Potential of yeast secretory vesicles in biodelivery systems

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feature Potential of yeast secretory vesicles in biodelivery systems

Membranous vesicular organelles (MVOs), such as secretory vesicles and exosomes, perform a variety of biological functions ranging from secretion to cellular communication in eukaryotic cells. Exosomes, particularly those of mammalian cells, have been widely studied as potential carriers in human therapeutic applications. However, no study has yet demonstrated the use of yeast secretory vesicles for such applications. Therefore, we explore here the current state of knowledge on yeast secretory vesicles and their potential use in therapeutic delivery systems. We focus on the characteristics shared by exosomes and yeast secretory vesicles to provide insights into the use of the latter as delivery vehicles. From this perspective, we speculate on the potential application of post-Golgi vesicles (PGVs) in the biomedical field.

Introduction Q2 The existence of phospholipid bilayer MVOs in

eukaryotic cells facilitates the exchange of materials between cells and the extracellular milieu. For instance, endosomes are carriers involved in importing extracellular materials by endocytosis, whereas secretory vesicles and exosomes serve to export materials from the cells to the extracellular milieu via exocytosis [1,2]. Exocytosis mediates cell–cell communication through the transfer of signaling molecules between cells. Conversely, these molecules can be assimilated by recipient cells through endocytosis of MVOs. In eukaryotic cells, exocytosis can occur through two different mechanisms: the classical pathway and the nonclassical pathway [3]. In the yeast Saccharomyces cerevisiae, the welldocumented classical pathway involves the

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translocation of proteins across membranous organelles (e.g., the endoplasmic reticulum, Golgi complex, and vacuoles) to the extracellular surface by means of secretory vesicles (Ø 50–150 nm) as carriers (i.e., via internal transport carriers). Among them, PGVs are MVOs that export cargo proteins to the extracellular milieu after binding and fusing with the plasma membrane [2,4] (Fig. 1a). Here, we focus on PGVs and compare their biological function and characteristics to those of exosomes from mammalian cells. Even though exosomes are widely studied as drug delivery vehicles, their application is still in development. Thus, we also explore the potential of PGVs as drug carriers.

Exosomes: biogenesis and characteristics Exosomes (Ø 50–120 nm) are MVOs that serve as extracellular carriers in the nonclassical pathway.

Exosomes carry proteins and/or nucleic acids, particularly miRNAs and mRNAs, from one cell to other cells [1,5–10]. They serve as shuttle vectors and act as mediators of intercellular communication, immune responses, and antigen presentation [5–8]. Exosomes are evolutionarily conserved from lower to higher eukaryotic organisms (i.e., from fungi to mammals). The biogenesis of exosomes begins during the last stage of endocytosis, where the endocytic membrane undergoes budding to form intraluminal vesicles (ILVs). ILVs accumulate within the original endocytic membrane, which at this stage is named the multivesicular body. These bodies then fuse with either the lysosomes for degradation or with the plasma membrane for the extracellular release of ILVs (i.e., exosomes) [1,5–8]. Exosomes are released from cells either constitutively or upon activation of a secretion

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Q1 Gurusamy Kutralam-Muniasamy1, Luis B. Flores-Cotera1 and Fermin Perez-Guevara1,2, [email protected], [email protected]

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(a)

