Methods and compositions related to the use of low HLB surfactants in the production of synthetic nanocarriers containing rapalogs

Abstract

To provide synthetic nanocarrier compositions comprising a hydrophobic carrier material in combination with a rapalog, such as rapamycin, which may exhibit improved initial sterile filterability.SOLUTION: The invention provides a composition comprising synthetic nanocarriers comprising: a hydrophobic carrier material; a rapalog; and a non-ionic surfactant with a hydrophilic-lipophilic balance (HLB) less than or equal to 10; where the amount of the non-ionic surfactant with an HLB less than or equal to 10 satisfies 0.01 wt.%≤(weight of the non-ionic surfactant with an HLB less than or equal to 10) / (weight of the hydrophobic carrier material)≤20 wt.%.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119 of U.S. Provisional Application Nos. 62 / 075,864 (filed November 5, 2014) and 62 / 075,866 (filed November 5, 2014), each of which is incorporated by reference herein in its entirety. [Background technology]

[0002] FIELD OF THE INVENTION The present invention relates to synthetic nanocarriers, and related compositions and methods, comprising a hydrophobic carrier material, a rapalog, and a nonionic surfactant with a hydrophilic-lipophilic balance (HLB) value of 10 or less. Summary of the Invention

[0003] SUMMARY OF THE INVENTION Provided herein are synthetic nanocarriers that can inhibit or reduce an immune response to an antigen, such as a co-administered antigen. The synthetic nanocarriers comprise a hydrophobic carrier material, a rapalog, and a non-ionic surfactant with a hydrophilic-lipophilic balance (HLB) value of 10 or less. It has surprisingly been discovered that the use of such non-ionic surfactants in formulations during synthetic nanocarrier formation can result in synthetic nanocarriers with improved stability and performance. In one aspect, a composition is provided that includes a synthetic nanocarrier comprising a hydrophobic carrier material, a rapalog, and a nonionic surfactant with a hydrophilic-lipophilic balance (HLB) value of 10 or less, wherein the amount of nonionic surfactant with an HLB value of 10 or less is ≧0.01 wt % but ≦20 wt % nonionic surfactant with an HLB value of 10 or less / hydrophobic carrier material.

[0004] In one embodiment of any one of the compositions or methods provided herein, the weights are the recipe weights of the ingredients to be combined during synthetic nanocarrier formulation. In one embodiment of any one of the compositions or methods provided herein, the weights are the weights of the ingredients in the resulting synthetic nanocarrier composition. In one embodiment of any one of the compositions or methods provided herein, the weights are the recipe weights of the ingredients to be combined during synthetic nanocarrier formulation. In one embodiment of any one of the compositions or methods provided herein, the weights are the weights of the ingredients in the resulting synthetic nanocarrier composition. In another aspect, a kit is provided that includes any one of the compositions provided herein.In one embodiment of any one of the provided kits, the composition is for use in any one of the methods provided herein.In one embodiment of any one of the provided kits, when the composition does not include an antigen, the kit further includes an antigen.In one embodiment of any one of the provided kits, the composition and the antigen are contained in separate containers.In one embodiment of any one of the provided kits, the composition and the antigen are contained in the same container.In one embodiment of any one of the provided kits, the kit further includes instructions for use in one embodiment of any one of the provided kits, and the instructions for use include a description of any one of the methods provided herein.

[0005] In another aspect, provided is a method comprising administering any one of the compositions provided herein to a subject. In one embodiment of any one of the provided methods, when the composition does not comprise an antigen, the method further comprises administering an antigen to the subject. In one embodiment of any one of the provided methods, the antigen is comprised in a different synthetic nanocarrier. In one embodiment of any one of the provided methods, the antigen is not coupled to any synthetic nanocarrier. In one embodiment of any one of the provided methods, the administering is by intradermal, intramuscular, intravenous, intraperitoneal, or subcutaneous administration.

[0006] In another aspect, there is provided a method for producing a synthetic nanocarrier comprising a nonionic surfactant with an HLB value of 10 or less and a rapalog, comprising obtaining or providing a hydrophobic carrier material, obtaining or providing a nonionic surfactant with an HLB value of 10 or less, obtaining or providing a rapalog, and combining the hydrophobic carrier material, the nonionic surfactant with an HLB value of 10 or less, and the rapalog to form the synthetic nanocarrier, wherein the amount of the nonionic surfactant with an HLB value of 10 or less in the synthetic nanocarrier is ≧0.01 wt % but ≦20 wt % nonionic surfactant with an HLB value of 10 or less / hydrophobic carrier material. In one embodiment of any one of the methods for producing provided herein, the method further comprises dissolving a hydrophobic carrier material, a nonionic surfactant having an HLB of 10 or less, and a rapalog in a solvent; obtaining or providing another surfactant; forming a first and then a second O / W emulsion with the dissolved hydrophobic carrier material, the nonionic surfactant having an HLB of 10 or less, and the rapalog, and the other surfactant; mixing the first and second O / W emulsions; and evaporating the solvent. In one embodiment of any one of the methods for producing provided herein, the solvent is dichloromethane, ethyl acetate, chloroform, or propylene carbonate. In one embodiment of any one of the methods for producing provided herein, the method further comprises filtering the resulting composition. In one embodiment of any one of the methods for producing provided herein, filtering comprises filtering through a 0.22 μm filter.

[0007] In another aspect, provided is a composition produced by any one of the methods for producing provided herein. The method for producing can be any one of the methods for producing provided, such as one of the methods shown in the examples. In one embodiment of any one of the compositions or methods provided herein, the synthetic nanocarrier composition is first sterile filterable through a 0.22 μm filter. In one embodiment of any one of the compositions or methods provided herein, the nonionic surfactant having an HLB of 10 or less is a nonionic surfactant having an HLB of less than 10. In one embodiment of any one of the compositions or methods provided herein, the nonionic surfactant having an HLB of 10 or less is a nonionic surfactant having an HLB of less than 9. In one embodiment of any one of the compositions or methods provided herein, the nonionic surfactant having an HLB of 10 or less is a nonionic surfactant having an HLB of less than 8. In one embodiment of any one of the compositions or methods provided herein, the nonionic surfactant having an HLB of 10 or less is a nonionic surfactant having an HLB of less than 7. In one embodiment of any one of the compositions or methods provided herein, the nonionic surfactant having an HLB of 10 or less is a nonionic surfactant having an HLB of less than 6. In one embodiment of any one of the compositions or methods provided herein, the nonionic surfactant having an HLB of 10 or less is a nonionic surfactant having an HLB of less than 5.

[0008] In one embodiment of any one of the compositions or methods provided herein, the nonionic surfactant having an HLB of 10 or less comprises a sorbitan ester, a fatty alcohol, a fatty acid ester, an ethoxylated fatty alcohol, a poloxamer, a fatty acid, cholesterol, a cholesterol derivative, or a bile acid or salt. In one embodiment of any one of the compositions or methods provided herein, the nonionic surfactant having an HLB of 10 or less comprises SPAN 40, SPAN 20, oleyl alcohol, stearyl alcohol, isopropyl palmitate, glycerol monostearate, BRIJ 52, BRIJ 93, Pluronic P-123, Pluronic L-31, palmitic acid, dodecanoic acid, glyceryl tripalmitate, or glyceryl trilinoleate. In one embodiment of any one of the compositions or methods provided herein, the nonionic surfactant having an HLB of 10 or less is SPAN 40.

[0009] In one embodiment of any one of the compositions or methods provided herein, the nonionic surfactant having an HLB of 10 or less is encapsulated in the synthetic nanocarrier, is present on the surface of the synthetic nanocarrier, or both. In one embodiment of any one of the compositions or methods provided herein, the amount of nonionic surfactant with an HLB of 10 or less is ≧0.1 but ≦15 wt.% nonionic surfactant with an HLB of 10 or less / hydrophobic carrier material. In one embodiment of any one of the compositions or methods provided herein, the amount of nonionic surfactant with an HLB of 10 or less is ≧1 but ≦13 wt.% nonionic surfactant with an HLB of 10 or less / hydrophobic carrier material. In one embodiment of any one of the compositions or methods provided herein, the amount of nonionic surfactant with an HLB of 10 or less is ≧1 but ≦9 wt.% nonionic surfactant with an HLB of 10 or less / hydrophobic carrier material.

[0010] In one embodiment of any one of the compositions or methods provided herein, the hydrophobic carrier material comprises one or more hydrophobic polymers or lipids. In one embodiment of any one of the compositions or methods provided herein, the hydrophobic carrier material comprises one or more hydrophobic polymers, wherein the one or more hydrophobic polymers comprise a polyester. In one embodiment of any one of the compositions or methods provided herein, the polyester comprises PLA, PLG, PLGA, or polycaprolactone. In one embodiment of any one of the compositions or methods provided herein, the hydrophobic carrier material comprises or further comprises PLA-PEG, PLGA-PEG, or PCL-PEG. In one embodiment of any one of the compositions or methods provided herein, the amount of hydrophobic carrier material in the synthetic nanocarrier is 5-95% by weight hydrophobic carrier material / total solids. In one embodiment of any one of the compositions or methods provided herein, the amount of hydrophobic carrier material in the synthetic nanocarrier is 60-95% by weight hydrophobic carrier material / total solids.

[0011] In one embodiment of any one of the compositions or methods provided herein, the amount of rapalog is ≧6 but ≦50% by weight rapalog / hydrophobic carrier material. In one embodiment of any one of the compositions or methods provided herein, the amount of rapalog is ≧7 but ≦30% by weight rapalog / hydrophobic carrier material. In one embodiment of any one of the compositions or methods provided herein, the amount of rapalog is ≧8 but ≦24% by weight rapalog / hydrophobic carrier material. In one embodiment of any one of the compositions or methods provided herein, the rapalog is encapsulated in a synthetic nanocarrier. In one embodiment of any one of the compositions or methods provided herein, the rapalog is rapamycin. In one embodiment of any one of the compositions or methods provided herein, the composition further comprises an antigen. In one embodiment of any one of the compositions or methods provided herein, the antigen is admixed with the synthetic nanocarrier in the composition.

[0012] In one embodiment of any one of the compositions or methods provided herein, the mean of the particle size distribution obtained using dynamic light scattering of the synthetic nanocarriers is greater than 120 nm in diameter. In one embodiment of any one of the compositions or methods provided herein, the diameter is greater than 150 nm. In one embodiment of any one of the compositions or methods provided herein, the diameter is greater than 200 nm. In one embodiment of any one of the compositions or methods provided herein, the diameter is greater than 250 nm. In one embodiment of any one of the compositions or methods provided herein, the diameter is less than 300 nm. In one embodiment of any one of the compositions or methods provided herein, the diameter is less than 250 nm. In one embodiment of any one of the compositions or methods provided herein, the diameter is less than 200 nm. In one embodiment of any one of the compositions or methods provided herein, the composition further comprises a pharmaceutically acceptable carrier.

[0013] In one embodiment of any one of the compositions or methods provided, the rapalog is rapamycin, the hydrophobic carrier material comprises PLA or PLGA and PLA-PEG, the non-ionic surfactant with an HLB of 10 or less is sorbitan monopalmitate, and the diameter of the synthetic nanocarriers is as provided in any one of the compositions provided herein, such as any one of the example compositions. In one embodiment of any one of the compositions or methods provided, the rapalog is rapamycin, the hydrophobic carrier material comprises PLA or PLGA and PLA-PEG, the non-ionic surfactant with an HLB of 10 or less is sorbitan monopalmitate, and the diameter of the synthetic nanocarriers and / or the wt % of rapamycin are as provided in any one of the compositions provided herein, such as any one of the example compositions. In one embodiment of any one of the compositions or methods provided, the rapalog is rapamycin, the hydrophobic carrier material comprises PLA or PLGA and PLA-PEG, the nonionic surfactant with an HLB of 10 or less is sorbitan monopalmitate, the diameter of the synthetic nanocarrier is as provided in any one of the compositions provided herein, such as any one of the example compositions, and / or the weight percent of rapamycin is as provided in any one of the compositions provided herein, such as any one of the example compositions, and / or the amount of polymer(s) is as provided in any one of the compositions provided herein, such as any one of the example compositions.In one embodiment of any one of the compositions or methods provided, the rapalog is rapamycin, the hydrophobic carrier material comprises PLA or PLGA and PLA-PEG, the nonionic surfactant with an HLB of 10 or less is sorbitan monopalmitate, the diameter of the synthetic nanocarrier is as provided in any one of the compositions provided herein, such as any one of the example compositions, and / or the weight percent of rapamycin is as provided in any one of the compositions provided herein, such as any one of the example compositions, and / or the amount of polymer(s) is as provided in any one of the compositions provided herein, such as any one of the example compositions, and / or the amount of sorbitan monopalmitate is as provided in any one of the compositions provided herein, such as any one of the example compositions.

[0014] In another aspect, any one of the compositions provided herein, such as any one of the example compositions, is provided. In another aspect, a method for producing any one of the compositions or kits provided herein is provided. In one embodiment, the method for producing includes any one of the steps of the methods provided herein, such as any one of the steps of the methods provided in the examples. In another aspect, there is provided a use of any one of the compositions or kits provided herein for the manufacture of a medicament for promoting immune tolerance in a subject. In another embodiment of any one of the uses provided herein, the use is for achieving any one of the methods provided herein. In another aspect, any one of the compositions or kits provided herein can be for use in any one of the methods provided herein. In another aspect, a method for manufacturing a medicament intended to promote immune tolerance is provided. In one embodiment, the medicament comprises any one of the compositions provided herein. [Brief explanation of the drawings]

[0015] Brief description of the drawings [Figure 1] Figure 1 shows results demonstrating the tolerance-inducing ability of nanocarriers and KLH (keyhole limpet hemocyanin) co-administered with rapamycin (RAPA). Mouse serum was analyzed for antibodies to KLH after each KLH challenge. [Figure 2] Figure 2 shows results demonstrating sustained antibody titer reduction by nanocarriers with low HLB surfactants. The acronym "tSIP" refers to the nanocarriers as described. [Figure 3] Figure 3 shows results demonstrating the efficacy of synthetic nanocarriers + KLH compared to free rapamycin + KLH in mice. Anti-KLH EC50 (symbols represent geometric mean ± 95% CI) in antibody titers on days 35 and 42 (after two or three KLH-only challenges) for mice treated or not with synthetic nanocarriers + KLH. The acronym "NC" refers to the nanocarrier as described. [Figure 4] Figure 4 shows the treatment protocol for Example 7. The acronym "NC" refers to nanocarriers as described. [Figure 5] Figure 5 shows the antigen specificity of synthetic nanocarriers + KLH in mice. Anti-OVA EC50 in antibody titers on day 65 for mice treated or not with synthetic nanocarriers + KLH (bars represent geometric mean ± 95% CI). The acronym "NC" refers to the nanocarrier as described.

