Bioconjugation of Calcium Phosphosilicate Nanoparticles For Selective Targeting of Cells in Vivo

Abstract

Non-aggregating resorbable calcium phosphosilicate nanoparticles (CPNPs) are bioconjugated to targeting molecules that are specific for particular cells. The CPNPs are stable particles at normal physiological pH. Chemotherapy and imaging agents may be integrally formed with the CPNPs so that they are compartmentalized within the CPNPs. In this manner, the agents are protected from interaction with the environment at normal physiological pH. However, once the CPNPs have been taken up, at intracellular pH, the CPNPs dissolve releasing the agent. Thus, chemotherapeutic or imaging agents are delivered to specific cells and permit the treatment and/or imaging of those cells. Use of the bioconjugated CPNPs both limits the amount of systemic exposure to the agent and delivers a higher concentration of the agent to the cell. The methods and principals of bioconjugating CPNPs are taught by examples of bioconjugation of targeting molecules for breast cancer, pancreatic cancer, and leukemia.

We claim: 
     
         1 . A resorbable non-agglomerated calcium phosphosilicate CPNP (CPNP) coupled to a targeting moiety specific to one or more biomolecules. 
     
     
         2 . The CPNP of  claim 1  further comprising a dye, a radioactive material, a magnetic material, a therapeutic agent or any combination thereof incorporated into the bulk of the CPNP. 
     
     
         3 . The CPNP of  claim 2  wherein the CPNP is coupled to a targeting moiety by a non-covalent bond. 
     
     
         4 . The CPNP of  claim 3  wherein the CPNP is coupled to the targeting moiety by an avidin-biotin linkage. 
     
     
         5 . The CPNP of  claim 4  of wherein the targeting moiety comprises an antibody, a peptide, a ligand, a receptor, or any combination thereof. 
     
     
         6 . The CPNP of  claim 5  wherein the antibody comprises an anti-CD71 antibody. 
     
     
         7 . The CPNP of  claim 5  wherein the peptide comprises a holotransferrin. 
     
     
         8 . The CPNP of  claim 5  wherein the peptide comprises a gastrin. 
     
     
         9 . The CPNP of  claim 8  wherein the gastrin comprises pentagastrin. 
     
     
         10 . The CPNP of  claim 2  wherein the CPNP is coupled to a targeting moiety by a covalent bond. 
     
     
         11 . The CPNP of  claim 10  wherein the CPNP is coupled to the targeting moiety by a polyethylene glycol-maleimide linkage. 
     
     
         12 . The CPNP of  claim 11  wherein the targeting moiety comprises an antibody, a polypeptide, a ligand, a receptor, or any combination thereof. 
     
     
         13 . The CPNP of  claim 11  wherein the polypeptide comprises a gastrin. 
     
     
         14 . The CPNP of  claim 13  wherein the gastrin comprises gastrin-10. 
     
     
         15 . A method of preparing a CPNP coupled to a targeting moiety comprising:
 a) reacting a calcium phosphosilicate CPNP having a carboxylate surface functionalization with a carbodiimide and avidin to yield an avidinylated calcium phosphosilicate CPNP; and   b) reacting a biotinylated targeting moiety with the avidinylated CPNP.   
     
     
         16 . The method of  claim 15  wherein the carboxylate surface functionalization comprises a citrate. 
     
     
         17 . The method of  claim 15  wherein the carbodiimide comprises 1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride. 
     
     
         18 . The method of  claim 15  of wherein the biotinylated targeting moiety comprises an antibody, a polypeptide, a ligand, a receptor, or any combination thereof. 
     
     
         19 . The method of  claim 18  wherein the biotinylated antibody comprises an anti-CD71 antibody. 
     
     
         20 . The method of  claim 18  wherein the biotinylated polypeptide comprises a holotransferrin. 
     
     
         21 . The method of  claim 18  wherein the polypeptide comprises a gastrin. 
     
     
         22 . The method of  claim 21  wherein the gastrin comprises pentagastrin. 
     
     
         23 . The method of  claim 15  further comprising incorporating into the CPNP a fluorescent dye, a radioactive material, a magnetic material, or any combination thereof. 
     
