Andrzej Marcinkowski

Self-assembled filomicelles prepared from polylactide/poly(ethylene glycol) block copolymers for anticancer drug delivery.

Bioresorbable filomicelles present many advantageous as drug delivery systems e.g., long circulation time and high loading efficiency. The aim of this study was to develop polylactide/poly(ethylene glycol) (PLA/PEG) filomicelles for drug delivery applications. A series of PLA/PEG diblock copolymers were synthesized using non-toxic initiator, and characterized by means of NMR and GPC. Analysis of morphology of micelles determined by TEM revealed that apart from the weight fraction also the molar mass of PEG and the stereochemistry of PLA block must be considered for tailoring micellar structures. The CMC was found to be dependent on the length and structure of the hydrophobic block. It was observed that the drug loading properties could be improved by selection of appropriate copolymer and encapsulation method. Slower release of paclitaxel was observed for mPEG5000 initiated copolymers than mPEG2000 initiated copolymers. Moreover, the influence of the length of hydrophobic block and its stereoisomeric form on drug release rate was evidenced. Therefore, PLA/PEG filomicelles with good stability, high drug loading capacity and sustained drug release appear most attractive for drug delivery applications.

Forensic Engineering of Advanced Polymeric Materials. Part 1 –Degradation Studies of Polylactide Blends with Atactic Poly[(R,S)-3-hydroxybutyrate] in Paraffin

Classical forensic polymer engineering concerns a study of failure in polymer products. This area of science comprises fracture of plastic products, or any other reason why such a product fails in service, or fails to meet its specification.1 So far, most of the reported case studies concern ex-post investigations of traditional polymeric materials such as PE, PP, PS, PVC, ABS or their thermoplastic composites. Little is known about forensic engineering of novel and advanced polymeric materials. Thus, there is a real gap in this area of knowledge. Furthermore, the ex-ante studies are needed to define and minimise the potential failure of novel polymer products before specific applications. Environmental stress cracking (ESC) is one of the most common causes of unexpected brittle failure of thermoplastic (especially amorphous) polymers. The rate of ESC is dependent on many factors, including, for example, the polymer’s chemical composition, bonding, crystallinity, surface roughness, molar mass and residual stress. It also depends on the chemical nature of liquid media and the temperature of the system. Thus, evaluation and understanding of the structure, properties and behaviour of advanced polymer materials for perspective practical applications is needed, especially in the field of compostable polymer packages of long-life products, such as cosmetics or household chemicals. Because of the slow environmental degradation of traditional packaging, the use of conventional plastics is accompanied by significant ecological problems. To reduce plastic waste from the packaging industry, biodegradable polymers have become interesting alternatives, for packages with a long shelf life.2 The most important environmentally friendly plastics are generally based on aliphatic polyesters, such as poly(lactic acid), poly(3-hyForensic Engineering of Advanced Polymeric Materials. Part 1 – Degradation Studies of Polylactide Blends with Atactic Poly[(R,S)-3-hydroxybutyrate] in Paraffin

Reversibly PEGylated nanocarrier for cisplatin delivery.

A star-shaped copolymer bearing a shell of poly(ethylene glycol) (PEG) chains was designed as a carrier of cisplatin. The proposed strategy was based on synthesis of a PEGylating agent and the incorporation of cisplatin as a reversible linker for PEG modification of the star macromolecules. The attachment of PEG chains to the stars and their release under physiological conditions, as well as the changes in particle size and mobility upon drug loading, was evidenced by diffusion ordered NMR spectroscopy (DOSY). The results demonstrated that PEGylation reduced inter-stars cross-linking and increased the stability of the nanocolloidal solution. The formation of PEG shell resulted in higher drug payload and improved drug release profile of the nanoconjugates. The in vitro bioassay in a panel of human tumor cell lines confirmed that the PEGylated conjugates exhibited superior growth inhibitory activity compared to the cisplatin-loaded nonPEGylated carrier.

Multidrug PLA-PEG filomicelles for concurrent delivery of anticancer drugs-The influence of drug-drug and drug-polymer interactions on drug loading and release properties.

