Purposing plant-derived exosomes-like nanovesicles for drug delivery: patents and literature review

ABSTRACT

Introduction

How can biotechnology and organic agriculture be fused and promoted simultaneously to overcome the main challenges in drug delivery systems. The role of organic agriculture in future human health treatment still represents a binary organic–conventional question. However, exosomes-like nanoparticles define a new organic path that plants and vegetables can release. In this review, we concisely propose plant-derived exosome-like nanovesicles and discuss their most important biological and pharmacological roles, representing a new tool for drug delivery.

Areas covered

Plant-derived exosomes-like nanovesicles; nature farming; green manufacturing practice; drug delivery; organic agriculture.

Expert opinion

There is growing interest in the potential use of plant-derived exosomes-like nanovesicles for various diagnostic and therapeutic applications that should translate into a supplement to current nano‐pharmaceuticals. Despite their clinical potential, the lack of sensitive preparatory and analytical technologies for plant-derived exosomes-like nanovesicles poses a barrier to clinical translation. An increasing number of articles are recently published on new analytical platforms to address these challenges in cross-comparison with conventional assay methods. This review also mentions two patents from ExoLab-Italia on plant-derived exosome-like nanovesicles, respectively, on plant-derived exosome-like nanovesicles’ ability to naturally deliver a series of potentially therapeutic molecules and a novel approach to upload them with therapeutic molecules.

KEYWORDS:

• Exosomes
• plant
• organic agriculture
• biodelivery
• drug delivery
• antioxidants

1. Introduction

Pharmaceutical studies have supported projects addressing identifying nanoparticles to deliver the active ingredients of current medications to increase their efficacy and reduce the undesirable side effects often caused by chemical or biological products for decades [1–3]. Since the pioneered times, the idea has been to build synthetic nanoparticles such as polymer nanoparticles, solid lipid nanoparticles (SLNs), crystal nanoparticles, and liposomes to enclose therapeutic molecules to avoid their degradation before reaching the target site. Each of these nanoparticles has advantages and disadvantages as a drug delivery vehicle. They have been designed to encapsulate hydrophilic molecules (siRNA, RNA, and DNA) and hydrophobic bioactive (proteins, phenolic peptides, and antibodies). However, we do not overlook that these are ‘artificial systems’ as they are obtained by chemical synthesis. This poses a substantial limitation for their clinical application because synthetic nanoparticles have two significant rules: each of their constituents must be evaluated for potential in vivo toxicity before clinical application [4], and the production scale is limited and expensive [5]. To overcome these limitations, organic agriculture may represent a useful source, such as plant-derived extracellular vesicles (PDEVs), a family of vesicles sizing from micro to nano that can be found extracellularly and represent a promising nanomedicine tool. They are isolated from different edible sources, such as fruits and vegetables, starchy roots and tubers, nuts and seeds, and fresh and dried plants. Simultaneously to the studies on PDEVs, plant-derived exosome-like nanovesicles (PDENs) have attracted considerable attention owing to their high reproducibility and promising results. Exosomes are a subtype of extracellular vesicles (EVs) formed by an endosomal route. They have gained attention as potential nano-drug delivery systems (NDDSs) as they are characterized by various desirable properties such as small size (typically 30–150 nm in diameter) [1,2,4–6], biocompatibility, and high stability.

They connect cells, tissues, compartments, and organs in physiological and pathological conditions. In this interconnecting activity, exosomes serve as natural transporters of a wide array of molecules and bioactive, including nucleic acids, proteins, and lipids. This, in turn, means they have a natural attitude to transfer molecular cargoes from a donor to acceptor cells or donor to acceptor tissues. Moreover, it has been shown that exosomes can deliver a series of compounds, including chemotherapeutics [4], theranostic molecules [5], and nanoparticles [6], at least in part due to their participation in the scavenging framework of our body [4,6]. The scientific community is still interested in the field focused on isolation and purifying exosomes from different human cells and biological fluids sources, including their morphological and functional characterization; nevertheless, PDENs have been gaining attention also for their simple accessibility, less cost-effectiveness, scalability, and their origin from edible and therapeutic plants. Therefore, in the light of available literature on the status of PDENs application without pretending to represent a systemic review of the issue, this manuscript aims to define a timely examination of relevant research progress that is significant for the continuous improvement of PDENs as NDDS.

