Self-assembly of a dual functional bioactive peptide amphiphile incorporating both matrix metalloprotease substrate and cell adhesion motifs

We describe a bioactive lipopeptide that combines the capacity to promote the adhesion and subsequent self-detachment of live cells, using template-cell-environment feedback interactions. This self-assembling peptide amphiphile comprises a diene-containing hexadecyl lipid chain (C 16 e) linked to a matrix metalloprotease-cleavable sequence, Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, and contiguous with a cell-attachment and signalling motif, Arg-Gly-Asp-Ser. Biophysical characterisation revealed that the PA self-assembles into 3 nm diameter spherical micelles above a critical aggregation concentration (cac). In addition, when used in solution at 5 – 150 nM (well below the cac), the PA is capable of forming ﬁ lm coatings that provide a stable surface for human corneal ﬁ broblasts to attach and grow. Furthermore, these coatings were demonstrated to be sensitive to metalloproteases expressed endogenously by the attached cells, and consequently to elicit the controlled detachment of cells without compromising their viability. As such, this material constitutes a novel class of multi-functional coating for both fundamental and clinical applications in tissue engineering.


Introduction
One of the main challenges in developing new biomaterials is the creation of a suitable environment in which cells can be seeded, grown, induced to differentiate towards the formation of complex tissues, and eventually detach from the surface leaving the synthetic component behind. Such an environment must be suitable for proper cell adhesion and survival, allowing nutrient and metabolite diffusion, and presenting adequate biochemical and mechanical cues. Presently, considerable efforts are being made towards the development of materials that incorporate bioactive moieties for use in tissue engineering and regenerative medicine. [1][2][3] However, few of these materials have reached the market, possibly due to the difficulty to transfer and implement such technologies and/or methodologies between research groups with very distinct sets of expertise.
In this context, the use of simple, synthetic biomimetic molecules has gained considerable popularity. For example, bio-inspired peptides now have important applications in many natural processes relevant to skin care and wound healing including inammation, cell proliferation, angiogenesis (growth of new blood vessels), and melanogenesis (production of the skin pigment melanin). 4 In these applications, presentation of the active peptide motif is important, as is delivery across the stratum corneum (outer layer of the epidermis containing keratin-rich cells) to the basement membrane. In order to enhance bioactivity, biocompatibility and delivery, lipid chains may be attached to peptides to create peptide amphiphiles (PAs).
One type of PA comprises a lipidic hydrophobic tail connected to a hydrophilic peptide segment. This conguration leads to self-assembled structures with a hydrophobic core surrounded by a peptide functionalized corona. 5 Self-assembly most commonly leads to nanobril or nanotape structures in which peptide epitopes are presented on the bril surface. [5][6][7][8][9][10][11][12] These nanostructures are being developed for diverse applications in bio-nanotechnology and regenerative medicine. 7,8,13 Stupp and coworkers have undertaken seminal work to elucidate the principles of self-assembly of PAs, [14][15][16][17] and explored several important applications in bioengineering, including the use of PA nanobers as scaffolds for bio-mineralization to regenerate bone, 18,19 in differentiation of progenitor 20 or stem cells, 21 or tissue scaffolding 22 including cartilage regeneration. 23 Other groups have examined the use of PAs for applications including cancer therapy, [24][25][26][27][28] and drug release systems, 29-31 among others. PAs enable the presentation of bioactive sequences such as cell adhesion motifs RGD 18 or RGDS, 32 protease substrates, [33][34][35] or even cell growth factors such as TGF-b1 (ref. 23) at the surface of the nanostructures. In general, the peptide amphiphiles have biologically relevant lipid chain lengths, in particular palmitoyl (hexadecyl, C 16 ).
We have recently been investigating the self-assembly of several series of bio-derived lipopeptides. For example, we showed that the PA C 16 -KTTKS, 36 known as Matrixyl® (the trade name is registered to Sederma SA, Le Perray-en-Yvelines, France), 37 is able to stimulate cells to produce collagen, 38 consistent with its use in cosmeceutical applications. The selfassembly of this and two related PAs used in cosmeceuticals was investigated. 39 We have also examined the self-assembly in aqueous solution of three lipopeptides derived from Bacillus subtilis which show antimicrobial activity. 40 Ourselves and others have examined the self-assembly of PAs containing a saturated C 16 lipid chain and an RGD or RGDS-containing headgroup (with tri-glycine, GGG spacers). 41,42 We found that these PAs aggregate into extended nanotapes, and we also investigated co-assembly with C 16 -ETTES. 42 We also prepared bioactive lms from these PAs, which were shown to provide stable adhesive coatings on patterned hydrophobic surfaces composed of striated polytetrauoroethylene (PTFE), signicantly enhancing proliferation of aligned cells and increasing the complexity of the produced tissue. 43 Very recently, we have developed PA coatings for cell and tissue therapies with a selfrelease action due to the incorporation within the peptide headgroup of matrix metalloprotease (MMP) substrate involved in remodelling the extracellular matrix as well as the RGDS cell adhesion motif. The peptide headgroup was attached to a simple (unsaturated) hexadecyl lipid chain. 34 In the present paper, we describe the synthesis and selfassembly of a novel PA that incorporates the same bifunctional peptide headgroup. The PA also contains a diene-containing hexadecyl lipid chain in order to ultimately prepare polymerizable PA lms with potential applications in wound healing, although this is not examined herein. The PA is denoted C16e-MMPRGDS, the term C16e indicating the diene-functionalized lipid chain. The peptide headgroup incorporates the N-terminal sequence Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln (TPGPQGIAGQ) was derived from the matrix metalloprotease (MMP) cleavage site of collagen type-I and functions as a specic proteolysis target for several MMPs, namely MMP1 and 2, 44 with predicted cleavage between the Gly and Ile residues. 45 The C-terminal sequence is the integrindependent cell-adhesion motif Arg-Gly-Asp-Ser (RGDS) found in several cell adhesion molecules and used extensively in biomaterial research to elicit cell attachment, promote survival, and control cell encapsulation and phenotype. 2 As well as reporting on the synthesis and self-assembly of this PA, here we also show that the PA is cytocompatible, and is capable of performing as a stable but bio-degradable cell-adhesive coating template. Thus, the application of polymerizable PA lms notwithstanding, the development of a contiguous MMP cleavage and cell adhesion peptide sequence facilitates the attachment of broblastic cells followed by their subsequent release (from the synthetic PA component) under endogenous enzyme control, combining to form a smart biomaterial for the generation of time-dependent cell release systems for future medical applications.

