Development of different nanosystems for drugs and siRNA delivery

Cancer is one of the leading causes of death in the world. Over the past several decades, the development of engineered nanosystems for targeted drug delivery have received great attention thanks to their possibility to overcome the limitations of classical cancer chemotherapy including poor solubility, targeting incapability, nonspecific action and, consequently, systemic toxicity. In this contest, four different models of nanocarriers have been analysed and compared for their capacity to target tumour tissue and to release the therapeutic agent in a controlled way: INU-EDA-P,C-DOXO; PHEA-EDA-P,C-DOXO; PVP-siRNA and RGO-siRNA. Inulin and PHEA were conjugated to the antineoplastic drug doxorubicin through a citraconylamide bridge used as a pH sensitive spacer and their different action in normal and tumour cells was compared by viability assay, fluorescence microscopy and flow cytometric analysis. The data revealed a higher effect against the cancer cells probably due to the higher capability to enter the cancer cells respect to the healthy ones, but also due to a partial drug release in tumour microenvironment, that presents a lower pH (6.5) respect to normal tissue (7.4). Because of the better efficiency of PHEA-EDA-P,C-DOXO, its preferential uptake into tumour cells was further demonstrated through co-culture experiments. Moreover, cellular internalization mechanism studies suggested a model in which the polymer would enter the cells through a caveolae-mediated endocytosis and would be steered toward lysosomal compartment where the drug would be released and become free to go into the nucleus. On the other hand, siRNA delivery was investigated comparing other two kind of nanosystems: PVP and Graphene nano-complexes conjugated with siRNA against Egr-1 (a transcription factor involved in the progress of the tumour) and Bcl-2 (a protein related with the apoptotic pathway in cancer cells). They are very different complexes, each with specific properties that would influence differently their ability as siRNA delivery system. Data relative to PVP nanogel showed it as a good candidate for siRNA delivery because it binds siRNA without any functional alteration or degradation. Moreover, the addition of a glutathione sensitive linker permitted a higher amount of siRNA released through a controlled way. Even graphene nanosystems have displayed good features as nanocarriers thanks to their biocompatibility and their ability to be internalized by cells. Furthermore, their bidimensional nature would permit to conjugate a large amount of oligonucleotides. All the complexes analysed showed specific features that make them good candidates for drug or siRNA delivery. The choice of the best nanosystem is depending on the target site and on the therapeutic agent conjugated. PHEA nanopolymers present better characteristics for doxorubicin delivery compared with those of Inulin. On the other hand, the hydrogel nature of PVP and the bidimensional structure of graphene can minimize the typical degradation problems of the oligonucleotides and, therefore, offer the best conditions for siRNA delivery. Moreover, the possibility to release biological molecules in controlled way (mediated by pH or Glutathione) and to recognize the specific tumour target allow to overcome the typical limits of the classic cancer therapy.Abstract Cancer is one of the leading causes of death in the world. Over the past several decades, the development of engineered nanosystems for targeted drug delivery have received great attention thanks to their possibility to overcome the limitations of classical cancer chemotherapy including poor solubility, targeting incapability, nonspecific action and, consequently, systemic toxicity. In this contest, four different models of nanocarriers have been analysed and compared for their capacity to target tumour tissue and to release the therapeutic agent in a controlled way: INU-EDA-P,C-DOXO; PHEA-EDA-P,C-DOXO; PVP-siRNA and RGO-siRNA. Inulin and PHEA were conjugated to the antineoplastic drug doxorubicin through a citraconylamide bridge used as a pH sensitive spacer and their different action in normal and tumour cells was compared by viability assay, fluorescence microscopy and flow cytometric analysis. The data revealed a higher effect against the cancer cells probably due to the higher capability to enter the cancer cells respect to the healthy ones, but also due to a partial drug release in tumour microenvironment, that presents a lower pH (6.5) respect to normal tissue (7.4). Because of the better efficiency of PHEA-EDA-P,C-DOXO, its preferential uptake into tumour cells was further demonstrated through co-culture experiments. Moreover, cellular internalization mechanism studies suggested a model in which the polymer would enter the cells through a caveolae-mediated endocytosis and would be steered toward lysosomal compartment where the drug would be released and become free to go into the nucleus. On the other hand, siRNA delivery was investigated comparing other two kind of nanosystems: PVP and Graphene nano-complexes conjugated with siRNA against Egr-1 (a transcription factor involved in the progress of the tumour) and Bcl-2 (a protein related with the apoptotic pathway in cancer cells). They are very different complexes, each with specific properties that would influence differently their ability as siRNA delivery system. Data relative to PVP nanogel showed it as a good candidate for siRNA delivery because it binds siRNA without any functional alteration or degradation. Moreover, the addition of a glutathione sensitive linker permitted a higher amount of siRNA released through a controlled way. Even graphene nanosystems have displayed good features as nanocarriers thanks to their biocompatibility and their ability to be internalized by cells. Furthermore, their bidimensional nature would permit to conjugate a large amount of oligonucleotides. All the complexes analysed showed specific features that make them good candidates for drug or siRNA delivery. The choice of the best nanosystem is depending on the target site and on the therapeutic agent conjugated. PHEA nanopolymers present better characteristics for doxorubicin delivery compared with those of Inulin. On the other hand, the hydrogel nature of PVP and the bidimensional structure of graphene can minimize the typical degradation problems of the oligonucleotides and, therefore, offer the best conditions for siRNA delivery. Moreover, the possibility to release biological molecules in controlled way (mediated by pH or Glutathione) and to recognize the specific tumour target allow to overcome the typical limits of the classic cancer therapy

