Improved ultraparticles

Abstract

The present disclosure relates to improved supraparticles loaded with high levels of a payload and methods for producing the same. Such supraparticles can be used in a range of therapeutic applications, for example, to improve the growth or survival of cells and / or to treat a disease.

Description

Technical Field

[0001] This disclosure relates to improved ultraparticles loaded with high-level payloads and methods for producing said ultraparticles. Such ultraparticles can be used in a range of therapeutic applications. Background of the Invention

[0003] Mesoporous silica is a porous material with extremely high surface area that has been widely used for encapsulating various compounds and for template synthesis of various nanostructured materials. While this type of porous material has attracted attention in a wide variety of applications such as drug delivery, various problems such as payload and sustained drug delivery remain unresolved.

[0004] It is desirable to provide new materials of this type and new methods for preparing such materials, as it is anticipated that said new materials may possess many interesting properties.

[0005] Therefore, improved ultraparticles are needed. Summary of the Invention

[0006] This disclosure is based on the accidental generation of ultraparticles capable of carrying high levels of payload. The inventors have also determined that the ultraparticles of this disclosure appear to have a unique pore size, which could explain the increased payload level. Therefore, in a first example, this disclosure relates to a composition comprising ultraparticles containing at least 1.5 μg of payload. In another example, this disclosure relates to a composition comprising ultraparticles containing at least 5 μg of payload. In yet another example, this disclosure relates to a composition comprising ultraparticles containing at least 8 μg of payload. In yet another example, this disclosure relates to a composition comprising ultraparticles containing at least 10 μg of payload. In yet another example, this disclosure relates to a composition comprising ultraparticles containing between 1.5 μg and 10 μg of payload.

[0007] In one example, the superparticles comprise pores with a diameter of at least 60 nm. In another example, the superparticles comprise pores with a diameter of at least 100 nm. In yet another example, the superparticles comprise pores with a diameter of at least 60-200 nm. In yet another example, the superparticles comprise pores with a diameter of at least 50-100 nm.

[0008] In another example, the superparticles have a disordered porous structure.

[0009] In one example, the superparticles consist of nanoparticles with a bimodal pore structure. In another example, the bimodal pore structure of the nanoparticles has a large pore size greater than 30 nm.

[0010] In another example, the payload is a small molecule, peptide, or protein. For example, the payload could be an antibody. In one example, the payload is a stem cell, virus, nucleic acid, neurotrophic factor, anticancer agent, neuroprotective agent, or a combination thereof. In one example, the payload is a neurotrophic factor. In another example, the payload is a neurotrophic protein. In one example, the neurotrophic factor has an isoelectric point between 9 and 10. In another example, the payload is neurotrophin-3. In yet another example, the payload is BDNF or neurotrophin-3.

[0011] In another example, the superparticles contain at least two, at least three, at least four, or at least five different payloads. In another example, the superparticles contain at least two payloads, one of which is a neurotrophic factor. In yet another example, the superparticles contain at least two payloads, one of which is a neurotrophic protein.

[0012] In other instances, the compositions disclosed herein comprise at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten superparticles. In these instances, the superparticles may contain different payloads.

[0013] In one example, superparticles can be produced by electrospraying a composition containing nanoparticles into a dicationic aqueous solution. In one example, the composition containing nanoparticles further comprises alginate or a polysaccharide derivative of said alginate. Therefore, in one example, superparticles can be produced by electrospraying a composition containing nanoparticles and alginate or a polysaccharide derivative of said alginate into a dicationic aqueous solution. The superparticles produced in these examples can be loaded with a payload of 1.5 μg to 15 μg, such as neurotrophic protein. In these examples, the superparticles may also be calcined before loading the payload to remove alginate. In one example, calcination is performed at about 650°C. In one example, the calcination lasts for about 6 to about 30 hours. In one example, the alginate is sodium alginate. Therefore, in one example, this disclosure covers compositions containing the superparticles disclosed herein, wherein the superparticles are manufactured by electrospraying a composition containing nanoparticles and alginate or a polysaccharide derivative of said alginate into a dicationic aqueous solution. In this example, the superparticles may be calcined to remove alginate. In one example, the calcination was carried out at approximately 650°C. In another example, the calcination lasted for approximately 6 to approximately 30 hours.

[0014] The inventors have also identified that, in some aspects, the ultraparticles disclosed herein can release their payload over a relatively long period of time. Furthermore, in some aspects, the ultraparticles disclosed herein can be provided in various formulations to manipulate the release of the payload. For example, the inventors have identified in certain instances that the ultraparticles according to this disclosure can release their respective payloads over an extended duration. They have also identified that, in some aspects, the ultraparticles disclosed herein can be provided in various formulations to accelerate or decelerate the release of the payload. Thus, in one instance, the ultraparticles or compositions disclosed herein can be provided in the form of a sustained-release formulation. In one instance, the sustained-release formulation is a foam or gel. In one instance, the compositions disclosed herein comprise glycogen hydrogels. In another instance, the compositions disclosed herein comprise alginate [(C6H8O6)] n [or a polysaccharide derivative thereof.] In another example, the compositions disclosed herein comprise sodium alginate [Na(C6H8O6)]. n [or the polysaccharide derivative of sodium alginate.]

[0015] In one instance, the compositions disclosed herein are provided in the form of an accelerated-release formulation. In one instance, the accelerated-release formulation is a foam or gel. In one instance, the gel or foam is thermally reversible. For example, the accelerated formulation may comprise the ultraparticles disclosed herein and a PF127-based hydrogel.

[0016] In another instance, this disclosure covers the use of compositions as defined herein in the manufacture of pharmaceutical agents for treating a disease or condition. In one instance, the disease or condition is hearing loss. In one instance, the hearing loss is characterized as sensorineural hearing loss (SNHL), age-related hearing loss, or noise-induced hearing loss.

[0017] In another instance, this disclosure covers a method for promoting the survival of spiral ganglion neurons in the ear of a subject, the method comprising administering a composition as defined herein to the subject. For example, the composition may be administered to the ear of the subject.

[0018] In another instance, this disclosure covers a method for treating a condition in which sustained delivery of a therapeutic payload is desired in a subject, the method comprising administering a composition as defined herein to the subject. In one instance, the composition is administered to the subject's ear. In another instance, the composition is administered intraperitoneally. In another instance, the composition is administered to the central nervous system. In another instance, the composition is administered via intraventricular delivery. In yet another instance, the composition is administered in a scaffold that retains the ultraparticles.

[0019] In another instance, this disclosure covers a pillbox that includes the compositions defined herein when used to treat a disease or condition. In one instance, the condition is hearing loss. In another instance, the pillbox also includes a cochlear implant.

[0020] The inventors have also determined that electrospraying a composition comprising nanoparticles and alginate can produce ultraparticles capable of carrying high levels of payload. Therefore, in one instance, this disclosure also covers a composition comprising nanoparticles and alginate when used to manufacture ultraparticles as defined herein. In one instance, the alginate is sodium alginate.

[0021] In another example, this disclosure covers a method for manufacturing ultraparticles, the method comprising electrospraying a composition comprising nanoparticles and alginate or a polysaccharide derivative of said alginate into a dicationic aqueous solution. In one example, the method further comprises subjecting the ultraparticles to calcination to remove the alginate. In one example, the calcination is carried out at about 650°C. In one example, the calcination is carried out for about 6 to about 30 hours.

[0022] Unless otherwise expressly stated, any embodiment described herein should be considered as imposing necessary modifications on any other embodiment.

[0023] This disclosure is not limited to the specific embodiments described herein, which are intended for illustrative purposes only. As described herein, functionally equivalent products, compositions, and methods are clearly within the scope of this invention.

[0024] Throughout this specification, unless otherwise specifically stated or the context otherwise requires, references to a single step, a composition of substances, a group of steps, or a group of compositions of substances shall be construed as covering one or more (i.e., one or more) of those steps, compositions of substances, groups of steps, or groups of compositions of substances.

[0025] This disclosure is described below with reference to the following non-limiting embodiments and the accompanying drawings. Attached Figure Description

[0026] Figure 1 Procedure for preparing MS-SP.

[0027] Figure 2 ζ-potential of MS-SP as a function of pH. ζ-potential was measured in 10 mM sodium acetate buffer (pH 4), 10 mM phosphate buffer (pH 6), 10 mM HEPES buffer (pH 8), and 10 mM sodium bicarbonate buffer (pH 10).

[0028] Figure 3Confocal microscopy images of MS-SP loaded with FITC-lysozyme (green). a, b) MS-SP at different magnifications. c) Inside a fragmented MS-SP loaded with FITC-lysozyme.

[0029] Figure 4 Drug loading was performed using different FITC lysozyme loading concentrations after an incubation period of 3 days (72 hours). a) FITC lysozyme to pore-free MS-SP alg (Black circle), small hole MS-SP alg (Blue square) and MS-SP alg (a) Comparison of adsorption amount in red triangles with FITC-lysozyme loading concentration. b) Comparison of FITC-lysozyme adsorption amount in non-pore MS-SP (black circle), well-done MS-SP (blue square), and MS-SP (red triangle) with FITC-lysozyme loading concentration. c) MS-SP alg The FITC lysozyme loading efficiency and d) MS-SP are presented. The data presented are the averages of three replicates using five SPs, and the error bars represent the standard deviation.

[0030] Figure 5 FITC-lysozyme loading and release behavior from MS-SPs with different diameters. a) FITC-lysozyme loading, b) in vitro FITC-lysozyme release characteristics, c) FITC-lysozyme release values ​​at each individual time point, d) in vitro FITC-lysozyme release standard curves from MS-SPs with diameters of 200, 550, 650, and 1000 μm.

[0031] Figure 6 Cell viability of human glioblastoma cells (U87MG cell line) incubated with different amounts of MS-SP for 48 hours. Ethanol was added to cells with the inserted fragment to prepare a negative (cytotoxic) control, and untreated cells represented 100% viability. Data presented are the mean of four samples, and error bars represent standard deviation.

[0032] Figure 7 In vitro cumulative drug release characteristics. a) From MS-SP (red circle) and MS-SP alg (Black triangle) In vitro FITC-lysozyme (0.2 mg / mL) -1 Drug loading (3 days) release. The data presented are the average of three replicates using ten SPs each, and the error bars represent the standard deviation. b) In vitro FITC-lysozyme (1.0 mg / mL) from MS-SP -1(a) Drug loading for 3 days) Release. (b) The inset in the illustration shows the in vitro FITC-lysozyme release characteristics starting from day 6. (c) From MS-SP (1.0 mg / mL) -1 The in vitro BDNF release (drug loading for 3 days) was measured. BDNF ELISA (Abcam) was used to measure BDNF concentration.

[0033] Figure 8 .MS-SP alg The values ​​of FITC-lysozyme released by MS-SP at each individual time point (loading conditions: 0.2 mg / mL loaded with 100 μL). -1 FITC lysozyme (3-day loading time). The data presented are the average of three replicates using ten superparticles each, and the error bars represent the standard deviation.

[0034] Figure 9 .MS-SP values ​​of FITC-lysozyme released at each individual time point (loading conditions: 100 μL of 1.0 mg / mL loaded over 3 days). -1 (FITC-lysozyme). Drug loading was 6.49 ± 0.48 μg per ultraparticle. The data presented are the average of three replicates using ten MS-SPs each, and the error bars represent standard deviations.

[0035] Figure 10 Absolute values ​​of BDNF drug in vitro at each individual time point (loading conditions: 1.0 mg / mL over 3 days). -1 (BDNF). The data presented are the average of three replicates each using an MS-SP, and the error bars represent the standard deviation.

[0036] Figure 11 a) MicroBCA assay was used to measure BDNF from MS-SP. b) Values ​​of BDNF released at each individual time point using MS-SP. Drug load was 6.82 μg per ultraparticle. The data presented are the average of three replicates, and the error bars represent the standard deviation.

