A polypeptide nanofilm made by layer-by-layer (LbL) self-assembly was built on a surface that mimics nonwoven, a material commonly used in wound dressings. Poly-L-lysine (PLL) and poly-L-glutamic acid (PLGA) are the building blocks of the nanofilm, which is intended as an enzymatically degradable lid for release of bactericides to chronic wounds. Chronic wounds often carry infection originating from bacteria such as
Self-assembly techniques have become increasingly popular as a tool to create controlled release systems for drug delivery. One such technique is layer-by-layer (LbL) self-assembly, which was pioneered by Decher [
Clean (acid etching) and ready to use 41 nm gold sensors (XanTec bioanalytics, Düsseldorf, Germany) were tailored to imitate a regenerated cellulose or nonwoven surface using a 2.0 mM mixture of 60 mol% 11-hydroxy-1-undecanethiol and 40 mol% 1-undecanethiol (Sigma Aldrich) in 99.5% ethanol. The sensors were immersed in the alkane thiol solution for 16–20 hours before rinsing in 99.5% ethanol and drying with nitrogen gas before use. Gold substrates used for a second or third time were cleaned in a Plasma Etch situated in a cleanroom. Chosen effect, gas, time/cycle, and amount of cycles were 100 W, O2, 1 min 10 sec/cycle, and two cycles. Contact angle measurements of the substrates were used as a control of the cleanliness.
Poly-L-lysine (PLL) (MW = 30–70 kDa) and poly-L-glutamic acid (PLGA) (MW = 50–100 kDa) were purchased from Sigma Aldrich. A 0.1 mM buffer solution was prepared from Tris-HCl (Sigma Aldrich) and ultrapure water (Milli-Q resistivity > 18 MΩ cm). NaCl (Fluka) (0.1 M) was added to the 0.1 mM Tris-HCl solution followed by addition of either PLL or PLGA in an amount corresponding to 1 mg/mL. Both polyelectrolyte solutions were placed on a magnetic stirrer for 1 hr before use. All end solutions had a pH of 7.4. The SAM covered gold surfaces were used for layer-by-layer (LbL) assembly of PLL/PLGA multilayers. Three PLL/PLGA bilayers were deposited by immersion into each polypeptide solution, respectively, with rinsing steps using Tris-HCl buffer after each immersion. The first PLL layer was immersed in solution for 30 mins allowing a stable cationic foundation for the rest of the build-up, whereafter the deposition and rinsing were performed with 10 min intervals. All (PLL/PLGA)3 samples were dried in N2 gas and placed for ellipsometry measurements in air and ambient temperature. Also, all samples were stored in air and remeasured after 24 hours to make sure that the samples were as dry as possible.
Self-assembly with the LbL technique can be described by creating an overcompensated surface charge by depositing one charged polymer on a surface which in turn enables further deposition of an oppositely charged polymer and so on [
Endoproteinase Glu-C from
Ellipsometry was performed at ambient temperature on an instrument from Beaglehole Instruments (New Zealand). More information about the measuring procedure can be found in the Supporting Information (see Supplementary Material available online at
Three PLL/PLGA bilayers were assembled directly on the alkane thiol model surface without the use of a primer. A primer such as poly(ethylene imine) is often used for LbL assemblies of polypeptides [
The ellipsometry raw data was exported to the TFCompanion software and models were created using raw data originating from the gold substrates modified by alkane thiols as the starting point. Both polypeptides are weak polyelectrolytes and LbL films from such polymers have been reported not to be rigid [
Measured data for
After the ellipsometry measurement the (Au-SAM)-(PLL/PLGA)3 substrate was immersed in buffer solution and allowed to swell for at least 30 minutes before use. The samples were then moved to a new container and a solution of either trypsin (bovine) or V8 glutamyl endopeptidase (
The dry nanofilm thickness of
The bacterial protease V8 behaved differently. The thickness of the nanofilm did not change much on exposure to the solution of V8 enzyme, as can also be seen from Figure
The (PLL/PLGA)3 nanofilm was measured with ellipsometry to study the thickness in its dry state. When comparing with the film’s wet and dry thicknesses, it is clear that about 60% of the wet film consists of water. This result is in accordance with previously reported values from similar systems despite the fact that in the present investigation the polypeptides were adsorbed directly to a tailored gold surface imitating nonwoven and not to a surface treated with a primer such as PEI, which is the normal procedure. This indicates that the character of the film without primer is similar to that with primer. This is practically important in our case because a nondegradable primer is highly unwanted in a wound care dressing. The enzymatic degradation of the nanofilm was also monitored by ellipsometry. Bovine trypsin adsorbed at the polypeptide surface but there were no indications of an enzymatic degradation of the LbL film even after sequential addition of the peptidase. Also the V8 glutamyl endopeptidase from
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work has been performed within the VINN Excellence Center SuMo Biomaterials, a center with financial support from the Swedish governmental funding agency Vinnova and from eight companies: AkzoNobel, AstraZeneca, Bohus Biotech, Lantmännen, Mölnlycke Health Care, SCA Hygiene Products, Södra Cell, and Tetrapak. The authors are grateful to Mölnlycke Health Care and the research school BIOSUM for economic support. They would also want to thank Dr. Stefan Meyer for helping with software and Dr. Natalie Plank for valuable help with the plasma treatment. This article is dedicated to the memory of Professor Pablo Etchegoin.