Plant-derived Edible Nanoparticles and miRNAs: Emerging Frontier

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Plant-derived Edible Nanoparticles and miRNAs: Emerging Frontier for Therapeutics and Targeted Drug-delivery Siavash Iravani, and Rajender S. Varma ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b00954 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on March 29, 2019

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ACS Sustainable Chemistry & Engineering

Plant-derived Edible Nanoparticles and miRNAs: Emerging Frontier for Therapeutics and Targeted Drug-delivery

Siavash Iravani1* and Rajender S. Varma2*

1Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Hezar

Jarib Street, 81746-73461, Isfahan, Iran 2Regional

Centre of Advanced Technologies and Materials, Department of Physical Chemistry,

Faculty of Science, Palacký University in Olomouc, Šlechtitelů 27, 783 71 Olomouc, Czech Republic. *Corresponding Authors: Rajender S. Varma, E-mail: [email protected] Siavash Iravani, E-mail: [email protected]

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Abstract Rigorous and timely studies have been conducted to develop natural and green nanomedicines wherein plant-derived edible nanoparticles (PDENs) have a great potential to be applied for targeted therapeutic delivery systems, because of their exclusive characteristics, namely desirable nanoparticle morphologies, environmentally safe and non-hazardous nature, appropriate tissue-specific targeting, and eminent potential for industrial pharmaceutical production. These nanoparticles can be exploited for drug transportation with distinctive benefits for treatment of diseases like cancers. In this article, the properties and applications of PDENs are discussed with latest developments including the recent trends in plant-derived edible exosomelike nanoparticles and miRNAs. Keywords: Plant-derived nanoparticles; miRNAs; Edible nanoparticles; Therapeutic delivery; Colon cancer; Drug delivery systems; Tumor cells


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Introduction In phytoformulative investigations, scientific studies have converged on the development of novel pharmaceutical delivery systems, such as proliposomes, liposomes, nanoparticles (NPs) comprising solid lipids, nanoemulsion, and drug transportation systems based on proteins lipids. In this field, improvements of bioavailability, stability, solubility, pharmacological activity, toxicity reduction, refining the distribution of tissue macrophages, protracted delivery, and fortification from physicochemical degradation are some of the important attributes. Advancement of a biomimetic and bio-inspired approaches to nanostructures is one of the vital challenges for the phyto- and nano-scientists.1 In the field of nanomedicine, various benefits of deploying plants as natural and renewable resources in green nano-factories is an ideal strategy for the production of pharmaceutical and nanomaterials of medicinal value. As an example, gliadin NPs were applied as nanocarriers for oral anticancer or lipophilic medications.3 Plant-derived nanostructures obtained from corn, wheat, and soybeans contain readily available proteins which are sustainable, biodegradable, and significantly less allergenic when compared to animal proteins (e.g., bovine collagen); these nanostructured entities can be applied as conveyance systems for antitumor medications.1-3 Advancements in bio-inspired and bio-mimetic substances and systems, adaptive and hierarchically-structured biomaterials, nanomaterials, three dimensional (3D) composites, and biocompatible materials are now garnering increased attention. Moreover, biomaterials with selective multi-functional properties can be applied in assorted processes namely catalysis, adsorption, separation, sensing, imaging and bio-sensing, and they will continue to dominate in near future. In recent years, greener nanotechnology and nanofabrication have been actively pursued for large-scale production of well-defined NPs and bio-nanomaterials with specific and 3 ACS Paragon Plus Environment


tailored functionalization. Despite the progress made, substantial challenges still persists that should be attended for maximum benefits from these plant-based nano-systems.2,3 In the fields of nanotechnology, pharmaceutical and food sciences, bio-nanostructures as innovative nano-carriers of bioactive materials for targeting delivery are very important. In fact, these industries continually attempt to uncover the numerous hidden attributes of these biocompatible and innocuous nanobased structures. Thus, they protect hydrophilic- and lipophilic-enriched bioactive entities from degradation, and preserving their effectiveness. However, in view of the anticipated behavior, specifically in the gastrointestinal (GI) tract, the physicochemical properties of nano-based structures in terms of charge and size might change dramatically. Critical challenges in the elaboration of these structures, especially when they are in contact with living systems, are to comprehend the procedures transpiring at their surfaces and their appropriate characterizations. Consequently, it is very important to grasp their possible toxicological effects, delivery and absorption behaviors.4 Indeed, plant-derived edible nanoparticles (PDENs) are membrane vesicles with nanosized structures discharged by edible plants, including ginger, lemon, grapefruit, and broccoli (Figure 1).5 These NPs might have intrinsic therapeutic activities against specific diseases, and for example, in the treatment of inflammatory bowel disease (IBD) and cancer; moreover, such renewable materials may be non-toxic and can be mass-produced. Nowadays, researchers are on the lookout for selecting and characterizing precise PDENs which have these inherent activities and scrutinize their intrinsic targeting. Plant-derived edible lipid nanoparticles (PDLNs) might serve as natural drug transporters efficiently distributing medications to exact location of the body.5


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Figure 1. PDENs and their biomedical applications PDENs show unique benefits, including suitable and desirable morphologies, safety, and desired tissue targeting, coupled with substantial possibility for large-scale preparation.6 Various characterization procedures have demonstrated that these nano-sized vehicles have common structural features with exosomes of mammalian origin. It has been reported that PDENs might be implicated in communications of plant cell-cell, and could possibly regulate the native immunity of plants, and might also transport lipids, mRNAs, microRNAs (miRNAs), and proteins into cells.7,8 Mainly, the miRNA-centered therapeutics include two tactics, including miRNA inhibition-synthetic single-stranded RNAs (anti-miRNAs), which cause the upregulation of the particular proteins by antagonizing the action of endogenous miRNA, and miRNA enhancementsynthetic miRNAs (called miRNA mimics), deployed for mimicking endogenous miRNAs and thus securing the identical role by prohibiting the mediating/translation the decay of target mRNAs.9-11 There are some important challenges in this regard, including predominantly, insignificant stability, ineffective delivery and off-target outcomes. Thus, there are some exigent 5 ACS Paragon Plus Environment


issues pertaining to the valuable delivery of therapeutic miRNA molecules. Dendrimers, polyethylenimine (PEI)-centered transporter systems, and poly(lactide-co-glycolide) -based entities as the lipid- and polymer-based vectors have been applied as delivery systems. Moreover, natural materials, including protamine, chitosan, and atelocollagen were applied for RNA transportation systems.12,13 In the field of natural nanocarriers, it has been shown that in addition to miRNA-based therapeutics, exosome-like NPs derived from edible plants could be applied for model cargo drug transportation.14-17 Consequently, natural PDENs can be used as innovative carriers for the delivery of miRNAs, and these diet-derived plant miRNAs can regulate endogenous gene expression in animals. It has been demonstrated that by systematic administration of unmodified exosomes to the animals, they are rapidly cleared by renal system or transport their load to undesirable tissues subsequent to accumulation in the liver,.18,19 The exosomes directing ability can be positively accelerated by presenting selective peptides or ligands on the exosome surfaces which might target the recipient cell holding associated receptor. Such aiming peptides might lose their steering competence and affinity or could be degraded.20-23 In this overview, the isolation, characterization, salient properties and applications of PDENs are discussed, as promising and suitable NPs for drug delivery and treatment of diseases including colon cancer, brain tumors, IBD, etc. Further, we discuss the potential of the edible plantderived exosome-like NPs (EPDENs) and miRNAs. Plants and nanotechnology Plants are green, sustainable and renewable platforms and are incredible resource for NPs as they have immense benefits as natural nano-factories.1 The plant-based NPs can be utilized for delivery of molecules as directed medication transportation systems and may be explored for


