Search

Intratumoral drug delivery is a promising approach for the treatment of glioblastoma multiforme (GBM)., Intratumoral drug delivery is a promising approach for the treatment of glioblastoma multiforme (GBM). However, drug washout remains a major challenge in GBM therapy. Our strategy, aimed at reducing drug clearance and enhancing site-specific residence time, involves the local administration of a multi-component system comprised of nanoparticles (NPs) embedded within a thermosensitive hydrogel (HG). Herein, our objective was to examine the distribution of NPs and their cargo following intratumoral administration of this system in GBM. We hypothesized that the HG matrix, which undergoes rapid gelation upon increases in temperature, would contribute towards heightened site-specific retention and permanence of NPs in tumors. BODIPY-containing, infrared dye-labeled polymeric NPs embedded in a thermosensitive HG (HG–NPs) were fabricated and characterized. Retention and distribution dynamics were subsequently examined over time in orthotopic GBM-bearing mice. Results demonstrate that the HG–NPs system significantly improved site-specific, long-term retention of both NPs and BODIPY, with co-localization analyses showing that HG–NPs covered larger areas of the tumor and the peri-tumor region at later time points. Moreover, NPs released from the HG were shown to undergo uptake by surrounding GBM cells. Findings suggest that intratumoral delivery with HG–NPs has immense potential for GBM treatment, as well as other strategies where site-specific, long-term retention of therapeutic agents is warranted.

The high drug loading capacity, cytocompatibility and easy functionalization of ordered mesoporous carbons (OMCs) make them attractive nanocarriers to treat several pathologies. OMCs&rsquo, efficiency could be further increased by embedding them into a hydrogel phase for an in loco prolonged drug release. In this work, OMCs were embedded into injectable thermosensitive hydrogels. In detail, rod-like (diameter ca. 250 nm, length ca. 700 nm) and spherical (diameter approximately 120 nm) OMCs were synthesized by nanocasting selected templates and loaded with ibuprofen through a melt infiltration method to achieve complete filling of their pores (100% loading yield). In parallel, an amphiphilic Poloxamer&reg, 407-based poly(ether urethane) was synthesized (Mn&macr, 72 kDa) and solubilized at 15 and 20% w/v concentration in saline solution to design thermosensitive hydrogels. OMC incorporation into the hydrogels (10 mg/mL concentration) did not negatively affect their gelation potential. Hybrid systems successfully released ibuprofen at a slower rate compared to control gels (gels embedding ibuprofen as such), but with no significant differences between rod-like and spherical OMC-loaded gels. OMCs can thus work as effective drug reservoirs that progressively release their payload over time and also upon encapsulation in a hydrogel phase, thus opening the way to their application to treat many different pathological states (e.g., as topical medications).

The development of new bio-based inks is a stringent request for the expansion of additive manufacturing towards the development of 3D-printed biocompatible hydrogels. Herein, methacrylated carboxymethyl cellulose (M-CMC) is investigated as a bio-based photocurable ink for digital light processing (DLP) 3D printing. CMC is chemically modified using methacrylic anhydride. Successful methacrylation is confirmed by 1H NMR and FTIR spectroscopy. Aqueous formulations based on M-CMC/lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator and M-CMC/Dulbecco&rsquo, s Modified Eagle Medium (DMEM)/LAP show high photoreactivity upon UV irradiation as confirmed by photorheology and FTIR. The same formulations can be easily 3D-printed through a DLP apparatus to produce 3D shaped hydrogels with excellent swelling ability and mechanical properties. Envisaging the application of the hydrogels in the biomedical field, cytotoxicity is also evaluated. The light-induced printing of cellulose-based hydrogels represents a significant step forward in the production of new DLP inks suitable for biomedical applications.

3D tissue-engineered models are promising tools in the screening and evaluation of drugs and therapies as well as in the investigation of the molecular mechanisms involved in disease onset and progression. In this context, we describe our efforts in soft tissue replication, to design in vitro models that have the potential to provide better insight into the development of ageing process and related pathologies, with particular reference to the cardiovascular field.

Nerve guidance channels facilitate nerve regeneration and represent an attractive alternative to nerve graft. Actually, nano- and microstructured biomaterials for nerve reconstruction have gained much attention, thanks to recent discoveries about topography effects on cell behavior and morphology. Electrospun fibres have been proposed as filler or structural component for nerve guidance channels, principally due to their similarity with extracellular matrices which facilitate nerve regeneration. Among several tested biomaterials, gelatin has been used to prepare fibres able to support Schwann cell migration and neurite outgrowth. In this work, the effects of gelatin fibre size on axon elongation and Schwann cell migration have been tested using dorsal root ganglia cultures. Moreover, we analyzed how fibres might affect the expression of specific neuronal subtype markers in sensory neuron cultures and how the combined effect of substrate and biological cues affects neurite growth and gene expression. Data show that fibre topography differentially affects both neurite outgrowth and gene expression and suggest that fibre size and topography associated to specific growth factor exposure might be used to select neuron subpopulations and favor the axonal growth of specific neurons. Anat Rec, 301:1668-1677, 2018. © 2018 Wiley Periodicals, Inc.

The replication method is a widely used technique to produce bioactive glass (BG) scaffolds mimicking trabecular bone. However, these scaffolds usually exhibit poor mechanical reliability and fast degradation, which can be improved by coating them with a polymer. In this work, we proposed the use of custom-made poly(urethane)s (PURs) as coating materials for 45S5 Bioglass®-based scaffolds. In detail, BG scaffolds were dip-coated with two PURs differing in their soft segment (poly(ε-caprolactone) or poly(ε-caprolactone)/poly(ethylene glycol) 70/30 w/w) (PCL-PUR and PCL/PEG-PUR) or PCL (control). PUR-coated scaffolds exhibited biocompatibility, high porosity (ca. 91%), and improved mechanical properties compared to BG scaffolds (2–3 fold higher compressive strength). Interestingly, in the case of PCL-PUR, compressive strength significantly increased by coating BG scaffolds with an amount of polymer approx. 40% lower compared to PCL/PEG-PUR- and PCL-coated scaffolds. On the other hand, PEG presence within PCL/PEG-PUR resulted in a fast decrease in mechanical reliability in an aqueous environment. PURs represent promising coating materials for BG scaffolds, with the additional pros of being ad-hoc customized in their physico-chemical properties. Moreover, PUR-based coatings exhibited high adherence to the BG surface, probably because of the formation of hydrogen bonds between PUR N-H groups and BG surface functionalities, which were not formed when PCL was used.

