Composite exosome compositions and applications thereof

Abstract

In one aspect, composite exosomal compositions are described herein employing a linker for coupling small molecules to the exosome. In some embodiments, a composite composition comprises an exosome, and at least one small molecule coupled or attached to the exosome via a linker, the linker at least partially disposed in the exosomal membrane.

Description

[0001] COMPOSITE EXOSOME COMPOSITIONS AND APPLICATIONS THEREOF

[0002] RELATED APPLICATION DATA

[0003] The present application claims priority pursuant to Article 8 of the Patent Cooperation Treaty to United States Provisional Patent Application Serial Number 63 / 542,843 filed October 6, 2023, which is incorporated herein by reference in its entirety.

[0004] FIELD

[0005] The present invention relates to exosome compositions and, in particular, to exosomes for delivery of small molecules to sites of diseased tissue.

[0006] BACKGROUND

[0007] Exosomes are membrane vesicles secreted by cells and implicated in intercellular communication. Exosomes generally have a size of 40 nm to 150 nm and carry various biomolecular species, including proteins, lipids, and nucleic acids. Such biomolecular cargo contained in the exosomes, which can reprogram and alter various signaling pathways, can be transferred to neighboring or distant cells through the uptake / endocytosis of exosomes.

[0008] The ability of exosomes to carry biomolecular cargo has attracted research and development centered on employing exosomes for drug delivery. At present, there are over two dozen biotechnology companies in various stages of pre-clinical and clinical development of exosome-based products, including exosomes being evaluated as cancer vaccines in human trials. However, loading pharmaceutical compositions into exosomes is very difficult and can be highly inefficient, leading to low yields and high losses of expensive drugs. Pharmaceutical compositions struggle to integrate with exosomes due to substantial differences in hydrophobic character.

[0009] SUMMARY

[0010] In view of the foregoing disadvantages, new strategies are required for employing exosomes for the delivery of pharmaceutical compositions. In one aspect, composite exosomal compositions are described herein employing a linker for coupling small molecules to the exosome. In some embodiments, a composite composition comprises an exosome, and at least one small molecule coupled or attached to the exosome via a linker, the linker at least partially disposed in the exosomal membrane.

[0011] Turning now to specific components, exosomes employed in the composite compositions can comprise any exosome consistent with the technical objectives described herein. In some embodiments, for example, the exosome can be sourced from monocytes and / or differentiated monocytes (macrophages and dendritic cells). Macrophages can include Ml polarized macrophages and / or M2 polarized macrophages. Additional sources of exosomes can include stem cell derived exosomes, milk (bovine) derived exosomes, and plant derived exosomes. Additional sources of exosomes include mesenchymal stem cells

[0012] As described herein, at least one small molecule is coupled or attached to the exosome via a linker, the linker at least partially disposed in the exosomal membrane. The linker comprises chemical species operable to insert into the exosomal membrane, thereby anchoring the small molecule to the exosome. In some embodiments, the linker comprises one or more aliphatic chains or moieties. Suitable aliphatic chains, in some embodiments, are G> - C30, such as C10 - C20. Length of the aliphatic chain can be varied as desired to the extent the aliphatic linker anchors the small molecule to the exosome via insertion or interaction with the exosomal membrane. Suitable aliphatic chains can be linear or branched and optionally contain one or more points of unsaturation. In some embodiments, the linker comprises two or more aliphatic chains or tails. Additionally, in some embodiments, the linker can comprise one or more cyclic, heterocyclic, aryl or heteroaryl moieties. Such moieties can comprise plurality of fused or unfused ring structures.

[0013] The small molecule coupled to the exosome via the linker, in some embodiments, is a pharmaceutical compound or synthetic compound exhibiting bioactivity. For example, the small molecule can exhibit anticancer, antibacterial, antiviral, or anti-inflammatory activity. A nonlimiting list of small molecule classes for use in composite exosomal compositions described herein is provided in Table 1.

[0014] Table 1 - Small Molecule Classifications

[0015] Additionally, Table 2 provides several physical properties of various small molecules for use in composite exosomal compositions described herein, including logP, solubility, and other parameters.

