Standardization Approaches for Extracellular Vesicle Loading with Oligonucleotides and Biologics.

Extracellular vesicles (EVs) are widely recognized for their potential as drug delivery systems. EVs are membranous nanoparticles shed from cells. Among their natural features are their ability to shield cargo molecules against degradation and enable their functional internalization into target cells. Especially biological or bio-inspired large molecules (LMs), like nucleic acids, proteins, peptides, and others, may profit from encapsulation in EVs for drug delivery purposes. In the last years, a variety of loading protocols are explored for different LMs. The lack of standardization in the EV drug delivery field has impeded their comparability so far. Currently, the first reporting frameworks and workflows for EV drug loading are proposed. The aim of this review is to summarize these evolving standardization approaches and set recently developed methods into context. This will allow for enhanced comparability of future work on EV drug loading with LMs.

Investigating 3R In Vivo Approaches for Bio-Distribution and Efficacy Evaluation of Nucleic Acid Nanocarriers: Studies on Peptide-Mimicking Ionizable Lipid.

Formulations based on ionizable amino-lipids have been put into focus as nucleic acid delivery systems. Recently, the in vitro efficacy of the lipid formulation OH4:DOPE has been explored. However, in vitro performance of nanomedicines cannot correctly predict in vivo efficacy, thereby considerably limiting pre-clinical translation. This is further exacerbated by limited access to mammalian models. The present work proposes to close this gap by investigating in vivo nucleic acid delivery within simpler models, but which still offers physiologically complex environments and also adheres to the 3R guidelines (replace/reduce/refine) to improve animal experiments. The efficacy of OH4:DOPE as a delivery system for nucleic acids is demonstrated using in vivo approaches. It is shown that the formulation is able to transfect complex tissues using the chicken chorioallantoic membrane model. The efficacy of DNA and mRNA lipoplexes is tested extensively in the zebra fish (Danio rerio) embryo which allows the screening of biodistribution and transfection efficiency. Effective transfection of blood vessel endothelial cells is seen, especially in the endocardium. Both model systems allow an efficacy screening according to the 3R guidelines bypassing the in vitro-in vivo gap. Pilot studies in mice are performed to correlate the efficacy of in vivo transfection.

Adjustable Thermo-Responsive, Cell-Adhesive Tissue Engineering Scaffolds for Cell Stimulation through Periodic Changes in Culture Temperature.

A three-dimensional (3D) scaffold ideally provides hierarchical complexity and imitates the chemistry and mechanical properties of the natural cell environment. Here, we report on a stimuli-responsive photo-cross-linkable resin formulation for the fabrication of scaffolds by continuous digital light processing (cDLP), which allows for the mechano-stimulation of adherent cells. The resin comprises a network-forming trifunctional acrylate ester monomer (trimethylolpropane triacrylate, or TMPTA), N-isopropyl acrylamide (NiPAAm), cationic dimethylaminoethyl acrylate (DMAEA) for enhanced cell interaction, and 4-acryloyl morpholine (AMO) to adjust the phase transition temperature (Ttrans) of the equilibrium swollen cross-polymerized scaffold. With glycofurol as a biocompatible solvent, controlled three-dimensional structures were fabricated and the transition temperatures were adjusted by resin composition. The effects of the thermally induced mechano-stimulation were investigated with mouse fibroblasts (L929) and myoblasts (C2C12) on printed constructs. Periodic changes in the culture temperature stimulated the myoblast proliferation.

Biodegradable macromers for implant bulk and surface engineering.

Macromers, polymeric molecules with at least two functional groups for cross-polymerization, are interesting materials to tailor mechanical, biochemical and degradative bulk and surface properties of implants for tissue regeneration. In this review we focus on macromers with at least one biodegradable building block. Manifold design options, such as choice of polymeric block(s), optional core molecule and reactive groups, as well as cross-co-polymerization with suitable anchor or linker molecules, allow the adaptation of macromer-based biomaterials towards specific application requirements in both hard and soft tissue regeneration. Implants can be manufactured from macromers using additive manufacturing as well as molding and templating approaches. This review summarizes and discusses the overall concept of biodegradable macromers and recent approaches for macromer processing into implants as well as techniques for surface modification directed towards bone regeneration. These aspects are reviewed including a focus on the authors' contributions to the field through research within the collaborative research project Transregio 67.

