two reactive groups on a single monomer could be the
same or different from one another. However, for ease of processing
they will generally be the same.
[0044] In one embodiment, the halide reactive groups are both
Br.
[0045] In one embodiment, the first monomer has formula XV:
##STR00012##
where RG represents a reactive group as defined anywhere herein and
X1 represent a hydrophilic group as described anywhere herein.
[0046] Preferably, X1 represents OH.
[0047] A single monomer may have only two or more than two reactive
groups selected from the reactive groups defined above.
[0048] Preferably, the ratio of first monomer:second monomer in the
polymer feed is in the range 52:48 to 48:52, more preferably about
50:50.
[0049] The molecular weight distribution of the modified
polyalkyleneimine polymer produced according to the method of the
second aspect preferably is in the range of from 1 to 100 kDa, more
preferably 5 to 30 kDa. Molecular weight distribution can be
measured by size exclusion chromatography.
[0050] One general reaction scheme for a polycondensation reaction
is shown below:
##STR00013##
where d is the degree of polymerisation; RG represents a reactive
group selected from --Cl, Br, --I, -tosyl and -mesyl; and Z1, Y1
and Y2 are as defined anywhere herein.
[0051] The product of the method according to the second aspect may
be linear polymers, branched polymers or a mixture of linear and
branched polymers.
[0052] During the polycondensation reaction, some branching may
occur. Branching occurs by the replacement of H by another
covalently bonded chain of that polymer.
[0053] Suitable solvents in which to carry out the polycondensation
reaction will be known to a person skilled in this art and include
methanol or a mixture of methanol and water.
[0054] A suitable temperature at which to carry out the
polycondensation reaction will be known to a person skilled in this
art. A suggested temperature is about 10.degree. C. lower than the
reflux temperature of the reaction mixture, preferably about
50.degree. C.
[0055] The polymer product of the polycondensation reaction may be
purified by one or more suitable purification techniques, for
example by visking dialysis, as is known in the art.
[0056] The purified polymer product may be concentrated by one or
more suitable techniques, for example by freeze drying, as is known
in the art.
[0057] The polyanionic agent is preferably an anionic polymer,
which may be an organic or inorganic polymer. In one arrangement
the anionic polymer comprises a polyphosphate, which may be a
heterophosphate or a homophosphate. The heterophosphate may
comprise a nucleic acid in the form of an oligo or polynucleotide
such as DNA or a synthetic analogue thereof, GNA, TNA and LNA being
typical examples. It is preferred that the polycationic agent is
not lipidic, especially where a nucleic acid is used as the
polyanionic agent according to the invention.
[0058] Where the polyphosphate comprises a homophosphate, this may
be an inorganic phosphate such as sodium polyphosphate
(NaPO.sub.3).sub.x.
[0059] Alternatively, the anionic polymer may comprise a
polyoxometalate. Polyoxometalates are polyatomic ions, usually
anions, which comprise three or more transition metal oxyanions
linked together with shared oxygen atoms to form large, closed
3-dimensional frameworks. The metal atoms that make up the
frameworks are sometimes called addenda atoms and the framework may
comprise one or more different addenda atoms. These addenda atoms
are typically group 5 or group 6 transition metals and may be
present in the framework in high oxidation states. Examples of the
transition metal atoms include molybdenum and tungsten. The
framework of the polyoxometalates may optionally incorporate one or
more heteroatoms such as phosphorus. As discussed in further detail
below, ammonium molybdate tetrahydrate, sodium phosphotungstate
tribasic hydrate and sodium phosphomolybdate hydrate are all useful
in the present invention, especially sodium polytungstate.
[0060] Compositions according to the invention may be used to
deliver a wide range of peptides or proteins as discussed herein.
Such peptides or proteins include enzymes such as
.beta.-galactosidase, antibodies and inert proteins such as bovine
serum albumin. It is possible according to the invention to deliver
polypeptides of different pIs and different molecular masses, as
discussed further in the examples set out below.
[0061] The invention may be used to deliver peptides or proteins to
a variety of cells, including cells in suspension, adherent cells
and primary cells. The invention may be used to deliver to cells in
vitro or in vivo.
