Mucosa-penetrating liposomes for esophageal local drug delivery.

Local drug delivery to the esophagus is hampered by rapid transit time and poor permeability of the mucosa. If some strategies aimed to improve the residence time have been proposed, non-invasive approaches to increase the drug penetration in the mucosa have not been described so far. Herein, we designed mucosa-penetrating liposomes to favor the penetration and retention of curcumin (CURC) in the esophagus. A novel mucosa penetrating peptide (MPP), SLENKGP, was selected by Phage Display and conjugated to pegylated liposomes at different PEG and MPP's surface densities. Pegylation assured a long residence time of liposomes (at least 30 min) in the esophagus in vivo, but it did not favor the penetration of CURC in the mucosa. MPP-decorated liposomes instead delivered a significant higher amount of CURC in the mucosa compared to naked pegylated liposomes. Confocal microscopy studies showed that naked pegylated liposomes remain confined in the superficial layers of the mucosa whereas MPP-decorated liposomes penetrate the whole epithelium. In vitro, MPP reduced the interaction of PEG with mucin, meanwhile favoring the paracellular penetration of liposomes across epithelial cell multilayers. In conclusion, pegylated liposomes represent a valid approach to target the esophagus and the surface functionalization with MPP enhances their penetration in the mucosa.

Endothelial cellular senescence and tau accumulation: An interplay full of opportunities?

Recent research has shown that tau protein can be passed to neighboring cells, leading to cellular senescence in the endothelial cells present in the central nervous system (CNS). This discovery could potentially open new doors for testing novel therapeutic compounds that specifically target senescent cells (senolytics) or for identifying new biomarkers that can enable early detection of tauopathies and dementia.

Nanoparticle delivery through the BBB in central nervous system tuberculosis.

Recent advances in Nanotechnology have revolutionized the production of materials for biomedical applications. Nowadays, there is a plethora of nanomaterials with potential for use towards improvement of human health. On the other hand, very little is known about how these materials interact with biological systems, especially at the nanoscale level, mainly because of the lack of specific methods to probe these interactions. In this review, we will analytically describe the journey of nanoparticles (NPs) through the brain, starting from the very first moment upon injection. We will preliminarily provide a brief overlook of the physicochemical properties of NPs. Then, we will discuss how these NPs interact with the body compartments and biological barriers, before reaching the blood-brain barrier (BBB), the last gate guarding the brain. Particular attention will be paid to the interaction with the biomolecular, the bio-mesoscopic, the (blood) cellular, and the tissue barriers, with a focus on the BBB. This will be framed in the context of brain infections, especially considering central nervous system tuberculosis (CNS-TB), which is one of the most devastating forms of human mycobacterial infections. The final aim of this review is not a collection, nor a list, of current literature data, as it provides the readers with the analytical tools and guidelines for the design of effective and rational NPs for delivery in the infected brain.

Gold and silver nanoparticles in Alzheimer's and Parkinson's diagnostics and treatments.

Neurodegenerative diseases (NDs) impose substantial medical and public health burdens on people worldwide and represent one of the major threats to human health. The prevalence of these age-dependent disorders is dramatically increasing over time, a process intrinsically related to a constantly rising percentage of the elderly population in recent years. Among all the NDs, Alzheimer's and Parkinson's are considered the most debilitating as they cause memory and cognitive loss, as well as severely affecting basic physiological conditions such as the ability to move, speak, and breathe. There is an extreme need for new and more effective therapies to counteract these devastating diseases, as the available treatments are only able to slow down the pathogenic process without really stopping or resolving it. This review aims to elucidate the current nanotechnology-based tools representing a future hope for NDs treatment. Noble metal nano-systems, that is, gold and silver nanoparticles (NPs), have indeed unique physicochemical characteristics enabling them to deliver any pharmacological treatment in a more effective way within the central nervous system. This can potentially make NPs a new hope for reversing the actual therapeutic strategy based on slowing down an irreversible process into a more effective and permanent treatment.

Shape-Driven Response of Gold Nanoparticles to X-rays.

