Fibrin nanoparticles and uses thereof

Abstract

The present invention provides fibrin nanoparticles having a mean diameter of 200-2000 nm. The present invention provides also methods for preparing fibrin particles of various sizes, including fibrin nanoparticles and fibrin microbeads. The present invention further provides compositions comprising fibrin particles and an agent, wherein the agent is coupled to the amine or carboxy moieties on the surface of the fibrin particles. In addition the present invention provides a composition comprising fibrin nanoparticles and an agent, wherein the agent is admixed with the fibrin nanoparticles. Still further, the present invention provides a method for introducing an agent into a cell, a method for isolating stem and/or progenitor cells from a biological sample, as well as a composition comprising fibrin particles bound to stem and/or progenitor cells.

What is claimed:  
     
         1 . Isolated fibrin nanoparticles having a mean diameter of 200-2000 nm.  
     
     
         2 . The fibrin nanoparticles of  claim 1 , having a density of 1.32±0.2 g/ml.  
     
     
         3 . A method for preparing fibrin nanoparticles comprising the steps of: (i) mixing an aqueous solution comprising fibrinogen, thrombin and Factor XIII in an oil emulsion at a temperature of 50-80° C., without the addition of an exogenous chemical cross-linking agent, until fibrin microbeads are formed in the mixture; (ii) homogenizing the mixture to form fibrin nanoparticles having a mean diameter of 200-2000 nm in the mixture; and (iii) isolating fibrin nanoparticles having a mean diameter of 200-2000 nm from the mixture.  
     
     
         4 . The method of  claim 3 , wherein the aqueous solution comprising fibrinogen, thrombin and Factor XIII is prepared by contacting thrombin with purified fibrinogen containing endogenous Factor XIII.  
     
     
         5 . The method of  claim 3 , wherein the aqueous solution comprising fibrinogen, thrombin and Factor XIII is prepared by combining thrombin with a cryoprecipitate containing endogenous fibrinogen and endogenous Factor XIII.  
     
     
         6 . The method of  claim 3 , wherein the aqueous solution comprising fibrinogen, thrombin and Factor XIII is prepared by adding separate amounts of fibrinogen, Factor XIII and thrombin.  
     
     
         7 . The method of  claim 3 , wherein ratio of fibrinogen:thrombin:Factor XIII in the aqueous solution is 5-100 mg/mL:1-100 U/mL:1-50 U/mL.  
     
     
         8 . The method of  claim 3 , wherein ratio of fibrinogen:thrombin:Factor XIII in the aqueous solution is 20-40 mg/mL:5-10 U/mL:2-20 U/mL.  
     
     
         9 . The method of  claim 3 , wherein the aqueous solution further comprises at least one agent.  
     
     
         10 . The method of  claim 9 , wherein the agent is a drug, protein, peptide, nucleic acid, saccharide, antigen, hapten, alginate, biodegradable polymer, polyethylene glycol or sugar.  
     
     
         11 . The method of  claim 3 , wherein the aqueous solution comprising fibrinogen, Factor XIII and thrombin is introduced into the oil within about 30 seconds after preparing the aqueous solution.  
     
     
         12 . The method of  claim 3 , wherein the oil in the oil emulsion is selected from the group consisting of corn oil, olive oil, soy oil, castor oil, mineral oil and combinations thereof.  
     
     
         13 . The method of  claim 12 , wherein the oil is corn oil.  
     
     
         14 . The method of  claim 3 , wherein the mixture in step (i) is mixed for about 3-9 hours.  
     
     
         15 . The method of  claim 3 , wherein the fibrin nanoparticles having a mean diameter of 200-2000 nm are isolated by filtration and sedimentation.  
     
     
         16 . The method of  claim 3 , wherein the fibrin nanoparticles isolated in step (iii) have a density of 1.32±0.2 g/mL.  
     
     
         17 . A method for preparing fibrin particles comprising the steps of: (i) mixing an aqueous solution comprising fibrinogen, thrombin and Factor XIII, without the addition of an exogenous chemical cross-linking agent, in a manner to obtain a fibrin clot; (ii) incubating the fibrin clot at ambient temperature for a period of time sufficient to obtain a cross-linked fibrin clot; (iii) mixing the cross-linked fibrin clot in oil at a temperature of 50-80° C. to obtain fibrin particles; (iv) homogenizing the fibrin particles to obtain fibrin particles of the desired size; and (v) isolating the fibrin particles of the desired size.  
     
