JP2009286808A - Composition and method for targeted delivery of biologically-active factors - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition and method for targeted delivery in vitro and in vivo of a therapeutic agent. <P>SOLUTION: The composition and method are provided for targeted delivery of biologically-active factors such as a cytokinin, growth factor, chemotherapeutics, nucleic acid and curative agent. The composition can be used to deal with disorder and pathology in organisms. The composition and method are for the enhancement of immunological response in humans or animals. The enhancement optionally produces stimulation or suppression of the immunological response. Other compositions and methods include a composition and method used for the decrease of toxicity of vaccines or drugs. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

(Related application)
This application is a continuation-in-part of US patent application Ser. No. 08 / 966,940 (filed Nov. 11, 1997) and is entitled “Methods and Compositions for Targeted Therapeutic Delivery”, US Provisional Application No. 60 / 075,811 (filed February 24, 1998); US provisional application number 60 / 086,696 (filed May 26, 1998); and US provisional application (numbers not yet assigned, November 1998) Claims priority of 6th application).

(Technical field)
The present invention relates to compositions and methods for targeted delivery of biologically active factors (eg, cytokines, growth factors, chemotherapeutic agents, nucleic acids, and therapeutic agents). Furthermore, the present invention relates to methods and compositions for enhancing immune responses in humans or animals. Such enhancement can result in stimulation or suppression of the immune response. Other compositions and methods of the invention include compositions and methods used for reducing vaccine and drug toxicity.

(Background of the Invention)
The introduction of therapeutic agents into specific target cells has long been a challenge for scientists. The challenge of specific targeting of therapeutic agents has long been a problem of biological treatment. The challenge is to provide a sufficient amount of the therapeutic agent to the target cells of the organism without providing too much exposure of the rest of the organism to the therapeutic agent. Such challenges are seen in drug delivery. Therapeutic agent formulations take advantage of chemical differences in the active agent, such as hydrophobic or hydrophilic, that target the active agent to the wild. In addition, size considerations such as the ability to cross the blood brain barrier limit the targeting of therapeutic agents.

  One method that has been used with limited success is to target cells bearing a specific receptor and provide antibodies to that receptor that act as carriers for the therapeutic agent. The therapeutic agent can be an agent that is cytotoxic or the therapeutic agent can be a radioactive moiety that causes cell death. The problems inherent in this technology are the isolation of a specific receptor, the production of antibodies that have selective activity against that receptor, and no cross-reactivity with other similar epitopes, and the therapeutic agent against the antibody. Radiolabel or attachment. The problems associated with such limited therapeutic delivery are that the therapeutic agent can never be released inside the targeted cell, and that the therapeutic agent is not releasably bound to the antibody. Thus, once the therapeutic agent is delivered to the site, it is not fully active or cannot have any activity.

  Another example of targeted delivery with limited success is the specific targeting of selected gene sequences to cells. Many techniques have been attempted for inserting genes into cells. Such techniques include sedimentation techniques, viral techniques, direct insertion using micropipettes and gene “guns”, and most crudely exposure of nucleic acids to cells. A widely used precipitation technique involves calcium phosphate and is used as a coprecipitate with DNA to form insoluble particles. The purpose is that at least some of these particles become internalized into the host cell by generalized cellular endocytosis. This results in the expression of a new or exogenous gene. This technique has low efficiency for putting exogenous genes into cells, where gene translation is obtained. Gene internalization is nonspecific with respect to which cells are transfected. This is because all exposed cells can internalize exogenous genes and there is no dependence on any particular recognition site for endocytosis. Although this technique is widely used in vitro, the use of this technique in vivo is not contemplated due to the lack of specificity of target cell selection and low uptake by highly differentiated cells. Furthermore, the use of this technique in vivo is limited by the insoluble nature of the precipitated nucleic acid.

  Another similar technique involves the use of DEAE-Dextran to transfect cells in vitro. DEAE-Dextran is detrimental to cells and results in nonspecific insertion of nucleic acids into cells. This method is not wise in vivo.

  Other techniques for transfecting cells or for providing exogenous gene entry into cells are also limited. The use of viruses as vectors has several applicability for in vitro and in vivo introduction of exogenous genes into cells. There is always the danger that the presence of viral proteins will produce undesirable effects in in vivo use. In addition, viral vectors can be limited in terms of the size of exogenous genetic material that can be delivered to cells.

  Exogenous gene delivery has also been used with liposomes that have captured nucleic acids. Liposomes are capsules surrounded by membranes that can be filled with various substances, including nucleic acids. Liposome delivery does not provide constant delivery to cells due to irregular packing of liposomes. Furthermore, liposomes cannot be targeted to specific cell types. Liposomes suffer from disruption problems and thus leakage of nucleic acids at undesirable sites through in vivo or in vitro systems.

  A powerful technique for inserting exogenous nucleic acid involves piercing the cell membrane with a micropipette or gene gun and inserting exogenous DNA into the cell. These techniques work well for some procedures, but are not widely applicable. These are highly labor intensive and require highly skilled manipulation of recipient cells. These are not techniques that are single procedures that work well in vivo. There is no specificity for a particular cell to receive the nucleic acid across the cell selection or cells in the target zone. Electroporation using electrical methods that alter the cell membrane has been successful in vitro for inserting genes into cells. Again, this technique cannot be used for in vivo applications.

  There have been several attempts at targeted delivery of DNA for specific cells that depend on the presence of receptors for glycoproteins. This delivery system used a polycation (eg, polylysine) that was non-covalently bound to DNA and also covalently bound to a ligand. Such use of covalent attachment of the polycation to the ligand does not allow degradation of the delivery system once the cell internalization mechanism has begun. This large complexed delivery system is covalently linked together and is very different from the way nucleic acids are found in cells in nature.

  Targeted delivery of specific factors to specific cells is important in the selective activation or control of the immune system. At present, there are only crude technologies for immunosuppression or activation. The immune system is a complex interactive system of the body that includes a wide variety of components including cellular, cellular factors that interact with stimuli from cells, both inside and outside the body. In addition to its direct effects, the immune system response is also affected by other systems in the body, including the nervous system, respiratory system, circulatory system, and digestive system.

  One aspect of the immune system that is known to be better is the ability to respond to foreign antigens that are presented by invasion of organisms, cellular changes within the body, or derived from vaccination. . Some of the first type of cells that respond to such activation of the immune system are phagocytic cells and natural killer cells. Phagocytes include, among other cells, monocytes, macrophages, and polymorphonuclear neutrophils. These cells generally can bind to a foreign antigen, internalize it and destroy it. They also produce soluble molecules that mediate other immune responses such as inflammatory responses. Natural killer cells can recognize and destroy certain virally infected fetal and tumor cells. Other factors of the immune response include both complement pathways that can respond independently to foreign antigens or can act in concert with cells or antibodies.

  One aspect of the immune system that is important for vaccination is the specific response of the immune system to specific pathogens or foreign antigens. Part of the response involves the establishment of “memory” for that foreign antigen. Upon the second exposure, the memory function allows for a quicker and generally greater response to the foreign antigen. Lymphocytes, in conjunction with other cells and factors, play a major role in both memory function and response.

  Vaccines have long been used to stimulate an immune response that protects an organism. Aluminum compounds have been used to form water-insoluble antigenic substances for vaccination purposes. Metals are also used in capsule polysaccharide metal complex vaccines. Such use is limited to the use of conjugates for the prevention and treatment of bacterial diseases. Selected metals have also been used as components of stable adjuvant emulsion compositions. It is known in the art that aluminum in the form of a monostearate or in the form of a hydroxide salt of a fatty acid is an emulsifying agent or stabilizer for emulsions in vaccine compositions.

  Targeted delivery of specific therapeutic agents or biologically active agents for the treatment of disease or pathology is not currently available. Existing therapies for the treatment of diseases and pathological conditions, including but not limited to genetic diseases, congenital diseases, and acquired diseases include biologicals that have a wide range of effects throughout the body. Require administration of active agent doses: bacterial disease, viral disease, cancer, immunodeficiency disease, autoimmune disease, psychiatric disease, cardiovascular disease, reproductive dysfunction, somatic cell growth dysfunction Stress-related diseases, muscular dystrophy, osteoporosis, ophthalmic diseases, allergies, and transplant rejection. These therapeutic agents are not specifically targeted to affected organs due to the direct delivery of biologically active agents.

  For example, current treatments for cancer include the administration of chemotherapeutic agents and other biologically active factors such as cytokines and immune factors. Systemic administration of chemotherapeutic agents results in organ damage, loss of sensation such as taste and perception, and toxicity and adverse side effects such as hair loss. Many chemotherapeutic agents are designed to kill rapidly dividing cells that indiscriminately affect the hematopoietic and gastrointestinal systems, causing changes in blood and immune cells, causing vomiting, gastric tightness, and weight loss . Administration of immune factors (eg, cytokines) to the systemic system causes activation of unwanted immune responses and inhibition of other immune functions. Such therapeutic agents provide treatment for the condition but then occur with a wide array of side effects that must be treated. Furthermore, bolus administration of the drug may not be optimal due to rapid clearance.

  Other types of treatment of biological conditions, including diseases, can use nucleic acids. Examples of such therapeutic treatments include gene replacement, antisense gene therapy, triplex gene therapy, and ribozyme based therapy. However, in order to be successful, effective means for delivery of therapeutic agents to specific cell types and locations and across cell membranes, nuclear membranes, and other membranes are required.

  Altering gene activity can be accomplished in a number of ways. For example, oligonucleotides (eg, antisense compounds) that are complementary to a particular genetic message or viral sequence have been shown to have, for example, inhibitory effects on viruses. By creating an antisense nucleic acid composition that hybridizes to the targeted RNA message of a cell or virus, translation of the message into a protein can be interrupted or prevented. In this manner, gene activity can be modulated.

  The ability to inactivate specific genes provides great therapeutic benefits. In tissue culture, antisense oligonucleotides inhibited infection by herpes virus, influenza virus, and human immunodeficiency virus causing AIDS. It may also be possible to target antisense oligonucleotides to mutant oncogenes. Antisense technology also retains the ability to regulate growth and development. However, for gene therapy to work, antisense therapeutic compounds must be delivered to the targeted site.

  Another type of gene therapy uses sense nucleic acids to modify gene activity. A defective gene is replaced or supplemented by administration of a nucleic acid that is less susceptible to the defect. For example, an administered normal nucleic acid can be a DNA molecule that is inserted into a chromosome or can be present in extracellular DNA and produce functional RNA, which in turn yields the desired gene product. . In this manner, gene defects and defects in the production of gene products can be corrected.

  Still further gene therapy has the ability to increase the normal gene complement of cells. For example, a gene therapy technique known as intracellular immunization allows the presence of the desired gene product in the cell. The desired gene product is then expressed on the surface of the cell, recognized by the body as foreign, and cells expressing the gene product are eliminated by the body's own immune system. However, this approach is currently unsuitable due to the lack of an effective gene delivery system that facilitates targeted delivery of such therapeutic agents to specific sites.

  Gene therapy can also be used as a method of drug delivery in vivo. For example, if a gene encoding a therapeutic compound can be delivered to endothelial cells, the gene product has facilitated access to the bloodstream. Currently, one method for gene delivery into a cell is to remove the cell from the body, expose the cell to nucleic acid ex vivo, and then reintroduce the cell into the body. Alternatively, gene therapy has been achieved in vivo by injection of naked DNA, DNA-containing liposomes, and viral or bacterial DNA-containing vectors. Retroviral vectors can be used to deliver genes ex vivo to isolated cells, which are then returned to the patient by infusion. However, retroviral vectors have several disadvantages: they can deliver genes only to dividing cells, random integration of genes to be delivered, unwanted genetic modifications Potentially causing and possibly returning to an infectious wild type retrovirus form.

  Triplex DNA technology is another form of gene therapy that utilizes oligonucleotides and compounds that specifically bind to specific regions of double-stranded DNA, thereby inactivating the targeted gene. The advantage of triplex DNA technology is that only a single copy of the oligonucleotide or compound is required to modify gene expression because the binding is at the DNA level rather than the mRNA level. However, the disadvantage of triple-stranded DNA technology is that oligonucleotides or compounds are not only cell membranes, but also microbial membranes (when treating microbial infections) or nuclear membranes (eukaryotic genes of foreign DNA integrated into chromosomal DNA). To change function or expression).

  Another gene therapy technique relates to the therapeutic use of ribozymes for the treatment of genetic disorders. Ribozymes are catalytic RNA molecules that consist of a hybridizing region and an enzymatic region. Ribozymes can be engineered to specifically bind and cleave the targeted region of the nucleic acid sequence in the future, or otherwise alter the sequence to alter its expression or translation into a gene product. It can be modified enzymatically.