(b) Blood vessel

Saccharomyces cerevisiae

Tumor cells Normal cells

v-SNARE

Therapeutic cargo

t-SNARE

Exosomes

Protein cargo

Membrane anchor

PGVs Membrane anchor

Tumor-specific ligand Tumor-specific receptor

Extracellular Cytoplasm

Plasma membrane

Extracellular

Cell membrane

Cytoplasm

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FIGURE 1

Q5 Comparative schema of cargo transfer mechanisms by membranous vesicular organelles (MVOs). A key and lock mechanism allows for the recognition between an MVO and a membrane, and their subsequent fusion process. Some proteins located on the surface of MVOs are analogous to keys, which are highly specific in their interaction with the corresponding locks (the protein counterpart on the membrane). v-soluble NSF attachment receptor protein (SNAREs; keys) present on the outer membrane of post-Golgi vesicles (PGVs) interact with t-SNAREs (locks) located on the inner plasma membrane during exocytosis, thereby allowing the fusion and subsequent cargo transfer (a). Likewise, in the case of cellular uptake of exosomes via endocytosis, the ligands (keys) located on the exosomal membrane interact with specific receptor proteins located on the membrane of recipient cells (locks) (b). The unavailability of one of the respective partners prevents the recognition and fusion of MVOs. Consequently, the release of cargo does not occur. Overall, the same principles of recognition, fusion, and cargo release are operative with both MVOs. Modified from [4] (a) and [1,6,35] (b). Features  PERSPECTIVE

pathway. The mechanisms involved in the packaging of cargo into exosomes and their transport across the cellular membrane have been described both in vivo and in vitro, but remain to be fully elucidated [6]. The exosomes generated from different cell types have particular functions and distinctive sizes, shapes, compositions, and degrees of hydrophobicity (summarized in Table 1). In terms of lipid composition, the exosomal membranes are enriched with lipid rafts [glycosylphosphatidylinositol (GPI)-anchored proteins and flotillin], cholesterol, ganglioside GM-3, sphingomyelin, phosphatidylcholine, and phosphatidylserine [1,6,8]. The exosomal proteins are mainly involved in cell–cell communication, vesicular transport, carbohydrate metabolism, and protein synthesis and turnover [1,6,10,11]. To date, proteomic studies have not yet identified any sorting signal for the recruitment of proteins into exosomes [8].

Exosomes as natural delivery vehicles The primary role of exosomes in intercellular communication has motivated great interest in the potential utility of these MVOs as nanovehicles for targeted biodelivery applications [6,12–15]. In particular, exosomes isolated from 2

mammalian cell cultures have been reported as practical drug delivery vehicles [6,7,12,13] (Table 2). Therapeutic cargo intended for delivery have been loaded into exosomes in several ways: (i) in vitro and ex vivo antigen pulsing with dendritic cell (DC)-derived exosomes [16–19]; (ii) drugs such as curcumin have been encapsulated into exosomes by mixing [20,21]; and, most recently, (iii) small interfering RNAs (siRNAs) of therapeutic interest have been loaded into exosomes through electroporation or chemically by using lipofectamine reagent [22–24]. However, recent work shows that electroporation is less efficient than previously believed [25]. Therefore, the practical application of exosomes may yet be limited by the lack of efficient techniques for loading them with the desired therapeutics. Exosomes interact with target cells through at least two mechanisms: (i) protein–protein interactions (i.e., receptor versus ligand) [6,26,27]); and (ii) lipid–lipid interactions (i.e., fusion between cell plasma membrane and exosomes leading to the cellular uptake of the exosome) [28]. As far as the protein–protein interaction is concerned, the therapeutic cargo loaded into exosomes is delivered to the recipient cells following interaction between the membrane proteins on exosomes (ligands) and

those of target cells (receptors) [6,24,29,30]. In other words, suitable interaction between exosomal ligands and receptors on the recipient cells is needed for the binding and the uptake of exosomes (Fig. 1b). Some proteins that are anchored to exosomes are cell specific [e.g., epithelial cell adhesion molecule (ECAM) in ovarian cancer], whereas others are ubiquitous to all exosome types [e.g., heat shock protein 90 (Hsp90), Alix, and endosomal sorting complexes required for transport (ESCRT)], regardless of their cell origin [5,6,14]. The identified tissuespecific surface proteins of exosomes have an intrinsic highly selective homing specificity that circumvents the need for manipulation to achieve target specificity [21]. Moreover, the potential use of exosomes in biomedical applications has been dramatically improved by the use of genetic engineering [12–16]. Extensive efforts currently focus on engineering the specific ligands identified on the surface membranes of exosomes to improve their targeting properties [12,13,23]. This has mainly been achieved by conjugating a targeting ligand with a membrane protein of exosomes, such as lysosomal-associated membrane protein-2B (Lamp2B). This strategy enables exosomes derived from a particular cell source to be targeted