2. Extracellular vesicles: from the endogenous sources to overcome artificial nanoparticles restrictions

Several advances in efficiency, specificity, gene expression duration, and safety have increased the number of synthetic nanoparticles entering clinical trials from 2014 to 2022 [7]. All currently available vector systems need to fulfill systems’ properties because of challenges that have arisen and need to be overcome to be ineffectiveness efficient. This led to a research focus on a suitable ideal vector delivery system. Recently, progress in biological or bioinspired drug carriers has been growing expeditiously. The EVs field is among the continually expanding areas of interest, particularly in exosomes. Synthesized exogenously siRNAs, miRNA mimics, and anti-miRNA oligonucleotides can be loaded into the exosomes via lipofection, electroporation, calcium chloride, sonication, co-incubation, or Saponin permeabilization [8]. In addition, the exosomes’ membranes can protect the loaded RNAs from RNase and phagocytosis [9], avoiding the endosomal pathway and enhancing the transfection efficiency of siRNAs [8]. As a natural delivery system for all living species, exosomes are an optimal tool for overcoming several limitations associated with other carrier systems [8]. For example, adeno-associated virus (AAV), a non-enveloped virus that can be engineered to deliver DNA to target cells, can be enveloped with exosomes providing a robust transgene expression in vivo [10] into the central nervous system (CNS). Orefice N.S et al. monitored the in vivo spreading of exosomes-enveloped AAVs after stereotactic administration in real-time using a probe-based confocal laser endomicroscopy. The results reported that AAVs’ exosome envelopment spread more from the injection site to the contralateral side. At the same time, the unenveloped AAVs were mainly located in the ipsilateral hemisphere [9]. Despite our knowledge supports that EVs play a relevant role as a versatile mediator of cell-to-cell communication by transferring cargoes from donor to acceptor cells, specifically transporting and delivering into recipient cells messenger molecules including proteins, lipids, DNA, and RNA, the EVs-content delivery process within the acceptor cell has yet to be unambiguously demonstrated, let alone quantified and remains debated.

3. Plant-derived exosomes-like nanovesicles cargo-loading methods

A current issue is selecting donor cells and determining the appropriate method for cargo loading into the exosome. Several cancer models have been employed to assess the advantages and disadvantages of the cargo-loaded exosome. Different strategies have been devised to load exosomes with exogenous cargoes [11,12]. These include co-incubation, electroporation, sonication, chemical transfection, freeze-thaw method, and extrusion. Of course, co-incubation appears to be the more physiological of the above methods. However, this method is characterized by two critical limits: (i) molecules/cargos can diffuse into the vesicles through an unforced passage through the target cell membrane, and (ii) the loading efficiency is low. Nevertheless, it has been used to introduce miRNA and transfection reagents into plant-derived nanovesicles [13,14]. Some other attempts have been made using agents able to perturb the exosomes membrane, such as polyethylene glycol coating (PEGylation) [15] or heparin-cRGD (tripeptide Arg-Gly-Asp motif) peptide conjugates (or tris (2-carboxyethyl) phosphine (TCEP) [16–20]. Lastly, some attempts have been made to use electroporation to upload molecules within the exosomes [21–23]. This has proven a valuable method to efficiently upload nanovesicles by only temporarily perturbing the vesicle membrane without any permanent damage. No less important is that while the artificial nanoparticles are ‘empty’ at the moment of loading, the natural nanovesicles are not ‘empty’; most of all, they already contain nucleic acids. This may represent at the same time an advantage, e.g. for the increased potentiality to transfer the material within target cells, but also a disadvantage because of needing an ad hoc procedure far from being standardized. Of course, the experience of loading natural EVs with different cargoes must be borrowed from the technical platforms exploited for loading artificial nanoparticles. Considering the advantages and disadvantages of each cargo loading method, which might depend on the specific nanovesicles of different natures to obtain the best vector with the best uploading method, the criteria making a topic still open for debate.

4. Plant-derived plant-derived exosomes-like nanovesicles from different green source

PDENs derived from nontoxic plants can overcome the shortcomings of existing nanosized delivery systems due to their origin, natural composition, and the opportunity to extract them in bulk from green sources [24]. Grapefruit is among the primary plant food sources studied due to its use in the delivery of nanomedicines. It was shown that grapefruit-derived nanocarriers (GNV) were very effective in providing a variety of therapeutic agents, including DNA expression vectors, siRNA, and antibodies, using both in vitro and in vivo conditions [25]. GNV has also been used as a drug delivery system for therapeutic agents in different cells where they did not show cytotoxicity or induce an inflammatory cytokine response [26]. Another study showed that ginger-derived nanovesicles (GDNs) were selectively absorbed by intestinal macrophages and improved DSS-induced rat colitis [27]. The authors also confirmed that GDNs are biodegradable and stable over a wide range of pH values suggesting that they could be developed as a new oral drug delivery system. Furthermore, their findings show that GDNs modulated the immune responses in the gut and maintained intestinal macrophage homeostasis [26]. The therapeutic potential of GDNs was further demonstrated by the inhibition of tumor growth in both CT26- and SW620-induced tumors in mice models [27]. The authors concluded that these nano-vectors were less toxic than nanoparticles from synthetic lipids. In addition, a critical aspect observed by Zhang et al. is that plant-derived nanoparticles do not cross the placental barrier after being injected intravenously into pregnant mice, suggesting they could be helpful as drug delivery systems [28]. At the same time, Quesenberry et al. assessed nanoparticles derived from edible plants (grapes, grapefruit, ginger, and carrots) with anti-cancer properties [29] inhibiting the growth of the chronic myeloid leukemia (CML) xenograft model in vivo. Furthermore, these nanoparticles exert their anticancer activity by stimulating the apoptotic mechanism of TNF-related apoptosis-inducing ligand (TRAIL) as an alternative method for cancer detection [30]. Furthermore, these nanoparticles acted as an antioxidant agent due to small RNAs, citrate, and vitamin C content, based on an in vitro study using mesenchymal stromal cells (MSCs) [31]. In this context, it is essential to underline that an electrostatic interactions system employed for nanovesicles from ginger to deliver drugs [27,32] and grapefruit nanovesicles charged with paclitaxel have been successfully administered intranasally in murine models [21]. This evidence fosters the proof of concept supporting PDENs as a new, very efficient, and side-effect-free approach in the nano delivery of therapeutic molecules. Additionally, there is an enthusiastic general interest in using plant-derived nanovesicles for drug delivery [30], which have shown to not undergo filter organ sequestration, with limited or no systemic toxicity [33]. In addition, compared to the currently available drug delivery systems, plant-derived nanovesicles have multiple advantages, such as low immunogenicity and stability in the gastrointestinal tract [34,35].

