U.S. patent application number 10/767329 was filed with the patent office on 2004-08-05 for protein and peptide delivery to mammalian cells in vitro.
Invention is credited to Budker, Vladimir G., Ekena, Kirk, Monahan, Sean D., Nader, Lisa.
Application Number | 20040151766 10/767329 |
Document ID | / |
Family ID | 32776182 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040151766 |
Kind Code |
A1 |
Monahan, Sean D. ; et
al. |
August 5, 2004 |
Protein and peptide delivery to mammalian cells in vitro
Abstract
Compositions and methods for delivery of proteins and peptides
to mammalian cells in vitro are described. Specifically,
polypeptide-surfactant complexes formed from noncovalent
hydrophobation of polypeptides and reversible hydrophobic
modification of polypeptides are described. The compositions can be
used to delivery positively charged, negatively charged and charge
neutral polypeptides to cells.
Inventors: |
Monahan, Sean D.; (Madison,
WI) ; Budker, Vladimir G.; (Middleton, WI) ;
Ekena, Kirk; (Middleton, WI) ; Nader, Lisa;
(Madison, WI) |
Correspondence
Address: |
Mark K. Johnson
Mirus
505 South Rosa Road
Madison
WI
53719
US
|
Family ID: |
32776182 |
Appl. No.: |
10/767329 |
Filed: |
January 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60443645 |
Jan 30, 2003 |
|
|
|
Current U.S.
Class: |
424/450 |
Current CPC
Class: |
A61K 47/24 20130101;
A61K 47/543 20170801; A61K 47/51 20170801; A61K 47/6949 20170801;
A61K 9/127 20130101; B82Y 5/00 20130101; A61K 47/22 20130101 |
Class at
Publication: |
424/450 |
International
Class: |
A61K 009/127 |
Claims
We claim:
1. A composition for intracellular delivery of a polypeptide,
comprising: a dried polypeptide-surfactant complex wherein the
surfactant is associated with the polypeptide via a noncovalent
bond.
2. The composition of claim 1 wherein the surfactant contains a
hydrophobic alkyl chain of 4 to 30 carbon atoms.
3. The composition of claim 2 wherein the surfactant additionally
contains a functional group selected from the list consisting of:
membrane active compounds, cell penetrating compounds, cell
targeting signals, interaction modifiers, steric stabilizers.
4. The composition of claim 1 wherein the complex is associated
with one or more lipids.
5. The composition of claim 4 wherein the composition consists of a
liposome.
6. The composition of claim 4 wherein the one or more of the lipids
contains a functional group selected from the list consisting of:
membrane active compounds, cell penetrating compounds, cell
targeting signals, interaction modifiers, steric stabilizers.
7. The composition of claim 2 wherein the complex is dissolved in
an organic or organic/aqueous solvent.
8. The composition of claim 7 wherein the dissolved complex is
added to one or more lipids.
9. A composition for intracellular delivery of a polypeptide,
comprising: a polypeptide-surfactant complex wherein the surfactant
is associated with the polypeptide via a covalent bond.
10. The composition of claim 9 wherein the surfactant contains a
functional group selected from the list consisting of: membrane
active compounds, cell penetrating compounds, cell targeting
signals, interaction modifiers, steric stabilizers.
11. The composition of claim 9 wherein the complex is
dehydrated.
12. The composition of claim 9 wherein the complex is associated
with one or more lipids.
13. The composition of claim 12 wherein the lipids form a
liposome.
14. The composition of claim 12 wherein the complex additionally
contains a functional group selected from the list consisting of:
membrane active compounds, cell penetrating compounds, cell
targeting signals, interaction modifiers, steric stabilizers.
15. The composition of claim 9 wherein the surfactant consists of
an alkyl chlorosilane.
16. The composition of claim 15 wherein the silane is selected from
the group consisting of compounds of general formula: 7wherein
R.sub.1, R.sub.2, and R.sub.3 are independent and are selected from
the group consisting of halogen, alkyl, and aryl.
17. The composition of claim 9 wherein the surfactant consists of a
surfactant-chelator.
18. The composition of claim 17 wherein the surfactant-chelator is
selected from the list consisting of: molecules of general formula
I 8and molecules of general formula II 9wherein R is an alkyl
group.
19. The composition of claim 9 wherein the surfactant consists of
an amphipathic maleic anhydride derivative.
20. A process for the reversible hydrophobic modification of a
polypeptide, comprising: forming a polypeptide-surfactant complex
wherein the surfactant is selected from the list consisting alkyl
chlorosilane, surfactant-chelator and amphipathic maleic
anhydride.
21. A process for delivering a polypeptide to a cell comprising: a)
associating a polypeptide with a surfactant via noncovalent
interaction to form a polypeptide-surfactant complex; b)
dehydrating the complex to form a polypeptide-surfactant dried salt
complex; c) dissolving the dried salt complex with an organic or
organic/aqueous solvent; and, d) contacting the cell with the
dissolved complex of step c).
22. The process of claim 21 wherein the one or more lipids are
added to the dissolved complex prior to contacting the cell with
the complex.
23. The process of claim 22 wherein the dissolved complex consists
of a liposome.
24. The process of claim 22 wherein the dissolved complex is dried
and rehydrated in aqueous solvent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of prior provisional
application 60/443,645 filed on Jan. 30, 2003.
FIELD OF INVENTION
[0002] The present invention relates to methods and formulations
for the delivery of peptides and proteins to cells.
BACKGROUND OF THE INVENTION
[0003] Recent advances in the areas of medicine and biotechnology
have led to the increased isolation and development of biologically
active and therapeutically or diagnostically useful peptides and
proteins. However, peptides and proteins generally have limited
stability (half-life) physiologically, and are rapidly degraded.
Additionally, peptides and proteins can have difficulty in
efficiently interacting with or crossing cell membranes. Peptides
and proteins are generally comprised of both charged and uncharged
amino acid residues that impart structure and solubility to the
peptide or protein. However, this charge can hinder membrane
binding and membrane transport in cells. Therefore the development
of methods for delivering peptides and proteins to cells is a
current and continuing need in the areas of research and
therapeutics.
[0004] Hydrophobic modification of compounds is known to increase
binding of the compound to the cellular membranes. Recently,
several examples of hydrophobic modification of peptides and
proteins have been described in the literature (Storch et al. 1996;
Kamyshny et al. 1997; Wang et al. 1997; Wang et al. 1999; Schreier
et al. 2000; Wang et al. 2000; Wang et al. 2002; Wang et al. 2003).
Hydrophobic modification entails the covalent attachment of one or
more hydrophobic substituents to the polypeptide, usually through a
nitrogen atom on the polypeptide. This modification can be
accomplished through the use of chemically stable or chemically
labile groups. If the attachment group is chemically labile, the
bond can cleave at a certain rate under physiological conditions in
order to allow the hydrophobic substituents to separate from the
polypeptide. Following delivery of the hydrophobized polypeptide,
several internalization pathways are possible. For example the
complex can bind to the cellular membrane and either be
endocytosed, directly transverse the membrane, or be trapped in or
on the membrane. The complex can also be endocytosed and then cross
internal membranes. In addition to the previously mentioned
possibilities, if the hydrophobic group is labile, the hydrophobic
group may cleave from the polypeptide in or near the cell. The
polypeptide can then enter the cell via the mechanisms that are
possible for the internalization of an unmodified polypeptide.
[0005] Several compound classes have been shown to be effective
linkers for the hydrophobation, including simple acylation.
Acylation with fatty acids or a variety of cyclic anhydrides are
examples. Cyclic anhydrides have been shown a great deal of
interest due to the lability of the linkage following the
modification of the polypeptide. For example, maleic anhydrides
have been previously utilized for reversible amine modification.
The resulting maleamic acids are known to be stable under basic
conditions, but hydrolyze rapidly under acidic conditions.
Hydrophobic modification has even shown promise for the oral
delivery of protein complexes. For example, Wang et al. has
described the oral delivery of salmon calcitonin through the use of
a reversible hydrophobation of the protein (Wang et al. 2003).
[0006] In addition to hydrophobation, several additional methods
have been developed for the delivery of polypeptides to cells.
Liposomal and micellar delivery, polymer conjugates, and systems
involving combinations of polymers and lipids have all been used
for the delivery of polypeptides to cells (Trubetskoy et al. 1993;
Bijsterbosch et al. 1994; Rao et al. 1997; Yoshikawa et al. 1997;
Capan et al. 1999; Schibli et al. 1999a; Schibli et al. 1999b;
Montserret et al. 2000; Rao et al. 2000; Betz et al. 2001; Futaki
et al. 2001; Gupta et al. 2001; Kisel et al. 2001; Nagy et al.
2001; Wang et al. 2001; Zelphati et al. 2001; Caliceti, et al.
2003; Copland et al. 2003; Mahato et al. 2003; Tiyaboonchai et al.
2003; Yang et al. 2003). Currently, there are several kits
available from a variety of manufactures for the delivery of
polypeptides to cell in vitro based on cationic lipids. However,
the liposomal delivery of polypeptides has not been shown to be
general. Since charge is the controlling factor in the binding of
the peptide with the lipid, widely variable results would be
expected based on the charge of the peptide. In the molecular
conjugate area, several examples of increased serum half lives have
been demonstrated following conjugations with a polymer. In all of
these areas, there is a great deal of examples in which the
lipid/liposome, micelle, and polymer conjugates are targeted to a
cell utilizing some type of polypeptide acting as a targeting
ligand.
SUMMARY OF THE INVENTION
[0007] In a preferred embodiment, we describe methods for delivery
of proteins (positively charged, negatively charged and charge
neutral) and peptides (negatively charged, positively charged and
charge neutral) into mammalian cells in vitro. The techniques
involve noncovalent hydrophobation and reversible hydrophobic
modification.
[0008] The present invention relates to new methods and
formulations for the delivery of polypeptides to cells. In a
preferred embodiment, the present invention encompasses the
noncovalent interaction of a charged polypeptide with a surfactant
of opposite charge to form a polypeptide-surfactant mixture. The
polypeptide-surfactant mixture is then dried to form a dried salt
complex. Forming a dried salt complex comprises: lyophilizing the
polypeptide-surfactant mixture. The dried salt complex is then
dissolved in an appropriate organic solvent or in an
organic/aqueous solvent mixture. The resultant complexes are then
contacted with the cell for delivery of the polypeptide to a
cell.
[0009] In a preferred embodiment of the invention, the dried salt
complex is dissolved in an appropriate organic solvent or in an
organic/aqueous solvent mixture, and mixed with lipids or
liposomes. In this embodiment, the lipid(s) and liposomes can
posses additional functionality, for example, membrane active
compounds, cell penetrating compounds, cell targeting signals,
interaction modifiers, steric stabilizers. Additionally, the
lipid(s) and liposomes can posses reactive groups to which membrane
active compounds, cell penetrating compounds, cell targeting
signals, interaction modifiers, steric stabilizers can be attached.
The mixture is then applied to cells.
[0010] In another preferred embodiment of the invention, the dried
salt complex is dissolved in an appropriate organic solvent or in
an organic/aqueous solvent mixture, mixed with lipid(s), and dried
to a film. The resulting film is hydrated with an aqueous solution,
vortexed, bath sonicated, and then applied to cells. In this
embodiment, the lipid(s) and liposomes can posses additional
functionality, for example, membrane active compounds, cell
penetrating compounds, cell targeting signals, interaction
modifiers, steric stabilizers. Additionally, the lipid(s) and
liposomes can posses reactive groups to which membrane active
compounds, cell penetrating compounds, cell targeting signals,
interaction modifiers, steric stabilizers can be attached.
[0011] In a preferred embodiment we describe the reversible
modification of ammonium salts, such as in amine containing
polypeptides, with compounds of general formula I 1
[0012] wherein R is an alkyl group. Compounds of this structure are
able to chelate amines on a polypeptide. Additional substituents
can be present on any carbon atom of the system as long as the
crown ether retains the ability to chelate to an amine. Additional
substituents include steric groups, targeting groups, and polymers.
