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Ilaria Ottonelli,1,* Elisa Adani,2,* Andrea Bighinati,2 Sabrina Cuoghi,1 Giovanni Tosi,1,3 Maria Angela Vandelli,1 Barbara Ruozi,1 Valeria Marigo,2,3 Jason Thomas Duskey1 1Nanotech Lab, Te.Far.T.I., Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 2Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 3Center for Neuroscience and Neurotechnology, Modena, Italy*These authors contributed equally to this workCorrespondence: Jason Thomas Duskey, Nanotech Lab, Te.Far.T.I., Department of Life Sciences, University of Modena and Reggio Emilia, Via Giuseppe Campi 103, Modena, MO, Italy, 41125, Tel +390592058573, Email jtduskey@unimore.it Valeria Marigo, Department of Life Sciences, University of Modena and Reggio Emilia, Via Giuseppe Campi 287, Modena, MO, 41125, Italy, Tel +390592055392, Email vmarigo@unimore.itPurpose: In recent years, microfluidic technologies have become mainstream in producing gene therapy nanomedicines (NMeds) following the Covid-19 vaccine; however, extensive optimizations are needed for each NMed type and genetic material. This article strives to improve LNPs for pDNA loading, protection, and delivery, while minimizing toxicity.Methods: The microfluidic technique was optimized to form cationic or neutral LNPs to load pDNA. Classical “post-formulation” DNA addition vs “pre” addition in the aqueous phase were compared. All formulations were characterized (size, homogeneity, zeta potential, morphology, weight yield, and stability), then tested for loading efficiency, nuclease protection, toxicity, and cell uptake.Results: Optimized LNPs formulated with DPPC: Chol:DOTAP 1:1:0.1 molar ratio and 10 μg of DOPE-Rhod, had a size of 160 nm and good homogeneity. The chemico-physical characteristics of cationic LNPs worsened when adding 15 μg/mL of pDNA with the “post” method, while maintaining their characteristics up to 100 μg/mL of pDNA with the “pre” addition remaining stable for 30 days. Interestingly, neutral LNPs formulated with the same method loaded up to 50% of the DNA. Both particles could protect the DNA from nucleases even after one month of storage, and low cell toxicity was found up to 40 μg/mL LNPs. Cell uptake occurred within 2 hours for both formulations with the DNA intact in the cytoplasm, outside of the lysosomes.Conclusion: In this study, the upcoming microfluidic technique was applied to two strategies to generate pDNA-LNPs. Cationic LNPs could load 10x the amount of DNA as the classical approach, while neutral LNPs, which also loaded and protected DNA, showed lower toxicity and good DNA protection. This is a big step forward at minimizing doses and toxicity of LNP-based gene therapy.Keywords: gene therapy, lipid nanoparticles, microfluidics, lipoplexes, DNA delivery

Lorella Pascolo,1 Barbara Bortot,1 Nuria Benseny-Cases,2 Alessandra Gianoncelli,3 Giovanni Tosi,4 Barbara Ruozi,4 Clara Rizzardi,5 Eleonora De Martino,1 Maria Angela Vandelli,4 Giovanni Maria Severini11Institute for Maternal and Child Health, Istituto di Ricovero e Cura a Carattere Scientifico Burlo Garofolo, Trieste, Italy; 2European Synchrotron Radiation Facility, Polygone Scientifique Louis Néel, Grenoble, France; 3Elettra-Sincrotrone Trieste, Area Science Park, Basovizza, Trieste, Italy; 4Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 5Department of Anatomical Pathology, Department of Pathology and Forensic Medicine, University of Trieste, Trieste, ItalyAbstract: Poly-lactide-co-glycolide (PLGA) is one of the few polymers approved by the US Food and Drug Administration as a carrier for drug administration in humans; therefore, it is one of the most used materials in the formulation of polymeric nanoparticles (NPs) for therapeutic purposes. Because the cellular uptake of polymeric NPs is a hot topic in the nanomedicine field, the development of techniques able to ensure incontrovertible evidence of the presence of NPs in the cells plays a key role in gaining understanding of their therapeutic potential. On the strength of this premise, this article aims to evaluate the application of synchrotron radiation-based Fourier transform infrared spectroscopy (SR-FTIR) spectromicroscopy and SR X-ray fluorescence (SR-XRF) microscopy in the study of the in vitro interaction of PLGA NPs with cells. To reach this goal, we used PLGA NPs, sized around 200 nm and loaded with superparamagnetic iron oxide NPs (PLGA-IO-NPs; Fe3O4; size, 10–15 nm). After exposing human mesothelial (MeT5A) cells to PLGA-IO-NPs (0.1 mg/mL), the cells were analyzed after fixation both by SR-FTIR spectromicroscopy and SR-XRF microscopy setups. SR-FTIR-SM enabled the detection of PLGA NPs at single-cell level, allowing polymer detection inside the biological matrix by the characteristic band in the 1,700–2,000 cm-1 region. The precise PLGA IR-signature (1,750 cm-1 centered pick) also was clearly evident within an area of high amide density. SR-XRF microscopy performed on the same cells investigated under SR-FTIR microscopy allowed us to put in evidence the Fe presence in the cells and to emphasize the intracellular localization of the PLGA-IO-NPs. These findings suggest that SR-FTIR and SR-XRF techniques could be two valuable tools to follow the PLGA NPs’ fate in in vitro studies on cell cultures.Keywords: PLGA-NPs, cell targeting, SR-FTIR, SR-XRF, imaging