Osteogenic Potential of Mesenchymal Stem Cells from Adipose Tissue, Bone Marrow and Hair Follicle Outer Root Sheath in a 3D Crosslinked Gelatin-Based Hydrogel.

Bone transplantation is regarded as the preferred therapy to treat a variety of bone defects. Autologous bone tissue is often lacking at the source, and the mesenchymal stem cells (MSCs) responsible for bone repair mechanisms are extracted by invasive procedures. This study explores the potential of autologous mesenchymal stem cells derived from the hair follicle outer root sheath (MSCORS). We demonstrated that MSCORS have a remarkable capacity to differentiate in vitro towards the osteogenic lineage. Indeed, when combined with a novel gelatin-based hydrogel called Osteogel, they provided additional osteoinductive cues in vitro that may pave the way for future application in bone regeneration. MSCORS were also compared to MSCs from adipose tissue (ADMSC) and bone marrow (BMMSC) in a 3D Osteogel model. We analyzed gel plasticity, cell phenotype, cell viability, and differentiation capacity towards the osteogenic lineage by measuring alkaline phosphatase (ALP) activity, calcium deposition, and specific gene expression. The novel injectable hydrogel filled an irregularly shaped lesion in a porcine wound model displaying high plasticity. MSCORS in Osteogel showed a higher osteo-commitment in terms of calcium deposition and expression dynamics of OCN, BMP2, and PPARG when compared to ADMSC and BMMSC, whilst displaying comparable cell viability and ALP activity. In conclusion, autologous MSCORS combined with our novel gelatin-based hydrogel displayed a high capacity for differentiation towards the osteogenic lineage and are acquired by non-invasive procedures, therefore qualifying as a suitable and expandable novel approach in the field of bone regeneration therapy.

Spray-Dried Nanoparticle-in-Microparticle Delivery Systems (NiMDS) for Gene Delivery, Comprising Polyethylenimine (PEI)-Based Nanoparticles in a Poly(Vinyl Alcohol) Matrix.

Nucleic acid-based therapies rely on efficient formulations for nucleic acid protection and delivery. As nonviral strategies, polymeric and lipid-based nanoparticles have been introduced; however, biological efficacy and biocompatibility as well as poor storage properties due to colloidal instability and their unavailability as ready-to-use systems are still major issues. Polyethylenimine is the most widely explored and promising candidate for gene delivery. Polyethylenimine-based polyplexes and their combination with liposomes, lipopolyplexes, are efficient for DNA or siRNA delivery in vitro and in vivo. In this study, a highly potent spray-dried nanoparticle-in-microparticle delivery system is presented for the encapsulation of polyethylenimine-based polyplexes and lipopolyplexes into poly(vinyl alcohol) microparticles, without requiring additional stabilizing agents. This easy-to-handle gene delivery device allows prolonged nanoparticle storage and protection at ambient temperature. Biological analyses reveal further advantages regarding profoundly reduced cytotoxicity and enhanced transfection efficacies of polyethylenimine-based nanoparticles from the nanoparticle-in-microparticle delivery system over their freshly prepared counterparts, as determined in various cell lines. Importantly, this nanoparticle-in-microparticle delivery system is demonstrated as ready-to-use dry powder to be an efficient device for the inhalative delivery of polyethylenimine-based lipopolyplexes in vivo, as shown by transgene expression in mice after only one administration.

Dual-Functional Hydrazide-Reactive and Anhydride-Containing Oligomeric Hydrogel Building Blocks.

