Chitosan-PNIPAM Thermogel Associated with Hydrogel Microspheres as a Smart Formulation for MSC Injection.

Regenerative medicine based on cell therapy has emerged as a promising approach for the treatment of various medical conditions. However, the success of cell therapy heavily relies on the development of suitable injectable hydrogels that can encapsulate cells and provide a conducive environment for their survival, proliferation, and tissue regeneration. Herein, we address the medical need for cyto- and biocompatible injectable hydrogels by reporting on the synthesis of a hydrogel-forming thermosensitive copolymer. The copolymer was synthesized by grafting poly(N-isopropylacrylamide-co-carboxymethyl acrylate) (PNIPAM-COOH) onto chitosan through amide coupling. This chemical modification resulted in the formation of hydrogels that exhibit a sol-gel transition with an onset at approximately 27 °C, making them ideal for use in injectable applications. The hydrogels supported the survival and proliferation of cells for several days, which is critical for cell encapsulation. Furthermore, the study evaluates the addition of collagen/chitosan hybrid microspheres to support the adhesion of mesenchymal stem cells within the hydrogels. Altogether, these results demonstrate the potential of the PNIPAM-chitosan thermogel for cell encapsulation and its possible applications in regenerative medicine.

Size-tunable lipid vectors for controlled local delivery of siRNA from gene activated matrix.

Tissue engineering aims to restore or replace different types of biological tissues through the association of cells, biologic factors and biomaterials. Currently, stem cells arise as a major cell source for many therapeutic indications, and their association with 3D scaffolds allow increasing regenerative medicine efficiency. In this context, the use of RNA interference to enhance or control stem cell differentiation into the desired phenotype appears as a promising strategy. However, achieving high transfection efficiency of cells in a 3D structure requires the use of a vector allowing for the spatiotemporally controlled release of the genetic material from these scaffolds. In this study, we report a new siRNA nanovector, called solvent exchange lipoplexe formulation (SELF), which has a tunable size, is stable over time in cell culture conditions and possess a high efficiency to transfect primary human mesenchymal stromal cells (hMSC). We associated SELFs with porous 3D collagen microspheres and demonstrated that the loading capacity and release kinetics were different depending on the size of the associated SELF. Interestingly, these different release profiles resulted in differences in the transfection kinetics of hMSCs. This original and unique type of gene activated matrix, with adaptable release kinetics, could be of interest for long-term and/or sequential transfection profiles of stem cells in 3D culture. STATEMENT OF SIGNIFICANCE: This work combines the use of human mesenchymal stromal cell (hMSC) and gene therapy for tissue engineering. Here, a gene-activated matrix was elaborated with collagen microspheres supporting hMSCs and acting as a reservoir for transfection vectors. This injectable GAM allows for the local and sustained delivery of nucleic acids, hence long-lasting transfection of the supported cells. With the original synthesis protocol presented herein, the size of the nanocarriers can be easily adapted, resulting in different siRNA release profiles from the microspheres. Most interestingly, different siRNA release profiles gave rise to different cell transfection profiles as assessed by the downregulation of a target gene. This highlights the versatility of the system and its suitability for various pathophysiological needs in regenerative medicine.

Degradable double hydrophilic block copolymers and tripartite polyionic complex micelles thereof for small interfering ribonucleic acids (siRNA) delivery.

Polymer vectors for gene therapy have been largely investigated as an alternative to viral vectors. In particular, double hydrophilic block copolymers (DHBCs) have shown potential in this domain, but to date studies mainly focus on non-degradable copolymers, which may be a restriction for further development. To overcome this limitation, we synthesized a DHBC (PEG43-b-PCL12(COOH)6.5) composed of a poly(ethylene glycol) (PEG) non-ionic and bioeliminable block and a degradable carboxylic acid-functionalized poly(ε-caprolactone) (PCL) block. The potential of this DHBC as an original vector for small interfering ribonucleic acids (siRNA) to formulate tripartite polyionic complex (PIC) micelles with poly(lysine) (PLL) was evaluated. We first studied the impact of the charge ratio (R) on the size and the zeta potential of the resulting micelles. With a charge ratio R = 1, one formulation with optimized physico-chemical properties showed the ability to complex 75% of siRNA. We showed a stability of the micelles at pH 7.4 and a disruption at pH 5, which allowed a pH-triggered siRNA release and proved the pH-stimuli responsive character of the tripartite micelles. In addition, the tripartite PIC micelles were shown to be non-cytotoxic below 40 µg/mL. The potential of these siRNA vectors was further evaluated in vitro: it was found that the tripartite PIC micelles allowed siRNA internalization to be 3 times higher than PLL polyplexes in murine mesenchymal stem cells, and were able to transfect human breast cancer cells. Overall, this set of data pre-validates the use of degradable DHBC as non-viral vectors for the encapsulation and the controlled release of siRNA, which may therefore constitute a sound alternative to non-degradable and/or cytotoxic polycationic vectors.

