The researchers found a less toxic way to administer drugs using natural lipids in plants

University of Louisville researchers have found a less toxic way to deliver medicines by using the natural lipids in plants, particularly grapefruit and ginger.

The resulting intellectual property portfolio consisting of 12 patent families, invented by Huang-Ge Zhang, Ph.D., of UofL’s James Graham Brown Cancer Center and Department of Microbiology and Immunology, has been licensed to Boston-based Senda BioSciences, a Flagship Pioneering company. UofL’s technology is part of Senda’s efforts to develop novel drug delivery platforms to solve the challenges of transferring therapeutics across biological barriers and throughout the body.

The UofL technologies use exosomes, which are very small fragments of living, edible plant cells, to transport various therapeutic agents, including anti-cancer drugs, DNA/RNA and proteins such as antibodies.

These exosomes help ensure the drug is properly absorbed by the body.

Current practice is to use nanoparticles or liposomes made from synthetic materials to deliver these medicines.

However, these materials are more expensive to produce in large quantities and can cause adverse health effects, such as cell toxicity and chronic inflammation.

The UofL edible-plant-derived exosomes don’t have these problems, Zhang said, since they come from natural, readily available sources. More importantly, these exosomes have anti-inflammatory effects.

“Our exosomes come from fruit or other edible plants – something good for you, that you buy in the grocery store and that humans have eaten forever,” said Zhang, an endowed professor of microbiology and immunology who holds the Founders Chair in Cancer Research. “And, they don’t require synthetic formulation.”

The exosomes made from fruit lipids also can be modified to target and deliver medications to specific cell types within the body – like homing missiles, Zhang said.

For example, the exosomes could be engineered to deliver a cancer therapeutic directly to cancer cells.

Zhang originally experimented with other fruits, including tomatoes and grapes. His epiphany came while eating a grapefruit – he realized his breakfast was chock-full of natural lipids that could be harvested to make exosomes at a larger scale.

The results of that work later were published in multiple scientific journals, including Nature Communications, and Cell Host & Microbe, and now are exclusively licensed to Sen-da Biosciences.

“These technologies could make a real difference in drug delivery, improving access and costs while reducing side effects, ” said Guillame Pfefer, CEO of Senda Biosciences. “We look forward to working with UofL to further develop these innovations and get them to market.”

Senda Biosciences holds an exclusive license to several UofL fruit-based drug delivery technologies, including technologies focused on the regulation of gut microbiota, through the UofL Commercialization EPI-Center, which works with industry and startups to commercialize university technologies. The EPI-Center team worked closely with Zhang and Senda to develop and grow the partnership.

“This is the kind of outcome we want for all our technologies,” said Holly Clark, Ph.D., deputy director of the Commercialization EPI-Center, who manages Zhang’s intellectual property portfolio.

“We’ve built a great working relationship between our innovator and our commercialization partner, Senda, and together, they will advance this suite of technologies for market.”

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Cancer is considered a multifactorial disease that causes millions of global deaths per year. The identification of selective treatment able to kill only tumor cells is hard to find, since cancer arises from an organism’s own healthy cells that have developed abnormal properties, resulting in uncontrolled cell growth [1].

Currently, cancer treatment strategies are based on chemotherapy using cytotoxic drugs. Systemic administration of these agents exhibited several issues, including poor specificity, low efficacy, high toxicity and induction of drug resistance. Pharmacokinetics, physiochemical properties, and poor selectivity of chemotherapeutics contribute to reduced therapeutic efficacy.

Moreover, the limited biological barrier penetration of cytotoxic agents prevents successful site-specific accumulation [2], requiring repeated administration which may lead to the acquisition of drug resistance. Furthermore, recent studies have demonstrated that the interaction between heterogeneous tumor cell populations and the microenvironment can induce therapeutic resistance [3].

Consequently, new approaches have been explored to increase the effectiveness of anti-tumor therapeutics systemically administered by enhancing therapeutic agent’s permeability and selectivity [4]. Among these, nanotechnology has been employed for drug delivery to improve therapeutic potency to cancerous cells while sparing healthy tissues.

