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WO2010148395A1 - Polymérosomes poreux paramagnétiques et leurs utilisations - Google Patents

Polymérosomes poreux paramagnétiques et leurs utilisations Download PDF

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Publication number
WO2010148395A1
WO2010148395A1 PCT/US2010/039348 US2010039348W WO2010148395A1 WO 2010148395 A1 WO2010148395 A1 WO 2010148395A1 US 2010039348 W US2010039348 W US 2010039348W WO 2010148395 A1 WO2010148395 A1 WO 2010148395A1
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Prior art keywords
nanoparticle
porous
agent
polymersome
paramagnetic ion
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Andrew Tsourkas
Zhiliang Cheng
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University of Pennsylvania Penn
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University of Pennsylvania Penn
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/06—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules
    • A61K51/065—Macromolecular compounds, carriers being organic macromolecular compounds, i.e. organic oligomeric, polymeric, dendrimeric molecules conjugates with carriers being macromolecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1217—Dispersions, suspensions, colloids, emulsions, e.g. perfluorinated emulsion, sols
    • A61K51/1234—Liposomes
    • A61K51/1237—Polymersomes, i.e. liposomes with polymerisable or polymerized bilayer-forming substances

Definitions

  • the invention relates to porous polymerosoms and their use in diagnosis and prognosis of diseases. Specifically, the invention relates to porous polymerosoms comprising lanthanides and their uses.
  • Magnetic resonance (MR) imaging procedures have become common practice in diagnostic clinical medicine owing to their ability to provide high-resolution three- dimensional images of soft tissue.
  • Many of these diagnostic procedures utilize intravenous MR contrast agents, such as gadolinium (Gd), to improve tissue contrast and to provide important information about perfusion, vascular permeability, and extracellular volume.
  • Gd gadolinium
  • the recent development of targeted paramagnetic contrast agents promises to even further expand the utility of diagnostic MR imaging by providing a mechanism to probe the molecular profile of tissues.
  • Gd gadolinium
  • Rl longitudinal relaxivities
  • the Rl of Gd-based contrast agents is dependent on two key features, the water-exchange rate between bulk water and water bound to the P-70442-PC
  • the rotational correlation lifetime is generally increased through conjugation of Gd chelates to macromolecular objects.
  • Some examples include dendrimers, polymers, liposomes, micelles, emulsions, and silica nanoparticles.
  • it is not only the relaxivity per Gd that defines the effectiveness of the contrast agent but also the number of chelated Gd per nanoparticle. These two parameters can be represented as the relaxivity per nanoparticle.
  • Nanovesicles have been transformed into effective paramagnetic contrast agents by encapsulating chelated Gd within the aqueous core. However, they are found to be detrimental for MR imaging applications because of slow water exchange rate through the lipid bilayer on the relaxivity of encapsulated Gd. Additionally, there are problems of leakages of encapsulated Gd through the membrane pores.
  • the invention provides a porous nanoparticle comprising: a porous polymersome shell; and a paramagnetic ion operably linked to a core therein, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell.
  • the invention provides a contrast agent for magnetic resonance (MR) imaging, the contrast agent comprising: a porous nanoparticle that comprises a porous polymersome shell and a paramagnetic ion operably linked to a core therein, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell.
  • MR magnetic resonance
  • the invention provides a blood pool agent, comprising: a porous nanoparticle that comprises a porous polymersome shell and a paramagnetic ion P-70442-PC
  • said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell.
  • the invention provides an imaging agent for macrophage infiltration comprising: a porous nanoparticle that comprises a porous polymersome shell and a paramagnetic ion operably linked to a core therein, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell.
  • the invention provides an agent for visualizing phagocytic cells comprising: a porous nanoparticle that comprises a porous polymersome shell and a paramagnetic ion operably linked to a core therein, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell.
  • the invention provides a method for producing a nanoparticle, whereby the nanoparticle is prodeuced by a hydration process, an electroformation process, or a method for forming vesicles with amphiphilic molecules.
  • the invention provides a method for producing a porous nanoparticle comprising the steps of: dissolving a non-filler polymer and a compatible filler in a solvent; removing the solvent, thereby forming a dehydrated filler-impregnated film; adding a paramagnetic ion operably linked to a molecular agent, to the dehydrated filler- impregnated film, whereby the molecular agent is capable of preventing the paramagnetic ion from leaking out of the porous nanoparticle; incubating the filler-impregnated dehydrated film and the paramagnetic ion operably linked to the molecular agent in the presence an aqueous solution, thereby forming vesicles; reducing the vesicle size; and removing the filler, creating a porous shell.
  • the invention provides a method for obtaining a magnetic resonance image (MRI), in a subject, the method comprising administering to said subject an emulsion comprising nanoparticles, said nanoparticles comprising a porous polymersome shell operably linked to a targeting ligand; and a paramagnetic ion operably linked to a core therein, whereby said core comprises a molecular agent that prevents said paramagnetic ion P-70442-PC
  • the targeting ligand is specific for a marker of a disease in said subject.
  • the invention provides a method for diagnosing atherosclerotic disease or predisposition thereto in a subject, comprising the step of administering to said subject an emulsion of porous nanoparticles, said nanoparticles comprising a porous polymersome shell operably linked to a VCAM-I targeting ligand; and a paramagnetic ion operably linked to a molecular agent, whereby the molecular agent is capable of preventing said paramagnetic ion from leaking out of said porous nanoparticle; obtaining a magnetic resonance image at an area of interest in the subject; and comparing the observed image with a reference image obtained from the same area of interest.
  • Figure 1 shows a schematic diagram illustrating approach used to synthesize paramagnetic porous polymersome.
  • Vesicles consisting of a diblock copolymer PBdEO and phospholipid POPC at a molar ratio of 85: 15 were prepared.
  • Generation 2 dendrimer conjugated Gd chelates were encapsulated within the aqueous interior during vesicle formation.
  • Crosslinking of PBdEO within the vesicle bilayer was induced by free radical polymerization
  • Pores were formed in the polymersome bilayer by extracting the POPC with surfactant Triton X-IOO; P-70442-PC
  • Figure 2 shows microscopy images of giant vesicles composed of diblock copolymers and lipids.
  • the vesicles were fluorescently-labeled by incorporating the phospholipid rhodamine- PE into the membrane bilayer.
  • (a) Phase contrast image (b) Fluorescence image. Uniform fluorescence is seen in individual giant vesicles. The bar represents 25 um;
  • the measuring angle of DLS is 90 °;
  • Figure 4 shows an evaluation of pore formation within cross-linked polymersomes following surfactant solubilization.
  • the water-soluble, low-molecular-weight dye carboxyfluorescein was encapsulated into chemically cross-linked polymersomes with and without 15% phospholipid.
  • Figure 5 shows the retention of generation 2 dendrimer within cross-linked porous polymersomes.
  • TRITC labeled dendrimer was encapsulated into chemically cross-linked polymersomes containing 15% phospholipid.
  • the emission spectrum of vesicles treated with Triton X-100 was nearly identical to that of the vesicles treated by buffer indicating that the dendrimer is retained within the porous vesicles;
  • Figure 6 shows relaxivity determination for Gd-dendrimer conjugates encapsulated within cross-linked porous polymersomes. Comparisons are made to Gd-dendrimer conjugates encapsulated within cross-linked polymersomes that do not contain pores and to Gd-DTPA. Measurements were acquired at 1.41 T (60 MHz) at 40 0 C;
  • Figure 7 is a schematic diagram illustrating approach used to prepare paramagnetic porous vesicles. Nanovesicles were formed through the co-assembly of diblock inert copolymer PBdEO and biodegradable copolymer PEOCL. Gd-DTPA-labeled Generation 3 dendrimer P-70442-PC
  • Pores were encapsulated within the aqueous interior during vesicle formation. Pores were subsequently formed in the polymersome bilayer by hydrolysis of the caprolactone block.
