Mucus permeating nanocarriers for controlled drug delivery

The purpose of this research project was to investigate the development of feasible mucus penetrating drug delivery systems in order to enhance the bioavailability of many therapeutic macromolecules. Mucus poses a dynamic semi-permeable barrier, which enables the exchange of nutrients, water, gases etc., while it simultaneously constitutes an impermeable barrier to most bacteria and pathogens. However, mucus elaborate architecture can also lead to the immobilization of macromolecules, hindering their passage and further absorption through the epithelium. Therefore, the scientific interest has focused on the development of functional nanocarriers, which has the ability to overcome this critical barrier and enhance the oral delivery of many macromolecular drugs.In the present study, mucus permeating self-nanoemulsifying drug delivery systems (SNEDDS) were developed as vehicles for the controlled oral delivery of insulin and PEP356 peptide. The design of this delivery system consisted of several combinations of oil/surfactant/cosurfactant in order to evaluate the self-nanoemulsifying performance of various excipients. An extensive experimental study was performed in order to assess the physicochemical properties of SNEDDS (i.e., average droplet size, in vitro cytotoxicity, in vitro permeability etc.) and select the most promising candidates for the incorporation of insulin or PEP356 peptide. Hydrophobic ion pairing was applied for the synthesis of protein/peptide complexes with an anionic phospholipid (DMPG) in order to improve drug solubility in SNEDDS. Drug-loaded SNEDDS were evaluated with respect to drug loading, release of the drug and drug protection against enzymatic degradation. The results demonstrated that the developed SNEDDS could be considered as novel delivery systems for the oral administration of peptides and proteins.Additionally, functional mucus permeating nanoparticles were synthesized, using poly(lactic-co-glycolic acid) (PLGA) polymer modified with polyethylene glycol (PEG). A promising hypothesis prevails that coating particles with a high PEG density can efficiently reduce particle-mucus adhesive interactions, since the hydrophilic PEG can effectively shield the hydrophobic core of the PLGA polymer. Accordingly, various PLGA-PEG copolymers were synthesized via the carbodiimide method and were evaluated in terms of the final PEG:PLGA molar ratio. Subsequently, PLGA-PEG nanoparticles were synthesized by the double emulsion method and were evaluated in comparison with unmodified PLGA nanoparticles (i.e., average size, zeta-potential, stability, in vitro cytotoxicity, in vitro permeability etc.). PLGA-PEG nanoparticles presented improved characteristics compared to unmodified nanoparicles with respect to their mucus permeability. The results showed that these functional nanoparticles exhibited higher mucus penetration rates in comparison with blank PLGA nanoparticles, indicating the reduction of adhesive interactions with mucus due to their high PEG surface coverage.Finally, a mucolytic approach was also performed in order to further enhance the permeability of PLGA-PEG nanoparticles. Mucolytic agents tend to disrupt the barrier properties of the mucus layer, thus affecting its bulk viscoelasticity. Accordingly, the mucolytic agent, 4-mercaptobenzoic acid (4-MBA), was encapsulated into PLGA-PEG nanoparticles in order to improve their transmucosal delivery compared to blank PLGA-PEG nanoparticles. Unfortunately, the results showed a fast initial release of 4-MBA, which is considered undesirable in order to facilitate improved retention time of drug carriers and evoke the local only disruption of mucus.