MICROBUBBLE COMPLEXES AND METHODS OF USE
[0001] The invention relates generally to novel binding of therapeutic agents to
albumin microbubble pharmaceuticals using an attachment of albumin affinity ligands to
the agents. The binding provides a method of microbubble-assisted delivery of
therapeutic agents to targeted cells or tissue of interest, either in vitro or in vivo.
[0002] Ultrasound-mediated destruction of microbubbles carrying drugs has been
found to be useful as a noninvasive drug delivery system. Drugs or other therapeutic
agents can be incorporated into the microbubbles in a number of different ways,
including binding of the drug to the microbubble shell and attachment of ligands. For
example, perfiuorocarbon-filled microbubbles are sufficiently stable for circulating in the
vasculature as blood pool agents; they act as carriers of these agents until the site of
interest is reached. Ultrasound applied over the skin surface can then be used to burst the
microbubbles at this site, causing localized release of the drug or therapeutic agents on
site specific locations.
[0003] More specifically, albumin microbubbles have been used and delivered to a
specific organ target by site-directed acoustic ultrasound. Albumin is a major protein in
blood, and its natural physiological role is to bind and carry a wide variety of
lipophilic/poorly soluble ligands throughout the body. These ligands, which may have an
affinity to albumin, include fatty acids and other biosynthetic and catabolic products that
are hydrophobic in nature. As such albumin microbubbles have been used to carry a
variety of therapeutic agents based on proteins and other biologies including,
oligonucleotides (ODN) and polynucleotides such as antisense ODN, with sequences
complementary to a specific targeted messenger RNA (mRNA) sequence. These
microbubble-nucleic acid complexes may be generated from unmodified ODN that are
mixed with albumin or lipid components during microbubble shell formation or
alternatively, the complex formation can be performed by mixing preformed
microbubbles with an ODN of interest. In both cases, the ODN acts as a mechanistic
intervention in the processes of gene translation or an earlier processing event. The
advantage of this approach is the potential for gene-specific actions which should be
reflected in a relatively low dose and minimal non-targeted side effects.
[0004] However, a key barrier to translating the potent biology of ODN into
drugs is known to be at the level of drug delivery with efficacy and safety. For example,
ODN delivery with chemical formulation, viral vectors, and particle delivery have been
hampered with clinical safety related problems before therapeutic efficacy can be
attained. Furthermore, the use of albumin microbubbles as a carrier of ODNs such as
siRNA is limited due to the limited binding of the ODNs to the albumin microbubble as
well as the stability of the albumin-ODN complex. Due to negative shell surface
potential of albumin, the negatively charged shorter nucleic acids do not bind very well to
the microbubble and gene transfection efficiencies using these complexes are generally
suboptimal.
[0005] Thus there is a need to improve the binding of the therapeutic agents to the
microbubble as well as improving the stability and efficacy of the microbubble complex.
[0006] Furthermore there is a need to reduce toxicity in the selective delivery of
highly cytotoxic drugs. Non-targeted delivery of these drugs can cause systemic toxicity
and has prevented the use of many of these drugs all together or at higher doses required
for good efficacy. Attempts to deliver these as pro-drugs in many cases have reduced this
problem, however, selective uptake in the targeted tissue is not always easy to achieve as
most of the uptake mechanisms in the diseased tissue are also present in the normal
tissue. Enhancing the uptake of these drugs in selective tissues by non-natural
mechanisms as disclosed herein, therefore can add considerable value.
[0007] Provided herein are novel compositions and methods for increased binding of
therapeutic drugs to microbubbles using the affinity of ligand-therapeutic compositions
towards the albumin shell.
[0008] Systemic circulation of the microbubbles carrying the therapeutic composition
can be easily visualized through ultrasound imaging. Therapeutic agent is released from
the microbubbles using a trigger of high energy pulsed ultrasound specific to the site of
treatment. The cavitation of microbubbles causes sonoporation of the neighboring
cells/tissue.
[0009] In one embodiment a microbubble complex is disclosed comprising a
microbubble having an outer shell comprising a mixture of native and denatured albumin
and a hollow core encapsulating a perfiuorocarbon gas, a therapeutic agent selected
from the group comprising a small molecule chemotherapeutic agent, a peptide, a
carbohydrate, or an oligonucleotide, and a bifunctional linker having one end attached to
the therapeutic agent and the other attached to a ligand through reaction of a reactive
group on the ligand. The ligand is bound to the outer shell of the microbubble through
hydrophobic interactions.
[0010] In another embodiment a method is for delivering the aforementioned
microbubble complex to a tissue target is disclosed. The method comprising the steps of
providing the microbubble complex, administrating the microbubble complex to a subject
wherein the subject is the source of the tissue target, and administering ultrasonic energy
to the subject, wherein the ultrasound energy is sufficient to cause cavitation of the
microbubble complex in the tissue target.
[001 1] These and other features, aspects, and advantages of the present invention
will become better understood when the following detailed description is read with
reference to the accompanying figures wherein:
[0012] FIG. 1 is a representation of non-covalent binding of siRNA-ligand to
albumin microbubbles.
[0013] FIG. 2 is representative micrograph of a gel shift assay for a mixture of
cholesterol conjugated siRNA (2 pmoles) and varying amounts of Optison (0, 9, 22 and
46 pmoles for i, ii, iii and iv respectively).
[0014] FIG. 3 is a. representative micrograph of a gel shift assay for Cy3-siRNA
(4 pmoles) mixed with varying concentrations of either Optison or native HSA which
shows no shift in gel assay.
[0015] FIG. 4 is a. representative micrograph of a gel assay for Cy3-cholesterolsiRNA
(2 pmoles) mixed with varying concentrations of either Optison, native HSA or
denatured HSA.