  • the measuring angle of DLS was 90 °.
  • Figure 9 shows kinetics of CF release from nanovesicles prepared with PBdEO/PEOCL at molar ratios of ( ⁇ ) 75: 25, ( ⁇ ) 85:15 and (A) 100:0.
  • the vesicles were incubated in PBS buffer (0.1 M, pH 5.1) and temperature 37 0 C. At the end of each experiment, total CF fluorescence was determined by the addition Triton X-100.
  • Figure 10 shows vesicle diameters as a function of storage time at PBS buffer (0.1 M, pH 5.1) and 37 0 C.
  • Vesicles prepared using PBdEO/PEOCL at a molar ratio of 75:25 (A) and vesicles prepared from PBdEO only (•) were evaluated.
  • Figure 11 shows retention of generation 3 dendrimer within biodegradable porous polymersomes.
  • FITC labeled dendrimer was encapsulated into polymersomes containing 25% PEOCL and incubated in pH 5.1 buffer at 37 0 C .
  • the vesicles were centrifuged on a Microcon centrifugal filtering device with a 50 kDa MWCO membrane (Millipore) on various days and the liquid that flowed through the filter (i.e. diffusate) was tested for fluorescence. For comparison, the liquid that did not flow through the filter (retentate) was also measured for fluorescence.
  • Figure 12 shows relaxivity determination for Gd-dendrimer conjugates encapsulated within porous polymersomes.
  • Polymersomes were formed using 75%PBdEO and 25% PEOCL. This was followed by hydrolysis of the polycaprolactone block. Tl measurements were acquired at 1.41 T (60 MHz) at 40 0 C. For comparison, Tl measurements were also made for Gd-dendrimer conjugates and Gd-DTPA encapsulated within non-porous PBdEO polymersomes.
  • Figure 13 shows the cell viability of NIH 3T3 cells incubated with nanovesicles. Nanovesicles were incubated with NIH 3T3 cells at various Gd concentrations for 4 hours. Viability was measured and normalized to cells grown in the absence of any particles based on MTT assay.
  • FIG. 14 shows Gd concentration in the blood at various times following the intravenous injection of Gd-encapsulated porous polymersomes into C57BL/6 mice.
  • Figure 15 shows magnetic resonance images of C57BL/6 mice at various time points following the intravenous injection of Gd-encapsulated porous polymersomes.
  • the local hyperintensity generated by the polymersomes was visualized using a 4.7 T small animal MR. Images of the (A) kidney and (B) bladder were acquired pre-injection and 2, 4 and 24 hr post- injection.
  • Figure 16 shows measurements of MR signal-to-noise ratio (SNR) in the kidney (gray) and spleen (black) at various times following intravenous injection of Gd-encapsulated porous polymersomes. All measurements were normalized to the pre-injection ratio.
  • SNR signal-to-noise ratio
  • FIG 17 shows biodistribution of gadolinium in various organs isolated from C57BL/6 mice, 4 and 24 hrs after intravenous injection of Gd-encapsulated porous polymersomes.
  • ICP-MS inductively coupled plasma mass spectroscopy
  • the invention relates to porous polymerosoms and their use in diagnosis and prognosis of diseases. Specifically, the invention relates to porous polymerosoms comprising lanthanides and their uses.
  • a porous nanoparticle comprising: a porous polymersome shell; and a paramagnetic ion operably linked to a core therein, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell.
  • a contrast agent for magnetic resonance (MR) imaging comprising a porous nanoparticle that comprises a porous polymersome shell and a paramagnetic ion operably linked to a core therein, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell.
  • a blood pool agent comprising: a porous nanoparticle that comprises a porous polymersome shell and a paramagnetic ion operably linked to a core therein, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell.
  • an imaging agent for macrophage infiltration comprising: a porous nanoparticle that comprises a porous polymersome shell and a paramagnetic ion operably linked to a core therein, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell.
  • an agent for visualizing phagocytic cells comprising: a porous nanoparticle that comprises a porous polymersome shell and a paramagnetic ion operably linked to a core therein, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell.
  • a method for producing a nanoparticle whereby the nanoparticle ir prodeuced by a hrdration process, electroformation, or a method for forming vesicles with amphiphilic molecules.
  • a method for producing a porous nanoparticle comprising the steps of: dissolving a non- filler polymer and a compatible filler in a solvent; removing the solvent, thereby forming a dehydrated filler-impregnated film; adding a paramagnetic ion operably linked to a molecular agent, to the dehydrated filler-impregnated film, whereby the molecular agent is capable of preventing the paramagnetic ion from leaking out of the porous nanoparticle; incubating the filler-impregnated dehydrated film and the paramagnetic ion operably linked to the molecular agent in the presence an aqueous solution, thereby forming vesicles; reducing the vesicle size; and removing the filler, creating a porous shell.
  • a method for obtaining a magnetic resonance image (MRI), in a subject comprising administering to said subject an emulsion comprising nanoparticles, said nanoparticles comprising a porous polymersome shell operably linked to a targeting ligand; and a paramagnetic ion operably linked to a core therein, whereby said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell, whereby the targeting ligand is specific for a marker of a disease in said subject.
  • MRI magnetic resonance image
  • a method for diagnosing atherosclerotic disease or predisposition thereto in a subject comprising the step of administering to said subject an emulsion of porous nanoparticles, said nanoparticles comprising a porous polymersome shell operably linked to a VCAM-I targeting ligand; and a paramagnetic ion operably linked to a molecular agent, whereby the molecular agent is capable of preventing P-70442-PC
  • the paramagnetic ion from leaking out of the porous nanoparticle obtaining a magnetic resonance image at an area of interest in the subject; and comparing the observed image with a reference image obtained from the same area of interest.
  • porous polymerosoms and their use in diagnosis and prognosis of diseases.
  • porous polymerosoms comprising lanthanides and their uses, for example, as an imaging agent, a contrast agent, a blood pool agent, an imaging agent for macropage infiltration, an agent for visualizing phagocytic cells.
  • MR contrast agents in contrast to other imaging modalities (i.e. x-ray, nuclear, and ultrasound), the effect of MR contrast agents is not seen directly on the image, but rather it is their effect on proton relaxation, normally water protons, that is observed. In another embodiment, it is the change in relaxation rate of water protons in the presence of a paramagnetic ion, such as lanthanides (e.g. Gd) that is detectable by MR and is responsible for enhancing image contrast.
  • a paramagnetic ion such as lanthanides (e.g. Gd)
  • Tl and T2 refer to the relaxation time of protons in the longitudinal and transverse plane respectively and [M] is the concentration of contrast agent or metal ion.
  • Gadolinium is typically used as a Tl agent because they are generally more effective at reducing the longitudinal relaxation rate.
  • Optimal relaxivity of a contrast agents is dependent in one embodiment, on two key features, the water-exchange rate and the rotational correlation lifetime. In one embodiment, faster water exchange rates are preferred, which means that the paramagnetic ion, such as lanthanides (e.g. Gd), should efficiently relax the water that comes in contact with it and the relaxed water should exchange rapidly with the bulk water so that many water molecules can be relaxed by a single paramagnetic ion.
  • the paramagnetic ion such as lanthanides (e.g. Gd)
  • the rotational correlation lifetime is increased in one embodiment, through conjugation of the paramagnetic ion, such as lanthanides (e.g. Gd) chelate to macromolecular objects.
  • the paramagnetic ion such as lanthanides (e.g. Gd) chelate to macromolecular objects.
  • self-assembled nanoparticle systems comprised of amphiphilic molecules (i.e. liposomes, micelles, and emulsions) are particularly attractive due to their unique structure wherein the hydrophobic domain serves in one embnodiment, as a natural carrier environment for hydrophobic drugs and the exterior surface provides a platform for attaching targeting ligands.