[0016] Table 2 - Small Molecule Physical Properties

[0017] aComputed by PubChem 2.1 (PubChem release 2021.05.07)bComputed by XLogP33.0 (PubChem release 2021.05.07)c(ALogPS 3.0) ochem.eu / homedMelting Point prediction (best Estate) ochem.eu / homeeWater solubility model based on logP and Melting Point ochem.eu / home

[0018] In some embodiments, small molecules of composite compositions described herein have a molecular weight less than 2,000 g / mol or less than 1,000 g / mol. The small molecule can be covalently bound to the linker. In some embodiments, the small molecule can be bound to the linker via a moiety selected from the group consisting of carbamate, ester, amide, disulfide, immine, enamine, beta amino ester, malemeide, and diazo. Specific identity of the moiety binding the small molecule to the linker can be dependent on several considerations including, but not limited, to specific chemical identity of the small molecule and the local biological environment in which the composite exosome will be disposed For example, the moiety binding the small molecule to the linker can be cleaved by one or more species or conditions in the local environment. In some embodiments, the moiety is cleaved by an enzyme or other protein species in the local biological environment. Alternatively, the moiety can be cleaved by pH conditions (acidic or basic) of the local biological environment. The local biological environment can be within a cell or in the intercellular environment.

[0019] Cleaving the moiety releases the small molecule into the local biological environment. Coupling the small molecule to linker and subsequent cleavage of the small molecule does not alter or substantially alter the biological activity and / or structure of the small molecule. Further, release kinetics of the small molecule into the local biological environment, in some embodiments, can be tailored via selection of the linker identity. In some embodiments, shorter linker structures, such as shorter aliphatic chains, can demonstrate a faster release profile of the small molecule. The shorter aliphatic chains, for example, do not anchor as deeply in the exosomal membrane, thereby facilitating accelerated release kinetics relative to longer aliphatic chains. Table 3 provides some physical properties of the linker.

[0020] Table 3 - Physical Properties of the Linker (represented as alcohol) aComputed by PubChem 2.1 (PubChem release 2021.05.07)bComputed by XLogP33.0 (PubChem release 2021.05.07)c(ALogPS 3.0) ochem.eu / homedMelting Point prediction (best Estate) ochem.eu / homeeWater solubility model based on logP and Melting Point ochem.eu / home

[0021] Table 4 provides several physical properties of the small molecule resiquimod functionalized with various linkers. Table 4 - Physical Properties of Small Molecule Coupled to Linker

[0022] aComputed by PubChem 2.1 (PubChem release 2021.05.07)c(ALogPS 3.0) ochem.eu / homedMelting Point prediction (best Estate) ochem.eu / homeeWater solubility model based on logP and Melting Point ochem.eu / home Species of small molecules coupled to a linker, in some embodiments, do not self-assemble in the aqueous phase or aqueous-based phase. Non-self-assembly can facilitate anchoring of the small molecule to the exosome via the linker.

[0023] In some embodiments, a single species of small molecule is coupled to the exosome via the linker. Alternatively, a plurality of different small molecule species can be coupled to the same exosome via linkers In such embodiments, the linking moieties binding the differing small molecule species to the exosome can be selected to release the small molecules into the desired biological environment simultaneously or in a staggered manner.

[0024] In another aspect, dispersions are described herein. A dispersion, in some embodiments, comprises an aqueous or aqueous-based continuous phase, and a dispersed phase comprising composite particles, the composite particles comprising an exosome, and at least one small molecule coupled to the exosome via a linker, the linker at least partially inserting into the exosomal membrane. Composite particles comprising the exosome, linker and small molecule can have any composition, structure, and / or properties described hereinabove. In some embodiments, the composite particles have an average size of 50 nm to 150 nm or 90 nm to 100 nm. In some embodiments, the composite particles have the same composition, including the same small molecule. In other embodiments, composite particles can have differing compositions, including differing small molecules. Additionally, the small molecule can be present in the dispersion in any desired amount. In some embodiments, for example, the small molecule is present at a concentration of 0.5-2 mg / ml or greater than 2 mg / ml.

[0025] In another aspect, methods of making composite compositions are described herein. In some embodiments, a method comprises providing an exosome, and inserting a linker into the exosomal membrane, the linker coupled to a small molecule. The exosome, linker and small molecule can have any composition, structure, and / or properties described herein above. In some embodiments, the linker is coupled to the small molecule prior to insertion in the exosomal membrane. Incorporation efficiency of the small molecules with the exosomes in the production of a dispersion described herein above, in some embodiments, can be at least 60%, at least 70%, least 80, or at least 90%. Incorporation efficiency, in some embodiments, can range from 60- 100%, in some embodiments.

[0026] In some embodiments, for example, aliphatic derivatives of small molecule immunomodulators are dissolved to near saturation in organic solvent (typically ethanol). The aliphatic derivative refers to the small molecule modified with an aliphatic linker, as described herein. A small amount (typically 30 uL) (no more than 10% final volume) of the small molecule derivative solution is added to exosomes in PBS (typically 300 uL) (typically 8.0E1 1 exosomes / mL). Samples are incubated for 30 minutes at room temperature to allow for insertion and incorporation of API into the exosomal membranes. Exosomes loaded with drug are separated from unloaded drug on a CL-6B size exclusion column. HPLC is used to analyze the amount of API loaded into the exosomes. Exosomes loaded with API are eluted in PBS.