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TABLE 1

The biophysical and biochemical characteristics of mammalian exosomes, fungal exosomes, and PGVs. Characteristics

Vesicle subtype Mammalian exosomes

Fungal exosomes

PGVs

Physical (size, sedimentation, and shape)

40–100 nm; 100 000 g; cup shaped; pH, 5.0–5.5

Similar to exosomes

50–150 nm; 100 000 g; sphere shaped; pH, 5.3–5.6

Intracellular origin

Multivesicular bodies

Multivesicular bodies

Shed from trans-Golgi network

Sources

Immune cells: B cells, DCs, T cells, mast cells. Tumorous cells: basophilic leukemia, bladder cancer, brain tumor, breast/colorectal/lung/stomach cancer, medulloblastoma, melanoma, mesothelioma, nasopharyngeal carcinoma, and pancreatic adenocarcinoma

Pathogenic fungal species: Paracoccidioides brasiliensis, Sporothrix schenckii, Candida albicans, Candida parapsilosis, Malassezia sympodialis, and Histoplasma capsulatum

Nonpathogenic yeast strains: Saccharomyces cerevisiae mutants (sec4-2, sec6-4, sec4-8, sec23-1, exo70-35, and exo70–38)

Modified from [5,6,8]

Modified from [11]

[44,46,49–58]

Refs

toward distinctive recipient cells, provided that the targeting exosome ligand is aligned in an appropriate position and orientation to match the desired receptor [24,31–35]. As far as the lipid–lipid interaction is concerned, Parolini et al. [28] have shown that this interaction depends on microenvironmental conditions, such as low pH, and that this might occur largely when electrostatic charges exist in cells and exosomes. This suggests that the existence of these favorable conditions must be verified when new potential vehicles are studied for the transfer of therapeutic molecules. Targeting of exosomes for the delivery of therapeutic cargo has been successful (Table 2); however, the applicability of exosomes as therapeutic delivery systems is limited by two factors. First, the protein content of exosomes varies according to the cell type [e.g., exosomes from B lymphocytes, DCs, mast cells, and intestinal epithelial cells are enriched in major histocompatibility complex (MHC) class I and II, whereas exosomes of other origins are not) [5]. Moreover, tumor-derived exosomes can carry immunosuppressors that can inactivate T lymphocytes or natural killer cells, or promote the differentiation of regulatory T lymphocytes or myeloid cells, resulting in the suppression of immune responses [6,26,27]. In allogeneic exosomal

therapy, the presence of MHC proteins and virulence factors, such as glucuronoxylomannan and Hsp70, can cause undesirable immunological reactions [36–38]. In addition, some exosomes are able to deliver viral proteins or miRNAs that can be potentially translated and active in target cells [7,39], that is, exosomes might have some pathogenic potential. For instance, exosomes released from lymphoblastoid cell lines are loaded with the viral oncoprotein latent membrane protein 1 (LMP1) and Epstein– Barr virus-miRNAs [40] (for retroviruses, see [41]). Moreover, Federici et al. [42] have shown that exosomes released by human cells are capable of carrying the anticancer drug cisplatin in native form, contributing to the extracellular elimination of this anticancer drug, and accordingly reducing the effectiveness of treatment. Therefore, careful attention should also be paid to the selection of mammalian cell lines for the production of exosomes. Accordingly, not all exosomes are suitable as delivery devices; thus, thoughtful selection of exosomes is also necessary to avoid adverse immunological effects [31]. Indeed, the adaptive immune responses resulting from the use of engineered exosomes as delivery vehicles have to be studied in more detail [35]. Second, the establishment of cell cultures is expensive and labor intensive. From

