Irradiated extracellular vesicles and methods of making and using same

Abstract

Compositions for treating cancer and other conditions, with methods of use and manufacture, that advantageously employ a therapeutically effective amount of extracellular vesicles (EVs) harvested from light irradiated cells. In some implementations, the EVs are bacterial extracellular vesicles (BEVs), and the BEVs are cultured from Lactobacillus brevis (Lacto). The cancer may be ovarian cancer, colon cancer, prostate cancer, lung cancer, breast cancer, colorectal cancer, brain cancer, hepatocellular carcinoma, or pancreatic cancer. Results herein show in particular that red-light irradiated Lacto-EVs can robustly amplify immunotherapy efficacy in cancer.

Description

[0001] IRRADIATED EXTRACELLULAR VESICLES AND METHODS OF MAKING AND USING SAME TECHNICAL FIELD

[0002] The present disclosure relates generally to compositions and methods for treating various medical conditions, particularly cancer, using extracellular vesicles which are photobiomodulatory conditioned to enhance production, delivery, and bioactivity.

[0003] BACKGROUND

[0004] Immunotherapy has been considered a breakthrough in cancer treatment, but despite this success, most patients fail to respond to treatment. Overall response rates have been shown to be about 20 to 40%. Growing evidence indicates that the gut microbiota can contribute to responsiveness to immunotherapy. The gut represents a key site for immune regulation, and the delicate balance between immune surveillance and tolerance can fluctuate in response to changes in the microbiota, subsequently-influencing systemic immunity- and affecting extra-intestinal diseases. There is increased evidence for the role played by the microbiome and / or their products on influencing the host immune cells in health and disease. Microbial therapy has had several prominent successes, and numerous viral and bacterial candidates are progressing through clinical trials. The specific microorganism interactions with the host immune cells are being explored for cancer therapy. However, the use of live and attenuated bacteria in cancer immunotherapy suffers from uncontrolled and strong immune response that may outweigh the benefits of immune activation, raising significant safety concerns.

[0005] Extracellular vesicles (EVs) are small membrane-bound particles released by various cell types including bacteria that contain proteins, nucleic acids, and lipids. They play crucial roles in intercellular communication by transferring bioactive molecules between cells. Both gram-negative and gram-positive bacteria release EVs as a means of intercellular communication. Microbiota-derived bacterial extracellular vesicles (BEVs) can enter the systemic circulation and disseminate to distant organs. Probiotics are microorganisms that provide a health benefit upon consumption. Probiotic bacteria have been shown to have anti cancer properties against various cancer types. BEVs derived from probiotics can help delay the progression of gastrointestinal ailments. Compared to their parent bacteria, BEVs are non-replicative and safer to use than their parent bacteria, given their non-infectious nature and better safety profile. The small sizes of the BEVs enable passive targeting of the tumor potentially via the enhanced permeability and retention effect. BEVs also exhibit high stability to a wide range of temperatures and treatments and carry many of the same immunogenic surface- and membrane-associated components found in their parental bacteria. Many bacterial products, such as toxins, peptides, and enzymes, have been studied for cancer therapy.

[0006] Light therapy, or more particularly red light therapy involving exposure to low-level red or near-infrared light, known as photobiomodulation (PBM), has gained attention for its potential therapeutic benefits across various facets of human health. However, the impact of red light on gut microbiota-derived EVs has not been investigated. The discovery and characterization of BEVs derived from Lactobacillus brevis (Lacto-EVs or R-Lacto-EVs that are red light irradiated) species and their therapeutic effects on cancer viability is described in the present application. Notably, BEVs derived from red light-exposed Lactobacillus brevis exhibit significantly augmented therapeutic efficacy against cancers. Additionally, other bacteria types may also have anticancer effects, including but not limited to gram-negative and / or anaerobic light-irradiated bacteria which may generate EVs that have cancer effects.

[0007] SUMMARY

[0008] In accordance with one embodiment, there is provided a composition for treating cancer comprising a therapeutically effective amount of light irradiated extracellular vesicles (EVs). In some implementations, the EVs are bacterial extracellular vesicles (BEVs), and the BEVs are cultured from Lactobacillus brevis. The cancer may be ovarian cancer, colon cancer, prostate cancer, lung cancer, breast cancer, colorectal cancer, brain cancer (e.g., glioblastoma), hepatocellular carcinoma, or pancreatic cancer. In various embodiments, the light irradiated EVs are exposed to red light for 12-48 hours, and the light irradiated EVs can be filtered. The irradiated EVs can comprise exosomes 30-150 nm and / or microvesicles 100-1,000 nm isolated from mesenchymal stromal cells, endothelial cells, fibroblasts, keratinocytes, or combinations thereof. In some implementations, the irradiated EVs are included with a buffer, with the buffer containing a non-thiol antioxidant at 0.1 -5 mM. The irradiated EVs, particularly probiotic BEVs, can be included in a dietary supplement and / or a yogurt for administration.

[0009] In accordance with another embodiment, there is provided a therapeutically effective amount of irradiated bacterial extracellular vesicles (BEVs). The BEVs can be advantageously irradiated with red light, and may be cultured from probiotic bacteria, including Lactobacillus brevis.

[0010] In accordance with yet another embodiment, there is a method of manufacturing a composition for treating cancer. The method includes irradiating cells and / or isolated extracellular vesicles (EVs) with light to form irradiated EVs, and changing an EV capability metric for the irradiated EVs by 20% or more relative to a nonirradiated control, the concentration of extracellular vesicles (EVs) released from the irradiated cells. The irradiated EVs are exposed to light with a wavelength of 620-1050 nm. In some embodiments, the irradiated EVs are exposed to light with a wavelength in the range of 620-680 nm, 780-960 nm. or 900-950 nm. In some embodiments, wavelength is a line wavelength selected from one of 630 ± 10 nm, 660 ± 10 nm, 808 ± 10 nm, 830 ± 10 nm, or 940 ± 10 nm. An irradiance for the irradiating step can be 1-200 mW / cm2, or more particularly, 5-50 mW / cm2. A fluence for the irradiating step can be 0.1 - 100 J / cm2, or more particularly, 0.5-10 J / cm2.

[0011] In various embodiments, the irradiating step irradiates isolated EVs that are suspended in an aqueous medium. An EV suspension depth can be about 2-10 mm with active mixing. An irradiance uniformity is preferably less than 10% coefficient of variation (CV). To enhance EV viability, a temperature increase during the irradiating step is less than or equal to 2°C. In some methods, a temperature during the irradiating step is maintained between 2-10°C, and an electromagnetic radiation during the irradiation step is pulsed at 10-1,000 Hz with a duty cycle of 10-50%.

[0012] In various embodiments, the EV capability metric is a concentration of EVs, a target-cell uptake measurement, a functional bioassay measurement, a zeta-potential magnitude shift, a nanoparticle tracking analysis (NTA) size distribution, and / or a cargo change metric. In some implementations, more than one EV capability metric may be used in conjunction with other EV capability metric to carry out the disclosed methodology. In specific examples: the concentration of EVs increases by 50% as compared with the nonirradiated control; the target-cell uptake measurement is determined by flow cytometry or single-particle tracking, with the target-cell uptake measurement being 120% or more; the functional bioassay measurement quantifies the fibroblast scratch closure amount, and the fibroblast scratch closure amount is 120% or more; the functional bioassay measurement quantifies the TGF-P decrease amount, and the TGF-P decrease amount is less than or equal to 80%; the zeta-potential magnitude shift is greater than or equal to 2 mV; the NTA size distribution is preserved within ±15 nm; and the cargo change metric is an increase in miR-29b, with the increase in miR-29b being 30% or more. In an advantageous implementation to help maintain the bioavailability of the irradiated EVs, the EV capability metric change persists for 24-72 hours when stored at 2-8°C.

[0013] In one embodiment, the irradiated EVs are exposed to light with a wavelength of 520-560 nm, with an irradiance for the irradiating step being 1 -20 mW / cm2, a fluence during the irradiation step being 0.1-3 J / cm2, while limiting lipid peroxidation.

[0014] In various embodiments, the irradiating step is conducted in a light-conditioning system comprising a light source, a photodiode dosimeter, and a mixing element. The light-conditioning system can include a sample vessel with a 2-10 mm optical path, with the light source being an LED array emitting 600-1050 nm wavelength light with a uniform irradiance having a coefficient of variation (CV) less than 10%. The lightconditioning system can include closed-loop thermometry and a controller configured to maintain a change in temperature of less than or equal to 2°C.

[0015] In accordance with yet another embodiment, there is provided a method of treating cancer in a subject, comprising the step of providing a therapeutically effective amount of red-light irradiated extracellular vesicles derived from Lactobacillus brevis (Lacto-EVs). The concentration of Lacto-EVs can be 1.0xl08-1.0xl010particles / ml, or more preferably, 6.0xl08-1.0xl09particles / ml. The method may further include the step of priming the subject by enhancing miR-214 production. The Lacto-EVs are advantageously irradiated with red light and filtered. In one implementation, the concentration of Lacto-EVs is 0.1 xlO11-IxlO11particles per dose, with a dosage rate of 1-3 times weekly for 1-6 weeks. The method may also include the step of priming the subject by enhancing miR-214 production. The treatment method may also include combination immunotherapy and / or chemotherapy treatment. The providing step can include topical application, intradermal administration, subcutaneous administration, perilesional administration, intraneural administration, or intravenous administration. The providing step may also include providing the irradiated EVs in a yogurt for oral administration.

[0016] It was discovered that brief, non-thermal red or near-infrared illumination of isolated EVs reproducibly increases functional readouts in target cells without deleterious shifts in size distribution or oxidation indices. Useful wavelength bands include 620-680 nm, 780-860 nm, and 900-950 nm. In certain embodiments the radiation is pulsed (10-1,000 Hz; 10-50% duty), yielding greater augmentation at matched fluence.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Preferred example embodiments will hereinafter be described in conjunction with the appended drawings, wherein like designations denote like elements, and wherein:

[0019] FIG. 1 illustrates size distribution of naive Lacto-EVs assessed using Nanosight analysis;

[0020] FIG. 2 illustrates size distribution of red light-radiated Lacto-EVs assessed using Nanosight analysis;

[0021] FIG. 3 is a representative TEM image showing protruded and released EVs from a red-light irradiated Lacto-EV (R-Lacto-EV);

[0022] FIG. 4 is a representative TEM image showing the ultrastructure of isolated R-Lacto EVs;

[0023] FIG. 5 is a Western blot showing that R-Lacto-EVs were positive to lipoteichoic acid (LTA), but not positive to cellular EV marker proteins. CD63 and CD9;

[0024] FIG. 6 depicts optical density (OD) values of hepatocellular carcinoma cells alongside normal liver cells using a naive Lactobacillus EV treatment, and for each X- axis entry the OD values for control (no EV treatment), naive Lactobacillus EVs (lxl0A08 p / mL). and naive Lactobacillus EVs (lxl0A09 p / mL) are represented from left to right;

[0025] FIG. 7 illustrates the cell viability of hepatocellular carcinoma cells alongside normal liver cells using a naive Lactobacillus EV treatment, and for each X-axis entry the OD values for control (no EV treatment), naive Lactobacillus EVs (lxl0A08 p / mL), and naive Lactobacillus EVs (lxl0A09 p / mL) are represented from left to right;