Synthesis of hexadecadienoic acid
The basic method for synthesizing the diene-containing lipid chain was taken and modied from ref. 46, the reaction scheme is illustrated in Scheme 1. A mixture of 2.22 mL dodecanal, 4.88 mL triethyl 4-phosphonocrotonate, 0.46 g LiOH$H 2 O and 15 g 4Å molecular sieves in 50 mL of tetrahydrofuran were reuxed under nitrogen gas for 12 hours. The resulting solution was crudely cleaned up through a short silica column using diethyl ether as the elution solution and the main yellow fraction was collected. This main fraction was concentrated on a rotary evaporator. Flash chromatography with a silica column and an elution solution of 19 : 1 hexane : ethylacetate was performed and fractions collected. Thin layer chromatography on silica plates was used to identify the contents of the fractions and those containing the product were pooled and concentrated on a rotary evaporator. To this concentrated solution 15 mL of a 1 : 1 solution of ethanol and 20% NaOH was added and heated to 70 C and stirred for 3 hours. To this solution 15 mL of saturated cold NaCl was added and placed at 4 C for 2 hours. The precipitated solution was then ltered and the resultant cake placed in a desiccator to nish drying. The resultant material was identied by mass spectrometry and was produced in 72% yield in relation to the starting material.

Peptide synthesis
Peptides were synthesised using standard Merrield solid phase peptide synthesis methods with 5 times molar excess of each Fmoc-amino acid at each couple step and 4.75 times excess of HBTU and DIPEA with a coupling time of 3 hours each. All synthesis was conducted on standard Wang resins with the last amino acid pre-coupled. Deprotection and cleavage of the nal peptides from the resin was undertaken using the cleavage mixture of TFA/thioanisole/water/phenol/TIS (82.5 : 5:5 : 5:2.5 v/v). The crude peptides were puried using a Supelco Discovery Bio wide pore C18-10, 15 cm Â 10 mm column with buffer A as acetonitrile, 0.01 M HCl and buffer B H 2 O, 0.01 M HCl. The following gradient was used in all purications; 0-30% buffer A over 5 minutes, 30-54% buffer A over 9 minutes, 54-90% buffer A over 5 minutes, 90% buffer A for 3 minutes and 90-0% buffer A over 3 minutes. Elution of peptides from the column was detected using a UV detector at 214 nm, and all the main peptide peaks came off in the region of the second gradient. Multiple injections were pooled and freeze dried to give the pure peptide. Mass spectrometry was used to conrm the identity of the peaks in the chromatogram. Soft Matter Paper 600 mL aliquot of each concentration was deposited into each well of an ultra-low attachment 6 well plate (Costar, Corning, USA) which was then allowed to evaporate overnight to form a dry lm on the bottom of the well. The lm containing wells were then washed and rinsed with phosphate buffer saline (PBS) three times. Human corneal stromal broblasts (hCSFs) extracted from corneal rings were seeded at a density of 1.5 Â 10 4 cells per cm 2 onto each PA coated well to assess cell attachment, and cultured in 2 mL of serum-free medium (SFM, composed of DMEM/F12 with 1 mM ascorbic acid, 1% of ITS supplement (Sigma-Aldrich, USA), 1% penicillin/streptomycin), supplemented with 5 Â 10 À6 M of alltrans retinoic acid (RA) to minimize the expression of matrix metalloproteases from hCSFs. 47 RA was prepared and dissolved in DMSO as a stock concentration of 5 Â 10 À2 M, sterile-ltered, and stored in a À80 C freezer until use. Medium was replaced every other day. Cell proliferation assays were assessed over the course of seven days, and cell quantication was performed on day 1, 3, 5, and 7 to determine the number of cells growing on the PA lms. The number of live cells was determined using the Alamar blue assay, by incubating the cells in 1 : 10 reasazurin:SFM solution for 4 hours, and measuring the uorescence of 100 mL aliquots on a plate reader, in triplicate, using excitation/emission wavelengths of 530-560/590 nm, respectively. The signal from the triplicates was averaged and a standard curve was used to extrapolate uorescence signals to cell numbers. Cell viability was assessed at the end of the culture period using the live/dead cell double staining kit (Merck Millipore, Germany), with live (calcein-positive) and dead (propidium iodide-positive) cells imaged in an Axiovert 200 microscope (Zeiss GMBH, DE). Cells were counted from ten uorescence micrographs collected from random areas from each culture condition replicate. Data was analysed using two-way ANOVA followed by Bonferroni post-hoc tests, with differences deemed signicant at p < 0.05. All experiments were performed in triplicate, and using cells isolated from three independent donors.

Cell culture
Coating stability and bio-degradability C 16 e-MMPRGDS lms were prepared from PA solutions at 50 nM as described above, imaged by phase-contrast microscopy, and then incubated in fresh serum-free medium (SFM) and cellconditioned media at 37 C for seven days. Conditioned media was produced by culturing hCSFs for ve days with SFM alone or supplemented with RA, to either obtain matrix metalloprotease (MMP)-enriched (+MMP) or MMP-deprived (ÀMMP) conditioned medium, respectively. 48 Changes in the shape and morphology of the lms was evaluated aer incubation by reimaging coatings using a Nikon Eclipse inverted microscope (Nikon, JP) coupled with a Jenoptik CCD camera (Jenoptik AG, DE). All experiments were performed in triplicate.

Circular dichroism (CD)
Spectra were recorded using a Chirascan spectropolarimeter (Applied Photophysics, UK). CD was performed on a solution of C 16 e-MMPRGDS in water which was then placed in a cover slip cuvette (0.01 mm thick). Spectra are presented with absorbance A < 2 at any measured point with a 0.5 nm step, 1 nm bandwidth and 1 second collection time per step at 20 C, taking four averages.

Fourier transform infra-red (FTIR) spectroscopy
Spectra were measured on a Nicolet Nexus spectrometer with DTGS detector. FTIR data was measured for a 1 wt% solution of C 16 e-MMPRGDS dissolved in D 2 O. Samples were sandwiched between two CaF 2 plate windows (spacer 0.025 mm). Spectra were scanned 128 times over the range of 4000-900 cm À1 . Data was corrected by baseline subtraction.

X-ray diffraction (XRD)
X-ray diffraction was performed on stalks prepared by suspending drops of PA solution (1 wt% in water) between the ends of wax-coated capillaries, and allowing them to dry. The stalk was mounted vertically onto the four axis goniometer of a RAXIS IV++ X-ray diffractometer (Rigaku) equipped with a rotating anode generator. The XRD data was collected using a Saturn 992 CCD camera.