The invention is also meant to encompass the delivery to cells of
the modified polypeptide by mixing the complex with lipid(s) or by
hydrating the lipid(s) with a solution containing the modified
polypeptide. The lipid(s) can posses additional functionality, for
example, membrane active compounds, cell penetrating compounds,
cell targeting signals, interaction modifiers, steric stabilizers.
Additionally, the lipid(s) can posses reactive groups to which
membrane active compounds, cell penetrating compounds, cell
targeting signals, interaction modifiers, steric stabilizers can be
attached.
[0013] In yet another aspect, the present invention encompasses the
reversible modification of ammonium salts, such as in amine
containing polypeptides, with compounds of general formula II 2
[0014] wherein R is an alkyl group. Compounds of this structure are
able to chelate amines on a polypeptide. Additional substituents
can be present on any carbon atom of the system as long as the
crown ether retains the ability to chelate to an amine. Additional
substituents include steric groups, targeting groups, and polymers.
The invention is also meant to encompass the delivery to cells of
the modified polypeptide, by mixing the complex with lipid(s) or by
hydrating the lipid(s) with a solution containing the modified
polypeptide. The lipid(s) can posses additional functionality, for
example, membrane active compounds, cell penetrating compounds,
cell targeting signals, interaction modifiers, steric stabilizers.
Additionally, the lipid(s) can posses reactive groups to which
membrane active compounds, cell penetrating compounds, cell
targeting signals, interaction modifiers, steric stabilizers can be
attached.
[0015] In a preferred embodiment, the present invention encompasses
the reversible modification of polypeptides with compounds of
general formula III 3
[0016] wherein R.sub.1, R.sub.2, and R.sub.3 are independent and
are selected from the group consisting of halogen, alkyl, or aryl.
The invention is meant to encompass the delivery to cells of the
modified polypeptide by mixing the complex with lipid(s) or by
hydrating the lipid(s) with a solution containing the modified
polypeptide. The lipid(s) can posses additional functionality, for
example, membrane active compounds, cell penetrating compounds,
cell targeting signals, interaction modifiers, steric stabilizers.
Additionally, the lipid(s) can posses reactive groups to which
membrane active compounds, cell penetrating compounds, cell
targeting signals, interaction modifiers, steric stabilizers can be
attached.
[0017] In a preferred embodiment, the present invention encompasses
the reversible modification of polypeptides with compounds of
general formula IV 4
[0018] in which R is selected from the group consisting of alkyl,
aryl, aralkyl, a steric group, or a targeting group, and R' is
selected from the group of hydrogen, alkyl, or aryl. The invention
is meant to encompass the delivery to cells of the modified
polypeptide, by mixing the complex with lipid(s) or by hydrating
the lipid(s) with a solution containing the modified polypeptide.
The lipid(s) can posses additional functionality, for example,
membrane active compounds, cell penetrating compounds, cell
targeting signals, interaction modifiers, steric stabilizers.
Additionally, the lipid(s) can posses reactive groups to which
membrane active compounds, cell penetrating compounds, cell
targeting signals, interaction modifiers, steric stabilizers can be
attached.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1. Peptide delivery to mammalian cells. (A) NES peptide
control, 2 .mu.g NES peptide incubated with HeLa cells according to
technique #1. (B) formulation #56, technique #1. (C) modification
#8, formulation #30, technique #1. (D) PosPep control, 2 .mu.g
PosPep incubated with HeLa cells according to technique #1. (E)
formulation #57, technique #1. (F) modification #10, formulation
#12, technique #1. (G) NePep control, 2 .mu.g NegPep peptide
incubated with HeLa cells according to technique #2. (H)
formulation #2, technique #2. Formulations are described in example
4, techniques are described in example 6.
[0020] FIG. 2. Protein delivery to mammalian cells. (A)
FITC-labeled BSA control, FITC-labeled BSA incubated with HeLa
cells according to technique #2. (B) Modification # 26, technique
#2. (C) Formulation #58, Technique #1. (D) Formulation #59,
Technique #1. Formulations are described in example 4, techniques
are described in example 6.
[0021] FIG. 3. Illustration of pH sensitive reversible
hydrophobation of polypeptide using amphipathic maleic anhydride
derivatives.
DETAILED DESCRIPTION OF THE INVENTION
[0022] In the present invention, we describe methods for delivery
of proteins (positively charged, negatively charged and charge
neutral) and peptides (negatively charged, positively charged or
charge neutral) into mammalian cells in vitro. Noncovalent
hydrophobation and reversible hydrophobic modification of
polypeptide are described. In addition novel compounds are
described which can by used for the hydrophobation of
polypeptides.
[0023] It is known in the literature that hydrophobation (also
referred to as lipidation) of compounds and macromolecular carrier
systems can increase cellular interactions (Storch et al. 1996;
Kamyshny et al. 1997; Wang et al. 1997; Wang et al. 1999; Schreier
et al. 2000; Wang et al. 2000; Wang et al. 2002; Wang et al. 2003).
The present invention describes methods for the noncovalent
hydrophobation of polypeptides. For the intent of this description,
polypeptide encompasses the terms peptide and protein.
Additionally, the invention details systems for the reversible
hydrophobic modification of polypeptides. By adjusting the molar
ratio of reagents utilized in the hydrophobic modification (both
for the noncovalent hydrophobation and for the reversible
modification), the methods provide for tailoring the hydrophobation
level of polypeptide in order to optimize cellular delivery.
[0024] In one embodiment, the formation of a dried salt complex is
described. The dried salt complex can be formed from through
lyophilization of a polypeptide-surfactant complex or
polypeptide-surfactant mixture. This dried salt complex is soluble
in organic solvents or in mixed organic/aqueous solvents. A
polypeptide-surfactant complex is the complex obtained from mixing
a polypeptide with a surfactant of opposite charge. The polypeptide
can be as the free acid or base or as the acid or base addition
salt. If the polypeptide is the acid or base addition salt, then
the surfactant employed for the complex formation should be the
corresponding acid or base addition salt. If the polypeptide is the
free acid or base, then the surfactant employed for the complex
formation should be the corresponding free acid or base. The
polypeptide-surfactant complex can be between one or more charged
groups on a polypeptide with one or more surfactant molecules of
opposite charge.
[0025] The polypeptide-surfactant complex can be prepared for
example, from the positively charged amine groups on a polypeptide
when interacted with a negatively charged surfactant. Conversely, a
polypeptide-surfactant complex can be prepared from the negatively
charged carboxylate groups on a polypeptide when interacted with a
positively charged surfactant. A cationic polypeptide is a
polypeptide containing a net positive charge. The polypeptide can
contain units that are charge positive, charge neutral, or charge
negative, however, the net charge of the polypeptide must be
positive. An anionic polypeptide is a polypeptide containing a net
negative charge. The polypeptide can contain units that are charge
negative, charge neutral, or charge positive, however, the net
charge on the polypeptide must be negative. A neutral polypeptide
is a polypeptide containing a net neutral charge. The polypeptide
can contain units that are charge negative, charge neutral, or
charge positive, however, the net charge on the polypeptide must be
negative. The term zwitterionic refers to the product (salt) of the
reaction between an acidic group and a basic group that are part of
the same molecule.
[0026] For positively charged polypeptides, the
polypeptide-surfactant complex can, in principle, be prepared from
any surfactant in which the pKa of the surfactant is lower than the
pKa of the amine groups on the polypeptide. Examples of negatively
charged surfactants include carboxylic acid containing surfactants
(or pharmaceutically acceptable salts of carboxylic acids),
phosphoric acid containing surfactants (or pharmaceutically
acceptable salts of phosphoric acids, i.e. phosphates), and
sulfuric acid containing surfactants (or pharmaceutically
acceptable salts of sulfuric acids, i.e. sulfates). For example,
oleic acid, oleic acid sodium salt, palmitic acid and its salts,
pamitoleic acid and its salts, and sodium dodecylsulfate.
[0027] The polypeptide-surfactant complex can also be prepared from
the negatively charged groups on a polypeptide (carboxylic acid,
phosphates, or sulfates) interacting with a positively charged
surfactant. In principle, the polypeptide-surfactant complex can be
prepared from any surfactant in which the pKa of the surfactant is
higher than the pKa of the acidic group(s) on the polypeptide. For
example, a negatively charged polypeptide can be interacted with
cetyltrimethyl-ammonium bromide in order to complex the negatively
charged groups of a polypeptide. Examples of positively charged
surfactants include, but are not limited to cetyltrimethyl-ammonium
bromide, cetylpyridinium bromide, dodecylpyridinium chloride,
dodecyltrimethylammonium bromide, and cetyldimethylethylammonium
bromide.
[0028] A surfactant refers to a compound that contains a polar
group (hydrophilic) and a non-polar (hydrophobic) group on the same
molecule. The hydrophobic group of the surfactant is most
preferably an alkyl chain of 4 to 30 carbon atoms, and can contain
sites of unsaturation. A variety of methods other than those
described here can be envisioned for preparing the
polypeptide-surfactant complex, and for drying the complex to
afford the dried salt complex and are meant to be included within
the scope of this invention.
[0029] Another embodiment of the present invention involves the
association of the dried salt complex with one or more lipids. By
association in this case we mean that the dried salt complex is
mixed with the lipid or with liposomes. For example, the dried salt
complex can be dissolved in an organic solution and added to an
organic solution containing lipid(s). The resulting mixture can be
dried to a film as is typically done in the art with liposomal
preparations, and hydrated with an aqueous solution to generate the
liposomes. Alternatively, the dried salt complex can be dissolved
in an organic solution (for example ethanol) and added to lipid(s)
in an organic solution (for example ethanol). The resulting
solution can be utilized directly for liposomal formation as is
typically done in the art for liposomal preparations.
Alternatively, the dried salt complex can be dissolved in an
organic solution or organic/aqueous solution mixture and added to a
lipid film to rehydrate the film or added directly to
liposomes.
[0030] Another aspect of the present invention encompasses the
formation of a polypeptide-surfactant complexes by the interaction
of the polypeptide with a surfactant-chelator. In the present
invention, a crown ether chelator possessing a hydrophobic group is
complexed with the ammonium salt of a polypeptide in order to
associate a hydrophobic group with the polypeptide. Crown ethers,
and more specifically 18-crown-6 ethers, are known to chelate to
ammonium salts (Greene et al. 1999). Inclusion of a hydrophobic
group off of the crown ether, as in general formula I and II,
allows for a noncovalent association of the hydrophobic group to a
polypeptide amine group. In the present invention the alkyl groups
in general formula I are preferably from 3-30 carbons in length,
can contain unsaturated carbons, and can be branched. In the
present invention the alkyl groups in general formula II are
preferably from 3-30 carbons in length, can contain unsaturated
carbons, amide groups, and esters, and can include branching. The
association of the hydrophobic group to the polypeptide is
transient since the ammonium salt can be displaced from the crown
ether. The formed polypeptide-surfactant complex can be dried to
afford a dried salt complex. However, for polypeptide-surfactant
complexes formed using chelators of general formula I or II, the
invention is not limited to the dried salt complex. Additional
groups can be present on any carbon atom of the chelators of
general formula I and II, so long as the crown ether can still
chelate to the ammonium salt. Other groups include for example,
membrane active compounds, cell penetrating compounds, cell
targeting signals, interaction modifiers, steric stabilizers.
Additionally, the chelator can posses reactive groups to which
membrane active compounds, cell penetrating compounds, cell
targeting signals, interaction modifiers, steric stabilizers can be
attached.
