Development of Radiopharmaceuticals for NPY Receptor-5 (Y5) Nuclear Imaging in Tumors by Synthesis of Specific Agonists and Investigation of Their Binding Mode.

The neuropeptide-Y (NPY) family acts through four G protein-coupled receptor subtypes in humans, namely, Y1, Y2, Y4, and Y5. A growing body of evidence suggest the involvement of the NPY system in several cancers, notably the Y5 subtype, thus acting as a relevant target for the development of radiopharmaceuticals for imaging or targeted radionuclide therapy (TRT). Here, the [cPP(1-7),NPY(19-23),Ala31,Aib32,Gln34]hPP scaffold, further referred to as sY5ago, was modified with a DOTA chelator and radiolabeled with 68Ga and 111In and investigated in vitro and in vivo using the MCF-7 model. For in vivo studies, MCF-7 cells were orthotopically implanted in female nude mice and imaging with small animal positron emission tomography/computed tomography (µPET/CT) was performed. At the end of imaging, the mice were sacrificed. A scrambled version of sY5ago, which was also modified with a DOTA chelator, served as a negative control (DOTA-[Nle]sY5ago_scrambled). sY5ago and DOTA-sY5ago showed subnanomolar affinity toward the Y5 (0.9 ± 0.1 and 0.8 ± 0.1 nM, respectively) and a single binding site at the Y5 was identified. [68Ga]Ga-DOTA-sY5ago and [111In]In-DOTA-sY5ago were hydrophilic and showed high specific internalization (1.61 ± 0.75%/106 cells at 1 h) and moderate efflux (55% of total binding externalized at 45 min). On µPET/CT images, most of the signal was depicted in the kidneys and the liver. MCF-7 tumors were clearly visualized. On biodistribution studies, [68Ga]Ga-DOTA-sY5ago was eliminated by the kidneys (∼60 %ID/g). The kidney uptake is Y5-mediated. A specific uptake was also noted in the liver (5.09 ± 1.15 %ID/g vs 1.13 ± 0.21 %ID/g for [68Ga]Ga-DOTA-[Nle]sY5ago_scrambled, p < 0.05), the lungs (1.03 ± 0.34 %ID/g vs 0.20 %ID/g, p < 0.05), and the spleen (0.85 ± 0.09%ID/g vs 0.16 ± 0.16%ID/g, p < 0.05). In MCF-7 tumors, [68Ga]Ga-DOTA-sY5ago showed 12-fold higher uptake than [68Ga]Ga-DOTA-[Nle]sY5ago_scrambled (3.43 ± 2.32 vs 0.27 ± 0.15 %ID/g, respectively, p = 0.0008) at 1 h post-injection. Finally, a proof-of-principle tissular micro-imaging study on a human primary cancer sample showed weak binding of [111In]In-DOTA-sY5ago in prostatic intra-neoplasia and high binding in the ISUP1 lesion while normal prostate was free of signal.

Impact of membrane lipid polyunsaturation on dopamine D2 receptor ligand binding and signaling.

Increasing evidence supports a relationship between lipid metabolism and mental health. In particular, the biostatus of polyunsaturated fatty acids (PUFAs) correlates with some symptoms of psychiatric disorders, as well as the efficacy of pharmacological treatments. Recent findings highlight a direct association between brain PUFA levels and dopamine transmission, a major neuromodulatory system implicated in the etiology of psychiatric symptoms. However, the mechanisms underlying this relationship are still unknown. Here we demonstrate that membrane enrichment in the n-3 PUFA docosahexaenoic acid (DHA), potentiates ligand binding to the dopamine D2 receptor (D2R), suggesting that DHA acts as an allosteric modulator of this receptor. Molecular dynamics simulations confirm that DHA has a high preference for interaction with the D2R and show that membrane unsaturation selectively enhances the conformational dynamics of the receptor around its second intracellular loop. We find that membrane unsaturation spares G protein activity but potentiates the recruitment of ß-arrestin in cells. Furthermore, in vivo n-3 PUFA deficiency blunts the behavioral effects of two D2R ligands, quinpirole and aripiprazole. These results highlight the importance of membrane unsaturation for D2R activity and provide a putative mechanism for the ability of PUFAs to enhance antipsychotic efficacy.