[0016] FIG. 5 is a graphical representation of binding properties of cholesterolsiRNA
to Optison and native HSA; (A) Relative fluorescence of cholesterol-siRNA
bands is measured from the gel shift assay (B) fraction bound of siRNA calculated from
relative fluorescence and plotted against the albumin concentration.
[0017] FIG. 6 is a graphical representation of the uptake of siRNA by U-87 tumor
cells in opticell is quantified by measuring cell cy3-fluorescence.
[0018] FIG. 7 includes micrographs of fluorescence images showing a comparison of
siRNA transfection between a lipid transfection reagent (RNAifect) and Optison.
[0019] FIG. 8 is a graphical representation of the mean cell fluorescence values and
standard error of siRNA transfection between a lipid transfection reagent (RNAifect) and
Optison.
[0020] FIG. 9 is a graphical representation of the fraction bound of fluoresceinmyristate
to Optison and native HSA as calculated from anisotropy values.
[0021] FIG. 10 A and B are the fluorescein bound to Optison (0, 8, 40 and 200
pmoles for i, ii, iii and iv respectively) and visualized on the gel as dark bands for
fluorescein-myristate (63 pmoles) and fiuorescein-stearate (180 pmoles ) respectively.
The following detailed description is exemplary and not intended to limit the invention of
the application and uses of the invention. Furthermore, there is no intention to be limited
by any theory presented in the preceding background of the invention or descriptions of
the drawings.
[0022] The invention relates generally to microbubble-assisted delivery of a
therapeutic agent to cells or tissue of interest, either in vitro or in vivo.
[0023] In certain embodiment the therapeutic agent may be comprised of a small
molecule chemotherapeutic agent, a peptide, a carbohydrate, or an oligonucleotide; and a
bifunctional linker having one end attached to the therapeutic agent and the other
attached to a ligand through reaction of a reactive group on the ligand, and wherein the
ligand is bound to the outer shell of the microbubble. In certain embodiment the
therapeutic agent may be an oligonucleotide (ODN). Oligonucleotides refers to nucleic
acid polymers that are formed by bond cleavage of longer nucleic acids or are
synthesized using building blocks, protected phosphoramidites of natural or chemically
modified nucleosides or, to a lesser extent, of non-nucleosidic compounds. The length of
the oligonucleotide may vary from a short nucleic acid polymer of fifty or fewer base
pairs to more than 200 base pairs. As used herein, ODN also refers to polynucleotides
having more than 200 base pairs. Also included are antisense ODN which refer to single
strands of DNA or RNA that are complementary to a chosen sequence. In the case of
antisense RNA, antisense RNA prevents protein translation of certain messenger RNA
strands by binding to them. Antisense DNA can be used to target a specific,
complementary (coding or non-coding) RNA. Also included are small interfering RNA
(siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of
double-stranded RNA molecules, typically 20-25 nucleotides in length, that play a variety
of roles in biology including the RNA interference (RNAi) pathway, where it interferes
with the expression of a specific gene, as an antiviral mechanism, or in shaping the
chromatin structure of a genome.
[0024] In certain embodiments, the therapeutic agent may be a cytotoxin. As used
herein cytotoxin refers to a substance that has a toxic effect on cells. For example a
cytotoxin may cause undergo necrosis, in which they lose membrane integrity and die as
a result of cell lysis. In other examples a cytotoxin may be associated with antibodydependent
cell mediated cytotoxicity wherein a cell is marked by an antibody and acted
upon by certain lymphocytes.
[0025] Examples of cytotoxic agents are listed in Goodman and Gilman's "The
Pharmacological Basis of Therapeutics," Tenth Edition, McGraw-Hill, New York, 2001.
These include taxol; nitrogen mustards, such as mechlorethamine, cyclophosphamide,
melphalan, uracil mustard and chlorambucil; ethylenimine derivatives, such as thiotepa;
alkyl sulfonates, such as busulfan; nitrosoureas, such as carmustine, lomustine, semustine
and streptozocin; triazenes, such as dacarbazine; folic acid analogs, such as methotrexate;
pyrimidine analogs, such as fluorouracil, cytarabine and azaribine; purine analogs, such
as mercaptopurine and thioguanine; vinca alkaloids, such as vinblastine and vincristine;
antibiotics, such as dactinomycin, daunorubicin, doxorubicin, bleomycin, mithramycin
and mitomycin; enzymes, such as L-asparaginase; platinum coordination complexes,
such as cisplatin; substituted urea, such as hydroxyurea; methyl hydrazine derivatives,
such as procarbazine; adrenocortical suppressants, such as mitotane; hormones and
antagonists, such as adrenocortisteroids (prednisone), progestins (hydroxyprogesterone
caproate, medroprogesterone acetate and megestrol acetate), estrogens (diethylstilbestrol
and ethinyl estradiol), antiestrogens (tamoxifen), and androgens (testosterone propionate
and fiuoxymesterone).
[0026] Drugs that interfere with intracellular protein synthesis, protein synthesis
inhibitors, can also be coupled to the ligand; such drugs are known to these skilled in the
art and include puromycin, cycloheximide, andribonuclease.
[0027] In one embodiment, the protein, which the therapeutic agent acts as an
inhibitor for, includes, but is not limited to, enzymes, soluble and serum proteins, proteins
expressed on a surface of a cell, non-immunoglobulin proteins, intracellular proteins, and
segment of proteins that are or can be made water-soluble, either individually or in
combinations thereof as well as any derivatives of the proteins.