Radiotherapy (RT) involves delivering X-ray beams to the tumor site to trigger DNA damage. In this approach, it is fundamental to preserve healthy cells and to confine the X-ray beam only to the malignant cells. The integration of gold nanoparticles (AuNPs) in the X-ray methodology could be considered a powerful tool to improve the efficacy of RT. Indeed, AuNPs have proven to be excellent allies in contrasting tumor pathology upon RT due to their high photoelectric absorption coefficient and unique physiochemical properties. However, an analysis of their physical and morphological reaction to X-ray exposure is necessary to fully understand the AuNPs' behavior upon irradiation before treating the cells, since there are currently no studies on the evaluation of potential NP morphological changes upon specific irradiations. In this work, we synthesized two differently shaped AuNPs adopting two different techniques to achieve either spherical or star-shaped AuNPs. The spherical AuNPs were obtained with the Turkevich-Frens method, while the star-shaped AuNPs (AuNSs) involved a seed-mediated approach. We then characterized all AuNPs with Transmission Electron Microscopy (TEM), Uv-Vis spectroscopy, Dynamic Light Scattering (DLS), zeta potential and Fourier Transform Infrared (FTIR) spectroscopy. The next step involved the treatment of AuNPs with two different doses of X-radiation commonly used in RT, namely 1.8 Gy and 2 Gy, respectively. Following the X-rays' exposure, the AuNPs were further characterized to investigate their possible physicochemical and morphological alterations induced with the X-rays. We found that AuNPs do not undergo any alteration, concluding that they can be safely used in RT treatments. Lastly, the actin rearrangements of THP-1 monocytes treated with AuNPs were also assessed in terms of coherency. This is a key proof to evaluate the possible activation of an immune response, which still represents a big limitation for the clinical translation of NPs.

Caffeic Acid-Grafted PLGA as a Novel Material for the Design of Fluvastatin-Eluting Nanoparticles for the Prevention of Neointimal Hyperplasia.

Drug-eluting nanoparticles (NPs) administered by an eluting balloon represent a novel tool to prevent restenosis after angioplasty, even if the selection of the suitable drug and biodegradable material is still a matter of debate. Herein, we provide the proof of concept of the use of a novel material obtained by combining the grafting of caffeic acid or resveratrol on a poly(lactide-co-glycolide) backbone (g-CA-PLGA or g-RV-PLGA) and the pleiotropic effects of fluvastatin chosen because of its low lipophilic profile which is challenging for the encapsulation in NPs and delivery to the artery wall cells. NPs made of such materials are biocompatible with macrophages, human smooth muscle cells (SMCs), and endothelial cells (ECs). Their cellular uptake is demonstrated and quantified by confocal microscopy using fluorescent NPs, while their distribution in the cytoplasm is verified by TEM images using NPs stained with an Ag-PVP probe appositely synthetized. g-CA-PLGA assures the best control of the FLV release from NP sizing around 180 nm and the faster SMC uptake, as demonstrated by confocal analyses. Interestingly and surprisingly, g-CA-PLGA improves the FLV efficacy to inhibit the SMC migration, without altering its effects on EC proliferation and migration. The improved trophism of NPs toward SMCs, combined with the excellent biocompatibility and low modification of the microenvironment pH upon polymer degradation, makes g-CA-PLGA a suitable material for the design of drug-eluting balloons.

A Multiscale Study of Phosphorylcholine Driven Cellular Phenotypic Targeting.

Phenotypic targeting requires the ability of the drug delivery system to discriminate over cell populations expressing a particular receptor combination. Such selectivity control can be achieved using multiplexed-multivalent carriers often decorated with multiple ligands. Here, we demonstrate that the promiscuity of a single ligand can be leveraged to create multiplexed-multivalent carriers achieving phenotypic targeting. We show how the cellular uptake of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-(diisopropylamino)ethyl methacry-late) (PMPC-PDPA) polymersomes varies depending on the receptor expression among different cells. We investigate the PMPC-PDPA polymersome insertion at the single chain/receptor level using all-atom molecular modeling. We propose a theoretical statistical mechanics-based model for polymersome-cell association that explicitly considers the interaction of the polymersome with the cell glycocalyx shedding light on its effect on the polymersome binding. We validate our model experimentally and show that the binding energy is a nonlinear function, allowing us to tune the interaction by varying the radius and degree of polymerization. Finally, we show that PMPC-PDPA polymersomes can be used to target monocytes in vivo due to their promiscuous interaction with SRB1, CD36, and CD81.