The impact of lipid polyunsaturation on the physical and mechanical properties of lipid membranes.

The lipid composition of cellular membranes and the balance between the different lipid components can be impacted by aging, certain pathologies, specific diets and other factors. This is the case in a subgroup of individuals with psychiatric disorders, such as schizophrenia, where cell membranes of patients have been shown to be deprived in polyunsaturated fatty acids (PUFAs), not only in brain areas where the target receptors are expressed but also in peripheral tissues. This PUFA deprivation thus represents a biomarker of such disorders that might impact not only the interaction of antipsychotic medications with these membranes but also the activation and signaling of the targeted receptors embedded in the lipid membrane. Therefore, it is crucial to understand how PUFAs levels alterations modulate the different physical properties of membranes. In this paper, several biophysical approaches were combined (Laurdan fluorescence spectroscopy, atomic force microscopy, differential scanning calorimetry, molecular modeling) to characterize membrane properties such as fluidity, elasticity and thickness in PUFA-enriched cell membranes and lipid model systems reflecting the PUFA imbalance observed in some diseases. The impact of both the number of unsaturations and their position along the chain on the above properties was investigated. Briefly, data revealed that PUFA presence in membranes increases membrane fluidity, elasticity and flexibility and decreases its thickness and order parameter. Both the level of unsaturation and their position affect these membrane properties.

An original approach to measure ligand/receptor binding affinity in non-purified samples.

Several biochemical and biophysical methods are available to determine ligand binding affinities between a biological target and its ligands, most of which require purification, labelling or surface immobilisation. These measurements, however, remain challenging in regards to membrane proteins, as purification processes require their extraction from their native lipid environment, which may in turn impact receptor conformation and functionality. In this study, we have developed a novel experimental procedure using microscale thermophoresis (MST) directly from cell membrane fragments, to determine different ligand binding affinities to a membrane protein, the dopamine D2 receptor (D2R). In order to achieve this, two main challenges had to be overcome: determining the concentration of dopamine D2R in the crude sample; finding ways to minimize or account for non-specific binding of the ligand to cell fragments. Using MST, we were able to determine the D2R concentration in cell membrane fragments to approximately 36.8 ± 2.6 pmol/mg. Next, the doses-responses curves allowed for the determination of KD, to approximately 5.3 ± 1.7 nM, which is very close to the reported value. Important details of the experimental procedure have been detailed in this paper to allow the application of this novel method to various membrane proteins.

The role of the lipid environment in the activity of G protein coupled receptors.

G protein coupled receptors (GPCRs) are a class of membrane proteins that sense extracellular signals ranging from light to odorants and small molecules and activate intracellular signaling pathways that control important physiological responses. Being composed of 7 transmembrane helices linked by extracellular and intracellular loops, the great majority of the sequence of these receptors is embedded in the lipid membrane. Therefore, it is expected GPCR structure and function to be impacted by the surrounding lipid environment and the lipid membrane physico-chemical and mechanical properties. A large number of examples from the literature is provided to highlight the role of the lipid nature (lipid headgroup, membrane polyunsaturation and cholesterol) and membrane physical and mechanical properties (curvature elastic stress, membrane thickness and hydrophobic mismatch, fluidity) in the activity of different GPCRs. In addition, lipids are important co-factors being identified in very specific locations in several GPCR structures. GPCRs and G proteins can also be lipid post-translationally modified and such events can significantly impact membrane binding, trafficking and signaling. These aspects are all treated in this review. Understanding how the lipid can modulate GPCR activity is important not only from a fundamental point of view but also due to the fact that certain pathologies, where GPCRs are central targets, have been associated with important lipid imbalance. Establishing a link between the lipid pathological imbalance and the receptor functioning in such environment is thus essential as it can open avenues to potentially innovative therapeutic strategies.

