WO2013059617A9 - Compositions de liposome et leurs procédés d'utilisation - Google Patents

Compositions de liposome et leurs procédés d'utilisation Download PDF

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WO2013059617A9
WO2013059617A9 PCT/US2012/061061 US2012061061W WO2013059617A9 WO 2013059617 A9 WO2013059617 A9 WO 2013059617A9 US 2012061061 W US2012061061 W US 2012061061W WO 2013059617 A9 WO2013059617 A9 WO 2013059617A9
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liposomes
liposome
brain
cell
transferrin
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PCT/US2012/061061
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WO2013059617A1 (fr
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Jagdish Singh
Gitanjali SHARMA
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Ndsu Research Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome

Definitions

  • BBB blood brain barrier
  • Gene therapy may prove to be highly valuable in treatment of these CNS disorders.
  • efficient delivery of a desired gene to brain is hampered by the BBB, which is composed of tightly packed cerebral microvascular endothelial cells in conjunction with astrocytes and pericytes.
  • the BBB protects and regulates the homeostasis of brain.
  • it also limits the transport of small molecules, particularly biopharmaceutical drugs such as several proteins, peptides, genes and interference RNA into the brain, thereby limiting the treatment of many brain diseases.
  • Viruses are equipped with different molecular mechanisms for overcoming these hurdles and can therefore, serve as efficient vectors for delivery of desired gene.
  • the severe immune response and cytotoxicity associated with these viruses have reduced their potential application as gene delivery vectors.
  • the present invention provides liposomes and liposomal formulations.
  • the liposomes include a brain-targeting polypeptide conjugated to a phospholipid and a cell-penetrating polypeptide conjugated to a phospholipid.
  • the liposomes are useful in the delivery of therapeutic agents to a brain cell or brain tumor cell.
  • the liposomes may be loaded with one or more therapeutic agent, for example, polypeptides, nucleic acids, or small molecules.
  • the present invention also provides compositions comprising the liposomes of the invention.
  • the present invention provides methods for delivering a therapeutic agent to a target cell, for example, a brain cell or brain tumor cell, by contacting the cell with a liposome of the invention.
  • contacting may be accomplished by administering the liposome at a distance from the target cell.
  • a brain cell or cells of a vertebrate may be contacted with the liposomes by administering the liposomes parenterally, for example, intravenously.
  • the methods of the invention may be used to treat a subject in need of treatment by administering to the subject a liposome containing a therapeutic agent effective to treat the subject.
  • FIG 1 is schematic representation showing introduction of nucleic acid into liposomes.
  • FIG 2 shows H NMR spectra of PEG-DOPE phospholipid (FIG 2A) and PEG- DOPE conjugated to poly-L-arginine (FIG 2B).
  • FIG 3 shows atomic force microscopy (AF ) images of liposomes.
  • FIG 4 is a series of confocal microscopic images showing uptake of liposomes labeled with rhodamine B sulfonyl (Rh-PE) by human embryonic kidney 293 (HEK293) (FIG 4A) and rat endothelial cells (FIG 4B).
  • Rh-PE rhodamine B sulfonyl
  • FIG 5 is a series of confocal microscopic images showing uptake of liposomes labeled with labeled with indocarbocyanine dye (Dil) by mouse brain endothelial cells (bEnd3).
  • FIG 6 is a graph showing percent cell viability of bEnd3 cells and glial cells following uptake of blank liposomes, transferrin poly-L-arginine (Tf-PR) liposomes, or FuGENE® HD reagent.
  • FIG 7 shows confocal images of primary rat glial cells transfected with FuGENE® green fluorescent protein (GFP)-lipoplexes HD reagent (FIG 7A, left panel)) or Tf-PR liposomes encapsulating chitosan-GFP polyplexes (FIG 7A, left panel), and flow cytometric analysis of the transfected cells (FIG 7A and FIG 7B, right panels).
  • GFP green fluorescent protein
  • FIG 8 is a graph showing sodium fluorescein permeability of bEnd3 cells and a combination of bEnd3 cells and glial cells of an in vitro BBB model.
  • FIG 9 compares the permeability coefficients of various liposome formulations by cells in an in vitro BBB model.
  • FIG 10 shows percent uptake of various liposome formulations by cells in an in vitro BBB model as a function of time.
  • FIG 11 shows a graphical representation of formation of liposomes and delivery of liposomes to brain
  • FIG 12 shows a graphical representation of percent hemolysis by different liposomes (FIG12A) and a photograph of Eppendorf tubes containing supernatant from RBCs exposed to different liposomes at varying concentrations (FIG 12B).
  • FIG 13 shows confocal microscopic images of red blood cells (RBCs) exposed to (A) Plain (B) Tf (C) Tf-PR liposomes at 600 nmoles of lipids, (D) PBS, (E) Tf-PR- liposomes at 800 nmoles of lipids, or (F) Triton X 100.
  • RBCs red blood cells
  • FIG 14 shows the biodistribution of DiR- labeled liposomes in different organs of an adult rat as a function of time.