[0037] Figure 12 a) MS-SP (red circle) and MS-SP in the PF127 hydrogel system alg (Black square) In vitro FITC-lysozyme release characteristics. b) For Figure (a) (loading 100 μL of 0.2 mg mL) -1 FITC-lysozyme, the value of FITC-lysozyme released at each individual time point. (MS-SP and MS-SP) algIn the experiments, the drug loadings were 1.89 ± 0.08 μg per superparticle and 1.93 ± 0.03 μg per superparticle, respectively. The data presented are the averages of three replicates using ten superparticles each, and the error bars represent the standard deviation.

[0038] Figure 13 In vitro release characteristics at 50% loading (3.65 μg / SP)

[0039] Figure 14 Degradation of MS-SP after 150 days of in vitro drug release studies. a) SEM image of MS-SP after 150 days of incubation in PBS (pH 7.4) at 37°C. b, c) SEM images of the surface structure of MS-SP after 150 days of incubation in PBS (pH 7.4) at 37°C.

[0040] Figure 15 The amount (%) and total amount (μg) of neurotrophin-3 are shown. One week after implantation, each SP contained approximately 2 μg of the initial loading of neurotrophin-3 (approximately 40%). Detailed Implementation

[0041] General technologies and selected definitions

[0042] Unless otherwise explicitly defined, all technical and scientific terms used herein should be regarded as having the same meaning as commonly understood by a person skilled in the art (e.g., physiology, clinical research, molecular biology, high surface integral quanta, electrospraying, and biochemistry).

[0043] Unless otherwise stated, the techniques used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described and illustrated in the literature of the following resources: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); T.A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); D.M. Glover and B.B. Hames (ed.), and F.M. Ausubel et al. (ed.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updated versions to date); Ed. Harlow and David. Lane (ed.), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory (1988); and J.E. C. Olgan et al. (ed.), Current Protocols in Immunology, John Wiley and Sons (1984). Wiley & Sons (including all updated versions to date).

[0044] As used in this specification and the appended claims, the terms in the singular and singular forms "a / an" and "the" optionally include plural referents, for example, unless the content expressly indicates otherwise. Thus, for example, reference to "a superparticle" includes multiple superparticles.

[0045] Unless otherwise stated, as used herein, the term “about” means + / - 10%, more preferably + / - 5%, more preferably -1% of the specified value.

[0046] The term “and / or” (e.g., “X and / or Y”) should be understood to mean “X and Y” or “X or Y”, and should be regarded as providing explicit support for both meanings or either meaning.

[0047] Throughout this specification, the word “comprise” or variations thereof such as “comprises” or “comprising” should be understood to imply inclusion of the stated element, integer or step, or a group of elements, integers or steps, rather than exclusion of any other element, integer or step, or a group of elements, integers or steps.

[0048] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural processes of the treated individual or cells in a clinicopathological process. Exemplary desired effects of treatment include alleviating symptoms associated with the condition being treated. In the case of treating hearing loss, desired therapeutic effects include reducing the rate of hearing loss and improving or alleviating hearing loss. An individual is considered successfully "treated," for example, when one or more symptoms associated with the condition are alleviated or eliminated.

[0049] A “therapeutic effective dose” refers to the minimum amount required to achieve a measurable improvement in a particular condition. A therapeutic effective dose may also include at least the minimum amount required to achieve a measurable improvement in the subject’s condition. The effective dose may be provided in one or more administrations. The therapeutic effective dose may vary depending on the severity of the condition being treated and also on the weight, age, ethnic background, sex, health, and / or physical condition of the subject being treated. Typically, the effective dose will fall within a relatively broad range (e.g., a “dosage” range) that can be determined through routine testing and experimentation by a medical practitioner. The therapeutic effective dose may be administered in a single dose or in one or more repeated doses during the treatment period.

[0050] Payload

[0051] According to this disclosure, superparticles may contain a variety of payloads. A “payload” can be any agent that can be used to treat a condition. Examples of agents include biological products such as polynucleotides, antibodies, monoclonal antibodies, antibody fragments, antibody-drug conjugates, proteins, bioactive proteins, fusion proteins, recombinant proteins, peptides, polypeptides, synthetic polypeptides, vaccines, therapeutic sera, viruses, polynucleotides, cells (such as stem cells or portions thereof), and small molecules.

[0052] Exemplary viral payloads may include appropriately modified retroviruses, adenoviruses (AdV), adeno-associated viruses (AAV), or recombinant forms such as recombinant adeno-associated virus (rAAV) and its derivatives, such as self-complementary AAV (scAAV) and non-integrating AV. Other exemplary therapeutic viral payloads may include herpes simplex virus (HSV), lentivirus, vaccinia virus, and vesicular stomatitis virus (VSV). For example, the therapeutic viral payload may include AAV. Various AAV serotypes are known and may be suitable viral payloads. In one example, the AAV is serotype 2. In another example, the AAV is serotype 1. In other examples, the AAV is serotype 3, 4, 7, 8, 9, 10, 11, 12, or 13.

[0053] In one example, the small molecule is a neurotransmitter. The term "neurotransmitter" is used in the context of this disclosure to refer to a substance that transmits signals across chemical synapses from one cell to another. Generally, neurotransmitters transmit signals across chemical synapses from one neuron to a target cell, such as another neuron, muscle cell, or glandular cell. In another example, the small molecule is a receptor agonist. The term "agonist" is used in the context of this disclosure to refer to a substance that elicits a physiological response when combined with a receptor. In another example, the small molecule is a receptor antagonist. The term "antagonist" is used in the context of this disclosure to refer to a substance that interferes with or inhibits the physiological function of a receptor.

[0054] Exemplary polynucleotides include antisense polynucleotides, double-stranded DNA (dsDNA), or double-stranded RNA (dsRNA). In one example, the dsDNA or dsRNA is an aptamer. In another example, the dsRNA is siKNA, miRNA, or shRNA.

[0055] Exemplary antibodies and fragments thereof include human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, or single-domain antibodies. In one example, the antibody may be a bispecific antibody-drug conjugate or a biosimilar antibody. Other exemplary polypeptides include cytokines, chemokines, hormones, and coagulation factors. In one example, the polypeptide is an enzyme. Exemplary enzymes include proteases, lipases, asparaginases, iliprotamases, tissue plasminogen activators, collagenases, glutaminases, hyaluronidases, streptokinases, uricases, urokinases, or nucleases, such as programmable nucleases. In one example, the enzyme may be a DNA methyltransferase. In one example, the enzyme may be a programmable nuclease that targets the introduction of genetic modifications into a gene or its regulatory region. For example, the programmable nuclease may be an RNA-guided engineered nuclease (RGEN). In one example, the RGEN is derived from an archaea genome or may be a recombinant form thereof. In another example, the RGEN may be derived from a bacterial genome or a recombinant form thereof. In another example, the RGEN originates from a type I (CRISPR)-cas (CRISPR-related) system. In another example, the RGEN originates from a type II (CRISPR)-cas (CRISPR-related) system. In yet another example, the RGEN originates from a type III (CRISPR)-cas (CRISPR-related) system. In one example, the nuclease originates from either a type I or type II RGEN.

[0056] In another example, the therapeutic payload is a DNA methylation inhibitor, a histone acetyltransferase inhibitor, or a histone deacetylase inhibitor.

[0057] In another example, the therapeutic payload may be an antigen that stimulates an immune response in a subject. Exemplary antigens include proteins, peptides, polysaccharides, or oligosaccharides (free or conjugated to a protein carrier) or mixtures thereof. Other exemplary antigens include cells or portions thereof, or viral particles or portions thereof.

[0058] In one instance, the therapeutic payload is an antitumor agent.

[0059] Other exemplary therapeutic payloads include agonists or antagonists targeting membrane receptors in the inner ear. Such therapeutic payloads can modulate neurotransmission. In another example, the therapeutic payload is a neurological agent that can be used to treat one or more neurological conditions such as Alzheimer's disease, Parkinson's disease, epilepsy, or multiple sclerosis.

[0060] In one instance, the payload is a “neurotrophic factor.” The term neurotrophic factor, in the context of this disclosure, refers to a molecule that enhances the growth or survival potential of any cell, including those from the auditory system. For example, neurotrophic factors covered by this disclosure can enhance the growth or survival of cells from the auditory system or synaptic connections of said cells located in the inner ear, middle ear, or vestibular system. Exemplary cells include spiral ganglion neurons (SGN), hair cells (including inner ear hair cells and outer ear hair cells), cochlear glial cells, and Schwann cells. Exemplary synaptic connections include connections between hair cells, and between hair cells and SGN or other neurons discussed above. Other examples include neuronal cell bodies or their synaptic connections in Rosenthal's canal, neurons or synaptic connections in the upper and middle cochlear regions, and / or nerve fibers in the skeletal spiral lamina.

[0061] Exemplary neurotrophic factors may include agents discussed above that have known therapeutic efficacy for directly or indirectly enhancing the survival of cells from the auditory system and / or synaptic connections of said cells.

[0062] In one instance, the neurotrophic factor is a neurotrophic peptide. Exemplary neurotrophic peptides include brain-derived neurotrophic factor (BDNF), nerve growth factor, neurotrophin-3, neurotrophin-4, members of the ciliary neurotrophic factor (CNTF) family such as CNTF, leukemia inhibitory factor (LIF), interleukin-6 (IL-6), glial maturation factor (GMF), insulin-like growth factor-1 (IGF-1), neuroregulatory protein 1, neuroregulatory protein 2, neuroregulatory protein 3 and neuroregulatory protein 4, vascular endothelial growth factor (VEGF), members of the glial cell-derived neurotrophic factor (GDNF) family such as GDNF, neurorankin (NRTN), artemin (ARTN) and persephin (PSPN), liver glycosides such as A1, A2, A3, A4, A5, B1, B2 and B3, insulin-like growth factor-1 (IGF-1), and interleukins such as IL-11.

[0063] In one instance, the neurotrophic factor is selected from the group consisting of: brain-derived neurotrophic factor (BDNF), nerve growth factor, neurotrophin-3, neurotrophin-4, ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), and IL-11.

[0064] One cause of degeneration of spiral ganglion neurons is the loss of endogenous supply of neurotrophic proteins. Therefore, in one instance, the neurotrophic factor is a neurotrophic protein. The term "neurotrophic protein" is used in the context of this disclosure to refer to proteins that induce neuronal survival, development, and / or function and / or synaptic connections. Exemplary neurotrophic proteins have been discussed above and include BDNF, nerve growth factor, neurotrophin-3, and neurotrophin-4. Therefore, in one instance, the superparticle contains BDNF. In another instance, the superparticle contains nerve growth factor. In another instance, the superparticle contains neurotrophin-3. In another instance, the superparticle contains neurotrophin-4. In one instance, the superparticle may contain at least two different neurotrophic proteins. In other instances, the superparticle may contain at least three or four different neurotrophic proteins.

[0065] Therapeutic efficacy can be improved by applying superparticles containing a variety of different therapeutic payloads. Thus, in one instance, the superparticles may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different therapeutic payloads.

