Kerstin T. Mader

Local examination of skin diffusion using FTIR spectroscopic imaging and multivariate target factor analysis.

In the context of trans-dermal drug delivery it is very important to have mechanistic insight into the barrier function of the skins stratum corneum and the diffusion mechanisms of topically applied drugs. Currently spectroscopic imaging techniques are evolving which enable a spatial examination of various types of samples in a dynamic way. ATR-FTIR imaging opens up the possibility to monitor spatial diffusion profiles across the stratum corneum of a skin sample. Multivariate data analyses methods based on factor analysis are able to provide insight into the large amount of spectroscopically complex and highly overlapping signals generated. Multivariate target factor analysis was used for spectral resolution and local diffusion profiles with time through stratum corneum. A model drug, 4-cyanophenol in polyethylene glycol 600 and water was studied. Results indicate that the average diffusion profiles between spatially different locations show similar profiles despite the heterogeneous nature of the biological sample and the challenging experimental set-up.

Polyurethane scaffold with in situ swelling capacity for nucleus pulposus replacement

Nucleus pulposus (NP) replacement offers a minimally invasive alternative to spinal fusion or total disc replacement for the treatment of intervertebral disc (IVD) degeneration. This study aimed to develop a cytocompatible NP replacement material, which is feasible for non-invasive delivery and tunable design, and allows immediate mechanical restoration of the IVD. A bi-phasic polyurethane scaffold was fabricated consisting of a core material with rapid swelling property and a flexible electrospun envelope. The scaffold was assessed in a bovine whole IVD organ culture model under dynamic load for 14 days. Nucleotomy was achieved by incision through the endplate without damaging the annulus fibrosus. After implantation of the scaffold and in situ swelling, the dynamic compressive stiffness and disc height were restored immediately. The scaffold also showed favorable cytocompatibility for native disc cells. Implantation of the scaffold in a partially nucleotomized IVD down-regulated catabolic gene expression, increased proteoglycan and type II collagen intensity and decreased type I collagen intensity in remaining NP tissue, indicating potential to retard degeneration and preserve the IVD cell phenotype. The scaffold can be delivered in a minimally invasive manner, and the geometry of the scaffold post-hydration is tunable by adjusting the core material, which allows individualized design.

ATR-FTIR spectroscopy and spectroscopic imaging of solvent and permeant diffusion across model membranes.

The uptake and diffusion of solvents across polymer membranes is important in controlled drug delivery, effects on drug uptake into, for example, infusion bags and containers, as well as transport across protective clothing. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy has been used to monitor the effects of different solvents on the diffusion of a model compound, 4-cyanophenol (CNP) across silicone membrane and on the equilibrium concentration of CNP obtained in the membrane following diffusion. ATR-FTIR spectroscopic imaging of membrane diffusion was used to gain an understanding of when the boundary conditions applied to Ficks second law, used to model the diffusion of permeants across the silicone membrane do not hold. The imaging experiments indicated that when the solvent was not taken up appreciably into the membrane, the presence of discrete solvent pools between the ATR crystal and the silicone membrane can affect the diffusion profile of the permeant. This effect is more significant if the permeant has a high solubility in the solvent. In contrast, solvents that are taken up into the membrane to a greater extent, or those where the solubility of the permeant in the vehicle is relatively low, were found to show a good fit to the diffusion model. As such these systems allow the ATR-FTIR spectroscopic approach to give mechanistic insight into how the particular solvents enhance permeation. The solubility of CNP in the solvent and the uptake of the solvent into the membrane were found to be important influences on the equilibrium concentration of the permeant obtained in the membrane following diffusion. In general, solvents which were taken up to a significant extent into the membrane and which caused the membrane to swell increased the diffusion coefficient of the permeant in the membrane though other factors such as solvent viscosity may also be important.

