Particle structures comprising sterols and saponins

Abstract

The present invention pertains to complexes comprising sterols and saponins. The complexes are capable of binding a genetic determinant including a polynucleotide. The complexes may further comprise a lipophilic moiety, optionally a lipophilic moiety comprising a contacting group and/or a targeting ligand, and/or a saccharide moiety. The complexes may further comprise an immunogenic determinant and/or an antigenic determinant and/or a medicament and/or a diagnostic compound. The complexes may in even further embodiments be encapsulated by an encapsulation agent including a biodegradable microsphere. The present invention also pertains to pharmaceutical compositions and methods of treatment of an individual by therapy and/or surgery, methods of cosmetic treatment, and diagnostic methods practised on the human or animal body.

1 . A particle structure comprising:
 (i) a sterol comprising a gonane skeleton structure, wherein the sterol is anionic or neutral;   (ii) a saponin isolated from a plant species selected from the group consisting of  Quillaja saponaria, Saponaria officinalis, Gypsophila struthium, Aesculus hippocastanum, Silene jenisseenis, Acanthophyllum squarrosum , and combinations thereof, wherein said sterol and said saponin interact with each other; and   (iii) a cationic sterol comprising a gonane skeleton structure and at least one moiety carrying a positive charge at pH 7.0.   
     
     
         2 . The particle structure of  claim 1 , wherein said saponin comprises the following formula: 
       
         
           
           
               
               
           
         
         wherein R 1  is —C—O—R 4 , wherein R 4  is a linear or branched saccharide comprising at least one monosaccharide moiety selected from the group consisting of D-glucose, D-galactose, D-glucuronic acid, D-galacturonic acid, L-rhamnose, L-arabinose, D-xylose, D-fucose, D-apiose, D-ribose, D-allose, D-quinovose, and combinations thereof; and 
         wherein R 2  is —COOR 5 , —C═O, or —C—OH, wherein R 5  is —H or a linear or branched saccharide, said saccharide comprising at least one monosaccharide moiety selected from the group consisting of D-glucose, D-galactose, D-glucuronic acid, D-galacturonic acid, L-rhamnose, L-arabinose, D-xylose, D-fucose, D-apiose, D-ribose, D-allose, D-quinovose, and combinations thereof; and 
         wherein R 3  at each of positions C1, C2, C11, C15, C16, C21, C22, C23, C24, C26, C27, C29, and C30 is, independent of the other positions, —H, —OH; ═O; —COOH; —COOMe; or ══. 
       
     
     
         3 . The particle structure of  claim 2 , wherein R 5  is a linear or branched saccharide, said saccharide comprising at least one monosaccharide, wherein said at least one monosaccharide is substituted with acyl moieties selected from a group consisting of 3,5-dihydroxy-6-methyloctanoic acid, 3,5-dihydroxy-6-methyloctanoic acid, 5-O-α-L-rhamno-pyranosyl-(1→2)-α-L-arabino-furanoside, and 5-O-α-L-arabino-furanoside. 
     
     
         4 . The particle structure of  claim 2 , wherein R 1  is a branched trisaccharide. 
     
     
         5 . The particle structure of  claim 2 , wherein R 3  of said saponin is —OH at C16, ═O at C23, and —H at positions C1, C2, C11, C15, C21, C22, C24, C26, C27, C29, and C30. 
     
     
         6 . The particle structure of  claim 5 , wherein R 5  is a linear or branched saccharide, said saccharide comprising at least one monosaccharide, wherein said at least one monosaccharide is substituted with acyl moieties selected from a group consisting of 3,5-dihydroxy-6-methyloctanoic acid, 3,5-dihydroxy6-methyloc-tanoic acid, 5-O-α-L-rhamno-pyranosyl-(1→2)-α-L-arabino-furanoside, and 5-O-α-L-arabino-furanoside. 
     