[0016] Detailed Description of the Invention Before describing the present invention in detail, it is to be understood that this invention is not limited to specifically exemplified materials or process parameters, as these may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to limit the use of alternative terminology to describe the invention. All publications, patents, and patent applications cited herein, whether before or after the event, are hereby incorporated by reference in their entirety for all purposes. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. For example, a reference to a "polymer" includes a mixture of two or more such molecules or a mixture of a single polymer species of different molecular weights; a reference to a "synthetic nanocarrier" includes a mixture of two or more such synthetic nanocarriers or a plurality of such synthetic nanocarriers, etc.

[0017] As used herein, the term "comprise" or variations thereof, such as "comprises" or "comprising," should be read to indicate the inclusion of any recited integer (e.g., a feature, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., features, elements, characteristics, properties, method / process steps, or limitations), but not the exclusion of any other integer or group of integers. Thus, as used herein, the term "comprising" is inclusive and does not exclude additional, unrecited integers or method / process steps. In any one embodiment of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of." The phrase "consisting essentially of" is used herein to require the specified integer(s) or steps, as well as those that do not materially affect the characteristics or functionality of the claimed invention. As used herein, the term "consisting of" is used to indicate the presence of the enumerated integer (e.g., feature, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., feature, element, characteristic, property, method / process step, or limitation) alone.

[0018] A. Introduction It has been found that synthetic nanocarriers containing hydrophobic carrier materials combined with rapalogs such as rapamycin can be initially difficult to sterile filter through a 0.22 μm filter. The use of such filters in the production of synthetic nanocarriers is important because the filters can remove bacteria to a desired level, resulting in a more sterile composition, a beneficial feature for compositions used for in vivo administration. Surprisingly, due to the nature of the two hydrophobic components of such synthetic nanocarriers, it has been discovered that incorporating a nonionic surfactant such as sorbitan monopalmitate into the synthetic nanocarrier can result in synthetic nanocarriers with improved filterability, and in some embodiments, improved rapalog loading and therefore efficacy when administered to a subject. Consequently, it has been found that the use of a nonionic surfactant with a hydrophilic-lipophilic balance (HLB) of 10 or less can result in more effective loading of the rapalog (preferably a hydrophobic one), while also improving initial sterile filterability.

[0019] As shown in the examples, many such surfactants increased the throughput of synthetic nanocarrier formulations when first passed through a 0.22 μm filter. It has also been found that synthetic nanocarriers with such surfactants can provide higher loading of rapalogs, such as hydrophobic rapalogs, and provide sustained antigen-specific tolerance in subjects. While the use of surfactants with a higher hydrophilic-lipophilic balance (HLB) is typical for suspending or dissolving hydrophobic agents in solvents and introducing such agents into the oil-soluble phase during nanocarrier formulation, it may not support optimized production of highly loaded hydrophobic rapalogs, such as hydrophobic rapalogs, in nanocarriers. Instead, nonionic surfactants such as sorbitan monopalmitate (SPAN 40) (HLB 6.7) with an HLB of ≦10 represent a surfactant class that aids optimization.

[0020] Accordingly, provided are synthetic nanocarriers, compositions thereof, and related methods comprising a hydrophobic carrier material, a rapalog (preferably hydrophobic), and a non-ionic surfactant with a hydrophilic-lipophilic balance (HLB) of 10 or less. Such compositions may exhibit improved initial sterile filtration capabilities, and in some embodiments, improved loading and performance of rapalogs, such as hydrophobic rapalogs, and may be used in methods for reducing unwanted antigen-specific immune system activation and / or promoting antigen-specific tolerance. The present invention will now be described in more detail below.

[0021] B. Definition "Administering" or "administration" or "administer" means providing a material to a subject in a pharmacologically useful manner. The term is intended to include administering in some embodiments. "Causing to be administered" means directly or indirectly causing, encouraging, facilitating, assisting, inducing, or directing another party to administer a material. "Mixed" refers to mixing one component, such as an antigen, with another, such as a synthetic nanocarrier, in a composition. The components to be mixed are made or obtained separately and placed together. As a result, the components are not coupled to each other except for possible non-covalent interactions that may occur when placed together in a composition. An "effective amount" in the context of a composition or dose for administration to a subject refers to the amount of the composition or dose that induces one or more desired responses in the subject, such as the generation of an antigen-specific tolerogenic immune response. In some embodiments, an effective amount is a pharmacodynamically effective amount. Thus, in some embodiments, an effective amount is the amount of any of the compositions or doses provided herein that induces one or more desired therapeutic effects and / or immune responses as provided herein. This amount may be for in vitro or in vivo purposes. For in vivo purposes, the amount may be one that a clinician would consider may have a clinical benefit for a subject in need of antigen-specific immune tolerance. Any one of the compositions as provided herein may be an effective amount.

[0022] An effective amount can involve reducing the level of an unwanted immune response, although in some embodiments, it involves preventing an unwanted immune response altogether. An effective amount can also involve delaying the onset of an unwanted immune response. An effective amount can also be an amount that causes a desired therapeutic endpoint or a desired therapeutic result. In other embodiments, an effective amount can involve enhancing the level of a desired response, such as a therapeutic endpoint or result. An effective amount preferably results in a tolerogenic immune response in the subject against the antigen. Achievement of any of the foregoing can be monitored by routine methods. The effective amount will naturally depend on the particular subject being treated; the severity of the symptom, disease, or disorder; individual patient parameters, including age, health, size, and weight; the duration of treatment; the nature of concurrent treatment (if any); the specific route of administration, and similar factors within the knowledge and skill of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. The use of the maximum dose, that is, the maximally safe dose according to sound medical judgment, is generally preferred. However, it will be understood by those skilled in the art that a patient may require a lower or tolerated dose for medical reasons, psychological reasons, or indeed any other reason.

[0023] Generally, the dose of a component in a composition of the present invention refers to the amount of the component. Alternatively, the dose can be administered based on the number of synthetic nanocarriers that provide the desired amount. "Antigen" refers to a B cell antigen or a T cell antigen. "Antigen type(s)" refers to molecules that share the same or substantially the same antigenic characteristics. In some embodiments, the antigen may be a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, polysaccharide, or contained or expressed in a cell. In some embodiments, when the antigen is not well defined or characterized, etc., the antigen may be contained in a cell or tissue preparation, cell debris, cell exosomes, conditioned medium, etc. "Antigen-specific" refers to any immune response that is due to the presence of an antigen or a portion thereof, or that produces a molecule that specifically recognizes or binds to the antigen. For example, if the immune response is antigen-specific antibody production, antibodies that specifically bind to the antigen are produced. As another example, if the immune response is the proliferation and / or activity of antigen-specific B cells or CD4+ T cells, the proliferation and / or activity is due to recognition of the antigen or a portion thereof, alone or in complex with an MHC molecule, B cell, etc.

[0024] "Average," as used herein, refers to the arithmetic mean unless otherwise specified. "Encapsulating" means to surround at least a portion of a substance within a synthetic nanocarrier. In some embodiments, the substance is completely enclosed within the synthetic nanocarrier. In other embodiments, most or all of the encapsulated substance is not exposed to the local environment outside the synthetic nanocarrier. In other embodiments, only 50%, 40%, 30%, 20%, 10%, or 5% (wt / wt) is exposed to the local environment. Encapsulation differs from absorption, which places most or all of the substance on the surface of the synthetic nanocarrier, leaving the substance exposed to the local environment outside the synthetic nanocarrier. In any one embodiment of the compositions or methods provided herein, a rapalog and / or a nonionic surfactant with a hydrophilic-lipophilic balance (HLB) value of 10 or less is encapsulated within a synthetic nanocarrier.

[0025] "Hydrophobic carrier material" refers to any pharmaceutically acceptable carrier capable of delivering one or more molecules (e.g., a rapalog and a nonionic surfactant with an HLB of 10 or less) and having hydrophobic characteristics. Such materials include materials that can form, or be part of, a synthetic nanocarrier and can contain or be loaded with one or more molecules (e.g., a rapalog and a nonionic surfactant with an HLB of 10 or less). Generally, the carrier material enables delivery of one or more molecules (e.g., a rapalog and a nonionic surfactant with an HLB of 10 or less) to a target site or target cell, controlled release of the one or more molecules, and other desired activities. Examples of hydrophobic carrier materials that can be used to form synthetic nanocarriers include, without limitation, materials containing hydrophobic components, such as hydrophobic polymers such as polyesters or lipids. "Hydrophobic" refers to a material that does not substantially participate in hydrogen bonding to water. Such materials are generally nonpolar, primarily nonpolar, or neutral in charge. Carrier materials suitable for the compositions described herein can be selected based on their hydrophobicity on several levels. Thus, a hydrophobic carrier material is one that is hydrophobic overall and can consist entirely of hydrophobic components, such as hydrophobic polymers or lipids, although in some embodiments, the hydrophobic carrier material is hydrophobic overall and includes hydrophobic components, such as hydrophobic polymers or lipids, in combination with non-hydrophobic components.

[0026] "Initially sterile filterable" means that the nanocarrier has not previously been filtered through a filter, such as a 0.22 μm filter, but is still sterile filtered at a rate of at least 50 grams of nanocarriers / m² of filter membrane surface area. 2"Throughput" refers to a composition of synthetic nanocarriers that can be filtered at a throughput rate of 10 mL. In some embodiments of any one of the compositions or methods provided herein, the throughput rate is determined by taking a 9 mL volume of synthetic nanocarrier suspension and placing it into a 10 mL syringe with any one of the filters as provided herein. The synthetic nanocarrier suspension is then pushed through the filter until no further suspended material passes through the filter. The throughput rate can then be calculated based on the material pushed through the filter and the suspended material remaining in the syringe. In some embodiments of any one of the compositions or methods provided herein, the initially sterile-filterable composition is non-sterile and / or not suitable for in vivo administration (i.e., not substantially pure and contains soluble components that are never desirable for in vivo administration). In other embodiments of any one of the compositions or methods provided herein, the initially sterile-filterable composition comprises synthetic nanocarriers that have been produced but have not been further processed to produce a medical-grade material. In some embodiments of any one of the compositions or methods provided herein, the initially sterile filterable composition has not been previously filtered through a filter, such as a 0.22 μm filter, but has at least 60, 70, 80, 90, 100, 120, 130, 140, 160, 200, 250, 300, 350, 500, 750, 1000, or 1500 grams of nanocarriers / m of filter membrane surface area. 2 The 0.22 μm filter can be any filter with a pore size of 0.22 μm. Such filters can be made from a variety of materials, such as polyethylene sulfone, polyvinylidene fluoride, mixed cellulose esters, solvent-free cellulose acetate, regenerated cellulose, nylon, etc. Specific examples of filters include Millipore SLGPM33R, Millipore SLGVM33RS, Millipore SLGSM33SS, Sartorius 16534, Sartorius 17764, Sartorius 17845, etc.

[0027] "Maximum dimension of a synthetic nanocarrier" refers to the largest dimension of a nanocarrier measured along any axis of the synthetic nanocarrier. "Minimum dimension of a synthetic nanocarrier" refers to the smallest dimension of a synthetic nanocarrier measured along any axis of the synthetic nanocarrier. For example, for a spherical synthetic nanocarrier, the largest and smallest dimensions of the synthetic nanocarrier will be substantially the same and will be the size of its diameter. Similarly, for a cubic synthetic nanocarrier, the smallest dimension of a synthetic nanocarrier will be the smallest of its height, width, or length, while the largest dimension of the synthetic nanocarrier will be the largest of its height, width, or length. In embodiments, based on the total number of synthetic nanocarriers in the sample, the smallest dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample is 100 nm or greater. In embodiments, based on the total number of synthetic nanocarriers in the sample, the largest dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample is 5 μm or less. Preferably, based on the total number of synthetic nanocarriers in a sample, the minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample is greater than 110 nm, more preferably greater than 120 nm, more preferably greater than 130 nm, and even more preferably greater than 150 nm. The aspect ratio of the maximum and minimum dimensions of synthetic nanocarriers can vary depending on the embodiment. For example, the aspect ratio of the maximum dimension:minimum dimension of synthetic nanocarriers can vary from 1:1 to 1,000,000:1, preferably from 1:1 to 100,000:1, more preferably from 1:1 to 10,000:1, more preferably from 1:1 to 1000:1, even more preferably from 1:1 to 100:1, and even more preferably from 1:1 to 10:1.

[0028] Preferably, based on the total number of synthetic nanocarriers in a sample, at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample have a maximum dimension of 3 μm or less, more preferably 2 μm or less, more preferably 1 μm or less, more preferably 800 nm or less, more preferably 600 nm or less, and even more preferably 500 nm or less. In preferred embodiments, based on the total number of synthetic nanocarriers in a sample, at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample have a minimum dimension of 100 nm or more, more preferably 120 nm or more, more preferably 130 nm or more, more preferably 140 nm or more, and even more preferably 150 nm or more. Measurements of synthetic nanocarrier dimensions (e.g., effective diameters) may, in some embodiments, be obtained by suspending synthetic nanocarriers in a liquid (usually aqueous) medium and using dynamic light scattering (DLS) (e.g., using a Brookhaven ZetaPALS instrument). For example, a synthetic nanocarrier suspension can be diluted from an aqueous buffer into purified water to achieve a final concentration of approximately 0.01-0.5 mg / mL of the synthetic nanocarrier suspension. The diluted suspension can be prepared directly in or transferred to a cuvette suitable for DLS analysis. The cuvette can then be placed in the DLS, allowed to equilibrate to a controlled temperature, and then scanned for a time sufficient to obtain a stable, reproducible distribution based on appropriate inputs for the viscosity of the medium and the refractive index of the sample. The effective diameter or mean of the distribution is then reported. Determining the effective size of high aspect ratio or non-spherical synthetic nanocarriers may require enhancement techniques, such as electron microscopy, to obtain more accurate measurements. The "dimension" or "size" or "diameter" of a synthetic nanocarrier refers to the mean of the particle size distribution obtained, for example, using dynamic light scattering.

[0029] As used herein, a "nonionic surfactant having an HLB value of 10 or less" or a "low HLB surfactant" refers to a nonionic, amphiphilic molecule having a structure comprising at least one hydrophobic tail and a hydrophilic head, or having a hydrophobic group or region and a hydrophilic group or region. The tail portion of a surfactant generally consists of a hydrocarbon chain. Surfactants can be classified based on the charge characteristics of the hydrophilic head portion or group or region. As used herein, "HLB" refers to the hydrophilic-lipophilic balance or hydrophile-lipophile balance of a surfactant and is a measure of the hydrophilic or lipophilic nature of the surfactant.