     
         18 . The method of  claim 17 , wherein the aqueous solution comprising fibrinogen, thrombin and Factor XIII is prepared by contacting thrombin with purified fibrinogen containing endogenous Factor XIII.  
     
     
         19 . The method of  claim 17 , wherein the aqueous solution comprising fibrinogen, thrombin and Factor XIII is prepared by combining thrombin with a cryoprecipitate containing endogenous fibrinogen and endogenous Factor XIII.  
     
     
         20 . The method of  claim 17 , wherein the aqueous solution comprising fibrinogen, thrombin and Factor XIII is prepared by adding separate amounts of fibrinogen, Factor XIII and thrombin.  
     
     
         21 . The method of  claim 17 , wherein ratio of fibrinogen:thrombin:Factor XIII in the aqueous solution is 5-100 mg/mL:1-100 U/mL:1-50 U/mL.  
     
     
         22 . The method of  claim 17 , wherein ratio of fibrinogen:thrombin:Factor XIII in the aqueous solution is 20-40 mg/mL:5-10 U/mL:2-20 U/mL.  
     
     
         23 . The method of  claim 17 , wherein the fibrinogen is shaken before being mixed with the thrombin.  
     
     
         24 . The method of  claim 17 , wherein the aqueous solution comprising fibrinogen, thrombin and Factor XIII further comprises at least one agent.  
     
     
         25 . The method of  claim 24 , wherein the agent is a drug, protein, peptide, nucleic acid, saccharide, antigen, hapten, alginate, biodegradable polymer, polyethylene glycol or sugar.  
     
     
         26 . The method of  claim 17 , wherein the fibrin clot is incubated in step (ii) for about 45 minutes to about 2 hours.  
     
     
         27 . The method of  claim 17 , wherein the oil in step (iii) is selected from the group consisting of corn oil, olive oil, soy oil, castor oil, mineral oil and combinations thereof.  
     
     
         28 . The method of  claim 27 , wherein the oil is corn oil.  
     
     
         29 . The method of  claim 17 , wherein the cross-linked fibrin clot is mixed in oil in step (iii) for about 3-9 hours.  
     
     
         30 . The method of  claim 17 , wherein the fibrin particles are homogenized in step (iv) to obtain fibrin nanoparticles having a mean diameter of 200-2000 nm.  
     
     
         31 . The method of  claim 30 , wherein the fibrin nanoparticles having a mean diameter of 200-2000 nm are isolated by filtration and sedimentation.  
     
     
         32 . The method of  claim 31 , wherein the isolated fibrin nanoparticles have a density of 1.32±0.2 g/mL.  
     
     
         33 . The method of  claim 17 , wherein the fibrin particles are homogenized in step (iv) to obtain fibrin microbeads having a mean diameter of 30-300 μm.  
     
     
         34 . The method of  claim 33 , wherein the fibrin microbeads having a mean diameter of 30-300 μm are isolated by filtration and sedimentation.  
     
     
         35 . A method for preparing fibrin particles comprising the steps of: (i) mixing an aqueous solution comprising fibrinogen, thrombin and Factor XIII, without the addition of an exogenous chemical cross-linking agent, in a manner to obtain a fibrin clot; (ii) incubating the fibrin clot at ambient temperature for a period of time sufficient to obtain a cross-linked fibrin clot; (iii) heating the cross-linked fibrin clot at a temperature of 50-80° C. to obtain fibrin particles; (iv) homogenizing the fibrin particles to obtain fibrin particles of the desired size; and (v) isolating the fibrin particles of the desired size.  
     
     
         36 . The method of  claim 35 , wherein the aqueous solution comprising fibrinogen, thrombin and Factor XIII is prepared by contacting thrombin with purified fibrinogen containing endogenous Factor XIII.  
     
     
         37 . The method of  claim 35 , wherein the aqueous solution comprising fibrinogen, thrombin and Factor XIII is prepared by combining thrombin with a cryoprecipitate containing endogenous fibrinogen and endogenous Factor XIII.  
     
