Brain-penetrable multifunctional system and its use

Abstract

The present invention provides a BBB-permeable multifunctional system for synchronous delivery of separate active substances to the brain. The multifunctional system is based on an inorganic core particle that is conjugated to a first active agent via a first polymer linker, to a second active agent via a second polymer linker, and to a brain-internalizing transporter moiety via a third polymer linker. The preparation process of the multifunctional system, the pharmaceutical composition comprising the multifunctional system, and its use in treatment and / or diagnosis methods are further provided.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention is in the field of brain-targeted delivery systems for therapeutic and diagnostic applications. [Background technology]

[0002] 2. Background of the Invention In particular, a significant problem in the treatment of brain-related diseases, including neurodegenerative disorders and diseases, as well as brain tumors, is the difficulty of delivering important therapeutic and diagnostic agents to the brain through the blood-brain barrier (BBB). The BBB is a highly selective semipermeable boundary that separates the circulating blood from the central nervous system (CNS). The BBB primarily serves as a protective barrier for the brain, preventing the transfer of various elements, including hormones, neurotransmitters, or neurotoxins, from the bloodstream into the central nervous system. Specific and selective transporters present on the BBB supply glucose, free fatty acids, amino acids, vitamins, minerals, and electrolytes to the CNS, but nearly all high molecular weight (MW) drugs and more than 98% of low molecular weight drugs cannot cross the BBB.

[0003] To overcome the limitations associated with the BBB, various strategies have been explored. Among the many strategies, the exploitation of the transcytosis transport pathway of endogenous receptors expressed in brain capillary endothelium represents a promising approach to cross the cellular barrier. Based on this transport pathway, various nanomaterial-based drug delivery systems have been developed.

[0004] US Patent No. 10,182,986 is directed to a method of delivering nanoparticles across the blood-brain barrier to the brain of a subject by administering to the subject nanoparticles having a nanoparticle core and a targeting agent.

[0005] Ruan, Shaobo et al. (Biomaterials 37(2015):425-435) provided a gold nanoparticle-based delivery system loaded with doxorubicin (DOX) via an acid-responsive linker, hydrazone, and functionalized with angiopep-2, a specific ligand of low-density lipoprotein receptor-related protein-1 (LRP1), which could mediate the system to target glioma cells through the blood-brain barrier.

[0006] Shilo, Malka et al. (Nanoscale 6.4 (2014): 2146-2152) developed a technique directed at transporting insulin-targeted gold nanoparticles (INS-GNPs) through the blood-brain barrier for imaging and therapeutic applications.

[0007] Recent advances in cell biology have led to a paradigm shift in the treatment of various intractable diseases from the "one drug, one target" approach to combination therapy and multi-targeting drug approaches. However, although drug combinations may theoretically be therapeutically effective in treating various diseases, their clinical success is limited due to differences in the pharmacokinetics and tissue distribution of each drug in the combination.

[0008] To overcome these limitations, various approaches have been developed. Bispecific antibodies (bsAbs) are artificial proteins that combine the specificities of two antibodies in one molecule, interfering with multiple surface receptors or ligands simultaneously. bsAbs can also bring targets into close proximity to support the formation of protein complexes on one cell or induce cell-cell contacts.

[0009] Recently, nanoparticles have emerged as a promising platform for co-delivery of multiple drugs. Zhang, Tian et al. (Advanced healthcare materials 8.18(2019):1900543) provide a multi-targeted nanoparticle that delivers synergistic drugs to brain metastases of triple-negative breast cancer cells and tumor-associated macrophages through the blood-brain barrier.

[0010] Dixit et al. (Molecular pharmaceutics 12.9(2015):3250-3260) disclosed bi-receptor targeted theranostic nanoparticles for localized delivery and activation of photodynamic therapy drugs in glioblastoma.

[0011] However, there remains an unmet need for a multifunctional system that allows for the simultaneous and synchronized delivery of multiple bioactive agents to the brain to improve the effectiveness of the treatment of brain-related diseases or disorders. Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention provides a multifunctional system for co-delivery of at least two distinct active agents into the brain. The present invention further provides a method for preparing the multifunctional system and its use for the treatment of brain-related diseases or disorders. [Means for solving the problem]

[0013] The multifunctional system of the present invention is based on a core particle conjugated to a first active agent and a second active agent via a first polymer linker and a second polymer linker, respectively, and to a brain-internalizing transporter moiety via a third polymer linker.

[0014] The active agent conjugated to the core particle can include various types of therapeutic and / or diagnostic molecules of interest. In particular, it has been found that two different antibodies, or an antibody and a small molecule drug, which have poor BBB permeability in their original form, can be conjugated to a single core particle and further conjugated to insulin as a brain-internalizing transporter moiety, and efficiently penetrate the mouse brain after intravenous administration.

[0015] Moreover, by using two different fluorescently labeled antibodies conjugated to core particles, the antibodies are found to co-localize in specific brain regions.Therefore, the multifunctional system of the present invention not only facilitates the brain penetration of active agents, but also provides synchronous distribution of active agents in the brain.Advantageously, the synchronous distribution of different therapeutic agents can significantly improve the therapeutic efficacy of drug combinations.

[0016] The present invention is further based in part on the discovery that the relative lengths of the first, second and third polymer linkers have a significant effect on the penetration of the multifunctional system through the BBB. In particular, efficient BBB penetration can be achieved by using polymer linkers of different sizes to conjugate insulin and active agents to the core particle.

[0017] One beneficial feature of the system of the present invention is that the activity of the therapeutic agent conjugated to the delivery system remains intact, so that it does not necessarily have to be detached from the nanoparticle after crossing the BBB, for example, by using a cleavable linker. Alternatively, the delivery system may contain different therapeutic agents, at least one of which is not cleavable. Effect of the Invention

[0018] According to one aspect, (a) an inorganic particle bound to at least (i) a first linear polymer linker; (ii) a second linear polymer linker; and (iii) a third linear polymer linker; (b) a first biologically active molecule conjugated to a first linear polymer linker; (c) a second biologically active molecule conjugated to a second linear polymer linker; and (d) a brain-internalizing transporter moiety conjugated to a third linear polymer linker. A multifunctional particle comprising: the third linear polymer linker has a length substantially different from the lengths of the first and second linear polymer linkers; the molecular weight (MW) of the third polymer linker differs from the molecular weights of the first and second polymer linkers by at least about 1000 Da; Multifunctional particles are provided in which the first bioactive molecule is different from the second bioactive molecule.

[0019] According to some embodiments, the length of the third linear polymer linker is substantially longer than the length of the first and second linear polymer linkers.

[0020] According to some embodiments, the first, second and third polymer linkers are non-cleavable under physiological conditions.

[0021] According to some embodiments, the multifunctional particle comprises an additional polymer linker that is cleavable under physiological conditions. According to some embodiments, the cleavable polymer linker is conjugated to a chemotherapeutic drug or a toxin.

[0022] According to some embodiments, the molecular weight of the first and second linear polymer linkers is in the range of 1,000 to 10,000 Da, and the molecular weight of the third linear polymer linker is in the range of 2,000 to 11,000 Da. In certain embodiments, the molecular weight of the third linear polymer linker is higher than the molecular weight of the first and second linear polymer linkers.

[0023] According to some embodiments, the third linear polymer linker is composed of repeating monomer units, and at least one of the first and second linear polymer linkers is composed of the same repeating monomer units as the third linear polymer linker, and the third linear polymer linker has a different number of repeating monomer units than at least one of the first and second linear polymer linkers. In certain embodiments, the first, second and third linear polymer linkers are composed of the same repeating monomer units, and the third linear polymer linker has a different number of repeating monomer units than that of the first and second linear polymer linkers.

[0024] According to some embodiments, the first polymer linker and the second polymer linker are the same.

[0025] According to some embodiments, the first and second linear polymer linkers are attached to the inorganic particle via sulfide bonds, and the first and second biologically active molecules are conjugated to the respective linear polymer linkers via amide bonds.

[0026] According to some embodiments, the first and second bioactive molecules are independently selected from the group consisting of antibodies, peptides, small molecules, oligonucleotides, antisense RNA, and any fragments or combinations thereof. In certain embodiments, both the first and second bioactive molecules are antibodies or fragments thereof. In other embodiments, the first bioactive molecule is an antibody or fragment thereof and the second bioactive molecule is a small molecule.

[0027] According to some embodiments, the third linear polymer linker constitutes about 10 mol % to 40 mol % of the total polymer linkers attached to the inorganic particles.

[0028] According to some embodiments, each of the first and second linear polymer linkers independently constitutes between about 5 mol % and 40 mol % of the total polymer linkers attached to the inorganic particles.

[0029] According to some embodiments, the first, second, and third linear polymer linkers independently comprise a polymer selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof. According to certain embodiments, at least one of the first, second, and third linear polymer linkers is a polyether. In some exemplary embodiments, the polyether is polyethylene glycol (PEG). Polyethylene glycol, according to some embodiments, is selected from the group consisting of thiolated PEG acid (HS-PEG-COOH), thiolated PEG amine (HS-PEG-NH 2), and thiolated PEG thiol (SH-PEG-SH), with the thiolated end attached to the inorganic particle and the acid, amine or other thiol end conjugated to a brain internalizing transporter moiety or respective bioactive molecule. In another embodiment, the polyethylene glycol is SH-PEG-SH, with one thiolated end attached to the inorganic particle and the other thiol end conjugated to a chemotherapeutic drug or toxin. According to some embodiments, the multifunctional particle further comprises a fourth polymer linker attached to the inorganic particle, which serves a monofunctional purpose in capping the terminal functional groups on the particle and helps to allow distance between the molecules conjugated to the particle, hereafter understood to be interchangeable with the term "cap". According to some embodiments, the fourth polymer linker comprises a polymer selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof. In some exemplary embodiments, the fourth polymer linker comprises a polyether, and the polyether is methoxypolyethylene glycol (mPEG).

[0030] According to some embodiments, the inorganic particles are nanoparticles selected from the group consisting of metal nanoparticles, metal oxide nanoparticles, ceramic nanoparticles, and any combination thereof. The metal, according to some embodiments, is selected from the group consisting of gold, silver, platinum, iron, and any combination thereof. The metal oxide can be selected from the group consisting of iron oxide, magnesium oxide, nickel oxide, cobalt oxide, aluminum oxide, zinc oxide, copper oxide, manganese oxide, and any combination thereof. In some particular embodiments, the inorganic particles are selected from the group consisting of gold nanoparticles, iron(III) oxide nanoparticles, and iron(II,III) oxide nanoparticles. In further particular embodiments, the inorganic particles are gold nanoparticles.

[0031] According to some embodiments, the inorganic particles are nanoparticles having a diameter of 10 to 160 nm.

[0032] According to some embodiments, the brain-internalizing transporter moiety is selected from the group consisting of insulin, an antibody specific for the insulin receptor, transferrin, an antibody specific for the transferrin receptor, a polypeptide that specifically binds to the transferrin receptor, a polypeptide that specifically binds to the insulin receptor, insulin-like growth factor 1 (IGF-1), an antibody specific for IGF-1, a polypeptide that specifically binds to the insulin-like growth factor receptor 1, apolipoprotein A1, B, or E, lactoferrin, angiopep-2, low density lipoprotein, an antibody specific for the low density lipoprotein receptor or lipoprotein receptor-related protein, a polypeptide that specifically binds to the low density lipoprotein receptor or lipoprotein receptor-related protein, an antibody specific for the diphtheria toxin receptor, a polypeptide that specifically binds to the diphtheria toxin receptor, a BBB-permeable cell penetrating peptide (CPP), and any combination thereof. In certain embodiments, the brain-internalizing transporter moiety is insulin or a derivative, analog, conjugate, or fragment thereof.

[0033] According to some embodiments, the multifunctional particle further comprises a third bioactive molecule, which is conjugated to a linear polymer linker that is attached to the inorganic particle. According to some embodiments, the third bioactive molecule is a chemotherapeutic moiety or a toxin, which is conjugated to the particle by a SH-PEG-SH linker. According to some embodiments, the linker is cleavable, and the chemotherapeutic moiety or the toxin is released in the brain.

[0034] According to some particular embodiments, the inorganic particles are gold nanoparticles, the first linear polymer linker and the second linear polymer linker are each independently a thiolated PEG3500 acid or a thiolated PEG3500 amine, the third linear polymer linker is a thiolated PEG5000 acid or a thiolated PEG5000 amine, the brain-internalizing transporter moiety is insulin, and the chemotherapeutic agent is conjugated via a cleavable thiolated PEG3500 thiol linker.

[0035] According to some exemplary embodiments, the inorganic particles are gold nanoparticles, the first linear polymer linker and the second linear polymer linker are each independently a thiolated PEG3500 acid or a thiolated PEG3500 amine, the third linear polymer linker is a thiolated PEG5000 acid or a thiolated PEG5000 amine, and the brain-internalizing transporter moiety is insulin.

[0036] According to some exemplary embodiments, the inorganic particles are gold nanoparticles, the first linear polymer linker is a thiolated PEG1000 acid or a thiolated PEG1000 amine, the second linear polymer linker is a thiolated PEG3500 acid or a thiolated PEG3500 amine, the third linear polymer linker is a thiolated PEG5000 acid or a thiolated PEG5000 amine, and the brain-internalizing transporter moiety is insulin.

[0037] According to another aspect, there is provided a process for preparing a multifunctional particle according to the various embodiments described above, comprising the consecutive steps of: (a) partially coating the surface of an inorganic particle with a first linear polymer linker, followed by conjugating the first linear polymer linker to a first biologically active molecule; (b) partially coating the surface of an inorganic particle with a second linear polymer linker, followed by conjugating said second linear polymer linker to a second biologically active molecule; and (c) partially coating the surface of an inorganic particle with a third linear polymer linker, followed by conjugating said third linear polymer linker to a brain-internalizing transporter moiety, wherein steps (a), (b) and (c) can be performed in any order.

[0038] According to some aspects and embodiments, a process for preparing a multifunctional particle includes the steps of: (a) partially coating a surface of an inorganic particle with a first linear polymer linker and a second linear polymer linker, followed by conjugating the first linear polymer linker and the second linear polymer linker to a first biologically active molecule and a second biologically active molecule, wherein the first linear polymer linker and the second linear polymer linker are identical and the first biologically active molecule is different from the second biologically active molecule; and (b) partially coating a surface of the inorganic particle with a first linear polymer linker and a second linear polymer linker, followed by conjugating the first linear polymer linker and the second linear polymer linker to a first biologically active molecule and a second biologically active molecule, wherein the first linear polymer linker and the second linear polymer linker are identical and the first biologically active molecule is different from the second biologically active molecule; A process is provided that includes the sequential steps of partially coating a surface with a third linear polymer linker, followed by conjugating the third linear polymer linker to a brain internalizing transporter moiety, wherein the length of the third linear polymer linker is substantially different from the length of the first and second linear polymer linkers, and the molecular weight of the third polymer linker differs from the molecular weight of the first and second polymer linkers by at least about 1000 Da, and wherein steps (a) and (b) can be performed in any order.

[0039] According to some embodiments, a first polymer linker has a first functional end group configured to bind to a first biologically active molecule, a second polymer linker has a second functional end group configured to bind to a second biologically active molecule, and a third polymer linker has a third functional end group configured to bind to a brain-internalizing transporter moiety, and at least two of the first, second and third functional end groups are identical.

[0040] According to some embodiments, the process further comprises partially coating the surface of the inorganic particles with a fourth polymer linker, said fourth polymer linker being a monofunctional linker used to cap functional groups on the particles and allow distance between molecules conjugated to the particles.

[0041] According to some embodiments, each of the first linear polymer linker and the second linear polymer linker is added in an amount suitable to cover 5% to 40% of the surface of the inorganic particle, and the third linear polymer linker is added in an amount suitable to cover 5% to 40% of the surface of the inorganic particle.

[0042] According to some embodiments, the particles are gold nanoparticles (GNPs) and the process for preparing multifunctional gold nanoparticles comprises: (a) dissolving HAuCl 4 (b) reduction of the GNPs; (b) simultaneous incubation of the reduced GNPs with one type of monofunctional linker and two different types of heterofunctional linkers; (c) activation of the GNPs to obtain free COOH groups; (d) conjugation of a transporter or other moiety; and (d) conjugation of two different biologically active molecules by incubation with a solution containing a mixture thereof.

[0043] According to some embodiments, the monofunctional linker is mPEG-SH. According to certain embodiments, the monofunctional linker is mPEG6000-SH or PEG5000-SH, and is added so as to cover about 80 to 90% of the particle surface.

[0044] According to some embodiments, the heterofunctional linker is COOH-PEG-SH. According to some embodiments, one heterofunctional linker is COOH-PEG5000-SH and is added at a concentration that covers about 15% of the particle surface. According to some embodiments, another heterofunctional linker is COOH-PEG3500-SH and is added at a concentration that covers about 5% of the particle surface.

[0045] According to some embodiments, activation of the GNPs is carried out by mixing the GNPs with (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC).

[0046] According to some embodiments, the transporter is insulin and its conjugation is carried out by incubation with activated GNPs at a concentration of about 50-500 IU / ml for 1-5 hours.

[0047] According to some embodiments, the two biologically active molecules are incubated overnight with the activated GNPs at a concentration of 1-50 mg / ml.

[0048] Analysis of the GNPs is performed after each step using methods known in the art.

[0049] According to some embodiments, analysis of the GNPs is performed using dynamic light scattering (DLS).

[0050] According to some embodiments, quantification of the bioactive molecules and transporters (e.g., insulin) attached to the PEG groups on the GNPs is performed by enzyme-linked immunosorbent assay (ELISA) of the supernatant containing the unbound proteins left after centrifugation precipitation of the GNPs.

[0051] According to yet another aspect, there is provided a pharmaceutical composition comprising a multifunctional particle according to the various embodiments set out above and a pharma- ceutically acceptable carrier, excipient, or diluent.

[0052] According to some embodiments, the pharmaceutical composition is for use in the prevention, treatment, and / or monitoring of a brain-related disease or disorder in a subject in need thereof.

[0053] According to some embodiments, the pharmaceutical composition is for use in the simultaneous delivery of at least two biologically active molecules to the brain of a subject.

[0054] According to some embodiments, the pharmaceutical composition is formulated for at least one of intravenous (IV), intranasal (IN), intraperitoneal (IP) and intrathecal (IT) administration. According to some embodiments, the pharmaceutical composition is for use in preventing, treating and / or monitoring a brain-related disease or disorder in a subject in need thereof.

[0055] According to a further aspect, there is provided a method for simultaneously delivering at least two biologically active molecules to the brain of a subject, comprising administering to the subject a pharmaceutical composition according to various embodiments presented hereinabove. According to some embodiments, upon administration, the at least two biologically active molecules show synchronous distribution in the brain.

[0056] According to another aspect, there is provided a method for preventing, treating and / or monitoring a brain-related disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutical composition according to the various embodiments herein above.

[0057] According to some embodiments, the pharmaceutical composition is administered to the subject by at least one of intravenous (IV), intranasal (IN), intraperitoneal (IP) and intrathecal (IT) administration.

[0058] According to some embodiments, the method further comprises imaging the subject's brain, thereby evaluating the accumulation of multifunctional particles in the subject's brain.Imaging can be performed using any imaging method or system known in the art, including but not limited to imaging systems selected from the group consisting of computed tomography imaging (CT), X-ray imaging, magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), ultrasound (US), and any combination thereof.

[0059] According to some embodiments, the brain-related disease or disorder is primary brain cancer or secondary brain cancer. According to some embodiments, the brain cancer is a primary solid tumor. According to some embodiments, the brain tumor comprises a metastasis. According to some embodiments, the metastasis is from a tumor originating from a tissue other than the brain. According to some embodiments, the metastasis originates from a cancer selected from the group consisting of breast cancer, lung cancer, melanoma, renal cancer and colorectal cancer. According to some embodiments, the metastasis is a breast cancer metastasis.

[0060] According to some embodiments, the brain-related disease or disorder is primary brain cancer or secondary brain cancer; the inorganic particles are radiosensitizers; and the method further comprises radiation therapy.