[0026] FIG. 8 shows OD values of glioblastoma cell lines from the MTT assay that were treated with Lacto-EVs, and for each X-axis entry the OD values for control (no EV treatment) are shown on the left and naive Lactobacillus EVs (lxl0A09 p / mL) are shown on the right;

[0027] FIG. 9 displays cell viability of glioblastoma cell lines treated with Lacto-EVs in conjunction with Temozolomide (TMZ), and for each X-axis entry the cell viability percentage for control (no EV treatment) is shown on the left and naive Lactobacillus EVs (lxl0A09 p / mL) is shown on the right;

[0028] FIG. 10 illustrates cell viability of screened carcinoma cell lines following treatment with Lacto-EVs, and for each X-axis entry the cell viability percentage is represented in the following order from left to right: control (no EV treatment, media only), control (no EV treatment, PBS only), naive Lactobacillus EVs (lxl0A09 p / mL, collection 1), naive Lactobacillus EVs (lxl0A09 p / mL, collection 2), naive Lactobacillus EVs (lxl0A09 p / mL, collection 3), and naive Lactobacillus EVs (lxl0A09 p / mL, collection 4);

[0029] FIG. 11 displays cell viability of screened carcinoma cell lines treated with bacterial extracellular vesicles (BEVs) isolated from different preparations of Escherichia coli, and for each X-axis entry the cell viability percentage is represented in the following order from left to right: control (no EV treatment, media only), control (no EV treatment, PBS only), naive Escherichia coli EVs (lxl0A09 p / mL, collection 1), naive Escherichia coli EVs (lxl0A09 p / mL, collection 2), naive Escherichia coli EVs (lxl0A09 p / mL, collection 3), and naive Escherichia coli EVs (lxl0A09 p / mL, collection 4); FIG. 12 illustrates cell viability of screened carcinoma cell lines following treatment with Lacto-EVs, and for each X-axis entry’ the cell viability percentage is represented in the following order from left to right: control (no EV treatment, media only), control (no EV treatment, PBS only), naive Lactobacillus EVs (lxl0A09 p / mL, collection 1), naive Lactobacillus EVs (lxl0A09 p / mL, collection 2), naive Lactobacillus EVs (lxl0A09 p / mL, collection 3), and naive Lactobacillus EVs (lxl0A09 p / mL, collection 4);

[0030] FIG. 13 displays cell viability of screened carcinoma cell lines treated with BEVs isolated from different preparations of Escherichia coli, and for each X-axis entry the cell viability percentage is represented in the following order from left to right: control (no EV treatment, media only), control (no EV treatment, PBS only), naive Escherichia coli EVs (lxl0A09 p / mL, collection 1), naive Escherichia coli EVs (lxl0A09 p / mL, collection 2), naive Escherichia coli EVs (lxl0A09 p / mL, collection 3), and naive Escherichia coli EVs (lxl0A09 p / mL, collection 4);

[0031] FIG. 14 depicts OD values of hepatocellular, prostate, and ovarian carcinoma cells treated with human cerebral endothelial cell exosomes that express miR-214 (hCEC-Exo-214) or control solutions over a three-day period, and for each X-axis entry the OD values are represented in the following order from left to right: control I (nonprime, no EV treatment, media only), control II (non-prime, no EV treatment, PBS only), control I. P. (miR-214 primed, no EV treatment, media only), and control II. P. (miR-214 primed, no EV treatment, PBS only);

[0032] FIG. 15 depicts cell viability of hepatocellular, prostate, and ovarian carcinoma cells treated with hCEC-Exo-214 or control solutions over a three-day period, and for each X-axis entry the cell viability percentage is represented in the following order from left to right: control I (non-prime, no EV treatment, media only), control II (non-prime. no EV treatment, PBS only), control I. P. (miR-214 primed, no EV treatment, media only), and control II. P. (miR-214 primed, no EV treatment, PBS only);

[0033] FIG. 16 illustrates cell viability of cancer cells treated with various doses of Lacto-EVs without prior hCEC-Exo-214 pretreatment, and for each X-axis entry the cell viability percentage is represented in the following order from left to right: control I (non-prime, no EV treatment, media only), control II (non-prime, no EV treatment, PBS only), naive Lactobacillus EVs (lx!0A06 p / mL), naive Lactobacillus EVs (lxl0A07 p / mL). naive Lactobacillus EVs (lxl0A08 p / mL), and naive Lactobacillus EVs (lxl0A09 p / mL);

[0034] FIG. 17 illustrates cell viability' of cancer cells pretreated with hCEC-Exo-214 for three days before receiving different doses of Lacto-EVs for an additional five days, and for each X-axis entry the cell viability percentage is represented in the following order from left to right: control I. P. (miR-214 primed, no EV treatment, media only), control II P. (miR-214 primed, no EV treatment, PBS only), naive Lactobacillus EVs (miR-214 primed, lxl0A06 p / mL), naive Lactobacillus EVs (miR-214 primed, lxl0A07 p / mL), naive Lactobacillus EVs (miR-214 primed, lxl0A08 p / mL), and naive Lactobacillus EVs (miR-214 primed, lxl0A09 p / mL);

[0035] FIG. 18 displays optical density at 600 nm (OD600) values of Lactobacillus brevis subjected to 24 hours of red light radiation, and for each X-axis entry the OD values are represented in the following order from left to right: control (blank media only), naive Lactobacillus brevis EVs, and Lactobacillus brevis red light EVs;

[0036] FIG. 19 represents cell viabilities of liver cancer cells treated with varying concentrations of red light-exposed Lacto-EVs or naive Lacto-EVs, and for each X-axis entry the cell viability percentage is represented in the following order from left to right: control II (no EV treatment, PBS only), control I (no EV treatment, media only), naive Lactobacillus EVs (1.0xl0A08 p / mL), naive Lactobacillus EVs (3.0xl0A08 p / mL), naive Lactobacillus EVs (6.0xl0A08 p / mL), naive Lactobacillus EVs (1.0xl0A09 p / mL), red light Lactobacillus EVs (1.0xl0A08 p / mL), red light Lactobacillus EVs (3.0xl0A08 p / mL), red light Lactobacillus EVs (6.0xl0A08 p / mL), and red light Lactobacillus EVs (1.0xl0A09 p / mL);

[0037] FIG. 20 illustrates cell viabilities of breast cancer cells treated with varying concentrations of red light-exposed Lacto-EVs or naive Lacto-EVs, and for each X-axis entry the cell viability percentage is represented in the following order from left to right: control II (no EV treatment, PBS only), control I (no EV treatment, media only), naive Lactobacillus EVs (1.0xl0A08 p / mL), naive Lactobacillus EVs (3.0xl0A08 p / mL), naive Lactobacillus EVs (6.0xl0A08 p / mL), naive Lactobacillus EVs (1.0xl0A09 p / mL), red light Lactobacillus EVs (1.0xl0A08 p / mL), red light Lactobacillus EVs (3.0xl0A08 p / mL), red light Lactobacillus EVs (6.0xl0A08 p / mL), and red light Lactobacillus EVs (1.0xl0A09 p / mL);

[0038] FIG. 21 shows cell viabilities of ovarian, colon, prostatic, and pancreatic cancer cell lines treated with varying concentrations of red light-exposed Lacto-EVs or naive Lacto-EVs. and for each X-axis entry the cell viability percentage is represented in the following order from left to right: control II (no EV treatment, PBS only), control I (no EV treatment, media only), naive Lactobacillus EVs (1.0xl0A08 p / mL), naive Lactobacillus EVs (3.0xl0A08 p / mL), naive Lactobacillus EVs (6.0xl0A08 p / mL), naive Lactobacillus EVs (1.0xl0A09 p / mL), red light Lactobacillus EVs (1.0xl0A08 p / mL), red light Lactobacillus EVs (3.0xl0A08 p / mL), red light Lactobacillus EVs (6.0xl0A08 p / mL), and red light Lactobacillus EVs (1.0xl0A09 p / mL);

[0039] FIG. 22 shows the percentage decrease in cell viability in the red light Lacto-EV group relative to the naive Lacto-EV group, and for each X-axis entry the percentage decrease is represented in the following dosage order from left to right: 1.0xl0A08 p / mL, 3.0xl0A08 p / mL, 6.0xl0A08 p / mL, and 1.0xl0A09 p / mL;

[0040] FIG. 23 illustrates the impact of both filtered and non-filtered Lacto-EVs on the viability of normal cells, and for each X-axis entry the cell viability percentage is represented in the following order from left to right: control (no EV treatment, PBS), non-filtered naive Lactobacillus EVs (6.0xl0A08 p / mL), non-filtered naive Lactobacillus EVs (1.0xl0A09 p / mL), non-filtered red light Lactobacillus EVs (6.0xl0A08 p / mL), non-filtered red light Lactobacillus EVs (1.0xl0A09 p / mL), filtered naive Lactobacillus EVs (6.0xl0A08 p / mL), filtered naive Lactobacillus EVs (1.0xl0A09 p / mL), filtered red light Lactobacillus EVs (6.0xl0A08 p / mL), and filtered red light Lactobacillus EVs (1.0xl0A09 p / mL);

[0041] FIG. 24 demonstrates the inhibitory effect of filtered Lacto-EVs on carcinoma cell viability, and for each X-axis entry7the cell viability7percentage is represented in the following order from left to right: control (no EV treatment, media), control (no EV treatment, PBS), filtered naive Lactobacillus EVs (6.0xl0A08 p / mL), filtered naive Lactobacillus EVs (1.0xl0A09 p / mL), filtered red light Lactobacillus EVs (6.0xl0A08 p / mL), and filtered red light Lactobacillus EVs (1.0xl0A09 p / mL); FIG. 25 schematically illustrates an experimental protocol of R-Lacto-EV treatment for triple negative breast cancer (TNBC);

[0042] FIG. 26 is representative bioluminescent imaging (BLI) showing tumor sizes of individual mice treated with PBS and R-Lacto-EVs at 5 weeks after the treatment was initiated;

[0043] FIG. 27 is a graph showing the longitudinal BLI measurements showing that R-Lacto-EVs significantly reduced tumor growth compared to PBS;

[0044] FIG. 28 is a graph showing the caliper measurements indicating that R-Lacto-EVs significantly reduced tumor growth compared to PBS;

[0045] FIG. 29 is a graph showing that, compared to PBS, tumor bearing mice treated with R-Lacto-EVs significantly gained body weight;

[0046] FIG. 30 shows the tumor weight analysis at the end of the 5 week treatment period;

[0047] FIG. 31 is a graph illustrating that at the end of the experiment, tumor weight analysis showed that R-Lacto-EVs resulted in an 81% reduction of tumor weight compared to tumors from the PBS group;

[0048] FIG. 32 shows representative histopathological images of lung tissues showing that R-Lacto-EVs reduce lung metastasis;

[0049] FIG. 33 is a quantitative analysis of lung tissues showing that all tumor bearing mice treated with PBS had lung metastasis;