[0028] In a particular embodiment, the protein includes such as, but not limited to,
cysteine proteases, glutathione S transferase, epoxide hydrolase (EH), thiolase,
NAD/NADP-dependent oxidoreductase, enoyl coA hydratase, aldehyde dehydrogenase,
hydroxypyruvate reductase, tissue transglutaminase (tTG), formiminotransferase
cyclodeaminase (FTCD), aminolevulinate -dehydratase (ADD), creatin kinase,
carboxylesterase (LCE), monoacylglycerol (MAG) lipase, metalloproteases (MP),
phosphotases (protein tyrosine phosphotases, PTP), proteosome, FK506 binding protein
(FKBP12), mammalian target of Rapamycin (mTOR; alternatively known as FKBPrapamycin
binding domain (FRB)), serine hydrolase (superfamily), ubiquitin-binding
protein, -galactosidase, nucleotide binding enzymes, protein kinases, GTP-binding
proteins, cutinase, adenylosuccinate synthase, adenylosuccinate lyase, glutamate
dehydrogenase, dihydrofolate reductase, fatty acid synthase, aspartate transcarbamylase,
acetylcholinesterase, HMG cholate reductase, and cyclo-oxygenase (COX-1 and COX-2),
either individually or in combinations thereof. Also included are any derivatives of any
of the proteins.
[0029] In another example, the protein is substantially free of a cofactor.
"Substantially free of a cofactor" includes proteins that do not require any additional
cofactor, chemical, chemical modification, or physical modification to be naturally stable
under physiological conditions and room temperature and pressure in solution or as a
solid, and can bind its corresponding ligand in vivo.
[0030] In one embodiment, an albumin microbubble may be utilized to carry a
therapeutic agent in systemic delivery. Tissue targeted ultrasound acoustic energy may
then be used to cavitate the albumin microbubble and deliver the therapeutic agent into
the intracellular environment. For example the microbubble complex may be
administered intravenously or into the peritoneum (intraperitoneally) of a subject whose
cells or tissues are to be targeted. Once the microbubble complex is carried through the
subject to the targeted cell, the ultrasound acoustic energy is delivered. In certain
embodiments, visualization of the targeted cells may occur prior to delivering the
ultrasound while in still other embodiments visualization may be performed in real time
and the cavitation monitored.
[0031] In certain embodiments, the albumin outer shell of the microbubble is
comprised of both native and denatured albumin held together by mostly cysteine to
cysteine bonds. In certain embodiments, the primary composition of the albumin shell is
mostly in the native form wherein the denatured portion allows for increased cysteine
bond attachments. In certain embodiments the relative amount of denatured albumin to
native albumin ranges from approximately 0.5 to 30wt%. In other embodiments the
relative amount is in the range of approximately 1% to 15wt%. The mixture of native and
denatured albumin provides a balance of shell elasticity needed for cavitation, with
increased reactive binding sites on the microbubble surface. The microbubbles may be
formed by sonication of perfiuorocarbon gas in the presence of pre -heated albumin
solution, or alternatively by mixing of the gas and the pre -heated albumin using high
shear forces. A small part of the albumin molecules rearrange during sonication of pre
heated albumin solution and crosslinking occurs through disulfide linkages between
albumin molecules. These albumin molecules are believed to be similar in structure to an
F form of albumin which has more hydrophobic residues exposed. This allows increased
binding sites for hydrophobic interactions.
[0032] In certain embodiments, the microbubble may be filled with an insoluble
perfiuorocarbon gas, such as but not limited to, perfluoromethane, perfiuoroethane,
perfluoropropane, perfluorobutane, perfluoropentane, or a combination thereof. In
certain embodiments, the microbubbles are about 1 to about 5 microns in diameter, the
size being optimized to allow circulation through the blood stream.
[0033] In certain embodiments, the therapeutic-microbubble complexes comprise a
therapeutic agent modified with a linker having a reactive group capable of binding with
a ligand having affinity towards albumin. As such, the therapeutic agent may be coupled
to albumin though the ligand.
[0034] The linker includes any linking moiety that attaches the ligand to the
therapeutic agent through a first moiety. The linker can be as short as one carbon or a
long polymeric species such as polyethylene glycol, tetraethylene glycol (TEG),
polylysine or other polymeric species normally used in the pharmaceutical industry for
modulating pharmacokinetic and biodistribution characteristics of therapeutic agents.
Other linkers of varying length include C1-C250 length with one or more heteroatoms
selected from O, S, N, P, and optionally substituted with halogen atoms. In a particular
embodiment, the linker comprises at least one of an oligomeric or polymeric species
made of natural or synthetic monomers, oligomeric or polymeric moiety selected from a
pharmacologically acceptable oligomer or polymer composition, an oligo- or poly- amino
acid, peptide, saccharide, a nucleotide, and an organic moiety with 1-250 carbon atoms,
either individually or in combination thereof. The organic moiety with 1-250 carbon
atoms may contain one or more heteroatoms such as O, S, N or P and optionally
substituted with halogen atoms at one or more places.
[0035] The first moiety may simply be an extension of the linker, formed by the
reaction of a reactive species on the linker with a reactive group on the therapeutic agent.
Examples of reactive species and the reactive group include, but are not limited to,
activated esters (such as N-hydroxysuccinimide ester, pentafluorophenyl ester), a
phosphoramidite, an isocyanate, an isothiocyanate, an aldehyde, an acid chloride, a
sulfonyl chloride, a maleimide an alkyl halide, an amine, a phosphine, a phosphate, an
alcohol or a thiol with the proviso that the reactive species and reactive group are
matched to undergo a reaction yielding covalently linked conjugates.
[0036] In certain embodiments, the reactive group may be a primary amine
functionality, and as such, the amine modified therapeutic agent may be conjugated to the
affinity ligand through reaction of a carboxyl moiety of the ligand. In certain other
embodiments, the reactive group may be an alcohol attached to the ligand through a
phosphate group.