The purpose of this work was the preparation and characterization of polymeric membranes for the selective recognition of saccharides using molecular imprinting technology associated to phase inversion. A system able to bind saccharides with high selectivity is particularly important in the pharmaceutical sector, since some of these compounds are constituents of molecules which can exert serious toxic effects even at very low concentrations. Two polymeric matrices were prepared using poly(ethylene-co-vinyl alcohol) copolymers, with an ethylene molar content of 32% and 44%, and imprinted with two different saccharide molecules: maltose and 2-keto 3-deoxy-D-manno-octulosonate (KDO). Matrices imprinted against maltose and KDO showed an easy template extraction, high binding capability and satisfactory selectivity, particularly for matrix with an ethylene molar content of 44%.

Electrospun membranes have been widely used as scaffolds for soft tissue engineering due to their extracellular matrix-like structure. A mussel-inspired coating approach based on 3,4-dihydroxy-DL-phenylalanine (DOPA) polymerization was proposed to graft gelatin (G) onto poly(lactic-co-glycolic) acid (PLGA) electrospun membranes. PolyDOPA coating allowed grafting of gelatin to PLGA fibers without affecting their bulk characteristics, such as molecular weight and thermal properties. PLGA electrospun membranes were dipped in a DOPA solution (2 mg/mL, Tris/HCl 10 mM, pH 8.5) for 7 h and then incubated in G solution (2 mg/mL, Tris/HCl 10 mM, pH 8.5) for 16 h. PLGA fibers had an average diameter of 1.37 &plusmn, 0.23 &micro, m. Quartz crystal microbalance with dissipation technique (QCM-D) analysis was performed to monitor DOPA polymerization over time: after 7 h the amount of deposited polyDOPA was 71 ng/cm2. After polyDOPA surface functionalization, which was, also revealed by Raman spectroscopy, PLGA membranes maintained their fibrous morphology, however the fiber size and junction number increased. Successful functionalization with G was demonstrated by FTIR-ATR spectra, which showed the presence of G adsorption bands at 1653 cm&minus, 1 (Amide I) and 1544 cm&minus, 1 (Amide II) after G grafting, and by the Kaiser Test, which revealed a higher amount of amino groups for G functionalized membranes. Finally, the biocompatibility of the developed substrates and their ability to induce cell growth was assessed using Neonatal Normal Human Dermal Fibroblasts.

Defining the best combination of cells and biomaterials is a key challenge for the development of tendon tissue engineering (TE) strategies. Adipose-derived stem cells (ASCs) are ideal candidates for this purpose. In addition, controlled cell-based products adherent to good manufacturing practice (GMP) are required for their clinical scale-up. With this aim, in this study, ASC 3D bioprinting and GMP-compliant tenogenic differentiation were investigated. In detail, primary human ASCs were embedded within a nanofibrillar-cellulose/alginate bioink and 3D-bioprinted into multi-layered square-grid matrices. Bioink viscoelastic properties and scaffold ultrastructural morphology were analyzed by rheology and scanning electron microscopy (SEM). The optimal cell concentration for printing among 3, 6 and 9 &times, 106 ASC/mL was evaluated in terms of cell viability. ASC morphology was characterized by SEM and F-actin immunostaining. Tenogenic differentiation ability was then evaluated in terms of cell viability, morphology and expression of scleraxis and collagen type III by biochemical induction using BMP-12, TGF-&beta, 3, CTGF and ascorbic acid supplementation (TENO). Pro-inflammatory cytokine release was also assessed. Bioprinted ASCs showed high viability and survival and exhibited a tenocyte-like phenotype after biochemical induction, with no inflammatory response to the bioink. In conclusion, we report a first proof of concept for the clinical scale-up of ASC 3D bioprinting for tendon TE.

In the era of personalised medicine, novel therapeutic approaches raise increasing hopes to address currently unmet medical needs by developing patient-customised treatments. Three-dimensional (3D) bioprinting is rapidly evolving and has the potential to obtain personalised tissue constructs and overcome some limitations of standard tissue engineering approaches. Bioprinting could support a wide range of biomedical applications, such as drug testing, tissue repair or organ transplantation. There is a growing interest for 3D bioprinting in the orthopaedic field, with remarkable scientific and technical advances. However, the full exploitation of 3D bioprinting in medical applications still requires efforts to anticipate the upcoming challenges in translating bioprinted products from bench to bedside. In this review we summarised current trends, advances and challenges in the application of 3D bioprinting for bone and cartilage tissue engineering. Moreover, we provided a detailed analysis of the applicable regulations through the 3D bioprinting process and an overview of available standards covering bioprinting and additive manufacturing.

Piezoelectric ceramic nanomaterials have recently attracted attention in the biomedical field thanks to their interesting electrical properties in response to mechanical stimulation (and vice versa) combined with a good biocompatibility and the ability to promote the regeneration of electrically sensitive tissues. In tissue engineering approaches, in order to obtain smart scaffolds these materials must be combined with other biomaterials for processing through conventional as well as non-conventional technologies. In this work, a novel composite electrospun membrane was produced by combining extracellular matrix-like gelatin nanofibers with barium titanate nanoparticles (BTNPs). The electrospinning process was optimized to achieve a high BTNP load, reducing the formation of aggregates which could alter the morphology and stability of the membrane. A complete morphological, mechanical and chemical–physical characterization of the composite membranes was performed, confirming the integration of the BTNPs into the polymer fibers. Furthermore, the biocompatibility of the developed membranes was assessed using a sarcoma osteogenic cell line (SaOS-2).