Glutathione-sensitive nanogels for drug release

Nanogels (NGs) synthesized by pulsed electron-beam irradiation of semi-dilute poly (N-vinyl pyrrolidone) (PVP) aqueous solutions, at relatively low energy per pulse and doses within the sterilization dose range, represent a very interesting family of polymeric nanocarriers. Ionizing irradiation-induced crosslinking of PVP allows to control particle size, and surface chemistry of the polymer nanoparticles without making use of catalysts, organic solvents or surfactants, and with beneficial effects onto the purity and hence biocompatibility of the final products obtained. Furthermore, the availability of reactive functional groups, either generated by the radiation or purposely grafted via copolymerisation with suitable functional monomers enables the conjugation of therapeutics drug, that make them suitable nanocarriers for biomedical applications. In particular, we have developed a carboxyl-functionalized nanogel variant for glutathione-mediated delivery of a chemotherapeutic agent, Doxorubicin. The drug is linked to the nanoparticles through a linker containing a cleavable disulphide bridge, aminoethyldithiopropionic acid (AEDP). In vitro drug release experiments have shown that glutathione can induce the release of Doxorubicin, through the reduction of the disulfide bridge. These results suggest that such redox-responsive nanoparticles can deliver doxorubicin into the nuclei of tumor cells, thus inducing inhibition of cell proliferation, and provide a favourable platform to construct nanoscalar drug delivery systems for cancer therapy

Skin to skin interactions. Does the infant massage improve the couple functioning?

Transition to parenthood is a critical stage of life due to several changes the couple has to handle. A large body of studies described how transition to parenthood can be linked to the onset of depressive symptoms, as well as the perception of a low social support, and an increased stress, representing a risk for the early mother—baby relationship. Infant Massage (IM) emerged as an helpful tool to improve maternal skills in interacting with the baby, and leading toward a decreasing of post partum symptoms. However, a growing body of literature highlights that men also may experience post-partum diseases, representing an additional risk for the development of the baby. To date, no study observed the impact of the infant massage on both partners. The aim of the current qualitative research is to observe the impact of the IM on a single couple of parents at childbirth. Pre (Time 1) and post-intervention (Time 3) procedure has been established to observe the changes occurring over the time in the couple. In particular, each member of the couple filled out the EPDS, the BDI-II, the MSPSS and the PSI-SF both at Time 1 and at Time 3. The treatment (Time 2) was represented by the IM training, and lasted 4 weeks. Findings revealed a decrease in depressive symptoms in both partners, as well as an improvement of their perception of stress related to parental role. No changes has been detected with respect to the perception of social support. The IM seems to be a helpful approach to prevent the establishment of pathological conditions in new parents. Although no direct measures on the child were used, the current qualitative data seem to suggest that the IM may represent a valuable tool to prevent the onset of early negative outcomes of the baby. Further investigations and empirical data are needed to improve the knowledge in this field