Cryo-transmission electron microscopy (cryo-TEM)
Imaging was carried out using a eld emission cryo-electron microscope (JEOL JEM-3200FSC), operating at 200 kV voltage. Images were taken in bright eld mode and using zero loss energy ltering (omega type) with the slit with of 20 eV. Micrographs were recorded using a Gatan Ultrascan 4000 CCD camera. The specimen temperature was maintained at À187 C during the imaging. Vitried specimens were prepared using an automated FEI Vitrobot device using Quantifoil 3.5/1 holey carbon copper grids with the hole size of 3.5 mm. Just prior to use grids were plasma cleaned using a Gatan Solarus 9500 plasma cleaner and then transferred into an environmental chamber of a FEI Vitrobot having room temperature and 100% humidity. Thereaer 3 ml of sample solution (2 wt% concentration) was applied on the grid and it was blotted twice for 5 seconds and then vitried in a 1/1 mixture of liquid ethane and propane at À180 C. The most viscous gel (5 wt% concentration) was blotted 4 times for 5 s. The grid with vitried sample solution was maintained at liquid nitrogen temperature and then cryo-transferred to the microscope.

Fluorescence spectroscopy
The uorescence of pyrene was excited at 335 nm at room temperature, and emission spectra were recorded from 350 to 450 nm, using a 10.0 mm Â 5.00 mm quartz cell in a Varian Model Cary Eclipse spectrouorimeter. Excitation and emission bandwidths of 2.5 nm were used throughout the experiments. The concentration of pyrene in water was 1.3 Â 10 À5 M. The same pyrene solution was used to dilute each peptide sample to avoid any dilution effect on pyrene uorescence due to the addition of subsequent peptide amphiphile aliquots.
Small-angle and X-ray scattering (SAXS) Solution SAXS measurements was performed on the bioSAXS beamline BM29 at the ESRF, Grenoble, France. A solution of 1 wt% C 16 e-MMPRGDS was loaded in PCR tubes in an automated sample changer. This was injected into a xed capillary tube for the SAXS measurements. SAXS data was collected using a Pilatus 1 M detector. The sample-detector distance was 2.84 m. The X-ray wavelength was 0.99Å.

SAXS data modelling
The cryo-TEM and SAXS data for 1 wt% C 16 -MMPRGDS at pH 2 indicate that it forms spherical micelles. The SAXS data was modelled using the soware SASt. 49 A spherical shell model with a Gaussian polydispersity in outer radius was used. The model is based on scattering from a sphere with an outer radius R and an inner radius nR. The intensity is written as The Gaussian distribution function used to describe polydispersity in radius R is dened as follows: The parameters used to t the data were; outer radius (R 0 ), inner radius (vR 0 ), scattering contrast relative to the core (mDh), scattering contrast of shell (Dh), and Gaussian polydispersity parameters N and s. A constant background was applied, BG.