  A variety of biologically active factors have been isolated from humans or animals that have been reported to have therapeutic efficacy. These compounds include, but are not limited to, potent biologically active molecules such as cytokines and growth factors. However, when these various factors are isolated and purified from natural sources or genetically engineered materials and then injected into humans or animals, they frequently cause severe side effects and are desirable. It has been found to exhibit unsurpassed toxicity. Because of this toxicity, it has been difficult to use the compounds therapeutically. Furthermore, it has been difficult to generate antibodies to molecules using active compounds as antigens. Moreover, the toxicity of such molecules is associated with higher concentrations or presence at sites where factors are not commonly found when used in the treatment of disease. If such a powerful factor can be specifically targeted to the site where the factor is needed without exposure to other sites causing side effects, the problems associated with its systemic administration can be overcome.

  There is a very high need for compositions and methods for targeted delivery systems. These delivery systems can be used for targeted delivery to specific cells or organs of genetic material such as genes, polynucleotides, and antisense oligonucleotides that can be used in gene therapy. More particularly, there is a need for compositions that can facilitate the transport of genetic compounds and other drugs and therapeutic compounds across the cell membrane. What is also needed is a delivery system for the delivery of therapeutic agents and biological agents that affect cells or the surrounding environment to specific cell types and organs.

  Due to the safety issues associated with the use of attenuated vaccines and the low efficacy of inactivated vaccines, new methods of vaccination (eg, selective activation of immune cell components and methods of enhancing vaccine efficacy) There is a need in the art for compositions and methods to be provided. There is also a need in the art for compositions and methods for enhancing the immune system that stimulate both humoral and cell-mediated responses. There is a further need in the art to modulate the selectivity of the immune response and to manipulate the various components of the immune system to produce the desired response. Furthermore, there is a need for methods and compositions that can accelerate and develop immune responses for a faster response in activation. There is an increasing need for the ability to vaccinate both human and animal populations with vaccines that provide protection at only one dose.

  What is needed is a composition and method for target specific delivery of therapeutic agents only to target cells. It is preferred for some administrations and treatments where the therapeutic agent is internalized by the targeted cells. Once inside the cell, the therapeutic agent should be sufficiently released from the transport system so that the therapeutic agent is active. For example, if the therapeutic agent is an exogenous nucleic acid containing gene, it should be transcribed and translated and expressed if necessary. Such compositions and methods should be able to efficiently deliver therapeutic agents to target cells. What is also needed are compositions and methods that can be used in both in vitro and in vivo systems.

  There is also a need for therapeutically effective compositions that have reduced toxicity and that can be used in therapy for a wide range of immune, cancer, viral and bacterial diseases. Furthermore, there is a need for compositions that can reduce the toxicity of biologically active compositions that are toxic when found at very high concentrations or in the wrong location.

For example, the present invention provides the following items.
1. A targeted delivery composition comprising at least one effector molecule bound to a platform or at least one cell-specific targeting molecule.
(Item 2) The targeted delivery composition of item 1, wherein the effector molecule is attached to the platform a) direct binding to the platform; b) to an integration molecule attached to the platform. C) a less specific binding to an integrated molecule bound to the platform; and d) a binding of a complementary binding member.
(Item 3) The targeted delivery composition of item 1, comprising: a) direct binding to the platform; b) specific binding to an integration molecule bound to the platform; c) the A composition comprising a cell-specific targeting molecule bound in a manner comprising: less specific binding to an integration molecule bound to the platform; and d) binding of a complementary binding member.
4. The targeted delivery composition of claim 2, wherein the effector molecule comprises a therapeutic agent and a biologically active agent.
(Item 5) The targeted delivery composition according to item 1, wherein the cell-specific targeting molecule is interleukin-1 ("IL-1"), interleukin-2 ("IL -2 "), interleukin-3 (" IL-3 "), interleukin-4 (" IL-4 "), interleukin-5 (" IL-5 "), interleukin-6 (" IL-6 ") ”), Interleukin-7 (“ IL-7 ”), interleukin-8 (“ IL-8 ”), interleukin-10 (“ IL-10 ”), interleukin-11 (“ IL-11 ”) , Interleukin-12 ("IL-12"), interleukin-13 ("IL-13"), interleukin-15 ("IL-15"), interleukin-16 ("IL-16"), interleukin-16 Leukin-1 7 ("IL-17"), interleukin-18 ("IL-18"), lipid A, phospholipase A2, endotoxin, staphylococcal enterotoxin B and other toxins, type I interferon, type II interferon, tumor necrosis Factor (“TNFα”), transforming growth factor-β (“TGF-β”), lymphotoxin, migration inhibitory factor, granulocyte-macrophage colony stimulating factor (“CSF”), monocyte-macrophage CSF, granulocyte CSF, Vascular epidermal growth factor (“VEGF”), angiogenin, transforming growth factor (“TGFα”), heat shock proteins, blood group carbohydrate moieties, Rh factors, fibroblast growth factors, and other inflammatory regulatory proteins and Immunoregulatory protein, growth hormone, insulin, glucagon, parathyroid hormone Hormones, luteinizing hormone, follicle stimulating hormone, and luteinizing hormone releasing hormone, cell surface receptors, antibodies, nucleic acids, nucleotides, DNA, RNA, sense nucleic acids, antisense nucleic acids, cancer cell specific antigens; MART, MAGE, BAGE, and HSP; Mutant p53; Tyrosinase; Autoimmune antigen; Receptor protein, glucose, glycogen, phospholipid, and monoclonal and / or polyclonal antibodies, and basic fibroblast growth factor .
6. The targeted delivery composition of claim 1, wherein the platform is a colloidal metal.
7. The targeted delivery composition of claim 1, wherein the composition comprises at least one effector molecule and at least one cell-specific targeting molecule.
8. The targeting composition of claim 7, wherein at least one of the effector molecule or the cell-specific targeting molecule is on the platform, a) directly to the platform B) specific binding to an integration molecule bound to the platform; c) less specific binding to an integration molecule bound to the platform; and d) binding of a complementary binding member. Compositions that are combined in a manner.
9. A method for targeted delivery of an effector molecule to a cell, comprising administering at least one effector molecule bound to the platform and at least one cell-specific targeting molecule. A method comprising the steps of:
10. The method of claim 9, wherein the composition further comprises a molecule that incorporates.
11. The method of claim 9, wherein the molecule is administered in vivo.
12. The method of claim 9, wherein the molecule is administered in vitro.
(Summary of the Invention)
The present invention includes methods and compositions for targeted delivery of therapeutic agents to specific cells. The therapeutic agent is taken up by a particular cell for a particular receptor on the cell and is preferably internalized into the cell by receptor-mediated endocytosis. Thus, in a mixture of different cell types, the therapeutic agent is only internalized by cells having the selected receptor, and cells lacking that receptor are not affected.

  The methods and compositions of the present invention provide a new and versatile approach to targeted cell delivery systems. Preferred embodiments of the invention include a delivery structure or platform, and the composition to be delivered or a cell-specific targeting molecule is attached to the delivery structure or platform. For example, in one embodiment, one member of the binding group is bound to the delivery platform, and the complementary member of the binding group is bound to or selected from a therapeutic agent or effector molecule. Bound to a cell-specific ligand or targeting molecule. In a more preferred embodiment, one of the complementary members of the binding group binds to a cell-specific ligand, a targeting molecule, and another of the complementary members of the binding group is Bound to a therapeutic agent or effector molecule.

  The delivery system of the present invention provides a novel treatment involving targeted combinatorial therapeutics. The present invention includes compositions and methods for targeted drug delivery to specific cells in vitro or in vivo. The delivery structure or platform preferably provides a surface for reversible binding, but the application may include irreversible binding of elements to be administered to an organism or cell. In one preferred embodiment, the element to be bound is a member of a linking group. The binding group member may be selected from all such known pairs of binding groups including, but not limited to, antibody / antigen; enzyme / substrate; and streptavidin / biotin. The member of the binding group can then be bound to a cell specific ligand or therapeutic agent. The cell-specific ligand or targeting molecule includes any cell-specific marker, which is specific for one or a few cell types, or is widely expressed in cell types, organs, or systems Containing markers. Therapeutic agents include, but are not limited to, biologically active agents, formulations, radioactive agents, or agents such as agents that are detrimental to cells, nucleic acids, or other compounds that can affect cellular activity. Not.

  Other embodiments of such compositions comprise a delivery structure or platform to which members of the linking group are reversibly bound. Preferred embodiments of the invention include colloidal gold, such as a platform that can bind to a member of a binding group, and the targeting molecule and effector molecule bind to the member of the binding group, preferably a receptor-mediated cell. Endocytosis is used to create a targeted gene delivery system that provides internal delivery of therapeutic agents. In a more preferred embodiment, the linking group is streptavidin / biotin and the targeting molecule is a cytokine and the effector molecule is a therapeutic agent. Embodiments of the invention can also include the step of binding effector molecules or targeting molecules in a less specific manner, such as by using polycations.

  In the present invention, the method includes the preparation and administration of a targeted delivery system and the delivery of the targeted delivery system to selected cells. In the present invention, the therapeutic agent of the composition is intended to be active or effective at the cellular site. Such activity can be in any form known to those skilled in the art and can be cell death, functional protein production, cellular product production, enzymatic activity, cellular product transport, cell membrane component or nuclear component production. Including, but not limited to. The method of delivery to the targeted cells can be a method such as a method used for in vitro techniques such as addition to cell culture or medium, or a method used for in vivo administration. . In vivo administration can include routes of administration such as those used for direct application to cells or delivery of therapeutic agents to humans, animals, or other organisms.

Another preferred embodiment of the present invention is a method and composition wherein the biologically active agent or therapeutic agent is bound directly to the delivery platform (eg, binding of the cytokine composition to colloidal gold). including. Such compositions can be used to reduce the toxicity associated with administration of a therapeutic agent or can be used in a vaccine formulation. The composition of the present invention comprises a mixture of colloidal metals (eg colloidal gold (HAuCl ₄ ) combined with a substance that is normally toxic to humans or animals, or a substance that can generate an immune response), wherein The composition is less toxic or not toxic when administered to humans or animals. Such methods and compositions may be used in the treatment of bacterial infections, viral infections, cancers, and immune disease therapies, including but not limited to autoimmune diseases such as rheumatoid arthritis and acquired immune deficiencies. One advantage of the present invention is that a smaller amount of biologically active agent can be administered than in previously known methods (this agent is targeted directly to the site or the agent is delivered to the delivery platform). (Because it is less toxic for general administration due to its binding). Biologically active agents bound to the platform can also act as a fixed source of releasable biologically active factors, a constant release deposit.

  Targeted delivery of the present invention is a method for simultaneous and / or sequentially targeted stimulation of specific components of the immune system by a composition of the present invention that enhances or modifies the immune response to the composition And a composition. Components of the immune system include, but are not limited to, cells such as B cells, T cells, antigen presenting cells, phagocytic cells, macrophages, and other immune cells. Since several cell types of the immune system can all have the same cell marker, a “component-specific targeting molecule” can target several cell types or tissue types.

  Another aspect of the invention provides that antigens and vaccines increase the efficacy of inducing an immune response. The present invention includes compositions and methods for stimulation of specific individual components of the immune system with a composition by co-presentation of an antigen with an immune component-specific targeting molecule. Targeted activation of immune components can occur in vivo or in vitro.

  The present invention includes compositions and methods for the co-stimulation of many different individual immune components through the presentation of specific component stimulating compositions. One embodiment of such a composition includes a delivery platform with an antigen in combination with a component-specific immunostimulatory agent. The present invention also includes compositions and methods for continuous stimulation of the immune system by providing component stimulating compositions at one or more steps in the interacting factor and cellular immune response cascade.

  In a preferred embodiment, the method comprises continuous administration of the component stimulating composition. The composition may comprise the same component-specific immunostimulatory agent given at different times or in different modes of administration (eg, orally once and second time by injection). In another preferred embodiment, the method comprises sequential administration of different component-specific immunostimulatory agents. For example, the first component-specific immunostimulator stimulates the initiation process of the immune response, followed by administration of the second component-specific immunostimulator and subsequently stimulates the subsequent process of the immune response. The present invention administers multiple component-specific immunostimulants that initiate several pathways of the immune system, followed by the same or other component-specific immunostimulators for subsequent and sustained immune responses. Intended to be administered.

  Furthermore, it is contemplated in the present invention that the compositions and methods described herein can be used for stimulating an immune response or suppressing an immune response. Administration of component-specific immunostimulants for suppression of the immune response can be used to control autoimmune diseases or organ rejection.