Galvanic Deposition of Calcium Phosphate/Bioglass Composite Coating on AISI 316L

Calcium phosphate/Bioglass composite coatings on AISI 316L were investigated with regard to their potential role as a beneficial coating for orthopedic implants. These coatings were realized by the galvanic co-deposition of calcium phosphate compounds and Bioglass particles. A different amount of Bioglass 45S5 was used to study its effect on the performance of the composite coatings. The morphology and chemical composition of the coatings were investigated before and after their aging in simulated body fluid. The coatings uniformly covered the AISI 316L substrate and consisted of a brushite and hydroxyapatite mixture. Both phases were detected using X-ray diffraction and Raman spectroscopy. Additionally, both analyses revealed that brushite is the primary phase. The presence of Bioglass was verified through energy-dispersive X-ray spectroscopy, which showed the presence of a silicon peak. During aging in simulated body fluid, the coating was subject to a dynamic equilibrium of dissolution/reprecipitation with total conversion in only the hydroxyapatite phase. Corrosion tests performed in simulated body fluid at different aging times revealed that the coatings made with 1 g/L of Bioglass performed best. These samples have a corrosion potential of −0.068V vs. Ag/AgCl and a corrosion current density of 8.87 × 10−7 A/cm2. These values are better than those measured for bare AISI 316L (−0.187 V vs. Ag/AgCl and 2.52 × 10−6 A/cm2, respectively) and remained superior to pure steel for all 21 days of aging. This behavior indicated the good protection of the coating against corrosion phenomena, which was further confirmed by the very low concentration of Ni ions (0.076 ppm) released in the aging solution after 21 days of immersion. Furthermore, the absence of cytotoxicity, verified through cell viability assays with MC3T3-E1 osteoblastic cells, proves the biocompatibility of the coatings

A novel enzyme blend for efficient tissue dissociation and primary cells isolation

Tissue dissociation/primary cell isolation and cell harvesting are principal appli- cations for enzymes in tissue culture research and cell biology studies. The goal of a cell isolation procedure is to maximize the yield of functionally viable dissoci- ated cells. Among the parameters which affect the outcome of any particular dissociating procedure there are enzyme(s) used and related impurities presents in crude enzyme preparation. ABIEL srl recently produced the recombinant collagenase class I (Col G) and II (Col H) from Clostridium histolyticum (PCT WO 2011/073925 A9). The enzymes were produced in Escherichia coli and purified by affinity chromatography. The method of production adopted allows absolute control of the final composition of these enzymes, as well as their stability, purity, activity, absence of toxicity and higher reproducibility of batches. The two collagenases produced separately have been used in conjunction according to precise proportions to dissociate calvaria, liver, pancreas, retina of the BALB/c mouse; and bovine hoof. The analyses carried out on all isolated cell populations suggest that the cells maintain the structural and functional integrity of specific tissues/organs originating. Recombinant Col G and Col H enzymes produced by ABIEL are promising in the context of the tissue/cells dissociation, with the aim to make innovation in the fields of tissue engineering and transplantation medicine

multifunctional bioinstructive 3d architectures to modulate cellular behavior

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Functionalized Poly(N-isopropylacrylamide)-Based Microgels in Tumor Targeting and Drug Delivery

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

Recent developments and applications of smart nanoparticles in biomedicine

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

Collagen-Based Scaffolds for Chronic Skin Wound Treatment

Chronic wounds, commonly known as ulcers, represent a significant challenge to public health, impacting millions of individuals every year and imposing a significant financial burden on the global health system. Chronic wounds result from the interruption of the natural wound-healing process due to internal and/or external factors, resulting in slow or nonexistent recovery. Conventional medical approaches are often inadequate to deal with chronic wounds, necessitating the exploration of new methods to facilitate rapid and effective healing. In recent years, regenerative medicine and tissue engineering have emerged as promising avenues to encourage tissue regeneration. These approaches aim to achieve anatomical and functional restoration of the affected area through polymeric components, such as scaffolds or hydrogels. This review explores collagen-based biomaterials as potential therapeutic interventions for skin chronic wounds, specifically focusing on infective and diabetic ulcers. Hence, the different approaches described are classified on an action-mechanism basis. Understanding the issues preventing chronic wound healing and identifying effective therapeutic alternatives could indicate the best way to optimize therapeutic units and to promote more direct and efficient healing

Recovery of Bioactive Compounds from Marine Organisms: Focus on the Future Perspectives for Pharmacological, Biomedical and Regenerative Medicine Applications of Marine Collagen

Marine environments cover more than 70% of the Earth’s surface and are among the richest and most complex ecosystems. In terms of biodiversity, the ocean represents an important source, still not widely exploited, of bioactive products derived from species of bacteria, plants, and animals. However, global warming, in combination with multiple anthropogenic practices, represents a serious environmental problem that has led to an increase in gelatinous zooplankton, a phenomenon referred to as jellyfish bloom. In recent years, the idea of “sustainable development” has emerged as one of the essential elements of green-economy initiatives; therefore, the marine environment has been re-evaluated and considered an important biological resource. Several bioactive compounds of marine origin are being studied, and among these, marine collagen represents one of the most attractive bio-resources, given its use in various disciplines, such as clinical applications, cosmetics, the food sector, and many other industrial applications. This review aims to provide a current overview of marine collagen applications in the pharmacological and biomedical fields, regenerative medicine, and cell therapy