Synthesis and Assessment of the In Vitro and Ex Vivo Activity of Salicylate Synthase (Mbti) Inhibitors as New Candidates for the Treatment of Mycobacterial Infections.

Tuberculosis (TB) causes millions of deaths every year, ranking as one of the most dangerous infectious diseases worldwide. Because several pathogenic strains of M. tuberculosis (Mtb) have developed resistance against most of the established anti-TB drugs, new therapeutic options are urgently needed. An attractive target for the development of new anti-TB agents is the salicylate synthase MbtI, the first enzyme of the mycobacterial siderophore biochemical machinery, absent in human cells. In this work, a set of analogues of 5-(3-cyanophenyl)furan-2-carboxylic acid (I), the most potent MbtI inhibitor identified to date, was synthesized, characterized, and tested to further elucidate the structural requirements for achieving an efficient MbtI inhibition and potent antitubercular activity. The structure-activity relationships (SAR) discussed herein evidenced the importance of the side chain linked to the phenyl moiety to improve the in vitro antimycobacterial activity. In detail, 1f emerged as the most effective analogue against the pathogen, acting without cytotoxicity issues. To deepen the understanding of its mechanism of action, we established a fluorescence-based screening test to quantify the pathogen infectivity within host cells, using MPI-2 murine cells, a robust surrogate for alveolar macrophages. The set-up of the new assay demonstrates significant potential to accelerate the discovery of new anti-TB drugs.

Anti-spike antibodies and neutralising antibody activity in people living with HIV vaccinated with COVID-19 mRNA-1273 vaccine: a prospective single-centre cohort study.

BACKGROUND: Vaccines against COVID-19 are a powerful tool to control the current SARS-CoV-2 pandemic. A thorough description of their immunogenicity among people living with HIV (PLWHIV) is necessary. We aimed to assess the immunogenicity of the mRNA-1273 vaccine among PLWHIV. METHODS: In this prospective cohort, adult PLWHIV outpatients were enrolled during the Italian vaccination campaign. Enrolment was allowed irrespective of ongoing combination antiretroviral therapy (ART), plasma HIV viral load and CD4+ T cell count. A two-dose regimen of mRNA-1273, with administrations performed 28 days apart, was employed. The primary outcomes were anti-spike (anti-S) antibody titres and neutralising antibody activity, assessed 28 days after completing the vaccination schedule. A convenient sample of individuals not affected by HIV was also collected to serve as control (referred as healthy-donors, HDs). FINDINGS: We enrolled 71 PLWHIV, mostly male (84·5%), with a mean age of 47 years, a median CD4+ T cell count of 747·0 cells per µL and a median HIV viral load <50 copies/mL. COVID-19-experienced PLWHIV displayed higher anti-S antibody titres (p=0·0007) and neutralising antibody activity in sera (p=0·0007) than COVID-19-naïve PLWHIV. When stratified according to CD4+ T cell count (<350 cells/µL, 350-500 cells/µL, >500 cells/µL), anti-S antibody titres (6/71, median 2173 U/mL [IQR 987-4109]; 7/71, 5763 IU/mL [IQR 4801->12500]; 58/71, 2449 U/mL [IQR 1524-5704]) were not lower to those observed among HDs (10, median 1425 U/mL [IQR 599-6131]). In addition, neutralising antibody activity, stratified according to the CD4+ T cell count (6/71, median 1314 [IQR 606-2477]; 7/71, 3329 IU/mL [IQR 1905-10508]; 58/71, 1227 U/mL [IQR 761-3032]), was like those displayed by HDs (10, median 2112 U/mL [IQR 719-8889]). INTERPRETATION: In our cohort of PLWHIV with well-controlled ART, stable viral suppression and robust CD4+ T cell count, inoculation with mRNA-1273 vaccine given 4 weeks apart produced detectable humoral immune response, similar to individuals without HIV infection, supporting vaccination in PLWHIV. FUNDING: This study was partially supported by Italian Ministry of Health Ricerca Corrente 2021, by Intesa San Paolo COVID-19 emergency 2020 funds, and by Fondazione Cariplo Grant (INNATE-CoV).