[0062] Kits according to the invention may be supplied with
additional containers containing suitable buffers or other reagents
for use with the polyanionic and polycationic agents according to
the invention. Instructions for use of the kit may also be supplied
therewith.
[0063] In a further aspect the present invention provides a method
for delivering a peptide or protein into a target cell, which
method comprises contacting the peptide or protein with a
polycationic agent and a polyanionic agent to form a complex and
contacting the complex with the target cell so as to deliver the
peptide or protein thereto, wherein; [0064] (1) the polyanionic
agent comprises a polyphosphate or a polyoxometalate; and/or [0065]
(2) the polycationic agent comprises a polyalkyleneimine.
[0066] According to the method of the invention, it is possible to
contact the polyanionic agent with the polycationic agent and with
the peptide or protein in any order. In a preferred embodiment, the
polyanionic agent is contacted with the peptide or protein prior to
contact with the polycationic agent. It is thought that the peptide
or protein may form a complex with the polyanionic agent and this
complex then interacts with the polycationic agent to form a
delivery complex. Complexes according to the invention are
advantageously formed by ionic interactions.
[0067] According to the invention it is possible to deliver the
peptides or proteins to cells in vitro, for example in a molecular
biology application such as in the study of the role of a protein
in the regulation of different cellular processes.
[0068] In a further aspect the compositions of the invention may be
used for therapeutic or diagnostic applications as for example in
the manufacture of a medicament. According to this aspect, for
example, a therapeutic protein or peptide may be delivered to a
subject in vivo.
[0069] Thus, there is further provided use of a composition for the
manufacture of a medicament for delivering a peptide or protein
into a cell, which composition comprises a polycationic agent and a
polyanionic agent, wherein; [0070] (1) the polyanionic agent
comprises a polyphosphate or a polyoxometalate; and/or [0071] (2)
the polycationic agent comprises a polyalkyleneimine.
[0072] In this way, the medicament acts a vehicle for delivering
the therapeutic peptide or protein and would need to be prepared to
a level of purity and stability suitable for administration to a
subject.
[0073] There is further provided a product comprising a peptide or
protein, a polycationic agent, and a polyanionic agent as a
combined preparation for use in medicine, wherein; [0074] (1) the
polyanionic agent comprises a polyphosphate or a polyoxometalate;
and/or [0075] (2) the polycationic agent comprises a
polyalkyleneimine.
[0076] In this further aspect, the combined preparation of the
invention typically comprises either a kit for delivering a
therapeutic peptide or therapeutic protein in which the peptide or
protein, polyanionic agent and polycationic agent are stored in
separate containers; or a unitary composition in which each
component is present so as to form a complex for administration to
the subject.
[0077] The invention will now be described in further detail, by
way of example only, with reference to the accompanying drawings in
which:
[0078] FIG. 1 shows a comparison between embodiments of the
invention and the prior art as requested by transfection efficiency
and mean fluorescence intensity when delivering labelled antibody
to HeLa cells;
[0079] FIG. 2 shows a comparison between embodiments of the
invention and the prior art as measured by distribution of
transduced .beta.-galactosidase in cytoplasmic and membrane protein
fractions;
[0080] FIG. 3 shows a comparison between different polyanions in
relation to the enhancement of cationic polymer mediated protein
transfection as measured by transfection efficiency and mean
fluorescence intensity;
[0081] FIG. 4 shows a comparison between different polyanions in
the enhancement of protein transfection efficiency by polycations
pHP and LPEI, as measured by transfection efficiency and mean
fluorescence intensity;
[0082] FIG. 5a shows polyanion enhancement of pHP-mediated peptide
and inert protein transfection as measured by transfection
efficiency and mean fluorescence intensity;
[0083] FIG. 5b shows the enhancement of transfection by polyanion
PWp of proteins having different pI as measured by transfection
efficiency and mean fluorescence intensity;
[0084] FIG. 6 shows polyanion enhancement of pHP-mediated protein
transfection into primary and suspension cells as measured by
transfection efficiency, toxicity and mean fluorescence intensity;
and
[0085] FIG. 7 shows the effect of component mixing on protein
transfection as measured by transfection efficiency, toxicity and
mean fluorescence intensity.
