Self-rolling tubes based on poly(nisopropylacrylamide)/polycaprolactone

Transcription

1 Practical work Self-rolling tubes based on poly(nisopropylacrylamide)/polycaprolactone bilayers Motivation and aim of the experiment Self-rolling tubes based on polymer bilayers seem to be perspective materials for cells encapsulation. It can be successfully implemented for controlled delivery of cells in living organisms as well as for scaffold design for tissue engineering. In this experiment reversible rolling and unrolling of thermoresponsive polymer tubes and reversible capture and release of yeast cells will be demonstrated. 1) At the first part of experiment patterned poly(n-isopropylacrylamide)/polycaprolactone (PNIPAM/PCL) bilayer films will be fabricated by the means of dip coating (alternatively spin coating) and photolithography. All samples will be characterised using ellipsomerty, which allow us to control layer thicknesses in bilayer system as well as to determine swelling degree of PNIPAM. 2) At the second part tubes formation, their properties and behaviour and the possibility of cells encapsulation will be observed using light microscopy. Basics Methods: Polymer thin films preparation using spin coating and dip coating Photolithography Thin film thickness determination using ellipsometry (in air and in liquid) Light microscopy (bright-field and dark-field) Materials: Stimuli responsive polymers: thermoresponsive behaviour of PNIPAM Self-rolling of bilayer polymer films Description of the experimental part Polymers For sample preparation two polymers will be used. Poly(N-isopropylacrilamide) copolymer containing 1 mol% of 4-acryloylbenzophenone comonomer (PNIPAM-ABP) is a thermoresponsive polymer with Lower Critical Solution Temperature (LCST) of 28 C. Copolymerization of NIPAM with benzophenone derivative provides sensitivity to UV light and thus a possibility to crosslink PNIPAM upon UV irradiation. Polycaprolactone (PCL) is a water insoluble biodegradable and biocompatible polymer. Crosslinking of PCL will be achieved by adding of a small amount (4 wt.%) of pure 4-hydroxybenzophenone to the polymer. Principles of self-rolling tubes formation Self-rolled tubes are thin bilayer which is able to roll due to internal stress produced as a result of thermal expansion, lattice mismatch or swelling. In our case self-rolling behaviour is due to unequal swelling of polymer layers (Scheme 1). First polymer layer of PNIPAM (external polymer) being thermoresponsive, swells in water at the temperature below LCST and shrink at the temperature above LCST. Second polymer layer of PCL (internal polymer) 1

2 is hydrophobic and thus doesn t swells in water. Difference in swelling behaviour of two polymers leads to the tubes formation when patterned bilayer film is placed in water at the temperature below LCST. Scheme 1. Principles of tubes formation Sample preparation and characterisation Thin films of PNIPAM-ABP (approx. 300 nm) will be deposed on hydrophilized silicon wafer substrate from ethanol solution using dip coating (alternatively spin coating). Next, thin films (approx nm) of PCL will be deposed from toluene solution, which is selective solvent, on the top of PNIPAM-ABP film. Samples with two different thickness ratio r=th(pnipam)/th(pcl) should be obtained. On the each step of deposition thicknesses of obtained polymer films will be controlled by the means of nulling ellipsometry. The polymer bilayer will be patterned by irradiation with UV light through a mask. UV irradiation activates benzophenone fragments, which produce free radicals and leads to crosslinking in irradiated areas. Uncrosslinked polymers can be washed out in chloroform, which is common solvent for PNIPAM-ABP and PCL (Scheme 2). Scheme 2. Sample preparation 2

3 Another type of samples to be prepared is thin films of PNIPAM-ABP (approx nm) deposed on aminopropyltrimethoxy silane (APS) modified wafers. These films shouldn t be patterned, but just crosslinked. The samples will be used for analysis of the degree of swelling of crosslinked PNIPAM-ABP in PBS buffer ph 7.4 at the different temperatures (above and bellow LCST). Thicknesses of polymer films in dry and swollen states will be measured using nulling ellipsometry. Self-rolling tubes formation Self-rolling tubes formation will be observed using light microscopy. Previously obtained sample with patterned bilayer film should be placed on the microscope stage with Peltier element and heated up to temperature above LCST of PNIPAM-ABP (40 C). Put a droplet of PBS buffer ph 7.4 on the top of the sample and cover it with a cover slip. Using Peltier element, decrease the temperature below LCST (20 C) and observe the tube formation. This observation will be made in a bright-field mode of the microscope. Subsequent increasing and decreasing of the temperature will demonstrate reversibility of tubes formation. Tubes diameter will be measured using a dark-field mode of the microscope. Diameters of the tubes, formed from the films with different thickness ratio of the polymer films, should be compared. Finally, a possibility to encapsulate yeast cells within the tubes will be demonstrated. For this purpose the bakery yeast cells will be adsorbed on patterned sample from PBS ph = 7.4 buffer at the elevated temperature. After temperature decrease, cells might be captured within the formed tubes. Protocol Submission of the individual protocol within one month after practical work. The protocol should contain brief introduction, experimental part, results und discussion and the list of used literature. Protocol shouldn t be more then 20 pages. P.S. It would be very nice of you to write in English. 3

4 Preparation for the experiment Self-rolling tubes based on Poly(N-isopropylacrylamide)/Polycaprolactone bilayers. Recommended literature and control questions Nota bene: Please, read all recommended literature carefully, there will be a test before the experiment. This test will be based on the control questions and the mark for the test will influence the resulting mark for the practical work. You can find all recommended literature in supplementary materials and in Internet when going to the recommended web pages (see links). Stimuli responsive polymers and self-folding polymer films 1) What are stimuli responsive polymers? 2) Which stimuli are typically used in biomedical applications of stimuli responsive polymers? 3) Poly(N-isopropylacrylamide) (PNIPAM): structure formula, value of lower critical solution temperature (LCST), origin of thermoresponsive behaviour. 4) What are self-rolling polymer bilayers? 5) What is the driving force of Poly(N-isopropylacrylamide)/Polycaprolactone selfrolling tubes formation? Alarcon CdH., Pennadam S., Alexander C. Stimuli responsive polymers for biomedical applications. Chem.Soc.Rev., 2005, 34, Ionov L. Soft microorigami: self-folding polymer films. (article manuscript, see in supplementary materials) Zakharchenko S., Puretskiy N., Stoychev G., Stamm M., Ionov L. Temperature controlled encapsulation and release using partially biodegradable thermo-magnetosensitive self-rolling tubes. Soft Matter, 2010, 6, Sample fabrication 1) What are the main stages of dip coating process? How does film thickness depends on the withdrawal speed? 2) What are the main stages of spin coating process? How does film thickness depends on the spin speed? 3) Which types of film defects can be observed in spin coating process? What are the possible reasons of these defects? 4) What is the difference between positive and negative resist photolithography? 5) What is the mechanism of photochemical cross-linking of polymers with benzophenone derivatives? 3.1, G. T. Hermanson, Bioconjugate techniques, 2008, Second edition, Elsevier Inc., p. 205 Ellipsometry 1) Which basic elements does any ellipsometer include? 2) Which states of light polarisation exist? 4

5 3) Which characteristics can be directly measured in nulling ellipsometry? Basic equation of ellipsometry. G. E. Jellison Jr, Encyclopedia of Spectroscopy and Spectrometry, 2000, Academis Press, Ligth microscopy 1) How can one calculate total magnification of the compound microscope? 2) What is Köhler illumination? 3) What is the principle of bright-field microscopy? 4) What are advantages of dark-field microscopy in comparison with bright-field microscopy? R. Wayne Light and video microscopy, 2008, Academic press, p ,