The design of supramolecular (SM) hydrogels based on host-guest complexes represents an effective strategy to develop drug delivery systems. In this work, we designed SM hydrogels based on α-cyclodextrin and high-molar mass amphiphilic poly(ether urethane)s (PEUs, ) based on Poloxamer® 407 and differing in their chain extender. The successful formation of poly(pseudo)rotaxanes and their supramolecular interactions were chemically demonstrated. Then, self-healing (80-100% mechanical recovery) supramolecular hydrogels were developed by mixing PEU and α-cyclodextrin solutions at different concentrations. Stability in physiological-like environment and mechanical properties improved with increasing α-cyclodextrin content (9-10% w/v), meanwhile gelation time decreased. A synergistic effect of poly(pseudo)rotaxanes crystals and PEU micellar structures on gel properties was observed: the first were predominant at low PEU concentrations (1-5% w/v), while the latter prevailed at high PEU concentrations (7-9% w/v). Increasing PEU concentration led to gels with increased dissolution rate, not-fully developed networks and slight cytotoxicity, meanwhile residence time in aqueous media improved (>7 d). At low PEU concentrations (1-5% w/v), cytocompatible gels (100% cell viability) were obtained, which maintained their shape in aqueous medium up to 5 d and completely dissolved within 7 d. PEU chemical composition affected PEU/α-cyclodextrin interactions, with longer gelation time and lower mechanical properties in gels based on PEU with pendant functionalities. Gels progressively released a model molecule (fluorescein isothiocyanate-dextran) within 3-4 days with no initial burst release. We thus demonstrated the suitability of custom-made PEUs as constituent of SM hydrogels with α-cyclodextrin and the high potential of the resulting systems for drug delivery applications.

Poly(L-lactic) acid (PLLA) has been widely employed in tissue engineering due to its mechanical properties, biodegradability and biocompatibility. The layer-by-layer (LbL) technique was here proposed as a simple method to impart bioactivity to the surface of PLLA substrates. Aminolysis treatment was applied to introduce amino groups on the surface of PLLA solvent cast films. Then, PLLA films were coated with heparin (HE)/chitosan (CH) multilayer by the LbL technique. Each functionalization step was characterized through physico-chemical and morphological analyses. Aminolysis treatment increased film surface wettability (64.8° ± 2.4° against 74.6° ± 1.3° for untreated PLLA) due to the formation of surface amino groups, which were quantified by acid orange colorimetric assay (0.05 nmol/mm

Polymer nanoparticles (nps) have gained growing interest as carriers for anticancer drugs as they can target tumour tissues by both passive and active pathways. While the passive targeting mechanisms mainly rely on the small size of the carriers, active targeting requires surface modifications of the polymer core in order to introduce specific functionalities to actively recognize cancer cells. The present work proposes an innovative method for the preparation of surface-functionalized nps based on the use of biodegradable polyester- and polyester/ether-urethanes (PURs) embedding amino functionalities. Two polyurethanes were prepared, one based on just poly(ϵ-caprolactone) diol (PCL-PUR) and the other based on both PCL diol and poly(ethylene glycol) (PEG) (70/30 ratio, PCL-PEG-PUR). Nanoparticles of small size ranging between 150 and 200 nm and negative ζ potential (ranging from −18 mV to −27 mV) were obtained. Functional groups were exposed post nps preparation as confirmed by X-ray photoelectron spectroscopy, ninhydrin assay and 1H NMR, which evidenced a 24% tert-butyloxycarbonyl cleavage for PCL-PUR-NH2 nps and 29% for PCL-PEG-PUR-NH2 nps. The monoclonal antibody Herceptin (HER), which targets HER-2 receptors, was coupled through ethyl(dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) mediated chemistry. The optimal HER:NH2 ratio was determined to be 1:16 for the PEG-containing PUR and 1:8 for PCL-PUR. HER-nps maintained the intrinsic cytotoxicity of the antibody, as shown by the ca 50% decrease of HER-2-expressing HeLa cell viability. The results indicate that our protocol for surface functionalization of PUR nps, based on surface exposure of previously inserted functional groups followed by covalent coupling of biomolecules, is suitable for the preparation of nps for active recognition of target cells. © 2016 Society of Chemical Industry

Poloxamer P407 (P407) is a Food and Drug Administration approved triblock copolymer; its hydrogels show fast dissolution in aqueous environment and weak mechanical strength, limiting their in vivo application. In this work, an amphiphilic poly(ether urethane) (NHP407) was synthesized from P407, an aliphatic diisocyanate (1,6-hexanediisocyanate) and an amino acid derived diol (N-Boc serinol). NHP407 solutions in water-based media were able to form biocompatible injectable thermosensitive hydrogels with a lower critical gelation temperature behavior, having lower critical gelation concentration (6% w/v versus 18% w/v), superior gel strength (G′ at 37 °C about 40 000 Pa versus 10 000 Pa), faster gelation kinetics (

Localised controlled release of simvastatin from porous freeze-dried chitosan-gelatin (CH-G) scaffolds was investigated by incorporating simvastatin loaded poly-(dl-lactide-co-glycolide) acid (PLGA) microparticles (MSIMs) into the scaffolds. MSIMs at 10% w/w simvastatin loading were prepared using a single emulsion-solvent evaporation method. The MSIM optimal amount to be incorporated into the scaffolds was selected by analysing the effect of embedding increasing amounts of blank PLGA microparticles (BL-MPs) on the scaffold physical properties and on the in vitro cell viability using a clonal human osteoblastic cell line (hFOB). Increasing the BL-MP content from 0% to 33.3% w/w showed a significant decrease in swelling degree (from 1245±56% to 570±35%). Scaffold pore size and distribution changed significantly as a function of BL-MP loading. Compressive modulus of scaffolds increased with increasing BL-MP amount up to 16.6% w/w (23.0±1.0kPa). No significant difference in cell viability was observed with increasing BL-MP loading. Based on these results, a content of 16.6% w/w MSIM particles was incorporated successfully in CH-G scaffolds, showing a controlled localised release of simvastatin able to influence the hFOB cell proliferation and the osteoblastic differentiation after 11 days.