[0050] FIG. 34 shows that the mouse treated with R-Lacto-EVs showed lung metastasis with significantly fewer nodules;

[0051] FIG. 35 shows representative histopathological images of liver tissues showing that R-Lacto-EVs reduce liver metastatic tumors;

[0052] FIG. 36 is a quantitative analysis showing that mice treated with R-Lacto-EVs had significantly fewer liver metastatic tumors; FIG. 37 is a Western blot showing that R-Lacto-EVs significantly increased CD3 and CD4 T-cells and IL-2 cytokine as well as cleaved caspase 3 in tumor tissues compared to PBS;

[0053] FIG. 38 shows the quantitative analysis of FIG. 36, and for each cell type / protein, the PBS results are shown on the left, and the R-Lacto-EV results are shown on the right;

[0054] FIG. 39 is the immunohistochemistry analysis showing that R-Lacto-EVs substantially increased CD3 and CD8 T cells in tumor tissues where lipoteichoic acid (LTA) was positive;

[0055] FIG. 40 is a multiplex cytokine array analysis showing R-Lacto-EV impact on serum levels;

[0056] FIG. 41 is a multiplex cytokine array analysis showing an increase in anti-cancer cytokines in the tumor with R-Lacto-EV treatment; and

[0057] FIG. 42 shows representative images of six non-small cell lung cancer (NSCLC) cell lines that were treated with control medium or R-Lacto-EVs at 1.0xl0A9 p / ml with the corresponding quantitative MTT data, and for each X-axis entry7the cell viability7percentage is represented in the following order from left to right: control I (no EV treatment, media only), control II (no EV treatment, PBS only), R-Lacto-EVs (1.0xl0A08 p / mL), R-Lacto-EVs (3.0x10A08 p / mL), R-Lacto-EVs (6.0x10A08 p / mL), and R-Lacto-EVs (1.0xl0A09 p / mL), with ***p<0.0001.

[0058] DETAILED DESCRIPTION

[0059] Definitions

[0060] “Carrier,” “pharmaceutically acceptable carrier,” “excipient,” and / or “pharmaceutically acceptable excipient,” as used herein refers to a delivery system or adjuvant. A carrier delivery system functions to increase circulation time in the subject, increase solubility to enhance bioavailability, prevent protease or nuclease degradation, and / or allow drug targeting to specific cell types. Carrier generally refers to a substance that aids the administration of an active agent to a cell, an organism, or a subject, such as an excipient or adjuvant that can be included in the compositions of the disclosure and that causes no significant adverse toxicological effect on the subject. Non-limiting examples of pharmaceutically acceptable carriers include water, Poly(LC), CpG, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors and colors, liposomes, dispersion media, microcapsules, cationic lipid carriers, isotonic and absorption delaying agents, and the like. The carrier may also be a buffer that includes substances for providing the formulation with stability, sterility and isotonicity (e.g., antimicrobial preservatives, antioxidants, chelating agents and buffers), for preventing the action of microorganisms (e.g. antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and the like) or for providing the formulation with an edible flavor etc. In an advantageous implementation, the carrier is an agent that facilitates the delivery' of extracellular vesicles (EVs). One of skill in the art will recognize that other pharmaceutical carriers may be employed.

[0061] The term “extracellular vesicles" or “EVs” may include isolated EVs, cell-based treatment, or treating with the content of the EVs (e.g. cargo material including exosomes). Accordingly, exosome-based treatment may include treatment with EVs that contain exosomes. In a preferred embodiment, EVs and / or their exosomes are isolated for treatment purposes. In one specific implementation, EVs include exosomes (-30-150 nm) and microvesicles (-100-1,000 nm). However, bacteria and other prokary otic cells may not generate exosomes, and in those embodiments, EVs may include both small and large EVs, which in some implementations, may be filtered based on size (e.g., small EVs would be about 100-150 nm or less, and large filtered out EVs would be greater than 100-150 nm in one particular implementation).

[0062] The term “filtering” refers to a process in which EVS, cells, and / or supernatant materials are passed through a size-based separation device. In one example, a pore membrane is used for filtering, the pore membrane having a pore size between about 0.1-0.5 pm. In one more particular example, a double filtering method is used with a 0.22 pm pore membrane.

[0063] “Light irradiation” and “irradiating” or “exposing” or “photomodulating” or “light-conditioning” as used herein, refers to delivering 0.1-100 J / cm2 at 600-1050 nm. In one specific implementation, irradiation is a non-thermal exposure, wherein the change in temperature during the irradiation process is less than or equal to 2°C. In one example, red light or red light irradiation, as used herein, generally refers to exposure of cells or cellular materials to red light (620-750 nm) for a period of time sufficient to impact the size of the EVs or increase production of EVs from the cells. In one implementation, red light exposure is longer than 12 hours, preferably 24 hours or longer. The irradiation can be used to augment the EVs from their naturally occurring state, with ‘‘augmenting” generally referring to a 20% or more change in an EV capacity metric as compared to a nonirradiated control, as detailed further herein (e.g., concentration, target-cell uptake, wound-closure assay, reporter-gene output). Irradiated EVs may include irradiated EVs that have previously been isolated from cells and / or irradiated cells and then isolating EVs from the irradiated cells.

[0064] The terms “tumor”, “tumor causing cancer,” and “cancer” are used interchangeably and refer to a cell or population of cells whose growth, proliferation or survival is greater than growth, proliferation or survival of a normal counterpart cell. The cell or population of cells in a tumor or cancer possess abnormal growth, and ty pically the growth is uncontrolled.

[0065] As used herein, the terms “treatment” or “treating” refer to an approach for obtaining a beneficial or desired result, preferably a beneficial or desired clinical result. Such beneficial or desired clinical results include, but are not limited to: (1) curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, interfering with, or affecting a condition (e.g., a disease), the symptoms of the condition, or the biological manifestations of the condition; (2) interfering with one or more points in the biological cascade that leads to or is responsible for the condition; (3) preventing, delaying, or slowing the onset or progression of the symptoms, complications, biological manifestations, and / or biochemical indicia of a disease or condition; or (4) otherwise arresting or inhibiting further development of the disease, condition, or disorder. The compositions and methods of the present disclosure are suitable for obtaining beneficial or desired results such as reducing the proliferation of (or destroying) cancerous cells or other diseased cells, reducing metastasis of cancerous cells found in cancers, shrinking the size of the tumor, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and / or prolonging survival of treatment subjects. Treatment can include administration of one or more therapeutic agents with the purpose to achieve beneficial or desired results that are at least partially based on the methods described herein.

[0066] The term "prevention" as used herein refers to a prophylactic approach intended to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof.

[0067] As used herein, the term "‘subject” is intended to include human and non-human animals. The terms “subject” and “patient” are used interchangeably and can refer to human patients, as well as non-human primates or experimental animals such as rabbits, dogs, cats, rats, mice, and other animals. Preferred subjects of the present disclosure include mammals, or more particularly, human patients in need of a treatment for a disease or disorder. A subject of the present disclosure may be a patient suffering from cancer, such as liver cancer, breast cancer, prostate cancer, lung cancer, ovarian cancer, pancreatic cancer, or colon cancer.

[0068] As used herein, “about” means within acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, “about” can mean a range of up to 20%. When particular values are provided in the application, unless otherwise stated, the meaning of “about” should be assumed to be within acceptable error range for that particular value.

[0069] As used herein, an “effective amount” is defined as the amount required to confer a therapeutic effect on the treated patient, and is typically determined based on age, surface area, weight, and condition of the patient. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich et al., Cancer Chemother. Rep.. 50: 219 (1966). Body surface area may be approximately determined from height and weight of the patient. See, e g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, New York, 537 (1970). The terms “for example,” “for instance,” “such as,” and “like,” and the verbs “comprising,” “having,” “including,” and their other verb forms, when used in conjunction with a listing of one or more compounds or other items, are each to be construed as open-ended, meaning that the listing is not to be considered as excluding other, additional compounds or items. Other terms are to be construed using their broadest reasonable meaning unless they are used in a context that requires a different interpretation. In addition, the term “and / or” is to be construed as an inclusive OR. Therefore, for example, the phrase “A, B, and / or C” is to be interpreted as covering all the following: “A”; “B”; “C”; “A and B”; “A and C”; “B and C”; and “A, B, and C”.

[0070] Any numerical range disclosed herein encompasses the and lower limits and each intervening value, unless otherwise specified. Other than in working examples, or where otherwise indicated, numerical values (such as numbers expressing quantities of ingredients, reaction conditions) as used in the specification may be modified by the term “about”. Accordingly, unless indicated to the contrary, such numbers are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding techniques.

[0071] While the numerical parameters setting forth the scope of the disclosed subject matter are approximations, the numerical values set forth in the working examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in its respective testing measurements.

[0072] Unless defined otherwise, the meanings of technical and scientific terms as used herein are those commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs.

[0073] Description

[0074] Described herein is the discover}’ and characterization of bacterial extracellular vesicles derived from Lactobacillus brevis (Lacto-EVs) species and their therapeutic effects on cancer viability. Notably, bacterial extracellular vesicles (BEVs) derived from red light-irradiated cells may exhibit significantly augmented therapeutic efficacy against cancers, particularly with respect to anti-tumor effects. The photomodulated BEVs and photomodulation methods described herein are potentially capable of enhancing these anti-tumor effects, without harm, or causing more minimal harm, to non-cancerous cells. The treatments described herein may be particularly efficacious with liver cancer, breast cancer, prostate cancer, lung cancer, ovarian cancer, pancreatic cancer, or colon cancer.

[0075] In one embodiment, Lacto-EVs were extracted from the culture media of Lactobacillus brevis through a process of differential ultracentrifugation. Lactobacillus brevis strain 14869, ATCC was used. The Lacto-EVs’ characteristics were elucidated using Nanosight analyses and transmission electron microscopy (TEM). FIG. 1 illustrates size distribution of naive Lacto-EVs assessed using Nanosight analysis. Based on the Nanosight assay, Lacto-EVs that are between 90-200 nm in size may have a concentration between 0 and 3xlO11particles / ml. Specifically, Lacto-EVs that are about 134 nm in size may have a concentration of about 3.0 xlO11particles / ml.

[0076] FIGS. 2-5 characterize red light-exposed Lacto-EVs (R-Lacto-EVs), with FIG. 2 showing size distribution of red light-irradiated Lacto-EVs assess using Nanosight analysis. FIG. 3 is a representative TEM image showing the ultrastructure of isolated R-Lacto-EVs, and FIG. 4 is a representative TEM image showing the ultrastructure of isolated R-Lacto-EVs. Lactobacillus brevis strain BP 14 (ATCC #14869) was cultured aerobically (5% CO2) in Lactobacilli broth medium at 30°C for 3 days. The cultured Lactobacilli (Lacto) were irradiated with red-light. The Lacto were irradiated with red light at a low-level irradiance (e.g., 30mW / cm2) for two consecutive days, for 8 hours / day. Transmission electron microscopy (TEM) showed that red-light irradiated Lacto exhibited EV protrusion on the surface and release of EVs, as shown in FIGS. 3 and 4. After the irradiation, bacteria and debris were removed by centrifugation (4,000 rpm, 20 minutes) and were filtered through a 0.22 pm filter to obtain bacteria-free supernatant. Two liters of the supernatant were further filtered using a 100 kDa filter membrane with a Sartorius Vivaflow 200 tangential ultrafiltration system to harvest the red-light irradiated R-Lacto-EVs. According to the Minimal Information for Studies of Extracellular Vesicles 2023 (MISEV2023) guidelines, R-Lacto-EVs were vigorously characterized. FIGS. 2-4 show the sizes and morphology of R-Lacto-EVs as measured by TEM and nanoparticle tracking analysis (NTA), respectively. As shown in FIG. 5, Western blot analysis showed that R-Lacto-EVs were positive for lipoteichoic acid (LTA) — a well-established Gram-positive bacterial EV marker protein (MISEV2023) but were negative for cellular EV marker proteins (CD63 and CD9). These experiments have helped to illuminate the structural differentiations between R-Lacto-EVs as compared to naturally occurring EVs.