  Accordingly, it is an object of the present invention to provide multipurpose methods and compositions for influencing cells with therapeutic agents.

  Another object of the present invention is to provide methods and compositions for targeted delivery of therapeutic agents in vitro and in vivo.

  Yet another object of the present invention is methods and compositions for targeted delivery of therapeutic agents to cells having specific receptors.

  It is a further object of the present invention to provide methods and compositions for delivery of therapeutic agents to cells by receptor mediated endocytosis.

  Yet another object of the present invention is to provide methods and compositions comprising targeted delivery systems that can use specific targeting molecules to bind and deliver selected therapeutic agents.

  It is an object of the present invention to provide compositions and methods that can reduce the toxic effects of biologically active factors.

  Yet another object of the present invention is to provide methods and compositions for vaccinating humans or animals using normally toxic biologically active agents.

  A further object of the present invention is to provide methods and compositions for the sustained release of biologically active factors.

  A still further object of the present invention is to provide a reliable and easy method and composition for enhancing an immune response.

  Another object of the present invention is to provide methods and compositions for improving vaccine efficacy.

  Another object of the present invention is to provide vaccines and vaccinations that provide effective protection with administration of only one dose.

  Yet another object of the present invention is to provide compositions and methods for targeted stimulation of individual immune components in a particular manner.

  Another object of the present invention is to provide methods and compositions comprising component-specific immunostimulants that can affect specific components of the immune system.

Yet another object of the present invention is to provide methods and compositions for suppressing immune responses.
These and other objects, features and advantages of the present invention will become apparent after review of the detailed description of the preferred embodiments below.

FIG. 1 is a schematic diagram of a preferred embodiment of the present invention. FIG. 2 is a graph showing the saturable binding kinetics of the delivery platform with TNF-α. FIG. 3 illustrates the effect of colloidal gold on the generation of a mouse anti-mouse IL-6 antibody response. FIG. 4 shows the mouse anti-mouse IL-6 antibody response in IL-6 activated cells bound to colloidal gold in immunoreactivity versus the response activated with IL-6 or colloidal gold alone. Illustrate the increase. FIG. 5 illustrates the efficiency with which gold binds to IL-6. FIG. 6 illustrates the retention of IL-1 biological activity after treatment with gold. FIG. 7 illustrates the effect of time on the release of IL-2 from colloidal gold. FIG. 8 illustrates the effect of dilution on the release of TNFα from colloidal gold. FIG. 9 illustrates in vivo release of IL-2 bound to colloidal gold. FIGS. 10a-d illustrate the internalization of colloidal gold-bound IL-1β by MCF-7 cells. FIGS. 10a-d illustrate the internalization of colloidal gold-bound IL-1β by MCF-7 cells. FIGS. 10a-d illustrate the internalization of colloidal gold-bound IL-1β by MCF-7 cells. FIGS. 10a-d illustrate the internalization of colloidal gold-bound IL-1β by MCF-7 cells. FIG. 11 illustrates the effect on immunoreactivity of colloidal gold bound to TNFα, IL-6, and IL-1β. FIG. 12 illustrates in vitro internalization of the EGF / CG / IL-1β complex by macrophages. FIG. 13 illustrates in vitro internalization of EGF / CG / TNF-α complex by dendritic cells. FIG. 14 illustrates in vitro internalization of the EGF / CG / IL-6 complex by B cells. FIG. 15 illustrates in vitro internalization of the EGF / CG / IL-2 complex by T cells. FIG. 16 illustrates in vitro internalization of an EGG / Histone / DNA / Colloidal Gold chimera by human breast cancer cells MCF-7. FIG. 17 illustrates control transfection using EGF / Histone / DNA / Colloidal gold chimera and human breast cancer cells HS-578-T.

(Detailed explanation)
The present invention relates to compositions and methods for targeted delivery of therapeutic agents to cells. More particularly, the invention includes a delivery structure or platform composition to which another element is attached. A preferred embodiment of the present invention includes colloidal metal as a platform. This platform can bind members of the linking group to which the targeting molecule and effector molecule bind to create a targeted gene delivery system. Such a system preferably uses cellular receptor-mediated endocytosis to achieve internal delivery of the therapeutic agent. In the most preferred embodiment, the linking group is streptavidin / biotin and the targeting molecule is a cytokine and the effector molecule is a therapeutic agent. Embodiments of the invention may also include the step of binding effector molecules or targeting molecules in a less specific manner without using a binding partner, for example by using polycations or proteins. . Other embodiments of the invention include direct binding or association of effector molecules or targeting molecules to a delivery structure or platform.

  The present invention includes methods and compositions for targeted delivery of therapeutic agents using a colloidal metal as a platform. Such colloidal metals can reversibly or irreversibly bind molecules that interact with either the therapeutic agent or the targeting molecule. Whether the integrating molecule can be a specific binding molecule (eg, a member of a binding pair) or a non-specific integrating molecule that binds with lower specificity. Either. An example of such a less specific binding is the binding of a nucleic acid by a polycationic molecule (eg, polylysine or histone). The present invention contemplates the use of incorporation molecules (eg, polycationic elements) known to those skilled in the art, including but not limited to polylysine, protamine sulfate, histones, or asialoglycoproteins.

  Members of binding pairs include any such binding pair known to those of skill in the art including, but not limited to, antibody-antigen pairs, enzyme-substrate pairs; receptor ligand pairs; and streptavidin-biotin. New binding partners can be specifically designed. An essential feature of a binding partner is the specific binding between one of the binding pairs and the other member of the binding pair such that the binding partners can be specifically linked. Another desired feature of the binding member is that one member of the pair can also bind to or be bound to either the effector molecule or the targeting molecule, and the other member binds to the delivery platform. It is that.

  While not wishing to be bound by any particular theory, it is theorized that changes in pH that occur during endosome formation in receptor-mediated endocytosis can be utilized for internal cell delivery of therapeutic agents. When the colloidal metal composition is at a neutral pH, the composition is a sol. As the pH decreases, the colloidal metal composition precipitates and aggregates. The reduced pH includes a range of pH values from less than about 7 to about 2. A preferred reduced pH is that found in endosomes, which is about pH 4 to about pH 6, most preferably pH 5.6. Aggregation of metal particles in the endosome effectively reduces the surface area of the metal particles for binding, which results in the release of binding material from the metal carrier.

  The delivery platform is preferably a colloidal metal composition. For example, in a preferred embodiment, the delivery platform is colloidal gold particles that have a negative charge at approximately neutral pH. This negative charge prevents the attraction and binding of other negatively charged molecules. In contrast, positively charged molecules are attracted and bound to colloidal gold particles. Such positively charged molecules include polycations, including but not limited to polylysine, histone, protamine sulfate, and asialoglycoprotein. The histones contemplated in the present invention can be a mixture of different histones, one specific type of histone, or a combination of specific histones. Positively charged molecules can also include members of binding pairs (eg, antibody-antigen or streptavidin / biotin). After binding of the positively charged molecule to the delivery platform, a complementary binding member or effector molecule can be bound.

  An effector molecule is a molecule for cell or target selection, known as a targeting molecule herein, and a molecule that provides an effect once delivered to a target, therapeutic agent, or biologically active agent Including both. The association between the effector molecule and the metallic platform can be either through specific means using members of the binding pair and their specific binding, or through less specific means. For example, less specific means include, but are not limited to, ionic interactions.

  Use of the specific binding properties of the binding member includes binding of one of the binding pair member to the therapeutic agent and the complementary binding member to the metallic platform. Binding of one binding member to the therapeutic agent is accomplished through means well known in the art and exemplified in the binding of biotinylated therapeutic agents. The biotin can be coupled through methods such as protein binding, or can be incorporated into nucleic acids through synthesis using biotinylated nucleosides. The binding of biotin to the therapeutic agent can be accomplished using chemical linkage (eg, providing a specific linker or adding an active group to the therapeutic agent or targeting molecule).

  It is the use of such binding members that provides one aspect of the flexibility of the present invention. Any desired therapeutic agent can bind to one of the members of the binding pair. The complementary binding member then associates with the metallic platform. Thus, any therapeutic agent can be delivered using the metallic platform of the present invention. In addition, any targeting molecule can bind to one of the members of the binding group.

  In order to provide specificity to the selection of cells or targets for delivery of therapeutic agents, cell-specific targeting molecules can also be bound to a metallic platform. Such cell-specific targeting molecules include any molecule that binds to a structure on a cell, including but not limited to receptors found in cell membranes. Such cell-specific targeting molecules also include receptors or portions of receptors that can bind to molecules found in the cell membrane or not included in the cell membrane. Examples of such cell-specific targeting molecules include but are not limited to: interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin -3 ("IL-3"), interleukin-4 ("IL-4"), interleukin-5 ("IL-5"), interleukin-6 ("IL-6"), interleukin-7 ("IL-7"), interleukin-8 ("IL-8"), interleukin-10 ("IL-10"), interleukin-11 ("IL-11"), interleukin-12 (" IL-12 "), interleukin-13 (" IL-13 "), interleukin-15 (" IL-15 "), interleukin-16 (" IL-16 "), interleukin-17 (" IL- " 7 "), interleukin-18 (" IL-18 "), lipid A, phospholipase A2, endotoxin, staphylococcus enterotoxin B and other toxins, type I interferon, type II interferon, tumor necrosis factor (" TNFα "), Transforming growth factor-β (“TGF-β”), lymphotoxin, migration inhibitory factor, granulocyte macrophage colony stimulating factor (“CSF”), monocyte macrophage CSF, granulocyte CSF, vascular epidermal growth factor (“VEGF”) , Angiogenin, transforming growth factor (“TGFα”), heat shock proteins, blood group carbohydrate moieties, Rh factors, fibroblast growth factors and other inflammatory and immunoregulatory proteins, hormones (eg, growth) hormone, Insulin, glucagon, parathyroid hormone, luteinizing hormone, follicle stimulating hormone, and luteinizing hormone releasing hormone), cell surface receptor, antibody, nucleic acid, nucleotide, DNA, RNA, sense nucleic acid, antisense nucleic acid, cancer cell-specific antigen For example, MART, MAGE, BAGE, and HSP; mutant p53; tyrosinase; autoimmune antigen; receptor protein, glucose, glycogen, phospholipid, and monoclonal and / or polyclonal antibodies, and basic fibroblast growth factor.

  The elements that couple to the delivery device or platform can be joined by any method. Such elements include, but are not limited to, molecules that incorporate, molecules that specifically target, therapeutic agents, or biologically active agents. The following example illustrates a binding method for a molecule that incorporates, but the method is not limited to this element alone, and any element contemplated by the present invention can be bound in this method. Incorporating molecules (less specific binding molecules such as either a polycationic component or a specific binding molecule that is a member of a binding pair) can be bound to the colloidal metal by any method. One preferred method is to reconstitute the incorporating molecule in water at a pH of about 1-3 pH units above the isoelectric point pI of the incorporating molecule. Then about 100-1,000 μg, preferably 150-800 μg, most preferably 200-500 μg of incorporating molecules are incubated with the colloidal metal. The duration of incubation is not critical, but is preferably about 2-48 hours, more preferably 18-36 hours.

  After incubation, the colloidal metal platform bound to the integrating molecule is stabilized as needed by incubating overnight with a 1% by volume 1-100% solution of polyethylene glycol (PEG). Alternatively, cysteine, phospholipid, Brij58, or sulfhydryl-containing compounds can be used to stabilize the colloidal metal platform. This solution is then centrifuged and the resulting pellet is optionally in a 1% human serum albumin (HSA) solution with cysteine, phospholipid, sulfhydryl-containing compound, or in protein reconstitution buffer. Stabilized by incubation with Typically, between 90% and 95% of the components are bound by this method as determined by immunoassay measurements of unbound biologically active factors in the supernatant.

  The binding of elements in this manner changes the physical properties of the colloidal metal. Prior to bonding, the colloidal metal cannot be filtered through a 0.22 micron filter. After binding, the colloidal complex is easily filtered. This difference suggests that the metal no longer exists as a colloid but exists as an ionic solution.

  The amount of colloidal metal used in the present invention is between about 0.001 mg / ml and 1.0 mg / ml, and more preferred amounts of colloidal metal are about 0.01 mg / ml and 0.1 mg / ml. between ml. The amount of the composition according to the invention to be administered in vivo or in vitro will vary according to the desired application, the molecule to be delivered, the cells targeted for delivery, and the mode of administration.