[0027] In a further aspect, methods of treating disease are described herein. In some embodiments, a method comprises administering to a patient in need thereof a dispersion comprising an aqueous or aqueous-based continuous phase, and a dispersed phase comprising composite particles, the composite particles comprising an exosome, and at least one small molecule coupled to the exosome via a linker, the linker at least partially inserting into the exosomal membrane. The composite particles can have any composition, structure, and / or properties described hereinabove. The small molecule component of the composite particles can be selected to treat various diseases including autoimmune disorders, such as rheumatoid arthritis, TLR4 / 7 / 8 / 9 mediated inflammatory diseases, lupus, and / or multiple sclerosis. Additional diseases can include allergic conditions, lymphomas, 1BD, leukemia, cardiovascular disease, atherosclerosis, COPD, and ALS. Various cancers may also be treated with compositions described herein, including medulloblastoma and glioblastoma. In some embodiments, compositions described herein can be employed to treat neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease. Dispersions comprising composite particles can be administered by any desired technique, including intravenous, oral, subcutaneous, ocular, or inhalation.

[0028] These and other embodiments are further described in the following detailed description.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1A illustrates the chemical structure of resiquimod and various carbamate and amide derivatives for anchoring with the exosomal membrane, according to some embodiments.

[0031] FIG. IB characterizes TNF-Alpha expression in RAW264.7 macrophages (derivatives and resiquimod at 5 ug / mL resiquimod molar equivalent; INF-y and IL-4 are included as positive and negative controls, respectively). FIG. 1C provides loading efficiencies of the resiquimod derivatives via insertion of the aliphatic linkers into the exosomal membrane.

[0032] FIG. ID provides exosome vs. mouse plasma partition constant, Keq = [drug in plasma] / [dmg in exosome], (Keq=0 means no release of drug from exosome to serum).

[0033] FIG. 2A characterizes brain accumulation of naked BDNF or BDNF in ExoBDNF in healthy or brain-inflamed mice at 10 min. n = 4, **p < 0.01 by one-way ANOVA and post Newman-Keuls multiple comparison test.

[0034] FIG. 2B characterizes the distribution of BDNF or BDNF in ExoBDNF in brain parenchyma at 10 min in healthy mice determined by capillary depletion assay, n = 5,&&p < 0.01 and&&&p < 0.001 by paired two-tailed t-test, p < 0.001 by unpaired two-tailed t-test.

[0035] FIG. 3 illustrates tumor tissue accumulation of DIL-exosomes 24 h after administration in G-Smo mice. Gray arrows show blood vessels, white arrows show examples of labelled tumor cells.

[0036] FIG. 4 shows the reaction of resiquimod with hexadecyl chloroformate to yield an alkyldecorated resiquimod compound.

[0037] FIG. 5 characterizes the resiquimod-C16 carbamate derivative using HNMR. Detected proton peaks are labeled on the spectrum with peak areas approximately corresponding to the expected number of protons.

[0038] FIG. 6 displays HPLC results of resiquimod derivatives, with retention times on the Cl 8 reversed phase column measured as an approximation of relative hydrophobicity compared to the parent drug.

[0039] FIG. 7 illustrates additional API synthesized with C16 carbamate ligands according to the procedure shown in FIG. 4.

[0040] FIG. 8 characterizes a palbociclib-C16 carbamate derivative using HNMR. Detected proton peaks are labeled on the spectrum with peak areas approximately corresponding to the expected number of protons.

[0041] FIG. 9 shows a synthesis scheme for RA-16, a resiquimod pro-drug with amide linker to C16.

[0042] FIG. 10 illustrates a general scheme for loading aliphatic pro-drugs into exosomes, with RC16 displayed as an exemplary pro-drug. FIG. 11 displays graphical representations of the effects of exosome concentration and drug concentration on drug loading. The left panel shows the changes in loading efficiency when exosome concentration varies with constant RC16 loading. When the concentration of exosomes (“high”) was reduced by half (“low”), so was the loading efficiency. In the right panel, different amounts of RC16 was added to the same number of exosomes (0.3 mL of 8.0E11 exosomes / mL), and final RC16 concentrations and loading efficiencies were measured.

[0043] FIG. 12 displays graphical representations of loading efficiency of different resiquimod derivatives and confirmation of drug loading by in vitro drug release methods. In the left panel, loading efficiencies were measured for different derivatives, where 300 pg of the derivatives were added to 0.3 mL of 8.0E11 exosomes / mL. In the right panel, exosomes loaded with RC16 derivative were placed in dialysis devices and dialyzed against 5% serum in PBS The RC16- loaded exosomes were compared to free RC16 release from dialysis device over 24 hours.