[0061] Further embodiments and the full scope of the present invention will become apparent from the detailed description set forth below. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0062] [Figure 1]FIG. 1 depicts a schematic diagram of a gold nanoparticle (GNP; 1) bound to: (i) a first polymer linker (2) conjugated to a first antibody (3); (ii) a second polymer linker (4) conjugated to a second antibody (5); (iii) a third polymer linker (6) conjugated to insulin (7); and (iv) a monofunctional polymeric capping moiety (8). [Diagram 2] FIG. 2 is a bar graph showing quantification of the amount of gold (Au) (mg) found in the brain of mice 8 hours after intravenous administration of IgG1&Ins-GNP, Iba1&Ins-GNP, and IgG1&Iba1&Ins-GNP as measured by inductively coupled plasma optical emission spectroscopy (ICP-OES). [Diagram 3] Figure 3 shows immunocytochemistry fluorescence (IHC-F, double staining) images of cortical brain sections obtained from untreated mice (control, left) or mice intravenously injected with IgG1&Iba1&Ins-GNPs (middle) or mice intravenously injected with free fluorescently labeled antibodies (right). The top images show Iba1 labeling and 4',6-diamidino-2-phenylindole (DAPI) staining; the middle images show IgG1 labeling and DAPI staining; and the bottom images show merged images of IgG1 labeling and Iba1 labeling (without DAPI labeling). [Figure 4] Figure 4 shows immunohistochemical fluorescence (IHC-F, double staining) images of brain sections from the medulla region obtained from untreated mice (control, left), or mice intravenously injected with IgG1&Iba1&Ins-GNPs (middle) or free fluorescently labeled antibodies (right). The top images show Iba1 labeling and DAPI staining; the middle images show IgG1 labeling and DAPI staining; and the bottom images show merged images of IgG1 labeling and Iba1 labeling (without DAPI labeling). [Figure 5A] FIG. 5A: Quantification of the amount of Au (mg) found in mouse brain tissue 8 hours after intravenous administration of cisplatin (cisPt) and insulin (Ins) particles, cisPt&Ins-GNPs, IgG1&Ins-GNPs, cisPt&IgG1&Ins-GNPs or free cisplatin as measured by ICP-OES analysis. [Figure 5B] FIG. 5B: Quantification of the amount of Pt (mg) found in mouse brain tissue 8 hours after intravenous administration of cisPt&Ins-GNPs, free cisplatin, or cisPt&IgG1&Ins-GNPs as measured by ICP-OES analysis. [Figure 6] FIG. 6: Schematic diagram of a gold nanoparticle (GNP; 1) bound to: (i) a first polymer linker (2) conjugated to insulin (4); (ii) a second polymer linker (3) conjugated to a first antibody (5) and a second antibody (6); and (iii) a capping polymer moiety (7). [Figure 7] Figure 7A: MRI scan results of the brain of mice inoculated with breast cancer BT474 cells and treated with free antibody (Free Ab) or bifunctional GNPs (GNP-Ab). Treatment compositions (40 mg / kg Ab) were IP injected once a week for 4 consecutive weeks. [Figure 7B] FIG. 7B: Top - Images of excised brains examined by ICP-OES for GNP penetration and tumor accumulation. Brains were excised from mice bearing breast cancer BT474 cells and treated with free antibody (Free Ab), or with bifunctional GNPs (GNP-Ab), or left untreated. Bottom - Cross-sectional images of brains from mice treated with GNP antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] Detailed Description of the Invention The present invention provides a general BBB-permeable platform for synchronously delivering separate active substances to the brain.In particular, the present invention provides a multi-functional system for simultaneously co-delivering at least two separate active ingredients into the brain, the preparation process of the system, the pharmaceutical composition comprising said system, and their use for therapeutic and diagnostic applications.

[0064] The multifunctional delivery system is based on a core particle conjugated to a first active agent via a first polymer linker, to a second active agent via a second polymer linker, and to a brain-internalizing transporter moiety via a third polymer linker. Each of the first and second active agents can be a bioactive molecule (e.g., a drug) or a labeling molecule, and can include different types of molecules, such as, but not limited to, polypeptides, antibodies, peptides, oligonucleotides, and small molecules. Without being bound by any theory or mechanism, it is hypothesized that the brain-internalizing transporter moiety facilitates the penetration of the entire conjugate system through the BBB into the brain. Advantageously, improved treatment and / or diagnostic effects can be achieved by simultaneously delivering two or more distinct active agents with different cellular targets and / or different mechanisms of action on a single vehicle. As such, the delivery system of the present invention can be useful for the treatment and / or diagnosis of a wide range of brain-related diseases or disorders.

[0065] The present invention is based in part on the surprising discovery that two distinct antibodies, which in their original free form have poor BBB permeability, can be conjugated to a single core particle, which is further conjugated to insulin as a brain-internalizing transporter moiety, to efficiently penetrate the mouse brain and further co-localize in specific brain regions after intravenous administration. Furthermore, the multifunctional system has been shown to be suitable for the simultaneous delivery of different types of active agents. In particular, it has been shown that antibodies and small molecule drugs can be conjugated to a single core particle and delivered together to the mouse brain.

[0066] Any insulin molecule or insulin analog, derivative, conjugate or fragment capable of binding to an endogenous receptor (e.g., insulin receptor) expressed in human brain capillary endothelium (BBB) ​​may be used as a transporter according to the present invention. Insulin molecules that may be used according to the present invention include, but are not limited to, mammalian insulin, human insulin, recombinant insulin produced by any method known in the art, natural and isolated insulin, fast-acting, rapid-acting and short-acting insulin and analogs, intermediate-acting insulin and analogs, and long-acting insulin and analogs. Any active fragment of the above-mentioned insulin molecule may be used as long as it is capable of binding to an endogenous receptor expressed in human brain capillary endothelial cells and aiding in the transport of the molecule through the BBB.

[0067] According to the principle of the present invention, the first active agent and the second active agent are not loaded or encapsulated in the particle core, but are conjugated to the outer surface of the core particle via a polymer linker.Importantly, the activity of the active agent is maintained despite being conjugated to the core particle, so that the agent does not necessarily need to be separated from the system during BBB penetration.

[0068] As a diagnostic, this approach allows for early and accurate detection of brain-related diseases or disorders in some embodiments. For example, the multifunctional particle includes one or more bioactive molecules that target the system to diseased or damaged cells in the brain, and the core particle is or includes an imaging agent that allows tracking the particle in vivo using a suitable imaging modality.

[0069] As a treatment, in some embodiments, this approach allows for the delivery of efficient combinations of therapeutic agents. In some embodiments, the combination of separate therapeutic agents on a single platform results in optimized synergistic effects of the drug combination. Different active agents can be targeted to the same or different biological entities, such as intracellular receptors.

[0070] In some embodiments, a combination of therapeutic and diagnostic uses is possible, for example, by using a therapeutically active agent that is conjugated to a core particle, e.g., a gold nanoparticle, that constitutes or includes an imaging contrast agent.

[0071] Multifunctional system According to one embodiment, there is provided a multifunctional system for simultaneous delivery of separate active agents to the brain, comprising: (a) a core particle attached to at least: (i) a first polymer linker, (ii) a second polymer linker, and (iii) a third polymer linker; (b) a first active agent conjugated to a first polymer linker; (c) a second active agent conjugated to a second polymer linker; and (d) a brain-internalizing transporter moiety conjugated to a third polymer linker. wherein the first active agent is different from the second active agent.

[0072] According to another embodiment, (a) a core particle attached to at least: (i) a first polymer linker, (ii) a second polymer linker, and (iii) a third polymer linker; (b) a brain-internalizing transporter moiety conjugated to a third polymer linker; Including, A multifunctional system is provided in which the first and second polymer linkers each have a free functional end group configured for conjugating a first and a second active agent, where the first active agent is different from the second active agent.

[0073] In some embodiments, the length of the third polymer linker is substantially different from the length of at least one of the first polymer linker and the second polymer linker, In some embodiments, the length of the third polymer linker is substantially longer than the length of at least one of the first polymer linker and the second polymer linker.

[0074] In some embodiments, the first active agent and the second active agent are independently selected from a biologically active molecule and a labeling molecule.

[0075] The term "multifunctional system" may be used interchangeably herein with the terms "multifunctional particle" and "co-delivery system" and refers to a system capable of achieving at least two purposes or performing a single sophisticated function by incorporating at least two functional units. The system of the present invention incorporates multiple functional units with distinct purposes, including at least a first and a second active agent with distinct targets and / or distinct activities, as well as a brain-internalizing transporter moiety that acts as a molecular Trojan horse to deliver the system across the BBB.

[0076] As used herein, the term "co-delivery" can be used interchangeably with the term "simultaneous delivery" and means that two distinct active agents are delivered simultaneously in a single composition to their target, e.g., the subject's brain or a specific region in the subject's brain. In some embodiments, "co-delivery" means synchronous delivery, i.e., when administered, the distinct active agents exhibit synchronized pharmacokinetics and biodistribution. In some related embodiments, the two active agents exhibit synchronized distribution in the brain. As used herein, the term "synchronous distribution" means that the two active agents are co-localized in the same brain region / cell. In some embodiments, the two active agents accumulate in the same brain region.

[0077] In some embodiments, particularly when the first and second active agents are both therapeutic agents, synchronized pharmacokinetics and biodistribution results in synergistic effects and improved therapeutic responses of the drug combination.

[0078] The terms "delivery" and "delivered" encompass both delivery of active agent(s) by cleaving said active agent(s) from a delivery system (e.g., by using a cleavable linker) and delivery of active agent(s) while conjugated to a delivery system (e.g., by covalent conjugation). Advantageously, the composition of the multifunctional system of the present invention does not interfere with the functionality of the active agent, so that it is not necessary to cleave the active agent from the system. According to some embodiments, the multifunctional system comprises a first and a second non-cleavable linker linking the active molecule and a cleavable linker linking the chemotherapy drug or toxin.

[0079] As used herein, the term "distinct" means that a first active agent molecule is distinguishably different from a second active agent molecule. The term "distinct" should be understood to encompass different molecules of the same type, such as two antibodies with different specificities, and two different molecules that are targeted to the same or distinct biological entities. Furthermore, the term "distinct" should be understood to encompass different molecules that contain similar specificities. For example, whole antibodies (e.g., IgG) and fragments of said antibodies (e.g., Fc / Fab regions) are considered to be separate active agents.

[0080] As used herein, the term "core particle" refers to a particle that constitutes the central portion of the co-delivery system. In some embodiments, the core particle is a nanoparticle. The term "nanoparticle" refers to a particle having a diameter of 1-1000 nm.

[0081] In some embodiments, the core particle is selected from the group consisting of metal particles, metal oxide particles, metal carbide particles, lipid particles, carbon-based particles, ceramic particles, polymer particles, and liposomes. Each possibility represents a separate embodiment of the present invention. In some embodiments, the core particle is an inorganic particle. In some embodiments, the inorganic particle is selected from the group consisting of metal particles, metal oxide particles, and ceramic particles. In some embodiments, the inorganic particle is selected from the group consisting of metal particles and metal oxide particles. In some embodiments, the inorganic particle is a metal particle. In other embodiments, the inorganic particle is a metal oxide particle. In certain embodiments, the inorganic particle is selected from gold particles and iron oxide particles.

[0082] In some embodiments, the metal particles are magnetic particles. In some embodiments, the inorganic particles are magnetic particles. In some embodiments, the magnetic particles are contrast agents for magnetic resonance imaging (MRI). Any magnetic particles suitable for use as MRI contrast agents can be used in the compositions and methods of the present invention. The magnetic particles can be formed, at least in part, from any material that is affected by a magnetic field. Examples of suitable materials include, but are not limited to, magnetite, hematite, ferrite, and materials that include one or more of iron, cobalt, manganese, nickel, chromium, gadolinium, neodymium, dysprosium, samarium, erbium, iron carbide, iron, or combinations thereof.

[0083] In some embodiments, the inorganic particles are contrast agents for computed tomography (CT) or X-ray imaging. In some embodiments, the inorganic particles are metal particles that can be used as CT or X-ray imaging contrast agents. As will be apparent to those skilled in the art, any metal and / or combination of metals suitable for use in CT or X-ray imaging can be used in the metal particles of the present invention in embodiments related to diagnostic use. In some embodiments, the metals that can be used to form the particles of the present invention are heavy metals or metals with high Z numbers. Examples of suitable metals include, but are not limited to, gold, silver, platinum, palladium, cobalt, iron, copper, tin, tantalum, vanadium, molybdenum, tungsten, osmium, iridium, rhenium, hafnium, thallium, lead, bismuth, gadolinium, dysprosium, holmium, and uranium, or combinations thereof.

[0084] In some embodiments, the multifunctional particle comprises: (a) (i) a first linear polymer linker; (ii) a second linear polymer linker; and (iii) an inorganic particle attached to a third linear polymer linker; (b) a first biologically active molecule conjugated to a first linear polymer linker; (c) a second biologically active molecule conjugated to a second linear polymer linker; and (d) a brain-internalizing transporter moiety conjugated to a third linear polymer linker. It essentially consists of The length of the third linear polymer linker is substantially different from the length of the first and second linear polymer linkers, the first bioactive molecule is different from the second bioactive molecule, and the inorganic particle is a contrast agent that can be detected by an imaging modality selected from computed tomography imaging (CT), X-ray imaging, magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), ultrasound (US), and any combination thereof. Advantageously, in such an embodiment, the multifunctional particle can be used in diagnostic applications without the need to conjugate a labeling molecule as an active agent.

[0085] According to some embodiments, the inorganic particles are metal particles selected from the group consisting of gold particles, silver particles, platinum particles, iron particles, copper particles, and mixtures or combinations thereof. Each possibility represents a separate embodiment. In some embodiments, the metal particles are gold (Au) particles.

[0086] In some embodiments, the inorganic particles are metal oxide particles. In some embodiments, the metal oxide particles are iron oxide (Fe 2 O 3 or Fe 3 O 4 ), magnesium oxide, nickel oxide, cobalt oxide, aluminum oxide, zinc oxide, copper oxide, and manganese oxide, or any combination thereof. Each possibility represents a separate embodiment of the present invention. In some embodiments, the metal oxide particles comprise an iron oxide selected from iron (III) oxide and iron (II,III) oxide. In some embodiments, the metal oxide particles are iron oxide particles, and the iron oxide is selected from iron (III) oxide and iron (II,III) oxide.

[0087] In some embodiments, the core particle is selected from the group consisting of a lipid particle, a carbon-based particle, a ceramic particle, a polymer particle, and a liposome.

[0088] In some embodiments, the core particle is a radiosensitizer. As used herein, the term "radiosensitizer" refers to an agent that makes cells, particularly cancer cells, more sensitive to radiation therapy. Typically, materials with high atomic numbers, such as gold (Z=79), enhance radiation sensitivity. Thus, gold nanoparticles are an example of a core particle that is a radiosensitizer.

[0089] According to some embodiments, the core particle has a diameter of 1-200 nm, 1-180 nm, 1-160 nm, 1-140 nm, 1-120 nm, 1-100 nm, 1-90 nm, 1-80 nm, 1-70 nm, 1-60 nm, 1-50 nm, 1-40 nm, 2-100 nm, 2-60 nm, 2-50 nm, 2-40 nm, 2-30 nm, 2-20 nm, 2-10 nm, 3-100 nm, 3-60 nm, 3-80 nm, 3-90 nm, 3-120 nm, 3-140 nm, 3-160 nm, 3-200 nm, 3-300 nm, 3-40 nm, 3-50 nm, 3-60 nm, 3-70 nm, 3-80 nm, 3-90 nm, 3-100 nm, 3-120 nm, 3-140 nm, 3-160 nm, 3-180 nm, 3-200 nm, 3-300 nm, 3-40 nm, 3-50 nm, 3-60 nm, 3-70 nm, 3-8 ...200 nm, 3-300 nm, 3 0nm, 3~50nm, 3~40nm, 3~30nm, 3~20nm, 4~100nm, 4~60nm, 4~50nm, 4~40nm, 5~200nm, 6~190nm, 7~180nm, 8 ~170nm, 10~160nm, 20~160nm, 10~150nm, 10~140nm, 10~120nm, 10~110nm, 10~100nm, 10~90nm, 10~80nm, 12~70nm, 14~60nm, 15~50nm, 15~40nm, 15~30nm, 20~30nm, 15~30nm, 20~90nm, 20~80nm, 20~70nm, 20~60m , 20~50nm, 20~40nm, 20~30nm, 30~70nm, 30~60nm, 40~60nm, 10~200nm, 20~200nm, 30~200nm, 40~200nm, 5 Nanoparticles having a diameter of 0-200 nm, 60-200 nm, 70-200 nm, 80-200 nm, 90-200 nm, 100-200 nm, 110-190 nm, 120-170 nm, 130-160 nm, 100-160 nm, 80-160 nm, 60-160 nm, 40-160 nm, 20-160 nm, 10-160 nm, 20-150 nm or 30-150 nm. Each possibility represents a separate embodiment of the present invention.According to some embodiments, the core particle is a nanoparticle having a diameter of at least 1 nm, at least 2 nm, at least 3 nm, at least 4 nm, at least 5 nm, at least 10 nm, at least 12 nm, at least 15 nm, at least 18 nm, at least 20 nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm, at least 45 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, at least 120 nm, at least 130 nm, at least 140 nm, or at least 150 nm. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the core particle is a nanoparticle having a diameter of at most 5 nm, at most 10 nm, at most 15 nm, at most 20 nm, at most 30 nm, at most 40 nm, at most 50 nm, at most 60 nm, at most 70 nm, at most 80 nm, at most 90 nm, at most 100 nm, at most 120 nm, at most 140 nm, at most 160 nm, at most 180 nm, or at most 200 nm. Each possibility represents a separate embodiment of the present invention.

[0090] According to some embodiments, the multifunctional particles, i.e., the entire co-delivery system, may be 5-500 nm, 6-400 nm, 8-300 nm, 10-300 nm, 10-200 nm, 10-180 nm, 10-160 nm, 10-150 nm, 10-100 nm, 20-90 nm, 20-80 nm, 20-70 nm, 20-60 nm, 25-100 nm, 25-90 nm, 25-80 nm, 25-70 nm, 25-60 nm, 25-50 nm, 25-60 nm, 25-70 nm, 25-80 nm, 25-90 nm, 25-80 nm, 25-70 nm, 25-60 nm, 25-50 nm, 25-80 nm, 25-90 nm, 25-80 nm, 25-90 nm, 25-80 nm, 25-70 nm, 25-60 nm, 25-50 nm, 25-60 nm, 25-70 nm, 25-80 nm, 25-80 nm, 25-9 ...90 nm, 25-80 nm, 25-90 nm, 25-60 nm, 25-50 nm, 25-50 nm, 25-60 nm, 25-70 nm, 25-80 nm, 25-80 nm, 30-60 nm, 40-200 nm, 40-150 nm, 40-120 nm, 40-100 nm, 40-80 nm, 40-60 nm, 50-300 nm, 50-250 nm, 50-200 nm, 50-180 nm, 50-150 nm, 60-200 nm, 70-180 nm, 80-180 nm, 90-170 nm, 100-160 nm, 100-200 nm, 150-200 nm or 150-180 nm. According to some embodiments, the multifunctional particles have a diameter of 2-200 nm, 1-100 nm, 1-150 nm, 1-200 nm, 2-50 nm, 2-100 nm, 2-150 nm, 4-50 nm, 4-100 nm, 4-150 nm, or 4-200 nm. Each possibility represents a separate embodiment of the invention. According to some embodiments, the multifunctional particles have a diameter of at least 1 nm, at least 2 nm, at least 5 nm, at least 10 nm, at least 15 nm, at least 20 nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm, at least 45 nm, at least 50 nm, at least 55 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, at least 120 nm, at least 130 nm, at least 140 nm, at least 150 nm, at least 160 nm, at least 180 nm, or at least 200 nm. Each possibility represents a separate embodiment of the present invention.According to some embodiments, the multifunctional particles have a diameter of at most 5 nm, at most 20 nm, at most 30 nm, at most 40 nm, at most 50 nm, at most 60 nm, at most 70 nm, at most 80 nm, at most 90 nm, at most 100 nm, at most 110 nm, at most 120 nm, at most 130 nm, at most 140 nm, at most 150 nm, at most 180 nm, at most 200 nm, at most 250 nm, at most 300 nm, at most 350 nm, at most 400 nm, at most 450 nm, or at most 500 nm. Each possibility represents a separate embodiment of the present invention.

[0091] As used herein, the term "diameter" of a particle / nanoparticle can be used interchangeably with the term "size" of a particle / nanoparticle and refers to the maximum linear distance between two points on the surface of the described particle / nanoparticle. The term "diameter" as used herein encompasses the size of spherical particles and the size of non-spherical particles and can refer to the actual size of a particle or its hydrodynamic diameter, including the contribution from a solvated sphere. Any method known in the art can be used to determine the size of a particle, such as transmission electron microscopy (TEM), scanning electron microscopy (SEM), and dynamic light scattering (DLS). The term "diameter" can refer to the average diameter of a plurality of particles measured by any of the above techniques.

[0092] The core particle is coated with a polymer layer comprising at least three polymers: a first polymer linker having a functional end group capable of binding to a first active agent, a second polymer linker having a functional end group capable of binding to a second active agent, and a third polymer linker conjugated to a brain internalizing transporter moiety. In some embodiments, the first polymer linker is conjugated to the first active agent.