an economic point of view, large-scale cultures of mammalian cells to produce exosomes is undesirable because of the requirements for complex growth media, long culture periods, and the relatively low cell densities attained. In addition, the yield of exosomes varies among cell types, and the specific factors underlying this variation are not yet understood [14,34,43]. Consequently, the current applications and limitations of exosomes as drug delivery carriers prompted us to evaluate the potential use of PGVs for targeting. To this end, two key requirements must be fulfilled [34,35]: (i) the successful isolation of PGVs; and (ii) the anchorage of selected ligands to the membranes of PGVs for specific cell targeting. These two issues are discussed below, taking into account the structural components, composition, and secretion mechanisms of PGVs. In this context, the knowledge accumulated from studies of exosomes in delivery applications can serve as an important foundation for the development of PGVs as therapeutic vehicles.

Secretory mutants: a source of yeast PGVs The secretion process of Saccharomyces cerevisiae is rapid, constitutive, and involves the transport of proteins packed in PGVs, which serve as intracellular carriers en route to the

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Protein profile

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Applications of exosomes for therapeutic purposes. Type

Source

Target

Membrane receptor

Ligand

Delivery cargo

Effects

Native exosomes

DCs

Cytotoxic T lymphocytes

N/A

N/A

Tumor peptide

Suppresses growth of murine tumors

[16]

SRDC cell line (CD8a + CD4 DC cell line)

Syngeneic and allogeneic mice

N/A

N/A

Toxoplasma gondii-derived antigens

Protective responses against T. gondii infections

[17]

Bone marrow-derived (bm)DCs

BALB/c mice

N/A

N/A

Diphtheria toxin (DT)

Induces immunoglobulin G (IgG)2b and IgG2a responses specific to DT

[18]

SRDC cell line

CBA/J mice

N/A

N/A

T. gondii-derived antigens

Strong humoral and cellular responses

[19]

Mouse lymphoma cell line (EL-4) and murine macrophage cell line (RAW 264.7)

Monocyte-activated myeloid cells

N/A

N/A

Curcumin, an anti-inflammatory drug

Induces apoptosis

[20]

Glioblastoma cells (GL26)

Microglial cells

N/A

N/A

Curcumin, JSI124

Therapeutic approach for brain inflammatory-related diseases

[21]

Peripheral blood

Monocytes and lymphocytes

N/A

N/A

Mitogen-activated protein kinase 1 siRNA

Gene silencing

[22]

HeLa and HT1080 cells

HeLa and HT1080 cells

N/A

N/A

siRNAs of RAD51 and RAD52

Post-transcriptional gene silencing resulted in massive reproductive cell death

[23]

DCs

Neurons, microglia, and oligodendrocytes of brain

Lamp2b

Neuron-specific RVG peptide

BACE siRNA, a therapeutic target in Alzheimer’s disease

Knockdown of BACE1 gene

[24]

HEK293 cells

Breast cancer cells

PDGFR

GE11 peptide

Let-7a miRNA

Tumor suppression

[31]

Fibrosarcoma cell line (MCA101 C57Bl/6)

Antigen-presenting cells

CIC2 domain of lactadherin

N/A

A model antigen, chicken egg ovalbumin

Antitumor immune responses

[32]

JAWSII and bmDCs

Antigen-presenting cells

CIC2 domain of lactadherin

N/A

Extracellular domain of carcinoembryonic antigen (CEA) and HER2

Enhances antigen-specific responses

[33]

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Modified exosomes

Refs

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TABLE 2

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extracellular milieu. The continuous secretion in wild type strains of S. cerevisiae prevents the intracellular build-up of PGVs (i.e., the low number of intracellular PGVs makes recovery difficult). Several proteins, such as Sec1-23, Ypt31/32, Snc1/2, Exo70, actin, and myosin, are required for the vesicular transport of proteins from the cytoplasm to the cell surface. Novick et al. [44,45] demonstrated that any functional defects in these proteins that mediate the secretion process could disrupt vesicular transport and result in the intracellular accumulation of vesicles. These authors obtained secretory mutants, defective in sec proteins, which accumulate vesicles at different stages of the secretion process in response to a temperature shift from a growth-permissive (25 8C) to a nongrowth permissive condition (37 8C). In the latter condition, it was shown that proteins associated to secretion and vesicles were still synthesized actively in the secretory mutants. In contrast to wild type strains, a subset of secretory mutants, known as late sec mutant strains (i.e., defective in sec1–10 and sec15) accumulate a substantial number of intracellular PGVs [44,45]. After cell disruption, the accumulated PGVs can be separated from cellular debris [i.e., microsomes, vacuole and Golgi fragments, and coat protein (COP)-I/II vesicles] by ultracentrifugation [46,47]. Therefore, these late sec mutant strains have been used for the successful isolation of PGVs (Table 1) and constitute an exceptional source of PGVs for potential application.

