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Polysaccharides are long chains of monosaccharides linked by glycosidic bonds. They are widely utilized in biomedical applications, for tissue engineering and wound management, and as excipients of pharmaceutical formulations. In cancer therapy, the development of nanoscale drug delivery systems aims at addressing issues related to the low efficacy of chemotherapeutics and protein drugs due to poor solubility and stability, and to off-target effects that severely affect patients’ body conditions. Biocompatibility, availability of functional groups, amenability of chemical derivatisation and multifunctional conjugation with drugs and targeting ligands make polysaccharide nanoparticles interesting drug delivery devices for cancer treatment. Targeting and controlling the drug release at the tumour site are promising approaches to maximise the anti-tumour effects and reduce side-effects. Xyloglucan is a highly branched, hydrophilic polysaccharide composed of a -D-glucan main chain and -D-xylose branches, partially substituted by -D-galactoxylose. Xyloglucan extracted from tamarind seeds (TS-XG) is commercially available, non-toxic, biodegradable and FDA-approved as food additive. Due to the high molecular weight and tendency to associate in ribbon-like aggregates, aqueous colloidal dispersions of xyloglucan are characterised by relatively large nanoparticles with a broad size distribution. Gamma irradiation of either the colloidal dispersion of xyloglucan (XG) or the solid powder is a very effective and clean methodology to resize the molecular weight of the polysaccharide and reduce the particle size of its colloidal dispersions. When xyloglucan is irradiated as aqueous dispersion, irradiation causes extensive chain scission, with a dramatic reduction of molecular weight already at low doses (50-500 Gy). In contrast, when it is irradiated in the solid state, higher doses are required to change the molecular weight (20-40 kGy), with moisture playing a protective role. Selected systems have been conjugated to doxorubicin (DOX), a powerful anticancer drug that is extensively applied in the clinical treatment of human malignancies, such as leukemia and cancer of the liver, ovary and breast. This drug causes severe peripheral toxicity that poses limits to the maximum dosage that can be administered, and the emergence of multidrug resistance. DOX has been bound to irradiated XG through a cleavable bond and then further loaded with DOX by self-assembly. Xyloglucan contains galactose, which can be recognised and internalised by asialoglycoprotein receptor (ASGP-R). In order to increase the targeting efficiency, XG has been also conjugated to folic acid. Drug release efficiency and tumour cells selectivity are under investigation.

Over the last decades, nanotechnology applied in medicine (nanomedicine) has sparked great interest from the scientific community, thanks to the possibility to engineer nanostructured materials, including nanoparticles (NPs), for a specific application. Their small size confers them unique properties because they are subject to physical laws in the middle between classical and quantum physics. This review is proposed to explain better how to design a specific NP and clarify the relationship between the type, size, and shape of NPs and the specific medical applications. NPs are classified into inorganic (metallic NPs, quantum dots, carbon-based nanostructures, mesoporous silica NPs) and organic (liposomes and micelles, dendrimers, and polymer NPs). Here, we report an accurate description of the potential of each NPs type focusing on their multiple areas of application, including theranostics drug delivery, imaging, tissue engineering, antimicrobial techniques, and nanovaccines. All these features make NPs a promise to revolutionize the new era of nanomedicine.

