Patent Publication Number: US-7723311-B2

Title: Delivery of bioactive substances to target cells

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority of U.S. Provisional Patent Application Ser. No. 60/479,381 filed Jun. 18, 2003. 
    
    
     STATEMENT OF GOVERNMENT INTEREST 
     This invention was partially funded by the Government under a grant from Naval Medical Center San Diego (NMCSD) under contract NCRADA-NMCSD-03-110. The Government has certain rights to portions of the invention. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates generally to the delivery of bioactive substances to target cells within a body, and more particularly, to the delivery of genetic material to the inner ear sensory cells of the inner ear using superparamagnetic nanoparticles. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to a method of introducing a bioactive substance into a target cell within a body. The bioactive substance is associated with a superparamagnetic nanoparticle. The method comprises introducing the bioactive substance and the nanoparticle into the body and moving the bioactive substance and the nanoparticle into the target cell using a controllable external magnetic field. The controllable external magnetic field is adapted to move the nanosphere in three dimensions. 
     The present invention further includes a method for introducing a bioactive substance into a target cell within a body wherein the bioactive substance is supported within a nanosphere. The nanosphere comprises at least one superparamagnetic nanoparticle and an outer bioerodable shell. The outer bioerodable shell supports the nanoparticle and the bioactive substance. The method comprises introducing the nanosphere into the body and moving the nanosphere into the target cell using a controllable external magnetic field. The controllable external magnetic field is adapted to move the nanosphere within the body in three dimensions. 
     Still yet, the present invention includes a system for introducing a bioactive substance into a target cell within a body. The system comprises a superparamagnetic nanoparticle, a biocompatible shell covering the nanoparticle and a magnetic field generator. The biocompatible shell is adapted to bond the bioactive substance with the nanoparticle. The magnetic field generator is adapted to move the nanoparticle to the target cell in three dimensions. 
     Further still, the present invention includes a method for introducing a bioactive substance into a target cell within a body wherein the bioactive substance is supported within a nanosphere. The nanosphere comprises a superparamagnetic nanoparticle and a bioerodable matrix. The bioerodable matrix supports the nanoparticle and the bioactive substance. The method comprises introducing the nanosphere into the body and moving the nanosphere into the target cell. The nanosphere is moved into the target cell using a controllable magnetic field adapted to move the nanosphere within the body in three dimensions. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  is a diagrammatic illustration of the present invention showing the use of a magnetic field to move superparamagnetic nanoparticles and their associated bioactive substance through a non-target cell and into a target cell.  FIG. 1  further shows the release of the bioactive substance from the nanoparticle inside the target cell. 
         FIG. 2  is a diagrammatic illustration of a nanoparticle having a biocompatible shell comprised of silica. The nanoparticle is shown bound to a bioactive substance via a covalent bond. 
         FIG. 3  is a diagrammatic representation of nanosphere delivery system constructed in accordance with the present invention. The nanosphere of  FIG. 3  comprises a plurality of nanoparticles each having a biocompatible shell. The nanoparticles are encapsulated within an outer biocompatible shell. The nanosphere is shown having a bioactive substance comprising a genetic material bonded to the outer biocompatible shell. 
         FIG. 4  is a diagrammatic representation of an alternative embodiment of a nanosphere constructed in accordance with the present invention. The nanosphere of  FIG. 4  comprises a plurality of silica coated nanoparticles bonded to a bioerodable polymer. The bioerodable polymer is shown supporting a genetic material. 
         FIG. 5  is an illustration of a human ear showing the movement of nanospheres through the round window membrane and into the inner ear. The magnetic field generator is shown in a plurality of positions to illustrate guided movement of the nanospheres by moving the magnetic field generator. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Delivery of bioactive substances, such as drugs or genetic material, to specific cells allows for the treatment of diseases and conditions that affect the human body. Several methods and systems have been developed to accomplish delivery of the bioactive substance. However, there remains an ongoing need for improved delivery methods and systems. 