  Other methods and uses for the preparation of colloidal compositions are found in related patent applications. These patent applications (USS 08/966, 940 and 60 / 075,811, 60 / 086,696) are hereby incorporated by reference in their entirety.

  The effector molecule is then bound to the metallic platform via the incorporating molecule. Effector molecules can be either cell-specific targeting molecules or therapeutic agents. The effector molecule can be bound directly to the integrating molecule (eg, histone) or can be bound through the specific interaction of the binding member. Where the method involves specific interactions with binding members, one of the binding members functions as an integrating molecule bound to the platform, and the complementary binding member binds to the effector molecule. For example, one embodiment of the present invention comprises a streptavidin and biotin binding member, which functions as an integrating molecule and associates with a colloidal metal platform. This biotin is bound to the effector molecule. In a further preferred embodiment, the biotin is bound to a cell-specific targeting molecule (eg, TNF-α). The biotin is also conjugated to a therapeutic agent (eg, a truncated form of the toxin that does not contain an extracellular binding domain).

  One embodiment of the present invention includes the use of streptavidin coupled to colloidal gold as a platform for developing and customizing targeted combination therapies. The invention includes therapeutic agent targeting and ligand-mediated endocytosis (RME) processes using ligand / receptor binding. This embodiment uses streptavidin conjugated to colloidal gold to allow the biotinylated ligand and therapeutic agent to coexist. In essence, by using streptavidin bound to colloidal gold, the problem of binding to two chemically distinct parts on a single colloidal gold particle is eliminated. Rather, the target and therapeutic chemistry is now based on well-characterized binding of biotin to streptavidin. A representative diagram of this embodiment is shown in FIG.

Some embodiments of the invention
The compositions and methods of the present invention include modifications and combinations of the mixtures of elements disclosed herein, as well as methods for their binding ability and activity. For example, one embodiment of the present invention includes either cell-specific targeting molecules directly bound to a metallic platform, and less specific binding by molecules that specifically bind to or incorporate a metallic platform. Containing a therapeutic agent bound via One embodiment of the present invention is directed to cell-specific targeting via either a therapeutic agent directly bound to a colloidal metal platform and a less specific binding by a molecule that specifically binds or incorporates. Including molecules that Another embodiment of the present invention is the binding of both cell-specific targeting molecules and therapeutic agents via less specific binding by molecules that specifically bind to or incorporate a metallic platform, or cells Includes direct binding of therapeutic agents to specific targeting molecules and metallic platforms. Still further embodiments of the invention include cell-specific targeting molecules bound to a metallic platform via binding by a binding member, and therapeutic agents bound to the metallic platform via a less specific binding means including. Contrasting embodiments of the present invention include cell-specific targeting molecules bound to metallic platforms via binding by molecular binding for less specific integration, and metallic platforms by complementary binding member binding Including therapeutic agents. Other combinations and modifications of such embodiments are contemplated as part of the present invention.

  The elements (including but not limited to targeting molecules, incorporating molecules, and effector molecules, such as but not limited to therapeutic agents and biologically active agents) that are bound to the delivery platform can be combined in any combination Can be combined. For example, two effector molecules can be bound to the platform such that the target and effector complex can be generated by the binding of cytokines and cytokine receptors to colloidal metal particles. Alternatively, gene delivery systems can be generated by binding nucleic acid components and cytokine receptors to colloidal metal particles. A therapeutic system can be generated by binding chemotherapeutic agents and cytokine receptors to colloidal metal particles. In addition, antibodies against cancer antigens can be targeting molecules and can be bound to colloidal metals with chemotherapeutic agents. In delivering chemotherapeutic agents to target cells (eg, cancer cells), low concentrations of chemotherapeutic agents can be used, thereby reducing the systemic toxicity of the chemotherapeutic agent.

  Another embodiment of the invention is a method for binding one molecule to a cell surface receptor while releasing a second molecule to the extracellular surface proximal to the target cell. The use of binding of the active agent to the same colloidal metal particles. Such slow release stores are useful for delivering biologically active molecules to their specific site of action.

  Yet another embodiment of the present invention is a method of using a specific molecular transport mechanism for one of these components that traverses the biological barrier and then carries the second component or group with the first component. Using two or more elements to the same delivery platform (eg, colloidal metal particles). For example, one of the components coupled to the delivery platform can be glucose. Glucose has a specific blood brain transport system and is bound to colloidal metals along with effector molecules (eg, biologically active molecules). Glucose active transport across the blood-brain barrier serves as a conduit for any relevant biologically active factor that cannot by itself and spontaneously cross this biological barrier. These compositions then serve as vehicles for delivery of therapeutic molecules to areas that are not normally available for the agent delivered (eg, the brain).

(Preferred delivery platform)
Colloidal metals are preferred delivery platforms, although other materials that function in a similar manner are intended to be included in the present invention. While not wishing to be bound by any particular theory, the role of colloidal metal, more preferably colloidal gold, in the present invention does not only act as a docking site for the associated element. On the contrary, its role can be as important as the role of the binding element. For example, certain drugs become active only after being internalized by cells. Drugs that use RME as a method of internalization often remain trapped in membrane organelles and ultimately are destroyed in endosomes, which are membrane organelles. Drug inactivation is often the result of a decrease in endosomal pH. It is this physiological change in endosomal pH that recognizes the value of colloidal gold. We have observed that protein-bound colloidal gold undergoes a physical change when exposed to an acidic, ie endosomal-like environment. In a very simple experiment, colloidal gold combined with a saturating concentration of BSA was dialyzed against MES buffer at two different pHs (7.4 and 5.6). The results of this experiment showed that colloidal gold dialyzed against physiological (ie 7.4) pH remained in its colloidal state. However, the same preparation dialyzed against acidic pH formed very large aggregates. By forming such aggregates, colloidal gold facilitates intracellular release of putative drugs by dissolving endosomes.

(Methods and compositions for administration)
The compositions of the invention can be administered to in vitro or in vivo systems. In vivo administration may include direct application to target cells or administration routes such as those used by pharmaceuticals including, but not limited to: oral, rectal, transdermal, ocular (intravitreal or For intratumoral (including intracameral), nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intratracheal, and epidural) administration Contains appropriate formulations. The formulations can exist in convenient unit dosage forms and can be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier or excipient. In general, formulations are prepared by uniformly and intimately bringing into association the composition with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

(Definition)
The terms “toxic response” and “toxicity” as used herein include, but are not limited to, the following reactions of animals or humans: fever; edema (including brain edema); psychosis; autoimmune disease; Bleeding; shock (hemorrhagic shock); sepsis; cachexia; or death.

  The terms “biologically active agent” and “therapeutic agent” include, but are not limited to: interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), Interleukin-3 ("IL-3"), Interleukin-4 ("IL-4"), Interleukin-5 ("IL-5"), Interleukin-6 ("IL-6"), Interleukin -7 ("IL-7"), interleukin-8 ("IL-8"), interleukin-10 ("IL-10"), interleukin-11 ("IL-11"), interleukin-12 ("IL-12"), interleukin-13 ("IL-13"), interleukin-15 ("IL-15"), interleukin-16 ("IL-16"), interleukin-17 (" I -17 "), interleukin-18 (" IL-18 "), lipid A, phospholipase A2, endotoxin, staphylococcus enterotoxin B and other toxins, type I interferon, type II interferon, tumor necrosis factor (" TNFα ") , Transforming growth factor-β (“TGF-β”), lymphotoxin, migration inhibitory factor, granulocyte macrophage colony stimulating factor (“CSF”), monocyte macrophage CSF, granulocyte CSF, vascular epidermal growth factor (“VEGF”) ), Angiogenin, transforming growth factor (“TGFα”), heat shock protein, carbohydrate group of blood group, Rh factor, fibroblast growth factor, hormones such as growth hormone, insulin, glucagon, vice versa Thyroid hormone Luteinizing hormone, follicle stimulating hormone, and luteinizing hormone releasing hormone, cell surface receptors, antibodies, chemotherapeutic agents, and other inflammatory and immunomodulating proteins, nucleic acids, nucleotides, DNA, RNA, sense nucleic acids, antisense nucleic acids, Cancer cell specific antigens; such as MART, MAGE, BAGE, and HSP; and immunotherapeutic drugs such as AZT, pharmaceuticals and other therapeutic drugs.

  Examples of therapeutic agents and organisms to be treated are found in the table below. This table is not limiting in that other therapeutic agents are contemplated by the present invention.

The term “a cell-specific targeting molecule” includes, but is not limited to: interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-3. ("IL-3"), interleukin 4 ("IL-4"), interleukin-5 ("IL-5"), interleukin-6 ("IL-6"), interleukin-7 ("IL -7 "), interleukin-8 (" IL-8 "), interleukin-10 (" IL-10 "), interleukin-11 (" IL-11 "), interleukin-12 (" IL-12 ") )), Interleukin-13 (“IL-13”), interleukin-15 (“IL-15”), interleukin-16 (“IL-16”), interleukin-17 (“IL-17”) Interleukin-18 ("IL-18"), lipid A, phospholipase A2, endotoxin, staphylococcus enterotoxin B and other toxins, type I interferon, type II interferon, tumor necrosis factor ("TNFα"), transforming growth factor -Β (“TGF-β”), lymphotoxin, migration inhibitory factor, granulocyte macrophage colony stimulating factor (“CSF”), monocyte macrophage, CSF, granulocyte CSF, vascular epidermal growth factor (“VEGF”), angiogenin , Transforming growth factor (“TGFα”), heat shock protein, carbohydrate group of blood group. Rh factor, fibroblast growth factor and other inflammatory and immunoregulatory proteins, hormones such as growth hormone, insulin, glucagon, parathyroid hormone, luteinizing hormone, follicle stimulating hormone, and luteinizing hormone releasing hormone, Cell surface receptors, antibodies, nucleic acids, nucleotides, DNA, RNA, sense nucleic acids, antisense nucleic acids, cancer cell specific antigens; eg, MART, MAGE, BAGE, and HSP; mutant p53; tyrosinase; autoimmune antigen; Glucose, glycogen, phospholipid, and monoclonal and / or polyclonal antibodies, and basic fibroblast growth factor.

The term “colloidal metal” as used herein includes any water-insoluble metal particle or metallic compound (hydrosol) dispersed in liquid water. The colloidal metal may be selected from Group IA, IB, IIB and IIIB metals of the periodic table, and transition metals, particularly Group VIII transition metals. Preferred metals are gold, silver, aluminum, ruthenium, zinc, iron, nickel and calcium. Other suitable metals may also include the following in various oxidation states: lithium, sodium, magnesium, potassium, scandium, titanium, vanadium, chromium, manganese, cobalt, copper, gallium, strontium, niobium, molybdenum, palladium, Indium, tin, tungsten, rhenium, platinum and gadolinium. The metal is preferably provided in an ionic state (eg A1 ³⁺ , Ru ³⁺ , Zn ²⁺ , Fe ³⁺ , Ni ²⁺ and Ca ²⁺ ions) derived from a suitable metal compound. Preferred metals are in the form of gold, especially Au ³⁺ . A particularly preferred form of colloidal gold is HAuCl ₄ (EY Laboratories, Inc., San Mateo, California). Another preferred metal is silver, particularly in sodium borate buffer having a concentration between about 0.1% and 0.001%, most preferably in about 0.01% solution. is there. Preferably, the color of such colloidal silver solutions is yellow and the colloidal particles are in the range of 1-40 nanometers. Such metal ions can be present alone or in the complex with other inorganic ions.

Some embodiments of the invention
(Methods and compositions for gene therapy)
The present invention includes methods and compositions for targeted delivery of exogenous nucleic acid or genetic information to specific cells. Exogenous genetic information is taken up by a particular cell by a particular receptor on the cell and is preferably internalized into the cell by receptor-mediated endocytosis. Thus, in a mixture of different cell types, exogenous nucleic acid is internalized only by cells having the selected receptor and does not affect cells that lack the receptor.

  The present invention includes compositions and methods for in vitro or in vivo transfection of specific cells. One embodiment of such a composition comprises a nucleic acid bound to a polycation bound to a colloidal metal. Preferred embodiments of the present invention include colloidal gold as a platform, which colloidal gold binds to targeting molecules and effector molecules and is targeted using cellular receptor-mediated endocytosis. Gene delivery systems can be generated and transfection can be achieved. In a more preferred embodiment, the targeting molecule is a cytokine and the effector molecule is exogenous genetic material such as DNA or RNA. This embodiment also includes incorporating molecules such as polycations.