[0066] For example, a hyperparticle may contain at least two different neurotrophic factors. In other instances, a hyperparticle may contain at least three, four, or five different neurotrophic factors. In these instances, various combinations of neurotrophic factors such as neurotrophic proteins are considered. Exemplary combinations of neurotrophic factors include BDNF and nerve growth factor; BDNF and neurotrophin-3; BDNF and neurotrophin-4; BDNF and CNTF; BDNF and GDNF; BDNF and IL-11; neurotrophin-3 and neurotrophin-4; neurotrophin-3 and CNTF; neurotrophin-3 and CNTF; neurotrophin-3 and GDNF; neurotrophin-3 and IL-11; neurotrophin-4 and CNTF; neurotrophin-4 and GDNF; neurotrophin-4 and IL-11; CNTF and GDNF; CNTF and IL-11; GDNF and IL-11; BDNF, neurotrophin-3 and neurotrophin-4; BDNF, neurotrophin-3 and CNTF; BDNF, neurotrophin-3 and GDNF; BDNF, CNTF and GDNF; BDNF, CNTF and IL-11. 1; Neurotrophin-3, neurotrophin-4 and CNTF; Neurotrophin-3, neurotrophin-4 and GDNF; Neurotrophin-3, CDNF and GDNF; Neurotrophin-3, neurotrophin-4 and IL-11; Neurotrophin-4, CNTF and GDNF; Neurotrophin-4, CNTF and IL-11; BDNF, neurotrophin-3, neurotrophin-4 and CNTF; BDNF, neurotrophin-3, neurotrophin-4 and GDNF; BDNF, neurotrophin-3, CNTF and GDNF; BDNF, neurotrophin-4, CNTF and GDNF; BDNF, BDNF, neurotrophin-3, neurotrophin-4 and IL-11; Neurotrophin-3, neurotrophin-4, CNTF and GDNF; Neurotrophin-3, neurotrophin-4, CNTF and IL-11.

[0067] Various ear interventions (such as surgical procedures and the implantation of hearing devices) can lead to side effects such as tissue damage, inflammation, and / or infection of the middle and inner ear. Biological responses to such side effects can indirectly affect the growth or survival potential of cells from the auditory system and / or the synaptic connections of said cells. Therefore, in one instance, neurotrophic factors contribute to tissue repair, reduce inflammation, and / or decrease infection. Thus, other exemplary neurotrophic factors include steroids or antioxidants. Other exemplary neurotrophic factors include antibodies or other binding proteins, such as anti-tropomyosin receptor kinase (TrK)B, anti-TrK C, or binding proteins that interact with the p75 neurotrophin receptor. For example, p75 neurotrophin receptor antagonists. In another instance, neurotrophic factors include nucleic acids. For example, neurotrophic factors may include gene therapy, silent RNA (such as siRNA or miRNA), or expression constructs (such as DNA plasmids containing the target nucleic acid). In one instance, the neurotrophic factor is an expression construct containing a nucleic acid encoding opsin.

[0068] Other exemplary combinations of neurotrophic factors include steroids, antioxidants, antibodies, or nucleic acids, and at least one, at least two, at least three, at least four, or at least five different neurotrophic factors. For example, the hyperparticles may contain steroids such as dexamethasone or prednisolone, and any one or more of BDNF, nerve growth factor, neurotrophin-3, neurotrophin-4, and GDNF. In another example, the hyperparticles may contain an expression vector comprising opsin and any one or more of BDNF, nerve growth factor, neurotrophin-3, neurotrophin-4, and GDNF. In yet another example, the hyperparticles may contain antibodies such as antigen myosin receptor kinase (TrK)B or anti-TrK C, and any one or more of BDNF, nerve growth factor, neurotrophin-3, neurotrophin-4, GDNF, and IL-11.

[0069] In one instance, the superparticle may contain at least two, at least three, at least four, or at least five different therapeutic payloads, wherein at least one therapeutic payload is a neurotrophic factor. For example, the superparticle may contain at least three different therapeutic payloads, two of which are neurotrophic factors. In another instance, the superparticle may contain at least four different therapeutic payloads, three of which are neurotrophic factors. In these instances, the therapeutic payload does not need to enhance the growth or survival potential of cells from the auditory system, but rather provides another therapeutic benefit. For example, the therapeutic payload may suppress the immune system of a subject after superparticle administration. In another instance, the superparticle may contain a payload and a neurotrophic factor that reduce the survival of cells or synaptic connections of cells from the auditory system. In this instance, the neurotrophic factor may mitigate the reduction in the survival of cells or synaptic connections of cells from the auditory system caused by the therapeutic payload. In one example, the superparticles contain antitumor agents, including cisplatin or related compounds; antibiotics, including aminoglycosides such as tobramycin or related compounds; loop diuretics such as furosemide; antimetabolites such as methotrexate; salicylates such as aspirin or radioactive fractions; and neurotrophic factors. For this purpose, the superparticles may contain antibiotics and any one or more of BDNF, nerve growth factor, neurotrophin-3, neurotrophin-4, GDNF, and IL-11.

[0070] Depending on the application site, the payload may need to diffuse from the middle ear to the inner ear or vestibular system. This can occur via diffusion of the payload across the round or oval window. Therefore, in one instance, the superparticle may contain molecules that facilitate the diffusion of the payload across the round and oval windows to the inner ear and / or vestibular system.

[0071] In one example, the payload has an isoelectric point greater than 7. In another example, the payload has an isoelectric point greater than 8. In another example, the payload has an isoelectric point greater than 9. In another example, the payload has an isoelectric point greater than 10. In another example, the payload has an isoelectric point between 7 and 10. In another example, the payload has an isoelectric point between 7 and 9. In another example, the payload has an isoelectric point between 8 and 10. In another example, the payload has an isoelectric point between 9 and 10.

[0072] In the context of this disclosure, a superparticle containing a payload may be referred to as a loaded superparticle. There are no particular limitations on the method of producing loaded superparticles, as long as the resulting superparticles can carry a payload of at least 1.5 μg. Preferably, the resulting superparticles can deliver the payload to the ear of a subject. Exemplary loading methods are summarized in Wang et al. (2009) J. Mater. Chem. 19, 6451 and include payload encapsulation and embedding. In a non-limiting example, superparticles can be loaded by contacting the superparticles with an aqueous solution of the payload and then incubating for a period of time. The payload solution may contain an excess amount of the payload to be loaded onto the superparticles, and the incubation may occur at room temperature. Agitation of the solution containing the superparticles and the payload can be used to enhance the loading of the payload.

[0073] Those skilled in the art will understand that the required level of payload may be affected by the payload itself and the indication to be treated according to this disclosure.

[0074] ultraparticles

[0075] The term "superparticle" ("SP") is used in the context of this disclosure to refer to aggregated particles comprising a porous network. The porous network provides superparticles with a large pore volume and surface area to carry payloads. A large pore volume and surface area are advantageous because they increase the amount of payload that the superparticle can carry. In one example, the superparticles according to this disclosure are aggregated nanoparticles.

[0076] In one instance, the ultraparticles according to this disclosure contain at least 1.5 μg of payload. In another instance, the ultraparticles contain at least 2.0 μg of payload.

[0077] In another example, the superparticles according to the invention contain at least 2.5 μg of payload. In another example, the superparticles contain at least 2.6 μg of payload. In another example, the superparticles contain at least 2.7 μg of payload. In another example, the superparticles contain at least 2.7 μg of payload. In another example, the superparticles contain at least 2.8 μg of payload. In another example, the superparticles contain at least 2.9 μg of payload. In another example, the superparticles contain at least 3.0 μg of payload. In another example, the superparticles contain at least 3.1 μg of payload. In another example, the superparticles contain at least 3.2 μg of payload. In another example, the superparticles contain at least 3.3 μg of payload. In another example, the superparticles contain at least 3.4 μg of payload. In another example, the superparticles contain at least 3.5 μg of payload. In another example, the superparticles contain at least 3.6 μg of payload. In another example, the superparticles contain at least 3.7 μg of payload. In another example, the superparticle contains at least 3.8 μg of payload. In another example, the superparticle contains at least 3.9 μg of payload. In another example, the superparticle contains at least 4.0 μg of payload. In another example, the superparticle contains at least 4.5 μg of payload. In another example, the superparticle contains at least 5.0 μg of payload. In another example, the superparticle contains at least 5.5 μg of payload. In another example, the superparticle contains at least 6.0 μg of payload. In another example, the superparticle contains at least 6.5 μg of payload. In another example, the superparticle contains at least 7.0 μg of payload. In another example, the superparticle contains at least 7.5 μg of payload. In another example, the superparticle contains at least 8.0 μg of payload. In another example, the superparticle contains at least 8.5 μg of payload. In another example, the superparticle contains at least 9.0 μg of payload. In another example, the superparticle contains at least 9.5 μg of payload. In another example, the superparticle contains at least 10 μg of payload. In another example, the superparticle contains at least 10.5 μg of payload. In another example, the superparticle contains at least 11 μg of payload. In another example, the superparticle contains at least 11.5 μg of payload. In another example, the superparticle contains at least 12 μg of payload. In another example, the superparticle contains at least 15 μg of payload. In another example, the superparticle contains at least 20 μg of payload.

[0078] For example, ultraparticles may contain at least 6 μg of payload.

[0079] In another example, the ultraparticle may contain a payload between approximately 2.5 and 10 μg. In another example, the ultraparticle may contain a payload between approximately 3 and 10 μg. In another example, the ultraparticle may contain a payload between approximately 4 and 10 μg. In another example, the ultraparticle may contain a payload between approximately 5 and 10 μg. In another example, the ultraparticle may contain a payload between approximately 6 and 10 μg. In another example, the ultraparticle may contain a payload between approximately 5 and 15 μg. In another example, the ultraparticle may contain a payload between approximately 5 and 20 μg. In another example, the ultraparticle may contain a payload between approximately 8 and 20 μg. In another example, the ultraparticle may contain a payload between approximately 8 and 15 μg.

[0080] For example, ultraparticles may contain a payload between approximately 6 and 8 μg.

[0081] In one instance, the ultraparticles according to this disclosure contain at least 1.5 μg of neurotrophic factor. In another instance, the ultraparticles contain at least 2.0 μg of neurotrophic factor.

[0082] In another example, the superparticles contain at least 2.5 μg of neurotrophic factor. In another example, the superparticles contain at least 3.0 μg of neurotrophic factor. In another example, the superparticles contain at least 3.5 μg of neurotrophic factor. In another example, the superparticles contain at least 4.0 μg of neurotrophic factor. In another example, the superparticles contain at least 5.0 μg of neurotrophic factor. In another example, the superparticles contain at least 6.0 μg of neurotrophic factor. In another example, the superparticles contain at least 7.0 μg of neurotrophic factor. In another example, the superparticles contain at least 8.0 μg of neurotrophic factor. In another example, the superparticles contain at least 9.0 μg of neurotrophic factor. In another example, the superparticles contain at least 10 μg of neurotrophic factor. In another example, the superparticles contain at least 10.5 μg of neurotrophic factor. In another example, the superparticles contain at least 11 μg of neurotrophic factor. In another example, the superparticles contain at least 11.5 μg of neurotrophic factor. In another example, the superparticles contain at least 12 μg of neurotrophic factor. In another example, the superparticles contain at least 15 μg of neurotrophic factor. In yet another example, the superparticles contain at least 20 μg of neurotrophic factor.

[0083] For example, ultraparticles may contain at least 6 μg of neurotrophic factors.

[0084] In another example, the superparticles may contain between about 2.5 and 10 μg of neurotrophic factor. In another example, the superparticles may contain between about 5 and 10 μg of neurotrophic factor. In another example, the superparticles may contain between about 6 and 10 μg of neurotrophic factor. For example, the superparticles may contain between about 6 and 8 μg of neurotrophic factor. In another example, the superparticles may contain between about 5 and 15 μg of neurotrophic factor. In another example, the superparticles may contain between about 5 and 20 μg of neurotrophic factor. In another example, the superparticles may contain between about 8 and 20 μg of neurotrophic factor. In another example, the superparticles may contain between about 8 and 15 μg of neurotrophic factor.

[0085] In one instance, the ultraparticles according to this disclosure contain at least 1.5 μg of neurotrophic protein. In another instance, the ultraparticles contain at least 2.0 μg of neurotrophic protein.

