WO2013030838A2 - Systèmes de cristaux liquides dendrimère-lyotropique (llc) - Google Patents

Systèmes de cristaux liquides dendrimère-lyotropique (llc) Download PDF

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WO2013030838A2
WO2013030838A2 PCT/IL2012/050336 IL2012050336W WO2013030838A2 WO 2013030838 A2 WO2013030838 A2 WO 2013030838A2 IL 2012050336 W IL2012050336 W IL 2012050336W WO 2013030838 A2 WO2013030838 A2 WO 2013030838A2
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Prior art keywords
dendrimer
ppi
llc
compound
mesophase
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PCT/IL2012/050336
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English (en)
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WO2013030838A3 (fr
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Nissim Garti
Dima Libster
Liron BITAN-CHERBACHOVSKY
Abraham Aserin
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Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.
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Publication of WO2013030838A2 publication Critical patent/WO2013030838A2/fr
Publication of WO2013030838A3 publication Critical patent/WO2013030838A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1635—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • This invention relates to novel dendrimer-lyotropic liquid crystal (LLC) systems and uses thereof.
  • Dendrimers have been studied as potential drug vehicles or scaffolds [1,2].
  • the beneficial properties of dendrimers have been known to be their low polydispersity and nanosized scale that permit their high penetration through cell membranes and their ability to mimic biomolecules [3].
  • the presence of terminal groups on the dendrimer's surface offers an excellent platform for the attachment of targeting groups, solubility modifiers, and stealth moieties, which reduce immunological interaction.
  • the three- dimensional structure of dendrimers consisting of an interior core, interior layer, and exterior surface, possesses the ability to encapsulate bioactive molecules in several sites; these include the central core and the flexible space created by the voids in the several interior layers.
  • the drugs may be chemically attached or physically absorbed on the dendrimers' surface or on its core [4,5].
  • Dendrimers have also been known to be therapeutically effective against Prion disease and Alzheimer's disease, HIV, cancer, and other diseases. Additionally, dendrimers have been shown to prevent formation of amyloid fibrils, destabilize amyloid aggregates, and prevent viral adhesion and replication.
  • Lyotropic liquid crystals are formed by polar lipids and certain surfactant mixing with water [6-10]. LLCs which are based on monoglycerides are self- assembled into a large variety of morphologies that exist between isotropic liquid and solid crystalline. Various mesophases can be formed, while the most significant are the lamellar, cubic, and hexagonal structures. The mesophases can be type I (oil-in-water, O/W) or type II (water-in-oil, W/O). These types of LLCs have been shown to provide sustained release of guest molecules with a range of physicochemical properties [11], thermodynamic stability, and improved solubility of drugs and bioactive molecules [8, 12-17]. REFERENCES
  • the major three lyotropic mesophases are the lamellar, hexagonal (and reverse hexagonal) and cubic (reverse cubic) mesophases. All three mesophases are known to solubilized, in their aqueous cores, hydrophilic materials, e.g., bioactives, and in their interface lypophillic materials. While mesophases based on glycerol monooleate (GMO), such as lamellar and cubic, can be formed at room temperature, the more thermodynamically favored hexagonal mesophase cannot.
  • GMO glycerol monooleate
  • the hexagonal mesophase is preferable over the other mesophases at least in its ability to solubilize biological materials such as peptides or protein-like compounds, due to the special arrangement of the elongated aqueous channels of the hexagonal mesophase.
  • the release of materials from the hexagonal mesophases may be affected to control the time of release, rate of release and efficiency of release of materials from the hexagonal mesophases.
  • the release is controllable by incorporating one or more dendrimers in the mesophase.
  • the hexagonal mesophases may be formed from the lamellar or cubic mesophases by direct conversion thereof, in the presence of dendrimers, to the hexagonal mesophases.
  • the presence of dendrimers not only permits the conversion into the hexagonal mesophase and the effective packing of the dendrimers and active materials within the hexagonal mesophases, but also permits reversal of the hexagonal mesophases back to the initial lamellar or cubic mesophases.
  • the reversal from the hexagonal to the lamellar and cubic mesophases results in an effective and on-demand release of the materials contained within the hexagonal mesophases to the environment containing the mesophase system.
  • the release profile/pattern of the content of the hexagonal mesophase e.g., dendrimers and optionally active materials
  • the presence of dendrimer(s) permits such conversions, thus enhancing the release.
  • the present invention is directed at a system, comprising a dendrimer, optionally in the presence of an active material (e.g., a drug), and a lyotropic liquid crystal (LLC), wherein the dendrimer is solubilized in the aqueous domains of the LLC; the LLC being selected from lamellar (L a ), reverse bicontinuous cubic (Q), and reverse hexagonal (Hn) LLCs.
  • L a lamellar
  • Q reverse bicontinuous cubic
  • Hn reverse hexagonal
  • the systems of the invention have demonstrated, e.g., the ability to deliver their content across cell membranes with high efficacy and without causing any damage to living tissues.
  • the invention also demonstrates the potential use of the systems of the invention in on-demand delivery of drugs and other actives across cell membranes.
  • the Den-LLC also comprises at least one active material, such as a drug, wherein said active material may or may not be chemically associated with the dendrimer component of said Den-LLC, as further disclosed hereinbelow.
  • a “dendrimer” (or a “dendrimer compound”) is a molecule or macromolecule having a branched arrangement of repeating structural units (“mers”), forming a substantially two-dimensional or three-dimensional symmetry.
  • the dendrimer may be in the general form of cone, an ellipse or in the form of a sphere.
  • the dendrimer employed in the systems of the invention comprises a polyvalent core covalently bonded to at least two dendritic branches, and extends through two or more generations (the generation is indicated by the letter "G")-
  • the dendrimer used in accordance with the invention is a sphere or circular-in shape dendrimer.
  • the dendrimer is a second- generation dendrimer, designated "G2". In further embodiments, the dendrimer is a G3 dendrimer, a G4 dendrimer or a higher generation dendrimer.
  • At least one of the terminal groups on the surface of the dendrimer has one or a plurality of functional group moiety permitting chemical modifications and chemical reactions at the terminal groups.
  • These functional groups may be selected from amino groups, hydro xyl groups (such as sugars and polyols), thiol groups, carboxylic acid groups, ester groups, sulfonic acid groups, and the like.
  • the dendrimer has a plurality of amino groups as terminal groups.
  • the dendrimer is selected from polyamidoamine (PAMAM) dendrimers, PAMAM (EDA) dendrimers, poly(propyleneimine) (PPI) dendrimers, polylysine dendrimers, and others.
  • the dendrimer is PPI. In some embodiments of the invention, the dendrimer is a second-generation (G2) poly(propyleneimine) (PPI) dendrimer.
  • G2 poly(propyleneimine)
  • a “lyotropic liquid crystal” or “LLC” in short, is a physical state of matter, which comprises two or more incompatible components, e.g., a surfactant and water, exhibiting concentration-dependent liquid -crystalline properties upon increase in a liquid phase.
  • the LLC state is formed through a segregation of two or more incompatible components at the nanometer scale, such that at the lyotropic phase, the phase is composed of lipophilic molecules and the tails of surfactant molecules to provide fluidity to the system.
  • lipophilic molecules e.g.
  • the LLC state is one or more of a lamellar mesophase (L a ), a reverse bicontinuous cubic mesophase (Q) and a reverse hexagonal mesophase (3 ⁇ 4).
  • the LLC systems employed in accordance with the present invention are based on glycerol monooleate (GMO, also known as monoolein, monoglyceride) or similar mono-glycerol-esters of poly unsaturated fatty acids, such as glycerol mono-linoleate, glycerol mono-linolenate, glycerol mono DHA (monodocosahexaenoyl glycerol), glycerol mono EPA (monoeicosapentaenoyl glycerol) and glycerol mono-archidate and others.
  • GMO glycerol monooleate
  • DHA monodocosahexaenoyl glycerol
  • glycerol mono EPA monoeicosapentaenoyl glycerol
  • the LLC systems are characterized by a high internal ordering and symmetry as well as by a vast interfacial area and the existence of both hydrophobic and hydrophilic domains.
  • the LLC systems are based on GMO (namely GMO is a component of the system).
  • the lamellar mesophase (“L a ”) may be obtained, as known in the art, by hydrating GMO with a given and restricted amount of water (between 10 and 20 wt%), at room temperature or slightly elevated temperatures while shaking the mixture.
  • the reverse bicontinuous cubic mesophase for example Ia3d or Pn3m, and/or other cubic mesophases, such as gyroid or primitive may be obtained, as known in the art, by mixing specific amount of water with GMO (between 20 and 40 wt% water) or similar at room temperature or slightly elevated temperatures.