Hemp-sprouts are emerging as a new class of attractive functional food due to their numerous health benefits when compared to other sprout species. Indeed, the high content of beneficial components including polyphenols and flavonoids makes this type of food a promising and successful market. However, the available literature on this topic is limited and often conflicting as regards to the content of phytocannabinoids. High-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) was applied in an untargeted metabolomics fashion to extracts of hemp seeds, sprouts and microgreens of nine different genotypes. Both unsupervised and supervised multivariate statistical analysis was performed to reveal variety-specific profiles of phytocannabinoids with surprisingly remarkable levels of phytocannabinoids even in chemotype V samples. Furthermore, a targeted HPLC-HRMS analysis was carried out for the quantitative determination of the major phytocannabinoids including CBDA, CBD, CBGA, CBG, CBCA, CBC, THCA, and trans-Δ 9 -THC. The last part of the study was focused on the evaluation of the enantiomeric composition of CBCA in hemp seeds, sprouts and microgreens in the different varieties by HPLC-CD (HPLC with online circular dichroism). Chiral analysis of CBCA showed a wide variability of its enantiomeric composition in the different varieties, thus contributing to the understanding of the intriguing stereochemical behavior of this compound in an early growth stage. However, further investigation is needed to determine the genetic factors responsible for the low enantiopurity of this compound., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Enzyme-based therapy has garnered significant attention for its current applications in various diseases. Despite the notable advantages associated with the use of enzymes as therapeutic agents, that could have high selectivity, affinity, and specificity for the target, their application faces challenges linked to physico-chemical and pharmacological properties. These limitations can be addressed through the encapsulation of enzymes in nanoplatforms as a comprehensive solution to mitigate their degradation, loss of activity, off-target accumulation, and immunogenicity, thus enhancing bioavailability, therapeutic efficacy, and circulation time, thereby reducing the number of administrations, and ameliorating patient compliance. The exploration of novel nanomedicine-based enzyme therapeutics for the treatment of challenging diseases stands as a paramount goal in the contemporary scientific landscape, but even then it is often not enough. Combining an enzyme with another therapeutic (e.g., a small molecule, another enzyme or protein, a monoclonal antibody, or a nucleic acid) within a single nanocarrier provides innovative multidrug-integrated therapy and ensures that both the actives arrive at the target site and exert their therapeutic effect, leading to synergistic action and superior therapeutic efficacy. Moreover, this strategic approach could be extended to gene therapy, a field that nowadays has gained increasing attention, as enzymes acting at genomic level and nucleic acids may be combined for synergistic therapy. This multicomponent therapeutic approach opens opportunities for promising future developments. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies., (© 2024 Wiley Periodicals LLC.)

Since its discovery as one of the main components of cannabis and its affinity towards the cannabinoid receptor CB1, serving as a means to exert its psychoactivity, Δ 9 -tetrahydrocannabinol (Δ 9 -THC) has inspired medicinal chemists throughout history to create more potent derivatives. Initially, the goal was to synthesize chemical probes for investigating the molecular mechanisms behind the pharmacology of Δ 9 -THC and finding potential medical applications. The unintended consequence of this noble intent has been the proliferation of these compounds for recreational use. This review comprehensively covers the most exhaustive number of THC-like cannabinoids circulating on the recreational market. It provides information on the chemistry, synthesis, pharmacology, analytical assessment, and experiences related to the psychoactive effects reported by recreational users on online forums. Some of these compounds can be found in natural cannabis, albeit in trace amounts, while others are entirely artificial. Moreover, to circumvent legal issues, many manufacturers resort to semi-synthetic processes starting from legal products extracted from hemp, such as cannabidiol (CBD). Despite the aim to encompass all known THC-like molecules, new species emerge on the drug users' pipeline each month. Beyond posing a significantly high public health risk due to unpredictable and unknown side effects, scientific research consistently lags behind the rapidly evolving recreational market., (© 2024. The Author(s).)

Small interfering RNA (siRNA) holds great potential to treat many difficult-to-treat diseases, but its delivery remains the central challenge. This study aimed at investigating the suitability of polymer-lipid hybrid nanomedicines (HNMeds) as novel siRNA delivery platforms for locoregional therapy of glioblastoma. Two HNMed formulations were developed from poly(lactic-co-glycolic acid) polymer and a cationic lipid: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol). After characterization of the HNMeds, a model siRNA was complexed onto their surface to form HNMed/siRNA complexes. The physicochemical properties and siRNA binding ability of complexes were assessed over a range of nitrogen-to-phosphate (N/P) ratios to optimize the formulations. At the optimal N/P ratio of 10, complexes effectively bound siRNA and improved its protection from enzymatic degradation. Using the NIH3T3 mouse fibroblast cell line, DOTAP-based HNMeds were shown to possess higher cytocompatibility in vitro over the DC-Chol-based ones. As proof-of-concept, uptake and bioefficacy of formulations were also assessed in vitro on U87MG human glioblastoma cell line expressing luciferase gene. Complexes were able to deliver anti-luciferase siRNA and induce a remarkable suppression of gene expression. Noteworthy, the effect of DOTAP-based formulation was not only about three-times higher than DC-Chol-based one, but also comparable to lipofectamine model transfection reagent. These findings set the basis to exploit this nanosystem for silencing relevant GB-related genes in further in vitro and in vivo studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

In this work, a heart-cut bidimensional achiral-chiral liquid chromatography method coupled to high-resolution mass spectrometry was developed for the separation of the main carboxylated phytocannabinoids, namely cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), cannabichromenic acid (CBCA), and cannabicyclolic acid (CBLA), and decarboxylated derivatives, namely cannabidiol (CBD), Δ 9 -tetrahydrocannabinol (Δ 9 -THC), cannabichromene (CBC), and cannabicyclol (CBL), and the evaluation of their enantiomeric composition in extracts of different Cannabis sativa L. varieties. Optimal conditions for the chiral analysis of CBC- and CBL-type compounds were found with methanol and water (95:5, v/v, with 0.1% formic acid, 1.5 mL/min) on an amylose-based chiral stationary phase. These settings also allowed to evaluate the parameters responsible for CBC and CBCA racemization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing ("early treatment") or reversing ("late treatment") HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic ("early treatment") or symptomatic ("late treatment") stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments ("2 cycle treatment") lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. DATA AVAILABILITY: This study does not include data deposited in public repositories. Data are available on request to the corresponding authors., Competing Interests: Declaration of Competing Interest The authors report no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cannabis is a multifaceted plant with numerous therapeutic properties on one hand, and controversial psychotropic activities on the other hand, which are modulated by CB1 endocannabinoid receptors. Δ9-Tetrahydrocannabinol (Δ9-THC) has been identified as the main component responsible for the psychotropic effects, while its constitutional isomer cannabidiol (CBD) has shown completely different pharmacological properties. Due to its reported beneficial effects, Cannabis has gained global popularity and is openly sold in shops and online. To circumvent legal restrictions, semi-synthetic derivatives of CBD are now frequently added to cannabis products, producing "high" effects similar to those induced by Δ9-THC. The first semi-synthetic cannabinoid to appear in the EU was obtained through cyclization and hydrogenation of CBD, and is known as hexahydrocannabinol (HHC). Currently, there is limited knowledge regarding HHC, its pharmacological properties, and its prevalence, as it is not commonly investigated in routine toxicological assays. In this study, synthetic strategies were explored to obtain an excess of the active epimer of HHC. Furthermore, the two epimers were purified and individually tested for their cannabinomimetic activity. Lastly, a simple and rapid chromatographic method employing a UV detector and a high-resolution mass spectrometer was applied to identify and quantify up to ten major phytocannabinoids, as well as the HHC epimers, in commercial cannabis samples., (© 2023. The Author(s).)