     
         7 . The particle structure of  claim 6 , wherein R 5  is a branched saccharide, said saccharide comprising seven monosaccharides, wherein said at least one monosaccharide is substituted with acyl moieties selected from a group consisting of 3,5-dihydroxy-6-methyloctanoic acid, 3,5-dihydroxy-6-methyloc-tanoic acid, 5-O-α-L-rhamno-pyranosyl-(1→2)-α-L-arabino-furanoside, and 5-O-α-L-arabino-furanoside. 
     
     
         8 . The particle structure of  claim 7 , wherein the saponin is QuilA. 
     
     
         9 . The particle structure of  claim 8 , wherein the saponin is substantially pure. 
     
     
         10 . The particle structure of  claim 1 , wherein the saponin is isolated from  Quillaja saponaria.    
     
     
         11 . The particle structure of  claim 10 , wherein the saponin is selected from the group consisting of QA1, QA2, QA3, QA4, QA5, QA6, QA7, QA8, QA9, QA10, QA11, QA12, QA13, QA14, QA15, QA16, QA17, QA18, QA19, QA20, QA21, QA22, and combinations thereof. 
     
     
         12 . The particle structure of  claim 1 , wherein said sterol of (i) is selected from the group consisting of cholesterol, lanosterol, lumisterol, stigmasterol, sitosterol, ergosterol, thiocholesterol, nordihydro-epi-andosterol, and combinations thereof. 
     
     
         13 . The particle structure of  claim 1 , wherein said cationic sterol of (iii) comprises the following formula: 
       
         
           
           
               
               
           
         
         wherein X is 
         —OOC(CH 2 ) 3 N + (CH 3 ) 3 , 
         —OOC(CH 2 ) 2 COO(CH 2 ) 2 N + (CH 3 ) 3 , 
         —OCONH(CH 2 ) 2 N(CH 3 ) 2 ; 
         —OCONH(CH 2 ) 2 N + (CH 3 ) 3 Cl − ; 
       
       
         
           
           
               
               
           
         
       
     
     
         14 . The particle structure of  claim 13 , wherein X is —OCONH(CH 2 ) 2 N(CH 3 ) 2 . 
     
     
         15 . The particle structure of  claim 13 , wherein X is —OCONH(CH 2 ) 2 N + (CH 3 ) 3 Cl − . 
     
     
         16 . The particle structure of  claim 1 , further comprising a bioactive agent. 
     
     
         17 . The particle structure of  claim 16 , wherein the bioactive agent is selected from the group consisting of DNA, RNA, a contrast agent, an immunogenic determinant, an antigenic determinant, a chemotherapeutic agent, a peptide, a nucleic acid of either natural or synthetic origin, and combinations thereof. 
     
     
         18 . The particle structure of  claim 17 , wherein the bioactive agent is an immunogenic determinant. 
     
     
         19 . The particle structure of  claim 1  further comprising a targeting ligand selected from the group consisting of proteins, antibodies, antibody fragments, hormones, hormone analogues, glycoproteins, lectins, peptides, polypeptides, amino acids, sugars, monosaccharides, polysaccharides, carbohydrates, vitamins, steroids, steroid analogs, cofactors, nucleosides, nucleotides, nucleotide acid constructs, polynucleotides, optionally constructs or polynucleotides of either natural or synthetic origin, and combinations thereof. 
     
     
         20 . The particle structure of  claim 19 , wherein the targeting ligand is a carbohydrate. 
     
     
         21 . The particle structure of  claim 1 , further comprising a compound selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, triglycerides, fatty acids, and hydrophobic amino acid residues, and combinations thereof. 
     
     
         22 . The particle structure of  claim 21 , wherein said compound is phosphatidylcholine. 
     