5. Plant-derived plant-derived exosomes-like nanovesicles as drug transporters: a world of possibilities awaits

Researchers must surmount numerous challenges when aiming to bloom new therapeutic approaches, including issues with delivery, tissue specificity, potential off-target effects, safety, toxicity, and large-scale production costs. Addressing these challenges, in the last decade, studies have moved to PDENs representing themselves as avant-garde innocuous, robust, and feasible carriers for nanomedicine [36,37]. This new approach was because PDENs may exert the same function due to the many analogies with their human counterpart. Like human nanovesicles, PDENs have shown their natural ability to deliver chemical molecules [38].

The relevance of PDENs in interspecies communication is derived from their content in biomolecules (lipids, proteins, and miRNAs), absence of toxicity, and easy internalization by mammalian cells, as well as their anti-inflammatory, immunomodulatory, and regenerative properties. In addition, due to their lipid structure (like the plasma membrane structure), PDENs protect their content against external agents. Indeed, the high level of nanovesicles’ lipid membrane resistance to external physical stimuli has been shown [39]. The occurrence of common identities between PDENs and EVs is not a surprise based on the contribution of EVs to PDENs. A recent comparative analysis of nanovesicles isolated with the same procedure either from a disrupted leaf or the extracellular apoplastic space of A. thaliana reported similarities and differences among both vesicle types [40,41]. However, although they share some characteristics, they differ in size, density distribution, and protein content. The study identified 1438 distinct proteins in the Arabidopsis leaf nanovesicle and 787 in the apoplast EVs samples. The presence of diverse membrane and soluble proteins derived from specific subcellular origins was observed in EVs and PDENs [40,41]; only the EVs proteome showed significant relative enrichment of accessions associated with extracellular function and cell wall localization. Notably, the plant-derived EVs and human plasma EVs proteomes shared more similarities than the two plant nanovesicle proteomes [25]. Emerging designs are considering the possibilities for PDENs content and release manipulation and suggest the exciting avenue of using engineered PDENs to deliver bioactive compounds.

6. Plant-derived exosomes-like nanovesicles: aspirant natural drug delivery?

By analogy with artificial nanoparticles, exosomes are the best candidate for ‘natural drug delivery.’ In the last decade, many attempts have been made to account for the ability of exosomes to deliver molecules of various origins. Of course, the most investigated area was cancer therapy using drug-loaded exosomes. We learn much about exosome composition, function, isolation, and characterization. However, two facts are emerging that have hampered the use of human exosomes as an ideal delivery system for therapeutic molecules or at least placed exosomes under severe discussion: (i) they belong to a scavenging framework of our organism for unwanted or toxic material; (ii) they change in number and variety under disease condition. Some scientific reports still show as exosomes released by both normal and tumor cells may contain drugs (e.g. cisplatin) [4,5] or nanoparticles (e.g. gold nanoparticles) [6] administered to the cells. Of interest, all the exosomes deliver are in their native form, therefore, fully active. This, on the one hand, supports the evidence that exosomes represent the way through which cells may eliminate unwanted material extracellularly; on the other hand, it is dreadful to realize the versatile role of exosomes containing various molecular cargo in their native form, which may travel and ending in potentially all the organs and compartments of our body, promoting the release of partially undesirable cargo. This suggests that exosomes are always like a double-edged knife: on one edge, they may serve as an ideal nano vector for potentially all the therapeutic molecules; on the other, they continuously deliver toxic molecules that our macrophage/histiocytic apparatus have scavenged. We also know that exosomes increase in number and decrease in size when tumor cells are cultured in the acidic milieu mimicking the tumor microenvironment [42], mirrored in the tumor plasma of tumor patients [43]. A further problem of human nanovesicles is that industrial production is heavily hampered, but the difficulties of obtaining vast amounts of exosomes from factories of normal human cells. Thus, while promising, the future of drug delivery cannot be nanovesicles released by human cells, at least at the commercial level.

However, through the studies on human exosomes, we obtained proof that natural nanovesicles can deliver molecules of different natures, such as drugs [4,5] and theranostic nanoparticles [6]. Moreover, human nanovesicles are natural carriers for bioactive molecules [44] and therapeutic antibodies [39]. Therefore, a series of preclinical investigations were performed with human and plant-derived nanovesicles that will represent a reference for clinical trials’ use of natural nanovesicles for drug delivery (Table 1 and Table 2). The results are highly encouraging and represent a real hope to increase the efficacy and reduce the toxicity of approved and new therapeutic compounds. However, some clinical trials use human and plant-derived nanovesicles (Table 3). An interesting observation is that the clinical trials employing plant-derived nanovesicles use the oral route of administration, representing the natural way human beings introduce fruits and vegetables.

Table 1. Pre-clinical evidence on the use of human nanovesicles as a drug delivery system.

