Oral TNF-α siRNA delivery via milk-derived exosomes for effective treatment of inflammatory bowel disease.

Oral administration facilitates the direct delivery of drugs to lesions within the small intestine and colon, making it an ideal approach for treating patients with inflammatory bowel disease. However, multiple physical barriers impede the delivery of oral RNA drugs through the gastrointestinal tract. Herein, we developed a novel oral siRNA delivery system that protects nucleic acids in extreme environments by employing exosomes derived from milk to encapsulate tumor necrosis factor-alpha (TNF-α) siRNA completely. The remarkable structural stability of milk-derived exosomes (M-Exos), as opposed to those from HEK293T cells, makes them exceptional siRNA carriers. Results demonstrate that milk exosomes loaded with TNF-α siRNA (M-Exo/siR) can effectively inhibit the expression of TNF-α-related inflammatory cytokines. Moreover, given that milk exosomes are composed of unique lipids with high bioavailability, orally administered M-Exo/siR effectively reach colonic tissues, leading to decreased TNF-α expression and successful alleviation of colitis symptoms in a dextran sulfate sodium-induced inflammatory bowel disease murine model. Hence, milk-derived exosomes carrying TNF-α siRNA can be effectively employed to treat inflammatory bowel disease. Indeed, using exosomes naturally derived from milk may shift the current paradigm of oral gene delivery, including siRNA.

Post-insertion technique to introduce targeting moieties in milk exosomes for targeted drug delivery.

BACKGROUND: Recently, increased attention has been given on exosomes as ideal nanocarriers of drugs owing to their intrinsic properties that facilitate the transport of biomolecular cargos. However, large-scale exosome production remains a major challenge in the clinical application of exosome-based drug delivery systems. Considering its biocompatibility and stability, bovine milk is a suitable natural source for large-scale and stable exosome production. Because the active-targeting ability of drug carriers is essential to maximize therapeutic efficacy and minimize side effects, precise membrane functionalization strategies are required to enable tissue-specific delivery of milk exosomes with difficulty in post-isolation modification. METHODS: In this study, the membrane functionalization of a milk exosome platform modified using a simple post-insertion method was examined comprehensively. Exosomes were engineered from bovine milk (mExo) with surface-tunable modifications for the delivery of tumor-targeting doxorubicin (Dox). The surface modification of mExo was achieved through the hydrophobic insertion of folate (FA)-conjugated lipids. RESULTS: We have confirmed the stable integration of functionalized PE-lipid chains into the mExo membrane through an optimized post-insertion technique, thereby effectively enhancing the surface functionality of mExo. Indeed, the results revealed that FA-modified mExo (mExo-FA) improved cellular uptake in cancer cells via FA receptor (FR)-mediated endocytosis. The designed mExo-FA selectively delivered Dox to FR-positive tumor cells and triggered notable tumor cell death, as confirmed by in vitro and in vivo analyses. CONCLUSIONS: This simple and easy method for post-isolation modification of the exosomal surface may be used to develop milk-exosome-based drug delivery systems.

RNAi therapies: Expanding applications for extrahepatic diseases and overcoming delivery challenges.

The era of RNA medicine has become a reality with the success of messenger RNA (mRNA) vaccines against COVID-19 and the approval of several RNA interference (RNAi) agents in recent years. Particularly, therapeutics based on RNAi offer the promise of targeting intractable and previously undruggable disease genes. Recent advances have focused in developing delivery systems to enhance the poor cellular uptake and insufficient pharmacokinetic properties of RNAi therapeutics and thereby improve its efficacy and safety. However, such approach has been mainly achieved via lipid nanoparticles (LNPs) or chemical conjugation with N-Acetylgalactosamine (GalNAc), thus current RNAi therapy has been limited to liver diseases, most likely to encounter liver-targeting limitations. Hence, there is a huge unmet medical need for intense evolution of RNAi therapeutics delivery systems to target extrahepatic tissues and ultimately extend their indications for treating various intractable diseases. In this review, challenges of delivering RNAi therapeutics to tumors and major organs are discussed, as well as their transition to clinical trials. This review also highlights innovative and promising preclinical RNAi-based delivery platforms for the treatment of extrahepatic diseases.