Plasmon Waveguide Resonance: Principles, Applications and Historical Perspectives on Instrument Development.

Plasmon waveguide resonance (PWR) is a variant of surface plasmon resonance (SPR) that was invented about two decades ago at the University of Arizona. In addition to the characterization of the kinetics and affinity of molecular interactions, PWR possesses several advantages relative to SPR, namely, the ability to monitor both mass and structural changes. PWR allows anisotropy information to be obtained and is ideal for the investigation of molecular interactions occurring in anisotropic-oriented thin films. In this review, we will revisit main PWR applications, aiming at characterizing molecular interactions occurring (1) at lipid membranes deposited in the sensor and (2) in chemically modified sensors. Among the most widely used applications is the investigation of G-protein coupled receptor (GPCR) ligand activation and the study of the lipid environment's impact on this process. Pioneering PWR studies on GPCRs were carried out thanks to the strong and effective collaboration between two laboratories in the University of Arizona leaded by Dr. Gordon Tollin and Dr. Victor J. Hruby. This review provides an overview of the main applications of PWR and provides a historical perspective on the development of instruments since the first prototype and continuous technological improvements to ongoing and future developments, aiming at broadening the information obtained and expanding the application portfolio.

Design, synthesis, and biological evaluation of a multifunctional neuropeptide-Y conjugate for selective nuclear delivery of radiolanthanides.

BACKGROUND: Targeting G protein-coupled receptors on the surface of cancer cells with peptide ligands is a promising concept for the selective tumor delivery of therapeutically active cargos, including radiometals for targeted radionuclide therapy (TRT). Recently, the radiolanthanide terbium-161 (161Tb) gained significant interest for TRT application, since it decays with medium-energy ß-radiation but also emits a significant amount of conversion and Auger electrons with short tissue penetration range. The therapeutic efficiency of radiometals emitting Auger electrons, like 161Tb, can therefore be highly boosted by an additional subcellular delivery into the nucleus, in order to facilitate maximum dose deposition to the DNA. In this study, we describe the design of a multifunctional, radiolabeled neuropeptide-Y (NPY) conjugate, to address radiolanthanides to the nucleus of cells naturally overexpressing the human Y1 receptor (hY1R). By using solid-phase peptide synthesis, the hY1R-preferring [F7,P34]-NPY was modified with a fatty acid, a cathepsin B-cleavable linker, followed by a nuclear localization sequence (NLS), and a DOTA chelator (compound pb12). In this proof-of-concept study, labeling was performed with either native terbium-159 (natTb), as surrogate for 161Tb, or with indium-111 (111In). RESULTS: [natTb]Tb-pb12 showed a preserved high binding affinity to endogenous hY1R on MCF-7 cells and was able to induce receptor activation and internalization similar to the hY1R-preferring [F7,P34]-NPY. Specific internalization of the 111In-labeled conjugate into MCF-7 cells was observed, and importantly, time-dependent nuclear uptake of 111In was demonstrated. Study of metabolic stability showed that the peptide is insufficiently stable in human plasma. This was confirmed by injection of [111In]In-pb12 in nude mice bearing MCF-7 xenograft which showed specific uptake only at very early time point. CONCLUSION: The multifunctional NPY conjugate with a releasable DOTA-NLS unit represents a promising concept for enhanced TRT with Auger electron-emitting radiolanthanides. Our research is now focusing on improving the reported concept with respect to the poor plasmatic stability of this promising radiopeptide.

Direct Monitoring of GPCR Reconstitution and Ligand-Binding Activity by Plasmon Waveguide Resonance.