SOURCE	COMPOUND/MOLECULE	OUTCOME	REFERENCE
Adipose tissue	Thymoquinone	Exosomes loaded with thymoquinone efficiently decrease breast cancer cell viability in vitro	[61]
Cerebrospinal fluid from brain-injured patients	siRNA against apoptosis speck-like protein containing a caspase recruitment domain (ASC)	Efficient silencing of ASC lead to a significant decrease in caspase-1 activation and blockage of inflammasome activation in rats after spinal cord injury	[62]
Human peripheral blood	miR-21 and miR-21 inhibitor	miR-21 inhibitor-exosomes decrease cardiac fibrosis at the infarcted and border zones in C57BL/6 mice with myocardial infarction	[63]
In vitro regular cell lines	Doxorubicin	Anti-tumor effect	[64]
	Doxorubicin	Exosomes loaded with doxorubicin exhibited higher cytotoxicity against osteosarcoma compared to free doxorubicin.	[65]
	Stimulator of Interferon Genes	growth reduction in immunocompetent mice implanted with subcutaneous B16F10 tumors	[66]
In vitro tumor cell lines	Black bean phytochemicals	Antiproliferative activity in a tumor cell line in vitro	[67]
	Cationic bovine serum albumin (CBSA) conjugated siS100A4	Suppression of postoperative lung metastasis in triple-negative breast cancer	[68]
	Cas9-/sgRNA-expressing plasmids	inhibition of cancer cell proliferation via apoptosis induction in SKOV3 xenograft mice	[69]
	Curcumin and Indocyanine	Inhibition of tumor growth in vitro and in vivo	[70]
	Doxorubicin	Antiproliferative effect and tumor growth inhibition	[71]
	Doxorubicin	Cytotoxic effect in HT1080 and HeLa cells and inhibition of tumor growth in vitro	[72]
	Doxorubicin and gold nanorods	Excellent tumor targeting and synergistic photothermal chemotherapy	[73]
	Doxorubicin and miRNA	Apoptosis induction and anti-tumor effect in vitro and in vivo	[74]
	Fluorouracil and miR-21 inhibitor	Cell proliferation inhibition of 5-FU-resistant HCT-116 cells and anti-tumor effect without adverse effects xenograft-tumor model	[75]
	Gemcitabine	Cell viability reduction of Panc-1 cells in vitro and anti-tumor effect in Panc-1 xenograft BALB/c nude mice	[76]
	Paclitaxel	Potent cytotoxic effect in prostate cell lines	[77]
	Paclitaxel	Cytotoxic effect in tumor cell line	[78]
Uterine fluid	Gonadotropin	In vitro improvement of endometrial receptivity	[79]

Table 2. Pre-clinical evidence on the use of plant-derived nanovesicles as a drug delivery system.

SOURCE Pre-clinical evidence on the use of plant-derived nanovesicles as a drug delivery system	COMPOUND/MOLECULE	OUTCOME	REFERENCE
Acerola (Malpighia emarginata)	miR-340 & mir-146	Orally administrated miRNA/vesicles downregulated miRNA’s target gene	[14]
Aloe (Aloe vera)	Indocyanine green	Vesicles loaded with indocyanine green inhibited melanoma growth	[19]
Cabbage (Brassica oleracea)	miR-184 & doxorubicin	Therapeutic drugs encapsulated in cabbage-derived vesicles suppressed cell tumor growth.	[13]
Ginger (Zingiber officinale)	Doxorubicin	Lipid re-assembled ginger nanovesicles are loaded with doxorubicin; loaded nanovesicles exert apoptotic effects in vitro and reduce tumor growth in vivo	[27]
	siRNA-CD98	Orally administered siRNA-CD98/GDLVs were targeted explicitly to colon tissues and efficiently reduced CD98 expression.	[89]
	survivin siRNA	After intravenous administration, the complex folic acid-ginger vesicles-siRNA inhibited tumor growth in a murine xenograft model.	[32]
	6-school	In vivo oral administration of 6-shogaol in ginger-derived vesicles enhanced the bioavailability of 6-shogaol in colon tissues compared to oral administration of free compound; moreover 6-shogaol/vesicles showed a more potent regenerative effect compared to free 6-shogaol	[80]
Grapefruit (Citrus paradisi)	anti-Stat3 inhibitor (JSI-124)	Reduction of Stat 3 expression in mice intranasally treated with grapefruit vesicles loaded with JSI-124	[26]
	doxorubicin	Efficient targeting of in vivo glioma and anti tumor effect	[17]
	HSP70 & BSA	Grapefruit vesicles ameliorated the uptake of exogenous proteins (HSP70 and BSA) both in vitro and in vivo	[81]
	inflammatory chemokine receptor	Grapefruit-derived nanovector coated with inflammatory-related receptor-enriched membranes of activated leukocytes (IGNVs) are enhanced for homing to inflammatory tumor tissues.	[82]
	miR-17	Intranasally administrated grapefruit nanovesicles deliver miR17 to mice brain tumors	[83]
	miR-18	Grapefruit-derived lipids carrying miR18 inhibit liver metastasis through induction of M1 macrophages	[84]
Lemon (Citrus limon)	Doxorubicin	Lemon vesicles loaded with doxorubicin can efficiently enter in doxorubicin resistant cells and exert a potent anti-tumor effect	[16]

UC: Ultracentrifugation, imDCs: Immature-dendritic cells, SC: Subcutaneous, ID: Intradermal, MSCs: Mesenchymal stem cells, UF: Ultrafiltration, DF: Diafiltration, EM: Electron microscopy, CTL: Cytotoxic T lymphocyte, GM-CSF: Granulocyte-macrophage colony-stimulating factor, NK: Natural killer, MHC: Major histocompatibility complex, ICAM-1: Intercellular adhesion molecule 1, LAMP-3: Lysosomal-associated membrane protein 3, HSP: Heat shock protein, rIFN-γ: Recombinant interferon-γ, EV: Extracellular vesicles IV: Intravenous, EV: Extracellular vesicles

Table 3. Clinical trials using human or plant-derived source for either drug-delivery or unmodified (source: clinicaltrials.gov).