Accordingly, recent investigations have demonstrated the use of nanoparticles (NPs) as a promising platform for the delivery of therapeutic agents. NPs provide better therapeutic efficacy through the improvement of loading and release parameters [5,6], biocompatibility and drug circulation-time [7,8,9] and, finally, the controlled and targeted delivery of drugs to target sites [10–14].

In this manner, NPs significantly decrease adverse health effects and simultaneously increase tumor selectivity [15]. Efficient delivery of NPs is also the result of the enhanced permeability and retention effect (EPR) [16]. Indeed, due to high permeability and poor lymphatic drainage, the tumor vasculature is highly abnormal thus promoting the extravasation of NPs into tumors. Unfortunately, despite promising therapeutic applications, nanotechnology has provided only modest improvements in patient survival.

NPs have showed several limitations due to their suboptimal properties, including premature drug release during NPs synthesis, storage or circulation in blood and lack of specificity for the tumor. These issues result in an inability to reach and effectively penetrate tumors [17].

Additionally, after administration, NPs can interact with the immune system (i.e. uptake by macrophages known as Mononuclear Phagocytic System (MPS)) resulting in a strong adverse response to the treatment [18,19]. Currently, only a small number of NPs such as polyethylene glycol (PEG)-conjugated liposomal doxorubicin (i.e., Doxil/Caelyx) and liposomal irinotecan (i.e., Onivyde) have seen Food and Drug Administration-approval for cancer therapy [20].

PEG-conjugation of NPs prevents MPS recognition and exploits EPR-mediated tumor accumulation [21]. Nevertheless, Gabizon A.A. et al. have demonstrated that repeated injections of PEGylated liposomes are associated with the production of anti-PEG antibodies, which extend blood clearance and reduce the efficacy of these formulations [22].

In addition, biological barriers can hinder NP penetration reducing bioavailability and limiting therapeutic efficacy [2]. In conclusion, the improvement of the therapeutic index of injectable nanocarriers is strictly connected to their ability to: 1) circulate in the bloodstream while avoiding the opsonization process; 2) escape immune surveillance; 3) preserve their cargo; 4) deliver drug into tissue desired sites; 5) overcome the biological barriers; 6) penetrate target cell membranes and 7) minimize accumulation at undesired sites.

Recently, to overcome these NP limitations, exosomes have emerged as a promising platform for cancer treatment, providing a viable alternative to NPs [23]. In this review, we describe exosomes and their application in drug delivery. We will pay special attention to the use of exosome-like NPs as drug delivery systems for anti-cancer treatment.

EXOSOMES

Exosomes are extracellular vesicles in the range of 30–150 nm, secreted by almost all cell types in both physiological and pathological conditions. Their origin begins from multi vesicular bodies (MVB) and their release occurs upon MVB fusion with plasma membrane (Figure 1) [24].

Exosomes are highly heterogeneous with diverse molecular compositions and contain a variety of cargoes. However, although their heterogeneity can often be dependent on the origin cell type, exosomes derived from the same cell type have also expressed different molecular compositions. The composition of the exosome surface is decorated by several cell-specific antigens, including fusion proteins, adhesion molecules, and integrins which address the selective targeting of those cells [25–27].

Exosomes circulate in the bio-fluids and transport messages from one cell to another; these messages include several molecules, such as lipids, proteins, DNA, mRNA, non-coding RNA and various metabolites [28] that can modify the behavior of recipient cells [29] both at short- and long-distance cell communication. The cargo internalization in recipient cells occurs mainly by endocytosis but it has also been demonstrated that exosomes are able to transfer their payload by fusion with the plasma membrane of the recipient cells. [30]. It is known that exosome fusion with recipient cells occurs preferentially in acidic conditions, explaining the reason why tumors may have a better uptake than normal cells [31].