     
         23 . The particle structure of  claim 1 , further comprising a compound selected from the group consisting of a quaternary ammonium compound, a dialkyldimethylammonium compound, dioctadecyldimethyl ammonium chloride, dioctadecyldimethyl ammonium bromide, dioctadecyl/octadienyldimethyl ammonium chloride, dioctadecyl/octadienyld-imethyl ammonium bromide, dimethyldioctadecylammonium bromide (DDAB), dodecyltrimethylammonium bromide, a hexadecyltrimethylammonium compound, mixed alkyltrimethylammonium bromide (Cetrimide per BP); a tetradecyltrimethylammonium compound, and a combination of said compounds, wherein said compound is capable of associating with said bioactive agent. 
     
     
         24 . The particle structure of  claim 1 , wherein a ratio between the sterol of (i) and the cationic sterol of (iii) is less than 1000:1 to more than 1:4. 
     
     
         25 . The particle structure of  claim 1 , comprising:
 (i) cholesterol;   (ii) QuilA;   (iii) DC-Chol;   (iv) phosphatidylcholine; and   (v) a bioactive agent.   
     
     
         26 . The particle structure of  claim 1 , comprising:
 (i) cholesterol;   (ii) QuilA;   (iii) DC-Chol;   (iv) phosphatidylcholine; and   (v) a targeting ligand.   
     
     
         27 . The particle structure of  claim 1 , wherein said particle has a spherical shape and a diameter in the range of from 25 nm to 75 nm. 
     
     
         28 . The particle structure of  claim 1 , wherein said particle is in the form of a cage-like matrix. 
     
     
         29 . The particle structure of  claim 1 , characterized by a zeta potential of more positive than about −50 mV. 
     
     
         30 . The particle structure of  claim 29 , characterized by the zeta potential of about −40 mV to about −10 mV. 
     
     
         31 . A method of preparing a particle structure, comprising mixing together:
 (i) a sterol comprising a gonane skeleton structure, wherein the sterol is anionic or neutral; with   (ii) a saponin isolated from a plant species selected from the group consisting of  Quillaja saponaria, Saponaria officinalis, Gypsophila struthium, Aesculus hippocastanum, Silene jenisseenis, Acanthophyllum squarrosum , and combinations thereof;   (iii) at least one organic solvent; and   (iv) a cationic sterol comprising a gonane skeleton structure and at least one moiety carrying a positive charge at pH 7.0;   wherein the ingredients i) to iv) may be mixed simultaneously, or sequentially, in any order.   
     
     
         32 . The method of  claim 31 , further comprising the addition to the mixture of one or more bioactive agents. 
     
     
         33 . The method of  claim 31 , further comprising addition to the mixture of a compound selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, triglycerides, fatty acids, hydrophobic amino acid residues, and combinations thereof. 
     
     
         34 . The method of  claim 31 , further comprising:
 (v) removing surplus reactants and/or purifying the prepared complexes.   
     
     
         35 . The method of  claim 31 , wherein said sterol of (i) is selected from the group consisting of cholesterol, lanosterol, lumisterol, stigmasterol, sitosterol, ergosterol, thiocholesterol, nordihydro-epi-andosterol, and combinations thereof. 
     
     
         36 . The method of 31, wherein the cationic sterol of (iii) comprises the following formula: 
       
         
           
           
               
               
           
         
         wherein X is 
         —OOC(CH 2 ) 3 N + (CH 3 ) 3 , or 
         —OOC(CH 2 ) 2 COO(CH 2 ) 2 N + (CH 3 ) 3 , or 
         —OCONH(CH 2 ) 2 N(CH 3 ) 2 ; or 
         —OCONH(CH 2 ) 2 N + (CH 3 ) 3 Cl − ; or 
       
       
         
           
           
               
               
           
         
       
     
     
         37 . The method 31, wherein the solvent is present in a (vol/vol) amount of 25% (vol/vol), 10% (vol/vol), 4% (vol/vol), 1% (vol/vol), or 0.1% (vol/vol). 
     
     
         38 . A vaccine composition comprising the particle structure of  claim 1 .