Results
We investigated the self-assembly of C 16 e-MMPRGDS, a PA which can potentially act as a scaffold to support cell attachment, growth and subsequent release of cell-derived tissue from the initial scaffold. Cryo-TEM images were obtained for a 1 wt% solution of PA dissolved in water as illustrated by a representative example shown in Fig. 1. This image reveals that C 16 e-MMPRGDS self-assembles into very small spherical micelles with a diameter of approximately 3 nm. A similar size of lipopeptide micelles has been found in our previous work, 40,50,51 and the radius is consistent with the length of the lipopeptide molecules. Interestingly, we observed that the micelles pack hexagonally in some areas with a lattice spacing of 6.7 nm (as shown in Fig. S1 †). The hexagonal ordering is tentatively ascribed to enhanced packing of micelles within ultrathin regions of the cryo-TEM lm. This is consistent with the fact that (bulk) SAXS measurements (to be discussed shortly) show no evidence of hexagonal ordering.
Small-angle X-ray scattering (SAXS) was employed to further probe the C 16 e-MMPRGDS spherical micelle structures, complementing the cryo-TEM images and providing information on the internal structure of the micelles. A synchrotron SAXS prole for a 1 wt% solution of C 16 e-MMPRGDS is shown in Fig. 2a. The form factor prole was tted to a spherical micelle model using the modelling soware SASt. 49 The tting parameters (listed in full in Table 1) obtained from the t indicate an outer radius of 3.3 nm, which is in good agreement with the cryo-TEM images discussed above. The cryo-TEM (Fig. S1 †) revealed that the spherical micelles adopt a hexagonal arrangement in some areas, but since hexagonal ordering is not obtained by SAXS this is believed, as mentioned above, to be an artefact arising from the sample preparation during vitrication. Temperature-dependent SAXS was performed for a xed concentration (1 wt% solution) of the PA upon heating, from 5 C to 55 C (i.e. across the melting point for the lipid chain 52,53 ) as shown in Fig. 2b. The temperature-dependent SAXS curves are similar to one another indicating that the spherical micelle structure is retained upon heating even across the lipid chain melting temperature (40-45 C). The SAXS proles for all three temperatures were tted to the form factor for a core-shell spherical micelle (Fig. 2b). The parameters for the ts are listed in Table 1. The data reveals that at 5 C the micelles have a radius of less than 3 nm and upon heating, an increase in radius of the micelles is observed with the largest increase from 5 C to 30 C. The core-shell contrast decreases with increasing temperature, possibly due to some solubilisation of water within the lipid interior of the micelle.   Spectroscopic techniques were employed to elucidate the secondary structure of C 16 e-MMPRGDS. Circular dichroism (CD) spectroscopy measurements were performed to investigate secondary structure, the spectra being shown in Fig. 3a. A minimum at 230 nm was observed at room temperature, which suggests a lack of ordered structure. 54,55 Temperature-dependent CD reveals no overall change in structure upon heating and cooling in the range of 20-90 C. The FTIR spectrum obtained at room temperature in the amide I 0 region shown in Fig. 3b indicates a disordered conformation, as a main peak at approximately 1650 cm À1 is observed. [56][57][58] Thus, based on the data from both spectroscopic methods, this PA is characterised by a disordered secondary structure, with weak hydrogen bonding within the assembly which favours spherical micelle formation. 8,53 To further probe the spherical micelle nanostructure, bre XRD was employed using a stalk dried from a 1 wt% solution of C 16 e-MMPRGDS. The two-dimensional pattern was found to be isotropic, so it was integrated to one-dimensional proles. The meridional and equatorial 1D intensity proles are shown in Fig. 3c. A single peak is observed with a d spacing of 4.39Å (calculated using d ¼ 2p/q). A b-sheet structure is characterized by a b-strand spacing of 4.7Å in the meridional direction along with a spacing of 10-13Å in the equatorial direction, associated with the spacing of b-sheets. [59][60][61] As no such peaks are present in the intensity prole a b-sheet structure can be ruled out and instead the observed peak is ascribed to the packing of the lipid chain, as it is broad and also unoriented. The XRD data is in good agreement with the spectroscopy data revealing a lack of intermolecular hydrogen bonded b-sheet structure and is consistent with the presence of spherical micelles which are unable to accommodate a b-sheet arrangement of PA molecules. The critical aggregation concentration (CAC) of C 16 e-MMPRGDS was determined at room temperature using a uorescence assays with pyrene. The uorescence vibrational structure of pyrene is sensitive to its environmental hydrophobicity and has been successfully used previously to determine the CAC of amphiphilic molecules. 