  • FIG 15 shows ex-vivo imaging of different organs isolated from rats at 24h time point post intravenous injection of liposomes (FIG 14A); and ex-vivo images of brains isolated from rats at 24h time point (FIG 14B).
  • FIG 16 is a graph comparing transfection efficiencies of Tf-PR liposomes and Tf-liposomes, in different organs (FIG 15A) and brain (FIG 15B) after intravenous injection of ⁇ -gal plasmid encapsulating liposomes at dose of 50 ⁇ g of DNA/rat.
  • FIG 17 shows images of different tissues after transfection with ⁇ -gal plasmid encapsulating liposomes.
  • Described in the examples below are surface-modified liposomes comprising phospholipids having a transferrin linked to at least some of the phospholipids, which allows targeting of endothelial cells of the BBB by the liposomes. Additionally, the surfaces of the liposomes were modified with poly-L-arginine coupled to phospholipids to improve delivery of molecular cargo into cells.
  • the ability of liposomes to deliver to brain cells nucleic acids encoding a protein was assessed by delivering a nucleic acid comprising a reporter sequence, i.e., a DNA sequence encoding GFP. The DNA was complexed with a chitosan.
  • the liposomes may be used to deliver therapeutic agents including polypeptides
  • nucleic acids e.g., microRNA, siRNA, polynucleotides encoding polypeptides potentially affording therapeutic benefit
  • conventional pharmaceutical agents small molecules or drugs, including, without limitation, chemotherapeutic, antineoplastic, antiangiogenic, and antipsychotic agents.
  • nucleic acids may be complexed to chitosan, which is a biodegradable, non-toxic linear polysaccharide with gene delivery properties.
  • a suitable promoter i.e., a promoter that allows expression in the target cell.
  • suitable promoters may include, for example, inducible promoters or constitutive promoters.
  • polypeptide is used broadly to describe, any biopolymer comprising at least three amino acid residues linked by peptide bonds, and encompasses molecules of any chain length ranging from short chain (e.g., three or more amino acids) peptides to full length proteins.
  • a brain-targeting polypeptide includes polypeptides that correlate with increased ability of liposomes containing the brain-targeting polypeptide to cross the BBB or with increased uptake of liposomes by brain cells or brain tumor cells, relative to that of liposomes lacking the brain-targeting polypeptide.
  • transferrin may be as the targeting polypeptide to target endothelial cells of the BBB. It is known that there is a high density of transferrin receptors on the surface of brain capillaries. Liposomes having receptor binding peptide sequences of transferrin conjugated to phospholipids may also be used to target liposomes to brain cells or brain tumor cells.
  • polypeptides comprising a transferrin receptor targeting subfragment of transferrin such as HAIYPRH (SEQ ID NO:1), THRPPMWSPVWP (SEQ ID NO:2), or THRPPMWSPVWPGGGKL (SEQ ID NO:3) may be useful in the compositions and methods of the invention.
  • antibodies directed to transferrin receptors may be useful in the compositions and methods of the invention, e.g., the monoclonal antibody 0X26 could be used to modify the liposomes in combination with a cell penetrating polypeptide.
  • Other proteins that may be useful as brain targeting polypeptides in the compositions and methods of the invention include, without limitation, insulin, lactoferrin, leptin, insulin-like growth factor-1 and insulin-like growth factor-2.
  • poly-L-arginine was coupled to phospholipids and located on the surface of liposomes to enhance uptake of exogenous molecular cargo by cells.
  • liposomes could be modified to include other cell penetrating peptides (CPPs) linked to phospholipids in liposomes may be used.
  • CPPs cell penetrating peptides
  • suitable cell penetrating peptides include, but are not limited to, Penetratin (pAntp), HIV TAT, MAP, Transportan, Transportan 10, R7 peptide, pVEC, MPG peptide, KALA peptide, Buforin 2, FHV-coat, BMV-Gag, CADY, and MPG.
  • Poly-D-arginine may also be used as a cell-penetrating polypeptide.
  • the nucleic acids may be complexed with chitosan, a biodegradable, non-toxic linear polysaccharide with gene delivery properties, to enhance delivery of the nucleic acids. Complexation of nucleic acids with chitosan helps to reduce degradation of the encapsulated nucleic acid at the acidic pH of endosomes.
  • the chitosan may optionally include a hydrophobically-modified low molecular weight chitosan as described in US Provisional Application 61/549,972, filed October 21 , 2011 , which is incorporated by reference in its entirety.
  • Tf-Pr- liposomes having a near zeta potential (12.48j ⁇ 3.2 mV were found to be suitable for delivery to across the BBB.
  • liposomes may have other combinations of targeting polypeptides and CPPs.
  • these dual modified liposomes may have any zeta potential that allows delivery across the BBB.
  • the liposomes may have zeta potentials in the range of from about 0 mV to about 20 mV, or from about 0 mV to about 18 mV, or from about 0 mV to about 16 mV.
  • liposomes having a 1 :1 ratio of transferrin to poly-L- arginine are effective in delivery to cells.