Biomimetic hydrogels are advanced biomaterials that have been developed following different synthetic routes. Covalent postfabrication functionalization is a promising strategy to achieve efficient matrix modification decoupled of general material properties. To this end, dual-functional macromers were synthesized by free radical polymerization of maleic anhydride with diacetone acrylamide (N-(1,1-dimethyl-3-oxobutyl)acrylamide) and pentaerythritol diacrylate monostearate. Amphiphilic oligomers (Mn < 7.5 kDa) with anhydride contents of 7-20% offered cross-linking reactivity to yield rigid hydrogels with gelatinous peptides (E = 4-13 kPa) and good cell adhesion properties. Mildly reactive methyl ketones as second functionality remained intact during hydrogel formation and potential of covalent matrix modification was shown using hydrazide and hydrazine model compounds. Successful secondary dihydrazide cross-linking was demonstrated by an increase of hydrogel stiffness (>40%). Efficient hydrazide/hydrazine immobilization depending on solution pH, hydrogel ketone content as well as ligand concentration for bioconjugation was shown and reversibility of hydrazone formation was indicated by physiologically relevant hydrazide release over 7 days. Proof-of-concept experiments with hydrazido-functionalized hyaluronan demonstrated potential for covalent aECM immobilization. The presented dual-functional macromers have perspective as reactive hydrogel building blocks for various biomedical applications.

Dual-Component Gelatinous Peptide/Reactive Oligomer Formulations as Conduit Material and Luminal Filler for Peripheral Nerve Regeneration.

Toward the next generation of nerve guidance conduits (NGCs), novel biomaterials and functionalization concepts are required to address clinical demands in peripheral nerve regeneration (PNR). As a biological polymer with bioactive motifs, gelatinous peptides are promising building blocks. In combination with an anhydride-containing oligomer, a dual-component hydrogel system (cGEL) was established. First, hollow cGEL tubes were fabricated by a continuous dosing and templating process. Conduits were characterized concerning their mechanical strength, in vitro and in vivo degradation and biocompatibility. Second, cGEL was reformulated as injectable shear thinning filler for established NGCs, here tyrosine-derived polycarbonate-based braided conduits. Thereby, the formulation contained the small molecule LM11A-31. The biofunctionalized cGEL filler was assessed regarding building block integration, mechanical properties, in vitro cytotoxicity, and growth permissive effects on human adipose tissue-derived stem cells. A positive in vitro evaluation motivated further application of the filler material in a sciatic nerve defect. Compared to the empty conduit and pristine cGEL, the functionalization performed superior, though the autologous nerve graft remains the gold standard. In conclusion, LM11A-31 functionalized cGEL filler with extracellular matrix (ECM)-like characteristics and specific biochemical cues holds great potential to support PNR.

Rat Osteosarcoma Cells as a Therapeutic Target Model for Osteoregeneration via Sclerostin Knockdown.

There are various conceptually different strategies to improve bone regeneration and to treat osteoporosis, each with distinct inherent advantages and disadvantages. The use of RNA interference strategies to suppress the biological action of catabolic factors or antagonists of osteogenic proteins is promising, and such strategies can be applied locally. They are comparably inexpensive and do not suffer from stability problems as protein-based approaches. In this study, we focus on sclerostin, encoded by the SOST gene, a key regulator of bone formation and remodeling. Sclerostin is expressed by mature osteocytes but also by late osteogenically differentiated cells. Thus, it is difficult and requires long-term cultures to investigate the effects of SOST silencing on the expression of osteogenic markers using primary cells. We, therefore, selected a rat osteosarcoma cell line, UMR-106, that has been shown to express SOST and secrete sclerostin in a comparable fashion as late osteoblasts and osteocytes. We investigated the effects of differentiating supplements on SOST expression and sclerostin secretion in UMR-106 cells and found that addition of 100 ng/ml of bone morphogenetic protein (BMP)-2 strongly induced sclerostin secretion, whereas dexamethasone inhibited secretion. Effects of silencing SOST in UMR-106 cells cultured in various differentiation media including BMP-2 and/or dexamethasone were determined next with the aim to find promising test conditions for a readout system for the evaluation of future small interfering RNA release formulations for local induction of bone formation. We found a direct correlation between attenuated SOST expression and an increase in the osteogenic potential of UMR-106 cells. The combination of SOST silencing and BMP-2 could synergistically improve osteogenic factors. A lowered proliferation rate in silenced groups may indicate a faster switch to differentiation.