Spinal cord injury (SCI) is characterized by a cascade of events that lead to sensory and motor disabilities. To date, this condition is irreversible, and no cure exists. To improve myelin repair and limit secondary degeneration, we developed a multitherapy based on nanomedicines (NMeds) loaded with the promyelinating agent triiodothyronine (T3), used in combination with systemic ibuprofen and mouse nerve growth factor (mNGF). Poly-L-lactic-co-glycolic acid (PLGA) NMeds were optimized and loaded with T3 to promote sustained release. In vitro experiments confirmed the efficacy of T3-NMeds to differentiate oligodendrocyte precursor cells. In vivo rat experiments were performed in contusion SCI to explore the NMed biodistribution and efficacy of combo drugs at short- and long-term post-lesion. A strong anti-inflammatory effect was observed in the short term with a reduction of type M1 microglia and glutamate levels, but with a subsequent increase of TREM2. In the long term, an improvement of myelination in NG2-IR, an increase in MBP content, and a reduction of the demyelination area were observed. These data demonstrated that NMeds can successfully be used to obtain more controlled local drug delivery and that this multiple treatment could be effective in improving the outcome of SCIs.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor, associated with low long-term survival. Nanoparticles (NPs) developed against GBM are a promising strategy to improve current therapies, by enhancing the brain delivery of active molecules and reducing off-target effects. In particular, NPs hold high potential for the targeted delivery of chemotherapeutics both across the blood-brain barrier (BBB) and specifically to GBM cell receptors, pathways, or the tumor microenvironment (TME). In this review, the most recent strategies to deliver drugs to GBM are explored. The main focus is on how surface functionalizations are essential for BBB crossing and for tumor specific targeting. We give a critical analysis of the various ligand-based approaches that have been used to target specific cancer cell receptors and the TME, or to interfere with the signaling pathways of GBM. Despite the increasing application of NPs in the clinical setting, new methods for ligand and surface characterization are needed to optimize the synthesis, as well as to predict their in vivo behavior. An expert opinion is given on the future of this research and what is still missing to create and characterize a functional NP system for improved GBM targeting.

Curcumin is a well-known antioxidant used as traditional medicine in China and India since ages to treat variety of inflammatory ailments as a food supplement. Curcumin has antitumor properties with neuroprotective effects in Alzheimer's disease. Curcumin elevates brain-derived neurotrophic factor (BDNF) and dopamine (DA) levels in the brain indicating its role in substance abuse. Methamphetamine (METH) is one of the most abused substances in the world that induces profound neurotoxicity by inducing breakdown of the blood-brain barrier (BBB), vasogenic edema and cellular injuries. However, influence of curcumin on METH-induced neurotoxicity is still not well investigated. In this investigation, METH neurotoxicity and neuroprotective effects of curcumin nanodelivery were examined in a rat model. METH (20 mg/kg, i.p.) neurotoxicity is evident 4 h after its administration exhibiting breakdown of BBB to Evans blue albumin in the cerebral cortex, hippocampus, cerebellum, thalamus and hypothalamus associated with vasogenic brain edema as seen measured using water content in all these regions. Nissl attaining exhibited profound neuronal injuries in the regions of BBB damage. Normal curcumin (50 mg/kg, i.v.) 30 min after METH administration was able to reduce BBB breakdown and brain edema partially in some of the above brain regions. However, TiO 2 nanowired delivery of curcumin (25 mg/kg, i.v.) significantly attenuated brain edema, neuronal injuries and the BBB leakage in all the brain areas. BDNF level showed a significant higher level in METH-treated rats as compared to saline-treated METH group. Significantly enhanced DA levels in METH-treated rats were also observed with nanowired delivery of curcumin. Normal curcumin was able to slightly elevate DA and BDNF levels in the selected brain regions. Taken together, our observations are the first to show that nanodelivery of curcumin induces superior neuroprotection in METH neurotoxicity probable by enhancing BDNF and DA levels in the brain, not reported earlier., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

A drawback in the development of treatments that can reach the retina is the presence of barriers in the eye that restrain compounds from reaching the target. Intravitreal injections hold promise for retinal delivery, but the natural defenses in the vitreous can rapidly degrade or eliminate therapeutic molecules. Injectable hydrogel implants, which act as a reservoir, can allow for long-term drug delivery with a single injection into the eye, but still suffer due to the fast clearance of the released drugs when traversing the vitreous and random diffusion that leads to lower pharmaceutic efficacy. A combination with HA-covered nanoparticles, which can be released from the gel and more readily pass through the vitreous to increase the delivery of therapeutic agents to the retina, represents an advanced and elegant way to overcome some of the limitations in eye drug delivery. In this article, we developed hybrid PLGA-Dotap NPs that, due to their hyaluronic acid coating, can improve in vivo distribution throughout the vitreous and delivery to retinal cells. Moreover, a hydrogel implant was developed to act as a depot for the hybrid NPs to better control and slow their release. These results are a first step to improve the treatment of retinal diseases by protecting and transporting the therapeutic treatment across the vitreous and to improve treatment options by creating a depot system for long-term treatments.