Indication	Year, phase, patients	DETAILS	RESULTS/ STATUS	REFERENCE
Malignant ascites and pleural effusion	2000, Phase 1, (n = 15)	Source: Tumor-derived Dose: Not reported Administration: Perfused to the pleural or peritoneal cavity, 4 times/week Manipulation: Loaded with chemotherapeutic drugs	Unknown status	[45]
Malignant pleural effusion	January 2016, Phase 2, (n = 90)	Source: Malignant pleural effusion, Dose: Not reported Administration: Not reported Manipulation: Loaded with methotrexate	Recruiting	[46]
Metastatic pancreatic cancer	March 2020, Phase 1, (n = 28)	Source: MSCs, allogeneic, Dose: Not reported Administration: IV on days 1, 4, and 10. Treatment repeated every 14 days for up to 3 courses Manipulation: KrasG12D siRNA (iExosomes)	Not yet recruiting	[47]
Bronchopulmonary dysplasia	June 2019, Phase 1, (n = 18)	Source: MSCs Dose: −200 pmol phospholipid/kg Administration: Intravenous Manipulation: Not specified (UNEX-42)	Recruiting	[48]
Type 1 diabetes	April 2014, Phase 1, (n = 20)	Source: MSCs allogeneic Dose: (1.22–1.51)×106 cells/kg Administration: Day 0 and 7, Intravenous Manipulation: Unmodified	Unknown	[49]
Macular holes	March 2017, Phase 1, (n = 44)	Source: MSCs allogeneic Dose: 50 μg or 20 μg Administration: Dripped into vitreous cavity Manipulation: Unmodified	Recruiting	[50]
Acute ischemic stroke	April 2019, Phase 1/2, (n = 5)	Source: MSCs allogeneic Dose: 200 μg Administration: Stereotaxic injection Manipulation: Enriched by miR-124	Not yet recruiting	[51]
Colon cancer	January 2011, Phase 1, (n = 35)	Source: Plant-derived Dose: Not reported Administration: Tablets taken daily for 7 days Manipulation: Loaded with curcumin	Active, not recruiting	[52]
Radiation- and chemotherapy-induced oral mucositis	August 2012, Phase 1, (n = 60)	Source: Grape derived Dose: Not reported Administration: Oral administration daily for 35 days Manipulation: Unmodified	Active, not recruiting	[53]
Insulin resistance and chronic inflammation in polycystic ovary syndrome	May 2018, not applicable	Source: Plant-derived (ginger and/or aloe) Dose: Not reported Administration: Not reported Manipulation: Unmodified	Not yet recruiting	[54]
Melanoma	2000, Phase 1, (n = 15)	Source: imDC, autologous Dose: 4 × 1013 or 1.3 × 1013 MHC Class II molecules Administration: SC (90% of the volume) and ID (10%) injections weekly for 4 weeks Purification: 500-kDa concentration and UC with D2O/sucrose cushion Characterization: CD81 tetraspanin Bioactivity: SEE test of potency Exosome manipulation: Pulsed with MAGE 3 tumor peptides	No Grade II toxicity; No detected MAGE3-specific CD4+ and CD8 + T cells	[55]
Non-small cell lung cancer	Not reported. Phase 1, (n = 4)	Source: imDC, autologous Dose: 1.3 × 1013 MHC Class II molecules Administration: SC (90% of the volume) and ID (10%) injections weekly for 4 weeks Purification: 500-kDa concentration and UC with D2O/sucrose cushion Characterization: Not reported Bioactivity: MHC Class II molecules; ELISPOT for peptide-specific immune response Exosome manipulation: Pulsed with MAGE-A3, -A4, -A10, and MAGE-3DPO4 tumor peptide	Well-tolerated and only Grade 1–2 adverse events; MAGE-specific T-cell responses in 1/3 patients; increased NK lytic activity in 2/4 patients	[56]
Non-small cell lung cancer	May 2010, Phase 2, (n = 22)	Source: Mature- dendritic (mDCs), autologous (induced by rIFN-γ) Dose: 8.5 × 1011–1.0 × 1013 MHC Class II molecules Administration: Four ID at 1-week intervals Purification: UF; DF, and UC through a 1.21 g/mL sucrose cushion Characterization: Exosome marker: Tetraspanin Bioactivity: Activation of LT11 cells Function: MHC class II molecules and CD40, CD86, and ICAM-1/CD54 Exosome manipulation: Pulsed with MAGE-A1, -A3, NY-ESO-1, Melan-A/MART1, MAGE-A3-DP04, EBV tumor peptides	One patient had Grade 3 hepatotoxicity; boosting the NK cell arm of antitumor immunity.	[57,58]
Colon cancer	Not reported, Phase 1, (n = 40)	Source: Ascites, autologous Dose: 100–500 μg of protein Administration: Four SC at weekly intervals Purification: Differential centrifugation + sucrose/D2O density gradient UC Characterization: Exosome marker: HSPs (including HSC70, HSP70, and HSP90), CD80, ICAM-1, CD71), and LAMP-3; EM Bioactivity: Function: Tumor-associated carcinoembryonic antigen, MHC-I, and MHC-II molecules Exosome manipulation: ±GM-CSF	Safe, well-tolerated; tumor-specific antitumor CTL response in exosome plus GM-CSF group	[59]
Chronic kidney disease	April 2014, Phase 2/3, (n = 40)	Source: MSCs, allogeneic Dose: 100 μg/kg/dose Administration: Two doses of MSC-EVs, intraarterial and intravenous injections Purification: UC at 100,000 × g Characterization: CD9, CD63; EM Bioactivity: CD45, CD73 Exosome manipulation: Unmodified	Safe, well-tolerated; improved kidney function; decreased inflammation	[60]