[0037] The ligand, also named the affinity ligand herein, includes fatty acids,
steroids, small aromatic compounds or a combination thereof. Examples of albumin
binding molecules may be found in US patent application publication number
2010/0172844, published July 8, 2010.
[0038] For example in certain embodiments the affinity ligand is a fatty acid,
including but not limited to myristoyl, lithocolic-oleyl, docosanyl, lauroyl, steoroyl,
palmitoyl, oleoyl, or linoleoyl. In other embodiments, the affinity ligand is a lipophilic
molecule such as a steroid or modified steroid including, cholesterol, cholic acid,
lithocholic acid, or chenodeoxycholic acid. In other embodiments, the affinity ligand is a
high affinity molecule selected from 4-p-iodophenyl-butyric acid and analogs or
derivatives thereof. In still other embodiments, the therapeutic agent comprises siR A,
the linker comprises tetraethylene glycol, and the ligand comprises cholesterol.
[0039] In certain embodiments, the therapeutic-albumin complexes may be prepared
either by sonicating ligand-modified therapeutic agent with albumin or lipid in the
presence of perfluorocarbon or by mixing preformed bubbles with ligand modified
therapeutic agents. In certain embodiments, these molecules may be attached to
therapeutic agents of interest during therapeutic agent synthesis. For example the
phosphoramidites of cholesterol may be used to incorporate cholesterol during DNA or
RNA synthesis on a nucleic acid synthesizer, or post synthesis by incorporating a reacting
moiety.
[0040] In certain embodiments, the therapeutic agent is a modified ODN which may
be prepared enzymatically by using modified nucleoside triphosphates; modified either
with the ligand itself or with a reactive functionality for post synthesis modification with
the ligand. Ligand attachment may be at one or both termini, internal to the nucleic acid
sequence or at multiple positions depending upon the ODN use. In certain embodiments,
where siRNA is the ODN, attachment may be through the 3' OH position.
[0041] In certain embodiments, in addition to the ligand, the therapeutic agent,
including where the therapeutic agent is ODN, may be selectively modified to protect
from nucleases. In certain embodiments, stabilizing modification may include
phosphorothioate modification, or 2'-OMe modification.
[0042] In certain embodiments, the microbubble complexes may be incubated with
cells or the tissue of interest or injected into the body, preferably intravenously, and then
cavitated with ultrasound energy at desired site and at a predetermined time or during live
imaging.
[0043] In certain embodiments, the microbubble complex may be viewed during
systemic travel in the blood circulation under normal ultrasound diagnostic imaging.
When the bubble arrives on tissue target, in this case the tumor, a series of pulsed
acoustic energy waves are sent to the tumor. This creates inertial cavitation on the
microbubble, which collapses the microbubble. Cavitation of the microbubble occurs
where the acoustic energy is maximally located. This direction is achieved on the
ultrasound probe by parameters related to mechanical index force, optimal ultrasound
acoustic distance, and dimensions of the ultrasound acoustic sweep. The force generated
can then potentially form micropores within the cellular plasma membrane. Typically the
pulsed energy is administered at a frequency of about 0.5 to about 5MHz.
[0044] These micropores, along with the microjetting force created under inertial
cavitation, may facilitate the entrance of ODN into the cellular cytoplasmic environment.
For example when the ODN is siRNA, siRNA in the intracellular environment will utilize
the host machinery to silence mRNA and later protein synthesis. Similarly, where
mRNA message acts as an angiogenesis promoting proteins including vascular
endothelial growth factor (VEGF), the reduction of VEGF expression in a tumor may halt
or slow tumor growth. After microbubble cavitation the dense gas of the microbubble
center is exhaled out of the body and the albumin shell is metabolized and excreted via
the liver elimination pathway.
[0045] In an exemplary embodiment, a bolus of the microbubble complex may be
mixed to an optimal ratio from previous therapeutic investigations. Once the mixture of
the complex is established, the bolus drug is injected systemically by venous route.
[0046] For example in the use of a siRNA-microbubble bolus, the bolus may be
monitored in the first pass blood kinetics. The microbubble resonance and thus enhanced
ultrasound contrast may be monitored with an ultrasound probe using low diagnostic
levels of acoustic energy. During circulation the bolus arrives on organ target.
Cardiovascular tissue perfusion may assist in delivering the bolus into deep microvessels
with small lumen diameters. By supplying pulsed acoustic energy, sufficient energy may
be provided for the microbubble to undergo inertial cavitation. Once the microbubble
cavitation is complete the siRNA contents may be delivered across the plasma membrane
and into the diseased cell. While in the cytoplasmic intracellular environment the siRNA
can have a therapeutic effect.
[0047] During microbubble cavitation siRNA entrance into the cell may occur by
various mechanisms. For example the siRNA may enter the cell by a : a microjetting
force from the collapsing microbubble which can push siRNA into the cytoplasm.
Alternatively the mechanism may include microjetting energy or sonoluminescence
energy which creates temporary micropores within the plasma membrane to allow for
passive diffusion of the siRNA into the cell, or during microbubble resonance before
actual cavitation the microbubble bumping into the plasma membrane may push the
siRNA in the cell.
[0048] As such, the mechanism of microbubble delivery has potential applications in
the treatment of a wide variety of diseases, which can include cancer, inflammatory,
infectious, cardiovascular, metabolic, autoimmune, and central nervous system diseases.
Many of these diseases cannot currently be effectively treated by virtue of targeting
molecular mechanisms not accessible to conventional small molecule drugs and
monoclonal antibodies.