Assessment of hepatic steatosis based on needle biopsy images from deceased donor livers.

BACKGROUND: Assessment of hepatic steatosis (HS) before transplantation requires the pathologist to read a graft biopsy. A simple method based on the evaluation of images from tissue samples with a smartphone could expedite and facilitate the liver selection. This study aims to assess the degree of HS by analysing photographic images from liver needle biopsy samples. METHODS: Thirty-three biopsy-images were acquired with a smartphone. Image processing was carried out using ImageJ: background subtraction, conversion to HSB colour space, segmentation of the biopsy area, and evaluation of statistical features of Hue, Saturation, Brightness, Red, Green, and Blue channels on the biopsy area. After feature extraction, correlations were made with gold standard HS percentage assessed at two levels (frozen-section vs glass-slide). Sensitivity, specificity, and accuracy were calculated for each feature. RESULTS: Correlations were found for H, S, R. The sensitivity, specificity, and accuracy of the final classifier based on the K* algorithm were 94%, 92%, 94%. LIMITATIONS: Accuracy assessment was performed considering macrovesicular steatosis on specimens with mostly < 30% HS. CONCLUSIONS: The steatosis assessment based on needle biopsy images, proved to be an effective and promising method. Deep learning approaches could also be experimented with a larger set of images.

Pre-Drawn Syringes of Comirnaty for an Efficient COVID-19 Mass Vaccination: Demonstration of Stability.

Moving towards a real mass vaccination in the context of COVID-19, healthcare professionals are required to face some criticisms due to limited data on the stability of a mRNA-based vaccine (Pfizer-BioNTech COVID-19 Vaccine in the US or Comirnaty in EU) as a dose in a 1 mL-syringe. The stability of the lipid nanoparticles and the encapsulated mRNA was evaluated in a "real-life" scenario. Specifically, we investigated the effects of different storing materials (e.g., syringes vs. glass vials), as well as of temperature and mechanical stress on nucleic acid integrity, number, and particle size distribution of lipid nanoparticles. After 5 h in the syringe, lipid nanoparticles maintained the regular round shape, and the hydrodynamic diameter ranged between 80 and 100 nm with a relatively narrow polydispersity (<0.2). Samples were stable independently of syringe materials and storage conditions. Only strong mechanical stress (e.g., shaking) caused massive aggregation of lipid nanoparticles and mRNA degradation. These proof-of-concept experiments support the hypothesis that vaccine doses can be safely prepared in a dedicated area using an aseptic technique and transferred without affecting their stability.

Engineering Polymeric Nanosystems against Oral Diseases.

Nanotechnology and nanoparticles (NPs) are at the forefront of modern research, particularly in the case of healthcare therapeutic applications. Polymeric NPs, specifically, hold high promise for these purposes, including towards oral diseases. Careful optimisation of the production of polymeric NPs, however, is required to generate a product which can be easily translated from a laboratory environment to the actual clinical usage. Indeed, considerations such as biocompatibility, biodistribution, and biodegradability are paramount. Moreover, a pre-clinical assessment in adequate in vitro, ex vivo or in vivo model is also required. Last but not least, considerations for the scale-up are also important, together with an appropriate clinical testing pathway. This review aims to eviscerate the above topics, sourcing at examples from the recent literature to put in context the current most burdening oral diseases and the most promising polymeric NPs which would be suitable against them.

Tuning cell behavior with nanoparticle shape.

We investigated how the shape of polymeric vesicles, made by the exact same material, impacts the replication activity and metabolic state of both cancer and non-cancer cell types. First, we isolated discrete geometrical structures (spheres and tubes) from a heterogeneous sample using density-gradient centrifugation. Then, we characterized the cellular internalization and the kinetics of uptake of both types of polymersomes in different cell types (either cancer or non-cancer cells). We also investigated the cellular metabolic response as a function of the shape of the structures internalized and discovered that tubular vesicles induce a significant decrease in the replication activity of cancer cells compared to spherical vesicles. We related this effect to the significant up-regulation of the tumor suppressor genes p21 and p53 with a concomitant activation of caspase 3/7. Finally, we demonstrated that combining the intrinsic shape-dependent effects of tubes with the delivery of doxorubicin significantly increases the cytotoxicity of the system. Our results illustrate how the geometrical conformation of nanoparticles could impact cell behavior and how this could be tuned to create novel drug delivery systems tailored to specific biomedical application.