     
         38 . The method of  claim 35 , wherein the aqueous solution comprising fibrinogen, thrombin and Factor XIII is prepared by adding separate amounts of fibrinogen, Factor XIII and thrombin.  
     
     
         39 . The method of  claim 35 , wherein ratio of fibrinogen:thrombin:Factor XIII in the aqueous solution is 5-100 mg/mL:1-100 U/mL:1-50 U/mL.  
     
     
         40 . The method of  claim 35 , wherein ratio of fibrinogen:thrombin:Factor XIII in the aqueous solution is 20-40 mg/mL:5-10 U/mL:2-20 U/mL.  
     
     
         41 . The method of  claim 35 , wherein the aqueous solution comprising fibrinogen, thrombin and Factor XIII further comprises at least one agent.  
     
     
         42 . The method of  claim 41 , wherein the agent is a drug, protein, peptide, nucleic acid, saccharide, antigen, hapten, alginate, biodegradable polymer, polyethylene glycol or sugar.  
     
     
         43 . The method of  claim 35 , wherein the fibrin clot is incubated in step (ii) for about 45 minutes to about 2 hours.  
     
     
         44 . The method of  claim 35 , wherein the cross-linked fibrin clot is heated in step (iii) for about 3-9 hours.  
     
     
         45 . The method of  claim 35 , wherein the fibrin particles are homogenized in step (iv) to obtain fibrin nanoparticles having a mean diameter of 200-2000 nm.  
     
     
         46 . The method of  claim 45 , wherein the fibrin nanoparticles having a mean diameter of 200-2000 nm are isolated by filtration and sedimentation.  
     
     
         47 . The method of  claim 46 , wherein the isolated fibrin nanoparticles have a density of 1.32±0.2 g/mL.  
     
     
         48 . The method of  claim 35 , wherein the fibrin particles are homogenized in step (iv) to obtain fibrin microbeads having a mean diameter of 30-300 μm.  
     
     
         49 . The method of  claim 48 , wherein the fibrin microbeads having a mean diameter of 30-300 μm are isolated by filtration and sedimentation.  
     
     
         50 . A method for preparing fibrin particles comprising the steps of: (i) introducing an aqueous solution comprising fibrinogen, thrombin and Factor XIII, without the addition of an exogenous chemical cross-linking agent, into a spray dryer; (ii) spray drying the mixture to obtain spray dried fibrin particles; (iii) heating the spray dried particles at a temperature of 50-80° C. to remove water; and (iv) isolating fibrin particles of the desired size.  
     
     
         51 . The method of  claim 50 , wherein the aqueous solution comprising fibrinogen, thrombin and Factor XIII is prepared by contacting thrombin with purified fibrinogen containing endogenous Factor XIII.  
     
     
         52 . The method of  claim 50 , wherein the aqueous solution comprising fibrinogen, thrombin and Factor XIII is prepared by combining thrombin with a cryoprecipitate containing endogenous fibrinogen and endogenous Factor XIII.  
     
     
         53 . The method of  claim 50 , wherein the aqueous solution comprising fibrinogen, thrombin and Factor XIII is prepared by adding separate amounts of fibrinogen, Factor XIII and thrombin.  
     
     
         54 . The method of  claim 50 , wherein ratio of fibrinogen:thrombin:Factor XIII in the aqueous solution is 5-100 mg/mL:1-100 U/mL:1-50 U/mL.  
     
     
         55 . The method of  claim 50 , wherein ratio of fibrinogen:thrombin:Factor XIII in the aqueous solution is 20-40 mg/mL:5-10 U/mL:2-20 U/mL.  
     
     
         56 . The method of  claim 50 , wherein the aqueous solution further comprises at least one agent.  
     
     
         57 . The method of  claim 56 , wherein the agent is a drug, protein, peptide, nucleic acid, saccharide, antigen, hapten, alginate, biodegradable polymer, polyethylene glycol or sugar.  
     
     
         58 . The method of  claim 50 , wherein spray dryer has a chamber temperature of 150-230° C.  
     
     
         59 . The method of  claim 50 , wherein spray dryer has an outlet temperature of 85-120° C.  
     