[0044] FIG. 13 displays graphical representations of recovery of exosomes after purification and protein binding of resiquimod derivatives. In the left panel, exosome recovery after loading and purification was determined for RC16 and other derivatives. Exosome concentration was measured using nanoparticle tracking analysis before and after drug-loading. In the right panel, equilibrium dialysis was used to measure protein binding of API after a 4 hour incubation against human plasma.

[0045] FIG. 14 displays the loading efficiency of Palbociclib-C16 derivative loaded into exosomes. Palbociclib-C16 was loaded in to exosomes in a modified procedure of FIG. 10, and loading efficiency was measured for parent API and the Cl 6 derivative.

[0046] FIG. 15 is a graphical representation of stability of the RC 16 derivative in biological matrices. RC16 (100 pg / mL) was incubated in plasma from mice, rats, and humans for 48 hours and aliquots were taken to measure the concentrations of resiquimod (parent drug) and RC16.

[0047] FIG. 16 displays graphical representations of the effects of exosomes derived from different sources on monocytes and macrophages. Exosomes were isolated from bone polarized macorphages (either M0, Ml, or M2), and bone marrow derived monocytes or macrophages from Balb / c mice were exposed to the exosomes. TNF-a expression was measured after 24 hour exposure for monocytes (left panel) and macrophages (right panel). FIG. 17 is a graphical representation of exosome accumulation in animal brains. DiR fluorescently labeled exosomes were injected into 10 day old pups both with and without medulloblastoma tumors in the Gsmo mouse model of medulloblastoma. At 24 hours, the full body of the mice were imaged and fluorescence was measured in the head, normalized to the amount of injected fluorescence in the exosomes. Exosomes from MO, Ml, and M2 macrophages were injected and compared.

[0048] FIG. 18 is a graphical representation of exosome accumulation in healthy and diseased animal brains. DiR fluorescently labeled exosomes were injected into 10 day old pups both with and without medulloblastoma tumors in the Gsmo mouse model of medulloblastoma. At 24 hours, the full body of the mice were imaged and fluorescence was measured in the head, normalized to the amount of injected fluorescence in the exosomes Exosomes from MO, M 1 , and M2 macrophages were injected and compared for healthy and tumor mice.

[0049] FIG. 19 is a graphical representation of RC16 and resiquimod treatment in a 4T1 model of triple negative breast cancer. Balb / c mice were inoculated with 500,000 4T1 tumor cells in the left mammary fat pad. Once tumors reached 100 mm3, mice were injected with saline, 5 mg / kg resiquimod equivalent of resiquimod, or RC16 by subcutaneous injection on days 0, 3, 7, and 10. Tumor sizes were measured with calipers.

[0050] FIG. 20 is a graphical representation of Ml and M2 Exo-RC16 treatment in a 4T1 model of triple negative breast cancer. Balb / c mice were inoculated with 500,000 4T1 tumor cells in the left mammary fat pad. Once tumors reached 100 mm3, mice were injected with saline, 5 mg / kg Ml Exo-RC16, or M2 Exo-RC16 by subcutaneous injection on days 0, 3, 7, and 10. Tumor sizes were measured with calipers.

[0051] DETIALED DESCRIPTION

[0052] Embodiments described herein can be understood more readily by reference to the following detailed description and examples. Methods, devices, and features described herein, however, are not limited to the specific embodiments presented in the detailed description and examples. It should be recognized that these embodiments are merely illustrative of the principles of the present disclosure. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the disclosure. EXAMPLE 1 - Composite Exosomal Compositions

[0053] A series of new hydrophobically modified carbamate prodrugs of resiquimod operable to anchor the drug in the exosome membrane (FIG. 1A) were developed. In compounds 1-4 alkyl or cholesteryl groups are linked to resiquimods’ functionally important amino group via carbamate link that is cleavable by CES in cells. The biological activity of new prodrugs was confirmed by measuring the TNF-Alpha expression in RAW264.7 macrophages (FIG. IB). Raw macrophage cell line was treated with 5 ug / mL resiquimod equivalent of each synthesized derivative as well as an INF-y positive control and IL-4 negative control. TNF-Alpha expression downstream of TLR activation was measured by ELISA after 24 hour exposure to derivatives. RC6 and RC10 derivatives had decreased activation compared to resiquimod, as did the cholesterol derivative. Of all the prodrugs the RC16 stimulated the highest fold-change in TNF- Alpha expression which was similar to that of resiquimod (FIG. IB). This derivative was also best loaded in exosomes (FIG. 1C). The prodrug was completely retained in exosomes in the serum-partitioning assay (FIG. ID). Based on the activity and loading data we selected RC 16 as the lead compound for these studies.