Nanoparticles (NPs) are paving the way for improved treatments for difficult to treat diseases diseases; however, much is unknown about their fate in the body. One important factor is the interaction between NPs and blood proteins leading to the formation known as the "protein corona" (PC). The PC, consisting of the Hard (HC) and Soft Corona (SC), varies greatly based on the NP composition, size, and surface properties. This highlights the need for specific studies to differentiate the PC formation for each individual NP system. This work focused on comparing the HC and SC of three NPs with different matrix compositions: a) polymeric NPs based on poly(lactic- co -glycolic) acid (PLGA), b) hybrid NPs consisting of PLGA and Cholesterol, and c) lipidic NPs made only of Cholesterol. NPs were formulated and characterized for their physico-chemical characteristics and composition, and then were incubated in human plasma. In-depth purification, identification, and statistical analysis were then performed to identify the HC and SC components. Finally, similar investigations demonstrated whether the presence of a targeting ligand on the NP surface would affect the PC makeup. These results highlighted the different PC fingerprints of these NPs, which will be critical to better understand the biological influences of the PC and improve future NP designs., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors.)

Cannabidiolic acid (CBDA) and trans-Δ 9 -tetrahydrocannabinolic acid (trans-Δ 9 -THCA) are known to be the major phytocannabinoids in Cannabis sativa L., along with their decarboxylated derivatives cannabidiol (CBD) and trans-Δ 9 -tetrahydrocannabinol (trans-Δ 9 -THC). The cis isomer of Δ 9 -THC has been recently identified, characterized and quantified in several Cannabis sativa varieties, which had been heated (decarboxylated) before the analysis. Since decarboxylation alters the original phytocannabinoids composition of the plant, this work reports the identification and characterization of the carboxylated precursor cis-Δ 9 -THCA. The compound was also synthesized and used as analytical standard for the development and validation of a liquid chromatography coupled to high resolution mass spectrometry-based method for its quantification in ten Cannabis sativa L. samples from different chemotypes. The highest concentrations of cis-Δ 9 -THCA were found in CBD-rich varieties, lower levels were observed in cannabigerol (CBG)-rich varieties (chemotype IV) and in those varieties with a balanced level of both CBD and THC (chemotype III), while its levels were not detectable in cannabichromene (CBC)-rich varieties (chemotype VI). The presence of the cis isomer of THC and THCA raises the question on whether to include or not this species in the calculation of the total amount of THC to classify a cannabis variety as a drug-type or a fiber-type (hemp)., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Glioblastoma Multiforme (GBM) is a devastating disease with a low survival rate and few efficacious treatment options. The fast growth, late diagnostics, and off-target toxicity of currently used drugs represent major barriers that need to be overcome to provide a viable cure. Nanomedicines (NMeds) offer a way to overcome these pitfalls by protecting and loading drugs, increasing blood half-life, and being targetable with specific ligands on their surface. In this study, the FDA-approved polymer poly (lactic-co-glycolic) acid was used to optimise NMeds that were surface modified with a series of potential GBM-specific ligands. The NMeds were fully characterised for their physical and chemical properties, and then in vitro testing was performed to evaluate cell uptake and GBM cell specificity. While all targeted NMeds showed improved uptake, only those decorated with the-cell surface vimentin antibody M08 showed specificity for GBM over healthy cells. Finally, the most promising targeted NMed candidate was loaded with the well-known chemotherapeutic, paclitaxel, to confirm targeting and therapeutic effects in C6 GBM cells. These results demonstrate the importance of using well-optimised NMeds targeted with novel ligands to advance delivery and pharmaceutical effects against diseased cells while minimising the risk for nearby healthy cells.

L-Kynurenine (KYN) and kynurenic acid (KYNA) are products of the metabolism of L-tryptophan (TRP) in the central nervous system of animals, but they are not commonly found in plants. In particular, KYNA is known for its interesting pharmacological properties (anti-oxidative, anti-inflammatory, hypolipidemic, and neuroprotective), which suggest a potential functional food ingredient role. The three compounds were identified in samples of Cannabis sativa L. by means of high-performance liquid chromatography coupled to high-resolution mass spectrometry using an untargeted metabolomics approach. Their concentrations were evaluated using a targeted metabolomics method in three organs of the plant (roots, stem, and leaves) in soil at two different growth stages and in hydroponics conditions. The distribution of TRP, KYN and KYNA was found tendentially higher in leaves compared to stem and roots and changed over time. Moreover, the levels of KYNA found in this study are unprecedentedly high compared to those found so far in other plant species, suggesting that Cannabis sativa L. could be a promising alternative source of this metabolite., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Tunneling nanotubes (TNTs), discovered in 2004, are thin, long protrusions between cells utilized for intercellular transfer and communication. These newly discovered structures have been demonstrated to play a crucial role in homeostasis, but also in the spreading of diseases, infections, and metastases. Gaining much interest in the medical research field, TNTs have been shown to transport nanomedicines (NMeds) between cells. NMeds have been studied thanks to their advantageous features in terms of reduced toxicity of drugs, enhanced solubility, protection of the payload, prolonged release, and more interestingly, cell-targeted delivery. Nevertheless, their transfer between cells via TNTs makes their true fate unknown. If better understood, TNTs could help control NMed delivery. In fact, TNTs can represent the possibility both to improve the biodistribution of NMeds throughout a diseased tissue by increasing their formation, or to minimize their formation to block the transfer of dangerous material. To date, few studies have investigated the interaction between NMeds and TNTs. In this work, we will explain what TNTs are and how they form and then review what has been published regarding their potential use in nanomedicine research. We will highlight possible future approaches to better exploit TNT intercellular communication in the field of nanomedicine.

Reactive oxygen species (ROS)-sensitive drug delivery systems (DDS) specifically responding to altered levels of ROS in the pathological microenvironment have emerged as an effective means to enhance the pharmaceutical efficacy of conventional nanomedicines, while simultaneously reducing side effects. In particular, the use of the biocompatible, biodegradable, and non-toxic ROS-responsive thioketal (TK) functional group in the design of smart DDS has grown exponentially in recent years. In the design of TK-based DDS, different technological uses of TK have been proposed to overcome the major limitations of conventional DDS counterparts including uncontrolled drug release and off-target effects. This review will focus on the different technological uses of TK-based biomaterials in smart nanomedicines by using it as a linker to connect a drug on the surface of nanoparticles, form prodrugs, as a core component of the DDS to directly control its structure, to control the opening of drug-releasing gates or to change the conformation of the nano-systems. A comprehensive view of the various uses of TK may allow researchers to exploit this reactive linker more consciously while designing nanomedicines to be more effective with improved disease-targeting ability, providing novel therapeutic opportunities in the treatment of many diseases.