DETAILED DESCRIPTION OF THE INVENTION
[0086] Transfection efficiency of Polyhydroxypropyleneimine (pHP)
was initially tested on HeLa cells using Alexa Fluor (AF)
488-labeled antibody (goat IgG) as a control protein. Different
polyanions: DNA, sodium polyphosphate (pP) and sodium polytungstate
(pW) were tested as additives aiming to improve complex formation.
Commercial protein transfection reagents Chariot (Ambion) and
ProJect (Pierce) were used as positive controls. The transfection
efficiency was evaluated using three criteria: the percent of AF488
positive cells, the percent of dead cells (toxicity) and the mean
fluorescence intensity (MFI).
[0087] The results show that antibody cannot internalize into the
cell on its own (FIG. 1). The amount of AF488-positive cells
increased to 65% when cationic polymer pHP was used in complex with
the antibody. The percent of transfected cells was even higher (up
to 85-95%) when different polyanions (DNA, pP or pW) were added
into the mixture, suggesting that polyanions have positive effect
for protein delivery. Polyanions alone, on the other hand, have no
effect on protein entry into the cell (FIG. 3). Comparison of
obtained antibody transfection results with two commonly used
commercial protein transfection reagents--Chariot and Project,
reveals very similar transfection efficiencies (85% for Chariot and
80% for Project). However, when comparing the MFI values, the
polytungstate evidently is more effective and mediates the biggest
amount of protein (MFI .about.370) being delivered into the cell,
which is significantly higher than that shown for Chariot or
ProJect (MFI.about.100 and 150, respectively).
[0088] To determine cellular localization of transduced proteins,
the cells were transfected with .beta.-galactosidase using the same
compositions and protocols as described above. The cells were
further fractionated using ProteoJET.TM. Membrane Protein
Extraction Kit (Fermentas) in order to separate membrane and
cytosolic proteins. Enzymatic activity of .beta.-galactosidase was
estimated in both fractions (FIG. 2). Results show that majority of
.beta.-gal activity was detected in the cytosolic fraction for all
pHP and Project-mediated transfections, while very little or no
.beta.-gal activity was detected in the membrane fraction of
pHP-transfected cells, suggesting that cationic polymer (with or
without polyanions) positions transduced proteins exclusively
inside the cell. For Project, however, considerable amount of
.beta.-gal activity was found in the membrane, indicating that
equivalent amount of protein after transfection remains stuck
within or on the surface of the cellular membrane. For
Chariot-mediated transfection, significantly more
.beta.-galactosidase was found in the membrane fraction than in the
cytosol. In conclusion, pHP-polyanion mixture facilitates highly
efficient protein transduction resulting primarily in cytosolic
protein localization inside the cell.
[0089] To examine if other polyanions contribute to cationic
polymer-mediated protein transduction, we tested sodium
phosphomolybdate hydrate (pMoP), ammonium molybdate tetrahydrate
(pMo), as well as sodium phosphotungstate tribasic hydrate (pWP)
along with previously used polyanions: DNA, pP and pW (FIG. 3). The
results show that all analyzed polyanions enhance protein
transfection to a similar level of 80-95%. The MFI data, however,
singled out polytungstates (with or without hetero atoms) as the
most potent enhancers (MFI.about.1200). The polyoxometalates (POMs)
carrying hetero atom (pWP and pMoP) apparently performed slightly
worse than POMs without hetero atom (pW and pMo).
[0090] To further investigate if polyanions have positive effect in
combination with other polycations used in protein transfections, a
popular cationic polymer--LPEI was tested along with pHP in
fluorescently labelled antibody transfections. Polyanions--DNA, pP
and pW were used to assist protein packaging prior to complexation
with LPEI (FIG. 4). The results show that polyanions enhance
LPEI-mediated protein transfection as efficiently as pHP-mediated
transfection. The MFI values increase from 10 units (protein-LPEI)
to 25, 60 and 130 units upon addition of DNA, pP or pW,
respectively. The results suggest that negatively charged
polyanions may interact with positively charged regions of the
antibody and consequently facilitate protein-polyanion interaction
with the positively charged polycation.
