A Simple, Semi-Quantitative Acyl Biotin Exchange-Based Method to Detect Protein S-Palmitoylation Levels.

Protein S-palmitoylation is a reversible post-translational lipidation in which palmitic acid (16:0) is added to protein cysteine residue by a covalent thioester bond. This modification plays an active role in membrane targeting of soluble proteins, protein-protein interaction, protein trafficking, and subcellular localization. Moreover, palmitoylation is related to different diseases, such as neurodegenerative pathologies, cancer, and developmental defects. The aim of this research is to provide a straightforward and sensitive procedure to detect protein palmitoylation based on Acyl Biotin Exchange (ABE) chemistry. Our protocol setup consists of co-immunoprecipitation of native proteins (i.e., CD63), followed by the direct detection of palmitoylation on proteins immobilized on polyvinylidene difluoride (PVDF) membranes. With respect to the conventional ABE-based protocol, we optimized and validated a rapid semi-quantitative assay that is shown to be significantly more sensitive and highly reproducible.

Protein Biocargo and Anti-Inflammatory Effect of Tomato Fruit-Derived Nanovesicles Separated by Density Gradient Ultracentrifugation and Loaded with Curcumin.

Plant-derived nanovesicles (PDNVs) have become attractive alternatives to mammalian cell-derived extracellular vesicles (EVs) both as therapeutic approaches and drug-delivery vehicles. In this study, we isolated tomato fruit-derived NVs and separated them by the iodixanol density gradient ultracentrifugation (DGUC) into twelve fractions. Three visible bands were observed at densities 1.064 ± 0.007 g/mL, 1.103 ± 0.006 g/mL and 1.122 ± 0.012 g/mL. Crude tomato PDNVs and DGUC fractions were characterized by particle size-distribution, concentration, lipid and protein contents as well as protein composition using mass spectrometry-based proteomics. Cytotoxicity and anti-inflammatory activity of the DGUC fractions associated to these bands were assessed in the lipopolysaccharide (LPS)-stimulated human monocytic THP-1 cell culture. The middle and the low-density visible DGUC fractions of tomato PDNVs showed a significant reduction in LPS-induced inflammatory IL-1ß cytokine mRNA production. Functional analysis of proteins identified in these fractions reveals the presence of 14-3-3 proteins, endoplasmic reticulum luminal binding proteins and GTP binding proteins associated to gene ontology (GO) term GO:0050794 and the regulation of several cellular processes including inflammation. The most abundant middle-density DGUC fraction was loaded with curcumin using direct loading, sonication and extrusion methods and anti-inflammatory activity was compared. The highest entrapment efficiency and drug loading capacity was obtained by direct loading. Curcumin loaded by sonication increased the basal anti-inflammatory activity of tomato PDNVs.

High-Yield Separation of Extracellular Vesicles Using Programmable Zwitterionic Coacervates.

Programmable coacervates based on zwitterionic polymers are designed as dynamic materials for ion exchange bioseparation. These coacervates are proposed as promising materials for the purification of soft nanoparticles such as liposomes and extracellular vesicles (EVs). It is shown that the stimulus-responsiveness of the coacervates and the recruitment of desired molecules can be independently programmed by polymer design. Moreover, the polymeric coacervates can recruit and release intact liposomes, human EVs, and nanoalgosomes in high yields and separate vesicles from different types of impurities, including proteins and nucleic acids. This approach combines the speed and simplicity of precipitation methods and the programmability of chromatography with the gentleness of aqueous two-phase separation, thereby guaranteeing product stability. This material represents a promising alternative for providing a low-shear, gentle, and selective purification method for EVs.

A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications.

Tumor growth and metastasis strongly rely on cell-cell communication. One of the mechanisms by which tumor cells communicate involves the release and uptake of lipid membrane encapsulated particles full of bioactive molecules, called extracellular vesicles (EVs). EV exchange between cancer cells may induce phenotype changes in the recipient cells. Our work investigated the effect of EVs released by teratocarcinoma cells on glioblastoma (GBM) cells. EVs were isolated by differential centrifugation and analyzed through Western blot, nanoparticle tracking analysis, and electron microscopy. The effect of large EVs on GBM cells was tested through cell migration, proliferation, and drug-sensitivity assays, and resulted in a specific impairment in cell migration with no effects on proliferation and drug-sensitivity. Noticeably, we found the presence of the EGF-CFC founder member CRIPTO on both small and large EVs, in the latter case implicated in the EV-mediated negative regulation of GBM cell migration. Our data let us propose a novel route and function for CRIPTO during tumorigenesis, highlighting a complex scenario regulating its effect, and paving the way to novel strategies to control cell migration, to ultimately improve the prognosis and quality of life of GBM patients.