  • amphiphilic molecules i.e. liposomes, micelles, and emulsions
  • Amphiphilic nanoparticles are easily transformed in another embodiment into paramagnetic contrast agents using the methods described herein, by either encapsulating chelated Gd within the hollow core (in the case of liposomes) or through the immobilization of chelated Gd onto the outer membrane surface.
  • Liposomes have been extensively investigated as a possible carrier of Gd-chelates to enhance the contrast efficacy and to change the pharmacokinetic properties of the contrast agent. Liposomes present a very desirable platform for Gd-based nanoparticles because very high concentrations of Gd can be encapsulated within the lipid bilayer or immobilized on the liposome membrane.
  • liposomes with encapsulated Gd-chelates have previously been used for contrast-enhanced MR imaging, they suffer from several shortcomings. Most notably, the relaxivity of the encapsulated Gd is reduced due to the slow flux of water across the vesicle bilayer. Although, this can be partially overcome by increasing the surface-to-volume ratio (i.e. decreasing the size of the vesicle), even liposomes 100 nm in diameter have been shown to exhibit a relaxivity (per Gd) that is 62% lower than free chelated Gd.
  • Gd loading will not be restricted by the available surface area, but it will also free up more sites on the membrane surface for attaching targeting ligands. It is well accepted that target affinity increases as the number of targeting ligands per nanoparticle increases. Additional benefits of using polymersomes over phospholipids is that they are extremely stable and often exhibit extremely long circulation times (-20 hours). P-70442-PC
  • these novel vesicles are composed of three key features each in its own discrete embodiment: (1) a stabilized polymer shell; (2) a porous membrane structure that allows for improved water flux across the bilayer, and (3) a molecular agent operably linked to a lanthanide, such as Gd complex in one embodiment, that is loaded inside the porous vesicles for MRI detection., in other discrete embodiments provided in the methods for diagnosis and prognosis described herein.
  • the molecular agent that is capable of preventing the lanthanide from leaking out of the polyme shell.
  • a porous nanoparticle comprising: a porous polymersome shell; and a paramagnetic ion operably linked a core therein, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell.
  • the term, "operably linked" refers to the paramagnetic ion and the core being arranged so that they function in concert for their intended purposes.
  • the porous polymersome described herein is used in the methods described herein.
  • the paramagnetic ion operably linked to a core and is used in the nanoparticles described herein is a lanthanide, or a lanthanide-chelated agent in another embodiment, or their combination in yet another discrete embodiment.
  • the lanthanide used in the methods and porous polymersomes described herein is Gd 3+ .
  • the lanthanide used as the paramagnetic ion described and used herein is Eu 3+ , Tm 3+ , Dy 3+ or Yb 3+ , each a discrete embodiment of the paramagnetic ion used herein.
  • a porous nanoparticle comprising: a porous polymersome shell; and a paramagnetic ion operably linked to a core therein, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell.
  • the paramagnetic ion is gadolinium (Gd), a gadolinium-chelated agent or their combination.
  • the porosity is P-70442-PC
  • the size of the nanoparticle is between about 20 and about 400 nm.
  • the labelled core is entrapped or otherwise encapsulated within the porous polymersome.
  • Porcity refers in one embodiment, to the fractional volume (dimension-less) that is not occupied by solid material.
  • porosity refers in one embodiment, to the fractional volume of the diblock copolymer that is not occupied by by removing a filler material.
  • porosity referrs to the void volume that includes the interstitial volume between swollen nanoparticle shell-forming polymer plus any volume within swollen nanoparticle shell-forming polymer (i.e., internal porosity)) that is not occupied by the diblock copolymer or the filler or the paramagnetic lanthanide or the molecular agent that prevents the paramagnetic ion from leaking out of the porous polymersome shell, or other solid components (e.g., fibers).
  • a porous nanoparticle comprising: a porous polymersome shell; and a paramagnetic ion operably linked a core therein, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell.
  • said paramagnetic ion can be attached to a molecular agent that is sufficiently large so as to prevent said paramagnetic ion from being leaked out of said porous polymersome shell.
  • the molecular agent examples include, but are not limited to, a dendrimer, a polymer, a macromolecule, a peptide, a protein, a diblock copolymer, a bulky chelating agent, a nucleic acid, a polylysine, a dextran, or a similar macromolecule known in the art.
  • dendrimers refer to polymers of spherical or other three-dimensional shapes that have precisely defined compositions and that possess a precisely defined molecular weight.
  • the dendrimers used in the methods and compositions described herein are synthesized as water-soluble macromolecules through appropriate selection of internal and external moieties.
  • dendritic macromolecules are characterized by a highly branched, layered structure with a multitude of chain ends.
  • the polymersome shell further comprises a phospholipid.
  • the phospholipid is phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), or their combination.
  • PC phosphatidylcholine
  • PI phosphatidylinositol
  • PS phosphatidylserine
  • PE phosphatidylethanolamine
  • the polymersome is comprised of a di-block copolymer.
  • the di-block copolymer is PEG-PLA, or PEG-PCL, PEO-PPO, PEO-PHEMA, PEO-PMMA, PEO-PMAA, PEO-PMOXZ, PEO-PtBuA, PEO-PMA, PBdEO, PEO-PEE, PS- PEO, PB-PEO or their combination in other discrete embodiments of the di-block copolymer used in the methods and compositions described herein.
  • the block polymer is tactic, atactic or syndiotactic and comprises in another embodiment, no less than about 1,000 paramagnetic ions.
  • the polymersome is cross-linked and the cross-link density is, in another embodiment, no less than 5%.
  • the a molecular agent is conjugated to the paramagnetic ion, wherein the molecular agent is capable of preventing the paramagnetic ion from leaking out of the polymersome shell.
  • the porous polyersome is functionalized with a targeting agent. In other embodiments, no functionalization of the porous polyersome is needed.
  • the paramagnetic porous polymersomes may be used as a blood pool agent, or to report on macrophage infiltration, or to look at other phagocytic cells. It is known in the art that these applications do not require targeting agents, although for improved phagocytic uptake alternatives to PEO may be desirable for the hydrophilic block(e.g. PAA).
  • the di-block copolymers used in the methods described herein are tactic, or atactic, syndiotactic or their compbination in other didcrete embodiments.
  • the di-block copolymer is degradable, for example, bio-degradable.
  • the polymersome comprises a porous nanovesicle.
  • the porous nanovesicle is a stable nanovesicle that is capable of cross-linking to another molecule.
  • the porous nanovesicle comprises a non-filler phospholipid.
  • the porous nanovesicle comprises an amphiphilic molecule as a non-filler.
  • the polymersome comprises a composition comprising a drug.
  • the composition may be encapsulated within the hydrophobic domain.
  • the composition may be encapsulated within the hydrophilic domain.
  • the composition can degrade or get destabilized in circulation such that the paramagnetic ion operably linked to said core is cleared by renal filtration (e.g., through the kidneys).
  • a magnetic resonance imaging agent comprising the nanoparticle described hereinabove.
  • the nanoparticle described hereinabove is used in the methods described herein.
  • a magnetic resonance imaging agent comprising, in another embodiment, a nanoparticle, comprising the porous nanoparticle of any one of the embodiments described supra, operably linked to a ligand, wherein the ligand is specific for a pre- selected marker.
  • the marker is a marker of a cancer, or a cardiovascular disease, apoptosis or their combination in other discrete embodiments of the markers used in the methods described herein.
  • the invention provides a contrast agent for magnetic resonance (MR) imaging, the contrast agent comprising the porous nanoparticle of the invention.
  • the invention provides a blood pool agent, comprising the porous nanoparticle of the invention.
  • the invention provides an imaging agent for macrophage infiltration comprising the porous nanoparticle of the invention.
  • the invention provides an agent for visualizing phagocytic cells comprising the porous nanoparticle of the invention.
  • the invention provides a method for producing a nanoparticle, whereby the nanoparticle ir prodeuced by a hrdration process, electroformation, or a method for forming vesicles with amphiphilic molecules.
