HK1251462B - Microbubble complexes and methods of use - Google Patents

Description

Microbubble complex and its usage

This application is a divisional application. The original application was filed on September 18, 2012, with application number 201280045451.6 (PCT/EP2012/068288) and the invention title was "Microbubble Complex and Method of Use".

Technical Field

This invention generally relates to novel combinations of therapeutic agents with albumin microbubble drugs, utilizing the attachment of albumin affinity ligands to the therapeutic agent. This combination provides a method for delivering the therapeutic agent, in vitro or in vivo, to target cells or tissues of interest with the assistance of microbubbles.

Background Technology

Ultrasound-mediated disruption of drug-carrying microbubbles has been found to be useful for non-invasive drug delivery systems. Drugs or other therapeutic agents can be incorporated into microbubbles in a variety of ways, including binding the drug to the microbubble shell and attaching ligands. For example, perfluorocarbon-filled microbubbles circulate stably in blood vessels as pooling agents; they act as carriers of these agents until they reach the site of interest. Ultrasound can then be applied to the skin surface to rupture the microbubbles at that site, resulting in the local release of the drug or therapeutic agent at a site-specific location.

More specifically, albumin microvesicles have been used and delivered to specific organ targets via site-directed acoustic ultrasound. Albumin, the main protein in blood, has a natural physiological function of binding to and carrying a wide variety of lipophilic/weakly soluble ligands throughout the body. These ligands, which have an affinity for albumin, include fatty acids and other hydrophobic biosynthetic and catabolite products. Therefore, albumin microvesicles have been used to carry various protein-based and other biological agents, including oligonucleotides (ODNs) and polynucleotides such as antisense ODNs, which have sequences complementary to the specifically targeted messenger RNA (mRNA) sequences. These microvesicle-nucleic acid complexes can be generated from unmodified ODNs mixed with albumin or lipid components during microvesicle shell formation, or by mixing pre-formed microvesicles with the ODN of interest. In both cases, the ODN acts as a mechanical intervention in gene translation or earlier processing events. The advantage of this approach is the potential for gene-specific action, which should manifest as relatively low doses and minimal off-target side effects.

However, a key obstacle to translating the effective biology of ODNs into drugs is known to be the level of efficient and safe drug delivery. For example, ODN delivery using chemical agents, viral vectors, and microparticles has been hampered by chemical safety concerns before therapeutic effects can be achieved. Furthermore, the use of albumin microvesicles as ODNs, such as siRNA vectors, is limited due to the limited binding of ODNs to albumin microvesicles and the stability of albumin-ODN complexes. Because of the negative shell surface potential of albumin, negatively charged short nucleic acids do not bind very well to microvesicles, and gene transfection efficiency with these complexes is generally suboptimal.

Therefore, it is necessary to improve the binding of therapeutic agents with microbubbles and to improve the stability and efficacy of microbubble complexes.

Furthermore, there is a need to reduce the toxicity of selectively delivering highly cytotoxic drugs. Non-targeted delivery of these drugs can cause systemic toxicity, preventing the administration of many of these drugs in combination or at higher doses required for good efficacy. While attempts to deliver these drugs as prodrugs have mitigated this problem in many cases, selective absorption in targeted tissues is not always readily achieved because most absorption mechanisms in diseased tissues also exist in normal tissues. Therefore, improving the absorption of these drugs in selective tissues through non-natural mechanisms as disclosed herein could be of significant value.

Summary of the Invention

This article provides novel compositions and methods for increasing the binding of therapeutic drugs to microvesicles by utilizing the affinity of ligand-therapeutic compositions for albumin shells.

Systemic circulation of microbubbles carrying therapeutic compositions can be easily visualized using ultrasound imaging. The therapeutic agent is released from the microbubbles using a trigger of high-energy pulsed ultrasound specific to the treatment site. Cavitation of the microbubbles leads to sonoporation in adjacent cells/tissues.

In one embodiment, a microbubble complex is disclosed, comprising microbubbles (having a shell containing a mixture of natural and denatured albumin and a hollow core encapsulating perfluorocarbon gas), a therapeutic agent (selected from small molecule chemotherapeutic agents, peptides, carbohydrates, or oligonucleotides), and a bifunctional linker (one end of which is connected to the therapeutic agent and the other end of which is reactively connected to the ligand via a reactive group on a ligand). The ligand binds to the shell of the microbubble through hydrophobic interactions.

In another embodiment, a method for delivering the aforementioned microbubble complex to a tissue target is disclosed. The method includes the steps of providing the microbubble complex, administering the microbubble complex to a subject (where the subject is the source of the tissue target), and administering ultrasound energy to the subject (where the ultrasound energy is sufficient to cause cavitation of the microbubble complex within the tissue target).

Attached Figure Description

These and other features, aspects, and advantages of the invention will be better understood when the following detailed description is read with reference to the accompanying drawings, wherein:

Figure 1 shows the non-covalent binding of siRNA-ligand to albumin microvesicles.

Figure 2 shows representative photomicrographs of gel migration assays of mixtures of cholesterol-conjugated siRNA (2 picomol) and different amounts of Optison (i, ii, iii and iv: 0, 9, 22 and 46 picomol, respectively).