  • the liposomes may have transferrin to poly-L-arginine ratios in the range of from about 1 :1 to about 8:2 (e.g., 1 : 1 , 7:3, 6:4, 8:2).
  • poly-L-arginine and transferrin may be conjugated to PEG-DSPE-COOH or PEG-DOPE.
  • any suitable phospholipid may be used in the liposomes of the invention.
  • N-glutaryl PE was also tested and found to be suitable for the conjugation of poly-L-arginine and transferrin to the liposomes, as confirmed by NMR and microBCA assay, respectively.
  • liposomes made with PEG-DSPE-COOH or PEG-DOPE exhibited greater stability, and the results with transfection studies obtained using conjugated transferrin and poly-L- arginine were more reliable, easier to perform and reproducible.
  • compositions and methods of the invention are not intended to be limited to any particular phospholipid, and different linker phospholipids (e.g., DSPE-PEG-COOH, N- Glutaryl PE or DSPE-PEG-maleimide) may be used for conjugating the targeting or cell penetrating polypeptides to the liposomes.
  • linker phospholipids e.g., DSPE-PEG-COOH, N- Glutaryl PE or DSPE-PEG-maleimide
  • Delivery of a liposome to a cell may be accomplished by contacting a cell with the liposome, which encompasses both direct and indirect contacting.
  • contacting a cell encompasses causing a cell to be contacted by administering the liposomes to an organism.
  • Any suitable mode of administration may be used, including, without limitation, oral and parenteral administration.
  • novel liposomal formulations described herein have been tested for uptake by primary rat brain endothelial cells and for transfection in primary glial cells. Doses of liposomes required to confer therapeutic benefit may vary depending on the liposome components and the therapeutic agent. Suitable doses for transfection may be determined, for example, by using a series of geometrically diluted concentrations, as detailed below. The formulated liposomes were being evaluated for their ability to cross the BBB using both in-vitro and in-vivo models, using the approach summarized in FIG 1.
  • liposomes were formulated using post-insertion technique (1).
  • Poly-L arginine was coupled to the phospholipid: 1 ,2-dioleoyl-sn-glycero- 3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (PEG-DOPE) in 1 : 1 molar ratio as described previously (2).
  • PEG-DOPE polyethyleneglycol
  • one gram of poly-L-arginine (Molecular weight; 5000- 5000 Da) was dissolved in 25 ml of 50mM sodium tetraborate buffer. The resulting solution was stirred vigorously at room temperature for about 30 minutes and subsequently passed through sterile 0.2 ⁇ filters.
  • PEG-DOPE was then added to the solution in stoichiometric amounts with Poly-L-arginine.
  • the clear micellar suspension was then stirred at room temperature for 6 hours.
  • the synthesized product was dialyzed against deionized water using dialysis tubing (Spectra/Por 2 dialysis membrane; molecular weight cut off: 12-14 kDa, Spectrum Laboratories) and freeze dried.
  • the conjugation of poly-L-arginine to the lipid was confirmed using H NMR (Mercury Varian 500 MHz spectrometer at 25°C) technique (FIG 2).
  • Peaks at 1.60 and 1.80 were assigned to the CH 2 groups of arginine side chains; an additional peak at 3.18 in the PR-PEG-lipid spectra was assigned to the (t, 2H, -CH2-N-, methoxy PEG linked to arginine; peak at 3.68-3.69 was assigned to 77H, PEG side chain of the lipid and the peak at 1.27 was assigned to the lipid side chain.
  • the PEGylated phospholipid conjugated to PR was then combined with the other lipids, with the following lipid composition: 1 ,2-dioleoyl-sn-glycero3- phosphoethanolamine (DOPE): 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP): PR-lipid: 3 -Hydroxy-5-cholestene 3-hemisuccinate (CHEM) 45:45:4:2 mol percent, in chloroform:methanol (2:1) solution and dried on rotavapor to form a thin film of lipids.
  • DOPE 1,2-dioleoyl-sn-glycero3- phosphoethanolamine
  • DOTAP 1 ,2-dioleoyl-3-trimethylammonium-propane
  • PR-lipid 3 -Hydroxy-5-cholestene 3-hemisuccinate
  • CHEM 45:45:4:2 mol percent
  • lipid film was then hydrated with HEPES Buffered Saline (pH 7.3) to form poly-L- arginine coupled liposomes (total lipid content of the liposomes was ⁇ / ⁇ of the liposomal suspension. The remaining 4% of the lipid content was formed by transferrin conjugated lipid).
  • chitosan DNA polyplexes were added to the hydration buffer at N/P ratio of 5. Transferrin was added to these liposomes via post-insertion technique.
  • Transferrin was conjugated to the lipid 1 ,2-distearoyl-s/7-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol) 2000] (DSPE-PEG-COOH) as described earlier (3). Briefly, DSPE-PEG-COOH (4 mole % of the total phospholipid content) was suspended in HEPES buffered saline (pH 5.0) to form micelles.