Στην παρούσα διδακτορική διατριβή διεξάγεται η πειραματική μελέτη της σύνθεσης καινοτόμων συστημάτων μεταφοράς βιομορίων, τα οποία έχουν την ικανότητα να διαπερνούν τη βλέννα. Ο βλεννογόνος είναι υπεύθυνος για την παραγωγή της βλέννας, η οποία αποτελεί την πρώτη γραμμή άμυνας του οργανισμού. Η βλέννα αποτελεί ένα δυναμικό, ημιπερατό στρώμα, το οποίο επιτρέπει τη μεταφορά ουσιών, όπως θρεπτικών συστατικών, νερού και αερίων, ενώ ταυτόχρονα αποτρέπει την είσοδο βακτηρίων και παθογόνων μικροοργανισμών. Ωστόσο, η βλέννα αποτρέπει την είσοδο όχι μόνο των παθογόνων παραγόντων, αλλά και των βιομορίων, αποτρέποντας τη διέλευση τους μέσω της βλέννας και την περαιτέρω απορρόφηση τους από τα επιθηλιακά κύτταρα. Για το λόγο αυτό, το επιστημονικό ενδιαφέρον έχει στραφεί στην εφαρμογή καινοτόμων τεχνικών για τη σύνθεση λειτουργικών νανοφορέων, οι οποίοι επιτρέπουν την αποτελεσματική χορήγηση βιομορίων μέσω των βλεννωδών μεμβρανών.Στην παρούσα μελέτη αναπτύχθηκαν διάφοροι τύποι νανοφορέων ικανών να διαπερνούν τη βλέννα, εφαρμόζοντας διάφορες καινοτόμες στρατηγικές. Πιο συγκεκριμένα, αναπτύχθηκαν αυτο-νανογαλακτωματοποιούμενα συστήματα μεταφοράς φαρμάκων (SNEDDS), τα οποία χρησιμοποιήθηκαν ως φορείς για την ελεγχόμενη στοματική χορήγηση της ινσουλίνης και του πεπτιδίου PEP356. Κατά τον πειραματικό σχεδιασμό των SNEDDS, επιλέχθηκαν διάφοροι συνδυασμοί ελαίου/επιφανειοδραστικής ουσίας/συνεπιφανειοδραστικής ουσίας και μελετήθηκε η ικανότητα αυθόρμητου σχηματισμού νανογαλακτώματος. Στη συνέχεια, ακολούθησε ο χαρακτηρισμός των SNEDDS και επιλέχτηκαν οι βέλτιστοι συνδυασμοί. Επιπροσθέτως, εφαρμόστηκε η μέθοδος της υδρόφοβης ιοντικής σύζευξης για τη σύνθεση συμπλόκων ινσουλίνης ή PEP356 με ανιονικό φωσφολιπίδιο (DMPG), με στόχο τη μείωση της υδροφιλικότητάς τους. Εν συνεχεία, πραγματοποιήθηκε η ενσωμάτωση των συμπλόκων στα SNEDDS και ακολούθησε η μελέτη του ρυθμού αποδέσμευσης καθώς και ο έλεγχος της προστασίας από τα SNEDDS έναντι της δράσης ενζύμων του εντέρου. Τα αποτελέσματα απέδειξαν τη σύνθεση ενός καινοτόμου συστήματος μεταφοράς φαρμάκων για την αποτελεσματική στοματική χορήγηση θεραπευτικών βιομορίων.Επιπροσθέτως, πραγματοποιήθηκε η σύνθεση νανοσωματιδίων με «ολισθηρή» επιφάνεια, χρησιμοποιώντας το πολυμερές πολυ(γαλακτικό-γλυκολικό) οξύ (PLGA) και το πολυμερές πολυαιθυλενογλυκόλη (PEG). Τα τελευταία χρόνια, έχει επικρατήσει ότι η επιφανειακή κάλυψη των σωματιδίων με PEG έχει ως αποτέλεσμα τη μείωση των αλληλεπιδράσεων προσκόλλησης, οι οποίες δημιουργούνται μεταξύ των σωματιδίων και της βλέννας. Συνεπώς, πραγματοποιήθηκε η σύνθεση διαφόρων τύπων συμπολυμερών μέσω της αντίδρασης του καρβοδιιμιδίου και ακολούθησε η αξιολόγησή τους. Εν συνεχεία, παρασκευάστηκαν νανοσωματίδια PLGA-PEG με τη μέθοδο του διπλού γαλακτώματος και πραγματοποιήθηκε ο πλήρης χαρακτηρισμός τους. Αξίζει να σημειωθεί ότι τα νανοσωματίδια PLGA-PEG παρουσίασαν βελτιωμένα χαρακτηριστικά σε σχέση με τα νανοσωματίδια PLGA και πιο συγκεκριμένα εμφάνισαν αυξημένη ικανότητα διάχυσης μέσα στη βλέννα, υποδεικνύοντας την ικανοποιητική μείωση των αλληλεπιδράσεων λόγω της υψηλής επιφανειακής κάλυψης με PEG.Τέλος, πραγματοποιήθηκε η σύνθεση νανοσωματιδίων PLGA-PEG, που φέρουν το βλεννολυτικό παράγοντα 4-μερκαπτοβενζοϊκό οξύ (4-MBA) με στόχο την περαιτέρω ενίσχυση της ικανότητάς τους να διαπερνούν τη βλέννα σε σχέση με τα νανοσωματίδια PLGA-PEG. Η στρατηγική αυτή στηρίζεται στη βασική αρχή της τοπικής και επιλεκτικής διάρρηξης της βλέννας με τη χρήση βλεννολυτικών παραγόντων. Συνεπώς, πραγματοποιήθηκε ο εγκλεισμός του 4-MBA στα νανοσωματίδια PLGA-PEG με στόχο την αποτελεσματική διάχυση τους μέσα στη βλέννα. Δυστυχώς, τα αποτελέσματα έδειξαν ότι ο εγκλεισμός του 4-MBA στα νανοσωματίδια PLGA-PEG δεν επέτρεψε την ελεγχόμενη αποδέσμευσή του, ευννοώντας πιθανή διάρρηξη του συνολικού βλεννώδους ιστού, οδηγώντας πιθανόν σε ανεπιθύμητες για τον οργανισμό παρενέργειες