  In the present invention, this method involves the preparation of a targeted gene delivery system and the delivery of the targeted gene delivery system to cells for transfection. It is contemplated in the present invention that the nucleic acid of the composition is ultimately translated and expressed by the cell. Such expression can be in any form known to those of skill in the art and includes, but is not limited to, functional proteins, cell product production, enzyme activity, cell product transport, cell membrane component production or nuclear components. Not. The method of delivery to the targeted cell can be a method such as a method used for in vitro techniques such as addition to cell culture or a method used for in vivo administration. In vivo administration can include direct administration to cells or routes of administration as used for humans, animals or other organisms.

  Figures 16 and 17 show experiments using embodiments of the present invention. FIG. 16 demonstrates successful gene delivery targeted by the receptor in MCF-7, a human breast cancer cell. EGF / histone chimeras were constructed on 40 nm colloidal gold. During precipitation and resuspension, plasmid DNA encoding the pSVβ-galactosidase gene was incubated and re-centrifuged as described above. The presence of the enzyme (β-galactosidase) was detected by incubating the cells with the substrate X-gal. Blue-green staining indicates cells in which the gene product is present, while pink staining indicates the presence of an EGF / histone / DNA / colloidal gold chimera.

  FIG. 17 shows a control transfection using an EGF / histone / DNA / colloidal gold chimera. Human breast cancer cells, HS-578-T, were treated with the same chimera as described in FIG. 16 at the same time as MCF-7 cells. The cells were then treated with X-gal to detect the presence of the transfected gene. FIG. 17 clearly shows that HS-578-T cells could bind to the chimera but could not be internalized, as found by black staining. Thus, the absence of blue-green color development indicates no transfection.

(Immune stimulation)
The present invention relates to compositions and methods for enhancing immune responses and increasing vaccine efficacy through simultaneous or sequential targeting of specific immune components. More particularly, specific immune components including, but not limited to, antigen presenting cells (APCs) (eg, macrophages and dendritic cells) and lymphocytes (eg, B cells and T cells) include one or more components Provided individually by specific immunostimulants. Particularly preferred embodiments provide for the activation of immune responses using specific antigens in combination with component specific immunostimulants. As used herein, a component-specific immunostimulatory agent refers to an agent that is specific for and capable of producing that component of the immune system, so that this component has activity in the immune response . This agent can result in several different components of the immune system and this ability can be used in the methods and compositions of the invention. The agent can be naturally occurring or can be produced and manipulated through molecular biology techniques or protein receptor manipulation.

  Activation of this component in the immune response can result in stimulation or suppression of other components of the immune response, leading to overall stimulation or suppression of the immune response. For ease of expression, stimulation of the immune component is described herein, but all responses of the immune component are due to the term stimulation, including but not limited to stimulation, suppression, rejection and feedback activity. It is understood that it is intended.

  The resulting immune component can have multiple activities, leading to both suppression and stimulation of the feedback mechanism or initiation or suppression. The present invention is not limited by the examples of immunological responses detailed herein, but contemplates component-specific effects in all aspects of the immune system.

  Activation of individual components of the immune system can be simultaneous, sequential or any combination thereof. In one embodiment of the method of the invention, the multi-component specific immunostimulant is administered simultaneously. In this method, the immune system is stimulated simultaneously with four separate preparations, each of which comprises a composition comprising a component-specific immunostimulatory agent. Preferably, the composition comprises a component specific immunostimulator associated with a colloidal metal. More preferably, the composition comprises a component-specific immunostimulatory agent associated with a particle of one size or colloidal metal of different size particles and an antigen. Most preferably, the composition comprises a colloidal metal and antigen of one size particle or a component specific immunostimulator associated with a colloidal metal and antigen of a different size particle.

  The inventors have found that specific component-specific immunostimulants can be used to provide stimulation, up-regulation, effects specific to individual immune components. For example, interleukin-1β (IL-1β) specifically stimulates macrophages, while TNF-α (tumor necrosis factor α) and Flt-3 ligand specifically stimulate dendritic cells. Heat killed Mycobacterium butyricum and interleukin-6 (IL-6) are specific stimulators of B cells, and interleukin-2 (IL-2) is a specific stimulator of T cells. Compositions containing such component-specific immunostimulatory agents provide specific activation of macrophages, dendritic cells, B cells and T cells, respectively. For example, macrophages are activated when a composition comprising IL-1β, a component-specific immunostimulatory agent, is administered. A preferred composition is IL-1β associated with a colloidal metal, and the most preferred composition is an IL associated with a colloidal metal and an antigen (an antigen to provide a macrophage response specific for that antigen). −1β.

  Many elements of the immune response are required for effective vaccination. Embodiments of the costimulation method include 1) IL-1β for macrophages, 2) TNF-α and Flt-3 ligands for dendritic cells, 3) IL-6 for B cells, and 4) for T cells. Administration of four separate preparations of a composition of a component-specific immunostimulant comprising IL-2. The composition of the component-specific immunostimulatory agent can be administered by any route known to those skilled in the art and can use the same route or different routes depending on the desired immune response.

  In another embodiment of the methods and compositions of the invention, the individual immune components are activated simultaneously. For example, this continuous activation can be divided into two phases, a primer phase and an immune phase. The induction phase includes stimulation of APC (preferably macrophages and dendritic cells), while the immunization phase includes stimulation of lymphocytes (preferably B cells and T cells). In each of the two stages, activation of individual immune components can be simultaneous or sequential. For continuous activation, the preferred method of activation is activation of macrophages, then dendritic cells, then B cells, then T cells. The most preferred method is combined sequential activation, where there is simultaneous activation of macrophages and dendritic cells, followed by simultaneous activation of B cells and T cells. This is an example of a multicomponent specific immunostimulatory method and composition for initiating several pathways of the immune system.

  The methods and compositions of the present invention can be used to enhance the effectiveness of any type of vaccine. This method enhances the effectiveness of the vaccine by targeting specific immune components for activation. A composition comprising a colloidal metal and a component-specific immunostimulator associated with an antigen is used to increase contact between the antigen and the specific immune component. Examples of diseases for which vaccines are currently available include cholera, diphtheria, hemophilus, hepatitis A, hepatitis B, influenza, measles, meningitis, mumps, pertussis, pressure ulcers, pneumococcal pneumonia, polio, rabies , Rubella, tetanus, tuberculosis, typhoid fever, varicella-zoster, whooping cough, and yellow fever.

  The combination of route of administration and packaging used to deliver the antigen to the immune system is a powerful tool in designing the desired immune response. The present invention includes methods and compositions including various packaging methods (eg, including liposomes, microcapsules or microspheres) that can provide long-term release of the immunostimulatory composition. These packaging systems act as an internal reservoir for holding the antigen and gradually release the antigen for activation of the immune system. For example, liposomes can be filled with a composition comprising an antigen and a component-specific immunostimulator associated with a colloidal metal. Further combinations are colloidal gold particles interspersed with viral particles that are candidates for active vaccines or packaged to contain DNA for putative vaccines. The gold particles then also contain cytokines that can be used to target the virus to specific immune cells. In addition, fusion protein vaccines that target two or more potential vaccine candidates can be made and vaccines for two or more applications can be generated. The particles can also include an immunogen that has been chemically modified by the addition of polyethylene glycol that can slowly release the substance.

  Antigen / component-specific immunostimulant / metal complexes are gradually released from liposomes, recognized as foreign by the immune system, and activate the immune system by specific components directed to component-specific immunostimulants Make it. The cascade of immune responses is activated more rapidly by the presence of component-specific immunostimulants and the immune response occurs more quickly and more specifically.

  Other methods and compositions contemplated in the present invention include the use of immunostimulatory / colloidal metal complexes specific for antigens / components where the colloidal metal particles have different sizes. Continuous administration of component-specific immunostimulatory agents can be achieved in a single dose administration through the use of these different sized colloidal metal particles. One dose contains the antigen and four independent component-specific immunostimulators, each complexed with colloidal metal particles of different sizes. Thus, co-administration provides continuous activation of immune components to obtain a more effective vaccine and further protection against this population. Other types of such single dose administration with continuous activation may be provided by a combination of different sized colloidal particles and liposomes, or liposomes loaded with different sized colloidal metal particles.

  The use of such a vaccination system as described above is very important in providing a vaccine that can be administered in one dose. Single dose administration is important in the treatment of animal populations such as livestock or wild populations of animals. Single dose administration is important in the treatment of populations that resist health care, such as the poor, homeless, rural residents or people in developing countries with inadequate health care. Many people in all countries are not available for preventive types of health care such as vaccination. The reappearance of infectious diseases such as tuberculosis has increased the demand for vaccines that can be given once and still provide effective protection over a long period of time. The compositions and methods of the present invention provide such effective protection.

  The methods and compositions of the invention can also be used to treat diseases in which an immune response occurs by stimulating or suppressing components that are part of the immune response. Examples of such diseases include Addison's disease, allergy, anaphylaxis, Breton syndrome, cancer (including solid tumors and blood born tumors), eczema, Hashimoto's thyroiditis, polymyositis, dermatomyositis, 1 Type 2 diabetes, acquired immune deficiency syndrome, transplant rejection (eg, kidney, heart, pancreas, lung, bone and liver transplantation), Graves disease, multiple endocrine autoimmune diseases, hepatitis, microscopic polyarteritis, nodular Polyarteritis, pemphigus, primary biliary cirrhosis, pernicious anemia, coeliac disease, antibody-mediated nephritis, glomerulonephritis, rheumatic diseases, systemic lupus erythematosus, rheumatoid arthritis, seronegative spondylitis, rhinitis , Sjogren's syndrome, systemic sclerosis, sclerosing cholangitis, Wegener's granulomatosis, herpes zoster, psoriasis, vitiligo Multiple sclerosis, encephalomyelitis, Giant-Barre syndrome, myasthenia gravis, Lambert-Eaton syndrome, sclera, episclera, uveitis, chronic mucocutaneous candidiasis, urticaria, transient infant low Gamma globulinemia, myeloma, X-linked IgM excess syndrome, Viscott-Aldrich syndrome, telangiectasia ataxia, autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune neutropenia , Waldenstrom's macroglobulinemia, amyloidosis, chronic lymphocytic leukemia and non-Hodgkin's lymphoma.

  The composition of the present invention comprises a component-specific immunostimulatory agent. The composition may comprise one component-specific immunostimulator or a multi-component specific immunostimulator. A preferred embodiment of this composition includes a component-specific immunostimulator associated with a colloidal metal. More preferred embodiments include component-specific immunostimulators associated with colloidal metals, and other elements (antigens, receptor molecules, nucleic acids, pharmaceuticals, specifically for targeting the effects of component-specific immunostimulants) Including, but not limited to, chemotherapeutic agents and carriers. The compositions of the invention can be delivered to the immune component in any manner. In one embodiment, the antigen and component-specific immunostimulatory agent is bound to the colloidal metal in such a manner that a single colloidal metal particle is bound to both the antigen and the immunostimulatory agent.

(Summary)
In one disclosed embodiment of the invention, the specific immune components that are stimulated are macrophages, dendritic cells, B cells and T cells. In a preferred embodiment, the composition contains a component-specific immunostimulatory agent. In a more preferred embodiment, the composition comprises a component specific immunostimulator associated with a colloidal metal. In another disclosed embodiment, the antigen and component-specific immunostimulatory agent is bound to a colloidal metal (eg, colloidal gold) and the resulting chimeric molecule is presented to the immune component.

  The present invention includes the presentation of antigen and component-specific immunostimulatory agents on a variety of different delivery platforms or carrier combinations. For example, a preferred embodiment involves the administration of a component-specific immunostimulatory agent in a liposome carrier and an antigen associated with colloidal gold. Further combinations are colloidal gold particles interspersed with virus particles that are active vaccine candidates or packaged to contain DNA for a putative vaccine. The gold particles then also contain cytokines that can be used to target the virus to specific immune cells. Such an embodiment provides an internal vaccine preparation that gradually releases antigen to the immune system for a long-term response. This type of vaccine is particularly beneficial for a single dose of vaccine. All types of carriers are contemplated in the present invention, including but not limited to liposomes and microcapsules.

(Reduction of toxicity and vaccine administration)
The present invention includes compositions and methods for administration of common toxic bioactive factors to humans or animals. In general, a composition according to the present invention comprises a mixture of colloidal metals combined with a substance that is normally toxic to humans or animals capable of generating an immune response, wherein the composition comprises a human or animal When administered to humans or animals, it is less toxic or non-toxic. The composition optionally includes a pharmaceutically acceptable carrier, such as an aqueous solution, or an excipient, buffer, antigen stabilizer or bactericidal carrier. Oils such as paraffin oil may also be included in the composition if desired.