Figure 3 shows representative photomicrographs of gel migration assays of Cy3-siRNA (4 picomol) mixed with different concentrations of Optison or natural HSA (which does not migrate in the gel assay).

Figure 4 shows representative photomicrographs of gel assays of Cy3-cholesterol-siRNA (2 picomol) mixed with different concentrations of Optison, natural HSA, or denatured HSA.

Figure 5 is a representative diagram of the binding characteristics of cholesterol-siRNA with Optison and natural has; (A) relative fluorescence of cholesterol-siRNA bands measured by gel migration assay; (B) binding fraction of siRNA calculated by relative fluorescence and plotted against albumin concentration.

Figure 6 is a representative diagram of siRNA absorbed by U-87 tumor cells in Opticell by measuring cy3-fluorescence in cells.

Figure 7 includes a photomicrograph showing fluorescence images comparing siRNA transfection with the lipid transfection reagent (RNAifect) and Optison.

Figure 8 is a representative graph showing the average cellular fluorescence value and standard error of siRNA transfection between the lipid transfection reagent (RNAifect) and Optison.

Figure 9 is a representative graph of the binding fractions of fluorescein-myristate with Optison and natural HSA calculated using anisotropy values.

Figure 10 A and B show fluorescein bound to Optison (i, ii, iii and iv are 0, 8, 40 and 200 picomoles, respectively). fluorescein-myristate (63 picomoles) and fluorescein-stearate (180 picomoles) appear as black bands on the gel.

Detailed Implementation

The following detailed description is exemplary and is not intended to limit the invention of this application or its application. Furthermore, it is not intended to be limited by any theory set forth in the foregoing background or accompanying drawings of this invention.

The present invention generally relates to the delivery of therapeutic agents to cells or tissues of interest in vitro or in vivo with the assistance of microbubbles.

In some embodiments, the therapeutic agent may comprise a small molecule chemotherapeutic agent, peptide, carbohydrate, or oligonucleotide; and a bifunctional linker with the therapeutic agent attached at one end and the ligand attached at the other end via a reactive group on a ligand, wherein the ligand binds to the shell of the microvesicle. In some embodiments, the therapeutic agent may be an oligonucleotide (ODN). An oligonucleotide refers to a nucleic acid polymer formed by the breaking of bonds in a long nucleic acid, or synthesized using a protected phosphoramidite as a building block of a natural or chemically modified nucleoside or, in rare cases, a non-nucleoside compound. The length of an oligonucleotide can vary from short nucleic acid polymers of 50 or fewer base pairs to greater than 200 base pairs. As used herein, ODN also refers to a polynucleotide having greater than 200 base pairs. Antisense ODNs are also included, which refer to a single strand of DNA or RNA complementary to a selected sequence. In the case of antisense RNA, the antisense RNA prevents the protein translation of certain messenger RNA strands by binding to them. Antisense DNA can be used to target specific complementary (coding or non-coding) RNA. This also includes small interfering RNA (siRNA), sometimes called short interfering RNA or silent RNA, which is a class of double-stranded RNA molecules, typically 20-25 nucleotides long, that play a variety of roles in biology (including the RNA interference (RNAi) pathway, in which it interferes with the expression of specific genes), serving as an antiviral mechanism, or playing a role in the formation of genomic chromatin structure.

In some embodiments, the therapeutic agent may be a cytotoxin. As used herein, a cytotoxin refers to a substance that has a toxic effect on cells. For example, cytotoxins can cause necrosis, in which cells lose membrane integrity and die due to cell lysis. In other instances, cytotoxins may be accompanied by antibody-dependent cell-mediated cytotoxicity, in which cells are labeled with antibodies and act through certain lymphocytes.

Examples of cytotoxic agents are listed in Goodman and Gilman's "The Pharmacological Basis of Therapeutics," 10th edition, McGraw-Hill, New York, 2001. These include paclitaxel; nitrogen mustards, such as nitrogen mustard, cyclophosphamide, melphalan, uracil nitrogen mustard, and chlorambucil nitrogen mustard; ethyleneimine derivatives, such as thiotepa; alkyl sulfonates, such as busulfan; nitrosoureas, such as carmustine, lomustine, semustine, and streptozocin; triazines, such as dacarbazine; folic acid analogs, such as methotrexate; pyrimidine analogs, such as fluorouracil, cytarabine, and azalipine; purine analogs, such as mercaptopurine and thioguanine; vinca alkaloids, such as vincristine and vinblastine; and antibiotics, such as vinblastine. Drugs such as daunorubicin, doxorubicin, bleomycin, sclerosomycin, and mitomycin; enzymes such as L-asparaginase; platinum coordination complexes such as cisplatin; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine; adrenocortical inhibitors such as mitotane; hormones and antagonists such as corticosteroids (prednisone), progesterone (hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (diethylstilbestrol and ethinylestradiol), estrogen antagonists (tamoxifen), and androgens (testosterone propionate and fluorometholone).

Drugs that interfere with intracellular protein synthesis, and protein synthesis inhibitors, may also be conjugated with ligands; such drugs are known to those skilled in the art, including puromycin, cycloheximide, and ribonuclease.