PGVs from secretory mutants: size distribution and biochemical characteristics Wild type strains of S. cerevisiae produce PGVs ranging in diameter from 50 to 70 nm [48]. The mutant strains defective in vesicular transport are able to accumulate PGVs of different sizes. For instance, the extensively studied late sec mutants and other yeast mutants, such as exo7035 and exo70-38, are able to accumulate PGVs in the range of 80–100 nm [49]. The ypt31/32 mutant accumulates both small (50–80 nm) and large (100–150 nm) PGVs [50], whereas the sla2p yeast mutant strain accumulates vesicles in the range of 40–60 nm [51]. Regardless of their size, all of the PGVs recovered to date are spherical and have similar lipid and protein compositions. Forsmark et al. [52] reported the biochemical composition of PGVs from mutant strains sec6-4, sec23-1, and sro7. In all cases, the dominant lipid constituents of the PGV membrane were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sterol, and sphingolipids

[47]. Table 1 shows the protein types identified inside and anchored on the membrane surface of PGVs. Regardless of the particular mutant considered, the proteins found within PGVs were shown to be typical cell wall or integral membrane proteins, whereas others were cytosolic [52]. Thus, the PGVs recovered from different mutant strains carry similar protein types with variations only at the quantitative level. Previous findings with other sec mutant strains have shown similar results [52,53]. However, a recent study suggested that the specific proteins carried by PGVs vary depending on the carbon source and culture conditions applied, such as salinity or temperature [52].

Membrane proteins of PGVs: anchors for targeting ligands Several of the PGV membrane surface proteins, including Ypt31/32, Sec4p, Sec15p, Snc1p/2p, and Sso 1p/2p, have motifs or domains required for membrane anchoring [i.e., a transmembrane domain (TMD) or a prenylated motif ] [54–61]. Several studies have evaluated the use of PGV membrane protein domains for anchorage. For example, Ossig et al. [59] substituted the Cterminal cysteines of the Sec4p protein with the membrane anchor of Snc2p to verify Sec4p protein translocation onto the membrane of PGVs. Similarly, Grote et al. [60] replaced the TMD of the Snc protein with the geranylgeranyl anchor and verified Snc protein localization onto PGVs. The immobilization of a bacterial polyhydroxyalkanoate synthase onto PGVs by using the known anchorage domains of Snc2p was recently reported [61]. Importantly, this latter study is a proof of concept that the immobilization of heterologous proteins onto PGVs is feasible in practice. This also demonstrates that the anchorage domains of membrane proteins, or even whole membrane proteins, could be used to integrate heterologous proteins (particularly ligands) onto the membrane surface of PGVs. Some other proteins available on the membrane of PGVs could be used to fuse targeting ligands, including; Sso1p/2p, lipid rafts (GPI-anchored proteins), cell wall proteins (e.g., Pma1p or Bgl2p) and plasma membrane proteins (e.g., FusMidp). The PGVs produced by S. cerevisiae secretory mutants could have significant advantages over exosomes produced by mammalian cells with respect to eventual mass production of MVOs: (i) yeast cells grow faster; (ii) yeast culture media are simpler and cheaper; (iii) yeast cultures are already established and more amenable for industrial-scale applications; and (iv) the production of PGVs from yeast will be cheaper than that of exosomes from mammalian cells.