[0077] The effects of Lacto-EVs on carcinoma cell viability were tested with a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Eighteen cancer cell lines were examined, encompassing breast cancer (MDA-MB-468, MDA-MB-231), lung cancer (A549), prostate cancer (PC3), colon cancer (HCT-116), liver cancer (F2), ovarian cancer (OVCAR-3, A2780 Parental, A2780 Cisplatin), pancreatic tumor cells (PANC-1), hepatocellular carcinoma (HepG2, Hep3B), and glioblastoma (GBM, HF2354 Parental, HF2354 TMZ, HF2587 Parental, HF2587 TMZ). Additionally, normal liver cells (THLE-02) and fibroblasts (NIH-3T3) served as control cell lines. Cells were seeded at a density of 800 cells per well in 96-well plates and cultured for 24 hours. Subsequently, extracellular vesicles (EVs) were administered and the cells were incubated for a further five days. On day five, the MTT reagent was diluted in serum-free Eagle's Minimum Essential Medium (EMEM), added to each well, and incubated for 3 hours. Following completion of the incubation period, the MTT solution was aspirated and 150 pL of dimethyl sulfoxide (DMSO) was added to each well, followed by 30 minutes of plate shaking. Finally, the optical density (OD) values were measured using a spectrum reader.

[0078] Lacto-EVs, particularly those that have been subjected to the irradiation methods disclosed herein, may reduce the cell viability of pan cancers. Further, the antitumor effects associated with BEVs derived from specific probiotic bacteria may be cell-type specific and dose-dependent. FIG. 6 depicts OD values of hepatocellular carcinoma cells alongside normal liver cells using a naive Lactobacillus EV treatment. FIG. 7 illustrates the cell viability of hepatocellular carcinoma cells alongside normal liver cells using a naive Lactobacillus EV treatment. Normal liver cell line THLE-02 (CRL-2706) and hepatocellular cell lines HepG2 (HB-8065) and Hep3B (HB-8064) were used. Each cell line included a control with no EV treatment, treatment with naive Lactobacillus EVs administered at a concentration of IxlO8particles / ml, and treatment with naive Lactobacillus EVs administered at a concentration of 1x109particles / ml. The OD values and cell viability of each cell line exposed to each treatment were determined. Lacto-EVs administered at a concentration of IxlO9particles / ml to hepatocellular cell lines may significantly reduce the OD values of the cell lines. For example, Lacto-EVs administered at a concentration of IxlO9particles / ml to cell line HepG2 produced an OD value of 0.1847 and a cell viability of 40.2826%, which represents a decrease of the OD value by 60%. Lacto-EVs administered at a concentration of IxlO9particles / ml to cell line Hep3B produced an OD value of 0.1720 and a cell viability of 36.6281%, which represents a decrease of the OD value by 64%.

[0079] However, Lacto-EVs administered at a lower concentration of IxlO8particles / ml may not significantly decrease the OD values of hepatocellular carcinoma cells. For example. Lacto-EVs administered at a concentration of IxlO8particles / ml to cell line HepG2 produced an OD value of 0.4625 and a cell viability of 100.8592%. Lacto-EVs administered at a concentration of IxlO8particles / ml to cell line Hep3B produced an OD value of 0.4674 and a cell viability of 99.5273%, which represents a slight decrease of the OD value.

[0080] Conversely, Lacto-EVs administered to the normal liver cell line may produce no notable alteration in OD values. For example, Lacto-EVs administered at a concentration of IxlO8particles / ml to cell line THLE-02 produced an OD value of 0.4527 and a cell viability of 98.7780%. Lacto-EVs administered at a concentration of IxlO9particles / ml to cell line THLE-02 produced an OD value of 0.4520 and a cell viability of 98.6427%.

[0081] Furthermore, treatment with Lacto-EVs at a concentration of 1 * 109particles / ml resulted in reductions of 35% (HF2354 Parental), 20% (HF2354 TMZ), 40% (HF2587 Parental), and 23% (HF2587 TMZ) in the OD values of glioblastoma cell lines compared to cells treated with a PBS solution and these values can be increased with treatment involving red light-irradiated BEVs in particular. This demonstrates the capability of Lacto-EVs to selectively reduce the viability of liver cancer and glioblastoma cells.

[0082] FIG. 8 shows OD values of glioblastoma cell lines treated with Lacto-EVs in conjunction with Temozolomide (TMZ). FIG.9 displays cell viability of glioblastoma cell lines treated with a Lacto-EVs in conjunction with TMZ. TMZ is a chemotherapy agent. The viability of other cancer types was tested including breast cancers (MDA- MB-468, MDA-MB- 231), lung cancer (A549), prostate cancer (PC3), colon cancer (HCT-116), liver cancer (F2), ovarian cancer (OVCAR-3, A2780 Parental), and pancreatic tumor cells (PANC-1). Each cell line included a control with no EV treatment and treatment with naive Lactobacillus EVs administered at a concentration of IxlO9particles / ml. The OD values and cell viability of each cell line exposed to each treatment were determined.

[0083] Lacto-EVs administered at a concentration of IxlO9particles / ml may suppress the viability of various cancer ty pes including breast cancers, lung cancer, prostate cancer, colon cancer, liver cancer, ovarian cancer, and pancreatic cancer. Notably, the viability of prostate cancer, ovarian cancer, and liver cancer cells may be inhibited by over 50% compared to a control group without EV treatment which can be further enhanced with the red light-irradiation methods disclosed herein. For example, Lacto-EVs administered at a concentration of IxlO9particles / ml to cell line prostate cancer (PC3) produced an OD value of 0.3113 and a cell viability of 24.88%. which represents a decrease of the OD value by 76%. Lacto-EV s administered at a concentration of lx 109particles / ml to cell line ovarian cancer (OVCAR-3) produced an OD value of 0.2652 and a cell viability of 19.14%, which represents a decrease of the OD value by 81%. Lacto-EVs administered at a concentration of IxlO9particles / ml to cell line liver cancer (F2) produced an OD value of 0.5373 and a cell viability of 49.45%, which represents a decrease of the OD value by 51%.

[0084] To further validate the anti-tumor potential of Lacto-EVs and their impact on a broader spectrum of cancer cell types, Lacto-EVs from different preparations of Lactobacillus brevis were examined and the data may be reliable in assessing dosages for red light-irradiated BEVs. FIG. 10 illustrates cell viability of screened carcinoma cell lines in screen group A following treatment with Lacto-EVs. FIG. 12 illustrates cell viability of screened carcinoma cell lines in screen group B following treatment with Lacto-EVs. Screen group A included carcinoma cell lines breast cancers (MDA-MB-468, MDA-MB- 231), lung cancer (A549), prostate cancer (PC3), colon cancer (HCT-116), liver cancer (F2), ovarian cancer (OVCAR-3, A2780 Parental, A2780 Cisplatin), pancreatic tumor cells (PANC-1), and fibroblasts (NIH-3T3). Screen group B included normal liver cells (THLE-02), hepatocellular carcinoma (HepG2, Hep3B), and glioblastoma (HF2354 Parental, HF 2354 Temozolomide, HF2587 Parental, HF2587 Temozolomide). Each cell line included control A with no EV treatment (media only), control B with no EV treatment (PBS only), and treatment with naive Lactobacillus EVs administered at a concentration of IxlO9particles / ml (collections 1-4). The cell viability of each cell line exposed to each treatment was determined.

[0085] Treatment with Lacto-EVs, at a concentration of IxlO9particles / ml, from various bacterial preparations may lead to a substantial reduction in OD values in prostate cancer, ovarian cancer, and hepatocellular carcinoma, compared to cells treated with media only or a PBS solution. A substantial reduction in OD values may include a reduction in OD values of over 50%. For example, Lacto-EVs administered at a concentration of IxlO9particles / ml to cell line prostate cancer (PC3) produced a cell viability of 27.64% to 29.67%, which represents a decrease of the OD value by approximately 71%. Lacto-EVs administered at a concentration of IxlO9particles / ml to cell line ovarian cancer (OVCAR-3) produced a cell viability of 26.47% to 26.88%, which represents a decrease of the OD value by approximately 73%. Lacto-EVs administered at a concentration of IxlO9particles / ml to cell line ovarian cancer (A2780 Cisplatin-resistant) produced a cell viability of 45.52% to 47.17%, which represents a decrease of the OD value by approximately 54%. Lacto-EVs administered at a concentration of IxlO9particles / ml to cell line hepatocellular carcinoma (HepG2) produced a cell viability of 47.22% to 48.56%, which represents a decrease of the OD value by approximately 52%. Lacto-EVs administered at a concentration of IxlO9particles / ml to cell line hepatocellular carcinoma (Hep3B) produced a cell viability of 46.24% to 48.14%, which represents a decrease of the OD value by approximately 53%.

[0086] Lacto-EVs may not alter the OD values of normal cell lines. For example. Lacto-EVs administered at a concentration of IxlO9particles / ml to cell line normal liver cells (THLE-02) produced a cell viability of 99.48% to 100.70%.

[0087] FIG. 11 displays cell viability of screened carcinoma cell lines in screen group A treated with bacterial extracellular vesicles (BEVs) isolated from Escherichia coli. FIG. 13 displays cell viability of screened carcinoma cell lines in screen group B treated with bacterial extracellular vesicles (BEVs) isolated from Escherichia coli. Screen group A included carcinoma cell lines breast cancers (MDA-MB-468, MD A-MB- 231 ), lung cancer (A549), prostate cancer (PC3), colon cancer (HCT-116), liver cancer (F2), ovarian cancer (OVCAR-3, A2780 Parental, A2780 Cisplatin), pancreatic tumor cells (PANC-1), and fibroblasts (NIH-3T3). Screen group B included normal liver cells (THLE-02), hepatocellular carcinoma (HepG2, Hep3B), and glioblastoma (HF2354 Parental, HF 2354 Temozolomide, HF2587 Parental, HF2587 Temozolomide). Each cell line included control A with no EV treatment (media only), control B with no EV treatment (PBS only), and treatment with naive Escherichia coli EVs administered at a concentration of IxlO9particles / ml (collections 1-4). The cell viability' of each cell line exposed to each treatment was determined.