[0093] In some embodiments, the second polymer linker is conjugated to a second active agent. In some embodiments, the core particle comprises an additional polymer linker having a functional end group capable of binding to a chemotherapeutic agent or a toxin. According to some embodiments, the additional polymer linker is cleavable. According to some embodiments, the cleavable polymer linker is an SH-PEG-SH linker.

[0094] The term "coated" as used herein is intended to mean that a layer, e.g. a polymer layer comprising a plurality of polymer moieties, is chemically attached to the surface of a core particle, thereby at least partially covering said core particle. A "particle coated with a polymer layer" means that each polymer moiety in the polymer layer is chemically attached to the particle via a functional end group, e.g. a thiol group, of said polymer moiety. The chemical attachment can be covalent, semi-covalent or non-covalent.

[0095] The term "polymer moiety" may be used interchangeably with the term "polymer" and refers to a molecule containing two or more repeating subunits linked in a linear, branched, hyperbranched, dendritic, or cyclic arrangement, or any combination thereof. In some embodiments, the term "polymer moiety" refers to a molecule containing at least three repeating subunits linked in a linear, branched, hyperbranched, dendritic, or cyclic arrangement, or any combination thereof. Examples of subunits include alkylenes, arylenes, heteroalkylenes, amino acids, nucleic acids, sugars, and the like. Examples of polymer moieties include, but are not limited to, poly(ethylene glycol) groups, poly(ethylene amine) groups, and poly(amino acid) groups. The terms "polymer moiety" and "polymer" also encompass polymer linkers. As used herein, the term "polymer linker" refers to a polymer moiety that originally contains at least one functional / reactive group that allows for attachment to a substance, such as a particle. In some embodiments, the polymer linker is a bifunctional polymer having at least two functional / reactive groups that allow for attachment to at least two substances, thereby linking between the at least two substances. In some embodiments, the polymer linker is a monofunctional polymer having one functional / reactive group that allows for binding to one substance, e.g., a core particle. As used herein, it should be understood that the terms "monofunctional", "bifunctional", "functional" and the like refer to the polymer linker in its original form prior to attachment to the core particle and / or the brain-internalizing transporter moiety or respective active agent.

[0096] In some embodiments, the core particle is attached to a first polymer linker. In some embodiments, the core particle is attached to a second polymer linker. In some embodiments, the core particle is attached to a third polymer linker. In some embodiments, the core particle is attached to a first, second and third polymer linker.

[0097] The term "attached" can be used interchangeably with the term "conjugate". In some embodiments, the bond is covalently conjugated. The terms "covalently attached", "covalently attached", "covalently linked" and "covalent bond" are used interchangeably herein and refer to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently attached drug coating refers to a drug coating that forms a chemical bond with the functionalized material surface, as compared to attachment to the surface via other means, such as adhesion or electrostatic interactions. It will be appreciated that a drug (e.g., a polymer) that is covalently attached to a surface can also be attached via other means in addition to covalent attachment.

[0098] In some embodiments, the polymer moiety and / or linker is attached to the outer surface of the core particle via a chemical attachment selected from the group consisting of covalent attachment, semi-covalent attachment, and non-covalent attachment. Each possibility represents a separate embodiment of the present invention. In some embodiments, the polymer moiety and / or linker is attached to the outer surface of the core particle via semi-covalent attachment. As used herein, the term "semi-covalent attachment" refers to a coordinate bond in which the shared electron pair forming the bond originates from the same atom. In the present disclosure, semi-covalent attachment can occur between metal particles, e.g., gold particles, and thiol groups.

[0099] In some embodiments, at least one of the first, second and third polymer linkers is a linear polymer linker. In some embodiments, the first polymer linker is a linear polymer linker. In some embodiments, the second polymer linker is a linear polymer linker. In some embodiments, the third polymer linker is a linear polymer linker. In some embodiments, the first polymer linker and the second polymer linker are linear polymer linkers. In some embodiments, the first and third polymer linkers are linear polymer linkers. In some embodiments, the second and third polymer linkers are linear polymer linkers. In some embodiments, the first, second and third polymer linkers are linear polymer linkers. In some embodiments, the linear polymer linker is a bifunctional linear polymer having two types of functional / reactive groups at both ends of the linear polymer. In some embodiments, each of the first, second and third polymer linkers is independently a linear bifunctional polymer linker having two types of functional / reactive groups on both ends of said linear polymer.

[0100] As used herein, the term "linear" polymer / polymer linker refers to a polymer / polymer linker in which, in some embodiments, at least 80% of the monomer units are linked linearly, i.e., in the form of a single polymer chain. In further embodiments, the term "linear" polymer / polymer linker refers to a polymer / polymer linker in which at least 90% of the monomer units are linked linearly. In further embodiments, the term "linear" polymer / polymer linker refers to a polymer / polymer linker in which about 100% of the monomer units are connected linearly. As used herein, the term "single polymer chain" refers to a polymer chain comprising monomers that are connected such that the monomer units are bonded to each other through two atoms, one on each monomer unit.

[0101] In some embodiments, the multifunctional system further comprises an additional polymer moiety attached to the core particle. In some embodiments, the additional polymer moiety is a linear polymer. In some embodiments, the additional polymer moiety is a monofunctional polymer used as a capping to close or inactivate functional groups on the particle and allow distance between linkers carrying bioactive molecules. In some embodiments, the additional polymer moiety is a monofunctional polymer linker. The additional polymer moiety is thus, in some embodiments, a fourth polymer linker attached to the core particle. In some embodiments, the core particle is attached to the first, second, third and fourth polymer linkers. In some embodiments, the fourth polymer linker is monofunctional, i.e., has a single functional end group originally configured to conjugate the polymer linker to the core particle and to be used as a capping moiety. In some embodiments, the fourth polymer linker is a linear monofunctional polymer.

[0102] In some embodiments, the first polymer linker comprises a polymer selected from the group consisting of, but not limited to, polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof, with each possibility representing a separate embodiment of the present invention.

[0103] As used herein, the term "derivative" refers to a compound that has the same core structure as, or is closely similar to, a parent compound, but has chemical or physical modifications, such as different or additional groups, including, but not limited to, alkoxy groups, carboxy groups, amine groups, methoxy groups, and thiol groups.

[0104] In some embodiments, the first polymer linker comprises a polyether. In some embodiments, the first polymer linker is a polyether. In some embodiments, the polyether is polyethylene glycol (PEG) or a derivative thereof.

[0105] Where appropriate, the abbreviation (PEG) is used in combination with a numerical suffix indicating the average molecular weight of the PEG. A form of PEG or PEG species is a PEG or PEG derivative having the designated average molecular weight.

[0106] As used herein, "PEG or derivatives thereof" refers to any compound that includes at least one polyethylene glycol moiety. PEGs exist in linear and branched forms, including multi-arm and / or grafted polyethylene glycols. The term "PEG derivatives" as used herein refers to PEGs that are modified by alkylation of the terminal hydroxyl group. In some embodiments, the terminal hydroxyl group is alkylated with a linear or branched C1-C6 alkyl. PEGs may further include functional groups. PEGs may be monofunctional, bifunctional, or multifunctional polyethylene glycols.

[0107] Exemplary functional groups include, but are not limited to, the following: hydroxyl, carboxyl, thiol, amine, phosphate, phosphonate, sulfate, sulfite, sulfonate, sulfoxide, sulfone, amide, ester, ketone, aldehyde, cyano, alkyne, azide, and alkene, or combinations thereof.

[0108] In some embodiments, the first polymer linker comprises a thiol (-SH) end group. In some embodiments, the first polymer linker is chemically attached to the core particle via the thiol (-SH) end group. In some embodiments, the first polymer linker is conjugated to the first active agent via an amide bond. In some embodiments, the core particle is bound to the first polymer linker via a sulfide bond, and the first active agent is conjugated to the first polymer linker via an amide bond. In some embodiments, the core particle is an inorganic particle, bound to the first polymer linker via a sulfide bond, and the first active agent is conjugated to the first polymer linker via an amide bond. In some embodiments, the first polymer linker in the co-delivery system has the structure -SR-CONH-, where R is a polymer chain made of repeating monomer units. In other embodiments, the first polymer linker in the co-delivery system has the structure -SR-NHCO-, where R is a polymer chain made of repeating monomer units. In some embodiments, the first polymer linker is a thiolated PEG acid (HS-PEG-COOH) or a thiolated PEG amine (HS-PEG-NH 2 ) is selected from HS and COOH / NH 2 It should be understood that the end group refers to the polymer linker prior to conjugation with the core particle and active agent. In some embodiments, the thiol group is chemically attached to the core particle and the acid or amine group is covalently conjugated to the first active agent. In some embodiments, the first polymer linker in the co-delivery system has a structure selected from -S-PEG-C(O)- and -S-PEG-NH-.

[0109] In some embodiments, the first polymer linker is a non-cleavable linker. In some embodiments, the first polymer linker is non-cleavable under physiological conditions.

[0110] As used herein, the term "non-cleavable" refers to a stable bond that is not acid or base sensitive, is not sensitive to reducing or oxidizing agents, and is not sensitive to enzymes that may be found in cells or in the circulatory system. In some embodiments, the non-cleavable polymer linker lacks a pH-sensitive hydrazone. In some embodiments, the non-cleavable polymer linker lacks a disulfide bond. In some embodiments, the non-cleavable polymer linker lacks an ester bond. The term "polymer linker is non-cleavable" is understood to be meant to include the bond between the core particle and the polymer linker; the bond between each polymer linker and each active agent; or the bond between each polymer linker and the brain-internalizing transporter moiety, as well as any bond within the polymer linker itself.

[0111] Additionally, in some embodiments, the particle comprises a chemotherapeutic agent or a toxin attached via a cleavable linker, for example, an SH-PEG-SH linker.

[0112] In some embodiments, the first polymer linker has a molecular weight (MW) of 2,000 to 7,000 Daltons (Da). In some embodiments, the first polymer linker has a molecular weight (MW) of 500 to 10,000 Da, 1,000 to 10,000 Da, 600 to 9,500 Da, 700 to 9,000 Da, 800 to 8,500 Da, 800 to 6,000 Da, 800 to 5,000 Da, 800 to 4,000 Da, 800 to 3,000 Da, 800 to 2,000 Da, 900 to 8,000 Da, 1,000 to 7,000 Da, 1,500 to 6,500 Da, 2,000 to 6,0 00Da, 3,000~6,000Da, 4,000~6,000Da, 1,000~2,000Da, 1,000~3,000Da, 1,000~4,000Da, 1,000~5,000Da, 1,000~7,000 Da, 3,400~7,000Da, 2,000~3,000Da, 2,000~5,000Da, 2,000~7,000Da, 2,000~10,000Da, 3,000~3,400Da, 3,000~4,000Da The first polymer linker has a molecular weight (MW) within a range selected from the group consisting of 3,000-5,000 Da, 3,000-7,000 Da, 3,000-10,000 Da, 5,000-7,000 Da, 5,000-10,000 Da, and 7,000-10,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the first polymer linker has a MW of at least 1,000 Da, at least 1,500 Da, at least 2,000 Da, at least 2,500 Da, at least 3,000 Da, at least 3,400 Da, at least 4,000 Da, at least 5,000 Da, at least 6,000 Da, at least 7,000 Da, or at least 8,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the first polymer linker has a MW of up to 2,000 Da, up to 3,000 Da, up to 4,000 Da, up to 5,000 Da, up to 6,000 Da, up to 7,000 Da, or up to 10,000 Da. Each possibility represents a separate embodiment.

[0113] In some embodiments, the second polymer linker comprises a polymer selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof, with each possibility representing a separate embodiment of the present invention.

[0114] In some embodiments, the second polymer linker comprises a polyether. In some embodiments, the second polymer linker is a polyether. In some embodiments, the polyether is polyethylene glycol (PEG) or a derivative thereof.

[0115] In some embodiments, the second polymer linker comprises a thiol (-SH) end group. In some embodiments, the second polymer linker is chemically attached to the core particle via a thiol (-SH) end group. In some embodiments, the second polymer linker is conjugated to the second active agent via an amide bond. In some embodiments, the core particle is bound to the second polymer linker via a sulfide bond, and the second active agent is conjugated to the second polymer linker via an amide bond. In some embodiments, the core particle is an inorganic particle, bound to the second polymer linker via a sulfide bond, and the second active agent is conjugated to the second polymer linker via an amide bond. In some embodiments, the second polymer linker in the co-delivery system has the structure -SR-CONH-, where R is a polymer chain made of repeating monomer units. In other embodiments, the second polymer linker in the co-delivery system has the structure -SR-NHCO-, where R is a polymer chain made of repeating monomer units. In some embodiments, the second polymer linker is a thiolated PEG acid (HS-PEG-COOH) or a thiolated PEG amine (HS-PEG-NH 2) is selected from HS and COOH / NH 2 It should be understood that the end group refers to the polymer linker prior to conjugation with the core particle and active agent. In some embodiments, the thiol group is chemically attached to the core particle and the acid or amine group is covalently conjugated to the second active agent. In some embodiments, the second polymer linker in the co-delivery system has a structure selected from -S-PEG-C(O)- and -S-PEG-NH-.

[0116] In some embodiments, the second polymer linker is a non-cleavable linker. In some embodiments, the second polymer linker is non-cleavable under physiological conditions.

[0117] In some embodiments, the second polymer linker has a molecular weight (MW) of 2,000 to 7,000 Da. In some embodiments, the second polymer linker is 500 to 10,000 Da, 600 to 9,500 Da, 700 to 9,000 Da, 800 to 8,500 Da, 800 to 6,000 Da, 800 to 5,000 Da, 800 to 4,000 Da, 800 to 3,000 Da, 800 to 2,000 Da, 900 to 8,000 Da, 1,000 to 7,000 Da, 1,500 to 6,500 Da, 2,000 to 6,000 Da, 3,000 to 6,000 Da, 4,000 to 6,000 Da, 1,000 to 2,000 Da, 1,000 to 3,000 Da, 1,0 and 7,000-10,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the second polymer linker has a MW of at least 1,000 Da, at least 1,500 Da, at least 2,000 Da, at least 2,500 Da, at least 3,000 Da, at least 3,400 Da, at least 4,000 Da, at least 5,000 Da, at least 6,000 Da, at least 7,000 Da, or at least 8,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the second polymer linker has a MW of up to 2,000 Da, up to 3,000 Da, up to 4,000 Da, up to 5,000 Da, up to 6,000 Da, up to 7,000 Da, or up to 10,000 Da. Each possibility represents a separate embodiment.

[0118] According to some embodiments, the first polymer linker and the second polymer linker comprise different polymers. According to some embodiments, the first polymer linker and the second polymer linker are different polymers. In some embodiments, the first polymer linker and the second polymer linker comprise the same polymer. In some embodiments, the first polymer linker and the second polymer linker are the same.

[0119] In some embodiments, the first polymer linker and the second polymer linker comprise the same polymer selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof. In some embodiments, both the first polymer linker and the second polymer linker comprise PEG. In some embodiments, both the first polymer linker and the second polymer linker are PEG. In some embodiments, both the first polymer linker and the second polymer linker comprise thiolated PEG. In some embodiments, the first polymer linker and the second polymer linker comprise thiolated PEG acids (HS-PEG-COOH) or thiolated PEG amines (HS-PEG-NH 2 In some embodiments, the first polymer linker and the second polymer linker comprise a thiolated PEG acid (HS-PEG-COOH) or a thiolated PEG amine (HS-PEG-NH 2 In some embodiments, both the first polymer linker and the second polymer linker are thiolated PEG acids (HS-PEG-COOH). In some embodiments, both the first polymer linker and the second polymer linker are thiolated PEG amines (HS-PEG-NH 2 ).

[0120] In some embodiments, the third polymer linker comprises a polymer selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof, with each possibility representing a separate embodiment of the present invention.

[0121] In some embodiments, the third polymer linker comprises a polyether. In some embodiments, the third polymer linker is a polyether. In some embodiments, the polyether is polyethylene glycol (PEG) or a derivative thereof.

[0122] In some embodiments, the third polymer linker comprises a thiol (-SH) end group. In some embodiments, the third polymer linker is chemically attached to the core particle via a thiol (-SH) end group. In some embodiments, the third polymer linker is conjugated to the brain-internalizing transporter moiety via an amide bond. In some embodiments, the core particle is conjugated to the third polymer linker via a sulfide bond, and the brain-internalizing transporter moiety is conjugated to the third polymer linker via an amide bond. In some embodiments, the core particle is an inorganic particle and is conjugated to the third polymer linker via a sulfide bond, and the brain-internalizing transporter moiety is conjugated to the third polymer linker via an amide bond. In some embodiments, the third polymer linker in the co-delivery system has the structure -SR-CONH-, where R is a polymer chain made of repeating monomer units. In other embodiments, the third polymer linker in the co-delivery system has the structure -SR-NHCO-, where R is a polymer chain made of repeating monomer units. In some embodiments, the third polymer linker is a thiolated PEG acid (HS-PEG-COOH) or a thiolated PEG amine (HS-PEG-NH 2 ) is selected from HS and COOH / NH 2 It should be understood that the terminal group refers to the polymer linker prior to conjugation with the core particle and the brain-internalizing transporter moiety. In some embodiments, the thiol group is chemically attached to the core particle and the acid or amine group is covalently conjugated to the brain-internalizing transporter moiety. In some embodiments, the third polymer linker in the co-delivery system has a structure selected from -S-PEG-C(O)- and S-PEG-NH-.

[0123] In some embodiments, the third polymer linker has a molecular weight (MW) of 2,000 to 7,000 Da. In some embodiments, the third polymer linker is 2,000 to 10,000 Da, 2,000 to 9,500 Da, 2,000 to 9,000 Da, 2,000 to 8,500 Da, 2,000 to 6,000 Da, 2,000 to 5,000 Da, 2,000 to 4,000 Da, 2,000 to 3,000 Da, 2,000 to 8,000 Da, 2,000 to 7,000 Da, 2,000 to 6,500 Da, 2,000 to 6,000 Da, 3,000 to 6,000 Da, 4,000 to 6,000 Da, 2,000 to 3,000 Da, 2,000 and MW in a range selected from the group consisting of: 2,000-4,000 Da, 2,000-5,000 Da, 2,000-7,000 Da, 2,000-11,000 Da, 2,000-3,000 Da, 2,000-5,000 Da, 2,000-7,000 Da, 2,000-10,000 Da, 3,000-10,000 Da, 3,000-7,000 Da, 3,000-5,000 Da, 3,000-3,400 Da, 3,400-7,000 Da, 5,000-7,000 Da, 5,000-10,000 Da, and 7,000-10,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the third polymer linker has a MW of at least 2,000, at least 2,500 Da, at least 3,000 Da, at least 3,400 Da, at least 4,000 Da, at least 5,000 Da, at least 6,000 Da, at least 7,000 Da, or at least 8,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the third polymer linker has a MW of up to 2,000 Da, up to 3,000 Da, up to 4,000 Da, up to 5,000 Da, up to 6,000 Da, up to 7,000 Da, or up to 10,000 Da. Each possibility represents a separate embodiment.

[0124] In some embodiments, the third polymer linker is a non-cleavable linker. In some embodiments, the third polymer linker is non-cleavable under physiological conditions.

[0125] In some embodiments, at least one of the first, second and third polymer linkers, or additional polymer linkers, comprises a cleavable linker. In some embodiments, at least one of the first and second polymer linkers comprises a cleavable linker. In some embodiments, each of the first and second polymer linkers comprises an independently cleavable linker. According to some embodiments, the cleavable linker is SH-PEG-SH. According to some embodiments, the cleavable linker comprises a bond that is susceptible to cleavage by an endogenous molecule located or expressed in the brain. In some embodiments, the cleavable linker is PEG succinimidyl succinate (PEGSS). According to some embodiments, the endogenous molecule is glutathione. According to some embodiments, the endogenous molecule is selected from the group consisting of a protease, a nuclease, a hydronium ion, and a reducing agent. In some embodiments, the endogenous molecule is selected from neuroserpin and serpin B. Each possibility represents a separate embodiment. According to some embodiments, a cleavable linker connects the chemotherapeutic molecule or toxin to the multifunctional particle.

[0126] Any chemotherapeutic molecule or toxin known in the art to have anti-cancer activity can be used in the multifunctional delivery system of the present invention. Chemotherapeutic molecules include irinotecan, deruxtecan, emtansine, mitoxantrone, topoisomerase inhibitors, vinca-derived spindle poisons: vinblastine, vincristine, vinorelbine (taxol), paclitaxel, docetaxel; alkylating agents: mechlorethamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide; methotrexate; 6-mercaptopurine; 5-fluorouracil, cytarabine, gemcitabine; podophyllonitrile, cyclophosphamide ... Examples include, but are not limited to, toxins: etoposide, topotecan, dacarbazine; antibiotics: doxorubicin (adriamycin), bleomycin, mitomycin; nitrosoureas: carmustine (BCNU), lomustine, epirubicin, idarubicin, daunorubicin; inorganic ions: cisplatin, carboplatin; interferons, asparaginase; hormones: tamoxifen, leuprolide, flutamide, and megestrol acetate. Each possibility represents a separate embodiment of the present invention.