[0086] In another example, the superparticle contains at least 2.5 μg of neurotrophic protein. In another example, the superparticle contains at least 3.0 μg of neurotrophic protein. In another example, the superparticle contains at least 3.5 μg of neurotrophic protein. In another example, the superparticle contains at least 4.0 μg of neurotrophic protein. In another example, the superparticle contains at least 5.0 μg of neurotrophic protein. In another example, the superparticle contains at least 6.0 μg of neurotrophic protein. In another example, the superparticle contains at least 7.0 μg of neurotrophic protein. In another example, the superparticle contains at least 8.0 μg of neurotrophic protein. In another example, the superparticle contains at least 9.0 μg of neurotrophic protein. In another example, the superparticle contains at least 10 μg of neurotrophic protein. In another example, the superparticle contains at least 10.5 μg of neurotrophic protein. In another example, the superparticle contains at least 11 μg of neurotrophic protein. In another example, the superparticles contain at least 11.5 μg of neurotrophic protein. In another example, the superparticles contain at least 12 μg of neurotrophic protein. In another example, the superparticles contain at least 15 μg of neurotrophic protein. In yet another example, the superparticles contain at least 20 μg of neurotrophic protein.

[0087] For example, ultraparticles may contain at least 6 μg of neurotrophic protein.

[0088] In another example, the superparticles may contain between about 2.5 and 10 μg of neurotrophic protein. In another example, the superparticles may contain between about 5 and 10 μg of neurotrophic protein. In another example, the superparticles may contain between about 6 and 10 μg of neurotrophic protein. For example, the superparticles may contain between about 6 and 8 μg of neurotrophic protein. In another example, the superparticles may contain between about 5 and 15 μg of neurotrophic protein. In another example, the superparticles may contain between about 5 and 20 μg of neurotrophic protein. In another example, the superparticles may contain between about 8 and 20 μg of neurotrophic protein. In another example, the superparticles may contain between about 8 and 15 μg of neurotrophic protein.

[0089] In one example, the superparticles according to this disclosure contain at least 1.5 μg of payload, wherein said payload has an isoelectric point between 9 and 10. In another example, the superparticles contain at least 2.0 μg of payload, wherein said payload has an isoelectric point between 9 and 10.

[0090] In another example, the superparticle contains at least 2.5 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticle contains at least 3.0 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticle contains at least 3.5 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticle contains at least 4.0 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticle contains at least 5.0 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticle contains at least 6.0 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticle contains at least 7.0 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticle contains at least 8.0 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticle contains at least 9.0 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticle contains at least 10 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticle contains at least 10.5 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticle contains at least 11 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticle contains at least 11.5 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticle contains at least 12 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticle contains at least 15 μg of payload, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticles contain a payload of at least 20 μg, wherein the payload has an isoelectric point between 9 and 10.

[0091] For example, the superparticles may contain at least 6 μg of payload, wherein the payload has an isoelectric point between 9 and 10.

[0092] In another example, the superparticles may contain a payload between about 2.5 and 10 μg, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticles may contain a payload between about 5 and 10 μg, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticles may contain a payload between about 6 and 10 μg, wherein the payload has an isoelectric point between 9 and 10. For example, the superparticles may contain a payload between about 6 and 8 μg, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticles may contain a payload between about 5 and 20 μg, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticles may contain a payload between about 5 and 15 μg, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticles may contain a payload between about 8 and 20 μg, wherein the payload has an isoelectric point between 9 and 10. In another example, the superparticles may contain a payload between about 8 and 15 μg, wherein the payload has an isoelectric point between 9 and 10. For example, the payload may be a neurotrophic protein having an isoelectric point between 9 and 10.

[0093] In one instance, superparticles with the aforementioned payload can be provided in an alginate hydrogel.

[0094] The superparticles of this disclosure may carry the payloads exemplified above and may have pore sizes selected from the examples discussed below. In one example, the superparticles are microporous. The term "microporous" is used in the context of this disclosure to refer to particles having a pore size of less than about 2 nm. For example, microporous superparticles may have a pore size of about 0.5 nm to about 2 nm. In other examples, microporous superparticles have pore sizes of about 1 nm to about 2 nm and about 1.5 nm to about 2 nm. In another example, the superparticles are mesoporous. The term "mesoporous" is used in the context of this disclosure to refer to particles having pores with a diameter between about 2 nm and about 50 nm. For example, mesoporous superparticles may have a pore size of about 2 nm to about 50 nm. In other examples, mesoporous superparticles have pore sizes of about 2 nm to about 40 nm and about 2 nm to about 30 nm. In another example, the superparticles are macroporous. The term "macroporous" is used in the context of this disclosure to refer to particles having a pore size greater than about 50 nm. For example, macroporous ultraparticles can have pore sizes of about 50 nm to about 500 nm. In other examples, macroporous ultraparticles have pore sizes of about 50 nm to about 250 nm, about 50 nm to about 150 nm, and about 50 nm to about 100 nm.

[0095] In one example, the superparticles consist of microporous nanoparticles. In another example, the superparticles consist of nanoparticles with pore sizes from about 0.5 nm to about 2 nm. In other examples, the superparticles consist of nanoparticles with pore sizes from about 1 nm to about 2 nm, and from about 1.5 nm to about 2 nm. In yet another example, the superparticles consist of mesoporous nanoparticles. In one example, the superparticles consist of nanoparticles with pore sizes from about 2 nm to about 50 nm. In other examples, the superparticles consist of nanoparticles with pore sizes from about 2 nm to about 40 nm, and from about 2 nm to about 30 nm. In yet another example, the superparticles consist of macroporous nanoparticles. In one example, the superparticles consist of nanoparticles with pore sizes from about 50 nm to about 95 nm. In other examples, the superparticles consist of nanoparticles with pore sizes from about 50 nm to about 85 nm, from about 50 nm to about 75 nm, and from about 50 nm to about 65 nm.

[0096] In another example, the superparticles consist of nanoparticles with a bimodal pore structure. The term "bimodal" is used in the context of this disclosure to refer to particles comprising multiple pore sizes, typically smaller and larger. For example, the superparticles may comprise nanoparticles with both mesopores and macropores. In one example, such superparticles consist of nanoparticles with pores ranging from 2 nm to 95 nm. In another example, the superparticles consist of bimodal nanoparticles with pores ranging from 10 nm to 95 nm. In yet another example, the superparticles consist of bimodal nanoparticles with pores ranging from 15 nm to 95 nm.

[0097] In other examples, the superparticles may include pore sizes ranging from 1 nm to 200 nm. In other examples, the superparticles consist of bimodal nanoparticles having a smaller pore size of about 1 nm to about 5 nm and a larger pore size of about 10 nm to about 50 nm. In another example, the superparticles consist of bimodal nanoparticles having a smaller pore size of about 2 nm to about 4 nm and a larger pore size of about 15 nm to about 40 nm. In another example, the superparticles consist of bimodal nanoparticles having a smaller pore size of about 2 nm to about 3 nm and a larger pore size of about 4 nm to about 40 nm. In other examples, the superparticles consist of bimodal nanoparticles having a smaller pore size of about 10 nm to about 50 nm and a larger pore size of about 70 nm to about 95 nm. In another example, the superparticles consist of bimodal nanoparticles having a smaller pore size of about 15 nm to about 40 nm and a larger pore size of about 80 nm to about 95 nm.

[0098] In one example, the superparticles consist of microparticles. In another example, the superparticles consist of microporous microparticles. In one example, the superparticles consist of microparticles with a pore size of about 0.5 nm to about 2 nm. In other examples, the superparticles consist of microparticles with a pore size of about 1 nm to about 2 nm, or about 1.5 nm to about 2 nm. In another example, the superparticles consist of mesoporous microparticles. In one example, the superparticles consist of microparticles with a pore size of about 2 nm to about 50 nm. In other examples, the superparticles consist of microparticles with a pore size of about 2 nm to about 40 nm, or about 2 nm to about 30 nm. In another example, the superparticles consist of macroporous microparticles. In one example, the superparticles consist of microparticles with a pore size of about 50 nm to about 500 nm. In other examples, the superparticles consist of microparticles with a pore size of about 50 nm to about 250 nm, about 50 nm to about 150 nm, or about 50 nm to about 100 nm.

[0099] In another example, the superparticles consist of microparticles with a bimodal pore structure. For example, the superparticles may include microparticles with both mesopores and macropores. In one example, such superparticles consist of microparticles with pores in the range of 2 nm to 500 nm. In another example, the superparticles consist of bimodal microparticles with pores in the range of 10 nm to 250 nm. In yet another example, the superparticles consist of bimodal microparticles with pores in the range of 15 nm to 150 nm.

[0100] In other examples, the superparticles consist of bimodal microparticles having a smaller pore size of about 1 nm to about 5 nm and a larger pore size of about 10 nm to about 50 nm. In another example, the superparticles consist of bimodal microparticles having a smaller pore size of about 2 nm to about 4 nm and a larger pore size of about 15 nm to about 70 nm. In yet another example, the superparticles consist of bimodal microparticles having a smaller pore size of about 2 nm to about 3 nm and a larger pore size of about 15 nm to about 65 nm. In other examples, the superparticles consist of bimodal microparticles having a smaller pore size of about 10 nm to about 30 nm and a larger pore size of about 15 nm to about 200 nm. In yet another example, the superparticles consist of bimodal microparticles having a smaller pore size of about 15 nm to about 40 nm and a larger pore size of about 15 nm to about 150 nm. In yet another example, the superparticles consist of bimodal microparticles having a smaller pore size of about 20 nm to about 30 nm and a larger pore size of about 100 nm to about 120 nm.

[0101] In another example, the ultraparticles are loaded with at least 3 μg of the aforementioned effective load and consist of bimodal nanoparticles having a smaller pore size of about 2 nm to about 4 nm and a larger pore size of about 15 nm to about 40 nm. In yet another example, the ultraparticles are loaded with at least 5 μg of the aforementioned effective load and consist of bimodal nanoparticles having a smaller pore size of about 2 nm to about 4 nm and a larger pore size of about 15 nm to about 40 nm.

[0102] In another example, the ultraparticles are loaded with at least 8 μg of the aforementioned payload and consist of bimodal nanoparticles having a smaller pore size of about 2 nm to about 4 nm and a larger pore size of about 15 nm to about 40 nm. In yet another example, the ultraparticles are loaded with at least 10 μg of the aforementioned payload and consist of bimodal nanoparticles having a smaller pore size of about 2 nm to about 4 nm and a larger pore size of about 15 nm to about 40 nm. In these examples, the ultraparticles may be provided in the form of an alginate hydrogel.

[0103] In another example, the superparticles are composed of microparticles and nanoparticles. In this example, the microparticles and nanoparticles may have the pore sizes mentioned above. For example, the superparticles may be composed of microparticles and nanoparticles with a bimodal pore structure.

[0104] In one example, the superparticles may have a substantially uniform pore size. In another example, the superparticles have a variable pore size. In this example, the pore size may be variable, but falls within a specific size range. For example, the superparticles may be primarily mesoporous. In another example, the superparticles may be primarily macroporous. In another example, the superparticles consist of nanoparticles with a substantially uniform pore size. In another example, the superparticles consist of bimodal nanoparticles with a substantially uniform small pore size and a substantially uniform large pore size. In another example, the superparticles consist of microparticles with a substantially uniform pore size. In another example, the superparticles consist of bimodal microparticles with a substantially uniform small pore size and a substantially uniform large pore size.

[0105] In one instance, the aforementioned superparticles may have an ordered pore structure. These superparticles have pores with regular three-dimensional spacing. In another instance, the aforementioned superparticles may have a disordered pore structure. These superparticles have pores with irregular three-dimensional spacing. In yet another instance, the superparticles have a combination of both ordered and disordered pore structures.

[0106] Those skilled in the art will recognize that the pore size of ultraparticles can be measured by, for example, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and X-ray computed tomography. Those skilled in the art can identify ultraparticles having the pore sizes exemplified above by measuring the width of the widest point across the three-dimensional structure of the ultraparticle. In one instance, the widest point, or pore, may be located on the surface of the ultraparticle.