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additional biomedical applications. Plant-derived nanostructures frequently have superior level of convolution which matches the erudition of manmade engineered biomaterials. Furthermore, altering the well-structured plant-based biomaterials into purposeful resources using synthetic approaches can exploit the inherent native morphology. Plant nanostructures, nanostructures from plant biomolecules, and nanostructures produced using plant biomolecules can be used for drug transportation systems, energy production, environmental phytoremediation, or nanofabrication via bio-templating. Further, the distinct application of plant nanostructures incorporated within carbonaceous and polymeric biomaterials has revealed higher level of biodegradability, signifying the great potential of these natural biocomposites in the field of biomedicine and bio-sensing.24, 25 Plants and plant-derived NPs can be applied as simple, safer, eco-friendlier, low-cost, and relatively less toxic materials for treatment of diseases like cancers, when compared to the conventional treatment methods. For instance, in the field of cancer, phytochemicals are selective in their functions and act specifically on tumor cells without affecting normal cells.26 The growing incidence of diseases like cancer and higher associated costs for the treatment, existing limitations of the conventional therapy, and high toxicity of present anticancer drugs present a serious challenge to researchers for designing and developing eco-friendly, biocompatible and costeffective strategies in a greener way. It appears that phytomolecules such as curcumin, epigallocatechin, isothiocyanates, gossypol, sulforaphane, garcinol, etc. with high biodegradability and biocompatibility have contributed to enhanced efficacy in treatment of diseases.26

Isolation and characterization of PDENs Differential ultracentrifugation coupled with density gradient centrifugation is considered the standard method for the isolation of PDENs wherein plants are crushed using a mixer; larger 7 ACS Paragon Plus Environment


particles and plant fibers are separated from the ensuing juice via low-speed centrifugation. While high-speed centrifugation is applied to slab the exosome-like NPs, medium-speed centrifugation is deployed to take away unbroken organelles and bulky bits. The angle of rotor sedimentation and g force rotor type affect the amount and characteristic of the attained NPs besides sedimenting the proteins and/or protein/RNA aggregates and other vesicles. Thereafter, a sucrose density gradient step is exercised to secede the PDENs from impurities of varying masses. Ultracentrifugation is inadequate in securing extra cleansed plant edible NPs which can be achieved by gradient ultracentrifugation, although additional processing time period to required (~one to five hours).27-29 Indeed, the contents of PDENs, especially, lipids, RNAs and proteins, vary from mammalian exosomes counterparts30 as exemplified by grape exosome-like NPs (GENs) from grapes that encompass 28 detected proteins and 96 miRNAs.16 Lipidomic findings demonstrated that GENs are augmented with phosphatidylethanolamines (PE) (26%) and phosphatidic acids (PA) (53%) (Figure 2).16 Remarkably, elevated proportions of PA in GENs could ensue via activation of GEN phospholipase D in the course of extractive procedure for GEN lipids. Nevertheless, this was refuted as shown by lipidomic investigation of lipids obtained from GENs or intact grapes irrespective of the use or nonuse of 75% isopropanol in phosphate-buffered saline (PBS) as extractive media; GEN PA proportion remained unchanged in the absence (49.1 ± 3.8%, n = 5) or presence (47.2 ± 5.2%, n = 5) of 75% isopropanol. Furthermore, PA proportion in intact grape was abundantly inferior (18.2 ± 1.9%) compared to GEN (47.2 ± 5.2%) complement obtained from the identical batch of grapes. Additionally, the nucleic acids existence was analyzed in GENs which showed considerable quantities of RNAs being identified by agarose gel electrophoresis indicating RNAse treatments cause the degradation of GEN RNAs. Further, GEN miRNA profile by MS analysis have shown that GENs encompassed miRNAs.16 8 ACS Paragon Plus Environment

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Figure 2. Sucrose gradient ultracentrifugation and differential centrifugation for identifying and characterizing isolated GENs. a) Gradient band showed by arrow. b) Sizing of particles. c) Zetasizer measurement of charge. d) Summary of lipids in GENs. (e) Agarose gel runs. Reproduced from Ref 16 (An open access article). Mammalian exosomes, in contrast, characteristically comprise in excess of 1,000 proteins and about 100-300 miRNAs. Cholesterol and sphingomyelin are the main contents of mammalian exosomes lipid profile, and they contain only little amounts phosphatidylethanolamine and PA. On the other hand, GENs comprise 98% phospholipids (approximately, 50% of that being PA) and only miniscule amount (~2%) of usual plant lipids. The comprehended intermingling of PA with



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rapamycin (mTOR), is well-known to activate proliferation and cell growth.31 Additionally, the induction of inter-vesicular fusion is conjectured owing to PA being extremely fusogenic when calcium is present.32 High-throughput small RNA sequencing results help scrutinize the miRNAs present in coconut water,

33

and the alterations in miRNA contents amongst mature and immature coconut

water were evaluated. Thus, coconut endosperm comprised 14 new miRNAs and a collection of 47 recognized miRNAs from 25 families. Prospective miRNA target genes could be detected in the human genome via inquiry by target gene prediction software; miRNAs levels, as analyzed by real-time PCR, were elevated in mature coconut water relative to immature counterpart. Furthermore, NPs akin to exosomes were sequestered from coconut water. After ultracentrifugation,

using

fluorescence

staining

with

1,1′-dioctadecyl-3,3,3′,3′-

tetramethylindocarbocyanine perchlorate revealed existence of structural particles in the coconut water specimens. Further size analysis of the exosome-like NPs originating from coconut water, by dynamic light scattering (DLS) and scanning electron microscopy (SEM) , revealed that the detected particles have mean diameters of 60 and 13 nm, respectively.33 Grapefruit-derived exosome-like NPs (GDNs) bearing ~ 137 proteins and a contrasting lipid profile from GENs, were detected;17 they are apparently loaded with higher levels of phosphatidylcholine (29%) and phosphatidylethanolamine (46%). Various proteins are accountable for controlling the carbohydrate/lipid metabolism as per the mass spectrometry (MS)based protein profiling of GDNs. Such plant-derived exosome-like NPs alterations could play critical functions in interspecies communication including extended stretch of communication through the whole digestive tract of mammals.17 In another high-performance liquid chromatography (HPLC) analytical study, broccoli-derived nanoparticle lipids showed that 10 ACS Paragon Plus Environment