[0127] According to some embodiments, the chemotherapeutic agent is selected from alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthracenedione-substituted ureas, methylhydrazine derivatives, adrenal cortical suppressants, corticosteroids, progestins, estrogens, antiestrogens, androgens, antiandrogens, and gonadotropin-releasing hormone analogs. According to another embodiment, the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil (5-FU), leucovorin (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel, and docetaxel. One or more chemotherapeutic agents can be used with the multifunctional delivery system of the present invention. Each possibility represents a separate embodiment of the present invention.

[0128] According to some embodiments, the toxin is selected from microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors and RNA polymerase inhibitors. According to additional embodiments, the toxin is selected from the group consisting of MMAE, MMAF, saporin, DM4, DM1, SN38, calicheamicin, DXd, PBD, duocarmycin, sandramycin, alpha-amanitin, chaetocin, CYT997, daunorubicin, 17-AAG, agrochelin A, doxorubicin, methotrexate, colchicine, cordycepin, epothilone B, hygrolysin, herboxidien, ferulenol, curubrin, paclitaxel, englerin A, taltoburin, triptolide, cryptophycin, and nemorubicin. Each possibility represents a separate embodiment of the present invention.

[0129] According to some embodiments, the multifunctional particle further comprises a cleavage molecule inducer. According to some embodiments, the cleavage molecule inducer is selected from the group consisting of N-acetyl-l-cysteine ​​(NAC), glutathione monoester, gamma-glutamylcysteine, gamma-glutamylcysteine ​​synthetase, and glutathione synthetase. Each possibility represents a separate embodiment.

[0130] In some embodiments, the endogenous molecule is glutathione and the cleavage molecule inducer is selected from the group consisting of N-acetyl-l-cysteine ​​(NAC), glutathione monoester, gamma-glutamylcysteine, gamma-glutamylcysteine ​​synthetase, and glutathione synthetase.

[0131] According to some embodiments, at least one of the first polymer linker and the second polymer linker is different from the third polymer linker. In some embodiments, at least one of the first polymer linker and the second polymer linker comprises the same polymer as the third polymer linker. In some embodiments, the first polymer linker, the second polymer linker and the third polymer linker comprise the same polymer. In further embodiments, the first polymer linker is composed of repeating monomer units and the third polymer linker is composed of the same repeating monomer units as the first linear polymer linker. In some related embodiments, the first linear polymer linker has a different number of repeating monomer units than the third linear polymer linker. In some embodiments, the second polymer linker is composed of repeating monomer units and the third polymer linker is composed of the same repeating monomer units as the second linear polymer linker. In some related embodiments, the second linear polymer linker has a different number of repeating monomer units than the third linear polymer linker. In some embodiments, the first and second polymer linkers are identical and composed of repeating monomer units, and the third polymer linker is composed of the same repeating monomer units as the first and second linear polymer linkers. In some related embodiments, the first and second linear polymer linkers have a different number of repeating monomer units than the third linear polymer linker.

[0132] In some embodiments, the first, second and third polymer linkers comprise the same polymer selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, poly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives and combinations thereof. In some embodiments, the first, second and third polymer linkers comprise PEG. In some embodiments, the first, second and third polymer linkers are PEG. In some embodiments, the first, second and third polymer linkers comprise thiolated PEG. In some embodiments, the first, second and third polymer linkers comprise thiolated PEG acids (HS-PEG-COOH) or thiolated PEG amines (HS-PEG-NH 2 In some embodiments, the first, second and third polymer linkers comprise a thiolated PEG acid (HS-PEG-COOH) or a thiolated PEG amine (HS-PEG-NH 2 In some embodiments, the first, second and third polymer linkers are thiolated PEG acids (HS-PEG-COOH). In some embodiments, the first, second and third polymer linkers are thiolated PEG amines (HS-PEG-NH 2 ).

[0133] In some embodiments, a first active agent is covalently conjugated to a first polymer linker through a first functional end group of the linker, a second active agent is covalently conjugated to a second polymer linker through a second functional end group of the linker, and a brain internalizing transporter moiety is covalently conjugated to a third polymer linker through a third functional end group of the linker. Exemplary functional end groups include, but are not limited to, thiol groups, carboxyl groups, and amine groups. In some embodiments, at least two of the first functional end group, the second functional end group, and the third functional end group are the same. In some embodiments, the first functional end group and the second functional end group are the same. In some embodiments, the first functional end group and the third functional end group are the same. In some embodiments, the second functional end group and the third functional end group are the same. In some embodiments, the first functional end group, the second functional end group, and the third functional end group are the same.

[0134] In some embodiments, the first functional end group and the second functional end group are different. In some embodiments, the first functional end group and the third functional end group are different. In some embodiments, the second functional end group and the third functional end group are different.

[0135] In some embodiments, the first, second and third polymer linkers are linear. According to the principles of the present invention, the length of the third polymer linker is substantially different from the length of at least one of the first and second polymer linkers. In some embodiments, the length of the third polymer linker is substantially different from the length of the first polymer linker. In some embodiments, the length of the third polymer linker is substantially different from the length of the second polymer linker. In some embodiments, the length of the third polymer linker is substantially different from the length of both the first and second polymer linkers. In some embodiments, the length of the first polymer linker is substantially similar to the length of the second polymer linker, and the length of the third polymer linker is substantially different from the length of both the first and second polymer linkers.

[0136] In some embodiments, the term "length" of a polymer segment or linker refers to the length of the polymer, which depends on the number of monomers incorporated therein, the length of each monomer unit, the polymer chain structure (e.g., whether the polymer is linear or branched), spatial conformation, deformation of valence angles (or bond angles), and the degree of stretching or coiling.

[0137] The length of a polymer can be calculated as known in the art, for example, as described in Introduction to Physical Polymer Science, 4th Edition, LHSperling, 1st Edition: November 4, 2005, Chapter 3. Furthermore, as known in the art, various computational modeling methods can be used to evaluate the length of a polymer, which can be performed using, inter alia, Hyperchem, ACD / 3D, MOE 2010.10, or Chem 3D software. Physical characterization methods, such as, for example, light scattering, can also be used to evaluate the length of a polymer. It should be understood that when evaluating the difference in the length of polymer linkers, the same length definition (or length measurement method) must be used for the polymer linkers being compared.

[0138] The term "length" when referring to a linear polymer can refer to different definitions of length. According to some embodiments, the term "length" refers to the displacement length, also referred to herein as the "end-to-end" length, which is the distance between the two ends of the polymer chain of a coiled polymer. The end-to-end length can be calculated, for example, by the Flory radius: (chemical 1) F=an3 / 5 Formula I (where F=Flory radius, a=monomer size, n=degree of polymerization) It can be expressed as:

[0139] According to some embodiments, the term "length" refers to the contour length, which is the distance between the two ends of a polymer chain when the polymer is stretched. The contour length can be considered as the maximum possible displacement length. The contour length (also referred to herein as the "old contour length") can be calculated by dividing the MW of the polymer by the MW of the monomer unit and multiplying by the length of the monomer unit. To take into account the bond angle, the contour length (also referred to herein as the "new contour length") can be calculated by dividing the MW of the polymer by the MW of the monomer unit and multiplying by the length of the monomer unit and further multiplying by the cosine of ((bond angle theta-180) / 2).

[0140] As described hereinabove, the length of a linear polymer can be estimated based on its molecular weight and the chemical structure of the monomer unit. To evaluate the difference between polymer linkers containing the same polymer (i.e., composed of the same type but with different numbers of monomer units), the molecular weight of the polymer linker can be conveniently used. Thus, in some embodiments, the molecular weight of the third polymer linker is substantially different from the molecular weight of at least one of the first and second linear polymer linkers. In some embodiments, the molecular weight of the third polymer linker is substantially different from the molecular weight of the first polymer linker. In some embodiments, the molecular weight of the third polymer linker is substantially different from the molecular weight of the second polymer linker. In some embodiments, the molecular weight of the third polymer linker is substantially different from the molecular weight of the first polymer linker and the second polymer linker.

[0141] As used herein, the term "substantially different" refers to a difference of at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 15%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%. Each possibility represents a separate embodiment of the invention. The term "substantially higher" means that a first value is higher than a second value, and the difference between the first value and the second value is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 15%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%. Each possibility represents a separate embodiment of the invention.

[0142] In some embodiments, the molecular weight of the monomer unit of the third polymer linker is substantially similar to the molecular weight of the monomer unit of at least one of the first and second polymer linkers. As used herein, the term "substantially similar" refers to a similarity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%. Each possibility represents a separate embodiment of the present invention.

[0143] In some embodiments, the third polymer linker and at least one of the first and second polymer linkers comprise similar polymers. In some embodiments, the third linear polymer linker is composed of repeating monomer units, and at least one of the first and second linear polymer linkers is composed of the same repeating monomer units as the third linear polymer linker, and the third linear polymer linker has a different number of repeating monomer units than the first and second linear polymer linkers. In some embodiments, the third polymer linker and at least one of the first and second polymer linkers are similar except for the length of the third and the first and / or second polymer linkers.

[0144] In some embodiments, the third polymer linker and / or the first and second polymer linkers are at least about 100 Da, at least about 150 Da, at least about 200 Da, at least about 250 Da, at least about 300 Da, at least about 350 Da, at least about 400 Da, at least about 450 Da, at least about 500 Da, at least about 550 Da, at least about 600 Da, at least about 650 Da, at least about 700 Da, at least about 800 Da, at least about 900 Da, at least about 1000 Da, at least about 150 Da, at least about 200 Da, at least about 250 Da, at least about 300 Da, at least about 350 Da, at least about 400 Da, at least about 450 Da, at least about 500 Da, at least about 550 Da, at least about 600 Da, at least about 650 Da, at least about 700 Da, at least about 1000 Da, at least about 15 ... The respective molecular weight difference is at least about 750 Da, at least about 800 Da, at least about 850 Da, at least about 900 Da, at least about 950 Da, at least about 1000 Da, at least about 1100 Da, at least about 1200 Da, at least about 1300 Da, at least about 1400 Da, at least about 1500 Da, at least about 1600 Da, at least about 1700 Da, at least about 1800 Da, at least about 1900 Da, or at least about 2000 Da. Each possibility represents a separate embodiment of the present invention.

[0145] In some embodiments, the difference between the length of the third polymer linker and the length of at least one of the first polymer linker and the second linear polymer linker is configured to allow exposure of the brain-internalizing transporter moiety on the outer surface of the co-delivery system facing the BBB. It should be understood that the active agent is not enclosed or encapsulated within the core particle, but rather is attached to its outer surface via the polymer linker, similar to the brain-internalizing moiety attached to the surface of the same core particle via the polymer linker. Without being bound by theory or mechanism of action, it is contemplated that attaching the brain-internalizing transporter moiety via a polymer chain having a similar length to the first and / or second polymer linker may prevent sufficient exposure of the brain-internalizing moiety on the outer surface of the co-delivery system, thereby restricting the system from passing through the BBB.

[0146] Furthermore, without being bound by theory or mechanism of action, it is contemplated that active agents that are not enclosed or encapsulated within the core particle remain accessible and active despite being bound to the multifunctional system. Advantageously, the specific composition of the multifunctional system of the present invention, which ensures the formation of conjugate particles with a specific hierarchical structure, not only allows the delivery of a combination of various types of active agents, but also does not interfere with the functionality of the active agents and does not necessarily require cleavage of the link between at least one of the active agents and the core particle after crossing the BBB.

[0147] Thus, in some embodiments, the molecular weight of the third polymer linker is higher than the molecular weight of at least one of the first and second polymer linkers. In further embodiments, the molecular weight of the third polymer linker is higher than the molecular weight of both the first and second polymer linkers. In some embodiments, the molecular weight of the third polymer linker is higher than the molecular weight of the first and / or second polymer linkers, provided that the molecular weight of the first and / or second polymer linkers is less than 4950 Da. In some embodiments, the molecular weight of the third polymer linker is higher than the molecular weight of the first and / or second polymer linkers, provided that the molecular weight of the first and / or second polymer linkers is less than 4900 Da. In some embodiments, the molecular weight of the third polymer linker is higher than the molecular weight of the first and / or second polymer linkers, provided that the molecular weight of the first and / or second polymer linkers is less than 4800 Da. In some embodiments, the molecular weight of the third polymer linker is higher than the molecular weight of the first and / or second polymer linker, provided that the molecular weight of said first and / or second polymer linker is less than 4780 Da. In some embodiments, the third polymer linker is a PEG derivative having a molecular weight of about 5000 Da, and at least one of the first and second polymer linkers is a PEG derivative having a molecular weight of about 3500 kDa. In some embodiments, the third polymer linker is a PEG derivative having a molecular weight of about 5000 Da, and both the first and second polymer linkers are PEG derivatives having a molecular weight of about 3500 kDa.

[0148] In some embodiments, the third polymer linker has a molecular weight higher than that of at least one of the first and second polymer linkers. In some embodiments, the MW of the polymer linker is directly dependent on the relative molecular weight of the active molecule and the brain-localizing portion. In some embodiments, the first active molecule has a higher MW than the brain-localizing portion, and the first polymer linker has a lower MW than the third polymer linker. In some embodiments, the second active molecule has a higher MW than the brain-localizing portion, and the second polymer linker has a lower MW than the third polymer linker.

[0149] In some embodiments, the third polymer linker is longer than the first and / or second polymer linker. In some embodiments, the third polymer linker has a longer end-to-end distance than the first and / or second polymer linker. In some embodiments, the third polymer linker has a longer contour distance than the first and / or second polymer linker.

[0150] In some embodiments, the third polymer linker has a lower MW than the MW of at least one of the first and second polymer linkers. In some related embodiments, the MW of at least one of the first and second polymer linkers is at least about 4000 Da. In further related embodiments, the difference between the MW of the first polymer linker and the MW of at least one of the first and second polymer linkers is at least about 2000 Da. Without being bound by theory or mechanism of action, it is contemplated that the significantly longer first and / or second linker allows the folding (or higher rate of coiling) of the polymer chain, so that the actual distance between each active agent and the core particle is smaller than the distance between the brain-internalizing portion and the core particle, and the active agent is at least partially shielded by the brain-internalizing portion exposed on the surface of the multifunctional particle during BBB penetration. In some related embodiments, the end-to-end distance of the third polymer linker is greater than the end-to-end distance of the first and / or second polymer linker despite the higher MW of said first and / or second polymer linker.

[0151] In some embodiments, the distance between the first active agent and the core particle and the distance between the second active agent and the core particle are smaller than the distance between the brain-internalizing portion and the core particle. In some embodiments, at least one end group of the third polymer linker is similar to at least one end group of the first polymer linker. In some embodiments, at least one end group of the third polymer linker is similar to at least one end group of the second polymer linker. In some embodiments, the two end groups of the third polymer linker are similar to the two end groups of the first polymer linker. In some embodiments, the two end groups of the third polymer linker are similar to the two end groups of the second polymer linker. In some embodiments, the two end groups of the first polymer linker are similar to the two end groups of the second polymer linker.

[0152] In some embodiments, the core particle is linked to an additional fourth polymer. In some embodiments, the polymer is a monofunctional polymer linker. In some embodiments, the core particle is coated with a polymer layer comprising a first polymer linker, a second polymer linker, a third polymer linker and an additional fourth polymer linker, the additional polymer linker being monofunctional and used to cap the functional groups on the particle, allowing sufficient distance between the other linkers and the active molecule and the transporter. The terms "fourth polymer" and "fourth polymer linker" can be used interchangeably. In some embodiments, the fourth polymer functions as a spacer moiety. In some embodiments, the fourth polymer linker is a linear polymer linker. In some embodiments, the fourth polymer is selected from the group consisting of polyethers, polyacrylates, polyanhydrides, polyvinyl alcohols, polysaccharides, apoly(N-vinylpyrrolidone), polyglycerin (PG), poly(N-(2-hydroxypropyl)methacrylamide), polyoxazolines, poly(amino acid)-based hybrids, recombinant polypeptides, derivatives, and combinations thereof.

[0153] As used herein, the term "monofunctional" means that the polymer before being conjugated to the core particle has only one type of functional group that is configured to conjugate the polymer to the core particle.Therefore, the monofunctional polymer linker is not conjugated, cannot conjugate any moiety other than the core particle, and is used as a capping moiety.

[0154] In some embodiments, the fourth polymer comprises the same monomer units as the first and / or second polymer. In some embodiments, the fourth polymer comprises the same monomer units as the third polymer linker. In some embodiments, the first, second, third and fourth polymers comprise the same monomer units. In some embodiments, the fourth polymer is attached to the core particle via a thiol end group of the polymer. In some embodiments, the fourth polymer is a polyether. In some embodiments, the polyether is methoxypolyethylene glycol (mPEG) or a derivative thereof. In some embodiments, the mPEG is thiolated (mPEG-SH) and the thiolated mPEG is attached to the core particle via a thiol end group.

[0155] In some embodiments, the fourth polymer has a MW of 1,000-7,000 Da. In some embodiments, the fourth polymer has a MW of 500-1,000 Da, 500-3,000 Da, 500-7,000 Da, 500-10,000 Da, 1,000-3,000 Da, 1,000-4,000 Da, 1,000-5,000 Da, 1,000-7,000 Da, 1,000-10,000 Da, 3,000-5,000 Da, 3,000-7,000 Da, 3,000-10,000 Da, or 7,000-10,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the fourth polymer has a MW of at least 1,000 Da, at least 2,000 Da, at least 3,000 Da, at least 4,000 Da, at least 5,000 Da, at least 6,000 Da, at least 7,000 Da, or at least 8,000 Da. Each possibility represents a separate embodiment. According to some embodiments, the fourth polymer has a MW of up to 1,000 Da, up to 2,000 Da, up to 3,000 Da, up to 4,000 Da, up to 5,000 Da, up to 6,000 Da, up to 7,000 Da, or up to 10,000 Da. Each possibility represents a separate embodiment.

[0156] In some embodiments, the length of the fourth polymer is substantially similar to the length of at least one of the first polymer linker, the second polymer linker, and the third polymer linker. In some embodiments, the length of the fourth polymer is substantially similar to the length of the first polymer linker. In some embodiments, the length of the fourth polymer is substantially similar to the length of the second polymer linker. In some embodiments, the length of the fourth polymer is substantially similar to the length of the third polymer linker. In some embodiments, the length of the fourth polymer is substantially similar to the length of the polymer linker (first, second, or third) whose length is longer than the length of at least one of the other polymer linkers. In some embodiments, the molecular weight of the fourth polymer is substantially similar to the molecular weight of the polymer linker (first, second, or third) that has a higher molecular weight than at least one of the other polymer linkers. In some embodiments, the MW of the fourth polymer is substantially similar to the MW of the first polymer linker. In some embodiments, the MW of the fourth polymer is substantially similar to the MW of the second polymer linker. In some embodiments, the MW of the fourth polymer is substantially similar to the MW of the third polymer linker.

[0157] Without being bound by theory or mechanism of action, the efficacy of the co-delivery system of the present invention also depends on the molar ratio of the different polymer linkers, which dictates the density of the brain-internalizing transporter moieties and the active agent within the co-delivery system.

[0158] In some embodiments, the first polymer linker comprises about 5-70 mol%, 5-60 mol%, 5-40 mol%, 8-60 mol%, 10-60 mol%, 10-55 mol%, 10-50 mol%, 10-40 mol%, 10-30 mol%, 10-25 mol%, 10-20 mol%, 15-60 mol%, 15-55 mol%, 15-50 mol%, 15-45 mol%, 15-40 mol%, 15-50 mol%, 15-60 mol%, 15-55 mol%, 15-50 mol%, 15-45 mol%, 15-40 mol%, 15-50 mol%, 15-60 mol%, 15-70 mol%, 15-80 mol%, 15-80 mol%, 15-90 mol%, 15-10 ... %. Each possibility represents a separate embodiment of the present invention. In some embodiments, the first polymer linker comprises at least 2 mol%, at least 4 mol%, at least 5 mol%, at least 6 mol%, at least 8 mol%, at least 10 mol%, at least 12 mol%, at least 15 mol%, at least 18 mol%, at least 20 mol%, at least 25 mol%, at least 30 mol%, at least 35 mol%, at least 40 mol%, at least 50 mol%, or at least 60 mol% of the total polymer attached to the core particle, with each possibility representing a separate embodiment.