Simultaneous monitoring of drug and solvent diffusion across a model membrane using ATR-FTIR spectroscopy

Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy has been used to simultaneously follow the diffusion of model drugs and solvent across polydimethylsiloxane (silicone) membrane. Three model drugs, cyanophenol (CNP), methyl nicotinate (MN) and butyl paraben (BP) were selected to cover a range of lipophilicities. Isostearyl isostearate (ISIS) was chosen as the solvent because its large molecular weight should facilitate observation of whether the drug molecules are able to diffuse through the membrane independently of the solvent. The diffusion of the three drugs and the solvent was successfully described by a Fickian model. The effects of parameters such as the absorption wavelength used to follow diffusion on the calculated diffusion coefficient were investigated. Absorption wavelength which affects the depth of penetration of the infrared radiation into the membrane did not significantly affect the calculated diffusion coefficient over the wavelength range tested. Each of the model drugs was observed to diffuse independently of the solvent across the membrane. The diffusion of a CNP-ISIS hydrogen bonded complex across the membrane was also monitored. The relative diffusion rates of the solute and solvent across the membrane can largely be accounted for by the molecular size of the permeant.

The effects of esterified solvents on the diffusion of a model compound across human skin: An ATR-FTIR spectroscopic study

Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy has been used to investigate the effects of three fatty acid esters on skin permeation. Propylene glycol diperlargonate (DPPG), isopropyl myristate (IPM) and isostearyl isostearate (ISIS) were selected as pharmaceutically relevant solvents with a range of lipophilicities and cyanophenol (CNP) was used as a model drug. The resultant data were compared with that obtained when water was used as the solvent. The diffusion of CNP, DPPG and IPM across epidermis was successfully described by a Fickian model. When ISIS was used as a solvent Fickian behaviour was only obtained across isolated stratum corneum suggesting that the hydrophilic layers of the epidermis interfere with the permeation of the hydrophobic ISIS. The diffusion coefficients of CNP across epidermis in the different solvents were not significantly different. Using chemometric data analysis diffusion profiles for the solvents were deconvoluted from that of the skin and modelled. Each of these solvents was found to diffuse at a faster rate across the skin than CNP. DPPG considerably increased the concentration of CNP in the stratum corneum in comparison with the other solvents indicating strong penetration enhancer potential. In contrast IPM produced a similar CNP concentration in the stratum corneum to water with ISIS resulting in a lower CNP concentration suggesting negligible enhancement and penetration retardation effects for these two solvents respectively.

Heterodimeric BMP-2/7 for nucleus pulposus regeneration—In vitro and ex vivo studies

Intervertebral disc (IVD) degeneration is the leading trigger of low back pain, which causes disability and leads to enormous healthcare toll worldwide. Biological treatment with growth factors has evolved as potential therapy for IVD regeneration. Bone morphogenetic protein 2 (BMP‐2) and BMP‐7 have shown promise in this regard. In the current study, we evaluated the effect of BMP‐2/7 heterodimer for disc regeneration both in vitro and in organ culture. Nucleus pulposus (NP) cells isolated from bovine caudal disc were cultured in a fibrin‐hyaluronan (FBG‐HA) hydrogel for up to 14 days. BMP‐2/7 heterodimer covalently incorporated within the hydrogel up‐regulated the aggrecan and type II collagen gene expression, and glycosaminoglycan synthesis of NP cells. The activity of the BMP‐2/7 heterodimer was dose dependent. The higher dose of BMP‐2/7 was further assessed in an IVD whole organ system. After 14 days of culture with cyclic dynamic load, the BMP‐2/7 heterodimer delivered into the nucleotomized region showed potential to stimulate the gene expression and synthesis of proteoglycan in the remaining NP tissue after partial nucleotomy. The gene expression level of type I collagen and alkaline phosphatase in the native disc tissue were not affected by BMP‐2/7 treatment, indicating no adverse fibroblastic or osteogenic effect on the disc tissue. Intradiscal delivery of BMP‐2/7 heterodimer may be a promising therapeutic approach for NP regeneration. The current IVD whole organ partial nucleotomy model may be utilized for screening of other biomaterials or drugs to treat early degenerative disc disorders.