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sulforaphane was augmented in NPs relative to microparticles obtained from broccoli extracts; insignificant amount of sulforaphane existed in broccoli extracts.34 Immature dendritic cells derived from monocyte, differentiated for 7 days with IL-4 and granulocyte-macrophage colonystimulating factor (GM-CSF) with or without sulforaphane have been evaluated. Adding GM-CSF and sulforaphane made the bone marrow cells differentiation with inferior CD11c expression and an advanced PD-L1expression (a marker related to controlling dendritic cells) than did addition of GM-CSF by itself.34 Broccoli-derived NPs (BDNs) were isolated after a 100,000×g centrifugation for about one hour, and the supernatant was gathered from well-beaten broccoli; BDNs were obtained from the supernatant via a column filtration system. The BDNs size distribution (~ 18 to 118 nm) was evaluated by nanosizer and affirmed by electron microscopy. These ensuing NPs possessed a negative zeta potential value (approximately, −39 to −2.6 mV) as measured via Zeta potential.34 It is possible to apply plant-derived vesicles as nanovectors in biomaterials delivery systems. Protein bio-cargo in citrus juice sac cell-originated vesicles have been examined35 wherein fractions of nano-sized and micro-sized vesicles were obtained from C. aurantium, C. limon, Citrus sinensis, and C. paradisi, analyzed, and protein loads were contrasted using labelfree quantitative shotgun proteomics; each sample had 600-800 identifiable proteins. High expression levels of heat shock proteins, clathrin heavy chain, fructose-bisphosphate aldolase 6 and glyceraldehyde-3-phosphate dehydrogenase, among others were detected in all tests whereas aquaporin was extremely expressed solely in the nanovesicle fractions. Further, large amount of diverse enzymes, comprising oxidoreductases and hydrolases, were ubiquitous in citrus fruit sac cell-originated vesicles.35



Transmission electron microscopy (TEM) was applied for characterizing PDENs although this characterization method does not provide more mechanical or biochemical data, but, it is the important method applied for ultrastructural subcellular evaluation.36-38 TEM analytical methods entails well dispersed exosome-like NPs in a deionized water-soluble solution being spotted onto a carbon-layered grid, fixed using 1% glutaraldehyde, and visualized after staining with 2% phosphotungstic acid. For instance, various PDENs structures emanating from tomato, grapefruit, carrot and ginger have been described.39 Moreover, atomic force microscopy (AFM), a very highresolution type of scanning probe microscopy (SPM), was applied for analyzing the individual PDENs in terms of their size and structure. Indeed, AFM has proven resolution of fractions of a nanometer, and is thousand times superior to the typical diffraction limit of the optical microscopy.40-42 NPs derived from edible ginger (GDNPs) chiefly amassed at the 8/30% (band 1) and 30/45% (band 2) boundaries of the sucrose gradient as shown in figure 3; a weak band being identified as well at the 45/60% borders (band 3).43 The zeta potentials and size distributions of GDNPs were evaluated using photon correlation spectroscopy (PCS) using Brookhaven apparatus. The average size discerned ranged ~ 292.5 nm for NPs detected in band 1, 232 nm from band 2, and 220 nm from band 3. A zeta potential denomination of about -12 mV at pH 6 (the pH of the duodenum-jejunum) was detected for GDNPs originating from bands 1 (GDNPs 1) and 2 (GDNPs 2) whereas a zeta potential close to zero (about -2.1 mV) was ascribed for band 3 NPs (GDNPs 3). It was revealed that exclusively the first two, GDNPs 1 and GDNPs 2, could tolerate freeze/thaw cycles and were extremely stable at room temperature over seven days with the exception of GDNPs 3; thus, GDNPs 3, because of their low instability and yield, were not examined any longer. Furthermore, TEM and AFM were applied for the characterization of GDNPs 1 and 12 ACS Paragon Plus Environment

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GDNPs 2, and the ensuing information affirmed that the GDNPs integrity and size were coherent with those gauged by PCS method.43

Figure 3. Sucrose gradient ultracentrifugation depicts three bands (A); Visualization and characterization of GDNPs using TEM (B, D) and AFM (C, E) are shown. Reproduced with permission from Ref 43.

DLS (quasi-elastic light scattering or photon correlation spectroscopy), is applied for evaluating the small particles size-distribution in suspension, and PDENs sizes and zeta potentials.39 In fact, the scattered light intensity fluctuations are due to the Brownian movement of the particles, and are correlated to acquire data regarding particle size. As highly sensitive and being unobtrusive, DLS needs only tiny sample amounts and has been extensively deployed in various disciplines and considered a benchmark for rapid and precisely evaluating the NPs size 13 ACS Paragon Plus Environment


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distribution in suspensions;44-46 nanoparticle size is an important factor for its therapeutic effectiveness. It has been reported that the obtained PDENs from edible plants (such as ginger, grapefruit, tomato, and garlic) have an average sizes of ~400 nm for grape NPs, and 250 nm for ginger and grapefruit NPs. Carrot NPs, on the other hand, has two wider distributions, ~100 nm and ~1,000 nm. PDENs also show negative zeta potentials (~49.2 to 1.52 mV) of somewhat low value at pH 6, demonstrating that they display mutual repulsion and lacking aggregation tendency.47 Biomedical applications of PDENs: Therapeutics and targeted drug-delivery PDENs have shown appropriate property profile including rather low immunogenicity, elevated internalization rate, confirmed GI stability, and potential to surmount the blood-brain barrier but interestingly, they do not traverse the placental barrier.5 Some important examples of PDENs and their biomedical applications are mentioned in Table 1. In general, the present chemotherapeutics as applied against cancer diseases have several disadvantages, including toxicity and the lack of specificity. Actually, various polymeric NPs such as organic and inorganic biomaterials (micelles) were utilized as nanocarriers for anticancer drugs.48 In one study, activated leukocytes were used to coat grapefruit-derived lipid NPs (GDNVs).49 As a result, it was demonstrated that the ensuing particles could be targeted to inflamed tumor as delivery systems for doxorubicin (Dox). Various mouse models of inflammation-driven disorder were deployed, and consequently, it was shown that such receptorenriched membranes had better targeting success relative to GDNVs.49 Table 1. Some important biomedical applications of PDENs PDENs

Target

Drugs 14 ACS Paragon Plus Environment

Reference

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Grapefruit

Ginger

Lemon Broccoli

Colon cancer Brain tumor Colon cancer Colon cancer Brain tumor Colon

Chronic myeloid leukemia Colon

Dox, curcumin JSI-124 (Cucurbitacin I) Paclitaxel Luciferase gene siRNA miR17 miRNA, gingerol, and shogaol Dox siRNA-CD98

49

Proteins Sulforaphane

54

50

51 43

52 53

34

Actually, lipids are used as favored carriers for drug delivery, but synthetic lipids and liposomes NPs can generate adverse consequences including inflammasome activation, apoptosis, and cell stress.55 PDLNs are applied as suitable agents for transporting different curative agents. The application of safe or harmless naturally-occurring nanovectors prepared from PDLNs was reported as feasible substitute method for drug transportation, in vivo.50 The possibility of preparing naturally nanovectors from ginger NPs derived lipids was reported; ensuing lipid NPs lacked cytotoxicity or adverse effects on intestinal barrier functions, indicating that they might be valuable for efficient drug hauling, in vivo.50 Grapefruit-derived nanovectors could move DNA expression vectors, chemotherapeutic materials, proteins, and siRNAs to various types of cells.50 Grapefruit-derived nanovectors have been used to coat liver medicinal materials and folic acid (FA), and it was reported that the mentioned approach meaningfully enhanced the aiming efficiency to target cells which express folate receptors. It has been reported that grapefruit-derived nanovectors were not as toxic as NPs prepared from synthetic lipids and did not traverse the placental barrier upon intravenous injection to pregnant mice, indicating that they may be valuable in hauling drugs. Furthermore, Grapefruit-derived nanovectors with FA-augmented exteriors have been applied for transporting paclitaxel (PTX) to the tumor location, intravenously. Marked 15 ACS Paragon Plus Environment