Development of Coprocessed Chitin-Calcium Carbonate as Multifunctional Tablet Excipient for Direct Compression, Part 2: Tableting Properties.

The use of multifunctional excipients is gaining interest as it simplifies formulations by replacing the need of multiple monofunctional excipients. In previous work, coprocessed chitin-calcium carbonate (CC) showed to have good potential as a multifunctional excipient for fast disintegrating tablets produced by direct compression. It allowed for good tablet strength, enhanced powder flowability, and higher true and bulk densities with fast disintegrating properties. The objective of this work is to gain insight on CC tableting properties under different tablet manufacturing conditions (different lubrication levels, compression speeds, and dwell times) and in formulations with drug models: ibuprofen and paracetamol. Results showed that CC exhibited good tabletability, compressibility, and compactibility profiles. CC does not require the addition of lubricant and can be used at high compression speeds and different dwell times. When included in formulations with ibuprofen and paracetamol at different percentages, CC enhanced tablets strength and promoted fast disintegration and drug dissolution. In conclusion, this study shows that CC can be used as a multifunctional excipient (filler-disintegrant-binder) for fast disintegrating tablets produced by direct compression.

Development of Coprocessed Chitin-Calcium Carbonate as Multifunctional Tablet Excipient for Direct Compression.

Owing to the increasing interest in multifunctional excipients for tableting, coprocessing of individual excipients is regularly used to produce excipients of improved multifunctionality superior to individual excipients or their physical mix. The use of chitin as an excipient in tablet formulation is limited because of certain drawbacks such as poor flowability and low true density. The objective of this work is to improve these properties through coprocessing of chitin with calcium carbonate (CaCO3) by precipitating CaCO3 on chitin particles using different methods. In addition, optimization of the coprocessed chitin was carried out to improve the excipient's properties. Physicochemical (CaCO3 content, true density, X-ray diffraction, infrared spectroscopy, and scanning electron microscopy) and functional testing (swelling force, flowability, tensile strength, deformation mechanism, and disintegration time) were used to characterize the coprocessed product. Results showed that the calcite CaCO3 polymorph is precipitated on the chitin surface and that it interacts with chitin at carbonyl- and amide-group level. In addition, the coprocessed excipient has an improved true density and powder flowability, with CaCO3 forming single layer on the chitin particles surface. Tableting studies showed that the coprocessed powder exhibited an intermediate deformation behavior between CaCO3 (most brittle) and chitin (most plastic). Tablets showed acceptable tensile strength and rapid disintegration (2-4 s). These results show the potential use of coprocessed chitin-CaCO3 as a multifunctional excipient for fast disintegration of tablets produced by direct compression.

Electric-field-induced shape transition of nematic tactoids.

The occurrence of new textures of liquid crystals is an important factor in tuning their optical and photonics properties. Here, we show, both experimentally and by numerical computation, that under an electric field chitin tactoids (i.e., nematic droplets) can stretch to aspect ratios of more than 15, leading to a transition from a spindlelike to a cigarlike shape. We argue that the large extensions occur because the elastic contribution to the free energy is dominated by the anchoring. We demonstrate that the elongation involves hydrodynamic flow and is reversible: the tactoids return to their original shapes upon removing the field.

Tripartite polyionic complex (PIC) micelles as non-viral vectors for mesenchymal stem cell siRNA transfection.