Isolation of Extracellular Vesicles From Microalgae: A Renewable and Scalable Bioprocess.

Extracellular vesicles (EVs) play a crucial role as potent signal transducers among cells, with the potential to operate cross-species and cross-kingdom communication. Nanoalgosomes are a subtype of EVs recently identified and isolated from microalgae. Microalgae represent a natural bioresource with the capacity to produce several secondary metabolites with a broad range of biological activities and commercial applications. The present study highlights the upstream and downstream processes required for the scalable production of nanoalgosomes from cultures of the marine microalgae Tetraselmis chuii. Different technical parameters, protocols, and conditions were assessed to improve EVs isolation by tangential flow filtration (TFF), aiming to enhance sample purity and yield. The optimization of the overall bioprocess was enhanced by quality control checks operated through robust biophysical and biochemical characterizations. Further, we showed the possibility of recycling by TFF microalgae cells post-EVs isolation for multiple EV production cycles. The present results highlight the potential of nanoalgosome production as a scalable, cost-effective bioprocess suitable for diverse scientific and industrial exploitations.

Extracellular Vesicles From Microalgae: Uptake Studies in Human Cells and Caenorhabditis elegans.

Extracellular vesicles (EVs) are lipid membrane nano-sized vesicles secreted by various cell types for intercellular communication, found in all kingdoms of life. Nanoalgosomes are a subtype of EVs derived from microalgae with a sustainable biotechnological potential. To explore the uptake, distribution and persistence of nanoalgosomes in cells and living organisms, we separated them from a culture of the chlorophyte Tetraselmis chuii cells by tangential flow filtration (TFF), labelled them with different lipophilic dyes and characterized their biophysical attributes. Then we studied the cellular uptake of labelled nanoalgosomes in human cells and in C. elegans, demonstrating that they enter the cells through an energy dependent mechanism and are localized in the cytoplasm of specific cells, where they persist for days. Our data confirm that nanoalgosomes are actively uptaken in vitro by human cells and in vivo by C. elegans cells, supporting their exploitation as potential nanocarriers of bioactive compounds for theranostic applications.

Rapid Characterization and Quantification of Extracellular Vesicles by Fluorescence-Based Microfluidic Diffusion Sizing.

Extracellular vesicles (EVs) are emerging as promising diagnostic and therapeutic tools for a variety of diseases. The characterization of EVs requires a series of orthogonal techniques that are overall time- and material-consuming. Here, a microfluidic device is presented that exploits the combination of diffusion sizing and multiwavelength fluorescence detection to simultaneously provide information on EV size, concentration, and composition. The latter is achieved with the nonspecific staining of lipids and proteins combined with the specific staining of EV markers such as EV-associated tetraspanins via antibodies. The device can be operated as a single-step immunoassay thanks to the integrated separation and quantification of free and EV-bound fluorophores. This microfluidic technique is capable of detecting and quantifying components associated to EV subtypes and impurities and thus to measure EV purity in a time scale of minutes, requiring less than 5 µL of sample and minimal sample handling before the analysis. Moreover, the analysis is performed directly in solution without immobilization steps. Therefore, this method can accelerate screening of EV samples and aid the evaluation of sample reproducibility, representing an important complementary tool to the current array of biophysical methods for EV characterization, particularly valuable for instance for bioprocess development.

Impact of the flame retardant 2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) in THP-1 macrophage-like cell function via small extracellular vesicles.