  • the hydration process comprises the steps of: dissolving a non-filler polymer and a compatible filler in a solvent; removing the solvent, thereby forming a dehydrated filler-impregnated film; adding a paramagnetic ion operably linked to a molecular agent, to the dehydrated filler-impregnated film, whereby the molecular agent is capable of preventing the paramagnetic ion from leaking out of the porous nanoparticle; incubating the filler-impregnated dehydrated film and the paramagnetic ion operably linked to the molecular agent in the presence an aqueous solution, thereby forming vesicles; reducing the vesicle size; and removing the filler, creating a porous shell.
  • a method of producing a porous nanoparticle comprising the steps of: 1) dissolving a di-block copolymer and a compatible filler in a solvent, 2) removing the solvent, thereby forming a dehydrated filler-impregnated film, 3) adding a paramagnetic ion operably linked to a molecular agent to the dehydrated filler-impregnated film, whereby the molecular agent is capable of preventing the paramagnetic ion from leaking out of the porous nanoparticle, 4) incubating the filler- impregnated dehydrated film and the paramagnetic ion operably linked to the molecular agent in the presence an aqueous solution, thereby forming vesicles. 5) reducing the vesicle size 6) Cross-linking the di-block copolymer 6) removing the filler, creating a porous shell.
  • the ompatible filler used in the methods described herein is a non-polymerizable phospholipid or a degradable polymer/phospolipid composition in another discrete embodiment.
  • any appropriate filler material may be used in the compositions and methods described herein, so long as it can be removed leaving a void in the shell, which in certain embodiment, will allow free exchange of water between the internal space of the polymersome and the environment.
  • the void created by the removal of the filler material allow for free diffusion, referring in another embodiment to the diffusion of water molecules in and out of the internal space of the polymersome, without having to dissolve in the outer shell.
  • the porous vesicle membrane described in the nanoparticles and methods described herein leads in one embodiment to a significant improvement in the water-exchange rate of the encapsulated Gd due to the faster flux of water across the bilayer.
  • the relaxivity of the nanoparticle is improved due in another embodiment, to the slower rotational correlation lifetime of the encapsulated Gd-dendrimers.
  • the polymersomes possess an unobstructed outer surface that is used for attaching targeting ligands and a hydrophobic domain that in one embodiment, presents a natural carrier environment for hydrophobic drugs. Therefore, the porous polymersomes described herein provide in one embodiment, a powerful new platform for combined targeted drug delivery and MR imaging.
  • the step of reducing the vesicle size in the methods provided herein is done using an extrusion means, a jet-impingement means, a sonicating means, a milling means, and emulsion precipitation means, or their combination.
  • the step of removing the filler described hereinabove and is used in the methods provided herein comprises contacting the reduced sized, and in another embodiment, cross linked shell to an agent capable of removing the filler without affecting the shell's integrity.
  • the agent used in the methods provided herein is a solvent, or an enzyme, an acid, or their combination in other discrete embodiments of the agent used to remove the Phospholipids, thereby creating the porosity uin one embodiment.
  • the filler is removed by heat, light, or combination thereof.
  • the methods , nanoparticles and polymersomes described hereinabove are used in the methods of obtaining a pathology-specific magnetic resonance image (MRI) of a subject.
  • MRI magnetic resonance image
  • a method of obtaining a magnetic resonance image (MRI), in a subject comprising administering to said subject an emulsion comprising nanoparticles, said nanoparticles comprising a porous polymersome shell operably linked to a targeting ligand; and a paramagnetic ion operably linked to a core therein, whereby the targeting ligand is specific for a marker of a disease in said subject, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell.
  • MRI magnetic resonance image
  • a method of obtaining a pathology- specific magnetic resonance image (MRI) of a subject comprising administering to said subject an emulsion comprising nanoparticles, said nanoparticles comprising a porous polymersome shell operably linked to a targeting ligand; and a paramagnetic ion operably linked to a core therein, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell, whereby the targeting ligand is specific for a marker of the pathology of said subject.
  • MRI magnetic resonance image
  • the targeting ligand is coupled covalently to the polymersome.
  • the targeting ligand used in the methods described herein is a small P-70442-PC
  • the targeting ligand is specific for a marker of a pathology of interest.
  • a method of obtaining a breast- cancer specific magnetic resonance image (MRI) of a subject comprising administering to said subject an emulsion comprising nanoparticles, said nanoparticles comprising a porous polymersome shell operably linked to an antibody, or a fragment thereof, specific against a carcinoembryonic antigen (CEA); and a paramagnetic ion operably linked to a core therein, wherein said core comprises a molecular agent that prevents said paramagnetic ion from leaking out of said porous polymersome shell, thereby delivering a breast-cancer specific magnetic resonance image (MRI) of a subject.
  • MRI breast- cancer specific magnetic resonance image
  • a method an diagnosing atherosclerotic disease or predisposition thereto in a subject comprising the step of administering to said subject an emulsion of porous nanoparticles, said nanoparticles comprising a porous polymersome shell operably linked to a VCAM-I targeting ligand; and a paramagnetic ion operably linked to a molecular agent, whereby the molecular agent is capable of preventing the paramagnetic ion from leaking out of the porous nanoparticle; obtaining a magnetic resonance image at an area of interest in the subject; and comparing the observed image with a reference image obtained from the same area of interest.
  • the marker of the pathology of said subject is a marker of a cancer, or a cardiovascular disease, apoptosis or their combination in other discrete embodiments of the methods described herein.
  • the cancer marker for which a specific ligand is operably linked to the shell of the polymersome described herein is a Transferrin receptor, or c-MET, ⁇ v- ⁇ 3 integrins, EGFR, Her2/neu, PSA, a member of the MUC-type mucin family, a member of the epidermal growth factor receptor (EGFR) family, a carcinoembryonic antigen (CEA), a MAGE (melanoma antigen) gene family antigen, a T/Tn antigen, a hormone receptor, a Cluster Designation/Differentiation (CD) antigen, a tumor suppressor gene, a cell cycle regulator, an oncogene, an oncogene receptor, a proliferation marker, an adhesion molecule, a P-70442-PC
  • VEGF vascular endothelial growth factor
  • the pathology of interest, sought to be specifically imaged is cardiovascular disease and the cardiovascular disease marker is VCAM-I, or integrin glcyoprotein Ilb/IIIa, myosin, e-selectin, or their combination in other discrete embodiments of the CVD markers for which a specific ligand is operably linked to the shell of the polymersome described herein.
  • a method an diagnosing atherosclerotic disease or predisposition thereto in a subject comprising the step of administering to said subject an emulsion of porous nanoparticles, said nanoparticles comprising a porous polymersome shell operably linked to a VCAM-I targeting ligand; and a paramagnetic ion operably linked to a molecular agent, whereby the molecular agent is capable of preventing said paramagnetic ion from leaking out of said porous nanoparticle; obtaining a magnetic resonance image at an area of interest in the subject; and comparing the observed image with a reference image obtained from the same area of interest.
  • the pathology of interest, sought to be specifically imaged is cardiovascular disease and the cardiovascular disease marker is VCAM-I wherein the VCAM-I targeting ligand is a peptide represented by the sequence VHSPNKK set forth in SEQ ID. No. 1, its peptidomimetic molecule, a nucleotide encoding it or a combination thereof.
  • the porous polymersome shell used in the methods described herein is impregnated with a compatible filler, capable of binding the VCAM-I targeting ligand, which, in another embodiment, is represented by the sequence VHSPNKK set forth in SEQ ID. No. 1, its peptidomimetic molecule, a nucleotide encoding it or a combination thereof.
  • the nucleotide sequence encoding the amino-acid sequence represented by SEQ ID No. 1 is gugcacucccccaacaagaag (SEQ ID No. 2), or its analog.