Reduced periodontal support, deriving from chronic inflammatory conditions, such as periodontitis, is one of the main causes of tooth loss. The use of dental implants for the replacement of missing teeth has attracted growing interest as a standard procedure in clinical practice. However, adequate bone volume and soft tissue augmentation at the site of the implant are important prerequisites for successful implant positioning as well as proper functional and aesthetic reconstruction of patients. Three-dimensional (3D) scaffolds have greatly contributed to solve most of the challenges that traditional solutions (i.e., autografts, allografts and xenografts) posed. Nevertheless, mimicking the complex architecture and functionality of the periodontal tissue represents still a great challenge. In this study, a porous poly(&epsilon, caprolactone) (PCL) and Sr-doped nano hydroxyapatite (Sr-nHA) with a multi-layer structure was produced via a single-step additive manufacturing (AM) process, as a potential strategy for hard periodontal tissue regeneration. Physicochemical characterization was conducted in order to evaluate the overall scaffold architecture, topography, as well as porosity with respect to the original CAD model. Furthermore, compressive tests were performed to assess the mechanical properties of the resulting multi-layer structure. Finally, in vitro biological performance, in terms of biocompatibility and osteogenic potential, was evaluated by using human osteosarcoma cells. The manufacturing route used in this work revealed a highly versatile method to fabricate 3D multi-layer scaffolds with porosity levels as well as mechanical properties within the range of dentoalveolar bone tissue. Moreover, the single step process allowed the achievement of an excellent integrity among the different layers of the scaffold. In vitro tests suggested the promising role of the ceramic phase within the polymeric matrix towards bone mineralization processes. Overall, the results of this study demonstrate that the approach undertaken may serve as a platform for future advances in 3D multi-layer and patient-specific strategies that may better address complex periodontal tissue defects.

Recent progresses in tissue engineering are directed towards the development of technologies able to provide personalized scaffolds recreating the defect shape in a patient specific manner. To achieve this ambitious goal, 3D bioprinting can be combined with a suitable bioink, able to create a physiological milieu for cell growth. In this work, a novel chitosan-based hydrogel was developed combining photocrosslinking and thermo-sensitive properties. Commercial chitosan (CS) was first methacrylated and then mixed with β glycerol phosphate salt (β-GP) to impart a thermally induced phase transition. The absence of cytotoxic degradation products and the excellent biocompatibility of the developed hydrogel was confirmed through in vitro tests using different cell lines (NIH/3T3, Saos-2, SH-SY5Y). Cellularized 3D structures were obtained though 3D bioprinting technologies confirming the processability of the developed hydrogels and its unique biological properties.

The characteristic poor stability and high fluid permeability of Poloxamer®- based gels have severely limited their biomedical application. In this work, Poloxamer 407 was used as building block to synthesize a poly(ether urethane) (PEU), which aqueous solutions formed gels with improved stability and mechanics compared to Poloxamer itself. PEU chains formed micelles in aqueous solution (diameter ~40 nm at 25°C) and systems with PEU content higher than 5±1% w/v underwent a temperature- driven gelation. Gel properties were tuned acting on PEU concentration in the starting solutions, with compositions within the range 8-18% w/v showing high potential for biomedical applications (gelation at 37°C within 3-10 minutes, residence time from few days to many weeks, injectability). Model proteins (bovine serum albumin, horseradish peroxidase) were encapsulated in mild conditions and their release was modulated by gel composition (on day 3, approx. 85, 65 and 55% of encapsulated payload released from gels with 8, 15 and 18% w/v concentration). Released peroxidase retained approx. 30-40% of its activity up to 2 days, a key aspect for biomolecules in the drug delivery field.

Articular cartilage (AC) lacks the ability to self-repair and cell-based approaches, primarily based on using chondrocytes and mesenchymal stem cells (MSCs), are emerging as effective technology to restore cartilage functionality, because cells synergic functionality may support the maintenance of chondrogenic phenotype and promote extracellular matrix regeneration. This work aims to develop a more physiologically representative co-culture system to investigate the influence of MSCs on the activity of chondrocytes. A thermo-sensitive chitosan-based hydrogel, ionically crosslinked with β–glycerophosphate, is optimised to obtain sol/gel transition at physiological conditions within 5 minutes, high porosity with pores diameter in vitro mechanical integrity with compressive and equilibrium Young’s moduli of 37 kPa and 17 kPa, respectively. Live/dead staining showed that after 1 and 3 days in culture, the encapsulated MSCs into the hydrogels are viable and characterised by round-like morphology. Furthermore chondrocyte spheroids, seeded on top of gels that contained either MSCs or no cells, show that the encapsulated MSCs stimulate chondrocyte activity within a gel co-culture, both in terms of maintaining the coherence of chondrocyte spheroids, leading to a larger quantity of CD44 (by immunofluorescence) and a higher production of collagen and glycosaminoglycans (by histology) compared with the mono-culture.

In this study we examined the potential of novel biodegradable polymers of polyesterurethane (PU), and its PEGylated (PU-PEG) form as nanocarriers of Infliximab (INF), to treat inflammation in an in-vitro epithelial model. Nanoparticles (NPs) formulated were of average size of 200–287 nm. INF loading of NPs (INF-NPs) resulted in an increase in size and zeta potential. No cytotoxicity was observed for any of the NPs. Cellular interaction and uptake of PU NPs were similar compared with polycaprolactone (PCL) NPs and significantly higher to Poly(lactic-co-glycolic) acid (PLGA) NPs. Cellular interaction was higher for corresponding PEG-NPs. INF-PU and INF-PU-PEG NPs showed a rapid rate and extent of recovery of the epithelial barrier function in inflamed Caco-2 cell monolayers and decreased cytokine levels in inflamed monocytes. Results obtained in this study are promising and the potential of PU and PU-PEG NPs for drug delivery and targeting to treat gastrointestinal inflammation warrants further investigation.