In the context of regenerative medicine, the use of RNA interference mechanisms has already proven its efficiency in targeting specific gene expression with the aim of enhancing, accelerating or, more generally, directing stem cell differentiation. However, achievement of good transfection levels requires the use of a gene vector. For in vivo applications, synthetic vectors are an interesting option to avoid possible issues associated with viral vectors (safety, production costs, etc.). Herein, we report on the design of tripartite polyionic complex micelles as original non-viral polymeric vectors suited for mesenchymal stem cell transfection with siRNA. Three micelle formulations were designed to exhibit pH-triggered disassembly in an acidic pH range comparable to that of endosomes. One formulation was selected as the most promising with the highest siRNA loading capacity while clearly maintaining pH-triggered disassembly properties. A thorough investigation of the internalization pathway of micelles into cells with tagged siRNA was made before showing an efficient inhibition of Runx2 expression in primary bone marrow-derived stem cells. This work evidenced PIC micelles as promising synthetic vectors that allow efficient MSC transfection and control over their behavior, from the perspective of their clinical use.

PeakForce QNM AFM study of chitin-silica hybrid films.

Chitin-silica hybrid thin films, prepared through the colloidal self-assembly of chitin nanorods and siloxane oligomers, have been studied for the first time by PeakForce QNM AFM mode to explore their structure and mechanical behaviour. The change in structure and mechanical properties of chitin-silica hybrids is mainly driven by the relative quantities in chitin nanorods and silica, expressed as the chitin volume fraction ϕchi. The coating of the chitin polysaccharide by silica leads to an increase of the nanorods diameter and films surface roughness at small ϕchi values. The DMT (Derjaguin-Muller-Toporov) modulus increased both at small ϕchi due to a large amount of silica and at very high ϕchi→1 due to an incomplete tip penetration between nanorods. The local parallel orientation of nanorods observed at different ϕchi values resulted in a modulus increase due to an enhancement of the cohesion between nanorods.

Chitin's Functionality as a Novel Disintegrant: Benchmarking Against Commonly Used Disintegrants in Different Physicochemical Environments.

Disintegrants are used as excipients to ensure rapid disintegration of pharmaceutical tablets and further ensure proper dissolution of the active pharmaceutical ingredient. This study investigates disintegration mechanisms of chitin and common disintegrants. Swelling assessment (swelling force and swelling ratio) in different media, and compaction behavior (pure or mixed with other excipients) tabletability, deformation (Heckel modeling), and compact disintegration times were investigated on the tested disintegrants (alginic acid calcium salt, crospovidone, sodium starch glycolate, croscarmellose sodium, and chitin). Results show that the physicochemical properties of the disintegration medium such as pH and ionic strength, as well as other formulation ingredients, affect the disintegrant functionalities. Heckel analysis using the mean yield pressure "Py" shows that alginic acid calcium salt is the most brittle among the studied disintegrants, while crospovidone has the most plastic deformation mechanism, followed by chitin. Chitin showed good tabletability and disintegration properties that were not influenced by the physicochemical formulation environment. Chitin is largely available and easily modifiable and thus a promising material that could be used as a multifunctional excipient in tablet formulation.

Nanoparticles as contrast agents for brain nuclear magnetic resonance imaging in Alzheimer's disease diagnosis.

Nuclear Magnetic Resonance Imaging (MRI) of amyloid plaques is a powerful non-invasive approach for the early and accurate diagnosis of Alzheimer's disease (AD) along with clinical observations of behavioral changes and cognitive impairment. The present article aims at giving a critical and comprehensive review of recent advances in the development of nanoparticle-based contrast agents for brain MRI. Nanoparticles considered for the MRI of AD must comply with a highly stringent set of requirements including low toxicity and the ability to cross the blood-brain-barrier. In addition, to reach an optimal signal-to-noise ratio, they must exhibit a specific ability to target amyloid plaques, which can be achieved by grafting antibodies, peptides or small molecules. Finally, we propose to consider new directions for the future of MRI in the context of Alzheimer's disease, in particular by enhancing the performances of contrast agents and by including therapeutic functionalities following a theranostic strategy.

Non-viral gene activated matrices for mesenchymal stem cells based tissue engineering of bone and cartilage.

