Patent Publication Number: US-2010119610-A1

Title: Packaged pegylated gold nanoparticles

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority under 35 U.S.C. §120 to provisional application Ser. No. 61/113,812 filed Nov. 12, 2008, herein incorporated by reference in its entirety. 
    
    
     BACKGROUND OF THE INVENTION 
     Gold particles and particularly gold nanoparticles of spheroidal or rod shape are of significant interest for many uses such as surface enhanced Raman spectroscopy (SERS) and for thermal treatment of cancer tumors. Many other uses also exist, particularly for gold nanoparticles, and in particular hydrophilic polyethylene glycol coated gold particles, i.e., analytical techniques, bioassays, etc. 
     One particular problem that has existed for such particles is presentation of the packaged particle in a manner that provides polyethylene glycol nanoparticles (PGNP) that can be stored for long periods of time without adverse operability affects. As used herein adverse operability affects means that after storage the nanoparticles can be used for their intended purpose without sacrificing their utility. 
     In accordance with one of the objects of the invention as embodied and broadly described herein, the invention provides packaged stable products and compositions for shipping, storing and ready for use of shaped nanoparticles. The nanoparticles may be either spheres or rods in shape. 
     In another aspect the invention relates to packaged polyethylene glycol coated nanoparticles of gold (PGNP) of increased storage stability. That is to say they may be stored up to two weeks without sedimentation; they may be stored at room temperature as opposed to requiring refrigeration at 4° C.; and they may be stored without biological growth for up to two weeks. 
     In a further aspect the invention relates to a unique packaging system for PGNP particles whether shaped as spheres or rods. 
     Additional advantages of the invention will be set forth in part in the description which follows, and in part will be apparent from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and obtained by means of the elements in combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. 
     BRIEF SUMMARY OF THE INVENTION 
     Gold nanoparticles conjugated to polyethylene glycol and active binding molecules such as antibodies, proteins, lectins and DNA are suspended in a water vehicle at a concentration from 10 7  to 10 15  of gold conjugated nanoparticle, and then placed in a non-air permeable sealed container such as a centrifuge tube and then the centrifuge tube itself is sealed in a film package that is non-permeable to air. The package can be stored at temperatures up to 70° C. for two weeks and will successfully resist biological growth. Cold temperature storage is thus avoided. The stability also exists over a wide range of pH, provides stability in up to two molar salt solutions, and provides particles that do not sediment out for long periods of time. Finally the particles are operable as neutral, negative or positively charged particles. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a perspective view of the fully packaged hydrophilic polyethylene glycol coated gold nanoparticles. 
         FIG. 2  is a view of the PGNP particle filled centrifuge tube which is placed inside the polyethylene package of  FIG. 1 . 
         FIG. 3  is a cross section through the filled sealed centrifuge tube shown in  FIG. 2 . 
     
    
    
     DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT 
     The applicant has developed and optimized packaged nanoparticles which may be either nanospheres or nanorods, specifically for use in biological, preclinical and in vivo applications. Preferably the particles are nanorods coated with a dense layer of hydrophilic polyethylene glycol (PEG) polymers that shield the gold surface of the nanoparticle and give the particles ultra long circulation times in vivo. In comparison with other commercially available PEG nanoparticles, those of the present invention have longer circulation times, greater packaged stability and overall superior product performance in vivo. 
     Typically gold nanoparticles, even those covered with hydrophilic polyethylene glycol need to be stored at colder temperatures, i.e., 4° C.; and often after standing they must be resuspended using sonication or vortexing before they can be used. These packaged particles also have good centrifugation properties and sedimentation properties. For purposes of comparison, Table 1 below shows comparative properties of prior art packaged GNPS and comparative properties of the PGNP gold particles of the present invention. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Comparative Data 
               
            
           
           
               
               
               
            
               
                   
                 Adsorptive 
                   
               
               
                   
                 gnps (prior art) 
                 PGNPs of invention 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                 pH 
                 5 to 9 
                 3 to 11 
               
               
                 Temp 
                 Always refrig to 
                 Room temp storage up to 70° C. 
               
               
                   
                 4° C. 
                 w/o biological growth for 2 weeks 
               
               
                 Salt (saline) 
                 Up to 100 mM 
                 1-2 M 
               
               
                 Chemicals 
                 No solvents 
                 Most solvents 
               
               
                 Centrifugation 
                 2 times 
                 Up to 10 times 
               
               
                 Charge control 
                 None 
                 Can be made −, +, or neutral 
               
               
                 Sedimentation 
                 80 nm spheres 
                 Did not sediment after two weeks 
               
               
                   
                 sediment in 3 days 
               
               
                   
               
            
           
         
       
     
     As best seen in  FIG. 1 , the gold particles  10  are collected inside of a centrifuge tube  12  and sealed, typically in phosphate buffer solution having less than 0.0001% ascorbic acid. The particles are gold nanoparticles which may vary in size from 10 nm nanoparticles to 100 nm nanoparticles. They usually are placed in a water vehicle at a concentration of from about 10 7  to 10 15  gold particles. Preferably to 10 8  to 10 12  concentration. Gold particles may be spheres or rods, depending upon use. For particular advantages of nanorods see co-pending application, international publication WO/2006/065762-A2, the disclosure of which is incorporated herein by reference. 
     For the PGNP particles  10  packaged inside of the centrifuge tube  12  (which itself is sealed inside polyethylene package  14 ) specifications may be as follows: 
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Specifications 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 In batch size variation 
                 &lt;10% CV 
               