  • micellar suspension was then treated with 360 ⁇ of both N-(3- dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) (0.5 M in H 2 0) and Sulpho-N-hydroxysuccinimide (NHS) (0.5 M in H 2 0) per ⁇ of the phospholipid.
  • EDC N-(3- dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride
  • NHS Sulpho-N-hydroxysuccinimide
  • Unconjugated protein was removed by passing the suspension through sephadex G-100 column pre- equilibrated with HEPES Buffered saline (pH 7.3). The resulting transferrin micelles were stirred overnight with the poly-L-arginine conjugated liposomes at room temperature to form transferrin and poly-L-arginine coupled liposomes. The protein content of the liposomes was evaluated using micro BCA assay (Uncoupled blank liposomes and poly-L-arginine coupled liposomes were used as controls). Almost, 60% of the transferrin was observed to be coupled to the liposomes using micro BCA assay.
  • Atomic force Microscopy The shape and surface of the liposomes was evaluated using atomic force microscopy (AFM DI-3100 instrument, Veeco, MN, USA).
  • the liposomal samples were diluted with HEPES buffered saline (pH 7.3). A small drop (1 ⁇ ) of the liposomal suspension was placed onto the surface of freshly cleaved mica film and air dried. The samples were observed in non-contact tapping mode at a scanning frequency of 1 Hz.
  • AFM images indicate the formation of spherical liposomal vesicles (FIG 3).
  • Size and zeta potential The average hydrodynamic particle size and zeta potential of Tf-PR-liposomes were evaluated in suspension form using Zetasizer Nano ZS 90 (Malvern instruments, Malvern, UK) at 25°C and were observed to be in the range of 196.2 ⁇ 2.5 nm and 5.19 ⁇ 0.7mV (mean ⁇ S.E.) respectively.
  • Tf-Pr-liposomes The uptake of Tf-Pr-liposomes was evaluated in HEK 293 cells and primary rat brain endothelial cells.
  • the liposomes were labeled fluorescently with 1 ,2-dioleoyl-s ?-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl (Rh-PE) and were incubated with both cell types (6x10 5 cells/35 mm culture dish) in serum free media.
  • Rh-PE 0.5 mole%) was incorporated into the lipid mixture and dried to form a thin film.
  • the lipid film was hydrated with buffer to form liposomes followed by insertion of transferrin coupled lipid as described above (preparation of liposomes). Following cell uptake, the liposomes were removed and cells were washed with phosphate buffered saline, pH 7.3. HEK 293 cells showed an efficient uptake in about 20 minutes and rat endothelial cells showed an uptake in about 2 hours (FIG 4). These preliminary results indicate the potential of liposomes to be taken up by the rat brain endothelial cells. Because brain endothelial cells have a higher concentration of protein caveolin-1 , which facilitates transcytosis, the liposomes may be transcytosed into the brain.
  • Cell uptake of Dil labeled liposomes in bEnd3 cells was performed using the same technique as described above. Rh-PE (0.5mol%) was replaced with the fluorescent dialkylindocarbocyanine dye, Dil (Invitrogen) (0.5 mol%) and the nuclei of the cells were labeled with DAPI.
  • the brain endothelial cells (primary culture) used previously were replaced with the bEnd3 cell line from ATCC as the primary cells were isolated from the surface capillaries of the brain (emerging from aortic artery).
  • Rh-PE was replaced with the indocarbocyanine dye, Dil, to simulate the in-vivo experimental conditions.
  • the liposomes, for in-vivo tracking, will be labeled with the commonly used molecular tracer, Dil dye.
  • Rh-PE has previously been found to have in-vivo cytotoxic effects. The results for the uptake studies are shown in FIG 5.
  • Cytotoxicity of the liposomes The cytotoxicities of the formulated liposomes and the commercially available FuGENE® HD Transfection reagent were evaluated using MTT assay (4). Because the liposomes will be targeted for delivery across BBB composed of tightly sealed endothelial cells in colloquium with glial cells, the cytotoxicity was evaluated in both these cells. The cell viabilities were evaluated at varying concentrations (100nM, 200nM, 400nM and 600nM) prepared by serial dilutions of liposomes in serum free media. These viabilities (not shown here) decreased with increasing the concentration of phospholipids.
  • Transfection efficiency of liposomes Next, the ability of Tf-Pr-liposomes to efficiently deliver the desired gene to the primary culture of glial cells isolated from rat brain was evaluated. The transfection potential of these liposomes was evaluated in the glial cell cultures using GWiz-GFP plasmid (Aldevron, Fargo). The cells were seeded onto 35 mm culture dishes precoated with poly-l-lysine at density of 6x10 6 cells/dish and cultured in DMEM-High containing 10% Fetal Bovine Serum (FBS) at 37°C in 5% C0 2 until approximately 80% confluent.
  • FBS Fetal Bovine Serum
  • Liposomal formulations containing GWiz-GFP-chitosan complexes were then added to these cells in serum free media. After 5 hours, the media containing liposomes was removed and the cells were further incubated for a total of 48 hours in serum containing media. The cells were then analyzed for GFP expression (FIG 7) using confocal laser scanning microscope (Olympus, FV5-PS0). Quantitative evaluation was performed using flow cytometric analysis. The transfection efficiency using liposomes was observed to be higher than the transfection efficiency observed using marketed formulation , FuGENE® HD Transfection reagent.