[0159] In some embodiments, the second polymer linker comprises about 5-70 mol%, 5-60 mol%, 5-40 mol%, 8-60 mol%, 10-60 mol%, 10-55 mol%, 10-50 mol%, 10-40 mol%, 10-30 mol%, 10-25 mol%, 10-20 mol%, 15-60 mol%, 15-55 mol%, 15-50 mol%, 15-45 mol%, 15-40 mol%, 15-40 mol%, 15-5 ...0 mol%, 15-40 mol%, 15-50 mol%, 15-60 mol%, 15-50 mol%, 15-40 mol%, 15-50 mol%, 15-50 mol%, 15-60 mol%, 15-60 mol%, 15-50 mol%, 15-40 mol%, 15-50 mol%, 15-50 mol%, 15-60 mol%, 15-70 mol%, 15-80 mol%, 15-80 mol%, 15-90 mol%, 15-90 mol%, 15-100 mol%, 15-100 mol%, 15-100 mol%, 15-100 mol%, 15-100 mol%, 15-100 mol%, 15-100 mol%, 15-100 mol%, 15-100 mol%, %, 15-30 mol%, 15-25 mol%, 15-20 mol%, 2-10 mol%, 2-20 mol%, 2-50 mol%, 2-60 mol%, 2-70 mol%, 5-10 mol%, 5-20 mol%, 5-70 mol%, 10-20 mol%, 10-50 mol%, 10-70 mol%, 20-50 mol%, 20-40 mol%, 30-50 mol%, 30-60 mol%, 30-70 mol%, 50-60 mol%, or 50-70 mol%. Each possibility represents a separate embodiment of the present invention. In some embodiments, the second polymer linker comprises at least 2 mol%, at least 4 mol%, at least 5 mol%, at least 6 mol%, at least 8 mol%, at least 10 mol%, at least 12 mol%, at least 15 mol%, at least 18 mol%, at least 20 mol%, at least 25 mol%, at least 30 mol%, at least 35 mol%, at least 40 mol%, at least 50 mol%, or at least 60 mol% of the total polymer attached to the core particle, with each possibility representing a separate embodiment.

[0160] In some embodiments, the third polymer linker represents about 5-70 mol%, 5-60 mol%, 5-40 mol%, 8-60 mol%, 10-60 mol%, 10-55 mol%, 10-50 mol%, 10-40 mol%, 10-30 mol%, 10-25 mol%, 10-20 mol%, 15-60 mol%, 15-55 mol%, 15-50 mol%, 15-45 mol%, 15-40 mol%, 15-50 mol%, 15-60 mol%, 15-55 mol%, 15-50 mol%, 15-45 mol%, 15-40 mol%, 15-50 mol%, 15-60 mol%, 15-70 mol%, 15-80 mol%, 15-80 mol%, 15-90 mol%, 15-10 ... %. Each possibility represents a separate embodiment of the present invention. In some embodiments, the third polymer linker comprises at least 2 mol%, at least 4 mol%, at least 5 mol%, at least 6 mol%, at least 8 mol%, at least 10 mol%, at least 12 mol%, at least 15 mol%, at least 18 mol%, at least 20 mol%, at least 25 mol%, at least 30 mol%, at least 35 mol%, at least 40 mol%, at least 50 mol%, or at least 60 mol% of the total polymer attached to the core particle, with each possibility representing a separate embodiment.

[0161] In some embodiments, the fourth polymer comprises about 5-90 mol%, 5-85 mol%, 5-80 mol%, 10-80 mol%, 20-78 mol%, 25-75 mol%, 30-75 mol%, 40-75 mol%, 50-75 mol%, 60-75 mol%, 60-70 mol%, 60-80 mol%, 5-60 mol%, 10-60 mol%, 10-55 mol%, 10-50 mol%, 10-40 mol%, 15-60 mol%, 15-55 mol%, 15-50 mol%, 15-45 mol%, or 15-40 mol% of the total polymer bound to the core particle. Each possibility represents a separate embodiment of the invention. In some embodiments, the fourth polymer comprises 60-80 mol% of the total polymer bound to the core particle. In some embodiments, the fourth polymer comprises 50-80 mol% of the total polymer attached to the core particle. In some embodiments, the fourth polymer comprises at least 2 mol%, at least 4 mol%, at least 5 mol%, at least 6 mol%, at least 8 mol%, at least 10 mol%, at least 12 mol%, at least 15 mol%, at least 18 mol%, at least 20 mol%, at least 25 mol%, at least 30 mol%, at least 35 mol%, at least 40 mol%, at least 45 mol%, at least 50 mol%, at least 55 mol%, at least 60 mol%, at least 65 mol%, or at least 70 mol% of the total polymer attached to the core particle. Each possibility represents a separate embodiment.

[0162] In some embodiments, the first polymer linker constitutes about 5-45 mol % of the total polymer attached to the core particle, the second polymer linker constitutes about 5-45 mol % of the total polymer attached to the core particle, the third polymer linker constitutes about 10-45 mol % of the total polymer attached to the core particle, and the fourth polymer constitutes about 40-80 mol % of the total polymer attached to the core particle.

[0163] In some embodiments, the first polymer linker constitutes about 10-40 mol % of the total polymer attached to the core particle, the second polymer linker constitutes about 10-40 mol % of the total polymer attached to the core particle, the third polymer linker constitutes about 10-40 mol % of the total polymer attached to the core particle, and the fourth polymer constitutes about 40-70 mol % of the total polymer attached to the core particle.

[0164] In some embodiments, the first polymer linker and the second polymer linker comprise about 10%-60 mol%, 10-50 mol%, 10-45 mol%, 10-40 mol%, 10-30 mol%, or 10-20 mol% of the total polymer linkers attached to the core particle, with each possibility representing a separate embodiment of the present invention.

[0165] It is understood that the mole % of each polymer is dependent upon the other polymers attached to the core particle such that the total mole % of polymers does not exceed 100%.

[0166] In some embodiments, the (w / w / w / w) ratio of the first polymer linker, the second polymer linker, the third polymer linker, and the fourth polymer is from 5:5:5:85 to 20:20:30:30.

[0167] According to the principles of the present invention, the co-delivery system comprises a brain-internalizing transporter moiety conjugated to a third polymer linker. The term "brain-internalizing transporter moiety" may be used interchangeably with the term "brain-internalizing moiety" herein and refers to a molecule that can specifically bind to a receptor or surface protein expressed by a cellular component of the BBB. The three major cellular elements of the brain microvasculature that collectively form the BBB are brain endothelial cells, astrocyte endfeet, and pericytes (PCs). In some embodiments, the brain-internalizing transporter moiety can bind to a receptor or surface protein expressed by brain endothelial cells. In some embodiments, the brain-internalizing transporter moiety can bind to a receptor or surface protein expressed by astrocyte foot processes. In some embodiments, the brain-internalizing transporter moiety can bind to a receptor or surface protein expressed by pericytes (PCs). Without being bound by theory or mechanism, it is hypothesized that the brain-internalizing moiety facilitates transport of the entire codelivery system across the BBB, possibly via receptor-mediated transcytosis (RMT) or receptor-mediated endocytosis (RME) mechanisms.

[0168] In some embodiments, the brain-internalizing moiety is selected from, but is not limited to, insulin, an antibody specific for insulin receptor, or a portion of such an antibody, e.g., a Fab fragment, transferrin, an antibody specific for transferrin receptor, or a portion of such an antibody, a polypeptide that specifically binds to transferrin receptor, a polypeptide that specifically binds to insulin receptor, insulin-like growth factor 1, an antibody specific for insulin-like growth factor receptor 1, or a portion of such an antibody, a polypeptide that specifically binds to insulin-like growth factor receptor 1, apolipoprotein A1, B, or E, lactoferrin, angiopep-2, an antibody specific for low-density lipoprotein receptor or lipoprotein receptor-related protein, a polypeptide that specifically binds to low-density lipoprotein receptor or lipoprotein receptor-related protein, an antibody specific for diphtheria toxin receptor, or a portion of such an antibody, a polypeptide that specifically binds to diphtheria toxin receptor, and a BBB-permeable cell-penetrating peptide (CPP). Each possibility represents a separate embodiment of the invention. As used herein, the term "cell-penetrating peptide (CPP)" refers to a peptide with enhanced ability to cross cell membrane bilayers without causing significant lethal membrane damage. The term "BBB-permeable CPP" refers to a cell-permeable peptide that can cross the membrane of BBB cells and thus penetrate into the brain (Zou, Li-li et al. Current neuropharmacology 11.2 (2013): 197-208., and Stalmans, Sofie et al. PloS one 10.10 (2015): e0139652).

[0169] Other cellular proteins known in the art that can promote transcytosis can also be used as brain-internalizing moieties. In some embodiments, the brain-internalizing moiety is selected from the group consisting of insulin, transferrin, low density lipoprotein, apolipoprotein A1, B, or E, and lactoferrin. Each possibility represents a separate embodiment of the invention. In some embodiments, the brain-internalizing moiety is selected from the group consisting of insulin and transferrin. In some embodiments, the brain-internalizing moiety is insulin. In some embodiments, the molecular weight (MW) of the brain-internalizing moiety is about 5 kilodaltons (kD).

[0170] According to the principles of the present invention, the first polymer linker is conjugated to a first active agent, and the second polymer linker is conjugated to a second active agent. As used herein, the term "active agent" refers to an agent intended to be delivered to the brain of a subject and can be used as a therapeutic agent, a targeting agent, or a diagnostic agent. In some embodiments, each of the first active agent and the second active agent is independently selected from a bioactive molecule and a labeling molecule. According to some embodiments, the first active agent is characterized by poor BBB permeability. According to some embodiments, the second active agent is characterized by poor BBB permeability. According to some embodiments, the first active agent and the second active agent are characterized by poor BBB permeability. According to some embodiments, upon crossing the BBB, the first and / or second active agent can further target the co-delivery system to specific regions in the brain, such as the hippocampus, striatum, medulla oblongata, cerebellum, and cortex. According to some embodiments, upon crossing the BBB, the first and / or second active agent can target the nanodelivery system to specific cell populations in the brain, such as glioma (or other tumor) cells, microglial cells, astrocytes and neuronal cells.

[0171] In some embodiments, each of the first active agent and the second active agent is independently selected from the group consisting of, but not limited to, a small molecule, a polymer, an oligonucleotide, an antisense RNA, a peptide, a chemical reagent, a toxin, and any combination thereof. In some embodiments, each of the first active agent and the second active agent is independently selected from the group consisting of a polymer, a peptide, a toxin, and a small molecule. In some embodiments, each of the first active agent and the second active agent is independently selected from the group consisting of a polypeptide, an antibody, a peptide, and a small molecule. Each possibility represents a separate embodiment of the present invention.

[0172] The oligonucleotide molecule according to the present invention may comprise any DNA or RNA molecule, which may be natural, synthetic or modified.The oligonucleotide may be single-stranded or double-stranded.The oligonucleotide of the present invention may comprise, but is not limited to, small interfering RNA (siRNA), microRNA (miRNA), double-stranded RNA (dsRNA), antisense RNA or DNA, aptamer oligonucleotide, peptide nucleic acid (PNA), sugar ring modified oligonucleotide, nucleoside organothiophosphate (PS) analog, CpG oligonucleotide, and DNA enzyme.

[0173] In some embodiments, the first active agent and the second active agent are of the same type selected from small molecules, antibodies, oligonucleotides, antisense RNA, and peptides. In some related embodiments, the first polymer linker and the second polymer linker are the same.

[0174] In some embodiments, the first and / or second active agent is a bioactive molecule. In some embodiments, the bioactive molecule is contiguous with the respective polymer linker. As used herein, the term "bioactive molecule" refers to a compound or molecule that can induce or modify a biological response in a system or bind to a specific cell receptor / marker, thereby targeting the system to a specific cell. In some embodiments, the bioactive molecule is a therapeutic agent. In some embodiments, the bioactive molecule has a therapeutic application. In some embodiments, the bioactive molecule has a diagnostic application. In some embodiments, the bioactive molecule has both a therapeutic application and a diagnostic application. In some embodiments, the bioactive molecule comprises a small molecule, a polymer, an oligonucleotide, an antisense RNA, a peptide, a chemical reagent, or any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0175] In some embodiments, the first active agent and / or the second active agent is a macromolecule. The term "macromolecule" as defined herein refers to a very large molecule, generally formed by polymerization of monomers. In some embodiments, the macromolecule is a polypeptide or a protein. In some embodiments, the macromolecule is an enzyme. In some embodiments, the macromolecule is an antibody or a fragment thereof. In some specific embodiments, the antibody is selected from the group consisting of anti-IgG1, anti-IbA1, anti-HER2+ (trastuzumab & pertuzumab), anti-EGFR (cetuximab), anti-GD2, and checkpoint inhibitor antibodies, such as anti-PD-1, anti-PD-L1 and anti-CTLA-4, or fragments thereof.

[0176] As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that contain at least one binding domain formed from the folding of polypeptide chains with a three-dimensional binding space with an internal surface shape and charge distribution complementary to the antigenic determinant characteristic of the antigen. Antibodies typically have a tetrameric form, containing two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain. The variable regions of each light / heavy chain pair form the antibody binding site. Antibodies can be oligoclonal, polyclonal, monoclonal, chimeric, camelized, CDR-grafted, multispecific, bispecific, catalytic, humanized, fully human, anti-idiotypic, and antibodies that can be labeled in soluble or binding form, alone or in combination with other amino acid sequences, as well as fragments, variants or derivatives thereof, including epitope-binding fragments. Antibodies can be from any species. The term antibody also includes binding fragments, including, but not limited to, Fv, Fab, Fab', F(ab')2 single chain antibodies (svFC), dimeric variable regions (diabodies) and disulfide-linked variable regions (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Antibody fragments may or may not be fused to another immunoglobulin domain, including, but not limited to, an Fc region or a fragment thereof. Those skilled in the art will further recognize that other fusion products may be generated, including, but not limited to, scFv-Fc fusions, variable region (e.g., VL and VH)-Fc fusions and scFv-scFv-Fc fusions.

[0177] In some embodiments, the first active agent and / or the second active agent is an antibody. In some embodiments, the antibody is an antibody that specifically binds to a receptor present on the surface of a target cell in the brain. In some embodiments, the antibody is an antibody that specifically binds to a receptor present on cells in a specific brain region. In some embodiments, the antibody is an antibody that specifically binds to a receptor present on the surface of a diseased cell in the brain. In some embodiments, the antibody is a bispecific antibody. In some embodiments, both the first active agent and the second active agent are bispecific antibodies. In some embodiments, the first active agent and / or the second active agent is an antibody that has therapeutic activity against a brain-related disease or disorder.

[0178] Exemplary antibodies include, but are not limited to, anti-HER2+ (trastuzumab & pertuzumab), anti-EGFR (cetuximab), checkpoint inhibitor antibodies (anti-PD-1, anti-PD-L1, anti-CTLA-4), and anti-GD2.

[0179] In some embodiments, the antibody has a molecular weight (MW) of 100-120 kD, 100-150 kD, 100-200 kD, 100-250 kD, 150-200 kD, 150-250 kD, 200-250 kD. Each possibility represents a separate embodiment. In some embodiments, the antibody has a MW of at least 100 kD, at least 110 kD, at least 120 kD, at least 130 kD, at least 140 kD, at least 150 kD, at least 160 kD, at least 180 kD, at least 200 kD, at least 250 kD. Each possibility represents a separate embodiment. In some embodiments, the antibody has a MW of 150-200 kD. In some embodiments, the antibody has a MW of 130-180 kD. In some embodiments, the antibody has a MW of 140-160 kD.

[0180] In some specific embodiments, the antibodies have a MW of 150-200 kD and each polymer linker comprises a PEG with a MW of at least 1,000 Da, at least 2,000 Da, at least 2,500 Da, or at least 3,000 Da. In some embodiments, the antibodies have a MW of 150-200 kD and each polymer linker comprises a PEG with a MW of up to 2,000 Da, up to 2,500 Da, up to 3,000 Da, up to 3,500 Da, up to 4,000 Da, up to 5,000 Da, or up to 6,000 Da. In some embodiments, the antibodies have a MW of 150-200 kD and each polymer linker comprises a PEG with a MW of 1,000 Da to 4,000 Da. In some such embodiments, the brain-internalizing moiety is insulin having a MW of 5-6 kD and the third polymer linker comprises a PEG having a MW of at least 4,000 Da.

[0181] In some embodiments, the first active agent and / or the second active agent is a peptide. In some embodiments, the peptide can specifically bind to a receptor present on the surface of a target cell in the brain. In some embodiments, the peptide can specifically bind to a receptor present on a cell in a specific brain region. In some embodiments, the peptide can specifically bind to a receptor present on the surface of a diseased cell in the brain. In some embodiments, the peptide has therapeutic activity against brain-related diseases or disorders.

[0182] As used herein, the term "peptide" refers to any polymeric compound produced by amide bond formation between the α-carboxyl group of one D- or L-amino acid and the α-amino group of another D- or L-amino acid.

[0183] In some embodiments, the first active agent and / or the second active agent is a small molecule. In some embodiments, the small molecule can specifically bind to a receptor present on the surface of a target cell in the brain. In some embodiments, the small molecule can specifically bind to a receptor present on a cell in a specific brain region. In some embodiments, the small molecule can specifically bind to a receptor present on the surface of a diseased cell in the brain. In some embodiments, the small molecule has therapeutic activity against a brain-related disease or disorder.

[0184] The term "small molecule" as used herein refers to a synthetic or naturally occurring organic or inorganic molecule that generally has a molecular weight of less than 1000 Da. Any fragment of a peptide, protein, or polypeptide, including native sequences and variants that fall within the above-mentioned molecular weight range, is also encompassed by the term "small molecule."

[0185] In some embodiments, the first and / or second active agent is a therapeutic agent effective in treating a brain-related disease or disorder. In some embodiments, the first and / or second active agent is an antibody used in treating or diagnosing a brain-related disease. In some embodiments, the first and / or second active agent is a small molecule used in treating or diagnosing a brain-related disease. In some embodiments, the small molecule is selected from cisplatin, lapatinib, neratinib, and tucatinib. Each possibility represents a separate embodiment of the present invention.

[0186] In some embodiments, at least one of the first active agent and the second active agent is a labeled molecule. The term "labeled molecule" as used herein refers to a molecule capable of producing a signal detectable by an appropriate detection means, including but not limited to radioactive and fluorescent molecules. In some embodiments, the labeled molecule has diagnostic applications. In some embodiments, the labeled molecule is a diagnostic agent. In some embodiments, the labeled molecule comprises a small molecule, a macromolecule, an oligonucleotide, an antisense RNA, a peptide, or any combination thereof. In some embodiments, the labeled molecule is a small molecule. In some embodiments, the labeled molecule is an antibody.

[0187] In some embodiments, the first and / or second active agent is a small molecule having a MW of less than 1,000 Daltons (Da). In some embodiments, the small molecule has a MW of 10-50 Da, 10-100 Da, 10-500 Da, 10-1,000 Da, 50-100 Da, 50-500 Da, 50-1,000 Da, 100-300 Da, 100-500 Da, 100-800 Da, 100-1,000 Da, 500-800 Da, 500-1,000 Da, or 800-1,000 Da. Each possibility represents a separate embodiment. In some embodiments, the small molecule has a MW of less than 1,000 Da, less than 900 Da, less than 800 Da, less than 700 Da, less than 600 Da, less than 500 Da, less than 400 Da, less than 300 Da, less than 200 Da, less than 100 Da. Each possibility represents a separate embodiment. In some embodiments, the small molecule has a MW of 100 Da or more, 200 Da or more, 300 Da or more, 400 Da or more, 500 Da or more, 600 Da or more, 700 Da or more, 800 Da or more, 900 Da or more. Each possibility represents a separate embodiment. In some specific embodiments, the first and / or second active agent is a small molecule and each polymer linker comprises a PEG having a MW of up to 3,000 Da, up to 2,500, up to 2,000 Da, up to 1,500, or up to 1,000 Da. Each possibility represents a separate embodiment.

[0188] In some embodiments, the first and / or second active agent is an antisense RNA.In some embodiments, the first and / or second active agent is a drug.

[0189] According to the principles of the present invention, the multifunctional system allows for the synchronized co-delivery of two active agents into the brain. In some embodiments, at least one of the first active agent and the second active agent has poor BBB penetration in its original free form. In some embodiments, both the first active agent and the second active agent have poor BBB penetration in their original free form.

[0190] In some embodiments, each of the first and second active agents is a therapeutic agent having therapeutic activity against a brain-related disease or disorder. One advantage of the co-delivery system is that it may induce synergistic effects. With regard to the co-delivery of different active agents, the therapeutic outcome may be either additive (i.e., the outcome expected by combining the effects of each drug individually) or synergistic (i.e., the combination produces a significant benefit over that expected by adding the individual effects). In some embodiments, the combination of the first and second active agents produces an additive therapeutic effect. In other embodiments, the combination of the first and second active agents produces a synergistic therapeutic effect.

[0191] In some embodiments, the first active agent is a therapeutic agent and the second active agent is a targeting agent capable of binding to specific surface receptors or ligands, thus targeting the system to specific brain regions or specific cell populations within the brain, thereby providing enhanced and focused treatment. In some related embodiments, the second active agent further has therapeutic activity against a brain-related disease or disorder.

