Cytotoxicity and bioactivity assessments for Cu2+ and La3+ doped high-silica sol-gel derived bioglasses: The complex interplay between additive ions revealed.

We show the influence of two functional ions (Cu2+ and La3+ ), incorporated into a quaternary (Si, Ca, Na, P) sol-gel derived bioactive glass system, on its particle size, cytotoxicity, and bioactivity. By doping the parent glass with the two ions in singular or combined forms, 15 doped glasses were prepared by a rapid sol-gel technique. The influence of the combined doping on the particle size and cell viability was successfully evaluated by the aid of signal-to-noise-ratio (S/N), using Taguchi analysis. This allowed us to analyze the complex interplay of effects between these ions, and the marked differences in biocompatibility between the three cell types studied. Cu addition had a significant effect on reducing the glass particle size, while both increased density. Cell viability was significantly improved for some doping combinations, demonstrating that while combined Cu-La doping was beneficial for biocompatibility with lymphoblasts, individual high-Cu or low-La doping was better with fibroblasts, and either high-Cu or low-La doping, or certain combined Cu-La combinations, were the optimum for osteoblasts. However, the bioactivity of doped samples was generally similar to that of the parent glass, although both La, and particularly Cu, did appear to aid dissolution of ions when immersed in SBF, act as glass modifiers, and encourage HAp crystallization. The results reveal that potential synergistic benefits can be obtained by combining the effects on the mean particle size, density, cytotoxicity, and bioactivity of the glasses. The greatly improved biocompatibility of some of the doped glasses makes them promising candidates for biomedical applications.

Therapeutic PCL scaffold for reparation of resected osteosarcoma defect.

Osteosarcomas are highly malignant tumors, which develop rapid growth and local infiltration, inducing metastases that spread primarily in the lung. Treatment of these tumors is mainly based on pre- and post-operative chemotherapy and surgery of the primary tumor. Surgical resection though, generates bone defects. Reparation of these weaknesses presents formidable challenges to orthopedic surgery. Medicine regenerative grafts that act as both tumor therapy with constant local drug delivery and tissue regeneration may provide a new prospect to address this need. These implants can provide sustained drug release at the cancer area, decreasing systemic second effects such as inflammation, and a filling of the resected tissues with regenerative biomaterials. In this study microporous poly-ε-caprolactone (PCL) scaffolds have been developed for sustained local release of anti-inflammatory drug dexamethasone (DXM), used as drug model, in cancer medicine regenerative field. The microporous PCL matrix of the scaffolds supported the attachment, proliferation and osteogenic differentiation of osteoblast-like cells, while the polyelectrolyte multilayers, anchored to the inner pore surfaces, sustained locally DXM release. These microporous scaffolds demonstrate the ability to deliver DXM as a localized tumor therapy and to promote proliferation and differentiation of osteoblast-like cells in vitro.

mRNA delivery using non-viral PCL nanoparticles.

Messenger RNA (mRNA) provides a promising alternative to plasmid DNA as a genetic material for delivery in non-viral gene therapy strategies. However, it is difficult to introduce mRNA in vivo mainly because of the instability of mRNA under physiological conditions. Here, mRNA-protamine complex encapsulated poly(ε-caprolactone) (PCL) nanoparticles (NPs) are proposed for the intracellular delivery of mRNA molecules. The nanoparticles with a size of about 247 nm in diameter have a core-shell structure with an mRNA-containing inner core surrounded by PCL layers, providing high stability and stealth properties to the nanoparticles. The partial neutralization of the negatively charged mRNA molecules with the cationic protamine allows one to modulate the release kinetics in a pH-dependent manner. At pH 7.4, mimicking the conditions found in the systemic circulation, only 25% of the mRNA is released after 48 hours post incubation, whereas at pH 5.0, recreating the cell endosomal environment, about 60% of the mRNA molecules are released within the same time window post incubation. These NPs show no cytotoxicity to NIH 3T3 fibroblasts, HeLa cells and MG63 osteoblasts up to 8 days of incubation. Given the stability, preferential release behavior, and well-known biocompatibility properties of PCL nanostructures, our non-viral PCL nanoparticles are a promising system that simultaneously resolved the two major problems of mRNA introduction and the instability, opening the door to various new therapeutic strategies using mRNA.