[0107] The superparticles of this disclosure can be characterized by the distance between their particles. In one example, the average distance between the particles can be in the range of about 80 nm to about 400 nm. In another example, the average distance between the colloidal particles is in the range of about 90 nm to about 300 nm, and about 100 nm to...

[0108] In one instance, the pores of the superparticle are interconnected. For example, a superparticle may contain a series of interconnected pores. In another instance, the pores of the superparticle are not interconnected. In yet another instance, the superparticle has a combination of both interconnected and non-interconnected pores.

[0109] The ultraparticles of this disclosure are characterized by the volume of their pores. In one example, the pore volume of the ultraparticles is approximately 0.5 mL / g. -1 Approximately 10 mL g -1 Within a certain range. In another example, the ultraparticles had a size of approximately 0.8 mL g. -1 Approximately 5 mL g -1 Approximately 1 mL g -1 To approximately 2.5 mL g -1 Approximately 1.5 mL g -1 Approximately 2 mL g -1 The pore volume.

[0110] The superparticles disclosed herein may have a hollow core or a toroidal core. In one instance, the internal volume of the superparticle core is at least about 45%, at least about 55%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the total volume of the superparticle. Exemplary methods for producing hollow-core superparticles include acid core processes, such as those described in US 4,468,498; and ester core processes, such as those described in US 5,157,084 and 5,521,253.

[0111] The superparticles disclosed herein are characterized by the surface area of ​​their structure. In one example, the surface area of ​​the superparticles is approximately 500 m². 2 g -1 Approximately 1500m 2 g -1 Within this range. In another example, the ultraparticles have a size between approximately 5500 m. 2 g-1 With approximately 1250m 2 g -1 Approximately 600m 2 g -1 With 1000m 2 g -1 Approximately 600m 2 g -1 With 700m 2 g -1 The surface area between them. In one example, the surface area of ​​the superparticles is approximately 600 m². 2 g -1 In one instance, the surface area of ​​the superparticles was approximately 620 m². 2 g -1 .

[0112] The ultraparticles according to this disclosure may be characterized by specific in vitro release characteristics. In one instance, the ultraparticle release payload lasts for at least 50 days. In another instance, the ultraparticle release payload lasts for at least 100 days. In yet another instance, the ultraparticle release payload lasts for at least 150 days.

[0113] In another instance, the ultraparticles release an initial burst of payload, followed by sustained release of the payload. For example, the ultraparticles release a high level of payload for approximately 3 to 10 days, followed by sustained release characteristics for 30 days or longer. In another instance, the ultraparticles release a high level of payload for approximately 8 to 10 days, followed by sustained release characteristics for 50 days or longer.

[0114] In one instance, the initial burst release of the payload is 1.2 μg / day lasting for at least 2 days. In another instance, the initial burst release of the payload is 1.3 μg / day lasting for at least 3 days. In yet another instance, the initial burst release of the payload is 1.4 μg / day lasting for 2 to 4 days. One instance of a sustained release characteristic is at least 0.04 μg / day. Another instance of a sustained release characteristic is at least 0.06 μg / day. Other instances of sustained release characteristics are at least 0.07 μg / day and at least 0.1 μg / day.

[0115] In another example, the ultraparticles release at least 2 μg of payload for at least 20 days. In another example, the ultraparticles release at least 2 μg of payload for at least 30 days. In another example, the ultraparticles release at least 2 μg of payload for at least 40 days. In another example, the ultraparticles release at least 4 μg of payload for at least 20 days. In another example, the ultraparticles release at least 4 μg of payload for at least 30 days. In another example, the ultraparticles release at least 4 μg of payload for at least 40 days.

[0116] In another example, the ultraparticles release at least 2 μg of neurotrophic factor for at least 20 days. In another example, the ultraparticles release at least 2 μg of neurotrophic factor for at least 30 days. In another example, the ultraparticles release at least 2 μg of neurotrophic factor for at least 40 days. In another example, the ultraparticles release at least 4 μg of neurotrophic factor for at least 20 days. In another example, the ultraparticles release at least 4 μg of neurotrophic factor for at least 30 days. In another example, the ultraparticles release at least 4 μg of neurotrophic factor for at least 40 days. For example, the ultraparticles release at least 2 μg of BDNF for at least 20 days. In other examples, the ultraparticles can release at least 2 μg of BDNF for at least 40 days. In another example, the ultraparticles can release at least 4 μg of BDNF for at least 20 days. In another example, the ultraparticles can release at least 4 μg of BDNF for at least 40 days.

[0117] Those skilled in the art can use various methods to measure the in vitro release characteristics of ultraparticles. Examples of such methods are discussed in Example 7 below. For instance, the ultraparticles disclosed herein may be loaded with a labeled payload, such as FITC-lysozyme, before incubation in a solution such as PBS. Intermittent measurements of fluorescence can be used to determine the level of payload released over time (e.g., μg).

[0118] In one example, the superparticles of this disclosure are generated from nanoparticles with diameters between about 1 nm and 100 nm. In another example, the superparticles are generated from microparticles with diameters between about 0.1 μm and 100 μm. In yet another example, the superparticles are generated from both nanoparticles and microparticles.

[0119] Exemplary particles forming the superparticles of this disclosure include organic particles, inorganic particles, metallic particles, or combinations thereof. Exemplary organic particles include polymer particles such as polyglycolic acid (PGA), polylactic acid (PLA), poly(methacrylic acid), poly(ethylacrylic acid), polyacrylic acid (PAA), poly(N-isopropylacrylamide), poly(N,N-dimethylacrylamide), polyamide, poly-2-hydroxybutyrate (PHB), gelatin, polycaprolactone (PCL), and poly(lactic-co-glycolic acid) copolymer (PLGA). Exemplary inorganic particles include mineral fillers such as heavy fillers or high-density fillers, pigments, clay, and other synthetic particles. Other exemplary inorganic particles include minerals such as barite, hematite, magnesium oxide, and inorganic oxides including titanium dioxide, calcium oxide, zinc oxide, magnesium oxide, cerium oxide, zirconium dioxide, and silicon dioxide. In one example, the material is silicon dioxide (i.e., silica). Therefore, in one example, the superparticles may be referred to as silica superparticles. Exemplary metal particles include gold, silver, and copper. In one instance, the superparticles may contain the same type of particles. For example, the superparticles may consist essentially of silica particles. In another instance, the superparticles may contain different types of particles; for example, silica and clay particles. In still other instances, the superparticles may contain at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different types of particles.

[0120] In one instance, the superparticles comprise a polyelectrolyte or a polyelectrolyte material. Examples of such superparticles are disclosed in WO 2006 / 037160. In this instance, the polyelectrolyte may be a positively charged polyelectrolyte (or capable of being positively charged), a negatively charged polyelectrolyte (or capable of being negatively charged), or have zero net charge.

[0121] The superparticles of this disclosure can have various shapes. For example, the superparticles can have a spherical shape. Exemplary spherical shapes include spheres and ovals. In another instance, the superparticles have a non-spherical shape. Exemplary non-spherical shapes include dumbbells, hemispheres, disks, tetrahedrons, fibers, cylindrical spheres, and irregular shapes. In one instance, the superparticles can have an ordered structure. For example, the superparticles can comprise an ordered array.

[0122] The spherical ultraparticles of this disclosure can be characterized by their diameter. For example, the ultraparticles of this disclosure have a diameter greater than 100 μm. For example, the ultraparticles of this disclosure may have diameters of at least about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, about 600 μm, about 650 μm, about 700 μm, about 750 μm, about 800 μm, about 850 μm, about 900 μm, about 950 μm, and about 1000 μm. For example, the ultraparticles may have a diameter of about 550 μm. This diameter of the ultraparticles is advantageous because it allows for high drug loading while facilitating delivery via the inner ear canal. In other examples, the superparticles may have diameters between about 150 μm and about 1000 μm, about 200 μm and about 900 μm, and about 300 μm and about 800 μm. In another example, the superparticles may have diameters between about 400 μm and about 600 μm. In another example, the superparticles may have diameters between about 450 μm and about 550 μm. In another example, the superparticles may have diameters between about 520 μm and about 580 μm. In another example, the superparticles may have diameters between about 460 μm and about 540 μm. In another example, the superparticles may have diameters between about 470 μm and about 530 μm. In another example, the superparticles may have diameters between about 480 μm and about 520 μm. In another example, the superparticles may have diameters between about 490 μm and about 510 μm. In other instances, the superparticles may be characterized by their width across the widest point of their three-dimensional structure. For example, superparticles may have a width consistent with the diameter illustrated above.

[0123] In another example, the surface of the superparticles is modified by adding functional portions to enhance the load-bearing capacity. Any number of functional portions can be added to the surface of the superparticles, with the selection of functional portions chosen to complement the loaded load. In one example, a portion such as 3-aminopropyltriethoxysilane (APTS) is grafted onto the surface of the silica superparticles. This introduces amine functional groups that can interact with any carboxyl groups present on the load. In another example, the superparticles are modified to carry an overall net charge that can enhance the load-bearing capacity (Tan et al. Adv. Mater. (2012) 24, 3362-3366). For example, the surface of the superparticles can be modified to carry an overall net positive charge, thereby enhancing the load-bearing capacity of a payload carrying an overall net negative charge. In another example, the surface of the superparticles is modified to carry an overall net negative charge, thereby enhancing the load-bearing capacity of a payload carrying an overall net positive charge.

[0124] The superparticles disclosed herein may also include cross-linked functional portions. For example, the functional portions may be cross-linked via a chemical reaction. In one instance, polyglycolic acid (PGA) molecules are cross-linked via a chemical reaction between PGA molecules and cystamine (Tan et al. Adv. Mater. (2012) 24, 3362-3366).

[0125] preparation

[0126] Ultraparticles can be formulated into pharmaceutical compositions suitable for administration to a subject. Exemplary pharmaceutical compositions may provide ultraparticles alone or in combination with pharmaceutically acceptable carriers, diluents, or excipients. In these compositions, the ultraparticles are provided in an amount sufficient to deliver a therapeutically effective amount to the subject. Depending on the specific route of administration, a variety of acceptable carriers known in the art may be used, such as those described in Remington's Pharmaceutical Sciences (Mack Publishing Co., NJUSA, 1991).

[0127] Exemplary pharmaceutical compositions may also comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, emulsions, and sterile powders that are reconstituted into sterile injectable solutions or dispersions only prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or mediators include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils, and injectable organic esters such as ethyl oleate. Appropriate flowability may be maintained, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Such compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants, or antimicrobial and antifungal agents.

[0128] In another instance, the ultraparticles may be incorporated into sustained-release or targeted delivery systems. For example, the inventors have identified that providing an alginate hydrogel to the ultraparticles disclosed herein slows the release of the payload from the ultraparticles. Such sustained-release systems may include foams, gels, drops, and sprays for topical application, or reservoirs for injection or injection cannulas. Exemplary sustained-release systems include polymer matrices, liposomes, and microspheres. Polymer matrices include reservoir-based systems in which the ultraparticles are encapsulated in a porous polymer coating (Yang and Pierstorff (2012) JALA.17, 50-58); and monolithic matrix systems in which the ultraparticles are embedded in a polymer matrix (Langer R (1990) Science.249, 1527-1533). Liposomes may be biodegradable amphiphilic drug delivery systems formulated using phospholipids and cholesterol. Microspheres may be formulated using biodegradable and biocompatible polymers. In another instance, the sustained-release system may be polysaccharide-based. For example, the ultraparticle formulation may contain anionic polysaccharides. In one example, the polysaccharide may be alginate or a derivative thereof. For example, the ultraparticles may contain alginate hydrogels. Various alginate derivatives are known in the art. Examples include sodium alginate, potassium alginate, and calcium alginate. Other examples include barium alginate and strontium alginate. In one example, the alginate is sodium alginate. Thus, in one example, this disclosure covers formulations comprising the above-mentioned ultraparticles and sodium alginate. Alginate is available from a variety of sources (e.g., Sigma, FMC Health and Nutrition). In another example, the polysaccharide is an unbranched polysaccharide, such as glycosaminoglycans. For example, the polysaccharide may be hyaluronic acid.

