Light-activated loading of therapeutic molecules into modified extracellular vesicles for targeted delivery to cells

Abstract

Methods and compositions for light-activated loading of therapeutic compositions into yeast-generated extracellular vesicles (EVs) and their methods of use in treating one or more diseases or conditions.

Description

[0001] LIGHT-ACTIVATED LOADING OF THERAPEUTIC MOLECULES INTO MODIFIED EXTRACELLULAR VESICLES FOR TARGETED DELIVERY TO CELLS CROSS-REFERENCE TO RELATED APPLICATIONS This International PCT application claims the benefit of and priority to International PCT application PCT / US2023 / 076329 filed October 7, 2023, the specification, claims and drawings of which are incorporated herein by reference in their entirety. SEQUENCE LISTING The instant application contains contents of the electronic sequence listing (90355-00082- Sequence-Listing.xml; Size: 26,317 bytes; and Date of Creation: April 4, 2024) is herein incorporated by reference in its entirety. TECHNICAL FIELD The present invention is directed to novel methods, systems, and compositions to facilitate light activated loading of therapeutic compositions into yeast-generated extracellular vesicles (yEVs or EVs) and their methods of use in treating one or more disease or conditions. BACKGROUND One of the major challenges facing the use of therapeutic molecules and compounds, also sometimes referred to as a drugs, therapeutics, or therapeutic compositions, to control diseases and genetic disorders is an effective means to deliver the drug to a targeted tissue while avoiding degradation or its elimination from the body. Recently, lipid encased nanovesicles have proven to be an effective means to deliver therapeutic molecules to human cells while protecting their cargo from degradation. The use of lipid nanovesicles to deliver mRNA to cells has proven to be an efficient means to vaccinate humans against SARS-CoV-2. However, the artificial lipids frequently used to make these nanovesicles are not tissue or cell specific, and are not well tolerated by humans limiting their applications to non-therapeutic purposes. An alternative, natural lipid nanovesicle that has also been shown to package RNA, proteins and other small molecules are extracellular vesicles (EVs) or exosomes produced and released from the surface of human cells. Most eukaryotic EVs are well tolerated but have limitations due to the heterogeneity of cell types from which they originate resulting in broad distributions of particle size and heterogeneous cargo. Furthermore, EVs or exosomes produced in human cell lines can potentially be contaminated by non-target RNA species and human pathogens such as viruses. In addition, each of these delivery platforms must be stored at ultralow temperatures and are costly to produce An alternative platform for producing more uniform EVs that package therapeutic molecules is the production of EVs in single celled eukaryotic organisms (non-human) having uniform genetic traits. Examples of single-celled organisms that can be engineered to deliver therapeutic molecules include yeast, such as Saccharomyces cerevisiae and Saccharomyces boulardii. Significantly, these yeast strains are well tolerated by humans, being used in the manufacture of food and beverages consumed by humans. Significantly, S. boulardii has been safely used as a human probiotic for over 65 years. More specifically, S. boulardii has many traits that are ideal to produce EVs for delivery of therapeutics to humans. S. boulardii produces EVs that package RNA, proteins, and other small molecules. In addition, the genome of S. boulardii is available and S. boulardii can be genetically engineered and / or have its genome edited for the purpose of modifying EV targeting or cargo. Analyses of the transcriptome of S. boulardii whole cells indicates that there are no RNA species that have greater than 11 nucleotides similarity to human transcripts and there is only a single RNA species that has any complementary to a human transcript, a non-coding RNA species. This observation contrasts with human exosomes that contain 100s of RNA species that could have off-target effects. S. boulardii can also be grown at large scale in fermenters and has a shelf life of over a year at room temperature when freeze-dried. Finally, it has been observed that yeast extracellular vesicles can be delivered to mice orally as well as by injection and have access to all major organs in the mouse body. These traits open the possibility of using S. boulardii as a complete, single celled system for the large-scale production and packaging of therapeutic molecules into EVs for delivery to humans. Several traditional methods have been developed to load therapeutic compositions into EVs for delivery to target cells or tissues. These methods for encapsulating cargo into EVs can be roughly divided into two types: cell-based loading methods and non-cell-based loading methods. In the cell-based loading approach, cargos are usually delivered into the donor cells first. After being packaged into EVs, the cargos can be secreted and collected in an EV-carrying manner for therapeutic use. Non-cell-based loading approaches involve directly loading chemicals or biomolecules into isolated EVs through electroporation, sonication, incubation, and / or transfection. However, each of these non-cell based methods involve specific technical and commercial limitations. For example, passive incubation loading typically includes low loading efficiencies, while transfection relies on transfection efficiency, which is always variable, and may further alter the structure of the EVs. Methods such as electroporation, sonication, and freeze thaw disrupt the EVs structure and increase EV instability and can further result in undesired EV aggregation and low yields. Finally, current cell, and non-cell based methods of EV loading lack the ability to precisely, selectively, and uniformly load each EV so that it can be more effectively dosed for therapeutic applications. As such, there exists a long-felt need for an efficient, and commercially viable, method to load therapeutic compositions precisely and uniformly into EV structures, all while causing minimal disruption to their structure and aggregation patterns. SUMMARY OF THE INVENTION The present invention is directed to novel methods, systems, and compositions for actively loading a xenobiotic, such as a therapeutic compound into an EV, wherein the EV can deliver the therapeutic composition to a target cell thereby treating a disease or condition or generating some physiological or other effect. In another aspect, the present invention includes systems and compositions to genetically modify yeast cells, and preferably a Saccharomyces yeast cell, to express one or more heterologous fusion peptides configured to facilitate light-driven active transport of target compositions, such as therapeutic compositions into an EV derived from said yeast cell. In one preferred aspect the current invention includes novel constructs for the heterologous expression of one or more fusion peptides in yeast adapted to facilitate for light-driven active transport of xenobiotic compositions into a yeast EV. In another aspect, the present invention includes systems and compositions for light-driven loading of a xenobiotic, and preferably a hydrophobic xenobiotic compound into a Saccharomyces-generated extracellular vesicle (EV). In a preferred aspect, this embodiment can include generating an EV having a heterologous membrane bound fusion peptide. In this preferred aspect, the fusion peptide includes a first domain comprising membrane targeting domain or motif, which can include an EV membrane protein anchor and / or a protein extracellular vesicle sorting motif (p-ESM), a second domain comprising a proton driven xenobiotic transporter, and a third domain comprising a light-dependent proton pump. The membrane targeting domain can be configured to orient the proton driven xenobiotic transporter and / or the light-dependent proton pump such that when a pH gradient is established across the membrane of the EV in response to light energy being applied to the yEV, the transporter will actively and selectable transport a xenobiotic from the external environment into the lumen, or internal portion of the EV by the transporter. In a quantity of EV can be loaded with one or more xenobiotics, and preferably therapeutic compounds and isolated. These isolated EVs can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition that can be administered to a subject in need thereof to treat a disease or condition. In another aspect, the present invention includes a genetically modified yeast cell configured to express a heterologous nucleotide, operably linked to a promoter, encoding a fusion peptide. In this preferred aspect, the fusion peptide includes a first domain comprising membrane targeting domain, which can include an EV membrane protein anchor and / or a protein extracellular vesicle sorting motif (p-ESM), a second domain comprising a proton driven xenobiotic transporter, and a third domain comprising a light-dependent proton pump. The fusion peptide of the invention can be expressed in the yeast cell and incorporated into a yeast-derived EV through the membrane targeting domain. The EVs having the fusion peptide can be isolated and light energy introduced, optionally in the presence of one or more co-factors that can interact with the light-dependent proton pump causing the active transport of proton into the lumen of the EV forming a pH gradient across its membrane. In this preferred aspect, the yeast-derived EVs of the invention can be contacted with a xenobiotic, such that the xenobiotic is transported into the lumen of the EV by the transporter driven by the artificial gradient created by the light-dependent proton pump. Additional aspects of the invention will be evident from the specification, figures, and claims provided herein. BRIEF DESCRIPTION OF THE FIGURES FIG 1. Upper, yEV membrane protein model of the truncated Sur7-AbeM fusion protein. Truncated Sur7 is indicated in red and the AbeM protein in blue. Lower, AbeM-pESM protein with C-terminal yEV protein sorting motif used to localize the AbeM protein into the yEV membrane. FIG 2. Fluorescence excitation and emission spectra of doxorubicin in solution FIG 3. Calibration curves for doxorubicin absorbance (A) and fluorescence (B) are generated from data shown in Figure 2. Despite a very low doxorubicin quantum yield, fluorescence is detected at a very low concentration of the drug (~ 2 µM). The doxorubicin fluorescence signal was detected at ^ 550 nm using an excitation ^ of 470 nm. Since the scattering of concentrated exosome suspensions is very high, proper detection of the doxorubicin signal requires spectrum taking and background subtraction. FIG 4. The efficiency of cellular uptake of free doxorubicin (DX) vs doxorubicin loaded into yEVs (EVs-DX) by electroporation (A) or by AbeM-mediated uptake (B). FIG 5. Properties of yEVs before and after acidification treatment. FIG 6. Oregon Green fluorescence decay from yEVs equilibrated at pH 3 and suspended in pH 8 buffer. FIG 7. Procedure for generating yEV transmembrane proton gradient and pH-dependent drug loading. Figure 8. Relative uptake of doxorubicin into wild type (WT) and engineered (Sur7-Tr and Sur7-Full) as a function of yEV lumenal pH. FIG 9. Determination of the optimal yEV lumenal pH for doxorubicin uptake. FIG 10. Final comparison of doxorubicin uptake into various constructs. FIG 11. Protein structure of Saccharomyces cerevisiae T-SNARE EV membrane localized protein SSO2 predicted by Alphafold. FIG 12. Protein structure of Haloterrigena turkmenica rhodopsin predicted by Alphafold. Figure 13. Human glycophorin A protein structure predicted by Alphafold. FIG 14. AbeM protein structure predicted by Alphafold. FIG 15. yEV membrane protein model of the truncated Sur7-AbeM fusion protein. The truncated Sur7 is shown at N-terminus, following by the AbeM protein, single-helix of glycophorin (GpA), and microbial delta rhodopsin with retinal cofactor at C-terminus. The microbial delta rhodopsin is a light-driven proton-pumping protein from Haloterrigena turkmenica (Halo). FIG 16A-B. pH changes solution of yEVs upon laser illumination. (A) Illustration of Sur7- anchor fusion constructs with microbial delta rhodopsin, a light-driven proton-pumping protein from H. turkmenica (Halo) (SEQ ID NO.’s 23 and 24). (B) Changes in proton concentration at 30 min after laser illumination. FIG 17A-B. pH changes in solutions of yEVs upon laser illumination. The left panel (A) illustrates the AbeM fusions with microbial delta rhodopsin, a protein that pumps protons upon exposure to light (SEQ ID NO.25 and 26). (B) The right panel shows the time-dependent changes in proton concentration after laser illumination. FIG 18. Doxorubicin uptake into yEVs expressing AbeM-Halo fusions is light-dependent. Laser-illuminated wild-type yEVs grown in the presence of all-trans-retinal served as negative control. FIG 19A-B. yEVs expressing AbeM-Halo fusions may uptake fluorescent derivative of antibiotic vancomycin. (A) Laser-illuminated wild-type yEVs grown in the presence of all-trans- retinal served as negative control. (B) Uptake of vancomycin-FL is pH-dependent. Results for yEVs expressing S7tr-AbeM-Halo (SEQ ID NO. 25). Similar results were obtained for AbeM- GpA-Halo (SEQ ID NO.26) yEVs. FIG 20. Doxorubicin uptake into yEVs expressing Sur7tr-AbeM-Halo fusion (SEQ ID NO.26) depends on light intensity. FIG 21. Concentration-dependent drug uptake into yEVs expressing AbeM-Halo fusions A. Uptake of doxorubicin into AbeM-Halo (SEQ ID NO.4) and Sur7tr-AbeM-Halo (SEQ ID NO. 3) constructs is about the same. Solid lines represent the approximations of the data sets with the Hill equation. B. Uptake of Vancomycin-FL into Sur7tr-AbeM-Halo (SEQ ID NO.3), saturation concentration is unreachable. DETAILED DESCRIPTION OF THE INVENTION While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims. All references cited herein are incorporated in their entirety by reference. The terminology used herein is for describing particular embodiments and is not intended to be limiting. As used herein, the singular forms “a,” “and” and “the” include plural referents unless the content and context clearly dictate otherwise. Thus, for example, a reference to “a” or “the” marker may include a combination of two or more such markers. Unless defined otherwise, all scientific and technical terms are to be understood as having the same meaning as commonly used in the art to which they pertain. For the purposes of the present invention, the following terms are defined above. The present invention is directed to EVs derived from Saccharomyces, such as Saccharomyces cerevisiae or Saccharomyces boulardii engineered to actively load therapeutic compositions into an EV that can be administered to a subject in need thereof to treat a disease or condition. In one aspect, the invention describes novel systems, methods and compositions actively loading therapeutic compositions, such as biological and non-biological therapeutic molecules, or conjugates of biological and / or non-biological compositions into yeast EVs using a light driven proton pump to drive transport by a proton-xenobiotic antiporter into a yeast EVs. In one aspect, the invention describes novel systems, methods and compositions actively loading therapeutic compositions, such as biological and non-biological therapeutic molecules, or conjugates of biological and / or non-biological compositions into yeast EVs using a pH gradient to drive transport of therapeutic compositions, such as one or more xenobiotics, by an AbeM proton antiporter, into a yeast EVs. In one aspect, the invention describes systems and methods for loading a xenobiotic compound into a Saccharomyces-generated extracellular vesicle (EV) engineered to include a heterologous membrane bound xenobiotic transporter. In a preferred embodiment, a yeast cell, and preferably a S. boulardii yeast cell can be transformed by an expression vector, operably linker to a promoter, encoding a heterologous fusion peptide having a first domain comprising a first membrane targeting domain and a second domain comprising a proton driven xenobiotic transporter. In one preferred embodiment, the membrane targeting domain includes an EV membrane protein anchor. In this embodiment, the fusion peptide of the invention is configured such that the EV membrane protein is adapted to bind to the EV membrane and orient the xenobiotic transporter in relation to the EV membrane such that the target xenobiotic, such as a therapeutic compound is actively and specifically loaded from the external EV environment into the EV. In a preferred embodiment, the fusion peptide of the invention includes the amino acid sequence according to SEQ ID NO.19, or a sequence having at least 80% sequence homology with SEQ ID NO.19, or a fragment thereof. In another preferred embodiment, the membrane targeting domain includes a protein extracellular vesicle sorting motif (p-ESM). As used herein, a p-ESM includes a protein sequence in a polypeptide that forms a structural motif that causes the corresponding polypeptide to be preferentially packaged when introduced to a yeast-derived EV. Exemplary p-ESMs are described in U.S. Patent Application No.63 / 536,478, and specific p-ESMs of the invention are provided in Table 1, Table 1A; see also SEQ ID NO’s.5-458, and in a preferred embodiment SEQ ID NO’s. 1-24, all of which are incorporated herein by specific reference. In a preferred embodiment, the fusion peptide of the invention includes the amino acid sequence according to SEQ ID NO.21, or a sequence having at least 80% sequence homology with SEQ ID NO.21, or a fragment thereof. In one preferred embodiment, the fusion peptide of the invention includes a proton driven xenobiotic transporter such as an AbeM protein (SEQ ID NO.4), or fragment thereof. As further described in the examples below, in this embodiment, the AbeM transporter can be coupled, preferably through a linker, and preferably a peptide linker according to SEQ ID NO.20, with a second domain comprising a Sur7 protein that has been engineered to orient the AbeM transporter in relation to the outer membrane of the EV to pump the xenobiotics into the EV in response to a pH gradient between the internal and external EV environments. In a preferred embodiment, this modified Sur7 protein can include a truncated Sur7 protein according to SEQ ID NO. 17, or a sequence having at least 80% sequence homology with SEQ ID NO.17, or a fragment thereof. In another preferred embodiment, the fusion peptide of the invention includes the proton driven xenobiotic transporter, again such as an AbeM protein (SEQ ID NO.4), or fragment thereof that is with a second domain comprising a p-ESM, that in a preferred embodiment preferentially directs the fusion peptide to a yEV, and can further orient the xenobiotic transporter in relation to the outer membrane of the EV such that it pumps a xenobiotic into the EV in response to a pH gradient between the internal and external EV environments. In a preferred embodiment, this modified p-ESM protein can include a peptide according to SEQ ID NO.22, or a sequence having at least 80% sequence homology with SEQ ID NO. 2, or a fragment thereof. As noted above, additional exemplary p-ESMs are described in U.S. Patent Application No. 63 / 536,478, all of which are incorporated herein by specific reference. As noted above, in one preferred embodiment, the EV of the invention can be configured to include acidic lumenal pH. In a preferred embodiment, this can be accomplished through one or more proton pump, while in other preferred embodiments, this can further be accomplished by incubating the EVs in an acidic environment, such as an acidic buffer solution. In this embodiment, the rage of the acidic solution, as well as the desired lumenal pH can be determined based on the target xenobiotic, quantity of EVs, as well as the desired dose to be actively loaded into the EV. In a preferred embodiment, the acidic solution can have a pH of approximately 3, which can generate an EV lumenal pH of approximately 4 after incubation. Naturally, this is an exemplary embodiment as the pH solution and lumenal pH can encompass a variety of pH ranges. Next, the EV having an acidic lumenal pH can be contacted with an environment having a pH that is higher, or more basic that the lumenal pH generating a pH gradient across the membrane of the EV, which energetically drives the transporter to import a target xenobiotic from the external environment into the EV. In a preferred embodiment, the step of generating a pH gradient across the membrane of the EV includes contacting the EV with a transfer solution, sometimes referred to as a transfer media, having a pH that is higher than the lumenal pH. Again, while a range of pH gradients are contemplated within the scope of the invention, in one embodiment the transfer solution has a pH of approximately 8. As further described in the examples below, the xenobiotic can be contacted with the EV, for example by being added to the transfer media prior to, or after generating the pH gradient. The amount of xenobiotic to be loaded can be dependent on the transported used, the physical characteristic of the xenobiotic to be transported as well as the pH gradient generated to drive the active transport process. The loaded EVs of the invention can further be isolated from the transfer media and combined with a pharmaceutically acceptable carrier forming a pharmaceutical composition as generally defined herein. A therapeutically effective amount of the pharmaceutical composition of the invention can be administered to a xenobiotic to a subject in need thereof, the subject preferably being a mammal, and more preferably being a human or human cell. In another preferred aspect, the invention includes a genetically modified yeast cell configured to express a heterologous fusion peptide for actively loading a therapeutic composition into an EV. In this preferred embodiment, an exemplary EV membrane protein anchor, a light- dependent proton pump, optionally a transmembrane domain, which may be included depending on the orientation of the transporter, for example a proton driven drug transporter in the EV membrane; a proton-driven drug transporter and a linker protein. In another preferred embodiment, heterologous fusion peptide for actively loading a therapeutic composition into an EV may include an EV-localized Saccharomyces cerevisiae SSO2 membrane protein, which may be a full-length or a truncated portion extending from position M1 to D161 of SEQ ID NO.1; 2) bacteriorhodopsin derived from Haloterrigena turkmenica 3) a human glycophorin A transmembrane protein to allow for the proper functional orientation of C-terminal protein fusions domains including Halo and AbeM, 4) an AbeM protein from Acinetobacter baumannii and; 5) a linker peptide adapted to allow proper conformational folding of the N- and C-terminal protein fusions. All nucleotide sequence encoding the fusion peptide, or other heterologously expressed peptides, can be codon optimized for expression in yeast, and preferably Saccharomyces boulardii. In another aspect, retinal required for Halo function may be produced by the yeast cell, or added exogenously to EVs to reconstitute light-driven proton pumping. In this embodiment, a yeast cell, and preferably a Saccharomyces yeast cell, can be engineered to heterologously express retinal by the co-expression of genes of the beta-carotene biosynthesis pathway including the crtYB, crtI and crtE genes (or from other beta-carotene producing organisms) and the animal beta- carotene cleavage enzyme, beta-carotene 15,15'-dioxygenase which produces trans retinal. In another preferred aspect, the invention includes novel methods and compositions for loading xenobiotics into EVs produced by yeast. In this embodiment, a target xenobiotic is glycosylated forming a xenobiotic glycoside which can be actively transported the across the membrane of the EV into its lumen through a proton driven drug transporter. The glycosidic bond between the xenobiotic and the glycosyl moiety can be hydrolyzed via a glycosidase anchored to the lumenal side of the EV membrane protein. In another preferred aspect, the invention includes a genetically modified yeast cell configured for the heterologous expression of one or more xenobiotic deconjugating enzymes in a genetically modified yeast cell. In this aspect, a xenobiotic deconjugating enzyme may be expressed in the yeast cell and anchored on the lumenal side of the EV, to an EV membrane protein to regenerate the parent deconjugated compound and to reduce back-flow of the conjugate, such as a xenobiotic conjugate, through its membrane transporter. In another aspect, the EVs of the invention may be modified to display surface ligands that target delivery of the EVs to unique receptors displayed on the surfaces of targeted cell types. The receptor-specific ligands may be fused or anchored to EV membrane proteins and displayed on the EV exterior surface. Examples of such EVs displaying surface ligands for directed transport to target cell receptors are described by Sayre et al., in PCT / US2022 / 014958, which is incorporated herein by reference. The present invention includes systems and compositions to genetically modify yeast cells, and preferably a Saccharomyces yeast cell, to express one or more heterologous peptides configured to facilitate the active transport of target compositions, such as preferably therapeutic compositions, into an EV derived from said yeast cell. In one preferred embodiment, a yeast cell, and preferably a Saccharomyces yeast cell, is engineered to express one or more heterologous fusion proteins including a light-driven proton pump, and a proton driven xenobiotic or xenobiotic conjugate transporter linked to an EV membrane protein. This chimeric protein is configured to orient the fusion peptide of the light- driven proton pump in the EV membrane such that it leads to the light-driven acidification of the EV lumen and the vectorial transport of a target composition, such as xenobiotics or drugs into the EV. The active transport of the target compositions is driven by proton efflux through bacterial and / or eukaryotic small molecule transporters, including xenobiotic transporters. In another preferred embodiment, a yeast cell, and preferably a Saccharomyces yeast cell, is engineered to express one or more heterologous fusion proteins including a bacteriorhodopsin, and preferably a bacteriorhodopsin according to SEQ ID NO.2, or a fragment of variation thereof; light-driven proton pump; a proton-driven xenobiotic or xenobiotic conjugate (e.g., glycosides, glucuronic acid, sulfate or glutathione conjugates) transporter linked to an EV membrane protein that is configured to orient the bacteriorhodopsin in the EV membrane, leading to the light-driven acidification of the EV lumen. The vectorial transport of a target composition, such as a xenobiotic or drug, into the EV driven by proton efflux through bacterial and / or eukaryotic small molecule transporters including xenobiotic transporters from the following transporter families: Multidrug and toxic compound extrusion family (MATE); resistance / nodulation / cell division (RND); small multidrug resistance family (SMR); Major facilitator antiporter (MFS); and / or ATP-dependent transporters (ABC). Methods and systems for a light-activated xenobiotic transport system are described. In one preferred embodiment, a Saccharomyces-generated extracellular vesicle (EV) be isolated, for example from a genetically modified yeast cell configured to express a heterologous nucleotide sequence, operably linked to a promoted, expressing a fusion peptide forming light-dependent xenobiotic transport system having: a light-dependent proton pump; a proton driven xenobiotic transporter; and a membrane targeting domain. In this embodiment, the fusion peptide can be anchored to the membrane of the EV and orientated such that in response to light energy the light- dependent proton pump generates a pH across the membrane of the EV causing a xenobiotic positioned outside the EV to be transported into the lumen of the EV by the action of the transporter. As noted above, the fusion peptide forming the light-activated xenobiotic transport system can further include a transmembrane domain configured to orient the light-dependent proton pump relative to the EV membrane so as to allow proton transport into the lumenal space. As shown in Figure 17, the transmembrane domain can preferably include a transmembrane-helix of glycophorin A (GpA) or odd numbered transmembrane protein that is positioned between the light- dependent proton pump and the proton driven xenobiotic transporter. In one embodiment, the transmembrane domain is selected from a peptide having an amino acid sequence according to SEQ ID NO’s 3, 28, or a fragment thereof, while in alternative embodiments the transmembrane domain is selected from a peptide having an amino acid sequence having between 80% to 99% sequence homology with SEQ ID NO’s 3, 28, or a fragment thereof. The fusion peptide forming the light-activated xenobiotic transport system includes a light- dependent proton pump, which can preferably include a bacteriorhodopsin peptide. In a preferred embodiment, the bacteriorhodopsin peptide can be derived from a bacteria, such as Haloterrigena turkmenica. In a preferred embodiment, the light-dependent proton pump includes a peptide, and preferably a bacteriorhodopsin peptide derived from Haloterrigena turkmenica (Halo) according to SEQ ID NO. 2, or a fragment thereof. In a preferred embodiment, the light-dependent proton pump includes a Halo peptide according to SEQ ID NO.2, or a sequence having between 80% to 99% sequence identity with SEQ ID NO.2. As described below and in the Example, the bacteriorhodopsin peptide in response to light energy, and in the presence of a retinal, and preferably a trans-retinal co-factor can cause the unidirectional transport of protons across the EV membrane into the lumenal space generating a pH gradient which in turn drives the proton driven xenobiotic transporter causing an externally positioned xenobiotic to be actively transported into the lumenal space of the EV. Retinal required for the bacteriorhodopsin function may be produced by the yeast cell, or added exogenously to EVs to reconstitute light-driven proton pumping. In this embodiment, a yeast cell, and preferably a Saccharomyces yeast cell, can be engineering to heterologously express retinal by the co-expression of genes of the beta-carotene synthesis pathway including, the crtYB (SEQ ID NO. 14), crtI (SEQ ID NO. 15) and crtE (SEQ ID NO. 16), or genes from other beta- carotene producing organisms, and the animal beta-carotene cleavage enzyme, beta-carotene 15,15'-dioxygenase which produces trans retinal. In another preferred embodiment, the fusion peptide forming the light-activated xenobiotic transport system includes