Smart design and in vitro testing of nanoparticles for microenvironmentally-triggered extracellular drug release

In the field of nanotechnology, one of the most operative research areas is nanomedicine, which applies nanotechnology to highly specific medical interventions for the prevention, diagnosis and treatment of diseases. Currently, the major issue that nanomedicine needs to face is the smart design and production of nanoparticles (NPs) based drug delivery systems for cancer therapy. Highly efficient drug delivery based on nanoparticles could potentially reduce the drug dose needed to achieve therapeutic benefit, thus reducing the side effects associated with the systemic delivery of drugs, whit great benefit to the patient. Indeed, a site-specific delivery of the active compound can be obtained manipulating NP surface by attaching ligands, such as peptides, antibodies or aptamers. Moreover, both passive and active targeting of the drug can be easily obtained by manipulating NP size and surface characteristics. NPs can also control and sustain the release of a drug during transport to, or at, the site of localization, altering drug distribution and subsequent clearance. At present, a new family of nanovectors, defined as stimuli-responsive nanocarriers (SRNs), is emerging. The key point in their mechanism of action lay in the fact that a specific cellular or extracellular endogenous stimulus of chemical, biochemical, or physical origin can modify NP conformation thus promoting the release of the active agent in a specific biological environment [1] [2]. In particular, a large variety of enzymes, such as proteases, glucuronidase, or carboxylesterases can be used as biochemical triggers. Generally the proteases, that are extracellularly expressed, such as the matrix metalloproteases (MMPs), are up-regulated in tumour microenvironment and are responsible for the proteolysis of the extracellular matrix (ECM) and of the basement membranes along with tissue remodelling and metastasis invasion. Since that, they are commonly identified as biomarkers of malignant tissues [3]. In the light of these considerations, Chapter.1 points out a smart approach in NPs design that takes benefits from the MMPs over-expression at tumour site, in order to produce a stimuli-responsive nanocarrier that allows a site specific drug release. To this aim, we proposed the use of a novel nanoparticle able to carry safely doxorubicin (Dox) at tumour tissues, and to respond to MMP-2 enzyme. The produced NPs are made up of a biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) – block – PEG copolymer (namely PELGA), blended with a TAP (Tumour Activated Pro-drug) composed by a MMP-2-sensitive peptide bound to Dox at the C-terminus and to PLGA molecule at the N-terminus. These NPs are named PELGA-TAP NPs. The presence of the MMP-2 enzyme in situ, leads to the destruction of the bond between the peptide and the Dox, with the consequent diffusion and accumulation of the drug in the extracellular environment. This mechanism allows the drug delivery only in presence of an endogenous stimulus that comes from the very nature of the tumour tissue itself. Furthermore, the same NPs were prepared without the presence of the peptide sequence, as negative control, and were named PELGA-Dox. Spheroids of U87 (Human Glioma cells) and HDF (Human Dermal Fibroblast) cells were used as in vitro models of tumour and healthy tissue, respectively, to demonstrate NPs ability to “sense” the differences in the expression levels of endogenous MMP-2 enzymes [4]. Since the production process and effectiveness of PELGA-TAP and PELGA-Dox NPs was well established and consolidate, in Chapter.2 we tested them in a new three-dimensional microtissue (3D µTP) model, which is an in vitro tissue equivalent proposed by Brancato et al. [5]. They fabricate µTPs with the aim to replicate in vitro the composition and the functionalities of the tumour microenvironment. In this work they clearly show that µTPs better recapitulate the important differences existing in vivo between normal and cancer-activated stroma representing a more suitable system to mimic in vitro the tumour microenvironment. In particular, the 3D model was developed using normal fibroblasts (NF) and human epithelial cell lines (MCF10), or cancer-activated fibroblasts (CAF) and human breast adenocarcinoma cells (MCF7), to produce healthy and cancer microtissues, respectively. In this scenario, PELGA-TAP and PELGA-Dox NPs were tested in terms of Dox release on these µTPs in order to further validate their efficacy and selective drug release in a more realistic in vitro model, which better resemble tumour microenvironment, closer to the in vivo conditions [6]. Moreover, Chapter.3 shows an upgrade of the PELGA-TAP NP presented above. The approach used for the production of the nanocarrier takes advantages from the layer by layer polymer deposition technique developed and optimized by Vecchione et al. [7]. This technique allows the production of a very stable nanocarrier able to load large amounts of hydrophobic drugs and prevents their systemic leakage. The delivery system we proposed is a crosslinked polyelectrolytes nanocapsule (NC) based on an oil-core and a matrix metalloproteases-2-sensitive shell. MMP-2 enzymes catalyse the disassembly of the NC, which is stabilized by a MMP-2-cleavable peptide sequence as cross-linker. Also in this case, the drug release occurs in a spatially-controlled fashion upon an endogenous stimulus coming from the very nature of the tumour itself. The same NC was also produced with a scrambled peptide sequence as negative control. These NCs were tested on a spheroidal in vitro model, in order to proof their selective shell destabilization and consequent stimuli-responsive drug release in tumour microenvironment. Spheroids of U87 and HDF were used as models of tumour and healthy tissue, respectively. Cell viability was evaluated by means of Alamar Blue Assay. Moreover, the selective disassembly of the NC shell was followed using confocal microscopy and colocalization analyses were also performed. Finally, in Chapter.4 preliminary studies aimed to point out the advantages of an extracellular drug delivery are presented

