Chemical engineering

The polymeric materials group at the Department of Chemical Engineering of the University of Coimbra, Portugal

The Chemical Process Engineering and Forest Products Research Centre (CIEPQPF) was established in 1994 with the objective of creating a structured framework for the research activities in the Chemical Engineering Department (DEQ) of the University of Coimbra. As part of its mission, CIEPQPF supports scientific and technical training in the field of chemical engineering, both at undergraduate and graduate levels, while also strengthening the provision of services and participation in activities involving the surrounding society. In this context, the research developed at the centre has been concerned in particular with the needs of the Portuguese industry, having increased the number of projects in collaboration with domestic industries as well as the provision of services to them.

In this article, we are going to outline the activities of the centre related to the area of polymeric materials and biomaterials. We have extensive experience in the preparation and characterisation of polymeric materials targeted for biomedical applications.

Application

The term ‘biomaterial’ has been defined as “a material used in a medical device intended to interact with biological systems”. The application of natural and synthetic polymers to medical problems has increased substantially in the last 20 years. There are numerous applications of these materials in medicine, e.g. orthopaedics, dialysis, ophthalmology, surgery, cardiology and controlled delivery systems. To be used as biomaterials, the polymers need to meet certain requirements, namely in biocompatibility, bio-acceptability and biodegradability. In this case, we can consider two important groups: biostable polymers and biodegradable polymers.

In order to be used as biomaterials, these polymeric materials may have to be modified either chemically or physically.
The polymeric materials group is interested in the development of new polymeric materials to be applied in several areas, mainly as biomaterials, drug delivery systems and biosensors.

Development

In the last few years, the team has developed synthetic and natural-based polymers for biotechnology and industrial applications. As the result of all this work, the group has published around 200 papers in international journals and secured three patents. The team has got all the facilities for the preparation and characterisation of polymeric materials, surfaces and nanoparticles. These include DSC, DMTA, NMR, FTIR, SEM, SEC, multi-SEC, equipment for plasma treatment, equipment for surface energy determination, electrospinning and all the equipment for particle size determination. Molecular modelling/simulation methods, along with high-performance computing platforms, have been applied in both supporting the experimental results and studying the new target materials at different length/time scales.

The polymeric materials group has 25 years’ experience in the preparation and characterisation of polymeric materials for applications in biotechnology and biomaterials. In this group, the following areas have been developed: biosensors, injectable hydrogels, drug delivery systems, nanoparticles and nanocapsules, polyester synthesis, living radical polymerisation, surface modifications, biological adhesives, tissue engineering, polymeric scaffolds and immobilisation of enzymes. More recently, some priority work has been done on the development of bio-adhesives, surface modification and polymer-liposome complexes (PLC) for drug delivery.

With respect to surface modification, different techniques are applied in our group, including plasma techniques followed by polymerisation; etching; spray coating; dip coating; graft copolymerisation at the surface; UV, gamma radiation and laser treatment; and the use of self-assembly coatings. We also use techniques which involve the use and immobilisation of biological molecules or cells on the surface of the materials.
Another area of research in which we are actively working is the development of biological (surgical) adhesives, namely based on photocrosslinkable polymers. For that purpose, biodegradable polymers are modified in order to introduce carbon double bonds, which may then be used to crosslink the polymeric matrix. This reaction is performed under UV irradiation and in the presence of a suitable and biocompatible photoinitiator. Recently, antibiotics have been immobilised in the bio-adhesive structure to improve their biological performance.
We are also interested in the development of scaffolds for tissue engineering applications by combining electrospun fibre mats of biodegradable polyesters with photocurable protein-based hydrogels.

PLCs

Different stimuli-responsive polymers with low molecular weight and narrow polydispersity with a cholesterol end-group were synthesised by atom transfer radical polymerisation (ATRP). The polymers were later incorporated into a liposomal formulation, and a polymer-liposome complex was obtained. This work reported promising results regarding the development of stable liposomes with stimuli-responsive properties that can be loaded with therapeutic drugs. Three main characteristics were afforded by the presence of the polymers in the formulation of the liposomes: stabilisation at neutral pH, increased uptake by phagocytic and non-phagocytic cells, and release of content in mild acidic conditions.