  • the in-vitro model of BBB was constructed using a combination of bEnd3 cells (American Type Culture Collection; ATCC®, Manassas, VA, US) and primary glial cell culture. Glial cells (1.5 x 10 4 /cm 2 ) were seeded on the bottom side of collagen-coated polyethylene terepthalate (PET) membrane (0.4 ⁇ pore -size) of transwell inserts (BD BioCoatTM, BD Biosciences, NC, USA). The cells were allowed to adhere firmly to the membrane for overnight.
  • bEnd3 cells American Type Culture Collection; ATCC®, Manassas, VA, US
  • Glial cells 1.5 x 10 4 /cm 2
  • PET collagen-coated polyethylene terepthalate
  • transwell inserts BD BioCoatTM, BD Biosciences, NC, USA
  • the endothelial cells were seeded on the inside or the luminal surface of culture inserts placed in 6-well plates containing DMEM High (HyClone®, Thermo Scientific, Utah, USA) with 10% fetal bovine serum (FBS) and 1% Psf (penicillin- streptomycin-fungizone).
  • DMEM High HyClone®, Thermo Scientific, Utah, USA
  • FBS fetal bovine serum
  • Psf penicillin- streptomycin-fungizone
  • the cells were checked under the microscope for confluency and morphology. Evaluation of barrier integrity: The barrier integrity of the in-vitro models was evaluated by measuring the flux Na-F across the barrier layer based on the previously reported method (7, 9).
  • Cell culture inserts with both glial and endothelial cells and with only endothelial cells were transferred to 6 well plates containing 1.5 ml of 1x Hepes buffered saline (pH 7.4) in the lower or the abluminal compartment. In the luminal compartment of the inserts, the culture medium was replaced with 1 ml of the buffer containing 10pg/ml Na-F. The culture inserts were transferred to new wells containing Hepes buffer at specific time intervals of 5, 15, 30 and 45 min.
  • Transport of Dil-labeied liposomes across in-vitro model The transport of four types of Dil-labeled liposomes PEGylated uncoupled liposomes (Blank), transferrin coupled liposomes (Tf-liposomes), poly-L-arginine coupled liposomes (PR-liposomes) and transferrin and poly-L-arginine coupled liposomes (Tf-PR-liposomes) was measured across the endothelial and glial monolayers cell culture inserts. To mimic the in-vivo environment, flux of these liposomes was evaluated in sterile phosphate buffered saline (PBS, pH 7.4) containing 5 % FBS.
  • PBS sterile phosphate buffered saline
  • the inserts were transferred to 6- well plates containing1.5 ml of PBS in the lower compartment.
  • the culture medium inside the inserts was replaced with 1.0 ml of fresh serum containing buffer followed by the addition of liposomal suspensions (100nM) to this compartment.
  • the inserts were transferred at 15 min, 30 min, 60 min, 2 h, 4 h and 8 h to new wells with serum-PBS.
  • the concentration of the liposomes in the upper and the lower compartment were determined by measuring the fluorescence intensity of the dye molecule in the samples using fluorescence SpectraMax® M5 Multi-mode microplate reader (Molecular devices, CA, USA; excitation wavelength: 553 nm, emission wavelength: 570 nm).
  • P app for each liposomal formulation was calculated, according to Gaillard (2000) by using the following equation (1):
  • Paa P dQ/dt. 1/A.C 0 .60 (cm/sec)
  • P t is the permeability of the total system
  • Pf is the permeability for the cell free membrane
  • P e is the permeability of the endothelial cell barrier layer.
  • Tf-PR liposomes were prepared using a thin film hydration and post-insertion technique. Briefly, the primary amino group of poly-L-arginine was coupled to the linker phospholipid, DSPE- PEG-COOH via EDC/NHS reaction to form poly-arginine coupled lipid (DSPE-PEG- PR). The PR-coupled lipid was then combined with other phospholipids DOPE/DOTAP/DSPE-PEG-PR/cholesterol to form PR-liposomes using thin film hydration technique.
  • Transferrin was coupled to DSPE-PEG-COOH to form Tf- micelles, which were stirred with PR-liposomes at 60°C for 1 h to form the bi-functional, Tf-PR-liposomes.
  • the liposomes were characterized for hydrodynamic size and zeta potential using dynamic light scattering technique.
  • the coupling efficiencies of transferrin and poly-L-arginine were determined using micro bicinchonic acid assay and H NMR, respectively.
  • Stability studies for liposomes Stability of a delivery vector, during storage and under biological conditions, is an important parameter governing the activity of the associated therapeutic agent. Physical and colloidal factors like size distribution and over all charge can be used for determining the stability of liposomal formulations. Therefore, the stability of plain, Tf, and Tf-PR-liposomes in HEPES buffer, pH 7.4 was evaluated under storage conditions of 4°C for 30 days. Also, the stability of the formulation was investigated under simulated in vivo conditions by incubating the liposomes with 10% fetal bovine serum (FBS) at 37°C for 60 minutes.