Use of Sugar Dispensers to Disrupt Ant Attendance and Improve Biological Control of Mealybugs in Vineyard

Planococcus ficus (Signoret) and Pseudococcus comstocki (Kuwana) (Hemiptera: Pseudococcidae) are economically important pests occurring in vineyards, causing severe economic losses for growers and compromising bunch production. The partial effectiveness of insecticides used in controlling mealybug infestations as well as their high impact on the environment and on human health have led to the research of alternative and sustainable control methods, including biological control. Several natural enemies are reported to be effective against mealybugs, but their activity may be hindered by tending ants. These social insects are known to exhibit a mutualistic relationship with mealybugs, resulting in extremely aggressive behavior against beneficial insects. Consequently, this study explored a method to mitigate ant attendance by means of sugar dispensers in order to improve ecosystem services, as well as decrease mealybug infestation in vineyards. Field trials were carried out in four commercial vineyards of Northern Italy infested by mealybugs, in which Anagyrus vladimiri Triapitsyn (Hymenoptera: Encyrtidae) and Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) were released as biological control agents. Our results showed that sugar dispensers reduced ant activity and mealybug infestation, leading to a significant enhancement of ecosystem services. The technique showed a great potential in boosting biological control against mealybugs in field conditions, though the field application seemed to be labour intensive and needs to be replicated for a multi-year evaluation

Design of biodegradable bi-compartmental microneedles for the stabilization and the controlled release of the labile molecule collagenase for skin healthcare.

Polymeric microneedles (MNs) have emerged as a novel class of drug delivery system thanks to their ability in penetrating the skin with no pain, encapsulate active proteins and in particular, proposed bicompartimental MNs can tune protein release

Cell Membrane-Coated Oil in Water Nano-Emulsions as Biomimetic Nanocarriers for Lipophilic Compounds Conveyance

Recently, we developed ultra-stable oil in water nano-emulsions (O/W NEs), able to carry both internal and external cargos (Somes), such as lipophilic compounds and hydrophilic coatings, respectively, that we call here NEsoSomes. O/W NEs are an excellent bioengineering tool for drug and molecules delivery, due to their ability to dissolve a large number of hydrophobic compounds and protect them from hydrolysis and degradation under biological conditions. At present, no report is available on the combination of cell membrane coatings with such nanocarriers, probably due to their typical instability feature. Since then, we have reported, for the first time, a new cell membrane (CM)-coated nanomaterial composed of membranes extracted from glioblastoma cancer cells (U87-MG) deposited on NEsoSomes, through a liquid–liquid interface method, to produce highly controllable membrane caked nano-capsules, namely CM-NEsoSomes. CM-NEsoSomes were physically characterized by dynamic light scattering (DLS) over time and their correct morphology was analyzed by confocal and transmission electron microscopy (TEM) microscopy. Moreover, CM-NEsoSomes biocompatibility was tested on the healthy model cell line, performing cell cytotoxicity and uptake assay, showing nanocarriers uptake by cells with no induced cytotoxicity

Use of Sugar Dispensers to Disrupt Ant Attendance and Improve Biological Control of Mealybugs in Vineyard

Planococcus ficus (Signoret) and Pseudococcus comstocki (Kuwana) (Hemiptera: Pseudococcidae) are economically important pests occurring in vineyards, causing severe economic losses for growers and compromising bunch production. The partial effectiveness of insecticides used in controlling mealybug infestations as well as their high impact on the environment and on human health have led to the research of alternative and sustainable control methods, including biological control. Several natural enemies are reported to be effective against mealybugs, but their activity may be hindered by tending ants. These social insects are known to exhibit a mutualistic relationship with mealybugs, resulting in extremely aggressive behavior against beneficial insects. Consequently, this study explored a method to mitigate ant attendance by means of sugar dispensers in order to improve ecosystem services, as well as decrease mealybug infestation in vineyards. Field trials were carried out in four commercial vineyards of Northern Italy infested by mealybugs, in which Anagyrus vladimiri Triapitsyn (Hymenoptera: Encyrtidae) and Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) were released as biological control agents. Our results showed that sugar dispensers reduced ant activity and mealybug infestation, leading to a significant enhancement of ecosystem services. The technique showed a great potential in boosting biological control against mealybugs in field conditions, though the field application seemed to be labour intensive and needs to be replicated for a multi-year evaluation

Engineered PLGA-PVP/VA based formulations to produce electro-drawn fast biodegradable microneedles for labile biomolecule delivery