[0091] To demonstrate that polyanions are able to enhance
transfection of any type protein, a number of proteins of different
size, pI value or function were chemically conjugated to FITC and
examined using the same conditions as those used for antibody
transfections described above. Successful delivery of 5 kDa peptide
(FIG. 5a), 12 kDa cytochrome C, 18 kDa .beta.-lactoglobulin (FIG.
5b), 66 kDa BSA, 97 kDa amyloglucosidase, as well as earlier tested
116 kDa .beta.-galactosidase (FIG. 2) and 150 kDa IgG (FIG. 1, 3,
4), confirm that polyanions enhance transfection of any size
protein carried by cationic polymer pHP. The MFI values primarily
depend on the size of the protein, i.e. the extent of FITC
labelling. Smaller proteins had lower number of FITC molecules and,
as a result, their fluorescence was weaker. The transfection of
proteins bearing different pI (amyloglucosidase--pI 3.5,
.beta.-lactoglobulin pI 5.5, cytochrome C pI 10.5) gave similar
results (FIG. 5b), all three proteins were delivered with
.about.90% efficiency. The amount of polyanion used in this case,
depended on the pI of the protein: less polyanion was needed for
transfection of amyloglucosidase (0.5 .mu.l), more for transfection
of cytochrome C (1.0 .mu.l). Overall, the results show, that
polyanion-polycation combination enhances the transfection of
proteins with (i) different size, (ii) different pI and (iii)
different function.
[0092] For the final evaluation of polyanion exerted effect on the
pHP-mediated protein transfection the experiments were carried on
different cell types: primary human lung fibroblasts (primary
cells--usually difficult to transfect), HeLa S3 (loosely adherent
cell line), and Jurkat T cell lymphoma cells (suspension cell
line--known to be very difficult to transfect by chemical methods).
The results showed that irrespective of the cell type used, the
transfection efficiencies reached 90% (FIG. 6). The fluorescence
level in strongly adherent HLF cells was the highest (MFI
.about.1500), while in semiadherent or suspension cells, HeLa S3
and Jurkat, the MFI was 350 and 500, respectively, suggesting that
the extent of macromolecule uptake depends on the cell type. Cell
size in this experiment should be taken into consideration as well,
since HLF cells are significantly bigger, can internalize more
material than HeLa S3 or Jurkat cells, and thus fluoresce more
intensively than smaller cells. In conclusion, the polyanion (here,
pWP) grouping with protein prior to complexation with polycation
(pHP) facilitates efficient protein delivery into the primary,
adherent and suspension cell types.
[0093] Evaluation of polyanion-protein-polycation complex formation
after different component mixing schedule and its influence on
transfection efficiency was carried out in order to determine the
best possible way to form protein-pHP complexes and to ensure the
most efficient cargo transport through the cellular membrane. The
results apparently were very similar, no matter how the components
were mixed together (FIG. 7), the transfection efficiencies ranged
from 80 to 95%. Slightly lower MFI values (200 units) suggested
that protein should not be the last element added into the mix, but
rather mixed with either polycation or polyanion first.
Example 1
Analysis of the Protein Transfection Using pHP
[0094] Transfection of HeLa (Human cervical carcinoma-derived cell
line) cells was carried out as follows: one day before the
transfection experiment, the cells were seeded in a 24-well tissue
culture plate at the density of 5.times.10.sup.4 cells per well in
the total volume of 1 ml DMEM culture medium supplemented with 10%
FBS. The cells were incubated at 37.degree. C. in a CO.sub.2
incubator until they reached 70-80% confluency (usually within 24
h). On the day of transfection, the growth medium was removed and
replaced with 0.5 ml of warm serum-free medium. Alexa Fluor
488-labeled goat IgG antibody (1 .mu.g) was diluted in 100 .mu.l of
0.15M NaCl solution and mixed with different amounts of polyanions:
DNA (1 .mu.g pUC18), sodium polyphosphate (10 mM pP--1 .mu.l) or
sodium polytungstate (10 Mm pW--3 .mu.l). Cationic polymer pHP (1
.mu.l) was added into the protein-polyanion mixture and vortexed
immediately for few seconds to ensure even distribution of the
material. The complexes were allowed to form for 15-20 min at room
temperature and added to the cell culture in a drop-wise manner.