[0088] Treatment with control EVs isolated from Escherichia coli may not significantly affect the OD values of the tested cancer cell lines. Accordingly, Lacto-EVs are preferable to at least Escherichia coli lines, but it is certainly possible to enhance the EVs from other bacterial types with red light irradiation as well.

[0089] In one embodiment, priming of carcinoma cells with exosomes derived from human cerebral endothelial cells enriched with miR-214 (hCEC-Exo-214) may sensitize carcinoma cells to the anti-tumor effect of Lacto-EVs. hCEC-Exo-214 may display potent therapeutic efficacy against hepatocellular carcinoma cells. There may be a synergistic selective anti -tumor effect of Lacto-EV s, preferably red light-irradiated BEVs, in conjunction with hCEC-Exo-214 treatment. To evaluate whether Lacto-EVs augment the therapeutic impact of miR-214 on hepatocellular, prostate, and ovarian carcinoma cells, the cells were transfected with miR-214 mimics for 72 hours and subsequently treated with Lacto-EV s at concentrations of 1 x 108and 1 x 109particles / ml for an additional five days.

[0090] FIG. 14 depicts OD values of hepatocellular, prostate, and ovarian carcinoma cells treated with hCEC-Exo-214 or control solutions over a three-day period. FIG. 15 depicts cell viability' of hepatocellular, prostate, and ovarian carcinoma cells treated with hCEC-Exo-214 or control solutions over a three-day period. The cell lines that were used include hepatocellular carcinoma (HepG2, Hep3B), normal liver cells (THLE-02), prostate cancer (PC3), and ovarian cancer (OVCAR-3). Each cell line included control 1 with no primer or exosome treatment (media only), control II with no primer or exosome treatment (PBS only), control I. P with miR-214 primer at a concentration of IxlO9particles / ml and no exosome treatment (media only), and control II. P. with miR-214 primer at a concentration of IxlO9particles / ml and no exosome treatment (PBS only). The OD values and cell viability7of each cell line exposed to each treatment were determined. Treatment with hCEC-Exo-214 alone at a concentration of 1×109particles / ml may not induce a significant reduction in the OD values of hepatocellular, prostate, or ovarian cancer cells. However, this priming impact of enhancing miR-214 prior to treatment of red light-irradiated BEVs may be an advantageous treatment method for patients with hepatocellular, prostate, or ovarian cancers.

[0091] FIG. 16 illustrates cell viability of cancer cells treated with various doses of Lacto-EVs without prior hCEC-Exo-214 pretreatment. The cell lines that were used include hepatocellular carcinoma (HepG2, Hep3B), normal liver cells (THLE-02), prostate cancer (PC3), and ovarian cancer (OVCAR-3). Each cell line included control 1 with no primer or EV treatment (media only), control II with no primer or EV treatment (PBS only), naive Lactobacillus EVs administered at a concentration of 1x106particles / ml, naive Lactobacillus EVs administered at a concentration of IxlO7particles / ml, naive Lactobacillus EVs administered at a concentration of IxlO8particles / ml, and naive Lactobacillus EVs administered at a concentration of IxlO9particles / ml. The cell viability of each cell line exposed to each treatment was determined.

[0092] Treatment with Lacto-EVs at a dose of 1×109particles / ml may markedly decrease the OD values compared to lower doses of Lacto-EVs (1×106, 1×107, and 1×108particles / ml), as well as bacterial media and PBS alone.

[0093] FIG. 17 illustrates cell viability of cancer cells pretreated with hCEC-Exo-214 for three days before receiving different doses of Lacto-EVs for an additional five days. The cell lines that were used include hepatocellular carcinoma (HepG2, Hep3B), normal liver cells (THLE-02), prostate cancer (PC3), and ovarian cancer (OVCAR-3). Each cell line included control I. P with miR-214 primer and no EV treatment (media only), control II. P. with miR-214 primer and no EV treatment (PBS only). miR-214 primed naive Lactobacillus EVs administered at a concentration of IxlO6particles / ml, miR-214 primed naive Lactobacillus EVs administered at a concentration of IxlO7particles / ml, miR-214 primed naive Lactobacillus EVs administered at a concentration of IxlO8particles / ml, and miR-214 primed naive Lactobacillus EVs administered at a concentration of IxlO9particles / ml. The cell viability of each cell line exposed to each treatment was determined. Compared to treatment with Lacto-EVs alone, the combination of Lacto-EVs with hCEC-Exo-214 priming may result in further reductions in the cell viability’ of HepG2, Hep3B, PC3, and OVCAR-3 carcinoma cell lines. Furthermore, our observations revealed that compared to treatment with Lacto-EVs alone, the combination of Lacto-EVs with hCEC-Exo-214 priming resulted in further reductions in the cell viability of HepG2 (58% in Lacto-EV alone vs. 66% in combination treatment). Hep3B (59% vs. 78%). PC3 (57% vs. 62%). and OVCAR-3 (65% vs. 82%) carcinoma cell lines. Moreover, these results indicate a potentially promising impact when enhancing miR-214 production prior to treatment with red light-irradiated BEVs.

[0094] Notably, this combination therapy may exhibit no effect on the OD values of the control normal liver cell. For example, the combination of hCEC-Exo-214 priming with Lacto-EVs administered at a concentration of IxlO6particles / ml to cell line normal liver cells (THLE-02) produced a cell viability’ of 99.30%. The combination ofhCEC-Exo-214 priming with Lacto-EVs administered at a concentration of IxlO7particles / ml to cell line normal liver cells (THLE-02) produced a cell viability of 102.23%. The combination of hCEC-Exo-214 priming with Lacto-EVs administered at a concentration of IxlO8particles / ml to cell line normal liver cells (THLE-02) produced a cell viability of 102.72%. The combination of hCEC-Exo-214 priming with Lacto-EVs administered at a concentration of IxlO9particles / ml to cell line normal liver cells (THLE-02) produced a cell viability of 100.80%.

[0095] Red light radiation in particular may enhance the anti-cancer effects of Lacto-EVs and may increase the generation of EVs from Lactobacillus brevis bacteria. Red light radiation may increase the release of BEVs from Lactobacillus brevis. To investigate the impact of red light on probiotics, Lactobacillus brevis cultures w ere subjected to red light exposure using a RedRush 360 device (manufactured by Red Therapy company, China) for 24 hours after a two-day culture period. The device was equipped with 120 x 3 Watt LEDs (60 x 660 nm and 60 x 850 nm). The bacterial cultures were subjected to the 660 nm red LEDs. Initially, bacterial proliferation was assessed by measuring the optical density7at 600 nm (OD600).

[0096] FIG. 18 displays OD600 values of blank media only, naive Lactobacillus brevis, and Lactobacillus brevis subjected to 24 hours of red light radiation. The OD600 value was measured on days 0-3. In one embodiment, bacterial cells are exposed to light having a wavelength of 620-750 nm for 12-48 hours, more preferably 24 hours or more, after the Lacto-EVs were isolated for treatment.

[0097] Lactobacillus brevis cultures exposed to red light were shown to have significantly increased OD600 values compared to those not subjected to radiation. For example, on day three, blank media only produced an OD600 value of 0.0517. On day three, naive Lactobacillus brevis produced an OD600 value of 1.7263. On day three, red-light exposed Lactobacillus brevis produced an OD600 value of 5.2003.

[0098] The size of Lacto-EVs may remain relatively unchanged following red light exposure. For example, Nanosight analysis indicated that the size of naive Lacto-EVs ranged from 50 to 200 nm. The size of red light exposed Lacto-EVs similarly ranged from 50 to 200 nm.

[0099] Lacto-EVs released from irradiated bacteria may have an increased concentration compared to those from non-radiated cultures. For example, naive Lacto-EVs released from non-irradiated cultures had a concentration of 3.53 xlO10particles / ml. Red light radiated Lacto-EVs had a concentration of 5.28* 1010particles / ml. In some implementations, the red light exposure may result in a 50% increase or more in concentration of released Lacto-EVs as compared to a nonirradiated control.

[0100] Red light may enhance the therapeutic efficacy of Lacto-EVs. Red light radiation may selectively enhance the therapeutic efficacy of killing cancer cells in a dose-dependent and cell-type-specific manner. Red light-exposed Lacto-EVs may have therapeutic potential on cancer viability7. To evaluate this potential, Lacto-EVs derived from Lactobacillus brevis subjected to red light radiation, along with control naive Lacto-EVs, were incubated at various concentrations (IxlO8, 3*108, 6*108, and IxlO9particles / ml) with cancer cell lines including hepatocellular, lung, breast, prostate, pancreatic, and colon carcinomas over a five-day period.

[0101] FIG. 19 represents cell viabilities of liver cancer cells treated with varying concentrations of red light-exposed Lacto-EVs or naive Lacto-EVs. The cell lines that were used include hepatocellular carcinoma (HepG2, Hep3B) and normal liver cells (THLE-02). Each cell line included control I with no EV treatment (media only), control II with no EV treatment (PBS only), naive Lactobacillus EVs administered at a concentration of IxlO8particles / ml, naive Lactobacillus EVs administered at a concentration of 3x108particles / ml. naive Lactobacillus EVs administered at a concentration of 6xl08particles / ml, naive Lactobacillus EVs administered at a concentration of IxlO9particles / ml, red light Lactobacillus EVs administered at a concentration of IxlO8particles / ml, red light Lactobacillus EVs administered at a concentration of 3xl08particles / ml, red light Lactobacillus EVs administered at a concentration of 6xl08particles / ml, and red light Lactobacillus EVs administered at a concentration of IxlO9particles / ml. The cell viability of each cell line exposed to each treatment was determined.

[0102] FIG. 20 illustrates cell viabilities of breast cancer cells treated with varying concentrations of red light-exposed Lacto-EVs or naive Lacto-EVs. The cell lines that were used include breast cancer MDA-MB-231 and MDA-MB-468. Each cell line included control I with no EV treatment (media only), control II with no EV treatment (PBS only), naive Lactobacillus EVs administered at a concentration of IxlO8particles / ml, naive Lactobacillus EVs administered at a concentration of 3xl08particles / ml, naive Lactobacillus EVs administered at a concentration of 6xl08particles / ml, naive Lactobacillus EVs administered at a concentration of IxlO9particles / ml, red light Lactobacillus EVs administered at a concentration of IxlO8particles / ml, red light Lactobacillus EVs administered at a concentration of 3xl08particles / ml, red light Lactobacillus EVs administered at a concentration of 6x108particles / ml, and red light Lactobacillus EVs administered at a concentration of IxlO9particles / ml. The cell viability of each cell line exposed to each treatment was determined.

[0103] FIG. 21 shows cell viabilities of ovarian, colon, prostatic, and pancreatic cancer cell lines treated with varying concentrations of red light-exposed Lacto-EVs or naive Lacto-EVs. The cell lines that were used include prostate cancer (PC3). ovarian cancer (OVCAR-3), pancreatic tumor cells (PANC-1), lung cancer (A549), and colon cancer (HCT-116, CRL-2221). Each cell line included control I with no EV treatment (media only), control II with no EV treatment (PBS only), naive Lactobacillus EVs administered at a concentration of 1x108particles / ml, naive Lactobacillus EVs administered at a concentration of 3x108particles / ml, naive Lactobacillus EVs administered at a concentration of 6x108particles / ml, naive Lactobacillus EVs administered at a concentration of IxlO9particles / ml, red light Lactobacillus EVs administered at a concentration of IxlO8particles / ml, red light Lactobacillus EVs administered at a concentration of 3x108particles / ml, red light Lactobacillus EVs administered at a concentration of 6x108particles / ml, and red light Lactobacillus EVs administered at a concentration of IxlO9particles / ml. The cell viability of each cell line exposed to each treatment was determined.