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Current exosome engineering guides future development of PGVs The remarkable similarities between exosomes and PGVs with respect to their nature, size, and morphology are summarized in Table 1. Importantly, the recognition mechanisms and fusion with the membrane for cargo transfer are also similar, as schematized in Fig. 1. However, the biological functions of these MVOs differ as well as their components (i.e., proteins and RNAs). For instance, the functional role of exosomes varies depending on the cell type. Contrarily, the functional role of PGVs is secretion in all the yeasts reported so far (S. cerevisiae, Kluyveromyces lactis, Pichia pastoris, Yarrowia lipolytica, and Schizosaccharomyces pombe). As long as PGVs are produced for this unique function, their composition is simpler than that of exosomes. As shown in Table 1, no component from PGVs has been assigned a function homologous to those of exosomes. PGV proteins take part in cell wall biogenesis, cellular transport, and carbohydrate and lipid metabolism. In this context, the absence of exosomal membrane proteins that are considered immunogenic on the PGVs might be an added advantage. Regarding recognition, the specific ligands located on membranes of exosomes define their targeting properties and provide specificity for the delivery of therapeutic cargo to target cells [13,24,31–33] (Table 2). In addition, delivery specificity can be enhanced by engineering exosomes, such that they carry ad-hoc ligands for desired target cells. By contrast, yeast PGVs are naturally unable to target mammalian cells due to the absence of mammalian ligands on their membranes. However, it is conceivable that ligands for specific receptors mediating cellular uptake could be anchored to the membranes of PGVs and serve well for effective targeting, similar to exosomes [13]. To this end, prior knowledge gained from engineering exosomes can be used advantageously to incorporate targeting ligands onto the membrane of PGVs. The known anchorage domains of membrane proteins of PGVs appear to have the same function as those of exosomes, that is, they serve as anchors. Accordingly, the anchorage domains of membrane proteins reported in PGVs could be fused with targeting ligands and sited with the desired orientation or topology on the membrane of PGVs (i.e., with the ligand facing the cytoplasm) [59–61]. In this way, proper placement of ligands on PGVs can be attained. PGVs and exosomes are analogous MVOs after their recovery. Intact PGVs have been recovered from S. cerevisiae cultures, without affecting the topological organization of the membrane

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TABLE 3

Yeast proteins present in the lumen of PGVs and their signal peptidesa.Source: Adapted from [62]. Proteins

Signal peptide

Galactosidase

MFAFYFLTACISLKGVFG:VSPSYNGLGL

Acid phosphatase

MFKSVVYSILAASLANA:GTIPLGKLAD

Carboxypeptidase Y

MKAFTSLLCGLGLSTTLAKA:ISLQRPL

Invertase

MKIYHIFSVCYLITLCAAATTAREEFF

Mating factor a-1

MLLQAFLFLLAGFAAKISA:SMTNETSDRP

Glucoamylase

MVGLKNPYTHTMQRPFLLAYLVLSLLFNSALGFPTALVPRGS

Mating factor alpha-2

MKFISTFLTFILAAVSVTASSDEDIAQVPA

a

The enlisted signal peptides could be of interest to target therapeutic proteins/peptides into PGVs.

proteins [36]. This suggests that ligands on the membranes of engineered PGVs remain intact after recovery and are functionally able to interact with receptors on the target recipient cells. The required ligand–receptor interaction for the recognition and fusion of engineered PGVs with the cell membrane must enable the release of cargo into the recipient cells. The similar lipid profile of the two MVOs, despite their different origins, could also facilitate the fusion of engineered PGVs with the membrane of recipient

cells, in a similar fashion as exosomes [5]; however, the existence of this kind of interaction remain to be documented. Therapeutic proteins and/or peptides of interest could be packed into PGVs by using known specific signal peptides of PGV proteins (Table 3) [62]; that is, the signal peptides from the latter proteins could be used to generate fusion tags with therapeutic proteins, to place the therapeutic protein inside intact PGVs. This strategy might help to circumvent external