Eye-Mimicked Neural Network Composed of Photosensitive Neural Spheroids with Human Opsin Proteins.

An in vitro model, composed of the short-wavelength human opsins and rhodopsins, is created. Two types of photosensitive neural spheroids are transfected for selective reaction under bluish-purple and green lights. These are employed to two devices with intact neuron and neural-spheroid to study the interaction. By photostimulation, the photosensitive spheroid initiated photoactivation, and the signal generated from its body is transmitted to adjacent neural networks. Specifically, the signal traveled through the axon bundle in narrow gap from photosensitive spheroid to intact spheroid as an eye-to-brain model including optic nerve. The whole process with photosensitive spheroid is monitored by calcium ion detecting fluorescence images. The results of this study can be applied to examine vision restoration and novel photosensitive biological systems with spectral sensitivity.

Exosome-guided direct reprogramming of tumor-associated macrophages from protumorigenic to antitumorigenic to fight cancer.

Highly immunosuppressive tumor microenvironment containing various protumoral immune cells accelerates malignant transformation and treatment resistance. In particular, tumor-associated macrophages (TAMs), as the predominant infiltrated immune cells in a tumor, play a pivotal role in regulating the immunosuppressive tumor microenvironment. As a potential therapeutic strategy to counteract TAMs, here we explore an exosome-guided in situ direct reprogramming of tumor-supportive M2-polarized TAMs into tumor-attacking M1-type macrophages. Exosomes derived from M1-type macrophages (M1-Exo) promote a phenotypic switch from anti-inflammatory M2-like TAMs toward pro-inflammatory M1-type macrophages with high conversion efficiency. Reprogrammed M1 macrophages possessing protein-expression profiles similar to those of classically activated M1 macrophages display significantly increased phagocytic function and robust cross-presentation ability, potentiating antitumor immunity surrounding the tumor. Strikingly, these M1-Exo also lead to the conversion of human patient-derived TAMs into M1-like macrophages that highly express MHC class II, offering the clinical potential of autologous and allogeneic exosome-guided direct TAM reprogramming for arming macrophages to join the fight against cancer.

Photosensitive Nanodiscs Composed of Human Photoreceptors for Refractive Index Modulation at Selective Wavelengths.

A photoreceptor on the retina acts as an optical waveguide to transfer an individual photonic signal to the cell inside, which is determined by the refractive index of internal materials. Under the photoactivation of photoreceptors making conformational and chemical variation in a visual cell, the optical signal modulation is demonstrated using an artificial photoreceptor-based waveguide with a controlling beam refraction. Two types of nanodiscs are made of human photoreceptor proteins, short-wavelength-sensitive opsin and rhodopsin, with spectral sensitivity. The refractive index and nonlinear features of those two photosensitive nanodiscs are investigated as fundamental properties. The photonanodiscs are photoactivated in such a way that allow refractive index tuning over 0.18 according to the biological function of the respective proteins with color-dependent response.

The Potential of Cell-Penetrating Peptides for mRNA Delivery to Cancer Cells.

In vitro transcribed mRNA for the synthesis of any given protein has shown great potential in cancer gene therapy, especially in cancer vaccines for immunotherapy. To overcome physiological barriers, such as rapid degradation by enzymatic attack and poor cellular uptake due to their large size and hydrophilic properties, many delivery carriers for mRNAs are being investigated for improving the bioavailability of mRNA. Recently, cell-penetrating peptides (CPPs) have received attention as promising tools for gene delivery. In terms of their biocompatibility and the ability to target specific cells with the versatility of peptide sequences, they may provide clues to address the challenges of conventional delivery systems for cancer mRNA delivery. In this study, optimal conditions for the CPP/mRNA complexes were identified in terms of complexation capacity and N/P ratio, and protection against RNase was confirmed. When cancer cells were treated at a concentration of 6.8 nM, which could deliver the highest amount of mRNA without toxicity, the amphipathic CPP/mRNA complexes with a size less than 200 nm showed high cellular uptake and protein expression. With advances in our understanding of CPPs, CPPs designed to target tumor tissues will be promising for use in developing a new class of mRNA delivery vehicles in cancer therapy.