     Targeted delivery of bioactive substances using nanospheres and/or nanoparticles to a specific site within a body provides advantages over systemic or oral administration of the bioactive substance to the body. For example, effective doses of bioactive substance may be delivered at varying doses to a desired target cell without exposing the entire body to adverse conditions or side effects. Further, the present method and system allows for the delivery of bioactive substance into sensitive or remote areas of the body in a non-invasive manner using an externally controlled magnetic field adapted to move the nanoparticle in three dimensions. 
     Viral agents have been used for targeted delivery of genetic material to specific cells within the body. A viral agent that has an affinity for the target cells is chosen to transport the genetic material to the target cells. However, the use of viruses to transport genetic material to specific cells presents difficulties such as infection of the host body, mutation of the virus, and incitement of harmful immunogenic reactions. Additionally, viruses are of such a size that there use may cause damaging trauma to the body by requiring invasive procedures. The present invention is useful in that it minimizes trauma to the body and can use non-immunogenic substances. 
     Turning now to the drawings in general and  FIG. 1  in particular, there is shown therein a system for introducing a bioactive substance  10  into a target cell  12  disposed within a body  14 . The bioactive substance  10  is shown bonded to a superparamagnetic nanoparticle  16 . The nanoparticle  16  may be covered by a biocompatible shell  18  ( FIG. 2 ) that is adapted to bond the bioactive substance  10  to the nanoparticle  16 . A magnetic field generator  20  is positioned outside the body  14  to move the nanoparticles  16  in three dimensions and into the target cell  12 . 
     To move the nanoparticle  16  and the bioactive substance  10  into the target cell  12  the magnetic field generator  20  generates a gradient, represented by arrows  22 , which attracts the nanoparticle to the magnetic field generator and into the target cell. The use of a magnetic field gradient  22  facilitates internalization of the nanoparticle  16  and bioactive substance  10  by the target cell  12 . Facilitating uptake of the nanoparticle  16  and bioactive substance  10  using the magnetic field generator may prevent premature release of the bioactive substance from the nanoparticle. Once the nanoparticle  16  and bioactive substance  10  are moved into the target cell  12 , the bond between them is broken and the bioactive substance may be released. 
     The magnetic field generator  20  may comprise a plurality of magnets (not shown) that are arranged such that a magnetic field is generated, within which numerous gradients  22  may be created to three-dimensionally direct the nanoparticles  16  to the target cell  12 . An alternative magnetic field generator may comprise an electromagnetic field generating coil that is movable in three dimensions and adapted to create a gradient  22  that moves the nanoparticle  16  through a non-target cell  24  and into the target cell  12 . It will be appreciated that the electromagnetic field generating coil may be moved by any means that permits three-dimensional movement of the nanoparticle  16  through the body  14 . In a preferred embodiment the electromagnetic field generating coil may be supported on the end of a robotic arm (not shown) that is programmed to move around the body  14  so that the nanoparticle  16  is directed in three dimensions to the target cell  12 . 
     Turning now to  FIG. 2 , there is shown therein the nanoparticle  16  and bioactive substance  10  of  FIG. 1  covalently bonded to one another. The nanoparticle  16  may be comprised of a ferrite such as magnetite and is preferably superparamagnetic. Because the nanoparticles  16  are superparamagnetic, the nanoparticles will only be attracted to the strongest side of the magnetic field gradient  22  and will not be attracted by other or similar nanoparticles when in a magnetic field. Thus, particle to particle interactions resulting in aggregation or other undesirable effects are minimized. Once the magnetic field is removed, the nanoparticles  16  lose their magnetic remanence. 