2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) is one of the most widespread environmental brominated flame-retardant congeners which has also been detected in animal and human tissues. Several studies have reported the effects of PBDEs on different health issues, including neurobehavioral and developmental disorders, reproductive health, and alterations of thyroid function. Much less is known about its immunotoxicity. The aim of our study was to investigate the effects that treatment of THP-1 macrophage-like cells with PBDE-47 could have on the content of small extracellular vesicles' (sEVs) microRNA (miRNA) cargo and their downstream effects on bystander macrophages. To achieve this, we purified sEVs from PBDE-47 treated M(LPS) THP-1 macrophage-like cells (sEVsPBDE+LPS) by means of ultra-centrifugation and characterized their miRNA cargo by microarray analysis detecting the modulation of 18 miRNAs. Furthermore, resting THP-1 derived M(0) macrophage-like cells were cultured with sEVsPBDE+LPS, showing that the treatment reshaped the miRNA profiles of 12 intracellular miRNAs. This dataset was studied in silico, identifying the biological pathways affected by these target genes. This analysis identified 12 pathways all involved in the maturation and polarization of macrophages. Therefore, to evaluate whether sEVsPBDE+LPS can have some immunomodulatory activity, naïve M(0) THP-1 macrophage-like cells cultured with purified sEVsPBDE+LPS were studied for IL-6, TNF-α and TGF-ß mRNAs expression and immune stained with the HLA-DR, CD80, CCR7, CD38 and CD209 antigens and analyzed by flow cytometry. This analysis showed that the PBDE-47 treatment does not induce the expression of specific M1 and M2 cytokine markers of differentiation and may have impaired the ability to make immunological synapses and present antigens, down-regulating the expression of HLA-DR and CD209 antigens. Overall, our study supports the model that perturbation of miRNA cargo by PBDE-47 treatment contributes to the rewiring of cellular regulatory pathways capable of inducing perturbation of differentiation markers on naïve resting M(0) THP-1 macrophage-like cells.

Nano-structured myelin: new nanovesicles for targeted delivery to white matter and microglia, from brain-to-brain.

Neurodegenerative diseases affect millions of people worldwide and the presence of various physiological barriers limits the accessibility to the brain and reduces the efficacy of various therapies. Moreover, new carriers having targeting properties to specific brain regions and cells are needed in order to improve therapies for the brain disorder treatment. In this study, for the first time, Myelin nanoVesicles (hereafter defined MyVes) from brain-extracted myelin were produced. The MyVes have an average diameter of 100-150 â€‹nm, negative zeta potential, spheroidal morphology, and contain lipids and the key proteins of the myelin sheath. Furthermore, they exhibit good cytocompatibility. The MyVes were able to target the white matter and interact mainly with the microglia cells. The preliminary results here presented allow us to suppose the employment of MyVes as potential carrier to target the white matter and microglia in order to counteract white matter microglia-related diseases.

2,2'4,4'-Tetrabromodiphenyl Ether (PBDE-47) Modulates the Intracellular miRNA Profile, sEV Biogenesis and Their miRNA Cargo Exacerbating the LPS-Induced Pro-Inflammatory Response in THP-1 Macrophages.

The 2,2'4,4'-tetrabromodiphenyl ether (PBDE-47) is one of the most prominent PBDE congeners detected in the environment and in animal and human tissues. Animal model experiments suggested the occurrence of PBDE-induced immunotoxicity leading to different outcomes and recently we demonstrated that this substance can impair macrophage and basophil activities. In this manuscript, we decided to further examine the effects induced by PBDE-47 treatment on innate immune response by looking at the intracellular expression profile of miRNAs as well as the biogenesis, cargo content and activity of human M(LPS) macrophage cell-derived small extracellular vesicles (sEVs). Microarray and in silico analysis demonstrated that PBDE-47 can induce some epigenetic effects in M(LPS) THP-1 cells modulating the expression of a set of intracellular miRNAs involved in biological pathways regulating the expression of estrogen-mediated signaling and immune responses with particular reference to M1/M2 differentiation. In addition to the cell-intrinsic modulation of intracellular miRNAs, we demonstrated that PBDE-47 could also interfere with the biogenesis of sEVs increasing their number and selecting a de novo population of sEVs. Moreover, PBDE-47 induced the overload of specific immune related miRNAs in PBDE-47 derived sEVs. Finally, culture experiments with naïve M(LPS) macrophages demonstrated that purified PBDE-47 derived sEVs can modulate macrophage immune response exacerbating the LPS-induced pro-inflammatory response inducing the overexpression of the IL-6 and the MMP9 genes. Data from this study demonstrated that PBDE-47 can perturb the innate immune response at different levels modulating the intracellular expression of miRNAs but also interfering with the biogenesis, cargo content and functional activity of M(LPS) macrophage cell-derived sEVs.