[0031] A polypeptide-surfactant complex formed from general formula
I and II can further be formulated or associated with lipids or
liposomes. By association in this case we mean that
polypeptide-surfactant complex formed from general formula I and II
is mixed with the lipid or with liposomes. For example, the
polypeptide-surfactant complex formed from general formula I and II
can be added to an organic solution containing lipid(s). The
resulting mixture can be dried to a film and hydrated with an
aqueous solution to afford the liposomes. Alternatively, a
polypeptide-surfactant complex formed from general formula I and II
can be added to a lipid film to rehydrate the film, or can be added
directly to liposomes. The lipid(s) and liposomes can posses
additional functionality selected from the list consisting of:
membrane active compounds, cell penetrating compounds, cell
targeting signals, interaction modifiers, steric stabilizers.
Additionally, the lipid(s) and liposomes can posses reactive groups
to which such groups can be attached.
[0032] The present invention also involves systems for the
reversible hydrophobic modification of polypeptides in order to
broaden or enhance their ability to be delivered to cells through a
variety of formulations. As outlined, the hydrophobic modification
can increase the ability to formulate the polypeptide in a form for
delivery to a cell. However, the reversibility is an important
aspect of the experiment in that the hydrophobic modification is
able to separate from the polypeptide under physiological
conditions, thus allowing the natural (unmodified) polypeptide to
be regenerated. Two systems are described for use in the reversible
modification of polypeptides.
[0033] As such, in one aspect of the invention, the invention
encompasses the reversible hydrophobic modification of polypeptides
by alkyl chlorosilanes to form the reversible hydrophobic
polypeptide complex. Silylchlorides are known to react with a wide
variety of organic functional groups (Greene et al. 1999) to afford
silylated derivatives. The reaction of an amine and a silylchloride
generates a silazane. Silazanes are generally very hydrolytically
labile, making them useful as temporary hydrophobation systems.
Upon hydrolysis, the original amine is regenerated together with a
silanol or silyl ether. The present invention encompasses the
modification of polypeptides with silyl chlorides of general
formula III 5
[0034] wherein R.sub.1, R.sub.2, and R.sub.3 are independent and
are selected from the group consisting of halogen, alkyl, or aryl.
More specifically, R.sub.1, R.sub.2, and R.sub.3 are independent
and are selected from the group consisting of halogen (chloride or
bromide), alkyl (from 1-30 carbons, can contain unsaturation, and
can be branched for example in a tert butyl or isopropyl group),
aryl (phenyl, or substituted phenyl ring), and other functional
groups. The reversible hydrophobic polypeptide complex can be dried
or lyophilized to afford a dried reversible hydrophobic polypeptide
complex.
[0035] Hydrophobation of a polypeptides through reversible
hydrophobic modification may also be achieved by reacting amino
groups on the polypeptide with hydrophobic amides derived from
2-propionic-3-methylmale- ic anhydride to afford the reversible
hydrophobic polypeptide complex (Naganawa et al. 1994; Hermanson
1996; Reddy et al. 2000; Dinand et al. 2002; Rozema et al. 2003).
The present invention encompasses the reversible modification of
polypeptides with compounds of general formula IV 6
[0036] in which R is selected from the group consisting of alkyl,
aryl, a steric group, or a targeting group, and R' is selected from
the group of hydrogen, alkyl (from 1-30 carbons, can contain
unsaturation, and can be branched for example in a tert butyl or
isopropyl group), or aryl (phenyl, or substituted phenyl ring). The
reversible hydrophobic polypeptide complex can be dried or
lyophilized to afford a dried reversible hydrophobic polypeptide
complex.
[0037] Maleic anhydrides react with amines on the polypeptide to
form maleamic acids. This reaction is reversible. Maleamic acids
are known to be stable under basic conditions, but hydrolyze under
acidic conditions. In acidic conditions, the amide bond formed
during the reaction between the amine and the anhydride is cleaved
to yield the original unmodified amine and the maleic anhydride
(FIG. 3)
[0038] Another embodiment of the present invention involves the
association of the reversible hydrophobic polypeptide complex with
one or more lipids, or with liposomes. By association in this case
we mean that dried reversible hydrophobic polypeptide complex is
mixed with the lipid or with liposomes. For example, the dried
reversible hydrophobic polypeptide complex can be dissolved in an
organic solution and added to an organic solution containing
lipid(s). The resulting mixture can be dried to a film as is
typically done in the art with liposomal preparations and hydrated
with an aqueous solution to form the liposomes. Alternatively, the
dried reversible hydrophobic polypeptide complex can be dissolved
in an organic solution and added to an organic solution containing
lipid(s). The resulting solution can be utilized directly for
liposomal formation as is typically done in the art with liposomal
preparations. Alternatively, the dried reversible hydrophobic
polypeptide complex can be dissolved in an organic solution or in
an organic/aqueous solution mixture and added to the lipid film to
rehydrate the film, or can be added directly to liposomes. The
lipid(s) and/or liposomes can posses additional functional groups.
Additionally, the lipid(s) and liposomes can posses reactive groups
to which functional groups can be attached.
[0039] Another embodiment of the present invention involves new
methods for the application of lipid formulations onto cells in
vitro. Traditionally, liposomal formulations have been widely
utilized in the area of drug delivery and gene therapy. We were
unable to observe cytoplasmic delivery of polypeptides into cells
with a number of liposomes when using typical formulations:
formation of liposomes in the presence of the polypeptide, dilution
of the liposomes into culture media and incubation of the liposomes
with cells for several hours. Incubation of cells with higher
concentrations of liposomes/micelles for typical periods of time
(one hour or longer) is frequently toxic to cells. In the present
invention, more concentrated polypeptide formulations are added to
cells in smaller volumes for shorter periods of time. Following a
short incubation cell growth media is added. We show that mixing of
liposomes with cells at high concentrations for short incubation
times provides for delivery of polypeptides. This method departs
from typical delivery systems where the polypeptide formulations
are first diluted with media before incubation with cells at
37.degree. C. for several hours.
[0040] Definitions
[0041] Chemical Bond--A chemical bond is a covalent or noncovalent
bond.
[0042] Covalent Bond--A covalent bond is a chemical bond in which
each atom of the bond contributes one electron to form a pair of
electrons. A covalent bond can also mean a coordinate or dative
bond.
[0043] Noncovalent Bond--A noncovalent bond or ionic bond is a bond
in which electrons are transferred to atoms to afford charged
atoms. Atoms of opposite charge can form an interaction.
[0044] Hydrophobation--Hydrophobation, or hydrophobic modification,
is the act of associating a compound that possesses a hydrophobic
group, such as a surfactant, with another compound via a chemical
bond.
[0045] Noncovalent Hydrophobation--Noncovalent Hydrophobation or
noncovalent hydrophobic modification is the formation of a
noncovalent bond between a compound that possesses a hydrophobic
group, such as a surfactant, and another compound.
[0046] Amphiphilic and Amphipathic Compounds--Amphipathic, or
amphiphilic, compounds have both hydrophilic (water-soluble) and
hydrophobic (water-insoluble) parts.
[0047] Lipid--Any of a diverse group of organic compounds that are
insoluble in water, but soluble in organic solvents such as
chloroform and benzene. Lipids contain both hydrophobic and
hydrophilic sections. The term lipids are meant to include complex
lipids, simple lipids, and synthetic lipids.
[0048] Complex Lipids--Complex lipids are the esters of fatty acids
and include glycerides (fats and oils), glycolipids, phospholipids,
and waxes.
[0049] Simple Lipids--Simple lipids include steroids and
terpenes.
[0050] Synthetic Lipids--Synthetic lipids includes amides prepared
from fatty acids wherein the carboxylic acid has been converted to
the amide, synthetic variants of complex lipids in which one or
more oxygen atoms has been substituted by another heteroatom (such
as Nitrogen or Sulfur), and derivatives of simple lipids in which
additional hydrophilic groups have been chemically attached.
Synthetic lipids may contain one or more labile groups.
[0051] Fats--Fats are glycerol esters of long-chain carboxylic
acids. Hydrolysis of fats yields glycerol and a carboxylic acid--a
fatty acid. Fatty acids may be saturated or unsaturated (contain
one or more double bonds).
[0052] Oils--Oils are esters of carboxylic acids or are glycerides
of fatty acids.
[0053] Glycolipids--Glycolipids are sugar containing lipids. The
sugars are typically galactose, glucose or inositol.
[0054] Wax--Waxes are any of various solid or semisolid substances
generally being esters of fatty acids.
[0055] Fatty Acids--Fatty acids are considered the hydrolysis
product of lipids (fats, waxes, and phosphoglycerides).
[0056] Surfactant--A surfactant is a surface active agent, such as
a detergent or a lipid, which is added to a liquid to increase its
spreading or wetting properties by reducing its surface tension. A
surfactant refers to a compound that contains a polar group
(hydrophilic) and a non-polar (hydrophobic) group on the same
molecule. A cleavable surfactant is a surfactant in which the polar
group may be separated from the nonpolar group by the breakage or
cleavage of a chemical bond located between the two groups, or to a
surfactant in which the polar or non-polar group or both may be
chemically modified such that the detergent properties of the
surfactant are destroyed.
[0057] Detergent--Detergents are compounds that are soluble in
water and cause nonpolar substances to go into solution in water.
Detergents have both hydrophobic and hydrophilic groups
[0058] Micelle--Micelles are microscopic vesicles that contain
amphipathic molecules but do not contain an aqueous volume that is
entirely enclosed by a membrane. In micelles the hydrophilic part
of the amphipathic compound is on the outside (on the surface of
the vesicle). In inverse micelles the hydrophobic part of the
amphipathic compound is on the outside. The inverse micelles thus
contain a polar core that can solubilize both water and
macromolecules within the inverse micelle.
[0059] Liposome--Liposomes are microscopic vesicles that contain
amphipathic molecules and contain an aqueous volume that is
entirely enclosed by a membrane.
[0060] Microemulsions--Microemulsions are isotropic,
thermodynamically stable solutions in which substantial amounts of
two immiscible liquids (water and oil) are brought into a single
phase due to a surfactant or mixture of surfactants. The
spontaneously formed colloidal particles are globular droplets of
the minor solvent, surrounded by a monolayer of surfactant
molecules. The spontaneous curvature, H0 of the surfactant
monolayer at the oil/water interface dictates the phase behavior
and microstructure of the vesicle. Hydrophilic surfactants produce
oil in water (O/W) microemulsions (H0>0), whereas lipophilic
surfactants produce water in oil (W/O) microemulsions.
[0061] Hydrophobic Groups--Hydrophobic groups indicate in
qualitative terms that the chemical moiety is water-avoiding.
Typically, such chemical groups are not water soluble, and tend not
to form hydrogen bonds.
[0062] Hydrophilic Groups--Hydrophilic groups indicate in
qualitative terms that the chemical moiety is water-preferring.
Typically, such chemical groups are water soluble, and are hydrogen
bond donors or acceptors with water.
[0063] Charge, Polarity, and Sign--The charge, polarity, or sign of
a compound refers to whether or not a compound has lost one or more
electrons (positive charge, polarity, or sign) or gained one or
more electrons (negative charge, polarity, or sign).
[0064] Drying--Drying means removing the solvent from a sample, for
example, removing the solvent from a complex under reduced
pressure. Drying also means dehydrating a sample, or lyophilization
of a sample.
[0065] Salt--A salt is any compound containing ionic bonds; i.e.,
bonds in which one or more electrons are transferred completely
from one atom to another. Salts are ionic compounds that dissociate
into cations and anions when dissolved in solution and thus
increase the ionic strength of a solution.
[0066] Pharmaceutically Acceptable Salt--Pharmaceutically
acceptable salt means both acid and base addition salts.
[0067] Pharmaceutically Acceptable Acid Addition Salt--A
pharmaceutically acceptable acid addition salt is a salt that
retains the biological effectiveness and properties of the free
base, is not biologically or otherwise undesirable, and is formed
with inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid and the like, and
organic acids such as acetic acid, propionic acid, pyruvic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, mandelic acid, methanesulfonic
acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid,
trifluoroacetic acid, and the like.