[0030] The HLB of any one of the surfactants provided herein may be calculated using Griffin's method or Davie's method. For example, using Griffin's method, the HLB of a surfactant is the product of the molecular mass of the hydrophilic portion of the surfactant divided by the molecular mass of the entire surfactant, multiplied by 20. The HLB value is a scale from 0 to 20, where 0 corresponds to a completely hydrophobic (lipophilic) molecule, and 20 corresponds to a completely hydrophilic (lipophobic) molecule. In some embodiments, the HLB of any one of the surfactants of the compositions or methods provided herein is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (for example, as determined by Griffin's or Davie's method). Examples of such surfactants for use in any one of the compositions and methods provided herein include, without limitation, sorbitan esters such as SPAN 40 and SPAN 20; fatty alcohols such as oleyl alcohol and stearyl alcohol; fatty acid esters such as isopropyl palmitate and glycerol monostearate; ethoxylated fatty alcohols such as BRIJ 52 and BRIJ 93; poloxamers such as Pluronic P-123 and Pluronic L-31; fatty acids such as palmitic acid and dodecanoic acid; triglycerides such as glyceryl tripalmitate and glyceryl trilinoleate; cholesterol; cholesterol derivatives such as sodium cholesteryl sulfate and cholesteryl dodecanoate; and bile salts or acids such as lithocholic acid and sodium lithocholate.Further examples of such surfactants are sorbitan monostearate (SPAN 60), sorbitan tristearate (SPAN 65), sorbitan monooleate (SPAN 80), sorbitan sesquioleate (SPAN 83), sorbitan trioleate (SPAN 85), sorbitan sesquioleate (Arlacel 83), sorbitan dipalmitate, mono- and diglycerides of fatty acids, polyoxyethylene sorbitan trioleate (Tween 85), polyoxyethylene sorbitan hexaoleate (G 1086), sorbitan monoisostearate (Montane 70), polyoxyethylene alcohols, polyoxyethylene glycol alkyl ethers, polyoxyethylene (2) oleyl ether (BRIJ 93), polyoxyethylene cetyl ether (BRIJ 52), polyethylene glycol dodecyl ether (BRIJ 53), polyoxyethylene glycol alkyl ether (BRIJ 54), polyoxyethylene glycol alkyl ether (BRIJ 55), polyoxyethylene glycol alkyl ether (BRIJ 56), polyoxyethylene glycol alkyl ether (BRIJ 57), polyoxyethylene glycol alkyl ether (BRIJ 58), polyoxyethylene glycol alkyl ether (BRIJ 59 ... L4); 1-monotetradecanoyl-rac-glycerol; glyceryl monostearate; glycerol monopalmitate; ethylenediaminetetradecanoyltetraol (Tetronic 90R4, Tetronic 701), polyoxyethylene (5) nonylphenyl ether (IGEPAL CA-520), MERPOL A surfactant, MERPOL SE surfactant, and poly(ethylene glycol) sorbitol hexaoleate. Further examples will also be apparent to those skilled in the art.

[0031] "Pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to a pharmacologically inactive material used together with a pharmacologically active material to formulate a composition. Pharmaceutically acceptable excipients include various materials known in the art, including, but not limited to, sugars (e.g., glucose, lactose, etc.), preservatives such as antibacterial agents, reconstitution aids, coloring agents, saline (e.g., phosphate-buffered saline), and buffers. "Providing" means an activity or set of activities performed by an individual that supplies a needed item or set of items or method for the practice of the invention. The activity or set of activities may be undertaken either directly or indirectly by oneself. "Rapalog" refers to rapamycin and molecules (analogs) structurally related to rapamycin (sirolimus), preferably hydrophobic. Examples of rapalogs include, but are not limited to, temsirolimus (CCI-779), deforolimus, everolimus (RAD001), ridaforolimus (AP-23573), and zotarolimus (ABT-578). Additional examples of rapalogs can be found, for example, in WO Publication WO 1998 / 002441 and U.S. Patent No. 8,455,510, the disclosures of which are incorporated herein by reference in their entireties.

[0032] "Solvent" refers to a substance capable of dissolving a solute, such as any one or more of the components of a synthetic nanocarrier as provided herein. In some embodiments, the solvent is useful for forming synthetic nanocarriers, such as in an emulsion process (e.g., a double emulsion process). Examples of such solvents include dichloromethane, ethyl acetate, chloroform, and propylene carbonate. Examples also include solvent mixtures that are combinations of poorly water-soluble organic solvents with water-miscible solvents, such as acetone, ethanol, dimethyl sulfoxide, dimethylformamide, formamide, and the like. Further examples will be known to those skilled in the art. "Subject" means animals, including warm-blooded mammals such as humans and primates; birds; domestic or farm animals such as cats, dogs, sheep, goats, cows, horses, and pigs; laboratory animals such as mice, rats, and guinea pigs; fish; reptiles; zoo and wild animals; and the like.

[0033] "Surfactant" refers to a compound that can reduce the surface tension between two liquids or between a liquid and a solid. Surfactants may act as detergents, wetting agents, emulsifiers, foaming agents, and dispersants, and can be used in the formation of synthetic nanocarriers as provided herein. In some embodiments, the surfactant is a nonionic surfactant with an HLB value of 10 or less. "Synthetic nanocarrier(s)" means a discrete object not found in nature, possessing at least one dimension that is 5 microns or less in size. As provided herein, synthetic nanocarriers comprise hydrophobic carrier materials. Consequently, synthetic nanocarriers can be synthetic nanocarriers including, but not limited to, nanoparticles of hydrophobic polymers as well as lipid-based nanoparticles. Synthetic nanocarriers can be a variety of different shapes, including, but not limited to, spherical, cubic, conical, rectangular (ellipsoidal), cylindrical, donut-shaped, and the like. Synthetic nanocarriers in accordance with the present invention comprise one or more surfaces. In embodiments, synthetic nanocarriers may possess an aspect ratio of greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.

[0034] Synthetic nanocarriers in accordance with the invention having minimum dimensions of about 100 nm or less, preferably 100 nm or less, do not comprise surfaces with complement-activating hydroxyl groups, or alternatively comprise surfaces consisting essentially of moieties that are not complement-activating hydroxyl groups. In preferred embodiments, synthetic nanocarriers in accordance with the invention having minimum dimensions of about 100 nm or less, preferably 100 nm or less, do not comprise surfaces that substantially activate complement, or alternatively comprise surfaces consisting essentially of moieties that do not substantially activate complement. In more preferred embodiments, synthetic nanocarriers in accordance with the invention having minimum dimensions of about 100 nm or less, preferably 100 nm or less, do not comprise surfaces that substantially activate complement, or alternatively comprise surfaces consisting essentially of moieties that do not activate complement.

[0035] "Total solids" refers to the total weight of all components contained in a synthetic nanocarrier composition or suspension. In some embodiments of any one of the compositions or methods provided herein, the amount of total solids is determined as the total mass of dry nanocarriers per mL of suspension. This can be determined by gravimetric methods. "Weight %" refers to a 100-fold ratio of one weight to another. For example, weight % can be a 100-fold ratio of the weight of one component to another, or a 100-fold ratio of one component to the total weight of more than one component. Generally, weight % is measured as an average across a population of synthetic nanocarriers, or across the synthetic nanocarriers in a composition or suspension.

[0036] C. Compositions and Related Methods Provided herein are synthetic nanocarrier compositions with improved initial sterile filterability and, in some embodiments, improved rapalog loading and, therefore, efficacy when administered to a subject. Such synthetic nanocarriers comprise a hydrophobic carrier material and a rapalog, which is preferably hydrophobic. Surprisingly, it has been discovered that a certain class of surfactants, nonionic surfactants with a hydrophilic-lipophilic balance (HLB) of 10 or less, can provide improved initial sterile filterability and, in some embodiments, more effective loading of the rapalog in the synthetic nanocarriers. As shown in the examples, increased throughput of synthetic nanocarrier compositions when first passed through a 0.22 μm filter was found when a surfactant such as SPAN 40 was incorporated in the synthetic nanocarrier composition. As also shown in the examples, a number of such synthetic nanocarriers formulated with a nonionic surfactant with a hydrophilic-lipophilic balance (HLB) of 10 or less was also able to provide sustained antigen-specific tolerance in subjects.

[0037] Optimized amounts of nonionic surfactants with HLB values ​​of 10 or less in synthetic nanocarriers as provided herein have also been discovered. In some embodiments of any one of the compositions or methods provided herein, the amount of nonionic surfactants with HLB values ​​of 10 or less in the synthetic nanocarriers is ≧0.01 but ≦20 wt% (nonionic surfactant with HLB value of 10 or less / hydrophobic carrier material). In some embodiments of any one of the compositions or methods provided herein, the amount of nonionic surfactants with HLB values ​​of 10 or less in the synthetic nanocarriers is ≧0.1 but ≦15 wt%, ≧0.5 but ≦13 wt%, or ≧1 but ≦9 or 10 wt% (nonionic surfactant with HLB value of 10 or less / hydrophobic carrier material). In other embodiments of any one of the compositions or methods provided herein, the amount of nonionic surfactant with an HLB value of 10 or less in the synthetic nanocarrier is ≧0.01 but ≦17 wt%, ≧0.01 but ≦15 wt%, ≧0.01 but ≦13 wt%, ≧0.01 but ≦12 wt%, ≧0.01 but ≦11 wt%, ≧0.01 but ≦10 wt%, ≧0.01 but ≦9 wt%, ≧0.01 but ≦8 wt%, ≧0.01 but ≦7 wt%, ≧0.01 but ≦6 wt%, ≧0.01 but ≦5 wt% (nonionic surfactant with an HLB value of 10 or less / hydrophobic carrier material), etc. In still other embodiments of any one of the compositions or methods provided herein, the amount of nonionic surfactant with an HLB value of 10 or less in the synthetic nanocarrier is ≧0.1 but ≦15 wt%, ≧0.1 but ≦14 wt%, ≧0.1 but ≦13 wt%, ≧0.1 but ≦12 wt%, ≧0.1 but ≦11 wt%, ≧0.1 but ≦10 wt%, ≧0.1 but ≦9 wt%, ≧0.1 but ≦8 wt%, ≧0.1 but ≦7 wt%, ≧0.1 but ≦6 wt%, ≧0.1 but ≦5 wt% (nonionic surfactant with an HLB value of 10 or less / hydrophobic carrier material), etc.In still other embodiments of any one of the compositions or methods provided herein, the amount of nonionic surfactant with an HLB value of 10 or less in the synthetic nanocarrier is ≧0.5 but ≦15 wt%, ≧0.5 but ≦14 wt%, ≧0.5 but ≦13 wt%, ≧0.5 but ≦12 wt%, ≧0.5 but ≦11 wt%, ≧0.5 but ≦10 wt%, ≧0.5 but ≦9 wt%, ≧0.5 but ≦8 wt%, ≧0.5 but ≦7 wt%, ≧0.5 but ≦6 wt%, ≧0.5 but ≦5 wt% (nonionic surfactant with an HLB value of 10 or less / hydrophobic carrier material), etc. In still other embodiments of any one of the compositions or methods provided herein, the amount of nonionic surfactant with an HLB value of 10 or less in the synthetic nanocarrier is ≧1 but ≦9 wt%, ≧1 but ≦8 wt%, ≧1 but ≦7 wt%, ≧1 but ≦6 wt%, ≧1 but ≦5 wt% (nonionic surfactant with an HLB value of 10 or less / hydrophobic carrier material), etc. In still other embodiments of any one of the compositions or methods provided herein, the amount of nonionic surfactant with an HLB value of 10 or less in the synthetic nanocarrier is ≧5 but ≦15 wt%, ≧5 but ≦14 wt%, ≧5 but ≦13 wt%, ≧5 but ≦12 wt%, ≧5 but ≦11 wt%, ≧5 but ≦10 wt%, ≧5 but ≦9 wt%, ≧5 but ≦8 wt%, ≧5 but ≦7 wt%, ≧5 but ≦6 wt% (nonionic surfactant with an HLB value of 10 or less / hydrophobic carrier material), etc. In some embodiments of any one of the compositions or methods provided herein, the amount of nonionic surfactant with an HLB value of 10 or less in the synthetic nanocarrier is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 wt. % (nonionic surfactant with an HLB value of 10 or less / hydrophobic carrier material). Any one of the HLB values ​​provided herein may be determined using the Griffin or Davie method.

[0038] The optimized amount of hydrophobic carrier material in the synthetic nanocarrier composition was also determined. Preferably, in some embodiments of any one of the compositions or methods provided herein, the amount of hydrophobic carrier material in the synthetic nanocarrier composition is 5 to 95 wt% (hydrophobic carrier material / total solids). In other embodiments of any one of the compositions or methods provided herein, the amount of hydrophobic carrier material in the synthetic nanocarrier is 10 to 95, 15 to 90, 20 to 90, 25 to 90, 30 to 80, 30 to 70, 30 to 60, 30 to 50 wt% (hydrophobic carrier material / total solids), etc. In still other embodiments of any one of the compositions or methods provided herein, the amount of hydrophobic carrier material in the synthetic nanocarrier is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 wt% (hydrophobic carrier material / total solids).

[0039] Furthermore, the amount of rapalog, such as rapamycin, in the synthetic nanocarrier can also be optimized. Preferably, such an amount can result in an effective result (e.g., sustained antigen-specific tolerance) when the synthetic nanocarrier is administered to a subject. In some embodiments of any one of the compositions or methods provided herein, the synthetic nanocarrier comprises ≧6 but ≦50% by weight of rapalog / hydrophobic carrier material. In some embodiments of any one of the compositions or methods provided herein, the synthetic nanocarrier comprises ≧6 but ≦45% by weight, ≧6 but ≦40% by weight, ≧6 but ≦35% by weight, ≧6 but ≦30% by weight, ≧6 but ≦25% by weight, ≧6 but ≦20% by weight, or ≧6 but ≦15% by weight of rapalog / hydrophobic carrier material. In other embodiments of any one of the compositions or methods provided herein, the synthetic nanocarrier comprises ≧7 but ≦45% by weight, ≧7 but ≦40% by weight, ≧7 but ≦35% by weight, ≧7 but ≦30% by weight, ≧7 but ≦25% by weight, ≧7 but ≦20% by weight, or ≧7 but ≦15% by weight of rapalog / hydrophobic carrier material. In still other embodiments of any one of the compositions or methods provided herein, the synthetic nanocarrier comprises ≧8 but ≦24% by weight of rapalog / hydrophobic carrier material. In some embodiments of any one of the compositions or methods provided herein, the synthetic nanocarrier comprises 6, 7, 8, 9, 10, 12, 15, 17, 20, 22, 25, 27, 30, 35, 45, or more by weight of rapalog / hydrophobic carrier material.

[0040] The amount of a component or material as listed herein for any one of the compositions or methods provided herein can be determined using methods known to those of skill in the art or otherwise provided herein. For example, the amount of a nonionic surfactant with an HLB of 10 or less can be measured by extraction followed by quantification using an HPLC method. The amount of a hydrophobic carrier material can be determined using HPLC. Such determination of the amount can, in some embodiments, be followed by the use of proton NMR or other orthogonal methods, such as MALDI-MS, to determine the identity of the hydrophobic carrier material. Similar methods can be used to determine the amount of a rapalog in any one of the compositions provided herein. In some embodiments, the amount of the rapalog is determined using HPLC. Further examples of methods for determining the amount of a component or material are provided elsewhere herein, such as in the examples. For any one of the compositions or methods provided herein, the amount of a component or material can also be determined based on the formulated weight of the nanocarrier formulation. Accordingly, in some embodiments of any one of the compositions or methods provided herein, the amount of any one of the components provided herein is that of the component in the aqueous phase during formulation of the synthetic nanocarrier. In some embodiments of any one of the compositions or methods provided herein, the amount of any one of the components is that of the component in the produced synthetic nanocarrier composition and the result of the formulation process.