  • the compatible filler, capable of binding the VCAM-I targeting ligand is succinic acid
  • the targeting ligands cover a range of suitable moieties which bind to components of blood clots.
  • a component may itself be used to generate a ligand by using the component to raise antibodies or to select aptamers that are specific binding partners for the component.
  • a suitable ligand may be known in the art.
  • antibodies can be raised to desired components by conventional techniques and can be provided, in certain embodiments, as monoclonal antibodies or fragments thereof, or as single chain antibodies produced recombinantly.
  • the subject to be administered the compositions described herein is human, it may be desirable to humanize antibody-type ligands using techniques generally known in the art.
  • suitable proteins which bind to targets can be discovered through phage-display techniques or through the preparation of peptide libraries using other appropriate methods.
  • Selective aptamers which are able selectively to bind desired targets may also be prepared using known techniques such as SELEXTM. (Aptamers are oligonucleotides which are selected from random pools for their ability to bind selected targets.)
  • peptidomimetics which are small organic molecules intended to mimic peptides of known affinities can also be used as targeting agents.
  • targeting agents that bind to fibrin, as fibrin is a particularly characteristic element included in blood clots.
  • Antifibrin antibodies are particularly preferred, including fragments thereof, such as the F ab , F (ab') 2 fragments, single chain antibodies (F v ) and the like.
  • the targeting agent targets components of the blood clot other than fibrin, such as gpIIb/IIIa, clotting factors Xa and FXa and the like.
  • additional components of the emulsion can be bound to the nanoparticles in ways similar to those which are used to bind the ligands.
  • Other components which maybe coupled to the nanoparticles through entrapment in the coating layer include radionuclides. P-70442-PC
  • radionuclides include in another embodiment, " Tc.
  • the radioactive ions can be provided to the preformed emulsion in a variety of ways.
  • " Tc- pertechnate may be mixed with an excess of stannous chloride and incorporated into the preformed emulsion of nanoparticles, followed by removal of unbound " Tc-pertechnate by repeated centrifugation and washing.
  • Stannous oxinate can be substituted for stannous chloride.
  • commercially available kits such as the HM-PAO (exametazine) kit marketed as Ceretek® by Nikomed Amersham can be used. Means to attach various radioligands to the nanoparticles described herein are understood in the art.
  • paramagnetic metals useful in the MRI contrast agents described herein include rare earth metals, typically, lanthanum, ytterbium, gadolinium, europium, and the like. Iron ions may also be used.
  • the paramagnetic metals useful in the MRI contrast agents described herein include rare earth metals, typically, lanthanum, ytterbium, gadolinium, europium, and the like. Iron ions may also be used. Also included in the surface of the nanoparticle, in some embodiments described herein, are biologically active agents. These biologically active agents can be of a wide variety, including proteins, nucleic acids, pharmaceuticals, and the like.
  • antineoplastic agents include hormones, analgesics, anesthetics, neuromuscular blockers, antimicrobials or antiparasitic agents, antiviral agents, interferons, antidiabetics, antihistamines, antitussives, anticoagulants, and the like.
  • the inclusion of a chelating agent containing a paramagnetic ion makes the emulsion useful as a magnetic resonance imaging contrast agent. Because the particles comprise large amounts of fluorine, the addition of a paramagnetic ion is not necessary to make these particles useful for MRI.
  • the inclusion of biologically active materials makes them useful as drug delivery systems.
  • the inclusion of radionuclides makes them useful either as therapeutics for radiation treatment or as diagnostics for imaging or both. A multiplicity of such activities may be included; thus, images can be obtained of targeted tissues at the same time active substances are delivered to them.
  • 19 F magnetic resonance imaging can be used to track the location of the particles concomitantly with their additional functions described above.
  • encapsulating membrane is a vesicle in all respects except for the necessity of aqueous solution. Encapsulating membranes, by definition, compartmentalize by being semi- or selectively permeable to solutes, either contained inside or maintained outside of the spatial volume delimited by the membrane.
  • a vesicle is a capsule in aqueous solution, which also contains aqueous solution.
  • the interior or exterior of the capsule could also be another fluid, such as an oil or a gas.
  • a "capsule,” as the term is used herein, is the encapsulating membrane plus the space enclosed within the membrane.
  • membrane refers to a spatially distinct collection of molecules that defines a 2-dimensional surface in 3-dimensional space, and thus separates one space from another in at least a local sense. Such a membrane must also be semipermeable to solutes. It must also be sub-microscopic, as resulting from a process of self- assembly.
  • membranes can have fluid or solid properties, depending on temperature and on the chemistry of the amphiphiles from which it is formed. At some temperatures, the membrane can be fluid (having a measurable viscosity), or it can be solid- like, with an elasticity and bending rigidity.
  • the membrane can store energy through its mechanical deformation, or it can store electrical energy by maintaining a transmembrane potential. Under some conditions, membranes can adhere to each other and coalesce (fuse). Soluble amphiphiles can bind to, and intercalate within a membrane.
  • Polymersomes refers in certain embodiments to vesicles, which are assembled from synthetic polymers in aqueous solutions. Unlike liposomes, a polymersome does not include lipids or phospholipids as its majority component. Consequently, polymersomes can be thermally, mechanically, and chemically distinct and, in particular, more durable and resilient than the most stable of lipid vesicles. The polymersomes assemble during processes of lamellar swelling, e.g., by film or bulk rehydration or through an additional phoresis step, as described below, or by other known methods. Like liposomes, polymersomes form by "self assembly," a spontaneous, entropy-driven process of preparing a closed semi-permeable membrane.
  • the ability to impart structural stability into vesicle bilayer architectures is highly beneficial in biological applications that require nanoplatforms with long in vivo half-lives.
  • polymerized polymersomes provided a stable, alternative platform to liposomes.
  • materials may be "encapsulated" in the aqueous interior or intercalated into the hydrophobic membrane core of the polymersome vesicle of the present invention.
  • Numerous technologies can be developed from such vesicles, owing to the numerous unique features of the bilayer membrane and the broad availability of super-amphiphiles, such as block copolymers.
  • the synthetic polymersome membrane can exchange material in one embodiment with the "bulk," i.e., the solution surrounding the vesicles.
  • Each component in the bulk has a partition coefficient, meaning it has a certain probability of staying in the bulk, as well as a probability of remaining in the membrane.
  • Conditions can be predetermined so that the partition coefficient of a selected type of molecule will be much higher within a vesicle's membrane, thereby permitting the polymersome to decrease the concentration of a molecule, such as cholesterol, in the bulk.
  • phospholipid molecules have been shown to incorporate within polymersome membranes by the simple addition of the phospholipid molecules to the bulk.
  • polymersomes can be formed with a selected molecule, such as a hormone, incorporated within the membrane, so that by controlling the partition coefficient, the molecule will be released into the bulk when the polymersome arrives at a destination having a higher partition coefficient.
  • a selected molecule such as a hormone
  • the polymersomes of the present invention are formed from synthetic, amphiphilic copolymers.
  • An "amphiphilic” substance is one containing both polar (water-soluble) and hydrophobic (water-insoluble) groups.
  • Polymers are macromolecules comprising connected monomelic units. The monomelic units may be of a single type (homogeneous), or a variety of types (heterogeneous). The physical behavior of the polymer is dictated by several features, including the total molecular weight, the composition of the polymer (e.g., the relative concentrations of different monomers), the chemical identity of each monomeric unit and its interaction with a solvent, and the architecture of the polymer (whether it is single chain or branched chains). For example, in polyethylene glycol (PEG), which is a polymer of ethylene oxide (EO), the chain lengths which, when covalently attached to a phospholipid, P-70442-PC
  • the preferred class of polymer selected to prepare the polymersomes of the present invention is the "block copolymer.”