The recent discovery of the novel heptyl phytocannabinoids cannabidiphorol (CBDP) and Δ 9 -tetrahydrocannabiphorol (Δ 9 -THCP) raised a series of questions relating to the presence and abundance of these new unorthodox compounds in cannabis inflorescence or derived products. As fresh inflorescence contains mainly their acid precursors, which are not commercially available, an ad hoc stereoselective synthesis was performed in order to obtain cannabidiphorolic acid (CBDPA) and Δ 9 -tetrahydrocannabiphorolic acid (THCPA) to be used as analytical standards for quantitative purposes. The present work reports an unprecedented targeted analysis of both pentyl (C5) and heptyl (C7) CBD- and THC-type compounds in forty-nine cannabis samples representing four different chemotypes. Moreover, the ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry-based method was applied for the putative identification of other heptyl homologs of the most common phytocannabinoid acids, including cannabigerophorolic acid (CBGPA), cannabichromephorolic acid (CBCPA), cannabinophorolic acid (CBNPA), cannabielsophorolic acid (CBEPA), cannabicyclophorolic acid (CBLPA), cannabitriophorolic acid (CBTPA), and cannabiripsophorolic acid (CBRPA)., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Enzymes, as natural and potentially long-term treatment options, have become one of the most sought-after pharmaceutical molecules to be delivered with nanoparticles (NPs); however, their instability during formulation often leads to underwhelming results. Various molecules, including the Tween ® polysorbate series, have demonstrated enzyme activity protection but are often used uncontrolled without optimization. Here, poly(lactic-co-glycolic) acid (PLGA) NPs loaded with β-glucosidase (β-Glu) solutions containing Tween ® 20, 60, or 80 were compared. Mixing the enzyme with Tween ® pre-formulation had no effect on particle size or physical characteristics, but increased the amount of enzyme loaded. More importantly, NPs made with Tween ® 20:enzyme solutions maintained significantly higher enzyme activity. Therefore, Tween ® 20:enzyme solutions ranging from 60:1 to 2419:1 mol:mol were further analyzed. Isothermal titration calorimetry analysis demonstrated low affinity and unquantifiable binding between Tween ® 20 and β-Glu. Incorporating these solutions in NPs showed no effect on size, zeta potential, or morphology. The amount of enzyme and Tween ® 20 in the NPs was constant for all samples, but a trend towards higher activity with higher molar rapports of Tween ® 20:β-Glu was observed. Finally, a burst release from NPs in the first hour with Tween ® :β-Glu solutions was the same as free enzyme, but the enzyme remained active longer in solution. These results highlight the importance of stabilizers during NP formulation and how optimizing their use to stabilize an enzyme can help researchers design more efficient and effective enzyme loaded NPs.

Microfluidic technologies have recently been applied as innovative methods for the production of a variety of nanomedicines (NMeds), demonstrating their potential on a global scale. The capacity to precisely control variables, such as the flow rate ratio, temperature, total flow rate, etc., allows for greater tunability of the NMed systems that are more standardized and automated than the ones obtained by well-known benchtop protocols. However, it is a crucial aspect to be able to obtain NMeds with the same characteristics of the previously optimized ones. In this study, we focused on the transfer of a production protocol for hybrid NMeds (H-NMeds) consisting of PLGA, Cholesterol, and Pluronic ® F68 from a benchtop nanoprecipitation method to a microfluidic device. For this aim, we modified parameters such as the flow rate ratio, the concentration of core materials in the organic phase, and the ratio between PLGA and Cholesterol in the feeding organic phase. Outputs analysed were the chemico-physical properties, such as size, PDI, and surface charge, the composition in terms of %Cholesterol and residual %Pluronic ® F68, their stability to lyophilization, and the morphology via atomic force and electron microscopy. On the basis of the results, even if microfluidic technology is one of the unique procedures to obtain industrial production of NMeds, we demonstrated that the translation from a benchtop method to a microfluidic one is not a simple transfer of already established parameters, with several variables to be taken into account and to be optimized.

A sensitive and straightforward HPLC-UV method was developed for the simultaneous quantification of the two main impurities in "pure" commercial cannabigerol (CBG) samples. The identification of such impurities, namely cannabigerovarin (CBGV) and cannabigerobutol (CBGB), the propyl and butyl homologs of CBG, respectively, was accomplished employing the high-resolution mass spectrometry (HRMS) technique, and subsequently confirmed by comparison with the same compounds obtained by chemical synthesis. Complete spectroscopic characterization (NMR, FT-IR, UV, and HRMS) of both impurities is reported in the present work. The method was validated in terms of linearity, which was assessed in the range 0.01-1.00 μg/mL, sensitivity, selectivity, intra- and inter-day accuracy and precision, and short-term stability, which all satisfied the acceptance criteria of the ICH guidelines. Application of the method to the analysis of four commercial CBG samples highlighted a certain variability in the impurity profile that might be ascribed to the hemp variety of the starting plant material. With these new analytical standards in hand, it would be interesting to investigate their concentrations in different hemp varieties and expand the scope of a phytocannabinomics approach for a comprehensive profiling of this remarkable class of natural compounds., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Introduction: Cannabidiol (CBD), the nonintoxicating constituent of cannabis, is largely employed for pharmaceutical and cosmetic purposes. CBD can be extracted from the plant or chemically synthesized. Impurities of psychotropic cannabinoids Δ 9 -tetrahydrocannabinol (Δ 9 -THC) and Δ 8 -THC have been found in extracted CBD, thus hypothesizing a possible contamination from the plant. Materials and Methods: In this study, synthetic and extracted CBD samples were analyzed by ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry and the parameters that can be responsible of the conversion of CBD into THC were evaluated by an accelerated stability test. Results: In synthetic and extracted CBD no trace of THC species was detected. In contrast, CBD samples stored in the dark at room temperature on the benchtop for 3 months showed the presence of such impurities. Experiments carried out under inert atmosphere in the absence of humidity or carbon dioxide led to no trace of THC over time even at high temperature. Conclusions: The results suggested that the copresence of carbon dioxide and water from the air could be the key for creating the acidic environment responsible for the cyclization of CBD. These findings suggest that it might be appropriate to review the storage conditions indicated on the label of commercially available CBD., Competing Interests: No competing financial interests exist., (Copyright 2021, Mary Ann Liebert, Inc., publishers.)