     The nanoparticle  16  of  FIG. 2  is shown encapsulated in a biocompatible shell  18 . In a preferred embodiment of the present invention the biocompatible shell  18  may comprise silica (SiO 2 ) or titania (TiO 2 ). Encapsulation of the nanoparticle  16  in the biocompatible shell  18  hermetically seals the nanoparticle to help prevent corrosion of the nanoparticle and provides a surface charge to promote suspension of the nanoparticle in solution to facilitate uptake of the nanoparticle by non-target  24  and target cells  12 . The biocompatible shell  18  also provides a substrate for the attachment of amines  26  that can serve as linkers to other molecules. The biocompatible shell  18  of  FIG. 2  is shown to provide a covalent bond  30  such as a Sulfhydryl bond between the bioactive substance  10  and the nanoparticle  16 . 
     Turning now to  FIG. 3 , there is shown therein a diagrammatic representation of a nanosphere  32  prepared using the methods and systems described in co-pending U.S. patent application Ser. No. 10/724,563, the contents of which are incorporated herein by reference. The nanosphere  32  of  FIG. 3  comprises a plurality of superparamagnetic nanoparticles  16  supported within the nanosphere by an erodable polymer matrix (not shown). Each nanoparticle  16  may be encapsulated within the previously described biocompatible silica shell  18 . The nanosphere  32  has an outer shell  34  that may be adapted to support the bioactive substance  10 . The nanosphere generally has a diameter of less than 300 nanometers, and more preferably a diameter of 100 nanometers or less. 
     The nanoparticles  16  may be arranged within the outer shell  34  such that they have uniformly aligned magnetic moments  36 . Uniform alignment of the nanoparticles&#39; magnetic moments  36  increases the magnetic susceptibility of the nanosphere  32  thus providing more efficient transport of the nanosphere and the bioactive substance  10  through the body  14  and into the target cell  12 . 
     The outer shell  34  generally encapsulates the nanoparticle  16  and provides a support mechanism for the bioactive substance  10  so that it may be transported with the nanoparticles to the target cell  12 . In one embodiment the outer shell  34  may comprise a bioerodable polymer that is adapted to release an encapsulated bioactive substance  38 . In this embodiment, the outer bioerodable shell  34  may comprise any erodable synthetic or natural polymer that is biocompatible. Polylactides, polyglycolides and collagen have been found to be acceptable for use as the outer bioerodable shell  34  of the nanosphere  32 . 
     If the outer shell  34  comprises a bioerodable polymer, the nanosphere  32  may form a reservoir  40  that encapsulates the bioactive substance  38  and the nanoparticles  16  within the nanosphere. As the outer shell  34  is dissolved, the bioactive substance  38  is released from the nanosphere  32  and dispersed into the cytoplasm (not shown) of the target cell  12 . The inclusion of the erodable polymer matrix further aids in regulating release of the bioactive substance  38 . 
     Continuing with  FIG. 3 , the bioerodable polymer matrix may be used to entrap the bioactive substance  38  within the outer bioerodable shell  38 . As the outer bioerodable shell  34  and the erodable matrix dissolve the bioactive substance  38  is released at a rate dependent upon dissolution of the outer shell and the matrix. Thus, it is preferable that the erodable polymer matrix is non-toxic and capable of being consumed, metabolized or expelled by the target cell  12 . ( FIG. 1 .) An example of such an erodable polymer matrix is collagen. A tightly cross-linked matrix will exhibit a slow release rate providing low doses of bioactive substance  38  over longer periods of time. When no bioerodable matrix is present rapid release of the bioactive substance  38  can be expected. 
     Continuing with  FIG. 3 , the bioactive substance  10  may alternatively be supported on the outer shell  34 . In such cases the outer shell  34  may be formed from either the bioerodable polymer or a biostable polymer. By way of example, the outer shell  34  of the nanosphere  32  may comprise a silica matrix. The silica matrix may have a plurality of amine groups  26  attached to the outer surface  42  of the outer shell  34  that functionalize the nanosphere  32 . These amine groups  26  give the outer surface  42  of the shell  34  a net positive charge. A positively charged outer shell  34  has an affinity for bioactive substance  10  comprising genetic material that has a generally negative net charge. 