The study of G-protein-coupled receptor (GPCR ) mechanisms of activation and signaling often the isolation, purification, and reconstitution of GPCRs in model lipid membranes. GPCR reconstitution from a detergent-micelle system into model membranes is usually a laborious process whose success is tested after the whole process is over by rather indirect methods such as SDS-PAGE and western blotting or by biophysical approaches. Following that, protein activity is measured in yet a different experimental setup. Overall, the whole procedure is tedious, long, and often requires high quantities of material and the use of labeled proteins. Herein, a protocol is described to follow, in a single experimental setup, both GPCR reconstitution from detergent micelles into planar lipid bilayers and its ligand-binding activity using plasmon waveguide resonance. Alternatively, receptor/ligand interactions can also be investigated from cell membrane fragments overexpressing the receptor of interest, bypassing isolation and reconstitution procedures. The method is direct, sensitive, and does not rely on the use of any labeled material.

Structural insights into the AapA1 toxin of Helicobacter pylori.

BACKGROUND: We previously reported the identification of the aapA1/IsoA1 locus as part of a new family of toxin-antitoxin (TA) systems in the human pathogen Helicobacter pylori. AapA1 belongs to type I TA bacterial toxins, and both its mechanism of action towards the membrane and toxicity features are still unclear. METHODS: The biochemical characterization of the AapA1 toxic peptide was carried out using plasmid-borne expression and mutational approaches to follow its toxicity and localization. Biophysical properties of the AapA1 interaction with lipid membranes were studied by solution and solid-state NMR spectroscopy, plasmon waveguide resonance (PWR) and molecular modeling. RESULTS: We show that despite a low hydrophobic index, this toxin has a nanomolar affinity to the prokaryotic membrane. NMR spectroscopy reveals that the AapA1 toxin is structurally organized into three distinct domains: a positively charged disordered N-terminal domain (D), a single α-helix (H), and a basic C-terminal domain (R). The R domain interacts and destabilizes the membrane, while the H domain adopts a transmembrane conformation. These results were confirmed by alanine scanning of the minimal sequence required for toxicity. CONCLUSION: Our results have shown that specific amino acid residues along the H domain, as well as the R domain, are essential for the toxicity of the AapA1 toxin. GENERAL SIGNIFICANCE: Untangling and understanding the mechanism of action of small membrane-targeting toxins are difficult, but nevertheless contributes to a promising search and development of new antimicrobial drugs.

Ionpair-π interactions favor cell penetration of arginine/tryptophan-rich cell-penetrating peptides.

Cell-penetrating peptides (CPPs) internalization occurs both by endocytosis and direct translocation through the cell membrane. These different entry routes suggest that molecular partners at the plasma membrane, phospholipids or glycosaminoglycans (GAGs), bind CPPs with different affinity or selectivity. The analysis of sequence-dependent interactions of CPPs with lipids and GAGs should lead to a better understanding of the molecular mechanisms underlying their internalization. CPPs are short sequences generally containing a high number of basic arginines and lysines and sometimes aromatic residues, in particular tryptophans. Tryptophans are crucial residues in membrane-active peptides, because they are important for membrane interaction. Membrane-active peptides often present facial amphiphilicity, which also promote the interaction with lipid bilayers. To study the role of Trp and facial amphiphilicity in cell interaction and penetration of CPPs, a nonapeptide series containing only Arg, Trp or D-Trp residues at different positions was designed. Our quantitative study indicates that to maintain/increase the uptake efficiency, Arg can be advantageously replaced by Trp in the nonapeptides. The presence of Trp in oligoarginines increases the uptake in cells expressing GAGs at their surface, while it compensates for the loss of charge interactions from Arg and maintains similar peptide uptake in GAG-deficient cells. In addition, we show that facial amphiphilicity is not required for efficient uptake of these nonapeptides. Thermodynamic analyses point towards a key role of Trp that highly contributes to the binding enthalpy of complexes formation. Density functional theory (DFT) analysis highlights that salt bridge-π interactions play a crucial role for the GAG-dependent entry mechanisms.

Cholesterol impacts chemokine CCR5 receptor ligand-binding activity.