Isolation and Characterization of CD271⁺ Stem Cells Derived from Sheep Dermal Skin.

BACKGROUND: Mesenchymal stem cells (MSCs) have great promise in the field of regenerative medicine due to their differentiation potential into several lineages. Besides the bone marrow, MSCs can be obtained from the dermis, which represents a large stem cell reservoir in the skin. Sheep provide an appropriate large animal model for preclinical studies. In this study, we focused on the isolation and characterization of MSCs from sheep dermis as an alternative to bone marrow MSCs (bmMSCs). METHODS: Primary ovine cells were obtained from the dermis for comparison with bone marrow. CD271(+)/45(-) dermal MSCs (CD271-dMSCs), which were sorted by flow cytometry, and plastic-adherent bmMSCs were examined for morphology, proliferation and senescence-associated ß-galactosidase activity in both low and high oxygen conditions. CD271 expression on cultured cells was assessed by flow cytometry. Adipogenic and osteogenic potentials of CD271-dMSCs were evaluated by oil red O and von Kossa staining. Chondrogenic capacity of CD271-dMSCs and CD271(+)/CD45(-) bone marrow cells (CD271-bmMSCs) was detected using immunohistochemistry and measurement of sulfated glycosaminoglycans. RESULTS: The cell proliferation assay demonstrated no significant difference between CD271-dMSCs and bmMSCs under low oxygen conditions. Cultured CD271-dMSCs revealed much more CD271 expression compared to CD271-bmMSCs. CD271-dMSCs and CD271-bmMSCs showed basically similar expression of the cartilage-specific proteins aggrecan and collagen type II, although with a stronger staining in CD271-bmMSC-derived cultures. Remarkably, there was co-expression of CD271 and aggrecan during chondrogenic differentiation, suggesting an involvement of CD271 in chondrogenesis. CONCLUSION: Based on these findings, CD271-dMSCs might serve as an appropriate alternative cell source in preclinical research.

Gelatin-based biomaterial engineering with anhydride-containing oligomeric cross-linkers.

Chemically cross-linked gelatin hydrogels are versatile cell-adhesive hydrogel materials that have been established for a variety of biomedical applications. The most prominent cross-linker is glutaraldehyde, which, however, has been described to cause compatibility problems and loss of microscopic but relevant structural features. A recently developed oligomeric cross-linker that contains anhydride functionalities was evaluated as cross-linker for the fabrication of gelatin-based hydrogels and microparticles. In a fast curing reaction, hydrogels composed of gelatin and oligomeric cross-linker were fabricated with good conversion over a wide concentration range of constituents and with cross-linkers of different anhydride contents. Hydrogel properties, such as dry weight and mechanics, could be controlled by hydrogel composition and rheological properties correlated to elastic moduli from 1 to 10 kPa. The gels were shown to be cytocompatible and promoted cell adhesion. In soft formulations, cells migrated into the hydrogel bulk. Gelatin microparticles prepared by a standard water-in-oil emulsion technique were also treated with the novel oligomers, and cross-linking degrees matching those obtained with glutaraldehyde were obtained. At the same time, fewer interparticular cross-links were observed. Fluorescein-derivatized cross-linkers yielded labeled microparticles in a concentration-dependent manner. The oligomeric cross-linkers are presented as an efficient and possibly more functional and compatible alternative to glutaraldehyde. The engineered hydrogel materials hold potential for various biomedical applications.

Aging processes in dental thermoplastics - Thermoanalytical investigations and effects on Vickers as well as Martens hardness.