a light-driven proton pump, again such as a bacteriorhodopsin peptide (SEQ ID NO. 2), or fragment thereof coupled with a membrane targeting domain, which can include a protein extracellular vesicle sorting motif (p-ESM) that is configured to preferentially directs the fusion peptide to a EV, and can further orient the xenobiotic transporter in relation to the outer membrane of the EV such that it pumps a xenobiotic into the EV in response to a pH gradient between the internal and external EV environments. In a preferred embodiment, this modified p-ESM protein can include a peptide according to SEQ ID NO’s 31 or 22, or a sequence having between 80% to 99% sequence homology with SEQ ID NO.31 or 22, or a fragment thereof. As noted above, additional exemplary p-ESMs are described in U.S. Patent Application No. 63 / 536,478, all of which are incorporated herein by specific reference. In one preferred embodiment, the fusion peptide forming the light-activated xenobiotic transport system includes a proton driven xenobiotic transporter such as an AbeM protein (SEQ ID NO. 4), or fragment thereof, or a sequence having between 80%-99% . sequence homology with SEQ ID NO. 4, or a fragment thereof As further described in the examples below, in this embodiment, the AbeM transporter can be coupled, preferably through a linker, and preferably a peptide linker according to SEQ ID NO. 11, or 20, with a membrane targeting domain. In a preferred embodiment, the membrane targeting domain includes membrane protein anchor, which in a preferred embodiment described above can include Sur7 protein, or other yeast EV membrane peptide, which has been engineered to orient the transporter in relation to the outer membrane of the EV to pump the xenobiotics into the EV in response to a pH gradient between the internal and external EV environments. In a preferred embodiment, this modified Sur7 protein can include a truncated Sur7 (Sur7tr) protein according to SEQ ID NO’s 17 or 27, or a sequence having between 80% to 99% sequence homology with SEQ ID NO’s 17 or 27, or a fragment thereof. In one specific embodiment, a fusion peptide forming the light-activated xenobiotic transport system can include an amino acid sequence according to SEQ ID NO.25, or a sequence having between 80% to 99% sequence homology with SEQ ID NO.25, or a fragment thereof. In another specific embodiment, a yeast cell can be engineered to express a heterologous nucleotide sequence, operably linked to a promoter, encoding a fusion peptide forming the light-activated xenobiotic transport according to SEQ ID NO. 25, or a sequence having between 80% to 99% sequence homology with SEQ ID NO.25, or a fragment thereof. In one specific embodiment, a fusion peptide forming the light-activated xenobiotic transport system can include an amino acid sequence according to SEQ ID NO.26, or a sequence having between 80% to 99% sequence homology with SEQ ID NO.26, or a fragment thereof. In another specific embodiment, a yeast cell can be engineered to express a heterologous nucleotide sequence, operably linked to a promoter, encoding a fusion peptide forming the light-activated xenobiotic transport according to SEQ ID NO. 26, or a sequence having between 80% to 99% sequence homology with SEQ ID NO.26, or a fragment thereof. In another preferred embodiment, yeast transgene constructs can be cloned into yeast / E. coli shuttle vectors such as pRS416 and pRS426 for expression of the transgene constructs using the appropriate yeast gene promoter / terminator, such as TDH3p / CYC1t, all of the above being readily known and understood by those skilled in the art. In one aspect of the invention, an exemplary conjugate can include a drug-glycoside. Glycosides are glycoconjugates comprising a sugar covalently attached to another (non-sugar) molecule (the aglycone). Examples in nature include phytochemicals, where the glycoside form is used for storage or transport, and the aglycone is released from the sugar by hydrolysis. Drug glycosides can act as prodrugs, facilitating solubilization and absorption. The prodrugs are then hydrolyzed in the body by glycoside hydrolase enzymes, and the aglycone is then able to perform its therapeutic function. Glycosides can be formed enzymatically by the action of glycosyltransferase enzymes, or by chemical synthesis. Chemical synthesis allows for the glycosylation of a wide variety of API aglycones; enzymatic synthesis is constrained by the substrate specificity of the enzyme. Chemical synthesis via Fischer glycosidation or glycosylation, the Koenigs-Knorr reaction, or chloromethyl synthons are known in the art. Numerous glycoconjugate prodrugs have been described, from daunorubicin (leukemia chemotherapeutic) to propofol (anesthetic) and losartan (treatment of high blood pressure). In one example, the cancer chemotherapeutic 5-fluorouracil (5-FU) was used as the aglycone.5-FU is widely used for a number of cancers and is on the List of Essential Medicines of the World Health Organization. It is administered topically and intravenously; both routes lead to side effects, hence the interest in glycosylation of 5-FU for administration as a prodrug. Transport of glyco–5-FU across cell membranes into the cytosol is mediated by sodium dependent glucose transporter 1 (SGLT1), which normally occurs in the mammalian small intestine; it has been functionally expressed in the yeast Saccharomyces cerevisiae, where it displayed both influx and efflux of substrate, depending on the orientation of the substrate concentration gradient. Hydrolysis of glyco–5-FU has been demonstrated using beta-glucosidase from Agrobacterium sp. (ABG; EC 3.2.1.21, CAZy GH1), a protein of 459 residues (52.2 kDa). Co-expression of a xenobiotic deconjugating enzyme in yeast EVs linked to the lumenal side of an EV membrane allows the system to regenerate the parent xenobiotic and to reduce loss of xenobiotic conjugates (glycosides, glutathione conjugates, etc.) from the EVs and to make the xenobiotic biologically more active than its conjugate. In this example, yeast (Saccharomyces boulardi) was engineered to express sodium dependent glucose transporter 1 (SGLT1) (SEQ ID NO.13), and beta-glucosidase (ABG) (SEQ ID NO.12), and to target them to EVs by tethering them genetically to domains of EV-resident proteins. The chemotherapeutic agent 5-fluorouracil (5-FU) was chemically glycosylated. The purified glyco–5-FU was then administered to the engineered EVs. Transport of glyco–5-FU into the EVs via SGLT1, and its hydrolysis to 5-FU and glucose by ABG, were further demonstrated. In another embodiment, the invention is directed to genetically modified yeast cells adapted to heterologously express and present on the surface of EVs receptor-specific ligands anchored to EV membrane proteins as a fusion peptide. In a preferred embodiment, the anchor portion of the fusion peptide can include one of several EV membrane localized proteins including, Chr1 (GPI- anchored cell wall protein) (SEQ ID NO. 5), Sur7 (multi-pass membrane protein) (SEQ ID NO. 6) or Nce102 (non-classical export protein) (SEQ ID NO.7), or fragments or variants thereof. In another embodiment, cancer cell specific or enriched receptor ligands can be anchored to the EV membrane protein C- or N-terminus so that the ligands are displayed on the surface of the EV. More specifically, in one embodiment surface displayed ligands to target EVs to P-selectin receptors, which are over-expressed in blood vessels of a variety of cancers, can be selected. Specific embodiments may include one or more of the following: NRP1 receptor, follicle- stimulating hormone (FSH) receptor FSHR; epidermal growth factor (EGF) receptor. The examples of receptor-binding ligands used for fusion are C-end class peptides for binding NRP1 receptor (SEQ ID NO.8); AEYLR -small peptide binding EGFR (SEQ ID NO.9); small peptide FSH33-53 for binding FSHR (SEQ ID NO.10). In one embodiment, one or more of the EVs of the invention may include a pharmaceutical compositions. A “pharmaceutical composition” of the invention include a composition of the invention and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” as used herein, refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. The term, “pharmaceutically acceptable carrier” as used herein, includes any and all solvents, or a dispersion medium including, but not limited to, water, ethanol, a polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, stachyose, and the like), suitable mixtures thereof, and vegetable oils, coatings, isotonic and absorption delaying agents, liposomes, commercially available cleansers, and the like. Supplementary bioactive ingredients also can be incorporated into such carriers. As used herein “xenobiotic” refers to a chemical or other compound, whether biological or non-biological which is not a normal component of an organism which is exposed to. Exemplary xenobiotic compositions include, tkanamycin, gentamicin, ofloxacin, ciprofloxacin, erythromycin, chloramphenicol, DAPI, triclosan, acriflavine, Hoechst 33342, daunomycin, doxorubicin, trimethoprim, and / or rhodamine 6G among others. The term “endogenous” gene or protein means that said gene or protein is expressed from a gene naturally found in the genome of a eukaryotic cell. The term “heterologous” gene or protein means that said gene or protein is not expressed from a gene naturally found in the genome of a eukaryotic cell. As used herein, the term “gene” or “polynucleotide” refers to a single nucleotide or a polymer of nucleic acid residues of any length. The polynucleotide may contain deoxyribonucleotides, ribonucleotides, and / or their analogs and may be double-stranded or single stranded. A polynucleotide can comprise modified nucleic acids (e.g., methylated), nucleic acid analogs or non-naturally occurring nucleic acids and can be interrupted by non-nucleic acid residues. For example, a polynucleotide includes a gene, a gene fragment, cDNA, isolated DNA, mRNA, tRNA, rRNA, isolated RNA of any sequence, recombinant polynucleotides, primers, probes, plasmids, and vectors. Included within the definition are nucleic acid polymers that have been modified, whether naturally or by intervention. As used herein, the phrase “expression,” “gene expression” or “protein expression,” such as the level of includes any information pertaining to the amount of gene transcript or protein present in a sample, in a cell, in a patient, secreted in a sample, and secreted from a cell as well as information about the rate at which genes or proteins are produced or are accumulating or being degraded (e.g., reporter gene data, data from nuclear runoff experiments, pulse-chase data etc.). Certain kinds of data might be viewed as relating to both gene and protein expression. For example, protein levels in a cell are reflective of the level of protein as well as the level of transcription, and such data is intended to be included by the phrase “gene or protein expression information.” Such information may be given in the form of amounts per cell, amounts relative to a control gene or protein, in unitless measures, etc. The term “fusion protein” as used herein is used as it is in the art. Namely, the fusion proteins used in the methods and compositions of the present invention involve two separate proteins or protein domains that are linked by a covalent bond. In one embodiment, the covalent bond linking the two domains is an amine bond. In more specific embodiments, the anchor protein and the foreign protein is a fusion protein comprising a single-chain polypeptide. In even more specific embodiments, the single-chain polypeptide comprising the anchor protein and the foreign protein further comprises a linker peptide sequence. Any linker sequence can be used to covalently link the anchor protein and the foreign protein. As used herein, the term “peptide linker(s),” “linker(s),” or “linker moiety” refers to a peptide or polypeptide sequence, e.g., a synthetic peptide or polypeptide sequence, which connects two domains in a linear amino acid sequence of a polypeptide chain. In one embodiment, the polypeptides of invention are encoded by nucleic acid molecules that encode peptide linkers which either directly or indirectly connect the anchor protein and foreign protein which make up the construct. These linkers may be interposed between the anchor protein and foreign protein. If the linker connects two protein moieties contiguously in the linear polypeptide sequence, it is referred to as a “direct” linkage. In contrast, the linkers may link the first protein moiety, i.e., anchor protein or foreign protein, to a binding moiety which is, in turn, linked to the second protein moiety, i.e., anchor protein or foreign protein, thereby forming an indirect linkage. Linkers are typically located at the N or C terminus of the protein moieties. In one embodiment, the linker linking the anchor protein and the foreign protein is a peptide comprised of glycine (Gly)n, wherein n is an integer that is the same or higher than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. In another embodiment, the linker linking the anchor protein and the foreign protein is a gly-ser linker. As used herein, the term “gly-ser peptide linker” (GS) refers to a peptide comprising or consisting of glycine (G or Gly) and serine (Sor Ser) residues. Exemplary gly-ser peptide linkers comprise the amino acid sequence (Gly4 Ser)n or (Gly3 Ser)n. Another exemplary gly-ser peptide linker comprises the amino acid sequence S(Gly4 Ser)n wherein n is an integer that is the same or higher than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. In another embodiment, the linker linking the anchor protein and the foreign protein is a peptide comprising SEQ ID NO.11. As used herein, a “fragment” of a polypeptide refers to a single amino acid or a plurality of amino acid residues comprising an amino acid sequence that has at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 20 contiguous amino acid residues or at least 30 contiguous amino acid residues of a sequence of the polypeptide. A “variant” include a peptide having the same function as an identified peptide, while having a different sequence or being derived from a different organism than an identified peptide. As used herein, a “fragment” of poly- or oligonucleotide refers to a single nucleic acid or to a polymer of nucleic acid residues comprising a nucleic acid sequence that has at least 15 contiguous nucleic acid residues, at least 30 contiguous nucleic acid residues, at least 60 contiguous nucleic acid residues, or at least 90% of a sequence of the polynucleotide. In some embodiment, the fragment is an antigenic fragment, and the size of the fragment will depend upon factors such as whether the epitope recognized by an antibody is a linear epitope or a conformational epitope. Thus, some antigenic fragments will consist of longer segments while others will consist of shorter segments, (e.g., 5, 6, 7, 8, 9, 10, 11 or 12 or more amino acids long, including each integer up to the full length of the polypeptide). Those skilled in the art are well versed in methods for selecting antigenic fragments of proteins. The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,” “prevent” and grammatical equivalents (including “lower,” “smaller,” etc.) when in reference to the expression of any symptom in an untreated subject relative to a treated subject, mean that the quantity and / or magnitude of the symptoms in the treated subject is lower than in the untreated subject by any amount that is recognized as clinically relevant by any medically trained personnel. In one embodiment, the quantity and / or magnitude of the symptoms in the treated subject is at least 10% lower than, at least 25% lower than, at least 50% lower than, at least 75% lower than, and / or at least 90% lower than the quantity and / or magnitude of the symptoms in the untreated subject. The term “introducing,” “administered” or “administering”, as used herein, refers to any method of providing a composition of EVs to a patient such that the composition has its intended effect on the patient. In one embodiment, EVs may be introduced to a patient in vivo, while in other alternative embodiments, EVs may be introduced to subject cells in vitro which may then be administered to a patient in vivo. The term “patient,” or “subject” as used herein, is a human or animal and need not be hospitalized. For example, out-patients, persons in nursing homes are “patients.” A patient may comprise any age of a human or non-human animal and therefore includes both adult and juveniles (i.e., children). It is not intended that the term “patient” connote a need for medical treatment, therefore, a patient may voluntarily or involuntarily be part of experimentation whether clinical or in support of basic science studies. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome. As used herein, “expression cassette” refers to a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The peptides of the invention of the present invention may be chimeric. The expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. As used herein, a promoter region or promoter element refers to a segment of DNA or RNA that controls transcription of the DNA or RNA to which it is operatively linked. The promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter. In addition, the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences may be cis acting or may be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. To accomplish delivery of therapeutic compositions, such as small-molecule drugs, biologics, or other xenobiotics to target cells, the methods and compositions of the present invention comprise extracellular vesicles (EVs), and preferably EVs generated from Saccharomyces, such as S. boulardii or S. cerevisiae. EVs generated from Saccharomyces sometimes are also interchangeably referred to as Saccharomyces-generated extracellular vesicles, sometimes also referred to as SGEVs. The term extracellular vesicles refers to membranous vesicles released from cells. The extracellular vesicles of the methods and compositions of the invention are composed of lipid bilayers that can be loaded with, and carry cargo in its interior. The lipid bilayer of the EVs may also include proteins embedded therein. In some embodiments, the SGEVs of the compositions and methods of the present invention can be exosomes or ectosomes. As is well-known, exosomes are generally formed upon the endocytosis of multivesicular endosomes (MVEs) to form intraluminal vesicles (ILVs) which are subsequently released into the extracellular environment as exosomes, whereas ectosomes are assembled at and released from the plasma membrane. Often, the primary structural feature distinguishing ectosomes and ectosomes is diameter. In some embodiments, the diameter of the SGEVs are between about 30 nm to about 180 nm, between about 50 nm to about 200 nm, between about 75 nm to about 250 nm, between about 100 nm to about 300 nm, between about 125 nm to about 350 nm, between about 150 nm to about 400 nm, between about 175 nm to about 450 nm, between about 200 nm to about 500 nm, between about 250 nm to about 550 nm, between about 300 nm to about 600 nm, between about 350 nm to about between about 650 nm, between about 400 nm to about 700 nm, between about 450 nm to about 750 nm, between about 500 to about 800 nm, between about 550 nm to about 850 nm, between about 600 nm to about 900 nm, between about 650 nm to about 950 nm, between about 700 nm to about 1000 nm, between about 750 nm to about 1050 nm, between about 800 nm to about 1100 nm, between about 850 nm to about 1150 nm or between about 900 nm to about 1200 nm. Thus, exosomes may comprise components on their membrane surface, including but not limited to, proteins, glycoproteins, proteoglycans, carbohydrates, and lipids, which may be used to direct delivery of therapeutic compositions loaded into EVs. As understood by the disclosure herein, Saccharomyces is a single-celled organism, but the term “extracellular vesicle,” as it relates to the SGEVs, refers to vesicles that are secreted from Saccharomyces into the local environment, such as, but not limited to, cell culture medium or the gastro-intestinal tract of organisms that may have ingested or consumed or been administered the Saccharomyces, which secrete the vesicles containing a therapeutic composition. In one embodiment, the SGEVs are secreted from Saccharomyces cerevisiae or Saccharomyces boulardii. The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti- sense) strand. The coding sequence which encodes the peptides may be identical to the coding sequence shown in the sequence listing, or that of any of the deposited clones, or may be a different coding sequence which, as a result of the redundancy or degeneracy of the genetic code, encodes the same fusion proteins as shown in the sequence listing. The term “nucleotide sequence encoding a peptide” encompasses a nucleotide sequence which includes only coding sequences for the polypeptide, e.g., heterologous protein, as well as a polynucleotide which includes additional coding and / or non-coding sequences. Thus, for example, the polynucleotides of the present invention may encode for a peptide, e.g., a heterologous protein, or for a peptide having a prosequence or for a protein having both a prosequence and presequence. The polynucleotides of the present invention may also have the coding sequence fused in frame to, for example, a marker sequence which allows for identification of the polypeptide of the present invention. The marker sequence may be a GFP protein, a hexa-histidine tag to provide for purification of the fusion protein is used. The invention also relates to vectors, including but not limited to, expression vectors comprising the polynucleotides encoding the fusion proteins of the present invention. Types of vectors for expression for proteins and fusion proteins are well known in the art. In one embodiment, the vector is an expression vector for protein expression in Saccharomyces. Yeast expression vectors are commercially available from manufacturers. The present invention also relates to methods of making and using these Saccharomyces- generated EVs. In one embodiment, the methods of making the SGEVs of the present invention comprise introducing into the Saccharomyces the expression vector encoding one or more heterologous proteins and related systems which actively transport therapeutic compositions into the EV, or a fragment or variant thereof, of the present invention to generate a host Saccharomyces cell. The host cell is then cultured under conditions to permit protein production from the vector encoding the heterologous protein. In one embodiment, the host cells of the present invention are Saccharomyces cerevisiae or Saccharomyces boulardii. Culture conditions for culturing yeast host cells are well-known in the art. The continued culture of the host cell will permit production and secretion of the SGEVs into the cell culture environment, where they can be isolated from culture. Methods of isolating extracellular vesicles, such as exosomes, from cell culture media are well- known in the art and are reviewed in Li, P. et al., Theranostics, 7(3):789-804 (2017), which is incorporated by reference herein. Generally speaking, methods of isolating the SGEVs from culture include but are not limited to ultracentrifugation methods, size-based enrichment methods (e.g., tangential flow filtration, size- exclusion chromatography), immunoaffinity capture-based methods, precipitation methods, microfluidics-based methods or some combination thereof. The route of administration of the SGEVs includes, but is not limited to, topical, transdermal, intranasal, rectal, oral, subcutaneous, intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal as disclosed herein. In one example SGEV’s may be derived or isolated from a GRAS and / or probiotic yeast cell, such as Saccharomyces cerevisiae, and preferably Saccharomyces boulardii. For example, Saccharomyces boulardii probiotics, releasing wild type exosomes, have been shown to diminish disease severity by reducing the expression of inflammatory cytokines and stimulating the expression of anti- inflammatory cytokines in multiple organs including the lungs and cardiovascular system. Saccharomyces boulardii cells also have low immunogenicity and positively modulate host immune response in the presence of additional antigens. S. boulardii is well established for to allows the present inventors to engineer the S. boulardii strain for expression and loading of specific xenobiotics in exosomes. Cultivation of S. boulardii is fast, low-cost, and easy to scale up using established procedures. Finally, the lipids present in EVs are natural and thus not likely to be cytotoxic when used therapeutically unlike artificial lipids frequently used to package mRNA for vaccines. In specific embodiments, the oral administration of the SGEVs includes administering engineered yeast, producing the SGEVs, as a probiotic. As used herein, a probiotic is a microorganism, such as a bacteria or yeast, generally recognized as safe for human or animal consumption. The probiotics of the present invention may or may not have additional health benefits to the consumer. In specific embodiments of the present invention, the probiotic is a Saccharomyces cerevisiae or a Saccharomyces boulardii. The terms “comprises”, “comprising”, are intended to have the broad meaning ascribed to them in U.S. Patent Law and can mean “includes”, “including” and the like. The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention. Indeed, while this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. EXAMPLES Example 1: Overview and Experimental Rationale. As described above, S. boulardii produces extracellular vesicles (yEV) that may package RNA, proteins, and other small molecules. S. boulardii can also be genetically engineered and / or have its genome-edited to modify yEV targeting or cargo. To actively load therapeutic small molecular weight drugs into yeast extracellular vesicles Applicants have incorporated into the yEV membrane a multidrug / proton antiporter, AbeM. AbeM is a multidrug transporter of the acid- loving bacterium Acinetobacter baumannii and belongs to the MATE (multidrug and toxic efflux) class of multidrug transporters which also function as proton antiporters. In the presence of a transmembrane proton gradient, AbeM has been demonstrated to transport multiple drugs including, rhodamine G, ethidium bromide, various antibiotics, and doxorubicin. As described in Sayre et al, PCT / US2023 / 076329 to which the current application claim priority, Applicant demonstrated that expression of AbeM in yEVs supported drug uptake into the yEVs lumen upon generation of an artificial proton (H+) gradient. In one iteration, the AbeM gene was linked to a truncated Sur7 protein containing only the first 3 transmembrane helixes. According to the AbeM crystal structure, the N- and C-terminus of the functional AbeM protein are located on the cytoplasmic (high pH side) surface of the bacterial membrane. The AbeM orientation in the truncated Sur7 gene fusion with AbeM resulted in an AbeM orientation that supported transport of drugs from the outside of the yEV (high pH) to the inside (low pH) of the yEV. A second AbeM construct was linked to a protein yEV sorting motif obtained from the C- terminus of chitinase (Uniprot number A0A0L8VL70; see also SEQ ID NO.22) and located at the C-terminus of AbeM to localize the AbeM protein in the yEV membrane. Previously, Applicant further generated a transmembrane pH gradient by incubation of AbeM-expressing yEVs in slightly acid conditions followed by transfer into a drug-containing neutral pH solution. To generate a proton gradient inside yEVs without mechanical manipulation of the yEVs Applicants introduced a bacteriorhodopsin to transduce protons across the membrane driven by light. Bacteriorhodopsin is a seven transmembrane spanning protein containing retinal as a chromophore to absorb light. When bacteriorhodopsin is exposed to light, the retinal cofactor changes from all-trans to the 13-cis conformation. After returning to the ground state, bacteriorhodopsin undergoes a structural change that results in active ion (proton) movement across the bacteriorhodopsin. Thus, rhodopsin converts light energy into a proton (ion) driving force. In one embodiment, yEV localized microbial delta rhodopsin from Haloterrigena turkmenica (Halo) along with added retinal is used to generate a light-dependent pH gradient. To determine if Halo functions in the yEV membranes, two gene constructs expressing Halo as a Sur7 fusion protein were created. These constructs included a Sur7-Halo-Flag (SEQ ID NO.23). Fusion protein and an N-terminal Halo fused to a full-length Sur7 protein (SEQ ID NO.24) (Figure 16A). To fuse the AbeM drug transporter with the light-driven proton pump Halo, Applicant utilized already verified AbeM expressing constructs. Since it had previously been shown that Halo must have its N-terminus located on the lumenal side of the yEV membrane, Applicants separated AbeM and Halo with a single transmembrane-helix glycophorin A (GpA). Thus, creating a Sur7tr-AbeM-GpA-Halo fusion protein (SEQ ID NO. 25). In another iteration, Applicant incorporated a protein yEV-sorting motif (pESM) to direct the AbeM-HALO fusion protein into the yEV membrane. This construct is designated as AbeM-GpA-Halo-PESM (SEQ ID NO. 26). (Figure 17 A). Doxorubicin was used as a model drug for loading into yEVs because it is highly solubility in aqueous buffers and fluorescent. As another exemplary embodiment, Applicants used a fluorescent derivative of the antibiotic vancomycin – vancomycin-BODIPY-FL. Example 2: Materials and Methods. yEV isolation: Overnight cultures of Saccharomyces boulardii were diluted 100 times with YPD medium. Cultures were then grown for 24 h at 30 °C with shaking (200 rpm) in YPD supplemented with 10 µM all-trans-retinal (Cayman Chemical Company, #18449). For yEV isolation, cells and debris were removed by centrifugation at 3500 × g for 35 min. yEVs were then concentrated from the supernatant using a tangential flow filtration device (Pall) with 300 kD membrane to a yEV concentration of approximately 5x1011yEVs / mL. Isolated yEVs were aliquoted and stored in PBS buffer at −80 °C. yEV quantification: The concentration of yEVs was measured using Nanoparticle Tracking Analysis (NTA) with a Particle Metrix NTA. To obtain proper measurement concentrations, all samples were diluted in water to a final volume of 1.5 ml. The samples were then further diluted until a concentration of 140–200 particles / frame was achieved. For each measurement, 11 positions were scanned for three cycles under the following settings: Focus: autofocus; Camera sensitivity for all samples: 75; Shutter: 80; Scattering Intensity: 30; Cell temperature: 25 °C. The video recordings were analyzed by the equipment software to obtain the number of yEVs per ml. Buffer exchange of yEV preps: To quickly purify yEV preps free from any unincorporated