Assessing the physicochemical stability and intracellular trafficking of mRNA-based COVID-19 vaccines

: The emergence of SARS-CoV-2 in Wuhan, China in 2019 has had a profound impact on humanity in every facet. While vaccines against this viral pathogen have been approved a year later, limitations to this therapeutic intervention persist, such as drug sensitivity to transportation and storage conditions, as well as significant financial losses from non-injected resuspended vials. Our research delves into the effects of thermal denaturation (4 - 40 °C) and light irradiation (720 and 10460 kJ/m2) on the mRNA-based vaccines BNT162b2 from BioNTech/Pfizer and mRNA-1273 from Moderna. We also investigated vaccine stability following incubation in syringes to simulate potential interactions with silicon oil. By assaying the effects of these stressors via biochemical and biophysical methods, we aim to elucidate the physicochemical properties, integrity, and stability of these mRNA-based vaccines. Furthermore, the incorporation of a fluorophore into both vaccines allowed us to monitor their localization within cells and assess their capacity to evade vesicular transport mechanisms, thus evaluating the differences between the two formulations. A comprehensive understanding of the aforementioned attributes can enable the establishment of optimal storage and manipulation conditions for these vaccines, thereby ensuring their safe and efficacious application while minimizing the waste of functional and safe therapeutic agents

Light for Crime Investigation

The identification and quantification of material present and collected at a crime scene, including their comparison against known standards, are critical requirements in investigative analyses. Forensic analysts use a variety of tools and techniques to achieve this. In this lecture, light is presented as one of the key elements in crime investigation. Light of selected wavelengths and instruments based on light technology are powerful tools in crime investigation (light as a “friend”) for detecting DNA, spores, polymers, fibres, glass, gunshot residues and drugs of abuse. On the other hand, light can degrade samples of utility in investigative analyses, i.e., UV light breakage of DNA during DNA profiling, and sunlight may alter crime investigation results when outdoor samples are collected and then subjected to forensic analyses (light as a “foe”). At the same time, the adverse effects of light on evidence of materials of interest could open opportunities for development of additional markers not yet explored or enrichment of libraries of dedicated instruments by adding degradation products to reduce false negative or false positive results. Therefore, this lecture will critically review the available methods of crime scene investigation which exploit light, discussing their advantages and limitations

A Study on Photostability of Amphetamines and Ketamine in Hair Irradiated under Artificial Sunlight

Drugs incorporated into hair are exposed to the environment, and cosmetic and chemical treatments, with possible decreases in their content. Knowledge concerning the effect of sunlight on drug content in hair can be helpful to forensic toxicologists, in particular, when investigating drug concentrations above or below pre-determined cut-offs. Twenty authentic positive hair samples were selected which had previously tested positive for amphetamines and/or ketamine. Washed hair were divided into two identical strands, with the former exposed at 765 W/m2 (300–800 nm spectrum of irradiance) for 48 h in a solar simulator, and the latter kept in the dark. Hair samples were extracted and analyzed by liquid chromatography high-resolution mass spectrometry detection. The percentage of photodegradation was calculated for each analyte (i.e., amphetamine, methamphetamine, methylendioxyamphetamine, ketamine, and norketamine). In parallel, photodegradation processes of standard molecules dissolved in aqueous and organic solutions were studied. In 20 hair samples positive for the targeted analytes, exposure to artificial sunlight induced an appreciable decrease in drug concentrations. The concentration ranges in the non-irradiated hair samples were 0.01–24 ng/mg, and 65% of samples exhibited a decrease in post-irradiation samples, with reduction from 3% to 100%. When more drugs were present in the same hair sample (i.e., MDMA and ketamine) the degradation yields were compound dependent. A degradation product induced by irradiation of ketamine in aqueous and methanol solutions was identified; it was also found to be present in a true positive hair sample after irradiation. Ketamine, amphetamines, and their metabolites incorporated in the hair of drug users undergo degradation when irradiated by artificial sunlight. Only for ketamine was a photoproduct identified in irradiated standard solutions and in true positive irradiated hair. When decisional cut-offs are applied to hair analysis, photodegradation must be taken into account since sunlight may produce false negative results. Moreover, new markers could be investigated as evidence of illicit drug use