7. Perspectives and challenges: the promise of plant-derived exosomes-like nanovesicles as a new therapeutic tool

To date, we do not have sufficient clinical data supporting the ability of either human or PDENs to deliver therapeutic molecules with hopefully reduced systemic toxicity efficiently. Although clinical trials are still ongoing (Table 2), representing a significant advancement of PDENs as therapeutic nanocarriers, many challenges remain until they mature into clinical translation, primarily to the long-standing and expensive regulatory procedure to get to human use. PDENs are essential conveyors of information between cells through the transmission of various proteins, bioactive lipids, and genetic data to alter the phenotype and function of the recipient cells [24]; this is making them ‘intermediaries’ in numerous biological and pathological processes within plant-derived cells and cells of other species. However, we have to say that there still needs to be more clarity about whether communication to recipient cells is specific or stochastic. Moreover, their exploitation as nanotherapeutics or to deliver nucleic acids, protein, and other drug cargoes across various biological barriers has recently garnered much attention. In addition, with the use as a shuttle for therapeutic molecules, new evidence is emerging showing that nanovesicles from plants may have therapeutic potential due to their content in antioxidant bioactive but also plant-related nucleic acids (e.g. mRNAs and microRNAs).

The interest in PDENs is increasing in their potential applications as natural suppliers. Their structures include a series of antioxidants (ascorbic acid, glutathione, superoxide dismutase-1, catalase), lipids, proteins, nucleic acids, and secondary metabolites [28,32,85]. The PDENs content is protected from external stress and damage by a lipid-enriched membrane containing entirely bioavailable antioxidants, thus representing a valid alternative to the synthetic antioxidants currently available in pharmacies. Another key advantage is the high level of biocompatibility in daily-consumed foods. Increasing evidence is showing that plant-derived nanovesicles enter mammalian cells and mediate plant–animal cross-kingdom gene regulation. Notably, it is evidence that small plant RNAs packed in PDENs could survive in an active form in animals and exogenously modulate host cellular processes via genetic crosstalk, possibly leading to cancer suppression [32]. This indicates a potential medical application for PDENs in regulating fundamental biological processes in the human body. In addition, PDENs may have a role in regulating the immune response [86,87], facilitating tissue regeneration [88], and delivering therapeutic molecules of various origins [29,32,85,89,90]. These nanovesicles can be used as suppliers, dermo-cosmetic and regenerative medicine for the above reasons. The potentially tremendous impact of these numerous applications represents a fantastic opportunity for PDENs. Overall, by multidisciplinary associations in cell and molecular kinetics, engineering, investigation, and medicine, we believe a hopeful future for clinical translation of PDEVLNs-based research that further studies will need to be undertaken.

8. Plant-derived nanovesicles for delivery of antiviral therapy and vaccines

One of the major breakthroughs in the history of medicine is undoubtedly the discovery of vaccines. During the COVID-19 pandemic, we raised the question of how PDEVs might be employed against COVID-19. Stably transformed, despite the fact that green approach requires more time for the development of antigen-producing lines, nonetheless, this approach offers the possibility of developing oral vaccines in which the plant cell could act as the antigen delivery agent. In this scenario, we believe that PDEN-based vaccines may be considered the future of therapeutics owing to their role in inhibiting viral infection, triggering host immune response, and their involvement in disease progression. Notably, there are some standard features between viruses and exosomes in terms of biochemical composition, size, biomolecule transfer mechanisms, facilitation of entry into host cells, biogenesis, and multiplication of viruses in host cells. Furthermore, changes in exosome cargo during viral infection, such as the transfer of viral particles into uninfected cells and immune response modulation, have led researchers to characterize this subpopulation of EVs better and investigate their therapeutic potential in antigen presentation for safe vaccine design [91]. Multiple criteria should be considered to design an efficacious vaccine. Thanks to efficiently carrying cargo, exosomes act as natural delivery vehicles and constitute a specific and efficient delivery system in antigen presentation [36,91,92]. Therefore, it may also fall under the vaccine general-purpose criteria. An interesting preclinical study published recently in the Journal of Biological Chemistry [93] demonstrated that mRNA-loaded exosomes could successfully deliver multiple functional mRNAs supporting the further development of mRNA-loaded exosomes for use as vaccines and therapeutics. Moreover, characteristics of exosomes, such as high vascular permeability, stability, solubility, and bio‑distribution, make them ideal candidates for vaccines [94]. Numerous in vivo studies have been performed to evaluate the toxicity and immunogenicity of exosomes [95], highlighting that the critical features of EVs‑based vaccines, including their ability to induce poor immunogenicity, mean EVs population can be safely and efficiently used in vaccine development.