doxorubicin yEV preps were subjected to column gel-filtration using PCR Kleen™ Spin Columns from Bio-Rad (7326300). For this purpose, 50-80 mL of yEVs suspension was applied to the column, following the manufacturer's instructions. The columns were immediately spun at 800 x g for 2 minutes, and the samples were collected at the bottom of 1.5 mL centrifuge tubes. Fluorescent drugs quantification using plate reader: Absorbance and fluorescence emission measurements were performed using SpectraMax, M4 plate-reader (Molecular Devices). Calibration curves for both doxorubicin and vancomycin BODIPY-FL (Thermofisher, V34850) fluorescence were generated using serial dilutions of the compounds. The doxorubicin fluorescence signal was detected at 550 nm using an excitation of 470 nm. The Vancomycin BODIPY-FL fluorescence signal was detected at 525 nm using an excitation wavelength of 485 nm and a cutoff filter at 515 nm. Since the scattering of concentrated exosome suspensions is very high, proper detection of the doxorubicin signal requires background subtraction. Light sources: To generate a light-generated pH gradient, we used LED-based laser pointers with a wavelength of ~532 nm. Lasers having powers of either ~5 mW or ~100 mW were used. Measurement of the proton pumping activity of yEVs: To evaluate the proton pumping activity of yEVs, yEVs isolated from S. boulardii by ultrafiltration were used. The yEVs were washed extensively with distilled water. The proton pumping activity of the yEVs was measured by monitoring changes in the pH of the yEVs suspension. The concentration of yEVs was adjusted to 1010yEVs / ml using distilled water. Original pH of the samples was recorded. The pH values of the yEVs preps were measured at indicated time points over 2 hours after laser illumination using a pH meter (Sension+ PH31, Hach). Example 1: Light-driven proton transport in yEVs isolated from Halo-fusion protein expressing yeast strains. yEVs expressing Halo constructs harvested from yeast grown in the presence of all-trans- retinal appeared brightly pink, while wild-type yEVs produced under the same conditions were also slightly pink but noticeably lighter in color. To evaluate the efficiency of proton pumping by yEVs expressing Sur7-Halo (SEQ ID NO. 23) and Halo-Sur7 (SEQ ID NO. 24), Applicants measured the pH of yEV solutions before and after direct illumination with laser pointers as light source. Wild-type yEVs saturated with all- trans-retinal was used as a control. Before the experiments, all yEV samples were kept in dark conditions. A suspension of 5 mL of yEVs (1010particles / mL) was prepared in distilled water (pH 7) and illuminated for 2 minutes with a 5mW green laser pointer. Light-dependent changes in pH were measured at 0, 5, and 30 minutes after illumination. Applicant’s found that proton pumping by Halo-yEVs, produced an increase in pH in the external media that continued for approximately 30 minutes. We observed a 6-fold decrease in the concentration of protons in the suspension of the most active construct, Halo-Sur7 (SEQ ID NO.24) (Figure 16B). Reduced light-dependent pH changes were observed in the suspension of Sur7-Halo (SEQ ID NO. 23) yEVs, while there were no pH changes in the suspension of WT yEVs. Based on these observations, Applicant concluded that the light-driven proton-pumping microbial delta rhodopsin (Halo) is properly folded in the Halo-Sur7 (SEQ ID NO.24) fusion and preserves its functions in yEVs membranes. Example 2: Halo is active in both Sur7tr-AbeM-GpA-Halo and AbeM-GpA-Halo. Next, Applicant’s evaluated if the light-dependent pH changes occurred in yEVs isolated from engineered strains carrying both Halo and AbeM proteins. yEVs from both engineered strains were characterized by NTA instrument. No difference in size or Zeta-potential was observed between yEVs from wild-type or engineered strains to determine if light could drive generation of a proton gradient. Next, 1010yEVs / mL yEV suspensions were prepared and illuminated as described above. The external pH was monitored for 120 min. As shown in Figure 17, both constructs demonstrated time-dependent pH changes related to proton pumping into the yEVs. Construct Sur7tr-AbeM- GpA-Halo (SEQ ID NO.25) was most efficient. No pH changes were observed for the wild-type yEV suspension. Example 3: Accumulation of doxorubicin by Sur7tr-AbeM-GpA-Halo and AbeM-Halo yEVs required light exposure. To verify the applicability of Sur7tr-AbeM-GpA-Halo (SEQ ID NO.25) and AbeM-GpA- Halo (SEQ ID NO. 26) yEVs constructs for drug-uptake, light-treated (2 min, 5 mW light) and untreated yEVs suspension (1010yEVs / ml) were supplied with an equal volume of 100 µM solution of doxorubicin. Samples were incubated at 23 °C for 1 h. At the end of the incubation period unincorporated doxorubicin from yEV preps was removed by column gel-filtration. Purified samples were quantified by NTA analysis and loaded on a black 96-well microplate. As shown in Fig 18, no doxorubicin uptake was observed by wild-type yEVs; drug uptake of light- treated samples was 3-5 fold higher than of light untreated samples. Thus, efficient drug uptake of doxorubicin into Sur7tr-AbeM-GpA-Halo and AbeM-GpA-Halo yEVs is light-dependent and attributed to the light-dependent proton pumping activity of Halo. Example 4: Sur7tr-AbeM-GpA-Halo (Seq ID 3) and AbeM-Halo (Seq ID 4) yEVs light-dependent uptake of antibiotic vancomycin is pH dependent Since vancomycin is a known AbeM substrate, the very bright fluorescently labeled antibiotic vancomycin-BODIPY FL (briefly, vancomycin) could be a very convenient tool to study drug uptake into yEVs (Fig 19). Thus, Applicant’s verified the light-dependent uptake of vancomycin into the yEVs lumen. Light-treated yEVs were mixed with equal volume of 10 µM of vancomycin. Samples were incubated for 1 h at 23 °C and were treated as described above for doxorubicin (Fig.19A). The yEV lumenal vancomycin concentration in Sur7tr-AbeM-GpA-Halo and AbeM-GpA-Halo yEVs (SEQ ID NO’s 3 and 4) was significantly higher than in background of wild-type yEVs. Thus, Applicants established that AbeM-Halo constructs (SEQ ID NO’s 3 and 4) are capable of light-dependent uptake of another ligand, vancomycin. Next Applicant’s evaluated the potential impact of different external buffer pHs on drug uptake to determine the optimal pH for drug uptake. We chose to use a fluorescent vancomycin ligand for this purpose.10 µM vancomycin solutions were prepared in 50 mM buffers: glycine pH 3.0, MES pH 5.7, and HEPES pH 7.2. Vancomycin uptake experiments were performed as described above. Results are shown in Fig.19 B. Applicant concluded that a neutral pH external buffer was optimal for drug uptake. Example 5: Higher energy light results in higher drug uptake. Light intensity-dependent rates of drug uptake were further evaluated. In previous experiments Applicants used 5 mW light source and illuminated samples for 2 min. In addition, Applicants used a 100 mW light source for 5s, 30s, and 60s. The results are shown in Fig.20. The illumination with 100 mW light led to greater drug uptake compared to 5 mW illumination. Applicants concluded that 1 min exposure of Sur7tr-AbeM-Halo fusion (SEQ ID NO. 3) yEVs with a 100 mW light source is optimal for drug uptake. (Fig. 20) In separate experiments Applicants verified that 60s 100 mW light exposure produces similar results as 120s light exposure. Applicants elected to use 120s 100 mW light exposure in further experiments. Example 6: Light-dependent drug uptake by Sur7tr-AbeM-GpA-Halo (Seq ID 3) and AbeM-Halo (Seq ID 4 ) yEVs in optimized conditions. To determine the yield of drug-uptake under optimal conditions, Applicant’s prepared concentrated 1011yEVs / mL suspensions in neutral distilled water. Samples were light treated for 2 min using a 100 mW light source and mixed with serial dilutions of doxorubicin and vancomycin. Incubation in light was continued for 1 h at 23 °C. Results of doxorubicin drug uptake by Sur7tr- AbeM-GpA-Halo (SEQ ID NO.3) and AbeM-GpA-Halo (SEQ ID NO.4) yEVs preps are shown in Fig 21A. In agreement with previous experiments, doxorubicin uptake was slightly better for Sur7tr-AbeM-GpA-Halo. Results were fitted to Hill equation. The concentration of EF50%we obtained from regression fit was about the same for both constructs ~150 µM. Thus, the optimum concentration of doxorubicin for loading into yEVs is approximately 300 µM. Using this concentration leads to a concentration of 100-150 µM of the drug in the suspension of yEVs at a yEV concentration of 5x1011particles / ml. Thus, each yEV particle contain 100-150 doxorubicin molecules. Example 7: Loading of doxorubicin into yEVs by electroporation Electroporation for the loading of yEVs with doxorubicin was repeated four times with average loading yield 0.79 µg per 1010yEVs (SE=0.3). The total amount of recovered yEVs was measured on ZetaView NTA, and the drug loading efficiency was determined by fluorescence signal measurements at 470 / 560 nm Ex / Em on a SpectraMax M4. Example 8: Cellular uptake of free doxorubicin and doxorubicin loaded into yEVs Free doxorubicin at concentrations of 0.45 µg / ml or 4.5 µg / mL and of doxorubicin (0.45 µg / mL) loaded into yEVs was incubated with H1299 human lung cells for 2 hours at 37 C. Cells were then rinsed 3 times with fresh RPMI media to remove non-incorporated doxorubicin and images of cells were taken using an RFP filter set (excitation 545-580 nm, emission 610) using the same exposure settings for all samples. The fluorescent signal attributed to doxorubicin captured by cells was measured with ImageJ to compare the efficiency of doxorubicin uptake. Applicants observed that more than a 10-fold greater concentration of free doxorubicin was required to yield the same doxorubicin fluorescence signal intensity compared to doxorubicin delivered by yEVs (Figure 4) Example 5: Proton gradient formation To load doxorubicin into AbeM-expressing EVs, Applicants ensured the formation of the proton gradient across the yEV membrane. Applicants equilibrated yEVs with protons by overnight incubation with acid buffers. To preserve AbeM integrity a buffer pH equal to 3 or higher was used to acidify the yEV lumen. To create a yEV transmembrane pH gradient, Applicants did a very fast buffer exchange by spin-column gel filtration. As shown in figure below (Figure 5) it is apparent that this acidification treatment had no apparent impact on yEV particle size integrity or yield. To make sure that a proton gradient was generated, Applicants monitored proton release using the fluorescent pH indicator Oregon Green. At neutral pH, Oregon Green has high fluorescence which decreases at acid pH. To monitor the transmembrane fluorescence change, 75 µL of 50 nM of OG solution in 50 mM Tris-HCl pH 8.0 was mixed with 25 µl freshly column- exchanged proton-loaded yEVs in the well of a black microplate. Changes in fluorescence in response to proton release from acidified yEVs were monitored by fluorescence ( ^ex 485 nm, cutoff 495 nm, ^em 525 nm) using the kinetic mode of plate reader Spectramax M4. As shown in Figure 6, the pH gradient degrades slowly (> 2 h). Thus, Applicants incubated acid-equilibrated yEVs for a longer time, ~ 16 h. Example 9: Doxorubicin uptake into wild-type and Sur7-AbeM-expressing yEVs yEVs preps were prepared as described above as a water-suspension. The general scheme of procedure is presented in Figure 7. To create pH gradients, yEVs were equilibrated in buffers of acid pH overnight at room temperature. The following buffers were used for the acid equilibration of yEVs: 100 mM Glycine pH 3.0, 100 mM MES pH 4.0, and 100-mM potassium acetate, pH 5.4. After incubation samples were quickly transferred to 10 mM Tris pH 7.5 and immediately mixed with equal amounts of 400 mM Tris pH 8.0, 500 ^M doxorubicin. Following incubation for 16 h at room temperature unincorporated doxorubicin was removed by gel-filtration as described above. yEV preps were analyzed on doxorubicin content, aliquoted, and stored at – 80 °C. Example 10: Proper AbeM orientation in the membrane for drug pumping into yEVs To determine the working orientations of AbeM, Applicants used preps of yEVs in which AbeM was linked to the C-terminus of either a truncated (Tr) and full (Full) Sur7 protein that serves as an anchor for localization in the yEV membrane. Doxorubicin content was detected by fluorescence and concentrations were determined using the calibration curve. In this experiment, Applicants found that doxorubicin uptake was observed only in Tr constructs. No significant uptake of doxorubicin was observed in yEVs expressing the Full Sur7-AbeM protein fusion or in yEVs without AbeM. Thus, all future experiments were performed with the Tr construct. Example 11: Optimal pH for doxorubicin uptake To determine optimal conditions for doxorubicin uptake into Tr-yEVs, Applicants compared doxorubicin uptake using yEVs equilibrated at various acid pHs followed by transfer to approximately pH 8 media plus doxorubicin (500 µM). We observed that a yEV lumenal pH of approximately 4.0 was optimal for doxorubicin uptake. Example 12: Doxorubicin uptake by yEVs expressing the AbeM-p-ESM construct. Using the established pH gradient driven drug loading conditions, Applicants analyzed doxorubicin uptake into yEVs containing the AbeM-p-ESM protein. AbeM-p-ESM-yEVs were equilibrated at pH 4.0 for 16 h in MES pH 4. Using a spin column, buffer was exchanged into 100 mM Tris-HCl pH 8.0 supplemented with 250 µM doxorubicin. After incubation for 24 h, excess of doxorubicin was removed, and doxorubicin content was analyzed by fluorescence emission using a plate reader. As shown in Fig.10, AbeM-p-ESM-yEVs had 6-fold greater drug uptake than Tr-yEV and 50-fold greater doxorubicin uptake than Full-yEVs construct. Doxorubicin-loaded AbeM-PESM-yEVs are visibly colored and may easily detected by both spectrophotometry and fluorescence. Drug loading on AbeM-p-ESM-yEVs was also much faster. Applicants found that 50% of doxorubicin was loaded after 1 h incubation with drug. Each AbeM-p-ESM-yEV is estimated to contain 5-10 molecules of doxorubicin. Example 13: Results Summary Applicants demonstrate that yEVs expressing AbeM linked to the truncated Sur7 yEV membrane protein having three transmembrane spanning domains supports significant uptake of doxorubicin when the internal pH of the yEV lumen is approximately pH 4 and the external approximately pH is 8, unlike yEVs expressing AbeM linked to the Full Sur7 yEV membrane protein having 4 transmembrane spanning domains. Applicants describe conditions for generating a pH gradient for loading doxorubicin loading into yEVs expressing the truncated Sur7-AbeM construct. We note that pH 4 is more optimal than pH 3 or pH 5 in certain embodiments, however in specific other embodiments optimal pH can differ. AbeM-mediated and proton dependent drug uptake can be an effective means to deliver drugs such as doxorubicin by yEVs to live cells than by using free drugs in solution substantially reducing the potential for off-target side effects. Unlike electroporation mediated drug uptake into yEVs, AbeM-p-ESM mediated drug loading into yEVs is industrially scalable by pre-equilibrating a large volume of yEVs at pH 4.0 and then transferring the yEVs to pH 7.8 plus drug for 1 hour. The ability to efficiently deliver hydrophilic and / or hydrophobic drugs in yEVs in a targeted manner to specific organs and cells substantially reduces the potential for off-target side effects and can effectively lower the drug dosage required for effective disease management.