Combinatorial Intracellular Delivery Screening of Anticancer Drugs.

Conventional drug solubilization strategies limit the understanding of the full potential of poorly water-soluble drugs during drug screening. Here, we propose a screening approach in which poorly water-soluble drugs are entrapped in poly(2-(methacryloyloxyethyl phosphorylcholine)-poly(2-(diisopropylaminoethyl methacryate) (PMPC-PDPA) polymersomes (POs) to enhance drug solubility and facilitate intracellular delivery. By using a human pediatric glioma cell model, we demonstrated that PMPC-PDPA POs mediated intracellular delivery of cytotoxic and epigenetic drugs by receptor-mediated endocytosis. Additionally, when delivered in combination, drug-loaded PMPC-PDPA POs triggered both an enhanced drug efficacy and synergy compared to that of a conventional combinatorial screening. Hence, our comprehensive synergy analysis illustrates that our screening methodology, in which PMPC-PDPA POs are used for intracellular codelivery of drugs, allows us to identify potent synergistic profiles of anticancer drugs.

On the shuttling across the blood-brain barrier via tubule formation: Mechanism and cargo avidity bias.

The blood-brain barrier is made of polarized brain endothelial cells (BECs) phenotypically conditioned by the central nervous system (CNS). Although transport across BECs is of paramount importance for nutrient uptake as well as ridding the brain of waste products, the intracellular sorting mechanisms that regulate successful receptor-mediated transcytosis in BECs remain to be elucidated. Here, we used a synthetic multivalent system with tunable avidity to the low-density lipoprotein receptor-related protein 1 (LRP1) to investigate the mechanisms of transport across BECs. We used a combination of conventional and super-resolution microscopy, both in vivo and in vitro, accompanied with biophysical modeling of transport kinetics and membrane-bound interactions to elucidate the role of membrane-sculpting protein syndapin-2 on fast transport via tubule formation. We show that high-avidity cargo biases the LRP1 toward internalization associated with fast degradation, while mid-avidity augments the formation of syndapin-2 tubular carriers promoting a fast shuttling across.

Combinatorial entropy behaviour leads to range selective binding in ligand-receptor interactions.

From viruses to nanoparticles, constructs functionalized with multiple ligands display peculiar binding properties that only arise from multivalent effects. Using statistical mechanical modelling, we describe here how multivalency can be exploited to achieve what we dub range selectivity, that is, binding only to targets bearing a number of receptors within a specified range. We use our model to characterise the region in parameter space where one can expect range selective targeting to occur, and provide experimental support for this phenomenon. Overall, range selectivity represents a potential path to increase the targeting selectivity of multivalent constructs.

Real-time imaging of polymersome nanoparticles in zebrafish embryos engrafted with melanoma cancer cells: Localization, toxicity and treatment analysis.

BACKGROUND: The developing zebrafish is an emerging tool in nanomedicine, allowing non-invasive live imaging of the whole animal at higher resolution than is possible in the more commonly used mouse models. In addition, several transgenic fish lines are available endowed with selected cell types expressing fluorescent proteins; this allows nanoparticles to be visualized together with host cells. METHODS: Here, we introduce the zebrafish neural tube as a robust injection site for cancer cells, excellently suited for high resolution imaging. We use light and electron microscopy to evaluate cancer growth and to follow the fate of intravenously injected nanoparticles. FINDINGS: Fluorescently labelled mouse melanoma B16 cells, when injected into this structure proliferated rapidly and stimulated angiogenesis of new vessels. In addition, macrophages, but not neutrophils, selectively accumulated in the tumour region. When injected intravenously, nanoparticles made of Cy5-labelled poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate) (PEG-PDPA) selectively accumulated in the neural tube cancer region and were seen in individual cancer cells and tumour associated macrophages. Moreover, when doxorubicin was released from PEG-PDPA, in a pH dependant manner, these nanoparticles could strongly reduce toxicity and improve the treatment outcome compared to the free drug in zebrafish xenotransplanted with mouse melanoma B16 or human derived melanoma cells. INTERPRETATION: The zebrafish has the potential of becoming an important intermediate step, before the mouse model, for testing nanomedicines against patient-derived cancer cells. FUNDING: We received funding from the Norwegian research council and the Norwegian cancer society.