Alzheimer’s Disease (AD) is the most common cause of dementia, having a remarkable social and healthcare burden worldwide. Amyloid β (Aβ) and protein Tau aggregates are disease hallmarks and key players in AD pathogenesis. However, it has been hypothesized that microglia can contribute to AD pathophysiology, as well. Microglia are CNS-resident immune cells belonging to the myeloid lineage of the innate arm of immunity. Under physiological conditions, microglia are in constant motion in order to carry on their housekeeping function, and they maintain an anti-inflammatory, quiescent state, with low expression of cytokines and no phagocytic activity. Upon various stimuli (debris, ATP, misfolded proteins, aggregates and pathogens), microglia acquire a phagocytic function and overexpress cytokine gene modules. This process is generally regarded as microglia activation and implies that the production of pro-inflammatory cytokines is counterbalanced by the synthesis and the release of anti-inflammatory molecules. This mechanism avoids excessive inflammatory response and inappropriate microglial activation, which causes tissue damage and brain homeostasis impairment. Once the pathogenic stimulus has been cleared, activated microglia return to the naïve, anti-inflammatory state. Upon repeated stimuli (as in the case of Aβ deposition in the early stage of AD), activated microglia shift toward a less protective, neurotoxic phenotype, known as “primed” microglia. The main characteristic of primed microglia is their lower capability to turn back toward the naïve, anti-inflammatory state, which makes these cells prone to chronic activation and favours chronic inflammation in the brain. Primed microglia have impaired defence capacity against injury and detrimental effects on the brain microenvironment. Additionally, priming has been associated with AD onset and progression and can represent a promising target for AD treatment strategies. Many factors (genetics, environmental factors, baseline inflammatory status of microglia, ageing) generate an aberrantly activated phenotype that undergoes priming easier and earlier than normally activated microglia do. Novel, promising targets for therapeutic strategies for AD have been sought in the field of microglia activation and, importantly, among those factors influencing the baseline status of these cells. The CX3CL1 pathway could be a valuable target treatment approach in AD, although preliminary findings from the studies in this field are controversial. The current review aims to summarize state of the art on the role of microglia dysfunction in AD pathogenesis and proposes biochemical pathways with possible targets for AD treatment.

Alzheimer’s disease (AD) is the most common form of cognitive decline worldwide, occurring in about 10% of people older than 65 years. The well-known hallmarks of AD are extracellular aggregates of amyloid β (Aβ) and intracellular neurofibrillary tangles (NFTs) of tau protein. The evidence that Aβ overproduction leads to AD has paved the way for the AD pathogenesis amyloid cascade hypothesis, which proposes that the neuronal damage is sustained by Aβ overproduction. Consistently, AD cerebrospinal fluid (CSF) biomarkers used in clinical practice, including Aβ 1–42, Aβ 1–40, Aβ 42/40 ratio, and pTau, are related to the amyloid hypothesis. Recently, it was suggested that the Aβ deposition cascade cannot fully disclose AD pathogenesis, with other putative players being involved in the pathophysiology of the disease. Among all, one of the most studied factors is inflammation in the brain. Hence, biomarkers of inflammation and microglia activation have also been proposed to identify AD. Among them, CX3 chemokine ligand 1 (CX3CL1) has taken center stage. This transmembrane protein, also known as fractalkine (FKN), is normally expressed in neurons, featuring an N-terminal chemokine domain and an extended mucin-like stalk, following a short intra-cytoplasmatic domain. The molecule exists in both membrane-bound and soluble forms. It is accepted that the soluble and membrane-bound forms of FKN evoke differential signaling within the CNS. Given the link between CX3XL1 and microglial activation, it has been suggested that CX3CL1 signaling disruption could play a part in the pathogenesis of AD. Furthermore, a role for chemokine as a biomarker has been proposed. However, the findings collected are controversial. The current study aimed to describe the cerebrospinal fluid (CSF) levels of CX3XL1 and classical biomarkers in AD patients.

Coatings were grown on the AZ31 Mg alloy by a hard anodizing process in the hot glycerol phosphate-containing electrolyte. Anodizing conditions were optimized, maximizing corrosion resistance estimated by impedance measurements carried out in Hank's solution at 37 °C. A post anodizing annealing treatment (350 °C for 24 h) allowed us to further enhance the corrosion resistance of the coatings mainly containing magnesium phosphate according to energy-dispersive X-ray spectroscopy and Raman analyses. Gravimetric measurements revealed a hydrogen evolution rate within the limits acceptable for application of AZ31 in biomedical devices. In vitro tests demonstrated that the coatings are biocompatible with a preosteoblast cell line.