[0041] Synthetic nanocarriers as provided herein comprise a hydrophobic carrier material, such as a hydrophobic polymer or lipid. Thus, in some embodiments, synthetic nanocarriers provided herein comprise one or more lipids. In some embodiments, synthetic nanocarriers may comprise a lipid bilayer. In some embodiments, synthetic nanocarriers may comprise a lipid monolayer. In some embodiments, synthetic nanocarriers may comprise a core comprising a polymer matrix surrounded by a lipid layer (e.g., a lipid bilayer, a lipid monolayer, etc.). Further hydrophobic carrier materials include lipids (synthetic and natural), lipid-polymer conjugates, lipid-protein conjugates, and crosslinkable oils, waxes, lipids, etc. Further examples of lipid materials for use as hydrophobic carrier materials as provided herein can be found, for example, in PCT Publication Nos. WO2000 / 006120 and WO2013 / 056132, the disclosures of such materials being incorporated herein by reference in their entireties.

[0042] Accordingly, in some embodiments, the synthetic nanocarriers provided herein can be liposomes. Liposomes can be prepared using methods such as those described by Kim et al. (1983, Biochim. Biophys. Acta 728, 339-348); Liu et al. (1992, Biochim. Biophys. Acta 1104, 95-101); Lee et al. (1992, Biochim. Biophys. Acta 1103, 185-197), Brey et al. (US Patent Application Publication No. 20020041861), Hass et al. (US Patent Application Publication No. 20050232984), Kisak et al. (US Patent Application Publication No. 20050260260), and Smyth-Templeton et al. Such liposomes can be produced by standard methods such as those reported by U.S. Patent Application Publication No. 20060204566, the disclosures of which are incorporated herein by reference in their entirety. Hydrophobic carrier materials as provided herein include one or more hydrophobic polymers or units thereof. However, in some embodiments, while the hydrophobic carrier material is hydrophobic overall, the hydrophobic carrier material may also include polymers or units thereof that are not hydrophobic.

[0043] Accordingly, the hydrophobic carrier material as provided herein can include polyesters such as hydrophobic polyesters. Polyesters include copolymers containing lactic acid and glycolic acid units, such as poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide), collectively referred to herein as "PLGA"; and homopolymers containing glycolic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as "PLA". In some embodiments, exemplary polyesters include, for example, polyhydroxy acids; PEG copolymers, and copolymers of lactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers, PLGA-PEG copolymers, and derivatives thereof). In some embodiments, polyesters include, for example, poly(caprolactone), poly(caprolactone)-PEG copolymer, poly(L-lactide-co-L-lysine), poly(serine ester), poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

[0044] In some embodiments, the polymer of the hydrophobic carrier material may be PLGA. PLGA is a biocompatible and biodegradable copolymer of lactic acid and glycolic acid, and various forms of PLGA are characterized by the ratio of lactic acid to glycolic acid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lactic acid. The degradation rate of PLGA can be adjusted by changing the ratio of lactic acid to glycolic acid. In some embodiments, the PLGA used in accordance with the present invention is characterized by a lactic acid to glycolic acid ratio of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85.

[0045] Hydrophobic carrier materials as provided herein may include one or more polymers, or units thereof, that are non-methoxy-terminated pluronic polymers. A "non-methoxy-terminated polymer" refers to a polymer that has at least one terminus that terminates in a moiety other than methoxy. In some embodiments, the polymer has at least two termini that terminate in a moiety other than methoxy. In other embodiments, the polymer has no termini that terminate in methoxy. A "non-methoxy-terminated pluronic polymer" refers to a polymer other than a linear pluronic polymer that has methoxy at both termini. In some embodiments, the hydrophobic carrier material may comprise polyhydroxyalkanoates, polyamides, polyethers, polyolefins, polyacrylates, polycarbonates, polystyrenes, silicones, fluoropolymers, or units thereof. Further examples of polymers that may be included in the hydrophobic carrier materials provided herein include polycarbonates, polyamides, or polyethers, or units thereof. In other embodiments, the polymer of the hydrophobic carrier material may comprise poly(ethylene glycol) (PEG), polypropylene glycol, or units thereof.

[0046] In some embodiments, it is preferred that the hydrophobic carrier material comprises a biodegradable polymer. Thus, in such embodiments, the polymer of the hydrophobic carrier material may comprise a polyether or a unit thereof, such as poly(ethylene glycol) or polypropylene glycol. In addition, the polymer may comprise a block copolymer of a polyether and a biodegradable polymer, thereby making the polymer biodegradable. In other embodiments, the polymer does not solely comprise a polyether or a unit thereof, such as poly(ethylene glycol) or polypropylene glycol or a unit thereof. Other examples of polymers suitable for use in the present invention include, but are not limited to, polyethylene, polycarbonate (e.g., poly(1,3-dioxan-2-one)), polyanhydrides (e.g., poly(sebacic anhydride)), polypropyl fumerate, polyamides (e.g., polycaprolactam), polyacetals, polyethers, polyesters (e.g., polylactide, polyglycolide, polylactide-co-glycolide, polycaprolactone, polyhydroxy acids (e.g., poly(β-hydroxyalkanoates))), poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine, polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

[0047] Still other examples of polymers that may be included in the hydrophobic carrier material include acrylic polymers such as, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymers, poly(methyl methacrylate), poly(methacrylic anhydride), methyl methacrylate, polymethacrylate, poly(methyl methacrylate) copolymers, polyacrylamide, aminoalkyl methacrylate copolymers, glycidyl methacrylate copolymers, polycyanoacrylate, and combinations comprising one or more of the foregoing polymers.

[0048] In some embodiments, polymers of a hydrophobic carrier material can be combined to form a polymer matrix. A wide variety of polymers and methods for forming polymer matrices therefrom are known in the art. In some embodiments, synthetic nanocarriers comprising a hydrophobic polymer matrix create a hydrophobic environment within the synthetic nanocarrier. In some embodiments, the polymer may be modified with one or more moieties and / or functional groups. A variety of moieties or functional groups can be used in accordance with the present invention. In some embodiments, the polymer may be modified with acyclic polyacetals derived from polyethylene glycol (PEG), carbohydrates, and / or polysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Some embodiments may be made using the general teachings of U.S. Patent No. 5,543,158 to Gref et al. or WO Publication No. WO2009 / 051837 to Von Andrian et al.

[0049] In some embodiments, the polymer may be modified with a lipid or fatty acid group. In some embodiments, the fatty acid group may be one or more of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, or lignoceric acid. In some embodiments, the fatty acid group may be one or more of palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, alpha-linoleic acid, gamma-linoleic acid, arachidonic acid, gadoleic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, or erucic acid. In some embodiments, the polymer is preferably biodegradable. In some embodiments, the polymer according to the present invention comprises a polymer approved for human use by the U.S. Food and Drug Administration (FDA) under 21 CFR § 177.2600.

[0050] The polymer may be a natural or non-natural (synthetic) polymer. The polymer may be a homopolymer or a copolymer comprising two or more monomers. With respect to the sequence, the copolymer may be random, block, or may contain a combination of random and block sequences. Typically, the polymer according to the present invention is an organic polymer. In some embodiments, the polymer may be a linear or branched polymer. In some embodiments, the polymer may be a dendrimer. In some embodiments, the polymer may be substantially cross-linked to one another. In some embodiments, the polymer may be substantially free of cross-linking. In some embodiments, the polymer may be used in accordance with the present invention without undergoing a cross-linking step. It should further be understood that synthetic nanocarriers may comprise block copolymers, graft copolymers, blends, mixtures, and / or adducts of any of the foregoing and other polymers. Those skilled in the art will recognize that the polymers listed herein represent an exemplary, but not comprehensive, list of polymers that may be used in accordance with the present invention, provided that the polymer meets the desired criteria.

[0051] The properties of these and other polymers and methods for preparing them are well known in the art (e.g., U.S. Patents 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181). More generally, various methods for synthesizing certain suitable polymers are described in Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles of Polymerization by Odian, John Wiley & Sons, Fourth Edition, 2004; Contemporary Polymer Chemistry by Allcock et al., Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and U.S. Patents 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

[0052] A wide variety of synthetic nanocarriers can be used in accordance with the present invention. In some embodiments, synthetic nanocarriers are spherical or spherical-shaped. In some embodiments, synthetic nanocarriers are flat or plate-shaped. In some embodiments, synthetic nanocarriers are cubic or cuboid-shaped. In some embodiments, synthetic nanocarriers are ellipsoidal or oval. In some embodiments, synthetic nanocarriers are cylindrical, conical, or pyramidal. In some embodiments, it is desirable to use a population of synthetic nanocarriers that are relatively uniform with respect to size or shape, so that each synthetic nanocarrier has similar properties. For example, based on the total number of synthetic nanocarriers, at least 80%, at least 90%, or at least 95% of the synthetic nanocarriers may have a minimum or maximum dimension that falls within 5%, 10%, or 20% of the average diameter or average dimension of the synthetic nanocarriers.

[0053] Compositions according to the present invention may contain elements in combination with pharmaceutically acceptable additives, such as preservatives, buffers, saline, or phosphate-buffered saline. The compositions may be made using conventional pharmaceutical manufacturing and compounding techniques to create useful dosage forms. In embodiments, such compositions including synthetic nanocarriers are suspended in a sterile saline solution for injection along with a preservative. In some embodiments, any component of a synthetic nanocarrier as provided herein may be isolated. Isolated refers to an element that is separate from its native environment and present in a quantity sufficient to permit its identification or use. This means, for example, that the element may be purified by chromatography or electrophoresis. An isolated element may be substantially pure, but need not be. An isolated element may be admixed with pharmaceutically acceptable excipients in a pharmaceutical preparation, such that the element may comprise only a small weight percent of the preparation. An element is nevertheless isolated in that it is separated from substances that may be associated with biological systems, i.e., isolated from other lipids or proteins. Any of the elements provided herein may be isolated and included in a composition or used in a method in isolated form.

[0054] D. Methods of Making and Using the Compositions and Related Methods Synthetic nanocarriers may be prepared using a variety of methods known in the art. For example, synthetic nanocarriers can be formed by methods such as nanoprecipitation, flow focusing using fluid channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, grinding (including freeze-grinding), supercritical fluid (such as supercritical carbon dioxide) processing, fine emulsion procedures, microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, and other methods known to those skilled in the art. Alternatively or additionally, aqueous and organic solvent syntheses for monodisperse semiconducting, conductive, magnetic, organic, and other nanomaterials have been described (Pellegrino et al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; and Trindade et al., 2001, Chem. Mat., 13:3843). Additional methods are described in the literature (see, e.g., Doubrow, Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy," CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755; US Patents 5,578,325 and 6,007,845; P. Paolicelli et al., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles" Nanomedicine. 5(6):843-853 (2010)).

[0055] A variety of materials are available, including C. Astete et al., “Synthesis and characterization of PLGA nanoparticles” J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K. Avgoustakis “Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties and Possible Applications in Drug Delivery” Current Drug Delivery 1:321-333 (2004);C. Reis et al., “Nanoencapsulation I. Methods for preparation of drug-loaded polymeric nanoparticles” Nanomedicine 2:8- 21 (2006);P. Paolicelli et al., “Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853 (2010) may be used to encapsulate materials into synthetic nanocarriers as desired. Other methods suitable for encapsulating materials into synthetic nanocarriers may be used, including, but not limited to, those disclosed in U.S. Patent No. 6,632,671 to Unger, issued October 14, 2003.

[0056] In some embodiments, synthetic nanocarriers are prepared by a nanoprecipitation process or spray drying. The conditions used to prepare synthetic nanocarriers may be varied to produce particles of desired size or characteristics (e.g., hydrophobicity, hydrophilicity, external morphology, "stickiness," shape, etc.). The method of preparing synthetic nanocarriers and the conditions used (e.g., solvent, temperature, concentration, air flow rate, etc.) may depend on the materials included in the composition of the synthetic nanocarrier and / or carrier matrix. If synthetic nanocarriers prepared by any of the above methods have a size range outside the desired range, such synthetic nanocarriers can be sized, for example, using sieves.

[0057] In embodiments, synthetic nanocarriers may be combined with antigens or other compositions by admixture in the same vehicle or delivery system. The compositions provided herein may contain inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonate, acetate, or citrate) and pH adjusters (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citric acid or acetate, amino acids and their salts), antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80), and the like. 80, polyoxyethylene 9-10 nonylphenol, sodium desoxycholate), solution and / or cryo / lyo stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmolality adjusters (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsiloxane), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymer stabilizers and viscosity adjusters (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose), and cosolvents (e.g., glycerol, polyethylene glycol, ethanol).

[0058] The compositions according to the present invention may contain pharmaceutically acceptable additives. The compositions may be prepared using conventional pharmaceutical manufacturing and compounding techniques to create useful dosage forms. Suitable techniques for use in practicing the present invention may be found in Handbook of Industrial Mixing: Science and Practice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004 John Wiley & Sons, Inc.; and Pharmaceutics: The Science of Dosage Form Design, 2nd Ed. Edited by ME Auten, 2001, Churchill Livingstone. In an embodiment, the composition is suspended in sterile saline solution for injection together with a preservative. It should be understood that the compositions of the present invention can be made in any suitable manner, and that the present invention is in no way limited to compositions that can be produced using the methods described herein. Selection of an appropriate method of manufacture may require attention to the properties of the particular components involved.

[0059] In some embodiments, the composition is manufactured under sterile conditions or is initially or terminally sterilized. This can ensure that the resulting composition is sterile and non-infectious, thereby improving safety compared to non-sterile compositions. This provides a valuable safety measure, especially when the subject receiving the composition has an immunodeficiency, suffers from an infectious disease, and / or is prone to infectious diseases. In some embodiments, the composition may be lyophilized and stored in a suspension or as a lyophilized powder, depending on the formulation strategy to maintain activity for a long period of time. Administration according to the present invention may be by a variety of routes, including but not limited to intradermal, intramuscular, subcutaneous, intravenous and intraperitoneal routes. The compositions referred to herein may be manufactured and prepared for administration using conventional methods.

[0060] The compositions of the present invention can be administered in effective amounts, such as those described elsewhere herein. The dosage forms may contain varying amounts of components in accordance with the present invention. The amounts of components present in the dosage forms of the present invention can be varied according to their properties, the therapeutic benefit to be achieved, and other such parameters. In embodiments, dose ranging studies can be conducted to determine the optimal therapeutic amount present in the dosage form. In embodiments, the components are present in the dosage form in an amount effective to produce the desired effect and / or reduced immune response upon administration to a subject. It may be possible to determine the amount to achieve the desired result using conventional dose ranging studies and techniques in a subject. The dosage forms of the present invention may be administered at varying frequencies. In embodiments, at least one administration of the compositions provided herein is sufficient to produce a pharmacologically relevant response.