               
                   
                 Shape monodispersity 
                 95% nanorods 
               
               
                   
                 Surface charge (zeta) 
                 +0 mV typ 
               
               
                   
                 Axial size 
                 10 nm 
               
               
                   
                 Wavelength coverage 
                 700-850 nm 
               
               
                   
                 pH 
                 ~5 
               
               
                   
                   
               
            
           
         
       
     
     For use in in vivo heating of cancer tumors, 10 nm axial sized nanorods are used for wavelengths of 700 nm, 750 nm, 780 nm, 808 nm and 850 nm, since the 10 nm size offers the highest photo thermal conversion per gram. The packaged product can be available in highly concentrated solutions, for example 1 ml sizes, depending upon the size of the centrifuge tube utilized. Preferably the gold nanorods are shipped in PBS, with less than 0.0001% ascorbic acid, CTAB capping agent and unbound PEG. 
     In general, preferred PGNP particles are nanorods and may be made and packaged in accordance with the specifications shown in Table 3. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Data Sheet 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Part # 
                 Axial 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 LSPR 
               
               
                 Nanorod 
                 Di- 
                   
                 Peak 
                   
                 Peak 
                   
                   
                   
                   
                   
                   
                 LSPR 
                 SSPR 
                 Peak 
                 Line- 
               
               
                 (Axial 
                 am- 
                 Long 
                 LSPR 
                 OD 
                 SSPR 
                 OD 
                   
                 Wt 
                   
                   
                 Molar- 
                 Molar 
                 Molar 
                 LSPR 
                 width 
               
               
                 DiamPeak 
                 eter 
                 Size 
                 Wave 
                 LSPR 
                 Wave 
                 SSPR 
                   
                 conc. 
                   
                   
                 ity 
                 Ext. (M- 
                 Ext. (M- 
                 Accuracy 
                 80% 
               
               
                 LSPR) 
                 (nm) 
                 (nm) 
                 (nm) 
                 (AU) 
                 (nm) 
                 (AU) 
                 NPS/ml 
                 (μgml) 
                 Wt % 
                 PPM 
                 (pM) 
                 (cm−1) 
                 (cm−1) 
                 (nm) 
                 (nm) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 30-PM-850 
                 10 
                 45 
                 850 
                 50.0 
                 512 
                 12.50 
                 2.6E+13 
                 1786.7 
                 0.1786% 
                 1786 
                 43699 
                 1.14E+09 
                 2.86E+08 
                 +/−10 
                 100 
               
               
                 30-PM-808 
                 10 
                 41 
                 808 
                 50.0 
                 512 
                 12.50 
                 2.9E+13 
                 1805.6 
                 0.1806% 
                 1806 
                 48943 
                 1.02E+08 
                 2.55E+08 
                 +/−10 
                 75 
               
               
                 30-PM-780 
                 10 
                 38 
                 780 
                 50.0 
                 512 
                 12.50 
                 3.1E+13 
                 1745.8 
                 0.1746% 
                 1746 
                 50982 
                 9.81E+05 
                 2.45E+08 
                 +/−10 
                 65 
               
               
                 30-PM-750 
                 10 
                 35 
                 750 
                 50.0 
                 512 
                 12.50 
                 3.2E+13 
                 1670.7 
                 0.1671% 
                 1671 
                 53199 
                 9.40E+08 
                 2.35E+08 
                 +/−10 
                 50 
               
               
                 30-PM-700 
                 10 
                 29 
                 700 
                 50.0 
                 512 
                 12.50 
                 3.3E+13 
                 1483.6 
                 0.1484% 
                 1484 
                 55617 
                 8.99E+08 
                 3.25E+08 
                 +/−10 
                 40 
               
               
                   
               
               
                 LSPR = Longitudinal SPR peak 
               
               
                 SSPR = Axial SPR peak 
               
               
                 Shape monodispersity (% rods) &gt;95% 
               
               
                 Size variation +/−10% (both dimensions) 
               
               
                 Aspect ratio variation = Peak LSPR accuracy/96 
               
            
           
         
       
     
     The manner of treating the gold nanoparticles in order to conjugate them with polyethylene glycol is known, see Huff, Terry B., et al., “Controlling the Cellular Uptake of Gold Nanorods”,  Langmuir,  23:1596-1599 (2007). 
     Importantly, the packaged gold particles, whether nanorods or nanospheres, when prepared as herein described, and when packaged as herein described, have unusually advantageous properties in the sense of being capable of successful use, including in vivo use without adverse operability affects. That is to say, they resist adverse operability affects at pH&#39;s of from 3 to 11; they may be stored at temperatures up to 70° C. for 2 weeks; they are stable in one or more salt solutions; they have no adverse operability affects to particle charges and can be neutral/negative or positive; and there is no noticeable sedimentation in centrifuge tubes stored for up to two weeks after packaging; in fact they can be centrifuges up to ten times without significant sedimentation. For nanorods, the preferred particle size is 10 nm to 25 nm diameter on the short axis and on the long axis 20 nm to 80 nm. The spherical particles may range from 5 nm to 100 nm in diameter. 
     The package  14 , is preferably polyethylene film package, but other polyolefin film packages may also be used. 
     It will be apparent to those skilled in the art that various modifications and variations can be made in the description and the packaging herein without departing from the scope or spirit of the invention. Other embodiments of the invention will also be apparent and it is intended that specification and examples be considered as exemplary only with the true scope and spirit of the invention being defined by the following claims.