The Tissue Engineering (TE) strategy is widely focused on the development of perfusion bioreactors to promote the production of three-dimensional (3D) functional tissues. To optimize tissue production, it is worth investigating the engineering parameters of a bioreactor system for identifying a beneficial range of operation variables. Mathematical and numerical modeling of a perfusion bioreactor is capable to provide relevant insights into the fluid flow and nutrients transport while predicting experimental data and exploring the impact of changing operating parameters, such as fluid velocities. In this work, the hydrodynamic parameters and oxygen transport were investigated using mathematical equations and Computational Fluid Dynamics (CFD) analysis modeling on a novel perfusion bioreactor working as an airlift external loop. Mathematical models and numerical simulation were associated with experimental results at different configurations to evaluate the effect of the reactor parameters on its hydrodynamics. All predictions of gas holdup and liquid circulation velocity from the modeling were in good agreement with the experimental data. A Poly-L-lactic acid (PLLA) scaffold was produced by Thermally Induced Phase Separation (TIPS) and used as 3D support for biological tests in static and dynamic conditions to assess cell viability and proliferation inside the bioreactor. The whole set of fluid-dynamic and biological results showed that our bioreactor environment offers the benefits of an in vitro potential system to produce functional engineered tissues.

The perfusion of flow during cell culture induces cell proliferation and enhances cellular activity. Perfusion bioreactors offer a controlled dynamic environment for reliable in vitro applications in the tissue engineering field. In this work, to evaluate the effects of the operating parameters of a custom-made bioreactor, numerical simulations were performed to solve the fluid velocity profile inside the bioreactor containing multi-grid support that allows allocating of multiple seeded scaffolds at the same time. The perfusion system exhibited a uniform distribution of liquid velocities within the regions, suitable for cell growth on seeded scaffolds. The effects of the porous microstructure of scaffolds on the extracellular matrix deposition also play a crucial role during perfusion cultures. In the present study, a numerical simulation was implemented at the pore level of the scaffold for fluid flow through porous media during perfused culture. Micro-computed tomography was used to obtain the digital 3D image of the complex geometry of a PLLA scaffold, offering a detailed analysis from a volume-based methodology without simplifications of the results as for pore or Darcy's law-models. Predictions about the uniformity of the flow field through the scaffolds-bioreactor system have been assessed by quantifying the cell viability of a perfusion culture while using pre-osteoblastic cells seeded on 24 PLLA scaffolds for up to 6 days.

Over the past several decades, the development of engineered small particles as targeted and drug delivery systems (TDDS) has received great attention thanks to the possibility to overcome the limitations of classical cancer chemotherapy, including targeting incapability, nonspecific action and, consequently, systemic toxicity. Thus, this research aims at using a novel design of Poly(N-isopropylacrylamide) p(NIPAM)-based microgels to specifically target cancer cells and avoid the healthy ones, which is expected to decrease or eliminate the side effects of chemotherapeutic drugs. Smart NIPAM-based microgels were functionalized with acrylic acid and coupled to folic acid (FA), targeting the folate receptors overexpressed by cancer cells and to the chemotherapeutic drug doxorubicin (Dox). The successful conjugation of FA and Dox was demonstrated by dynamic light scattering (DLS), Fourier-transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), UV-VIS analysis, and differential scanning calorimetry (DSC). Furthermore, viability assay performed on cancer and healthy breast cells, suggested the microgels’ biocompatibility and the cytotoxic effect of the conjugated drug. On the other hand, the specific tumor targeting of synthetized microgels was demonstrated by a co-cultured (healthy and cancer cells) assay monitored using confocal microscopy and flow cytometry. Results suggest successful targeting of cancer cells and drug release. These data support the use of pNIPAM-based microgels as good candidates as TDDS.