In one embodiment, the protein in which the therapeutic agent acts as its inhibitor includes, but is not limited to, enzymes alone or in combination, soluble and serum proteins, proteins expressed on the cell surface, non-immunoglobulin proteins, intracellular proteins and segments of water-soluble or water-soluble proteins, as well as any derivatives of proteins.

In specific implementation schemes, proteins include, but are not limited to, cysteine proteases, glutathione S-transferases, epoxide hydrolases (EH), thiolases, NAD/NADP-dependent oxidoreductases, enoyl-CoA hydratases, aldehyde dehydrogenases, hydroxypyruvate reductases, tissue transglutaminase (tTG), imine methyltransferase cyclization deaminase (FTCD), aminopentanoate dehydratase (ADD), sarcosine kinases, carboxylesterases (LCE), monoacylglycerol (MAG) lipases, metalloproteinases (MP), phosphatases (protein tyrosine phosphatase, PTP), and protein... The following proteins are included: white body, FK506-binding protein (FKBP12), mammalian target of rapamycin (mTOR; or FKBP-rapamycin binding domain (FRB)), serine hydrolases (superfamily), ubiquitin-binding proteins, β-galactosidase, nucleotide-binding enzymes, protein kinases, GTP-binding proteins, cutinase, adenine succinate synthase, adenylate succinate lyase, glutamate dehydrogenase, dihydrofolate reductase, fatty acid synthase, aspartate transcarbamate, acetylcholinesterase, HMG-cholate reductase, and cyclooxygenases (COX-1 and COX-2). This also includes any derivatives of any protein.

In another example, the protein is essentially free of cofactors. “Essentially free of cofactors” includes proteins that do not require any additional cofactors, chemicals, chemical modifications, or physical modifications to be naturally stable under physiological conditions and at room temperature and under pressure in solution or as a solid, and that can bind to their corresponding ligands in vivo.

In one embodiment, albumin microbubbles can be used to carry therapeutic agents during systemic drug delivery. Tissue-targeted ultrasound energy can then be used to cavitate the albumin microbubbles and deliver the therapeutic agent into the intracellular environment. For example, the microbubble complex can be administered intravenously to a subject whose cells or tissues are to be targeted, or administered to the peritoneum of said subject (intraperitoneal). Once the microbubble complex has been carried to the target cells by the subject, ultrasound energy is delivered. In some embodiments, the target cells can be visualized prior to ultrasound delivery, while in other embodiments, visualization and monitoring of cavitation can be performed in real time.

In some embodiments, the albumin shell of the microbubble comprises both native and denatured albumin, held together primarily by cysteine-cysteine bonds. In some embodiments, the majority of the albumin shell is in its native form, with the denatured portion allowing for increased cysteine bond connections. In some embodiments, the relative amount of denatured albumin to native albumin is approximately 0.5–30 wt%. In other embodiments, the relative amount is approximately 1%–15 wt%. The mixture of native and denatured albumin provides a balance of shell elasticity required for cavitation, increasing reactive binding sites on the microbubble surface. Microbubbles can be formed by acoustic treatment with perfluorinated carbon gas in the presence of a preheated albumin solution, or by high shear force with a mixture of gas and preheated albumin. A small fraction of albumin molecules rearrange during acoustic treatment of the preheated albumin solution, cross-linking through disulfide bonds between albumin molecules. These albumin molecules are believed to be structurally similar to the F form of albumin, with more exposed hydrophobic residues. This allows for increased binding sites for hydrophobic interactions.

In some embodiments, the microbubbles may be filled with insoluble perfluorinated carbon gas, such as, but not limited to, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane, or combinations thereof. In some embodiments, the microbubbles have a diameter of about 1 to about 5 micrometers, optimized to allow passage through blood circulation.

In some embodiments, the therapeutic agent-microbubble complex comprises a therapeutic agent modified with a linker having a reactive group capable of binding to a ligand having an affinity for albumin. Therefore, the therapeutic agent can be coupled to albumin via the ligand.

The linker includes any connecting portion that links the ligand to the therapeutic agent via a first part. The linker can be as short as one carbon atom or as long as a polymer such as polyethylene glycol, tetraethylene glycol (TEG), polylysine, or other polymers commonly used in the pharmaceutical industry to modulate the pharmacokinetic and biodistribution characteristics of therapeutic agents. Other linkers of varying lengths include those with a C1-C250 length having one or more heteroatoms selected from O, S, N, and P, optionally substituted with halogen atoms. In specific embodiments, the linker comprises at least one of the following: an oligomer or polymer, alone or in combination, composed of natural or synthetic monomers; an oligomeric or polymeric moiety selected from pharmacologically acceptable oligomeric or polymeric components; an oligo- or poly-amino acid; a peptide; a sugar; a nucleotide; and an organic moiety having 1-250 carbon atoms. The organic moiety having 1-250 carbon atoms may contain one or more heteroatoms such as O, S, N, or P, optionally substituted with halogen atoms at one or more positions.