elevation of FA receptors in various tumor cells have been beneficially exploited by FA-enriched NPs; meaningfully enhanced homing effectiveness was discerned thereby reducing the tumor volumes in mice xenografted with CT26 or SW620 cells.50 In an interesting study, safe therapeutic approach for treating brain-linked disease via intranasal transportation has been reported51 wherein the potential of grapefruit-derived nanovectors to move miR17 for treating mouse brain tumors was shown; grapefruit-derived nanovectors coated with FA (FA-GNVs) were honed to a folate receptor positive brain tumor (GL26). Moreover, FA-GNVs coated polyethylenimine (FA-pGNVs) not merely enhanced the competence to transfer RNA, but the polyethylenimine toxicity was terminated by grapefruitderived nanovectors. Intranasal administration of miRNA 17 conveyed by FA-pGNVs made expeditious transportation of miR17 to brain which was taken up by GL-26 tumor cells selectively. It appears that mice that were intranasally doctored by FApGNV/miR17 showed slowed growth of brain tumor.51 It has been shown that orally administered GENs

to mice elicited stimulation of cells

proliferation in the intestinal epithelium, and was related to intestinal stem cell growth all over the intestine and colon.16 Furthermore, orally administered GENs were resistant to degradation by the stomach acidity, and the extremely active proteolytic enzymes residing along the intestinal tract. These new findings revealed that PDENs could be orally transfered to the intestine, where intestinal cells may grab them culminating in intestinal regeneration. Colonic tissues being aimed by PDENs endowed with anti-inflammatory properties might be an innovative natural and safe transportation system which could find application to deal with digestive tract diseases (e.g., IBD). Interspecies communication between gut host cells and plants was established because of GENs novel transport characteristics and bio-functions;16 apparently, these NPs can enter the intestinal 16 ACS Paragon Plus Environment

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mucus obstruction, be sieged by mouse intestinal stem cells (ISCs), and meaningfully trigger the activation of Lgr5hi ISCs via the Wnt/β-catenin conduit.16 It has been demonstrated that orally dispensed GENs had defensive influences on dextran sulfate sodium (DSS)-induced colitis mice through inducing ISCs proliferation and growth, which have an important function in controlling the intestinal epithelial cells (IEC) differentiation and are needed for refurbishment of intestinal tissue repair and homeostasis.16 In order to examine whether GENs could directly stimulate the ISCs, Lgr5-EGFPhi stem cells were isolated from crypts of Lgr5-EGFP-IRES-CreERT2 mice and then cultured in vitro in in the company of GENs (40 µg/ml); the NPs directly promoted the growth of Lgr5-EGFPhi ISCs, and accelerated the formation of organoid structure. This study suggests that liposome-like NPs (LLNs) built with GEN lipids were needed for ISCs targeting as well as the cells around Lgr5-EGFP+ cells (Figure 4).16



Figure 4. LLNs stimulated the production of ISCs, and accelerated the proliferation of Lgr5-EGFP+ when assembled with GEN lipids. a) Analysis of LLNs built with GENS lipids. b) LLNs in mouse intestine. c) LLNs built with lipids from GEN enhance the proliferation of intestine stem cells. d) Various gene expressions induced by LLNs built from GEN’s lipids. e) C57BL/6 intestinal tissues cultured for 6, 12 and 18 h. Reproduced from Ref 16 (An open access article). Edible NPs control the homeostasis of intestinal immune by steering dendritic cells (DCs) as was shown in a study using three mouse colitis models. Orally administered NPs obtained from broccoli extracts can be applied for protecting mice from colitis. BDN-facilitated stimulation of adenosine monophosphate-activated protein kinase (AMPK) in DCs played an important role in induction of tolerant DCs and prevention of DC activation. Transporting DCs pre-pulsed with 18 ACS Paragon Plus Environment

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complete BDN lipids, but not those that are depleted in sulforaphane, averted DSS-induced colitis in C57BL/6 (B6) mice, reinforcing the function of BDN sulforaphane in stimulation of DC tolerance.34 Mice nursed with only DSS showed slower weight loss, but BDN therapy inhibited DSS-induced weight loss. Thus, the administration of DSS extremely accelerated the expression of tumor necrosis factor alpha (TNF-α), interleukin (IL)-17A, and interferon gamma (IFN-γ), in the tissues of colon. On the other hand, treatment with BDN resulted in decrease of DSS-induced IFN-γ, IL-17A, and TNF-α, and but increment for of IL-10 (Figure 5).



Figure 5. BDN administration protected mice from intestinal inflammation (especially, induced colitis). A-D) BDN dispensation afforded protection to mice from DSS-promoted colitis. E) Weight to length ration of colon. F) Thickness of colon. G) Histologic profile on colon. H) FACS plots of staining. Reproduced from Ref 34 (An open access article).


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PDENs impacted the intestinal functions, and the influences of NPs from edible fruits on intestinal carriers have been examined56 using well-characterized apple derived NPs (APNPs) obtained by ultracentrifugation. Fluorescently labeled APNPs were internalized by human epithelial colorectal adenocarcinoma (Caco-2) cells, indicating that fruit-derived NPs could be adopted in vivo into intestinal epithelial cells. It was demonstrated that the level of mRNA expression of diverse carriers, such as organic-anion-carrying polypeptide (OATP) 2B1, were altered in APNP-treated Caco-2 cells; activities of OATP2B1and the expression of proteins were reduced by APNP contact. Intact APNPs were required for these procedures, for the simple reason that boiling or sonication rescinded the consequences. The detected reduction of OATP2B1 expression appeared to be facilitated by big molecules in the APNPs because the content of applederived tiny molecules in APNPs was insignificant. Further, it was revealed that the 3'-untranslated region of the OATP2B1 gene was needed for APNPs response, indicating that APNPs-derived miRNA may be implicated. These findings indicated that miRNA present in food could influence the intestinal carriers via NPs derived from foods, and these NPs can be valuable for transferring the large biomolecules to tissues in the intestine.56 Ulcerative colitis and Crohn's disease (as IBD) are long-lasting, returning inflammatory ailments of GI tract. Thus, influencing the intestinal macrophages role is deemed as a critical approach for treating IBD diseases.57 In one study, the selective uptake of grapefruit-derived nanovesicles by intestinal macrophages improved mouse colitis that was induced by DSS;17 their anti-inflammatory influences were produced by up-regulating heme oxygenase-1 (HO-1) expression and constraining TNF-α and IL-1b production in intestinal macrophages. It has been demonstrated that these biodegradable and biocompatible nanovesicles are unchanged across a wide ranging pH, thus suggesting their potential application in oral drug transportation systems. 21 ACS Paragon Plus Environment