In addition, exosomes have a wide variety of functions that certainly play a key role in both physiological and pathological processes. Tumor-derived (TD) exosomes are able to modulate tumor microenvironment components and affect immune system functions. TD exosomes have an immunosuppressive behavior, helping tumor cells to avoid immune system clearance [32]. Moreover, they play a key role in the cross-talk between tumor cells and the microenvironment, converting it into a tumor prone environment. Conversely, the stroma itself can release exosomes supporting tumor growth [33]. Another important function of exosomes is the contribution in cell motility and dissemination. In fact, they can modulate the extracellular matrix, drive hematopoietic cells towards an inflammatory phenotype and stimulate epithelial to mesenchymal transition in non-metastatic cells [34]. Exosomes are also involved in chemoresistance through various mechanisms such as: 1) actively export the drug out of the cells, reducing its concentration into the cytoplasm [35]; 2) transport drug efflux pumps; 3) modulate the sensitivity of other cells [36]; 4) deliver molecules (i.e. miRNA, pro-survival proteins, etc.) to other tumor cells or to the stroma; 5) induce drug resistance mechanisms increasing drug expulsion (Figure 2) [37].

Exosomes have high versatility in translational medicine. Indeed, they can be used for diagnosis, prognosis and treatment of cancer. TD exosomes represent non-invasive biomarker because they carry tumor biomarkers and antigens and can be isolated from different body fluids of cancer patients, such as saliva, plasma and urine [38]. Thanks to their ability to activate immune cell response and the major histocompatibility complex on their surface, exosomes are a favorable strategy for cancer vaccination, inducing a potent anti-tumor response. Moreover, immunogenicity of exosomes can be artificially increased by genetic modification or fusion with specific antigens [39].

USE OF EXOSOMES IN NANOMEDICINE

Exosomes represent an efficient drug delivery platform, due to their good biodistribution, biocompatibility and low immunogenicity. Exosomes present very good permeability and can cross most biological membranes [24]. Recent studies have reported that they can pass through the blood–brain barrier, demonstrating their potential in brain cancer treatment [40]. Moreover, a recent study showed that exosomes derived from fruit can efficiently deliver curcumin, a drug able to interfere with colon carcinogenesis. A phase I clinical trial has been undertaken to study the effects of curcumin delivered by fruit-derived exosomes fruit on treatment of colon cancer [41]. For all these reasons, exosomes are promising candidates for cancer treatment delivery. Currently, there are 33 clinical trials that involve exosomes as diagnostic/prognostic factors [clinicaltrials.gov].

Since interest in exosomes as nanocarriers has intensified, many techniques for their isolation and consequent loading have been developed [42–44]. Among the purification techniques, the most commonly employed protocols are based on: centrifugation [45,46], microfiltration [47,48], density gradient separation [45], immunoaffinity capture using antibodies specific for exosome surface proteins [45,49,50], and microfluidic [51,52]. On the other hand, the available methods to load drugs into exosomes include, but are not limited to, passive loading by electroporation [53,54], saponin membrane permeabilization [55], freeze/thaw cycles [56], sonication [57], and extrusion [58,59].

Alternatively, to keep the loading process as natural as possible, cells can be induced to incorporate the payload during exosome formation [60]. This approach has been employed to develop Mesenchymal Stromal Cell (MSC)-derived exosomes loaded with paclitaxel [60]. MSCs have been shown to possess innate tumor targeting abilities that successfully transfer to released exosome vesicles.

Furthermore, once loaded with an anti-cancer drug, MSC-derived exosomes exhibited inhibited tumor growth, thus demonstrating the potential of this platform [60]. Recently, a new endogenous method to produce labeled exosomes, has been described by Monopoli M.P. et al. This method allows the production of labeled exosomes presenting endogenous fluorescent molecules, previously internalized by the cells. This approach can be used also to produce drug-loaded exosomes [61].

Exosomes produced by many different cells have been explored for use in clinical applications [62–64]. TD exosomes have gained the interest of many groups for delivery of anticancer drugs [65] and for their use in immunotherapy [66]. The message contained in TD exosomes can be used not only to exchange information among cells, but also as a diagnostic and prognostic biomarker of cancer [67].

Taylor D.D. et al. have used the miRNA content of TD exosomes as a diagnostic biomarker of ovarian cancer [67], while Jin H. et al. identified early diagnostic biomarkers of pancreatic cancer by using differential proteomic analysis of TD exosomes [68]. A clear and complete characterization of the genomic and proteomic content of TD exosomes is of crucial importance and many efforts have been devoted toward this aim [69,70].