This study aimed to analyze the influence of drug-drug and drug-polymer interactions on drug loading and release properties of multidrug micelles. Three hydrophobic drugs-paclitaxel (Ptx), 17-AAG and rapamycin (Rap) were incorporated in poly(l-lactide)-poly(ethylene glycol) (PLA-PEG) filomicelles. Double loaded micelles containing Ptx and 17-AAG were used for the sake of comparison. (1)H NMR confirmed the effective incorporation of the various drugs in micelles, and HPLC allowed to determine the drug loading contents. FTIR was used to evaluate interactions between particular drugs and between drugs and copolymer. Ptx and 17-AAG present similar loading efficiencies in double loaded micelles probably due to interactions of drugs with each other and also with the copolymer. In contrast, unequal drug loading properties are observed for triple loaded micelles. Rapamycin shows very weak interactions with the copolymer, and displays the lowest loading efficiency. In vitro release of drugs from micelles was realized in pH 7.4 phosphate buffered saline at 37°C, and monitored by HPLC. Similar release profiles are observed for the three drugs: a strong burst followed by slower release. Nevertheless, Ptx release from micelles is significantly slower as compared to 17-AAG and Rap, probably due to interactions of NH and OH groups of Ptx with the carbonyl group of PLA. In vitro cytotoxicity of Ptx/17-AAG/Rap loaded micelles and a mixture of free drugs was determined. Drug loaded micelles exhibit advantageous effect of prolonged drug release and cytotoxic activity against Caco-2 cells, which makes them a promising solution for simultaneous drug delivery to solid tumors. Therefore, understanding of interactions within multidrug micelles should be a valuable approach for the development of concurrent delivery systems of anticancer drugs with tailored properties.

The Influence of Chain Microstructure of Biodegradable Copolyesters Obtained with Low-Toxic Zirconium Initiator to In Vitro Biocompatibility

Because of the wide use of biodegradable materials in tissue engineering, it is necessary to obtain biocompatible polymers with different mechanical and physical properties as well as degradation ratio. Novel co- and terpolymers of various composition and chain microstructure have been developed and applied for cell culture. The aim of this study was to evaluate the adhesion and proliferation of human chondrocytes to four biodegradable copolymers: lactide-coglycolide, lactide-co-ε-caprolactone, lactide-co-trimethylene carbonate, glycolide-co-ε-caprolactone, and one terpolymer glycolide-colactide-co-ε-caprolactone synthesized with the use of zirconium acetylacetonate as a nontoxic initiator. Chain microstructure of the copolymers was analyzed by means of 1H and 13C NMR spectroscopy and surface properties by AFM technique. Cell adhesion and proliferation were determined by CyQUANT Cell Proliferation Assay Kit. After 4 h the chondrocyte adhesion on the surface of studied materials was comparable to standard TCPS. Cell proliferation occurred on all the substrates; however, among the studied polymers poly(L-lactide-coglycolide) 85 : 15 that characterized the most blocky structure best supported cell growth. Chondrocytes retained the cell membrane integrity evaluated by the LDH release assay. As can be summarized from the results of the study, all the studied polymers are well tolerated by the cells that make them appropriate for human chondrocytes growth.

Bio)degradation studies of degradable polymer composites with jute in different environments

The introduction of new, environmentally friendly and sustainable plastics in the packaging and end-user industry is a solution to the problem of waste management. The degradation of polyesters in different environments could result from an enzymatic attack or simple hydrolysis, or both. The degree of degradation of blends containing polylactide and poly(3-hydroxybutyrate-co-4-hydroxybutyrate), PLA/P(3HB-co-4HB), and its composites with 20 %wt of jute incubated in distilled water at 70 ℃ (abiotic conditions) under industrial composting conditions (system KNEER) was investigated using a Zeiss optical microscope, an atomic force microscope, gel permeation chromatography, differential scanning calorimetry and thermal gravimetric analysis. PLA/P(3HB-co-4HB) was tested under laboratory composting conditions in order to verify whether biodegradation of this material occurs under industrial composting conditions. The addition of jute fibres did significantly reduce the disintegration of the composites during degradation.

Synthesis and characterisation of PEG-peptide surfaces for proteolytic enzyme detection.

AbstractPeptide surfaces were obtained by the covalent immobilisation of fluorescently labelled pentapeptides carboxyfluorescein–glycine–arginine–methionine–leucine–glycine, either directly or through a poly(ethylene glycol) (PEG) linker on modified silicon wafers. Each step during the preparation of the peptide surfaces was confirmed by several surface characterisation techniques. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy were used to determine the surface composition, the wafers philicity was measured by contact angle and atomic force microscopy was used to investigate the surface morphology. Exposure of the peptide surfaces to trypsin resulted in the release of a fluorescently labelled peptide product, which allowed the kinetics of the enzymatic reaction to be followed with the aid of fluorescence spectroscopy. The electrospray ionisation mass spectrometry analysis of the post-digestion solution confirmed that the pentapeptides attached to the solid support undergo specific trypsin hydrolysis at the C-terminus of the arginine residues. Detailed surface analyses before and after the enzyme action was performed using ToF-SIMS. Because of the limited accessibility of the short peptide directly attached to the surface, a quantitative yield of enzymatic hydrolysis was observed only in case when the peptide was bound through the PEG linker. The insertion of the PEG linker increased the number of immobilised peptides and the rate of enzymatic digestion which consequently improved the quality of the enzyme assays. The described approach may be used for different peptide sequences designed for other proteases. FigureMonitoring of trypsin hydrolysis on PEG-peptide surface