     It will be appreciated that the bioactive substance  10  or  38  may itself form the outer shell by attaching the bioactive substance directly to the silica coated nanoparticles  16  or alternatively to the previously described silica matrix. 
     The outer shell  34  of the nanosphere  32  may have a cell adhesion factor (not shown) supported on the outer surface  42  of the shell  34 . The use of cell adhesion factors enhances endocytosis of the bioactive substance  10  or  38  supported by the nanosphere  32  by the target cell  12 . ( FIG. 1 .) Thus, the cell adhesion factor may comprise a protein having an affinity for a predetermined type of cell. It will be appreciated that a wide array of cell adhesion factors may be used with nanospheres  32  of the present invention without departing from the spirit of the invention. 
     Turning now to  FIG. 4 , there is shown therein an alternative nanosphere  44  of the present invention that may be used to deliver the bioactive substance  10  to the target cell  12 . The nanosphere  44  of  FIG. 4  comprises a plurality of superparamagnetic nanoparticles  16  supported by a bioerodable polymer matrix  46 . In the present embodiment, the nanoparticles  16  are shown with the biocompatible shell  18 . The nanoparticles  16  may be supported by the bioerodable polymer matrix  46  so that they have substantially aligned magnetic moments  36 . The bioactive substance  10  is likewise supported by the bioerodable polymer matrix  46  so that the amount of bioactive substance  10  released from the nanosphere  44  and into the target cell  12  may be controlled over time. 
     Turning now to  FIG. 5 , there is shown therein an illustration of a human ear  48 . The ear  48  shown in  FIG. 5  comprises an outer ear  50 , a middle ear  52  and an inner ear  54 . The outer ear  50  has an ear canal  56  that is closed at one end by a tympanic membrane  58 , or eardrum. The middle ear comprises an ossicular chain that normally connects the ear drum  58  to a cochlea  60 . The ossicular chain includes a malleus  62 , an incus  64 , and a stapes  66 . A properly functioning ossicular chain transmits and amplifies sound vibrations from the ear drum  58  through the malleus  62 , incus  64  and stapes  66  to vibrate an oval window (not shown) of the inner ear  54 . Vibration of the oval window is transmitted to the fluid of the inner ear to cause movement of ear sensory cells within the cochlea  60  of the inner ear  54 . Electrical impulses from the ear sensory cells are sent from the cochlea  60  along an auditory nerve  68  to the brain of the mammal where the signals are processed for hearing. 
     Damage to the ear sensory cells, or hair cells, of the cochlea  60  is the leading cause of sensorineural hearing loss. Congenital conditions and/or exposure to injurious levels of noise may be the cause of damage to the hair cells. After the hair cells are initially damaged, a number of inner ear cell death programs are activated that result in eventual hair cell death and permanent hearing loss. However, the supporting cells may remain alive with the capacity to regenerate hair cells and restore hearing when triggered by the appropriate bioactive substance  10 . 
       FIG. 5  illustrates a method of moving magnetically responsive nanospheres  32  or  44 , as described herein, into the inner ear  54  for regeneration or repair of hair cells. Nanosphere  32  is used herein for illustration purposes, it will be appreciated that nanospheres having different constructions and configurations and individual nanoparticles  16  as previously described herein may be used to treat the target cells without departing from the spirit of the invention. The nanospheres  32  are placed near the round window membrane  70  of the inner ear  54  and pulled through the round window membrane using the gradient  22  generated by the magnetic field generator  20  in position A. Once inside the cochlea  60 , the nanospheres  32  are moved is three dimensions through the perilymph to hair cell supporting cells using the external magnetic field generator  20 . The diagrammatic magnetic field generator  20  is shown, in  FIG. 5 , in an alternative position B to facilitate movement of the nanosphere  32  through the basal turn  72  of the cochlea  60 . 