EXPERIMENTAL
Example 1 Microbubble-siRNA complex
[0049] FIG. 1 is a representation illustration of binding of siRNA to albumin
encapsulated microbubbles to form a microbubble-siRNA complex.
[0050] The target siRNA for VEGF silencing (vascular endothelial growth factor)
were synthesized by IDTDNA technologies. IDTDNA provided lipid modifications such
as cholesteryl TEG on the siRNA (Chol-siRNA) as well as dye conjugation.
Sense strand: 5*-Cy3/GCAUUUGUUUGUCCAAGAUmUmU/3 *-Lipid (SEQ ID NO: 1)
Antisense strand; 5'/mAmArA rUrCrU rUrGrG rArCrA rArArC rArArA rUrGrC/3'
(SEQ ID NO: 2)
[0051] Cyanine dye, Cy3 on the siRNA has an excitation wavelength of 550 nm and
a peak emission of 580 nm. The siRNA has been labeled with a cy3 dye for easy
visualization of siRNA during binding assays and other characterization techniques.
Optison™ (GE Healthcare, Chalfont St. Giles, United Kingdom, 10 mg/ml albumin) was
centrifuged; the top layer was discarded and the excess albumin solution in the bottom
was used for the binding studies. Lyophilized human serum albumin (HSA) powder
(Sigma Aldrich, St. Louis MO) was dissolved in IX phosphate buffered saline (PBS) to
make a stock solution of 10 mg/mL. Both Optison and native albumin solution dilutions
were prepared with IX PBS. Denatured HSA solution was prepared by heating native
HSA solution to 80 °C for 20 minutes.
Binding reaction:
[0052] The stock solutions of cy3-siRNA and cy3-siRNA-cholesterol, 20 mM were
prepared in RNAse free water and stored at -20 °C. 4 pmoles of cy3-siRNA and 2 pmoles
of cholesterol-siRNA solutions were mixed with varying amounts of Optison solution,
native HSA and denatured HSA solution, ranging from 0 to 50 pmoles. The reaction
buffer was IX PBS, pH 7.4. The reaction mixture was incubated under dark at 25 °C for
45 minutes. After incubation, ten mΐ of siR A mixtures was mixed with 2 mΐ of Novex®
Hi-Density TBE Sample Buffer (5X) (Invitrogen, Carlsbad, CA, USA).
Gel electrophoresis:
[0053] All the reactions were loaded onto precast 6% nondenaturing polyacrylamide
gels (Invitrogen, Carlsbad, CA, USA). The gel was run at 100 V for 45 min in 0.5X
Novex TBE Running buffer (Invitrogen, Carlsbad, CA, USA). The gels containing either
DNA, protein or both were imaged for cy3 fluorescence using a typhoon scanner
(Typhoon™9410, GE Healthcare).
Results:
[0054] The fluorescence of Cy3 attached to siRNA can be visualized as distinct
siRNA bands on the gel. When a mixture of siRNA and albumin solution was run on the
gel, the mobility of albumin bound-siRNA is slower than free-siRNA resulting in two
bands on the gel. In preliminary trials, sypro ruby stain from EMSA kit (Molecular
Probes, Eugene, OR, USA) was used to observe the albumin bands in the gel. An
example is shown in FIG. 2 which is a gel shift assay for a mixture of cholesterol
conjugated siRNA (2 pmoles) and varying amounts of Optison (0, 9, 22 and 46 pmoles
for i, ii, iii and iv respectively). Fluorescence imaging of the gel shows distinct bands for
siRNA (lower band sections) and albumin (upper band sections).
Cy3-siRNA
[0055] When the mixture of cy3-siRNA and native HSA/Optison was run on the gel,
there was no bound-siRNA visualized for increasing concentrations of albumin. There
was no significant binding of cy3-siRNA with either native HSA or Optison solution.
This is shown in FIG. 3 which is a fluorescence image of a gel for Cy3-siRNA (4 pmoles)
mixed with varying concentrations of either Optison or native HSA shows no shift in gel
assay. The dark bands on the gel are the cy3-fiuorescence on siRNA. There is no
significant binding of cy3-siRNA to both Optison and native HSA.
Chol-siRNA
[0056] FIG. 4 shows the gel images for binding of chol-siRNA with Optison, native
HSA and denatured HSA. Chol-siRNA bound to both native HSA and Optison solution,
while the binding significantly decreased for the same amount of denatured HSA. The
fluorescence intensity of siRNA in each lane was estimated manually by drawing a box
around the bands. Background, equivalent to the average intensity value of the gel, was
subtracted from the intensity value of each siRNA band. Fluorescence intensities of
bound siRNA over a wide range of albumin concentrations were calculated. Relative
fluorescence, R was calculated as:
R = (Fbound-Ffree)/ Ffree ( 1)
Fbound is fluorescence intensity of bound-siRNA band and Ffree is fluorescence
intensity of free-siRNA band. Relative fluorescence was plotted against albumin
concentration. This is shown in FIG. 5, which is a graphical comparison of binding
properties of cholesterol-siRNA to Optison and native HSA as described below.
[0057] At low albumin concentration ranging from 0 to 15 mM, linear dependence of
bound fluorescence on albumin concentration was visualized. FIG 5, graph A shows that
at this concentration range, the amount of chol-siRNA bound to Optison solution was
higher than the binding to native HSA. To estimate binding constants, higher
concentration of albumin was used to allow saturation of amount of siRNA bound to
albumin. Fraction bound, x is determined as:
x= (Fbound-Ffree)/ (Fsat-Ffree) (2)
Fsat is the fluorescence intensity of maximum bound-siRNA under saturation conditions.