[0129] In another example, the ultraparticles are provided in the form of a formulation that accelerates payload release. For example, the ultraparticles may be provided as a hydrogel based on Pluronic F127 (PF127) or the like. In one example, the ultraparticles may be provided as a PF127-based hydrogel.

[0130] In another instance, a superparticle formulation is provided to enhance diffusion across membranes (such as the tympanic membrane, oval window, or round window) within the subject's ear. For example, the superparticle formulation may contain artificial perilymph.

[0131] In another instance, the ultraparticles were incorporated or embedded in scaffolds that are compatible with the subject and degrade into products harmless to the subject. These scaffolds contained the ultraparticles to be transplanted into the subject.

[0132] A variety of different scaffolds can be successfully used in practicing this invention. Exemplary scaffolds include, but are not limited to, biodegradable scaffolds. Natural biodegradable scaffolds include collagen, fibronectin, and laminin scaffolds. Suitable scaffolds include polyglycolic acid scaffolds or synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid.

[0133] Treatment and application

[0134] This disclosure covers methods for treating a disease or condition, said methods including the administration of ultraparticles as defined herein. Thus, in one instance, a composition comprising ultraparticles according to this disclosure is administered to a subject in an amount effective in treating the subject's disease or condition. In one instance, said disease or condition is hearing loss. The term "hearing loss" is used in the context of this disclosure to refer to any reduction in a subject's ability to detect or process sound. Therefore, reference to hearing loss includes partial hearing loss or complete deafness.

[0135] In one instance, hearing loss is characterized as sensorineural hearing loss (SNHL). In the context of this disclosure, SNHL refers to hearing loss resulting from damage to the fragile sensory hair cells within the cochlea or loss of their synaptic connections with spiral ganglion neurons (SGNs) or dysfunction of Schwann cells in the cochlea. In one instance, hearing loss is characterized as age-related hearing loss. In another instance, hearing loss is noise-induced. In yet another instance, hearing loss is disease-induced or hereditary. In yet another instance, hearing loss is caused by exposure to otoxins, such as aminoglycosides.

[0136] In one instance, the subject is a mammal. In another instance, the subject is a human. For example, a human subject could be an adult. In one instance, a human subject is a child. Other exemplary mammalian subjects include companion animals such as dogs or cats, or livestock such as horses or cattle. Terms such as “subject,” “patient,” or “individual” are terms that may be used interchangeably in the context of this disclosure.

[0137] In one instance, ultraparticles according to the present disclosure are administered intraperitoneally. The present disclosure also covers methods for delivering a payload to the cells, tissues, or organs of a subject via the ear, the methods comprising administering ultraparticles according to the present disclosure to the subject's ear. In this instance, the method may deliver a payload from the inner ear, middle ear, and / or vestibular system of a subject to cells. In another instance, the method delivers a therapeutic payload to the nerve cells, neural tissue, or brain of a subject.

[0138] In one example, the composition is applied to the tympanic membrane. In this example, the composition may be formulated for topical application (e.g., drops, gel, foam, spray).

[0139] In another example, the composition is administered into the “middle ear” cavity. In the context of this disclosure, the term “middle ear” refers to the space between the tympanic membrane and the inner ear. Thus, the middle ear is located outside all inner ear tissues. For example, the composition can be administered into the middle ear via injection through the tympanic membrane. In this example, the ultraparticles can be administered as a reservoir-type injectable. In another example, an opening in the tympanic membrane can be created by the treating clinician to facilitate the entry of the composition into the middle ear. When the composition is administered into the middle ear, it can be applied to the round window and / or oval window.

[0140] In another instance, ultraparticles are applied to the inner ear. For example, ultraparticles may be applied to the cochlea. In one instance, ultraparticles may be applied to the basal turn of the cochlea. Surgical techniques for accessing the cochlea or other structures of the inner ear are known in the art. Exemplary techniques for surgical access to the human cochlea are described, for example, in Clark GM, et al., "Surgery for an improved multiple-channel cochlear implant", Ann Otol Rhinol Laryngol 93:204-7, 1984 and Clark GM, "Surgical and safety considerations of multichannel cochlear implants in children", Ear and Hearing Supplement 12:15S-24S, 1991.

[0141] The above discussion explored the combination of ultraparticles and ear interventions. In these examples, ear interventions can occur simultaneously with ultraparticle administration. For instance, a cochlear implant can be implanted concurrently with ultraparticles. However, the survival rate of spiral ganglion neurons may vary depending on the practicality of the cochlear implant. Therefore, it may be desirable to implant the cochlear implant after ultraparticle administration. For example, the cochlear implant can be implanted approximately one month, two months, three months, or six months after ultraparticle administration. In these examples, additional ultraparticles can be administered along with the cochlear implant.

[0142] In one instance, a first dose of the superparticles was applied to the subject's cochlea, and at least a second dose of the superparticles was applied to the subject's round window and / or oval window.

[0143] In one instance, a therapeutically effective amount is applied to the subject's ear. In another instance, multiple superparticles are applied to the subject's ear. For example, at least two, at least three, at least four, at least five, at least ten, or at least twenty superparticles may be applied to the subject's ear. In another instance, about one to ten superparticles are applied. In still other instances, about two to nine, about three to eight, about four to seven, or about five to six superparticles are applied to the subject's ear. In these instances, the superparticles may contain the same payload. Alternatively, in another instance, the superparticles may carry different payloads. For example, superparticles loaded with neurotrophic factors and superparticles loaded with steroids may be applied. In another instance, superparticles loaded with neurotrophic factors and superparticles loaded with antibiotics may be applied. In yet another instance, superparticles loaded with neurotrophic factors may be applied together with antibiotics.

[0144] Composition / Pharm Kit

[0145] The ultraparticles according to this disclosure may be provided in the form of a kit or package. For example, the compositions disclosed herein may be packaged in a suitable container along with written instructions for use in the treatment of inner ear disorders. In one instance, the compositions may be provided in a single-dose container such as an ear dropper or a pre-filled syringe.

[0146] In one example, the pillbox includes ultraparticles according to the present disclosure used in a method for treating inner ear disorders. In another example, the pillbox includes ultraparticles according to the present disclosure used in a method for treating hearing loss. In one example, the hearing loss is SNHL. In another example, the hearing loss is noise-induced hearing loss. In one example, the pillbox according to the present disclosure also includes a hearing aid or implant. Thus, in one example, the pillbox may include ultraparticles and a cochlear implant.

[0147] manufacture

[0148] This disclosure covers methods for generating ultraparticles capable of loading high levels of payload. In one example, the ultraparticles may be manufactured from a composition comprising nanoparticles and alginate or a polysaccharide derivative of said alginate. Various examples of nanoparticles are provided above. In one example, the nanoparticles have a bimodal porous structure. Various methods can be used to generate nanoparticles. One such method is described in Cui et al. 2015, ACS Nano, 9, 1571-1580.

[0149] In another example, the ultraparticles according to this disclosure are generated by electrospraying. Examples of electrospraying are summarized in Jaworek A., 2007 Powder Technology 1, 18-35. Examples of electrospraying are also illustrated below. In one example, this disclosure covers a method for manufacturing ultraparticles, the method comprising electrospraying a composition comprising nanoparticles and alginate or a polysaccharide derivative of said alginate into a dicationic aqueous solution.

[0150] Those skilled in the art will understand that electrospraying parameters can be optimized based on the type of solution used for electrospraying. For example, voltage and flow rate can be optimized to provide ultraparticles of the desired size. In one example, the flow rate is approximately 6-10 mL / h. -1 In another example, the flow rate was approximately 7-9 mL / h. -1 In another example, the flow rate was approximately 8 mL / h. -1 In one example, the voltage is between approximately 10 and 25 kV. In another example, the voltage is between approximately 11 and 20 kV. In yet another example, the voltage is between approximately 12 and 14 kV. In yet another example, the voltage is approximately 13 kV.

[0151] In one example, ultraparticles are generated by electrospraying a solution of nanoparticles. In one example, the concentration of nanoparticles in the solution is approximately 20 mg / ml. In another example, the concentration of nanoparticles in the solution is approximately 30 mg / ml. In another example, the concentration of nanoparticles in the solution is approximately 40 mg / ml. In another example, the concentration of nanoparticles in the solution is approximately 50 mg / ml. In yet another example, the concentration of nanoparticles in the solution is approximately 60 mg / ml.

[0152] In another example, ultraparticles are produced by electrospraying a solution of nanoparticles containing alginate or a derivative thereof. In one example, the nanoparticle solution consists of 5 mg / mL... -1 Preparation of alginate solution. In one example, the nanoparticle solution was prepared from 10 mg / mL. -1 Preparation of alginate solution. In one example, the nanoparticle solution was prepared from 20 mg / mL. -1 Preparation of alginate solution. In one example, the nanoparticle solution was prepared from 30 mg / mL. -1 The solution was prepared from alginic acid. In another example, the nanoparticle solution was prepared from 5 mg / mL. -1 Up to 30 mg / mL -1 The solution was prepared from alginic acid. In another example, the nanoparticle solution was prepared from 10 mg / mL. -1 Up to 30 mg / mL -1The solution was prepared from alginic acid. In another example, the nanoparticle solution was prepared from 20 mg / mL. -1 Up to 30 mg / mL -1 Preparation of alginate solution.

[0153] In one example, the nanoparticle solution consists of 5 mg / mL of water. -1 Preparation of alginate. In one example, the nanoparticle solution was prepared from 10 mg / mL of water. -1 Preparation of alginate. In one example, the nanoparticle solution was prepared from 20 mg / mL of water. -1 Preparation of alginate. In one example, the nanoparticle solution was prepared from 30 mg / mL of water. -1 Preparation of alginate. In another example, the nanoparticle solution was prepared from 5 mg / mL of water. -1 Up to 30 mg / mL -1 Preparation of alginate. In another example, the nanoparticle solution was prepared from 10 mg / mL of water. -1 Up to 30 mg / mL -1 Preparation of alginate. In another example, the nanoparticle solution was prepared from 20 mg / mL of water. -1 Up to 30 mg / mL -1 Preparation of alginic acid.

[0154] In another example, ultraparticles are generated by electrospraying a solution of nanoparticles containing alginate.

[0155] There are no particular limitations on alginate derivatives, as long as they form a gel at a defined temperature. In one example, the alginate derivative is a polysaccharide derivative. Alginate derivatives include various alginate forms. Examples include sodium alginate, potassium alginate, and calcium alginate. Other examples include barium alginate and strontium alginate. In one example, alginate is sodium alginate. In another example, ultraparticles are produced by electrospraying a solution of nanoparticles containing alginate.

[0156] Depending on the desired size and shape of the superparticles, alginates of various viscosities can be used to produce the superparticles according to this disclosure. For example, alginates with a viscosity of about 20 to 300 mPa*s can be used. In another example, the alginate has a viscosity of about 20 to 200 mPa*s. In one example, the alginate has a viscosity of 20 mPa*s. In another example, the alginate has a viscosity of 100 mPa*s. In yet another example, the alginate has a viscosity of 200 mPa*s.

[0157] In one example, ultraparticles are produced by electrospraying a composition containing nanoparticles into an aqueous solution. In one example, this is a dicationic aqueous solution. Exemplary dicationic components include Ca... 2+ and Ba 2+ For example, the aqueous solution may contain calcium chloride. In another example, the aqueous solution contains barium chloride.