Adipose derived microvascular fragments (ad-MVFs) consist of effective vascularization units able to reassemble into efficient microvascular networks. Because of their content in stem cells and related angiogenic activity, ad-MVFs represent an interesting tool for applications in regenerative medicine. Here we show that gentle dissociation of rat adipose tissue provides a mixture of ad-MVFs with a length distribution ranging from 33–955 μm that are able to maintain their original morphology. The isolated units of ad-MVFs that resulted were able to activate transcriptional switching toward angiogenesis, forming tubes, branches, and entire capillary networks when cultured in 3D collagen type-I hydrogel. The proper involvement of metalloproteases (MMP2/MMP9) and serine proteases in basal lamina and extracellular matrix ECM degradation during the angiogenesis were concurrently assessed by the evaluation of alpha-smooth muscle actin (αSMA) expression. These results suggest that collagen type-I hydrogel provides an adequate 3D environment supporting the activation of the vascularization process. As a proof of concept, we exploited 3D collagen hydrogel for the setting of ad-MVF–islet of Langerhans coculture to improve the islets vascularization. Our results suggest potential employment of the proposed in vitro system for regenerative medicine applications, such as the improving of the islet of Langerhans engraftment before transplantation.

During the last decades, biomaterials have been deeply studied to perform and improve coatings for biomedical devices. Metallic materials, especially in the orthopedic field, represent the most common material used for different type of devices thanks to their good mechanical properties. Nevertheless, low/medium resistance to corrosion and low osteointegration ability characterizes these materials. To overcome these problems, the use of biocoatings on metals substrate is largely diffused. In fact, biocoatings have a key role to confer biocompatibility properties, to inhibit corrosion and thus improve the lifetime of implanted devices. In this work, the attention was focused on Hydroxyapatite-Chitosan (HA/CS) and Hydroxyapatite-Polyvinylacetate (HA/PVAc) composites, that have been studied as biocoatings for 304 SS based devices. Hydroxyapatite was selected for its osteoconductivity thanks to its chemical structure similar to bones. Furthermore, Chitosan and Polyvinylacetate are largely used yet in medical field (e.g. antibacterial agent or drug deliver) and in this work were used to create a synergic interaction with hydroxyapatite to increase the strength and bioactivity of coating. Biocotings were obtained by galvanic deposition process that does not require an external power supply. It is a spontaneous electrochemical deposition in which materials with different standard electrochemical potential were short-circuited and immersed in an electrolytic solution. Electrons supply for the cathodic reaction in the noblest material comes from oxidation of the less noble material. SEM, EDS, XRD and RAMAN were performed for chemical-physics characterization of biocoatings. Polarization and impedance measurements have been carried out to evaluate corrosion behavior. Besides, in-vitro cytotoxicity assays have been done for the biological features.

The "microenvironment" where a tumor develops plays a fundamental role in determining its progression, the onset of metastasis and, eventually, its resistance to therapies. Tumor cells can be considered more or less invasive depending both on the nature of the cells and on the site where they are located. Commonly adopted laboratory culture protocols for the investigation of tumor cells take usually place on standard two-dimensional supports. However, such cultures do not allow for reproduction of the biophysical properties of the tumor’s microenvironment, thus causing the cells to lose most of their relevant characteristics. In this work MDA-MB 231 breast cancer cells were cultivated within Poly- l -Lactic Acid (PLLA) scaffolds produced via Thermally Induced Phase Separation (TIPS). Starting from a ternary solution (polymer-solvent-nonsolvent) we produced scaffolds with different morphologies, porosities and pore architectures. The influence of porosity and average pore size upon cell adhesion and growth were investigated by using Cell Counting Kit-8 (CCK-8) as cell viability test, a fluorescence assay staining cell with DAPI and Scanning Electron Microscopy (SEM). Our study demonstrates that the average pore size of the polymeric scaffolds influences both the cell adhesion and resulting morphology of the growing breast cancer cells. In particular, the reported data corroborate the evidence that an average pore size ranging from 40 to 50 μm induces tumor cell aggregation and the formation of the irregular tumor masses typically observed in-vivo. In addition, TIPS proved to be a suitable manufacturing technique for finely tuning the scaffolds’ architecture, relevant to developing the most effective microenvironment for an in-vitro tumor cells growth closely mimicking in-vivo conditions.