[0068] Pharmaceutically Acceptable Base Addition Salt--A
pharmaceutically acceptable base addition salt is a salt that
retains the biological effectiveness and properties of the free
acid, is not biologically or otherwise undesirable, and is prepared
from the addition of an inorganic organic base to the free acid.
Salts derived from inorganic bases include, but are not limited to,
sodium, potassium, calcium, lithium, ammonium, magnesium, zinc, and
aluminum salts and the like. Salts derived from organic bases
include, but are not limited to, salts of primary, secondary, and
tertiary amines, such as methylamine, triethylamine, and the
like.
[0069] Functional group--Functional groups include cell targeting
signals, nuclear localization signals, compounds that enhance
release of contents from endosomes or other intracellular vesicles
(releasing signals), and other compounds that alter the behavior or
interactions of the compound or complex to which they are attached.
Additionally, a functional group also means a chemical functional
group that can undergo further chemical reactions. Examples include
but are not limited to hydroxyl groups, amine groups, thiols,
carboxylic acids, aldehydes, and ketones.
[0070] Cell targeting signals--Cell targeting signals are any
signals that enhance the association of the biologically active
compound with a cell. These signals can modify a biologically
active compound such as drug or nucleic acid and can direct it to a
cell location (such as tissue) or location in a cell (such as the
nucleus) either in culture or in a whole organism. The signal may
increase binding of the compound to the cell surface and/or its
association with an intracellular compartment. A cell targeting
signal can be, but is not limited to, a protein, peptide, lipid,
steroid, sugar, carbohydrate, (non-expressing) polynucleic acid or
synthetic compound. Cell targeting signals such as ligands enhance
cellular binding to receptors. The ligand may seek a target within
the cell membrane, on the cell membrane or near a cell. Binding of
ligands to receptors typically initiates endocytosis. Chemical
groups that react with thiol, sulfhydryl, or disulfide groups on
cells can also be used to target many types of cells. Other
targeting groups include molecules that interact with membranes
such as lipids, fatty acids, cholesterol, dansyl compounds, and
amphotericin derivatives. In addition viral proteins could be used
to bind cells.
[0071] Nuclear localization signals--Nuclear localizing signals
enhance the targeting of a pharmaceutical into proximity of the
nucleus and/or its entry into the nucleus. Such nuclear transport
signals can be a protein or a peptide such as the SV40 large T
antigen NLS or the nucleoplasmin NLS. The nuclear transport
proteins themselves could also function as NLS's. Several NLS
peptides have been derived from the SV40 T antigen, hnRNP A1
protein, nucleoplasmin, c-myc, and M9 proteins.
[0072] Membrane active compounds--Membrane active polymers or
compounds are molecules that are able to alter membrane structure.
This change in structure can be shown by the compound inducing one
or more of the following effects upon a membrane: an alteration
that allows small molecule permeability, pore formation in the
membrane, a fusion and/or fission of membranes, an alteration that
allows large molecule permeability, or a dissolving of the
membrane. This alteration can be functionally defined by the
compound's activity in at least one the following assays: red blood
cell lysis (hemolysis), liposome leakage, liposome fusion, cell
fusion, cell lysis and endosomal release. More specifically
membrane active compounds allow for the transport of molecules with
molecular weight greater than 50 atomic mass units to cross a
membrane. This transport may be accomplished by either the total
loss of membrane structure, the formation of holes (or pores) in
the membrane structure, or the assisted transport of compound
through the membrane.
[0073] Cell penetrating compounds--Cell penetrating compounds,
which include cationic import peptides (also called peptide
translocation domains, membrane translocation peptides,
arginine-rich motifs, cell-penetrating peptides, and peptoid
molecular transporters) are typically rich in arginine and lysine
residues and are capable of crossing biological membranes. In
addition, they are capable of transporting molecules to which they
are attached across membranes. Examples include TAT peptide, VP22
peptide, and Drosophila antennapedia peptide. Cell penetrating
compounds are not strictly peptides. Short, non-peptide polymers
that are rich in amines or guanidinium groups are also capable of
carrying molecules crossing biological membranes. Like membrane
active peptides, cationic import peptides are defined by their
activity rather than by strict amino acid sequence
requirements.
[0074] Interaction Modifiers--An interaction modifier changes the
way that a molecule interacts with itself or other molecules
relative to molecule containing no interaction modifier. The result
of this modification is that self-interactions or interactions with
other molecules are either increased or decreased. For example cell
targeting signals are interaction modifiers which change the
interaction between a molecule and a cell or cellular component.
Polyethylene glycol is an interaction modifier that decreases
interactions between molecules and themselves and with other
molecules.
[0075] Steric Stabilizer--A steric stabilizer is a long chain
hydrophilic group that prevents aggregation by sterically hindering
particle to particle or polymer to polymer electrostatic
interactions. Examples include: alkyl groups, PEG chains,
polysaccharides, alkyl amines. Electrostatic interactions are the
noncovalent association of two or more substances due to attractive
forces between positive and negative charges.
[0076] Chelator--A Chelator is a polydentate ligand, a molecule
that can occupy more than one site in the coordination sphere of an
ion, particularly a metal ion, primary amine, or single proton.
Examples of chelators include crown ethers, cryptates, and
non-cyclic polydentate molecules. A crown ether is a cyclic
polyether containing (--X--(CR1-2)n)m units, where n=1-3 and m=3-8.
The X and CR1-2 moieties can be substituted, or at a different
oxidation states. X can be oxygen, nitrogen, or sulfur, carbon,
phosphorous or any combination thereof. R can be H, C, O, S, N, P.
The crown ether ring system is named as [(n+1)m crown m] for
X=oxygen, as [(n+1)m azacrown m] when X=nitrogen, as [(n+1)m
thiocrown m] when X=sulfur. In the case of two or more heteroatoms
present in the ring the heteroatom positions are specified. For
example, 12-crown-4, 4-aminobenzo-12-crown-4,
4-formylbenzo-12-crown-4, 4-hydroxybenzo-12-crown-4,
4-acryloylamidobenzo-12-crown-4, 4-vinylbenzo-12-crown-4,
15-crown-5, 4-aminobenzo-15-crown-5, 4-formylbenzo-15-crown-5,
4-hydroxybenzo-15-crown-5, 4-acryloylamidobenzo-15-crown-5,
4-vinylbenzo-15-crown-5, 18-crown-6, benzo-18-crown-6,
4-aminobenzo-18-crown-6, 4-formylbenzo-18-crown-6,
4-hydroxybenzo-18-crown-6, 4-acryloylamidobenzo-18-crown-6,
4-vinylbenzo-18-crown-6, (18-crown-6)-2,3,11,12-tetracarboxcylic
acid, 2-hydroxymethyl-18-crown-6, 2-aminomethyl-18-crown-6,
1-aza-18-crown-6, 16-crown-4, 20-crown-4, and 18-crown-6,
polyvinylbenzo 15-crown-5. A subset of crown ethers described as a
cryptate contain a second (--X--(C.sub.R1-2)n).sub.z strand where
z=3-8. The beginning X atom of the strand is an X atom in the
(--X--(C.sub.R1-2)n).sub.m unit, and the terminal 5CH.sub.2 of the
new strand is bonded to a second X atom in the
(--X--(C.sub.R1-2)n).sub.m unit. Non-cyclic polydentate molecules
containing (--X--(C.sub.R1-2)n).sub.m unit(s), where n=1-4 and
m=1-8. The X and C.sub.R1-2 moieties can be substituted, or at a
different oxidation states. X can be oxygen, nitrogen, or sulfur,
carbon, phosphorous or any combination thereof. For example
di(ethylene glycol), hexa(ethylene glycol) and other polyglycols,
tri(propylene glycol), ethylene diamine,
N,N,N',N'-tetramethyldiethyldiamine,
N,N,N',N'-ethylenediamine-tetraaceti- c acid, spermine, spermidine,
diethylenetriamine, 1,3-diaminopropane, phenanthroline,
1,2-bis(dimethylphosphino)-ethane,
1,4-bis(dicyclohexylphosphino)butane,
1,2-bis(phenylphosphino)-ethane,
1,4-bis(phenylphosphino)-butane.
EXAMPLES
Example 1
Peptide Synthesis
[0077] To illustrate the utility of the invention in delivering
peptides to cells, three different peptides were synthesized: a
negatively charged peptide, a positively charged peptide and a
charge neutral peptide. Peptide sequences were prepared using
standard FastMoc FMOC Chemistry on an ABI 433A Peptide Synthesizer.
Prior to deprotection and cleavage of the polypeptide from the
resin, lissamine was added to fluorescently label the N-terminus of
the polypeptide. The polypeptides were cleaved from the resin and
deprotected using TFA/TIPS/H.sub.2O (95/2.5/2.5) and purified by
reverse phase HPLC. Molecular weights were verified using as PE
SCIEX API150EX Mass Spectrometer.
[0078] NES sequence (NES; Seq ID 1)--RLQLPPLERLTLD
[0079] Positive sequence (PosPep; Seq ID 2)--GKNRGKSAQAKRLR
[0080] Negative Sequence (NegPep; Seq ID 3)--GEGMEEGEFSEA
[0081] These sequences are merely indicated as examples, and are
not intended to limit the scope of the invention in any way.
Example 2
Synthesis of Protein/Peptide Modification Reagents
[0082] 1. Preparation of 2-(3-dodecyl-propionamide)-3-methyl Maleic
Anhydride (CDM12):
[0083] To a solution of 2-(3-propionic acid)-3-methyl maleic
anhydride (11.1 mg, 0.060 mmol, Mirus Corporation) in
dichloromethane (1.2 mL) was added oxalyl chloride (26 .mu.L, 0.302
mmol, Aldrich Chemical Company) and diisopropylethylamine (10.5
.mu.L, 0.060 mmol, Aldrich Chemical Company). The resulting
solution was stirred at ambient temperature for 1 h and
concentrated under reduced pressure. The resulting residue was
resuspended in dichloromethane (1.2 mL) and dodecylamine (27.7
.mu.L, 0.121 mmol, Aldrich Chemical Company) was added. The
solution was stirred at ambient temperature for 12 h. The solution
was concentrated under reduced pressure, and taken up in EtOAc (5
mL). The solution was extracted 3.times.10 mL 1N HCl, washed
1.times.10 mL H.sub.2O, 1.times.10 mL brine, and concentrated to
afford 19.5 mg (92%) of 2-(3-dodecyl-propionamide)-3-methyl maleic
anhydride.
[0084] 2. Preparation of 2-(3-butyl-propionamide)-3-methyl Maleic
Anhydride (CDM4):
[0085] To a solution of 2-(3-propionic acid)-3-methyl maleic
anhydride (10.6 mg, 0.057 mmol, Mirus Corporation) in
dichloromethane (1.2 mL) was added oxalyl chloride (25 .mu.L, 0.288
mmol, Aldrich Chemical Company) and diisopropylethylamine (9.9
.mu.L, 0.057 mmol, Aldrich Chemical Company). The resulting
solution was stirred at ambient temperature for 1 h and
concentrated under reduced pressure. The resulting residue was
resuspended in dichloromethane (1.2 mL) and butylamine (11.3 .mu.L,
0.114 mmol, Aldrich Chemical Company) was added. The solution was
stirred at ambient temperature for 12 h. The solution was
concentrated under reduced pressure, and taken up in EtOAc (5 mL).
The solution was extracted 3.times.10 mL 1N HCl, washed 1.times.10
mL H.sub.2O, 1.times.10 mL brine, and concentrated to afford 9.8 mg
(72%) of 2-(3-butyl-propionamide)-3-met- hyl maleic anhydride.
Example 3
Polypeptide Modification
[0086] 1. Preparation of NegPep-CPB:
[0087] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added cetylpyridinium bromide (2.0 .mu.g,
0.005 .mu.mol, Aldrich Chemical Company). The resulting solution
was vortexes, frozen, and lyophilized.