Nanoalgosomes: Introducing extracellular vesicles produced by microalgae.

Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.

Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds.

Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 µg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and ß-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.

Can Be miR-126-3p a Biomarker of Premature Aging? An Ex Vivo and In Vitro Study in Fabry Disease.

Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by lysosomal accumulation of glycosphingolipids in a wide variety of cytotypes, including endothelial cells (ECs). FD patients experience a significantly reduced life expectancy compared to the general population; therefore, the association with a premature aging process would be plausible. To assess this hypothesis, miR-126-3p, a senescence-associated microRNA (SA-miRNAs), was considered as an aging biomarker. The levels of miR-126-3p contained in small extracellular vesicles (sEVs), with about 130 nm of diameter, were measured in FD patients and healthy subjects divided into age classes, in vitro, in human umbilical vein endothelial cells (HUVECs) "young" and undergoing replicative senescence, through a quantitative polymerase chain reaction (qPCR) approach. We confirmed that, in vivo, circulating miR-126 levels physiologically increase with age. In vitro, miR-126 augments in HUVECs underwent replicative senescence. We observed that FD patients are characterized by higher miR-126-3p levels in sEVs, compared to age-matched healthy subjects. We also explored, in vitro, the effect on ECs of glycosphingolipids that are typically accumulated in FD patients. We observed that FD storage substances induced in HUVECs premature senescence and increased of miR-126-3p levels. This study reinforces the hypothesis that FD may aggravate the normal aging process.

Antioxidant Bioprospecting in Microalgae: Characterisation of the Potential of Two Marine Heterokonts from Irish Waters.

Microalgae constitute a heterogeneous and diverse range of organisms capable of accumulating bioactive metabolites, making them promising feedstock for applications in the nutraceutical, functional food, animal feed, biofertilisation or biofuel sectors. There has been renewed interest in recent times in natural sources of antioxidants, particularly as health products and preserving agents. Microalgae strains isolated from aquatic habitats in Ireland were successfully brought into culture. The 91 strains were grown phototrophically in nutrient-enriched media to generate biomass, which was harvested and assessed for antioxidant potential. Extracts were screened for antioxidant activity using a modified volumetric Trolox-ABTS assay and the Folin-Ciocalteu method. Two heterokont marine strains of interest were further studied to ascertain variations in antioxidant capacity across different stages of batch culture growth. The antioxidant activity of extracts of bacillariophyte cf. Stauroneis sp. LACW24 and ocrophyte cf. Phaeothamnion sp. LACW34 increased during growth with a maximum being observed during the late stationary or early death phase (2.5- to 8-fold increases between days 20 and 27). Strains LACW24 and LACW34 contained 5.9 and 3.0 mg g-1 (DW) of the xanthophyll fucoxanthin, respectively. Extracts of strains also showed no cytotoxicity towards mouse cell lines. These results highlight the potential of these strains for biomass valorisation and cultivation upscaling and to be further considered as part of ongoing bioprospecting efforts towards identifying novel species to join the relatively narrow range of commercially exploited marine microalgae species.

Biomanufacturing of Tomato-Derived Nanovesicles.

Micro- and nano-sized vesicles (MVs and NVs, respectively) from edible plant resources are gaining increasing interest as green, sustainable, and biocompatible materials for the development of next-generation delivery vectors. The isolation of vesicles from complex plant matrix is a significant challenge considering the trade-off between yield and purity. Here, we used differential ultracentrifugation (dUC) for the bulk production of MVs and NVs from tomato (Solanum lycopersicum L.) fruit and analyzed their physical and morphological characteristics and biocargo profiles. The protein and phospholipid cargo shared considerable similarities between MVs and NVs. Phosphatidic acid was the most abundant phospholipid identified in NVs and MVs. The bulk vesicle isolates were further purified using sucrose density gradient ultracentrifugation (gUC) or size-exclusion chromatography (SEC). We showed that SEC using gravity column efficiently removed co-purifying matrix components including proteins and small molecular species. dUC/SEC yielded a high yield of purified vesicles in terms of number of particles (2.6 × 1015 particles) and protein quantities (6.9 ± 1.5 mg) per kilogram of tomato. dUC/gUC method separated two vesicle populations on the basis of buoyant density. Proteomics and in silico studies of the SEC-purified MVs and NVs support the presence of different intra- and extracellular vesicles with highly abundant lipoxygenase (LOX), ATPases, and heat shock proteins (HSPs), as well as a set of proteins that overlaps with that previously reported in tomato chromoplast.