     When the nanosphere reaches the hair cell supporting cell, the magnetic field generator  20  may be moved to an alternative position to facilitate magnetofection of the nanosphere into the cell. Once inside the hair cell supporting cell, the bioactive substance  10  is released into the cytoplasm of the target cell to begin repair or regeneration of the hair cells. The bioactive substance  10  released into the hair cells may comprise a genetic material such as the Hath-1 gene. The Hath-1 gene has been shown to stimulate regeneration of hair cells in mammals. See, “Robust Generation of New Hair Cells in the Mature Mammalian Inner Ear by Adenovirus Expression of Hath-1,” J. Shou, J. L. Zheng, W. Q. Gao, Molecular and Cellular Neuroscience 2003; 23:169-170, the contents of which are incorporated herein by reference. 
     The present invention also comprises a method for introducing a bioactive substance  10  into a target cell  12  within a body  14 . The bioactive substance  10  is generally associated with a superparamagnetic nanoparticle  16 . The bioactive substance  10  is introduced into the target cell  12  by introducing the bioactive substance and the nanoparticle  16  into the body  14  and moving the bioactive substance into the target cell. The bioactive substance  10  is moved into the target cell  12  using an externally controlled magnetic field that is adapted to move the nanoparticle  16  and bioactive substance through the body  14  and any non-target cells  24 . Movement of the nanoparticle  16  may comprise generating a gradient  22  in the external magnetic field. Preferably one of the nanoparticles  16  or nanospheres  32  or  44  as described herein may be used for this purpose. 
     In accordance with the method of present invention, the bioactive substance  10  may comprise genetic materials, such as DNA, RNA, plasmids, oligonucleotides or proteins, which are bonded to the biocompatible silica shell  18  that covers the nanoparticle  16 . The bond between the genetic material  10  and the silica shell  18  is adapted to release the genetic material after the nanoparticle  16  and genetic material are pulled into the target cell  12 . 
     In an exemplary application of the present method, the body  14  may comprise a mammal having a target cell  12  disposed within the cochlea  60  of the mammal&#39;s ear  50 . Thus, the externally controlled magnetic field may be used to move the genetic material  10  and nanoparticle  16  into the cochlea  16 , then to disperse the genetic material throughout the cochlea and across the cellular membrane (not shown) of the ear sensory cells. Once inside the target ear sensory cell  12 , the genetic material may be released from the nanoparticle  16  or nanosphere  32 . The genetic material  10  may then transfect the ear sensory cell or the supporting cell to cause repair or regeneration of the cells. 
     EXAMPLE PROCEDURE 
     Superparamagnetic nanoparticles having a silica shell were synthesized using the modified Massart procedures described in co-pending U.S. patent application Ser. No. 10/724,563. The nanoparticles were made of magnetite (Fe3O4) and synthesized to have a diameter of less than 30-50 nanometers. A two Molar iron (III) sulfate heptahydrate solution was prepared in two (2) Molar HCl and combined with one Molar iron (III) chloride hexahydrate aqueous solution. The solutions were mixed and washed in a 0.7 Molar ammonium hydroxide solution and rapidly stirred. The resulting precipitate was stirred for thirty (30) minutes then collected using a magnet. After multiple washes, the precipitate was re-suspended in 0.7 Molar ammonium hydroxide and peptized by the addition of one (1) Molar tetramethylammonium hydroxide aliquots. The volume of the resulting suspension was then taken to 250 ml for processing to add the silica shell to the nanoparticles. 
     To confirm the iron oxide phase and size of the magnetic nanoparticle, several uncoated magnetite particles were characterized using X-ray diffraction (XRD). XRD analysis revealed the presence of magnetite particles having an average diameter of ten (10) nanometers. The diameter of the magnetite particles was confirmed using Transmission Electron Microscopy (TEM). Further observation of the uncoated magnetite particles using High Resolution Transmission Electron Microscopy further established the existence of magnetite particles. 