The first part may simply be an extension of the connector, formed by the reaction of a reactant on the connector with a reactive group on the therapeutic agent. Examples of reactants and reactive groups include, but are not limited to, active esters (such as N-hydroxysuccinimide ester, pentafluorophenyl ester), phosphoramides, isocyanates, isothiocyanates, aldehydes, acyl chlorides, sulfonyl chlorides, maleimides, alkyl halides, amines, phosphines, phosphate esters, alcohols, or thiols, provided that the reactant and reactive group are matched to produce a reaction that yields a covalently linked conjugate.

In some embodiments, the reactive group can be a primary amine functional group, thus allowing the amine-modified therapeutic agent to conjugate with an affinity ligand via a reaction of the ligand's carboxyl moiety. In some other embodiments, the reactive group can be an alcohol linked to the ligand via a phosphate group.

Ligands, also referred to herein as affinity ligands, include fatty acids, steroids, small aromatic compounds, or combinations thereof. Examples of albumin-binding molecules can be found in U.S. Patent Application Publication No. 2010/0172844, published July 8, 2010.

For example, in some embodiments, the affinity ligand is a fatty acid, including but not limited to myristoyl, lithocolic-oleyl, docosyl, lauroyl, steoroyl, palmitoyl, oleoyl, or linolenic acid. 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 its analogues or derivatives. In still other embodiments, the therapeutic agent comprises siRNA, the linker comprises tetraethylene glycol, and the ligand comprises cholesterol.

In some embodiments, the therapeutic agent-albumin complex can be prepared by sonicating a ligand-modified therapeutic agent with albumin or lipids in the presence of perfluorinated carbon, or by mixing a pre-formed microbubble with a ligand-modified therapeutic agent. In some embodiments, these molecules can be attached to the therapeutic agent of interest during therapeutic agent synthesis. For example, phosphorous amides of cholesterol can be used to incorporate cholesterol during DNA or RNA synthesis on a nucleic acid synthesizer, or to incorporate cholesterol post-synthesis via the incorporation reaction moiety.

In some embodiments, the therapeutic agent is a modified ODN, which can be prepared by enzyme catalysis with a modified nucleoside triphosphate; post-synthetic modification with the ligand is performed using the ligand itself or by modifying reactive functional groups. Depending on the intended use of the ODN, the ligand linkage can be at one or both ends, within the nucleic acid sequence, or at multiple locations. In some embodiments, when the siRNA is an ODN, linkage can be performed via the 3'OH position.

In some embodiments, in addition to the ligand, the therapeutic agent (including when the therapeutic agent is an ODN) may be selectively modified to defend against nucleases. In some embodiments, stabilizing modifications may include phosphate thioester modifications or 2'-OMe modifications.

In some embodiments, the microbubble complex can be incubated with or injected into the body (preferably intravenously) with cells or tissues of interest, and then cavitated at the desired site with ultrasound energy during a predetermined time or on-site imaging.

In some implementations, microbubble complexes can be observed during a systemic journey through the bloodstream under normal ultrasound diagnostic imaging. When the bubbles reach the tissue target (in this case, a tumor), a series of pulsed acoustic energy waves are delivered to the tumor. This creates inertial cavitation on the microbubbles, causing them to collapse. Cavitation of the microbubbles occurs when the acoustic energy is maximally localized. This localization is accomplished on the ultrasound probe using parameters related to the mechanical exponential force, the optimal ultrasound acoustic distance, and the ultrasound acoustic scan size. The resulting force then has the potential to create micropores within the cell membrane. Pulsed energy at frequencies of approximately 0.5–5 MHz is typically delivered.

These micropores, along with the microjet force generated by inertial cavitation, can facilitate the entry of ODN into the cellular cytoplasm. For example, when the ODN is siRNA, the siRNA in the intracellular environment will utilize host mechanisms to silence mRNA and subsequent protein synthesis. Similarly, when mRNA information acts as angiogenesis-promoting proteins (including vascular endothelial growth factor (VEGF)), reduced VEGF expression in tumors can halt or slow tumor growth. After microbubble cavitation, the dense gas at the center of the microbubble is exhaled from the body, and the albumin shell is metabolized and excreted via the hepatic clearance pathway.

In an exemplary implementation, the microbubble complex can be mixed to an optimal ratio based on prior therapeutic studies. Once the mixture of complexes is established, the drug is administered via intravenous systemic injection.

For example, when using siRNA-microbubble injection, the injection can be monitored during first-pass hemodynamics. Microbubble resonance and the resulting enhanced ultrasound contrast can be monitored using an ultrasound probe with low-diagnostic acoustic energy. The injection reaches the organ target during circulation. Cardiovascular tissue perfusion can help deliver the injection into deep microvessels with small lumen diameters. By supplying pulsed acoustic energy, sufficient energy can be provided to the microbubbles for inertial cavitation. Once microbubble cavitation is complete, the siRNA contents can be delivered across the plasma membrane into diseased cells. Furthermore, siRNA can have therapeutic effects in the cytoplasmic intracellular environment.

During microbubble cavitation, siRNA can enter the cell through various mechanisms. For example, siRNA can enter the cell via: a) microjet force from collapsed microbubbles, which can propel siRNA into the cytoplasm. Alternatively, the mechanism may include microjet energy or sonoluminescence energy, which creates temporary micropores within the plasma membrane to allow passive diffusion of siRNA into the cell, or microbubbles impacting the plasma membrane during microbubble resonance before actual cavitation can push siRNA into the cell.