Mini-invasively injectable hydrogels are widely attracting interest as smart tools for the co-delivery of therapeutic agents targeting different aspects of tissue/organ healing (e.g., neo-angiogenesis, inflammation). In this work, copper-substituted bioactive mesoporous glasses (Cu-MBGs) were prepared as nano- and micro-particles and successfully loaded with ibuprofen through an incipient wetness method (loaded ibuprofen approx. 10% w/w). Injectable hybrid formulations were then developed by dispersing ibuprofen-loaded Cu-MBGs within thermosensitive hydrogels based on a custom-made amphiphilic polyurethane. This procedure showed almost no effects on the gelation potential (gelation at 37 &deg, C within 3&ndash, 5 min). Cu2+ and ibuprofen were co-released over time in a sustained manner with a significantly lower burst release compared to MBG particles alone (burst release reduction approx. 85% and 65% for ibuprofen and Cu2+, respectively). Additionally, released Cu2+ species triggered polyurethane chemical degradation, thus enabling a possible tuning of gel residence time at the pathological site. The overall results suggest that hybrid injectable thermosensitive gels could be successfully designed for the simultaneous localized co-delivery of multiple therapeutics.

Cancer therapy often relies on the combined action of different molecules to overcome drug resistance and enhance patient outcome. Combined strategies relying on molecules with different pharmacokinetics often fail due to the lack of concomitant tumor accumulation and, thus, to the loss of synergistic effect. Due to their ability to enhance treatment efficiency, improve drug pharmacokinetics, and reduce adverse effects, polymer nanoparticles (PNPs) have been widely investigated as co-delivery vehicles for cancer therapies. However, co-encapsulation of different drugs and probes in PNPs requires a flexible polymer platform and a tailored particle design, in which both the bulk and surface properties of the carriers are carefully controlled. In this work, we propose a core-shell PNP design based on a polyurethane (PUR) core and a phospholipid external surface. The modulation of the hydrophilic/hydrophobic balance of the PUR core enhanced the encapsulation of two chemotherapeutics with dramatically different water solubility (Doxorubicin hydrochloride, DOXO and Docetaxel, DCTXL) and of Iron Oxide Nanoparticles for MRI imaging. The outer shell remained unchanged among the platforms, resulting in un-modified cellular uptake and in vivo biodistribution. We demonstrate that the choice of PUR core allowed a high entrapment efficiency of all drugs, superior or comparable to previously reported results, and that higher core hydrophilicity enhances the loading efficiency of the hydrophilic DOXO and the MRI contrast effect. Moreover, we show that changing the PUR core did not alter the surface properties of the carriers, since all particles showed a similar behavior in terms of cell internalization and in vivo biodistribution. We also show that PUR PNPs have high passive tumor accumulation and that they can efficient co-deliver the two drugs to the tumor, reaching an 11-fold higher DOXO/DCTXL ratio in tumor as compared to free drugs. STATEMENT OF SIGNIFICANCE: Exploiting the synergistic action of multiple chemotherapeutics is a promising strategy to improve the outcome of cancer patients, as different agents can simultaneously engage different features of tumor cells and/or their microenvironment. Unfortunately, the choice is limited to drugs with similar pharmacokinetics that can concomitantly accumulate in tumors. To expand the spectrum of agents that can be delivered in combination, we propose a multi-compartmental core-shell nanoparticles approach, in which the core is made of biomaterials with high affinity for drugs of different physical properties. We successfully co-encapsulated Doxorubicin Hydrochloride, Docetaxel, and contrast agents and achieved a significantly higher concomitant accumulation in tumor versus free drugs, demonstrating that nanoparticles can improve synergistic cancer chemotherapy.

Hypothesis Wound healing is a complex process that often requires treatment with antibacterial agents to avoid infection, which affects the optimal tissue regeneration process. Ideal scaffolds for wound healing treatment should combine biomimetic features to ensure the tissue growth on properly designed extracellular matrix (ECM)-like scaffolds and antibacterial properties in order to avoid bacterial colonization. Experiments In this work, gelatin cross-linked nanofibers (GL-nanofibres), with diameters ranging from 200 to 300 nm, were prepared via a “green electrospinning technique” to mimic the structure and composition of the extracellular matrix (ECM), and promote the normal skin wound healing process. Nanofibres were doped with two antibacterial agents (gentamicin sulphate or silver nanoparticles) to achieve an antibacterial effect against Gram + and Gram- bacteria. Findings The ECM-mimicking structure of GL-nanofibres was not affected by the presence of the antibacterial agents, which were homogeneously distributed within the mats as shown by SEM and EDS. The antibacterial properties of the developed matrices were confirmed using 4 strains (S. aureus, S. epidermidis, P. aeruginosa and E. coli) while the biocompatibility of the developed substrates and their ability to induce cell growth was assessed using Neonatal Normal Human Dermal Fibroblasts (NHDF-Neo).

Inert polytetrafluoroethylene (PTFE) membranes for periodontal regeneration suffer from weak osteoconductive properties. In this work, a strategy for hydroxyapatite (HAp) coating on PTFE films through an adhesive layer of self-polymerized 3,4-dihydroxy-DL-phenylalanine (polyDOPA) was developed to improve surface properties. Physico-chemical and morphological analysis demonstrated the deposition of polyDOPA and HAp, with an increase in surface roughness and wettability. A discontinuous coating was present after 14 days in PBS and MC3T3-E1 cells proliferation and adhesion were improved. Results confirmed the potential application of polyDOPA/HAp-coated films for periodontal disease treatments.

Biodegradable poly(lactide-co-glycolide) (PLGA) nanoparticles, containing human parathyroid hormone (PTH (1–34)), prepared by a modified double emulsion-solvent diffusion-evaporation method, were incorporated in porous freeze-dried chitosan-gelatin (CH-G) scaffolds. The PTH-loaded nanoparticles (NPTH) were characterised in terms of morphology, size, protein loading, release kinetics and in vitro assessment of biological activity of released PTH and cytocompatibility studies against clonal human osteoblast (hFOB) cells. Structural integrity of incorporated and released PTH from nanoparticles was found to be intact by using Tris-tricine SDS-PAGE. In vitro PTH release kinetics from PLGA nanoparticles were characterised by a burst release followed by a slow release phase for 3–4 weeks. The released PTH was biologically active as evidenced by the stimulated release of cyclic AMP from hFOB cells as well as increased mineralisation studies. Both in vitro and cell studies demonstrated that the PTH bioactivity was maintained during the fabrication of PLGA nanoparticles and upon release. Finally, a content of 33.3% w/w NPTHs was incorporated in CH-G scaffolds, showing an intermittent release during the first 10 days and, followed by a controlled release over 28 days of observation time. The increased expression of Alkaline Phosphatase levels on hFOB cells further confirmed the activity of intermittently released PTH from scaffolds.