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[0003] SEQUENCE LISTING SEQ ID NO.1 Amino Acid T-SNARE EV membrane localized protein SSO2* Saccharomyces cerevisiae MSNANPYENNNPYAENYEMQEDLNNAPTGHSDGSDDFVAFMNKINSINANLSRYENIINQIDAQHKDLLT QVSEEQEMELRRSLDDYISQATDLQYQLKADIKDAQRDGLHDSNKQAQAENCRQKFLKLIQDYRIIDSNY KEESKEQAKRQYTIIQPEATDEEVEAAINDVNGQQIFSQALLNANRRGEAKTALAEVQARHQELLKLEKT MAELTQLFNDMEELVIEQQENVDVIDKNVEDAQQDVEQGVGHTNKAVKSARKARKNKIRCLIICFIIFAI VVVVVVVPSVVETRK * Transmembrane spanning amino acid sequences underlined *bold font aspartate 161 is the last amino acid encoded to generate a truncated three transmembrane spanning (TMS) protein. SEQ ID NO.2 Amino Acid bacteriorhodopsin (Halo)* Haloterrigena turkmenica MIPELEMYRLGFYVTAVATLVFLGWVAKKPAGTRRYYLPAPIVCGTLSLAYFGMSVELLRVMTPSGQPLP MTRYIDYFVATAIMVAVAGKVAGATRRQLIAIVVLTVGWVGISLGRYFLTGTTVLAATLGTVVVLGALIY MMVWPVTKRSGETAGERVLLYGKLRNLLILLWVAYLVVGVVSRQGIGLLDAFGGIFAGAYLDIATRIGFG LLLLRGPDAIAHLIDETGSNGGSGESGDEVTFTESSDDPGTDPDIEPAD * Transmembrane spanning amino acid sequences underlined SEQ ID NO.3 Amino Acid Glycophorin A transmembrane spanning domain (amino acids 92-114)* Homo Sapiens ITLIIFGVMAGVIGTILLISYG * Transmembrane spanning amino acid sequences underlined SEQ ID NO.4 Amino Acid AbeM protein sequence* Acinetobacter baumannii MSNVTSFRSELKQLFHLMLPILITQFAQAGFGLIDTIMAGHLSAADLAAIAVGVGLWIPVMLLFSGIMIA TTPLVAEAKGARNTEQIPVIVRQSLWVAVILGVLAMLILQLMPFFLHVFGVPESLQPKASLFLHAIGLGM PAVTMYAALRGYSEALGHPRPVTVISLLALVVLIPLNMIFMYGLGPIPALGSAGCGFATSILQWLMLITL AGYIYKASAYRNTSIFSRFDKINLTWVKRILQLGLPIGLAVFFEVSIFSTGALVLSPLGEVFIAAHQVAI SVTSVLFMIPLSLAIALTIRVGTYYGEKNWASMYQVQKIGLSTAVFFALLTMSFIALGREQIVSVYTQDI NVVPVAMYLLWFAMAYQLMDALQVSAAGCLRGMQDTQAPMWITLMAYWVIAFPIGLYLARYTDWGVAGVW LGLIIGLSIACVLLLSRLYLNTKRLSQT * Transmembrane spanning amino acid sequences underlined SEQ ID NO.5 Amino Acid Chr1 protein ligand anchor Saccharomyces boulardii MKVLDLLTVLSASSLLSTFAAAESTATADSTTAASSTASCNPLKTTGCTPDTALATSFSEDFSSSSKWFT DLKHAGEIKYGSDGLSMTLAKRYDNPSLKSNFYIMYGKLEVILKAANGTGIVSSFYLQSDDLDEIDIEWV GGDNTQFQSNFFSKGDTTTYDRGEFHGVDTPTDKFHNYTLDWAMDKTTWYLDGESVRVLSNTSSEGYPQS PMYLMMGIWAGGDPDNAAGTIEWAGGETNYNDAPFTMYIEKVIVTDYSTGKKYTYGDQSGSWESIEADGG SIYGRYDQAQEDFAVLANGGSISSSSTSSSTVSSSASSTVSSSVSSTVSSSASSTVSSSVSSTVSSSSSV SSSSSTSPSSSTATSSKTLASSSVTTSSSISSFEKQSSSSSKKTVASSSTSESIISSTKTPATVSSTTRS TVAPTTQQSSVSSDSPVQDKGGVATSSNDVTSSTTQISSKYTSTIQSSSSEASSTNSVQISNGADLAQSL PREGKLFSVLVALLALL SEQ ID NO.6 Amino Acid Sur7 protein ligand anchor Saccharomyces boulardii MVKVWNIVLRLVVLLFLAGNTLLLILMIISGATDHYPVNRFYWVQGNTTGIPNAGDETRWTFWGACLQDK DGSDTCTSNLAPAYPISPVDNFNTHINVPHQFISKRDAFYYLTRFSFCFFWIALAFVGVSFILYVLTWCS KMLSEMVLILMSFGFVFNTAAVVLQTAASAMAKNAFHDDHRSAQLGASMMGMAWASVFLCIVEFILLVFW SVRARLASTYSIDNSRYRTSSRWNPFHREKEQATDPILTATGPEDMQQSASIVGPSSNANPVTATAATEN QPKGINFFTIRKSHERPDDVSV SEQ ID NO.7 Amino Acid Nce102 protein ligand anchor Saccharomyces cerevisiae MLALADNILRIINFLFLVISIGLISSLLNTQHRHSSRVNYCMFACAYGIFTDSLYGVFANFIEPLAWPLV LFTLDFLNFVFTFTAGTVLAVGIRAHSCNNSSYVDSNKITQGSGTRCRQAQAAVAFLYFSCAIFLAKTLM SVFNMISNGAFGSGSFSKRRRTGQVGVPTISQV SEQ ID NO.8 Amino Acid C-terminal ligand for binding to NRP1 receptor Artificial RPAR SEQ ID NO.9 Amino Acid C-terminal ligand for binding to EGFR receptor Artificial AEYLR SEQ ID NO.10 Amino Acid C-terminal FSH ligand for binding to FSHR receptor Artificial YTRDLVYKDPARPKIQKTCTFGGG SEQ ID NO.11 Amino Acid Linker protein Artificial SGGGGSGG SEQ ID NO.12 Amino Acid ABG xenobiotic-glycoside deconjugating enzyme protein Agrobacterium sp. MTDPNTLAARFPGDFLFGVATASFQIEGSTKADGRKPSIWDAFCNMPGHVFGRHNGDIACDHYNRWEEDL DLIKEMGVEAYRFSLAWPRIIPDGFGPINEKGLDFYDRLVDGCKARGIKTYATLYHWDLPLTLMGDGGWA SRSTAHAFQRYAKTVMARLGDRLDAVATFNEPWCAVWLSHLYGVHAPGERNMEAALAAMHHINLAHGFGV EASRHVAPKVPVGLVLNAHSAIPASDGEADLKAAERAFQFHNGAFFDPVFKGEYPAEMMEALGDRMPVVE AEDLGIISQKLDWWGLNYYTPMRVADDATPGVEFPATMPAPAVSDVKTDIGWEVYAPALHTLVETLYERY DLPECYITENGACYNMGVENGQVNDQPRLDYYAEHLGIVADLIRDGYPMRGYFAWSLMDNFEWAEGYRMR FGLVHVDYQTQVRTVKNSGKWYSALASGFPKGNHGVAKG SEQ ID NO.13 Amino Acid Sodium dependent glucose transporter 1 (SGLT1) protein Homo Sapiens RTVGGFFLAGRSMVWWPIGASLFASNIGSGHFVGIAGTAAAGGIAIGGYEWNALIFVVVLGWLFVPIYVK AGVVTMPEYLRKRFGGKRIQVYLSVLSLIVYIFTKISADIFSGAIFIQLAIGLNLYLAIIILLAITALYT ITGGLAAVIYTDTLQTFIMVVGSFILMGFAFKEVGGYDAFMQKYMEAIPSNISYGNTTIDSSCYRPREDA FHIFRDPVTGDLPWPGLIFGLSILALWYWCTDQ SEQ ID NO.14 Amino Acid crtYB - Bifunctional lycopene cyclase / phytoene synthase Rhodotorula diobovata MGGYDYWLVHARWTIPPSVALWLVFRKLRTWRDVYKTCFLITIAVTATIPWDSYLIRNRIWSYPDSSVVG PTLFAIPYEEVFFFFVQTYLTSTLYAVLTRPIVHPVLLPRTPSEGRAVRWTGTALLCGVFALAWAKLEEG GEGTYLALIVGWVAPFLTLLWWVASTHIIAMPRSTLLLAIFAPTFFLWELDARALQRGTWVIEKGTKLGW DFRGLEIEEAVFFLLTNVMIVFGMAACDHCLAVHDLRSYDKGTSSVFPPLHDFGPILINSPDAKQAQRID DLRAAIEILSVHSKSFSTASMVFDGRLRLDLLALYAWCRVCDDLVDNASSVAAAEANIRMISSCLDLLYP PSTSTPTSHPVRISNEAIAAALPGLSEPERGSFRLLALLPITRPPLDELLDGFRTDLSFLAFAGEKAAGG KPSGGSTSIPSELPIKTDEDLLVYANNVASSVADLCVQLVWAHCATSVPEPEQRAILSAAREMGQALQLV NIARDVPADLEIHRIYLPGRGLDVPVADLSPDRRELLRRAREMAAHSRGAIERLPREARGGIRAACDVYL SIGGAVDKALDEGRVLERARVAKGTRAWKAWTAL SEQ ID NO.15 Amino Acid crtI - Phytoene desaturase (neurosporene-forming) protein sequence Rhodobacter sphaeroides MPSISPASDADRALVIGSGLGGLAAAMRLGAKGWRVTVIDKLDVPGGRGSSITQEGHRFDLGPTIVTVPQ SLRDLWKTCGRDFDADVELKPIDPFYEVRWPDGSHFTVRQSTEAMKAEVARLSPGDVAGYEKFLKDSEKR YWFGYEDLGRRSMHKLWDLIKVLPTFGMMRADRSVYQHAALRVKDERLRMALSFHPLFIGGDPFNVTSMY ILVSQLEKEFGVHYAIGGVAAIAAAMAKVIEGQGGSFRMNTEVDEILVEKGTATGVRLASGEVLRAGLVV SNADAGHTYMRLLRNHPRRRWTDAHVKSRRWSMGLFVWYFGTKGTKGMWPDVGHHTIVNAPRYKGLVEDI FLKGKLAKDMSLYIHRPSITDPTVAPEGDDTFYALSPVPHLKQAQPVDWQAVAEPYRESVLEVLEQSMPG IGERIGPSLVFTPETFRDRYLSPWGAGFSIEPRILQSAWFRPHNISEEVANLFLVGAGTHPGAGVPGVIG SAEVMAKLAPDAPRARREAEPAERLAAE SEQ ID NO.16 Amino Acid crtE Geranylgeranyl pyrophosphate synthase Agmenellum quadruplicatum MVVADAHTQGFSLAQYLQEQKTIVETALDQSLVITEPVTIYEAMRYSLLAGGKRLRPILCLAACEMLGGT AAMAMNTACALEMIHTMSLIHDDLPAMDNDDLRRGKPTNHKVYGEDIAILAGDALLSYAFEYVARTPDVP AERLLQVIVRLGQAVGAEGLVGGQVVDLESEGKTDVAVETLNFIHTHKTGALLEVCVTAGAILAGAKPEE VQLLSRYAQNIGLAFQIVDDILDITATAEELGKTAGKDLEAQKVTYPSLWGIEKSQAEAQKLVAEAIASL EPYGEKANPLKALAEYIVNRKN SEQ ID NO.17 Amino Acid Sur7 protein ligand anchor - truncated Saccharomyces boulardii MVKVWNIVLRLVVLLFLAGNTLLLILMIISGATDHYPVNRFYWVQGNTTGIPNAGDETRWTFWG ACLQDKDGSDTCTSNLAPAYPISPVDNFNTHINVPHQFISKRDAFYYLTRFSFCFFWIALAFVG VSFILYVLTWCSKMLSEMVLILMSFGFVFNTAAVVLQTAASAMAKNAFHDDHRS SEQ ID NO.18 Amino Acid Sur7 / GGGS3-linker / AbeM* Artificial MVKVWNIVLRLVVLLFLAGNTLLLILMIISGATDHYPVNRFYWVQGNTTGIPNAGDETRWTFWG ACLQDKDGSDTCTSNLAPAYPISPVDNFNTHINVPHQFISKRDAFYYLTRFSFCFFWIALAFVG VSFILYVLTWCSKMLSEMVLILMSFGFVFNTAAVVLQTAASAMAKNAFHDDHRSAQLGASMMGM AWASVFLCIVEFILLVFWSVRARLASTYSIDNSRYRTSSRWNPFHREKEQATDPILTATGPEDM QQSASIVGPSSNANPVTATAATENQPKGINFFTIRKSHERPDDVSVSGGGGSGGGGSGGGGSMS NVTSFRSELKQLFHLMLPILITQFAQAGFGLIDTIMAGHLSAADLAAIAVGVGLWIPVMLLFSG IMIATTPLVAEAKGARNTEQIPVIVRQSLWVAVILGVLAMLILQLMPFFLHVFGVPESLQPKAS LFLHAIGLGMPAVTMYAALRGYSEALGHPRPVTVISLLALVVLIPLNMIFMYGLGPIPALGSAG CGFATSILQWLMLITLAGYIYKASAYRNTSIFSRFDKINLTWVKRILQLGLPIGLAVFFEVSIF STGALVLSPLGEVFIAAHQVAISVTSVLFMIPLSLAIALTIRVGTYYGEKNWASMYQVQKIGLS TAVFFALLTMSFIALGREQIVSVYTQDINVVPVAMYLLWFAMAYQLMDALQVSAAGCLRGMQDT QAPMWITLMAYWVIAFPIGLYLARYTDWGVAGVWLGLIIGLSIACVLLLSRLYLNTKRLSQT *Sur7 sequence is normal font, AbeM sequence is in bold font. Peptide linker is in italics. SEQ ID NO.19 Amino Acid Sur7 truncated / GGGS3-linker / AbeM* Artificial MVKVWNIVLRLVVLLFLAGNTLLLILMIISGATDHYPVNRFYWVQGNTTGIPNAGDETRWTFWG ACLQDKDGSDTCTSNLAPAYPISPVDNFNTHINVPHQFISKRDAFYYLTRFSFCFFWIALAFVG VSFILYVLTWCSKMLSEMVLILMSFGFVFNTAAVVLQTAASAMAKNAFHDDHRSSGGGGSGGGG SGGGGSMSNVTSFRSELKQLFHLMLPILITQFAQAGFGLIDTIMAGHLSAADLAAIAVGVGLWI PVMLLFSGIMIATTPLVAEAKGARNTEQIPVIVRQSLWVAVILGVLAMLILQLMPFFLHVFGVP ESLQPKASLFLHAIGLGMPAVTMYAALRGYSEALGHPRPVTVISLLALVVLIPLNMIFMYGLGP IPALGSAGCGFATSILQWLMLITLAGYIYKASAYRNTSIFSRFDKINLTWVKRILQLGLPIGLA VFFEVSIFSTGALVLSPLGEVFIAAHQVAISVTSVLFMIPLSLAIALTIRVGTYYGEKNWASMY QVQKIGLSTAVFFALLTMSFIALGREQIVSVYTQDINVVPVAMYLLWFAMAYQLMDALQVSAAG CLRGMQDTQAPMWITLMAYWVIAFPIGLYLARYTDWGVAGVWLGLIIGLSIACVLLLSRLYLNT KRLSQT *Sur7 sequence is normal font, AbeM sequence is in bold font. Peptide linker is in italics. SEQ ID NO.20 Amino Acid Linker protein Artificial SGGGGSGGGGSGGGGS SEQ ID NO.21 Amino Acid AbeM / p-ESM* Artificial MSNVTSFRSELKQLFHLMLPILITQFAQAGFGLIDTIMAGHLSAADLAAIAVGVGLWIPVMLLF SGIMIATTPLVAEAKGARNTEQIPVIVRQSLWVAVILGVLAMLILQLMPFFLHVFGVPESLQPK ASLFLHAIGLGMPAVTMYAALRGYSEALGHPRPVTVISLLALVVLIPLNMIFMYGLGPIPALGS AGCGFATSILQWLMLITLAGYIYKASAYRNTSIFSRFDKINLTWVKRILQLGLPIGLAVFFEVS IFSTGALVLSPLGEVFIAAHQVAISVTSVLFMIPLSLAIALTIRVGTYYGEKNWASMYQVQKIG LSTAVFFALLTMSFIALGREQIVSVYTQDINVVPVAMYLLWFAMAYQLMDALQVSAAGCLRGMQ DTQAPMWITLMAYWVIAFPIGLYLARYTDWGVAGVWLGLIIGLSIACVLLLSRLYLNTKRLSQT TSQTTTTSIVSSASLQTAITSTLSPATKSSSVVSLQTATTSTLSPTTTSTSSGSTSSDSTA * AbeM in bold, pESM in normal font SEQ ID NO.22 Amino Acid p-ESM Artificial TSQTTTTSIVSSASLQTAITSTLSPATKSSSVVSLQTATTSTLSPTTTSTSSGSTSSDSTA SEQ ID NO.23 Amino Acid Sur7-Halo* Artificial MVKVWNIVLRLVVLLFLAGNTLLLILMIISGATDHYPVNRFYWVQGNTTGIPNAGDETRWTFWG ACLQDKDGSDTCTSNLAPAYPISPVDNFNTHINVPHQFISKRDAFYYLTRFSFCFFWIALAFVG VSFILYVLTWCSKMLSEMVLILMSFGFVFNTAAVVLQTAASAMAKNAFHDDHRSAQLGASMMGM AWASVFLCIVEFILLVFWSVRARLASTYSIDNSRYRTSSRWNPFHREKEQATDPILTATGPEDM QQSASIVGPSSNANPVTATAATENQPKGINFFTIRKSHERPDDVSVTSSGGGGSGGGGSGGGGS PRMIPELEMYRLGFYVTAVATLVFLGWVAKKPAGTRRYYLPAPIVCGTLSLAYFGMSVELLRVM TPSGQPLPMTRYIDYFVATAIMVAVAGKVAGATRRQLIAIVVLTVGWVGISLGRYFLTGTTVLA ATLGTVVVLGALIYMMVWPVTKRSGETAGERVLLYGKLRNLLILLWVAYLVVGVVSRQGIGLLD AFGGIFAGAYLDIATRIGFGLLLLRGPDAIAHLIDETGSNGGSGESGDEVTFTESSDDPGTDPD IEPAD *Sur7 sequence is in bold font, Halo sequence is in normal font. Linker peptide linker is in italics. SEQ ID NO.24 Amino Acid Halo-Sur7* Artificial MVPELEMYRLGFYVTAVATLVFLGWVAKKPAGTRRYYLPAPIVCGTLSLAYFGMSVELLRVMTP SGQPLPMTRYIDYFVATAIMVAVAGKVAGATRRQLIAIVVLTVGWVGISLGRYFLTGTTVLAAT LGTVVVLGALIYMMVWPVTKRSGETAGERVLLYGKLRNLLILLWVAYLVVGVVSRQGIGLLDAF GGIFAGAYLDIATRIGFGLLLLRGPDAIAHLIDETGSNGGSGESGDEVTFTESSDDPGTDPDIE PADTSTSSGGGGSGGGGSGGGGSPRMVKVWNIVLRLVVLLFLAGNTLLLILMIISGATDHYPVN RFYWVQGNTTGIPNAGDETRWTFWGACLQDKDGSDTCTSNLAPAYPISPVDNFNTHINVPHQFI SKRDAFYYLTRFSFCFFWIALAFVGVSFILYVLTWCSKMLSEMVLILMSFGFVFNTAAVVLQTA ASAMAKNAFHDDHRSAQLGASMMGMAWASVFLCIVEFILLVFWSVRARLASTYSIDNSRYRTSS RWNPFHREKEQATDPILTATGPEDMQQSASIVGPSSNANPVTATAATENQPKGINFFTIRKSHE RPDDVSV *Halo is normal font; Sur7 sequence is bold font, Peptide linker is in italics. SEQ ID NO.25 Amino Acid Sur7tr-AbeM-Halo* Artificial MVKVWNIVLRLVVLLFLAGNTLLLILMIISGATDHYPVNRFYWVQGNTTGIPNAGDETRWTFWG ACLQDKDGSDTCTSNLAPAYPISPVDNFNTHINVPHQFISKRDAFYYLTRFSFCFFWIALAFVG VSFILYVLTWCSKMLSEMVLILMSFGFVFNTAAVVLQTAASAMAKNAFHDDHRSDVSGGGGSGG GGSGGGGSTSPRMSNVTSFRSELKQLFHLMLPILITQFAQAGFGLIDTIMAGHLSAADLAAIAV GVGLWIPVMLLFSGIMIATTPLVAEAKGARNTEQIPVIVRQSLWVAVILGVLAMLILQLMPFFL HVFGVPESLQPKASLFLHAIGLGMPAVTMYAALRGYSEALGHPRPVTVISLLALVVLIPLNMIF MYGLGPIPALGSAGCGFATSILQWLMLITLAGYIYKASAYRNTSIFSRFDKINLTWVKRILQLG LPIGLAVFFEVSIFSTGALVLSPLGEVFIAAHQVAISVTSVLFMIPLSLAIALTIRVGTYYGEK NWASMYQVQKIGLSTAVFFALLTMSFIALGREQIVSVYTQDINVVPVAMYLLWFAMAYQLMDAL QVSAAGCLRGMQDTQAPMWITLMAYWVIAFPIGLYLARYTDWGVAGVWLGLIIGLSIACVLLLS RLYLNTKRLSQTGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLPRMIPELEMYRL GFYVTAVATLVFLGWVAKKPAGTRRYYLPAPIVCGTLSLAYFGMSVELLRVMTPSGQPLPMTRY IDYFVATAIMVAVAGKVAGATRRQLIAIVVLTVGWVGISLGRYFLTGTTVLAATLGTVVVLGAL IYMMVWPVTKRSGETAGERVLLYGKLRNLLILLWVAYLVVGVVSRQGIGLLDAFGGIFAGAYLD IATRIGFGLLLLRGPDAIAHLIDETGSNGGSGESGDEVTFTESSDDPGTDPDIEPAD * Sur7tr sequence is normal font, Peptide linker is in italics. AbeM sequence is in bold font. GpA is bold underlined. Halo is underlined. SEQ ID NO.26 Amino Acid AbeM-GpA-Halo-P-ESM* Artificial MSNVTSFRSELKQLFHLMLPILITQFAQAGFGLIDTIMAGHLSAADLAAIAVGVGLWIPVMLLF SGIMIATTPLVAEAKGARNTEQIPVIVRQSLWVAVILGVLAMLILQLMPFFLHVFGVPESLQPK ASLFLHAIGLGMPAVTMYAALRGYSEALGHPRPVTVISLLALVVLIPLNMIFMYGLGPIPALGS AGCGFATSILQWLMLITLAGYIYKASAYRNTSIFSRFDKINLTWVKRILQLGLPIGLAVFFEVS IFSTGALVLSPLGEVFIAAHQVAISVTSVLFMIPLSLAIALTIRVGTYYGEKNWASMYQVQKIG LSTAVFFALLTMSFIALGREQIVSVYTQDINVVPVAMYLLWFAMAYQLMDALQVSAAGCLRGMQ DTQAPMWITLMAYWVIAFPIGLYLARYTDWGVAGVWLGLIIGLSIACVLLLSRLYLNTKRLSQT TSGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRLPRMIPELEMYRLGFYVTAVATL VFLGWVAKKPAGTRRYYLPAPIVCGTLSLAYFGMSVELLRVMTPSGQPLPMTRYIDYFVATAIM VAVAGKVAGATRRQLIAIVVLTVGWVGISLGRYFLTGTTVLAATLGTVVVLGALIYMMVWPVTK RSGETAGERVLLYGKLRNLLILLWVAYLVVGVVSRQGIGLLDAFGGIFAGAYLDIATRIGFGLL LLRGPDAIAHLIDETGSNGGSGESGDEVTFTESSDDPGTDPDIEPADTRQTTTTSIVSSASLQT AITSTLSPATKSSSVVSLQTATTSTLSPTTTSTSSGSTSSDSTATR *AbeM sequence is in bold font. GpA is bold underlined. Halo is underlined. P-ESM is in italic. SEQ ID NO.27 Amino Acid Sur7tr- 2 Saccharomyces boulardii MVKVWNIVLRLVVLLFLAGNTLLLILMIISGATDHYPVNRFYWVQGNTTGIPNAGDETRWTFWG ACLQDKDGSDTCTSNLAPAYPISPVDNFNTHINVPHQFISKRDAFYYLTRFSFCFFWIALAFVG VSFILYVLTWCSKMLSEMVLILMSFGFVFNTAAVVLQTAASAMAKNAFHDDHRSDV SEQ ID NO.28 Amino Acid Glycophorin A transmembrane spanning domain - 2 Homo Sapiens GERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIRRL SEQ ID NO.29 Amino Acid Flag-Tag Artificial DYKDHDGDYKDHDIDYKDDDDK SEQ ID NO.30 Amino Acid p-ESM-2 Artificial TRQTTTTSIVSSASLQTAITSTLSPATKSSSVVSLQTATTSTLSPTTTSTSSGSTSSDSTATR