EVs preserve naïve antigen confirmation, and access to all organs via bodily fluids gives an added advantage compared with other delivery agents, such as lipid-based nanoparticles (LNPs) or viral vectors [37,96].

Therefore, engineered EVs can fulfill the criteria for an efficacious vaccine due to their efficient antigen‑presenting system and high biosafety.

Antiviral treatment is currently available for public use since the SARS‑CoV‑2 genomic sequence was identified;> 100 vaccine studies have been performed, ~50 of which have reached human experimentation, and selected vaccines are currently being administered [97]. Available SARS‑CoV‑2 vaccines are based on the classical approach of viral vectors, particularly adenoviruses [98–100]. Although adenovirus‑based vaccines are well‑characterized, they are limited by pre‑existing immunity of the virus vector employed in the vaccine design, which may restrict the immune response against COVID‑19 antigens, decreasing their efficacy [101]. Another essential concern is the community’s risk of re‑infection with emerging viruses due to a lack of long‑lasting immunity [102]. Thus, viral infection may become endemic (similar to the influenza virus endemic), necessitating yearly vaccination programs. In addition, multiple immunizations with such viral vectors are requested due to new coronavirus variants, if not compelling, which could lead to a more complicated form of the infection. Such adverse effects may pose more danger to an individual than the initial infection. Notably, specific recipients of the AstraZeneca COVID‑19 vaccine, which has received approval from the UK authorities, exhibited a rare blood‑clotting disorder; hence, the safety of these vaccines is highly questionable [103]. In 2020, the LANCET journal reported concerns about using a recombinant adenovirus type-5 (Ad5) vector for COVID-19, increasing the population at risk of HIV infection [104]. PDNVs-based vaccines may constitute an innovative alternative for an efficient virus‑free, human‑derived vaccine design, eliminating the adenovirus vector‑based vaccine drawbacks associated with preexisting immunity. Due to this advantage of EVs over virus‑based vectors, several biotechnology companies are focusing on vaccine development using EVs as a platform against SARS‑CoV‑2.

Immunologically, a vaccine targeting the mutation‑prone S protein and the more stable and conserved N, M, and E proteins are required to surmount the immune escape characteristics exhibited by SARS‑CoV‑2 variants [105]. Hence given this, PDNVs, notable PDENs-based vaccines displaying SARS‑CoV‑2 structural proteins, might be a novel approach to overcome the shortcomings of existing vaccines and contain escalating cases of COVID‑19. Furthermore, exosome-based vaccines comprising all four‑target antigens (S, M, E, and N proteins) induce strong Nab and T cell responses, thereby conferring prolonged immunity with no risk of reversion of vaccination‑induced virulence and pre‑existing immunity. Moreover, incorporating these immunogens into the exosomes, which are virus‑free and exhibit lower immunogenicity and higher absorption rate than exiting vehicles such as LNPs or adenoviruses, would fulfill the requirements of an ideal vaccine eliminates the need for booster doses [106] in the future time. These advantages of exosome‑based vaccines over conventional vaccines fit the necessity of a vaccine targeting SARS‑CoV‑2 and emerging SARS‑CoV‑2 variants. The main challenge envisioned for developing COVID-19 PDEVs-based vaccines will be, as is typical for all vaccines, testing their efficacy in large clinical trials to validate their safety while fulfilling the requirements of regulatory agencies. However, the fact that there are precedents of a plant-made biopharmaceutical approved for human use and plant-made vaccines against influenza under clinical trials (with promising safety and efficacy) is encouraging.

9. Conclusions

We want to emphasize as this review does not want to compare PDENs to the existing methods to deliver drugs; rather we would like to propose a new method that has all the potentiality to (i) represent a way to deliver drug with a high level of bioavailability; (ii) to be a natural way to deliver drugs and bioactives, being PDENs natural transporters of a broad array molecules; (iii) to be without possible side effects either systemic or at the organ levels, inasmuch as PDENs derive from organic fruits and vegetables that are routinely assumed by the human beings worldwide.