  The compositions of the invention can be used to vaccinate humans or animals against biologically active agents that are normally toxic when inoculated. Furthermore, the present invention can be used to treat certain diseases with cytokines or growth factors. By mixing a biologically active agent with a colloidal metal prior to administration to a human or animal, the toxicity of the biologically active agent is reduced or eliminated so that the agent has its therapeutic effect. It is possible to demonstrate. The combination of colloidal metal and such biologically active agent maintains or increases the therapeutic outcome while reducing toxicity, thereby improving efficacy. This is because higher concentrations of biologically active factors can be administered, or allows the use of a combination of biologically active factors. Therefore, the use of colloidal metals in combination with biologically active factors can result in higher concentrations of biologically active factors or previously unavailable toxic substances being administered to humans or animals. To be used to do.

  One embodiment of the present invention is the use of biologically active agents associated with colloidal metals as vaccine preparations. Among the many benefits of such a vaccine is the reduced toxicity of the normally toxic factor. A vaccine against a biologically active agent can be prepared by any method. One preferred method for preparing a vaccine against a biologically active agent is to mix the selected biologically active agent and the colloidal metal in a salt-free medium, preferably sterile water. That is. The salt-free medium can be buffered as necessary with, for example, a Tris buffer. In one embodiment of the invention, the colloidal metal solution is diluted 1: 1 with a solution of biologically active agent.

  This medium should preferably not contain sodium salts. The colloidal gold solution exhibits a light pink color and this color should not change when a solution of biologically active agent is added. If the colloidal gold solution changes from pink to purple, this indicates that gold has precipitated and cannot be reconstituted for effective immunity. The shelf life of this colloidal gold and biologically active agent mixture is about 24 hours.

  The mixture of biologically active agent and colloidal metal is then injected into the appropriate animal. For example, rabbits weighing between about 2-5 kg did not experience significant side effects after every 2 weeks of administration of a composition comprising colloidal gold and 1 mg of either IL-1 or IL-2 cytokines. . Since biologically active agents are non-toxic when administered according to the present invention, an optimal amount of antigen can be administered to the animal. The compositions according to the invention can be administered in a single dose or they can be administered in multiple doses spaced over an appropriate time scale to fully utilize the secondary immune response. For example, antibody titers are maintained by administering additional antigen once a month.

  The vaccine may further comprise a pharmaceutically acceptable adjuvant, including Freund's complete adjuvant, Freund's incomplete adjuvant, lipopolysaccharide, monophosphoryl lipid A, muramyl dipeptide, liposome containing lipid A, alum, mura Miltripeptide phosphatidylethanolamine, and keyhole limped hemocyanin include, but are not limited to. A preferred adjuvant is Freund's incomplete adjuvant, which is preferably diluted 1: 1 with a mixture of colloidal metal and biologically active agent.

  The amount of biologically active agent used in the present invention is between about 200 μg and 500 μg of bound protein in 25 ml of gold, which is then continuously concentrated to 200 μl. The ideal concentration to be administered in vivo is about 6.5 mg protein / kg body weight. The actual amount will vary depending on the particular patient and condition being treated. The amount of bound biologically active agent released in the body depends on the amount of protein initially bound to the colloidal metal and the total concentration of protein administered to the patient.

  The method of use of the composition comprises the step of administering to a human or animal an effective amount of the composition comprising a colloidal metal mixed with a biologically active agent, wherein the compound comprises It is less toxic or non-toxic when administered to humans or animals. The composition according to the invention can be administered as a vaccine against substances that are normally toxic or can be therapeutics, where the toxicity of normally toxic drugs is reduced, thereby over a longer period of time, Allows administration of larger amounts of drug.

  In the practice of the present invention, the process of administering the composition is not considered critical. The routes by which the composition may be administered according to the present invention include, but are not limited to, subcutaneous, intramuscular, intraperitoneal, oral and intravenous routes. A preferred route of administration is the intravenous route. Another preferred route of administration is intramuscular.

Interleukin-2 (IL-2) is known to show significant therapeutic results in the treatment of kidney cancer. However, its toxic side effects result in a significant number of patient deaths. In contrast, when IL-2 is mixed with colloidal gold, little or no toxicity is observed and a strong immune response results. The dose previously used for IL-2 treatment is an order of 21 × 10 ⁶ units of IL-2 per person weighing 70 kg per day (7 × 10 ⁶ units of IL-2 TID per 70 kg person) ). One unit is equal to about 50 picograms, two units are equal to about 0.1 nanograms, and thus 20 × 10 ⁶ units are equal to one milligram. In one embodiment of the invention, the amount of IL-2 given to the rabbit is about 1 mg per 3 kg rabbit. In effect, the study of the effects of administration of biologically active factors described herein included doses that were 20 times greater than those previously given to humans.

  In another embodiment, when IL-2 (1 mg per 3 kg animal) is administered to 3 rabbits every 3 days over a period of 2 weeks, all animals appear clinically ill and Two of them died from the obvious toxic effects of IL-2. When the same dose of IL-2 was combined with colloidal gold and then administered to 3 rabbits over the same 2 week period, no toxicity was observed and a significant antibody response occurred in all 3 animals. “Positive antibody response” as used herein is defined as a 3-4 fold increase in specific antibody responsiveness as determined by direct ELISA, comparing post-immune blood with pre-immune blood. Is done. Direct ELISA is performed by binding IL-2 onto the microtiter plate and determining the amount of IgG bound to IL-2 on the plate with goat anti-rabbit IgG conjugated with alkaline phosphatase. Is done. Therefore, it is considered that the biological effect of IL-2 remains. If a higher and more effective immune response is required because the toxic effects are minimized, higher concentrations of IL-2 can be administered if necessary.

  In another embodiment, the invention includes a method for treating a disease by administration of a composition comprising one or more biologically active agents bound to a colloidal metal. After administration, the biologically active agent is released from the colloidal metal. Without wishing to be bound by any theory, it is conceivable that the release is controlled not by a function of circulation time but by an equilibrium constant.

Colloidal metal: When a biologically active agent complex (“complex”) is incubated with cells for 25 days, only 5% of the biologically active factor is released from the colloidal metal. It was found that Thus, circulation time alone does not explain the mechanism by which biologically active factors are released from the complex in vivo. However, it has been found that the amount of biologically active released depends in part on the concentration of the complex in the body. When analyzing various dilutions of the complex (CytELIZA)
assay system CytImmune Sciences, Inc. , Collage Park, MD), it was found that a more dilute solution of the complex released significantly more biologically active agent. For example, essentially no release of biologically active factor was found in a 1: 100 dilution of the complex, but more than 35,000 pg of biologically active factor was present in the same sample complex Of 1: 100,000 dilution.

  Thus, the lower the concentration of the complex, the greater the amount of biologically active factor released. The higher the concentration of the complex, the lower the amount of biologically active factor released. Thus, due to the in vivo serial dilution of the complex with blood and extracellular fluid, the same therapeutic effect is achieved by administering to the patient a lower dose of biologically active agent than can be administered by previously known methods there's a possibility that.

  The amount of biologically active agent released from the colloidal metal: biologically active agent complex depends on the amount of biologically active agent initially bound to the colloidal metal. It has also been found. More biologically active agent is released in vivo from the complex with a higher amount of the first bound biologically active agent. Thus, one skilled in the art can control the amount of biologically active agent delivered by varying the amount of biologically active agent initially bound to the colloidal metal.

  These combined features allow a large amount of biologically active agent to be bound to the colloidal metal, thereby making the toxicity of the biologically active agent more toxic than if administered alone. Provides a way to lower. A small amount of colloidal metal: biologically active complex can then be administered to the patient, resulting in a slow release of the biologically active agent from the complex. This method provides an extended low dose biologically active / therapeutic agent for the treatment of diseases such as cancer and immune diseases.

  The compositions of the present invention are useful in the treatment of a number of diseases including cancers such as leukemia (including solid tumors and blood bearing tumors); autoimmune diseases such as rheumatoid arthritis; osteoporosis Hormone deficiency diseases such as: Hormonal abnormalities caused by hypersecretion such as acromegaly; Infectious diseases such as septic shock; Enzyme deficiency diseases (eg, phenylketonuria, inability to metabolize phenylalanine) Genetic diseases such as, and autoimmune diseases such as AIDS.

  The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations on the scope of the invention. On the contrary, means may be provided for various other embodiments, modifications, and equivalents thereof, after reading the description herein, the spirit and / or attachment of the present invention. It is clearly understood that themselves may be suggested to one of ordinary skill in the art without departing from the scope of the following claims.

Example I
This example demonstrates that colloidal gold neutralizes toxic substances and allows an antibody response if not neutralized. When IL-2 (1 mg per 3 kg animal) was administered to 3 rabbits every 3 days over a period of 2 weeks, all animals appeared clinically ill and 2 animals had obvious IL-2 Died of severe toxic effects. When the same dose of IL-2 is combined with colloidal gold and then administered to 3 rabbits over the same 2 week period, no toxicity is observed and a significant antibody response occurs in all 3 animals. A positive antibody response is defined as a 3-4 fold increase in specific antibody reactivity as determined by direct ELISA comparing post-immunized blood with pre-immunized blood. A direct ELISA is performed by binding IL-2 to a microtiter plate and determining the amount of IgG bound to IL-2 on the plate with goat anti-rabbit IgG conjugated to alkaline phosphatase.

Example II
This example further demonstrates that colloidal gold neutralizes otherwise toxic substances and allows for an antibody response. Endotoxin or lipid A (25, 50, and 100 μg per 35 mg mouse) is administered by subcutaneous injection every 4 days over a period of 2 weeks. For 10 mice, endotoxin is given in “neat” and the remaining 10 are mixed 1: 1 with endotoxin with colloidal gold. The injection volume is prepared by the addition of potassium carbonate / sodium citrate buffer (pH 6.5, diluted 1: 1). The same protocol is also used when lipid A is the test drug.

  Animals are checked 15 minutes, 30 minutes and 60 minutes after each injection and then checked hourly and daily. Surviving animals are tested for specific antibody responses to toxic substances injected simultaneously with either endotoxin or lipid A. Most animals injected with either endotoxin or lipid A in combination with colloidal gold survived, whereas animals injected with pure endotoxin or lipid A died during the 2-week study period. Furthermore, surviving animals had an antibody response to specific toxicants as determined by direct ELISA.

Example III
This example illustrates the effect of colloidal gold on cytokine activity in vivo. Groups of mice receive a dose of IL-2 that approximates the dose of IL-2 given to cancer patients undergoing immunotherapy. In previous experiments, 18 μg IL-2 tablets given to nude mice reduced the size of graft tumors, but animals were killed within 2 weeks. Paciotti, G .; F. , And Tamarkin, L .; , Interleukin-2 Differential Effects the Propagation of a Hormone-Dependent and a Hormone-Independent Human Breast Cancer Cell Line In Vitro and InC13. This is incorporated herein by reference.

  The effect of gold in a mouse model system is tested by the following procedure: a group of mice is treated with IL-2 alone, IL-2 mixed with colloidal gold, colloidal gold alone, or saline solution through an osmotic small pump. To do. Mice are treated for 7 days, after which they are sacrificed and their lymphocytes collected. The cells are stained for T cell markers or B cell markers using specific mouse monoclonal antibodies for flow cytometric analysis. Activated T cells and B cells are divided into T cell number, ratio of helper T cell to suppressor T cell, activated cellular IL-2 receptor, B cell number, and natural killer cell ("NK") number. Is determined by evaluating

  The few surviving animals treated with IL-2 alone showed an increase in T cell number and activity (as determined by IL-2 receptor). Virtually all animals survived IL-2 treatment in combination with colloidal gold, and these animals (as determined by activated B cells and total IgG, as measured by direct ELISA) both B It showed an increase in cell function, an increase in T cell function (as determined by T cell number and activity using the number of IL-2 receptors as an indicator of activity), and an increase in NK activity.

Example IV
The following biological experiments show that colloidal gold reduces the toxicity of lipopolysaccharide (LPS). LPS is the lipid / sugar part of the bacterial cell wall. When injected into animals, this molecule mimics many of the clinical responses of septic shock. Therefore, mice were injected with various amounts of LPS in the presence or absence of colloidal gold. Specifically, Balbic mice were administered either 100 μg or 400 μg LPS in the presence or absence of colloidal gold (WE coli 055: B5 strain; 10 mg / ml (water); Difco Labs) ). The pH of the 15 nm colloidal gold mixture (EY Labs) was adjusted to about 10, while the pH of LPS was adjusted to 8 with 0.1 N NaOH. Subsequently, the appropriate amount (ie 10 μl for a 100 μg dose and 40 μl for a 400 μg dose) was added to 500 μl of colloidal gold. This mixture was allowed to stand for 30 minutes and subsequently injected (intraperitoneally) into mice.