Chimeric lipid/block copolymer nanovesicles: Physico-chemical and bio-compatibility evaluation

Chimeric systems are mixed nanovectors composed by different in nature materials and exhibit new functionalities and properties. The particular chimeric nanovectors, formed by the co-assembly of low and high molecular weight amphiphiles, have the potential to be utilized as drug delivery platforms. We have utilized two lipids, l-α-phosphatidylcholine, hydrogenated (Soy)(HSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and a poly(oligoethylene glycol acrylate)-b-poly(lauryl acrylate) (POEGA-PLA) block copolymer, at different molar ratios, in aqueous media. Light scattering, differential scanning calorimetry (DSC) and imaging techniques (cryo-TEM, AFM) were employed in order to elucidate the structure and properties of the nanostructures, as well as the cooperativity between the components. DSC experiments showed considerable interaction of the block copolymer with the lipid bilayers and suggested an inhomogeneous distribution of the copolymer chains and lateral phase separation of the components. Vesicle formation was observed in most cases by cryo-TEM with a chimeric membrane exhibiting kinks, in accordance with DSC data. A series of biocompatibility experiments indicated good in vitro biological stability and low cytotoxicity in vivo of the novel nanocarriers. Finally, ibuprofen (IBU) was used as model drug in order to study the loading and the release properties of the prepared chimeric lipid/block copolymer vesicles.

Atomic force microscopy in the production of a biovital skin graft based on human acellular dermal matrix produced in-house and in vitro cultured human fibroblasts.

The most efficient method in III° burn treatment is the use of the autologous split thickness skin grafts that were donated from undamaged body area. The main limitation of this method is lack of suitable donor sites. Tissue engineering is a useful tool to solve this problem. The goal of this study was to find the most efficient way of producing biovital skin substitute based on in house produced acellular dermal matrix ADM and in vitro cultured fibroblasts. Sixty samples of sterilized human allogeneic skin (that came from 10 different donors) were used to examine the influence of decellularizing substances on extracellular matrix and clinical usefulness of the test samples of allogeneic human dermis. Six groups of acellular dermal matrix were studied: ADM-1 control group, ADM-2 research group (24 h incubation in 0.05% trypsin/EDTA solution), ADM-3 research group (24 h incubation in 0.025% trypsin/EDTA solution), ADM-4 research group (24 h incubation in 0.05% trypsin/EDTA solution and 4 h incubation in 0,1% SDS), ADM-5 research group (24 h incubation in 0.025% trypsin/EDTA solution and 4 h incubation in 0,1% SDS), and ADM-6 research group (24 h incubation in 0,1% SDS). Obtained ADMs were examined histochemically and by atomic force microscopy (AFM). ADMs were settled by human fibroblasts. The number of cultured cells and their vitality were measured. The obtained results indicated that the optimal method for production of living skin substitutes is colonization of autologous fibroblasts on the scaffold prepared by the incubation of human allogeneic dermis in 0.05% trypsin/EDTA.

Mixed lipid/polymer nanostructures: From advanced materials to drug delivery systems

The aim of this investigation was to study the alterations of the physicochemical characteristics of L-α-phosphatidylcholine, hydrogenated (Soy) (HSPC) and dipalmitoyl phosphatidyl choline (DPPC) liposomes, caused by the incorporation of a poly (oligoethylene glycol acrylate)-b-poly(lauryl acrylate) (POEGA-PLA) block copolymer at different molar ratios. We used Dynamic and Electrophoretic Light Scattering to determine the size and the ζ-potential; imaging techniques for investigate the structure and Static Light Scattering for quantifying the fractal morphology of the prepared nanosystems in situ. The size of mixed nanostructures became smaller with the incorporation of the block copolymer into the lipid membrane. The size of the prepared nanosystems ranged between 50-80nm. The fractal dimension (df) decreased significantly with the incorporation of block copolymer into liposomal bilayers. The morphology of DPPC:POEGA-PLA mixed nanostructures (with df equal to 1.8) is open (more loose). On the other hand, the morphology of HSPC: POEGA-PLA (with df equal to 2.1) is more compact and dense. The molar ratio of the POEGA-PLA did not alter the morphology of the mixed nanostructures, expect from HSPC:POEGA-PLA system. Finally, we studied the drug loading properties of the mixed nanostructures in order to examine their properties as advanced Drug Delivery nanosystems. Copyright