[0058] Fraction bound was plotted against increasing albumin concentrations , as
shown in FIG. 5 graph B, and the data points were fitted to the following equilibrium
equation;
x = n* [Albumin]/ (kd + [Albumin]) (3)
kd is the dissociation constant, n is the number of binding sites and [Albumin] is the total
albumin concentration for the respective samples. Equation 3 was solved using a non
linear fit to determine kd and n for binding of chol-siRNA to both Optison and native
HSA (Table 1). Microsoft Excel's solver tool was utilized for this non-linear fit, and the
sum of squared errors (SSE) was found to be 0.07 and 0.06 for Optison and native HSA
respectively. The binding constant of chol-siRNA was similar for both Optison and
native HSA.
Example 2 Delivery of siRNA to tumor cells
Cell Culture:
[0059] MATBIII rat mammary carcinoma and U-87 human glioblastoma cells were
cultured in McCoy's 5A Medium (modified) (IX) (Invitrogen, Carlsbad, CA, USA) and
Eagle's Minimum Essential Medium (EMEM) (ATCC, Manassas, VA) respectively. Both
the media solutions were supplemented with 10% heat deactivated fetal bovine serum
(FBS) (Fisher Scientific, Springfield, NJ) and 1% penicillin-streptomycin (Sigma
Aldrich, St Louis, MO). The cells were maintained at 37 °C in a humidified atmosphere
with 5% C02.
Sonication of substrate-attached cells:
[0060] MATB-III and U-87 cells were grown in 10 mL capacity Opticell units (Nalge
Nunc International, Rochester, NY) to 90% confluence. The media in OptiCell was
replaced with 10 mL fresh media containing 40 pmoles of either cy3-siRNA or
cholesterol-siRNA. The opticell was left in the incubator for 24 hours at 37 °C.
Separately, the cells were either treated with siRNA solution mixed with Optison
microbubbles (300 ) or a lipid transfection reagent (90 ) (RNAifect, Qiagen,
Valencia, CA). For siRNA/Optison mixtures, Vivid i imagers with a cardiac sector probe
(3S) was used to rupture the microbubbles and deliver the siRNA drug from
microbubbles. The opticell was immobilized in a water bath, and the ultrasound probe
was attached to a motion arm that spanned the entire length of the opticell. The tip of the
probe was immersed in water, and the distance between the probe and opticell surface
was 3 cm that allowed sonication of the entire width of the opticell. The microbubbles in
opticell were treated with a mechanical index, MI > 1.3 continuous sonication. The probe
moved at a speed of 1 m/s over the entire length of the opticell. After sonication, the cells
were incubated for 24 hours at 37 °C. Similarly, the cells treated with RNAifect were also
kept in the incubator for 24 hours. After incubation, the cells were imaged using a
fluorescence microscope (Zeiss Axio Imager.Zl, Carl Zeiss). The filter used for cy3 was
DsRed/Cy3 (546 ex/620 em). In the region of interest (ROI) in the fluorescent images,
cell fluorescence was measured and the mean values of cell fluorescence were calculated.
ImageJ was used to process the images and calculate fluorescence intensities.
[0061] The data are reported as mean + 1.0 standard error (SE) for N=4. The
statistical significance of the differences between the groups was evaluated using twosample
t-test and the statistical analyses were carried out using Minitab® 12 (Minitab
Inc, State College, PA USA).
Results:
[0062] The effect of the delivery system is illustrated in FIG. 6 for U-87 cells
incubated with either cy3-siRNA or chol-siRNA. The delivery of siRNA into the tumor
cells is represented by average cell cy3-fluorescence. For each group, mean fluorescence
values and standard errors are reported in. Cell sonication substantially enhanced cy3-
siRNA penetration into the cell. Due to the effects of sonoporation, average cell
fluorescence for Optison/ultrasound treated cells was 39% more than untreated cells. For
cholesterol-siRNA, there was a 53% increase in average cell fluorescence after treatment
with Optison/ultrasound. Significant differences between the groups were evaluated using
two sample t-tests (p =0.032 for cy3-siRNA and p=0.059 for cholesterol-siRNA).
[0063] Similarly for MATBIII cells, the effect of Optison/ultrasound treatment was
compared to a commercially available lipid transfection reagent. The cells were treated
with either Cy3-siRNA or chol-siRNA in combination with either RNAifect or
Optison/ultrasound delivery agents and the results are shown in FIGs. 7 and 8. FIG. 7
shows representative images of the cells after treatment. FIG. 8 reports the mean cell
fluorescence for all groups with standard errors represented as error bars. For cy3-siRNA,
the average cell fluorescence was higher for Optison/ultrasound treatment (two sample ttest,
p= 0.007). This is primarily due to sonoporation of the cells in the presence of
microbubbles. There was no significant difference between RNAifect and
Optison/ultrasound delivery of chol-siRNA into cells. Although the average cell
fluorescence was similar, the lipid transfection reagent was found to be toxic to the tumor
cells as evidenced by the irregular shape of the cells in FIG. 7. It should be noted that the
same amount of transfection reagent and siR A was used in both unmodified and
cholesterol-siRNA. While the transfection reagent was toxic in both the cases, it was
higher for chol-siRNA.
Example 3 Preliminary binding studies of therapeutic-fatty acid conjugates to
microbubbles were evaluated using fluorescein - fatty acid conjugates.
Conjugation method;
[0064] Fatty acid NHS ester (2 equivalents, 5.37 mg Myristic acid NHS ester or 6.38
mg Stearic acid NHS ester was taken in a 50:50 mixture of DMSO and dichloromethane
(100 ul) and mixed with a solution of Fluorescein cadaverine (FL-Cadaverine, 5 mg, 1
equivalent, in 50 ul DMSO). To this diisopropylethyl amine (3.8 equivalents) was added
and mixture was vortexed to give a clear solution. Samples were kept in the dark at room
temperature. After 4.5h, reaction was checked by HPLC and was found to be complete.