Nanotechnology is an emerging field of modern science based on the use of nanoparticles (NPs) with a huge potential in many sectors, including nanomedicine. Their small size confers them unique properties because they are subject to physical laws that are in the middle between classical and quantum physics. In this context, NPs project plays a pivotal role because the composition, size, shape and surface proprieties need to be carefully considered for their optimal design and application. As reported in this review, NPs are classified in inorganic (metallic NPs; quantum dots; carbon-based nanostructures; mesoporous silica nanoparticles) and organic (liposomes and micelles, dendrimers and polymer nanoparticles) ones. Here, we report an accurate description of the potential of each NPs type focusing on their multiple areas of application like theranostics drug delivery, imaging, tissue engineering, antimicrobial techniques and nanovaccines, and therefore they represent a promise to revolutionize the new era of nanomedicine, especially in cancer research.

Advancements in type 1 diabetes mellitus treatments have vastly improved in recent years. The move toward a bioartificial pancreas and other fully implantable systems could help restore patient's glycemic control. However, the long-term success of implantable medical devices is often hindered by the foreign body response. Fibrous encapsulation "walls off" the implant to the surrounding tissue, impairing its functionality. In this study we aim to examine how streptozotocin-induced diabetes affects fibrous capsule formation and composition surrounding implantable drug delivery devices following subcutaneous implantation in a rodent model. After 2 weeks of implantation, the fibrous capsule surrounding the devices were examined by means of Raman spectroscopy, micro-computed tomography (μCT), and histological analysis. Results revealed no change in mean fibrotic capsule thickness between diabetic and healthy animals as measured by μCT. Macrophage numbers (CCR7 and CD163 positive) remained similar across all groups. True component analysis also showed no quantitative difference in the alpha-smooth muscle actin and extracellular matrix proteins. Although principal component analysis revealed significant secondary structural difference in collagen I in the diabetic group, no evidence indicates an influence on fibrous capsule composition surrounding the device. This study confirms that diabetes did not have an effect on the fibrous capsule thickness or composition surrounding our implantable drug delivery device. Impact Statement Understanding the impact diabetes has on the foreign body response (FBR) to our implanted material is essential for developing an effective drug delivery device. We used several approaches (Raman spectroscopy and micro-computed tomography imaging) to demonstrate a well-rounded understanding of the diabetic impact on the FBR to our devices, which is imperative for its clinical translation.

During the last years biomaterials have been largely investigated in order to perform and improve biomedical devices. As regards orthopedic field, the most common equipment used (such as implants, bone grafts or screws) are constituted by metallic materials (steel and titanium alloys), characterized by low/medium resistance to corrosion and low osteointegration ability. Furthermore, these factors could produce local inflammations of the tissues surrounding the implants, increasing kinetics of corrosion phenomena. Scientific community has focused the attention on biocoatings interposed between metal and aggressive environment in order to inhibit corrosion. Furthermore, these coatings are able to decrease the ions release and to improve the lifetime of implanted devices. In this work, hydroxyapatite-chitosan composites were studied as coatings of 304 SS steel substrates. Hydroxyapatite was chosen for its bioactivity thanks to its chemical structure similar to bones that makes suitable for osteoconductive action. Chitosan is a polysaccharide derived from chitin, usually extracted from the shells of crustaceans, largely used in medical field as antibacterial agent and/or for drug deliver. Hydroxyapatite-chitosan composites were realized by an innovative method based on galvanic process that consists in a simple galvanic coupled of metals with different redox potentials, which is advantageous firstly because does not require external power supply.