[0158] In one example, ultraparticles are generated by electrospraying the composition, wherein the concentration of alginate in the composition is 5 mg / mL. -1 Up to 30 mg / mL -1 The concentration of nanoparticles in the composition is 20 mg / mL. -1 Up to 50 mg / mL -1 The voltage is 10kV to 25kV. In another example, ultraparticles are generated by electrospraying the composition, wherein the concentration of alginate in the composition is 10 mg / mL. -1 Up to 30 mg / mL -1 The concentration of nanoparticles in the composition is 30 mg / mL. -1 Up to 50 mg / mL -1 The voltage is 11 kV to 21 kV. In another example, ultraparticles are generated by electrospraying the composition, wherein the concentration of alginate in the composition is 20 mg / mL. -1 Up to 30 mg / mL -1 The concentration of nanoparticles in the composition is 35 mg / mL. -1 Up to 45 mg / mL -1 The voltage is 12kV to 14kV. In these examples, the flow rate can be 8 mL / h. -1 .

[0159] In another example, ultraparticles are generated by electrospraying the composition, wherein the concentration of alginate in the composition is 30 mg / mL. -1 The concentration of nanoparticles in the composition is 40 mg / mL. -1 The voltage is 13kV and the flow rate is 8mL / h. -1 .

[0160] In one example, ultraparticles manufactured using the methods defined herein are calcined to remove alginate or its derivatives. In one example, calcination is performed at approximately 500°C. In another example, calcination is performed at approximately 550°C. In another example, calcination is performed at approximately 600°C. In another example, calcination is performed at approximately 650°C. In another example, calcination is performed at approximately 700°C. In one example, the calcination lasts from approximately 6 to approximately 30 hours. In another example, the calcination lasts for approximately 10 hours. In another example, the calcination lasts for approximately 20 hours. In another example, the calcination lasts for approximately 30 hours.

[0161] Example

[0162] Example 1 - Nanoparticle generation

[0163] Mesoporous silica nanoparticles (MS-NPs) were produced using methods based on those described by Cui et al. 2015, ACS Nano, 9, 1571-1580. 1.1 g of cetyltrimethylammonium bromide (CTAB) was completely dissolved in 50 mL of Milli-Q with stirring. Subsequently, 4.3 g of poly(acrylic acid solution) (PAA, M...) was added at room temperature (25 °C) with vigorous stirring for 20 min. w =250kDa, a 35wt% solution in water), until a clear solution is obtained. Then, 3.5ml of ammonium hydroxide solution (28%-30%) is added to the solution under vigorous stirring to obtain an emulsion suspension. After stirring for 20 minutes, 4.46ml of tetraethyl orthosilicate (TEOS) is added. The solution is stirred for another 15 minutes, and then the mixture is transferred to a Teflon-sealed autoclave and placed at 90°C for 48 hours.

[0164] The synthesized MS-NP was washed once with ethanol, twice with water, and twice with ethanol, and finally dried at 90 °C. The organic template was removed by calcination at 550 °C for 30 hours.

[0165] Example 2 -Initial Ultraparticle Manufacturing Method-Method A

[0166] Mesoporous silica (MS) nanoparticles were prepared according to Wang et al. (2010) Chem Mater. 22, 3829-3831. MS nanoparticles were dispersed in Milli-Q water at a concentration of 5 wt% and briefly sonicated to form a stable colloidal suspension. Aliquots of the MS nanoparticle dispersion, ranging from 0.5 to 2.0 μL, were then applied to a flat surface pre-coated with a paraffin film. The droplets were dried under an airflow to assemble the MS nanoparticles into mesoporous silica superparticles (capillary-forced MS-SPs) via capillary forces. The size of the capillary-forced MS-SPs was controlled by the volume of the nanoparticle dispersion applied within the droplets.

[0167] Under capillary forces, MS colloids self-assemble into a compact structure to form MS-SP. The capillary-forced MS-SP was then removed from the paraffin film and transferred to a ceramic container, where it was annealed at 923 K to enhance its mechanical stability. The capillary-forced MS-SP was then loaded with a payload. Each particle was loaded with approximately 1.33 μg of protein. The capillary-forced MS-SP exhibited a bimodal pore structure (2-3 nm and 15-30 nm), and the macropores (spaces between densely packed nanoparticles) in the capillary-forced MS-SP were 100-200 nm.

[0168] Example 3 -Modified manufacturing method-Method B

[0169] Add 80 mg of MS-NP powder to 2 ml of sodium alginate solution (30 mg / mL in water). -1 The resulting solution was sonicated for 1 hour until MS-NP was uniformly distributed in the sodium alginate solution.

[0170] In order to form macroporous MS-SP ( L MS-SP), the sonicated solution is added to a 3 mL plastic syringe and placed in a syringe pump, then approximately 8 mL of h is used. -1 The flow rate will be used to electrospray the liquid into a calcium chloride solution bath (1 wt% prepared in water) (electrospray setup as follows) Figure 1 (As shown). Droplet size was controlled by applying an electric field between the end of the tube and the calcium chloride solution. Alginate beads (MS-SP) were collected from a calcium chloride bath. L MS-SP alg ), and load the effective payload. Compared with those particles produced by method A, for these particles ( L MS-SP showed significantly enhanced drug loading performance: approximately 7.8 μg of protein per particle, with improved mechanical stability.

[0171] Example 4 -Modified manufacturing method-Method C

[0172] MS-SP is generated using the method described in Example 3. L MS-SP alg Sodium alginate was removed by calcination at 650 °C for 30 hours. This step removed all organic components, leaving only mesoporous silica nanoparticles (MS-SP) and trace amounts of calcium chloride and sodium chloride. The MS-SP was then loaded with a drug loading capacity. A significantly enhanced drug loading capacity was observed for these particles (LMS-SP) compared to those produced by method A: approximately 7.8 μg of protein per particle.

[0173] Ultraparticles are also composed of non-porous nanoparticles (non-porous) N MS-SP alg ; N MS-SP alg ) and nanoparticles with pore sizes <2 nm (micropore MS- S SP alg ; S MS-SP alg The drug loading capacity was significantly reduced after removing alginate from these ultraparticles (Table 1).

[0174] Table 1 summarizes the maximum drug load of MS-SPs generated by methods B and C.

[0175] Table 1: MS-SP load generated by methods B and C

[0176] Particle type <![CDATA[q max (ug / particles)]]> <![CDATA[non-porous N MS-SP alg > <![CDATA[q max =8.215]]> <![CDATA[Non-porous N MS-SP]]> <![CDATA[q max =2.009]]> <![CDATA[Small hole S MS-SP alg > <![CDATA[q max =10.06]]> <![CDATA[Small hole S MS-SP]]> <![CDATA[q max =2.802]]> <![CDATA[Macroporous L MS-SP alg > <![CDATA[q ma x=9.245]]> <![CDATA[Macroporous L MS-SP]]> <![CDATA[q max =7.843]]>

[0177] Example 5 -Ultraparticle release properties

[0178] Lysozyme

[0179] Sterilized SP was mixed with 100 μL of FITC-lysozyme solution (Milli-Q water, 0.2 mg / mL). -1 They were incubated together to prepare SP FITC-loaded lysozyme for in vitro release studies. Lysozyme is a good model protein for mimicking the neurotrophic protein BDNF (because lysozyme is inexpensive and readily available, while BDNF is expensive) due to its similar physicochemical properties (lysozyme, M... w =14.3±0.5KDa, And pI = 11; BDNF, M w =13KDa, And pI = 10).

[0180] The in vitro release characteristics of MS-SP loaded with lysozyme were shown in Figure 4 Parts a) and b) and Figure 7 In section a), the release characteristics of MS-SPs produced by methods A and C are similar, although the MS-SPs produced by method C have a significantly higher load of labeled lysozyme. Compared to MS-SPs produced by methods A and C, the payload release of MS-SPs produced by method B is significantly reduced.

[0181] ζ potential

[0182] Then, MS-SP was loaded with fluorescein-labeled lysozyme (FITC-lysozyme). Figure 2 As shown, MS-SP exhibits a negative zeta potential ranging from approximately -7.6 mV to approximately -33.9 mV as the pH increases from 4 to 10. Therefore, positively charged lysozyme and BDNF can be loaded into MS-SP using electrostatic driving forces. (Confocal microscopy image) Figure 3 (a) and (b) show FITC-lysozyme loaded onto the surface of MS-SP. However, due to the very large size of MS-SP (hundreds of micrometers in diameter), standard laser scanning confocal microscopy is not suitable for imaging the internal structure of the SP. However, after rupturing the MS-SP with a scalpel, the interior could be imaged, and FITC-lysozyme was also observed in the porous internal structure of the MS-SP. Figure 3 c).

[0183] Additional assessment of load capacity

[0184] Then, FITC-lysozyme was used at different loading concentrations with a 3-day incubation period to study the loading capacity of different types of SPs. Figure 4 Generally, the drug loading increases with increasing FITC-lysozyme concentration. Results indicate that alginate-containing... N MS-SP alg , S MS-SP alg and L MS-SP alg Its loading capacity is higher than that of alginate-removed alginates. N MS-SP, S MS-SP and L MS-SP (at comparable concentrations). This is likely due to increased electrostatic interaction between positively charged FITC-lysozyme and negatively charged alginate near neutral pH. Furthermore, at low FITC-lysozyme loading concentrations (<0.4 mg / mL)... -1 )Down, L MS-SP alg With the same characteristics as the non-porous MS-SP alg and S MS-SPalg Similar drug loading capacity, but at higher concentrations (>0.4 mg / mL) -1 ),and N MS-SP alg and S MS-SP alg In comparison, more FITC-lysozyme can be loaded onto L MS-SP alg In the middle. For MS-SP with alginate removed, a similar trend was observed: with N MS-SP and S Compared to MS-SP L MS-SP can load more FITC-lysozyme. These results indicate that large porous structures (in...) L MS-SP and L MS-SP alg The diameter of the MS-SP is a key factor in increasing drug loading, likely by providing additional surface area and thus enabling loading capacity when both the outer particle surface and the inner particle region (in the SP structure) are fully saturated with drug. Additionally, the loading capacity also depends on the diameter of the MS-SP, with larger MS-SPs (1000 μm) exhibiting greater loading capacity than smaller MS-SPs (200 μm). Figure 5 ).

[0185] The experimentally determined FITC-lysozyme to N MS-SP and S MS-SP alg The maximum loading amounts in the samples were approximately 3 μg and 2 μg per SP, respectively, significantly lower than the approximately 15 μg per SP. L MS-SP (FITC-lysozyme concentration is 1.5 mg / mL) -1 Furthermore, the loading time was 3 days. In addition, the experimentally determined FITC-lysozyme... N MS-SP and S MS-SP alg The maximum loading amounts in the samples were approximately 2 μg and 1 μg per SP, respectively, which are also significantly lower than the approximately 10 μg per SP. L MS-SP (FITC-lysozyme concentration is 5.0 mg / mL) -1 And the load time is 3 days.

[0186] Drug loading efficiency of six different SPs, such as Figure 4 As shown in c and 4d, as the loading concentration of FITC-lysozyme increases, more FITC-lysozyme can be loaded into SP.

[0187] BDNF

[0188] MS-SP was sterilized by immersing it in 100 μl of ethanol (80 vol / vol%) for 4 hours, followed by rinsing six times with 100 μl of Milli-Q water. The MS-SP was then washed once in 0.1 M phosphate-buffered saline (PBS). MS-SP was loaded by placing it in an Eppendorf tube containing 15 μl of BDNF (Geneway, BDNF Human Protein, catalog #10-663-45078) solution (1 mg / mL BDNF) and incubating at room temperature for three days with occasional manual shaking. Notably, loading of approximately 10 μg of BDNF was achieved.