Electrospun fibrous substrates mimicking extracellular matrices can be prepared by electrospinning, yielding aligned fibrous matrices as internal fillers to manufacture artificial nerves. Gelatin aligned nano-fibers were prepared by electrospinning after tuning the collector rotation speed. The effect of alignment on cell adhesion and proliferation was tested in vitro using primary cultures, the Schwann cell line, RT4-D6P2T, and the sensory neuron-like cell line, 50B11. Cell adhesion and proliferation were assessed by quantifying at several time-points. Aligned nano-fibers reduced adhesion and proliferation rate compared with random fibers. Schwann cell morphology and organization were investigated by immunostaining of the cytoskeleton. Cells were elongated with their longitudinal body parallel to the aligned fibers. B5011 neuron-like cells were aligned and had parallel axon growth when cultured on the aligned gelatin fibers. The data show that the alignment of electrospun gelatin fibers can modulate Schwann cells and axon organization in vitro, suggesting that this substrate shows promise as an internal filler for the design of artificial nerves for peripheral nerve reconstruction.

Smart nanoscale drug delivery systems combine the advantage of the small size, to achieve passive targeting capacity and enhanced cell internalization, with the ability to undergo morphological or chemical changes in response to the target-site microenvironment, leading to accelerated release in the disease milieu. Such systems are designed to respond to a variety of different internal stimuli that characterize diseased tissues, the most common of which are pH, reductive environment, temperature, and enzyme concentration. For instance, tumor pH is substantially lower than normal tissue pH, and the concentration of certain enzymes, such as matrix metalloproteases, is increased to sustain tumor growth. Moreover, the high rate of cell proliferation and the lack of an efficient circulation system make the microenvironment highly reductive. All of these features can be exploited to maximize drug delivery at the target site, through the design of delivery systems that accelerate payload release under the specific target-site conditions. This chapter reviews the most common stimuli-sensitive polymer drug delivery systems, with a specific focus on tumor milieu-responsive nanoparticles.

Random and aligned gelatin (GL) and chitosan (CS) nano-fibres have been prepared by electrospinning tuning the collector rotation speed. The effect of fibre alignment on cell adhesion and proliferation was assessed in vitro by using different Schwann cell (SC) and neuronal models. Moreover, actin cytoskeleton organization, lamellipodia and filipodia formation, and axon outgrowth were evaluated. GL and CS fibres induced similar adhesion and proliferation rates. GL and CS random fibres promoted higher adhesion and proliferation rates induction in comparison to the aligned ones, although GL and CS fibres alignment resulted in SC and axon-oriented growth. Filipodia formation was higher on aligned fibres, suggesting that these substrates can promote higher cell migration in comparison to random ones. 50B11 (neuronal cell line) differentiation was higher on GL fibres, whereas no differences were observed in dorsal root ganglia explants model. These data suggest that both GL and CS fibres can be promising substrates to be used in peripheral nerve reconstruction. Copyright © 2016 John WileySons, Ltd.

Nerve guidance channels facilitate nerve regeneration and represent an attractive alternative to nerve graft. Actually, nano- and microstructured biomaterials for nerve reconstruction have gained much attention, thanks to recent discoveries about topography effects on cell behavior and morphology. Electrospun fibres have been proposed as filler or structural component for nerve guidance channels, principally due to their similarity with extracellular matrices which facilitate nerve regeneration. Among several tested biomaterials, gelatin has been used to prepare fibres able to support Schwann cell migration and neurite outgrowth. In this work, the effects of gelatin fibre size on axon elongation and Schwann cell migration have been tested using dorsal root ganglia cultures. Moreover, we analyzed how fibres might affect the expression of specific neuronal subtype markers in sensory neuron cultures and how the combined effect of substrate and biological cues affects neurite growth and gene expression. Data show that fibre topography differentially affects both neurite outgrowth and gene expression and suggest that fibre size and topography associated to specific growth factor exposure might be used to select neuron subpopulations and favor the axonal growth of specific neurons. Anat Rec, 301:1668-1677, 2018. © 2018 Wiley Periodicals, Inc.

Three-dimensional (3D) tissue models offer new tools in the study of diseases. In the case of the engineering of cardiac muscle, a realistic goal would be the design of a scaffold able to replicate the tissue-specific architecture, mechanical properties, and chemical composition, so that it recapitulates the main functions of the tissue. This work is focused on the design and preliminary biological validation of an innovative polyester urethane (PUR) scaffold mimicking cardiac tissue properties. The porous scaffold was fabricated by thermally induced phase separation (TIPS) from poly(e-caprolactone) diol, 1,4-butanediisocyanate, and l-lysine ethyl ester. Morphological and mechanical scaffolds characterization was accomplished by confocal microscopy, and micro-tensile and compression techniques. Scaffolds were then functionalized with fibronectin by plasma treatment, and the surface treatment was studied by x-ray photoelectron spectroscopy, attenuated total reflectance Fourier transform infrared spectra, and contact angle measurements. Primary rat neonatal cardiomyocytes were seeded on scaffolds, and their colonization, survival, and beating activity were analyzed for 14 days. Signal transduction pathways and apoptosis involved in cells, the structural development of the heart, and its metabolism were analyzed. PUR scaffolds showed a porous-aligned structure and mechanical properties consistent with that of the myocardial tissue. Cardiomyocytes plated on the scaffolds showed a high survival rate and a stable beating activity. Serine/threonine kinase (AKT) and extracellular signal-regulated kinases (ERK) phosphorylation was higher in cardiomyocytes cultured on the PUR scaffold compared to those on tissue culture plates. Real-time polymerase chain reaction analysis showed a significant modulation at 14 days of cardiac muscle (MYH7, prepro-ET-1), hypertrophy-specific (CTGF), and metabolism-related (SLC2a1, PFKL) genes in PUR scaffolds.