Plant-Derived Extracellular Vesicles as Therapeutic Nanocarriers

Mammalian exosomes have emerged as a promising class of functional materials, inspiring novel applications as therapeutic vehicles and nutraceutical compounds. Despite this, their immunogenicity has been an issue of controversy within the scientific community. Although, exosome-like vesicles, innately formed in plants and inherent to eukaryotic cell-derived vesicles, could soothe most of the concerns, they are notably underutilized as therapeutic modalities. This review highlights all efforts published so far, on the use of plant-derived extracellular vesicles (EVs) as therapeutic delivery systems. A summary of the physicochemical characteristics of plant-derived EVs is provided along with their main biological composition and in vitro/in vivo evidence of their therapeutic efficacy provided where available. Despite only a hand full of clinical trials being underway, concerning these vesicles, they arguably possess significant potential as nanodelivery systems of natural origin

A Review of the EU’s Regulatory Framework for the Production of Nano-Enhanced Cosmetics

Literature has suggested metallic nanomaterials (NMs) for a multitude of applications in cosmetic products, either as active ingredients or excipients. Alike most high-paced industrial sectors, cosmetology continues to capitalize on its unique properties/functions (e.g., as UV-filters, colorants, etc.), adding value to a wide range of products. However, as a result of their nano-scale, NMs do not always conform with the handling guidelines of their bulk counterparts, nor do conventional analytical methods account for their complex physicochemical and biological interactions. Among others, metallic nanoparticles have attracted the interest of many over the years due to their unique features, but possible precautions should be considered because of their bio-persistent nature. As a result, it is prevalent to consider a nano-specific framework, to regulate the use of NMs and the production of nano-enhanced cosmetics. To address this, we provide insight into the NMs that are currently used in the EU market, with a focus on metallic NMs, while analyzing the underlying legislation and relevant Opinions of the Scientific Committee on Consumer Safety (SCCS), from a scientific and commercial perspective. Even though the current Cosmetics Regulation (EU Regulation No 1223/2009) already entails specific provisions on NMs, cosmetic products incorporating unauthorized NMs have been repeatedly commercialized in the European Union. Considering the potential risks of NMs if they are mishandled, we provide an analysis of the risk assessment, as stated in Article 16 of the Cosmetics Regulation, to serve as a guideline for the future growth of nano-enhanced products. Based on the limited integration of metallic NMs along with multiple non-metallic NPs into cosmetic products, the attention of the community is directed towards coordinating efforts on the integration of metallic NMs into cosmetics

Antibacterial Effect of Colloidal Suspensions Varying in Silver Nanoparticles and Ions Concentrations

A lot of effort has been dedicated recently to provide a better insight into the mechanism of the antibacterial activity of silver nanoparticles (AgNPs) colloidal suspensions and their released silver ionic counterparts. However, there is no consistency regarding whether the antibacterial effect displayed at cellular level originates from the AgNPs or their ionic constitutes. To address this issue, three colloidal suspensions exhibiting different ratios of AgNPs/silver ions were synthesized by a wet chemistry method in conjunction with tangential flow filtration, and were characterized and evaluated for their antimicrobial properties against two gram-negative, Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), and two gram-positive, Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis), bacterial strains. The produced samples contained 25% AgNPs and 75% Ag ions (AgNP_25), 50% AgNPs and 50% Ag ions (AgNP_50), and 100% AgNPs (AgNP_100). The sample AgNP_100 demonstrated the lowest minimum inhibitory concentration values ranging from 4.6 to 15.6 ppm for all four bacterial strains, while all three samples indicated minimum bactericidal concentration (MBC) values ranging from 16.6 ppm to 62.5 ppm against all strains. An increase in silver ions content results in higher bactericidal activity. All three samples were found to lead to a significant morphological damage by disruption of the bacterial cell membranes as analyzed by means of scanning electron microscopy (SEM). The growth kinetics demonstrated that all three samples were able to reduce the bacterial population at a concentration of 3.1 ppm. SEM and growth kinetic data underline that S. epidermidis is the most sensitive among all strains against the investigated samples. Our results showed that all three AgNPs colloidal suspensions exhibited strong antibacterial properties and, thus, they can be applied in medical devices and antimicrobial control systems

Biological Activities of Citrus-Derived Extracellular Vesicles on Human Cells: The Role of Preservation

