Functionalized Gelatin/Polysaccharide Hydrogels for Encapsulation of Hepatocytes.

Liver diseases represent a considerable burden to patients and healthcare systems. Hydrogels play an important role in the engineering of soft tissues and may be useful for embedding hepatocytes for different therapeutic interventions or the development of in vitro models to study the pathogenesis of liver diseases or testing of drugs. Here, we developed two types of hydrogels by crosslinking hydrazide-functionalized gelatin with either oxidized dialdehyde hyaluronan or alginate through the formation of hydrazone bonds. Gel formulations were studied through texture analysis and rheometry, showing mechanical properties comparable to those of liver tissue while also demonstrating long-term stability. The biocompatibility of hydrogels and their ability to host hepatocytes was studied in vitro in comparison to pure gelatin hydrogels crosslinked by transglutaminase using the hepatocellular line HepG2. It was found that HepG2 cells could be successfully embedded in the hydrogels, showing no signs of gel toxicity and proliferating in a 3D environment comparable to pure transglutaminase cross-linked gelatin hydrogels used as control. Altogether, hydrazide gelatin in combination with oxidized polysaccharides makes stable in situ gelling systems for the incorporation of hepatocytes, which may pave the way for use in liver tissue engineering and drug testing.

Characterization of Controlled Release Starch-Nimodipine Implant for Antispasmodic and Neuroprotective Therapies in the Brain.

Parenteral depot systems can provide a constant release of drugs over a few days to months. Most of the parenteral depot products on the market are based on poly(lactic acid) and poly(lactide-co-glycolide) (PLGA). Studies have shown that acidic monomers of these polymers can lead to nonlinear release profiles or even drug inactivation before release. Therefore, finding alternatives for these polymers is of great importance. Our previous study showed the potential of starch as a natural and biodegradable polymer to form a controlled release system. Subarachnoid hemorrhage (SAH) is a life-threatening type of stroke and a major cause of death and disability in patients. Nimotop® (nimodipine (NMD)) is an FDA-approved drug for treating SAH-induced vasospasms. In addition, NMD has, in contrast to other Ca antagonists, unique neuroprotective effects. The oral administration of NMD is linked to variable absorption and systemic side effects. Therefore, the development of a local parenteral depot formulation is desirable. To avoid the formation of an acidic microenvironment and autocatalytic polymer degradation, we avoided PLGA as a matrix and investigated starch as an alternative. Implants with drug loads of 20 and 40% NMD were prepared by hot melt extrusion (HME) and sterilized with an electron beam. The effects of HME and electron beam on NMD and starch were evaluated with NMR, IR, and Raman spectroscopy. The release profile of NMD from the systems was assessed by high-performance liquid chromatography. Different spectroscopy methods confirmed the stability of NMD during the sterilization process. The homogeneity of the produced system was proven by Raman spectroscopy and scanning electron microscopy images. In vitro release studies demonstrated the sustained release of NMD over more than 3 months from both NMD systems. In summary, homogeneous nimodipine-starch implants were produced and characterized, which can be used for therapeutic purposes in the brain.

Development and Characterization of Novel In-Situ-Forming Oleogels.

PLGA-based in situ forming implants (ISFI) often require a high amount of potentially toxic solvents such as N methyl-Pyrrolidone (NMP). The aim of the present study was to develop lipid in-situ-forming oleogels (ISFOs) as alternative delivery systems. 12-Hydroxystearic acid (12-HSA) was selected as the oleogelling agent and three different oleoformulations were investigated: (a) 12-HSA, peanut oil (PO), NMP; (b) 12-HSA, medium-chain triglycerides (MCT), ethanol; (c) 12-HSA, isopropyl myristate (IPM), ethanol. The effects of the 12-HSA concentration, preparation method, and composition on the mechanical stability were examined using a texture analysis and oscillating rheology. The texture analysis was used to obtain information on the compression strength. The amplitude sweeps were analyzed to provide information on the gel strength and the risk of brittle fractures. The frequency sweeps allowed insights into the long-term stability and risk of syneresis. The syringeability of the ISFOs was tested, along with their acute and long-term cytotoxicity in vitro. The developed ISFOs have the following advantages: (1) the avoidance of highly acidic degradation products; (2) low amounts of organic solvents required; (3) low toxicity; (4) low injection forces, even with small needle sizes. Therefore, ISFOs are promising alternatives to the existing polymer/NMP-based ISFIs.