The chemokine CCR5 receptor is target of maraviroc, a negative allosteric modulator of CCR5 that blocks the HIV protein gp120 from associating with the receptor, thereby inhibiting virus cellular entry. As noted with other G-protein-coupled receptor family members, the role of the lipid environment in CCR5 signaling remains obscure and very modestly investigated. Controversial literature on the impact of cholesterol (Chol) depletion in HIV infection and CCR5 signaling, including the hypothesis that Chol depletion could inhibit HIV infection, lead us to focus on the understanding of Chol impact in the first stages of receptor activation. To address this aim, the approach chosen was to employ reconstituted model lipid systems of controlled lipid composition containing CCR5 from two distinct expression systems: Pichia pastoris and cell-free expression. The characterization of receptor/ligand interaction in terms of total binding or competition binding assays was independently performed by plasmon waveguide resonance and fluorescence anisotropy, respectively. Maraviroc, a potent receptor antagonist, was the ligand investigated. Additionally, coarse-grained molecular dynamics simulation was employed to investigate Chol impact in the receptor-conformational flexibility and dynamics. Results obtained with receptor produced by different expression systems and using different biophysical approaches clearly demonstrate a considerable impact of Chol in the binding affinity of maraviroc to the receptor and receptor-conformational dynamics. Chol considerably decreases maraviroc binding affinity to the CCR5 receptor. The mechanisms by which this effect occurs seem to involve the adoption of distinct receptor-conformational states with restrained structural dynamics and helical motions in the presence of Chol.

Dendron-Functionalized Surface: Efficient Strategy for Enhancing the Capture of Microvesicles.

Microvesicles (MVs) are used by various types of cells in the human body for intercellular communication, making them biomarkers of great potential for the early and non-evasive diagnosis of a spectrum of diseases. An integrated analysis including morphological, quantitative, and compositional studies is most desirable for the clinical application of MV detection; however, such integration is limited by the currently available analysis techniques. In this context, exploiting the phosphatidylserine (PS) exposure of MVs, we synthesized a series of dendritic molecules with PS-binding sites at the periphery. PS-dendron binding was studied at the molecular level using NMR approaches, whereas PS-containing membrane-dendron interaction was investigated in an aqueous environment using plasmon waveguide resonance spectroscopy. As a proof of concept, polyethylene terephthalate surface was functionalized with the synthetic dendrons, forming devices that can capture MVs to facilitate their subsequent analyses.

Biophysical Insight on the Membrane Insertion of an Arginine-Rich Cell-Penetrating Peptide.

Cell-penetrating peptides (CPPs) are short peptides that can translocate and transport cargoes into the intracellular milieu by crossing biological membranes. The mode of interaction and internalization of cell-penetrating peptides has long been controversial. While their interaction with anionic membranes is quite well understood, the insertion and behavior of CPPs in zwitterionic membranes, a major lipid component of eukaryotic cell membranes, is poorly studied. Herein, we investigated the membrane insertion of RW16 into zwitterionic membranes, a versatile CPP that also presents antibacterial and antitumor activities. Using complementary approaches, including NMR spectroscopy, fluorescence spectroscopy, circular dichroism, and molecular dynamic simulations, we determined the high-resolution structure of RW16 and measured its membrane insertion and orientation properties into zwitterionic membranes. Altogether, these results contribute to explaining the versatile properties of this peptide toward zwitterionic lipids.

Putative interaction site for membrane phospholipids controls activation of TRPA1 channel at physiological membrane potentials.

The transient receptor potential ankyrin 1 (TRPA1) channel is a polymodal sensor of environmental irritant compounds, endogenous proalgesic agents, and cold. Upon activation, TRPA1 channels increase cellular calcium levels via direct permeation and trigger signaling pathways that hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2 ) in the inner membrane leaflet. Our objective was to determine the extent to which a putative PIP2 -interaction site (Y1006-Q1031) is involved in TRPA1 regulation. The interactions of two specific peptides (L992-N1008 and T1003-P1034) with model lipid membranes were characterized by biophysical approaches to obtain information about affinity, peptide secondary structure, and peptide effect in the lipid organization. The results indicate that the two peptides interact with lipid membranes only if PIP2 is present and their affinities depend on the presence of calcium. Using whole-cell electrophysiology, we demonstrate that mutation at F1020 produced channels with faster activation kinetics and with a rightward shifted voltage-dependent activation curve by altering the allosteric constant that couples voltage sensing to pore opening. We assert that the presence of PIP2 is essential for the interaction of the two peptide sequences with the lipid membrane. The putative phosphoinositide-interacting domain comprising the highly conserved F1020 contributes to the stabilization of the TRPA1 channel gate.