[0104] FIG. 22 shows the percentage decrease in cell viability in the red light Lacto-EV group relative to the naive Lacto-EV group. The cell lines that were used include hepatocellular carcinoma (HepG2, Hep3B), normal liver cells (THLE-02), prostate cancer (PC3), ovarian cancer (OVCAR-3), pancreatic tumor cells (PANC-1), lung cancer (A549). breast cancer (MDA-MB-231, MDA-MB-468) and colon cancer (HCT-116, CRL-2221). The percentage decrease in cell viability in the red light Lacto-EV group relative to the naive Lacto-EV group was determined for Lactobacillus EVs administered at a concentration of IxlO8particles / ml, Lactobacillus EVs administered at a concentration of 3xl08particles / ml, Lactobacillus EVs administered at a concentration of 6xl08particles / ml, and Lactobacillus EVs administered at a concentration of IxlO9particles / ml. The percentage decrease of the naive group compared to the red light Lacto-EV group was determined using the formula (naive group cell viability - red light group cell viability) / naive group cell viability x 100%.

[0105] As shown in FIGS. 19-22 and Table 1 below, red light-treated Lacto-EVs administered at a concentration of 1 x 108particles / ml may not significantly alter the viability of the screened cancer cells compared to naive Lacto-EVs but for higher doses, red light-irradiated BEVs have been shown to be even more efficacious than nonirradiated BEVs.

[0106] Table 1

[0107]

[0108]

[0109] As shown in FIGS. 19 and 22 and Tables 2 and 3 below, red light-treated Lacto-EVs administered a concentration of 3x 108and 6xl08parti cles / ml exhibited a modest decrease in the viability' of liver cancer cells compared to naive Lacto-EVs. For example, naive Lacto-EVs administered at a concentration of 3x108parti cles / ml to cell line HepG2 produced a cell viability' of 68.043% and red light-treated Lacto-EVs administered at a concentration of 3x108parti cles / ml to cell line HepG2 produced a cell viability' of 59.186%, representing a 13.017% decrease in the viability of liver cancer cells. Naive Lacto-EVs administered at a concentration of 6xl08particles / ml to cell line HepG2 produced a cell viability of 62.451% and red light-treated Lacto-EVs administered at a concentration of 6x108particles / ml to cell line HepG2 produced a cell viability of 55.324%, representing a 11.414% decrease in the viability of liver cancer cells. Naive Lacto-EVs administered at a concentration of 3xl08particles / ml to cell line Hep3B produced a cell viability of 67.218% and red light-treated Lacto-EVs administered at a concentration of 3xl08particles / ml to cell line Hep3B produced a cell viability of 63.444%, representing a 5.614% decrease in the viability of liver cancer cells. Naive Lacto-EVs administered at a concentration of 6xl08particles / ml to cell line Hep3B produced a cell viability of 57.330% and red light-treated Lacto-EVs administered at a concentration of 6xl08particles / ml to cell line Hep3B produced a cell viability of 55.030%, representing a 4.012% decrease in the viability of liver cancer cells.

[0110] As shown in FIGS. 20 and 22 and Tables 2 and 3 below, red light-treated Lacto-EVs administered a concentration of 3×108and 6×108particles / ml exhibited a modest decrease in the viability of breast cancer cells compared to naive Lacto-EVs. For example, naive Lacto-EVs administered at a concentration of 3x108particles / ml to cell line MDA-MB-231 produced a cell viability of 50.795% and red light-treated Lacto-EVs administered at a concentration of 3xl08particles / ml to cell line MDA-MB-231 produced a cell viability of 45.851%, representing a 9.733% decrease in the viability of breast cancer cells. Naive Lacto-EVs administered at a concentration of 6xl08particles / ml to cell line MDA-MB-231 produced a cell viability of 45.554% and red light-treated Lacto-EVs administered at a concentration of 6x108particles / ml to cell line MDA-MB-231 produced a cell viability of 38.910%, representing a 14.584% decrease in the viability of breast cancer cells. Naive Lacto-EVs administered at a concentration of 3x108particles / ml to cell line MDA-MB-468 produced a cell viability of 49.046% and red light-treated Lacto-EVs administered at a concentration of 3x108particles / ml to cell line MDA-MB-468 produced a cell viability of 43.964%, representing a 10.363% decrease in the viability of breast cancer cells. Naive Lacto-EVs administered at a concentration of 6xl08particles / ml to cell line MDA-MB-468 produced a cell viability of 44.907% and red light-treated Lacto-EVs administered at a concentration of 6x108particles / ml to cell line MDA-MB-468 produced a cell viability of 39.839%, representing a 11.287% decrease in the viability of breast cancer cells.

[0111] As shown in FIGS. 21 and 22 and Tables 2 and 3 below, red light-treated Lacto-EVs administered a concentration of 3×108and 6×108particles / ml exhibited a modest decrease in the viability of prostatic cancer cells compared to naive Lacto-EVs. For example, naive Lacto-EVs administered at a concentration of 3xl08particles / ml to cell line PC3 produced a cell viability of 45.659% and red light-treated Lacto-EVs administered at a concentration of 3x108particles / ml to cell line PC3 produced a cell viability of 42.804%, representing a 6.254% decrease in the viability of prostatic cancer cells. Naive Lacto-EVs administered at a concentration of 6xl08particles / ml to cell line PC3 produced a cell viability of 42.532% and red light-treated Lacto-EVs administered at a concentration of 6xl08particles / ml to cell line PC3 produced a cell viability of 37.788%, representing a 11.153% decrease in the viability of prostatic cancer cells. As show n in FIGS. 21 and 22 and Tables 2 and 3 below-, red light-treated Lacto-EVs administered a concentration of 3xl08and 6xl08parti cles / ml exhibited a modest decrease in the viability of lung cancer cells compared to naive Lacto-EVs. For example, naive Lacto-EVs administered at a concentration of 3x108parti cles / ml to cell line A549 produced a cell viability of 54.981% and red light-treated Lacto-EVs administered at a concentration of 3xl08parti cles / ml to cell line A549 produced a cell viability of 50.269%, representing an 8.570% decrease in the viability of lung cancer cells. Naive Lacto-EVs administered at a concentration of 6xl08particles / ml to cell line A549 produced a cell viability of 50.963% and red light-treated Lacto-EVs administered at a concentration of 6xl08particles / ml to cell line A549 produced a cell viability of 43.972%, representing a 13.718% decrease in the viability of lung cancer cells.

[0112] As shown in FIGS. 21 and 22 and Tables 2 and 3 below red light-treated Lacto-EVs administered a concentration of 3xl08and 6xl08particles / ml may exhibit a modest decrease in the viability of ovarian cancer cells compared to naive Lacto-EVs. For example, naive Lacto-EVs administered at a concentration of 3xl08particles / ml to cell line OVCAR-3 produced a cell viability of 45.436% and red light-treated Lacto-EVs administered at a concentration of 3xl08particles / ml to cell line OVCAR-3 produced a cell viability of 40.380%, representing a ll.128% decrease in the viability of ovarian cancer cells. Naive Lacto-EVs administered at a concentration of 6x108particles / ml to cell line OVCAR-3 produced a cell viability of 41.536% and red light-treated Lacto-EVs administered at a concentration of 6xl08particles / ml to cell line OVCAR-3 produced a cell viability of 37.209%, representing a 10.419% decrease in the viability of ovarian cancer cells.

[0113] Table 2

[0114]

[0115]

[0116] Table 3

[0117]

[0118]

[0119] As shown in Table 4 below, high doses of red light-treated Lacto-EVs administered at a concentration of IxlO9parti cles / ml may significantly enhance the therapeutic efficacy compared to naive Lacto-EVs. The therapeutic efficacy may be enhanced by 14-61% compared to naive Lacto-EVs.

[0120] As shown in FIGS. 21 and 22 and Tables 1-4, red light-treated Lacto-EVs have the potential to markedly reduce colon cancer cell viability at moderate and high doses of EVs while naive Lacto-EVs and low doses may have no discernible effect on colon cancer cell viability. Colon cancer cells may be highly sensitive to treatment with red light-exposed Lacto-EVs. For example, naive Lacto-EVs administered at a concentration of 3xl08particles / ml to cell line CRL-2221 produced a cell viability of 100.241% and red light-treated Lacto-EVs administered at a concentration of 3xl08particles / ml to cell line CRL-2221 produced a cell viability of 57.862%, representing a 42.278% decrease in the viability of colon cancer cells. Naive Lacto-EVs administered at a concentration of 6xl08particles / ml to cell line CRL-2221 produced a cell viability of 100.626% and red light-treated Lacto-EVs administered at a concentration of 6x108particles / ml to cell line CRL-2221 produced a cell viability of 44.082%, representing a 56.193% decrease in the viability of colon cancer cells. Naive Lacto-EVs administered at a concentration of IxlO9particles / ml to cell line CRL-2221 produced a cell viability of 100.244% and red light-treated Lacto-EVs administered at a concentration of IxlO9particles / ml to cell line CRL-2221 produced a cell viability of 38.973%, representing a 61.122% decrease in the viability of colon cancer cells. Naive Lacto-EVs administered at a concentration of IxlO8particles / ml to cell line CRL-2221 produced a cell viability of 100.116% and red light-treated Lacto-EVs administered at a concentration of IxlO8particles / ml to cell line CRL-2221 produced a cell viability of 100.080%, representing a 0.036% decrease in the viability of colon cancer cells.

[0121] Additionally, red light-treated and naive Lacto-EVs may not significantly impact the OD values of normal liver cells. Table 4

[0122]

[0123] In some embodiments, compositions and methods involve filtering the EVs, which provided unexpectedly positive results for treating cancer cells while avoiding an undesirable impact on normal cells. To isolate or filter Lactobacillus derived extracellular vesicles (Lacto-EVs), Lactobacillus Brevis was grown in Lactobacillus MRS broth with shaking (200 rpm) overnight. The supernatant fraction was collected by centrifugation (6,000 g, 15 min, 4°C; and 10,000 g, 15 min, 4°C). Then, the supernatant was filtered through a 0.22 pM pore membrane (Coming, St. Louis, USA) to remove any remaining cells. After that, the media were ultracentrifuged at 100,000g for 2 hours at 4°C and the naive Lacto-EV pellets were resuspended in a minimal volume of PBS and stored at -80°C until use. To further enhance their safety, the naive Lacto-EVs or Lacto-EVs from red light-radiated Lactobacillus were passed through a 0.22 pm pore membranes again (filtered Lacto-EVs) to eliminate bacteria and foreign particles thoroughly. In some implementations, the pore membrane has a pore size between about 0.1-0.5 pm, but double filtering with a 0.22 pm helped more thoroughly isolate the Lacto-EVs.