  • GMO between 20 and 40 wt% water
  • WO 2005/063370 [21] and US applications derived therefrom disclose a cubic mesophase and are incorporated herein in their entirety.
  • the reverse hexagonal mesophase (“Hn”) consists of cylindrical columnar surfactant micelles arranged on a two-dimensional hexagonal lattice and is formed, as known in the art, in a binary mixture (namely without any active material or dendrimer) only at elevated temperatures.
  • WO 2010/150262 [20] and US applications derived therefrom disclose the hexagonal mesophase ("Hn") and are incorporated herein in their entirety.
  • All the mesophase structures can accommodate hydrophilic, hydrophobic and amphiphilic guest molecules, either within the aqueous compartments composed of dense packed, straight water-filled cylinders, or by direct interaction within the lipid hydrophobic moieties, orientated radially outward from the centers of the water rods
  • the present invention provides a "Den-LLC” system comprising dendrimer in a lyotropic liquid crystal (LLC) state selected from a lamellar mesophase (L a ), a reverse bicontinuous cubic mesophase (Q) or similar, and a reverse hexagonal mesophase (Hn).
  • the Den-LLC is a dendrimer solubilized in a mesophase selected from lamellar mesophase (herein designated "Den-La ), a reverse bicontinuous cubic mesophase (herein designated "Den-Q”) and a reverse hexagonal mesophase (herein designated "Den-Hn").
  • a dendrimer such as PPI-G2 solubilized in a LLC acts as a "water pump” and competes for water binding with other components of the LLC. This dehydration induces a unique structural shift from any initial Den-LLC system to a final Den-Hn system.
  • a Den-L a ⁇ Den-Hn and Den-Q ⁇ Den-Hn structural shifts were observed.
  • the final Den-Hn can be caused to transform back to the Den-Q states by increasing the water content of the LLCs systems or by reducing the concentration of the dendrimers in the system (upon release of the dendrimer or dendrimer associated with the active material).
  • the structural shifts (conversions, transitions) Den-L a ⁇ Den-Hn and Den-Q ⁇ Den-Hn may be affected by treating the Den-L a and Den-Q systems with any dehydrating agent, which would compete for water binding with the LLC components.
  • dehydrating agents may be selected from alcohol, propylenglycol (PG), glycerol (Gl), any other polyols or any other amino or thiol water-soluble compounds.
  • the invention provides Den-L a , Den-Q and Den-Hn systems, as well as phases comprising a mixture of Den-L a and Den-Hn, and a mixture of Den-Q and Den- Hn-
  • the "pure” systems comprise the single LLC mesophase only, while the “mixed” systems comprise two or more different LLC phases, at any ratio.
  • the mixed systems may comprise two or more Q mesophases such as diamond, giroid and primitive.
  • the mixed system comprises Den-L a and Den-Hn in a ratio selected from 99:1, 97:3, 94:6, 91 :9, 88: 12, 85:15, 82:18, 79:21, 76:24, 73:27, 70:30, 67:33, 64:36, 61 :39, 58:42, 55:45, 52:48, 49:51, 46:54, 43:57, 40:60, 37:63, 34:66, 31 :69, 28:72, 25:75, 22:78, 19:81 , 16:84, 13:87, 10:90, 7:93, 4:96, 1 :99, respectively.
  • the ratio between Den-L a and Den-Hn may be any other intermediate ratio.
  • the mixed system comprises Den-L a and Den-Hn wherein the ratio is 99:1, or 97:3, or 94:6, or 91 :9, or 88:12, or 85:15, or 82: 18, or 79:21 , or 76:24, or 73:27, or 70:30, or 67:33, or 64:36, or 61 :39, or 58:42, or 55:45, or 52:48, or 49:51, or 46:54, or 43 :57, or 40:60, or 37:63, or 34:66, or 31 :69, or 28:72, or 25:75, or 22:78, or 19:81, or 16:84, or 13 :87, or 10:90, or 7:93, or 4:96, or 1 :99, respectively.
  • the ratio between Den-L a and Den-Hn may be any other intermediate ratio.
  • the mixed system comprises Den-Q and Den-Hn in a ratio selected from 99:1, 97:3, 94:6, 91 :9, 88: 12, 85:15, 82:18, 79:21, 76:24, 73:27, 70:30, 67:33, 64:36, 61 :39, 58:42, 55:45, 52:48, 49:51, 46:54, 43:57, 40:60, 37:63, 34:66, 31 :69, 28:72, 25:75, 22:78, 19:81 , 16:84, 13:87, 10:90, 7:93, 4:96, 1 :99, respectively.
  • the ratio between Den-Q and Den-Hn may be any other intermediate ratio.
  • the mixed system comprises Den-Q and Den-Hn wherein the ratio is 99: 1, or 97:3, or 94:6, or 91 :9, or 88:12, or 85:15, or 82: 18, or 79:21 , or 76:24, or 73:27, or 70:30, or 67:33, or 64:36, or 61 :39, or 58:42, or 55:45, or 52:48, or 49:51, or 46:54, or 43 :57, or 40:60, or 37:63, or 34:66, or 31 :69, or 28:72, or 25:75, or 22:78, or 19:81, or 16:84, or 13 :87, or 10:90, or 7:93, or 4:96, or 1 :99, respectively.
  • the ratio between Den-Q and Den-Hn may be any other intermediate ratio.
  • the LLC systems comprise the at least one dendrimer in the LLC mesophase, the LLC mesophase being composed of GMO, at least one lipophilic compound and water.
  • the Den-LLC may additionally comprise at least one active material, e.g., a drug.
  • the at least one "lipophilic" compound is selected to interact with the GMO, forming together with water a ternary system, which may be extended to other multi- component systems having similarly 4, 5, 6 and further components, thus forming extended LLC systems.
  • the lipophilic component is selected to provide, maintain, enhance or diminish one or more structural property of the LLC system, such property may be selected amongst thermal stability, its cell-membrane fusion abilities, the stability of a chemical, e.g., the dendrimer or an active material, entrapped within the LLC mesophase or intercalated on its outer surface and maintain activity of labile chemicals solubilized in the system.
  • the at least one lipophilic compound employed may be an antioxidant or may be present in the LLC in addition to at least one antioxidant.
  • the lipophilic compound is selected to interact, typically via Van der Waals interactions, with hydrophobic residue of the GMO, to impose a structural modification on the LLC system.
  • the structural modification may involve modulation (increase or decrease) of one or more parameters such as the size of the spacing between the hydrophobic residues of the GMO, the lattice parameter, the radius and/or length of the cylindrical micelle (in the case of hexagonal mesophases) and others.
  • Such modifications enable efficient intercalation of the dendrimer and active materials within the LLC, e.g., cylindrical compartments of the hexagonal mesophase, and/or on their surface.
  • the lipophilic compounds may be selected from a triacylglycerol (TAGs), vitamin E (VE, alpha-tocopherol) and a phosphatidyl ester.
  • TAGs triacylglycerol
  • VE vitamin E
  • a phosphatidyl ester a phosphatidyl ester.
  • the at least one lipophilic compound is at least one TAG derived from a fatty acid having between 2 and 18 carbon atoms. No n- limiting examples include triacetin, tributyrin, tricaprylin, trilaurin, trimyristin and tristearin.
  • the lipophilic compound is an antioxidant, which may be selected from vitamin E (alpha-tocopherol); cholesterol; phytosterol; lycopene; beta- carotene; carnosic acid; beta-tocopherol; gamma-tocopherol; delta-tocopherol; epsilon- tocopherol, zeta 1 - tocopherol; zeta 2-tocopherol; eta-tocopherol and 1 -ascorbic acid 6- palmitate.
  • vitamin E alpha-tocopherol
  • cholesterol alpha-tocopherol
  • phytosterol lycopene
  • beta- carotene carnosic acid
  • beta-tocopherol gamma-tocopherol
  • delta-tocopherol delta-tocopherol
  • epsilon- tocopherol zeta 1 - tocopherol
  • zeta 2-tocopherol zeta 2-tocopherol
  • the antioxidant is vitamin E and the Den-LLC system is one or more of Den-GMO/vitaminE/water, Den-GMO/lipophilic/vitaminE/water, Den- GMO/vitaminE/alcohol/water and Den-GMO/lipophilic/vitaminE/alcohol/water, wherein the alcohol is ethanol.