Extracellular vesicle-guided in situ reprogramming of synovial macrophages for the treatment of rheumatoid arthritis.

Activation state of synovial macrophages is significantly correlated with disease activity and severity of rheumatoid arthritis (RA) and provides valuable clues for RA treatment. Classically activated M1 macrophages in inflamed synovial joints secrete high levels of pro-inflammatory cytokines and chemokines, resulting in bone erosion and cartilage degradation. Herein, we propose extracellular vesicle (EV)-guided in situ macrophage reprogramming toward anti-inflammatory M2 macrophages as a novel RA treatment modality based on the immunotherapeutic concept of reestablishing M1-M2 macrophage equilibrium in synovial tissue. M2 macrophage-derived EVs (M2-EVs) were able to convert activated M1 into reprogrammed M2 (RM2) macrophages with extremely high efficiency (>90%), producing a distinct protein expression pattern characteristic of anti-inflammatory M2 macrophages. In particular, M2-EVs were enriched for proteins known to be involved in the generation and migration of M2 macrophages as well as macrophage reprogramming factors, allowing for rapid and efficient driving of macrophage polarization toward M2 phenotype. After administration of M2-EVs into the joint of a collagen-induced arthritis mouse model, the synovial macrophage polarization was significantly shifted from M1 to M2 phenotype, a process that benefited greatly from the long residence time (>3 days) of M2-EVs in the joint. This superb in situ macrophage-reprogramming ability of EVs resulted in decreased joint swelling, arthritic index score and synovial inflammation, with corresponding reductions in bone erosion and articular cartilage damage and no systemic toxicity. The anti-RA effects of M2-EVs were comparable to those of the conventional disease-modifying antirheumatic drug, Methotrexate, which causes a range of toxic adverse effects, including gastrointestinal mucosal injury. Overall, our EV-guided reprogramming strategy for in situ tuning of macrophage responses holds great promise for the development of anti-inflammatory therapeutics for the treatment of various inflammatory diseases in addition to RA.

Potential of Colostrum-Derived Exosomes for Promoting Hair Regeneration Through the Transition From Telogen to Anagen Phase.

Human hair dermal papillary (DP) cells comprising mesenchymal stem cells in hair follicles contribute critically to hair growth and cycle regulation. The transition of hair follicles from telogen to anagen phase is the key to regulating hair growth, which relies heavily on the activation of DP cells. In this paper, we suggested exosomes derived from bovine colostrum (milk exosomes, Milk-exo) as a new effective non-surgical therapy for hair loss. Results showed that Milk-exo promoted the proliferation of hair DP cells and rescued dihydrotestosterone (DHT, androgen hormones)-induced arrest of follicle development. Milk-exo also induced dorsal hair re-growth in mice at the level comparable to minoxidil treatment, without associated adverse effects such as skin rashes. Our data demonstrated that Milk-exo accelerated the hair cycle transition from telogen to anagen phase by activating the Wnt/ß-catenin pathway. Interestingly, Milk-exo has been found to stably retain its original properties and efficacy for hair regeneration after freeze-drying and resuspension, which is considered critical to use it as a raw material applied in different types of alopecia medicines and treatments. Overall, this study highlights a great potential of an exosome from colostrum as a therapeutic modality for hair loss.

PDL1-binding peptide/anti-miRNA21 conjugate as a therapeutic modality for PD-L1high tumors and TAMs.

Upregulation of oncogenic miRNA21 (miR-21) plays a pivotal role in proliferation, migration and invasion of cancer cells. In addition to cancer cells, tumor-associated macrophages (TAMs) also have high abundance of miR-21, which accelerates malignant progression of tumors in the late stages of carcinogenesis. Despite of the pro-tumorigenic functions of miR-21 in TAMs and cancer cells, reliable therapeutic strategies to simultaneously inhibit miR-21 activity in both types of cell have not yet been developed. In this study, we designed a dual-targeting drug delivery system of miR-21 inhibitors that could bind to both tumor cells and macrophages with overexpressed PD-L1 receptors. This peptide-oligonucleotide conjugate (Pep-21) consists of a PDL1-binding peptide covalently linked with an anti-miR-21 inhibitor via click chemistry. Pep-21 was preferentially internalized in both cell types, consequently depleting endogenous miR-21. Our studies found that Pep-21 treatment reduced tumor cell migration, reprogrammed immunosuppressive M2-type TAMs into M1-type macrophages, and restrained tumor progression. Collectively, neutralization of miR-21 activity in both cancer cells and TAMs can be a promising strategy for effective antitumor responses.

