Nanocarriers having surface conjugated peptides and uses thereof for sustained local release of drugs

Abstract

Disclosed are biodegradable nanocarriers that have a net positive surface charge and zeta potential between about +2 to about +20 mV. The positive surface charge of the nanocarriers is provided by peptides that are covalently attached to the surface of the nanocarriers. The nanocarriers may comprise a drug and may be administered for localized and sustained delivery of the drug.

We claim: 
     
         1 . Nanocarriers that are biodegradable or that can be excreted and have a net positive surface charge and zeta potential between about +2 to about +20 mV, wherein the net positive surface charge is provided by peptides that are covalently attached to the surface of the nanocarriers. 
     
     
         2 . The nanocarriers of  claim 1 , wherein the nanocarriers comprise a polymeric carbohydrate and are transparent, and preferably have a diameter that permits for filter sterilizing. 
     
     
         3 . The nanocarriers of  claim 1 , wherein the nanocarriers comprise dextran which optionally is a condensed dextran hydrogel, chitosan, pullulan, or a dendrimer. 
     
     
         4 . The nanocarriers of  claim 1 , wherein the peptides comprise C-terminal amide groups. 
     
     
         5 . The nanocarriers of  claim 1 , wherein the peptides comprise at least 2 amino acids and no more than 11 amino acids. 
     
     
         6 . The nanocarriers of  claim 1 , wherein the amino acid sequence of the covalently attached peptides includes at least 2 L-Arginine residues, no more than 4 L-Arginine residues, and no other charged amino acids other than L-Arginine residues, and where the amount of covalently attached peptides produces a positive average zeta potential of the particles ranging from about +2 to about +20 mV. 
     
     
         7 . The nanocarriers of  claim 1 , wherein the amino acid sequence of the attached peptides comprises an amino acid sequence of a human protein that is secreted into the extracellular matrix or that is found in vitreous humor, blood, urine, or saliva, or has an amino acid sequence that is at least 75% identical with the corresponding sequence from the human protein that is secreted into the extracellular matrix or that is found in vitreous humor, blood, urine, or saliva. 
     
     
         8 . The nanocarriers of  claim 1 , wherein the peptides include at their N-terminus a neutral amino acid residue selected from the group consisting of sarcosine, or beta-alanine. 
     
     
         9 . The nanocarriers of  claim 1 , wherein the peptides are covalently attached to the nanocarriers via conjugation between hydroxyl groups on the surface of the nanocarriers and free amino groups N-terminally linked to the peptides the peptides. 
     
     
         10 . The nanocarriers of  claim 1 , wherein the peptides are covalently attached to the surface of the nanocarriers via carbamate conjugation between hydroxyl groups on the surface of the nanocarriers and an amino group appended to the peptide N-terminus, wherein the amino group is selected from the group consisting of a free alpha amino group on the peptides, a free beta, gamma, delta or epsilon amino acyl group on the peptides, as in epsilon aminocaproic acid, an amino-PEG(4-12) acyl amide of the peptide N-terminus, or a 3-pyrrolidyl-3-carboxylate amide an amino-PEG(4-12) acyl amide of the peptide N-terminus. 
     
     
         11 . The nanocarriers of  claim 1 , wherein the peptides are covalently attached to the surface of the nanocarriers via amide conjugation between carboxyl groups on the surface of the nanocarriers and an amino group appended to the peptide N-terminus, wherein the amino group is selected from the group consisting of a free alpha amino group on the peptides, a free beta, gamma, delta or epsilon amino acyl group on the peptides, as in epsilon aminocaproic acid, or an amino-PEG(4-12) acyl amide of the peptide N-terminus. 
     