  • the ratio between GMO to said at least one lipophilic compound is between 85:15 GMO/water (binary mixture), 65 :35 (binary), and 90:10 GMO/water/MCT (in ternary mixture) (wt%), respectively. In some embodiments, the ratio is between 70:30 and 90:10. In further embodiments, the ratio is 90:10.
  • the Den-LLC systems of the invention comprise between 10 and 35 weight percent (wt%) water. In some embodiments, the systems comprise between 13 and 23 wt% water, between 10 and 15 wt%, between 13 and 20 wt% water, between 15 and 20 wt% water or between 15 and 25 wt% water.
  • the systems of the invention comprise between 68 and 80 percent of GMO, between 7 and 10 percent of the at least one lipophilic compound and between 10 and 25 percent water; dendrimer concentration varying from 0.1-25% of the aquaes phase.
  • the Den-LLC systems comprise between 63 and 73 percent of GMO, between 12 and 14 percent of the at least one lipophilic compound and between 10 and 35 percent water; dendrimer concentration varying from 0.1-25% of the aqueous phase.
  • the Den-LLC systems of the present invention may also comprise at least one phospholipid.
  • the phospholipid is a glycerophospholipid being selected from mono-phosphatidyl glycerols, bis-phosphatidyl glycerols, and tris- phsophatidyl glycerols.
  • phospholipids are phosphatidyl choline (PC), dipalmitoylphosphatidylcholine, phosphatidyl ethanolamine (cephalin), phosphatidyl inositol, phosphatidyl serine, cardiolipin, plasmalogen, lysophosphatidic acid, phosphatidylinositol (3,4)-bisphosphate, phosphatidylinositol (3,5)-bisphosphate, phosphatidylinositol (4,5)-bisphosphate, phosphatidylinositol 4-phosphate, phosphatidylinositol (3,4,5)-trisphosphate, and phosphatidylinositol 3-phosphate.
  • PC phosphatidyl choline
  • cephalin phosphatidyl inositol
  • phosphatidyl serine phosphatidyl serine
  • cardiolipin cardiolipin
  • plasmalogen
  • the phosphatidyl ester is phosphatidyl choline (PC).
  • the Den-LLC systems of the invention may, in some embodiments, comprise an active material, such as a drug, a bioactive molecule, a macromolecule or a biomacromolecule.
  • the active material may be hydrophobic or hydrophilic in nature. The ability of the Den-LLC systems of the invention to solubilize one or more hydrophobic and/or hydrophilic materials permits the formation of a great variety of on- demand drug-delivery systems.
  • the term "active material” refers generally to a hydrophobic and/or a hydrophilic material having an effect on a living cell, in vitro or in vivo, upon interaction therewith, either by interacting with the surface of the living cell or by interacting with the cell following penetration thereinto.
  • the active material may be a drug (a therapeutic), a diagnostic agent (e.g., a contrasting agent), a bioactive molecule, a macromolecule or a biomacromolecule.
  • the active material is a vitamin, a peptide, a protein, a hormone, a sugar, an enzyme, a lipid, a nucleotide, a metal (such as gold, silver, and others), a metal oxide (such as silica, iron oxide), and others.
  • the bioactive molecule is selected from insulin, desmopressin, lysine vasopressin, somatostatin, Renin inhibitor, cytochrome C, myoglobin, lysozyme (LSZ), cyclosporine A (CSA), chemotripsinogen A.
  • it may be selected from Arixtra (Fondaparinux Sodium), low molecular weight heparin (LMWH), calcitonin, interferon beta la, follicle stimulating hormone (FSH) and other drugs.
  • the biomacromolecule may be selected from a natural amino acid, a natural amino acid sequence, an unnatural amino acid, an unnatural amino acid sequence, a peptide, a protein, an antibody, RNA, siRNA, DNA, and any fragments or derivatives thereof.
  • the sequences are selected from Mouse PARP-1 Target sequences CAGGCCAGCU, GGUCUUUAA,
  • the active material may be solubilized in the Den-LLC systems of the invention in such a way that there exists no or minimal chemical interaction between the dendrimer and the active material.
  • the active material and the dendrimer may be chemically associated to each other.
  • the term "chemically associated" or any lingual variation thereof refers to the force holding the two entities together, namely the dendrimer and the active material.
  • Such force may be any type of chemical or physical bonding interaction known to a person skilled in the art.
  • Non- limiting examples of such association interactions are covalent bonding, ionic bonding, coordination bonding, complexation, hydrogen bonding, van der Waals bonding, hydrophobicity-hydrophilicity interactions, etc.
  • the association is via covalent bonding. It should be understood to a person skilled in the art that in some cases the associative interactions between two atoms or two chemical entities may involve more than one type of chemical and/or physical interactions.
  • the invention provides a process for the preparation of a Den- LLC system, as described herein, the process comprising:
  • said period of time is between about 10 and 60 minutes. In other embodiments, said temperature is between about 15°C and 80°C.
  • the liquid mixture further comprises at least one active material.
  • the invention provides a process for forming a reverse hexagonal system comprising at least one dendrimer, the process comprising: obtaining a lamellar (L a ) or reverse bicontinuous cubic (Q) LLC system comprising (solubilizing) at least one dendrimer, and
  • said LLC system comprising at least one dendrimer with an amount of a dehydrating agent, as defined herein, until a conversion ensues from said lamellar (L a ) or reverse bicontinuous cubic (Q) system to said reverse hexagonal system.
  • the dehydrating agent is an additional amount of said at least one dendrimer. In other embodiments, the dehydrating agent is at least one other dendrimer.
  • the dehydrating agent is selected from propylene glycol, glycerol, sugars, sorbitol, mannitol, xylitol, lactytol, and any mono and low molecular weight mono-, or di- and poly-sugars.
  • a further aspect of the invention provides a composition comprising a Den-LLC system, as described herein.
  • the Den-LLC systems of the invention may be utilized for a great variety of applications, all of which are based on the ability of systems of the present invention to solubilize materials, e.g., active material, for the preparation of a variety of compositions.
  • the composition may be pharmaceutical compositions or non-pharmaceutical compositions such as a cosmetic composition, a food additive composition, an antioxidant composition, a preservative composition and others.
  • the composition is selected from a pharmaceutical composition, a cosmetic composition, a composition for diagnosis, and a food additive.
  • the pharmaceutical composition according to the invention may comprise one or more pharmaceutically acceptable carriers, for example, vehicles, adjuvants, excipients, or diluents; such carriers are well-known to those who are skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active compounds and one which has no detrimental side effects or toxicity under the conditions of use.
  • pharmaceutically acceptable carriers for example, vehicles, adjuvants, excipients, or diluents; such carriers are well-known to those who are skilled in the art and are readily available to the public.
  • the pharmaceutically acceptable carrier be one which is chemically inert to the active compounds and one which has no detrimental side effects or toxicity under the conditions of use.
  • compositions of the present invention are merely exemplary and are in no way limiting.
  • compositions suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions.
  • Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent.
  • Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.
  • the systems of the present invention can be made into aerosol formulations to be administered via inhalation.
  • aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the Den-LLC system may be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying
  • Oils which can be used in parenteral formulations, include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts
  • suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxy- ethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-P-aminopriopionates, and 2- alkyl- imidazoline quaternary ammonium salts, and (3) mixtures thereof.
  • the systems of the present invention may be made into injectable formulations.
  • the requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238- 250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4 th ed., pages 622-630 (1986).
  • compositions for diagnosis or in vitro use comprising Den-LLC according to the present invention; namely a LLC system, as defined above, incorporating at least one dendrimer, and optionally at least one active material.
  • a pharmaceutical composition comprising Den- LLC according to the present invention; namely a LLC, as defined above, incorporating at least one dendrimer, and optionally at least one active material.
  • the pharmaceutical compositions of the invention are provided for use as delivery means of the at least one compound across a cell membrane.
  • the at least one compound to be delivered across a cell membrane is said dendrimer.
  • the at least one compound is an active material.
  • the blood-brain barrier (BBB) is considered a major obstacle for the delivery of such compositions.
  • BBB blood-brain barrier
  • BCSFB blood-cerebrospinal fluid barrier
  • the Den-LLC systems of the invention have been proven as means for transporting active materials across the BBB.
  • the present invention provides a pharmaceutical composition and also a drug-delivery system for transporting an active material across the blood-brain barrier (BBB) or into a cell.
  • BBB blood-brain barrier
  • the at least one compound is a dendrimer. In other embodiments, the at least one compound is an active material.
  • compositions may be suitable for any type of administration, including oral, transdermal, aerosol, parenteral, subcutaneous, intravenous, intramuscular, interperitoneal, rectal, and vaginal administrations.