[0088] 2. Preparation of NegPep-CTAB:
[0089] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added cetyltrimethyl-ammonium bromide (1.0
.mu.g, 0.0027 .mu.mol, Aldrich Chemical Company). The resulting
solution was vortexes, frozen, and lyophilized.
[0090] 3. Preparation of NegPep-DPC:
[0091] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added dodecylpyridinium chloride (1.6
.mu.g, 0.0056 .mu.mol, Aldrich Chemical Company) and the solution
was vortexes. The solution was frozen and lyophilized.
[0092] 4. Preparation of NegPep-DAB:
[0093] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added dodecyltrimethylammonium bromide (1.7
.mu.g, 0.0055 .mu.mol, Aldrich Chemical Company) and the solution
was vortexes. The solution was frozen and lyophilized.
[0094] 5. Preparation of NegPep-CDAB:
[0095] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added cetyldimethylethylammonium bromide
(2.1 .mu.g, 0.0055 .mu.mol, Aldrich Chemical Company) and the
resulting solution was vortexes. The solution was frozen and
lyophilized.
[0096] 6. Preparation of PosPep-TOPPS:
[0097] To a solution of PosPep (2.0 .mu.g, 0.00095 .mu.mol) in
H.sub.2O (25 .mu.L) was added TOPPS (1.0 .mu.g, 0.0029 .mu.mol,
Aldrich Chemical Company) and the resulting solution was vortexes.
The solution was frozen and lyophilized.
[0098] 7. Preparation of PosPep-CDM12-0.45:
[0099] To a solution of PosPep (2.0 .mu.g, 0.00095 .mu.mol) in
H.sub.2O (25 .mu.L) was added CDM12 (0.15 .mu.g, 0.00043 .mu.mol)
and the resulting solution was vortexes. The mixture was frozen and
lyophilized.
[0100] 8. Preparation of NES-CDM12-1:
[0101] To a solution of NES peptide (10 .mu.g, 0.0048 .mu.mol) in
H.sub.2O:EtOH (20 .mu.L, 1:1) was added CDM12 (1.7 .mu.g, 0.0048
.mu.mol, 1 mg/mL solution in EtOH) and
diisopropylaminomethyl-polystyrene (3 mg, Fluka Chemical Company).
The resulting mixture was mixed for 30 min and centrifuged to
remove the solid support base.
[0102] 9. Preparation of NES-CDM12-2:
[0103] To a solution of NES peptide (10 .mu.g, 0.0048 .mu.mol) in
H.sub.2O:EtOH (20 .mu.L, 1:1) was added CDM12 (3.4 .mu.g, 0.0096
.mu.mol, 1 mg/mL solution in EtOH) and
diisopropylaminomethyl-polystyrene (3 mg, Fluka Chemical Company).
The resulting mixture was mixed for 30 min and centrifuged to
remove the solid support base.
[0104] 10. Preparation of PosPep-CDM12-1:
[0105] To a solution of PosPep (10 .mu.g, 0.0047 .mu.mol) in
H.sub.2O:EtOH (20 .mu.L, 1:1) was added CDM12 (1.7 .mu.g, 0.0047
.mu.mol, 1 mg/mL solution in EtOH) and
diisopropylaminomethyl-polystyrene (3 mg, Fluka Chemical Company).
The resulting mixture was mixed for 30 min and centrifuged to
remove the solid support base.
[0106] 11. Preparation of PosPep-CDM12-2:
[0107] To a solution of PosPep (10 .mu.g, 0.0047 .mu.mol) in
H.sub.2O:EtOH (20 .mu.L, 1:1) was added CDM12 (3.3 .mu.g, 0.0094
.mu.mol, 1 mg/mL solution in EtOH) and
diisopropylaminomethyl-polystyrene (3 mg, Fluka Chemical Company).
The resulting mixture was mixed for 30 min and centrifuged to
remove the solid support base.
[0108] 12. Preparation of NegPep-CDM12-1:
[0109] To a solution of NegPep (10 .mu.g, 0.0055 .mu.mol) in
H.sub.2O:EtOH (20 .mu.L, 1:1) was added CDM12 (2.2 .mu.g, 0.0055
.mu.mol, 1 mg/mL solution in EtOH) and
diisopropylaminomethyl-polystyrene (3 mg, Fluka Chemical Company).
The resulting mixture was mixed for 30 min and centrifuged to
remove the solid support base.
[0110] 13. Preparation of NegPep-CDM12-2:
[0111] To a solution of NegPep (10 .mu.g, 0.0055 .mu.mol) in
H.sub.2O:EtOH (20 .mu.L, 1:1) was added CDM12 (4.3 .mu.g, 0.011
.mu.mol, 1 mg/mL solution in EtOH) and
diisopropylaminomethyl-polystyrene (3 mg, Fluka Chemical Company).
The resulting mixture was mixed for 30 min and centrifuged to
remove the solid support base.
[0112] 14. Preparation of NES-MK10-1:
[0113] To a solution of NES peptide (10 .mu.g, 0.0048 .mu.mol) in
H.sub.2O:EtOH (20 .mu.L, 1:1) was added 2-dceyl-1,4,7,10,13,16
hexaoxacyclooctadecane (1.9 .mu.g, 0.0048 .mu.mol, Merck, 1 mg/mL
solution in EtOH). The resulting solution was mixed for 30 min
prior to use.
[0114] 15. Preparation of NES-MK10-2:
[0115] To a solution of NES peptide (10 .mu.g, 0.0048 .mu.mol) in
H.sub.2O:EtOH (20 .mu.L, 1:1) was added 2-dceyl-1,4,7,10,13,16
hexaoxacyclooctadecane (3.88 .mu.g, 0.0096 .mu.mol, Merck, 1 mg/mL
solution in EtOH). The resulting solution was mixed for 30 min
prior to use.
[0116] 16. Preparation of PosPep-MK10-1:
[0117] To a solution of PosPep peptide (10 .mu.g, 0.0047 .mu.mol)
in H.sub.2O:EtOH (20 .mu.L, 1:1) was added 2-dceyl-1,4,7,10,13,16
hexaoxacyclooctadecane (1.9 .mu.g, 0.0047 .mu.mol, Merck, 1 mg/mL
solution in EtOH). The resulting solution was mixed for 30 min
prior to use.
[0118] 17. Preparation of PosPep-MK10-2:
[0119] To a solution of PosPep peptide (10 .mu.g, 0.0047 .mu.mol)
in H.sub.2O:EtOH (20 .mu.L, 1:1) was added 2-dceyl-1,4,7,10,13,16
hexaoxacyclooctadecane (3.83 .mu.g, 0.0095 .mu.mol, Merck, 1 mg/mL
solution in EtOH). The resulting solution was mixed for 30 min
prior to use.
[0120] 18. Preparation of NegPep-MK10-1:
[0121] To a solution of NegPep peptide (10 .mu.g, 0.0055 .mu.mol)
in H.sub.2O:EtOH (20 .mu.L, 1:1) was added 2-dceyl-1,4,7,10,13,16
hexaoxacyclooctadecane (2.2 .mu.g, 0.0055 .mu.mol, Merck, 1 mg/mL
solution in EtOH). The resulting solution was mixed for 30 min
prior to use.
[0122] 19. Preparation of NegPep-MK10-2:
[0123] To a solution of NegPep peptide (10 .mu.g, 0.0055 .mu.mol)
in H.sub.2O:EtOH (20 .mu.L, 1:1) was added 2-dceyl-1,4,7,10,13,16
hexaoxacyclooctadecane (4.5 .mu.g, 0.011 .mu.mol, Merck, 1 mg/mL
solution in EtOH). The resulting solution was mixed for 30 min
prior to use.
[0124] 20. Preparation of NES-Tos-MK10-1:
[0125] To a solution of NES peptide (10 .mu.g, 0.0048 .mu.mol) in
H.sub.2O:EtOH (20 .mu.L, 1:1) was added sodium p-toluenesulfonate
(0.93 .mu.g, 0.0048 .mu.mol, Aldrich Chemical Company) as a 1 mg/mL
solution in H.sub.2O, followed by 2-dceyl-1,4,7,10,13,16
hexaoxacyclooctadecane (1.9 .mu.g, 0.0048 .mu.mol, Merck, 1 mg/mL
solution in EtOH). The resulting solution was mixed for 30 min
prior to use.
[0126] 21. Preparation of NES-Tos-MK10-2:
[0127] To a solution of NES peptide (10 .mu.g, 0.0048 .mu.mol) in
H.sub.2O:EtOH (20 .mu.L, 1:1) was added sodium p-toluenesulfonate
(1.9 .mu.g, 0.0096 .mu.mol, Aldrich Chemical Company) as a 1 mg/mL
solution in H.sub.2O, followed by 2-dceyl-1,4,7,10,13,16
hexaoxacyclooctadecane (3.88 .mu.g, 0.0096 .mu.mol, Merck, 1 mg/mL
solution in EtOH). The resulting solution was mixed for 30 min
prior to use.
[0128] 22. Preparation of PosPep-Tos-MK10-1:
[0129] To a solution of PosPep peptide (10 .mu.g, 0.0047 .mu.mol)
in H.sub.2O:EtOH (20 .mu.L, 1:1) was added sodium
p-toluenesulfonate (0.91 .mu.g, 0.0047 .mu.mol, Aldrich Chemical
Company) as a 1 mg/mL solution in H.sub.2O, followed by
2-dceyl-1,4,7,10,13,16 hexaoxacyclooctadecane (1.9 .mu.g, 0.0047
.mu.mol, Merck, 1 mg/mL solution in EtOH). The resulting solution
was mixed for 30 min prior to use.
[0130] 23. Preparation of PosPep-Tos-MK10-2:
[0131] To a solution of PosPep peptide (10 .mu.g, 0.0047 .mu.mol)
in H.sub.2O:EtOH (20 .mu.L, 1:1) was added sodium
p-toluenesulfonate (1.8 .mu.g, 0.0095 .mu.mol, Aldrich Chemical
Company) as a 1 mg/mL solution in H.sub.2O, followed by
2-dceyl-1,4,7,10,13,16 hexaoxacyclooctadecane (3.83 .mu.g, 0.0095
.mu.mol, Merck, 1 mg/mL solution in EtOH). The resulting solution
was mixed for 30 min prior to use.
[0132] 24. Preparation of NegPep-Tos-MK10-1:
[0133] To a solution of NegPep peptide (10 .mu.g, 0.0055 .mu.mol)
in H.sub.2O:EtOH (20 .mu.L, 1:1) was added sodium
p-toluenesulfonate (1.1 .mu.g, 0.0055 .mu.mol, Aldrich Chemical
Company) as a 1 mg/mL solution in H.sub.2O, followed by
2-dceyl-1,4,7,10,13,16 hexaoxacyclooctadecane (2.2 .mu.g, 0.0055
.mu.mol, Merck, 1 mg/mL solution in EtOH). The resulting solution
was mixed for 30 min prior to use.
[0134] 25. Preparation of NegPep-Tos-MK10-2:
[0135] To a solution of NegPep peptide (10 .mu.g, 0.0055 .mu.mol)
in H.sub.2O:EtOH (20 .mu.L, 1:1) was added sodium
p-toluenesulfonate (2.1 .mu.g, 0.0055 .mu.mol, Aldrich Chemical
Company) as a 1 mg/mL solution in H.sub.2O, followed by
2-dceyl-1,4,7,10,13,16 hexaoxacyclooctadecane (4.5 .mu.g, 0.011
.mu.mol, Merck, 1 mg/mL solution in EtOH). The resulting solution
was mixed for 30 min prior to use.