Claims

CLAIMS What is claimed is:

1. A light-activated xenobiotic transport system comprising: ^ a Saccharomyces-generated extracellular vesicle (EV), wherein the EV includes a fusion peptide includes: ^ a light-dependent proton pump; ^ a proton driven xenobiotic transporter; ^ a membrane targeting domain; and ^ wherein in response to light energy the light-dependent proton pump generates a pH across the membrane of the EV causing a xenobiotic to be transported into the lumen of the EV by the action of the transporter.

2. The system of claim 1, further comprising a transmembrane domain configured to orient the light-dependent proton pump relative to the EV membrane so as to allow proton transport into the lumenal space.

3. The system of claim 2, wherein the transmembrane domain positioned between the light- dependent proton pump and the proton driven xenobiotic transporter.

4. The system of claim 2, wherein the transmembrane domain comprises a transmembrane-helix of glycophorin A (GpA) or odd numbered transmembrane protein.

5. The system of claim 2, wherein the transmembrane domain is selected from a peptide having an amino acid sequence according to SEQ ID NO’s 3, 28, or a fragment thereof.

6. The system of claim 2, wherein the transmembrane domain is selected from a peptide having an amino acid sequence having between 80% to 99% sequence homology with SEQ ID NO’s 3, 28, or a fragment thereof.

7. The system of claim 1, wherein said Saccharomyces-generated EV is selected from a Saccharomyces cerevisiae generated EV, or a Saccharomyces boulardii EV.

8. The system of claim 1, wherein said EV is exposed to approximately 100 mW of light energy.

9. The system of claim 1, wherein the light-dependent proton pump comprises a bacteriorhodopsin peptide.

10. The system of claim 9, wherein said bacteriorhodopsin peptide is derived from Haloterrigena turkmenica.

11. The system of claim 1, wherein the light-dependent proton pump comprises a peptide according to SEQ ID NO.2, or a fragment thereof.

12. The system of claim 1, wherein the light-dependent proton pump comprises a peptide according to SEQ ID NO. 2, or a sequence having between 80% to 99% sequence identity with SEQ ID NO.

2.

13. The system of claim 9-12, further comprising a quantity of retinal.

14. The system of claim 13, wherein said retinal is endogenously produced in a yeast cell.

15. The yeast cell of claim 13, wherein said retinal added exogenously to interact with the light- dependent proton pump.

16. The system of claim 1, wherein said proton driven xenobiotic transporter comprises a transporter selected from: a multidrug and toxic compound extrusion family (MATE) transporter, a resistance / nodulation / cell division (RND) transporter, a small multidrug resistance family (SMR) transporter, a major facilitator antiporter (MFS) transporter, an ATP-dependent transporters (ABC) transporter, or a combination of the same.

17. The system of claim 1, wherein said proton driven xenobiotic transporter comprises peptide having an amino acid sequence according to SEQ ID NO. 4, or a sequence having at least 80% sequence homology with SEQ ID NO.4, or a fragment thereof.

18. The system of claim 1, wherein said membrane targeting domain comprises an EV membrane protein anchor.

19. The system of claim 18, wherein said EV membrane protein anchor comprises a truncated Sur7 protein.

20. The system of claim 18, wherein said EV membrane protein anchor is selected from a peptide having an amino acid sequence according to SEQ ID NO’s 17, 27, or a fragment thereof.