Coupled delivery of imatinib mesylate and doxorubicin with nanoscaled polymeric vectors for a sustained downregulation of BCR-ABL in chronic myeloid leukemia.

In this work, we have investigated the potential benefits of combining biodegradable pH sensitive core-shell PCL NPs loaded with IM and enzyme sensitive polyelectrolyte complexes (PECs) loaded with doxorubicin (DOX). Our in vitro studies confirmed the excellent antileukemic activity of dual drug loaded nanoparticles on CML cells. As compared with a drug alone, co-treatment with IM and DOX loaded in nanoparticles allowed a sustained downregulation of BCR-ABL and significant CML stem cell death. This furthermore showed that couple formulation of nanoparticles enhanced the drugs' kinetics and efficacy, combined with the pH sensibility of core-shell PCL NPs loaded with IM and enzyme sensitive polyelectrolyte complexes (PECs) loaded with DOX. Our study demonstrates that dual drug loaded nanoparticles work in a synergistic manner, lowering the dose and confirming that both drugs reach the target cell specifically, maximizing the cytotoxicity while minimizing the chances of cell resistance to any one drug.

Biocompatible and biodegradable fluorescent microfibers physiologically secreted by live cells upon spontaneous uptake of thiophene fluorophore.

Live cells can form multifunctional and environmentally responsive multiscale assemblies of living and non-living components. We recently reported the results of a unique approach to introduce supplementary properties, fluorescence in particular, into fibrillar proteins produced by live fibroblasts and extruded into the ECM. In this work, we demonstrate that the physiological secretion of fluorescent nanostructured microfibers upon the spontaneous uptake of the appropriate fluorophore extends to living cells derived by different tissue contexts. We also show that live cells seeded on fluorescent microfibers have a different fate in terms of the cellular morphology, cytoskeleton rearrangement and viability. These results suggest that the microfibers, which are biocompatible and biodegradable, can be used as multiscale biomaterials to direct the cell behaviour.

Uptake of imatinib-loaded polyelectrolyte complexes by BCR-ABL(+) cells: a long-acting drug-delivery strategy for targeting oncoprotein activity.

RATIONALE & AIM: Imatinib mesylate (IM), a selective tyrosine kinase inhibitor of the oncoprotein BCR-ABL, is the 'gold standard' for patients with chronic myeloid leukemia (CML) but the drug does not eliminate CML stem cells, leading to disease relapse on drug discontinuation. At present, much effort is focused on delivery carriers that can increase the intracellular retention and antileukemic impact of IM. We previously validated IM-loaded polyelectrolyte microcapsules as effective purging agents to eradicate BCR-ABL(+) cells from CML patient autografts. The aim is to develop controlled release carriers that can increase the intracellular retention and functionality of IM in leukemia cells. MATERIALS & METHODS: Herein, novel polyelectrolyte complexes were used as model carriers for IM in a CML cell line (KU812) and CD34(+) cells freshly isolated from patients. RESULTS & DISCUSSION: Polyelectrolyte complexes promoted a long-acting BCR-ABL kinase inactivation that was necessary to promote apoptosis at approximately twofold lower intracellular IM dose compared with the microscale formulation polyelectrolyte microcapsules. CONCLUSION: IM-loaded polyelectrolyte complexes can be used as more efficient delivery devices for overcoming drug resistance of BCR-ABL(+) leukemic cells.

Physiological formation of fluorescent and conductive protein microfibers in live fibroblasts upon spontaneous uptake of biocompatible fluorophores.