     
         60 . The method of  claim 50 , wherein the fibrin particles isolated in step (iv) are fibrin nanoparticles having a mean diameter of 200-2000 nm.  
     
     
         61 . The method of  claim 60 , wherein the fibrin nanoparticles having a mean diameter of 200-2000 nm are isolated by filtration and sedimentation.  
     
     
         62 . The method of  claim 61 , wherein fibrin nanoparticles have a density of 1.32±0.2 g/mL.  
     
     
         63 . The method of  claim 50 , wherein the fibrin particles isolated in step (iv) are fibrin microbeads having a mean diameter of 30-300 μm.  
     
     
         64 . The method of  claim 63 , wherein the fibrin microbeads having a mean diameter of 30-300 μm are isolated by filtration and sedimentation.  
     
     
         65 . A composition comprising fibrin nanoparticles and an agent, wherein the agent is admixed with the fibrin nanoparticles.  
     
     
         66 . The composition of  claim 65 , wherein the agent is a drug, protein, peptide, nucleic acid, saccharide, antigen, hapten, alginate, biodegradable polymer, polyethylene glycol or sugar.  
     
     
         67 . A composition comprising fibrin particles and an agent, wherein the agent is coupled to the amine or carboxy moieties on the surface of the fibrin particles.  
     
     
         68 . The composition of  claim 67 , wherein fibrin particles are fibrin nanoparticles having a mean diameter of 200-2000 nm.  
     
     
         69 . The composition of  claim 67 , wherein the fibrin particles are fibrin microbeads having a mean diameter of 30-300 μm.  
     
     
         70 . The composition of  claim 67 , wherein the agent is a drug, protein, peptide, nucleic acid, saccharide, antigen or hapten.  
     
     
         71 . A method for coupling an agent to a fibrin particle comprising contacting the fibrin particle with the agent under conditions permitting the formation of a complex between the agent and the fibrin particle, and isolating the complex.  
     
     
         72 . The composition of  claim 71 , wherein the fibrin particle is a fibrin nanoparticle having a diameter of 200-2000 nm.  
     
     
         73 . The composition of  claim 71 , wherein the fibrin particle is a fibrin microbead having a mean diameter of 30-300 μm.  
     
     
         74 . The method of  claim 71 , wherein the agent is a drug, protein, peptide, nucleic acid, saccharide, antigen or hapten.  
     
     
         75 . The method of  claim 71 , wherein the agent is bound to the amine or carboxy moieties on the surface of the fibrin particle.  
     
     
         76 . The method of  claim 71 , wherein the agent is coupled to the fibrin particle via a linker.  
     
     
         77 . The method of  claim 76 , wherein the linker is bound to the amine or carboxy moieties on the surface of the fibrin particle.  
     
     
         78 . A method for introducing an agent into a cell comprising contacting the cell with a composition comprising fibrin nanoparticles and an agent, wherein the agent is admixed with the fibrin nanoparticles or coupled to the amine or carboxy moieties on the surface of the fibrin nanoparticles, so that the nanoparticles and the agent are introduced into the cell.  
     
     
         79 . The method of  claim 78 , wherein the agent is a drug, protein, peptide, nucleic acid, antigen or hapten.  
     
     
         80 . The method of  claim 78 , wherein the agent is admixed with the fibrin nanoparticles.  
     
     
         81 . The method of  claim 78 , wherein the agent is coupled to the amine or carboxy moieties on the surface of the fibrin nanoparticles.  
     
     
         82 . The method of  claim 78 , wherein the cells are selected from the group consisting of primary endothelial cells, smooth muscle cells, dendritic cells, fibroblasts, chondrocytes and osteoblasts, osteogenic bone marrow derived progenitors, 3T3 carcinoma cell line and mouse mammary carcinoma cell line.  
     
     
         83 . A method for isolating stem and/or progenitor cells from a biological sample comprising stem and/or progenitor cells, said method comprising the steps of: contacting the biological sample with fibrin particles so that the fibrin particles bind to the stem and/or progenitor cells present in the biological sample, and isolating the fibrin particles bound to the stem and/or progenitor cells from the biological sample.  
     
     
         84 . A composition comprising fibrin particles bound to stem and/or progenitor cells.