Recent regenerative medicine and tissue engineering strategies for bone and cartilage repair have led to fascinating progress of translation from basic research to clinical applications. In this context, the use of gene therapy is increasingly being considered as an important therapeutic modality and regenerative technique. Indeed, in the last 20 years, nucleic acids (plasmid DNA, interferent RNA) have emerged as credible alternative or complement to proteins, which exhibited major issues including short half-life, loss of bioactivity in pathologic environment leading to high dose requirement and therefore high production costs. The relevance of gene therapy strategies in combination with a scaffold, following a so-called "Gene-Activated Matrix (GAM)" approach, is to achieve a direct, local and sustained delivery of nucleic acids from a scaffold to ensure efficient and durable cell transfection. Among interesting cells sources, Mesenchymal Stem Cells (MSC) are promising for a rational use in gene/cell therapy with more than 1700 clinical trials approved during the last decade. The aim of the present review article is to provide a comprehensive overview of recent and ongoing work in non-viral genetic engineering of MSC combined with scaffolds. More specifically, we will show how this inductive strategy can be applied to orient stem cells fate for bone and cartilage repair.

3D compartmented model to study the neurite-related toxicity of Aß aggregates included in collagen gels of adaptable porosity.

UNLABELLED: Insoluble deposits of ß-amyloid (Aß) are associated to neurodegenerative pathologies, in particular Alzheimer's Disease (AD). The toxicity of synthetic amyloid-like peptides has been largely demonstrated and shown to depend upon their aggregation state. However, standard 2D cell culture conditions are not well suited to study the role of the close vicinity of Aß aggregates and growing neurites in the degenerative process. Here, we have designed a compartmented set-up where model neural cells are differentiated on the surface of Aß-containing collagen matrices. The average pore size can be modulated, from below 0.2µm to more than 0.5µm by simple treatment with collagenase, to respectively hamper or permit neurite outgrowth towards the depth of the matrix. Dense Aß aggregates (Congo red and ThT-positive) were obtained inside the collagen matrix with a homogeneous distribution and dimensions similar to those observed in post-mortem brain slices from Alzheimer's patients. The aggregates are not toxic to cells when the pore size is small, in spite of relatively high concentrations of 0.05-0.62mg of peptide per gram of collagen (equivalent to 11.3-113µM). In contrast, on Aß-containing matrices with large pores, massive neural death is observed when the cells are seeded in the same conditions. It is the first time to our knowledge that Aß aggregates with a typical morphology of dense plaques are obtained within a porous biomimetic matrix, and are shown to be toxic only when accessible to differentiating cells. STATEMENT OF SIGNIFICANCE: Insoluble deposits of ß-amyloid (Aß) are associated to neurodegenerative pathologies, in particular Alzheimer's Disease (AD). In this study, we have formed Aß aggregates directly inside a biomimetic collagen matrix loaded with growth factors to induce the differentiation of PC12 or SH-SY6Y cells. For the first time, we show that when the contact between cells and Aß aggregates is allowed by opening up the matrix porosity, the close vicinity with aggregates induces neurite dystrophy. The compartmented 3D culture model developed and used in this study is a valuable tool to study the cytotoxicity of preformed dense Aß aggregates and proves that contact between the aggregates and neurons is required to induce neurodegenerative processes.

Mesoporous alumina from colloidal biotemplating of Al clusters.

A simple and green synthesis route was disclosed for the achievement of mesoporous alumina microparticles employing polysaccharide nanoparticles (α-chitin nanorods) as templates. Pore textures can be tuned by the cationic alumina precursor. Compared to small cations, the use of Al13 and Al30 oxo-hydroxo clusters leads to better defined and elongated mesopores. Electron microscopy and spectroscopic ((13) C, (27) Al NMR, XPS) measurements demonstrated that this is related to the effective coating of α-chitin nanorods by these pre-condensed colloids.

Electric-field alignment of chitin nanorod-siloxane oligomer reactive suspensions.

Uniaxially anisotropic chitin-silica nanocomposite solids have been obtained thanks to the electric field-induced macroscopic alignment of liquid-crystalline reactive cosuspensions. We demonstrate how chitin nanorods (260 nm long, 23 nm thick) can be aligned upon the application of an alternating current (ac) electric field, and within water-ethanol suspensions containing reactive siloxane oligomers (D(h) ∼ 3 nm). The alignment at the millimeter length scale is monitored by in situ small-angle X-ray scattering (SAXS) and polarized light optical microscopy. The composition and state (isotropic, chiral nematic) of the cosuspensions are proven to be determining factors. For nematic phases, the alignment is preserved when the electric field is switched off. Further solvent evaporation induces sol-gel transition, and uniaxially anisotropic chitin-silica nanocomposites are formed after complete drying of the aligned nematic suspensions. Here, the collective response of colloidal mesophases to external electric fields and the subsequent formation of ordered nanocomposite solids would represent a new opportunity for materials design.

