Some things old, new and borrowed: Delivery of dabrafenib and vemurafenib to melanoma cells via self-assembled nanomicelles based on an amphiphilic dendrimer

Two clinically approved anticancer drugs targeting BRAF in melanoma patients - dabrafenib (DAB) and vemurafenib (VEM) - have been successfully encapsulated into nanomicelles formed upon self-assembly of an amphiphilic dendrimer AD based on two C18 aliphatic chains and a G2 PAMAM head. The process resulted in the formation of well-defined (∼10 nm) core-shell nanomicelles (NMs) with excellent encapsulation efficiency (∼70% for DAB and ∼60% for VEM) and good drug loading capacity (∼27% and ∼24% for DAB and VEM, respectively). Dynamic light scattering (DLS), transmission electron microscopy (TEM), small-angle x-ray scattering (SAXS), nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), and molecular simulation (MS) experiments were used, respectively, to determine the size and structure of the empty and drug-loaded nanomicelles (DLNMs), along with the interactions between the NMs and their cargoes. The in vitro release data revealed profiles governed by Fickian diffusion; moreover, for both anticancer molecules, an acidic environment (pH = 5.0) facilitated drug release with respect to physiological pH conditions (pH = 7.4). Finally, both DAB- and VEM-loaded NMs elicited enhanced response with respect to free drug treatments in 4 different melanoma cell lines

Sustainable Immobilized Biocatalysts for Industrial Processes

The thesis is part of the EU-ITN INTERfaces project and the current project addressed the challenge of transforming immobilized enzymes into efficient and sustainable industrial biocatalysts. To achieve these objectives, a new bio-based crosslinking agent (2,5-Furandicarboxaldehyde, abbreviated as DFF) was developed with the aim to replace the fossil-based, volatile and toxic glutaraldehyde (GA). The efficiency of DFF was demonstrated in the hydrolysis of maltose catalyzed by glucoamylase from Aspergillus niger immobilized on an amino-functionalized poly(methyl methacrylate) carrier activated with DFF, which displayed high stability both in batch and in continuous flow settings, comparable to results observed for the GA-immobilized enzyme. The reactivity of DFF was also investigated, demonstrating that in water, unlike GA, it reacts with amines forming only stable imine bonds. The work on enzyme immobilization with glutaraldehyde led to a publication. In the second part of the project, rice husk was investigated as a renewable and inexpensive enzyme carrier. The lignocellulosic material was characterized using stereoscopical and SEM microscopy, porosimetry, contact angle measures. Its biodegradability in marine environment was also assessed. Rice husk was subject to delignification, in order to modify its hydrophilicity, and different pre-treatments were applied for introducing functional groups able to form covalent bonds with the enzymes. Glucoamylase and two industrially relevant lipases (TLL and CaLB) were immobilized using different immobilization techniques: (a) adsorption and crosslinking (with both DFF and GA); (b) covalent immobilization on rice husk functionalized by chemo- or enzymatic oxidation. The immobilization of lipases via adsorption and crosslinking displayed good hydrolytic activity. On the other hand, the immobilization of glucoamylase was unsuccessful with both tested techniques. This was attributed to the surface interactions between the highly glycosylated and hydrophilic enzyme and the rice husk, which presents the hydrophobic lignin on the surface. Moreover, the efficiency of samples of TLL physically immobilized on rice husk with the aid of binders was tested in the solvent-free interesterification of triglycerides, reaction used for the industrial production of cocoa-butter analogues. The tested formulations showed good stability and were able to successfully catalyze the interesterification reaction.The thesis is part of the EU-ITN INTERfaces project and the current project addressed the challenge of transforming immobilized enzymes into efficient and sustainable industrial biocatalysts. To achieve these objectives, a new bio-based crosslinking agent (2,5-Furandicarboxaldehyde, abbreviated as DFF) was developed with the aim to replace the fossil-based, volatile and toxic glutaraldehyde (GA). The efficiency of DFF was demonstrated in the hydrolysis of maltose catalyzed by glucoamylase from Aspergillus niger immobilized on an amino-functionalized poly(methyl methacrylate) carrier activated with DFF, which displayed high stability both in batch and in continuous flow settings, comparable to results observed for the GA-immobilized enzyme. The reactivity of DFF was also investigated, demonstrating that in water, unlike GA, it reacts with amines forming only stable imine bonds. The work on enzyme immobilization with glutaraldehyde led to a publication. In the second part of the project, rice husk was investigated as a renewable and inexpensive enzyme carrier. The lignocellulosic material was characterized using stereoscopical and SEM microscopy, porosimetry, contact angle measures. Its biodegradability in marine environment was also assessed. Rice husk was subject to delignification, in order to modify its hydrophilicity, and different pre-treatments were applied for introducing functional groups able to form covalent bonds with the enzymes. Glucoamylase and two industrially relevant lipases (TLL and CaLB) were immobilized using different immobilization techniques: (a) adsorption and crosslinking (with both DFF and GA); (b) covalent immobilization on rice husk functionalized by chemo- or enzymatic oxidation. The immobilization of lipases via adsorption and crosslinking displayed good hydrolytic activity. On the other hand, the immobilization of glucoamylase was unsuccessful with both tested techniques. This was attributed to the surface interactions between the highly glycosylated and hydrophilic enzyme and the rice husk, which presents the hydrophobic lignin on the surface. Moreover, the efficiency of samples of TLL physically immobilized on rice husk with the aid of binders was tested in the solvent-free interesterification of triglycerides, reaction used for the industrial production of cocoa-butter analogues. The tested formulations showed good stability and were able to successfully catalyze the interesterification reaction