OBJECTIVE: The influence of various aging protocols, representing and accelerating influences present in the dental context, on possible changes in the microstructure and mechanical properties of thermoplastics was investigated. In order to minimize the complexity of the systems, first pure polymers and then later the equivalent dental polymeric materials were analyzed. MATERIALS AND METHODS: Pure polymers (Poly(methyl methacrylate) - PMMA, Polyoxymethylene homopolymer - POM-H, Polyether ether ketone - PEEK, Nylon 12 - PA12, Polypropylene - PP) were analyzed before as well as after applying different aging protocols relevant to the oral environment (ethanol, thermocycling, alkaline and acidic setting) by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The thermoanalytical parameters used were glass transition temperature (Tg), melting peak and crystallization peak temperature (Tpm, Tpc) and decomposition behavior. In a second step selected commercially available dental products (Telio CAD - PMMAD, Zirlux Acetal - POMD, Juvora Natural Dental Disc - PEEKD) aged by the protocol that previously showed strong effects were examined and additionally tested for changes in their Vickers and Martens hardness by Mann-Whitney-U test. RESULTS: The combinations of pure polymers and viable aging protocols analyzed within this study were identified via TGA or DSC as PA12 & thermocycling, POM-H & denture cleanser/lactic acid/ethanol, PP & lactic acid. The dental polymeric materials PMMAD and POMD due to aging in lactic acid showed slight but significantly (p < 0.01) reduced Vickers and partly Martens hardness. PEEK showed the greatest material resistance within this study.

The pore size of PLGA bone implants determines the de novo formation of bone tissue in tibial head defects in rats.

PURPOSE: The influence of the pore size of biodegradable poly(lactic-co-glycolic acid) scaffolds on bone regeneration was investigated. METHODS: Cylindrical poly(lactic-co-glycolic acid) scaffolds were implanted into a defect in the tibial head of rats. Pore sizes of 100-300, 300-500, and 500-710 µm were tested and compared to untreated defects as control. Two and four weeks after implantation, the specimens were explanted and defect regeneration and de novo extracellular matrix generation were investigated by MRI, quantitative solid-state NMR, and mass spectrometry. RESULTS: The pore size of the scaffolds had a pronounced influence on the quantity of the extracellular matrix synthesized in the graft; most collagen was synthesized within the first 2 weeks of implantation, while the amount of hydroxyapatite increased in the second 2 weeks. After 4 weeks, the scaffolds contained large quantities of newly formed lamellar bone while the control defects were filled by inhomogenous woven bone. Best results were obtained for scaffolds of a pore size of 300-500 µm. CONCLUSION: Our analysis showed that the structure and dynamics of the regenerated extracellular matrix was very similar to that of the native bone, suggesting that biomineralization was significantly enhanced by the choice of the most appropriate implant material.

Utilizing 4D Printing to Design Smart Gastroretentive, Esophageal, and Intravesical Drug Delivery Systems.

The breakthrough of 3D printing in biomedical research has paved the way for the next evolutionary step referred to as four dimensional (4D) printing. This new concept utilizes the time as the fourth dimension in addition to the x, y, and z axes with the idea to change the configuration of a printed construct with time usually in response to an external stimulus. This can be attained through the incorporation of smart materials or through a preset smart design. The 4D printed constructs may be designed to exhibit expandability, flexibility, self-folding, self-repair or deformability. This review focuses on 4D printed devices for gastroretentive, esophageal, and intravesical delivery. The currently unmet needs and challenges for these application sites are tried to be defined and reported on published solution concepts involving 4D printing. In addition, other promising application sites that may similarly benefit from 4D printing approaches such as tracheal and intrauterine drug delivery are proposed.

Fabrication of 3D Printed, Core-and-Shell Implants as Controlled Release Systems for Local siRNA Delivery.

The development and clinical translation of small interfering RNA (siRNA) therapies remains challenging owing to their poor pharmacokinetics. 3D printing technology presents a great opportunity to fabricate personalized implants for local and sustained delivery of siRNA. Hydrogels can mimic the mechanical properties of tissues, avoiding the problems associated with rigid implants. Herein, a thermoresponsive composite hydrogel suitable for extrusion 3D-printing is formulated to fabricate controlled-release implants loaded with siRNA-Lipofectamine RNAiMAX complexes. A hydrogel matrix mainly composed of uncharged agarose to protect siRNA from decomplexation is selected. Additionally, pluronic F127 and gelatin are added to improve the printability, degradation, and cell adhesion to the implants. To avoid exposing siRNA to thermal stress during the printing process, a core-and-shell design is set up for the implants in which a core of siRNA-complexes loaded-pluronic F127 is printed without heat and enclosed with a shell comprising the thermoresponsive composite hydrogel. The release profile of siRNA-complexes is envisioned to be controlled by varying the printing patterns. The results reveal that the implants sustain siRNA release for one month. The intactness of the released siRNA-complexes is proven until the eighth day. Furthermore, by changing the printing patterns, the release profiles can be tailored.