     
         12 . The nanocarriers of  claim 1 , wherein the peptides have a formula:
 B—Z-AA0-(AA1) n -Y, where n is an integer between 1 and 10 where no less than two AA and no more than 4 AA are L-arginine, and all other AA are neutral (not lysine or glutaric acid or aspartic acid).   wherein:   B is present or absent, and when B is present, B is selected from the group consisting of amino-n-butoxy, amino-ethoxyethyloxy, amino-piperidyl (3, or 4)-oxy, amino-pyrrolidinyl (3)-oxy, amino-benzyl (3, or 4)-oxy, aminoethylamido-valeric acid (4)-oxy, amino-cyclohexyl (3, or 4)-oxy, and amino-cyclopentyl (3)-oxy;   Z is absent or present, and when Z is present, Z is a dicarboxylic acid, including suberic, adipic, glutaric, dimethylglutaric, succinic in half amide bond to AA0 or AA1, where the OH group of an above amino alcohol, B is bonded as an ester to the free dicarboxylic acid group in half-amide linkage to or AA1;   AA0 is present or absent, and when present, AA0 is selected from the group consisting of L-arginine, a naturally occurring amino acid with an uncharged side chain (e.g., sarcosine and glycine), beta-alanine, and L-proline;   AA1 is L-arginine or a naturally occurring amino acid with an uncharged side chain;   Y is an amide, a mono-substituted or di-substituted alkyl amide (e.g., methylamide, ethylamide, and dimethylamide), or a PEG (4-12) amide;   with the proviso that:
 the peptides have a net positive charge and their multiple linkage to carrier produces nanoparticles having zeta potential between about +2 to about +20 mV; when peptide comprises from about 10% to about 50% of the conjugate mass, whereby ester hydrolysis slowly reduces peptide content and zeta potential of the nanoparticle, leading to accelerating vitreal loss. 
   
     
     
         13 . The nanocarrier of  claim 12  in which the appended esterified dicarboxypeptide [Z-AA0-(AA1) n -Y], comprising from about 10% to about 50% of the total nanocarrier mass, is bio-active, as in anti-angiogenic peptide adipic-Sar-Tyr-Asn-Leu-Tyr-Arg-Val-Arg-Ser-amide (SEQ ID NO:6), thus acting simultaneously as a prodrug as well as an anchor to slow diffusion. 
     
     
         14 . The nanocarriers of  claim 12 , wherein B is an amino-PEG-carboxylic acid having between 4 and 12 ethylene glycol units in amide bond to AA0 or AA1, and Z is absent. 
     
     
         15 . The nanocarriers of  claim 1  in which amino-PEG-OH or amino-PEG-OMe of molecular weight ranging from 200 to 2,000 grams/mole are additionally appended to carriers through carbamate or amide bonds while comprising from about 10% to about 50% of the conjugate mass. 
     
     
         16 . The nanocarriers of  claim 1 , wherein the peptides are selected from the group consisting of: 
       
         
           
                 
               
                   (SEQ ID NO: 1) 
                 
                   NH 2 -PEG(8-12)-CO-Gly-Val-Ile-Thr-Arg-Ile-Arg-NH 2 ; 
                 
                     
                 
                   (SEQ ID NO: 3) 
                 
                   NH 2 -PEG(8-12)-CO-Arg-Arg-Ser-Ser-Arg-Arg-Trp-NH 2 ; 
                 
                     
                 
                   (SEQ ID NO: 4) 
                 
                   NH 2 -PEG(8-12)-CO-Tyr-Arg-Val-Arg-NH 2 ; 
                 
                     
                 
                   (SEQ ID NO: 5) 
                 
                   NH 2 -PEG(8-12)-CO-Arg-Arg-Tyr-Arg-Leu-NH 2 ; 
                 
                     
                 
                   (SEQ ID NO: 9) 
                 
                   NH 2 -PEG(8-12)-CO-Arg-Arg-Ser-Ser-Arg-Arg-NH 2 ; 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         and the N-terminal amino group is in covalently linked to a hydroxyl on the surface of the particles through an amide linkage or carbamate linkage. 
       
     
     
         17 . The nanocarriers of  claim 1 , wherein the peptides are selected from the group consisting of: 
       
         
           
                 
               
                   (SEQ ID NO: 4) 
                 
                   3-pyrrolidine-CONH-PEG(8)-CO-Tyr-Arg-Va1-Arg-Ser- 
                 
                   NH 2 ; 
                 
                   and 
                 
                     
                 
                   (SEQ ID NO: 5) 
                 
                   3-pyrrolidine-CONH-PEG(8)-CO-Arg-Arg-Tyr-Arg-Leu- 
                 
                   NH 2 . 
                 
             
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         18 . The nanocarriers of  claim 12 , wherein the appended esterified dicarboxypeptide [B—Z-AA0-(AA1) n -Y] comprising from about 10% to about 50% of the total nanocarriers mass is pyrrolidinyl (3)-oxy-adipic-Sar-Tyr-Asn-Leu-Tyr-Arg-Val-Arg-Ser-amide (SEQ ID NO:6). 
     
     
         19 . A pharmaceutical composition comprising the nanocarriers of  claim 1  and a suitable pharmaceutical carrier. 
     
     
         20 . A method comprising administering the pharmaceutical composition of  claim 19  to a subject in need thereof.