Development of Poly(sorbitol adipate)-g-poly(ethylene glycol) Mono Methyl Ether-Based Hydrogel Matrices for Model Drug Release.

Hydrogels were prepared by Steglich esterification and by crosslinking pre-synthesized poly(sorbitol adipate)-graft-poly(ethylene glycol) mono methyl ether (PSA-g-mPEG) using different-chain-length-based disuccinyl PEG. PSA and PSA-g-mPEG were investigated for polymer degradation as a function of time at different temperatures. PSA-g-mPEG hydrogels were then evaluated for their most crucial properties of swelling that rendered them suitable for many pharmaceutical and biomedical applications. Hydrogels were also examined for their Sol-Gel content in order to investigate the degree of cross-linking. Physical structural parameters of the hydrogels were theoretically estimated using the modified Flory-Rehner theory to obtain approximate values of polymer volume fraction, the molecular weight between two crosslinks, and the mesh size of the hydrogels. X-ray diffraction was conducted to detect the presence or absence of crystalline regions in the hydrogels. PSA-g-mPEG hydrogels were then extensively examined for higher and lower molecular weight solute release through analysis by fluorescence spectroscopy. Finally, the cytotoxicity of the hydrogels was also investigated using a resazurin reduction assay. Experimental results show that PSA-g-mPEG provides an option as a biocompatible polymer to be used for pharmaceutical applications.

Characterization of radicals in polysorbate 80 using electron paramagnetic resonance (EPR) spectroscopy and spin trapping.

Polysorbates are an important class of nonionic surfactants that are widely used to stabilize biopharmaceuticals. The degradation of polysorbate 20 and 80 and the related particle formation in biologics are heavily discussed in the pharmaceutical community. Although a lot of experimental effort was spent in the detailed study of potential degradation pathways, the underlying mechanisms are only sparsely understood. Besides enzymatic hydrolysis, another proposed mechanism is associated with radical-induced (auto)oxidation of polysorbates. To characterize the types and the origin of the involved radicals and their propagation in bulk material as well as in diluted polysorbate 80 solutions, we applied electron paramagnetic resonance (EPR) spectroscopy using a spin trapping approach. The prerequisite for a meaningful experiment using spin traps is an understanding of the trapping rate, which is an interplay of (i) the presence of the spin trap at the scene of action, (ii) the specific reactivity of the selected spin trap with a certain radical as well as (iii) the stability of the formed spin adducts (a slow decay rate). We discuss whether and to which extent these criteria are fulfilled regarding the identification of different radical classes that might be involved in polysorbate oxidative degradation processes. The ratio of different radicals for different scenarios was determined for various polysorbate 80 quality grades in bulk material and in aqueous solution, showing differences in the ratio of present radicals. Possible correlations between the radical content and product parameters such as the quality grade, the manufacturing date, the manufacturer, the initial peroxide content according to the certificate of analysis of polysorbate 80 are discussed.

Selection and Incorporation of siRNA Carrying Non-Viral Vector for Sustained Delivery from Gellan Gum Hydrogels.