  • Block copolymers are polymers having at least two, tandem, interconnected regions of differing chemistry. Each region comprises a repeating sequence of monomers. Thus, a “diblock copolymer” comprises two such connected regions (A-B); a “triblock copolymer,” three (A-B-C), etc. Each region may have its own chemical identity and preferences for solvent. Thus, an enormous spectrum of block chemistries is theoretically possible, limited only by the acumen of the synthetic chemist.
  • the block copolymer, used in the invention is degradable, for example, biodegradable.
  • a diblock copolymer in the "melt" phase, (pure polymer), may form complex structures as dictated by the interaction between the chemical identities in each segment and the molecular weight.
  • the interaction between chemical groups in each block is given by the mixing parameter or Flory interaction parameter, [chi], which provides a measure of the energetic cost of placing a monomer of A next to a monomer of B.
  • [chi] Flory interaction parameter
  • the segregation of polymers into different ordered structures in the melt is controlled by the magnitude of [chi] N, where N is proportional to molecular weight. For example, the tendency to form lamellar phases with block copolymers in the melt increases as [chi] N increases above a threshold value of approximately 10.
  • a linear diblock copolymer of the form A-B can form a variety of different structures. In either pure solution (the melt) or diluted into a solvent, the relative preferences of the A and B blocks for each other, as well as the solvent (if present) will dictate the ordering of the polymer material. In the melt, numerous structural phases have been seen for simple AB diblock copolymers.
  • the most common lamellae-forming amphiphiles also have a hydrophilic volume fraction between 20 and 50%. Such molecules form, in aqueous solutions, bilayer membranes with hydrophobic cores never more than a few nanometers in thickness.
  • the present invention relates to all super- amphiphilic molecules which have hydrophilic block fractions within the range of 20-50% by volume and which can achieve a capsular state.
  • the ability of amphiphilic and super-amphiphilic molecules to self-assemble can be largely assessed, without undue experimentation, by suspending the synthetic super- amphiphile in aqueous solution and looking for lamellar and vesicular structures as judged by simple observation under any basic optical microscope or through the scattering of light.
  • temperature can affect the stability of the thin lamellar structures, in part, by determining the volume of the hydrophobic portion.
  • the strength of the hydrophobic interaction which drives self-assembly and is required to maintain membrane stability, is generally recognized as rapidly decreasing for temperatures above approximately 50C.
  • Such vesicles generally are not able to retain their contents for any significant length of time under conditions of boiling water.
  • PEG poly(ethylene glycol)
  • PEO polyethylene oxide
  • Additional compositions that are within the scope described herein are mixtures of two or more PEO, polypropylene oxide (PPO), and PEG.
  • the vesicle is bioresorbable.
  • bioresorbable refers to a molecule, when degraded by chemical reactions, leads to substituents which can be used by biological cells as building blocks for the synthesis of other chemical species, or can be excreted as waste.
  • PEO-PEE polyethyleneoxide- polyethylethylene
  • PEO-PEE polyethyleneoxide- polyethylethylene
  • compartmentalized agent in some embodiments, is of therapeutic value within the human body.
  • the vesicle contains compartmentalized or covalently P-70442-PC
  • Some vesicles additionally comprising at least one of an emissive agent, a cytotoxic agent, a magnetic resonance imaging (MRI) agent, positron emission tomography (PET) agent, radiological imaging agent or a photodynamic therapy (PDT) agent compartmentalized within the hydrophobic vesicle membrane.
  • the MRI agent compartmentalized is within the hydrophobic vesicle membrane.
  • the PET agent compartmentalized within the hydrophobic vesicle membrane Some compositions have at least one radiological imaging agent compartmentalized within the hydrophobic vesicle membrane. Some compositions have at least one PDT agent compartmentalized within the hydrophobic vesicle membrane.
  • the agents are compartmentalized the aqueous polymersome interior.
  • the vesicles can additionally comprising at least one of a secondary emissive agent, a cytotoxic agent, a magnetic resonance imaging (MRI) agent, positron emission tomography (PET) agent, radiological imaging agent or a photodynamic therapy (PDT) agent compartmentalized within the hydrophobic vesicle membrane.
  • a secondary emissive agent is compartmentalized within the aqueous polymersome interior.
  • polymersomes described herein contain at least one visible- or near infrared-emissive agent that is dispersed within the polymersome membrane. Some emissive agents emit light in the 700-1100 nm spectral regime.
  • Certain emissive agents comprise a porphyrin moiety.
  • the emissive agent comprises at least two porphyrin moieties where the porphyrin moieties are linked by a hydrocarbon bridge comprising at least one unsaturated moiety.
  • Some emissive agents useful in the invention are porphycene, rubyrin, rosarin, hexaphyrin, sapphyrin, chlorophyl, chlorin, phthalocynine, porphyrazine, bacteriochlorophyl, pheophytin, texaphyrin macrocyclic -based components, or metalated derivatives thereof.
  • Useful emissive agents include emissive agents that are a laser dye, fluorophore, lumophore, or phosphor.
  • the term "about” means in quantitative terms plus or minus 5%, or in another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment plus or minus 20%.
  • subject refers in one embodiment to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae.
  • the subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice and humans.
  • subject does not exclude an individual that is normal in all respects.
  • PEO(1300)-b-PBD(2500) was purchased from Polymer Source (Dorval, Quebec, Canada).
  • l-palmitoyl ⁇ -oleoyl-sn-glycero-S-phosphocholine (POPC) and 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhod-PE) were obtained from Avanti Polar Lipids (Alabaster, AL).
  • PAMAM dendrimers ethylenediamine core, generation 2 were from Aldrich Chemical Co. as 20% w/v solutions in methanol.
  • CF 5-(and-6)-carboxyfluorescein
  • TRITC tetramethylrhodamine-5-(and-6)- isothiocyanate
  • DTPA dianhydride Diethylenetriaminepentaacetic acid dianhydride
  • III gadolinium
  • II potassium persulfate
  • sodium metabisulfite sodium metabisulfite
  • iron (II) sulfate heptahydrate were obtained from Sigma-Aldrich. All other chemical were used as received. All of the buffer solutions were prepared with DI water.
  • the purified PAMAM-DTPA conjugates were mixed with 100 mg GdCl 3 in 0.1 M citrate buffer (pH 5.6) for overnight at 42 0 C.
  • the unreacted Gd 3+ was removed by centrifugal filter devices (Amicon Ultra-4, 5000 MWCO, Millipore Corp.) while simultaneously changing the buffer to 0.1 M PBS buffer.
  • the purified PAMAM-DTPA-Gd conjugates were used for vesicle encapsulation.
  • Nanometer sized vesicles were prepared using the film hydration technique.
  • An aqueous solution (0.1 M PBS, pH 7.0) was added to dried PBdEO film with 15 % POPC or without POPC and incubated in a 65 0 C water bath for half hour and then sonicated for another 1 hour at the same temperature.
  • Samples were subjected to ten freeze-thaw-vortex cycles in liquid nitrogen and warm H 2 O (65 0 C), followed by extrusion 21 times through two stacked 100 nm Nuclepore polycarbonate filters using a stainless steel extruder (Avanti Polar Lipids).
  • Polybutadiene in the cores of the vesicle membranes was cross-linked by free radical polymerization in solution. Specifically, a weighed amount of potassium persulfate was added into polymersome solution and then stirred for 1 hour. The polymerization was initiated by injecting appropriate amounts of redox couple, Na 2 S 2 OsZFeSO 4 -TH 2 O, in the sequence of sodium metabisulfite and ferrous sulfate. All cross-linking polymerization were performed at fixed weight ratios of 1 :1:0.5:0.02 between the PBdEO, potassium persulfate, sodium metabisulfite, and ferrous sulfate.
  • Triton X-IOO was added into polymerized vesicles and incubated for 10 minutes with shaking. The final concentration of Triton X-IOO is 10 mM. Triton X-IOO was subsequently removed through repeated washing on centrifugal filter devices (Amicon Ultra-4, IOOK MWCO, Millipore Corp.).