Supplementing brain cholesterol is emerging as a potential treatment for Huntington's disease (HD), a genetic neurodegenerative disorder characterized, among other abnormalities, by inefficient brain cholesterol biosynthesis. However, delivering cholesterol to the brain is challenging due to the blood-brain barrier (BBB), which prevents it from reaching the striatum, especially, with therapeutically relevant doses. Here we describe the distribution, kinetics, release, and safety of novel hybrid polymeric nanoparticles made of PLGA and cholesterol which were modified with an heptapeptide (g7) for BBB transit (hybrid-g7-NPs-chol). We show that these NPs rapidly reach the brain and target neural cells. Moreover, deuterium-labeled cholesterol from hybrid-g7-NPs-chol is released in a controlled manner within the brain and accumulates over time, while being rapidly removed from peripheral tissues and plasma. We confirm that systemic and repeated injections of the new hybrid-g7-NPs-chol enhanced endogenous cholesterol biosynthesis, prevented cognitive decline, and ameliorated motor defects in HD animals, without any inflammatory reaction. In summary, this study provides insights about the benefits and safety of cholesterol delivery through advanced brain-permeable nanoparticles for HD treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Enzymes have gained attention for their role in numerous disease states, calling for research for their efficient delivery. Loading enzymes into polymeric nanoparticles to improve biodistribution, stability, and targeting in vivo has led the field with promising results, but these enzymes still suffer from a degradation effect during the formulation process that leads to lower kinetics and specific activity leading to a loss of therapeutic potential. Stabilizers, such as bovine serum albumin (BSA), can be beneficial, but the knowledge and understanding of their interaction with enzymes are not fully elucidated. To this end, the interaction of BSA with a model enzyme B-Glu, part of the hydrolase class and linked to Gaucher disease, was analyzed. To quantify the natural interaction of beta-glucosidase (B-Glu,) and BSA in solution, isothermal titration calorimetry (ITC) analysis was performed. Afterwards, polymeric nanoparticles encapsulating these complexes were fully characterized, and the encapsulation efficiency, activity of the encapsulated enzyme, and release kinetics of the enzyme were compared. ITC results showed that a natural binding of 1:1 was seen between B-Glu and BSA. Complex concentrations did not affect nanoparticle characteristics which maintained a size between 250 and 350 nm, but increased loading capacity (from 6% to 30%), enzyme activity, and extended-release kinetics (from less than one day to six days) were observed for particles containing higher B-Glu:BSA ratios. These results highlight the importance of understanding enzyme:stabilizer interactions in various nanoparticle systems to improve not only enzyme activity but also biodistribution and release kinetics for improved therapeutic effects. These results will be critical to fully characterize and compare the effect of stabilizers, such as BSA with other, more relevant therapeutic enzymes for central nervous system (CNS) disease treatments.

Central nervous system (CNS) compartments remain one of the most difficult districts for drug delivery. This is due to the presence of the blood-brain barrier (BBB) that hampers 90% of drug passage, dramatically requiring non-invasive treatment strategies. Here, for the first time, the use of opioid-derived deltorphin-derivative peptides to drive biodegradable and biocompatible polymeric (i.e. poly-lactide-co-glycolide, PLGA) nanomedicines delivery across the BBB was described. Opioid-derived peptides were covalently conjugated to furnish activated polymers which were further used for fluorescently tagged nanoformulations. Beyond reporting production, formulation methodology and full physico-chemical characterization, in vivo tests generated clear proof of BBB crossing and CNS targeting by engineered nanomedicines opening the research to further applications of drug delivery and targeting in CNS disease models., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The chemical analysis of cannabis potency involves the qualitative and quantitative determination of the main phytocannabinoids: Δ 9 -tetrahydrocannabinol (Δ 9 -THC), cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), etc. Although it might appear as a trivial analysis, it is rather a tricky task. Phytocannabinoids are present mostly as carboxylated species at the aromatic ring of the resorcinyl moiety. Their decarboxylation caused by heat leads to a greater analytical variability due to both reaction kinetics and possible decomposition. Moreover, the instability of cannabinoids and the variability in the sample preparation, extraction, and analysis, as well as the presence of isomeric forms of cannabinoids, complicates the scenario. A critical evaluation of the different analytical methods proposed in the literature points out that each of them has inherent limitations. The present review outlines all the possible pitfalls that can be encountered during the analysis of these compounds and aims to be a valuable help for the analytical chemist. Graphical abstract.

Glioblastoma (GBM) is the most frequent and aggressive primary tumor of the brain and averages a life expectancy in diagnosed patients of only 15 months. Hence, more effective therapies against this malignancy are urgently needed. Several diseases, including cancer, are featured by high levels of reactive oxygen species (ROS), which are possible GBM hallmarks to target or benefit from. Therefore, the covalent linkage of drugs to ROS-responsive molecules can be exploited aiming for a selective drug release within relevant pathological environments. In this work, we designed a new ROS-responsive prodrug by using Melphalan (MPH) covalently coupled with methoxy polyethylene glycol (mPEG) through a ROS-cleavable group thioketal (TK), demonstrating the capacity to self-assembly into nanosized micelles. Full chemical-physical characterization was conducted on the polymeric-prodrug and proper controls, along with in vitro cytotoxicity assayed on different GBM cell lines and "healthy" astrocyte cells confirming the absence of any cytotoxicity of the prodrug on healthy cells (i.e. astrocytes). These results were compared with the non-ROS responsive counterpart, underlining the anti-tumoral activity of ROS-responsive compared to the non-ROS-responsive prodrug on GBM cells expressing high levels of ROS. On the other hand, the combination treatment with this ROS-responsive prodrug and X-ray irradiation on human GBM cells resulted in an increase of the antitumoral effect, and this might be connected to radiotherapy. Hence, these results represent a starting point for a rationale design of innovative and tailored ROS-responsive prodrugs to be used in GBM therapy and in combination with radiotherapy., (Copyright © 2020 Oddone, Boury, Garcion, Grabrucker, Martinez, Da Ros, Janaszewska, Forni, Vandelli, Tosi, Ruozi and Duskey.)