The local controlled release of siRNA is an attractive and rational strategy to enhance and extend the effectiveness of gene therapy. Since naked and unmodified siRNA has a limited cell uptake and knockdown efficiency, the complexation of siRNA with non-viral carriers is often necessary for the delivery of bioactive RNA. We evaluated the performance of three different non-viral siRNA carriers, including DOTAP lipoplexes (DL), chitosan polyplexes (CP), and solid lipid complexes (SLC). The physicochemical properties of the siRNA-nanocarriers were characterized by dynamic light scattering and gel electrophoresis. After in vitro characterization, the carrier with the most appropriate properties was found to be the DL suspension, which was subsequently loaded into a gellan gum hydrogel matrix and examined for its drug load, stability, and homogeneity. The hydrogels microstructure was investigated by rheology to assess the impact of the rheological properties on the release of the siRNA nanocarriers. A controlled release of complexed siRNA over 60 days in vitro was observed. By comparing the results from fluorescence imaging with data received from HPLC measurements, fluorescence imaging was found to be an appropriate tool to measure the release of siRNA complexes. Finally, the bioactivity of the siRNA released from hydrogel was tested and compared to free DL for its ability to knockdown the GFP expression in a DLD1 colon cancer cell model. The results indicate controlled release properties and activity of the released siRNA. In conclusion, the developed formulation is a promising system to provide local controlled release of siRNA over several weeks.

Hydrogels Based on Oxidized Cellulose Sulfates and Carboxymethyl Chitosan: Studies on Intrinsic Gel Properties, Stability, and Biocompatibility.

Cellulose and chitosan are excellent components for the fabrication of bioactive scaffolds, as they are biocompatible and abundantly available. Their derivatives Ocarboxymethyl chitosan (CMChi) and oxidized cellulose sulfate (oxCS) can form in situ gelling, bioactive hydrogels, due to the formation of imine bonds for crosslinking. Here the influence of the degrees of sulfation (DS), oxidation (DO), and the molecular weight of oxCS on intrinsic and rheological properties of such hydrogels and their ability to support the survival and growth of human-adipose-derived stem cells (hADSC) is investigated. It is found that the pH of the hydrogels is generally slightly acidic, while their network density and E-modulus are found to be dependent on the DS and DO, which makes the properties of hydrogels tunable. Extensive studies show that hydrogels can be stable for up to 14 days and that their stability is largely dependent on the DO, molecular weight, and the components mixing ratio. Cytotoxicity studies of the hydrogel with hADSCs show biocompatible gels in dependence on the molecular weight and degree of oxidation with viable cells up to 14 days. These findings can help to develop specifically tailored hydrogels for tissue engineering applications to replace different types of connective tissue.

Tabotamp®, Respectively, Surgicel®, Increases the Cell Death of Neuronal and Glial Cells In Vitro.

Oxidized regenerated cellulose (ORC) is an approved absorbable hemostat in neurosurgery, and contains 18-21% carboxylic acid groups. This modification leads to a low pH in aqueous solutions. Therefore, the aim of study was to analyze the pH­dependent effects of the ORC Tabotamp® on astrocytes, Schwann cells, and neuronal cells in vitro to investigate whether Tabotamp® is a suitable hemostat in cerebral eloquent areas. The ORC­dependent pH value changes were measured with (i) a pH meter, (ii) electron paramagnetic resonance spectroscopy, using pH­sensitive spin probes, and (iii) with fluorescence microscopy. Cell lines from neurons, astrocytes, and Schwann cells, as well as primary astrocytes were incubated with increasing areas of Tabotamp®. Cytotoxicity was detected using a fluorescence labeled DNA­binding dye. In addition, the wounding extent was analyzed via crystal violet staining of cell layers. The strongest pH reduction (to 2.2) was shown in phosphate buffered saline, whereas culture medium and cerebrospinal fluid demonstrated a higher buffer capacity during Tabotamp® incubation. In addition, we could detect a distance­dependent pH gradient by fluorescence microscopy. Incubation of Tabotamp® on cell monolayers led to detachment of covered cells and showed increased cytotoxicity in all tested cell lines and primary cells depending on the covered area. These in vitro results indicate that Tabotamp® may not be a suitable hemostat in cerebral eloquent areas.