  Within 12 hours after injection, all mice showed clinical signs of suppression and loss of activity. Within 24 hours after injection, the 400 μg dose of control mice began to die. By 72 hours, all control mice at a dose of 400 μg had died, while 75% of the mice treated with gold were alive and began to show signs of clinical improvement (ie, exercise). In addition, mice at 100 μg dose treated with gold were more active throughout the 36 hours of observation.

(Example V)
The following experiment describes the use of colloidal gold as a putative adjuvant for mouse antibody production against mouse IL-6. This experiment was performed for two goals: First, colloidal gold is used as an adjuvant that produces an immune response to the “self-antigen” (ie, produces an immune response against mouse proteins using a mouse model). Second, because IL-6 is one of the cytokines thought to be involved in cancer cachexia, metastasis, and sepsis, the ability to produce antibodies in its own system An advantage in vaccine production over -6 and similar endogenous compounds may be demonstrated.

  In short, the experiment is as follows. Several mice were immunized with a colloidal gold / mouse IL-6 mixture as described above. After about 3 weeks, mice were sacrificed and trunk blood was collected and analyzed for the presence of antibodies to mouse IL-6 by direct ELISA, as described above. The determination of antibody titer of serum in mice immunized with mouse IL-6 in combination with colloidal gold as a result of direct ELISA is shown in FIG. FIG. 3 demonstrates that mice generated an antibody response to mouse IL-6. This therefore indicates that gold is useful in producing antibodies against endogenous (ie, its own) toxins and cytokines that are thought to be involved in sepsis, cancer cachexia and metastasis.

  Based on these results, colloidal gold may also be useful for producing monoclonal antibodies in transgenic mice through an immune response against “self-antigens”. Any colloidal gold binding antigen can be used for immunization of transgenic mice, which results in the production of monoclonal antibodies in vivo.

Example VI
The following experiments show that cytokines mixed with colloidal gold retain their biological activity. The model used for these experiments is one of the models well known in the art. Paciotti, G .; F. And L. Tamarkin, Interleukin 1 direct regulates harmony-dependent human breast cancer cell proliferation in vitro, Mol. Endocrinol. 2: 459-464, 1988; and Paciotti, G .; F. , And L. Tamarkin, Interleukin-1 differentially synchronized estrogens-dependent and eastogen-independent human breast cells in the GoI I-phase. This model is based on the ability of IL-1, an estrogen-responsive human breast cancer cell, a cytokine that directly inhibits the growth of MCF-7. Briefly, IL-1 alone inhibits the growth of these cells through well-characterized IL-1 receptors on the surface of these breast cancer cells.

The following experiment demonstrates the ability of IL-1 to retain its biological activity when mixed with colloidal gold by determining the ability of IL-1 to inhibit the growth of these cells. Approximately 8,000 MCF-7 cells were plated in 24-well tissue culture plates. The next day, 15 nm gold particles were centrifuged at 14,000 rpm for 10 minutes and resuspended in sterile water. Human IL-1α was reconstituted in water to ^provide an initial stock of 5 × 10 ⁻⁵ M in water. The pH of gold and IL-1 was adjusted to about 8.0 using 0.1 M NaOH. Prior to mixing, IL-1 was diluted to 2 × 10 ⁻⁶ M, 2 × 10 ⁻⁸ M, and 2 × 10 ⁻¹⁰ M working stocks containing 250 μl gold (final volume = 0.5 ml). ). The gold control consisted of 250 μl gold and 250 μl sterile water. Each working stock was then further diluted 1/20 in tissue culture medium to final concentrations of 10 ⁻⁷ M, 10 ⁻⁹ M and 10 ⁻¹¹ M. These solutions were then added directly to MCF-7 cells along with appropriate controls. These data shown in FIG. 6 are the number of cells at various days after the addition of IL-1 with or without gold.

(Example VII)
The following experiment shows the efficiency of gold binding to cytokines. This experiment demonstrates that when the protein is combined with gold and then centrifuged, the protein is removed from the solution. In this experiment, IL-6 standard from ARI's Cytokit-6 was used. Prior to mixing, the pH of the gold and cytokine solution was adjusted to pH 9 using 0.1 N NaOH. This protein was pre-incubated with either gold or water prior to use in the ARI diagnostic kit for IL-6. Following this incubation, the colloidal gold IL-6 mixture was centrifuged and the supernatant was used to generate a standard curve. As can be seen in FIG. 3, gold was very effective at binding virtually all IL-6 within the dose range of the assay. This binding removes IL-6 from the supernatant. Even at the highest final concentration of IL-6 (1000 ng / ml), gold removed about 90% of IL-6 from the solution. This amount is based on the 1000 ng / ml OD of the IL-6 / gold supernatant, which is similar to the IL-6 standard 100 ng / ml alone.

Example VIII
The following examples show physical changes when mixing gold colloidal fluid with IL-6, potential antigens for vaccines. Although the gold particles are about 15 nm in size, they cannot be filtered through a 0.22 μm syringe filter. We believe this is due to the nature of the gold particles in this colloidal mixture. It is theorized that gold as a colloidal mixed form aggregates larger than individual spheres. Individual particles are smaller than the pore size of the filter, but this aggregate is larger and therefore unfilterable. However, the inventors have observed that once colloidal gold is incubated with protein, it is easily filtered through a 0.22 μm filter. Thus, the binding of cytokines appears to change the physical interaction between gold particles, which allows each gold particle to act as a 15 nm particle, allowing the particles to be easily filtered. This experiment defines the characteristics of antigen binding to colloidal metals.

Example IX
Human IL-2 was reconstituted in water at pH 11. 200 μg of IL-2 was incubated with 25 ml of colloidal gold for 24 hours. The colloidal gold-bound IL-2 solution was then centrifuged at 14,000 rpm for 20 minutes in a microcentrifuge at room temperature. The supernatant was then removed from the pellet. The IL-2 complex was then placed in each well of a 24-well plate and incubated at 37 ° C. for 25 days. Samples were removed from the wells on days 4, 5, 7, 8, 9, 11, 13, 17, 21, 23, and 25. The sample was centrifuged to remove the colloidal gold: IL-2 complex. The supernatant was frozen. After the last sample was collected and frozen, all samples were analyzed for IL-2 at CytImmune Science, Inc. Batch analysis with Cyt ELISA-2 sandwich EIA. The result of this assay is FIG.

(Example X)
Human TNFα was reconstituted in water at pH 11. 200 μg of TNFα was incubated with colloidal gold for 24 hours. The TNFα: colloidal gold solution was then centrifuged at 14,000 rpm for 20 minutes in a microcentrifuge at room temperature. The supernatant was then removed from the pellet. The pellet is then reconstituted in 1 ml water and diluted with milk buffer to a final concentration of 1: 100, 1: 1,000, 1: 10,000 and 1: 100,000 and room temperature And incubated for 24 hours. Samples were then obtained from CytImmune Science, Inc. Analysis was performed using the Cyt ELISA TNFα assay. The results of this assay are in FIG.

  This experiment shows that as a result of this dilution, the colloid releases more cytokines bound to it. Thus, the binding and release of cytokines by colloidal gold exhibits equilibrium kinetics applicable to in vivo conditions including stepwise dilution of colloidal gold with blood and extracellular fluid.

Example XI
500 μg of recombinant IL-2 was dissolved in 0.5 ml of pH = 11 water. To this solution was then added 25 ml of colloidal gold by the method described in Example 9. Balb / C mice were injected (intraperitoneally) with either 0.1 ml of colloidal gold-bound IL-2 or 100 μg of neat IL-2. The mice (4 mice / group / time point) were sacrificed at various time points (0, 0.5, 1.0, 1.5, 2.0, 3.0 and 24 hours) after injection, Blood was collected. The obtained serum was analyzed for IL-2 using CytImmune Science, Inc. Analysis by competitive EIA. Throughout the time point tested, the release of IL-2 from colloidal gold appeared to have a bimodal pattern (FIG. 7). One explanation for this may be the equilibrium state of IL-2 released from colloidal gold to the abdomen chich (and subsequently equilibrated with the blood pool).

Example XII
50,000 MCF-7 cells were plated into each well of a 6-well plate in 2 ml of IMEM without phenol red containing 10% CSS. The cells were grown to 70-80% confluence. 200 μg IL-1β was bound to colloidal gold using the method described in Example 9. After binding, the complexed material was centrifuged and blocked with 1% HSA solution. 100 μl of colloidal gold-bound IL-1β complex was added to each well and incubated for 1-5 days. The cells were then washed with 10% charcoal peeled fetal bovine serum (FBS) -free phenol red-free IMEM. The binding of colloidal gold: IL-1β complex to MCF-7 cells was detected by visualization of colloidal gold on the cell surface using bright field and phase contrast microscopy. Visualization by fluorescence microscopy using rabbit anti-human IL-1β internalized into cells, where colloidal gold / IL-1β remaining on the cell surface is internalized into cells determined as a negative signal between fluorescence and bright field image did. The results of this assay are illustrated in FIG.

  FIG. 10a is a bright field display of untreated MCF-7 cells (control), and FIG. 10b shows background fluorescence derived from non-specific binding of FITC-bound antibody. FIG. 10b illustrates a bright field display for MCF-7 cells treated with colloidal gold-binding IL-1β, and FIG. 10d shows MCF-7 treated with colloidal gold-binding IL-1β complex. Figure 2 illustrates FITC fluorescence and complex internalization for cells. Some of the colloidal gold: IL-1b complexes in these figures bind to the cell membrane as indicated by the bright spots and some are internalized into the cells as indicated by the dark spots. The

  This example shows that the colloidal metal: biologically active agent complex can bind to a cell surface receptor and subsequently the complex is internalized into the cell.

Example XIII
50,000 MCF-7 cells were plated in 2 ml of IMEM without phenol red containing 10% CSS in each well of a 6-well plate. The cells were grown to 70-80% confluence. The cells were then treated with 0.5 μg / ml TNFα, 5.0 μg / ml TNFα, or 50 μg / ml TNFα, 0.5 μg / ml IL-1β, 5.0 μg / ml IL-1β, 50 μg. / Ml of either medium of IL-1β.

  100 μg of IL-1β was bound to colloidal gold using the method described in Example 9. After conjugation, the complexed material was centrifuged and blocked with 1% HSA solution. 100 μl of colloidal gold-bound IL-1β complex was added to each well and incubated for 24-48 hours. The cells were then washed with IMEM without phenol red containing 10% CSS. Binding of IL-1β: colloidal gold: TNFα di-cytokine to MCF-7 cells was detected by visualization of cell surface colloidal gold using bright field or phase contrast microscopy .

  These data show that colloidal gold can bind two or more biologically active factors simultaneously, which can bind to the cell membrane and provide a targeted drug delivery system. Allows generation of the desired complex.

Example XIV
100 μg each of IL-6, IL-1β and TNFα were simultaneously bound to the same colloidal gold solution using the method described in Example 9. After centrifugation and blocking with HSA solution, the sample was obtained from CytImmune Science, Inc. for TNF-α. Used as an unknown in the sandwich assay. In this assay, monoclonal antibodies were used to capture TNF-α in the sample. This monoclonal antibody bound to TNF-α was then detected using a rabbit anti-human TNF-α antibody followed by an enzyme-labeled goat anti-rabbit antibody.

  To demonstrate that the same colloidal gold particles bound to IL-6, IL-1β and TNFα, a colloidal form coated with TNFα, IL-6 and IL-1β simultaneously using a monoclonal antibody against TNF-α. Three sets of quadruplicate samples containing gold particles were captured. To demonstrate tri-cytokine particles, one set of samples uses a TNF-α polyclonal antibody, another set uses an IL-6 polyclonal antibody, and the other set uses an IL-1β polyclonal antibody. Detected. All samples were detected simultaneously using goat anti-rabbit antibody. As expected, colloidal gold-bound TNF-α was easily captured and detected using a TNF-α monoclonal / polyclonal antibody binding pair. Furthermore, the same sample showed a significant amount of immune response for IL-6 and IL-1β, which was only possible when three cytokines bound to the same particle. The results of this assay are illustrated in FIG.

  These data indicate that colloidal gold can bind two or more biologically active factors simultaneously, which can bind to the cell membrane and provide a targeted drug delivery system. To obtain the desired complex. These complexes of interest can also be used to generate immune responses against “self-antigens” and to generate multiple monoclonal antibodies for immunization of transgenic mice. For example, the complex of interest from this experiment can be used to induce simultaneous production of TNFα, IL-6 and IL-1β monoclonal antibodies.