A large shift in retention time was observed for both conjugates (Retention times FLCadaverine
4.7 min, FL-Cadaverine stearate 12.1 minute and FL-Cadaverine Myristate
9.9 min, column X-Bridge Shield RP 18, 4.6x50 mm column, particle size 5 um, gradient
method 0-100%B in 15 min and 100%B for 5 min, solvent A 0.1M TEAA, pH 7.0 and
solvent B 100% acetonitrile, flow rate 1 ml/min) as expected. Crude product was diluted
with DMSO to ~2 ml and purified on AKTA purifier using Xterra MS CI 8, 19x100 mm
column and a gradient of 0-1 00%B in 18.75 column volumes at a flow rate of 10 ml/min.
Solvent A and B were as described above for the analytical method. Product was
collected in multiple fractions and each fraction was analyzed by analytical HPLC. Only
the purest fraction in each case (-90% purity) was used for binding studies (starting
material itself was -86% pure, remaining likely a regioisomer with same spectral
properties). This fraction was concentrated to dryness at room temperature. Residue was
suspended in water (~2ml) and extracted with dichloromethane (3x2ml). Organic extracts
were combined, dried over anhydrous sodium sulfate and concentrated to dryness.
Fluorescence polarization assay
[0065] The stock solution of fluorescein-myristate was prepared in IX PBS. The
concentration of fluorescein was kept low for the fluorescence polarization assay, at 126
nM. Varying concentrations of either Optison or HSA solutions, ranging from 0 to 15 mM
albumin concentrations, were added to the fluorescein myristate solution. The reaction
buffer was IX PBS, pH 7.4. The reaction mixture was incubated under dark at 25 °C for
15 minutes. After incubation, the changes in raw anisotropy values of fluorescein were
measured using a microplate reader (SpectraMax 5, Molecular Devices, Sunnyvale, CA).
[0066] The samples were measured in Corning 96-well plates (black plate with a
clear bottom) (Sigma Aldrich, St Louis, MO). Fluorescein was excited at 470 nm, and
emission was measured at 540 nm. Fraction bound (x) was calculated using the same
equation as before (Equation 2), but replacing fluorescence values with anisotropy
values. The fraction bound calculated was then plotted against albumin concentration as
shown in FIG. 9. The data are reported as mean + 1.0 standard error (SE) for N=3.
Equation 3 was used to determine kd and n for fluorescein-myristate binding to both
Optison and native HSA (Table 2). This is represented also in FIG. 10 which shows the
fluorescein bound to Optison (0, 8, 40 and 200 pmoles for i, ii, iii and iv respectively) is
visualized on the gel as dark bands for fluorescein-myristate (FIG 10A) (63 pmoles) and
fluorescein-stearate (FIG. 10B) (180 pmoles ) .
[0067] When the fluorescein without the myristate conjugation was tested for its
binding properties to albumin, no significant changes in anisotropy was observed. It is
well known that the fatty acids have stronger binding properties than cholesterol, and is
also confirmed here with the lower dissociation constants, kd, observed for fluoresceinmyristate
conjugate (Table 1 and Table 2).
Table 1: Number of binding sites and dissociation constants for binding of cholesterolsiRNA
to Optison and native HSA
Optison Native HSA
Number of binding sites, n 1.16 1.13
Dissociation constant, kd mM) 6.4
Table 2 : Number of binding sites and dissociation constants for binding of fluoresceinmyristate
to Optison and native HSA
Optison Native HSA
umber of binding sites, n 1.03 1.02
Dissociation constant, d (mM) 0.238 0.378
[0068] Therefore, conjugating a fatty acid such as myristate to a therapeutic
compound can increase the binding of such therapeutic compounds to the albumin shell
microbubbles. The dissociation constant of fluorescein-myristate binding to Optison was
lower than that of binding to native HSA. This suggests better hydrophobic binding
properties of microbubble shell that has both native and partially denatured albumin.
Example 4
Stability of siRNA in vivo:
[0069] The stability of therapeutic compounds such as siRNA is very low once
injected into the body. A comparison between subcutaneous and tail-vein injection of a
mixture of albumin microbubbles and native siRNA (without conjugates) was studied.
[0070] Eleven to fourteen weeks (body weight ~ 30g) old nu/nu mice were obtained
from Charles River Laboratories (Wilmington, MA). Animals were housed in accordance
with the Guide for the Care and Use of Laboratory Animals as adopted by the National
Institutes of Health. Lewis lung carcinoma cells (LLC) were inoculated subcutaneously to
the right flank of anaesthetized mice (3.5 x 106 cells/100 mΐ/mouse).
[0071] On the fourth day after inoculation, the mice were treated with anti-VEGF
siRNA (Sigma Life Sciences, St. Louis, MO) - microbubble mixtures, a siRNA dose of
1.0 mg/kg for subcutaneous injections and a dose of 2.0 mg/kg for tail-vein injections.
The injection mixture contained 100 m of microbubble solution and 100 m of siRNA in
RNAse free water. After injection, the tumors were sonicated using Vivid i imagers with
a cardiac sector probe (3S). The energies were delivered in a pulsatile form with the peak
MI at 1.3. Control group did not receive any treatments.