[0054] EXAMPLE 2 - Exosomal Delivery to the Brain

[0055] To determine whether macrophage exosomes could deliver cargo to the brain, the macrophage exosomes were loaded them with the brain derived neurotrophic factor (BDNF). We further compared brain accumulation of free BDNF, and exosome BDNF (ExoBDNF) in healthy and brain-inflamed mice (FIG. 2A). The accumulation of ExoBDNF in healthy mice brain was slightly but not significantly increased compared to BDNF alone. Brain inflammation resulted in a trend to increase the brain accumulation of free BDNF, but the difference was not significant compared to the healthy mice. In contrast, accumulation of ExoBDNF in the brain-inflamed mice was significantly increased compared to the same formulation in the healthy animals (3.6-fold). Moreover, the brain accumulation of ExoBDNF was also superior to that of the BDNF alone in the inflamed brain (2.2-fold). Importantly in the capillary depletion experiment, 89% and 95% of the BDNF accumulated in the brain distributed to the brain parenchyma in the BDNF alone and ExoBDNF groups (FIG. 2B), suggesting that both BDNF and BDNF in ExoBDNF group can penetrate the BBB. In addition, brain accumulation of exosome formulated BDNF in the parenchyma fraction was significantly higher than BDNF alone in healthy mice.

[0056] To examine whether exosomes can accumulate in medulloblastoma, fluorescent- tagged exosomes were administered to G-Smo mice, which are engineered to develop SHH driven medulloblastoma. We labelled macrophage exosomes with DIL (l,T-dioctadecyl-3,3,3',3 - tetramethylindo-carbocyanine perchlorate) and administered them via intraperitoneal (IP) injection to 13 -day old G-Smo mice, then examined fluorescence in the brain tumors after 24 hours. Signal in the tumors was noted (FIG. 3) including tumor cells (white arrows show examples) and vasculature (red arrows), showing that exosomes can potentially deliver cargo to the tumor. Accordingly, exosomes modified with small molecules, as described herein, may find application in the treatment of various brain diseases, including brain cancers.

[0057] Various embodiments of the invention have been described in fulfillment of the various objects of the invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations thereof will be readily apparent to those skilled in the art without departing from the spirit and scope of the invention.

[0058] EXAMPLE 3 - Cl 6 Carbamate Derivative Synthesis and Characterization

[0059] In FIG. 4, Resiquimod (15 mg, 48 pmol, 1 eq.) was dissolved in 1.0 mL dry dichloromethane with triethyl-amine. After the addition of alkyl chloroformate (10 eq.) in 1.0 mL dry dichloromethane, the reaction mixture was stirred on the ice until no further conversion could be observed (3-12 hr), monitored via HPLC and TLC. The reaction mixture was washed three times with water (2.0 mL), dried over sodium sulfate, and was evaporated. Product was washed three times with hexanes (2.0 mL) to get rid of excess alkyl chloroformate. Product was then dried on rotary evaporator under vacuum.

[0060] The result was an approximately 95-100% reaction yield of alkyl-decorated resiquimod compound. The reaction yield was typically of greater than 98% purity at scales ranging from 15-100 mg starting material. Samples were prepared according to the scheme in FIG. 4 with different alkyl chloroformates and cholesterol chloroformate and run on LC-MS to determine molecular weight and expected elemental analysis. These experimentally determined molecular weights (resiquimod, 314.17 g / mol; RC6, 442.56 g / mol; RC10, 498.67 g / mol; RC16, 582.83 g / mol; and RChol, 727.05 g / mol) were in agreement with predicted values. The RC16 derivative was then dissolved in chloroform for an HNMR experiment, as shown in FIG. 5. All expected proton peaks were detected and labeled on the spectrum with peak areas approximately corresponding to the expected number of protons.

[0061] For characterization, all synthesized derivatives were solubilized to approximately 25 pg / mL in 90% acetonitrile and 10% water. Samples were injected on a C18 reversed phase column with a 90% acetonitrile / 10% water mobile phase In FIG. 6, retention times are measured as an approximation of relative hydrophobicity compared to the parent drug. UV absorbance at 320 nm is displayed on the chromatogram. Longer alkyl chains (and chloroform) increased the retention time on the C18 HPLC column compared to the parent drug. This allows the inference that relative hydrophobicity with the cholesterol derivative being the most hydrophobic of the new pro-drugs. No other peaks are observed outside the derivatives indicating the purity of the synthesized compounds.

[0062] EXAMPLE 4 - Additional API Synthesis with C16 Carbamate Ligands

[0063] In FIG. 7, additional API were synthesized with the C 16 carbamate ligand according to the same procedure in FIG. 4 originally developed for resiquimod. A carbamate bond was formed on both primary, secondary, and cyclic amines at the circled sites in FIG. 7. Purity and yield varied.