Therefore, microbubble delivery mechanisms have potential applications in treating a wide range of diseases, including cancerous, inflammatory, infectious, cardiovascular, metabolic, autoimmune, and central nervous system disorders. Many of these diseases are currently untreatable because conventional small molecule drugs and monoclonal antibodies do not achieve the target molecular mechanism.

experiment

Example 1: Microvesicle-siRNA complex

Figure 1 is a representative illustration of siRNA binding to albumin-encapsulated microvesicles to form a microvesicle-siRNA complex.

Target siRNAs for VEGF (vascular endothelial growth factor) silencing are synthesized using IDTDNA technology. IDTDNA provides lipid modifications, such as cholesterol TEG groups on the siRNA (Chol-siRNA), and dye conjugation.

Positive chain: 5'-Cy3/GCAUUUGUUUGUCCAAGAUmUmU/3'-lipid (SEQ ID NO: 1)

Antonym: 5'/mAmArA rUrCrU rUrGrG rArCrA rArArC rArArA rUrGrC/3' (SEQ ID NO: 2)

The cyanine dye Cy3 on siRNA exhibits an excitation wavelength of 550 nm and a peak emission at 580 nm. The siRNA was labeled with Cy3 dye for easy visualization during binding assays and other characterization techniques. Optison ^™ (GE Healthcare, Chalfont St. Giles, United Kingdom, 10 mg/mL albumin) was centrifuged; the supernatant was discarded, and the excess albumin solution at the bottom was used for binding studies. A 10 mg/mL stock solution was prepared by dissolving lyophilized human serum albumin (HSA) powder (Sigma-Aldrich, St. Louis MO) in 1× phosphate-buffered saline (PBS). Both Optison and native albumin solution dilutions were prepared using 1× PBS. Denatured HSA solution was prepared by heating the native HSA solution to 80°C for 20 minutes.

Combination reaction:

Prepare cy3-siRNA and cy3-siRNA-cholesterol in 20 μM stock solutions in RNase-free water and store at -20°C. Mix 4 picoseconds of cy3-siRNA and 2 picoseconds of cholesterol-siRNA solution with varying amounts of Optison solution, native HSA, and denatured HSA solution ranging from 0 to 50 picoseconds. The reaction buffer is 1×PBS, pH 7.4. Incubate the reaction mixture in the dark at 25°C for 45 minutes. After incubation, mix 10 μL of the siRNA mixture with 2 μL of Novex® Hi-Density TBE Sample Buffer (5X) (Invitrogen, Carlsbad, CA, USA).

Gel electrophoresis:

Load all reactants onto a pre-prepared 6% non-denaturing polyacrylamide gel (Invitrogen, Carlsbad, CA, USA). Run the gel at 100 V in 0.5× Novex TBE electrophoresis buffer (Invitrogen, Carlsbad, CA, USA) for 45 minutes. Image the gels containing DNA, protein, or both against cy3 fluorescence using a Typhoon scanner (Typhoon ^™ 9410, GE Healthcare).

result:

Cy3 fluorescence of siRNA showed different siRNA bands on the gel. When a mixture of siRNA and albumin solution was run on the gel, the albumin-bound siRNA migrated less rapidly than the siRNA-free siRNA, producing two bands on the gel. In preliminary experiments, the albumin bands in the gel were observed using sypro ruby staining from an EMSA kit (Molecular Probes, Eugene, OR, USA). An example is shown in Figure 2, which is a gel migration assay of a mixture of cholesterol-conjugated siRNA (2 picomoles) with different amounts of Optison (i, ii, iii, and iv were 0, 9, 22, and 46 picomoles, respectively). Fluorescence imaging of the gel showed different siRNA (lower band) and albumin (upper band) bands.

Cy3-siRNA

When a mixture of cy3-siRNA and native HSA/Optison was run on a gel, increasing the albumin concentration did not result in any binding of the siRNA. cy3-siRNA did not show significant binding with either native HSA or Optison solutions. This is shown in Figure 3, which is a fluorescence image of a gel containing Cy3-siRNA (4 picomol) mixed with different concentrations of Optison or native HSA, showing no movement during gel assays. The black bands on the gel are cy3-fluorescence on the siRNA. cy3-siRNA did not show significant binding with either Optison or native HSA.

Chol-siRNA

Figure 4 shows gel images of chol-siRNA binding to Optison, native HSA, and denatured HSA. Chol-siRNA binds to both native HSA and Optison solutions, but binding is significantly reduced with equal amounts of denatured HSA. The fluorescence intensity of siRNA in each lane was manually estimated by drawing a frame around the bands. The intensity value of each siRNA band was subtracted from the background (which equals the average intensity value of the gel). The fluorescence intensity of bound siRNA was calculated over a wide range of albumin concentrations. The relative fluorescence R was calculated as:

R = (F_bound - F_free) / F_free (1)

F_binding represents the fluorescence intensity of the bound siRNA band, and F_free represents the fluorescence intensity of the free siRNA band. The relative fluorescence was plotted against albumin concentration. This is shown in Figure 5, which is a graphical comparison of the binding properties of cholesterol-siRNA with Optison and native HSA, as described below.