[0072] After 24 hours from the treatment day, the mice were euthanized and the
tumors were extracted. The tumors were frozen immediately and stored at -20 °C. The
tumors were thawed out at room temperature on the day of VEGF measurement. The
tumors were then lysed in RIPA buffer (protease inhibitors added) using a lysing matrix
tube (Lysing matrix tube A, RP Biomedical). The lysate collected from the samples were
then diluted, and measured for total protein using a protein kit (Pierce BCA reagent
protein assay kit) and for VEGF using an ELISA kit (Mouse VEGF ELISA kit,
RayBiotech, Norcross, GA).
[0073] The results are reported in Table 3 as mean pg VEGF/ mg protein for control
and different treatment groups. The subcutaneous injection of 1.0 mg/kg siRNA -
microbubble mixture resulted in an approximately 39 % decrease in VEGF when
compared to the control group (two-sample t-test; p=0.0096). While there was only a
minor difference between the control group and 2.0 mg/kg tail-vein injection of siRNAmicrobubble
mixtures, this may be due to lack or less efficient binding of unmodified
siR A to the microbubble.
Table 3 : The effect of siRNA delivery to tumors; mean pg VEGF/mg total protein.
Conditions Mean pg VEGF/ mg protein Std error 95% C
Control 296. 19 24.89 48. 9
optison/siR NA S g 181.97 8 47.78
optison/siRNA TV 2 mg 255. 10 51.06 100.07
[0074] The invention may be embodied in other specific forms without departing
from the spirit or essential characteristics thereof. The foregoing embodiments are
therefore to be considered in all respects as illustrative rather than limiting on the
invention described herein. The scope of the invention is thus indicated by the appended
claims rather than by the foregoing description, and all changes that come within the
meaning and range of equivalency of the claims are therefore intended to be embraced
therein.
SEQUENCE LISTING
< 110> MOHAN, PRAVEENA
LIM, HAE WON
LOWERY, LISA
BURCZAK, JOHN DONALD
ROTHMAN, JAMES EDWARD
SOOD, ANUP
<120> MICROBUBBLE COMPLEXES AND METHODS OF USE
<130> 250944-1
<140> 13/235,890
<141> 201 1-09-19
<160> 2
<170> Patentln version 3.5
<210> 1
<21 1> 2 1
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 1
gcauuuguuu guccaagauu u 2 1
<210> 2
<21 1> 2 1
<212> RNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
oligonucleotide
<400> 2
aaaucuugga caaacaaaug c 2 1
Claims:
1. A microbubble complex comprising:
a microbubble having an outer shell comprising a mixture of native and denatured
albumin and a hollow core encapsulating a perfluorocarbon gas;
a therapeutic agent selected from a group comprising a small molecule
chemotherapeutic agent, peptide, carbohydrate, oligonucleotide, cytotoxin, protein
synthesis inhibitor, or combination thereof;
a bifunctional linker having one end attached to the therapeutic agent and the
other attached to a ligand through reaction of a reactive group on said ligand; and
wherein the ligand is bound to the outer shell of the microbubble through
hydrophobic interactions.
2. The complex of claim 1 wherein the therapeutic agent is an oligonucleotide.
3. The complex of claim 2 wherein the oligonucleotide is a naturally occurring or
modified DNA or R A.
4. The complex of claim 3 wherein the RNA is a small interfering RNA.
5. The complex of claim 1 wherein the bifunctional linker comprises a oligo- or
poly- amino acid, peptide, saccharide, nucleotide, organic moiety having approximately 1
to 250 carbon atoms, or a combination thereof.
6. The complex of claim 1 wherein the bifunctional linker comprises tetraethylene
glycol (TEG) or polyethylene glycol.
7. The complex of claim 1 wherein the reactive group comprises an activated ester,
phosphoramidite, isocyanate, isothiocyanate, aldehyde, acid chloride, sulfonyl chloride,
maleimide , alkyl halide, amine, phosphine, phosphate, alcohol or thiol.
8. The complex of claim 1 wherein the ligand is a fatty acid, steroid, or a
combination thereof.
9. The complex of claim 1 wherein the ligand is 4-iodophenyl butyric acid or an
analog or derivative thereof.
10. The complex of claim 1 wherein the amount of denature to native albumin is in
the range of approximately 0.5 to 30 wt %.
11. The complex of claim 1 wherein the therapeutic agent comprises siR A, the
linker comprises tetraethylene glycol, and the ligand comprises cholesterol.
12. A method for delivering a microbubble complex to a tissue target comprising the
steps of:
providing a microbubble complex, said complex comprising;
a microbubble having an outer shell comprising a mixture of native and
denatured albumin and a hollow core encapsulating a perfiuorocarbon gas;
a therapeutic agent selected from a group comprising a small molecule
chemotherapeutic agent, peptide, carbohydrate, oligonucleotide, cytotoxin, protein
synthesis inhibitor, or combination thereof;
a bifunctional linker having one end attached to the therapeutic agent and
the other attached to a ligand through reaction of a reactive group on said ligand; and
wherein the ligand is bound to the outer shell of the microbubble through
hydrophobic interactions;
administrating the microbubble complex to a subject wherein the subject is the
source of the tissue target; and
administering ultrasonic energy to the subject, wherein said energy is sufficient to
cause cavitation of the microbubble complex in the tissue target.
13. The method of claim 12 wherein the tissue target is in vivo and administrating the
microbubble complex comprises intravenous or intraperitoneal injection of the
microbubble complex.
14. The method of claim 12 further comprising the step of visualizing the
microbubble complex at the tissue target prior to administering the ultrasonic energy for
cavitation of the microbubble complex.
15. The method of claim 12 wherein the visualizing and administering the ultrasonic
energy are performed in real time.
16. The method of claim 16 wherein the tissue target is in vitro.
17. The method of claim 1 wherein the therapeutic agent comprises siRNA, the
linker comprises tetraethylene glycol, and the ligand comprises cholesterol.