[0192] In some embodiments, at least one of the first and second active agents is a molecule capable of intracellular targeting, i.e., a molecule that targets an intracellular macromolecule. In some related embodiments, the molecule is conjugated to the core particle via a cleavable linker.

[0193] Complex diseases are often multifactorial and are known to involve redundant or synergistic actions of disease mediators or upregulation of different receptors involving crosstalk between their signaling networks (Kontermann, R. In: MAbs. Taylor & Francis, 2012. p. 182-197). As a result, blocking multiple different pathological factors and pathways can lead to significant improvement in therapeutic efficacy. This result can be achieved by combining different drugs or using dual targeting strategies.

[0194] In some embodiments, both the first and second active agents are capable of binding to a specific surface receptor or ligand. Thus, in some embodiments, the multifunctional system of the present invention allows for the combination of the specificities of two different active agents, e.g., antibodies, in a single system to simultaneously interfere with different surface receptors or ligands in the brain. Without being bound by any theory or mechanism of action, it is hypothesized that dual-targeted particles (e.g., dual antibody particles) can bring different targets in the brain into close proximity to support protein complex formation on a cell or induce cell-cell contact. In some embodiments, the first and second active agents are antibodies, and at least one of the first and second active agents is a bispecific antibody. Thus, in some embodiments, the multifunctional system of the present invention allows for simultaneous interference with two or more targets. In some related embodiments, at least one of the first and second active agents further has therapeutic activity against a brain-related disease or disorder. In some embodiments, both the first and second active agents further have therapeutic activity against a brain-related disease or disorder.

[0195] In some embodiments, the first active agent is an antibody and the second active agent is selected from the group consisting of an antibody, a peptide, a small molecule, an oligonucleotide, an antisense RNA, and any fragment or combination thereof. In a related embodiment, the first active agent is an antibody and the second active agent is Fas Ligand (FasL) or another death-inducing receptor ligand. In some embodiments, the first active agent is an antibody and the second active agent is selected from the group consisting of a peptide, a small molecule, an oligonucleotide, an antisense RNA, and any fragment or combination thereof. Each possibility represents a separate embodiment of the present invention. In some embodiments, the first active agent is an antibody and the second active agent is a small molecule.

[0196] In some embodiments, each of the first active agent and the second active agent is an antibody or a fragment thereof. In some embodiments, each of the first active agent and the second active agent is an antibody or an active fragment thereof. In some embodiments, each of the first active agent and the second active agent is an antibody or an antigen-binding fragment thereof. In some related embodiments, the first active agent and the second active agent comprise different antibodies. In other related embodiments, the first active agent and the second active agent comprise or consist of different fragments of the same antibody. For example, in some embodiments, the first active agent comprises or consists of a Fab region of an antibody and the second active agent comprises or consists of an Fc region of the same antibody. In other embodiments, the first active agent comprises or consists of a whole antibody (e.g., IgG) and the second active agent comprises or consists of a fragment of the same antibody. For example, in some embodiments, the first active agent comprises or consists of a whole antibody (e.g., IgG) and the second active agent comprises or consists of an Fc region of the same antibody.

[0197] In some embodiments, the first active agent is a peptide and the second active agent is selected from the group consisting of an antibody, a peptide, a small molecule, an oligonucleotide, an antisense RNA, and any fragment or combination thereof, with each possibility representing a separate embodiment of the present invention.

[0198] In some embodiments, the first active agent is a small molecule and the second active agent is selected from the group consisting of an antibody, a peptide, a small molecule, an oligonucleotide, an antisense RNA, and any fragment or combination thereof, with each possibility representing a separate embodiment of the present invention.

[0199] In some embodiments, the first active agent is an oligonucleotide and the second active agent is selected from the group consisting of an antibody, a peptide, a small molecule, an oligonucleotide, an antisense RNA, and any fragment or combination thereof, with each possibility representing a separate embodiment of the present invention.

[0200] In some embodiments, the first active agent is an antisense RNA and the second active agent is selected from the group consisting of an antibody, a peptide, a small molecule, an oligonucleotide, an antisense RNA, and any fragment or combination thereof, each possibility representing a separate embodiment of the present invention.

[0201] In some embodiments, the core particle is a gold nanoparticle. In some embodiments, the first linear polymer linker is a thiolated PEG3500 acid or a thiolated PEG35000 amine. In some embodiments, the second linear polymer linker is a thiolated PEG3500 acid or a thiolated PEG3500 amine. In some embodiments, the third linear polymer linker is a thiolated PEG5000 acid or a thiolated PEG5000 amine. In some embodiments, the brain-internalizing transporter moiety is insulin.

[0202] In some embodiments, the core particle is a gold nanoparticle. In some embodiments, the first linear polymer linker is a thiolated PEG3500 acid or a thiolated PEG35000 amine. In some embodiments, the second linear polymer linker is a thiolated PEG1000 acid or a thiolated PEG1000 amine. In some embodiments, the third linear polymer linker is a thiolated PEG5000 acid or a thiolated PEG5000 amine. In some embodiments, the brain-internalizing transporter moiety is insulin.

[0203] In some embodiments, the multifunctional particle further comprises at least one additional active agent attached to the core particle via an additional polymer linker. The different possibilities of the at least one additional active agent and each polymer linker are similar to those described above for the first and second active agents, and the first and second polymer linkers.

[0204] In some embodiments, the present invention provides a plurality of multifunctional particles of any of the above embodiments.

[0205] Preparation process According to another aspect, there is provided a process for preparing the multifunctional particles of the present invention in all its embodiments described above, comprising the steps of: a) partially coating the surface of a core particle with a first polymer linker, followed by conjugating the first polymer linker to a first active agent; b) partially coating the surface of the core particle with a second polymer linker, followed by conjugating the second polymer linker to a second active agent; c) partially coating the surface of the core particle with a third polymer linker, followed by conjugating the third polymer linker to a brain-internalizing transporter moiety. Including, Processes are provided in which steps (a), (b) and (c) can be performed in any order.

[0206] The term "partial coating," as used herein, refers to the conjugation of a plurality of respective polymer linkers to the surface of a particle such that the plurality of linkers partially cover the surface of the particle at a density level that is lower than the saturation level of the bare particle.

[0207] Any method known in the art can be used to determine the amount of polymer required to achieve full density (i.e., 100%) coating of the particles, and accordingly the amount required for partial coating. For example, adding different amounts of polymer to the particle solution and measuring the concentration of free polymer in the supernatant after centrifugation is a widely used method. Alternatively, any characterization method that is sensitive to changes in coating density can be used, such as zeta potential and DLS. Furthermore, theoretical calculations can be performed to determine the amount of polymer required to achieve complete coating, depending on the surface area of ​​the particles. For example, thiol-PEG molecules can be applied to a 0.35 nm 2 solution on a gold nanoparticle surface. 2 It has been previously shown that the thiol-PEG linker occupies a footprint area of ​​100 nm (Qian, Ximeni et al. Nature biotechnology 26.1 (2008): 83-90). Therefore, the amount of thiol-PEG linker required to cover 100% of the surface of gold nanoparticles (GNPs) can be calculated based on the average diameter of the GNPs.

[0208] In some embodiments, each of the first polymer linker, the second polymer linker, and the third polymer linker comprises 5-70%, 5-60%, 5-40%, 8-60%, 10-60%, 10-55%, 10-50%, 10-40%, 10-30%, 10-25%, 10-20%, 15-60%, 15-55%, 15-50% of the surface of the core particle. , 15-45%, 15-40%, 15-30%, 15-25%, 15-20%, 2-10%, 2-20%, 2-50%, 2-60%, 2-70%, 5-10%, 5-20%, 5-70%, 10-20%, 10-50%, 10-70%, 20-50%, 20-40%, 30-50%, 30-60% or 30-70% coverage. Each possibility represents a separate embodiment of the present invention.

[0209] In some embodiments, step (a) comprises removing 5-70%, 5-60%, 5-40%, 8-60%, 10-60%, 10-55%, 10-50%, 10-40%, 10-30%, 10-25%, 10-20%, 15-60%, 15-55%, 15-50%, 15-45%, 15-40%, 15-30%, 15-40%, 15-50%, 15-60%, 15-70%, 15-40%, 15-40%, 15-50%, 15-40%, 15-50%, 15-60%, 15-60%, 15-50%, 15-45%, 15-40%, 15-30%, 15-40%, 15-50%, 15-50%, 15-60%, 15-60%, 15-50%, 15-45%, 15-40%, 15-30%, 15-50%, 15-50%, 15-40%, 15-50%, 15-50%, 15-60%, 15-60%, 15-60%, 15-50%, 15-40%, 15-50%, 15-50%, 15-50%, 15-6 ...60%, 15-60%, 15-60%, 15-60%, 15-60%, Including coating 5-25%, 15-20%, 2-10%, 2-20%, 2-50%, 2-60%, 2-70%, 5-10%, 5-20%, 5-70%, 10-20%, 10-50%, 10-70%, 20-50%, 20-40%, 30-50%, 30-60%, 30-70%, 50-60%, or 50-70%. Each possibility represents a separate embodiment of the present invention.

[0210] In some embodiments, step (b) comprises removing 5-70%, 5-60%, 5-40%, 8-60%, 10-60%, 10-55%, 10-50%, 10-40%, 10-30%, 10-25%, 10-20%, 15-60%, 15-55%, 15-50%, 15-45%, 15-40%, 15-30%, 15-40%, 15-50%, 15-60%, 15-70%, 15-40%, 15-40%, 15-50%, 15-40%, 15-50%, 15-60%, 15-60%, 15-50%, 15-45%, 15-40%, 15-30%, 15-40%, 15-50%, 15-50%, 15-60%, 15-60%, 15-50%, 15-45%, 15-40%, 15-30%, 15-50%, 15-50%, 15-40%, 15-50%, 15-50%, 15-60%, 15-60%, 15-60%, 15-50%, 15-40%, 15-50%, 15-50%, 15-50%, 15-6 ...60%, 15-60%, 15-60%, 15-60%, 15-40%, Including coating 5-25%, 15-20%, 2-10%, 2-20%, 2-50%, 2-60%, 2-70%, 5-10%, 5-20%, 5-70%, 10-20%, 10-50%, 10-70%, 20-50%, 20-40%, 30-50%, 30-60%, 30-70%, 50-60%, or 50-70%. Each possibility represents a separate embodiment of the present invention.

[0211] In some embodiments, step (c) comprises removing 5-70%, 5-60%, 5-40%, 8-60%, 10-60%, 10-55%, 10-50%, 10-40%, 10-30%, 10-25%, 10-20%, 15-60%, 15-55%, 15-50%, 15-45%, 15-40%, 15-30%, 15-40%, 15-50%, 15-60%, 15-70%, 15-40%, 15-40%, 15-50%, 15-40%, 15-50%, 15-60%, 15-60%, 15-50%, 15-45%, 15-40%, 15-30%, 15-40%, 15-50%, 15-50%, 15-60%, 15-50%, 15-40%, 15-50%, 15-50%, 15-60%, 15-60%, 15-50%, 15-40%, 15-50%, 15-50%, 15-60%, 15-60%, 15-50%, 15-40%, 15-50%, 15-50%, 15-50%, 15-6 ...60%, 15-60%, 15-50%, 15-40%, 15-50%, 15-50%, Including coating 5-25%, 15-20%, 2-10%, 2-20%, 2-50%, 2-60%, 2-70%, 5-10%, 5-20%, 5-70%, 10-20%, 10-50%, 10-70%, 20-50%, 20-40%, 30-50%, 30-60%, 30-70%, 50-60%, or 50-70%. Each possibility represents a separate embodiment of the present invention.

[0212] In some embodiments, steps (a)-(c) are performed sequentially in any order. A person skilled in the art will be able to determine the optimal order of steps depending on different parameters, such as the type of core particle, the specific polymer linker, the active agent used, the brain internalizing transporter moiety, etc. In some embodiments, the process further comprises centrifugation after each of steps (a), (b) and (c).

[0213] In some embodiments, the first polymer linker and the second polymer linker are the same. In some related embodiments, steps (a) and (b) are carried out simultaneously by partially co-coating the surface of the core particle with the first polymer linker and the second polymer linker, and then conjugating the first active agent and the second active agent to the polymer linker. In some related embodiments, the step of partially co-coating the surface of the core particle with the first polymer linker and the second polymer linker comprises coating 10-70%, 10-60%, 10-40%, 10-60%, 10-60%, 10-55%, 10-50%, 10-45%, 10-40%, 10-30%, 10-25%, 10-20%, 15-60%, 15-55%, 15-50%, 15-45%, 15-40%, 15-30%, 15-25%, 15-20%, 10-20%, 10-50%, 10-70%, 20-50%, 20-40%, 30-50%, 30-60%, 30-70%, 50-60%, or 50-70% of the surface of the core particle. Each possibility represents a separate embodiment of the invention. In further related embodiments, conjugating the first and second active agents to the polymer linker comprises adding a mixture of the first and second active agents in a desired molar ratio to the particle solution.

[0214] In some embodiments, the process further comprises partially coating the surface of the core particle with a fourth polymer linker. In some embodiments, the fourth polymer linker is a monofunctional linker.

[0215] According to a related embodiment, there is provided a process for preparing multifunctional particles, comprising the steps of: a) partially coating the surface of a core particle with a first polymer linker, followed by conjugating the first polymer linker to a first active agent; b) partially coating the surface of the core particle with a second polymer linker, followed by conjugating the second polymer linker to a second active agent; c) partially coating the surface of the core particle with a third polymer linker, followed by conjugating the third polymer linker to a brain-internalizing transporter moiety; and d) partially coating the surface of the core particle with a fourth polymer linker. wherein the fourth polymeric linker is a monofunctional linker that functions as a capping moiety, and steps (a), (b), (c) and (d) can be performed in any order.

[0216] In some embodiments, the particles are gold nanoparticles (GNPs) and the process comprises: (a) dissolving HAuCl 4 (b) reduction of the GNPs; (c) simultaneous incubation of the reduced GNPs with one monofunctional linker and two different heterofunctional linkers; (d) activation of the GNPs to obtain free COOH groups; (d) conjugation of a transporter or other moiety; and (d) successive steps of conjugation by incubating two different bioactive molecules with a solution containing a mixture of them.

[0217] In some embodiments, the monofunctional linker is mPEG-SH. According to a particular embodiment, the monofunctional linker is mPEG5000-SH or mPEG6000-SH, and is added so as to cover about 80-90% of the particle surface.

[0218] In some embodiments, the heterofunctional linker is COOH-PEG-SH. According to some embodiments, one heterofunctional linker is COOH-PEG5000-SH and is added at a concentration that covers about 15% of the particle surface. According to some embodiments, another heterofunctional linker is COOH-PEG3500-SH and is added at a concentration that covers about 5% of the particle surface.

[0219] In some embodiments, activation of the GNPs is carried out by mixing the GNPs with (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC).

[0220] In some embodiments, the transporter is insulin, and its conjugation is carried out by incubation with activated GNPs at a concentration of about 50-500 IU / ml for 1-5 hours.

[0221] In some embodiments, the two biologically active molecules are incubated overnight with the activated GNPs at a concentration of 1-50 mg / ml.

[0222] Analysis of the GNPs is performed after each step using methods known in the art, for example, dynamic light scattering (DLS).

[0223] In some embodiments, quantification of bioactive molecules and transporters (e.g., insulin) attached to PEG groups on the GNPs is performed by enzyme-linked immunosorbent assay (ELISA) of the supernatant containing unbound proteins remaining after centrifugation precipitation of the GNPs.

[0224] The core particles, first polymer linker, second polymer linker, third polymer linker, fourth polymer linker, brain internalizing transporter moiety, and first and second active agents suitable for use in the preparation process are as described herein above in connection with various aspects and embodiments of the co-delivery system.

[0225] Pharmaceutical Compositions In yet another aspect, a pharmaceutical composition is provided that includes a multifunctional particle according to various embodiments described hereinabove and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition includes a plurality of multifunctional particles according to various embodiments described hereinabove and a pharma- ceutically acceptable carrier.

[0226] As used herein, "pharmaceutical acceptable formulation", "pharmaceutical composition" or "pharmaceutical acceptable composition" may include any of a number of carriers, such as solvents, dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption retardants, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, such materials and combinations thereof, as known to those skilled in the art (Remington, 1990). Pharmaceutical compositions containing the particles of the present invention as active ingredients can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, Pa. (1990). See also Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, Philadelphia, Pa. (2005).

[0227] The composition may contain different types of carriers depending on whether it is administered in solid, liquid or aerosol form and whether it needs to be sterile for administration route such as injection. Those skilled in the art will be familiar with the techniques of making sterile solutions for injection or any other route of application. Sterile injectable solutions are prepared by incorporating the required amount of active compound in the appropriate solvent with various other ingredients well known to those skilled in the art.

[0228] Carriers, in total, may comprise from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0229] According to some embodiments, the pharmaceutical composition is formulated for systemic administration. According to some embodiments, the pharmaceutical composition is formulated for systemic administration selected from intravenous and intranasal administration. According to some embodiments, the pharmaceutical composition is formulated for intravenous administration. According to some embodiments, the pharmaceutical composition is formulated for intranasal administration. According to some embodiments, the pharmaceutical composition is formulated for intrathecal administration.

[0230] Compositions contemplated herein may take the form of a solution, suspension, emulsion, aerosol, combinations thereof, or any other pharma- ceutically acceptable composition generally known in the art.

[0231] In some embodiments, the carrier is a solvent. In a non-limiting example, the composition may be placed in a solvent. Such solvents include any suitable solvent known in the art, such as water, saline, phosphate buffered saline, etc.

[0232] The formulation of the composition may vary depending on the route of administration. For example, for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. Sterile aqueous media that can be used will be known to those of skill in the art in light of the present disclosure.

[0233] Supplementary active ingredients can also be incorporated into the composition. For human administration, preparations must meet sterility and general safety and purity standards as required by FDA Office of Biologics standards. Administration can be by any known route.

[0234] In certain embodiments, the pharmaceutical composition comprises at least about 0.001 g to about 1 g of particles disclosed herein per kilogram of subject. In certain embodiments, the pharmaceutical composition comprises at least about 0.001 g to about 0.5 g of particles disclosed herein per kilogram of subject.

[0235] Pharmaceutical compositions may contain various antioxidants to retard oxidation of one or more components. In addition, prevention of microbial action may be provided by preservatives such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof. The compositions must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that exotoxin contamination should be kept at a minimum safe level, e.g., less than 0.5 ng / mg protein.

[0236] In embodiments in which the composition is in liquid form, the carrier can be a solvent or dispersion medium including, but not limited to, water, ethanol, polyols (e.g., glycerin, propylene glycol, liquid polyethylene glycol, and the like), lipids (e.g., triglycerides, vegetable oils, liposomes), and combinations thereof. In many cases, it will be preferable to include isotonic agents, such as sugars, sodium chloride, or combinations thereof.

[0237] In other embodiments, nasal solutions or sprays, aerosols or inhalants may be used. Nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays.

[0238] Solid compositions for oral administration are also contemplated, in these embodiments, the solid compositions may include, for example, solutions, suspensions, emulsions, tablets, pills, capsules, sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, or combinations thereof.

[0239] Sterile injectable solutions are prepared by incorporating the active compound (e.g., nanoparticles) in the required amount in an appropriate solvent along with various other ingredients as enumerated above. Liquid media should be suitably buffered if necessary, and liquid diluents should first be rendered isotonic with sufficient saline or glucose prior to injection.

[0240] Dosage can be repeated as necessary, as determined by those skilled in the art.Therefore, in some embodiments of the methods described herein, a single dose is contemplated.In other embodiments, two or more doses are contemplated.When two or more doses are administered to a subject, the time interval between administrations can be any time interval determined by those skilled in the art.

[0241] Therapeutic and Diagnostic Uses of the Compositions According to some embodiments, there is provided a pharmaceutical composition comprising the multifunctional particles of the present invention for use in the simultaneous delivery of a first active agent and a second active agent to the brain of a subject in need thereof.

[0242] According to another aspect, the present invention provides a method for synchronously delivering a first active agent and a second active agent to the brain of a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising the multifunctional particles described above in all embodiments thereof.

[0243] According to some embodiments, the pharmaceutical composition is for use in treating a brain-related disease or disorder in need thereof. According to some embodiments, the pharmaceutical composition is for use in preventing a brain-related disease or disorder in a subject in need thereof. According to some embodiments, the pharmaceutical composition is for use in monitoring a brain-related disease or disorder in a subject in need thereof.

[0244] According to some aspects and embodiments, there is provided a method for treating a brain-related disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the invention.

[0245] According to some aspects and embodiments, there is provided a method for preventing a brain-related disease or disorder in a subject, comprising administering to the subject a pharmaceutical composition of the invention.