  • the composition is adapted for oral administration.
  • compositions of the invention may be formulated as immediate release formulations, sustained release formulations, as controlled release formulation, and as gradual (step-wise dose) release formulations.
  • the invention also provides an oral drug delivery system comprising a Den-LLC system, as defined above, and at least one dendrimer and optionally at least one active material, wherein the system is adapted to deliver the dendrimer and/or active material orally at a rate and/or concentration greater than the rate and/or concentration of the active material when contained in a system different from said system.
  • a Den-LLC system as defined above
  • the system is adapted to deliver the dendrimer and/or active material orally at a rate and/or concentration greater than the rate and/or concentration of the active material when contained in a system different from said system.
  • composition of the invention may be used for "on demand” drug delivery, namely compositions in which the active material is released from the Den-LLC upon a certain controllable trigger, particularly change in the concentration of water within the micelles of the hexagonal mesophase, which cause changes in the dendrimer concentration within the micelles.
  • changes in concentration may be induced by changes in temperature, pH, presence of surrogate materials, etc.
  • a non-limiting example may be changes in temperature, which induce changes in volume within the Den-LLC, resulting in an increase in the effective concentration of the dendrimer within the micelles.
  • Such an increase in concentration induces phase transformation of the LLC and subsequently the release of the active material from the LLC.
  • sustained release has its ordinary meaning as understood by the skilled in the art, namely the controlled release of a drug from a dosage form over an extended period of time.
  • sustained-release dosage forms are those that have a release rate that is substantially longer than that of a comparable immediate release form, i.e., the release of a drug from a dosage form in a relatively brief period of time after administration.
  • the sustained release profile may be characterized by any measure known in the art, such as t 2 , the time to achieve a certain level of the drug in the blood or serum, the period of time in which the desired level of drug is maintained in the blood or serum, etc.
  • release rate refers to a characteristic related to the amount of an active ingredient released per unit time as defined by in vitro or in vivo testing.
  • the pharmaceutical compositions comprising the Den- LLC system is adapted for cellular microinjection, in vitro or in vivo, namely the ability to temporarily deform or open the tight junctions of the cell membrane in order to allow insertion or transport of an active material into the cell.
  • the conversion of the lamellar (L a ) or reverse bicontinuous diamond cubic (Q) system to the reverse hexagonal system greatly enhances this process.
  • the invention is therefore also directed at providing a method of inserting a material (e.g., said dendrimer or an active material) into a cell, in vitro or in vivo, said method comprising providing a composition according to the present invention, and contacting said cell with said composition, whereby insertion occurs.
  • the invention also provides a method of treatment or prevention of a disease or disorder in a subject (human or non-human), comprising administering a Den-LLC system, as described herein, the system optionally comprising at least one active material suitable for treating or preventing said disease or disorder.
  • the Den-LLC is free of an active material and the dendrimer is selected to affect treatment or prevention of a disease or disorder.
  • a dendrimer or an active compound (if present) across a cell membrane or through the blood-brain barrier comprising:
  • Den-Hn mesophase with at least one cell membrane or the blood-brain barrier under conditions permitting a phase transformation into a lamellar mesophase (Den-La) or a reverse bicontinuous cubic mesophase (Den-Q) to thereby deliver the compound across the cell membrane or through the blood -brain barrier.
  • Den-La lamellar mesophase
  • Den-Q reverse bicontinuous cubic mesophase
  • a dendrimer or an active compound (if present) across a cell membrane or through the blood-brain barrier comprising:
  • Den-Hn mesophase with at least one cell membrane or the blood-brain barrier under conditions permitting a phase transformation into a lamellar mesophase (Den-La) or a reverse bicontinuous cubic mesophase (Den-Q) to thereby deliver the dendrimer and/or the active compound across the cell membrane or through the blood-brain barrier.
  • Den-La lamellar mesophase
  • Den-Q reverse bicontinuous cubic mesophase
  • a dendrimer or an active compound (if present) across a cell membrane or through the blood-brain barrier comprising:
  • the invention further provides a method for forming a reverse hexagonal LLC system comprising at least one dendrimer compound, the method comprising:
  • the "effective amount" for purposes herein is determined by such considerations as may be known in the art.
  • the amount must be effective to achieve the desired therapeutic effect as described above, depending, inter alia, on the type and severity of the disease to be treated and the treatment regime.
  • the effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount.
  • an effective amount depends on a variety of factors including the affinity of the ligand to the receptor, its distribution profile within the body, a variety of pharmacological parameters such as half life in the body, on undesired side effects, if any, on factors such as age and gender, etc.
  • treatment refers to the administering of a therapeutic amount of the composition of the present invention which is effective to ameliorate undesired symptoms associated with a disease, to prevent the manifestation of such symptoms before they occur, to slow down the progression of the disease, slow down the deterioration of symptoms, to enhance the onset of remission period, slow down the irreversible damage caused in the progressive chronic stage of the disease, to delay the onset of said progressive stage, to lessen the severity or cure the disease, to improve survival rate or more rapid recovery, or to prevent the disease from occurring or a combination of two or more of the above.
  • the administration of said effective amount may by simultaneously, sequentially and/or separately from the administration of any other active material.
  • spontaneous administration may permit one component in the combination to be administered within a certain time period (e.g., 5 minutes, 10 minutes or even a few hours) after the other, provided that the circulatory half-life concentration of the first administered component in a combination is concurrently present in therapeutically effective amounts with the other components administered thereafter.
  • the time delay between administration of the components may vary depending on the exact nature of the components and the formulation containing them, the interaction between the individual components, their respective half-lives, and on such other factors as easily recognized by the versed artesian.
  • any lingual variation thereof is used herein to mean that the time period between administering one component and the other is significant i.e., the first administered component may no longer be present (or is present in subclinical amounts) in the bloodstream in a therapeutically effective amount when the second (or subsequent) component is administered.
  • an oral drug delivery system for the delivery of a drug, said system comprising a drug and an GMO-based mesophase, wherein said drug is contained within said mesophase, the mesophase being adapted to deliver the drug at a rate and/or concentration greater than the rate and/or concentration of the drug when contained in a system lacking or absenting said mesophase.
  • kit or a commercial package containing at least complex drug delivery system as herein described, and instructions for use.
  • the at least complex drug delivery system may be present in the kit in separate compartments or vials and may be mixed with each other immediately before application.
  • the two active components namely an aqueous phase comprising the dendrimer and active material, and a surfactant phase
  • a surfactant phase namely an aqueous phase comprising the dendrimer and active material, and a surfactant phase
  • the kit may further comprise at least one carrier, diluent or solvent useful for the dissolution of the active components, the dilution thereof or generally for the preparation of the composition (pharmaceutical, cosmetic or food additive).
  • the composition may be prepared by the end user (the consumer or the medical practitioner) according to the instructions provided or the experience and/or training of the end-user.
  • the kit may also comprise measuring tools for measuring the weight, volume or concentration of each component (active components and/or carriers).
  • Fig. 1 is a binary phase diagram of the GMO-water system.
  • Figs. 2A-2B shows the X-ray diffraction patterns and lattice parameters of 85 wt% GMO and 15 wt% water+PPI-G2 in variant concentrations (0-30 wt% from the aqueous phase):
  • Fig. 2A X-ray diffraction patterns of (a) 0, (b) 10, and (c) 20 wt% PPI- G2 measured at 25°C. The arrows on traces indicate the locations of the 10, 11 , and 20 diffraction peaks.
  • Fig. 2B Lattice parameter (A) of (O) L a and ( ⁇ ) H n structures as a function of PPI-G2 concentration.
  • Figs. 3A-3D are schematic illustrations of the solubilization location and interactions of protonated PPI-G2 in the L a system.
  • Two major modifications in the L a system are indicated: a reduction in the lattice parameter (i.e., l.p.), and reduction in the CPP, which caused to L a ⁇ H transition.
  • Figs. 3A and 3B represent L a mesophases that were formed at 0 and 5 wt% PPI-G2 concentrations in the aqueous phase, respectively.
  • Fig. 3C represents the more significant reduction in lattice parameter and the existence of two phases: and H n , when 10 wt% PPI-G2 was solubilized.
  • Figs.4A-4B shows the X-ray diffraction patterns and lattice parameters of 65 wt% GMO and 35 wt% water+PPI-G2 in variant concentrations (0-30 wt% PPI-G2 from the aqueous phase:
  • Fig. 4 A shows the X-ray diffraction patterns of (a) 5 and (b) 20 wt% PPI-G2 measured at 25°C showing the Q and H n mesophases;
  • Fig. 4B shows the lattice parameter (A) of (O) Q and ( ⁇ ) H n .