Anionic porphyrin-grafted porous silicon nanoparticles for photodynamic therapy.

Non-toxic porous silicon nanoparticles carry porphyrin covalently attached to their surface inside breast cancer cells for a more efficient photodynamic effect.

Efficient mesoporous silica-titania catalysts from colloidal self-assembly.

Mesoporous silica-titania materials of tunable composition and texture, which present a high catalytic activity in the mild oxidation of sulfur compounds, have been obtained by combining the spray-drying process with the colloidal self-assembly of α-chitin nanorods (biopolymer acting as a template) and organometallic oligomers.

Characterization of phospholipid bilayer formation on a thin film of porous SiO2 by reflective interferometric Fourier transform spectroscopy (RIFTS).

Classical methods for characterizing supported artificial phospholipid bilayers include imaging techniques such as atomic force microscopy and fluorescence microscopy. The use in the past decade of surface-sensitive methods such as surface plasmon resonance and ellipsometry, and acoustic sensors such as the quartz crystal microbalance, coupled to the imaging methods, have expanded our understanding of the formation mechanisms of phospholipid bilayers. In the present work, reflective interferometric Fourier transform spectrocopy (RIFTS) is employed to monitor the formation of a planar phospholipid bilayer on an oxidized mesoporous Si (pSiO(2)) thin film. The pSiO(2) substrates are prepared as thin films (3 µm thick) with pore dimensions of a few nanometers in diameter by the electrochemical etching of crystalline silicon, and they are passivated with a thin thermal oxide layer. A thin film of mica is used as a control. Interferometric optical measurements are used to quantify the behavior of the phospholipids at the internal (pores) and external surfaces of the substrates. The optical measurements indicate that vesicles initially adsorb to the pSiO(2) surface as a monolayer, followed by vesicle fusion and conversion to a surface-adsorbed lipid bilayer. The timescale of the process is consistent with prior measurements of vesicle fusion onto mica surfaces. Reflectance spectra calculated using a simple double-layer Fabry-Perot interference model verify the experimental results. The method provides a simple, real-time, nondestructive approach to characterizing the growth and evolution of lipid vesicle layers on the surface of an optical thin film.

Nanostructuration of titania films prepared by self-assembly to affect cell adhesion.

Nanostructured and dense titania films prepared by evaporation-induced self-assembly (EISA) are shown to possess tunable topographical nanoscale features on the order 2-12 nm. Thermal treatment (calcination) induces a transition from amorphous titania to crystalline anatase that modifies the chemical and structural properties of the surfaces via the migration of matter. For nanostructured films, the nanoporous network changes from organized ellipsoidal pores, approximately 4 nm x 2 nm, to a grid-like structure with pores on the order of 12 nm, whereas dense films show a slight roughening of the surface. Cells seeded on templated films show measurable, statistically significant differences in morphology compared with cells seeded on dense films. Moreover, although crystallization of templated films results in surfaces that promote less well-spread cells with higher circularities, the opposite trend is observed for dense films. As such, these results represent a new method to tailor interfaces for biomaterial applications, using EISA to control material patterning on the nanoscale. This self-assembly based approach allows the patterning on size scales that are inaccessible by most traditional techniques while offering the added potential to package and control the release of bioactive molecules.

Onion phases as biomimetic confined media for silica nanoparticle growth.

Phospholipid onion phases were investigated as biomimetic media for the synthesis of silica in a confined environment. Stable multilamellar nanovesicles incorporating sodium silicate solutions could be obtained. Upon aging, silica condensation occurs in the onion interlayer space while preserving the initial multilamellar organization. The hybrid structure consists of an array of apparently unconnected silica nanoparticles in the 20-30 A size range packed in the vesicular 50 A interlayer space, suggesting that the silica growth was efficiently controlled by its confinement in the onion lamellar organization.