[0136] 26. Preparation of Albumin-dimethyloctadecylsilane:
[0137] Rhodamine-labeled Bovine Serum Albumin (BSA, Sigma Chemical
Company) was dissolved in H.sub.2O to a final concentration of 20
mg/mL. A 1 .mu.L (20 .mu.g) aliquot of the stock solution was taken
and diluted with H.sub.2O to a final concentration of 1 mg/mL. To 2
.mu.L of this solution was added 100 .mu.L of chloroform, followed
by chlorodimethyloctadecylsilane (250 .mu.g in 5 .mu.L chloroform,
Aldrich Chemical Company). The resulting solution was sonicated for
10 sec and dried under vacuum. The resulting film was hydrated with
50 .mu.L PBS.
Example 4
Formulations for Protein/Peptide Delivery
[0138] 1. Preparation of DOTAP-Cl/NegPep-CPB:
[0139] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added cetylpyridinium bromide (2.0 .mu.g,
0.005 .mu.mol, Aldrich Chemical Company). The resulting solution
was vortexes, frozen, and lyophilized. The freeze dried material
was brought up in CHCl.sub.3 (50 .mu.L). DOTAP chloride (8.5 .mu.L
of 1 .mu.g/.mu.L solution in CHCl.sub.3, 0.012 .mu.mol, Avanti
Polar Lipids, Inc) was added and the solution was vortexes. The
solution was dried into a film under N.sub.2 and placed under
vacuum overnight.
[0140] 2. Preparation of DOTAP-Cl/NegPep-CTAB:
[0141] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added cetyltrimethyl-ammonium bromide (1.0
.mu.g, 0.0027 .mu.mol, Aldrich Chemical Company). The resulting
solution was vortexes, frozen, and lyophilized. The freeze dried
material was brought up in CHCl.sub.3 (50 .mu.L). DOTAP (8.5 .mu.g,
0.012 .mu.mol, Avanti Polar Lipids, Inc) was added and the solution
was vortexes. The solution was dried into a film under N.sub.2 and
placed under vacuum overnight
[0142] 3. Preparation of DOTAP-Cl/NegPep-DPC:
[0143] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added dodecylpyridinium chloride (1.6
.mu.g, 0.0056 .mu.mol, Aldrich Chemical Company) and the solution
was vortexes. The solution was frozen and lyophilized. The freeze
dried material was brought up in CHCl.sub.3 (50 .mu.L). DOTAP (8.5
.mu.g, 0.012 .mu.mol, Avanti Polar Lipids, Inc) was added and the
solution was vortexes. The mixture was dried into a film under
N.sub.2 and placed under vacuum overnight
[0144] 4. Preparation of DOTAP-Cl/NegPep-DAB:
[0145] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added dodecyltrimethylammonium bromide (1.7
.mu.g, 0.0055 .mu.mol, Aldrich Chemical Company) and the solution
was vortexes. The solution was frozen and lyophilized. The freeze
dried material was brought up in CHCl.sub.3 (50 .mu.L). DOTAP (8.5
.mu.g, 0.012 .mu.mol, Avanti Polar Lipids, Inc) was added and the
resulting solution was vortexes. The mixture was dried into a film
under N.sub.2 and placed under vacuum overnight
[0146] 5. Preparation of DOTAP-Cl/NegPep-CDAB:
[0147] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added cetyldimethylethylammonium bromide
(2.1 .mu.g, 0.0055 .mu.mol, Aldrich Chemical Company) and the
resulting solution was vortexes. The solution was frozen and
lyophilized. The freeze dried material was brought up in CHCl.sub.3
(50 .mu.L). DOTAP (8.5 .mu.g, 0.012 .mu.mol, Avanti Polar Lipids,
Inc) was added and the resulting solution was vortexes. The mixture
was dried into a film under N.sub.2 and placed under vacuum
overnight
[0148] 6. Preparation of DOTAP-Cl/PosPep-TOPPS:
[0149] To a solution of PosPep (2.0 .mu.g, 0.00095 .mu.mol) in
H.sub.2O (25 .mu.L) was added TOPPS (1.0 .mu.g, 0.0029 .mu.mol,
Aldrich Chemical Company) and the resulting solution was vortexes.
The solution was frozen and lyophilized. The freeze dried material
was brought up in CHCl.sub.3 (50 .mu.L). DOTAP (8.5 .mu.g, 0.012
.mu.mol, Avanti Chemical Company) was added and the solution was
vortexes. The mixture was dried into a film under N.sub.2 and
placed under vacuum overnight.
[0150] 7. Preparation of DOTAP-Cl/PosPep-CDM12-0.45:
[0151] To a solution of PosPep (2.0 .mu.g, 0.00095 .mu.mol) in
H.sub.2O (25 .mu.L) was added CDM12 (0.15 .mu.g, 0.00043 .mu.mol)
and the resulting solution was vortexes. The mixture was frozen and
lyophilized. The freeze dried material was brought up in CHCl.sub.3
(50 .mu.L). DOTAP (8.5 .mu.g, 0.012 .mu.mol, Avanti Chemical
Company) was added and the solution was vortexes. The mixture was
dried into a film under N.sub.2 and placed under vacuum
overnight.
[0152] 8. Preparation of DOTAP-Cl/NES:
[0153] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of NES
(2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS (30 .mu.L). The
resulting mixture was vortexes, and allowed to hydrate for 15 min,
followed by vortexing and sonication (<5 sec).
[0154] 9. Preparation of DOTAP-Cl/NES-CDM12-1:
[0155] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
NES-CDM12-1 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS (30
.mu.L). The resulting mixture was vortexes, and allowed to hydrate
for 15 min, followed by vortexing and sonication (<5 sec).
[0156] 10. Preparation of DOTAP-Cl/NES-CDM12-2:
[0157] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
NES-CDM12-2 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS (30
.mu.L). The resulting mixture was vortexes, and allowed to hydrate
for 15 min, followed by vortexing and sonication (<5 sec).
[0158] 11. Preparation of DOTAP-Cl/PosPep:
[0159] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
PosPep (2 .mu.g) in H.sub.2O (2 .mu.L) and PBS (30 .mu.L). The
resulting mixture was vortexes, and allowed to hydrate for 15 min,
followed by vortexing and sonication (<5 sec).
[0160] 12. Preparation of DOTAP-Cl/PosPep-CDM12-1:
[0161] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
PosPep-CDM12-1 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS
(30 .mu.L). The resulting mixture was vortexes, and allowed to
hydrate for 15 min, followed by vortexing and sonication (<5
sec).
[0162] 13. Preparation of DOTAP-Cl/PosPep-CDM12-2:
[0163] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
PosPep-CDM12-2 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS
(30 .mu.L). The resulting mixture was vortexes, and allowed to
hydrate for 15 min, followed by vortexing and sonication (<5
sec).
[0164] 14. Preparation of DOTAP-Cl/NegPep:
[0165] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
NegPep (2 .mu.g) in H.sub.2O (2 .mu.L) and PBS (30 .mu.L). The
resulting mixture was vortexes, and allowed to hydrate for 15 min,
followed by vortexing and sonication (<5 sec).
[0166] 15. Preparation of DOTAP-Cl/NegPep-CDM12-1:
[0167] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
NegPep-CDM12-1 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS
(30 .mu.L). The resulting mixture was vortexes, and allowed to
hydrate for 15 min, followed by vortexing and sonication (<5
sec).
[0168] 16. Preparation of DOTAP-Cl/NegPep-CDM12-2:
[0169] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
NegPep-CDM12-2 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS
(30 .mu.L). The resulting mixture was vortexes, and allowed to
hydrate for 15 min, followed by vortexing and sonication (<5
sec).
[0170] 17. Preparation of DOTAP-Cl/NES-MK10-1:
[0171] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
NES-MK10-1 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS (30
.mu.L). The resulting mixture was vortexes, and allowed to hydrate
for 15 min, followed by vortexing and sonication (<5 sec).
[0172] 18. Preparation of DOTAP-Cl/NES-MK10-2:
[0173] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
NES-MK10-2 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS (30
.mu.L). The resulting mixture was vortexes, and allowed to hydrate
for 15 min, followed by vortexing and sonication (<5 sec).
[0174] 19. Preparation of DOTAP-Cl/PosPep-MK10-1:
[0175] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
PosPep-MK10-1 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS (30
.mu.L). The resulting mixture was vortexes, and allowed to hydrate
for 15 min, followed by vortexing and sonication (<5 sec).
[0176] 20. Preparation of DOTAP-Cl/PosPep-MK10-2:
[0177] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
NES-CDM12-2 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS (30
.mu.L). The resulting mixture was vortexes, and allowed to hydrate
for 15 min, followed by vortexing and sonication (<5 sec).
[0178] 21. Preparation of DOTAP-Cl/NegPep-MK10-1:
[0179] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
PosPep-MK10-2 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS (30
.mu.L). The resulting mixture was vortexes, and allowed to hydrate
for 15 min, followed by vortexing and sonication (<5 sec).
[0180] 22. Preparation of DOTAP-Cl/NegPep-MK10-2:
[0181] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
NegPep-MK10-2 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS (30
.mu.L). The resulting mixture was vortexes, and allowed to hydrate
for 15 min, followed by vortexing and sonication (<5 sec).
[0182] 23. Preparation of DOTAP-Cl/NES-Tos-MK10-1:
[0183] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
NES-Tos-MK10-1 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS
(30 .mu.L). The resulting mixture was vortexes, and allowed to
hydrate for 15 min, followed by vortexing and sonication (<5
sec).
[0184] 24. Preparation of DOTAP-Cl/NES-Tos-MK10-2:
[0185] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
NES-Tos-MK10-2 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS
(30 .mu.L). The resulting mixture was vortexes, and allowed to
hydrate for 15 min, followed by vortexing and sonication (<5
sec).
[0186] 25. Preparation of DOTAP-Cl/PosPep-Tos-MK10-1:
[0187] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
PosPep-Tos -MK10-1 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and
PBS (30 .mu.L). The resulting mixture was vortexes, and allowed to
hydrate for 15 min, followed by vortexing and sonication (<5
sec).
[0188] 26. Preparation of DOTAP-Cl/PosPep-Tos-MK10-2:
[0189] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
PosPep-Tos -MK10-2 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and
PBS (30 .mu.L). The resulting mixture was vortexes, and allowed to
hydrate for 15 min, followed by vortexing and sonication (<5
sec).
[0190] 27. Preparation of DOTAP-Cl/NegPep-Tos -MK10-1:
[0191] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
NegPep-Tos-MK10-1 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS
(30 .mu.L). The resulting mixture was vortexes, and allowed to
hydrate for 15 min, followed by vortexing and sonication (<5
sec).
[0192] 28. Preparation of DOTAP-Cl/NegPep-Tos-MK10-2:
[0193] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) was added to a micro centrifuge tube,
concentrated into a film under a stream of nitrogen, and then
placed under vacuum. To the lipid film was added a solution of
NegPep-Tos-MK10-2 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS
(30 .mu.L). The resulting mixture was vortexes, and allowed to
hydrate for 15 min, followed by vortexing and sonication (<5
sec).
[0194] 29. Preparation of DOTAP-Cl/Cholesterol/NES:
[0195] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) and Cholesterol (5 .mu.g, 1 .mu.g/.mu.L in
chloroform, Avanti Polar Lipids, Inc) was added to a micro
centrifuge tube, concentrated into a film under a stream of
nitrogen, and then placed under vacuum. To the lipid film was added
a solution of NES (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1) and PBS
(30 .mu.L). The resulting mixture was vortexes, and allowed to
hydrate for 15 min, followed by vortexing and sonication (<5
sec).
[0196] 30. Preparation of DOTAP-Cl/Cholesterol/NES-CDM12-1:
[0197] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) and Cholesterol (5 .mu.g, 1 .mu.g/.mu.L in
chloroform, Avanti Polar Lipids, Inc) was added to a micro
centrifuge tube, concentrated into a film under a stream of
nitrogen, and then placed under vacuum. To the lipid film was added
a solution of NES-CDM12-1 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L, 1:1)
and PBS (30 .mu.L). The resulting mixture was vortexes, and allowed
to hydrate for 15 min, followed by vortexing and sonication (<5
sec).