Polymersomes Eradicating Intracellular Bacteria.

Mononuclear phagocytes such as monocytes, tissue-specific macrophages, and dendritic cells are primary actors in both innate and adaptive immunity. These professional phagocytes can be parasitized by intracellular bacteria, turning them from housekeepers to hiding places and favoring chronic and/or disseminated infection. One of the most infamous is the bacteria that cause tuberculosis (TB), which is the most pandemic and one of the deadliest diseases, with one-third of the world's population infected and an average of 1.8 million deaths/year worldwide. Here we demonstrate the effective targeting and intracellular delivery of antibiotics to infected macrophages both in vitro and in vivo, using pH-sensitive nanoscopic polymersomes made of PMPC-PDPA block copolymer. Polymersomes showed the ability to significantly enhance the efficacy of the antibiotics killing Mycobacterium bovis, Mycobacterium tuberculosis, and another established intracellular pathogen, Staphylococcus aureus. Moreover, they demonstrated to easily access TB-like granuloma tissues-one of the harshest environments to penetrate-in zebrafish models. We thus successfully exploited this targeting for the effective eradication of several intracellular bacteria, including M. tuberculosis, the etiological agent of human TB.

Gastrointestinal Perforations in Adult Whole-Liver Transplant Patients: Clinical, Radiologic, and Histopathologic Analysis.

BACKGROUND: Gastrointestinal perforation (GIP) is a rare complication after adult liver transplant (LT) associated with high morbidity and mortality. Limited data are available about clinical risk factors and underlying pathogenic mechanisms. METHODS: The retrospective study included all GIP cases from a consecutive cohort of 361 LT recipients during the period 2005-2017. Clinical variables were investigated as potential risk factors for GIP, and radiologic and histopathologic evaluations were undertaken to identify any causative mechanism. RESULTS: A total of 22 patients developed at least 1 episode of GIP (prevalence 6.1%) at a median time of 18.5 [interquartile range, 12.5-28.5] days after LT. The perforations occurred in the small bowel (63.6%), transverse colon (27.3%), right colon (22.7%), left colon (9.1%), and stomach (9.1%). A total of 27.3% of patients developed multiple sites of GIP, and in 31% GIP recurred after curative surgery. The 30-day mortality rate after relaparotomy was 40%. A history of previous abdominal surgery (odds ratio, 2.5) and early post-LT relaparotomy due to other complications (odds ratio, 2.6) were significant risk factors for GIP. No thromboembolic or steno-occlusive complications of any splanchnic vessel were detected at computed tomography scan, while histopathology examination on perforated gastrointestinal segments excluded cytomegalovirus infection, graft-vs-host disease, and inflammatory bowel disease. In all the cases, ischemic necrosis with aspecific microangiopathy and microembolization were the pathologic features detected. CONCLUSIONS: GIP is a severe complication after LT with frequent multiple gastrointestinal involvement and recurrence after curative surgery. The pathologic underlying mechanism is usually microvascular ischemia. Clinical risk factors are history of previous abdominal surgery and early post-LT relaparotomy.

Molecular bionics - engineering biomaterials at the molecular level using biological principles.

Life and biological units are the result of the supramolecular arrangement of many different types of molecules, all of them combined with exquisite precision to achieve specific functions. Taking inspiration from the design principles of nature allows engineering more efficient and compatible biomaterials. Indeed, bionic (from bion-, unit of life and -ic, like) materials have gained increasing attention in the last decades due to their ability to mimic some of the characteristics of nature systems, such as dynamism, selectivity, or signalling. However, there are still many challenges when it comes to their interaction with the human body, which hinder their further clinical development. Here we review some of the recent progress in the field of molecular bionics with the final aim of providing with design rules to ensure their stability in biological media as well as to engineer novel functionalities which enable navigating the human body.