Moreover, exosomes can also be isolated from sources other than cells. Recently, it has been revealed that bovine milk-derived exosomes contain mRNAs and miRNAs [71–73]. Moreover, edible plant-derived exosomes are currently under investigation for their anti-inflammatory and anti-cancer properties [74,75].

EXOSOMES-LIKE NANOCARRIERS

Despite the great potential of exosomes as delivery systems and the presence of clinical trials on this platform, the lack of standardized protocols for the isolation of sufficient quantities represents a major obstacle for their implementation [76]. To overcome this obstacle, synthetic extracellular nanovesicles have been recently developed. These cell-derived nanovesicles are made up of a lipid bilayer enriched with membrane-associated proteins derived from cells of interest.

They are produced mainly by using common protocols developed for liposomes synthesis (Figure 3A) [10], thus allowing drug loading which is not readily achievable in naturally purified exosomes. Moreover, synthetic exosome-like nanocarriers offer excellent versatility for surface modifications with entire cell membrane patches or with only a few selected membrane proteins crucial for specific function and/or a targeting effect [22,77,78].

Using the thin layer evaporation method commonly used for liposome synthesis, our group developed a protocol for the fabrication of biomimetic exosome-like vesicles for targeting inflamed tissues [10]. These immune cell-derived nanovesicles, called leukosomes, demonstrated a natural targeting ability of immune cells toward inflamed tissues by preserving the topology of plasma membrane proteins.

Specifically, we demonstrated the successful implementation of critical adhesion proteins, lymphocyte function-associated antigen 1 (LFA-1) and macrophage-1 antigen (Mac-1), along with over 300 additional proteins involved in signaling, adhesion, immunity, and transport into a lipid vesicle [79]. LFA-1 and Mac-1 have been shown to be responsible for adhesion to intracellular adhesion molecule 1, a ligand for both proteins found on endothelial cells [80]. We previously demonstrated when either LFA-1 or Mac-1 was blocked on the particle surface, a significant reduction in endothelium accumulation was observed [77].

Indeed, being synthetic nanovesicles, leukosomes were easily loaded with several types of payloads with different chemical compositions, demonstrating the versatility of this platform [10]. Leukosomes can also be used as an imaging tool of inflamed vasculature. Specifically, leukosomes showcased superior accumulation in in vivo models of breast cancer tumor and vascular lesions (i.e. atherosclerotic plaque), demonstrating their potential for theranostic drug delivery [81]. The leukosome’s membrane proteins, which have been characterized by proteomic-based approaches [10,82], induce also the formation of a protein corona [83–85] that is responsible for the prolonged circulation time of these nanovesicles when compared to traditional liposomes [86]. In fact, when nanoparticles encounter biological fluids, they are rapidly encased in a layer of biomolecules, creating a crown around their surface [87]. This corona has been shown to alter the biological fate of nanoparticles, with a considerable effect observed for active targeting applications [87]. Many studies have investigated the composition, structural conformation and impact of the protein corona on cellular uptake, targeting, cytotoxicity of inorganic and lipid nanoparticles (for a complete review, please refer to references [85]).

Taken further, even less is known regarding the protein corona of extracellular vesicles (e.g., exosomes) and exosome-like nanovesicles. In theory, exosomes should not have any corona other than specific receptors for their surface antigens. Exosome-like nanovesicles may or may not have a protein corona according to the method/material employed for their synthesis. To the best of our knowledge, our work on the protein corona of exosome-like nanovesicles (i.e., leukosomes) represents the only such example in literature [88]. Our synthesis process implied the use of phospholipids commonly used for liposomes. We found many common proteins between the corona of liposomes and leukosomes. However, due to the different impact of the corona on cellular uptake, we speculated that those proteins were oriented differently between the two types of nanovesicles, thus stressing the importance of considering not only the identity of proteins, but also their orientation in the corona [88]. We envision that this interesting aspect of the protein corona of exosome-like nanovesicles, so far not explored, will become of major interest very soon.