[0124] To further evaluate the safety of Lacto-EVs, their effects on the viability of various normal human epithelial and fibroblast cells were assessed, including human liver epithelial cells (THLE-02, ATCC: CRL-2706), human prostate epithelial cells (ATCC: CRL-2221). human breast epithelial cells (MCF-10A, ATCC: CRL-10317), human hepatic stellate cells (Lx-2. Millipore: SCC064), and human fibroblasts (N20). The findings revealed a distinct difference in size distribution between non-filtered and filtered Lacto-EVs. Unlike the filtered Lacto-EVs, the non-filtered Lacto-EVs exhibited a heterogeneous population, with multiple subtypes and vary ing peak size distributions.

[0125] MTT assays demonstrated that a 2-day treatment with non-filtered Lacto-EVs significantly decreased the viability of human prostate epithelial cells (CRL-2221), human breast epithelial cells (MCF-10A), and human fibroblasts (N20). In contrast, filtered Lacto-EVs had no detrimental effects on the viability of any of the normal human epithelial or fibroblast cells tested (FIG. 23). Moreover, filtered Lacto-EVs were found to reduce the viability of multiple cancer cell lines (FIG. 24). These results indicate that while filtered and red light-exposed Lacto-EVs retain anti -tumor efficacy equal to or greater than that of non-filtered Lacto-EVs, they exhibit greater safety for normal human cells.

[0126] FIGS. 25-31 illustrate the ability of R-Lacto-EVs to significantly reduce tumor growth in triple negative breast cancer. Based on in vitro data showing that R-Lacto-EVs significantly reduced human and mouse TNBC cell viability with very robust anticancer effect at 1 * IO9particles / ml, the R-Lacto-EVs impact on TNBC progression was examined in a 4T1 TNBC mouse model, as this model recapitulates immunosuppressive tumor microenvironment and metastasis observed in TNBC patients. Briefly, the TNBC model was established by orthotopically implanting 5*1054T1 -luciferase-transfected cells into the mammary fat pads of female BALB / c mice (6- 8 weeks old), as schematically illustrated in FIG. 25. One-week post-implantation, the presence of TNBC tumors was confirmed by non-invasive bioluminescence imaging (BLI) (see FIG. 26). These tumor-bearing mice were then randomized into control and treatment groups. The mice in the control (n=6) and treatment (n=6) groups received 0.3 mL phosphate-buffered saline (PBS) and R-Lacto-EVs (IxlO10parti cles / inj ection), respectively, via intraperitoneal injection. PBS or R-Lacto-EVs were administered three times / week for 5 consecutive weeks. Tumor growth was non-invasively monitored weekly by BLI and caliper, and the animal body weights were recorded to assess systemic toxicity. It was found that compared to the control, the R-Lacto-EV treatment significantly reduced tumor growth from week 2 onward to week 5 when the experiments ended, as shown in FIGS. 27 and 28. Quantitative measurements of isolated tumor showed that R-Lacto-EVs significantly reduced tumor weight by 81% compared to PBS, as shown in FIGS. 30 and 31. During the five weeks of R-Lacto-EV treatment, animals did not significantly lose their body weight (see FIG. 29).

[0127] FIGS. 32-36 help illustrate that R-Lacto-EVs significantly reduce tumor metastasis in TNBC tumor bearing mice. In addition to significant reduction of tumor growth, histopathological analysis of lung and liver tissues harvested from the mice showed that all mice in the PBS group (n=6) had lung metastasis with abundant tumor nodules. However, only one out of 6 mice treated with R-Lacto-EVs had lung metastasis (see FIGS. 32 and 33). Quantification of lung tumor nodules revealed that compared to the control mice, R-Lacto-EV treated mice had a 99.3% reduction in lung tumor nodules (FIG. 34). For liver tissues, all mice in the control and R-Lacto-EV groups exhibited liver metastasis, however, compared to the control, R-Lacto-EVs significantly reduced the number of liver metastasis nodules by 91 % (see FIGS. 35 and 36).

[0128] With reference to FIGS. 37-39, results illustrate that R-Lacto-EVs increase tumor killing T cells in TNBC tumor bearing mice. Anti-tumor T cell exhaustion is one of the major challenges for immunotherapy. Using Western blot and immunohistochemistry approaches, it was found that R-Lacto-EV treatment significantly increased CD3 and CD4 T cells in tumor compared to PBS, as shown in FIGS. 37 and 38. Immunohistochemistry showed that R-Lacto-EVs substantially increased CD3 and CD8 T cells, and lipoteichoic acid (LTA), a marker protein of R-Lacto-EVs, positive cells within the tumor niche (see FIG. 39). Additionally, R-Lacto-EV treatment significantly increased IL-2 cytokine within tumor tissues (see FIGS. 37 and 38). Together, these data suggest that R-Lacto-EVs are taken up by tumor and its niche cells, and augment anti-cancer T cells, which could underscore the R-Lacto-EV therapeutic effect on TNBC.

[0129] Additionally, R-Lacto-EVs increase apoptotic protein and reduce proteins that mediate epithelial-mesenchymal transition (EMT) and metastasis in TNBC tumor bearing mice. As shown in FIGS. 37 and 38, Western blot analysis showed that R-Lacto-EVs significantly increased cleaved caspase 3, an apoptotic protein, and reduced focal adhesion kinase (FAK) that mediated tumor metastasis. R-Lacto-EVs also significantly decreased vimentin, which is known to regulate EMT in tumor.

[0130] As shown in FIGS. 40 and 41, R-Lacto-EVs can also serve to increase anticancer cytokines in the tumor but decrease blood cytokines, which was unexpected. Using a multiplex cytokine array kit (RAYBIOTECH, #AAM-INF-1), cytokine profiles were analyzed in serum and tumor tissues. In the serum, compared to PBS, R-Lacto-EVs significantly reduced levels of IL-6 by 71%, IL-1 P by 40%, TNF-a by 61%. and IL- 10 by 74% (see FIG. 40). In the tumor tissues, it was found that compared to PBS, R-Lacto-EVs significantly increased levels of IFN-y by 47%, TNF-a by 167%, and IL-2 by 35%, while IL- 10, an immunosuppressive cytokine, significantly decreased by 44% (see FIG. 41). These data strongly suggest that R-Lacto-EVs specifically elevate and decrease anti-tumor and immunosuppressive cytokines, respectively, within tumor tissues, but unexpectedly, do not trigger systemic cytokine responses. With reference to FIG. 42, the effects of R-Lacto-EVs on human non-small cell lung carcinoma (NSCLC) cell viability were examined in six NSCLC cell lines, including H-1650 and H-1975 with Epidermal Growth Factor Receptor (EGFR) mutation. H2228 and H3122 with Anaplastic Lymphoma Kinase (ALK) fusion, and H2030 and Calu-6 with Kirsten Rat Sarcoma viral oncogene (KRAS) mutation. R-Lacto-EVs were added to the cultured cells at l. Ox 108, 3.0 x 108, 6.0x 108, and l. Ox 109particles / ml. Five days after the treatment, R-Lacto-EVs at 6. Ox 108parti cles / ml significantly reduced viable cells by approximately 70% to 85% in H-1650, H-1975, and H2030 cells, while R-Lacto-EVs at l. Ox 109parti cles / ml significantly reduced viable cells by approximately 60% to 85% across all six cancer lines (FIG. 42). These data provide strong in vitro evidence that R-Lacto-EVs have a robust anti-lung cancer effect.

[0131] Together, these in vivo data provide strong evidence that R-Lacto-EVs, as opposed to naturally occurring EVs, can be used as a monotherapy to suppress TNBC progression and metastasis potentially by augmentation of anti-cancer T cells and cancer killing proteins and by reducing molecules that promote metastasis. Anti-cancer T cell exhaustion in the tumor niche underlies reduced efficacy of immunotherapy. Thus, it is expected that R-Lacto-EVs would also robustly amplify immunotherapy efficacy in cancer.

[0132] In some embodiments, there is a method of producing augmented extracellular vesicles (EVs), comprising exposing isolated EVs suspended in an aqueous medium to electromagnetic radiation having a wavelength of about 600-1,050 nm, at an irradiance of about 1-200 mW / cm2to deliver a fluence of about 0.1-100 J / cm2, under conditions in which the sample temperature increase is <2 °C, thereby increasing at least one prespecified EV capability metric by >20% relative to a sham-exposed or nonirradiated control. EVs isolated by ultracentrifugation or size-exclusion chromatography are diluted to 1-5x10" particles / mL in isotonic buffer. The suspension (optical path ~5 mm) is illuminated at 660 nm or 808 nm at 10-30 mW / cm2to 1-5 J / cm2while maintaining 2-10 °C (AT < 2 °C) with continuous mixing. Post-conditioning, EVs are sterile-filtered (0.22 pm) and stored 2-8 °C.

[0133] Nanoparticle tracking analysis confirms modal size within ±15 nm of preexposure; zeta potential magnitude shifts by >2 mV. Oxidation indices remain within assay-defined limits. Function is demonstrated by the following EV capability metrics: (i) increased uptake in recipient cells, including fibroblasts or neurons, (ii) accelerated fibroblast scratch closure or reduced TGF-P reporter activity, and / or (iii) cargo changes (e.g., miR-29b f >30%).

[0134] In some embodiments, green wavelength treated EVs may also have increased bioactive impact. In one example, 520-560 nm illumination at 1-10 mW / cm2to 0.1-3 J / cm2at 2- 10 °C is expected to produce hormetic augmentation when lipid peroxidation is constrained. In other embodiments, the wavelength is red (e g., 620-680 nm), near infrared I (e.g., 780-860 nm), or a near infrared II windowlet (e.g., 780-860 nm). The wavelength may be a line wavelength selected from 630 ± 10, 660 ± 10, 808 ± 10, 830 ± 10, or 940 ± 10 nm.

[0135] To help achieve adequate transformation of the EVs from their naturally occurring state, the irradiance is about 5-50 mW / cm2, and the fluence is about 0.5-10 J / cm2, with the radiation being pulsed at about 10-1,000 Hz with a duty cycle of about 10-50%, at fluence equal to continuous wave controls. The exposure is conducted at about 2-10 °C, AT < 2 °C. With an aqueous medium, the EV suspension depth is about 2-10 mm with active mixing achieving irradiance uniformity CV < 10%. The medium comprises a physiologic buffer optionally containing a non-thiol antioxidant at 0.1-5 mM.

[0136] To achieve the method, the EV capability metric can be selected from one or more of: (a) target-cell uptake (flow cytometry' or single-particle tracking), (b) functional bioassay (e.g.. fibroblast scratch closure, neurite outgrowth, TGF-P reporter decrease), (c) zeta-potential magnitude shift by >2 mV, (d) preservation of NTA modal size within ±15 nm with oxidation index within preset bounds, (e) defined cargo change (e.g., miR-29b ( >30%). Example thresholds include uptake index >1.2x, scratch closure at 24 h >1.2x, TGF-P reporter <0.8x, and zeta-potential magnitude change >2 mV. To promote treatment success and viability, the augmented EV capability metric persists for >24-72 h during storage at 2-8 °C. In one implementation, endotoxin is maintained at <5 EU / kg dose equivalent. The irradiated EVs are advantageously sterile-filtered, formulated in isotonic buffer for topical, local injection, or intravenous administration. Incorporation of the EVs w ith a pharmaceutically acceptable excipient, such as an isotonic buffer, can improve the formulation stability and delivery.