Therefore, methotrexate (MTX), an anti-inflammatory drug, was encapsulated into grapefruitoriginated nanovesicles wherein it showed less toxicity than free drug, MTX and exhibited remarkably higher curative effects for colitis in mice induced by DSS. It has been suggested that grapefruit-derived nanovesicles could be applied as intestinal immune modulators that elevate homeostasis of intestinal macrophages. These NPs have good prospects for being used as oral transportation systems for small drug biomolecule for reducing the inflammatory reactions in humans. PDENs from grapefruit, carrots, grape, and ginger have been obtained and characterized.39 Interestingly, these NPs were found to be comparable to exosomes originating from mammals with respect to their constructions and sizes. Findings gained from PDEN-transfected macrophages demonstrated that ginger PDENs specially stimulated the expression levels of the antiinflammatory cytokine, IL-10 and antioxidant, HO-1. It was also reported that PDENs from ginger, grapefruit, and carrot could stimulate nuclear translocation of nuclear factor-like 2 (nrf2);39 PDENs appear to have dominating influences on tempering Wnt/TCF/LEF action and have critical roles in gut homeostasis.58 The interspecies communication facilitated by PDENs might participate in the stimulations of antioxidants, anti-inflammatory cytokines, and Wnt signaling, that are all very important for keeping intestinal homeostasis. In another study, Teng et al.,59 reported that miR-18a coated by grapefruit-derived nanovector interceded the hindrance of liver metastasis that was dependent upon the stimulation of M1 (F4/80+IFNγ+IL-12+) macrophages. Exosome-alike NPs derived from plants might influence cancer development, and prohibit the cell proliferation in different cell lines bearing tumor.60 For instance, Raimondo et al.,54 reported vesicles fractions from Citrus limon L., attained by using various ultracentrifugation, where density ranged from 1,15 to 1,19 g/ml and was analyzed by a specific proteomic profile. It 22 ACS Paragon Plus Environment

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has been shown that the obtained nanovesicles prohibited cancer cell development in various tumor cell lines, by triggering a TRAIL- intervened in vitro apoptotic cell demise. Moreover, lemon nanovesicles suppressed chronic myeloid leukemia (CML) in vivo tumor progression by precisely targeting tumor and by triggering TRAIL-mediated apoptotic cell procedures. Consequently, it has been suggested that edible nanovesicles derived from plants could be applied for treating cancers.54 Researchers have successfully obtained particular nanoparticle fractions from edible ginger, natural-based entities which include fats, to prepare ginger-derived nano-lipids, called nanovectors. These FA nanovectors were examined as a transportation system for a chemotherapeutic drug, Dox applied for treating colon cancers; Dox was adeptly loaded onto the FA nanovectors, ably taken up by colon cancer cells, and while demonstrating outstanding biocompatibility, successfully prohibited the tumor development. Relative to accessible selection for transferring Dox, the FA nanovectors discharged the drug more expeditiously in acidic pH background which bears a resemblance to the tumor setting. This indicates that this transportation approach could reduce the extreme drug side effects of drug like Dox. It has been highlighted that FA nanovectors comprising lipids from edible ginger could change the existing architype of drug transportation away from synthetically prepared NPs to the application of naturally occurring nanovectors from edible plants.52 Moreover, researchers have reported that they were safe and could be prepared on a large scale.52 Furthermore, Zhang et al.,52 reported a nanovector obtained from lipids from ginger which could be used as a delivery system for Dox to heal the colon cancer. Consequently, apoptosis and viability assays and technology that senses electric cell-substrate impedance demonstrated that ginger-derived nanovectors had outstanding biocompatibility unlike cationic liposomes at the same concentrations. These nanovectors had potential of loading Dox with high effectiveness and revealed a better pH-dependent profile for drug-discharge than 23 ACS Paragon Plus Environment


accessible liposomal-Dox. Altered GDNVs upon conjugation with the steering ligand FA, aimed Dox delivery to in vivo colon-26 tumors and improved the chemotherapeutic hindrance of tumor proliferation contrasted with unmodified drug by itself.52 In another study, Zhang et al.,53 introduced an innovative siRNA delivery system circumventing the limitations of unnatural NPs, including non-specificity, potential side effects, and economic preparation for ulcerative colitis therapy. The NPs comprising lipids from edible ginger, labeled ginger-derived lipid vehicles (GDLVs) were produced from ginger lipids through hydration of a lipid film, a routinely applied approach for a liposome assembly. It appears that these GDLVs could be applied as valuable siRNA-transportation systems whilst possibly avoiding issues pertaining to the traditional produced NPs. Exclusive numbers of NPs have been isolated and characterized from edible ginger via ultrasonic dispersion and super high-speed centrifugation; DLS showed that ginger-derived NPs to be about 230 nm in size with a negative potential. These NPs contain a few membrane proteins, precise natural membrane lipids, and have miRNAs with varying amounts of 6-gingerol and 6shogaol. Lipids of ginger-derived NPs largely comprised of phosphatidic acid, digalactosyldiacyl glycerol and monogalactosyldiacyl glycerol although other lipids, namely phosphatidylinositol, phosphatidylcholine, phosphatidylserine, glycerolphosphatidylethanolamine, and phosphatidyl glycerol could be detected in ginger-derived NPs. The protein content of ginger-derived NPs mainly of cytosolic origin, including actin and proteolytic enzymes; few membrane channels/transporters proteins are also found. It was shown that ginger-derived NPs contained plentiful exclusive miRNAs (length of 15-27 nucleotides); miRNAs typically prompt gene silencing via fastening to positions in the 3′UTR of a aimed mRNA, thus subduing the synthesis of proteins and starting the degradation of mRNAs.61 Ginger-derived NPs have been transferred 24 ACS Paragon Plus Environment

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orally and examined in non-starved mice; when they reached the colon, they were equally taken up by IEC and mice macrophages with or without colitis.16,17,62 Consequently, administering the ginger-derived NPs orally to mice decreased severe and long-lasting inflammation, stimulated intestinal mucosa healing, and reduced colitis-associated cancer demonstrating that ginger-derived NPs could inhibit the development of tumors and chronic colitis. In addition, these NPs decreased the levels of cytokines involved in pro-inflammation (TNF-α, IL-6 and IL-1β) and accelerated the levels of anti-inflammatory cytokines (IL-10 and IL-22) in colitic mice, indicating that the gingerderived NPs kindle the intestine healing factors and impede intestine-damaging parameters.62 Controlling the orientation of RNA to adorn exosome of human origin with cell steering ligands for precise siRNA transportation to tumors has been reported.63 Moreover, the use of arrow tail RNA NPs for presenting ligands on ginger-derived exosome-like nanovesicles (GDENs) for tumor growth curtailment and conveyance of siRNA through intravenous dispensation, was demonstrated. Folic acid was adorned on the surface of GDENs for aimed delivery of surviving siRNA to KB cancer models; gene knockdown efficiency in vitro by FA-3WJ/GDENs/siRNA complex was found to be analogous to transfection.63 In another study, Zhang et al.,43 evaluated a precise numbers of GDNPs 2 and reported their premise for valuable targeting of colon after oral dispensation; NPs with negative zeta potential being of about 230 nm size . NPs consisted of sizeable amounts of ginger bio-constituents, namely 6-gingerol and 6-shogaol, excessive levels of lipids, a few proteins, and about 125 miRNAs. It was revealed that GDNPs 2 were chiefly taken up by macrophages and IEC, and were safe and harmless while they avoided chronic colitis and colitis-associated cancers, decreased acute colitis, and improved intestinal repair. Orally administered GDNPs 2 accelerated the IEC subsistence and growth, enhanced the antiinflammatory cytokines (IL-10 and IL-22) in colitis models and decreased the pro-inflammatory 25 ACS Paragon Plus Environment


cytokines (TNF-α, IL-6 and IL-1β), indicating that GDNPs 2 are able to endorse the healing effects and weaken the damaging parameters.43 Biomedical applications of plant-derived miRNA miRNAs are a group of molecules that are single-stranded and small (18-24 nt), but function as effective regulators of gene expression and have numerous vital tasks in organisms. These molecules are important in biomedicine and gotten the attention of scientific researchers from the pharmaceutical industry, because of their vital roles in numerous common pathological and pathogenic states of human; non-coding particles intercede post transcriptional gene expression via decrepitude by exonuclease action or, decapping, or deadenylation of the poly(A) tail or target mRNA translation hindrance.64-67 These particles regulate significant bio–processes, such as apoptosis, metabolism, developmental timing, hormone signaling, immune responses, cell differentiation, proliferation, among others.21, 68 On the other hand, membranous nanovesicles, exosomes, are small and have sizes about 30-100 nm which secreted by various mammalian cells;69,70 miRNA rich microvesicles and mRNA can be moved into remote or adjoining cells and have critical role in intercellular communication.71,72 Recently, plant-derived exosome-like NPs have been analyzed and reported to have structural similarities to those of mammalian exosomes.39,73 Furthermore, as mentioned in previous sections, it has been established that EPDENs were taken up in the GI tract of mammals and had the ability to intercede animal-plant intercellular messages.54,74 Recently, it has been reported that species to species transfer of miRNAs may occur and thus control the expression of genes in the receiver cells. The interesting findings showed that edible plant-derived stable miRNAs might transfer to the circulatory system of mammals’ and