[0061] Another aspect of the present disclosure relates to kits. In some embodiments, the kit comprises any one of the compositions provided herein. In some embodiments of any one of the provided kits, the kit comprises a rapalog, a hydrophobic carrier material, and a non-ionic surfactant with a hydrophilic-lipophilic balance (HLB) of 10 or less. In some embodiments of any one of the provided kits, the amounts of the rapalog, the hydrophobic carrier material, and the non-ionic surfactant with a hydrophilic-lipophilic balance (HLB) of 10 or less are each any one of the amounts provided herein for that component. In some embodiments of any one of the provided kits, the kit further comprises an antigen. In some embodiments of any one of the provided kits, the compositions or components thereof can be contained in separate containers in the kit or in the same container. In some embodiments of any one of the provided kits, the container is a vial or an ampoule. In some embodiments of any one of the provided kits, the compositions or components thereof are contained in a solution separate from the container, such that the compositions or components may be added to the container at a later time. In some embodiments of any one of the provided kits, the compositions or components thereof are in lyophilized form, each in separate containers or the same container, so that they may be reconstituted at a later time. In some embodiments of any one of the provided kits, the kit further comprises instructions for reconstitution, mixing, administration, etc. In some embodiments of any one of the provided kits, the instructions comprise a description of the methods described herein. The instructions can be in any suitable form, such as, for example, a printed insert or label. In some embodiments of any one of the provided kits, the kit further comprises one or more syringes or other device(s) capable of delivering the synthetic nanocarriers to a subject in vivo.

[0062] example Example 1 - Low HLB surfactant, SM, increases RAPA loading and filterability of synthetic nanocarriers Nanocarrier compositions containing the polymers PLA (intrinsic viscosity 0.41 dL / g) and PLA-PEG (5 kDa PEG blocks, intrinsic viscosity 0.50 dL / g) and the hydrophobic drug rapamycin (RAPA) were synthesized using a water-in-oil emulsion evaporation method with or without the addition of the low HLB surfactant sorbitan monopalmitate (SM). The organic phase was formed by dissolving the polymer and RAPA in dichloromethane. Emulsions were formed by homogenizing the organic phase in the aqueous phase containing the surfactant PVA using a probe-tip sonicator. The emulsion was then combined with a larger volume of aqueous buffer and mixed to allow for solvent dissolution and evaporation. The resulting nanocarriers were washed and filtered through a 0.22 μm filter. All compositions contained 100 mg of polymer. The RAPA content in the various compositions varied.

[0063] [Table 1]

[0064] For compositions without the surfactant SM (Samples 1, 2, and 3), some indications were observed as the amount of RAPA added increased, limiting the ability to fully incorporate RAPA into the nanocarrier composition. The difference in nanocarrier size between pre- and post-filtration in the absence of SM increased at higher RAPA loading levels, indicating the presence of larger particulates (individual particles or aggregates) that were removed during the washing and / or filtration process. This was also indicated by a decreased filtration throughput before clogging. Finally, adding increasing amounts of RAPA to nanocarrier compositions without SM did not result in increased RAPA loading (e.g., Sample 1 compared to Sample 3). This indicates that additional RAPA was separable from the bulk nanocarriers and was removed during the washing and / or filtration steps.

[0065] In contrast, compositions containing the surfactant SM readily incorporated increasing amounts of RAPA. Nanocarrier size was not affected by filtration. Increasing amounts of RAPA added to the compositions resulted in increased RAPA loading of the nanocarriers. Some reduction in filtration throughput was observed at the highest loading level (Sample 6), which may be due to the inherently larger nanocarrier size. Thus, the incorporation of SM served to increase the RAPA loading and filterability of the synthetic nanocarrier compositions.

[0066] Example 2 - SM and cholesterol increased the loading and filterability of RAPA Nanocarrier compositions were produced using the materials and methods described in Example 1. Nanocarriers containing polymer and RAPA were produced with varying RAPA loading levels. In addition, nanocarriers with higher RAPA loading levels were also produced using the additives, surfactants SM or cholesterol, at an additive:RAPA mass ratio of 3.2:1.

[0067] [Table 2]

[0068] Nanocarrier samples produced in the absence of additives (Samples 7 and 8) demonstrated that increasing RAPA loading beyond the apparent nanocarrier saturation point tended to lead to reduced filtration throughput. Addition of either SM or cholesterol resulted in greater RAPA loading while maintaining stability (Samples 9 and 10). To assess the composition's ability to induce immune tolerance, mice were intravenously injected with co-administered nanocarriers and KLH (keyhole limpet hemocyanin) at the same RAPA dose three times weekly and then challenged weekly with KLH alone. Mouse serum was then analyzed for antibodies against KLH after each KLH challenge (Figure 1).

[0069] Although all mice receiving RAPA nanocarrier treatment received the same dose of RAPA, the various groups demonstrated different degrees of tolerization to KLH. All five mice receiving the nanocarrier composition with the lowest loading (Sample 7) had quantifiable titers of anti-KLH antibodies after the third KLH challenge (day 40). Mice in this group developed reduced titers of anti-KLH antibodies compared with mice receiving only PBS, but exhibited the least tolerance compared with the other nanocarrier groups. Increasing the RAPA loading of the nanocarriers in the absence of additives (SM or cholesterol) (Sample 8) significantly improved tolerization, with only two of the five mice demonstrating quantifiable titers after the third KLH challenge (day 40). The composition containing cholesterol as an additive (Sample 10) resulted in four of five mice demonstrating significant anti-KLH antibody titers after only two challenges (at day 33), despite the high RAPA loading of the nanocarriers. The nanocarrier composition containing SM (Sample 9) demonstrated both high throughput (0.22 μm filtration throughput during production) and excellent tolerization, as only one of five mice developed quantifiable titers of anti-KLH antibodies after three KLH challenges (at day 40). The results of this study indicate that both additives (SM and cholesterol) enabled increased nanocarrier loading, consistent with tolerance-inducing performance and processing desirability, as indicated by filtration throughput. SM, a low HLB surfactant, provided the properties required to increase nanocarrier stability and demonstrated higher performance.

[0070] Example 3 - Effect of low HLB surfactants on RAPA loading and filterability material and method PLA with an intrinsic viscosity of 0.41 dL / g was purchased from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, AL 35211) (product code 100 DL 4A). PLA-PEG-OMe block copolymer with approximately 5,000 kDa methyl ether-terminated PEG blocks and an overall intrinsic viscosity of 0.50 DL / g was purchased from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, AL 35211) (product code 100 DL mPEG 5000 5CE). Rapamycin (product code SIROLIMUS) was purchased from Concord Biotech Limited (1482-1486 Trasad Road, Dholka 382225, Ahmedabad, India). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, 3.4-4.6 mPa·s viscosity) was purchased from EMD Chemicals Inc. (480 South Democrat Road, Gibbstown, NJ 08027) (product code 1.41350). Dulbecco's Phosphate Buffered Saline 1× (DPBS) was purchased from Lonza (Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland) (product code 17-512Q). Sorbitan monopalmitate was purchased from Croda International (300-A Columbus Circle, Edison, NJ 08837) (product code SPAN 40). Polysorbate 80 was purchased from NOF America Corporation (One North Broadway, Suite 912, White Plains, NY 10601) (product code Polysorbate 80 (HX2)). Sorbitan monolaurate (SPAN 20) was purchased from Alfa Aesar (26 Parkridge Rd, Ward Hill, MA 01835) (product code L12099).Sorbitan stearate (SPAN 60) was purchased from Sigma-Aldrich (3050 Spruce St. St. Louis, MO 63103) (product code S7010). Sorbitan monooleate (SPAN 80) was purchased from Tokyo Chemical Industry Co., Ltd. (9211 North Harborgate Street, Portland, OR 97203) (product code S0060). Octyl β-D-glucopyranoside was purchased from Sigma-Aldrich (3050 Spruce St. St. Louis, MO 63103) (product code O8001). Oleyl alcohol was purchased from Alfa Aesar (26 Parkridge Rd, Ward Hill, MA 01835) (product code A18018). Isopropyl palmitate was purchased from Sigma-Aldrich (3050 Spruce St. St. Louis, MO 63103) (product code W515604). Polyethylene glycol hexadecyl ether (BRIJ 52) was purchased from Sigma-Aldrich (3050 Spruce St. St. Louis, MO 63103) (product code 388831). Polyethylene glycol oleyl ether (BRIJ 93) was purchased from Sigma-Aldrich (3050 Spruce St. St. Louis, MO 63103) (product code 388866). Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic L-31) was purchased from Sigma-Aldrich (3050 Spruce St. St. Louis, MO 63103) (product code 435406). Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic P-123) was purchased from Sigma-Aldrich (3050 Spruce St. St. Louis, MO 63103) (product code 435465).Palmitic acid was purchased from Sigma-Aldrich (3050 Spruce St. St. Louis, MO 63103) (product code P0500). DL-α-palmitin was purchased from Sigma-Aldrich (3050 Spruce St. St. Louis, MO 63103) (product code M1640). Glyceryl tripalmitate was purchased from Sigma-Aldrich (3050 Spruce St. St. Louis, MO 63103) (product code T5888).

[0071] For Sample 11, a solution was prepared as follows: Solution 1: A mixture of polymer and rapamycin was prepared by dissolving PLA at 75 mg / mL, PLA-PEG-Ome at 25 mg / mL, and rapamycin at 16 mg / mL in dichloromethane. Solution 2: A Polysorbate 80 mixture was prepared by dissolving Polysorbate 80 at 80 mg / mL in dichloromethane. Solution 3: Polyvinyl alcohol was prepared at 50 mg / mL in 100 mM phosphate buffer (pH 8).

[0072] An O / W emulsion was prepared by combining Solution 1 (0.5 mL), Solution 2 (0.1 mL), dichloromethane (0.4 mL), and Solution 3 (3.0 mL) in a small glass pressure tube, vortexing for 10 seconds, and then sonicating for 1 minute at 30% amplitude using a Branson Digital Sonifier 250 while the pressure tube was immersed in an ice-water bath. The emulsion was then added to a 50 mL beaker containing DPBS (30 mL). A second O / W emulsion was prepared using the same materials and methods as above and then added to the same container containing the first emulsion and DPBS. This was then stirred at room temperature for 2 hours to evaporate the dichloromethane and form nanocarriers. A portion of the nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging it at 75,600 × g and 4 °C for 50 minutes, removing the supernatant, and resuspending the pellet in DPBS containing 0.25% (w / v) PVA. The washing procedure was repeated, and the pellet was then resuspended in DPBS containing 0.25% (w / v) PVA to yield a nanocarrier suspension with a set concentration of 10 mg / mL based on polymer. The nanocarrier suspension was then filtered using a 0.22 μm PES membrane syringe filter (Millipore product number SLGP033RB). The filtered nanocarrier suspension was then stored at -20°C.

[0073] For samples 12-25, solutions were prepared as follows: Solution 1: A mixture of polymer and rapamycin was prepared by dissolving PLA at 75 mg / mL, PLA-PEG-Ome at 25 mg / mL, and rapamycin at 16 mg / mL in dichloromethane. Solution 2: An HLB mixture was prepared by dissolving HLB surfactants at 5.0 mg / mL in dichloromethane. The HLB surfactants included SPAN 20, SPAN 40, SPAN 60, SPAN 80, octyl β-D-glucopyranoside, oleyl acid, isopropyl palmitate, BRIJ 52, BRIJ 93, Pluronic L-31, Pluronic P-123, palmitic acid, DL-α-palmitin, and glyceryl tripalmitate. Solution 3: Polyvinyl alcohol was prepared at 62.5 mg / mL in 100 mM phosphate buffer (pH 8).

[0074] An O / W emulsion was prepared by combining Solution 1 (0.5 mL), Solution 2 (0.5 mL), and Solution 3 (3.0 mL) in a small glass pressure tube, vortexing for 10 seconds, and then sonicating for 1 minute at 30% amplitude using a Branson Digital Sonifier 250 while immersing the pressure tube in an ice-water bath. The emulsion was then added to a 50 mL beaker containing DPBS (30 mL). A second O / W emulsion was prepared using the same materials and methods as above and then added to the same beaker containing the first emulsion and DPBS. This was then stirred at room temperature for 2 hours to evaporate the dichloromethane and form nanocarriers. A portion of the nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging at 75,600 × g and 4 °C for 50 minutes, removing the supernatant, and resuspending the pellet in DPBS containing 0.25% (w / v) PVA. The washing procedure was repeated, and the pellet was then resuspended in DPBS containing 0.25% (w / v) PVA to yield a nanocarrier suspension with a set concentration of 10 mg / mL based on polymer. The nanocarrier suspension was then filtered using a 0.22 μm PES membrane syringe filter (Millipore product number SLGP033RB). The filtered nanocarrier suspension was then stored at -20°C.

[0075] [Table 3]

[0076] The HLB was determined for most low HLB surfactants using publicly available information. For DL-α-palmitin, the HLB was calculated using the following formula: Mw = 330.5 g / mol, hydrophilic portion = 119.0 g / mol; HLB = 119.0 / 330.5*100 / 5 = 7.2. For glyceryl palmitate, the HLB was calculated using the following formula: Mw = 807.3 g / mol, hydrophilic portion h = 173.0 g / mol; HLB = 173.0 / 807.3*100 / 5 = 4.3. For isopropyl palmitate, the HLB was calculated using the following formula: Mw = 298.5 g / mol, hydrophilic portion = 44.0 g / mol; HLB = 44.0 / 298.5*100 / 5 = 2.9. For oleyl alcohol, the HLB was calculated using the following formula: Mw = 268.5 g / mol, hydrophilic portion = 17.0 g / mol; HLB = 17.0 / 268.5*100 / 5 = 1.3. Additionally, the loading of low HLB surfactants was measured by extraction followed by quantification by HPLC.

[0077] Prior to injection into animals, the bulk nanocarrier suspension was thawed in a room-temperature water bath for 30 minutes. The nanocarriers were diluted with DPBS to achieve the desired concentration of 278 μg / mL rapamycin. Six-week-old C57BL / 6 female mice were intravenously treated with nanocarriers (1.17 mL) mixed with 130 μL of 10× KLH (keyhole limpet hemocyanin) on days 0, 7, and 14. Mice were boosted with 200 μg KLH on days 21, 28, 35, and 42. Anti-KLH IgG titers (measured by ELISA) were read on days 40, 47, and 61. The results demonstrate that low HLB surfactants can result in substantial rapamycin loading and filterability of synthetic nanocarriers. Additionally, all of the nanocarriers with low HLB surfactants, as shown in Figure 2, resulted in reduced antibody titers for at least 40 and 47 days.