At low albumin concentrations in the 0-15 μM range, a linear correlation between binding fluorescence and albumin concentration was observed. Figure 5, plot A, shows that within this concentration range, the amount of chol-siRNA binding to Optison solution was higher than that binding to native HAS. To estimate the binding constant, higher concentrations of albumin were used to saturate the amount of siRNA binding to albumin. The binding fraction x was determined as follows:

x = (F_bound - F_free) / (F_saturated - F_free) (2)

F saturation is the fluorescence intensity of siRNA that binds to the maximum extent under saturation conditions.

As shown in curve B of Figure 5, the data points are plotted against the increase in albumin concentration using the fractional method, and 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 of each sample. Equation 3 was solved using nonlinear fitting to determine kd and n for the binding of chol-siRNA to both Optison and native HSA (Table 1). Using Microsoft Excel's solution tool, this nonlinear fitting revealed sum of squared errors (SSE) of 0.07 and 0.06 for Optison and native HSA, respectively. The binding constant of chol-siRNA was similar to that of Optison and native HSA.

Example 2: Delivery of siRNA to tumor cells

Cell culture:

MATBIII rat breast cancer cells and U-87 human glioblastoma cells were cultured in McCoy's 5AMedium (modified) (1×) (Invitrogen, Carlsbad, CA, USA) and Eagle's minimum essential medium (EMEM) (ATCC, Manassas, VA), respectively. Both medium solutions were supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Fisher Scientific, Springfield, NJ) and 1% penicillin-streptomycin (Sigma Aldrich, St. Louis, MO). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

Acoustic processing of substrate-connected cells:

MATB-III and U-87 cells were grown to 90% confluence in 10 mL Opticell units (Nalge Nunc International, Rochester, NY). The medium in the Opticell was replaced with 10 mL of fresh medium containing 40 picomol cy3-siRNA or cholesterol-siRNA. The opticcell was incubated at 37°C for 24 hours. Cells were individually treated with siRNA solutions mixed with Optison microbubbles (300 μL) or lipid transfection reagent (90 μL) (RNAifect, Qiagen, Valencia, CA). For siRNA/Optison mixtures, microbubbles were ruptured and siRNA drugs were delivered from the microbubbles using a Vivid i imager with a cardiac sector probe (3S). The opticcell was fixed in a water bath with an ultrasound probe attached to a moving arm spanning the full length of the opticcell. The probe tip was immersed in water, and the distance between the probe and the opticell surface was 3 cm, allowing acoustic treatment across the entire width of the opticell. Microbubbles in the opticell were treated with continuous acoustic treatment using a mechanical index MI > 1.3. The probe moved at a speed of 1 m/s along the entire length of the opticell. After acoustic treatment, the cells were cultured at 37°C for 24 hours. Similarly, cells treated with RNAifect were also cultured in an incubator for 24 hours. After culture, the cells were imaged using a fluorescence microscope (Zeiss Axio Imager. Z1, Carl Zeiss). The filter used for Cy3 was a DsRed/Cy3 (546 ex/620 em). Cell fluorescence was measured in the region of interest (ROI) of the fluorescence image, and the average cell fluorescence was calculated. The images were processed using ImageJ, and the fluorescence intensity was calculated.

Data were reported as mean + 1.0 standard error (SE), N=4. The statistical significance of differences between groups was evaluated using a two-sample t-test, and statistical analysis was performed using Minitab® 12 (Minitab Inc, State College, PA USA).

result:

The effects of the delivery system on U-87 cells incubated with cy3-siRNA or chole-siRNA are shown in Figure 6. Delivery of siRNA into tumor cells is expressed as mean cy3-fluorescence. Mean fluorescence values and standard errors for each group are reported. Cell acoustic treatment substantially enhances cy3-siRNA penetration into cells. Due to the acoustic aperture effect, the mean cellular fluorescence of Optison/ultrasound-treated cells was 39% higher than that of untreated cells. For chole-siRNA, mean cellular fluorescence increased by 53% after Optison/ultrasound treatment. Significance between groups was evaluated using a two-sample t-test (p = 0.032 for cy3-siRNA, p = 0.059 for chole-siRNA).

Similarly, for MATBIII cells, the effects of Optison/ultrasound treatment were compared with those of commercially available lipid transfection agents. Cells were treated with Cy3-siRNA or chole-siRNA in combination with RNAifect or Optison/ultrasound delivery agents, and the results are shown in Figures 7 and 8. Figure 7 shows representative images of the cells after treatment. Figure 8 reports the mean cell fluorescence for all groups, with standard errors expressed as error bars. For cy3-siRNA, the mean cell fluorescence was higher with Optison/ultrasound treatment (two-sample t-test, p = 0.007). This is mainly due to the acoustic pore effect of cells in the presence of microbubbles. There was no significant difference between RNAifect and Optison/ultrasound delivery when chole-siRNA was delivered into the cells. Although the mean cell fluorescence was similar, the lipid transfection agents were found to be toxic to tumor cells, as evidenced by the irregular shape of the cells in Figure 7. It should be noted that equal amounts of transfection agent and siRNA were used for both unmodified and cholesterol-siRNA. Although the transfection agents were toxic in both cases, chole-siRNA was more toxic.