Current therapeutic strategies to treat neurodegenerative diseases, such as alpha-synucleinopathies, aim at enhancing the amount of drug reaching the brain. Methods proposed, such as intranasal administration, should be able to bypass the blood brain barrier (BBB) and even when directly intracerebrally injected they could require a carrier to enhance local release of drugs. We have investigated the effect of a model synthetic hydrogel to be used as drug carrier on the amount of alpha-synuclein aggregates in cells in culture. The results indicated that alpha-synuclein aggregation was affected by the synthetic polymer, suggesting the need for testing the effect of any used material on the pathological process before its application as drug carrier.

During the past few decades, thiomers have attracted interest as responsive-polymers in the design of smart hydrogels, due to thiol reactivity to physical stimuli (i.e., pH, oxygen, UV/Vis irradiation). However, thiomer potentialities have been limited by their low thiolation degree and lack of multi-responsiveness to physical cues. In this work, a new class of thiomers with tailored thiolation degree and sensitivity to multiple physical stimuli was developed. To this aim, a polyurethane (33,000 Da) was synthesised from Poloxamer® 407, 1,6-diisocyanatohexane and N-Boc diethanolamine. Secondary amino groups were exposed through Boc-removal (yield 80%, assessed by 1H NMR) giving up to 4.5 × 1020 ± 1.8 × 1019 –NH units/gpolymer (Orange II Sodium Salt colorimetric assay) without polymer chemical degradation. Polymer thermo-responsiveness was proved by the increase in micelle hydrodynamic diameter upon system heating up to 45 °C and through estimated critical micellar temperatures. Thioglycolic acid grafting to exposed amines via carbodiimide chemistry was optimised to give up to 1.73 × 1019 thiols/gpolymer (Ellman's method), while minimising disulphide bond formation. Finally, the best storage conditions against oxidation were investigated by quantifying free thiols at different time points.

The development of suitable strategies to treat ageing-related pathologies has attracted the interest of researchers in view of the increasing life expectancy in the next decades. Osteoporosis is a worldwide disease with high prevalence in humans older than 50 that dramatically increases the risk of bone fractures with associated disabilities. The innovative use of new biomaterials as models of the healthy and osteoporotic bone matrix would be a new strategy to study the physiological conditions associated with osteoporosis and the connection between microenvironment changes and the bone ageing process. In this work, experimental bioactive glass substrates were coated with various polymer formulations in order to impart tunable surface features to the whole systems, which will act as models of the healthy and aged bone tissue once they have been colonized by cells.

Hydrogels are promising materials in regenerative medicine applications, due to their hydrophilicity, biocompatibility and capacity to release drugs and growth factors in a controlled manner. In this study, biocompatible and biodegradable hydrogels based on blends of natural polymers were used in in vitro and ex vivo experiments as a tool for VEGF-controlled release to accelerate the nerve regeneration process. Among different candidates, the angiogenic factor VEGF was selected, since angiogenesis has been long recognized as an important and necessary step during tissue repair. Recent studies have pointed out that VEGF has a beneficial effect on motor neuron survival and Schwann cell vitality and proliferation. Moreover, VEGF administration can sustain and enhance the growth of regenerating peripheral nerve fibres. The hydrogel preparation process was optimized to allow functional incorporation of VEGF, while preventing its degradation and denaturation. VEGF release was quantified through ELISA assay, whereas released VEGF bioactivity was validated in human umbilical vein endothelial cells (HUVECs) and in a Schwann cell line (RT4-D6P2T) by assessing VEGFR-2 and downstream effectors Akt and Erk1/2 phosphorylation. Moreover, dorsal root ganglia explants cultured on VEGF-releasing hydrogels displayed increased neurite outgrowth, providing confirmation that released VEGF maintained its effect, as also confirmed in a tubulogenesis assay. In conclusion, a gelatin-based hydrogel system for bioactive VEGF delivery was developed and characterized for its applicability in neural tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

Injectable hydrogels are becoming of increasing interest in the field of tissue engineering thanks to their versatile properties and to the possibility of being injected into tissues or devices during surgery. In peripheral nerve tissue engineering, injectable hydrogels having shear-thinning properties are advantageous as filler of nerve guidance channels (NGCs) to improve the regeneration process. In the present work, gelatin-based hydrogels were developed and specifically designed for the insertion into the lumen of hollow NGCs through a syringe during surgery. Injectable hydrogels were obtained using an agar-gelatin 20:80 weight ratio, (wt/wt) blend crosslinked by the addition of genipin (A/GL_GP). The physicochemical properties of the A/GL_GP hydrogels were analysed, including their injectability, rheological, swelling and dissolution behaviour, and their mechanical properties under compression. The hydrogel developed showed shear-thinning properties and was applied as filler of NGCs. The A/GL_GP hydrogel was tested in vitro using different cell lines, among them Schwann cells which have been used because they have an important role in peripheral nerve regeneration. Viability assays demonstrated the lack of cytotoxicity. In vitro experiments showed that the hydrogel is able to promote cell adhesion and proliferation. Two- and three-dimensional migration assays confirmed the capability of the cells to migrate both on the surface and within the internal framework of the hydrogel. These data show that A/GL_GP hydrogel has characteristics that make it a promising scaffold material for tissue engineering and nerve regeneration. Copyright © 2014 John Wiley & Sons, Ltd.

Biomaterials should be mechanically tested at both the nanoscale and macroscale under conditions simulating their working state, either in vitro or in vivo, to confirm their applicability in tissue engineering applications. In this article, polyester-urethane-based films and porous scaffolds produced by hot pressing and thermally induced phase separation respectively, were mechanically characterized at both the macroscale and nanoscale by tensile tests and indentation-type atomic force microscopy. All tests were conducted in wet state with the final aim of simulating scaffold real operating conditions. The films showed two distinct Young Moduli populations, which can be ascribed to polyurethane hard and soft segments. In the scaffold, the application of a thermal cooling gradient during phase separation was responsible for a nanoscale polymer chain organization in a preferred direction. At the macroscale, the porous matrices showed a Young Modulus of about 1.5 MPa in dry condition and 0.3 MPa in wet state. The combination of nanoscale and macroscale values as well as the aligned structure are in accordance with stiffness and structure required for scaffolds used for the regeneration of soft tissues such as muscles. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 162–169, 2015.