62,63 The CAC of C 16 e-MMPRGDS in water was estimated by observing the uorescence intensity at the I 1 (l ¼ 383 nm) peak which corresponds to the rst vibronic band of pyrene. 62,64 Fig. 4 shows that there is a break in the uorescence intensity (I 1 ) against concentration curve which denotes that C 16 e-MMPRGDS begins to selfassemble into spherical micelles above a concentration of 0.0065 wt% (4 mM). The self-assembly studies conducted in this research were well above this CAC, being performed at concentrations of 1 wt%.
To understand the applicability of C 16 e-MMPRGDS as a bioactive, cell-adhesive template, the PA was drop-spotted in solution at 5-150 nM (i.e. well below the CAC) onto culture plates and allowed to dry overnight to form continuous lm coatings. Data presented in Fig. 5 demonstrates that C 16 e-MMPRGDS coatings were stable in cell-culture compatible aqueous environment (in fresh serum-free medium for 7 days at 37 C and 5% CO 2 ). Moreover, the PA was susceptible to cleavage by MMPs, as shown by the degradation of the coating aer incubation in cell-conditioned culture medium (Fig. 5, +MMPs panels). This degradation was specic, as conditioned medium deprived of cell-derived MMPs failed to alter the coating morphology and shape (Fig. 5, ÀMMPs panels).
The potential of C 16 e-MMPRGDS as a material to create cytocompatible adhesive templates was further assessed by seeding human corneal stromal broblasts (hCSFs) onto the PA lms. To restrict cell growth to the PA coatings and minimise unspecic cell adhesion, coatings were produced onto ultra-low attachment culture plates. As evident from Fig. 6a, PA coatings were stable on low-attachment plates, creating dened boundaries between the coated and non-coated surfaces. Upon seeding, hCSFs adhered to the bioactive PA lms but not to the noncoated low-attachment plastic. This is especially evident at the edge of the lms, where a clear delineation between the lms   and non-coated low-attachment surface was observed (Fig. 6b). In addition, hCSFs were able to proliferate within the area of the C 16 e-MMPRGDS coatings ( Fig. 6c and 7), reaching semi-conuence while maintaining high viability levels (Fig. 6d). Quantication of cell numbers indicated that all tested concentrations of the PA were capable of supporting cell adhesion and growth at signicantly higher levels compared to the non-coated control surfaces (Fig. 7). Aer seven days in culture, the number of hCSFs decreased. This effect was not caused by a reduction in cell viability (as demonstrated in Fig. 6d) but was due to the detachment of cells from the underlying adhesive surface, a consequence of the progressive cleavage of the C 16 e-MMPRGDS molecules by the endogenous proteolytic activity of hCSFs. Thus cells could be released from the scaffold under enzymatic control. This notion is also supported by the absence of such detachment from RGDS-containing PA coatings lacking an enzyme-cleavable peptide sequence. 43 It is known that MMPs have a fundamental role in the proteolytic activity of cells, which can ultimately lead to the cleavage of ECM components and hence cell detachment. 48 Due to supplementation of retinoic acid (RA) to the culture medium at 5 Â 10 À6 M, hCSFs retain the capability to secrete small amounts of matrix metalloproteases, namely MMP1. 43 As the secreted MMP enzymes begin to accumulate in the culture supernatant, the C 16 e-MMPRGDS coatings start degrading, and the cells lose their anchoring support, detach from the surface, and oat away. Together, these results indicate that C 16 e-MMPRGDS with RA enriched media can provide a coating suitable for initial cell adhesion, proliferation, and survival, but permit the subsequent self-detachment of cells though its specic sensitivity to cell-secreted, endogenous proteases over a number of days in culture.