(b) Features  PERSPECTIVE

(c)

(a)

(d)

Vector expressing the engineered ligand gene

(e)

Secretory mutants accumulating engineered PGVs Tumor-specific ligand Therapeutic cargo

(g) Membrane anchor Engineered PGVs

(f)

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FIGURE 2

Q6 Schematic representation of the proposed model of production, harvesting, and perspectives of postGolgi vesicles (PGVs) as targeting delivery carriers. (a) Transformation of a yeast (Saccharomyces cerevisiae) secretory mutant with a vector carrying the fusion gene cassette encoding the engineered ligand protein (e.g., tumor-specific ligand fused to a PGV membrane anchor). (b) Production and accumulation of engineered PGVs by the transformed S. cerevisiae secretory mutants. (c) Yeast cell disruption. (d) Purification of the engineered PGVs. (e) Incorporation of therapeutic cargo into the engineered PGVs. (f, g) Validation in preclinical and clinical trials. 6

procedures for the incorporation of therapeutic proteins into PGVs. In addition, in vivo packing of a given therapeutic protein in the lumen of PGVs would help to prevent its degradation. Thus, this novel approach of packing therapeutic proteins into PGVs in vivo warrants future investigation. Alternatively, established techniques developed for incorporating therapeutic cargo into exosomes could also be useful for PGVs [20–24]. In Fig. 2, we propose a working strategy to develop PGVs as targeting delivery carriers. Over the past decade, the use of exosomes as delivery vehicles has contributed to a research shift from the use of cancerous cells as drug targets to producers of exosomes. Likewise, late sec mutants might undergo a similar shift, from tools used in fundamental vesicular transport research to sources for the mass production of PGVs. Therefore, based on the similarities between exosomes and PGVs described above, we propose that PGVs are suitable as delivery carriers. Similar to other therapeutic delivery vehicles, we envisage that PGVs will be tailored in the future to target different cell types.

Concluding remarks Despite the potential of PGVs as drug delivery carriers, there are some outstanding issues that need to be addressed before the practical development of their application. First, the immunological responses generated by MVOs need to be evaluated. Although the immunological responses elicited by exosomes have been investigated in some detail [35–38], no study to date has addressed the immunological responses to PGV components. Therefore, whether PGVs could trigger an immune response and how the immunological system of the host would respond to the protein content of PGVs remain two unresolved but important questions. These issues must be addressed, and strategies should be developed to resolve any undesirable effects. Second, detailed studies (both in vivo and in vitro) must be conducted to establish whether the ligands that are currently applied for exosome targeting could also be suitable in PGVbased drug delivery. In this case, the specific ligand must enable endocytosis of PGVs by the target cells and, consequently, the delivery of their cargo. The possibility of lipid–lipid interaction with the target cells and its significance must be established. Third, comparative studies should be conducted to determine the delivery characteristics of PGVs in relation to other delivery vehicles (e.g., exosomes, liposomes, or micelles), including efficiency of cargo delivery and stability. Such

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studies would provide the foundation for the implementation of in-depth technical and economical evaluations. In summary, early studies on S. cerevisiae late sec mutants along with the use of their vesicular constituents to anchor heterologous proteins led us to propose that PGVs could emulate exosomes in delivery applications. The available information on applications of exosomes in drug delivery provides significant groundwork for the development of PGVs as delivery vehicles to harness their potential for future therapeutic applications.

Acknowledgments G.K.M. acknowledges support from the Q3 scholarship grant of CONACYT (no. 239835). The

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protein/synaptobrevin gene family: genetic interactions with the RAS and CAP genes. Proc. Natl. Acad. Sci. U. S. A. 89, 4338–4342 57 Salminen, A. and Novick, P.J. (1989) The Sec15 protein responds to the function of the GTP binding protein, Sec4, to control vesicular traffic in yeast. J. Cell Biol. 109, 1023–1036 58 ten Klooster, J.P. and Hordijk, P.L. (2007) Targeting and localized signalling by small GTPases. Biol. Cell 99, 1–12

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