Chondrocyte transplantation has been successfully tested and proposed as a clinical procedure aiming to repair articular cartilage defects. However, the isolation of chondrocytes and the optimization of the enzymatic digestion process, as well as their successful in vitro expansion, remain the main challenges in cartilage tissue engineering. In order to address these issues, we investigated the performance of recombinant collagenases in tissue dissociation assays with the aim of isolating chondrocytes from bovine nasal cartilage in order to establish the optimal enzyme blend to ensure the best outcomes of the overall procedure. We show, for the first time, that collagenase H activity alone is required for effective cartilage digestion, resulting in an improvement in the yield of viable cells. The extracted chondrocytes proved able to grow and activate differentiation/dedifferentiation programs, as assessed by morphological and gene expression analyses.

Chondrocyte transplantation has been successfully tested and proposed as a clinical procedure aiming to repair articular cartilage defects. However, the isolation of chondrocytes and the optimization of the enzymatic digestion process, as well as their successful in vitro expansion, remain the main challenges in cartilage tissue engineering. In order to address these issues, we investigated the performance of recombinant collagenases in tissue dissociation assays with the aim of isolating chondrocytes from bovine nasal cartilage in order to establish the optimal enzyme blend to ensure the best outcomes of the overall procedure. We show, for the first time, that collagenase H activity alone is required for effective cartilage digestion, resulting in an improvement in the yield of viable cells. The extracted chondrocytes proved able to grow and activate differentiation/dedifferentiation programs, as assessed by morphological and gene expression analyses.

Medical devices, such as silicone-based prostheses designed for soft tissue implantation, often induce a suboptimal foreign-body response which results in a hardened avascular fibrotic capsule around the device, often leading to patient discomfort or implant failure. Here, it is proposed that additive manufacturing techniques can be used to deposit durable coatings with multiscale porosity on soft tissue implant surfaces to promote optimal tissue integration. Specifically, the “liquid rope coil effect”, is exploited via direct ink writing, to create a controlled macro open-pore architecture, including over highly curved surfaces, while adapting atomizing spray deposition of a silicone ink to create a microporous texture. The potential to tailor the degree of tissue integration and vascularization using these fabrication techniques is demonstrated through subdermal and submuscular implantation studies in rodent and porcine models respectively, illustrating the implant coating's potential applications in both traditional soft tissue prosthetics and active drug-eluting devices.

Tumor angiogenesis is a multiphasic process, having the extracellular matrix remodeling as critical step. Different classes of proteolytic enzymes in matrix digestion/remodeling are involved. The role of lytic enzymes and their activation mode have not been completely elucidated. Herein, the crosstalk between endothelia and tumor cells, by realization of bi- and three-dimensional endothelial and breast cancer cells co-cultures, were studied in vitro. Particularly, the effects of two tumor conditioned media (TCM) were assessed about endothelial proliferation, migration, and invasiveness. An increase in expression of pro-MMP9 was detected when endothelial cells were cultured in the presence of both TCM; such as an up-regulation of MMP1 and MMP14 and a down-regulation of MMP7. Moreover the increased MMP2 gene expression from one of them and the stimulation MMP3 synthesis from the other one were observed; an increases of β3-integrin, VEGFA, and DPP4 molecules were detected when endothelia cells are cultured with both TCM. The selection/characterization of elements present in conditioned media from breast cancer cells differently affect endothelial cells, make them potential effectors useful in breast cancer treatment.