Actually, PDENs have low toxicity and immunogenicity compared to exosomes secreted from mammalian cells and have shallow cholesterol content and thus have high stability and biocompatibility in the body. In addition, plant-derived exosomes are known to be effective in wound healing and skin regeneration due to their high level of effective bioactive. Meanwhile, various methods for purifying exosomes from living organisms have been set up, including centrifugation, ultracentrifugation, density gradient centrifugation, chromatography, filtration, ultrafiltration, tangential flow filtration, polymer-based precipitation, total exosome extraction kit, and immunoaffinity. On the other hand, there is a general need to tune the existing methods to obtain exosomes from cell supernatants and body fluids to those to date used to obtain exosomes from plants. We have two patents regarding (i) a novel protocol to obtain plant-derived nanovesicles from fruits and vegetables deriving from organic agriculture [107]; (ii) methods to upload molecules and bioactive in plant-derived exosomes [108]. In the first invention, on the one hand, a complex procedure starting from the whole fruit or vegetable leads to obtaining a fluid material that undergoes filtration and repeated rounds of centrifugations and ultracentrifugations to obtain high-purity exosomes. On the other hand, this method allows for obtaining high-purity exosomes from many raw plants, including either fruits or vegetables. This improves the conventional plant exosome purification process at the laboratory level and suggests an easy process for producing amounts suitable for the industrial level. However, this invention contains a very original approach that is aimed to propose new products destined to the market of suppliers and dermo-cosmetics that do not correspond to exosomes’ preparation obtained from single fruits or vegetables but rather to a mix of exosomes deriving from different fruits and vegetables with the purpose to get to the best combination suitable for health-oriented indications. The second invention proposes uploading molecules and bioactive within plant-derived exosomes through a natural procedure or electroporation with an ad hoc exosome-related approach. These two methods can be used for different needs since both have shown full efficacy without altering the integrity of the exosomes. Both systems have demonstrated that molecules of various natures, once uploaded in the plant-derived nanovesicles, remain pretty stable within them and are always more efficient in entering, persisting, and exerting their action against target cells than the free molecules. Finally, loading nucleic acids in PDENs would overcome most of these challenges, as exosomes are biocompatible, have a unique tropism, and, depending on their cellular origin, pose little toxicity or immunogenicity threat. Exosomes can either be loaded endogenously through transfecting or overexpressing payloads in the cell source, followed by purifying the EVs produced by these cells, or exogenously through direct loading of isolated EVs using mechanical means (i.e. electroporation, sonication, freeze-thaw, cell extrusion) or chemical means (i.e. lipofection or calcium chloride treatment). Based on the literature, most attempts to load nucleic acids involve short RNAs such as siRNA, miRNA, and ASOs, and these payloads are loaded through exogenous means, usually electroporation. Another promising inventor has developed a method for loading cargo into PDEVs. In particular, the technique allows nucleic acid cargo such as DNA to be loaded into extracellular vesicles such as red blood cell-derived extracellular vesicles or exosomes. The resultant loaded extracellular vesicles are helpful in therapy and research for delivering the cargo to target cells in vitro and in vivo. In one aspect of the present disclosure, an extracellular vesicle is provided with a load or a population of such extracellular vesicles. The cargo is preferably a nucleic acid. The nucleic acid may be a DNA, RNA, oligonucleotide, or polynucleotide. The nucleic acid is most preferably a DNA. The nucleic acid may be circular or circularized, or linear. The nucleic acid may be double or single-stranded, preferably double-stranded. In some aspects, nucleic acid is a circularized DNA, such as minicircle DNA, plasmid, or nano plasmid. With this article, we want to emphasize the extreme versatility of exosomes obtained from plants. They can exert their natural effects well due to the content of very stable and effective bioactive; on the other hand, they can serve as an effective delivery system for a broad spectrum of therapeutic molecules with no evidence of primary toxicity. A key point is the use of plants derived from organic agriculture; this exciting field is developing hastily to obtain exosomes to avoid the concentration of pesticides within exosomes, as it has been shown for exosomes deriving from intensive agriculture.

10. Expert opinion

Many medical conditions can be cured if the current medical systems work synchronously through integrated approaches. Natural products are the base of novel therapeutic compounds and pose minimum adverse effects. Recent advances in the PDENs have gained impetus; however, several safety and regulatory requirements must be satisfied before pharmaceutical manufacturing and novel supportive therapeutic strategies can be realized. Given the infancy of the PDEN -based therapeutics, there are understandably no assays for safety testing and limited information about localization and biodistribution profiles. The safety of the source from which the PDENs are derived must also be considered. Given the similarities with the biopharmaceutical properties of fruits, significant learning can be applied from the organic agriculture sector. From a clinical perspective, a successful clinical translation of PDENs requires further standardization and addressing some of the significant challenges associated with their reproducible manufacture. However, as with the cell therapy field, considerable emphasis must be placed on understanding the fundamental of PDENs to begin addressing some of the standardization and manufacturing challenges. This also involves understanding the scalable production of the source as well as the fundamental bioprocessing conditions required to enable the reproducible production and purification of therapeutically relevant PDENs.

Article highlights

• There is a need of new approaches to use in drug delivery. The new approach should have the purpose to improve the currently used systems to deliver drug

• The ultimate endpoints should be the increase the effectiveness of the delivered drugs and reduce the side effects of the current treatments

• Here we propose to fuse biotechnology and organic agriculture to overcome the main challenges in drug delivery systems.,

• Solid scientific evidence suggests that exosomes-like nanovesicles may represent a new tool for drug delivery, with a very high level of bioavailability.

• In fact, there is growing interest in the potential use of plant-derived exosomes-like nanovesicles for various diagnostic and therapeutic in the field of nano‐pharmaceuticals.

• A new analytical platforms is proposing efficient and reliable methods to both obtain scalable plant-derived exosome-like nanovesicles and to upload with therapeutic molecules.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Author contribution statement

ON Salvatore: writing and coordinating the manuscript. DR Rossella: editing of the manuscript and literature research. M Davide: editing of the manuscript and literature research. L Mariantonia: writing and manuscript editing. F Stefano: writing and coordinating the authors’ activities.

Additional information

Funding

This paper was funded by the Italian Ministry of Health.