Sustained Exosome-Guided Macrophage Polarization Using Hydrolytically Degradable PEG Hydrogels for Cutaneous Wound Healing: Identification of Key Proteins and MiRNAs, and Sustained Release Formulation.

Macrophages (Mφs) are characterized by remarkable plasticity, an essential component of chronic inflammation. Thus, an appropriate and timely transition from proinflammatory (M1) to anti-inflammatory (M2) Mφs during wound healing is vital to promoting resolution of acute inflammation and enhancing tissue repair. Herein, exosomes derived from M2-Mφs (M2-Exos), which contain putative key regulators driving Mφ polarization, are used as local microenvironmental cues to induce reprogramming of M1-Mφs toward M2-Mφs for effective wound management. As an injectable controlled release depot for exosomes, hydrolytically degradable poly(ethylene glycol) (PEG) hydrogels (Exogels) are designed and employed for encapsulating M2-Exos to maximize their therapeutic effects in cutaneous wound healing. The degradation time of the hydrogels is adjustable from 6 days or up to 27 days by controlling the crosslinking density and tightness. The localization of M2-Exos leads to a successful local transition from M1-Mφs to M2-Mφs within the lesion for more than 6 days, followed by enhanced therapeutic effects including rapid wound closure and increased healing quality in an animal model for cutaneous wound healing. Collectively, the hydrolytically degradable PEG hydrogel-based exosome delivery system may serve as a potential tool in regulating local polarization state of Mφs, which is crucial for tissue homeostasis and wound repair.

Bovine colostrum derived-exosomes prevent dextran sulfate sodium-induced intestinal colitis via suppression of inflammation and oxidative stress.

Despite the rise in the global burden of inflammatory bowel disease, there is a lack of safe and effective therapies that can meet the needs of clinical patients. In this study, we investigated the beneficial effects of bovine milk, especially colostrum-derived exosomes (Col-exo) in a murine model of ulcerative colitis induced by dextran sodium sulfate (DSS). Col-exo activated the proliferation of colonic epithelial cells and macrophages, and created an environment to relieve inflammation by effectively removing reactive oxygen species and regulating the expression of immune cytokines. Besides, Col-exo could pass through the gastrointestinal tract intact and efficiently deliver bioactive cargoes to the stomach, small intestine, and colon. Our results showed that oral gavage of Col-exo can alleviate colitis symptoms including weight loss, gastrointestinal bleeding, and chronic diarrhea by modulating intestinal inflammatory immune responses. Overall, bovine colostrum-derived exosomes with excellent structural and functional stability may offer great potential as natural therapeutics for the recovery of colitis.

Ultraefficient extracellular vesicle-guided direct reprogramming of fibroblasts into functional cardiomyocytes.

Direct lineage conversion holds great promise in the regenerative medicine field for restoring damaged tissues using functionally engineered counterparts. However, current methods of direct lineage conversion, even those using virus-mediated transgenic expression of tumorigenic factors, are extremely inefficient (~25%). Thus, advanced methodologies capable of revolutionizing efficiency and addressing safety concerns are key to clinical translation of these technologies. Here, we propose an extracellular vesicle (EV)-guided, nonviral, direct lineage conversion strategy to enhance transdifferentiation of fibroblasts to induced cardiomyocyte-like cells (iCMs). The resulting iCMs have typical cardiac Ca2+ transients and electrophysiological features and exhibit global gene expression profiles similar to those of cardiomyocytes. This is the first demonstration of the use of EVs derived from embryonic stem cells undergoing cardiac differentiation as biomimetic tools to induce cardiac reprogramming with extremely high efficiency (>60%), establishing a general, more readily accessible platform for generating a variety of specialized somatic cells through direct lineage conversion.