Plasma treatment is a widely applied, easy, fast, and highly reproducible surface modification technique. In this work powder plasma treatment was exploited to expose carboxylic groups along the backbone of a water soluble polymer. Specifically, a custom-made amphiphilic poly(ether urethane) containing Poloxamer&reg, 407 blocks (Mw = 54,000 Da) was first synthesized and its powders were plasma treated in the presence of Acrylic Acid vapor. To maximize &ndash, COOH group exposure while preventing polymer degradation, different Ar gas flow rates (i.e., 10, 30, and 50 sccm) were investigated. Upon gas flow increase, significant polymer degradation was observed, with a 35% molecular weight reduction at 50 sccm Ar flow rate. On the other hand, the highest number of exposed carboxylic groups (5.3 &times, 1018 &plusmn, 5.5 &times, 1017 units/gpolymer) was obtained by setting gas flow at 10 sccm. Hence, a gas flow of 10 sccm turned out to be the best set-up to maximize &ndash, COOH exposure while preventing degradation phenomena. Additionally, upon plasma treatment, no detrimental effects were observed in the thermoresponsiveness of polymer aqueous solutions, which was ensured by Poloxamer&reg, 407 blocks. Therefore, the newly developed technology here applied on an amphiphilic poly(ether urethane) could pave the way to the tailored design of a plethora of different multifunctional hydrogels.

The aim of the present study is to characterize the microstructure of composite scaffolds for bone tissue regeneration containing different ratios of chitosan/gelatin blend and bioactive glasses. Starting from realistic 3D models of the scaffolds reconstructed from micro-CT images, the level of heterogeneity of scaffold architecture is evaluated performing a lacunarity analysis. The results demonstrate that the presence of the bioactive glass component affects not only macroscopic features such as porosity, but mainly scaffold microarchitecture giving rise to structural heterogeneity, which could have an impact on the local cell-scaffold interaction and scaffold performances. The adopted approach allows to investigate the scale-dependent pore distribution within the scaffold and the related structural heterogeneity features, providing a comprehensive characterization of the scaffold texture.

The production of efficient heart patches for myocardium repair requires the use of biomaterials with high elastomeric properties and controllable biodegradability. To fulfil these design criteria we propose biodegradable poly(ester urethanes) and poly(ether ester urethanes) from poly(ɛ-caprolactone) (PCL) and poly(ethylene glycol) (PEG) as macrodiols, 1,4-diisocyanatobutane as diisocyanate, l-Lysine Ethyl Ester and Alanine-Alanine-Lysine (AAK) as chain extenders. This peptide was used to tune biodegradability properties, since the Alanine–Alanine sequence is a target for the elastase enzyme. Enzymatic degradation tests demonstrated the feasibility of tuning biodegradability properties due to the introduction of AAK peptide in polyurethane backbone. Two formulations have been processed into porous scaffolds by Thermally-Induced Phase Separation (TIPS). Scanning Electron Microscopy micrographs revealed promising microstructures, which were characterized by stretched and unidirectional pores and mimicked the striated muscle tissue. Tensile tests showed that, although scaffolds are characterized by lower mechanical properties than films, these substrates have suitable elastomeric behaviors and elastic moduli for contractile and soft tissue regeneration. Viability tests on cardiomyocytes revealed the best cell response for dense film and porous scaffold obtained from PCL and Lysine Ethyl Ester-based polyurethane, with an increased viability for the porous substrate, which is ascribable to the morphological features of its microstructure. Future works will be addressed to study the in vivo behavior of these constructs and to confirm their applicability for myocardial tissue engineering. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.

Nanomedicine formulations such as biodegradable nanoparticles (nps) and liposomes offer several advantages over traditional routes of administration: due to their small size, nanocarriers are able to selectively accumulate inside tumours or inflammatory tissues, resulting in improved drug efficacy and reduced side effects. To further augment targeting ability of nanoparticles towards tumour cells, specific ligands or antibodies that selectively recognise biomarkers over-expressed on cancer cells, can be attached to the surface either by chemical bond or by hydrophilic/hydrophobic interactions. In the present work, Herceptin (HER), a monoclonal antibody (mAb) able to selectively recognise HER-2 over-expressing tumour cells (such as breast and ovarian cancer cells), was absorbed on the surface of nanoparticles through hydrophilic/hydrophobic interactions. Nps were prepared by a modified single emulsion solvent evaporation method with five different polymers: three commercial polyesters (poly(ε-caprolactone) (PCL), poly (D,L-lactide) (PLA) and poly (D,L-lactide-co-.glycolide) (PLGA)) and two novel biodegradable polyesterurethanes (PURs) based on Poly(ε-caprolactone) blocks, synthesised with different chain extenders (1,4-cyclohexane dimethanol (CDM) and N-Boc-serinol). Polyurethanes were introduced as matrix-forming materials for nanoparticles due to their high chemical versatility, which allows tailoring of the materials final properties by properly selecting the reagents. All nps exhibited a small size and negative surface charge, suitable for surface functionalisation with mAb through hydrophilic/hydrophobic interactions. The extent of cellular internalisation was tested on two different cell lines: MCF-7 and SK-BR-3 breast cancer cells showing a normal and a high expression of the HER-2 receptor, respectively. Paclitaxel, a model anti-neoplastic drug, was encapsulated inside all nps, and release profiles and cytotoxicity on SK-BR-3 cells were also assessed. Interestingly, PUR nps were superior to commercial polyester-based nps in terms of higher cellular internalisation and cytotoxic activity on the tested cell lines. Results obtained warrants further investigation on the application of these PUR nps for controlled drug delivery and targeting.