Analyzing the impact of different excipients on drug release behavior in hot-melt extrusion formulations using FTIR spectroscopic imaging.

The drug release performance of hot-melt extrudate formulations is mainly affected by its composition and interactions between excipients, drug and the dissolution media. For targeted formulation development, it is crucial to understand the role of these interactions on the drug release performance of extrudate formulations. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging was used with an in-situ flow-cell device to analyze the impact of different excipients on drug release from extrudates. The compositions differed in the type of polymer (copovidone and Soluplus®), the salt or acid form of ibuprofen and the addition of sodium carbonate. For comparison, conventional USP (United States Pharmacopeia) Apparatus 2 dissolution studies were performed. FTIR imaging revealed that differences in the drug release rate were mainly due to drug-polymer interactions. Ibuprofen acid showed interactions with the matrix polymer and exhibited a slower drug release compared to non-interacting ibuprofen salt. Addition of sodium carbonate to the ibuprofen acid containing formulations enhanced the drug release rate of these systems by interfering with the drug-polymer interactions. In addition, drug release rates also depended on the polymer type, showing faster drug release rates for extrudate formulations containing copovidone compared to Soluplus®. FTIR imaging revealed that the stronger the drug-polymer interaction in the formulations, the slower the drug release. The addition of sodium carbonate improved release as it reduces drug-polymer interactions and allows for the formation of the more water-soluble ibuprofen salt.

A fast and reliable empirical approach for estimating solubility of crystalline drugs in polymers for hot melt extrusion formulations.

A novel empirical analytical approach for estimating solubility of crystalline drugs in polymers has been developed. The approach utilizes a combination of differential scanning calorimetry measurements and a reliable mathematical algorithm to construct complete solubility curve of a drug in polymer. Compared with existing methods, this novel approach reduces the required experimentation time and amount of material by approximately 80%. The predictive power and relevance of such solubility curves in development of amorphous solid dispersion (ASD) formulations are shown by applications to a number of hot-melt extrudate formulations of ibuprofen and naproxen in Soluplus. On the basis of the temperature-drug load diagrams using the solubility curves and the glass transition temperatures, physical stability of the extrudate formulations was predicted and checked by placing the formulations on real-time stability studies. An analysis of the stability samples with microscopy, thermal, and imaging techniques confirmed the predicted physical stability of the formulations. In conclusion, this study presents a fast and reliable approach for estimating solubility of crystalline drugs in polymer matrixes. This powerful approach can be applied by formulation scientists as an early and convenient tool in designing ASD formulations for maximum drug load and physical stability.

Non-invasive identification of proteoglycans and chondrocyte differentiation state by Raman microspectroscopy.

Proteoglycans (PGs) are crucial extracellular matrix (ECM) components that are present in all tissues and organs. Pathological remodeling of these macromolecules can lead to severe diseases such as osteoarthritis or rheumatoid arthritis. To date, PG-associated ECM alterations are routinely diagnosed by invasive analytical methods. Here, we employed Raman microspectroscopy, a laser-based, marker-free and non-destructive technique that allows the generation of spectra with peaks originating from molecular vibrations within a sample, to identify specific Raman bands that can be assigned to PGs within human and porcine cartilage samples and chondrocytes. Based on the non-invasively acquired Raman spectra, we further revealed that a prolonged in vitro culture leads to phenotypic alterations of chondrocytes, resulting in a decreased PG synthesis rate and loss of lipid contents. Our results are the first to demonstrate the applicability of Raman microspectroscopy as an analytical and potential diagnostic tool for non-invasive cell and tissue state monitoring of cartilage in biomedical research.

Non-contact, label-free monitoring of cells and extracellular matrix using Raman spectroscopy.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.(1-5) However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.(6) As every chemical vibration is assigned to a specific Raman band (wavenumber in cm(-1)), each biological sample features a typical spectral pattern due to their inherent biochemical composition.(7-9) Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.(1) Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).(10) Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 °C, 5% CO(2)) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 °C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy for the non-contact and non-destructive monitoring of collagen damage within tissues.

The non-destructive and label-free monitoring of extracellular matrix (ECM) remodeling and degradation processes is a great challenge. Raman spectroscopy is a non-contact method that offers the possibility to analyze ECM in situ without the need for tissue processing. Here, we employed Raman spectroscopy for the detection of heart valve ECM, focusing on collagen fibers. We screened the leaflets of porcine aortic valves either directly after dissection or after treatment with collagenase. By comparing the fingerprint region of the Raman spectra of control and treated tissues (400-1800 cm(-1)), we detected no significant differences based on Raman shifts; however, we found that increasing collagen degradation translated into decreasing Raman signal intensities. After these proof-of-principal experiments, we compared Raman spectra of native and cryopreserved valve tissues and revealed that the signal intensities of the frozen samples were significantly lower compared to those of native tissues, similar to the data seen in the enzymatically-degraded tissues. In conclusion, our data demonstrate that Raman microscopy is a promising, non-destructive and non-contact tool to probe ECM state in situ.

Raman spectroscopy: a noninvasive analysis tool for the discrimination of human skin cells.

Noninvasive monitoring of tissue-engineered (TE) constructs during their in vitro maturation or postimplantation in vivo is highly relevant for graft evaluation. However, traditional methods for studying cell and matrix components in engineered tissues such as histology, immunohistochemistry, or biochemistry require invasive tissue processing, resulting in the need to sacrifice of TE constructs. Raman spectroscopy offers the unique possibility to analyze living cells label-free in situ and in vivo solely based on their phenotype-specific biochemical fingerprint. In this study, we aimed to determine the applicability of Raman spectroscopy for the noninvasive identification and spectral separation of primary human skin fibroblasts, keratinocytes, and melanocytes, as well as immortalized keratinocytes (HaCaT cells). Multivariate analysis of cell-type-specific Raman spectra enabled the discrimination between living primary and immortalized keratinocytes. We further noninvasively distinguished between fibroblasts, keratinocytes, and melanocytes. Our findings are especially relevant for the engineering of in vitro skin models and for the production of artificial skin, where both the biopsy and the transplant consist of several cell types. To realize a reproducible quality of TE skin, the determination of the purity of the cell populations as well as the detection of potential molecular changes are important. We conclude therefore that Raman spectroscopy is a suitable tool for the noninvasive in situ quality control of cells used in skin tissue engineering applications.

Raman spectroscopy as a tool for quality and sterility analysis for tissue engineering applications like cartilage transplants.

At present, the production of tissue engineered cartilage requires the concurrent production of two identical transplants. One transplant is used for destructive quality control and the second one is implanted into the patient. A non-invasive characterization of such tissue engineering samples would be a promising tool to achieve a production process of just one transplant that is both implanted and tested. Raman spectroscopy is a method that satisfies this requirement by analyzing cells without lysis, fixation or the use of any chemicals. This pure optical technique is based on inelastic scattering of laser photons by molecular vibrations of biopolymers. Characteristic peaks in Raman spectra of cells could be assigned to typical biochemical molecules present in biological samples. For the analysis of chondrocytes present in cartilage transplants, the determination of the cell vitality as well as the discrimination of another cell type have been studied by Raman spectroscopy. Another bottleneck in such biological processes under GMP conditions is sterility control, as most of the commonly used methods require long cultivation times. Raman spectroscopy provides a good alternative to conventional methods in terms of time saving. In this study, the potential of Raman spectroscopy as a quality and sterility control tool for tissue engineering applications was studied by analyzing and comparing the spectra of cell and bacteria cultures.