[0198] 31. Preparation of DOTAP-Cl/Cholesterol/PosPep:
[0199] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) and Cholesterol (5 .mu.g, 1 .mu.g/.mu.L in
chloroform, Avanti Polar Lipids, Inc) was added to a micro
centrifuge tube, concentrated into a film under a stream of
nitrogen, and then placed under vacuum. To the lipid film was added
a solution of PosPep (2 .mu.g) in H.sub.2O (2 .mu.L) and PBS (30
.mu.L). The resulting mixture was vortexes, and allowed to hydrate
for 15 min, followed by vortexing and sonication (<5 sec).
[0200] 32. Preparation of DOTAP-Cl/Cholesterol/PosPep-CDM12-1:
[0201] DOTAP-Cl (10 .mu.g, 1 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) and Cholesterol (5 .mu.g, 1 .mu.g/.mu.L in
chloroform, Avanti Polar Lipids, Inc) was added to a micro
centrifuge tube, concentrated into a film under a stream of
nitrogen, and then placed under vacuum. To the lipid film was added
a solution of PosPep-CDM12-1 (2 .mu.g) in H.sub.2O:EtOH (4 .mu.L,
1:1) and PBS (30 .mu.L). The resulting mixture was vortexes, and
allowed to hydrate for 15 min, followed by vortexing and sonication
(<5 sec).
[0202] 33. Preparation of DOTAP-Cl/NegPep-CPB:
[0203] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added cetylpyridinium bromide (0.44 .mu.g,
0.0011 .mu.mol, Aldrich Chemical Company). The resulting solution
was vortexes, frozen and lyophilized. The freeze dried material was
brought up in CHCl.sub.3 (50 .mu.L). DOTAP-Cl (10 .mu.g of 1
.mu.g/.mu.L in CHCl.sub.3, 0.014 .mu.mol, Avanti Polar Lipids, Inc)
was added and vortexes. The mixture was dried into a film under
N.sub.2 and placed under vacuum overnight.
[0204] 34. Preparation of DDAB/NegPep-CPB:
[0205] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added cetylpyridinium bromide (0.44 .mu.g,
0.0011 .mu.mol, Aldrich Chemical Company). The resulting solution
was vortexes, frozen and lyophilized. The freeze dried material was
brought up in CHCl.sub.3 (50 .mu.L). Dodecyldimethylammonium
bromide (6.0 .mu.g of 1 .mu.g/.mu.L in EtOH, 0.015 .mu.mol, Aldrich
Chemical Company) was added and vortexes. The mixture was dried
into a film under N.sub.2 and placed under vacuum overnight.
[0206] 35. Preparation of Me.sub.3Sphing/NegPep-CPB:
[0207] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added cetylpyridinium bromide (0.44 .mu.g,
0.0011 .mu.mol, Aldrich Chemical Company). The resulting solution
was vortexes, frozen and lyophilized. The freeze dried material was
brought up in CHCl.sub.3 (50 .mu.L). N,N,N-Trimethylsphingosine
(5.0 .mu.g of 1 .mu.g/.mu.L in CHCl.sub.3, 0.015 .mu.mol, Avanti
Polar Lipids, Inc) was added and vortexes. The mixture was dried
into a film under N.sub.2 and placed under vacuum overnight.
[0208] 36. Preparation of DOTAP-Cl/NegPep-DPC:
[0209] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added dodecylpyridinium chloride (0.31
.mu.g, 0.0011 .mu.mol, Aldrich Chemical Company). The resulting
solution was vortexes, frozen and lyophilized. The freeze dried
material was brought up in CHCl.sub.3 (50 .mu.L). DOTAP-Cl (10
.mu.g of 1 .mu.g/.mu.L in CHCl.sub.3, 0.014 .mu.mol, Avanti Polar
Lipids, Inc) was added and vortexes. The mixture was dried into a
film under N.sub.2 and placed under vacuum overnight.
[0210] 37. Preparation of DDAB/NegPep-DPC:
[0211] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added dodecylpyridinium chloride (0.31
.mu.g, 0.0011 .mu.mol, Aldrich Chemical Company). The resulting
solution was vortexes, frozen and lyophilized. The freeze dried
material was brought up in CHCl.sub.3 (50 .mu.L).
Dodecyldimethylammonium bromide (6.0 .mu.g of 1 .mu.g/.mu.L in
EtOH, 0.015 .mu.mol, Aldrich Chemical Company) was added and
vortexes. The mixture was dried into a film under N.sub.2 and
placed under vacuum overnight.
[0212] 38. Preparation of Me.sub.3Sphing/NegPep-DPC:
[0213] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added dodecylpyridinium chloride (0.31
.mu.g, 0.0011 .mu.mol, Aldrich Chemical Company). The resulting
solution was vortexes, frozen and lyophilized. The freeze dried
material was brought up in CHCl.sub.3 (50 .mu.L).
N,N,N-Trimethylsphingosine (5.0 .mu.g of 1 .mu.g/.mu.L in
CHCl.sub.3, 0.015 .mu.mol, Avanti Polar Lipids, Inc) was added and
vortexes. The mixture was dried into a film under N.sub.2 and
placed under vacuum overnight.
[0214] 39. Preparation of DOTAP-Cl/NegPep-DDAB:
[0215] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added dodecyldimethylammonium bromide (0.45
.mu.g, 0.0011 .mu.mol, Aldrich Chemical Company). The resulting
solution was vortexes, frozen and lyophilized. The freeze dried
material was brought up in CHCl.sub.3 (50 .mu.L). DOTAP-Cl (10
.mu.g of 1 .mu.g/.mu.L in CHCl.sub.3, 0.014 .mu.mol, Avanti Polar
Lipids, Inc) was added and vortexes. The mixture was dried into a
film under N.sub.2 and placed under vacuum overnight.
[0216] 40. Preparation of DOTAP-Cl/NegPep-MC753:
[0217] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added MC753 (0.45 .mu.g, 0.0011 .mu.mol,
Mirus Corporation). The resulting solution was vortexes, frozen and
lyophilized. The freeze dried material was brought up in CHCl.sub.3
(50 .mu.L). DOTAP-Cl (10 .mu.g of 1 .mu.g/.mu.L in CHCl.sub.3,
0.014 .mu.mol, Avanti Polar Lipids, Inc) was added and vortexes.
The mixture was dried into a film under N.sub.2 and placed under
vacuum overnight.
[0218] 41. Preparation of DDAB/NegPep-MC753:
[0219] To a solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in
H.sub.2O (25 .mu.L) was added MC753 (1.4 .mu.g, 0.0034 .mu.mol,
Mirus Corporation). The resulting solution was vortexes, frozen and
lyophilized. The freeze dried material was brought up in CHCl.sub.3
(50 .mu.L). Dodecyldimethylammonium bromide (6.0 .mu.g of 1
.mu.g/.mu.L in EtOH, 0.015 .mu.mol, Aldrich Chemical Company) was
added and vortexes. The mixture was dried into a film under N.sub.2
and placed under vacuum overnight.
[0220] 42. Preparation of DOTAP-Cl/NegPep:
[0221] A solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in H.sub.2O
(25 .mu.L) was vortexes, frozen and lyophilized. The freeze dried
material was brought up in CHCl.sub.3 (50 .mu.L). DOTAP-Cl (10
.mu.g of 1 .mu.g/.mu.L in CHCl.sub.3, 0.014 .mu.mol, Avanti Polar
Lipids, Inc) was added and vortexes. The mixture was dried into a
film under N.sub.2 and placed under vacuum overnight.
[0222] 43. Preparation of DDAB/NegPep:
[0223] A solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in H.sub.2O
(25 .mu.L) was vortexes, frozen and lyophilized. The freeze dried
material was brought up in CHCl.sub.3 (50 .mu.L).
Dodecyldimethylammonium bromide (6.0 .mu.g of 1 .mu.g/.mu.L in
EtOH, 0.015 .mu.mol, Aldrich Chemical Company) was added and
vortexes. The mixture was dried into a film under N.sub.2 and
placed under vacuum overnight.
[0224] 44. Preparation of Me.sub.3Sphing/NegPep:
[0225] A solution of NegPep (2.0 .mu.g, 0.0011 .mu.mol) in H.sub.2O
(25 .mu.L) was vortexes, frozen and lyophilized. The freeze dried
material was brought up in CHCl.sub.3 (50 .mu.L).
N,N,N-Trimethylsphingosine (5.0 .mu.g of 1 .mu.g/.mu.L in
CHCl.sub.3, 0.015 .mu.mol, Avanti Polar Lipids, Inc) was added and
vortexes. The mixture was dried into a film under N.sub.2 and
placed under vacuum overnight.
[0226] 45. Preparation of DOTAP-Cl/PosPep-CDM12 (1:1):
[0227] To a solution of PosPep (2.0 .mu.g, 0.00095 .mu.mol) in
H.sub.2O (25 .mu.L) was added CDM12 (0.33 .mu.g, 0.00095 .mu.mol,
Mirus Corporation). The resulting solution was vortexes, frozen and
lyophilized. The freeze dried material was brought up in CHCl.sub.3
(50 .mu.L). DOTAP-Cl (10 .mu.g of 1 .mu.g/.mu.L in CHcl.sub.3,
0.014 .mu.mol, Avanti Polar Lipids, Inc) was added and vortexes.
The mixture was dried into a film under N.sub.2 and placed under
vacuum overnight.
[0228] 46. Preparation of DOTAP-Cl/PosPep-CDM12 (1.6):
[0229] To a solution of PosPep (2.0 .mu.g, 0.00095 .mu.mol) in
H.sub.2O (25 .mu.L) was added CDM12 (2.0 .mu.g, 0.0057 .mu.mol,
Mirus Corporation). The resulting solution was vortexes, frozen and
lyophilized. The freeze dried material was brought up in CHCl.sub.3
(50 .mu.L). DOTAP-Cl (10 .mu.g of 1 .mu.g/.mu.L in CHCl.sub.3,
0.014 .mu.mol, Avanti Polar Lipids, Inc) was added and vortexes.
The mixture was dried into a film under N.sub.2 and placed under
vacuum overnight.
[0230] 47. Preparation of DOTAP-Cl/PosPep-AOT:
[0231] To a solution of PosPep (2.0 .mu.g, 0.00095 .mu.mol) in
H.sub.2O (25 .mu.L) was added dioctylsulfosuccinate sodium salt
(0.42 .mu.g, 0.00095 .mu.mol, Aldrich Chemical Company). The
resulting solution was vortexes, frozen and lyophilized. The freeze
dried material was brought up in CHCl.sub.3 (50 .mu.L). DOTAP-Cl
(10 .mu.g of 1 .mu.g/.mu.L in CHCl.sub.3, 0.014 .mu.mol, Avanti
Polar Lipids, Inc) was added and vortexes. The mixture was dried
into a film under N.sub.2 and placed under vacuum overnight.
[0232] 48. Preparation of AOT/PosPep:
[0233] A solution of PosPep (2.0 .mu.g, 0.00095 .mu.mol) in
H.sub.2O (25 .mu.L) was vortexes, frozen and lyophilized. The
freeze dried material was brought up in CHCl.sub.3 (50 .mu.L).
Dioctylsulfosuccinate sodium salt (6.0 .mu.g of 1 .mu.g/.mu.L in
EtOH, 0.011 .mu.mol, Aldrich Chemical Company) was added and
vortexes. The mixture was dried into a film under N.sub.2 and
placed under vacuum overnight.
[0234] 49. Preparation of DOTAP-Cl/PosPep:
[0235] A solution of PosPep (2.0 .mu.g, 0.00095 .mu.mol) in
H.sub.2O (25 .mu.L) was vortexes, frozen and lyophilized. The
freeze dried material was brought up in CHCl.sub.3 (50 .mu.L).