The exosome-like NPs can be easily modified to induce the over-expression of a specific membrane protein before the membrane protein extraction. Using such approach, we developed specialized leukosomes to selectively target the inflamed tissue in inflammatory bowel disease (IBD) mouse model [88]. These exosome-like NPs, are immune cells-derived nanovesicles doped with the integrin α4β7, responsible for the T-cells homing to inflamed tissue in the gastrointestinal tract. Treatment of IBD mice with these specialized nanovesicles unloaded (i.e. with no therapeutics) resulted in a reduction of inflammation and in an enhanced intestinal repair thus showing an intrinsic anti-inflammatory action of the α4β7 leukosomes [88].

With a different synthesis approach, Jang S.C. et al. have developed cell-derived nanocarriers, coined as exosome-mimetic nanovesicles for targeted delivery of chemotherapeutics to tumors [53]. In an effort to overcome the often difficult task of obtaining sufficient exosomes from traditional cell culture methods, the authors produced exosome-like nanovesicles by consecutive extrusion passages of cells through membrane filters with diminishing size followed by density gradient ultracentrifugation (Figure 3B) [53]. These immune cell-derived nanovesicles induced in vitro death of TNF-α-stimulated endothelial cell. Moreover, when loaded with chemotherapeutics and injected, these vesicles exhibited a reduction in in vivo tumor growth, demonstrating the promise of a serial extrusion approach to mitigate the potential insufficient number of exosomes obtained from traditional methods Importantly, if the plasma membrane proteins are removed by trypsinization, the nanovesicles lose their efficacy both in vitro and in vivo, thus highlighting the crucial role of plasma proteins [53]. More recently, exosome-like nanovesicles have been obtained by microfluidic approaches which represent a novel and robust method to scale-up the synthesis process [89].

SURFACE MODIFICATIONS
Recent evidence indicated that changing the composition of the exosome surface permits modifications to interactions between exosomes and targeted cells. Membrane-engineering approaches represent a novel strategy for the development of advanced drug-carrier exosomes. Alvarez-Erviti L. et al. transfected dendritic cells, isolated from mice, in order to express the neuronal targeting ligand RVG coupled with the exosomal membrane protein Lamp2b.

The obtained exosomes demonstrated higher cargo delivery potential to the brain in a mouse model, suggesting a possible application in the treatment of glioblastoma [90]. Ohno S.I. et al. engineered human embryonic kidney cells with a pDisplay vector encoding for GE11 peptide [91]. Compared to epidermal growth factor (EGF), this ligand showed comparable binding ability to the receptor (EGFR) but present less mitogenic and neoangiogenic activity. To increase targeting efficacy and accumulation at tumor site, the authors demonstrated exosomes purified from the transfected cells, express GE11 on their membrane and present a higher ability to target EGFR-expressing breast cancer cells both in vitro and in vivo compared to naturally purified exosomes [91].

Modification of the exosome surface using tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a so-called “death receptor”, has also been shown to provide improved targeting potential in various cancers [92]. Use of this ligand has been shown to induce apoptosis in tumor cells while not effecting normal cells. However, the non-specific accumulation has led investigators to investigate the incorporation of TRAIL into exosome-like vesicles.

Evaluation of exosomes enhanced with TRAIL was found to promote apoptosis in myeloma- and melanoma-based tumors. Despite this, no significant effect on tumor volume was observed in vivo. Evaluation of the treatment of lymphoma tumor-bearing mice showed similar signs of apoptosis in vitro; however, an opposite effect was observed in vivo [93].

Next to the engineering of donor cells to produce ligand-conjugated or drug-loaded exosomes, other techniques have been developed to improve the targeting ability of exosomes by surface modification. Tian T. et al. developed an efficient method to conjugate multiple functional ligands to the surface of exosomes pre-isolated from culture medium or body fluids without affecting the exosome features or the bioactivity of the loaded cargo [40]

Through a “copper-free click chemistry” reaction the authors conjugated c(RGDyK) peptide to the surface of exosomes derived from MSC [40]. Functionalized exosomes, delivering curcumin, could target the ischemic brain region in a mouse model of cerebral artery occlusion and reduce inflammation. Since c(RGDyK) is a recognized tumor-targeting ligand, the use of these synthetic exosomes could be investigated as drug carriers in cancer treatment.