Biodegradable polymer microneedles (MNs) are recognized as non-toxic, safe and stable systems for advanced drug delivery and cutaneous treatments, allowing a direct intradermal delivery and in some cases a controlled release. Most of the microneedles found in the literature are fabricated by micromolding, which is a multistep thus typically costly process. Due to industrial needs, mold-free methods represent a very intriguing approach in microneedle fabrication. Electro-drawing (ED) has been recently proposed as an alternative fast, mild temperature and one-step strategy to the mold-based techniques for the fabrication of poly(lactic-co-glycolic acid) (PLGA) biodegradable MNs. In this work, taking advantage of the flexibility of the ED technology, we engineered microneedle inner microstructure by acting on the water-in-oil (W/O) precursor emulsion formulation to tune drug release profile. Particularly, to promote a faster release of the active pharmaceutical ingredient, we substituted part of PLGA with poly(1-vinylpyrrolidone-co-vinyl acetate) (PVP/VA), as compared to the PLGA alone in the matrix material. Moreover, we introduced lecithin and maltose as emulsion stabilizers. Microneedle inner structural analysis as well as collagenase entrapment efficiency, release and activity of different emulsion formulations were compared to reach an interconnected porosity MN structure, aimed at providing an efficient protein release profile. Furthermore, MN mechanical properties were examined as well as its ability to pierce the stratum corneum on a pig skin model, while the drug diffusion from the MN body was monitored in an in vitro collagen-based dermal model at selected time points

Influence of demographic and organizational factors on the length of hospital stay in a general medicine department

Unnecessary Length of Hospital Stay (LOS) has significant consequences on the economy of the national healthcare system. Numerous factors may influence LOS, such as bad management of resources, beds and surgery procedures. In this work we investigate, among the demographic, clinical and organizational variables, those most affecting the LOS, through the use of Multiple Linear Regression model. Data of 262 patients were collected from the hospital information system of the General Medicine Department of the University L.P-o Hospital “San Giovanni di Dio and Ruggi d’Aragona” of Salerno. The regression model has been tested and optimized by selecting the most appropriate predictors and by finding the best trade-off between the number of independent variables and the absence of multicollinearity in the data. Results show that the variables influencing LOS were the gender, the number of procedures and the discharge modality

Trend of the LOS for patients suffering from different kidney injuries

Maintenance of kidney health is a global priority. This reflects the vital role that the kidneys play. Indeed, although often underestimated, the kidneys, perform various functions essential for our survival as maintaining water homeostasis through the removal of excess fluids and being responsible for the removal of toxic substances. Furthermore, they are involved in the regulation of blood pressure, the production of erythropoietin, activation of vitamin D to maintain bone health and the acid-base balance control to maintain the blood pH within normal values. Kidney damage at date is a very common condition and its identification and management are necessary in order to prevent its progression, comorbidities outcomes, reduce the mortality risks improving patient safety and medicines management. In this scenario, we propose a case study at University Hospital “San Giovanni di Dio e Ruggi d’Aragona” in Salerno (Italy) in the period between October 2018 and December 2020 on 847 patients with different kidney injuries considering their age and the presence of related pathologies. Aim of our wok is a better understand of the length of hospital stay (LOS) in order to improve the prediction of the disease stage, the quality of life of patients reducing hospitalization and mortality. Furthermore, our final goal is to understand the key mechanism related to the managing of patients reducing the costs

Trend of the LOS for patients suffering from different kidney injuries

Maintenance of kidney health is a global priority. This reflects the vital role that the kidneys play. Indeed, although often underestimated, the kidneys, perform various functions essential for our survival as maintaining water homeostasis through the removal of excess fluids and being responsible for the removal of toxic substances. Furthermore, they are involved in the regulation of blood pressure, the production of erythropoietin, activation of vitamin D to maintain bone health and the acid-base balance control to maintain the blood pH within normal values. Kidney damage at date is a very common condition and its identification and management are necessary in order to prevent its progression, comorbidities outcomes, reduce the mortality risks improving patient safety and medicines management. In this scenario, we propose a case study at University Hospital “San Giovanni di Dio e Ruggi d’Aragona” in Salerno (Italy) in the period between October 2018 and December 2020 on 847 patients with different kidney injuries considering their age and the presence of related pathologies. Aim of our wok is a better understand of the length of hospital stay (LOS) in order to improve the prediction of the disease stage, the quality of life of patients reducing hospitalization and mortality. Furthermore, our final goal is to understand the key mechanism related to the managing of patients reducing the costs

Impact of diagnostic techniques on the length of stay in emergency medicine

Emergency medicine is a new discipline that is rapidly developing and spreading in medical practice. The central themes of emergency medicine are resuscitation, laboratory and diagnostic imaging. In the emergency department, different parameters can be associated with a prolonged Length of Stay (LOS) as, for example; the protact use of computed tomography (TAC), radiology techniques, the need for advice from external consultants (1). To improve the efficiency of the emergency department and the assessment effectiveness of the state of healthy patient, it is important to identify the factors that affect the LOS (2).This work was based on the evaluation of the impact of demographic factors, clinical information and diagnostic techniques on the LOS in emergency medicine (LOS-ED). The dataset was carried out at the Emergency Medicine Unit of the hospital “San Giovanni di Dio e Ruggi d’Aragona” of Salerno. Multiple Linear Regression model was optimized considering the hospital stay after diagnostic procedures (dLOS) as dependent variable