  • FBS fetal bovine serum
  • Hemolysis assay The cationic charge of poly-L-arginine has been reported to influence the membrane structure and cause lysis of erythrocytes.9 Therefore, the influence of dual modified liposomes, on the membrane integrity of erythrocytes, was evaluated using hemolysis assay. Blood was collected from an adult rat into tubes containing EDTA solution and centrifuged at 2000 rpm for 10 minutes. The pelleted erythrocytes were washed three times with phosphate buffered saline (PBS), pH 7.4. The erythrocyte count was performed using a hemocytometer.
  • PBS phosphate buffered saline
  • the liposomes at different concentrations in PBS, were added to a definite concentration of erythrocytes and incubated at 37°C for 60 min. The samples were centrifuged at 2000 rpm for 10 min and absorbance (A) of the supernatant was analyzed at 540 nm by spectrophotometric analysis. Triton X-100 and PBS treated erythrocytes were used as controls for 100% and 0% hemolysis value, respectively. The percent hemolysis was calculated as:
  • Hemolysis (%) A (experimental group) - A (PBS)/ A (Triton X-100) - A (PBS)
  • FIG 11. A schematic showing the formation of liposomes for use in in vivo studies and delivery of the liposomes to the brain of a rat is shown in FIG 11. All animal experiments were conducted as approved by the Institutional Animal Care and Use Committee (IACUC) at North Dakota State University. Adult Sprague- Dawley rats were used to evaluate the biodistribution, transfection efficiency and biocompatibility of liposomes. The animals were housed under controlled temperature conditions with 12 hour light and dark cycle and were allowed free access to food and water.
  • IACUC Institutional Animal Care and Use Committee
  • the rats were injected via tail vein with either Dioctadecyl Tetramethylindotricarbocyanine Iodide (DiR) labeled liposomes or ⁇ - Galactosidase ( ⁇ -gal) expressing plasmid encapsulating liposomes.
  • Animals injected with ⁇ -gal plasmid alone or with phosphate buffered saline (PBS), pH 7.4 were used as control.
  • PBS phosphate buffered saline
  • the supernatant was separated and stored in ice for further processing with the assay kit.
  • the homogenates extracted from the tissue samples were diluted with an equal volume of assay buffer containing the substrate, o-nitrophenyl- -D-galactopyranoside (ONPG) and incubated at 37°C for 60 min.
  • the tissues samples transfected with ⁇ -gal plasmid express the enzyme ⁇ - galactosidase.
  • the ⁇ -gal activity of tissues was quantified using ⁇ -gal assay kit (Promega). The transfection efficiency was determined by evaluating the enzymatic hydrolysis of the colorless substrate ONPG to the yellow colored product o-nitrophenol by ⁇ -gal enzyme.
  • the reaction was terminated by the addition of sodium carbonate and the absorbance was measured at 420 nm.
  • Control rats (without administration of DNA or liposomes) were similarly processed to quantify the endogenous activity of individual organs.
  • Hematoxylin-eosin staining The biocompatibility of the liposomes, in vivo, was evaluated by histological examination of tissue sections transfected with ⁇ -gal plasmid encapsulating liposomes.
  • the transfected tissue sections from different organs were embedded in Tissue-Tek® OCTTM Compound (Sakura Finetek Inc.) and snap frozen in dry ice.
  • the frozen tissues were sectioned using cryostat and fixed in 4% paraformaldehyde (in PBS, pH 7.4).
  • the slides were stained with Harris hematoxylin (Sigma-Aldrich), excess stain was washed in running tap water and the slides were differentiated in 1% acid alcohol for 10 seconds.
  • the stained tissue section was then rinsed in tap water and blued in 1.36% lithium carbonate solution.
  • the slides were then washed in running tap water, dehydrated in 95% alcohol and counter stained with eosin Y-phloxine B solution for 5-10 seconds.
  • the stained slides were washed in tap water, cleared in xylene and mounted with Cytoseal 60 (Thermo Fisher Scientific Inc.).
  • Statistical analysis Statistical data were processed using Microsoft Excel 2010 software and presented as mean ⁇ standard deviation of the mean (S.D.). The treatment groups were compared using two tailed student's t-test and analysis of variance.
  • the size and zeta potential of the synthesized Tf-Pr liposomes were ⁇ 200 nm and ⁇ 12.48 ⁇ 3.2 mV, respectively.
  • the atomic force microscopy demonstrated the formation of non-aggregated spherical liposomal vesicles.
  • the coupling efficiency of transferrin, as determined using micro BCA assay, was observed to be about 59% of transferrin added for coupling to the liposomes.
  • the liposomes were found to be stable on storage at 4°C for one month and on incubation at 37°C for 60 min in the presence of 5% FBS (no significant change in size or zeta potential was observed).