     Encapsulation of the nanoparticle with silica provides an anionic surface charge that promotes endocytosis as well as a substrate for attachment of amines adapted to link the bioactive substance to the nanoparticle. The suspension of magnetite nanoparticles was stirred and a 4 ml aliquot was taken up to 100 ml with distilled water. A solution of 0.54% sodium silicate was prepared at a pH of 10.5, and 4 ml of the sodium silicate was added to the magnetite nanoparticle suspension. The pH of the resulting suspension was adjusted to 10.0 and stirred for an extended period of time. After settling for several hours, the silica-coated nanoparticles were removed from the excess silica using a magnet to pull the particles out of the solution and by washing the precipitate several times with distilled water. 
     Several of the silica-coated nanoparticles were analyzed using TEM to determine the size and structure of the nanoparticles produced in the above procedure. Analysis of the coated nanoparticles revealed an average diameter of approximately sixteen (16) nanometers with a standard deviation of 2.3 nanometers. The presence of the silica shell and iron oxide core was confirmed by energy-dispersive X-ray spectrometry (“EDS”). 
     Silica-coated nanoparticles were then functionalized by the addition of amine groups to the surface of the silica shell. The nanoparticles were treated with 3-aminopropyl trimethoxy silane and a 1 ml aliquot of the resulting suspension was brought to a volume of 5 ml with distilled water. Additional 3-aminopropyl trimethoxy silane was added to the suspension to bring the final concentration to five percent (5%). The reaction system was stirred and the resulting nanoparticles were washed and collected. A Kaiser assay was performed on several of the functionalized nanoparticles to confirm the presence of amine groups on the surface of the silica-coated nanoparticles. 
     Fluorescein isothiocyanate (FITC) was used to label the nanoparticle for subsequent location of the nanoparticle using confocal microscopy. The particles were conjugated with FITC using standard protocols to attach the FITC to the amine functional groups. 
     Guinea pigs were anesthetized and positioned such that an experimental ear was facing upward and parallel to the operating table. A retro-articular incision was made to expose the temporal bone over the middle ear cavity. The middle ear space was opened using an otological surgical drill system (MicroCraft™, Xomed Inc., Jacksonville, Fla.) to expose the ossicular chain of the subjects. 
     The silica-coated magnetic nanoparticles were suspended in saline at a pH of 7.4 and sonicated for several minutes. Sonication was performed to disperse the nanoparticles before placement onto the ossicular epithelium. A volume of 50-75 microliters of the nanoparticle suspension was applied to the target cells in 25 microliter doses. The operative site was closed and the subjects recovered during application of an external magnetic field to the their heads. 
     An externally vectored magnetic force was applied to the heads of the experimental animals using an external magnet so that the nanoparticles were pulled downward into the epithelia of the incus and tympanic membrane. The magnet created a magnetic field of approximately 0.35 Tesla at one inch from the experimental incus and tympanic membrane. Each subject was exposed to the external magnetic field for 20 to 30 minutes and subsequently monitored for survival for several days. 
     Eight to fifteen days after surgery the subjects were anesthetized and euthanized. The experimental incus and tympanic membrane were dissected and prepared for observation. Confocal laser and epifluorescence microscopy were used to confirm the delivery of FITC-labeled nanoparticles into the epithelia of the incus and tympanic membrane. Florescence within the target cells of the incus and the tympanic membrane confirmed that the FITC-labeled nanoparticles had been internalized by the target epithelial cells of the incus and tympanic membrane. Control specimens, not subjected to the external magnetic field, showed reduced intracellular fluorescence showing that the external magnetic field facilitated internalization of the FITC-labeled nanoparticles by the target cells. 
     Various modifications can be made in the design and operation of the present invention without departing from the spirit thereof. Thus, while the principal preferred construction and modes of operation of the invention have been explained in what is now considered to represent its best embodiments, which have been illustrated and described, it should be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically illustrated and described.