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subsequent to homing on the target, hinders their precise production of proteins. This interesting hypothesis, according to some scientific population, might offer a basis for innovative therapeutic interventions while another segment are swayed by the “false positive” effect of reported investigations from which the findings were gathered.68 It has been shown that plant miRNA MIR168a from Oryza sativa was stable in human serum75 and it targeted the low-density lipoprotein receptor adaptor protein 1 (LDLRAP1) comprising mRNA and could decrease the level of LDLRAP1 protein in the liver and blood of rice-fed mice, that finally culminates in the enhancement of low-density lipoproteins (LDL) in their plasma.75 In another study, Wang et al.76 identified a large number of small RNAs coming from assorted species, which includes human plasma and dietary plants; the most plentiful miRNAs being originated from Zea mays and O. sativa. Consequently, RNAse A activities may be thwarted by small RNAs noticed in human plasma. It was reported that the small RNA molecules might be protected by particular lipids, proteins, and other particles thus preventing their degradation.76 Furthermore, by deploying northern blot methods and quantitative reverse transcriptase PCR (qRT-PCR) techniques, researchers have identified high basal levels of ten designated plant miRNAs in human plasma.77,78 It has been shown that miRNAs of plant origin were seized by the digestive tract of mammals and could target the mammalian genes. It appears that engineered edible plants can be used for producing mammalian tumor suppressor miRNAs as efficient, safe, harmless, and low-cost chemo-preventive approach in humans. Plants bioengineering for generation of miRNAs of any preferred sequences is an attractive technology, and applications of edible plants to prepare therapeutic miRNAs is extremely viable and has substantial prospects in clinical and basic uses to offer an economical way to presently accessible transportation approaches and synthetic RNA



preparations.78 A comprehensive review by Xie et al.79 discussed the miRNAs as bioactive components in medicinal plants. The plant-based miRNAs and their beneficial abilities were illustrated in colon cancer model in mice where the orally administered cocktail of three tumor suppressing, plant-mimicking, miRNAs, with methyl functionality at 20th position of the 30 nucleotide, decreased the colon tumor affliction.78 Moreover, it was revealed that woman’s sera contained plant miR159 and its level was contrariwise associated with the progression of breast cancers and morbidity.80 The majority of detected miR159 was plentiful in extracellular vesicles. It has been revealed in vitro trials that synthetic miR159 had the potential to reduce the growth of breast cancer cells by aiming the sequence of 30 untranslated region of transcription factor 7 mRNA. The miR159 mimic, on oral dispensation, significantly restrained the development of mice xerograph breast tumors.80 Different underlying mechanisms for exogenous miRNAs permeability in intestine are as follows:79 1. The epithelium of intestine, up on retention of RNA-encompassing complexes, convert them to engulfed exosomes. Some fused endosomes age into lysosomes and get degraded though some of them might amalgamate with endosomes and experience transcytosis which delivers macromolecules to the other side of a bio-barrier.81,82 2. The tissue of gut-associated lymphoid may be recipient of RNA carriers which are conveyed via M cells in intestinal Peyerʼs patches to macrophages; tissue immune cells have an important role in the circulation of RNA-containing complexes through the body.83,84 3. The exertion of bio-functions may follow as a consequence of assimilation of plant miRNAs packaged within exosome-like NPs which are taken up by macrophages or ISCs.16 28 ACS Paragon Plus Environment

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4.

These unprotected miRNAs from plants, on account of poor stability may endure only for a limited time, but could conceivably be taken up into the cytoplasm via transmembrane miRNA carriers or receptor-facilitated endocytosis.81-84

5. Emerging from the GI tract diseases, traditional medications, malnutrition, and stressensued modified intestinal permeability might accelerate the foreign miRNAs uptake.84 The dietary mediations can change the gut microbiota for treating and preventing some severe diseases. It appears that the integrity of the intestinal epithelial barrier is protected by intestinal luminal miRNAs. The exosome-like NPs from plants have been shown to be taken up by the gut microbiota and the RNAs contained therein modify the composition of microbiome and physiology of the host. Ginger exosome-like NPs (GELNs) were favorably taken up by Lactobacillaceae in a GELNs lipid-reliant manner and include miRNAs which aim various genes in Lactobacillus rhamnosus. It has been reported that some functions of GELNs-RNAs, namely induced generation of IL-22 associated with barrier function enhancement, could improve mouse colitis through IL-22-reliant mechanisms.85 In another study, fecal miRNA-facilitated gene regulation of inter-species facilitated the host regulation of the gut microbiota.86 These miRNAs were plentiful in human fecal models and mouse and exist inside extracellular vesicles. To establish whether the pathology, related to body weight (BW) loss or shortening of colon, was instigated by the shortfall of discharged fecal miRNAs or by inherent cellular failure because of the shortage of epithelial miRNA, Fecal RNA from Dicer1ΔIEC mice or WT was administered to Dicer1ΔIEC receiver mice by gavage for seven days before DSS therapy. Consequently, longer colon length, lesser BW loss, and not as much of harsh colon damages were instigated after transmitting WT fecal RNA to Dicer1ΔIEC mice (Figure 6).86



Figure 6. The association of deficiency of IEC miRNA with exacerbated DSS colitis and its rescue after WT fecal RNA replacement. A) Change of BW (percent). B0 Length of colon. C) Examination by histology after DSS dispensation. D) Representation of fecal RNA move and initiation of colitis. E) Length of colon. F and G) Analysis after 9 days DSS dispensation. Reproduced from Ref 86 (An open access article). In fact, various intracellular transporters of endogenously initiating miRNAs have been detected, embracing microvesicle compartments, that are membrane-originated vesicles discharged from different cell types.72,87 In view of their sizes, source, and mechanisms of preparation, they can be separated as endosomal membrane-derived exosomes, shedding vesicles 30 ACS Paragon Plus Environment