  • Figs. 5A-5C is a schematic presentation illustrating the solubilization location and interactions of protonated PPI-G2 in the Q (Pn3m) system. Two major modifications are indicated: a reduction in the lattice parameter, and reduction in the CPP, which caused the Q ⁇ H transition.
  • the unloaded Q mesophase (Fig. 5A); the gradual decrease in lattice parameter is shown and the subsequent formation of the H n mesophase in the presence of 10 and 30 wt% PPI-G2 (Figs. 5B-5C), respectively.
  • Figs. 6A-6B show the GMO/VE systems (at 90:10 ratio) containing 20 wt% aqueous phase with G2 in concentration range of 0-30 wt% from the aqueous phase: X- ray diffraction patterns of: (a) 0 wt%, (b) 5 wt%, (c) 10 wt%, (d) 15 wt%, (e) 20 wt%,
  • Figs. 7A-7C is a schematic presentation of the solubilization location and interactions of protonated PPI-G2 in the H system. Two trends are indicated: an increase of the lattice parameter at 10 wt% PPI-G2 (Figs. 7A-7B) and a reduction in the l.p. at 30 wt% of the dendrimer (Fig. 7C).
  • Fig. 8A shows the dynamic frequency sweep test for GMO (85 wt%) and aq. phase (15 wt%) containing: (G ( ⁇ ) and G" ( ⁇ )), ((G' (A) and G" ( ⁇ )), ((G ( ⁇ ) and G” (O)), and ((G' ( ⁇ ) and G” (O)) of 0, 20, 25, and 30 wt% PPI-G2 at 25°C;
  • Fig. 8B shows the complex viscosity ⁇ *) as a function of the applied oscillation frequency ( ⁇ ) GMO (85 wt%) and 15 wt% aq. phase containing: ( ⁇ ) 0, (A) 20, ( ⁇ ) 25, and (O) 30 wt% PPI-G2, all at 25 °C.
  • Fig. 9A shows the dynamic frequency sweep test for GMO (65 wt%) and aq. phase (35 wt%) containing: (G' ( ⁇ ) and G" ( ⁇ )), ((G' (+) and G" (-)), ((G (A) and G" ( ⁇ )), ((G' ( ⁇ ) and G” (O)) and (G' ( ⁇ ) and G” (O)) of 0, 5, 20, 25 and 30 wt% PPI-G2 at 25°C;
  • Fig. 9B shows the complex viscosity ⁇ * as a function of the applied oscillation frequency ( ⁇ ) GMO (65 wt%) and 35 wt% aq. phase containing: ( ⁇ ) 0, (+) 5, (A) 20, ( ⁇ ) 25, and ( ⁇ ) 30 wt% of PPI-G2, all at 25°C.
  • Fig. 10A shows the complex viscosity ⁇ * as a function of the applied oscillation frequency ( ⁇ ) GMO/VE 90/10 ratio (72 and 8 wt%) and 20 wt% aq. phase containing: ( ⁇ ) 0, (A) 10, (O) 25 and (O) 30 wt% of PPI-G2, all at 25°C;
  • Fig. 10B shows the complex viscosity ⁇ * as a function of the applied oscillation frequency ( ⁇ ) of 30 wt% PPI-G2 loaded systems: (A) GMO (85 wt%) and 15 wt% aq. phase, (O) GMO/VE 90/10 ratio (72 and 8 wt%) and 20 wt% aq. phase, (D)GMO (65 wt%) and 35 wt% aq. phase, all at 25°C.
  • Fig. 11 depicts z ("coordination number") as a function of PPI-G2 concentration in the three mesophases: (A) for the GMO+15 wt% aq. phase, ( ⁇ ) for the GMO+35 wt% aq. phase and ( ⁇ ) for GMO/VE (90/10) +20 wt% aq. phase.
  • Fig. 13A are experimental (black lines) and computed (grey lines) EPR spectra for the three mesophases: L a : GMO+15 wt% aq. phase; Q: GMO+35 wt% aq. phase; and H n : GMO/VE (90/10) +20 wt% aq. Phase;
  • Fig. 13B are Experimental (black lines) and computed (grey lines) EPR spectra (chosen as examples) for the three mesophases by adding different amounts of PPI-G2: L a (GMO+15 wt% aq. phase) + PPI-G2 10 %; Q (GMO+35 wt% aq. phase) + PPI-G2 20 %; and H n (GMO/VE (90/10) +20 wt% aq. phase) + PPI-G2 30 %.
  • Figs. 14A-14C are schematic representations illustrating the solubilization location and interactions of PPI-G2 at 25 wt% in different three H n mesophases, containing various aqueous phase concentrations: 15 %wt aq. phase, l.p. -44.6A (Fig. 14A); 35 %wt aq. phase, l.p. 46.4A (Fig. 14B); and 20 %wt aq. phase, l.p. 52.8A (Fig. 14C).
  • Fig. 15 shows Na-DFC skin permeation vs. time in the Na-DFC LLC systems
  • Fig. 16 shows the cumulative Na-DFC skin permeation vs. time in the Na-DFC- only, "blank” systems ( ⁇ ) and in the Na-DFC-PEN, “combined” systems ( ⁇ ).
  • Figs. 17A-17C shows the release of Na-DFC (0.05 wt%) from Hn mesophase as a function of time after the addition of increasing TLL contents (U/g): ( ⁇ ) 0, and ( ⁇ ) 33
  • Fig. 17B shows the release of Na-DFC (0.05 wt%) from Hn mesophase as a function of time after the addition of 33 U/g TLL concentration with increasing water phase contents (wt%) of: ( ⁇ ) 15, ( ⁇ ) 20, ( ⁇ ) 25.
  • Fig. 17A-17C shows the release of Na-DFC (0.05 wt%) from Hn mesophase as a function of time after the addition of increasing TLL contents (U/g): ( ⁇ ) 0, and ( ⁇ ) 33
  • Fig. 17B shows the release of Na-DFC (0.05 wt%) from Hn mesophase as a function of time after the addition of 33 U/g TLL concentration with increasing water phase contents (wt%) of: ( ⁇ ) 15, ( ⁇ ) 20, ( ⁇ ) 25
  • FIG. 17C shows the release of Na-DFC after the addition of 33 U/g TLL concentration from a Hn system as a function of time with increasing Na-DFC wt% contents of: ( ⁇ ) 0.05, ( ⁇ ) 0.1 , and ( ⁇ ) 0.5. Dashed lines are intended as a visual guide only.
  • Figs. 18A-18B show confocal microscopy images of MEF cells after 24 hours from seeding (Fig. 18A) and 24 hours of incubation with hexosomes vehicles in to which G2 dendrimers were solubilized (Fig. 18B), when 1/10 dilution ratio was used.
  • Figs. 19A-19B show confocal microscopy images of MDA231 mammary cancer cells after 24 hours from seeding and 24 hours of incubation with hexosomes vehicles in to which G2 dendrimers were solubalized, when 1/10 dilution ratio was used: before (Fig. 19A) and after (Fig. 19B) exchanging the GFP-siRNA medium.
  • Figs. 20A-20B Western blot results for PARP-1 (Fig. 20 A) and a-acting (Fig. 20B): (1) untreated; (2) commercial transfection reagent applied; (3) PARP-1 directed siRNA transfected by a commercial transfection reagent; (4) Den- LLC; (5) PARP-1 directed siRNA transfected by Den-LLC.
  • Monoolein, GMO distilled glycerol monooleate that consists of 97.1 wt% monoglycerides, 2.5 wt% diglycerides, and 0.4 wt% free glycerol (acid value 1.2, iodine value 68.0, melting point 37.5°C) was purchased from Riken (Tokyo, Japan).
  • D-a- tocopherol, vitamin E 5-96 (containing 1430 international units of vitamin E) was obtained from ADM (Decatur, IL, USA).
  • PPI second generation (>95% purity) was obtained from SyMO-Chem, The Netherlands.
  • the GMO-based Den-LLC samples were formed by adding the dendrimer aqueous phase (solution of PPI-G2 in distilled water at a buffered pH -8.6-8.7) to the GMO. At pH -8.6-8.7 the PPI dendrimer bears positive charges. Adding 15 and 35 wt% of water-PPI-G2 solution to 85 and 65 wt% of GMO formed the L a and the Q mesophases, respectively. In order to form the Hn mesophase 20 wt% of water-PPI-G2 solution was added to GMO and VE (vitamin E) in 90/10 wt% ratio.