  • Phospholipid-doped polymersomes were prepared using diblock copolymers and a pre-defined percent of phospholipid (e.g. 1-25%). Dendrimers that have been labeled with P-70442-PC
  • chelated Gd are encapsulated within the phopholipid-doped polymersomes. The polymersomes are then cross-linked and the phospholipids are extracted, leaving pores for enhanced diffusion of water in the polymersome bilayer. This nanoparticle overcomes many of the limitations associated with Gd-loaded liposomes. It should be noted that chelated Gd was attached to dendrimers to prevent their diffusion through the polymersome pore. Also, the attachment of chelated Gd to dendrimer results in improved Rl.
  • the paramagnetic porous polymersomes were synthesized as described above (15% phospholipid was used during synthesis) and characterized their relaxivity, Rl.
  • the Rl per Gd was found to be 7.2 mM-ls-1.
  • chelated alone possesses an Rl of 3.9 mM-ls-1 and dendrimer labeled with chelated Gd exhibits an Rl of 11.5 mM-ls-1.
  • Polymersomes that do not have pores but have encapsulated chelated Gd exhibit an Rl of 3.4 mM-ls-1.
  • Porous polymersomes are depicted in Figure 1. Preparation of the porous polymersomes required generation 2 dendrimers to first be labeled with Gd-DTPA. The Gd- labeled dendrimers were then encapsulated into vesicles assembled from an mixture of polymerizable amphiphillic diblock copolymer (PEO(1300)-b-PBD(2500), PBdEO) and nonpolymerizabe native phospholipid (l-palmitoyl-2-oleoyl-*n-glycero-3-phosphocholine, POPC).
  • PEO polymerizable amphiphillic diblock copolymer
  • PBdEO polymerizable amphiphillic diblock copolymer
  • POPC nonpolymerizabe native phospholipid
  • Nonpolymerizable phospholipids were integrated into the polymer bilayer during vesicle formation, by using a low molar percentage of fluorescent phospholipid during fabrication.
  • a classical swelling technique was also adopted to form giant vesicles that could be observed by microscopy.
  • a uniform distribution of the fluorescent lipid was evident at the resolution of the optical microscope. This result demonstrated the feasibility of forming polymer-lipid hybrid vesicles.
  • the diblock copolymers were cross-linked via free radical polymerization and the stability of the polymerized vesicles was examined by surfactant solubilization. Specifically, polymerized and unpolymerized vesicles were treated with Triton X-100 at a high molar ratio of Triton X-100-to-polymer. Treatment of unpolymerized vesicles with Triton X-100 led to complete vesicle dissolution (data not shown) as determined by dynamical light scattering (DLS). The vesicles dissolved and formed mixed micelles when the surfactant concentration exceeded its critical concentration.
  • DLS dynamical light scattering
  • each of these examples had Gd immobilized on the surface of the nanoparticle and thus do not take advantage of the large intraparticle volume.
  • This architecture limited the attachment of targeting ligands.
  • the attachment of Gd directly to the large nanoparticle did improve Gd relaxivity due to their slower rotational correlation lifetimes.
  • Example 3 Preparin2 and testin2 VCAM-I tar2 ⁇ ted parama2netic porous polymersomes.
  • VCAM-I vascular cell adhesion molecular-1
  • Paramagnetic porous polymersomes are functionalized with VCAM-I targeted peptides to demonstrate that the contrast agent exhibits affinity and specificity for activated endothelial cells in culture.
  • Polymersomes that possess a 10% content of succinic acid terminated (PEO(1300)-b-PBD(2500)) (Polymer Source) are synthesized.
  • VCAM-I binding peptide VHSPNKK, as set forth in SEQ ID NO: 1.
  • the peptide isattached using carbodiimide chemistry as previously described.
  • Human umbilical vein endothelial cells (HUVEC) that have been activated with TNF-alpha are then labeled with the VCAM-I -targeted polymersomes.
  • Paramagnetic porous polymersomes labeled with scrambled peptides are used as a negative control.
  • Cell binding is measured on a Bruker mq60 MR relaxometer as described previously. The specificity of polymersome binding is further confirmed by performing competitive inhibition studies with anti-VCAM-1 antibodies.
  • Example 4 Comparison of polymersome -Gd versus perfluorocarbon Gd.
  • Perfluorocarbon nanoparticles have Gd on outside limits of number of targeting moieties.
  • the polymersomes of the invention have the Gd on inside leaving surface free to conjugate with more targeting molecules, which results in higher affinity (target flexibility and contrast at target site).
  • the nanoparticle of the invention is half size (e.g., 125nm) that could allow better penetration, longer circulation.
  • the Gd in the perfluorocarbon particle exhibits a relaxivity that is 2.48 times higher (per Gd) than the nanoparticle of the invention
  • the nanoparticle of the invention can have 4.8 times more Gd, and therefore, the nanoparticle of the invention can exhibit roughly 2x the total contrast.
  • the nanoparticle of the invention can be optimized to yield even higher contrast. Specifically, encapsulation with a larger dendrimer (e.g. G4 rather than G2) can result in higher Rl because of slower rotational velocities. Contrast can be improved by increasing pore sizes or increase concentration of Gd-labeled dendrimer during encapsulation; put the maximum number of Gd on each dendrimer.
  • a larger dendrimer e.g. G4 rather than G2
  • Contrast can be improved by increasing pore sizes or increase concentration of Gd-labeled dendrimer during encapsulation; put the maximum number of Gd on each dendrimer.
  • Example 5 Gd-encapsulated Porous Polymersomes as Highly Efficient MRI Contrast
  • PEO(1300)-&-PBD(2500) (PBdEO) and PEO(2000)-£-PCL (2700) were purchased from Polymer Source (Dorval, Quebec, Canada).
  • PEO, PBD and PCL represent polyethylene oxide, polybutadiene, and polycaprolactane separately.
  • ethylenediamine core, generation 3 were from Aldrich Chemical Co. as 20% w/v solutions in methanol.
  • 5-(and-6)-carboxyfluorescein (CF) and fluorescein isothiocyanate (FITC) were obtained from Molecular Probes.
  • Diethylenetriaminepentaacetic acid dianhydride (DTPA dianhydride) and gadolinium (III) chloride were obtained from Sigma-Aldrich. All other chemicals were used as received. All of the buffer solutions were prepared with DI water.
  • the unreacted Gd 3+ was removed by centrifugal filter devices (Amicon Ultra-4, 5000 MWCO, Millipore Corp.) while simultaneously changing the buffer to 0.1 M PBS buffer. To ensure complete removal of unreacted Gd 3+ , the Gd content in the eluent was checked after each centrifugation until no Gd 3+ was detectable.
  • the purified PAMAM-DTPA-Gd conjugates were used for vesicle encapsulation.
  • Polymer vesicles were prepared by dissolving PBdEO/PEOCL (100 mg PBdEO) in chloroform and then removing the solvent using a stream of N 2 gas. After further drying under vacuum overnight, the residual polymer film was hydrated with an aqueous solution (0.1 M PBS, pH 7.0) in a 65 0 C water bath for half hour and then sonicated for another 1 hour at the same temperature. Samples were subjected to ten freeze-thaw-vortex cycles in liquid nitrogen and warm H 2 O (65 0 C), followed by extrusion 21 times through two stacked 100 nm Nuclepore polycarbonate filters using a stainless steel extruder (Avanti Polar Lipids).
  • PAMAM-DTPA-Gd For PAMAM-DTPA-Gd encapsulation, PAMAM-DTPA-Gd was added to the dried polymer films. Non-entrapped PAMAM-DTPA-Gd was removed through repeated washing P-70442-PC
  • Tl relaxation time of the eluent was checked after each centrifugation until no Gd was detectable, i.e. until the Tl -relaxation time was equivalent to that of sodium phosphate (pH 7.0) buffer (-1000 ms). Tl-relaxation times were determined using a Bruker mq60 MR relaxometer operating at 1.41 T (60 MHz).