As an example,1, 2 PLCs were prepared with different polymer:lipid molar ratios (0, 0.05 and 0.10). The incorporation of CHO-PDMAEMA strongly stabilised the liposomes at 37°C and pH7 at both polymer:lipid ratios assayed. A slight decrease of pH led to their strong destabilisation. Zeta potential, particle size distribution and polydispersity index showed that the incorporation of CHO-PDMAEMA onto the liposomes led to a decrease of about 50-60% in the liposome size and positively charged the lipid vesicles. The addition of trehalose to the PLCs increased the particle size when compared to the corresponding cases without trehalose.

The optimal polymer:lipid ratio and the stability of both bare liposomes and PLCs were evaluated at 37°C and at different pHs, as well as after storage at 4°C, -80°C and freeze-drying in the presence or absence of trehalose 250mM. Preservation is an important issue for systems to be used as pharmaceutical formulations. In fact, unstable liposomes during storage represent a serious limiting factor for their applicability as drug delivery systems. It has been reported that the main causes leading to liposomes’ instability include hydrolysis or oxidation of phospholipids and variations in the vesicle size (originated in fusion and/or aggregation) which lead to leakage. Freezing or freeze-drying procedures are generally used to maintain the long term stability of liposomes. The harmful nature of the preservation processes led to a strong decrease in the stability of PLCs. Therefore, the addition of trehalose to the suspension of liposomes stabilised PLC during storage.

The internalisation of PLCs by eukaryotic cells was also assessed to give a complete picture of the system. In vitro studies on Raw 264.7 and Caco-2/TC7 cells demonstrated an efficient incorporation of PLCs into the cells. PLCs with higher stability were the ones that showed a better cell uptake. Monocytic cells (Raw 264.7) incorporated a higher quantity of calcein than enterocyte-like cells (Caco-2/TC7) because of their phagocytic capacity. It is known that clathrin-mediated endocytosis plays a main role in the internalisation of liposomes by professional phagocytic cells, which are generally more efficient than epithelial cells in liposomal uptaking. It was also demonstrated that the calcein uptake by eukaryotic cells correlated well with the stability of liposomes. In fact, liposomes preserved at 4°C and -80°C (the most stable ones) were highly incorporated, whereas freeze-dried liposomes were not. Interestingly, the calcein uptake of bacterial liposomes previously exposed to +80°C for five minutes (lysed liposomes) dramatically dropped.

These results indicate that the integrity of the liposomes is essential for an efficient incorporation of the fluorescent probe into the cells. Membrane disruption of freeze-dried liposomes explains the poor calcein transfer observed in freeze-dried liposomes. Considering that physical-chemical properties of liposomes often limit their use in medical applications, the results obtained support the use of natural formulations potentially useful as stimuli-responsive drug delivery systems. Our findings may lead to the possibility of targeting active compounds to specific intracellular compartments, thus improving biological effects.

1 - Alves P, Hugo A A, Tymczyszyn E E, Ferreira A F, Fausto R, Pérez P F, Coelho J F J, Simões P N, and Gómez-Zavaglia A, (2013) Effect of cholesterol-poly(N,N-dimethylaminoethyl methacrylate) on the properties of stimuli-responsive polymer liposome complexes. Colloids and Surfaces B: Biointerfaces, 104: 254–261

2 - Alves P, Hugo A A, Szymanowski F, Tymczyszyn E E, Pérez P F, Coelho J F J, Simões P N, and Gómez-Zavaglia A, (2014) Stabilization of polymer lipid complexes prepared with lipids of lactic acid bacteria upon preservation and internalization into eukaryotic cells. Colloids and Surfaces B: Biointerfaces, http://dx.doi.org/10.1016/j.colsurfb.2014.09.043

Maria Helena Gil
Integrated member of CIEPQPF
Department of Chemical Engineering
University of Coimbra
tel: +351 239 798700

http://www.uc.pt/fctuc/deq/ciepqpf/