The Potential of Bovine Colostrum-Derived Exosomes to Repair Aged and Damaged Skin Cells.

In this study, we examined the potentially beneficial effects of bovine colostrum-derived exosomes on UV-induced aging and damage in three major resident skin cells including keratinocytes, melanocytes, and fibroblasts. The treatment with colostrum exosomes prevented the UV-induced generation of intracellular reactive oxygen species in epidermal keratinocytes. In UV-stimulated melanocytes, colostrum exosomes could also significantly reduce the production of the protective skin-darkening pigment melanin, which may help to reduce the risk of excessive melanin formation causing skin hyperpigmentation disorders. In the human dermal fibroblasts treated with colostrum exosomes, the expression of matrix metalloproteinases was suppressed, whereas increased cell proliferation was accompanied by enhanced production of collagen, a major extracellular matrix component of skin. Taken together, our findings indicate that bovine colostrum-derived exosomes having excellent structural and functional stability offer great potential as natural therapeutic agents to repair UV-irradiated skin aging and damage.

Harnessing the Natural Healing Power of Colostrum: Bovine Milk-Derived Extracellular Vesicles from Colostrum Facilitating the Transition from Inflammation to Tissue Regeneration for Accelerating Cutaneous Wound Healing.

As wound healing is an extremely complicated process, consisting of a cascade of interlocking biological events, successful wound healing requires a multifaceted approach to support appropriate and rapid transitions from the inflammatory to proliferative and remodeling phases. In this regard, here the potential use of bovine milk extracellular vesicles (EVs) to enhance wound healing is investigated. The results show that milk EVs promote fibroblast proliferation, migration, and endothelial tube formation. In particular, milk EVs derived from colostrum (Colos EVs) contain various anti-inflammatory factors facilitating the transition from inflammation to proliferation phase, as well as factors for tissue remodeling and angiogenesis. In an excisional wound mouse model, Colos EVs promote re-epithelialization, activate angiogenesis, and enhance extracellular matrix maturation. Interestingly, Colos EVs are further found to be quite resistant to freeze-drying procedures, maintaining their original characteristics and efficacy for wound repair after lyophilization. These findings on the superior stability and excellent activity of milk Colos EVs indicate that they hold great promise to be developed as anti-inflammatory therapeutics, especially for the treatment of cutaneous wounds.

Measurement of Environmentally Influenced Variations in Anthocyanin Accumulations in Brassica rapa subsp. Chinensis (Bok Choy) Using Hyperspectral Imaging.

Dietary supplements of anthocyanin-rich vegetables have been known to increase potential health benefits for humans. The optimization of environmental conditions to increase the level of anthocyanin accumulations in vegetables during the cultivation periods is particularly important in terms of the improvement of agricultural values in the indoor farm using artificial light and climate controlling systems. This study reports on the measurement of variations in anthocyanin accumulations in leaf tissues of four different cultivars in Brassica rapa var. chinensis (bok choy) grown under the different environmental conditions of the indoor farm using hyperspectral imaging. Anthocyanin accumulations estimated by hyperspectral imaging were compared with the measured anthocyanin accumulation obtained by destructive analysis. Between hyperspectral imaging and destructive analysis values, no significant differences in anthocyanin accumulation were observed across four bok choy cultivars grown under the anthocyanin stimulation environmental condition, whereas the estimated anthocyanin accumulations displayed cultivar-dependent significant differences, suggesting that hyperspectral imaging can be employed to measure variations in anthocyanin accumulations of different bok choy cultivars. Increased accumulation of anthocyanin under the stimulation condition for anthocyanin accumulation was observed in "purple magic" and "red stem" by both hyperspectral imaging and destructive analysis. In the different growth stages, no significant differences in anthocyanin accumulation were found in each cultivar by both hyperspectral imaging and destructive analysis. These results suggest that hyperspectral imaging can provide comparable analytic capability with destructive analysis to measure variations in anthocyanin accumulation that occurred under the different light and temperature conditions of the indoor farm. Leaf image analysis measuring the percentage of purple color area in the total leaf area displayed successful classification of anthocyanin accumulation in four bok choy cultivars in comparison to hyperspectral imaging and destructive analysis, but it also showed limitation to reflect the level of color saturation caused by anthocyanin accumulation under different environmental conditions in "red stem," "white stem," and "green stem." Finally, our hyperspectral imaging system was modified to be applied onto the high-throughput plant phenotyping system, and its test to analyze the variation of anthocyanin accumulation in four cultivars showed comparable results with the result of the destructive analysis.