  • the number of polymersomes in the purified sample was then calculated by determining the amount of polymer in each vesicle. For this calculation, the average diameter of each polymersome was taken to be 130 nm. Further, the average area occupied by single polymer molecules in a bilayer has previously been determined to be ⁇ 1 nm 2 , 31 and the thickness of the polymersome bilayer has previously been determined to be -10 nm. 38 The amount of Gd in the polymersome sample was measured by ICP-AES.
  • % CF released (I x - 1 0 )/( I t - 1 0 ) x 100
  • I Q is the fluorescence intensity of the vesicle suspension containing CF at the initial time
  • I x is the fluorescence intensity at any given time
  • / t is the fluorescence intensity after addition of an aqueous solution of Triton X-100 to the suspension.
  • the NIH 3T3 mouse fibroblast cells were cultured in Dulbecco's Modified Eagle's medium with 10% fetal bovine serum at 37 0 C under 5% CO 2 .
  • NIH 3T3 cells were seeded in 96- well plates at a density of 10, 000 cells per well. After incubation overnight (37 0 C, 5% CO 2 ), the medium in each well was aspirated off and loaded with 100 ⁇ L of fresh medium containing nanoparticles with different Gd 3+ concentration. After incubation for 4 h, the nanoparticle containing medium in each well was aspirated off and replaced with 100 ⁇ L of medium and 10 ⁇ L of MTT reagent. The cells were incubated for 2 to 4 hours, then 100 ⁇ L detergent reagent was added and left at room temperature in the dark for 2 hours. The absorbance at 570 nm was measured using a microplate reader.
  • IACUC Institutional Animal Care and Use Committee
  • Imaging was performed using a 4.7 T small animal horizontal bore Varian INOVA system. Gradients used in the magnet were 12 cm diameter at 25 G/cm. Imaging was performed using a custom-built 50 mm diameter send-receive birdcage volume coil. Following induction of a mouse to the plane of anesthesia using 4% isoflurane/oxygen, mice were fixed to an acrylic patient bed in the prone position and maintained on a 1% isoflurane/oxygen mixture. Body temperature was monitored with a platinum rectal probe connected to a small-animal monitoring system (SA Incorporated) and maintained using a stream of heated air.
  • SA Incorporated small-animal monitoring system
  • porous polymer nanovesicles that exhibit improved permeability to water flux and a large capacity to store chelated Gd within the aqueous core.
  • the porous polymersomes -130 nm in diameter, were produced through the aqueous assembly of the polymers, PEO(1300)-&-PBD(2500) (PBDEO) and PEO(2000)-£-PCL(2700) (PEOCL).
  • PBDEO PEO(1300)-&-PBD(2500)
  • PEO PEO(2000)-£-PCL(2700)
  • Gd-encapsulated porous polymersomes can be used as an effective blood pool agent, a targeted contrast agent, and/or a platform for combined targeted drug delivery and MR imaging.
  • Gd-encapsulated porous polymersomes have been developed as MRI contrast agents, as shown schematically in Figure 13.
  • the paramagnetic porous polymersomes were prepared by first encapsulating Gd-DTPA-labeled dendrimers into vesicles assembled from a mixture of two amphiphilic diblock copolymers, PBdEO and PEOCL, via thin film hydration. The mean diameter of the polymersomes was then reduced to 130 ⁇ 10 nm (S. D.) by P-70442-PC
  • vesicles with 15 mol % PEOCL only exhibited 50 % release of CF over the same period.
  • Vesicles formulated with 100 % PBdEO exhibited less than a 5 % release of the encapsulated dye over 4 days.
  • FITC-labeled dendrimer was encapsulated within the vesicle core and its release was quantified following suspension of the polymersomes in PBS buffer (0.1 M phosphate, pH 5.1) at 37°C. This P-70442-PC
  • any dendrimer that was released from the polymersome would be detectable in the flow-thru, while encapsulated dendrimer would remain in the retentate. Consistent with the encapsulation of FITC-labeled dendrimers within structurally stable polymersomes, no significant increase in fluorescence was detected in the flow-thru for at least 5 days following suspension in acidic media (Figure 17).
  • a similar assay was also conducted with polymersomes containing Gd-labeled dendrimers, where Tl measurements of the flow-thru were conducted to confirm the absence of Gd.
  • the Tl relaxation time of the flow-thru was similar to that of pure phosphate buffer, -1000 ms.
  • non-porous vesicles with Gd-labeled dendrimer encapsulated within the core had an Rl relaxivity of 3.4 mM "1 s "1 per Gd and non-porous vesicles with Gd-DTPA encapsulated within the core had an Rl of 1.7 mM "1 s "1 per Gd. Therefore, the generation of pores within the vesicle bilayer led to a 2.2-fold improvement in relaxivity per Gd and coupling of the Gd- DTPA to the dendrimer led to another 2-fold improvement in relaxivity. Combined, the paramagnetic porous polymersomes exhibited more than a 4.4-fold improvement in relaxivity per Gd compared with non-porous vesicles with encapsulated Gd-DTPA.
  • parmagnetic porous polymersomes In addition to the enhancement observed in the relaxivity per Gd, parmagnetic porous polymersomes also benefit tremendously from their capacity to carry very large payloads of chelated Gd within their aqueous cores. Specifically, it was estimated that there were approximately 38,947 Gd per polymersome. This measurement assumed an average P-70442-PC
  • the unique pharmacokinetic properties of the paramagnetic porous polymersomes may make them a favorable option as either a blood pool agent or as a targeted imaging agent.
  • MR contrast agents many large macromolecular complexes and nanoparticles are being evaluated as MR contrast agents, but their prolonged retention is often a major limitation for clinical use. It has previously been reported that uptake of gadolinium chloride in the liver could lead to the depletion of Kupffer cells, raising concerns about toxicity.
  • smaller MR contrast agents are rapidly excreted from the kidneys, they only provide a small window for imaging and have fewer opportunities to bind target receptors resulting in less accumulation at the target site.
  • the Gd-encapsulated porous polymersomes of the invention offer an extended circulation time compared with small MR contrast agents, but are still predominantly cleared by the kidney and thus would be more rapidly excreted from the body than conventional nanoparticles.
  • the porous polymersomes described here may provide a powerful new platform for combined targeted drug delivery and MR imaging.

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Abstract

La présente invention concerne des polymérosomes poreux et leur utilisation dans le diagnostic et le pronostic de maladies. En particulier, l'invention concerne des polymérosomes poreux qui comportent des lanthanides, et leurs utilisations.
PCT/US2010/039348 2009-06-19 2010-06-21 Polymérosomes poreux paramagnétiques et leurs utilisations Ceased WO2010148395A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105087499A (zh) * 2015-09-07 2015-11-25 江南大学 一株多溴联苯醚单克隆抗体杂交瘤细胞株及其应用
US11026891B2 (en) 2016-08-16 2021-06-08 Eth Zurich Transmembrane pH-gradient polymersomes and their use in the scavenging of ammonia and its methylated analogs
US11713376B2 (en) 2017-09-12 2023-08-01 Eth Zurich Transmembrane pH-gradient polymersomes for the quantification of ammonia in body fluids
US11999829B2 (en) 2017-09-12 2024-06-04 Eth Zurich Method of making a polymersome
US12139617B2 (en) 2017-12-22 2024-11-12 North Carolina State University Polymeric fluorophores, compositions comprising the same, and methods of preparing and using the same
WO2020142664A1 (fr) * 2019-01-03 2020-07-09 Ionpath, Inc. Compositions et réactifs pour imagerie par faisceau d'ions
CN113939740A (zh) * 2019-01-03 2022-01-14 离子路径公司 用于离子束成像的组合物和试剂
JP2022517193A (ja) * 2019-01-03 2022-03-07 アイオンパス， インク． イオンビームイメージングのための組成物および試薬

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