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(bud right from the surface of cell), and apoptotic forms discharged in reaction to apoptotic stimuli).88-90 Notably, these vesicles apparently defend the degradation of miRNAs by RNAses.72,87 Several causal mechanisms pertaining to the strength of the plant-derived miRNAs are as follows:79 1. The stability of the plant-derived miRNAs is augmented by 2′-O-methylation by preventing exonucleolytic digestion and uridylation. 2. RNA-binding proteins determine the stability of moving miRNAs such as nucleophosmin 1 and Argonaute proteins. 3. Indeed, the degradation of miRNAs can be prevented by high-density lipoproteins, exosomes and microvesicles as miRNA carriers. 4. The exclusive sequences and GC contents of plant-derived miRNAs affects their stabilities. 5. Several secondary metabolites and plant constituents comprising polyphenols have potent ribonuclease inhibitory effects, which might safeguard plant miRNAs from the enzymatic milieu of the digestive tract. 6. Plant-based lipids, polysaccharides, and proteins, may shield the degradation of miRNAs or miRNA carriers during processing and production. Exosome-like NPs were detected and illustrated to carry lipids, proteins, and miRNAs in several plants.16,17,68 It was reported that EPDENs might facilitate communication among species and trigger human genes expression.39 These NPs, with have anti-inflammatory effects, comprise unique natural ultrastructures from plants that are comparable to exosomes physically. Exosome-like NPs from four edible plants were obtained and analyzed, and eventually shown to containing lipids, proteins, and miRNAs. Findings from EPDENs31 ACS Paragon Plus Environment


transfected macrophages demonstrated that GELNs specially induce the antioxidation gene expression, the anti-inflammatory cytokine, IL-10 and heme oxygenase-1. Ginger, grapefruit, and carrot exosome-like NPs, on the other hand, endorsed nuclear factor like (erythroidderived 2) activation. Moreover, evaluation of the intestines of canonical Wnt-reporter mice, demonstrated that the numbers of β-galactosidase(+) intestinal crypts were augmented, indicating that EPDENs treatment of mice guides Wnt-mediated activation of the TCF4 transcription machinery in the crypts.39 In another study, nanovesicles emanating from eleven edible plants were sequestered and subjected the ensuing EPDENs small RNA libraries to Illumina for sequencing.30 418 miRNAs (32 to 127 per species) in totality were detected and characterized from the eleven samples. Functional evaluations and target estimate demonstrated that vastly expressed miRNAs were closely related to cancer-related conduits and inflammatory responses. Accordingly, it was indicated that miRNAs in EPDENs had the ability to adjust human mRNA.30 In addition, it has been described that plant exosome treating approaches can be applied as nano-toxic, safer, economical substitute for clinical wound restorative. The injury healing activity of Triticum aestivum exosomes was demonstrated using in vitro approaches wherein enhanced strengths up to 200 μg/mL of T. aestivum exosome yielded surprising proliferative and migratory properties on epithelial, endothelial, and dermal fibroblast cells. Moreover, though plant exosomes have augmented the formation of tube-like structure of the endothelial cells, Annexin V staining of apoptotic cells caused by a decrease in the apoptotic cell number with no distribution to the cell cycle evaluation.91


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Current status and future prospects There are several strategies, such as non-viral and viral transportation systems applied experimentally to establish of the utmost appropriate vector for treating diseases like various kinds of cancers. In addition to their advantages, they have some lingering challenging issues including tissue-specific aiming, possible toxicity, upscaling cost-effective preparation and harmful environmental effects.92,93 PDENs are non-toxic and safe, and can be controlled/altered for redirected steering, amenable for production in cost-effective manner and has the potentials for biomolecules targeting delivery. These NPs provide several distinctive characteristics as they are obtained from edible plants, and the human immune systems had adapted to stimuli from these edible plants. They have demonstrated significant safety and nontoxicity and have been applied for treating IBD and cancers.92, 93 The structures of the PDEN lipid bilayer are different from those of synthetic liposomes and exosomes from mammals. It appears that elevated contents of phospholipids and glycolipids and the dearth of cholesterol renders PDENs as suitable medicament transportation systems for pointing to the ailing tissue. These NPs include bioactive secondary metabolites and plentiful plant miRNAs, and therefore, they provide a unique library for discovery of pharmaceutics. The metabolic destiny of PDENs seemed to be harmonious with liposomes, as both are dispelled via the kidney and liver. Nevertheless, using some novel surface alterations and reconstitution, PDENs may be directed to selectively amass at difficult-to-get tumors. For instance, intranasal administrated FA-embedded grapefruit NPs transporting the drug, Cucurbitacin I, has been shown. These NPs were effectively distributed to the brain and lung of mice. After 72 hours, they remained stable without changes in the brain, but not in the lung, thus demonstrating their exclusive brain tumor-targeting ability.50 Furthermore, the intravenous injection of plasma membrane-covered grapefruit NPs has been reported to xenograft colon cancer and breast cancer 33 ACS Paragon Plus Environment


model mice. It was found that an activated leukocyte transferring conduit permitted these membrane-layered NPs to rapidly leave the peripheral rotation and accrue at the colon and breast cancer swollen locations for more than 24 hours.49 In nanomedicine, researchers have used the addition of chitosan or PEGylation as chemical modification to enhance the bioavailability of NPs to liposomes or aimed tissue and decrease their bio-distribution to non-target tissues. These alterations significantly upsurge the associated immunogenicity, implying that an immune response is stimulated and the particles clearance is accelerated upon subsequent inoculation. Thus, PDENs as natural NPs with tissue-specific aiming assets and non-toxic character and exosomes from mammals can be applied as alternatives for targeted drug delivery, as they may have immense abilities to be applied clinically.5,94 Plant derived exosome-like NPs, as promising substitution, show biocompatibility via oral and intranasal dispensation. Moreover, systemic transportation of siRNA using exosome-like NPs directly obtained from plants was demonstrated.5, 63 The applications of phytomedicine are being received well due to their high therapeutic values and compared to allopathic medicines these bio-compounds shows fewer side effects.95-100 A better understanding of phytopharmaceutics functions and kinetics should help in designing novel drug molecules and effective treatments. The utilization of plant-based NPs and other nanoparticle-embedded by-products is rather important as it brings forth a crucial symbiotic association between plant science and nanotechnology; the correlation does provide inherently greener approach towards nanotechnology that may well be termed, green nanotechnology. Although it has many applications, but there are only handful of studies in phyto-nanotechnology; this may be because of the complexity of plant systems and other limitations. Phytonanotechnology has tremendous potential in the synthesis of assorted NPs employing the extracts 34 ACS Paragon Plus Environment

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from different portions of plants such as leaves, seeds, flowers and roots. Plant-based nanomaterials have notable applications in pharmaceutics and medicine, mainly in novel drugs discovery, imaging, targeted delivery of pharmaceutics, diagnosis methods and in making effective nanodevices; these efforts may prove valuable for developing new advanced therapies to control various epidemic diseases.95-103 Conclusion and Perspective In conclusion, PDENs have shown some remarkable properties in the field of nanomedicine and they may be good candidates for some diseases like cancers. Chemotherapy without proper targeting is common therapeutic approach practiced today for patients with cancers, a strategy which has barely any ability to differentiate between normal cells and cancerous ones, culminating in inadequate therapeutic influence on tumor cells and highly toxic side effects on normal cells. Empowering chemotherapeutic pharmaceuticals to aim for cancerous cells can be an important improvement for treating cancers. Application of nanoscience for targeted transportation of pharmaceuticals has provided enormous opportunities to advance the cancer and tumor treatment processes. But, harmful environmental impact, concerns with economical and largescale preparation, and associated extreme costs are important barriers which need to be addressed for further clinical applications. PDENs, separated from edible plant cells, have no cytotoxicity and are free from immunotoxicity. As an example mentioned before, ginger-derived NPs are capable of targeting the colon, diminishing colorectal tumorigenesis by decreasing proinflammatory cytokine levels, inhibiting the proliferation of intestinal epithelial cells, and stimulating its apoptosis. The surface alterations of PDENs or the plant-derived nanolipids combinations with other nano-sized bioactive substances could possibly be applied for fine tuning the PDENs to aim at various cancer sites without causing unfavorable immunotoxicity. It appears 35 ACS Paragon Plus Environment