Example 3: Evaluation of the preliminary binding of therapeutic fatty acid conjugates to microbubbles using fluorescein fatty acid conjugates. Investigate.

Joining methods:

Fatty acid NHS esters (2 equivalents, 5.37 mg NHS myristate or 6.38 mg NHS stearate) were dissolved in a 50:50 mixture (100 μL) of DMSO and dichloromethane, and mixed with a fluorescein cadaverine solution (FL-cadaverine, 5 mg, 1 equivalent, in 50 μL DMSO). Diisopropylethylamine (3.8 equivalents) was added, and the mixture was vortexed to obtain a clear solution. The sample was kept in the dark at room temperature. After 4.5 hours, the reaction was examined by HPLC, and the reaction was found to be complete. As expected, large variations in retention times were observed for both conjugates (retention times: FL-cadaverine 4.7 min, FL-cadaverine stearate 12.1 min, and FL-cadaverine myristate 9.9 min; column: X-Bridge Shield RP 18, 4.6 × 50 mm, particle size 5 μm; gradient method: 0–100% B over 15 min, followed by 5 min at 100% B; solvent A: 0.1 M TEAA, pH 7.0; solvent B: 100% acetonitrile; flow rate: 1 mL/min). The crude product was diluted to ~2 mL with DMSO and purified on an AKTA purifier using an Xterra MS C18, 19 × 100 mm column with a gradient of 0–100% B over 18.75 column volumes at a flow rate of 10 mL/min. Solvents A and B are as described above regarding the analytical methods. Products from multiple fractions were collected and analyzed by analytical HPLC. Only the purest fraction (~90% purity) from each case was used for the combined study (the raw material itself was ~86% pure; the remainder may be regioisomers with similar spectral properties). This fraction was concentrated to dryness at room temperature. The residue was suspended in water (~2 ml) and extracted with dichloromethane (3 × 2 ml). The organic extracts were combined, dried over anhydrous sodium sulfate, and concentrated to dryness.

Fluorescence polarization measurement

A fluorescein-myristate stock solution was prepared in 1×PBS. A low concentration of fluorescein at 126 nM was maintained for fluorescence polarization assays. Optison or HSA solutions of varying concentrations (0–15 μM albumin) were added to the fluorescein-myristate solution. The reaction buffer was 1×PBS, pH 7.4. The reaction mixture was incubated in the dark at 25°C for 15 minutes. After incubation, changes in the original anisotropy of fluorescein were measured using a microplate reader (SpectraMax 5, Molecular Devices, Sunnyvale, CA).

Samples were measured in Corning 96-well plates (black plates with transparent bottoms) (Sigma Aldrich, St. Louis, MO). Fluorescein was excited at 470 nm and emission was measured at 540 nm. The binding fraction (x) was calculated using the same equation (Equation 2) as before, but with anisotropic values instead of fluorescence values. The calculated binding fractions were then plotted against albumin concentration, as shown in Figure 9. Data were reported as mean + 1.0 standard error (SE), N=3. Equation 3 was used to determine the kd and n of fluorescein-myristate binding to both Optison and native HSA (Table 2). This is also shown in Figure 10, which shows fluorescein bound to Optison (i, ii, iii and iv are 0, 8, 40 and 200 picomoles, respectively), which appear as black bands on the gel as fluorescein-myristate (Figure 10A) (63 picomoles) and fluorescein-stearate (Figure 10B) (180 picomoles).

When the binding properties of fluorescein without myristate conjugate to albumin were tested, no significant change in anisotropy was observed. It is well known that fatty acids have stronger binding properties than cholesterol, and this is also confirmed here, with a lower dissociation constant kd observed for the fluorescein-myristate conjugate (Tables 1 and 2).

Table 1: Number of binding sites and dissociation constants of cholesterol-siRNA with Optison and native HSA

Table 2: Number of binding sites and dissociation constants of fluorescein-myristate with Optison and natural HSA

Therefore, conjugating fatty acids, such as myristate, with therapeutic compounds can increase the binding of these compounds to albumin-shell microvesicles. The dissociation constant of fluorescein-myristate binding to Optison is lower than that binding to native HSA. This suggests that microvesicle shells containing both native and partially denatured albumin have better hydrophobic binding properties.

Example 4

In vivo stability of siRNA:

Therapeutic compounds such as siRNA exhibit very low stability once injected into the body. A comparison was conducted between subcutaneous and tail vein injections of albumin microvesicles and a mixture of natural siRNA (unconjugated).

11–14 weeks old (~30 g) nu/nu mice were obtained from Charles River Laboratories (Wilmington, MA). Animals were housed in captivity according to the guidelines for the care and use of laboratory animals adopted by the National Institutes of Health. Lewis lung cancer cells (LLC) were subcutaneously injected into the right abdomen of anesthetized mice (3.5 × ^10⁶ cells/100 μl/mouse).