[0137] The EVs produced by the methods herein may be used to treat cancer. Administration may include topical application, intradermal, subcutaneous, perilesional, intraneural. or intravenous. In one implementation, particularly with probiotic BEVs, the irradiated EVs are included in a dietary supplement and / or a food product such as yogurt for oral administration. In one particular example, the dosing regimen is 0.1-lxlO11parti cles / dose, once to three times weekly for 1-6 weeks. Augmented EVs can be administered topically or by injection to enhance wound closure and reduce fibrotic remodeling, or for neuro-repair, at 0.1-lxlO11parti cles / dose, I -3 / / week lor 1-6 weeks.

[0138] A bench-scale conditioning vessel can provide uniform irradiance (CV < 10%), closed-loop temperature control, and in-line dosimetry. This may be scaled up to a closed bioreactor format. A light-conditioning system can include: (a) a sample vessel with 2-10 mm optical path, (b) a LED array emitting 600-1,050 nm with uniform irradiance CV < 10%, (c) closed-loop thermometry and controller maintaining AT < 2 °C, (d) an in-line photodiode dosimeter, and (e) a mixing element to maintain homogeneity.

[0139] Without being bound by theory, red / NIR modulate membrane protein / lipid redox states and membrane fluidity, altering zeta potential and cargo presentation to promote uptake; pulsing may permit redox recovery and amplify signaling at equal fluence.

[0140] It is to be understood that the foregoing description is of one or more preferred exemplary embodiments of the invention. The invention is not limited to the particular embodiment(s) disclosed herein, but rather is defined solely by the claims below. Furthermore, the statements contained in the foregoing description relate to particular embodiments and are not to be construed as limitations on the scope of the invention or on the definition of terms used in the claims, except where a term or phrase is expressly defined above. Various other embodiments and various changes and modifications to the disclosed embodiment(s) will become apparent to those skilled in the art. All such other embodiments, changes, and modifications are intended to come within the scope of the appended claims.

Claims

CLAIMS1. A composition for treating cancer, comprising:a therapeutically effective amount of light irradiated extracellular vesicles (EVs) included with a pharmaceutically acceptable excipient.

2. The composition of claim 1, wherein the EVs are bacterial extracellular vesicles (BEVs).

3. The composition of claim 2, wherein the BEVs are cultured from a probiotic bacteria.

4. The composition of any one of claims 1 to 3. wherein the BEVs are cultured from Lactobacillus brevis.

5. The composition of any one of claims 1 to 4, wherein the composition is for treating cancer and the cancer is ovarian cancer, colon cancer, prostate cancer, lung cancer, breast cancer, colorectal cancer, brain cancer, hepatocellular carcinoma, or pancreatic cancer.

6. The composition of any one of claims 1 to 5, wherein the light irradiated EVs are exposed to red light for 12-48 hours.

7. The composition of any one of claims 1 to 6, wherein the light irradiated EVs are filtered.

8. The composition of claim 1 or claim 2, wherein the irradiated EVs comprise exosomes 30-150 nm and / or microvesicles 100-1,000 nm isolated from mesenchymal stromal cells, endothelial cells, fibroblasts, keratinocytes, or combinations thereof.

9. The composition of any one of claims 1 to 8, wherein the pharmaceutically acceptable excipient is a buffer.

10. The composition of claim 9, wherein the buffer contains a non-thiol antioxidant at 0.1-5 mM.

11. The composition of any one of claims 1 to 10, wherein the irradiated EVs are included in a yogurt.

12. A composition for treating cancer, comprising:a therapeutically effective amount of irradiated bacterial extracellular vesicles (BEVs) included with a pharmaceutically acceptable excipient.

13. The composition of claim 12, wherein the BEVs are irradiated with red light.

14. The composition of claim 12 or 13, wherein the BEVs are cultured from Lactobacillus brevis.

15. The composition of any one of claims 12 to 14. wherein the cancer is ovarian cancer, colon cancer, prostate cancer, lung cancer, breast cancer, colorectal cancer, brain cancer, hepatocellular carcinoma, or pancreatic cancer.

16. The composition of any one of claims 12 to 1, wherein the irradiated BEVs are exposed to red light for 12-48 hours.

17. The composition of any one of claims 12 to 16, wherein the irradiated BEVs are filtered.

18. A composition for treating a cancer, comprising:a therapeutically effective amount of irradiated and filtered extracellular vesicles (EVs) included with a pharmaceutically acceptable excipient.

19. A method of manufacturing a composition for treating cancer, comprising the steps of:irradiating cells and / or isolated extracellular vesicles (EVs) with light to form irradiated EVs; andchanging an EV capability metric for the irradiated EVs by 20% or more relative to a nonirradiated control.

20. The method of claim 19, wherein the irradiated EVs are exposed to light with a wavelength of 620-1050 nm.

21. The method of claim 20, wherein the irradiated EVs are exposed to light with a wavelength of 620-680 nm.

22. The method of claim 20, wherein the irradiated EVs are exposed to light with a wavelength of 780-960 nm.

23. The method of claim 20, wherein the irradiated EVs are exposed to light with a wavelength of 900-950 nm.

24. The method of claim 20, wherein the wavelength is a line wavelength selected from one of 630 ± 10 nm, 660 ± 10 nm, 808 ± 10 nm, 830 ± 10 nm, or 940 ± 10 nm.

25. The method of any of claims 19 to 24, wherein an irradiance for the irradiating step is 1-200 mW / cm226. The method of claim 25, wherein the irradiance is 5-50 mW / cm2.

27. The method of any of claims 19 to 26, wherein a fluence for the irradiating step is 0.1 - 100 J / cm228. The method of claim 27, wherein the fluence is 0.5-10 J / cm2.

29. The method of any of claims 19 to 28, wherein the irradiating step irradiates isolated EVs that are suspended in an aqueous medium.

30. The method of claim 29, wherein an EV suspension depth is about 2-10 mm with active mixing.

31. The method of claim 29, wherein an irradiance uniformity is less than 10% coefficient of variation (CV).

32. The method of any of claims 19 to 31, wherein a temperature increase during the irradiating step is less than or equal to 2°C.

33. The method of claim 32, wherein a temperature during the irradiating step is maintained between 2-10°C.

34. The method of any one of claims 19 to 33, wherein an electromagnetic radiation during the irradiation step is pulsed at 10-1,000 Hz with a duty cycle of 10-50%.

35. The method of any one of claims 13 to 34, wherein the EVs are bacterial extracellular vesicles (BEVs).

36. The method of claim 35, wherein the BEVs are cultured from Lactobacillus brevis.

37. The method of any one of claims 19 to 36, further comprising the step of filtering the EVs released from the irradiated cells.

38. The method of any one of claims 19 to 37, wherein the EV capability metric is a concentration of EVs.

39. The method of claim 38, wherein the concentration of EVs increases by 50% as compared with the nonirradiated control.

40. The method of any one of claims 19 to 37, wherein the EV capability metric a target-cell uptake measurement.

41. The method of claim 40, wherein the target-cell uptake measurement is determined by flow cytometry or single-particle tracking.

42. The method of claim 40, wherein the target-cell uptake measurement is 120% or more.

43. The method of any one of claims 19 to 37, wherein the EV capability metric is a functional bioassay measurement.

44. The method of claim 43, wherein the functional bioassay measurement quantifies a fibroblast scratch closure amount, a neurite outgrowth amount, or a TGF-P decrease amount.

45. The method of claim 43, wherein the functional bioassay measurement quantifies the fibroblast scratch closure amount, and the fibroblast scratch closure amount is 120% or more.

46. The method of claim 43. wherein functional bioassay measurement quantifies the TGF-P decrease amount, and the TGF-P decrease amount is less than or equal to 80%.

47. The method of any one of claims 19 to 37. wherein the EV capability metric is a zeta-potential magnitude shift.

48. The method of claim 47, wherein the zeta-potential magnitude shift is greater than or equal to 2 mV.

49. The method of any one of claims 19 to 37. wherein the EV capability metric is a nanoparticle tracking analysis (NTA) size distribution.

50. The method of claim 49, wherein the NTA size distribution is preserved within ±15 nm.

51. The method of any one of claims 19 to 37, wherein the EV capability metric is a cargo change metric.

52. The method of claim 51, wherein the cargo change metric is an increase in miR-29b.

53. The method of claim 52, wherein the increase in miR-29b is 30% or more.

54. The method of any one of claims 19 to 53, wherein the EV capability metric change persists for 24-72 hours when stored at 2-8°C.

55. The method of claim 19, wherein the irradiated EVs are exposed to light with a wavelength of 520-560 nm.

56. The method of claim 55, wherein an irradiance for the irradiating step is 1-20 mW / cm257. The method of claim 55 or 56, wherein a fluence during the irradiation step is 0.1-3 J / cm258. The method of any of claims 55 to 57, wherein the irradiating step is accomplished in conditions to limit lipid peroxidation.

59. The method of any one of claims 19 to 58, wherein the irradiating step is conducted in a light-conditioning system comprising a light source, a photodiode dosimeter, and a mixing element.

60. The method of claim 59. wherein the light-conditioning system includes a sample vessel with a 2-10 mm optical path.

61. The method of claim 60, wherein the light source is an LED array emitting 600-1050 nm wavelength light with a uniform irradiance having a coefficient of variation (CV) less than 10%.

62. The method of claim 60, wherein the light-conditioning system includes closed-loop thermometry and a controller configured to maintain a change in temperature of less than or equal to 2°C.

63. A method of treating cancer in a subject, the method comprising the step of: providing a therapeutically effective amount of red-light irradiated extracellular vesicles derived from Lactobacillus brevis (Lacto-EVs).

64. The method of claim 63, wherein the concentration of Lacto-EVs is 1.0x108-1.0x1010particles / ml.

65. The method of claim 64, wherein the concentration of Lacto-EVs is 6.0x108-1.0x109particles / ml.

66. The method of claim 63, wherein the concentration of Lacto-EVs is 0.1 xlO11-1x1011particles per dose, with a dosage rate of 1-3 times weekly for 1-6 weeks.

67. The method of any one of claims 63 to 66, further comprising the step of priming the subject by enhancing miR-214 production.

68. The method of any one of claims 63 to 67, wherein the Lacto-EVs are filtered.

69. The method of any one of claims 63 to 68, further comprising the step of administering an immunotherapy treatment.

70. The method of any one of claims 63 to 69, further comprising the step of administering a chemotherapy treatment.

71. The method of any one of claims 63 to 70, further comprising the step of priming with cerebral endothelial cell exosomes that express or overexpress miR-214 before providing the therapeutically effective amount of red-light irradiated extracellular vesicles derived from Lacto-EVs.

72. The method of any of claims 63 to 71, wherein the providing step includes topical application, intradermal administration, subcutaneous administration, perilesional administration, intraneural administration, or intravenous administration.

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