In this work a new copolymer of inulin (INU) derivatized with ethylendiamine (EDA) and retinoic acid (RA), named INU-EDA-RA, was synthetized, characterized and employed to produce micelles as carriers for topical administration of corticosteroids for the potential treatment of diseases of posterior eye segment. Spectroscopic analysis confirmed a molar derivatization degree of 11.30 and 4.30% in EDA and RA, respectively. INU-EDA-RA micelles are capable of strong mucoadhesive interactions which result time-independent and stable over time but concentration depending. Moreover micelles are able to encapsulate efficiently from 3 to 13% (w/w) of lipophilic drugs, as dexamethasone, triamcinolone and triamcinolone acetonide. Drug loaded micelles are stable for three months when stored as freeze-dried powders and able to release high amount of drug when compared to drug dissolution profiles from suspensions. Moreover, drug loaded micelles are compatible with different ocular cell lines that are also able to internalize fluorescent micelles. Finally, drug loaded micelles enhance drug fluxes and permeability coefficients across corneal epithelial cells, thus reducing drug loss due to retention inside the cells.

Polycaprolactone (PCL) biocomposite nanofiber scaffolds with different concentrations of graphene oxide (GO) and GO surface grafted with poly(ethylene glycol) (GO-g-PEG) were prepared by electrospinning. Morphological, mechanical as well as wettability characterizations of electrospun nanofibers were carried out. Results showed that the average diameter of PLA/GO electrospun nanofibers decreased upon increasing the filler content. Differently, the diameter increased while using GO-g-PEG. Both nanofillers enhanced the electrospun PCL hydrophilicity even if PCL/GO-g-PEG samples exhibited improved wettability. The Young moduli of the composite nanofiber mats were improved by adding GO, and it was further enhanced when GO was surface grafted with PEG, in particular at low concentrations. This suggests the improved dispersion of GO-g-PEG due to the surface grafting with PEG. Furthermore, in order to assess the potential applications of the electrospun nanofibers in tissue engineering, MC3T3-E1 cells were cultured on PCL and PCL/GO-g-PEG scaffolds, thus providing preliminary and comparative proliferation rate assays.

The present study analyzed the ability of primary rat astrocytes to colonize a porous scaffold, mimicking the reticular structure of the brain parenchyma extracellular matrix, as well as their ability to grow, survive and differentiate on the scaffold. Scaffolds were prepared using poly-L-lactic acid (PLLA) via thermally-induced phase separation. Firstly, the present study studied the effects of scaffold morphology on the growth of astrocytes, evaluating their capability to colonize. Specifically, two different morphologies were tested, which were obtained by changing the polymer concentration in the starting solution. The structures were characterized by scanning electron microscopy, and a pore size of 20 µm (defined as the average distance between the pore walls) was detected. For comparison, astrocytes were also cultured in the traditional 2D culture system that we have been using since 2003. Then the effects of different substrates, such as collagen I and IV, and fibronectin were analyzed. The results revealed that the PLLA scaffolds, coated with collagen IV, served as very good matrices for astrocytes, which were observed to adhere, grow and colonize the matrix, acquiring their typical morphology. In addition, under these conditions, they secreted extracellular vesicles (EVs) that were compatible in size with exosomes. Their ability to produce exosomes was also suggested by transmission electron microscopy pictures which revealed both EVs and intracellular structures that could be interpreted as multivesicular bodies. The fact that these cells were able to adapt to the PLLA scaffold, together with our previous results, which demonstrated that brain capillary endothelial cells can grow and differentiate on the same scaffold, could support the future use of 3D brain cell co-culture systems.

Poly(N-vinyl pyrrolidone)-based-nanogels (NGs), produced by e-beam irradiation, are conjugated with monoclonal antibodies (mAb) for active targeting purposes. The uptake of immuno-functionalized nanogels is tested in an endothelial cell line, ECV304, using confocal and epifluorescence microscopy. Intracellular localization studies reveal a faster uptake of the immuno-nanogel conjugate with respect to the ‘bare’ nanogel. The specific internalization pathway of these immuno-nanogels is clarified by selective endocytosis inhibition experiments, flow cytometry and confocal microscopy. Active targeting ability is also verified by conjugating a monoclonal antibody which recognizes the αvβ3 integrin on activated endothelial cells. Epifluorescence images of the ‘wound healing assay’ on ECV304 cells provide evidence of nanogels localization only in the target cells. Therefore, the immuno-nanogels produced have the potential to recognize specific cell types in heterogeneous systems, which makes them promising candidates for targeted drug delivery applications.