Recent Advances in Exosome-Based Drug Delivery for Cancer Therapy.

Exosomes are a class of extracellular vesicles, with a size of about 100 nm, secreted by most cells and carrying various bioactive molecules such as nucleic acids, proteins, and lipids, and reflect the biological status of parent cells. Exosomes have natural advantages such as high biocompatibility and low immunogenicity for efficient delivery of therapeutic agents such as chemotherapeutic drugs, nucleic acids, and proteins. In this review, we introduce the latest explorations of exosome-based drug delivery systems for cancer therapy, with particular focus on the targeted delivery of various types of cargoes.

A Trojan-Horse Strategy by In Situ Piggybacking onto Endogenous Albumin for Tumor-Specific Neutralization of Oncogenic MicroRNA.

MicroRNAs (miRNAs), a recently discovered class of noncoding RNAs, play pivotal roles in regulating fundamental biological processes by suppressing the expression of target genes. Aberrant miRNA expression is commonly correlated with human diseases, including cancers. Anti-miRNA oligonucleotides provide an innovative therapeutic strategy for silencing disease-associated miRNAs. However, the clinical application of anti-miRNA therapy has been limited by formulation challenges and physiological delivery barriers. Here, to provide the safe and effective tumor-targeted delivery of anti-miRNAs, we designed carrier-free maleimide-functionalized anti-miRNAs (MI-Anti-miRNAs) that enable "piggybacking" onto albumin in vivo. These functionalized MI-Anti-miRNAs covalently bind to cysteine-34 of endogenous albumin within minutes. In addition to resulting in a markedly extended blood circulation lifetime, this strategy allows MI-Anti-miRNAs to "hitchhike" to the tumor site. Importantly, in situ-generated albumin-Anti-miRNAs are capable of intracellularly internalizing highly negatively charged anti-miRNA molecules and knocking down target miRNAs. In particular, MI-Anti-miRNAs that targeted miRNA-21, which is involved in tumor initiation, progression, invasion, and metastasis in several types of cancer, successfully repressed miRNA-21 activity, resulting in a superior antitumor activity in both solid and metastatic tumor models without causing systemic toxicity. This endogenous albumin-piggybacking approach using MI-Anti-miRNAs provides a simple and broadly applicable platform strategy for the systemic delivery of anti-miRNA therapeutics.

Extracellular Vesicles as Potential Theranostic Platforms for Skin Diseases and Aging.

Extracellular vesicles (EVs), naturally secreted by cells, act as mediators for communication between cells. They are transported to the recipient cells along with cargoes such as nucleic acids, proteins, and lipids that reflect the changes occurring within the parent cells. Thus, EVs have been recognized as potential theranostic agents for diagnosis, treatment, and prognosis. In particular, the evidence accumulated to date suggests an important role of EVs in the initiation and progression of skin aging and various skin diseases, including psoriasis, systemic lupus erythematosus, vitiligo, and chronic wounds. This review highlights recent research that investigates the role of EVs and their potential as biomarkers and therapeutic agents for skin diseases and aging.

Extracellular Vesicles as Potential Therapeutics for Inflammatory Diseases.

Extracellular vesicles (EV) deliver cargoes such as nucleic acids, proteins, and lipids between cells and serve as an intercellular communicator. As it is revealed that most of the functions associated to EVs are closely related to the immune response, the important role of EVs in inflammatory diseases is emerging. EVs can be functionalized through EV surface engineering and endow targeting moiety that allows for the target specificity for therapeutic applications in inflammatory diseases. Moreover, engineered EVs are considered as promising nanoparticles to develop personalized therapeutic carriers. In this review, we highlight the role of EVs in various inflammatory diseases, the application of EV as anti-inflammatory therapeutics, and the current state of the art in EV engineering techniques.