[0078] Example 4 - Effect of low HLB surfactants on the filterability of synthetic nanocarriers material and method PLA-PEG-OMe block copolymer with approximately 5,000 Da methyl ether-terminated PEG blocks and an overall intrinsic viscosity of 0.50 DL / g was purchased from Evonik Industries (Rellinghauser Strasse 1-11, 45128 Essen, Germany) (product code 100 DL mPEG 5000 5CE). PLA with an intrinsic viscosity of 0.41 dL / g was purchased from Evonik Industries (Rellinghauser Strasse 1-11, 45128 Essen, Germany) (product code 100 DL 4A). Rapamycin was purchased from Concord Biotech Limited (1482-1486 Trasad Road, Dholka 382225, Ahmedabad, India) (product code SIROLIMUS). Everolimus was purchased from LC Laboratories (165 New Boston Street, Woburn, MA 01801) (product number E-4040). Temsirolimus was purchased from LC Laboratories (165 New Boston Street, Woburn, MA 01801) (product number T-8040). Sorbitan monopalmitate was purchased from Croda (315 Cherry Lane, New Castle, Delaware 19720) (product code SPAN 40). Dichloromethane was purchased from Spectrum (14422 S San Pedro Gardena, CA 90248-2027) (product number M1266).EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa·s) was purchased from EMD Chemicals Inc. (480 South Democrat Road, Gibbstown, NJ 08027) (product code 1.41350). Dulbecco's Phosphate Buffered Saline, 1×, 0.0095 M (PO₄), calcium and magnesium free, was purchased from BioWhittaker (8316 West Route 24, Mapleton, IL 61547) (product code #12001, Lonza DPBS). Emulsification was performed using a Branson Digital Sonifier 250 with a 1 / 8" tapered tip titanium probe.

[0079] The solutions were prepared as follows: Solution 1: A polymer mixture was prepared by dissolving PLA-PEG-OMe (100 DL mPEG 5000 5CE) at 50 mg per mL and PLA (100 DL 4A) at 150 mg per mL in dichloromethane. Solution 2: Rapamycin was dissolved at 160 mg per mL in dichloromethane. Solution 3: Everolimus was dissolved at 150 mg per mL in dichloromethane. Solution 4: Temsirolimus was dissolved at 150 mg per mL in dichloromethane. Solution 5: Sorbitan monopalmitate (SPAN 40) was dissolved at 50 mg per mL in dichloromethane. Solution 6: Dichloromethane was sterile filtered using a 0.2 μm PTFE membrane syringe filter (VWR part number 28145-491). Solution 7: A polyvinyl alcohol solution was prepared by dissolving polyvinyl alcohol (EMPROVE® Polyvinyl Alcohol 4-88) at 75 mg per mL in 100 mM phosphate buffer, pH 8. Solution 8: A mixture of polyvinyl alcohol and Dulbecco's phosphate buffered saline, 1×, 0.0095 M (PO4), was prepared by dissolving polyvinyl alcohol (EMPROVE® Polyvinyl Alcohol 4-88) at 2.5 mg per mL in Dulbecco's phosphate buffered saline, 1×, 0.0095 M (PO4) (Lonza DPBS).

[0080] For Sample 26, an O / W emulsion was prepared by combining Solution 1 (0.5 mL), Solution 2 (0.1 mL), Solution 5 (0.1 mL), and Solution 6 (0.30 mL) in a small glass pressure tube. The solutions were mixed by repeated pipetting. Solution 7 (3.0 mL) was then added, and the formulation was vortex-mixed for 10 seconds. The formulation was then sonicated for 1 minute at 30% amplitude while the pressure tube was immersed in an ice bath. The emulsion was then added to an open 50 mL beaker containing Lonza DPBS (30 mL). This was then stirred at room temperature for 2 hours to evaporate the dichloromethane and form nanocarriers. A portion of the nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging at 75,600 × g and 4 °C for 50 minutes, removing the supernatant, and resuspending the pellet in Solution 8. The washing procedure was repeated, and the pellet was then resuspended in Solution 8 to yield a nanocarrier suspension with a set concentration of 10 mg per mL based on polymer. The nanocarrier formulation was filtered using a 0.22 μm PES membrane syringe filter (Millex part number SLGP033RS). The mass of the nanocarrier solution filtration throughput was measured. The filtered nanocarrier solution was then stored at -20°C.

[0081] For Sample 27, an O / W emulsion was prepared by combining Solution 1 (0.5 mL), Solution 2 (0.1 mL), and Solution 6 (0.40 mL) in a small glass pressure tube. The solutions were mixed by repeated pipetting. Solution 7 (3.0 mL) was then added, and the formulation was vortex-mixed for 10 seconds. The formulation was then sonicated for 1 minute at 30% amplitude while the pressure tube was immersed in an ice bath. The emulsion was then added to a 50 mL open beaker containing Lonza DPBS (30 mL). This was then stirred at room temperature for 2 hours to evaporate the dichloromethane and form the nanocarriers. A portion of the nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube and centrifuging it at 75,600 × g and 4 °C for 50 minutes, removing the supernatant, and resuspending the pellet in Solution 8. Repeating the washing procedure and then resuspending the pellet in Solution 8 resulted in a nanocarrier suspension with a set concentration of 10 mg polymer per mL. The nanocarrier formulation was filtered using a 0.22 μm PES membrane syringe filter (Millex part number SLGP033RS). The mass of the nanocarrier solution filtered was measured. The filtered nanocarrier solution was then stored at -20°C.

[0082] For Sample 28, an O / W emulsion was prepared by combining Solution 1 (0.5 mL), Solution 3 (0.2 mL), Solution 5 (0.1 mL), and Solution 6 (0.20 mL) in a small glass pressure tube. The solutions were mixed by repeated pipetting. Next, Solution 7 (3.0 mL) was added, and the formulation was vortex mixed for 10 seconds. The formulation was then sonicated for 1 minute at 30% amplitude with the pressure tube immersed in an ice bath. The emulsion was then added to a 50 mL open beaker containing Lonza DPBS (30 mL). This was then stirred for 2 hours at room temperature to allow the dichloromethane to evaporate and form nanocarriers. A portion of the nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging it at 75,600 × g and 4 °C for 50 minutes, removing the supernatant, and resuspending the pellet in Solution 8. The washing procedure was repeated, and the pellet was then resuspended in Solution 8 to achieve a nanocarrier suspension with a set concentration of 10 mg polymer per mL. The nanocarrier formulation was filtered using a 0.22 μm PES membrane syringe filter (Millex part number SLGP033RS). The filtration throughput of the nanocarrier solution was measured. The filtered nanocarrier solution was then stored at -20°C.

[0083] For Sample 29, an O / W emulsion was prepared by combining Solution 1 (0.5 mL), Solution 3 (0.2 mL), and Solution 6 (0.30 mL) in a small glass pressure tube. The solutions were mixed by repeated pipetting. Next, Solution 7 (3.0 mL) was added, and the formulation was vortex mixed for 10 seconds. The formulation was then sonicated for 1 minute at 30% amplitude with the pressure tube immersed in an ice bath. The emulsion was then added to a 50 mL open beaker containing Lonza DPBS (30 mL). This was then stirred for 2 hours at room temperature to allow the dichloromethane to evaporate and form the nanocarriers. A portion of the nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging it at 75,600 × g and 4 °C for 50 minutes, removing the supernatant, and resuspending the pellet in Solution 8. The washing procedure was repeated, and the pellet was then resuspended in Solution 8 to achieve a nanocarrier suspension with a set concentration of 10 mg polymer per mL. The nanocarrier formulation was filtered using a 0.22 μm PES membrane syringe filter (Millex part number SLGP033RS). The filtration throughput of the nanocarrier solution was measured. The filtered nanocarrier solution was then stored at -20°C.

[0084] For Sample 30, an O / W emulsion was prepared by combining Solution 1 (0.5 mL), Solution 4 (0.2 mL), Solution 5 (0.1 mL), and Solution 6 (0.20 mL) in a small glass pressure tube. The solutions were mixed by repeated pipetting. Next, Solution 7 (3.0 mL) was added, and the formulation was mixed with a vortex for 10 seconds. The formulation was then sonicated at 30% amplitude for 1 minute with the pressure tube immersed in an ice bath. The emulsion was then added to a 50 mL open beaker containing Lonza DPBS (30 mL). This was then stirred at room temperature for 2 hours to allow the dichloromethane to evaporate and form the nanocarriers. A portion of the nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging it at 75,600 × g and 4 °C for 50 minutes, removing the supernatant, and resuspending the pellet in Solution 8. The washing procedure was repeated, and the pellet was then resuspended in Solution 8 to achieve a nanocarrier suspension with a set concentration of 10 mg polymer per mL. The nanocarrier formulation was filtered using a 0.22 μm PES membrane syringe filter (Millex part number SLGP033RS). The filtration throughput of the nanocarrier solution was measured. The filtered nanocarrier solution was then stored at -20°C.

[0085] For Sample 31, an O / W emulsion was prepared by combining Solution 1 (0.5 mL), Solution 4 (0.2 mL), and Solution 6 (0.30 mL) in a small glass pressure tube. The solutions were mixed by repeated pipetting. Next, Solution 7 (3.0 mL) was added, and the formulation was vortex mixed for 10 seconds. The formulation was then sonicated for 1 minute at 30% amplitude with the pressure tube immersed in an ice bath. The emulsion was then added to a 50 mL open beaker containing Lonza DPBS (30 mL). This was then stirred at room temperature for 2 hours to evaporate the dichloromethane and form nanocarriers. A portion of the nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging at 75,600 × g and 4 °C for 50 minutes, removing the supernatant, and resuspending the pellet in Solution 8. The washing procedure was repeated, and the pellet was then resuspended in Solution 8 to achieve a nanocarrier suspension with a set concentration of 10 mg polymer per mL. The nanocarrier formulation was filtered using a 0.22 μm PES membrane syringe filter (Millex part number SLGP033RS). The filtration throughput of the nanocarrier solution was measured. The filtered nanocarrier solution was then stored at −20° C.

[0086] Nanocarrier size was determined by dynamic light scattering. The amount of rapalog in the nanocarriers was determined by HPLC analysis. The total mass of dry nanocarriers per mL of suspension was determined gravimetrically. Filterability was assessed by the amount of filtrate that passed through the first filter. The data show that for a number of rapalogs, incorporation of SPAN 40 in synthetic nanocarriers resulted in an increase in the filterability of the synthetic nanocarrier compositions.

[0087] [Table 4]

[0088] Example 5 - Effect of SPAN 40 on the filtration capacity of synthetic nanocarriers containing non-polyester polymers material and method Rapamycin was purchased from Concord Biotech Limited (1482-1486 Trasad Road, Dholka 382225, Ahmedabad, India) (product code: SIROLIMUS). Poly(styrene)-block-poly(ethylene glycol) (PS-PEG) with methyl ether-terminated PEG blocks of approximately 700–1,100 Da was purchased from Sigma Aldrich (3050 Spruce St. St. Louis, MO 63103) (product number 686476). Poly(styrene)-block-poly(methyl methacrylate) (PS-PMMA) with a 1:1 methacrylate:styrene ratio (MW = 30,000 Da) was purchased from Sigma Aldrich (3050 Spruce St. St. Louis, MO 63103) (product number 749184). 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was purchased from Avanti Polar Lipids, Inc. (Avanti Polar Lipids, Inc. 700 Industrial Park Drive, Alabaster, Alabama 35007-9105) (product number 850345P). Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (P-123) with a 20:70:20 PEG:PPG:PEG ratio was purchased from Sigma Aldrich (3050 Spruce St., St. Louis, MO 63103) (product code Pluronic® P-123, product number 435465). Dichloromethane was purchased from Spectrum (14422 S San Pedro Gardena, CA 90248-2027) (product number M1266). Sorbitan monopalmitate was purchased from Croda (315 Cherry Lane New Castle Delaware 19720) (product code SPAN 40).EMPROVE® Polyvinyl Alcohol 4-88 (PVA), USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa·s) was purchased from EMD Chemicals Inc. (480 South Democrat Road, Gibbstown, NJ 08027) (product code 1.41350). Dulbecco's Phosphate Buffered Saline (DPBS), 1X, 0.0095 M (PO₄), without calcium or magnesium, was purchased from BioWhittaker (8316 West Route 24, Mapleton, IL 61547) (product code 17-512Q). Emulsification was performed using a Branson Digital Sonifier 250 with a 1 / 8" tapered tip titanium probe.

[0089] The solutions were prepared as follows: Solution 1: Polymer and rapamycin solution was prepared by dissolving 50 mg of PS-PEG per mL and 8 mg of rapamycin per mL in dichloromethane. Solution 2: Polymer and rapamycin solution was prepared by dissolving 50 mg of PS-PMMA (Mw = 30,000 Da) per mL and 8 mg of rapamycin per mL in dichloromethane. Solution 3: Lipid and rapamycin solution was prepared by dissolving 50 mg of DMPC per mL and 8 mg of rapamycin per mL in dichloromethane. Solution 4: Polymer and rapamycin solution was prepared by dissolving 50 mg of (P-123) per mL and 8 mg of rapamycin per mL in dichloromethane. Solution 5: Dichloromethane was sterile filtered using a 0.2 μm PTFE membrane syringe filter (VWR part number 28145-491). Solution 6: SPAN 40 was dissolved at 50 mg per mL in dichloromethane. Solution 7: PVA was dissolved at 62.5 mg per mL in 100 mM pH 8 phosphate buffer.

[0090] For Sample 28, an O / W emulsion was prepared by combining Solution 1 (1.0 mL) and Solution 5 (0.05 mL) in a small glass pressure tube pre-cooled in an ice-water bath for >4 minutes. Solution 7 (3.0 mL) was then added, and the pressure tube was vortexed for 10 seconds. The emulsion was then sonicated for 1 minute at 30% amplitude with the pressure tube immersed in an ice bath. The resulting nanoemulsion was then added to a 50 mL open beaker containing DPBS (15 mL), and a piece of aluminum foil was placed over the open beaker. A second emulsion was prepared using the same procedure, and a new portion of DPBS (15 mL) was added to the first emulsion in the same 50 mL beaker. The beaker was left uncovered, and the aluminum foil was discarded. This was then stirred at room temperature for 2 hours to allow the dichloromethane to evaporate and form nanocarriers. A portion of the nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging at 75,600 x g and 4°C for 50 minutes, removing the supernatant, and resuspending the pellet in DPBS. The washing procedure was repeated, and the pellet was then resuspended in DPBS to achieve a nanocarrier suspension with a set concentration of 10 mg per mL based on polymer. The nanocarrier formulation was filtered using a 0.22 μm PES membrane syringe filter (Millex part number SLGP033RS). The filtration throughput of the nanocarrier solution was measured. The filtered nanocarrier solution was then stored at -20°C.

[0091] Sample 29 was prepared similarly to Sample 28, using Solution 6 instead of Solution 5. Sample 30 was prepared similarly to Sample 28, using Solution 2 instead of Solution 1. Sample 31 was prepared similarly to Sample 30, using Solution 6 instead of Solution 5. Sample 32 was prepared similarly to Sample 28, using Solution 3 instead of Solution 1. Sample 33 was prepared similarly to Sample 32, using Solution 6 instead of Solution 5. Sample 34 was prepared similarly to Sample 28, using Solution 4 instead of Solution 1. Sample 35 was prepared similarly to Sample 34, using Solution 6 instead of Solution 5.

[0092] Nanocarrier size was determined by dynamic light scattering. The amount of rapamycin in the nanocarriers was determined by HPLC analysis. The total dry nanocarrier mass per mL of suspension was determined gravimetrically. Filtration capacity was assessed by the mass of the nanocarrier filtrate through an initial 33 mm PES membrane 0.22 μm syringe filter. The results below, although not optimized, demonstrate that inclusion of SPAN 40 in synthetic nanocarriers with non-polyester polymers can, in some embodiments, increase the filtration capacity of the synthetic nanocarriers.