  • VE vitamin E
  • the aqueous phase concentration in each mesophase was kept constant (15, 35, and 20 wt% for L a , Q, and Hn, respectively), while the PPI-G2 content in the aqueous phase was 0-30 wt%.
  • the sample was heated to ⁇ 70°C in sealed tubes under nitrogen (to avoid oxidation of the GMO) for ca. 15 min.
  • the samples were stirred and cooled to 25°C. It should be noted that as a result of PPI- G2 solubilization the concentrations of the water were decreased, keeping the weight ratio of GMO/VE (9:1) and GMO with aqueous phase constant.
  • the samples were inserted between two glass microscope slides and observed with a Nikon light microscope Eclipse 80i model equipped with cross-polarizers and attached to a digital Nikon DXM 1200C camera and PC-monitor. The samples were analyzed at room temperature.
  • SAXS Small-Angle X-ray Scattering
  • SAXS measurements were used to identify the structure of the LLC containing various quantities of dendrimer (0-30 wt% in the aqueous phase).
  • the scattering experiments were performed using Ni-filtered Cu K a radiation (0.154 nm) from a Seifert ID 3000 generator that operated at 40 kV, 40 mA at the Nanotechnology Institute, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
  • the spectra were shone through an evacuated compact Kratky camera (Anton PAAR).
  • a linear position- sensitive detector (MBRAUN) was used to record the scattering patterns.
  • the sample- to-detector distance was 280 mm and was calibrated using silver behenate.
  • Multi-Gaussian fitting has been utilized to resolve individual bands in the spectra.
  • the peaks were analyzed in terms of peak frequencies, widths at half-height, and areas.
  • Rheological measurements were performed using the Rheoscope 1 rheometer (Thermo -Haake, Düsseldorf, Germany).
  • a cone -plate sensor was used with a diameter of 35 mm, cone angle of 1 °, and a gap of 0.024 mm.
  • the linear viscoelastic range (LVR) of a material was determined before carrying out the oscillatory measurements.
  • the shear moduli were independent of stress up to a critical applied stress and generally were observed to fall off sharply beyond the values of 50, 227, and 600 Pa for the L a , Q, and 3 ⁇ 4, respectively (it should be noted that for 25 and 30 wt% of PPI-G2 in the L a mesophase, the value was 600 Pa).
  • the samples of L a , Q, and 3 ⁇ 4 possess linear viscoelastic properties up to about 50, 227, and 600 Pa, respectively.
  • the viscoelasticity measurements were generally performed at 25, 100, and 120 Pa for L a , Q, and 3 ⁇ 4, respectively (and 185 Pa for 25 and 30 wt% PPI-G2 in the L a mesophase).
  • the 5-DSA probe was first dissolved in chloroform at a concentration of 2.5xlO ⁇ 3 M as a stock solution. An appropriate quantity of the probe solution was placed in tubes, and the solvent then evaporated before preparing the three mesophases within these tubes. A low concentration of lxlO ⁇ 4 M probe in the examined LLC systems was used for all EPR studies. Such a low concentration has already been demonstrated to not perturb the nanostructures.
  • EPR spectra were recorded at room temperature using a Bruker EMX spectrometer operating at X band (9.5 GHz) using Pyrex capillary tubes ( ⁇ 1 mm inner diameter) as sample containers. The spectra were recorded 24 h after sample preparation. EPR experiment setup includes modulation amplitude of 1 G, MW frequency of 9.42 GHz, center magnetic field of 3355.00 G, sweep width of 200.00 G, resolution of 1024 points, time constant of 0.32 msec, and conversion time of 10.24 msec, with a coherent acquisition of 49 scans per each EPR spectrum.
  • MDA-MB-231 Human mammary triple negative cancer cells (MDA-MB-231), were supplied by ATCC, (Manassas, VA, USA), cultured in 6-well multi-dish plates (Nunc; Roskilde, Denmark) and Glass Bottom Culture Dishes (MatTek corporation Ashland, MA 01721 USA) # P35GC-0-14-C Poly-d-lysine coated. MDA-MB-231 cells were maintained in high glucose Dulbecco' s Modified Eagle Medium (DMEM) L-glutamine supplemented (#41965), with 10% FBS (fetal bovine serum), and 1 % Pen-Strep- Amphotericin B addition. All reagents were from Gibco UK. Mouse embryonic fibroblasts (MEFs) were generated in the laboratory of Dr. Dantzer (Illkirch, France) and cultured in the same medium as MDA MB-231 cells. These cells were used within 5 passages.
  • DMEM Dulbecco' s Mod
  • G2 poly(propylene imine) dendrimer
  • PPI poly(propylene imine) dendrimer
  • SAXS small- angle X-ray scattering
  • ATR-FTIR Attenuated Total Reflectance Fourier Transform Infrared
  • ATR-FTIR analysis reinforced the strong binding of PPI-G2 to water and the competition with GMO polar moieties.
  • Downward shift of the H-O-H functional groups of the water suggested an increase in the mean water- water H-bond angle resulting from binding PPI-G2 to the water network.
  • Examination of the hydroxyl groups of GMO at ⁇ - and ⁇ -C-OH positions revealed a partial interaction of hydrogen bonds with N-H functional groups of the protonated PPI-G2 and the hydroxyl groups of GMO.
  • the presented results may be relevant for the design of a potentially advanced drug delivery nanosystem, allowing administration of dendrimers as a therapeutic agent from the host LLCs.
  • PPI-G2 was solubilized into three types of lyotropic liquid crystals (LLC): the lamellar (L a ), the bicontinuous cubic (Q, Pn3m), and the reverse hexagonal (3 ⁇ 4) mesophases.
  • LLC lyotropic liquid crystals
  • the lamellar phase was formed when 15 wt% water was added to the molten GMO.
  • a typical lamellar texture (L a mesophase) was identified by the cross-polarized light microscope. While at the concentration of PPI-G2 ranged from 0 to 5 wt% the mesophase still retained lamellar symmetry; two phase region (L a and I3 ⁇ 4) was detected in the presence of 10-20 wt% PPI-G2.
  • the L a ⁇ H n transition was completed at 20 wt% PPI-G2 and only the hexagonal mesophase was observed.
  • the lattice parameter of the L a mesophase decreased by ⁇ 3A, while the lattice parameter of the H mesophase exhibited no significant change (Figs. 2B and 3A-3C).
  • the decrease in the lattice parameter with increasing concentration of PPI-G2 in the lamellar phase can be explained, without wishing to be bound by theory, either by dehydration of the lipid polar head-groups or by an increase in its hydrocarbon chains' mobility. Since the PPI- G2 is a water soluble dendrimer, it may interact with the water network and compete with the monoolein polar heads for water binding.
  • the detected L a ⁇ Hn transition was further elaborated by ATR-FTIR measurements.
  • ATR-FTIR Molecular level characterization by ATR-FTIR.
  • ATR-FTIR was used to elucidate the molecular interactions of PPI-G2 within the L a and Hn structures. The spectrum was analyzed in terms of two regions: the water-rich core and the water surfactant interface, which showed significant modifications in the ATR-FTIR analysis.
  • the H-O-H bending band at -1650 cm -1 was used to characterize the competitive water interactions with the GMO headgroups and the PPI-G2 in the water rich core.
  • the water-rich core The reorganization of water upon addition of PPI-G2 was detected by the downward shift of the water bending vibration frequency. This frequency was augmented from 1651 cm “1 in the absence of PPI-G2, through -1649 with 15 wt% PPI-G2, and more moderately to 1646 cm “1 with 30 wt% PPI-G2 (an overall downward shift of 5 cm "1 ). It is well documented that the water bending vibration frequency decreases when ionic solutes are introduced to the water as a result of the mean water-water H-bond angle in their first hydration shell [18]. Ionic solute groups generally increase the RMS (root mean square) H-bond angle by increasing the population of more distorted H bonds at the expense of the less distorted population. Since the pH of the PPI-G2 aqueous solution is ⁇ 9.85, most of the PPI-G2 terminal amine groups are protonated and have positive surface charges, which make them function as cationic solutes.
  • the C a O-0 stretching mode was detected at -1171 cm “1 .
  • Up to 10 wt% PPI-G2 the frequency decreased to 1170 cm “1 and when more than 15 wt% dendrimer was introduced to the system it finally reached 1169 cm “1 .
  • the transition to low frequency of the CO-0 band can be interpreted by a deviation from the dihedral angle of 180° in the C y -Cp-C a O-O-C segment, induced by torsional motions or by a small population of gauche conformers near the CO-0 bond. It has been shown that a lower frequency position of the CO-0 band corresponds to more disordered states of the lipids and thus is consistent with the fact that it increases the curvature.