We have recently reported initial results concerning an original approach to introduce additional properties into fibrillar proteins produced by live fibroblasts and extruded into the ECM. The key to such an approach was biocompatible, fluorescent and semiconducting synthetic molecules which penetrated spontaneously the cells and were progressively encompassed via non-bonding interactions during the self-assembly process of the proteins, without altering cell viability and reproducibility. In this paper we demonstrate that the intracellular secretion of fluorescent microfibers can be generalized to living primary and immortalized human/mouse fibroblasts. By means of real-time single-cell confocal microscopy we show that the fluorescent microfibers, most of which display helical morphology, are generated by intracellular coding of the synthetic molecules. We also describe co-localization experiments on the fluorescent microfibers isolated from the cell milieu demonstrating that they are mainly made of type-I collagen. Finally, we report experimental data indicating that the embedded synthetic molecules cause the proteins not only to be fluorescent but also capable of electrical conductivity.

Micropatterned polyelectrolyte nanofilms promote alignment and myogenic differentiation of C2C12 cells in standard growth media.

Alignment of skeletal myoblasts is considered a critical step during myotube formation. The C2C12 cell line is frequently used as a model of skeletal muscle differentiation that can be induced by lowering the serum concentration in standard culture flasks. In order to mimic the striated architectures of skeletal muscles in vitro, micro-patterning techniques and surface engineering have been proven as useful approaches for promoting elongation and alignment of C2C12 myoblasts, thereby enhancing the outgrowth of multi-nucleated myotubes upon switching from growth media (GM) to differentiative media (DM). Herein, a layer-by-layer (LbL) polyelectrolyte multilayer deposition was combined with a micro-molding in capillaries (MIMIC) method to simultaneously provide biochemical and geometrical instructive cues that induced the formation of tightly apposed and parallel arrays of differentiating myotubes from C2C12 cells maintained in GM media for 15 days. This study focuses on two different types of patterned/self-assembled nanofilms based on alternated layers of poly (allylamine hydrochloride) (PAH)/poly(sodium 4-styrene-sulfonate) (PSS) as biocompatible but not biodegradable polymeric structures, or poly-L-arginine sulfate salt (pARG)/dextran sulfate sodium salt (DXS) as both biocompatible and biodegradable surfaces. The influence of these microstructures as well as of the nanofilm composition on C2C12 skeletal muscle cells' differentiation and viability was evaluated and quantified, pointing to give a reference for skeletal muscle regenerative potential in culture conditions that do not promote it. At this regard, our results validate PEM microstructured devices, to a greater extent for (PAH/PSS)5-coated microgrooves, as biocompatible and innovative tools for tissue engineering applications and molecular dissection of events controlling C2C12 skeletal muscle regeneration without switching to their optimal differentiative culture media in vitro.

Modulation of alignment and differentiation of skeletal myoblasts by biomimetic materials.

Proliferation and fusion of myoblasts are needed for the generation and repair of multinucleated skeletal muscle fibers in vivo. We developed a novel cell culture technique that results in formation of myotubes, organized in parallel much like the arrangement in muscle tissue. Alignment and fusion of myoblasts into parallel arrays of multinucleated myotubes are critical in skeletal muscle tissue engineering and more micro-patterning techniques and surface engineering have been tested by switching differentiating myotubes from growth medium (GM) to differentiative media (DM). One of the goals of tissue engineering is to develop tools allowing in vitro construction and mimicking of the final tissue architectures. The fabrication of polyelectrolyte multilayers (PEMs) may represent a promising approach for recreating physiological nanometer-sized cell environments in vitro. In this study we describe a method for generating biomimetic microstructured surfaces that promote cell adhesion and differentiation of parallel arrays of mature C2C12 myotubes continuously maintained in GM for 7 days. The structure consists of a 'double-sheet' PDMS structure that provides 'compliant' or 'stiff' microdomains to guide the cell self-patterning, coupled to layer-by-layer (LbL) self-assembled multilayers of biocompatible polyelectrolytes that promote C2C12 myoblasts alignment and differentiation. Our findings have relevance to the interpretation of in vitro data as well as to the study of cellular interactions with biomaterials.

Cell self-patterning on uniform PDMS-surfaces with controlled mechanical cues.