An amphiphilic copolymer carrying high-dose doxorubicin (21% on a weight basis), PHEA–EDA–P,C–Doxo, was prepared by coupling doxorubicin with a biocompatible polyaminoacid through a pH-sensitive spacer. Additional derivatization with 4-pentynoic acid endows it with self-assembling properties by means of π–π stacking. These micelles can be triggered to promptly release drug in lysosomes (∼40% in 12 h) through pH-dependent micelle hydrolysis after uptake. In vitro tests on co-cultures of cancer (MDA-MB 231) and normal (HB-2) breast cells proved that the conjugate was selectively internalized into the former rather than normal cells, exploiting the caveolae-dependent endocytosis pathway, explaining the selective cytotoxic effect toward cancer cells. Intracellular trafficking study of MDA-MB 231 showed that the delivery of the endocytosed drug occurs through the direct fusion of caveosomes with late lysosomes, triggering a massive release in the cytoplasm, bringing about cell death. Dose-effectiveness and mechanistic data indicate that PHEA–EDA–P,C–Doxo is endowed with a distinctive combination of selectivity and pharmacological potency (EC50 13 μM, Emax = 77% and EC50 > 25 μM, Emax = 21% for cancer and healthy cells respectively) that makes it an excellent candidate for future preclinical studies.

siRNAs are very potent drug molecules, able to silence genes involved in pathologies development. siRNAs have virtually an unlimited therapeutic potential, particularly for the treatment of inflammatory diseases. However, their use in clinical practice is limited because of their unfavorable properties to interact and not to degrade in physiological environments. In particular they are large macromolecules, negatively charged, which undergo rapid degradation by plasmatic enzymes, are subject to fast renal clearance/hepatic sequestration, and can hardly cross cellular membranes. These aspects seriously impair siRNAs as therapeutics. As in all the other fields of science, siRNAs management can be advantaged by physical-mathematical descriptions (modeling) in order to clarify the involved phenomena from the preparative step of dosage systems to the description of drug-body interactions, which allows improving the design of delivery systems/processes/therapies. This review analyzes a few mathematical modeling approaches currently adopted to describe the siRNAs delivery, the main procedures in siRNAs vectors’ production processes and siRNAs vectors’ release from hydrogels, and the modeling of pharmacokinetics of siRNAs vectors. Furthermore, the use of physical models to study the siRNAs vectors’ fate in blood stream and in the tissues is presented. The general view depicts a framework maybe not yet usable in therapeutics, but with promising possibilities for forthcoming applications.

Direct isolation of primary cells from tissues and organs allows for the maintenance of important cell characteristics and properties for in vitro studies and a plethora of biomedical applications. Dissociation of cells from the organ of interest is possible due to the enzymatic activity of collagenases. The choice and the dose of these enzymes is the critical step to obtain the maximum number of cells with intact structure and function. In this contest, Abiel collagenases class I (Col G) and class II (Col H) were synthesised using recombinant DNA technologies and their ability to degrade collagen in cell isolation from different tissues was tested. Examples of cells isolated with these enzymes include Langerhans islets, hepatocytes, chondrocytes, osteoblasts, fibroblasts, and stem and retinal cells that be used for in vitro studies. In this contest, primary cardiomyocytes represent a perfect murine model to investigate numerous heart diseases. Herein, a protocol of cardiomyocyte extraction from rat heart, using a combination of Abiel collagenases supplemented with thermolysin protease, is proposed. The structure and the viability of isolated cells were tested over time by optical and fluorescence microscopy and viability assays. Further cellular structure characterisation was performed by western blot analysis, using specific cardiomyocytes markers. Isolation of viable primary heart cells with unaltered properties and functionality can potentially provide in vitro models to study heart function, arrhythmias, long Q-T syndrome and cardiotoxicity.