Another approach exploited the use of liposomes for the generation of synthetic exosomes. Using this technique, Sato Y.T. et al. developed engineered hybrid exosomes by fusing phospholipidic liposomes with tyrosin kinase receptor (HER2)-expressing exosomes purified from HER2 expressing cells [56]. These results strongly imply that not all the components of the exosome’s membrane are required for an efficient delivery to the target cells, suggesting synthetic mimetic exosomes as promising candidates for a tailored anti-cancer drug delivery approach.

PROS AND CONS OF USING PURIFIED VS. SYNTHETIC EXOSOMES

TD exosomes mirror most of the molecular features of the tumor cells from which they originate, making them the perfect biomarker and drug delivery tool for the diagnosis and treatment of cancer. The low immunogenicity, negative surface charge and a surface protein composition similar to cell membrane are all characteristics that help improve TD exosome cellular internalization within target tumor cells. In addition, these features minimize degradation and clearance by the immune system, contributing to reduced side effects.

As such, several attempts have been made in the use of TD exosomes as vehicles for small bioactive molecules and chemotherapeutics, i.e. doxorubicin [94] and paclitaxel [60]. Nevertheless, growing evidence has demonstrated that TD exosomes support tumor growth, progression and metastases formation. In non-small cell lung cancer [95], glioma [96] and gastric cancer cell lines [97] TD exosomes have been shown to enhance the tumor microenvironment resulting in cancer cell proliferation [98] and angiogenesis [99]. Harris D.A. et al. have demonstrated in three different breast cancer cell lines that, according to the cells of origin, exosomes promoted tumor cell invasiveness and metastatic potential through the expression of adhesion molecules and proteases such as urokinase plasminogen activator, vimentin, galectin 3-binding protein and annexin A1 [100].

In addition, it has been demonstrated that TD exosomes promote tumor escape from immune recognition. Indeed, exosomes triggered apoptosis of activated cytotoxic T cells through expression of ligands such as FASL, TRAIL and PSL2 [101], impaired dendritic cell differentiation from monocytes, and suppressed lymphoid activation signaling molecules [69]. In melanoma patients, Taylor D.D. et al. demonstrated that TD exosomes enhance the production of myeloid-derived suppressor cells, which play a key role in immune system modulation [102].

Conversely, synthetic exosomes produced by genetic modification of the exosome-producing tumor cells represent a promising tool to achieve a higher antitumor immune response. The expression induction of artificial neoantigens or neoepitopes on the surface of exosomes can evoke anti-tumor recognition by immune cells. Koyama Y. et al. previously demonstrated that early secretory antigen target-6 (ESAT-6) is a potent antigen able to induce immune response. They produced ESAT-6 carrier exosomes from genetically modified tumor cells and showed significant tumor growth reduction in mice treated with these exosomes [103]. A different approach has been exploited by Wang J. et al. [104].

These authors labeled donor cell membranes with biotin and exposed them to a potent anti-neoplastic drug. After cell functionalization with avidin, to improve the targeting efficiency, they obtained exosomes expressing both biotin and avidin on the membrane surface and encapsulated with the drug. These synthetic exosomes showed high target ability to tumor cells and receptor-mediated cellular uptake.

The production of synthetic exosomes has yielded to overcome another important aspect in the application of purified exosomes in cancer treatment. Indeed, although cells continuously produce exosomes, the recovery is often too small especially for clinical application [105]. To obtain the desired amount of material, it is necessary to begin with a large number of initial cells and effectively quantity the cell media.

Moreover, biological fluids contain a mixture of exosomes produced by all a variety of cell types. Due to this aspect and the exosome’s small size, the processes of purification and isolation are still challenging. To date several techniques have been developed to isolate and purify exosomes. However, most of them require multiple, complicated and time-consuming steps of ultracentrifugation and with a lack of specificity [40]. On the contrary, the production of synthetic exosomes can be scaled up and the assembling process is easily controllable, overcoming the difficulties of using naturally derived exosomes.

reference link :https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395517/