Novel inulin-based mucoadhesive micelles loaded with corticosteroids as potential transcorneal permeation enhancers.

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.

E-beam crosslinked nanogels conjugated with monoclonal antibodies in targeting strategies.

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 αvß3 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.

Multi-Functional Nanogels for Tumor Targeting and Redox-Sensitive Drug and siRNA Delivery.

(1) Background: A new family of nanosystems able to discern between normal and tumor cells and to release a therapeutic agent in controlled way were synthetized by e-beam irradiation. This technique permits to obtain biocompatible, sterile, carboxyl-functionalized polyvinylpyrrolidone (PVP-co-acrylic acid) nanogels (NGs); (2) Methods: Here, we performed a targeting strategy based on the recognition of over-expressed proteins on tumor cells, like the folate receptor. The selective targeting was demonstrated by co-culture studies and flow cytometry analysis, using folate conjugated NGs. Moreover, nanoparticles were conjugated to a chemotherapeutic drug or to a pro-apoptotic siRNA through a glutathione sensitive spacer, in order to obtain a controlled release mechanism, specific for cancer cells. The drug efficiency was tested on tumor and healthy cells by flow cytometric analysis, confocal and epifluorescence microscopy and cytotoxicity assay; the siRNA effect was investigated by RNAi experiment; (3) Results: The data obtained showed that the use of NGs permits a faster cargo release in cancer cells, in response to high cytosolic glutathione level, also improving their efficacy; (4) Conclusion: The possibility of releasing biological molecules in a controlled way and to recognize a specific tumor target allows overcoming the typical limits of the classic cancer therapy.

The nucleic acid-binding protein PcCNBP is transcriptionally regulated during the immune response in red swamp crayfish Procambarus clarkii.

Gene family encoding cellular nucleic acid binding proteins (CNBP) is well conserved among vertebrates; however, there is limited knowledge in lower organisms. In this study, a CNBP homolog from the red swamp crayfish Procambarus clarkii was characterised. The full-length cDNA of PcCNBP was of 1257 bp with a 5'-untranslated region (UTR) of 63 bp and a 3'-UTR of 331 bp with a poly (A) tail, and an open-reading frame (ORF) of 864 bp encoding a polypeptide of 287 amino acids with the predicted molecular weight of about 33 kDa. The predicted protein possesses 7 tandem repeats of 14 amino acids containing the CCHC zinc finger consensus sequence, two RGG-rich single-stranded RNA-binding domain and a nuclear localization signal, strongly suggesting that PcCNBP was a homolog of vertebrate CNBP. The PcCNBP transcript was constitutively expressed in all tested tissues of unchallenged crayfish, including hepatopancreas, gill, eyestalk, haemocytes, intestine, stomach and cuticle with highest expression in haemocytes, intestine, gills and hepatopancreas. The mRNA expression of PcCNBP in haemocytes was modulated at transcriptional level by different immune challenges, suggesting its involvement in the immune response of P. clarkii during both bacteria and viruses infection.

Minimalism in radiation synthesis of biomedical functional nanogels.

A scalable, single-step, synthetic approach for the manufacture of biocompatible, functionalized micro- and nanogels is presented. In particular, poly(N-vinyl pyrrolidone)-grafted-(aminopropyl)methacrylamide microgels and nanogels were generated through e-beam irradiation of PVP aqueous solutions in the presence of a primary amino-group-carrying monomer. Particles with different hydrodynamic diameters and surface charge densities were obtained at the variance of the irradiation conditions. Chemical structure was investigated by different spectroscopic techniques. Fluorescent variants were generated through fluorescein isothiocyanate attachment to the primary amino groups grafted to PVP, to both quantify the available functional groups for bioconjugation and follow nanogels localization in cell cultures. Finally, a model protein, bovine serum albumin, was conjugated to the nanogels to demonstrate the attachment of biologically relevant molecules for targeting purposes in drug delivery. The described approach provides a novel strategy to fabricate biohybrid nanogels with a very promising potential in nanomedicine.