[0064] The Palbociclib-C16 derivative was then dissolved in chloroform for an HNMR experiment, shown in FIG. 8. All expected proton peaks are detected and labeled on the spectrum with peak areas approximately corresponding to the expected number of protons.

[0065] EXAMPLE 5 -Synthesis ofRA-16

[0066] A new resiquimod derivative, RA-16, was synthesized using palmitoyl chloride to form an amide bond rather than a chloroformate, as shown in FIG. 9. The work-up remained the same as the procedure in FIG. 4, but the products were slight different. Resiquimod was in 2-fold excess rather than the C16 ligand. The result was an amide containing C16 resiquimod drug, which was synthesized with a poorly degradable amide linker for more stable compound.

[0067] EXAMPLE 6 - Aliphatic Pro-drug Loading

[0068] A general scheme for loading aliphatic pro-drugs into exosomes is shown in FIG. 10. Briefly, after synthesis of a pro-drug, the API is dissolved to a high concentration in ethanol (for example approximately 10 to 20 mg / mL). API solution is added to the exosomes (in PBS) to the desired amount of API. The solution is made to be 10% ethanol by addition of pure ethanol (if needed) and the concentration is not to exceed 10% ethanol. The solution is incubated at room temperature for 10 minutes and free, unloaded drug is separated from drug loaded exosomes on a CL-6B size exclusion column. CL-6B separation yields 100% exosome recovery after separation and purification from unloaded API.

[0069] In the embodiment displayed in FIG. 10, a resiquimod C16 conjugate (RC16) is first prepared as described in Example 3. The RC16 is dissolved in ethanol to 10 mg / mL. Next 350 pL of exosomes in PBS, 17.5 pL of ethanol, and 17.5 pL of RC16 (approximately 10% ethanol final concentration) are combined and incubated at room temperature for 10 minutes. The lipid- like structure enables insertion of the drug conjugate into the lipid-bilayer of the exosomes. Then, unloaded drug is separated from the drug-loaded exosomes using a CL-6B size exclusion column.

[0070] The effects of exosome concentration and drug concentration on drug loading were examined. Briefly, 0.3 mL of 8.0E11 exosomes / mL were incubated with 30 pL of RC16 (different amounts) in ethanol for 10 minutes (See FIG. 10 for loading methods). Loaded exosomes were recovered from column and RC16 derivative was quantified on HPLC. Final RC16 concentration and the loading efficiency (% of API added that is recovered loaded in exosomes) were then measured. In the left panel of FIG. 11, when the amount of RC16 added is held the same, and exosome concentration varies, the loading efficiency changes in proportion to the exosome concentrations. When the concentration of exosomes was reduced by half, so was the loading efficiency. In the right panel of FIG. 11, different amounts of RC16 were added to the same number of exosomes (0 3 mL of 8.0E11 exosomes / mL). In this case, the overall RC16 concentration increased with increasing drug added, and the loading efficiency reached 100% at high concentrations or RC16 indicating a possible cooperative effect in drug-loading.

[0071] The loading efficiency of different resiquimod derivatives was then evaluated and drug loading was confirmed by in vitro drug release methods. In the left panel of FIG. 12, 300 pg of different derivatives were added to 0.3 mL of 8.0E11 exosomes / mL and loading efficiency was measured. In the right panel of FIG. 12, the exosomes loaded with RC16 derivative were placed in dialysis devices and dialyzed against 5% serum in PBS This was compared to free RC16 release from dialysis device. As the length of the aliphatic chain in the resiquimod derivative increased, the loading efficiency increased as well. On the other hand, the cholesterol derivative was less effective and actually formed its own micelles. These micelles did NOT co-elute with exosomes on the CL-6B column during separation. RC16 loaded in to exosomes maintained a pro-longed release profile compared to free-drug over 24 hours with a significant amount (75%) remaining loaded in exosomes after 24 hours.

[0072] Recovery of exosomes after purification and protein binding of resiquimod derivatives was then evaluated. In the left panel of FIG. 13, exosome concentrations were measured using nanoparticle tracking analysis before and after drug-loading and the number of exosomes was compared. In the right panel of FIG. 13, equilibrium dialysis was used to measure protein binding of API after a 4 hour incubation against human plasma. Approximately 100% of exosomes were recovered from all combined RC16 separations performed as well as separations of all other derivatives combined As the aliphatic chain length increased, the percentage of API which was protein bound also increased, up to 100% for RC10, RC16, and RCholesterol.