(Example XV)
The following is a general experimental protocol after molecular binding to colloidal gold. This molecule was reconstituted in water. 200 μg of molecules were incubated with 25 mL of colloidal gold for 24 hours. The molecule: colloidal gold complex solution was then centrifuged at 14,000 rpm for 20 minutes in a microcentrifuge at room temperature. The supernatant was then removed from the pellet.

Example XVI
50 μg of epidermal growth factor (EGF) was bound to 25 ml of 40 nm colloidal gold particles at pH = 11.0. This solution was shaken on a rocking platform for 24 hours. 50 μg (added as 50 μl) of targeting cytokine (ie IL-1β for target macrophages, IL-2 for target T cells, IL-6 for target B cells, and TNFα or Flt-3 ligand for target dendritic cells Were added to the EGF / Au solution and shaken for an additional 24 hours. The solution was then centrifuged at 14,000 rpm to separate the colloidal gold binding material and the colloidal gold unbound material. The supernatant was removed and the pellet reconstituted in 1 ml water containing 1% human serum albumin.

Example XVII
EGF was bound to colloidal gold (CG) using the procedure of Example 2. Tumor necrosis factor-α (TNF-α) was then bound to the EGF / CG complex using the procedure of Example 1 to generate an EGF / CG / TNF-α chimera.

Example XVIII
EGF was bound to colloidal gold (CG) using the procedure of Example 2. Interleukin-6 (IL-6) was then bound to the EGF / CG complex using the procedure of Example 1 to generate an EGF / CG / IL-6 chimera.

Example XIX
EGF was bound to colloidal gold (CG) using the procedure of Example 2. Interleukin-2 (IL-2) was then bound to the EGF / CG complex using the procedure of Example 1 to generate an EGF / CG / IL-2 chimera.

(Example XX)
The buffy coat was separated from the whole blood sample as is well known in the art. 100-500 mL whole blood was collected into heparin. The blood was carefully overlaid onto 50% (v / v) Ficoll-Hypaque solution and centrifuged at 2700 rpm for 7 minutes. The buffy coat, or leukocyte collection at the serum / Ficoll interface, was collected using a Pasteur pipette and placed in 10 mL PBS containing 0.5 mg / mL heparin. This was centrifuged at 1500 rpm and the pellet was washed and recentrifuged. The cells were washed twice in PBS solution and centrifuged once more.

The cells were resuspended in RPMI containing either 10% fetal calf serum or normal human serum and cultured in 6-well plates at a cell density of 10 ⁶ cells / well. The cells were then stimulated with 50-100 μL of one or all antigen / cytokine mixtures.

  As shown in FIG. 12, only macrophages internalized the EGF / CG / IL-1β chimera, whereas only dendritic cells internalized the EGF / CG / TNF-α chimera (FIG. 13). Similarly, only B cells internalized the EGF / CG / IL-6 chimera (FIG. 14) and only T cells internalized the EGF / CG / IL-2 chimera (FIG. 15).

  As shown by this experiment, specific component-specific immunostimulators are specific for individual immune components. Thus, it is possible to target specific immune components using component-specific immunostimulatory factors. Thereby, this targeting enhances their immune response and results in increased activity in the overall immune response.

Example XXI
For this experiment, staphylococcal endotoxin B was used as a putative antigen / vaccine molecule. This is because there is evidence that toxin binding to colloidal gold reduces its toxicity. 500 μg of toxin was first bound to 250 ml of 40 nm colloidal gold particles. The colloidal liquid was then aliquoted. 50 μg of targeted cytokines (IL-1β, IL-2, IL-6 and TNFα) were added to one of the aliquots and reincubated for 24 hours. The toxin-AU-cytokine colloid was centrifuged at 14,000 rpm and the supernatant was removed. The pellet was reconstituted in 1 ml of water. The pellet was assayed for cytokine concentration by either sandwich or competitive ELISA. This was done to determine the amount of pure (unbound) cytokine injected into control animals receiving saline or toxin alone.

  This immunization plan involved the simultaneous or sequential administration of a pure toxin / cytokine mixture (as a composition control) or a toxin-Au-cytokine chimera. Five mice / group were injected on days 1, 5, and 9 with 2.5 μg of pure toxin or the same dose of toxin / cytokine mixture conjugated with colloidal gold. During the 14 day immunization period, two more groups of mice received pure toxin / cytokine or toxin-Au-cytokine according to the schedule provided in Table 1.

All groups were reinoculated on day 30 with 1 μg of pure toxin alone. Protective immunity was demonstrated by reducing or lacking the ability of pure toxins and induced morbidity. An important observation is that the toxin bound to colloidal gold greatly reduced the toxicity of the toxin. Second, the titer of serum antibody to the toxin was 10 times higher than the titer that received pure treatment only. However, the serum antibody of the animals that received the subsequent treatment was 100 times greater than the animals that received the pure treatment. Finally, upon re-inoculation with pure toxin, 100% of the animals treated with toxin were killed, while only 20% of the fatality rates were observed in the simultaneous group.

  Thus, the compositions and methods of the present invention can be used to increase the effectiveness of a vaccine.

Example XXII
This experiment demonstrates the delivery of the bacterial gene, β-galactosidase (b-gal), to human breast cancer cells MCF-7 using colloidal gold conjugated to EGF. For this experiment, β-gal plasmid DNA was directly bound to 40 nm-EGF paired with colloidal gold particles. 25μ by the method disclosed in the related patent application. Once free EGF was separated from the bound fraction, 5 μg of pSV β-galactosidase plasmid was incubated overnight with EGF / Au on a rocking platform. This material was centrifuged and the pellet was reconstituted to 1 ml with water at pH = 11. 100 μl was added to MCF-7 cells growing in 6-well culture plates.

EGF / DNA Au was incubated with cells for 48 hours to allow receptor-mediated endocytosis of molecular chimeras. Subsequently, the cells were washed twice with serum-free medium, twice with PBS and then fixed with a 0.25% glutaraldehyde solution (in PBS) for 15 minutes. The fixed cells were washed 4 times with PBS. β-galactosidase substrate (X-gal) as a 0.2% solution in a buffer containing ₂ mM MgCl ₂ , 5 mM K ₄ Fe (CN) ₆ .3H ₂ O and 5 mM K ₃ Fe (CN) ₆ , Added to. The cells and substrate solution were incubated overnight.

  Successful cellular gene transfection and expression is indicated by conversion of the X-gal substrate to a green product. Transfection is confirmed by the presence of green staining on the cells. In fact, the presence of staining indicates that the gene has been delivered and transcribed and that the biologically active enzyme has been translated.

  Only a few cells stained positive for β-gal enzyme activity were observed. This could be due to a number of factors including poor binding efficiency between DNA and colloidal gold, lysosomal capture and / or poor transcription efficiency.

Example XXIII
This experiment was designed to investigate whether the lack of transfection in Example 2 was due to poor binding of plasmid DNA to gold. To test this, gold chimeras were used as targeting proteins and DNA binding proteins such as histones (added as either a heterogeneous mixture of all isotypes or individual isotypes), polylysine or protamine sulfate. Produced and adsorbed to DNA. 25 μg of EGF was bound to 40 nm colloidal gold particles as described above. Subsequently, histone, polylysine or protamine sulfate was added to the EGF Au solution at various concentrations ranging from 0.1-100 μg / ml.

  Upon addition of the DNA binding moiety, we observed that the colloidal gold EGF solution became cottony and subsequently precipitated. The precipitate was sonicated and centrifuged and resuspended to a final volume of 1 ml in water. 15 μg of pSV β-galactosidase plasmid was incubated overnight on a rocking platform. This material was centrifuged again, the supernatant was removed, and 0.5 ml of water was added to the pellet. This pellet was sonicated again and 0.1 ml of the solution was grown in 24-well plates using phenol red-free IMEM supplemented with 10% charcoal exfoliated fetal calf serum. Added to any of the -578-T human breast cancer cells. The cells and gold / protein / DNA chimera were incubated with each other for 48 hours while it was taken up by the cells.

  As can be seen in FIG. 16, the β-galactosidase gene was effectively transfected into MCF-7 cells. This is because the green strain is found in almost all cells. More interestingly, HS578-T cells known to have EGF receptors only showed chimeric extracellular binding (see FIG. 17). This suggests that the EGF receptor system does not undergo internalization in this cell line.

(Example XXIV)
A dialysis cassette was used to show the effect of pH change on colloidal gold particles. A dialysis cassette (Pierce; Slide-A-Lyzer ^™ ; cut-off molecular weight 2000) was loaded with colloidal gold particles as described in the present invention. This cassette was then placed in one of two MES buffered solutions. MES is 2- [N-morpholino] ethanesulfonic acid. The pH of the primary solution was 7.4 and the pH of the secondary solution was 5.6. The cassette was dialyzed for 24 hours. Subsequently, the colloidal gold solution in the cassette was collected, placed in a 6-well culture plate, and photographed. After 24 hours at pH 7.4, the colloidal gold particles remain unchanged and remain suspended in the MES buffer. In the cassette with the pH of the MES buffer changed to 5.6, after 24 hours there was a black precipitate in the cassette, indicating the precipitation of colloidal gold particles. This example described potential changes in the state of colloidal gold when the pH of the surrounding environment was acidified. This is similar to endosomal acidification after internalization.

(Example XXV)
Streptavidin bound to colloidal gold showed saturable binding kinetics. For this experiment, 500 μg streptavidin was bound to 50 ml of 32 nm colloidal gold for 1 hour. Subsequently, 5 ml of stabilizing solution (5% PEG 1450, 0.1% BSA) was added to the tube and allowed to mix for an additional 30 minutes. The solution was centrifuged to remove unbound streptavidin and washed twice with 5 ml stabilization solution. After the final spin, the pellet was reconstituted to a volume of 5 ml with the stabilizing solution. A 1 ml aliquot was dispensed into a microcentrifuge tube. To these tubes, increasing amounts of biotinylated human TNFα were added. Biotinylated cytokines were incubated with streptavidin gold for 1 hour. This material was centrifuged at 10,000 rpm for 10 minutes. The resulting supernatant was collected and saved for TNF determination. The pellet from each tube was washed once with the stabilization solution and re-centrifuged. The spin-derived supernatant was discarded. The pellet was reconstituted to 1 ml with the stabilizing solution. Both pellet and initial supernatant were then assayed for TNF concentration using our CYTELISA ^™ TNF kit. Greater than 90% of biotinylated TNF immunoreactivity can be found in the pellet (FIG. 2), indicating that biotinylated TNF was captured by streptavidin bound to gold.

(Example XXVI)
This experiment was to evaluate the feasibility of streptavidin gold complex as a targeted drug delivery system. In order for this to occur, streptavidin-conjugated colloidal gold must bind to both the biotinylated targeting ligand and the biotinylated therapeutic agent. In order to examine this, the present inventors performed the following experiment.

  100 ml of 32 nm colloidal gold solution was bound using a saturating concentration of streptavidin. After 1 hour, the solution was centrifuged and washed as above. The colloidal gold bound to streptavidin was then bound using a subsaturating concentration of biotinylated cytokine. This material was vortexed and incubated for 1 hour at room temperature. The solution was then centrifuged and the pellet was incubated with a solution of biotinylated polylysine. After 1 hour incubation, the solution was recentrifuged and washed. After final spin and resuspension (final volume of solution was about 1 ml), 50 μg of β-galactosidase reporter gene was incubated with concentrated streptavidin / biotinylated cytokine / polylysine chimera for 1 hour. This material was centrifuged to remove unbound plasmid DNA. The final construct (biotin EGF-SAP-Au-biotin polylysine-DNA) was centrifuged at 14,000 rpm. The supernatant was assayed for the presence of DNA by determining its OD at 260 nm. We observed 0.95 to 0.25 reduction in OD at 260 nm of the supernatant after incubation of plasmid DNA with the biotin EGF-SAP-Au-biotin polylysine construct. This DNA was bound by biotin EGF-SAP-Au-biotin polylysine-DNA and centrifuged from solution into a pellet. These data indicate that a new drug delivery system has been developed using avidin binding to colloidal gold. Targeting and biotinylation of the delivery payload were then used as a method for binding these molecules to colloidal gold based on drug / gene delivery systems.

  It will be appreciated that the foregoing relates only to particular embodiments of the invention and that many modifications or changes deviate from the spirit and scope of the invention as set forth in the appended claims herein. It should be understood that it can be done without.

  This patent contains at least one color photograph. Copies of this patent containing color photographs will be provided by the US Patent Office upon request and payment of the necessary costs.

Claims (1)

The invention described herein.