Extracellular vesicles (EVs) have been identified as important mediators for cell-to-cell communication. Citrus-based EVs in particular offer an excellent platform for nutraceutical delivery systems, as their endemic cargo includes micronutrients (e.g., ascorbic acid), which contribute to their antioxidant capacity. Despite being extensively investigated as to their therapeutic and diagnostic potential, their cargo is inherently unstable and thus directly affected by their storage and preservation. In this study, EVs were isolated from citrus fruit using tangential flow filtration and evaluated for their physicochemical characteristics, antioxidant activity and effects on human cells. To assess how their isolation and preservation methods affect these properties, the EVs were tested immediately after isolation (from fresh and freeze-thawed juices) or following freeze-drying. A measurable biological effect of cryoprotection on citrus-derived EVs was evident, whether during or after isolation. This was more pronounced in the cell-based assays, ranging from −4% to +32% in human skin fibroblast proliferation. Nevertheless, the effects on human cancer cells varied depending on the cell line. Although these results should be considered preliminary observations, subject to further investigation, it is safe to state that any type of preservation is expected to impact the EVs’ biological activity

Synthesis and Characterization of Novel Copper Nanoparticles for the Control of Leaf Spot and Anthracnose Diseases of Olive

Olive crop is frequently treated with copper fungicides to combat foliar and fruit diseases such as olive leaf spot caused by Fusicladium oleagineum and anthracnose caused by Colletotrichum spp. The replacement of copper-based products with more eco-friendly alternatives is a priority. Metal nanoparticles synthesized in several ways have recently revolutionized crop protection with applications against important crop pathogens. In this study, we present the development of four copper-based nanoparticles (CuNP Type 1 to 4) synthesized with a wet chemistry approach. The CuNPs were characterized using Transmission Electron Microscopy, Dynamic Light Scattering, Laser Doppler Electrophoresis, and Attenuated Total Reflection measurements. In addition, the activity of the four CuNP types was tested in vitro and in planta against F. oleagineum and Colletotrichum spp. In vitro sensitivity measurements showed that for both pathogens, mycelial growth was the most susceptible developmental stage to the tested compounds. Against both pathogens, CuNP Type 1 and Type 2 were found to be more active in reducing mycelial growth compared to the reference commercial compounds of copper oxide and copper hydroxide. In planta experiments showed that CuNP Type 3 and CuNP Type 4 exhibited a strong protectant activity against both F. oleagineum and Colletotrichum acutatum with control efficacy values significantly higher than those achieved by the applications of either reference product

Respiratory Diseases Diagnosis Using Audio Analysis and Artificial Intelligence: A Systematic Review

Respiratory diseases represent a significant global burden, necessitating efficient diagnostic methods for timely intervention. Digital biomarkers based on audio, acoustics, and sound from the upper and lower respiratory system, as well as the voice, have emerged as valuable indicators of respiratory functionality. Recent advancements in machine learning (ML) algorithms offer promising avenues for the identification and diagnosis of respiratory diseases through the analysis and processing of such audio-based biomarkers. An ever-increasing number of studies employ ML techniques to extract meaningful information from audio biomarkers. Beyond disease identification, these studies explore diverse aspects such as the recognition of cough sounds amidst environmental noise, the analysis of respiratory sounds to detect respiratory symptoms like wheezes and crackles, as well as the analysis of the voice/speech for the evaluation of human voice abnormalities. To provide a more in-depth analysis, this review examines 75 relevant audio analysis studies across three distinct areas of concern based on respiratory diseases’ symptoms: (a) cough detection, (b) lower respiratory symptoms identification, and (c) diagnostics from the voice and speech. Furthermore, publicly available datasets commonly utilized in this domain are presented. It is observed that research trends are influenced by the pandemic, with a surge in studies on COVID-19 diagnosis, mobile data acquisition, and remote diagnosis systems

Antibacterial Activity of Copper Nanoparticles against <i>Xanthomonas campestris</i> pv. <i>vesicatoria</i> in Tomato Plants

Copper-based bactericides have appeared as a new tool in crop protection and offer an effective solution to combat bacterial resistance. In this work, two copper nanoparticle products that were previously synthesized and evaluated against major bacterial and fungal pathogens were tested on their ability to control the bacterial spot disease of tomato. Growth of Xanthomonas campestris pv. vesicatoria, the causal agent of the disease, was significantly suppressed by both nanoparticles, which had superior function compared to conventional commercial formulations of copper. X-ray fluorescence spectrometry measurements in tomato leaves revealed that bioavailability of copper is superior in the case of nanoparticles compared to conventional formulations and is dependent on synthesis rather than size. This is the first report correlating bioavailability of copper to nanoparticle efficacy