that PDENs can be applied as the next-generation transportation systems for well-aimed therapeutic management of malignant diseases, and delivery of biologically active substances. But there are some limitations and challenges that need to be resolved or addressed for industrialization or upscale production. For instance, any possible risks and adverse effects should be analyzed and evaluated, carefully. This exploratory course will accelerate the process of finding innovative strategies and methods for treating or preventing different malignant and harmful human diseases. Further, for applying as dietary supplements, dehydrated plant-derived materials may be used for the production of tablets and powders, and thus, determination of where and how these plant-derived bioactive materials are preserved (such as during desiccation process) is one of the main concerns. Additionally, preservation of the beneficial functions during extraction and purification procedures is crucial. Importantly, for applying PDENs for further biomedical and clinical applications, some extra processing may be necessary. For instance, lipids obtained from plant-derived extracellular vesicles should be assembled with liposome-like NPs to be taken up by intestinal cells; cost of the production in view of excessive time and effort may be helped by newer technological developments. The focus of researches has shifted to miRNA molecules in view of their participation in gene expression regulations. It appears that the miRNA passageway from edible plants to recipient cells can be attained by eating plant materials and mechanistically crushing them by oral activity and partly digesting by different enzymes in stomach or mouth. Throughout these procedures, plant-derived miRNAs can be discharged from smashed cells and moved to the small intestine. By incorporating them into specific vesicles or proteins, they can be discharged and transferred to the desired cells through the circulatory systems. It should be noticed that further investigations should


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be conducted for better assessment of the plant-derived miRNAs; medicinal value need to be established unequivocally. AUTHOR INFORMATION Corresponding Authors: E-mail: [email protected] (Rajender Varma) ORCID: 0000-0001-9731-6228 E-mail: [email protected] (Siavash Iravani) ORCID: 0000-0003-3985-7928

Conflict of Interest: Author declares no conflict of interest.

List of abbreviations: 1.

3D

Three dimensional

2.

AFM

Atomic force microscopy

3.

AMPK

Adenosine monophosphate-activated protein kinase

4.

APNPs

Apple-derived NPs

5.

BDNs

Broccoli-derived NPs

6.

BW

Body weight

7.

CML

Chronic myeloid leukemia

8.

DCs

Dendritic cells

9.

DLS

Dynamic light scattering

10.

Dox

Doxorubicin

11.

DSS

Dextran sulfate sodium 37 ACS Paragon Plus Environment


12.

EPDENs

Edible plant-derived exosome-like NPs

13.

FA

Folic acid

14.

FA-GNVs

Grapefruit-derived nanovectors coated with folic acid

15.

FSC

Forward scatter

16.

GDENs

Ginger-derived exosome-like nanovesicles

17.

GDLVs

Ginger-derived lipid vehicles

18.

GDNs

Grapefruit-derived exosome-like NPs

19.

GDNPs

NPs derived from edible ginger

20.

GDNVs

Grapefruit-derived lipid NPs

21.

GELNs

Ginger exosome-like NPs

22.

GENs

Grape exosome-like NPs

23.

GI

Gastrointestinal

24.

GM-CSF

Granulocyte-macrophage colony-stimulating factor

25.

HO-1

Heme oxygenase-1

26.

HPLC

High-performance liquid chromatography

27.

IBD

Inflammatory bowel disease

28.

IEC

Intestinal epithelial cells

29.

IFN-γ

Interferon gamma

30.

IL

Interleukin

31.

LDL

Low-density lipoproteins

32.

LDLRAP1

Low-density lipoprotein receptor adaptor protein 1

33.

LLNs

Liposome-like NPs

34.

LPC

Lyso-phosphatidylcholines

35.

LPE

Lyso-phosphatidylethanolamines

36.

LPG

Lyso-phosphatidylglycerol 38 ACS Paragon Plus Environment

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37.

LPLs

Lamina propria lymphocytes

38.

ISCs

Intestinal stem cells

39.

MG/DG

Mono/di/glycerols

40.

miRNAs

MicroRNAs

41.

MLN

Mesenteric lymph node

42.

MS

Mass spectrometry

43.

MTX

Methotrexate

44.

NPs

Nanoparticles

45.

nrf2

Nuclear factor-like 2

46.

OATP

Organic-anion-transporting polypeptide

47.

PA

Phosphatidic acids

48.

PBS

Phosphate-buffered saline

49.

PC

Phosphatidylcholines

50.

PCR

Polymerase chain reaction

51.

PCS

Photon correlation spectroscopy

52.

PDENs

Plant-derived edible nanoparticles

53.

PDLNs

Plant-derived edible lipid nanoparticles

54.

PE

Phosphatidylethanolamines

55.

PEI

Polyethylenimine

56.

PG

Phosphatidylglycerol

57.

PI

Phosphatidylinositol

58.

PS

Phosphatidylserine

59.

PTX

Paclitaxel

60.

qRT-PCR

Quantitative reverse transcriptase PCR

61.

SEM

Scanning electron microscopy 39 ACS Paragon Plus Environment


62.

SPM

Scanning probe microscopy

63.

TEM

Transmission electron microscopy

64.

TNF-α

Tumor necrosis factor alpha

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PHOTOGRAPHS AND BIOGRAPHIES

Siavash Iravani Dr. Iravani has worked on several academic research projects at the Isfahan University of Medical Sciences (Faculty of Pharmacy and Pharmaceutical Sciences), including green and ecofriendly synthesis of nanomaterials, plant-derived nanostructures, phytochemical analysis, 53 ACS Paragon Plus Environment


graphene-based nanocomposites, water treatment technologies, nanoparticles for drug delivery in cancer, nanocarriers, and drug nanoparticles. His previous experience, of more than seven years, centers on drug development and industrial pharmacy in various capacities including research and development, formulation, and quality control. Dr. Iravani has authored over 50 peer-reviewed scientific publications including twelve book chapters and one book.

Rajender S. Varma Prof. Varma (Highly Cited Researchers 2016-18; Publons Awardee) was born in India (Ph.D., Delhi University 1976). After postdoctoral research at Robert Robinson Laboratories, Liverpool, U.K., he was faculty member at Baylor College of Medicine and Sam Houston State University prior to joining the Sustainable Technology Division at the US Environmental Protection Agency in 1999. He has a visiting scientist’s position at Regional Centre of Advanced Technologies and Materials, Palacky University at Olomouc, Czech Republic. He has over 45 years of research experience in management of multidisciplinary technical programs ranging from natural products chemistry to development of more environmentally friendly synthetic methods using microwaves, ultrasound, etc. Lately, he is focused on greener approaches to assembly of nanomaterials and sustainable applications of magnetically retrievable nanocatalysts in benign media. He is a member of the editorial advisory board of several international journals, has published over 515 papers, and has been awarded 16 US Patents, 6 books, 26 book chapters and 3 encyclopedia contributions with 36,000 citations. 54 ACS Paragon Plus Environment

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Graphical Abstract: PDENs, with safer, sustainable and biodegradable attributes, are featured as tissue-specific therapeutic delivery systems.


Plant-derived Edible Nanoparticles and miRNAs: Emerging Frontier

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