[0073] In FIG. 14, the Palbociclib-C16 derivative loading efficiency was evaluated. Palbociclib- C16 was loaded according to the procedure described in FIG. 10. However, Palbociclib and Palbociclib-C16 were dissolved in DMSO and the solution was made to be 5% DMSO rather than 10% ethanol. Loading efficiency was measured for parent API and the C 16 derivative. Palbociclib was determined to load at 58% efficiency, while no palbociclib was detected in the parent drug loading attempt.

[0074] In FIG. 15, the stability of the RC16 derivative in biological matrices was investigated. RC16 (100 pg / mL) was incubated in plasma from mice, rats, and humans for 48 hours and aliquots were taken to measure the concentrations of resiquimod (parent drug) and RC16. Concentrations were measured on HPLC. RC16 was rapidly metabolized in rodent plasma by circulating carboxylesterases, but they were stable over 48 hours in human plasma which lacks such enzymes. Results indicate that the derivatives are capable of being metabolized to the parent API.

[0075] The modulation of monocytes and macrophages by exosomes derived from different sources was then determined. Exosomes were isolated from bone polarized macorphages (either M0, Ml, or M2) and bone marrow derived monocytes or macrophages from Balb / c mice were exposed to the exosomes. TNF-a expression was measured after 24 hour exposure in FIG. 16. In bone marrow derived monocytes (left panel of FIG. 16), Ml derived exosomes showed significantly increased activation compared to MO and M2 derived monocytes. There was no difference between bone marrow derived macrophages exposed to different exosomes (right panel of FIG. 16).

[0076] EXAMPLE 7 - Exosome Targeting

[0077] Exosome delivery to animals with medulloblastoma tumors was evaluated with fluorescence imaging to determine accumulation of exosomes of difference sources. In FIG. 17, exosomes were fluorescently labeled with a DiR lipophilic dye and injected into 10 day old pups both with and without medulloblastoma tumors in the Gsmo mouse model of medulloblastoma. At 24 hours, an IVIS imaging system was used to image full body of mice and fluorescence was measured in the head, normalized to the amount of injected fluorescence in the exosomes. Exosomes from MO, Ml, and M2 macrophages were injected and compared. In tumor bearing mice, M2 derived exosomes showed an increased signal compared to MO and Ml derived exosomes which was statistically significant compared to Ml exosomes. This data suggests that M2 exosomes may home better to the brain / tumors.

[0078] In FIG. 18, exosome delivery in healthy mice and mice with medulloblastoma tumors was evaluated. DiR exosomes were injected into 10 day old pups both with and without medulloblastoma tumors in the Gsmo mouse model of medulloblastoma. At 24 hours, the full body of mice was imaged and fluorescence was measured in the head, normalized to the amount of injected fluorescence in the exosomes. Exosomes from MO, Ml, and M2 macrophages were injected and compared for healthy and tumor mice. M2 derived exosomes showed increased accumulation in tumor bearing mice compared to healthy mice. MO and Ml did not show this preference indicating that M2 exosomes may have preferential targeting to tumors / sites of inflammation.

[0079] A 4T1 model of triple negative breast cancer was used to investigate API activity in FIG. 19 and exosome-loaded API activity in FIG. 20. Balb / c mice were inoculated with 500,000 4T1 tumor cells in the left mammary fat pad. Once tumors reached 100 mm3, mice were injected with saline, 5 mg / kg resiquimod equivalent of resiquimod, or RC16 by subcutaneous injection on days 0, 3, 7, and 10 (FIG. 19). Tumor sizes were measured with calipers. RC16 and Resiquimod both significantly reduced tumor burden compared to no treatment and saline treated mice. However, there was no difference in tumor burden between resiquimod and RC16 treated mice. These results show that RC16 is comparably active to resiquimod in vivo.

[0080] In FIG. 20, once tumors reached 100 mm3, mice were injected with saline, 5 mg / kg Ml exo-RC16, or M2 exo-RC16 by subcutaneous injection on days 0, 3, 7, and 10. Tumor sizes were measured with calipers. RC16 remained therapeutically active after loading into exosomes, but there was no difference between Ml and M2 derived exosomes loaded with RC16. Both significantly reduced the tumor burden compared to saline treated mice. When comparing to FIG. 19, the exosomes showed lower tumor burden than drug alone but not statistically significant. In this particular instance, there did not appear to be synergistic effects between exosome delivery and RC16 loading.

[0081] Various embodiments of the invention have been described in fulfillment of the various objects of the invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations thereof will be readily apparent to those skilled in the art without departing from the spirit and scope of the invention.

Claims

CLAIMS1. A composite composition comprising: an exosome; and at least one small molecule coupled to the exosome via a linker, the linker at least partially disposed in the exosomal membrane.