  • Tf-PR-liposomes Slight hemolysis was observed with the Tf-PR-liposomes at a concentration of 800 nmoles of phospholipids exposed to the same number of erythrocytes.
  • Microscopic examination revealed changes in the structure of erythrocyte membrane and morphological appearance with Tf-PR-liposomes at 800 nmoles of phospholipids. However, these concentrations were extremely high and not used under normal physiological conditions in animals.
  • Phosphate buffered saline was used as a negative control and did not show any damage to erythrocyte membrane under microscopic examination or release of hemoglobin on spectrophotometric measurement.
  • Triton X-100 used as a positive control demonstrated complete lysis of erythrocytes under microscopic examination and release of hemoglobin on spectrophotometric examination.
  • Tf-liposomes The decrease in the concentration of Tf-liposomes was lesser in liver, spleen and kidneys as compared to the plain and bi-functional liposomes thus indicating increased circulation and lower elimination of Tf-liposomes by the macrophage system.
  • the bi-functional Tf-PR- liposomes accumulated mainly in lungs and heart. Also, the occurrence of a cell penetrating peptide on the transferrin receptor targeted liposomes resulted in greater penetration of Tf-PR-liposomes into brain in comparison to the plain and Tf-liposomes.
  • the ⁇ -galctosidase activity of the tissues was determined using the ⁇ -gal assay kit. High levels of ⁇ -galactosidase activity were observed in liver and spleen which corresponds to the higher distribution of liposomes in these organs.
  • the bi-functional liposomes demonstrated significantly (p ⁇ 0.05) higher levels of enzyme activity in brain (1.49 ⁇ 0.22398 mU/mg of protein) as compared to the single ligand or naked DNA (FIG 16A).
  • the Tf-PR-liposomes also showed higher levels of enzyme activity in different organs which could be attributed to the higher penetration and cationic charge associated with the CPP coupled Tf-liposomes (FIG 16B).
  • the enzymatic activity varied from 23.38 ⁇ 2.58 mU/mg of protein in spleen, 12.67 ⁇ 2.98 mU/mg of protein in liver, 9.73 ⁇ 1.9 mU/mg of protein in lungs, 6.43 ⁇ 1.91 mU/mg of protein in kidneys to 4.54 ⁇ 1.3 mU/mg of protein in heart with the bi- functional liposomes.
  • the levels of enzyme activity in the organs were observed to be comparatively low with the Tf-liposomes varying from 16.9 ⁇ 1.98 mU/mg of protein in spleen, 9.29 ⁇ 2.33 mU/mg of protein in liver, 4.43 ⁇ 1.99 mU/mg of protein in lungs, 4.48 ⁇ 1.76 mU/mg of protein in kidneys to 2.32 ⁇ 0.81 mU/mg of protein in heart.
  • the enzyme activity induced by naked DNA in different tissues was close to the endogenous activity of the corresponding tissue thereby indicating practically no transfection with naked plasmid.
  • the present study underscores the significance of novel bi-functional liposomes in delivery of desired gene to brain.
  • Receptor targeted stealth liposomes functionalized with a cell penetrating peptide were synthesized and used to improve delivery of a desired gene to brain.
  • the results from the biodistribution studies demonstrate that the bi-functional liposomes accumulate in the brain in amount two fold higher than the single ligand liposomes and produce significantly (p ⁇ 0.05) higher transfection levels as compared to the Tf-liposomes or naked DNA.
  • the Tf-PR liposomes showed excellent biocompatibility with both blood and tissues at the administered dose.
  • the reported liposomal vesicles were synthesized to possess near neutral charge and it was therefore, essential to monitor the stability of the liposomes during storage and under simulated in vivo conditions.
  • the stability of the liposomes was examined under storage conditions of 4°C for 30 days and in the presence of serum (10% FBS) at 37°C for 60 minutes. No significant change in size or zeta potential values was observed under the tested conditions.
  • the occurrence of the stearic stabilizer, polyethylene glycol (PEG), reduces aggregation and improves stability of the near neutral liposomes during prolonged storage.
  • the presence of negatively charged protein, transferrin balances the cationic charge of the CPP and further reduces the destabilization of liposomes by decreasing the non-specific binding of serum proteins with the cationic peptide.
  • the near-neutral, bi-functional liposomes were intended for delivery into systemic circulation for targeting the brain endothelial cells. Therefore, the liposomes were evaluated for their compatibility with blood. Cationic macromolecules trigger the adsorption of plasma proteins followed by adhesion and activation of platelets, thereby leading to thrombosis, embolization and hemolysis. The presence of cationic peptides on liposomal surface can induce interactions with the erythrocyte membrane causing cell lysis and release of hemoglobin. Consistent with demonstrated in vitro biocompatibility of liposomes in brain endothelial and primary glial cells, a hemolysis assay performed confirmed in vivo biocompatibility of the liposomes.
  • the transferrin liposomes did not show any toxicity up to a concentration of 800nmoles of phospholipid/ .4x10 6 erythrocytes and the bi-functional liposomes were biocompatible at concentrations as high as 620 nmoles of phospholipids/1.4x10 6 erythrocytes.