  • PPI-G2 was solubilized into the binary Q bicontinuous cubic mesophase composed of GMO (65 wt%) and water (35 wt%). There was no change in the mesophase symmetry at 5 wt% PPI-G2 concentration (Fig, 4A, a). A reduction of the lattice parameter by -7.5 A from -125 A to -117.5 A was detected, as examined by SAXS analysis (Fig. 4B). When the concentration of PPI-G2 was further increased (10- 30 wt%), a Q ⁇ Hn transition occurred, according to microscopic images and to SAXS spectrum (Fig. 4A, b).
  • the CO-0 bond ester stretching mode of the GMO was examined and exhibited a similar trend to that of the lamellar mesophase.
  • the lower frequency position of the CO-0 band that obtained corresponds to more disordered states of the lipids and thus is consistent with the fact that the curvature of the system was increased as a result of Q ⁇ Hn transition.
  • This outcome is well correlated with the SAXS results that displayed a significant decrease in the lattice parameter at PPI-G2 concentrations above 10 wt%, reflecting an increase in the curvature and finally the structural shift of the Q to 3 ⁇ 4 mesophase.
  • PPI-G2 was directly incorporated to the hexagonal structures composed of GMO/VE (90/10 ratio), and water (20 wt%) at a concentration range of 5-30 wt%. According to the microscope observations and the SAXS measurements (Fig. 6A), the hexagonal symmetry was retained over all the examined concentration range.
  • ATR-FTIR analysis enabled us to get deeper insight into the molecular interaction between the components and was well-correlated with the SAXS measurements.
  • the three mesophases are: GMO+15 wt% aq. phase, GMO+35 wt% aq. phase, and GMO/VE (90/10) + 20 wt% aq. phase.
  • Fig. 8A shows the mechanical moduli (C and G”) as a function of frequency for the L a mesophase at 25 °C and 15 wt% water content.
  • the L a mesophase behaves as a plastic material, demonstrating relatively low values of the both moduli ( ⁇ 1000 Pa). While G dominates over a low frequency spectrum, G" increases smoothly until it reached the values of the elastic moduli. Incorporation of PPI-G2 at 20-30 wt% from the aqueous phase, which induced the described transition to the Hn mesophases, was identified by tracking the evolvement of the mechanical moduli (Fig. 8A). Both G' and G" of the Hn mesophases were found to be two orders of magnitude higher than those of the empty L a mesophase, implying more rigid samples. Increasing PPI-G2 concentration caused higher values of G', thereby inducing an increase in the elasticity of the samples.
  • the elastic modulus remained higher than the viscous modulus (G") in the whole examined frequency range, suggesting gel-like behavior and relaxation times longer than those reported for other Hn mesophases.
  • the dynamic moduli of the investigated Hn mesophases (13-22 wt% water content) were shown to comply with quasi-Maxwellian frequency dependence functions characterized by the longest relaxation time (tm a x), calculated as the reciprocal frequency at the crossover point of the storage and loss moduli (l/ ⁇ ). Therefore, the solubilization of PPI-G2 changed the rheological behavior of GMO-based Hn mesophases.
  • PPI-G2 induced formation of very rigid hexagonal structures, which did not obey quasi-Maxwellian frequency dependence, possessing low aqueous phase content (15 wt%), high curvature, and small lattice parameter (44 A). Since the major rheological properties of the different phases depend primarily on the topology of the water-lipid interface, it is suggested that the presence of the PPI-G2 led to changes in the GMO-water interface during the transformation into more "solid-like" behavior.
  • PPI-G2 was solubilized into the binary Q bicontinuous cubic phase mesophase composed of GMO (65 wt%) and water (35 wt%). While there was no change in the phase symmetry at 5 wt% PPI-G2, a reduction of the lattice parameter by -7.5 A from -125 A to -117.5 A was detected. When the concentration of PPI-G2 was further increased (10-30 wt%), a Q->3 ⁇ 4 transition occurred, according to SAXS measurements.
  • PPI-G2 was directly incorporated within the hexagonal structures composed of GMO/VE (90/10 ratio) and water (20 wt%) at a concentration range of 5-30 wt%.
  • the hexagonal symmetry was retained over all the examined concentration range; however, SAXS measurements demonstrated two trends in the behavior of the lattice parameter.
  • a minor increase in the lattice parameter values (-2.5 A) from 54.5 A to 55.3 A was recorded at low concentrations of PPI-G2 (up to 10 wt%).
  • the lattice parameter values exhibited a notable decrease of -4.5 A from 54 A to 52.5 A.
  • the Hn mesophases with lowest aqueous phase concentration (15 wt%) possessed the highest complex viscosity and solid-like response, compared to the Hn structures with aqueous phase of 20 wt% (intermediate complex viscosity and elasticity) and finally with 35 wt% aqueous phase (the weakest response).
  • these results are in contrast with the rheological behavior of empty hexagonal mesophases, which showed enhanced elasticity and higher complex viscosity with increasing aqueous phase concentration.
  • solubilization of the dendrimer dictated the opposite trend.
  • Bohlin's theory was implemented in order to obtain additional insight into the differences between the flow responses of these systems.
  • Bohlin's model which is also reported in the literature as the "weak- gel model", was used to assess the flow character and micro structure of the obtained mesophases using rheological data.
  • Bohlin' s theory it is presumed that a flowing substance is divided into several flow units that are responsible for the macroscopic flow observed, which is only the consequence of their cooperative rearrangements. These flow units are supposed to be reflected in the micro structure of the material, in the form of molecular aggregates.
  • the main parameter introduced in this theory is the "coordination number", z, which corresponds to the number of flow units interacting with each other to give the cooperative flow response.
  • the magnitude of complex modulus is expressed by Eq. (2):
  • A is a constant that can be interpreted as the "interaction strength" between the flow units, revealing the amplitude of cooperative interactions.
  • Bohlin demonstrated that the cooperative flow on the hexagonal phase has a six-fold coordination, identified with the columnar structure of the mesophase. For lamellar mesophase z is equal to 2, while for a cubic structure it could vary between 6 (simple cubic structure), 8 (body- centered structure), and 12 (face-centered structure). This theory has been used to determine the interaction among aggregates and crystalline domains in hexagonal liquid crystals with nonionic surfactants and block copolymers. The values for 3 ⁇ 4 are reported as -6.7.
  • PPI-G2 In the water-rich cubic samples (Q ⁇ Hn), PPI-G2 was mostly incorporated in the bulk water, resulting in relatively low z numbers that decreased from 4.7 at 10 wt% PPI-G2 content to 3.8 at 30 wt% PPI-G2. Notably, the decrease in z numbers was found to be linear with PPI-G2 concentration, similar to the lattice parameter of the cubic phase (data not shown). It could be inferred that the dehydration of the monoolein polar moieties by PPI-G2 is linearly proportional to both the drop in the lattice parameter and the number of the interacting flow units and the described weaker mechanical properties such as inferior dynamic moduli values and a more liquid-like response upon oscillated perturbation and shearing. Such structural flexibility may be extremely beneficial for controlling and tuning the drug release by dendrimer-inspired mesophase formation possessing the same geometry but different water content, lattice parameters, and rheological properties.
  • the measured mechanical moduli and complex viscosity of the mesophases were also presented as a function of PPI-G2 concentration at constant frequency of 0.24 rad/s (Fig. 12A). This data presentation was employed to comprehend concentration dependent impact of storage and loss moduli on the complex viscosity. Increase in the complex viscosity in the samples containing 15 wt% aqueous phase can be directly correlated to the measured increase of both G' and G". However, more drastic raise of the elastic moduli, compared to the loss moduli, seemed to be more dominant contribution to the overall behavior of ⁇ .
  • the LLC mesophases depend on physicochemical factors, including surfactant and cosurfactant structure, surfactant and water concentrations, temperature, pH, and solubilizate properties and concentration.
  • the EPR study by means of the 5-DSA spin probe provided useful information about microviscosity, micropolarity, and the order of these systems.
  • the probe's amphiphilic characteristic enables its localization between surfactant molecules reporting on the nearby environment of different structures.
  • the three LLC systems used were investigated using an amphiphilic probe 5- DSA, and EPR spectral parameters that are also found to be pH dependent.
  • the pH was 8.6, which is essential for the dendrimer- water solution.
  • the probe In this basic environment, the probe is ionized (carboxylate anion), and consequently it is pulled toward the water phase due to ion-dipole interactions between the water and the probe, increasing the hydration shell of the latter.