     
         24 . The method of  claim 23  wherein the dye comprises indocyanine green, cascade blue, Rhodamine WT, fluorescein, or any combination thereof. 
     
     
         25 . The method of  claim 15  further comprising incorporating into the CPNP a chemotherapeutic agent. 
     
     
         26 . A method of preparing a CPNP coupled to a targeting moiety comprising:
 a) reacting a CPNP comprising calcium phosphosilicate and citrate with a carbodiimide and a maleimide polyethylene glycol amine so as to give rise to a CPNP comprising a surface moiety having a maleimide terminal group; and   b) reacting a binding moiety having a terminal sulfhydryl group with the CPNP.   
     
     
         27 . The method of  claim 26  wherein the carbodiimide comprises ethyl-N-(3-dimethylaminopropyl)-N′ hydrochloride carbodiimide. 
     
     
         28 . The method of  claim 26  wherein the sulfydryl-terminated binding moiety comprises an antibody, a polypeptide, a ligand, a receptor, or any combination thereof. 
     
     
         29 . The method of  claim 34  wherein the sulfhydryl-terminated binding moiety comprises a polypeptide. 
     
     
         30 . The method of  claim 29  wherein the polypeptide comprises gastrin-10. 
     
     
         31 . The method of  claim 26  further comprising incorporating into the CPNP a fluorescent dye, a radioactive material, a magnetic material, or any combination thereof 
     
     
         32 . The method of  claim 31  wherein the dye comprises indocyanine green, cascade blue, Rhodamine WT, fluorescein, or any combination thereof. 
     
     
         33 . The method of  claim 26  further comprising incorporating into the CPNP a chemotherapeutic agent. 
     
     
         34 . A method of preparing a CPNP coupled to a targeting moiety comprising:
 a) activating citrate-functionalized CPNPs with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide;   b) reacting the CPNPs with sulfo-NHS (N-hydroxysuccinimide) to form a high-yield, semi-stable intermediate;   c) reacting PEG, with both amine and citrate functional groups, with the sulfo-NHS ester-containing CPSNPs;   d) repeating the process with the citrate-PEG functional terminals of the PEGylated CPSNPs to generate sulfo-NHS ester-containing PEGylated CPSNPs; and   e) reacting with targeted antibodies, at neutral pH, to form specifically targeted, PEGylated CPSNPs.   
     
     
         35 . The method of  claim 34  wherein the targeted antibody is anti-CD117. 
     
     
         36 . The method of  claim 34  wherein the targeted antibody is anti-CD96. 
     
     
         37 . A method of treating a disease by administering to a subject in need thereof a therapeutically effective amount of a composition comprising:
 a) bioconjugated CPNPs possessing targeting molecules complementary to one or more biomolecules present in a subject exhibiting a disease; and   b) a chemotherapeutic agent integral to the CPNPs.   
     
     
         38 . The method of  claim 37  wherein one or more of the targeting molecules comprises a polypeptide, an antibody, a ligand, a receptor, or any combination thereof bound to a polyethylene glycol-maleimide molecule. 
     
     
         39 . The method of  claim 37  wherein one or more of the CPNPs traverses the blood-brain barrier of the subject. 
     
     
         40 . The method of  claim 37  wherein one or more of the targeting molecules comprises a polypeptide, an antibody, a ligand, a receptor, or any combination thereof bound to an avidin-biotin complex. 
     
     
         41 . A method of assaying a subject for a disease by administering CPNPs comprising:
 a) a targeting molecule complementary to one or more biomolecules present in a subject exhibiting a disease state, and   b) a material that will indicate its location within the subject.   
     
     
         42 . The method of  claim 41  wherein the material that indicates its location within the subject is a fluorescent dye, a radioactive material, a magnetic material, or any combination thereof. 
     
     
         43 . The method of  claim 41  wherein the targeting molecule comprises a polypeptide, an antibody, a ligand, a receptor, or any combination thereof bound to a polyethylene glycol-maleimide molecule. 
     
     
         44 . The method of  claim 43  wherein one or more of the CPNPs traverses the blood-brain barrier of the subject. 
     
     
         45 . The method of  claim 41  wherein one or more of the targeting molecules comprises a polypeptide, an antibody, a ligand, a receptor, or any combination thereof, bound to an avidin-biotin complex.