Aging Processes and Their Influence on the Mechanical Properties of Printable Occlusal Splint Materials.

Three-dimensional (3D)-printed occlusal splints are becoming more prevalent in the treatment of tooth substance loss due to their fast and cost-effective production. The purpose of this in vitro study was to investigate whether the mechanical properties (tensile strength-TS, modulus of elasticity in tension-ME, and Vickers hardness-HV) vary between the materials (printed dimethacrylate-based resins: Keyprint KeySplint soft-KEY, Luxaprint Ortho Plus-LUX, V-Print splint-VPR, printed methacrylate-based resins Freeprint splint 2.0-FRE, and milled methacrylate-based material, CLEAR splint-CLE), and the influence of aging processes (extraoral storage conditions and nightly or daily use) was examined. The printed methacrylate-based resins (FRE, LUX, and VPR) had much higher TS (43.7-48.5 MPa compared to 12.3-13.3 MPa), higher ME (2.01-2.37 GPa compared to 0.43-0.72 GPa), and higher HV (11.8-15.0 HV compared to 3.3-3.5 HV) than both of the methacrylate-based resins (KEY and CLE) after the production process. Although the TS, ME, and HV of the printed dimethacrylate resins (FRE, LUX, and VPR) decreased significantly under humid conditions with possibly elevated temperatures (thermocycling as well as 37 °C), these mechanical properties were significantly higher than both methacrylate-based resins (KEY and CLE). Therefore, printed dimethacrylate resins should be used rather than methacrylate-based resins for high expected masticatory forces, low wall thicknesses, or very long wearing times (≥6 months).

Retention force, translucency, and microstructural properties of translucent temporary luting cements: An in vitro study.

The aim of the study was to investigate the retention behaviour (pull-off force include adhesive remnant index = ARI) as well as translucency of various temporary luting cements and use microstructure elucidation methods to formulate explanatory approaches to their mode of action. The retention force of the temporary luting cements Provicol QM Plus (P+), Provicol QM Aesthetic (Pae), Bifix Temp (BiT), and as a reference a glass ionomer cement Meron (M) with a direct (Structur 3/S3) or an indirect (Structure CAD/SCAD) resin-based composite restauration was investigated after accelerated aging (thermocycling). Additional investigation of the physical properties was performed regarding to translucency and surface free energy. The microstructure was evaluated by X-ray diffraction, thermogravimetric analysis, differential scanning calorimetry and micro X-ray computed tomography. All tested temporary luting cements showed different pull-off forces in the range between 3.0 and 16.8 N in combination with S3 or SCAD after thermocycling. Only BiT with S3 showed pull-off forces of 129.2 N and complete retention on the restoration (ARI = 0), which was significant (p < .001) to all other samples. High translucency (BiT > Pae > M > P+) was observed for materials with lower crystalline content and low residual mass (usally resulting from higher organic content). M showed the highest surface free energy with a predominantly polar fraction, while BiT had a predominantly dispersive fraction. The highest porosity was observed in the coronal region of the restoration. The results suggest that translucency of temporary luting cements can be increased with higher organic and lower cryristall content. All combinations of cements and temporary restorations (direct/indirect; with the exception of BiT/S3) showed pull-off forces below 17 N (equivalent to a weight force of ∼1.7 kg), which allows manual detachment of the restoration by the dentist.

Radiofluorination of an Anionic, Azide-Functionalized Teroligomer by Copper-Catalyzed Azide-Alkyne Cycloaddition.