Discovering new materials to aid in the therapeutic delivery of drugs is in high demand. PLGA, a FDA approved polymer, is well known in the literature to form films or nanoparticles that can load, protect, and deliver drug molecules; however, its incompatibility with certain drugs (due to hydrophilicity or charge repulsion interactions) limits its use. Combining PLGA or other polymers such as polycaprolactone with other safe and positively-charged molecules, such as chitosan, has been sought after to make hybrid systems that are more flexible in terms of loading ability, but often the reactions for polymer coupling use harsh conditions, films, unpurified products, or create a single unoptimized product. In this work, we aimed to investigate possible innovative improvements regarding two synthetic procedures. Two methods were attempted and analytically compared using nuclear magnetic resonance (NMR), fourier-transform infrared spectroscopy (FT-IR), and dynamic scanning calorimetry (DSC) to furnish pure, homogenous, and tunable PLGA-chitosan hybrid polymers. These were fully characterized by analytical methods. A series of hybrids was produced that could be used to increase the suitability of PLGA with previously non-compatible drug molecules., Competing Interests: The authors declare no conflicts of interest.

The butyl homologues of Δ 9 -tetrahydrocannabinol, Δ 9 -tetrahydrocannabutol (Δ 9 -THCB), and cannabidiol, cannabidibutol (CBDB), were isolated from a medicinal Cannabis sativa variety (FM2) inflorescence. Appropriate spectroscopic and spectrometric characterization, including NMR, UV, IR, ECD, and HRMS, was carried out on both cannabinoids. The chemical structures and absolute configurations of the isolated cannabinoids were confirmed by comparison with the spectroscopic data of the respective compounds obtained by stereoselective synthesis. The butyl homologue of Δ 9 -THC, Δ 9 -THCB, showed an affinity for the human CB1 ( K i = 15 nM) and CB2 receptors ( K i = 51 nM) comparable to that of (-)- trans -Δ 9 -THC. Docking studies suggested the key bonds responsible for THC-like binding affinity for the CB1 receptor. The formalin test in vivo was performed on Δ 9 -THCB in order to reveal possible analgesic and anti-inflammatory properties. The tetrad test in mice showed a partial agonistic activity of Δ 9 -THCB toward the CB1 receptor.

The treatment of diseases that affect the central nervous system (CNS) represents a great research challenge due to the restriction imposed by the blood-brain barrier (BBB) to allow the passage of drugs into the brain. However, the use of modified nanomedicines engineered with different ligands that can be recognized by receptors expressed in the BBB offers a favorable alternative for this purpose. In this work, a BBB-penetrating peptide, angiopep-2 (Ang-2), was conjugated to poly(lactic- co -glycolic acid) (PLGA)-based nanoparticles through pre- and post-formulation strategies. Then, their ability to cross the BBB was qualitatively assessed on an animal model. Proof-of-concept studies with fluorescent and confocal microscopy studies highlighted that the brain-targeted PLGA nanoparticles were able to cross the BBB and accumulated in neuronal cells, thus showing a promising brain drug delivery system.

(-)-Trans-Δ 9 -tetrahydrocannabinol (Δ 9 -THC) is the main compound responsible for the intoxicant activity of Cannabis sativa L. The length of the side alkyl chain influences the biological activity of this cannabinoid. In particular, synthetic analogues of Δ 9 -THC with a longer side chain have shown cannabimimetic properties far higher than Δ 9 -THC itself. In the attempt to define the phytocannabinoids profile that characterizes a medicinal cannabis variety, a new phytocannabinoid with the same structure of Δ 9 -THC but with a seven-term alkyl side chain was identified. The natural compound was isolated and fully characterized and its stereochemical configuration was assigned by match with the same compound obtained by a stereoselective synthesis. This new phytocannabinoid has been called (-)-trans-Δ 9 -tetrahydrocannabiphorol (Δ 9 -THCP). Along with Δ 9 -THCP, the corresponding cannabidiol (CBD) homolog with seven-term side alkyl chain (CBDP) was also isolated and unambiguously identified by match with its synthetic counterpart. The binding activity of Δ 9 -THCP against human CB 1 receptor in vitro (K i  = 1.2 nM) resulted similar to that of CP55940 (K i  = 0.9 nM), a potent full CB 1 agonist. In the cannabinoid tetrad pharmacological test, Δ 9 -THCP induced hypomotility, analgesia, catalepsy and decreased rectal temperature indicating a THC-like cannabimimetic activity. The presence of this new phytocannabinoid could account for the pharmacological properties of some cannabis varieties difficult to explain by the presence of the sole Δ 9 -THC.

The accumulation of amyloid β (Aβ) triggers a cascade of toxic events in Alzheimer's disease (AD). The KLVFF peptide can interfere with Aβ aggregation. However, the peptide suffers from poor bioavailability and the inability to cross the blood-brain barrier. In this work, we study the possibility of adopting nanomedicine to overcome KLVFF limits in biodistribution. We produced new engineered polymeric nanoparticles (NPs), and we evaluated the cellular toxicity of these NPs and validated that KVLFF peptides released by NPs show the same promising effects on AD pathology. Our results revealed the successful generation of KVLFF loaded NPs that, without significant effects on cell heath, are even more potent in reversing Aβ-induced pathologies compared to the free peptide. Therefore, NPs will significantly advance KVLFF treatment as a therapeutic option for AD., Competing Interests: The authors declare no conflict of interest.