DOTAP-Cl (10 .mu.g of 1 .mu.g/.mu.L in CHCl.sub.3, 0.014 .mu.mol,
Avanti Polar Lipids, Inc) was added and vortexes. The mixture was
dried into a film under N.sub.2 and placed under vacuum
overnight.
[0236] 50. Preparation of DOTAP-Cl/NES-Zwitt 3-12 (1-3):
[0237] To a solution of NES (2.0 .mu.g, 0.00095 .mu.mol) in
H.sub.2O (25 .mu.L) was added Zwittergent 3-12 (1.0 .mu.g, 0.0030
.mu.mol, Calbiochem). The resulting solution was vortexes, frozen
and lyophilized. The freeze dried material was brought up in
CHCl.sub.3 (50 .mu.L). DOTAP-Cl (10 .mu.g of 1 .mu.g/.mu.L in
CHCl.sub.3, 0.014 .mu.mol, Avanti Polar Lipids, Inc) was added and
vortexes. The mixture was dried into a film under N.sub.2 and
placed under vacuum overnight.
[0238] 51. Preparation of DOTAP-Cl/NES-Zwitt 3-12 (1:6):
[0239] To a solution of NES (2.0 .mu.g, 0.00095 .mu.mol) in
H.sub.2O (25 .mu.L) was added Zwittergent 3-12 (2.0 .mu.g, 0.0060
.mu.mol, Calbiochem). The resulting solution was vortexes, frozen
and lyophilized. The freeze dried material was brought up in
CHCl.sub.3 (50 .mu.L). DOTAP-Cl (10 .mu.g of 1 .mu.g/.mu.L in
CHCl.sub.3, 0.014 .mu.mol, Avanti Polar Lipids, Inc) was added and
vortexes. The mixture was dried into a film under N.sub.2 and
placed under vacuum overnight.
[0240] 52. Preparation of DOTAP-Cl/NES-Zwitt 3-14 (1:3):
[0241] To a solution of NES (2.0 .mu.g, 0.00095 .mu.mol) in
H.sub.2O (25 .mu.L) was added Zwittergent 3-14 (1.0 .mu.g, 0.0027
.mu.mol, Calbiochem). The resulting solution was vortexes, frozen
and lyophilized. The freeze dried material was brought up in
CHCl.sub.3 (50 .mu.L). DOTAP-Cl (10 .mu.g of 1 .mu.g/.mu.L in
CHCl.sub.3, 0.014 .mu.mol, Avanti Polar Lipids, Inc) was added and
vortexes. The mixture was dried into a film under N.sub.2 and
placed under vacuum overnight.
[0242] 53. Preparation of DOTAP-Cl/NES-Zwitt 3-14 (1:6):
[0243] To a solution of NES (2.0 .mu.g, 0.00095 .mu.mol) in
H.sub.2O (25 .mu.L) was added Zwittergent 3-14 (2.0 .mu.g, 0.0055
.mu.mol, Calbiochem). The resulting solution was vortexes, frozen
and lyophilized. The freeze dried material was brought up in
CHCl.sub.3 (50 .mu.L). DOTAP-Cl (10 .mu.g of 1 .mu.g/.mu.L in
CHCl.sub.3, 0.014 .mu.mol, Avanti Polar Lipids, Inc) was added and
vortexes. The mixture was dried into a film under N.sub.2 and
placed under vacuum overnight.
[0244] 54. Preparation of DOTAP-Cl/NES-CDM12 (1:2):
[0245] To a solution of NES (2.0 .mu.g, 0.00095 .mu.mol) in
H.sub.2O (25 .mu.L) was added CDM12 (0.67 .mu.g, 0.0019 .mu.mol,
Calbiochem). The resulting solution was vortexes, frozen and
lyophilized. The freeze dried material was brought up in CHCl.sub.3
(50 .mu.L). DOTAP-Cl (10 .mu.g of 1 .mu.g/.mu.L in CHCl.sub.3,
0.014 .mu.mol, Avanti Polar Lipids, Inc) was added and vortexes.
The mixture was dried into a film under N.sub.2 and placed under
vacuum overnight.
[0246] 55. Preparation of DOTAP-Cl/NES:
[0247] A solution of NES (2.0 .mu.g, 0.00095 .mu.mol) in H.sub.2O
(25 .mu.L) was vortexes, frozen and lyophilized. The freeze dried
material was brought up in CHCl.sub.3 (50 .mu.L). DOTAP-Cl (10
.mu.g of 1 .mu.g/.mu.L in CHCl.sub.3, 0.014 .mu.mol, Avanti Polar
Lipids, Inc) was added and vortexes. The mixture was dried into a
film under N.sub.2 and placed under vacuum overnight.
[0248] 56. Preparation of DOTAP-Cl/Cholesterol/NES:
[0249] DOTAP-Cl (10 .mu.g, 0.2 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) and Cholesterol (5 .mu.g, 0.1 .mu.g/.mu.L in
chloroform, Avanti Polar Lipids, Inc) was added to a micro
centrifuge tube and air dried to produce a lipid film. To the lipid
film was added a solution of NES (2 .mu.g) in PBS (30 .mu.L). The
resulting mixture was vortexes, and allowed to hydrate for 10-30
min, followed by brief gentle vortexing and bath sonication (5
sec).
[0250] 57. Preparation of DOTAP-Cl/Cholesterol/Oleic
Acid/PosPep:
[0251] DOTAP-Cl (10 .mu.g, 0.2 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) and Cholesterol (2.5 .mu.g, 0.05 .mu.g/.mu.L in
chloroform, Avanti Polar Lipids, Inc) and Oleic Acid (0.2 .mu.g,
0.004 .mu.g/.mu.l in chloroform, Sigma) was added to a micro
centrifuge tube and air dried to produce a lipid film. To the lipid
film was added a solution of PosPep (2 .mu.g) in PBS (30 .mu.L).
The resulting mixture was vortexes, and allowed to hydrate for
10-30 min, followed by brief gentle vortexing and bath sonication
(5 sec).
[0252] 58. Preparation of DOTAP-Cl/Cholesterol/IgG F(ab)
Fragment:
[0253] DOTAP-Cl (10 .mu.g, 0.2 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) and Cholesterol (2.5 .mu.g, 0.05 .mu.g/.mu.L in
chloroform, Avanti Polar Lipids, Inc) was added to a micro
centrifuge tube and air dried to produce a lipid film. To the lipid
film was added a solution of FITC-IgG F(ab) fragment in PBS (30
.mu.L). The resulting mixture was vortexes, and allowed to hydrate
for 10-30 min, followed by brief gentle vortexing and bath
sonication (5 sec).
[0254] 59. Preparation of DOTAP-Cl/Cholesterol/IgG:
[0255] DOTAP-Cl (10 .mu.g, 0.2 .mu.g/.mu.L in chloroform, Avanti
Polar Lipids, Inc) and Cholesterol (2.5 .mu.g, 0.05 .mu.g/.mu.L in
chloroform, Avanti Polar Lipids, Inc) was added to a micro
centrifuge tube and air dried to produce a lipid film. To the lipid
film was added a solution of cy3-IgG in PBS (30 .mu.L). The
resulting mixture was vortexes, and allowed to hydrate for 10-30
min, followed by brief gentle vortexing and bath sonication (5
sec).
[0256] Unless otherwise stated, dried polypeptide formulations were
hydrated by addition of 30-50 .mu.l PBS. Formulations were
incubated at RT for 20 min at RT with occasional gentle vortexing
and 5-10 sec bath sonication.
Example 5
Cell Growth
[0257] HeLa cells were maintained in DMEM containing 10% fetal
bovine serum. For polypeptide delivery, cells were plated in 6-well
plates containing glass cover slips at a density of 50,000-100,000
cells per well and grown for 24-48 hours before incubation with
polypeptide formulations.
Example 6
Peptide Delivery to Cells
[0258] Two different techniques were used for contacting
polypeptide formulations with cells. In both techniques, cells were
grown to 30-70% confluency and washed once with 2 ml 37.degree. C.
PBS prior to addition of polypeptide formulations.
[0259] Technique #1:
[0260] Peptide formulations were diluted by addition of 150 .mu.l
37.degree. C. PBS (180-200 .mu.l total volume). Formulations were
than added to cells on cover slips. Following a 5 min incubation at
room temperature (RT) 0.8 ml 37.degree. C. DMEM was added to each
well and cells were again incubated for 5 min at RT. Media was then
aspirated off cells and 2.0 ml DMEM containing 10% bovine serum was
added. Cells were than incubated 40-60 min at 37.degree. C. in a
humidified CO.sub.2 incubator.
[0261] Technique #2:
[0262] Peptide formulations were diluted by addition of 950 .mu.l
37.degree. C. OptiMEM (GIBCO). Formulations were then added to
cells on cover slips and incubated for 4 h at 37.degree. C. in a
humidified CO.sub.2 incubator.
[0263] Visualization of Polypeptide Delivery to Cells:
[0264] In order to visually analyze polypeptide delivery, cells
were washed 3.times. with PBS, fixed for 30 min at 4.degree. C. in
PBS containing 4% formaldehyde and washed 3.times. with PBS. Cover
slips were then mounted onto slides for fluorescence microscopy
using a Zeiss LSM510 laser confocal microscope. Rhodamine was
detected by exciting with a HeNe laser at 543 nm and detecting with
a 560 nm long pass filter. For control samples, peptides alone were
used under the same concentrations.
Example 7
Demonstration of Peptide Delivery
[0265] Typically, for introduction of DNA into cells, a cationic
lipid along with the helper lipid DOPE is used. The inclusion of
DOPE is believed to facilitate exit of the complex from cellular
endosomes. Similar strategies have also been employed in attempts
to deliver proteins into cells, i.e. cationic lipids with DOPE. We
were unable to observe cytoplasmic delivery of polypeptides into
cells with a number of liposomes when using typical formulations:
formation of liposomes (cationic lipid/DOPE at 2:1 molar ratio) in
the presence of the polypeptide, dilution of the liposomes in
culture media and incubation of the liposomes with cells for
several hours.
[0266] However, using our polypeptide modifications, formulations
and cell incubation techniques, we have successfully delivered
polypeptides to the cell interior, for examples see FIG. 1. The
diffuse cellular staining patterns demonstrate that the
polypeptides are cytoplasmically delivered. Endocytosis and then
entrapment within endosomes would have appeared as a punctate
staining pattern.
[0267] While the cationic lipid DOTAP was used in these examples,
the modifications, formulations, and techniques used here would
also be applicable to other cationic lipids.
Example 8
Demonstration of Protein Delivery
[0268] There is no receptor-specific or nonspecific interaction
between Bovine Serum Albumin (BSA) and most cell types. Therefore,
incubation of BSA with cells does not lead to cell association or
internalization of the protein (see FIG. 2A). Similarly, there is
also no association of most cell types with IgG's or IgG F(ab)
fragments (data not shown). However, using the methods detailed
above, we have successfully delivered all three of these proteins
to HeLa cells. FIG. 2B shows the uptake of BSA when the BSA is
modified according to modification method #26. Both IgG F(ab)
fragment and IgG were delivered to HeLa cells using formulations
#58 and #59 (FIG. 2, C and D). These modifications and formulations
could be used to deliver many different proteins to cells.
Sequence CWU 1
1
3 1 13 PRT Artificial synthetic peptide derived from HIV REV
nuclear export signal 1 Arg Leu Gln Leu Pro Pro Leu Glu Arg Leu Thr
Leu Asp 1 5 10 2 14 PRT Artificial synthetic positively charged
peptide 2 Gly Lys Asn Arg Gly Lys Ser Ala Gln Ala Lys Arg Leu Arg 1
5 10 3 12 PRT Artificial synthetic peptide derived from alpha
tubulin gene 3 Gly Glu Gly Met Glu Glu Gly Glu Phe Ser Glu Ala 1 5
10
* * * * *