21. The system of claim 1, wherein said EV membrane protein anchor is selected from a peptide having an amino acid sequence having between 80% to 99% sequence homology with SEQ ID NO’s 17, 27, or a fragment thereof.

22. The system of claim 1, wherein said membrane targeting domain comprises a protein extracellular vesicle sorting motif (p-ESM).

23. The system of claim 22, wherein said p-ESM domain is selected from a peptide having an amino acid sequence according to SEQ ID NO’s 22, 30, or fragment thereof.

24. The system of claim 22, wherein said p-ESM domain is selected from a peptide having an amino acid sequence having between 80% to 99% sequence homology with SEQ ID NO’s 22, 30, or a fragment thereof.

25. The system of claim 1, wherein said fusion peptide further comprises one or more linker peptides.

26. The system of claim 25, herein the linker comprises a peptide having an amino acid sequence according to SEQ ID NO’s 11, 20, 30, or a fragment thereof.

27. The system of claim 1, wherein said fusion peptide comprises an amino acid sequence according to SEQ ID NO.25, or a sequence having between 80% to 99% sequence homology with SEQ ID NO.25, or a fragment thereof.

28. The system of claim 1, wherein said fusion peptide comprises an amino acid sequence according to SEQ ID NO.26, or a sequence having between 80% to 99% sequence homology with SEQ ID NO.26, or a fragment thereof.

29. The system of claim 1, wherein the xenobiotic is selected from: a therapeutic compound, a drug, a biologic, a DNA or RNA oligonucleotide, an exonuclease, flavorings, fragrances, food additives, a dye, a probe, a chemical compound, anti-cancer compounds, antibiotics, or conjugated variants, or a combination of the same 30. The system of claim 1, wherein the xenobiotic is selected from 5-fluorouracil, kanamycin, gentamicin, ofloxacin, ciprofloxacin, erythromycin, chloramphenicol, DAPI, triclosan, acriflavine, Hoechst 33342, daunomycin, doxorubicin, trimethoprim, and / or rhodamine 6G.

31. A pharmaceutical composition comprising the Saccharomyces-generated extracellular vesicle (EVs) of any of claims 1- 30, and a pharmaceutically acceptable carrier.

32. A method treating a disease or condition with a xenobiotic, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 31 to a subject in need thereof.

33. The method of claim 31, wherein said subject is a human.

34. A yeast cell expressing a heterologous nucleotide, operably linked to a promoter, encoding a fusion peptide comprising: ^ a light-dependent proton pump; ^ a proton driven xenobiotic transporter; ^ a membrane targeting domain; and ^ wherein in response to light energy the light-dependent proton pump generates a pH across the membrane of an extracellular vesicle (EV) causing a xenobiotic to be transported into the lumen of the EV by the action of the transporter.

35. The yeast cell of claim 34, wherein the light-dependent proton pump comprises a bacteriorhodopsin peptide.

36. The yeast cell of claim 34, wherein said bacteriorhodopsin peptide is derived from Haloterrigena turkmenica.

37. The yeast cell of claim 34, wherein the light-dependent proton pump comprises a peptide according to SEQ ID NO.2, or a fragment thereof.

38. The yeast cell of claim 34, wherein the light-dependent proton pump comprises a peptide according to SEQ ID NO. 2, or a sequence having between 80% to 99% sequence identity with SEQ ID NO.

2.

39. The yeast cell of claim 35-38, further comprising a quantity of retinal added exogenously to interact with the light-dependent proton pump.

40. The yeast cell of claim 39, wherein said retinal is endogenously produced in the yeast cell through the heterologous expression of the genes for the biosynthesis of beta-carotene and beta- carotene 15,15'-dioxygenase which produces trans retinal.

41. The yeast cell of claim 39, wherein said genes for the biosynthesis of beta-carotene crtYB, crtI, crtE, or fragments of the same.

42. The yeast cell of claim 34, further comprising a transmembrane domain configured to orient the light-dependent proton pump relative to the EV membrane so as to allow proton transport into the lumenal space.

43. The yeast cell of claim 42, wherein the transmembrane domain positioned between the light- dependent proton pump and the proton driven xenobiotic transporter.

44. The yeast cell of claim 42, wherein the transmembrane domain comprises a transmembrane- helix of glycophorin A (GpA) or odd numbered transmembrane protein.

45. The yeast cell of claim 42, wherein the transmembrane domain is selected from a peptide having an amino acid sequence according to SEQ ID NO’s 3, 28, or a fragment thereof.

46. The yeast cell of claim 42, wherein the transmembrane domain is selected from a peptide having an amino acid sequence having between 80% to 99% sequence homology with SEQ ID NO’s 3, 28, or a fragment thereof.

47. The yeast cell of claim 34, wherein said yeast cell is selected from Saccharomyces cerevisiae, or Saccharomyces boulardii.

48. The yeast cell of claim 34, wherein said proton driven xenobiotic transporter comprises a transporter selected from: a multidrug and toxic compound extrusion family (MATE) transporter, a resistance / nodulation / cell division (RND) transporter, a small multidrug resistance family (SMR) transporter, a major facilitator antiporter (MFS) transporter, an ATP-dependent transporters (ABC) transporter, or a combination of the same.

49. The yeast cell of claim 34, wherein said proton driven xenobiotic transporter comprises peptide having an amino acid sequence according to SEQ ID NO. 4, or a sequence having at least 80% sequence homology with SEQ ID NO.4, or a fragment thereof.

50. The yeast cell of claim 34, wherein said membrane targeting domain comprises an EV membrane protein anchor.

51. The yeast cell of claim 50, wherein said EV membrane protein anchor comprises a truncated Sur7 protein.

52. The yeast cell of claim 50, wherein said EV membrane protein anchor is selected from a peptide having an amino acid sequence according to SEQ ID NO’s 17, 27, or a fragment thereof.

53. The yeast cell of claim 34, wherein said EV membrane protein anchor is selected from a peptide having an amino acid sequence having between 80% to 99% sequence homology with SEQ ID NO’s 17, 27, or a fragment thereof.

54. The yeast cell of claim 34, wherein said membrane targeting domain comprises a protein extracellular vesicle sorting motif (p-ESM).

55. The yeast cell of claim 54, wherein said p-ESM domain is selected from a peptide having an amino acid sequence according to SEQ ID NO’s 22, 30, or fragment thereof.

56. The system of claim 54, wherein said p-ESM domain is selected from a peptide having an amino acid sequence having between 80% to 99% sequence homology with SEQ ID NO’s 22, 30, or a fragment thereof.

57. The yeast cell of claim 34, wherein said fusion peptide further comprises one or more linker peptides.

58. The yeast cell of claim 57, herein the linker comprises a peptide having an amino acid sequence according to SEQ ID NO’s 11, 20, 30, or a fragment thereof.

59. The yeast cell of claim 34, wherein said fusion peptide comprises an amino acid sequence according to SEQ ID NO.25, or a sequence having between 80% to 99% sequence homology with SEQ ID NO.25, or a fragment thereof.

60. The yeast cell of claim 34, wherein said fusion peptide comprises an amino acid sequence according to SEQ ID NO.26, or a sequence having between 80% to 99% sequence homology with SEQ ID NO.26, or a fragment thereof.

61. A extracellular vesicle (EV) isolated from the cell of any of claims 34 to 60.

62. The EV of claim 61, wherein said EV s exposed to approximately 100 mW of light energy.

63. The system of claim 34, wherein the xenobiotic is selected from: a therapeutic compound, a drug, a biologic, a DNA or RNA oligonucleotide, an exonuclease, flavorings, fragrances, food additives, a dye, a probe, a chemical compound, anti-cancer compounds, antibiotics, or conjugated variants, or a combination of the same 64. The system of claim 34, wherein the xenobiotic is selected from 5-fluorouracil, kanamycin, gentamicin, ofloxacin, ciprofloxacin, erythromycin, chloramphenicol, DAPI, triclosan, acriflavine, Hoechst 33342, daunomycin, doxorubicin, trimethoprim, and / or rhodamine 6G.

65. A pharmaceutical composition comprising the isolated EV of any of claims 34 - 64, and a pharmaceutically acceptable carrier.

66. A method treating a disease or condition with a xenobiotic, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 65 to a subject in need thereof.

67. The method of claim 65, wherein said subject is a human.

68. A method of loading a xenobiotic compound into a Saccharomyces-generated extracellular vesicle (EV), the method comprising: ^ isolating an EV, wherein the EV includes a fusion peptide having: ^ a light-dependent proton pump; ^ a proton driven xenobiotic transporter; ^ a membrane targeting domain; and ^ applying light energy to the EV, wherein in response to light energy the light-dependent proton pump generates a pH across the membrane of the EV causing a xenobiotic to be transported into the lumen of the EV by the action of the transporter.

69. The method of claim 68, further comprising a transmembrane domain configured to orient the light-dependent proton pump relative to the EV membrane so as to allow proton transport into the lumenal space.

70. The method of claim 69, wherein the transmembrane domain positioned between the light- dependent proton pump and the proton driven xenobiotic transporter.

71. The method of claim 69, wherein the transmembrane domain comprises a transmembrane- helix of glycophorin A (GpA) or odd numbered transmembrane protein.

72. The method of claim 69, wherein the transmembrane domain is selected from a peptide having an amino acid sequence according to SEQ ID NO’s 3, 28, or a fragment thereof.

73. The method of claim 69, wherein the transmembrane domain is selected from a peptide having an amino acid sequence having between 80% to 99% sequence homology with SEQ ID NO’s 3, 28, or a fragment thereof.

74. The method of claim 68, wherein the light-dependent proton pump comprises a bacteriorhodopsin peptide.

75. The method of claim 74, wherein said bacteriorhodopsin peptide is derived from Haloterrigena turkmenica.

76. The method of claim 68, wherein the light-dependent proton pump comprises a peptide according to SEQ ID NO.2, or a fragment thereof.

77. The method of claim 68, wherein the light-dependent proton pump comprises a peptide according to SEQ ID NO. 2, or a sequence having between 80% to 99% sequence identity with SEQ ID NO.

2.

78. The method of claim 74-77, further comprising a quantity of retinal added exogenously to interact with the light-dependent proton pump.

79. The method of claim 78, wherein said retinal is endogenously produced in the yeast cell through the heterologous expression of the genes for the biosynthesis of beta-carotene and beta- carotene 15,15'-dioxygenase which produces trans retinal.

80. The method of claim 78, wherein said genes for the biosynthesis of beta-carotene crtYB, crtI, crtE, or fragments of the same.

81. The method of claim 68, wherein said Saccharomyces-generated EV is selected from a Saccharomyces cerevisiae generated EV, or a Saccharomyces boulardii EV.

82. The method of claim 68, wherein said proton driven xenobiotic transporter comprises a transporter selected from: a multidrug and toxic compound extrusion family (MATE) transporter, a resistance / nodulation / cell division (RND) transporter, a small multidrug resistance family (SMR) transporter, a major facilitator antiporter (MFS) transporter, an ATP-dependent transporters (ABC) transporter, or a combination of the same.

83. The method of claim 68, wherein said proton driven xenobiotic transporter comprises peptide having an amino acid sequence according to SEQ ID NO. 4, or a sequence having at least 80% sequence homology with SEQ ID NO.4, or a fragment thereof.

84. The method of claim 68, wherein said membrane targeting domain comprises an EV membrane protein anchor.

85. The method of claim 84, wherein said EV membrane protein anchor comprises a truncated Sur7 protein.

86. The method of claim 84, wherein said EV membrane protein anchor is selected from a peptide having an amino acid sequence according to SEQ ID NO’s 17, 27, or a fragment thereof.

87. The method of claim 68, wherein said EV membrane protein anchor is selected from a peptide having an amino acid sequence having between 80% to 99% sequence homology with SEQ ID NO’s 17, 27, or a fragment thereof.

88. The method of claim 68, wherein said membrane targeting domain comprises a protein extracellular vesicle sorting motif (p-ESM).

89. The method of claim 88, wherein said p-ESM domain is selected from a peptide having an amino acid sequence according to SEQ ID NO’s 22, 30, or fragment thereof.

90. The method of claim 88, wherein said p-ESM domain is selected from a peptide having an amino acid sequence having between 80% to 99% sequence homology with SEQ ID NO’s 22, 30, or a fragment thereof.

91. The method of claim 68, wherein said fusion peptide further comprises one or more linker peptides.

92. The method of claim 91, herein the linker comprises a peptide having an amino acid sequence according to SEQ ID NO’s 11, 20, 30, or a fragment thereof.

93. The method of claim 68, wherein said fusion peptide comprises an amino acid sequence according to SEQ ID NO.25, or a sequence having between 80% to 99% sequence homology with SEQ ID NO.25, or a fragment thereof.

94. The method of claim 68, wherein said fusion peptide comprises an amino acid sequence according to SEQ ID NO.26, or a sequence having between 80% to 99% sequence homology with SEQ ID NO.26, or a fragment thereof.

95. The method of claim 68, wherein the xenobiotic is selected from: a therapeutic compound, a drug, a biologic, a DNA or RNA oligonucleotide, an exonuclease, flavorings, fragrances, food additives, a dye, a probe, a chemical compound, anti-cancer compounds, antibiotics, or conjugated variants, or a combination of the same 96. The method of claim 68, wherein the xenobiotic is selected from 5-fluorouracil, kanamycin, gentamicin, ofloxacin, ciprofloxacin, erythromycin, chloramphenicol, DAPI, triclosan, acriflavine, Hoechst 33342, daunomycin, doxorubicin, trimethoprim, and / or rhodamine 6G.

97. A pharmaceutical composition comprising a Saccharomyces-generated extracellular vesicle (EVs) produced by the method of any of claims 68-96, and a pharmaceutically acceptable carrier.

98. A method treating a disease or condition with a xenobiotic, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 97 to a subject in need thereof.

99. The method of claim 98, wherein said subject is a human.

100. A kit comprising an EV containing a xenobiotic of any claim above, a container to hold the EV, and instructions for use.

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