This study describes the synthesis, radiofluorination and purification of an anionic amphiphilic teroligomer developed as a stabilizer for siRNA-loaded calcium phosphate nanoparticles (CaP-NPs). As the stabilizing amphiphile accumulates on nanoparticle surfaces, the fluorine-18-labeled polymer should enable to track the distribution of the CaP-NPs in brain tumors by positron emission tomography after application by convection-enhanced delivery. At first, an unmodified teroligomer was synthesized with a number average molecular weight of 4550 ± 20 Da by free radical polymerization of a defined composition of methoxy-PEG-monomethacrylate, tetradecyl acrylate and maleic anhydride. Subsequent derivatization of anhydrides with azido-TEG-amine provided an azido-functionalized polymer precursor (o14PEGMA-N3) for radiofluorination. The 18F-labeling was accomplished through the copper-catalyzed cycloaddition of o14PEGMA-N3 with diethylene glycol-alkyne-substituted heteroaromatic prosthetic group [18F]2, which was synthesized with a radiochemical yield (RCY) of about 38% within 60 min using a radiosynthesis module. The 18F-labeled polymer [18F]fluoro-o14PEGMA was obtained after a short reaction time of 2-3 min by using CuSO4/sodium ascorbate at 90 °C. Purification was performed by solid-phase extraction on an anion-exchange cartridge followed by size-exclusion chromatography to obtain [18F]fluoro-o14PEGMA with a high radiochemical purity and an RCY of about 15%.

Modern Photodynamic Glioblastoma Therapy Using Curcumin- or Parietin-Loaded Lipid Nanoparticles in a CAM Model Study.

Natural photosensitizers, such as curcumin or parietin, play a vital role in photodynamic therapy (PDT), causing a light-mediated reaction that kills cancer cells. PDT is a promising treatment option for glioblastoma, especially when combined with nanoscale drug delivery systems. The curcumin- or parietin-loaded lipid nanoparticles were prepared via dual asymmetric centrifugation and subsequently characterized through physicochemical analyses including dynamic light scattering, laser Doppler velocimetry, and atomic force microscopy. The combination of PDT and lipid nanoparticles has been evaluated in vitro regarding uptake, safety, and efficacy. The extensive and well-vascularized chorioallantois membrane (CAM) of fertilized hen's eggs offers an optimal platform for three-dimensional cell culture, which has been used in this study to evaluate the photodynamic efficacy of lipid nanoparticles against glioblastoma cells. In contrast to other animal models, the CAM model lacks a mature immune system in an early stage, facilitating the growth of xenografts without rejection. Treatment of xenografted U87 glioblastoma cells on CAM was performed to assess the effects on tumor viability, growth, and angiogenesis. The xenografts and the surrounding blood vessels were targeted through topical application, and the effects of photodynamic therapy have been confirmed microscopically and via positron emission tomography and X-ray computed tomography. Finally, the excised xenografts embedded in the CAM were analyzed histologically by hematoxylin and eosin and KI67 staining.

31P and 13C solid-state NMR spectroscopy to study collagen synthesis and biomineralization in polymer-based bone implants.

A combination of solid-state NMR spectroscopy and MRI was used to evaluate the formation of extracellular matrix in poly(D,L-lactide-co-glycolide) (PLGA) bone implants. Porous PLGA scaffolds were implanted into rat tibiae and analysed after 2, 4 or 8 weeks. MRI clearly delineated the implants within the cancellous bone. Differences in the trabecular structure of the implanted material and native bone were demonstrated. In addition, implants were analyzed by solid-state NMR spectroscopy under magic angle spinning. (13)C NMR spectra showed the unambiguous signature of collagen formed in the scaffolds, but also the characteristic signals of the PLGA matrix, indicating that resorption was not complete after 8 weeks. Furthermore, (31)P NMR spectroscopy detected the inorganic component of the matrix, which is composed of bioapatite. (31)P NMR spectra were quantified and this analysis revealed that the amount of inorganic extracellular matrix formed de novo was significantly lower than in native bone. This demonstrates that solid-state NMR spectroscopy, in particular in combination with MRI, can provide useful information on the composition and structure of the extracellular matrix, and serve as a tool to evaluate the quality of tissue engineering strategies.