[0093] [Table 5]

[0094] Example 6 - SPAN 40 significantly increases the filterability of synthetic nanocarriers containing polyester polymers material and method PLA (100 DL 4A) with an intrinsic viscosity of 0.41 dL / g was purchased from Evonik Industries AG (Rellinghauser Strasse 1-11, Essen, Germany) (product code 100 DL 4A). PLA-PEG-OMe block copolymer with approximately 5,000 Da methyl ether-terminated PEG blocks and an overall intrinsic viscosity of 0.50 DL / g was purchased from Evonik Industries AG (Rellinghauser Strasse 1-11, Essen, Germany) (product code 100 DL mPEG 5000 5CE). Rapamycin was purchased from Concord Biotech Limited (1482-1486 Trasad Road, Dholka 382225, Ahmedabad, India) (product code SIROLIMUS). EMPROVE® Polyvinyl Alcohol 4-88 (PVA), USP (85-89% hydrolysis, 3.4-4.6 mPa·s viscosity) was purchased from EMD Chemicals Inc. (480 South Democrat Road, Gibbstown, NJ 08027) (product code 1.41350). Dulbecco's Phosphate Buffered Saline 1× (DPBS) was purchased from Lonza (Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland) (product code 17-512Q). Sorbitan monopalmitate (SPAN 40) was purchased from Croda International (300-A Columbus Circle, Edison, NJ 08837) (product code Span 40). PLGA (5050 DLG 2.5A) with approximately 54 wt% lactide and 46 wt% glycolide and an intrinsic viscosity of 0.24 dL / g was purchased from Evonik Industries AG (Rellinghauser Strasse 1-11, Essen Germany) (product code 5050 DLG 2.5A).PLGA (7525 DLG 4A) with approximately 73 wt% lactide and 27 wt% glycolide and an intrinsic viscosity of 0.39 dL / g was purchased from Evonik Industries AG (Rellinghauser Strasse 1-11, Essen, Germany) (7525 DLG 4A). Polycaprolactone (PCL) with an average Mw of 14,000 Da and Mn of 10,000 Da was purchased from Sigma-Aldrich (3050 Spruce St., St. Louis, MO 63103) (product code 440752).

[0095] For samples 1, 3, 5 and 7, solutions were prepared as follows: Solution 1: PLA-PEG-Ome was dissolved at 50 mg per mL, Span 40 at 10 mg per mL, and rapamycin at 32 mg per mL in dichloromethane. Solution 2: 100 DL 4A was dissolved at 150 mg per mL in dichloromethane. Solution 3: 5050 DLG 2.5A was dissolved at 150 mg per mL in dichloromethane. Solution 4: 7525 DLG 4A was dissolved at 150 mg per mL in dichloromethane. Solution 5: PCL was dissolved at 150 mg per mL in dichloromethane. Solution 6: PVA was prepared at 75 mg per mL in 100 mM phosphate buffer (pH 8).

[0096] An O / W emulsion was prepared by transferring 0.5 mL of solution 1 to a thick-walled glass pressure tube. 0.5 mL of solution 2 was added to make Lot 1, 0.5 mL of solution 3 was added to make Lot 3, 0.5 mL of solution 4 was added to make Lot 5, and 0.5 mL of solution 5 was added to make Lot 7. The two solutions were then mixed by repeated pipetting. 3.0 mL of solution 6 was then added, and the tube was vortexed for 10 seconds. The pressure tube was then emulsified by sonication at 30% amplitude for 1 minute using a Branson Digital Sonifier 250 while immersed in an ice-water bath. The emulsion was then added to a 50 mL beaker containing 30 mL of DPBS. This was then stirred at room temperature for 2 hours to evaporate the dichloromethane and form nanocarriers. A portion of the nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging at 75,600 × g for 50 minutes, removing the supernatant, and resuspending the pellet in DPBS. The washing procedure was repeated, and the pellet was then resuspended in DPBS to yield a nanocarrier suspension with a set concentration of 10 mg / mL based on polymer. The nanocarrier suspension was then filtered using a 0.22 μm PES membrane syringe filter (Millipore Part Number SLGP033RB) and, if necessary, a 0.45 μm PES membrane syringe filter (PALL Part Number 4614) and / or a 1.2 μm PES membrane syringe filter (PALL Part Number 4656). The filtered nanocarrier suspension was then stored at -20°C.

[0097] Nanocarrier size was determined by dynamic light scattering. The amount of rapamycin in the nanocarriers was determined by HPLC analysis. Filterability was determined by comparing the weight of the flow-through of the first sterile 0.22 μm filter with the actual mass of nanocarriers that passed through the filter prior to blocking or the yield determined from the total amount through the first and only filter. The total mass of dry nanocarriers per mL of suspension was determined gravimetrically.

[0098] For samples 2, 4, 6 and 8, solutions were prepared as follows: Solution 1: A mixture of polymer and rapamycin was prepared by dissolving PLA-PEG-Ome at 50 mg per mL and rapamycin at 32 mg per mL in dichloromethane. Solution 2: 100 DL 4A was dissolved at 150 mg per mL in dichloromethane. Solution 3: 5050 DLG 2.5A was dissolved at 150 mg per mL in dichloromethane. Solution 4: 7525 DLG 4A was dissolved at 150 mg per mL in dichloromethane. Solution 5: PCL was dissolved at 150 mg per mL in dichloromethane. Solution 6: Polyvinyl alcohol was prepared at 75 mg per mL in 100 mM phosphate buffer (pH 8).

[0099] An O / W emulsion was prepared by transferring 0.5 mL of solution 1 to a thick-walled glass pressure tube. To this was added 0.5 mL of solution 2 to make lot 2, 0.5 mL of solution 3 to make lot 4, 0.5 mL of solution 4 to make lot 6, and 0.5 mL of solution 5 to make lot 8. The two solutions were then mixed by repeated pipetting. The addition, washing, filtration, and storage of the PVA solution were the same as above. Nanocarrier size was evaluated as above. The results show that the inclusion of SPAN 40 in synthetic nanocarriers significantly increased the filterability of synthetic nanocarriers comprising polyester polymers.

[0100] [Table 6]

[0101] Example 7 - Synthetic nanocarriers with low HLB surfactant and significant RAPA loading result in sustained antigen-specific tolerance material and method PLA with an intrinsic viscosity of 0.41 dL / g was purchased from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, AL 35211) (product code 100 DL 4A). PLA-PEG-OMe block copolymer with approximately 5,000 Da methyl ether-terminated PEG blocks and an overall intrinsic viscosity of 0.50 DL / g was purchased from Lakeshore Biomaterials (756 Tom Martin Drive, Birmingham, AL 35211) (product code 100 DL mPEG 5000 5CE). Rapamycin was purchased from Concord Biotech Limited (1482-1486 Trasad Road, Dholka 382225, Ahmedabad, India) (product code SIROLIMUS). Sorbitan monopalmitate was purchased from Sigma-Aldrich (3050 Spruce St., St. Louis, MO 63103) (product code 388920). EMPROVE® Polyvinyl Alcohol (PVA) 4-88, USP (85-89% hydrolyzed, 3.4-4.6 mPa·s viscosity) was purchased from EMD Chemicals Inc. (480 South Democrat Road, Gibbstown, NJ 08027) (product code 1.41350). Dulbecco's Phosphate Buffered Saline 1× (DPBS) was purchased from Lonza (Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland) (product code 17-512Q).

[0102] The solutions were prepared as follows: Solution 1: A mixture of polymer, rapamycin, and sorbitan monopalmitate was prepared by dissolving PLA at 37.5 mg / mL, PLA-PEG-Ome at 12.5 mg / mL, rapamycin at 8 mg / mL, and sorbitan monopalmitate at 2.5 in dichloromethane. Solution 2: Polyvinyl alcohol was prepared at 50 mg / mL in 100 mM phosphate buffer (pH 8).

[0103] An O / W emulsion was prepared by combining Solution 1 (1.0 mL) and Solution 2 (3 mL) in a small glass pressure tube and vortexing for 10 seconds. The formulation was then homogenized by sonication at 30% amplitude for 1 minute. The emulsion was then added to an open beaker containing DPBS (30 mL). A second O / W emulsion was prepared using the same materials and methods as above and then added to the same beaker containing the first emulsion and DPBS. The combined emulsion was then stirred at room temperature for 2 hours to evaporate the dichloromethane and form nanocarriers. A portion of the nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube and centrifuging at 75,600 × g and 4 °C for 50 minutes, removing the supernatant, and resuspending the pellet in DPBS containing 0.25% (w / v) PVA. The washing procedure was repeated, and the pellet was then resuspended in DPBS containing 0.25% (w / v) PVA to obtain a nanocarrier suspension with a set concentration of 10 mg / mL based on polymer. The nanocarrier suspension was then filtered using a 0.22 μm PES membrane syringe filter (Millipore product number SLGP033RB). The filtered nanocarrier suspension was then stored at -20°C. The nanocarrier size was determined by dynamic light scattering. The amount of rapamycin in the nanocarriers was determined by HPLC analysis. The total mass of dry nanocarriers per mL of suspension was determined gravimetrically.

[0104] [Table 7]

[0105] The ability of synthetic nanocarriers to induce sustained immune tolerance to free rapamycin versus the model antigen KLH was evaluated. Groups of naive C57BL / 6 mice (n = 10 per group) were intravenously dosed with PBS (Group 1), 50 μg (∼2 mg / kg) free rapamycin alone or in combination with KLH (Groups 2 and 3, respectively), or 50 μg rapamycin encapsulated in synthetic nanocarriers alone (Group 6) or in combination with KLH (Groups 7 and 8) on days 0, 7, and 14 (Figure 3). To determine the effects of chronic rapamycin administration, Group 4 received free rapamycin alone (50 μg / day) five times per week from Day 0 to Day 20, or in combination with KLH administered once per week (Group 5). All groups were subsequently challenged with 200 μg of KLH on days 21, 28, and 35. Serum was collected and anti-KLH antibody responses were measured on days 35 and 42 (after two and three injections, respectively). Efficacy was assessed as the EC50 anti-KLH antibody titer as determined by ELISA. Figure 4 illustrates the protocol.

[0106] Control PBS-treated mice developed high levels of anti-KLH antibodies on days 35 and 42 after two and three KLH challenge injections, respectively. Mice treated with free rapamycin (either weekly or daily) in the absence of KLH developed similar levels of anti-KLH antibodies to the PBS-treated group. Mice treated with synthetic nanocarriers alone or daily free rapamycin and KLH showed delayed responses compared to the PBS control group, but titers were boosted with each KLH challenge. These results indicate that treatment with synthetic nanocarriers alone does not induce long-term immunosuppression and that KLH administered with daily free rapamycin does not induce sustained immune tolerance, even at the fifth full weekly dose of rapamycin as administered in the synthetic nanocarrier.

[0107] In contrast, mice treated with synthetic nanocarriers containing significant amounts of rapamycin plus KLH (groups 7 and 8) developed little or no detectable anti-KLH antibodies, even after receiving three weekly KLH challenges after treatment (a total of six KLH injections). This indicates sustained immune tolerance. Both lots of synthetic nanocarriers were similarly effective. All groups except the group treated with synthetic nanocarriers plus KLH developed anaphylactic responses by day 42. These results indicate that tolerization to KLH induced by treatment with synthetic nanocarriers containing significant amounts of rapamycin and KLH prevented the development of hypersensitivity responses.

[0108] To evaluate the antigen specificity of tolerance to KLH, all animals were challenged with OVA + CpG (35 μg + 20 μg) in the hind paw (sc) on days 49 and 56. Figure 5 shows that all animals developed similar levels of titers to OVA. This demonstrates that co-administration of synthetic nanocarriers with antigen can generate immunological tolerance and that synthetic nanocarrier treatment does not induce long-term immunosuppression. These results demonstrate that nanocarrier-encapsulated, rather than free, rapamycin (when present in significant amounts) induced sustained and antigen-specific immune tolerance when co-administered with a target antigen.

[0109] Example 8 - SPAN 40 increases the filterability of the rapalogs, rapamycin and everolimus material and method PLA with an intrinsic viscosity of 0.41 dL / g was purchased from Evonik Industries AG (Rellinghauser Strasse 1-11, Essen, Germany) (product code 100 DL 4A). PLA-PEG-OMe block copolymer with approximately 5,000 Da methyl ether-terminated PEG blocks and an overall intrinsic viscosity of 0.50 DL / g was purchased from Evonik Industries AG (Rellinghauser Strasse 1-11, Essen, Germany) (product code 100 DL mPEG 5000 5CE). Rapamycin was purchased from Concord Biotech Limited (1482-1486 Trasad Road, Dholka 382225, Ahmedabad, India) (product code SIROLIMUS). Everolimus was purchased from LC Laboratories (165 New Boston St # T, Woburn, MA 01801) (product code E-4040). Temsirolimus was purchased from LC Laboratories (165 New Boston Street, Woburn, MA 01801) (product number T-8040). Deforolimus was purchased from MedChem Express (11 Deer Park Drive, Suite 102D, Monmouth Junction, NJ 08852) (product code HY-50908). EMPROVE® Polyvinyl Alcohol 4-88, USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa·s) was purchased from EMD Chemicals Inc. (480 South Democrat Road, Gibbstown, NJ 08027) (product code 1.41350). Dulbecco's phosphate buffered saline 1× (DPBS) was purchased from Lonza (Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland) (product code 17-512Q).Sorbitan monopalmitate was purchased from Croda International (300-A Columbus Circle, Edison, NJ 08837) (product code SPAN 40).

[0110] The solutions were prepared as follows: Solution 1: A mixture of polymer and rapamycin was prepared by dissolving PLA at 150 mg / mL and PLA-PEG-Ome at 50 mg / mL. Solution 2: A rapamycin solution was prepared at 100 mg / mL in dichloromethane. Solution 3: An everolimus solution was prepared at 100 mg / mL in dichloromethane. Solution 4: A temsirolimus solution was prepared at 100 mg / mL in dichloromethane. Solution 5: A deforolimus solution was prepared at 100 mg / mL in dichloromethane. Solution 6: A sorbitan monopalmitate solution was prepared by dissolving SPAN 40 in dichloromethane at 50 mg / mL. Solution 7: Polyvinyl alcohol was prepared at 75 mg / mL in 100 mM phosphate buffer (pH 8).