Cannabidiol (CBD), one of the two major active principles present in Cannabis sativa, is gaining great interest among the scientific community for its pharmaceutical, nutraceutical and cosmetic applications. CBD can be prepared either by chemical synthesis or extraction from Cannabis sativa (hemp). The latter is more convenient from several points of view, including environmental and economic, but mainly for the absence of harmful organic solvents generally employed in the chemical synthesis. Although CBD produced by hemp extraction is the most widely employed, it carries two major impurities. The first one is the already known cannabidivarin (CBDV), whereas the second one is supposed to be the butyl analog of CBD with a four-term alkyl side chain. In this work, we report the isolation by semi-preparative liquid chromatography and the unambiguous identification of this second impurity. A comprehensive spectroscopic characterization, including NMR, UV, IR, circular dichroism and high-resolution mass spectrometry (HRMS), was carried out on this natural cannabinoid. In order to confirm its absolute configuration and chemical structure, the stereoisomer (1R,6R) of the supposed cannabinoid was synthesized and the physicochemical and spectroscopic properties, along with the stereochemistry, matched those of the natural isolated molecule. According to the International Nonproprietary Name, we suggested the name of cannabidibutol (CBDB) for this cannabinoid. Lastly, an HPLC-UV method was developed and validated for the qualitative and quantitative determination of CBDV and CBDB in samples of CBD extracted from hemp and produced according to Good Manufacturing Practices regulations for pharmaceutical and cosmetic use., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cannabidibutol (CBDB), a novel butyl analog of cannabidiol, was identified as impurity of commercial cannabidiol (CBD) extracted from hemp (for full data and results interpretation see "Analysis of impurities of cannabidiol from hemp. Isolation, characterization and synthesis of cannabidibutol , the novel cannabidiol butyl analog" Citti et al, 2019). The compound was isolated from a CBD sample and subject to a full characterization. First, a complete spectroscopic characterization was performed by Nuclear Magnetic Resonance (NMR): in particular, 1 H-NMR, 13 C-NMR, COSY, HSQC and HMBC, which were followed by UV absorption and circular dichroism (CD) spectra. In order to confirm the structural identity and stereochemistry of the compound, a stereoselective synthesis of the trans isomer (1 R ,6 R ) was carried out and all the chemical and spectroscopic properties were analyzed. The synthesized compound was characterized by NMR ( 1 H-NMR, 13 C-NMR, COSY, HSQC and HMBC), Infra-Red spectroscopy (IR), UV and CD absorption, matching the results obtained for the natural isolated compound. With the analytical standard in hand, a simple high-performance liquid chromatography method coupled to UV detection (HPLC-UV) was developed and validated in house in terms of linearity, accuracy, precision, dilution integrity and stability. The present data might be useful to any researcher or industry that may run into a very common impurity of CBD extracted from hemp, so it can be easily compared with their own experimental data., (© 2019 The Author(s).)

Mucopolysaccharidosis type II (MPSII) is a lysosomal storage disorder due to the deficit of the enzyme iduronate 2-sulfatase (IDS), which leads to the accumulation of glycosaminoglycans in most organ-systems, including the brain, and resulting in neurological involvement in about two-thirds of the patients. The main treatment is represented by a weekly infusion of the functional enzyme, which cannot cross the blood-brain barrier and reach the central nervous system. In this study, a tailored nanomedicine approach based on brain-targeted polymeric nanoparticles (g7-NPs), loaded with the therapeutic enzyme, was exploited. Fibroblasts from MPSII patients were treated for 7 days with NPs loaded with the IDS enzyme; an induced IDS activity like the one detected in healthy cells was measured, together with a reduction of GAG content to non-pathological levels. An in vivo short-term study in MPSII mice was performed by weekly administration of g7-NPs-IDS. Biochemical, histological, and immunohistochemical evaluations of liver and brain were performed. The 6-weeks treatment produced a significant reduction of GAG deposits in liver and brain tissues, as well as a reduction of some neurological and inflammatory markers (i.e., LAMP2, CD68, GFAP), highlighting a general improvement of the brain pathology. The g7-NPs-IDS approach allowed a brain-targeted enzyme replacement therapy. Based on these positive results, the future aim will be to optimize NP formulation further to gain a higher efficacy of the proposed approach.

Hemp seed oil is well known for its nutraceutical, cosmetic and pharmaceutical properties due to a perfectly balanced content of omega 3 and omega 6 polyunsaturated fatty acids. Its importance for human health is reflected by the success on the market of organic goods in recent years. However, it is of utmost importance to consider that its healthy properties are strictly related to its chemical composition, which varies depending not only on the manufacturing method, but also on the hemp variety employed. In the present work, we analyzed the chemical profile of ten commercially available organic hemp seed oils. Their cannabinoid profile was evaluated by a liquid chromatography method coupled to high-resolution mass spectrometry. Besides tetrahydrocannabinol and cannabidiol, other 30 cannabinoids were identified for the first time in hemp seed oil. The results obtained were processed according to an untargeted metabolomics approach. The multivariate statistical analysis showed highly significant differences in the chemical composition and, in particular, in the cannabinoid content of the hemp oils under investigation.

The treatment of Alzheimer's disease (AD) is up to today one of the most unsuccessful examples of biomedical science. Despite the high number of literature evidences detailing the multifactorial and complex etiopathology of AD, no cure is yet present on the market and the available treatments are only symptomatic. The reasons could be ascribed on two main factors: (i) lack of ability of the majority of drugs to cross the blood-brain barrier (BBB), thus excluding the brain for any successful therapy; (ii) lack of selectivity and specificity of drugs, decreasing the efficacy of even potent anti-AD drugs. The exploitation of specifically engineered nanomedicines planned to cross the BBB and to target the most "hot" site of action (i.e., β-amyloid) is one of the most interesting innovations in drug delivery and could reasonably represent an promising choice for possible treatments and even early-diagnosis of AD. In this chapter, we therefore outline the most talented approaches in AD treatment with a specific focus on the main advantages/drawbacks and future possible translation to clinic application., (© 2019 Elsevier B.V. All rights reserved.)

Cannabidiol (CBD), for long time considered as a minor cannabinoid of Cannabis sativa, has recently gained much attention due to its antioxidant, anti-inflammatory, analgesic and anticonvulsant properties. A liquid chromatography coupled to mass spectrometry based method was developed for the quantitative determination of CBD and other cannabinoids (Δ 9 -tetrahydrocannabinol (THC), 11-hydroxy-THC and 11-nor-9-carboxy-THC) in rat brain samples after oral administration of a single high dose (50 mg/kg) of CBD. The main challenge of the present work was to study CBD pharmacokinetics in rat cortex: the identification of its metabolites and pharmacodynamics through the study of variations in endogenous compounds' concentrations following CBD administration. An untargeted metabolomics approach revealed the formation of some CBD metabolites that are not commonly found in other body tissues or fluids. Lastly, the changes in some endogenous compounds' concentrations were correlated with some of the pharmacological properties of this cannabinoid., (Copyright © 2018 Elsevier B.V. All rights reserved.)