Meccanismi farmacologici e fotobiologici nella fototossicit\ue0 da farmaco

Molti farmaci, assunti per via topica o sistemica, possono aumentare la sensibilit\ue0 ai raggi solari, scatenando manifestazioni cutanee di varia gravit\ue0, classificabili come reazioni fototossiche o fotoallergiche. I medicinali fotosensibilizzanti sono antibiotici di diverse classi (sulfamidici, tetracicline, chinolonici), antinfiammatori non steroidei, chemioterapici antitumorali, psicotropi (antidepressivi triciclici, neurolettici, antiepilettici, ansiolitici), antiaritmici, Ace-inibitori, diuretici, antistaminici, anestetici, antifungini e antivirali e, pi\uf9 recentemente, inibitori della HMG-CoA reduttasi (statine). Tali reazioni avverse dipendono da diversi fattori, come il dosaggio del farmaco assunto, la dose e la penetrazione della radiazione attivante, lo spessore dello strato corneo, il grado di pigmentazione e la presenza degli altri cromofori a livello cutaneo, lo stato immunologico della persona interessata. I meccanismi alla base delle reazioni di fotosensibilizzazione si distinguono in \u201cfotodinamici\u201d e \u201cnon fotodinamici\u201d, a seconda che coinvolgano o meno l\u2019ossigeno. In tutti i casi, l'assorbimento di raggi UV (principalmente UVA e UVB) produce uno stato eccitato del farmaco o di un suo metabolita, cui seguono due principali vie: una che procede attraverso la generazione di radicali liberi, in grado di danneggiare direttamente i componenti cellulari, o che possono a loro volta reagire con l\u2019ossigeno e generare specie reattive dell\u2019ossigeno (radicali perossidici, perossido di idrogeno, radicali ossidrilici); la seconda attraverso il trasferimento di energia o di un elettrone dal farmaco eccitato all\u2019ossigeno con la generazione, rispettivamente, di ossigeno singoletto o di anione superossido, che, a loro volta, provocano l'ossidazione di biomolecole e quindi il danno cellulare. Farmaci fotosensibilizzanti possono anche dar luogo alla formazione di prodotti stabili di fotodegradazione, responsabili essi stessi della fototossicit\ue0 in vivo. I bersagli molecolari coinvolti nelle reazioni di fotosensibilizzazione sono principalmente i lipidi, le proteine, il DNA e le membrane lisosomiali. Quando, in seguito alla modifica fotochimica delle proteine, si genera un antigene, le reazioni fotosensibilizzanti coinvolgono il sistema immunitario e sono di tipo fotoallergico, meno frequenti. Uno studio approfondito dei meccanismi farmacologici e fotobiologici di un farmaco fotosensibilizzante pu\uf2 essere dunque di grande utilit\ue0 per prevedere tale reazione avversa anche in molecole simili e per poter prendere le opportune precauzioni per il paziente: la scelta di un farmaco non fotosensibile, in rapporto al tipo di esposizione, storia clinica, stagionalit\ue0, o la protezione dall\u2019esposizione alle radiazioni solari, pu\uf2 infatti contribuire a ridurre questa reazione avversa

Photodegradation of drugs of abuse in hair

Hair analysis is a valuable tool in clinical and forensic toxicology to demonstrate drug exposure when cases of chronic intoxication, use, abuse, or single dose consumption need to be diagnosed in the context of facilitated crimes, withdrawal controls, doping controls, or workplace drug testing, with a large window from weeks to months/years for drug detection. However, scalp hair is exposed to sunlight and/or artificial light for many hours per day; hence, the action of light on hair could alter the content of drugs/illicit drugs and/or metabolites and the xenobiotics can gradually disappear from the hair shaft or be transformed into other compounds having different structure from the parent molecule. Thus, light exposure should be considered as a potential confounder in studies investigating xenobiotics in hair giving rise to reduced drug concentrations or even false negative results. On the other hand, the formation of new photodegradation products could lead to the identification of new markers of abuse useful in forensic evaluations. Although the importance of the potentially detrimental effect of light on the exogenous molecules present in the hair shaft is being recognized, very few studies are actually present in the literature about the photodecomposition of illicit drugs in this precious biological specimen

Photolysis of cocaine, morphine, D9-tetrahydrocannabinol and EDDP in water solution.

In the frame of an ongoing project aimed at understanding the mechanisms of photodegradation and characterizing the phototoxicological profile of drugs and illicit drugs and their photosensitizing role in the biological systems [1-4], the photostability towards UVA and UVB light of cocaine, \uf0449-tetrahydrocannabinol (THC), morphine and EDDP in water solution has been studied. The extent of photolysis and the kind of photoproducts formed have been studied by UV-Vis spectrophotometric analysis, HPLC chromatography, HRMS, and HRMS/MS analysis for accurate mass measurement of small molecules. HPLC analysis allowed us to calculate the yield of photolysis; in particular, Cocaine showed photolysis under high UV light doses, forming demethylated and hydroxylated photoproducts, and the inactive metabolite ecgonyne methyl ester, as already reported in the literature under solar irradiation and photocatalytic processes [5]. Morphine proved also to be unstable under UVB light and, in lower extent, under UVA. It forms various photoproducts, some of them corresponding to oxidation products or deriving from the addition of water to the 7,8 double bond. Also the absorption spectrum of EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine), the main inactive metabolite of methadone, changes under increasing light doses, with the appearance of a new band between 300 and 350 nm. Under both UVA and UVB irradiation, it forms various degradation products, one of them dehydrogenated and another one losing also the ethyl group. For another one we suppose the loss of one of the two aromatic rings. THC also showed photolability under both UVA and UVB light. Its photodegradation products are still under investigation. Since the photo excited drugs could photo react directly with biological substrates, produce free radicals and reactive oxygen species , or even give rise to toxic photoproducts, particularly oxidation compounds, the phototoxicological profile of illicit drugs and their photoproducts covers particular importance in the study of the toxicity/ phototoxicity of compounds in the aquatic environment