2. The composite composition of claim 1, wherein the linker is aliphatic.

3. The composite composition of claim 1, wherein the linker comprises one or more aliphatic chains.

4. The composite composition of claim 1, wherein the linker comprises one or more aromatic moieties.

5. The composite composition of claim 1, wherein the small molecule is covalently bound to the linker.

6. The composite composition of claim 5, wherein the small molecule is covalently bound to the linker via a carbamate moiety, ester moiety, amide moiety, or disulfide moiety.

7. The composition of claim 1, wherein a plurality of small molecules are coupled to the exosome via a plurality of linkers.

8. The composite of claim 1, wherein the small molecule is a pharmaceutical compound.

9. The composite of claim 8, wherein the small molecule has anticancer activity.

10. The composite of claim 8, wherein the small molecule has antiviral activity.

11. The composite of claim 1, wherein the small molecule induces Ml polarization or repolarization.

12. The composite of claim 1, wherein the small molecule induces M2 polarization or repolarization.

13. The composite of claim 1, wherein the exosome is sourced from monocytes, differentiated monocytes, differentiated macrophages, and T-cells14. A dispersion comprising: an aqueous or aqueous-based continuous phase; and a dispersed phase comprising composite particles, the composite particles comprising an exosome, and at least one small molecule coupled to the exosome via a linker, the linker at least partially inserting into the exosomal membrane.

15. The dispersion of claim 14, wherein the linker is aliphatic.

16. The dispersion of claim 14, wherein the linker comprises one or more aliphatic chains.

17. The dispersion of claim 14, wherein the linker comprises one or more aromatic moieties.

18. The dispersion of claim 14, wherein the small molecule is covalently bound to the linker.

19. The dispersion of claim 18, wherein the small molecule is covalently bound to the linker via a carbamate moiety, ester moiety, amide moiety, or disulfide moiety.

20. The dispersion of claim 14, wherein the small molecule is a pharmaceutical compound.

21. The dispersion of claim 20, wherein the small molecule has anticancer activity or antiviral activity.

22. The dispersion of claim 14, wherein the small molecule induces Ml polarization or repolarization.

23. The dispersion of claim 14, wherein the small molecule induces M2 polarization or repolarization.

24. The dispersion of claim 14, wherein the composite particles have an average size of 50 nm to 150 nm.

25. The dispersion of claim 14, wherein the composite particles have an average size of 90 nm to 100 nm.

26. The dispersion of claim 20, wherein the pharmaceutical compound is present at a concentration of 0.5-2 mg / ml or greater.

27. A method of making a composite composition comprising: providing an exosome; and inserting a linker into the exosomal membrane, the linker coupled to a small molecule.

28. The method of claim 27, wherein the linker is covalently bound to the small molecule.

29. The method of claim 27, wherein the linker is coupled to the small molecule prior to insertion in the exosomal membrane.

30. The method of claim 28, wherein the small molecule is covalently bound to the linker via a carbamate moiety, ester moiety, amide moiety, or disulfide moiety.

31. The method of claim 27, wherein the linker is aliphatic.

32. The method of claim 31, wherein the linker comprises one or more aliphatic chains.

33. The method of claim 27, wherein the linker comprises one or more aromatic moieties.

34. The method of claim 27, wherein the small molecule is a pharmaceutical compound.

35. The method of claim 34, wherein the small molecule has anticancer activity.

36. The method of claim 34, wherein the small molecule has antiviral activity.

37. The method of claim 27, wherein the small molecule induces Ml polarization or repolarization.

38. The method of claim 27, wherein the small molecule induces M2 polarization or repolarization.

39. The method of claim 27, wherein the exosome is sourced from monocytes, differentiated monocytes, differentiated macrophages, T-cells, stem cells, milk derived exosomes, and / or plant derived exosomes.

40. A method of treating a disease comprising: administering to a patient in need thereof a dispersion comprising an aqueous or aqueousbased continuous phase, and a dispersed phase comprising composite particles, the composite particles comprising an exosome, and at least one small molecule coupled to the exosome via a linker, the linker at least partially inserting into the exosomal membrane.

41. The method of claim 40, wherein the pharmaceutical compound has anticancer activity.

42. The method of claim 40, wherein the pharmaceutical compound has antiviral activity.

43. The method of claim 40, the exosome is sourced from monocytes, differentiated monocytes, differentiated macrophages, cancer cells, and T-cells.

44. The method of claim 40, wherein the pharmaceutical compound is covalently bound to the linker via a carbamate moiety, ester moiety, amide moiety, or disulfide moiety.

45. The method of claim 40 further comprising releasing the pharmaceutical compound via cleavage of the linker.

46. The method of claim 40, wherein the linker is bound to the pharmaceutical compound prior to disposition in the exosomal membrane.

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