[0246] According to some aspects and embodiments, a method for monitoring a brain-related disease or disorder in a subject in need thereof is provided, comprising administering a pharmaceutical composition of the present invention to the subject and imaging the subject's brain. In some related embodiments, the pharmaceutical composition comprises the multifunctional particles described above, the core particles being gold nanoparticles, and imaging is performed using CT to allow detection of the multifunctional particles in the brain. In other embodiments, the pharmaceutical composition comprises the multifunctional particles described above, in which at least one active agent is a labeling molecule, e.g., a fluorescent or radioactive molecule that allows detection by an appropriate imaging modality. In some embodiments, the method for monitoring a brain-related disease or disorder comprises repeated administrations and / or repeated imaging sessions.

[0247] As used herein, the term "brain-related disease or disorder" refers to any disease or disorder that causes dysfunction of the brain or any of its cells. Non-limiting examples of brain-related diseases and disorders include neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease and dementia; neuromuscular diseases such as amyotrophic lateral sclerosis (ALS) and motor neuron disease; neurodevelopmental diseases such as autism spectrum disorder and attention deficit hyperactivity disorder (ADHD); autoimmune brain-related diseases such as multiple sclerosis (MS); neuropsychiatric disorders such as schizophrenia, addictions such as drug addiction and smoking addiction, eating disorders, obsessive-compulsive disorder, various forms of depression, anxiety disorders, cognitive disorders and affective disorders; seizure disorders such as epilepsy; pain disorders such as migraine; cerebrovascular disorders including traumatic brain injury and stroke; brain-related cancers such as brain tumors and neuronal tumors, brain metastases, glioma, glioblastoma (GBM), and gliosarcoma (GS); neurodevelopmental diseases such as Huntington's disease, Kennedy disease, metabolic disorders, lysosomal storage diseases and Duchenne; and neuroinfectious diseases.

[0248] In some embodiments, the disease is a central nervous system disease. According to some embodiments, the disorder is a brain disorder.

[0249] In some embodiments, the pharmaceutical composition is for use in the treatment of a brain-related disease or disorder, hi some embodiments, the brain-related disease or disorder is selected from the group consisting of brain-related cancers, neurodegenerative disorders, neuromuscular diseases, neurodevelopmental diseases, autoimmune brain-related diseases, neuropsychiatric disorders, seizure disorders, pain disorders, cerebrovascular diseases, neurodevelopmental diseases, and neuroinfectious diseases.

[0250] In some embodiments, the brain-related disease is a brain-related cancer. As used herein, the term "brain-related cancer" encompasses both primary brain tumors (i.e., primary brain cancers) and metastatic brain tumors (i.e., secondary brain cancers). In some embodiments, the brain-related cancer is selected from the group consisting of, but not limited to, brain and nerve tumors, brain metastases, gliomas, glioblastomas (GBM), and gliosarcoma (GS). In some embodiments, the brain-related disease is a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, and dementia. In some embodiments, the brain-related disease is a neuromuscular disease. In some embodiments, the neuromuscular disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS) and motor neuron disease. In some embodiments, the brain-related disease is a neurodevelopmental disease. In some embodiments, the neurodevelopmental disease is selected from the group consisting of autism spectrum disorder and attention deficit hyperactivity disorder (ADHD). In some embodiments, the brain-related disease is multiple sclerosis (MS). In some embodiments, the brain-related disease is a neuropsychiatric disorder. In some embodiments, the neuropsychiatric disorder is selected from the group consisting of schizophrenia, addictions such as drug addiction and smoking addiction, eating disorders, obsessive-compulsive disorder, various forms of depression, anxiety disorders, cognitive disorders, and affective disorders. In some embodiments, the brain-related disease is a seizure disorder. In some embodiments, the seizure disorder is epilepsy. In some embodiments, the brain-related disease is a pain disorder. In some embodiments, the brain-related disease is a cerebrovascular disorder. In some embodiments, the cerebrovascular disorder is selected from traumatic brain injury and stroke. In some embodiments, the brain-related disease is a neurodevelopmental disease. In some embodiments, the neurodevelopmental disease is selected from the group consisting of Huntington's disease, Kennedy's disease, metabolic disorders, lysosomal storage diseases, and Duchenne. In some embodiments, the brain-related disease is a neuroinfectious disease.

[0251] In some embodiments, the brain-related disease is Alzheimer's disease. In some embodiments, the brain-related disease is Parkinson's disease. According to some embodiments, the brain-related disease is Huntington's disease, spinocerebellar ataxia, amyotrophic lateral sclerosis, Friedreich's ataxia, motor neuron disease (Lou Gehrig's disease), or spinal muscular atrophy. According to some embodiments, the brain-related disease is a prion disease.

[0252] As used herein, the term "subject" refers to any animal (e.g., mammal), including, but not limited to, humans, non-human primates, rodents, etc. (e.g., that is to be the recipient of a particular treatment). Typically, the terms "subject" and "patient" are used interchangeably, unless otherwise indicated herein.

[0253] In some embodiments, the subject is a human subject. In some embodiments, the subject is at risk for a brain-related disease, disorder, or condition. In some embodiments, the subject has been diagnosed with a brain-related disease, disorder, or condition. In some embodiments, the subject has been diagnosed with a brain-related genetic disorder. In some embodiments, the subject has been diagnosed with a brain-related cancer. In some embodiments, the subject is at risk for a neurodegenerative disease. In some embodiments, the subject has been diagnosed with a neurodegenerative disease. In some embodiments, the subject has been diagnosed with Alzheimer's disease. In some embodiments, the subject has been diagnosed with Parkinson's disease.

[0254] As used herein, a subject at risk of suffering from a disease, disorder, or condition is a subject who exhibits one or more signs or symptoms indicative of a disease, disorder, or condition, or a subject who is screened for a disease, disorder, or condition (e.g., during a routine physical examination). A subject at risk of suffering from a disease, disorder, or condition may have one or more risk factors. A subject at risk of suffering from a disease, disorder, or condition includes individuals who have not been previously tested for the disease, disorder, or condition. However, a subject at risk of suffering from a disease, disorder, or condition also includes individuals who have been given a preliminary diagnosis but have not undergone a confirmatory test (e.g., biopsy and / or tissue diagnosis) or whose stage of the disease, disorder, or condition is unknown. This term further includes people who have previously suffered from a disease, disorder, or condition (e.g., individuals who have gone into remission).

[0255] A subject at risk for suffering from a brain-related disease, disorder, or condition may be diagnosed as having a brain-related disease, disorder, or condition, or may be found to not have a brain-related disease, disorder, or condition.

[0256] As used herein, a subject diagnosed with a brain-related disease, disorder, or condition may be diagnosed using any suitable method, including, but not limited to, biopsy, x-ray, blood test, and the diagnostic methods of the present invention. A "preliminary diagnosis" is a diagnosis based solely on visual inspection (e.g., CT scan or presence of a mass) and antigen testing.

[0257] As used herein, the terms "treatment," "treating," or "ameliorating" a disease, disorder, or condition refer to alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. To be an effective treatment, a composition useful herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide an improvement in the quality of life of the patient or subject.

[0258] In some embodiments, the method further comprises imaging the subject's brain region, hi some embodiments, the imaging is performed using an imaging system selected from the group consisting of computed tomography imaging (CT), x-ray imaging, magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), ultrasound (US), and any combination thereof.

[0259] In some embodiments, imaging is performed to assess accumulation of the co-delivery system in the brain of the subject.

[0260] In some embodiments, the subject is suffering from a brain-related disease or disorder and imaging is performed to determine the stage of the disease or disorder, hi some embodiments, the subject suffering from a brain-related disease or disorder is treated with a drug and imaging is used to follow up on the treatment.

[0261] In some embodiments, the imaging step is performed 0.5 to 96 hours after the administering step. In some embodiments, the imaging step is performed 0.5 to 48 hours after the administering step. In some embodiments, the imaging step is performed 0.5 to 24 hours after the administering step. In some embodiments, the imaging step is performed 0.5 to 12 hours after the administering step. In some embodiments, the imaging step is performed 1 to 12 hours after the administering step. In some embodiments, the imaging step is performed 1 to 6 hours after the administering step. In some embodiments, the imaging step is performed within 96 hours after the administering step. In some embodiments, the imaging step is performed within 48 hours after the administering step. In some embodiments, the imaging step is performed within 24 hours after the administering step. In some embodiments, the imaging step is performed within 12 hours after the administering step. In some embodiments, the imaging step is performed within 6 hours after the administering step.

[0262] The administration of the composition to the subject can be performed using any method known to those of skill in the art. The mode of administration can vary depending on the application. For example, the mode of administration can vary depending on the particular cell, brain region, or subject to be imaged. For example, the administration of the composition can be performed intravenously, intracerebrally, intracranially, intrathecally, intraventricularly, into the substantia nigra or substantia nigra region, intradermally, intraarterially, intraperitoneally, intralesionally, intratracheally, intranasally, intramuscularly, intraperitoneally, subcutaneously, orally, topically, locally, by inhalation (e.g., aerosol inhalation), injection, infusion, intrathecal injection, transmucosal injection, intracarotid injection, continuous infusion, by local perfusion directly bathing the target cells, via a catheter, via lavage, or by other methods known to those of skill in the art, or any combination of the above.

[0263] In some embodiments, the pharmaceutical composition is administered to the subject by a systemic route of administration. In some embodiments, the systemic administration is selected from intravenous (IV) administration and intranasal (IN) administration. In some embodiments, the pharmaceutical composition is administered to the subject by intrathecal (IT) administration. In some embodiments, the particles are administered intravenously. In some embodiments, the particles are administered intranasally.

[0264] The effective amount of the pharmaceutical composition is determined based on the intended purpose and the subject to be treated. The amount administered may also vary based on the specific route of administration used. The composition is preferably administered in a safe and effective amount. As used herein, the term "safe and effective amount" refers to an amount of the composition sufficient for the intended purpose without undue adverse side effects (toxicity, irritation, or allergic reaction).

[0265] In some embodiments, the method further comprises using an additional treatment. In some embodiments, particularly, the brain-related disease is a brain-related cancer, and the additional treatment is selected from, but not limited to, surgery, radiation therapy, and chemotherapy. In certain embodiments, the additional treatment is radiation therapy.

[0266] In some embodiments, the core particles are radiosensitizers, and the method of treating brain-related cancer further comprises the step of directing ionizing radiation to the tumor cells (where the particles accumulate) to thereby obtain locally enhanced radiation therapy within the tumor cells. In some embodiments, the composition is used for thermal ablation of the tumor cells where the composition accumulates using infrared waves without causing damage to surrounding normal tissue or substantial toxicity to the subject. As used herein, "ablation" refers to the destruction of cells. Methods of irradiating tissue with metal particles to enhance the effects of radiation therapy are known in the art.

[0267] kit In some embodiments, the present invention provides a kit comprising one or more compositions disclosed herein. In some embodiments, the present invention provides a kit useful for the methods disclosed herein. For example, the kit may comprise a container having a sterile reservoir that contains any of the compositions disclosed herein. In some embodiments, the kit further comprises instructions. For example, the kit may comprise instructions (e.g., indications, dosage, method, etc.) for administering the composition to a subject. In yet another example, the kit may comprise instructions for applying the compositions and methods of the present invention to an imaging system, such as computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI).

[0268] The description of various embodiments of the present invention is presented for illustrative purposes, but is not intended to be exhaustive or limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terms used herein are selected to best explain the principles of the embodiments, practical applications or technical improvements to the technology found in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

[0269] Any concentration range, percentage range, or ratio range recited herein should be understood to include any integer concentration, percentage or ratio within that range, and fractions thereof, such as tenths and hundredths of the integers, unless otherwise indicated.

[0270] Any numerical range recited herein for any physical characteristic, such as polymer subunits, size or length, should be understood to include any integer within the recited range, unless otherwise indicated.

[0271] As used herein, the term "about" when in conjunction with a value refers to plus or minus 10% of the reference value. For example, a molecular weight of about 1000 Da refers to a molecular weight of 1000 Da±100 Da.

[0272] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a particle" includes a plurality of such particles, and a reference to "the particle" includes a reference to one or more particles. It should be further noted that the claims may be drafted to exclude optional elements. Thus, this statement is intended to serve as a predicate for use of exclusive terminology, such as "exclusively," "only," and the like, in connection with the recitation of claim elements or the use of a "negative" limitation.

[0273] The term "plurality" means "two or more" unless expressly specified otherwise.

[0274] When a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, A and B, A and C, B and C, and / or A, B and C, etc.). It will be further understood by one of ordinary skill in the art that virtually any choice of language and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0275] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments according to the invention are specifically embraced by the invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the invention and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0276] The following examples are intended to illustrate the methods of making and using the compounds and methods of the present invention, and are not to be construed as limiting in any way. The present invention will now be described in conjunction with specific embodiments thereof, but it is apparent that many modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such modifications and variations that fall within the spirit and broad scope of the appended claims.

[0277] Working Example Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques, which are fully explained in the literature. See, for example, Molecular Cloning: A laboratory Manual, Sambrook et al. (1989); Current Protocols in Molecular Biology, vols. I-III, Ausubel, RM (ed.) (1994); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988); Watson et al., Recombinant DNA, Scientific American Books, New York; Birren et al. (eds.), Genome Analysis: A Laboratory Manual Series, vols. 1-4, Cold Spring Harbor Laboratory Press, New York. New York (1998); methods described in U.S. Patents 4,666,828; 4,683,202; 4,801,531; 5,192,659; and 5,272,057; Cell Biology: A Laboratory Handbook, vols. I-III, Cellis, JE (ed.) (1994); Culture of Animal Cells-A Manual of Basic Technique, Freshney, Wiley-Liss, NY (1994), 3rd ed.; Current Protocols in Immunology, vols. I-III, Coligan JE(eds.) (1994); Stites et al. (eds.), Basic and Clinical Immunology (8th ed.), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds.), Strategies for Protein Purification and Characterization-A Laboratory Course Manual, CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

[0278] Example 1: Multifunctional gold nanoparticles (GNPs) co-deliver two antibodies to the brain FIG. 1 shows a schematic diagram of a non-limiting exemplary multifunctional particle showing a gold nanoparticle (GNP; 1) bound to: (i) a first polymer linker (2) conjugated to a first antibody (3); (ii) a second polymer linker (4) conjugated to a second antibody (5); (iii) a third polymer linker (6) conjugated to a brain-internalizing transporter moiety (e.g., insulin; 7); and (iv) a monofunctional polymer moiety (8).

[0279] Preparation and characterization of GNPs conjugated to anti-IgG1, anti-Iba1 and insulin (IgG1&Iba1&Ins-GNPs) GNP synthesis: 20 nm spherical GNPs were synthesized using HAuCl 4 It was prepared by reduction of 50% w / v HAuCl in 200 ml of distilled water with citric acid. 4 A total of 414 μl of solution was boiled in an oil bath on a heating plate with stirring. After boiling, 4.04 ml of 10% sodium citrate solution was added and the mixture was stirred with boiling for an additional 10 minutes. After the solution was removed from the plate and cooled to room temperature, the solution was centrifuged to precipitate the nanoparticles.

[0280] Conjugation of PEG5000 to GNPs: GNPs were first partially coated (60% of the particle surface) with mPEG-SH (approximately 5 kDa; 40% of the particle surface) and heterofunctional HS-PEG-COOH (approximately 5 kDa; 20% of the particle surface). The amount of mPEG-SH and HS-PEG-COOH required for partial coating was such that the thiol-PEG molecules were 0.35 nm thick on the gold nanoparticle surface. 2 The conjugation was carried out by adding a mixture of HS-PEG-COOH (193 μl, 50 mg / ml) and mPEG-SH (387 μl, 50 mg / ml) to the GNP solution and mixing for 2 hours. The solution was then ultracentrifuged at 15,000 RPM for 20 minutes and then again at 20,000 RPM for 15 minutes. The precipitate containing the PEG-coated GNPs (total 60% coating) was transferred to a vial.

[0281] Conjugation of insulin: To facilitate the transport of multifunctional GNPs through the BBB, insulin was covalently conjugated to the carboxyl groups of HS-PEG-COOH by adding an excess amount of insulin along with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl) and NHS (N-hydroxysulfosuccinimide sodium salt) on ice and mixed for 2 h. The solution was then centrifuged twice at 15,000 RPM for 30 min (kept at low temperature) and the lower phase containing Ins-PEG-GNPs was transferred to a vial.

[0282] Conjugation of PEG3500 to GNPs: To further conjugate the antibody to the GNPs, 271 μl of HS-PEG-COOH (approximately 3.5 kDa) solution (50 mg / ml) was added to the partially coated GNPs to coat the remaining 40% of the particle surface. The solution was then mixed for 2 hours at 4° C., followed by repeated centrifugation at 15,000 RPM for 30 minutes.

[0283] Conjugation of anti-IgG1 and anti-Iba1: Fluorescently labeled anti-Iba1 and fluorescently labeled anti-IgG1 were covalently conjugated to the free carboxyl groups of HS-PEG-COOH (approximately 3.5 kDa) by adding a 1:1 molar mixture of fluorescently labeled anti-Iba1 (Abl95032-Rb Mono&Hu IBA-1-647) and fluorescently labeled anti-IgG1 (mouse monoclonal IgG1 Alexa Fluor 488 isotype control clone 11711) with EDC and NHS. The solution was then stirred for 2 hours at 4°C, followed by centrifugation to remove unbound antibody until a final concentration of Au reached 25 mg / ml.

[0284] To confirm the chemical conjugation of the antibodies, the multifunctional dual antibody nanoparticles were imaged using a fluorescence microscope. Both anti-IgG1 and anti-Iba1 fluorescent signals were detected, indicating that the chemical conjugation was successful.

[0285] In control experiments, similar particles were prepared with only one conjugated antibody, i.e. anti-IgG1 or anti-Iba1. Percentage coverage of the different coating molecules in the control particles: 20% PEG5000 (conjugated to insulin), 20% PEG3500 (conjugated to the respective antibody) and 60% mPEG5000.

[0286] Delivery of dual antibody-conjugated GNPs to mouse brain Male BALB / c mice were divided into five treatment groups: Group 1: Saline injection (untreated, control; n=2) Group 2: Anti-IgG1 & insulin conjugated GNPs (IgG1 & Ins-GNPs; n = 2) Group 3: Anti-Iba1 & insulin conjugated GNPs (Iba1 & Ins-GNPs; n = 2) Group 4: Multifunctional dual antibody GNP (IgG1&Iba1&Ins-GNP; n=2) Group 5: Free anti-IgG1 antibody and anti-Iba1 antibody (free Ab; n=2).

[0287] Mice were intravenously injected with IgG1&Ins-GNPs (200μl, 25mg / ml), Iba1&Ins-GNPs (200μl 25mg / ml), IgG1&Iba1&Ins-GNPs (200μl 25mg / ml), or an equal amount of free fluorescently labeled antibodies (200μl solution containing 0.2mg anti-IgG1 and 0.25mg anti-Iba1). Eight hours after injection, mice were perfused to remove any particles / antibodies present in the blood vessels. Mouse brains were then removed and analyzed using ICP-OES (groups 2, 3 and 4; n=2 per group) or histological evaluation (groups 1, 4 and 5: n=2 per group).

[0288] Figure 2 shows the amount of gold in mouse brains measured by ICP-OES. After IV injection of IgG1&Ins-GNP, Iba1&Ins-GNP or IgG1&Iba1&Ins-GNP, significant amounts of gold were found in the mouse brain, indicating successful penetration through the BBB to the brain. Interestingly, the penetration rate of the multifunctional dual antibody GNP (IgG1&Iba1&Ins-GNP) was even higher than that of Iba1&Ins-GNP.

[0289] For histological evaluation of mouse brains (groups 1, 4, and 5), 7 μm frozen sections of the cerebral cortex and medulla were prepared and immunostained (IHC-F double staining). Fluorescent antibody signals were detected and photographed using a confocal microscope. All photographs for each antibody were taken under the same exposure conditions.

[0290] Figures 3 and 4 show representative immunohistochemical fluorescence (IHC-F) images of cerebral cortex sections (Figure 3) and medulla sections (Figure 4). Surprisingly, no antibody signals were observed in brain sections from mice administered free antibodies (group 5), whereas both anti-IgG1 and anti-Iba1 signals were detected in the brains of mice administered multifunctional brain-targeting GNPs conjugated with these antibodies (group 4). Furthermore, fused images show the colocalization of the two types of antibodies in the cerebral cortex and medulla, indicating that the distribution of the antibodies is synchronized in the brain.