  • This polar structure causes the doxyl group to sense the environment just below the surfactant heads, which are located closer to the aqueous phase.
  • the EPR spectra for the 5-DSA probe were recorded for the three unloaded mesophases.
  • the spectral line shape changes, and the analysis needs the use of a computational procedure that takes into account the relaxation process and the different interactions of the magnetic components.
  • the spectra were computed by means of the well established procedures.
  • the only parameters that were changed in the fitting procedure were: (1) the correlation time for the diffusion rotational motion of the probe, mainly the perpendicular component, x perp , which reports on the interactions occurring between the nitroxide group and the active sites of the environmental molecules.
  • An increase in x perp corresponds to an increase in the micro viscosity, i.e., a decrease in 5-DSA mobility.
  • the order parameter, S which reports on the order of a lipid layer where the probe is embedded. S may change from 0 (no order) to 1 (maximum order).
  • the analysis of the results provides two parameters about the probe's environment: micro viscosity and order.
  • PPI-G2 Loaded Mesophases By solubilizing PPI-G2 within the mesophases, the EPR spectra change is a function of the dendrimer concentration.
  • Fig. 13B shows, as examples, the experimental and computed spectra obtained for the L a , Q, and Hn mesophases by adding 10, 20, and 30% G2-PPI, respectively.
  • the microviscosity and order parameters obtained by computation at various dendrimer concentrations are listed in Table 2.
  • EPR spectra of three different PPI-G2 loaded and unloaded mesophases in pH 8.6.
  • the three mesophases are: GMO+15 wt% aq. phase, GMO+35 wt% aq. phase and GMO/VE
  • Na-DFC sodium diclofenac
  • a-DFC permeability through porcine skin was determined in vitro with a Franz diffusion cell system.
  • Whole ear porcine skin was excised from ears of domestic pigs and mounted on Franz cells (diffusion area of 0.635 cm 2 ) with the stratum corneum facing the donor compartments.
  • the receptor compartment was filled with PBS (phosphate buffer saline, pH 7.2).
  • the receptor phase was kept under constant stirring at 37 ⁇ 0.5°C.
  • 200 mg of the LLC formulations containing 1 wt% Na-DFC were applied to the surface of the stratum corneum. The results were compared to a commercial composition containing 1 % Na-DFC (Voltaren ® ) in cream form.
  • the LLC systems show improved penetration of Na- DFC. From the results it is evident that the cubic and lamellar mesophases demonstrate better release of the drug, and therefore phase transformation from the Hn into L a or Q mesophase is beneficial.
  • Hn mesophases were prepared by mixing weighed quantities of GMO and TAG while heating to 45°C. An appropriate quantity of preheated water at the same temperature was added, and the samples were stirred and cooled to 25°C.
  • PEN polyethylene phenylene
  • PEN polyethylene phenylene
  • PEN increased the overall penetrating amount of Na-DFC.
  • the effect of the peptide was explored by calculating the K p , the permeability coefficients of the different systems. These calculations are based on the slope of the cumulative curve on steady state (J ss ), at which linearity is observed.
  • the permeability coefficients (cm hr -1 ) reveal a 2.2 fold increase in the presence of PEN.
  • Kp*1000 was 0.20 in the "blank” system, whereas in the PEN loaded system it was 0.44 cm hr "1 .
  • PEN may be regarded as an effective enhancer for skin permeability of Na-DFC LLC-system. It shows a faster propagation of the drug and an overall improvement of the skin permeation coefficient for Na-DFC.
  • Systems comprising PEN and the Den-LLC show similar penetration results, indicating the ability to incorporate PEN into the Den-LLC systems of the invention for enhancement of membrane-crossing.
  • Table 3 LLC systems and Den-LLC systems with and without PPI-G2.
  • TAG is required only for the formation at room temperature of Hn mesophase absent the dendrimer. Once the dendrimer is introduced into solution, no TAG is required for the formation of the 3 ⁇ 4 mesophase at room temperature due to the solubilization of the PPI-G2 dendrimer.
  • Hn ternary reverse hexagonal mesophase
  • Thermomyces lanuginosa lipase was solubilized into Hn mesophase for the benefits of continuing lipolysis of the lipids, consequently disordering and decomposing the hexagonal mesophase and transforming it to cubic and/or lamellar mesophases, thereby enhancing the diffusion of the encapsulated drug.
  • Pure Hn mesophases were prepared while heating to -50 °C in sealed tubes under nitrogen to avoid oxidation.
  • the different concentrations of lipase solutions were prepared by diluting the TLL stock solution (100,000 U/g) into a 0.1 M phosphate buffer at pH 7.5.
  • D 2 0 was used instead of the water.
  • the hydrolysis reaction was initialized by homogenous delivery of the lipase to the sample by mixing the aqueous component solubilizing TLL.
  • Den- Hn mesophase was prepared by initially dissolving Hn mesophase, Na-DFC and the dendrimer (0.5 to 25 wt % of the aqueous phase) within the aqueous component containing the lipase.
  • the aqueous fraction was added to the lipid component while agitated to achieve homogeneous composition of the lipase and the drug contents within the Hn mesophase.
  • 10 ml of the aqueous phase containing no lipase or drug was added on top of the formed H n mesophase, remaining in excess.
  • the formed samples were stored at 25 °C and sampled periodically by UV/Vis spectroscopy (1.6 ml). The analyzed samples were returned to the reaction vessel, maintaining the initial volume of the samples.
  • UV/vis absorbance measurements demonstrated linearity as a function of the concentration of solubilized Na-DFC within the aqueous phase over the range of 0-45 ⁇ g/ml.
  • the hexagonal system (glycerol monooleate, tricaprylin, and water) preserved its symmetry within ca. 200 min.
  • this step about 40-60% molar of the lipids were hydrolyzed, and a gradual shrinking of the Hn cylinders (decrease of 8 A in lattice parameter) was detected.
  • the Hn mesophase gradually disintegrated and transformed to cubic and finally to lamellar (faster rate) and the release of a model drug (sodium diclofenac) was significantly enhanced, which was assumed to be lipolysis rate-controlled. After about 15 h the Hn mesophase was disintegrated into two dispersed immiscible phases.
  • the transfection reagents included X-tremeGENE siRNA transfection reagent (Roche Diagnostics, #04-476-093-001), and LLC as a transfection reagent comprising the LLC and PPI-G2 dendrimers.
  • GFP green fluorescent protein
  • Figs. 18A-18B demonstrate fluorescence which is indicative to the presence of siRNA in the MEF cells.
  • Figs. 19A-19B demonstrate fluorescence which is indicative to the presence of siRNA in the MDA231 cells: before (Fig. 19A) and after (Fig. 19B) exchanging the GFP-siRNA medium.
  • siRNA was transported using the LLC-Den into cell (MDA231 and MEF) and strong Cy3-siRNA florescent was detected indicating of efficient transfection and implying that the vehicle acts as a useful transfection reagent for siRNA (approximately 13.4 kDa) and is not toxic to the cells.
  • MEFs were homogenized, and pellets were re-suspended in lysis buffer containing 20 mM Tris-HCI pH 7.5, 10 mM KC1, 0.1 mM EDTA, 1.5mM MgCl 2 , 0.2% NP-40, 1 mM DTT, and 1 % protease inhibitor (Sigma cocktail). The homogenate was incubated on the ice for 5 min and centrifuged at 6000 rpm for 5 min at 4°C.

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Abstract

La présente invention concerne de nouveaux systèmes complexes de cristaux liquides dendrimère-lyotropique (LLC) et leurs utilisations.
PCT/IL2012/050336 2011-08-29 2012-08-29 Systèmes de cristaux liquides dendrimère-lyotropique (llc) WO2013030838A2 (fr)

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CN107257681A (zh) * 2014-12-23 2017-10-17 卡姆拉斯公司 控释配制品
CN117563570A (zh) * 2024-01-16 2024-02-20 西安金沃泰环保科技有限公司 一种用于蛋白吸附的树脂及其制备方法

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Publication number Priority date Publication date Assignee Title
WO2016092569A1 (fr) 2014-12-10 2016-06-16 Council Of Scientific & Industrial Research Composition micellaire inverse discontinue en phase fd3m cubique pour la libération prolongée de médicaments thérapeutiques
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CN117563570A (zh) * 2024-01-16 2024-02-20 西安金沃泰环保科技有限公司 一种用于蛋白吸附的树脂及其制备方法
CN117563570B (zh) * 2024-01-16 2024-03-15 西安金沃泰环保科技有限公司 一种用于蛋白吸附的树脂及其制备方法

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