[0189] Example 6 - Ultraparticle cytotoxicity

[0190] To investigate whether MS-SP could be used as a biocompatible drug carrier, in vitro cytotoxicity studies were conducted on MS-SP. Human glioblastoma cells (U87MG cell line) were co-incubated with MS-SP, and cell viability was assessed using an Alamar Blue assay. Figure 6 As shown, even when the number of MS-SPs increases to ten per well (1×10 per well), 4 MS-SP is also non-toxic when administered per cell. Typical in vivo treatment protocols for similar particle systems recommend administering 1-8 SPs per inner ear.

[0191] Example 7 -Drug release

[0192] Based on the loading capacity results, 10 cells were incubated in PBS (pH 7.4) at 37°C. L MS-SP and 10 MS-SP alg To conduct a FITC-lysozyme release study (loading time 3 days, loading concentration 0.2 mg / mL) -1 ).

[0193] By sterilizing the SP, then ten MS-SPs or ten MS-SPs alg Mix with 100 μL of low-concentration FITC lysozyme solution (0.2 mg / mL in Milli-Q water). -1The samples were incubated together to prepare a SP containing FITC-lysozyme for in vitro release studies. After three days, the supernatant was removed, and 100 μL of phosphate-buffered saline (PBS, pH 7.4) was added to each tube and incubated at 37°C. At defined time intervals (within 150 days), 90 μL of the solution was collected and replaced with fresh PBS. The concentration of FITC-lysozyme in the supernatant was then determined by measuring the fluorescence of the collected samples using an Infinite M200 microplate reader and a standard curve.

[0194] for L MS-SP and L MS-SP alg After loading in low-concentration solutions, the FITC-lysozyme loading was 1.89±0.02 μg and 1.93±0.01 μg per SP, respectively. Figure 7 As shown in figure a, >1 μg of FITC-lysozyme was released per MS-SP over a period of more than 150 days, while during this time, it was released from MS-SP alg A lower amount of FITC-lysozyme was released (approximately 0.4 μg per SP). The lower amount of FITC-lysozyme released from the latter may be due to the presence of alginate, which may potentially block the macropores of the primary silica particles or interact strongly with lysozyme, thereby preventing or inhibiting lysozyme release. Figure 8 The figures show the individual values ​​of FITC-lysozyme release measured at each time point (used for preparation). Figure 7 (The cumulative results shown).

[0195] As the loading concentration of the drug used to load SP increases, more drug can be loaded onto it. L In MS-SP. Therefore, the study investigated incubation at higher concentrations (1.0 mg / mL). -1 Drug release characteristics of FITC-lysozyme and BDNF at this concentration (1.0 mg / mL). -1 The FITC-lysozyme loading was 6.49 ± 0.48 μg per SP. The release characteristics of FITC-lysozyme were observed in two release phases: a burst release in the first three days, followed by sustained release over more than 110 days. Figure 7 b). During the burst phase, each SP released approximately 4.2 μg of FITC-lysozyme. After 3 days, approximately 2.3 μg of FITC-lysozyme (load - amount released during the burst) remained in the SP, followed by a more sustained release behavior. The individual values ​​of FITC-lysozyme released at each time point were tens to hundreds of nanograms per SP. Figure 9 ).like Figure 7As shown in Figure c, the in vitro BDNF release characteristics are similar to those of FITC-lysozyme, with a burst release observed in the first three days, followed by sustained release for over 40 days. After 40 days, the cumulative release of the SP was approximately 4.68 μg of drug, and the weight of each SP was approximately 0.05 mg. Therefore, the observed cumulative release was approximately 93.6 μg mg. -1 This represents a 4000-fold improvement compared to recent observations for nanoporous silica nanoparticles. Individual values ​​of BDNF released at each time point range from tens to hundreds of nanograms per SP. Figure 10 It was also determined by MicroBCA (). Figure 11 The amount of BDNF released was measured, and the results were similar.

[0196] To investigate potential methods for further modulating drug release behavior and aiding in inner ear surgery, MS-SP and MS-SP incorporated into PF127 hydrogel were tested. alg In vitro drug release studies were conducted. PF127 hydrogel forms a viscous liquid at low temperatures (e.g., 4°C) and a gel at body temperature (37°C). In these studies, most FITC-lysozyme was observed to be released from MS-SP and MS-SP by approximately day 14. alg (All in PF127 hydrogel) release ( Figure 12 This demonstrates accelerated release kinetics. Therefore, the ultraparticle-PF127 system is more suitable when short-term drug release (approximately 1 week) is required, while SP without PF127 hydrogel is more suitable for long-term (months) drug release.

[0197] In vitro experiments were also conducted when SP was loaded at approximately 50% to establish BDNF release kinetics over time. Consistent with data from lysozyme, BDNF release was more linear in the first 4 weeks (e.g., no burst release was observed). Figure 13 ).

[0198] Example 8 - Degradation of ultraparticles

[0199] Porous silica particles can degrade in the biological environment (e.g., to form silicic acid, which can be excreted in urine) and have attracted attention in various biomedical applications. To investigate the degradation rate, MS-SP was incubated for 150 days (PBS, pH 7.4, 37°C). Figure 14 It was observed that the diameter of MS-SP decreased by approximately 55%, and the shape / morphology of MS-SP also changed significantly. The morphology and size of the primary silica particles on the MS-SP surface both changed.

[0200] Example 9 - Pharmacokinetics

[0201] In vivo studies were conducted to determine the drug payload and clearance of neurotrophic proteins delivered in the implanted ear superparticles at 4 hours, 3 days, and 7 days post-implantation. The aim was to determine the amount of neurotrophic protein remaining in the cochlea over a period of time.

[0202] One SP containing radiolabeled neurotrophin-3 was implanted into each cochlea. The cochlea was harvested after 4 hours (n=5), 3 days (n=7), or 7 days (n=4). Gamma counts throughout the cochlea were measured to determine the clearance rate of neurotrophin-3 over time. Figure 15 The remaining amount (% of load) and total amount (μg) of neurotrophin-3 are shown. One week after implantation, each SP contained approximately 2 μg of the initial load of neurotrophin-3 (approximately 40%).

[0203] Further experiments (n=2) were conducted to examine the clearance rate of neurotrophin-3 after round window delivery. Three days post-implantation, measurements from the intact cochlea were used again to determine the clearance rate of neurotrophin-3 from the round window membrane. A similar level of clearance was observed using the round window compared to the intracochlear delivery site (47.4% of neurotrophin-3 remained after round window delivery, compared to 56% after intracochlear delivery).

[0204] These data indicate that prolonged release of neurotrophin-3 can be achieved, with high levels of neurotrophin-3 remaining even one week after treatment.

[0205] Those skilled in the art will understand that numerous variations and / or modifications may be made to the disclosure as illustrated in the specific embodiments without departing from the broadly described scope of this disclosure. Therefore, this embodiment is to be regarded as illustrative rather than restrictive in all respects.

[0206] This application claims priority to AU2017903828, filed September 20, 2017; AU2017903829, filed September 20, 2017; and AU 2018902513, filed July 11, 2018, the disclosures of which are incorporated herein by reference.

[0207] All publications discussed above are incorporated herein by reference in their entirety. Any discussion of documents, bills, materials, devices, articles, etc., already included in this specification is for the purpose of providing context for this disclosure only. It should not be construed as an endorsement that any or all such matters existed prior to the priority date of the claims of this application and formed part of the prior art or common knowledge in the field relating to this disclosure.

[0208] References

[0209] Clark GM,et al.(1984)Ann Otol Rhinol Laryngol 93:204-207

[0210] Clark GM,et al.(1991)Ear and Hearing Suppl.12:15S-24S

[0211] Cui et al.(2015)ACS Nano 9:1571-1580

[0212] Jaworek(2007)Powder Technology 176(1),18-35

[0213] Langer R(1990)Science.249,1527-1533

[0214] Remington′s Pharmaceutical Sciences(Mack Publishing Co.N.J.USA,1991)

[0215] Tan et al.(2012)Adv.Mater.24:3362-3366

[0216] Wang et al.(2009)J.Mater.Chem.19:6451-6464

[0217] Wang et al.(2010)Chem Mater.22:3829-3831

[0218] Yang and Pierstorff(2012)JALA.17,50-58

Claims

1. A composition comprising superparticles, wherein the superparticles comprise mesoporous silica nanoparticles and at least one payload, wherein the payload is at least 4 μg of neurotrophic factor, and wherein the superparticles are manufactured by electrospraying a nanoparticle solution comprising nanoparticles and alginate into a dicationic aqueous solution.

2. The composition of claim 1, wherein the ultraparticles contain at least 5 μg of payload.

3. The composition of claim 1, wherein the ultraparticles comprise a payload of 1.5 μg to 10 μg.

4. The composition according to any one of claims 1 to 3, wherein the ultraparticles comprise pores with a diameter of at least 60 nm.

5. The composition according to any one of claims 1 to 3, wherein the ultraparticles comprise pores with a diameter of at least 50 nm.

6. The composition according to claim 1, wherein the ultraparticles have a disordered porous structure.

7. The composition according to claim 1, wherein the ultraparticles are composed of nanoparticles having a bimodal pore structure.

8. The composition of claim 7, wherein the nanoparticles have a bimodal pore structure and a large pore size greater than 30 nm.

9. The composition according to claim 1, wherein the neurotrophic factor has an isoelectric point between 8 and 10.

10. The composition according to claim 1, wherein the neurotrophic factor is BDNF.

11. The composition according to claim 1, wherein the neurotrophic factor is neurotrophic protein-3.

12. The composition of claim 1, wherein the superparticles comprise at least two different neurotrophic factor payloads.

13. The composition of claim 12, wherein the two different neurotrophic factor payloads are BDNF and neurotrophin-3.

14. The composition according to claim 1, wherein the composition comprises at least two superparticles.

15. The composition of claim 14, wherein the composition comprises superparticles, each having a different payload.

16. The composition of claim 1, wherein the composition is formulated for sustained-release ultraparticles.

17. The composition of claim 16, wherein the composition comprises glycogen hydrogel.

18. The composition according to claim 1, wherein the composition comprises alginate [(C6H8O6)] n ].

19. The composition according to claim 18, wherein the composition comprises sodium alginate [Na(C6H8O6)] n ].

20. Use of the composition according to any one of claims 1 to 19 in the manufacture of a medicament for treating a disease or condition.

21. The use as claimed in claim 20, wherein the condition is hearing loss.

22. The use as claimed in claim 21, wherein the hearing loss is characterized as sensorineural hearing loss (SNHL), age-related hearing loss, or noise-induced hearing loss.

23. Use of the composition of any one of claims 1 to 19 in the preparation of a medicament for promoting the survival of spiral ganglion neurons in the ear of a subject.

24. The use of claim 23, wherein the drug provides sustained delivery of a therapeutic payload.

25. The use as described in claim 23 or claim 24, wherein the composition is applied to the ear of the subject.

26. The use as described in claim 23 or claim 24, wherein the composition is administered intraperitoneally.

27. The use as described in claim 23 or claim 24, wherein the composition is applied in a scaffold that retains the ultraparticles.

28. Use of the composition according to any one of claims 1 to 19 in the preparation of a medicine box for treating hearing loss.

29. The use as described in claim 28, wherein the medicine box further comprises a cochlear implant.

30. Use of a composition comprising nanoparticles and alginate in the preparation of ultraparticles according to any one of claims 1-19.

31. The use as described in claim 30, wherein the alginate is sodium alginate.

32. A method for manufacturing ultraparticles according to any one of claims 1-19, the method comprising electrospraying a composition comprising nanoparticles and alginate or a polysaccharide derivative of said alginate into a dicationic aqueous solution.

33. The method of claim 32, further comprising subjecting the ultraparticles to calcination to remove alginic acid.

34. The method of claim 33, wherein the calcination is carried out at about 650°C.

35. The method of claim 33 or 34, wherein the calcination is carried out for about 6 to about 30 hours.

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