The exploitation of cell-instructive scaffolds with uniform physical/chemical surfaces and controlled stiffness will be greatly useful in tissue engineering applications to resemble the extracellular matrix (ECM) or topographical appearance of native tissues. We herein describe a versatile and straightforward method to assemble a polydimethylsiloxane (PDMS)-composite structure in which a uniformly laminin-coated membrane is placed on top of a micropatterned substrate that applies a stiffness gradient. This 'double-sheet' structure provides soft or stiff microdomains that guide the self-patterning of different cell types [e.g. chronic myeloid leukemia (KU812), cervix carcinoma (HeLa), NIH 3T3 and BJ], thereby stimulating their cytoskeletal remodeling. More interestingly, we used these uniform PDMS surfaces with patterned rigidity for obtaining co-cultures of tumor blood cells (KU812) and adherent fibroblasts (NIH 3T3) with spatially-controlled distribution. Thus, beyond single-cell stiffening and mechanosensing, these surfaces should also be used as simple and feasible co-culture systems for mimicking and dissecting the bidirectional interactions between blood cells and specific stromal elements of their in vivo microenvironment.

Cell Uptake and Validation of Novel PECs for Biomedical Applications.

This pilot study provides the proof of principle for biomedical application of novel polyelectrolyte complexes (PECs) obtained via electrostatic interactions between dextran sulphate (DXS) and poly(allylamine hydrochloride) (PAH). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that DXS/PAH polyelectrolyte complexes were Monodispersed with regular rounded-shape features and average diameters of 250 nm at 2 : 1 weight ratios of DXS/PAH. Fluorescently labelled DXS and fluorescein-isothiocyanate- (FITC-)conjugate DXS were used to follow cell uptake efficiency of PECs and biodegradability of their enzymatically degradable DXS-layers by using confocal laser scanning microscopy (CLSM). Moreover, quantitative MTT and Trypan Blue assays were employed to validate PECs as feasible and safe nanoscaled carriers at single-cell level without adverse effects on metabolism and viability.

Imatinib-loaded polyelectrolyte microcapsules for sustained targeting of BCR-ABL+ leukemia stem cells.

AIM: The lack of sensitivity of chronic myeloid leukemia (CML) stem cells to imatinib mesylate (IM) commonly leads to drug dose escalation or early disease relapses when therapy is stopped. Here, we report that packaging of IM into a biodegradable carrier based on polyelectrolyte microcapsules increases drug retention and antitumor activity in CML stem cells, also improving the ex vivo purging of malignant progenitors from patient autografts. MATERIALS & METHODS: Microparticles/capsules were obtained by layer-by-layer (LbL) self-assembly of oppositely charged polyelectrolyte multilayers on removable calcium carbonate (CaCO(3)) templates and loaded with or without IM. A leukemic cell line (KU812) and CD34(+) cells freshly isolated from healthy donors or CML patients were tested. RESULTS & DISCUSSION: Polyelectrolyte microcapsules (PMCs) with an average diameter of 3 microm, fluorescently labelled multilayers sensitive to the action of intracellular proteases and 95-99% encapsulation efficiency of IM, were prepared. Cell uptake efficiency of such biodegradable carriers was quantified in KU812, leukemic and normal CD34(+) stem cells (range: 70-85%), and empty PMCs did not impact cell viability. IM-loaded PMCs selectively targeted CML cells, by promoting apoptosis at doses that exert only cytostatic effects by IM alone. More importantly, residual CML cells from patient leukapheresis products were reduced or eliminated more efficiently by using IM-loaded PMCs compared with freely soluble IM, with a purging efficiency of several logs. No adverse effects on normal CD34(+) stem-cell survival and their clonogenic potential was noticed in long-term cultures of hematopoietic progenitors in vitro. CONCLUSION: This pilot study provides the proof-of-principle for the clinical application of biodegradable IM-loaded PMC as feasible, safe and effective ex vivo purging agents to target CML stem cells, in order to improve transplant outcome of resistant/relapsed patients or reduce IM dose escalation.