On day 4 post-inoculation, mice were treated with an anti-VEGF siRNA (Sigma Life Sciences, St. Louis, MO)-microbubble mixture. The siRNA dose was 1.0 mg/kg administered subcutaneously and 2.0 mg/kg via tail vein injection. The injection mixture contained 100 μL of microbubble solution and 100 μL of siRNA in RNase-free water. Following injection, tumors were acoustically treated using a Vivid i imager with a heart-region probe (3S). Pulsed energy was delivered with a peak MI of 1.3. The control group received no treatment.

Twenty-four hours after treatment, mice were euthanized, and tumors were extracted. The tumors were immediately frozen and stored at -20°C. On the day of VEGF measurement, the tumors were thawed at room temperature. The tumors were then lysed in RIPA buffer (with added protease inhibitors) using lysis matrix tubes (lysis matrix tube A, RPBiomedical). The lysates collected from the samples were then diluted, and total protein was measured using a protein assay kit (Pierce BCA reagent protein assay kit), and VEGF was measured using an ELISA kit (mouse VEGF ELISA kit, RayBiotech, Norcross, GA).

The results are reported in Table 3 as mean pg VEGF/mg protein in the control and different treatment groups. Subcutaneous injection of 1.0 mg/kg siRNA-microbubble mixture resulted in a VEGF decrease of approximately 39% compared to the control group (two-sample t-test; p = 0.0096). Although there was only a small difference between the control group and the 2.0 mg/kg tail vein injection of siRNA-microbubble mixture, this may be due to the lack of effective binding or minimal effective binding of unmodified siRNA to microbubbles.

Table 3: The role of siRNA delivery to tumors; average pg VEGF/mg total protein.

This invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments should be considered illustrative in all respects, and not limiting of the invention described herein. Thus, the scope of the invention is indicated by the appended claims rather than by the foregoing description, and all changes falling within the equivalent meaning and scope of the claims are intended to be included therein.

sequence list

<110> MOHAN, PRAVEENA

LIM, HAE WON

LOWERY, LISA

BURCZAK, JOHN DONALD

Rothman, James Edward

SOOD, ANUP

<120> Microbubble Complex and Usage Methods

<130> 250944-1

<140> 13/235,890

<141> 2011-09-19

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<170> PatentIn version 3.5

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<223> Description of artificial sequences: Synthetic oligonucleotides

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<110> MOHAN, PRAVEENA

LIM, HAE WON

LOWERY, LISA

BURCZAK, JOHN DONALD

ROTHMAN, JAMES EDWARD

SOOD, ANUP

<120> Microbubble Complex and Usage Method

<130> 250944-1

<140> 13/235,890

<141> 2011-09-19

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> Description of artificial sequences: Synthetic oligonucleotides

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gcauuuguuu guccaagauu u                         21

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<220>

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Claims (10)

1. A microbubble complex comprising: Microbubbles have a shell containing a mixture of natural and denatured albumin and a hollow core encapsulating perfluorocarbon gas; Therapeutic agents, which are small interfering RNAs; A bifunctional connector, one end of which is connected to the therapeutic agent and the other end of which is reactively connected to a ligand via a reactive group on a ligand, wherein the bifunctional connector comprises tetraethylene glycol (TEG); and The ligand is bound to the shell of the microbubble via hydrophobic interactions, and the ligand is a steroid. 2. The complex of claim 1, wherein the reactive group comprises an active ester, phosphoramide, aldehyde, acyl chloride, maleimide, alkyl halide, amine, phosphine, or alcohol. 3. The complex of claim 1, wherein the reactive group comprises isocyanate, isothiocyanate, sulfonyl chloride, phosphate ester or thiol. 4. The complex of claim 1, wherein the amount of denatured albumin is 0.5-30 wt% more than the amount of natural albumin. 5. Use of microbubbles, therapeutic agents, and bifunctional connectors in the preparation of microbubble complexes for methods of delivering said therapeutic agents to tissue targets: The microbubbles have an outer shell containing a mixture of natural and denatured albumin and a hollow core encapsulating perfluorocarbon gas. The therapeutic agent is small interfering RNA; The bifunctional connector is connected at one end to the therapeutic agent and at the other end to a ligand via a reactive group on the ligand, wherein the bifunctional connector comprises tetraethylene glycol (TEG); and The ligand is bound to the shell of the microbubble via a hydrophobic interaction, and the ligand is a steroid. The method includes the following steps: Provide microbubble complexes; The microbubble complex is administered to a subject, wherein the subject is the source of the tissue target; and Ultrasonic energy is delivered to the subject, wherein the energy is sufficient to cause cavitation of the microbubble complex in the tissue target. 6. The use of claim 5, wherein the tissue target is in vivo, and administration of the microbubble complex includes intravenous or intraperitoneal injection of the microbubble complex. 7. The use of claim 5, further comprising the step of revealing the microbubble complex at a tissue target prior to applying ultrasonic energy to cavitate the microbubble complex. 8. The use of claim 7, wherein the display and the application of ultrasonic energy are performed in real time. 9. The use of claim 5, wherein the tissue target is in vitro. 10. The use of claim 5, wherein the ligand is cholesterol.