The cells were further incubated for 2 h at 37.degree. C. in a
CO.sub.2 incubator. To remove unincorporated complexes, the
cultures were rinsed with PBS, and the cells were analyzed by FACS
(Fluorescence Activated Cell Sorter). Transfections using Chariot
(Ambion) and ProJect (Pierce) reagents were carried out following
manufacturer suggested protocols.
Example 2
Analysis of the Protein Localization after Polyanion-Polycation
Mediated Transfection
[0095] HeLa cell transfection with .beta.-galactosidase (1 .mu.g)
was carried out using the same protocol and conditions as described
above. The cells were further fractionated using ProteoJET.TM.
Membrane Protein Extraction Kit (Fermentas) in order to separate
membrane and cytoplasmic proteins. The enzymatic activity of
.beta.-galactosidase in both fractions was estimated using
colorimetric assay.
Example 3
Analysis of Different Polyanions in pHP-Mediated Protein
Transfections
[0096] Several different polyanions were tested for their ability
to improve labelled-IgG transfection. Polyanions were grouped as
follows: (i) phosphates--heterophosphates (DNA) and homophosphates
(sodium polyphosphate--pP), (ii) POMs--without hetero atom (sodium
polytungstate, pW, or ammonium molybdate tetrahydrate, pMo) and
with hetero atom (sodium phosphotungstate tribasic hydrate, pWP,
and sodium phosphomolybdate hydrate, pMoP). HeLa cells were
prepared for transfection essentially as described in Example 1.
The amount of each polyanion used was: 3 .mu.l of pW, pWP or pMoP,
and 2 .mu.l of pMo (each 10 mM stock concentration), 0.5 .mu.l of
pP (30 mM stock concentration), 1 .mu.g of DNA.
Polyanion-Antibody-pHP mixtures were incubated for 15 min and added
to the cells in a drop-wise manner. Transfection results were
processed 2 h later using Guava Easy Cyte Plus flow cytometry
system (Millipore).
Example 4
Analysis of Polyanions in Different Polycation-Mediated Protein
Transfection
[0097] Cationic polymer LPEI (ExGen 500) was tested in Alexa Fluor
488-labeled goat IgG transfection using polyanions--DNA, pP and pW
to assist the protein packaging prior to complexation with LPEI.
Chinese hamster ovary cells (CHOk1) were prepared for transfection
essentially the same way as HeLa cells (example 1). The cells were
cultured in RPMI medium supplemented with 10% FBS, the transfection
was carried out in serum free RPMI medium. The complexes were
formed the same way as described in example 1 for pHP, the amount
of LPEI used--3.3 .mu.l.
Example 5
Analysis of Polyanions in Transfections of Proteins of Different
Size and pI
[0098] FITC-labeled proteins--5 kDa peptide, BSA, amyloglucosidase
(pI 3.5), .beta.-lactoglobulin (pI 5.5) and cytochrome-C(pI 10.5)
were transfected into HeLa cells following the procedure described
in Example 1. The amount of pWP used: 0.5 .mu.l for
amyloglucosidase and .beta.-lactoglobulin, 1 .mu.l for cytochrome
C.
Example 6
Analysis of the Polyanion Effect on Difficult to Transfect Cell
Lines
[0099] Comparison of protein transfer efficiency using pHP and
polyanions (pWP) was tested in suspension cell lines, HeLa S3 and
Jurkat (Human T cell lymphoma cell line), as well as in primary
cells HLF (human lung fibroblasts). Suspension cells were seeded at
the density of 2.times.10.sup.5 cells/well, HLF
5.times.10.sup.4/well 24 hours before the transfection.
Antibody-pHP complexes in 0.15 M NaCl solution were prepared as
described earlier.
Example 7
Analysis of the Component Mixing Order Effect on Protein
Transfection
[0100] HeLa cells were prepared for transfection as described in
Example 1. The antibody IgG (1 .mu.g)-pWP (1 .mu.l)-pHP (1 .mu.l)
complexes were prepared in 0.15 M NaCl following different
component mixing order: IgG+PA+pHP, PA+IgG+pHP, IgG+pHP+PA,
pHP+IgG+PA PA+pHP+IgG, and pHP+PA+IgG. Complexes were allowed to
form for 15 min and added to the cells in a drop-wise manner.
* * * * *