Michał Kawalec

Synthesis and antiproliferative properties of ibuprofen-oligo(3-hydroxybutyrate) conjugates

Synthesis of novel conjugates of the non-steroidal anti-inflammatory drug - ibuprofen with nontoxic oligo(3-hydroxybutyrate) (OHB) is described. Presented results indicate that anionic ring-opening polymerization of (R,S)-beta-butyrolactone initiated with an alkali metal salt of (S)-(+)-2-(4-isobutylphenyl)propionic acid (ibuprofen) may constitute a convenient method of conjugation of selected drugs with biodegradable OHB. Furthermore using the MTT cell proliferation assay we demonstrated that ibuprofen conjugated with OHB exhibited significantly increased, as compared to free ibuprofen, potential to inhibit proliferation of HT-29 and HCT 116 colon cancer cells. However, the conjugates of ibuprofen and OHB are less toxic as was shown in oral acute toxicity test in rats. Although the mechanism of antiproliferative activity of ibuprofen-OHB conjugates (Ibu-OHB) has to be established, we suggest that partially it can be related to more effective cellular uptake of the conjugate than the free drug. This assumption is based on the observation of much more efficient accumulation of a marker compound - OHB conjugated with fluorescein, in contrast to fluorescein sodium salt, which entered cells inefficiently. Further characterization of biological properties of the ibuprofen-OHB conjugates would provide insight into the mechanism of their antiproliferative effect and assess the potential relevance of their anticancer activity.

Polyhydroxybutyrate targets mammalian mitochondria and increases permeability of plasmalemmal and mitochondrial membranes.

Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle.

Oxidative degradation of poly(3-hydroxybutyrate). A new method of synthesis for the malic acid copolymers

The thermal stability of poly(3-hydroxybutyrate) (PHB) in an oxidation environment was investigated in bulk at temperatures ranging from 100 °C to 140 °C. The process carried out in pure oxygen resulted in PHB backbone degradation with resulting non-volatile products typical for regular PHB thermal degradation while thermal treatment of PHB in an oxygen/ozone mixture resulted in increased rate of polymer backbone scission. The non-volatile degradation product contained macromolecules with several types of terminal groups but also a part of the 3-hydroxybutyrate repeating units was transformed into 3-malic acid units. NMR and multi-stage MS characterization revealed the random distribution of 3-malic acid units in the oligomeric products as well as the content of the malic acid units being dependent on oxidation conditions.

Human procollagen type I surface-modified PHB-based non-woven textile scaffolds for cell growth: preparation and short-term biological tests

3D fine porous structures obtained by electrospinning a poly[(R,S)-3-hydroxybutyrate] (aPHB)/ poly[(R)-3-hydroxybutyrate] (PHB) (85/15 w/w) blend were successfully modified with human procollagen type I by simple immersion of the polyester scaffold in an aqueous solution of the protein. Effective modification of the scaffold with human procollagen I was confirmed by an immunodetection test, which revealed the presence of the procollagen type I as an outer layer even on inner structures of the porous matrixes. Biological tests of 3D fabrics made of the PHB blend provide support for the adhesion and proliferation of human fibroblasts, while their modification with procollagen type I increased the biocompatibility of the final scaffolds significantly, as shown by the notable increase in the number of attached cells during the early hours of their incubation. Based on these findings, human procollagen type I surface-modified aPHB/PHB scaffolds should be considered a promising material in regenerative medicine.

Crystallinity as a tunable switch of poly(L-lactide) shape memory effects

Materials with shape memory effect (SME) have already been widely used in the medical field. The interesting part of this group is represented by double function materials. The bioresorption and SME ability are common in polyesters implants. The first information about vascular stent made of bioresorbable polyester with SME was published in 2000. However, there are not many investigations about SME control of elements in the aspect of material processing. In the present work, the ability to control the shape memory (SM) of bioresorbable and semicrystalline poly(L-lactide) (PLLA) is investigated. The studies are based on the unexpected effect of material orientation which was demonstrated even at low percentage deformation in crystallized mould injected material. The presented studies revealed that the different degrees of crystallinity obtained during processing might be a useful switch to create a tailored SME for a specific application. The prepared samples of variable morphology revealed a possibility to control the value of material stress during permanent shape recovery. The degree of shape recovery of the prepared samples was also controlable. The highest stress value observed during permanent shape recovery reached 10MPa for the sample annealed 60min at 115°C even when the sample was only deformed in 8%. The other significant aspect of this work is to present the problem of slow crystallization of the material during and after processing (cooling rate) as well as the possibility of negative SME change during the shelf life of the fabric.

The impact of shape memory test on degradation profile of a bioresorbable polymer

The semicrystalline poly(L-lactide) (PLLA) belongs to the materials with shape memory effect (SME) and as a bioresorbable and biocompatible polymer it have found many applications in medical and pharmaceutical field. Assessment of the SME impact on the polymer degradation profile plays crucial role in applications such as drug release systems or in regenerative medicine. Herein, the results of in vitro degradation studies of PLLA samples after SME full test cycle are presented. The samples were loaded and deformed in two manners: progressive and non-progressive. The performed experiments illustrate also influence of the material mechanical damages, caused e.g. during incorrect implantation of PLLA product, on hydrolytic degradation profile. Apparently, degradation profiles are significantly different for the material which was not subjected to the deformation and the deformed ones. The materials after deformation of 50% (in SME cycle) was characterized by non-reversible morphology changes. The effect was observed in deformed samples during the SME test which were carried out ten times.