Characterisation of intervertebral discs using MID-IR spectroscopic imaging

Introduction Lower back pain affects millions of people worldwide, and has been linked to degenerative changes in the intervertebral disc (IVD) of the spine. In the “NPmimetic” project, a multidisciplinary team has come together to create a biomimetic nanopolymer based implant and develop a minimally invasive therapy to reconstruct and regenerate diseased nucleus pulposus (NP). The biodegradable nanofibers of the implant can also be designed to carry anti-inflammatory drugs, which can be released in situ promoting healing and preventing inflammation (http://npmimetic.com/). An IVD consists primarily of a proteoglycan-water gel embedded in a randomly arranged collagen network in the NP, and highly ordered collagen lamella in the annulus fibrosus (AF). Disc cells in the AF are elongated parallel to the collagen fibers and produce predominantly collagen I in the outer AF in response to deformation. NP cells are responsive to hydrostatic pressure and synthesize mostly proteoglycan and collagen II. In adults, the cell density in an IVD is very low and cell phenotypes can change in response to altered matrix and stress distribution.1 For successful regeneration, tissue integrity together with the right mechanical environment is essential for normal cell function. To develop suitable biomimetic implants a thorough characterization profile, that can be used as an aspirational target is important. In this study, we use Fourier transform infrared (FTIR) microscopic imaging to obtain chemical maps of control and in-vivo CABC-degenerated goat IVDs.2,3 Materials and Methods Goat IVDs were kindly provided from VU University Medical Centre (VUMC), Amsterdam. The IVDs were formalin fixed (10%, overnight), paraffin embedded, and 4 µm sections were mounted on custom- made reflective steel slides. FTIR microscopy in transflection mode was used to generate chemical distribution maps from unstained paraffin sections. One microscopic image covers only a very small area (350 × 350 µm) of an IVD sample (∼2.8 × 2.3 cm, transverse section). Bigger areas are measured by sequential sample movement and image acquisition covering a user-defined mosaic image area. In this example, an area of 80 × 64 images was measured resulting in a total of 10,720 IR spectra per IVD section. FTIR mosaic imaging generally generates many thousands of data points. A major challenge is handling and analyzing such large and chemically complex datasets to extract meaningful information. However, using iterative multivariate curve resolution (MCR) techniques on the reduced data matrix from principal component analysis analysis of second derivative spectra it is possible to deconvolute highly overlapping infrared peaks into single contributions of different molecular species. Results The chemical identity of the extracted component using an iterative MCR algorithm is determined by comparing the extracted spectral profiles with the spectral profiles of reference materials for proteoglycan and collagen. Spectral features matching typical proteoglycan and collagen spectral characteristics are observed. The distribution of the extracted MCR factors for collagen and proteoglycan are compared with consecutive IVD sections, which were stained using traditional histological methods; Masson trichrome staining for collagen and Alcian blue staining for proteoglycan. A good match can be observed between the distribution maps of collagen and proteoglycans derived from FTIR microscopic imaging measurements and the traditionally stained sections. Conclusion FTIR microscopic imaging in transflection mode has been successfully used to generate molecular images of the collagen and proteoglycan distribution of natural IVD material without the use of contrast-enhancing agents. The use of second derivative spectra together with the application of iterative MCR algorithms shows great potential to enhance the chemical specificity of FTIR mosaic imaging and opens up the possibility to distinguish between different proteclycans and proteins as well as their secondary structure, which usually are expressed only through minor peak shifts. Acknowledgments We thank the European Union (NPMIMETIC ref 246351) for 7th Framework Program funding. Disclosure of Interest None declared References Adams MA, Roughley PJ. What is intervertebral disc degeneration, and what causes it? Spine 2006;31(18):2151–2161 Hoogendoorn RJW, Wuisman PIJM, Smit TH, Everts VE, Helder MN. Experimental intervertebral disc degeneration induced by chondroitinase ABC in the goat. Spine 2007;32(17):1816–1825 Hoogendoorn RJW, Helder MN, Kroeze RJ, Bank RA, Smit TH, Wuisman PI. Reproducible long-term disc degeneration in a large animal model. Spine 2008;33(9):949–954