[0111] O / W emulsions were prepared by adding solution 1 (0.5 mL) to a thick-walled pressure tube. For lots 1, 3, 5, and 7, this was combined with solution 6 (0.1 mL) and dichloromethane (0.28 mL). Lot 1 was then combined with solution 2 (0.12 mL), lot 3 with solution 3 (0.12 mL), lot 5 with solution 4 (0.12 mL), and lot 7 with solution 4 (0.12 mL). In a similar manner, lots 2, 4, 6, and 8 were combined with dichloromethane (0.38 mL), then lot 2 was combined with solution 2 (0.12 mL), lot 4 with solution 3 (0.12 mL), lot 6 with solution 4 (0.12 mL), and lot 8 with solution 5 (0.12 mL). For each individual lot, the total volume of the organic phase was therefore 1 mL. The combined organic phase solution was mixed by repeated pipetting. Next, Solution 7 (3.0 mL) was added, and the pressure tube was vortexed for 10 seconds. Then, while the pressure tube was immersed in an ice-water bath, it was sonicated for 1 minute at 30% amplitude using a Branson Digital Sonifier 250. The emulsion was then added to a 50 mL beaker containing DPBS (30 mL). This was then stirred at room temperature for 2 hours, allowing the dichloromethane to rapidly evaporate and form nanocarriers. The nanocarrier suspension was transferred to a centrifuge tube and centrifuged at 75,600 × g and 4 °C for 50 minutes. The supernatant was removed, and a portion of the nanocarriers was washed by resuspending the pellet in DPBS containing 0.25% (w / v) PVA. The washing procedure was repeated, and the pellet was then resuspended in DPBS containing 0.25% (w / v) PVA, resulting in a nanocarrier suspension with a set concentration of 10 mg / mL based on the polymer. The nanocarrier suspension was then filtered using a 0.22 μm PES membrane syringe filter (Millipore part number SLGP033RB). The filtered nanocarrier suspension was then stored at −20° C. The results show that incorporation of SPAN 40 in synthetic nanocarriers increased the filterability of the rapalogs, rapamycin and everolimus.

[0112] [Table 8]

[0113] Example 9 - Demonstrating the effect of component amounts on rapamycin loading and filterability of synthetic nanocarriers material and method PLA-PEG-OMe block copolymer with approximately 5,000 Da methyl ether-terminated PEG blocks and an overall intrinsic viscosity of 0.50 DL / g was purchased from Evonik Industries (Rellinghauser Strasse 1-11, 45128 Essen, Germany) (product code 100 DL mPEG 5000 5CE). PLA with an intrinsic viscosity of 0.41 dL / g was purchased from Evonik Industries (Rellinghauser Strasse 1-11, 45128 Essen, Germany) (product code 100 DL 4A). Rapamycin was purchased from Concord Biotech Limited (1482-1486 Trasad Road, Dholka 382225, Ahmedabad, India) (product code SIROLIMUS). Sorbitan monopalmitate was purchased from Croda (315 Cherry Lane, New Castle, Delaware 19720) (product code SPAN 40). Dichloromethane was purchased from Spectrum (14422 S San Pedro Gardena, CA 90248-2027) (product code M1266). EMPROVE® Polyvinyl Alcohol 4-88 (PVA), USP (85-89% hydrolyzed, viscosity 3.4-4.6 mPa·s) was purchased from EMD Chemicals Inc. (480 South Democrat Road, Gibbstown, NJ 08027) (product code 1.41350). Dulbecco's Phosphate Buffered Saline (DPBS), 1×, 0.0095 M (PO), calcium and magnesium free, was purchased from BioWhittaker (8316 West Route 24, Mapleton, IL 61547) (Part Number #12001, Product Code Lonza DPBS). Emulsification was performed using a Branson Digital Sonifier 250 with a 1 / 8" tapered tip titanium probe.

[0114] The solutions were prepared as follows: Polymer Solution: A polymer mixture was prepared by dissolving PLA-PEG-OMe (100 DL mPEG 5000 5CE) and PLA (100 DL 4A) in dichloromethane at the mg per mL indicated, at a PLA-PEG:PLA ratio of 1:3. Rapamycin Solution: Rapamycin was dissolved in dichloromethane at the mg per mL indicated. SPAN 40 Solution: Sorbitan monopalmitate (SPAN 40) was dissolved in dichloromethane at the mg per mL indicated. CHCl Solution: Dichloromethane (CHCl) was sterile filtered using a 0.2 μm PTFE membrane syringe filter (VWR part number 28145-491). PVA solution: Polyvinyl alcohol solution was prepared by dissolving polyvinyl alcohol (EMPROVE® Polyvinyl 4-88) in 100 mM phosphate buffer (pH 8) at the mg per mL indicated. DPBS PVA solution: A mixture of polyvinyl alcohol and Dulbecco's phosphate buffered saline, 1×, 0.0095 M (PO4), was prepared by dissolving polyvinyl alcohol (EMPROVE® Polyvinyl Alcohol 4-88) at 2.5 mg per mL in Dulbecco's phosphate buffered saline, 1×, 0.0095 M (PO4) (Lonza DPBS).

[0115] O / W emulsions were prepared by combining polymer solution, rapamycin solution, SPAN 40 solution, and / or CHCl solution (total volume 1–2 mL) in a thick-walled glass pressure tube. The solutions were mixed by repeated pipetting. Next, PVA solution (3–6 mL) was added (either as a single emulsion with 1 mL of organic phase and 3 mL of aqueous PVA solution, or as two single emulsions prepared sequentially). The formulation was vortex-mixed for 10 seconds and then sonicated at 30% amplitude for 1 minute while the pressure tube was immersed in an ice bath. The emulsion was then added to an open 50 mL beaker containing Lonza DPBS (30 mL). This was then stirred at room temperature for 2 hours to evaporate the dichloromethane and form the nanocarriers. A portion of the nanocarriers was washed by transferring the nanocarrier suspension to a centrifuge tube, centrifuging at 75,600 x g and 4°C for 50 minutes, removing the supernatant, and resuspending the pellet in DPBS PVA solution. The washing procedure was repeated, and the pellet was then resuspended in DPBS PVA solution to yield a nanocarrier suspension with a set concentration of 10 mg per mL based on polymer. The nanocarrier formulation was filtered using a 0.22 μm PES membrane syringe filter (Millex part number SLGP033RS). The mass of the filtered nanocarrier solution was measured. The filtered nanocarrier solution was then stored at -20°C.

[0116] Filterability is the percentage of measured nanocarriers passing through one 33 mm PES membrane 0.22 μm syringe filter (Millipore, part number SLGP033RB) in g / m of filter membrane surface area. 2 is given as: The results demonstrate the amounts of various components in a number of synthetic nanocarriers that can result in the first sterile-filterable synthetic nanocarriers with amounts of rapamycin predicted to be effective in vivo.

[0117] [Table 9]

Claims

1. a hydrophobic carrier material; Rapalog, and Nonionic surfactants with a hydrophilic-lipophilic balance (HLB) of 10 or less A composition comprising a synthetic nanocarrier comprising: the amount of said nonionic surfactant is ≧0.01 wt. % but ≦20 wt. % of said nonionic surfactant / hydrophobic carrier material (by weight); Rapalogs include rapamycin (sirolimus), temsirolimus (CCI-779), deforolimus, everolimus (RAD001), ridaforolimus (AP-23573), and zotarolimus (ABT-578); the nonionic surfactant comprises sorbitan monopalmitate (SM), SPAN® 20, SPAN® 40, SPAN® 60, SPAN® 80, oleyl alcohol, isopropyl palmitate, BRIJ® 52, BRIJ® 93, Pluronic® P-123, palmitic acid, or glyceryl tripalmitate; Preferably, the composition is first sterile filterable through a 0.22 μm filter.

2. (i) the nonionic surfactant is a nonionic surfactant having an HLB value of less than 10; (ii) the nonionic surfactant has an HLB value of less than 9; (iii) the nonionic surfactant has an HLB value of less than 8; (iv) the nonionic surfactant has an HLB value of less than 7; (v) the nonionic surfactant is a nonionic surfactant with an HLB value of less than 6; or (vi) The composition according to claim 1, wherein the nonionic surfactant is a nonionic surfactant having an HLB value of less than 5.

3. The composition of claim 1, wherein the nonionic surfactant is SPAN® 40.

4. 4. The composition of any one of claims 1 to 3, wherein the nonionic surfactant is encapsulated in a synthetic nanocarrier, is present on the surface of a synthetic nanocarrier, or both.

5. (i) the amount of the nonionic surfactant is ≥ 0.1 wt% but ≤ 15 wt% of the nonionic surfactant / hydrophobic carrier material (by weight); (ii) the amount of the nonionic surfactant is ≧1 wt. % but ≦13 wt. % of the nonionic surfactant / hydrophobic carrier material (by weight); or (iii) The composition of any one of claims 1 to 4, wherein the amount of the nonionic surfactant is ≥ 1 wt% but ≤ 9 wt% of the nonionic surfactant / hydrophobic carrier material (by weight).

6. (i) the hydrophobic carrier material comprises one or more hydrophobic polymers or lipids; (ii) the hydrophobic carrier material comprises one or more hydrophobic polymers, wherein the one or more hydrophobic polymers comprise a polyester; Preferably, wherein the polyester comprises PLA, PLG, PLGA, or polycaprolactone; or (iii) the hydrophobic carrier material comprises one or more hydrophobic polymers, wherein the one or more hydrophobic polymers comprise a polyester; Preferably, the composition according to any one of claims 1 to 5, wherein the polyester comprises PLA, PLG, PLGA, or polycaprolactone, and wherein the hydrophobic carrier material comprises or further comprises PLA-PEG, PLGA-PEG, or PCL-PEG.

7. (i) The amount of hydrophobic carrier material in the synthetic nanocarrier is 5 to 95 wt.% hydrophobic carrier material / total solids; or (ii) The composition of any one of claims 1 to 6, wherein the amount of hydrophobic carrier material in the synthetic nanocarrier is 60 to 95% by weight, hydrophobic carrier material / total solids.

8. (i) the amount of rapalog is ≥ 6 wt% but ≤ 50 wt%, rapalog / hydrophobic carrier material (by weight); (ii) the amount of rapalog is ≥ 7 wt% but ≤ 30 wt% rapalog / hydrophobic carrier material (by weight); or (iii) The composition of any one of claims 1 to 7, wherein the amount of rapalog is ≥ 8 wt% but ≤ 24 wt%, rapalog / hydrophobic carrier material (by weight).

9. The composition of any one of claims 1 to 8, wherein the rapalog is encapsulated in a synthetic nanocarrier.

10. Obtaining or providing a hydrophobic carrier material; Obtaining or providing a nonionic surfactant having an HLB value of 10 or less; obtaining or providing rapalogs; combining a hydrophobic carrier material with said nonionic surfactant and a rapalog to form a synthetic nanocarrier; and filtering the resulting composition; wherein filtering comprises filtering through a 0.22 μm filter. a method for producing a synthetic nanocarrier comprising the nonionic surfactant and a rapalog, comprising: the amount of the nonionic surfactant in the synthetic nanocarrier is ≧0.01 wt.% but ≦20 wt.% (nonionic surfactant / hydrophobic carrier material by weight); Rapalogs include rapamycin (sirolimus), temsirolimus (CCI-779), deforolimus, everolimus (RAD001), ridaforolimus (AP-23573), and zotarolimus (ABT-578); The method, wherein the nonionic surfactant comprises sorbitan monopalmitate (SM), SPAN® 20, SPAN® 40, SPAN® 60, SPAN® 80, oleyl alcohol, isopropyl palmitate, BRIJ® 52, BRIJ® 93, Pluronic® P-123, palmitic acid, or glyceryl tripalmitate.

11. Dissolving a hydrophobic carrier material, a nonionic surfactant having an HLB value of 10 or less, and a rapalog in a solvent; Obtaining or providing another surfactant; forming a first O / W emulsion with the dissolved hydrophobic carrier material, the nonionic surfactant, and the rapalog, and another surfactant, and then forming a second O / W emulsion; mixing the first O / W emulsion with the second O / W emulsion; and Evaporating the solvent further comprising 11. The method of claim 10, wherein the solvent is preferably dichloromethane, ethyl acetate, chloroform, or propylene carbonate.

12. (i) the nonionic surfactant is a nonionic surfactant having an HLB value of less than 10; (ii) the nonionic surfactant has an HLB value of less than 9; (iii) the nonionic surfactant has an HLB value of less than 8; (iv) the nonionic surfactant has an HLB value of less than 7; (v) the nonionic surfactant is a nonionic surfactant with an HLB value of less than 6; or (vi) The method according to claim 10 or 11, wherein the nonionic surfactant has an HLB value of less than 5.

13. The method of claim 10 or 11, wherein the nonionic surfactant is SPAN® 40.

14. 12. The method of claim 10 or 11, wherein the nonionic surfactant is encapsulated in a synthetic nanocarrier, is present on the surface of a synthetic nanocarrier, or both.

15. (i) the amount of the nonionic surfactant is ≥ 0.1 wt% but ≤ 15 wt% of the nonionic surfactant / hydrophobic carrier material (by weight); (ii) the amount of the nonionic surfactant is ≧1 wt. % but ≦13 wt. % of the nonionic surfactant / hydrophobic carrier material (by weight); or 12. The method of claim 10 or 11, wherein the amount of nonionic surfactant is ≥ 1 wt. % but ≤ 9 wt. % of the nonionic surfactant / hydrophobic carrier material (by weight).

16. (i) the hydrophobic carrier material comprises one or more hydrophobic polymers or lipids; (ii) the hydrophobic carrier material comprises one or more hydrophobic polymers, wherein the one or more hydrophobic polymers comprise a polyester; Preferably, wherein the polyester comprises PLA, PLG, PLGA, or polycaprolactone; or (iii) the hydrophobic carrier material comprises one or more hydrophobic polymers, wherein the one or more hydrophobic polymers comprise a polyester; 12. The method of claim 10 or 11, wherein the polyester preferably comprises PLA, PLG, PLGA, or polycaprolactone, and wherein the hydrophobic carrier material comprises or further comprises PLA-PEG, PLGA-PEG, or PCL-PEG.

17. (i) The amount of hydrophobic carrier material in the synthetic nanocarrier is 5 to 95 wt.% hydrophobic carrier material / total solids; or (ii) The method of claim 10 or 11, wherein the amount of hydrophobic carrier material in the synthetic nanocarrier is 60 to 95% by weight, hydrophobic carrier material / total solids.

18. (i) the amount of rapalog is ≥ 6 wt% but ≤ 50 wt%, rapalog / hydrophobic carrier material (by weight); (ii) the amount of rapalog is ≥ 7 wt% but ≤ 30 wt% rapalog / hydrophobic carrier material (by weight); or (iii) The method of claim 10 or 11, wherein the amount of rapalog is ≥ 8 wt% but ≤ 24 wt%, rapalog / hydrophobic carrier material (by weight).

19. The method of claim 10 or 11, wherein the rapalog is encapsulated in a synthetic nanocarrier.

20. (i) further comprising an antigen; or (ii) the composition of any one of claims 1 to 9, further comprising an antigen, wherein the antigen is admixed with the synthetic nanocarrier in the composition.

21. 21. A composition according to any one of claims 1 to 9 and 20, wherein the mean of the particle size distribution of the synthetic nanocarriers, obtained using dynamic light scattering, is greater than 120 nm in diameter, preferably greater than 150 nm in diameter.

22. 22. The composition of any one of claims 1 to 9, 20 and 21, further comprising a pharmaceutically acceptable carrier.

23. A kit comprising the composition of any one of claims 1 to 9 and 20 to 22.

24. 23. A composition according to any one of claims 1 to 9 and 20 to 22 for use in modulating an immune response.

Images