[0291] Example 2: Preparation of multifunctional GNPs coated with insulin and two anti-Her2 antibodies, trastuzumab and pertuzumab As a non-limiting example, gold nanoparticles carrying different anti-HER2 antibodies and insulin were produced as outlined in Figure 6. The exemplary particles are coated with a polymer layer (2-3 in Figure 6) containing two types of polymer linkers (5-S-PEG-C(O)-, approx. 5 kDa and -S-PEGC(O)-, approx. 3.5 kDa), the first linker is conjugated to insulin (4) and the second linker is conjugated to two different anti-HER2 antibodies (5-6), namely trastuzumab and pertuzumab. An additional (7) polymer moiety (-S-PEG-O-CH3 approx. 6 kDa) is used as a capping to control the density of other moieties on the particle.

[0292] GNP synthesis HAuCl 4 Spherical GNPs of 20 nm were prepared by citrate reduction of 42.77% w / v HAuCl in 200 ml double distilled water (DDW). 4 A total of 414 μl was boiled in an oil bath on a heating plate with stirring. After boiling, 4.04 ml of 10% w / v trisodium citrate in DDW was added. The solution was removed from the oil bath and allowed to cool in a stirring chamber.

[0293] Conjugation of COOH-PEG5000-SH, COOH-PEG3500-SH, and mPEG6000-SH to GNPs GNPs were incubated with mPEG6000-SH (approximately 6 kDa; 80% of the particle surface), heterofunctional COOH-PEG5000-SH (approximately 5 kDa; 15% of the particle surface) and heterofunctional COOH-PEG3500-SH (approximately 5 kDa; 5% of the particle surface). The amount of PEG moieties required for the percentage of coating was 0.35 nm for thiol-PEG molecules on the gold nanoparticle surface. 2The conjugation was carried out by adding a mixture of COOH-PEG5000-SH (127 μl, 50 mg / ml in DDW), mPEG6000-SH (809 μl, 50 mg / ml in DDW) and COOH-PEG3500-SH (30 μl, 50 mg / ml in DDW) to the GNP solution and mixing overnight. The solution was then centrifuged at 50,000 G for 20 min, and the precipitate was then redispersed in DDW and centrifuged at 50,000 G for 20 min. The precipitate containing the PEG-coated GNPs was transferred to a vial.

[0294] Activation of GNPs was performed by mixing them with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl, 30 mg / ml in DDW, 100 μl) and sulfo-NHS (N-hydroxysulfosuccinimide sodium salt, 30 mg / ml in DDW, 100 μl), followed by centrifugation at 50,000 G for 20 min. The precipitate containing the activated COOH groups was transferred to a vial.

[0295] Conjugation of insulin to HS-PEG5000-COOH was then performed by adding insulin (195IU, 100IU / ml) to the GNP solution for 3 hours. The solution containing trastuzumab and pertuzumab (total 15mg) was then placed in 2ml of borate buffer (PH8, 0.1M) and then added to the GNP-insulin solution to conjugate the remaining COOH-PEG3500-SH with overnight mixing. The solution was then centrifuged at 10,000G for 20 minutes. The precipitate was subsequently redissolved in saline and then centrifuged at 10,000G for 20 minutes.

[0296] The antibody and insulin coated GNPs (Abs&Ins-GNPs) were characterized using dynamic light scattering (DLS) after each step of the preparation. The hydrodynamic size and zeta potential of the GNPs confirmed the successful coating.

[0297] Quantification of antibodies (Ab) and insulin attached to the PEG groups on the GNPs was tested by enzyme-linked immunosorbent assay (ELISA) testing the supernatant containing unbound protein remaining after centrifugation.

[0298] Example 3: Preparation and characterization of multifunctional GNPs coated with insulin, two types of multifunctional antibodies and a chemotherapeutic molecule This experiment demonstrates the conjugation to GNPs with four different linkers: SH-PEG3500-SH for chemotherapy molecules, COOH-PEG5000-SH for insulin as a transporter molecule, COOH-PEG3500-SH for two different antibodies and mPEG(5000-6000)-SH as a spacer and capping moiety.

[0299] GNP synthesis 20 nm spherical GNPs were dissolved in HAuCl 4 The solution was prepared by citrate reduction of 42.77% w / v HAuCl in 200 ml double distilled water (DDW). 4 A total of 414 μl was boiled in an oil bath on a heating plate with stirring. After boiling, 4.04 ml of 10% w / v trisodium citrate in DDW was added. The solution was removed from the oil bath and allowed to cool at room temperature with stirring.

[0300] GNPs were incubated with mPEG(5000-6000)-SH (approximately 5 kDa; 75% of the particle surface), heterofunctional COOH-PEG5000-SH (approximately 5 kDa; 15% of the particle surface), heterofunctional COOH-PEG3500-SH (approximately 3.5 kDa; 5% of the particle surface) and SH-PEG3500-SH (approximately 3.5 kDa; 5% of the particle surface). The amount of PEG moiety required for the percentage of coating was 0.35 nm for thiol-PEG molecules on the gold nanoparticle surface. 2The conjugation was carried out by adding a mixture of COOH-PEG5000-SH (96.92 μl, 50 mg / ml in DDW), mPEG5000-SH (512.51 μl, 50 mg / ml in DDW), COOH-PEG3500-SH (23y.14 μl, 50 mg / ml in DDW) and SH-PEG3500-SH (22.19 μl, 50 mg / ml in DDW) to the GNP solution and mixing overnight. The solution was centrifuged at 50,000 G for 20 min, then the precipitate was redispersed in DDW and centrifuged at 50,000 G for 20 min. The precipitate containing the PEG-coated GNPs was transferred to a vial.

[0301] Conjugation of chemotherapy molecules to the surface of thiol-terminated PEG was performed by adding the molecules to the NP solution with stirring overnight. The next day, the solution was centrifuged once at 50,000G for 20 min. The precipitate was redissolved in 18 ml and transferred to a vial. The amount of chemotherapy molecule used for conjugation was calculated according to the amount of particles in the reaction, which differs for each molecule used.

[0302] Activation of GNPs was performed by mixing them with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl, 30 mg / ml in DDW, 100 μl) and sulfo-NHS (N-hydroxysulfosuccinimide sodium salt, 30 mg / ml in DDW, 100 μl), followed by centrifugation at 50,000 G for 20 min. The precipitate containing the activated COOH groups was transferred to a vial.

[0303] Conjugation of insulin to HS-PEG5000-COOH was then performed by adding insulin (195 IU, 100 IU / ml) to the GNP solution for 3 hours. The solution containing antibodies Ab1 and Ab2 was then placed in 2 ml of borate buffer (PH8, 0.1M) and then added to the GNP-insulin solution to conjugate the remaining COOH-PEG3500-SH with overnight mixing. The solution was then centrifuged at 10,000G for 20 minutes. The precipitate was subsequently redissolved in saline and then centrifuged at 10,000G for 20 minutes.

[0304] The Abs&Ins-GNPs were characterized using dynamic light scattering (DLS) after each step of the preparation. The hydrodynamic size and zeta potential of the GNPs confirmed the successful coating.

[0305] Quantification of the antibodies and insulin attached to the PEG groups on the GNPs was tested by enzyme-linked immunosorbent assay (ELISA) testing the supernatant containing the unbound proteins remaining after centrifugation.

[0306] Further quantification of the antibody and insulin is performed by stripping the coating from the surface of the GNPs followed by quantification by HPLC using a UV spectrophotometer.

[0307] The conformation of the antibody and insulin presence on the GNPs is examined by cellular assays in BT474 cells and the presence of gold in mouse brain.

[0308] Example 4: Multifunctional GNPs for co-delivery of antibodies and small molecule drugs into the brain Preparation and characterization of GNPs conjugated to cisplatin, anti-IgG1 and insulin (CisPt&IgG1&Ins-GNPs) 20 nm spherical GNPs were prepared as described in Example 1.

[0309] Conjugation of mPEG5000 and PEG1000 to GNPs: GNPs were first partially coated (60% of the particle surface) with mPEG-SH (approximately 5 kDa, 40% of the particle surface) and heterofunctional HS-PEG-COOH (approximately 1 kDa, 20% of the particle surface). The amount of mPEG-SH and HS-PEG-COOH required for partial coating was such that the thiol-PEG molecules were 0.35 nm thick on the gold nanoparticle surface. 2 This was derived from theoretical calculations based on the knowledge that the PEG-coated GNPs occupy a footprint area of ​​100 μl (Qian, Ximei et al. Nature biotechnology 26.1 (2008): 83-90). Conjugation was performed by adding a mixture of HS-PEG-COOH (40 μl, 50 mg / ml) and mPEG-SH (387 μl, 50 mg / ml) to the GNP solution and mixing for 2 h. The solution was then ultracentrifuged at 15,000 RPM for 20 min and then again at 20,000 RPM for 15 min. The precipitate containing the PEG-coated GNPs (total 60% coating) was transferred to a vial.

[0310] Conjugation of cisplatin: Cisplatin (cisPt) was covalently conjugated to the carboxyl groups of HS-PEG-COOH (PEG1000) by adding an excess of cisplatin together with EDC and NHS, followed by mixing for 3 h at 4° C. The solution was then centrifuged at 14,000 g for 30 min at 4° C., and the lower phase containing cisplatin-GNPs was transferred to a vial.

[0311] Conjugation of PEG5000 and insulin: To further conjugate insulin to GNPs, HS-PEG-COOH (5 kDa) (193 μl, 50 mg / ml) was added to the partially coated GNPs to coat 20% of the particle surface. The solution was then mixed for 3 hours at 4° C., followed by centrifugation at 14,000 g for 30 minutes at 4° C. Insulin was then covalently conjugated to the free carboxyl groups of HS-PEG-COOH (5 kDa) by adding an excess of insulin along with EDC and NHS. The solution was then stirred for 3 hours at 4° C., followed by further centrifugation at 14,000 g for 30 minutes at 4° C.

[0312] Conjugation of PEG3500 and anti-IgG1 to GNPs: To further conjugate IgG1 antibody to GNPs, 135 μl of HS-PEG-COOH (approximately 3.5 kDa) solution (50 mg / ml) was added to the partially coated GNPs to coat the remaining 20% ​​of the particle surface. The solution was then mixed at 4° C. for 2 hours, followed by centrifugation at 14,000 RPM for 30 minutes. Anti-IgG1 was then covalently conjugated to the free carboxyl groups of HS-PEG-COOH (approximately 3.5 kDa) by adding 135 μg of anti-IgG1 antibody along with EDC and NHS. The solution was then stirred at 4° C. for 2 hours, followed by centrifugation to remove unbound antibody until a final concentration of Au reached 25 mg / ml.

[0313] For control experiments, GNPs conjugated with insulin and either anti-IgG1 or cisplatin (ie, IgG1&Ins-GNPs and cisplatin&Ins-GNPs) were prepared.

[0314] Delivery of cisPt&IgG1&Ins-GNP conjugate GNPs to mouse brain Male BALB / c mice were divided into four treatment groups: Group 1: Anti-IgG1 & insulin conjugated GNPs (IgG1 & Ins-GNPs; n = 2) Group 2:Cisplatin & insulin conjugate GNP (CisPt&Ins-GNP; n=2) Group 3: Multifunctional GNP (cisPt&IgG1&Ins-GNP;n=2) Group 4: Free cisplatin (n=2)

[0315] Mice were intravenously injected with IgG1&Ins-GNP (200μl, 25mg / ml), cisPt&Ins-GNP (200μl 25mg / ml), cisPt&IgG1&Ins-GNP (200μl 25mg / ml), or an equivalent amount of free cisplatin. Eight hours after injection, the mice were perfused to remove any particles present in the blood vessels. The mouse brains were then removed and analyzed using ICP-OES to measure the amount of Au and Pt that penetrated the brain (Figure 6A and Figure 6B, respectively).

[0316] As can be seen in Figures 5A and 5B, after IV injection of cisPt&IgG1&Ins-GNPs, significant amounts of both gold and platin were found in the mouse brain, indicating successful penetration of the multifunctional GNPs through the BBB. Surprisingly, Figure 6B shows that the amount of Pt found in the brain of mice administered either cisPt&Ins-GNPs or cisPt&IgG1&Ins-GNPs was significantly higher than the amount after administration of an equal dose of free cisplatin, indicating that the multifunctional GNP platform enhances the penetration of the small molecule cisplatin through the BBB.

[0317] Example 5: Effect of linker length on the ability of nanodelivery systems to cross the BBB Several types of gold nanoparticles conjugated to insulin, IgG1 antibody, and Iba1 antibody are synthesized as described in Example 1 using different combinations of PEG linkers as specified in Table 1.

[0318] [Table 1]

[0319] To examine the effect of linker length on the ability of the nanodelivery system to cross the BBB, the brain-targeting particles listed in Table 1 are injected intravenously into the tail vein of male Balb / C mice (200 μl of 30 mg / ml). Eight hours after injection, the mice are sacrificed and perfused. The brains are then removed and analyzed by ICP-OES or ICP-MS to quantify the amount of gold that has penetrated the BBB.

[0320] Interestingly, the highest brain penetration was achieved by GNPs with a first and second polymer linker shorter than the third polymer linker. It is hypothesized that to efficiently penetrate the brain through the BBB, insulin, which acts as a brain-internalizing moiety, must be exposed on the surface of the entire nanodelivery system (i.e., present on the outer surface of the particle). Since insulin is much smaller than an antibody (approximately 150 kDa vs. 5 kDa), it must be conjugated to a longer linker than the linker used to bind the antibody in order to remain exposed on the surface of the nanodelivery system and not be blocked by the antibody.

[0321] Example 6: Effect of dual antibody GNPs on cancer cells: In vitro study GNPs conjugated to insulin and two different anti-HER2 antibodies (trastuzumab and pertuzumab; Brockhoff et al., Cell Prolif. 2007, 40, 488-507 and Scheuer et al., Cancer Res. 2009, 69(24), 9330-9336) are prepared according to the protocol described in Example 1. For comparison, GNPs conjugated to insulin and a single antibody (either trastuzumab or pertuzumab) are also prepared.

[0322] The HER-2 positive breast cancer cell line BT474 is used to determine the effect of each antibody conjugated GNP as a monotherapy and their combined effect. The cells are treated with different concentrations of trastuzumab-GNP, pertuzumab-GNP, or trastuzumab & pertuzumab-GNP. Untreated cells serve as the control sample.

[0323] The effect of different treatments on the cells is examined using cell cycle arrest measurements, apoptosis and proliferation assays. Each treatment is performed in triplicate.

[0324] Example 7: In vivo brain efficacy of bispecific GNP complexes The effectiveness of the platform as a multifunctional drug carrier was tested. In this experiment, both trastuzumab and pertuzumab antibodies, which are considered as first-line treatments for breast cancer tumors and are administered together, were used. Both antibodies were conjugated to GNPs and their effectiveness was tested in a metastatic breast brain tumor mouse model. 150K breast cancer BT474 cells were inoculated into the brains of NOD-SCID mice according to the bregma coordinates: anterior 0.5, lateral 1.7, depth 3.5. Tumor growth was monitored weekly by BLI imaging. Three weeks after tumor inoculation, mice were divided into four groups according to tumor size measured by MRI; a control group receiving saline, a control group receiving a mixture of free antibodies, and a test group receiving bifunctional GNPs (both antibodies are conjugated to the same particle). The treatment composition (40 mg / kg Ab) was IP injected once a week for four consecutive weeks. At this point, an MRI scan was performed to measure the tumor size again. The results showed that the bifunctional particles slowed tumor growth compared to untreated mice or free antibody treatment (Figure 7a). The brains were then removed and particle penetration was confirmed by ICP-OES. Figure 7b shows three representative brains, where tumors are clearly visible in the brains of mice fed bifunctional GNPs, as the particles accumulated in the tumors are purple in color.

[0325] Example 8: Multifunctional GNPs in combination with radiotherapy for cancer treatment: In vivo studies Gold nanoparticles conjugated to anti-EGFR (CTX) and temozolomide (TMZ) are prepared according to the protocol of Example 2.

[0326] Mice (n=25) are divided into 5 treatment groups (n=5 per group) as shown in Table 2 below to examine the contribution of different factors to the performance of the treatments.

[0327] [Table 2]

[0328] Mice were inoculated with human U87 GBM cells (3 × 10 4 ~3×10 5 ) was injected intracranially; the injection site is 2 mm posterior and 1.5 mm lateral to the bregma. MRI imaging is used to confirm tumor development, which is measured at approximately 14 days after induction.

[0329] Mice in groups 2-3 are given standard treatment including intraperitoneal TMZ (10 mg / kg, 5 days). Intravenous CTX (1 mg / kg) is administered to mice in group 3. Mice in groups 4 and 5 are intravenously administered TMZ&CTX-GNPs containing equal amounts of TMZ and CTX (10 mg / kg and 1 mg / kg, respectively). The whole brain is irradiated with 6 MV X-rays in fractions (10 Gy for 5 days, 2 Gy / day).

[0330] Mice are sacrificed upon clinical progression or at the end of the study protocol, approximately 180 days after tumor injection.

[0331] Mice are monitored for survival and health during the follow-up period.

[0332] After sacrifice, the brains are analyzed by immunohistochemistry.

[0333] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. (a) an inorganic particle attached to at least (i) a first linear polymer linker; (ii) a second linear polymer linker; and (iii) a third linear polymer linker; (b) a first biologically active molecule conjugated to said first linear polymer linker; (c) a second biologically active molecule conjugated to the second linear polymer linker; and (d) a brain-internalizing transporter moiety conjugated to said third linear polymer linker. A multifunctional particle comprising: the third polymer linker is longer than the first and second polymer linkers, as measured by contour length or molecular weight; A multifunctional particle, wherein the first biologically active molecule is different from the second biologically active molecule.

2. A multifunctional particle as described in claim 1, wherein both the first and second biologically active molecules are antibodies or antibody fragments thereof.

3. A multifunctional particle as described in claim 2, wherein each of the individual antibodies has a molecular weight of 130 to 180 kD.

4. The multifunctional particle described in claim 3, wherein each of the antibodies is selected from the group consisting of anti-HER2+, anti-EGFR, anti-GD2, and checkpoint inhibitor antibodies, or fragments thereof.

5. A multifunctional particle as described in claim 4, wherein the molecular weight of the first and second linear polymer linkers is at least 3,400 Da.

6. The multifunctional particle of claim 5 , wherein the molecular weight of the third polymer linker is at least 1000 Da greater than the molecular weight of the first and second polymer linkers.

7. The multifunctional particle of claim 4 , wherein the first, second, and third polymer linkers are non-cleavable under physiological conditions.

8. The multifunctional particle of claim 7, wherein the first and second linear polymer linkers are attached to the inorganic particle via a covalent bond, optionally a sulfide bond, and the first and second biologically active molecules are conjugated to the respective linear polymer linkers via a covalent bond, optionally an amide bond.

9. 4. The multifunctional particle of claim 3, wherein the third linear polymer linker comprises about 10 mol % to 40 mol % of all polymer linkers attached to the inorganic particle.

10. 4. The multifunctional particle of claim 3, wherein each of the first and second linear polymer linkers independently constitutes about 5 mol % to 40 mol % of the total polymer linkers attached to the inorganic particle.

11. The multifunctional particle of claim 3 , wherein the inorganic particle comprises a metal selected from the group consisting of gold, silver, platinum, iron, and any combination thereof.

12. The multifunctional particle of claim 3 , wherein the brain-internalizing transporter moiety is insulin or an analog, derivative, conjugate, or fragment thereof.

13. further comprising a third bioactive molecule, said third bioactive molecule being conjugated to a linear polymer linker attached to said inorganic particle; and optionally The multifunctional particle of claim 1 , wherein the third biologically active molecule is a chemotherapeutic molecule and the linear polymer linker is cleavable under physiological conditions.

14. The multifunctional particle described in claim 1, wherein the inorganic particles are gold particles, the first linear polymer linker and the second linear polymer linker are each independently a thiolated PEG3500 acid or a thiolated PEG3500 amine, the third linear polymer linker is a thiolated PEG5000 acid or a thiolated PEG5000 amine, and the brain internalization transporter moiety is insulin.

15. The multifunctional particle of claim 1, wherein the inorganic particles are gold particles, the first linear polymer linker is a thiolated PEG1000 acid or a thiolated PEG1000 amine, the second linear polymer linker is a thiolated PEG3500 acid or a thiolated PEG3500 amine, the third linear polymer linker is a thiolated PEG5000 acid or a thiolated PEG5000 amine, and the brain internalization transporter moiety is insulin.

16. 10. A pharmaceutical composition comprising the multifunctional particle of claim 1 and a pharmaceutically acceptable carrier for use in a method for preventing, treating, and / or monitoring a brain-related disease or disorder in a subject in need thereof, said method comprising administering said pharmaceutical composition to said subject; Optionally, further comprising imaging the brain of the subject, thereby assessing accumulation of said multifunctional particles in the brain of said subject; Optionally, the brain-related disease or disorder is primary brain cancer or secondary brain cancer, and the secondary brain cancer is optionally selected from the group consisting of breast cancer, lung cancer, melanoma, renal cancer, and colorectal cancer.

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