Summary and discussion
The PA C 16 e-MMPRGDS is shown to self-assemble into small spherical micelles above a critical aggregation concentration (0.0065 wt% in water). The peptide adopts a disordered conformation within the corona of the micelles, as probed by CD and FTIR spectroscopies, as well as X-ray diffraction. The secondary structure is not altered upon heating across the lipid chain melting temperature. The PA supports the growth of hCSFs for up to ve days, whilst ensuring cell self-detachment at later time points.
A remarkable feature of this study is the formation of spherical micelles by the PA with a disordered secondary structure. This is in complete contrast to the secondary structure and self-assembly properties of the homologue with saturated lipid chain C 16 -MMPRGDS. 34 This latter PA adopts a b-sheet secondary structure and forms extended nanotape structures. In addition, this PA is not only capable of promoting the adhesion and growth of corneal or skin broblasts but can also stimulate them to deposit native extracellular matrix, thus forming layered tissues. These newly-shaped tissues governed their own release from the template via the action of endogenous proteases (MMPs), using the RA-supplementation system applied in the present study. However, the coatings formed by the saturated C 16 -MMPRGDS PA were less susceptible to the MMPs endogenously secreted by cells. 34 A possible explanation for this discrepancy is that the diene functionality in C 16e -MMPRGDS has a profound inuence not only on the self-assembly but also on the bio-accessibility of proteolytic enzymes to the functional motifs within the nanostructure. Indeed, these two properties may be correlated, since it is possible that the formation of spherical micelles with peptides in a disordered conformation would allow the cleavable peptide sequence to be presented in a less compact conguration compared to that within b-sheet based nanotapes, and consequently enable less sterically-hindered access to the active site from bulky enzymes. In this context, investigating the relationship between self-assembly, molecule accessibility, 65 and bioactivity will be of future interest in these and related PA systems. Moreover, these systems represent simple but highly versatile, highly-transferable multi-purpose materials with promising applications in both fundamental and clinical research.