2,5-Furandicarboxaldehyde as a bio-based crosslinking agent replacing glutaraldehyde for covalent enzyme immobilization

In the quest for a bio-based and safer substitute for glutaraldehyde, we have investigated 2,5 diformylfuran (DFF) as bifunctional crosslinking agent for the covalent immobilization of glucoamylase on aminofunctionalized methacrylic resins. Immobilization experiments and systematic comparison with glutaraldehyde at four different concentrations for the activation step showed that DFF leads to comparable enzymatic activities at all tested concentrations. Continuous flow experiment confirms a similar long term stability of the immobilized formulations obtained with the two crosslinkers. The NMR study of DFF in aqueous solution evidenced a much simpler behaviour as compared to glutaraldehyde, since no enolic forms can form and only a mono-hydrated form was observed. Unlike in the case of glutaraldehyde, DFF reacts covalently with the primary amino groups via imine bond formation only. Nevertheless, the stability of the covalent immobilization was confirmed also at acidic pH (4.5), most probably because of the higher stability of the imine bonds formed with the aromatic aldehydes. In terms of toxicity DFF has the advantage of being poorly soluble in water and, more importantly, poorly volatile as compared to glutaraldehyde, which displays severe respiratory toxicity. We have performed preliminary ecotoxicity assays using Aliivibrio fischeri, a marine bacterium, evidencing comparable behaviour (below the toxicity threshold) for both dialdehydes at the tested concentrations

Ipilimumab experience in heavily pretreated patients with melanoma in an expanded access program at the University Hospital of Siena (Italy).

AIM OF STUDY: To evaluate the feasibility of ipilimumab treatment for metastatic melanoma outside the boundaries of clinical trials, in a setting similar to that of daily practice. METHODS: Ipilimumab was available upon physician request in the Expanded Access Programme for patients with life-threatening, unresectable stage III/IV melanoma who failed or did not tolerate previous treatments and for whom no therapeutic option was available. Induction treatment with ipilimumab 10 mg/kg was administered intravenously every 3 weeks, for a total of 4 doses, with maintenance doses every 12 weeks based on physicians' discretion and clinical judgment. Tumors were assessed at baseline, Week 12, and every 12 weeks thereafter per mWHO response criteria, and clinical response was scored as complete response (CR), partial response (PR), stable disease (SD), or progressive disease. Durable disease control (DC) was defined as SD at least 24 weeks from the first dose, CR, or PR. RESULTS: Disease control rate at 24 and 60 weeks was 29.6% and 15%, respectively. Median overall survival at a median follow-up of 8.5 months was 9 months. The 1- and 2-year survival rates were 34.8% and 23.5%, respectively. Changes in lymphocyte count slope and absolute number during ipilimumab treatment appear to correlate with clinical response and survival, respectively. Adverse events were predominantly immune related, manageable, and generally reversible. One patient died from pancytopenia, considered possibly treatment related. CONCLUSION: Ipilimumab was a feasible treatment for malignant melanoma in heavily pretreated, progressing patients. A sizeable proportion of patients experienced durable DC, including benefits to long-term survival

A phase II study of the L19IL2 immunocytokine in combination with dacarbazine in advanced metastatic melanoma patients

Engineered cytokine products represent promising agents for the treatment of immunogenic tumors, such as malignant melanoma, in addition to immune checkpoint inhibitors. Here we describe the results of a controlled, randomized phase II clinical trial, aimed at assessing the therapeutic potential of L19IL2, a fully human fusion protein consisting of the L19 antibody specific to the alternatively spliced extra-domain B of fibronectin, fused to human interleukin-2 in advanced metastatic melanoma. In one arm, patients received dacarbazine (DTIC; 1000 mg/m$^{2}$ of body surface on day 1 of 21-day cycles) as single agent, while in two other arms L19IL2 (22.5 million international units of IL2 equivalents) was added, based on two different schedules of administration. In total, 69 patients with stage IV melanoma were enrolled (24 in the dacarbazine arm, 23 and 22 in the other combination arms, respectively) and 67 received treatment. Analyses of efficacy results show a statistically significant benefit in terms of overall response rate and median progression-free survival for patients receiving L19IL2 in combination with DTIC, compared to DTIC as single agent. In light of these results, further clinical investigations with L19IL2 (alone or in combination with other agents) are warranted

The Participatory Methodology Adopted to Develop an mHealth App as an Educational Tool to Promote Organizational Health Literacy at a Maternal and Child Health Hospital

Patients and caregivers’ low health literacy and unmet health information needs may compromise an equitable access and navigation through the healthcare system and positive health outcomes. The aim of this three-year study was to describe the participatory methodology, characterized by the application of a user-centred approach, used to develop a mobile health application (MHA) as an educational tool to promote organizational health literacy at a maternal and child health hospital. More in detail, the MHA was designed to support the information and education of two specific categories of hospital end-users, that is expectant and new parents and their children in the first 1000 days of life, and caregivers of children undergoing otolaryngology surgery during the perioperative process. The Information System Research framework informed the participatory methodology illustrated in the study, thus providing an evidence-based foundation, allowing for an exploration of end-users’ information needs as well as a user-centred designing and development of the MHA content, functionalities, and technical features