  • At very high concentrations of liposomes above 800 nmoles of phospholipids, hemolysis was evident (FIG 11 B). This can be explained by the increasing interactions of the liposomes with the same number of erythrocytes at higher phospholipid concentrations.
  • the liposomes at the administered dose 15.2 pmoles of phospholipids/kg body weight were considered to be non-toxic.
  • the hemolytic activity of the plain liposomes (FIG 11 A) was observed to be higher than the transferrin coupled or bi-functional liposomes thus confirming the reduction in liposomal interactions with the erythrocyte membrane in the presence of negatively charged protein.
  • the distribution of the liposomes in vivo was tracked by fluorescent labeling of the liposomes with DiR followed by NIR imaging of different organs (spleen, liver, heart, kidneys, lungs and brain) at specific time points.
  • the preliminary results from whole animal imaging indicated significant accumulation of the DiR labeled liposomes in brain after 12 hours. However, much less fluorescence was observed in the brain at 6 hour time point. As expected, a strong fluorescence signal was observed in the liver after injection and the signal intensity decreased gradually with time becoming very low after 48 hours.
  • plain liposomes showed a more rapid elimination from the organs as compared to the Tf- or bi-functional liposomes which might can be explained by the greater clearance of the plain liposomes in comparison to other liposomes.
  • the initial uptake of Tf-liposomes was lower in liver and spleen and their accumulation continued at 72 h indicating longer circulation time of Tf- liposomes as compared to other liposomes.
  • the accumulation of Tf-liposomes was more in the kidneys in appraisal to other liposomes thereby, indicating higher renal elimination of Tf-liposomes.
  • the cell penetrating property of the bi-functional liposomes, in combination with the receptor targeting effect of transferrin protein resulted in higher accumulation of Tf-PR- liposomes in the brain as compared to the single ligand transferrin receptor targeted, Tf-liposomes.
  • the bi-functional, Tf-PR-liposomes have demonstrated high transfection efficiencies in primary culture of glial cells, in vitro.
  • the presence of high serum concentrations and non-specific binding of the liposomes to extracellular components can interfere with the transfection efficiency, in vivo. Therefore, the transfection potential of bi-functional liposomes was evaluated in vivo, using chitosan- ⁇ -galactosidase polyplexes encapsulated in the aqueous core of the liposomes, followed by intravenous injection of the formulation.
  • the bi-functional liposomes resulted in increased level of ⁇ -galactosidase activity in brain as compared to other liposomal formulations.
  • the bi-functional liposomes were modified with transferrin protein for targeting the transferrin receptors on brain endothelial cells and cell penetrating peptide for improving the penetration across the barrier layer.
  • the results from the in vivo transfection studies further emphasized the significance of dual mechanism of liposomal transport that resulted in higher penetration across BBB and improved the expression of encapsulated gene in brain. High levels of expression were also observed in spleen and liver which can be rationalized by the greater distribution of different liposomal formulations to these organs.
  • the mechanism of uptake of the liposomes by brain endothelial cells has been demonstrated by this laboratory (10).
  • the liposomes are primarily transported across the BBB via receptor mediated transcytosis, and the presence of poly-L-arginine on the surface of receptor targeted liposomes further improves their penetration into brain.
  • the cationic charge of CPP improves gene expression in the targeted cells and tissues.
  • the cationic charge of CPPs conjugates can induce tissue necrosis and inflammation.
  • the biocompatibility of the liposomes was therefore, evaluated by histological examination of the transfected tissues.
  • the transfected tissues from different organs were sectioned and stained with hematoxylin-eosin followed by examining under color microscope. No inflammation or necrosis was observed in the tissues isolated from the animals administered with liposomes at dose of 15.2 pmoles of phospholipids/kg body weight.
  • the poly-ethylene-glycol chains and transferrin protein counterbalanced the cationic charge of poly-L-arginine peptides and reduced the negative effects of this cell penetrating peptide.
  • the dual-modified liposomes showed significantly (p ⁇ 0.05) higher accumulation in rat brain as compared to transferrin-liposomes. Also, the ⁇ -galactosidase activity of brain tissue transfected using dual-modified and transferrin-liposomes was about 1.5 and 0.95 mU of ⁇ -gal/mg of protein, respectively. Hemolysis assay of liposomes demonstrated that the dual-modified liposomes were biocompatible. The bi-functional liposomes can therefore serve as safe and efficient non-viral drug and gene delivery vectors.
  • Oxaliplatin encapsulated in PEG-coated cationic liposomes induces significant tumor growth suppression via a dual-targeting approach in a murine solid tumor model. 137: 8-14.
  • Pentosan polysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages.

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Abstract

L'invention concerne des liposomes qui comprennent un polypeptide ciblant le cerveau conjugué à un phospholipide et un polypeptide pénétrant à l'intérieur d'une cellule conjugué à un phospholipide, des formulations liposomales contenant les liposomes, et des procédés d'administration de liposomes à des cellules.
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