The Contribution of Differential Scanning Calorimetry for the Study of Peptide/Lipid Interactions.

Membrane-active peptides include a variety of molecules such as antimicrobial (AMP), cell-penetrating (CPP), viral, and amyloid peptides that are implicated in several pathologies. They constitute important targets because they are either at the basis of novel therapies (drug delivery for CPPs or antimicrobial activity for AMPs) or they are the agents causing these pathologies (viral and amyloid peptides). They all share the common property of interacting with the cellular lipid membrane in their mode of action. Therefore, a better understanding of the peptide/lipid (P/L) interaction is essential to help decipher their mechanism of action. Among the different biophysical methods that can be used to fully characterize P/L interactions, differential scanning calorimetry (DSC) allows determining the peptide effect on the lipid phase transitions, a property that reflects the P/L interaction mode. A general protocol for classical DSC experiments for P/L studies will be provided.

Study of G-Protein Coupled Receptor Signaling in Membrane Environment by Plasmon Waveguide Resonance.

Here we describe an experimental technique, termed plasmon waveguide resonance (PWR) spectroscopy that enables the characterization of molecular interactions occurring at the level of anisotropic thin films as lipid membranes and therein inserted or interacting molecules. PWR allows one to characterize such molecular interactions at different levels: (1) acquire binding curves and calculate dissociation constants; (2) obtain kinetic information; (3) obtain information about associated anisotropy changes and changes in membrane thickness; (4) obtain insight about lateral homogeneity (formation of domains). Points 1, 2, and 4 can be directly obtained from the data. Point 3 requires spectral fitting procedures so that the different optical parameters characterizing thin films as proteolipid membranes, namely refractive index and extinction coefficient for both p- (TM component of light that is parallel to the incident light) and s- (TE component of light that is perpendicular to the incident light) polarizations and thickness, can be determined. When applied to membrane proteins as the G-protein coupled receptor (GPCR) family, both ligand-induced conformational changes of the receptor can be followed as well as interactions with effectors (e.g., G-proteins). Additionally, by either altering the lipid composition in cellular membranes or specifically controlling its composition in the case of lipid model membranes with reconstituted proteins, the role of the lipid environment in receptor activation and signaling can be determined. Additionally, the eventual partition of receptors in different lipid microdomains (e.g., lipid rafts) can be followed. Such information can be obtained  ex cellulo with mammalian cell membrane fragments expressing the protein of interest and/or in vitro with lipid model systems where the protein under investigation has been reconstituted. Moreover, PWR can also be applied to directly follow the reconstitution of membrane proteins in lipid model membranes. The measurements are performed directly (no labeling of molecular partners), in real time and with very high sensitivity. Here we will discuss different aspects of GPCR activation and signaling where PWR brought important information in parallel with other approaches. The utility of PWR is not limited to GPCRs but can be applied to any membrane protein. PWR is also an excellent tool to characterize the interaction of membrane active molecules (as cell penetrating, antimicrobial, viral and amyloid peptides) with lipids. A brief section is dedicated to such applications, with particular emphasis on amyloid peptides. To finalize, as PWR is a homemade technology, ongoing instrument developments aiming at breaking current experimental limitations are briefly discussed, namely, the coupling of PWR with electrochemical measurements and the expansion of measurements from the visible to the infrared region.

Improved Detection of Plasmon Waveguide Resonance Using Diverging Beam, Liquid Crystal Retarder, and Application to Lipid Orientation Determination.

Plasmon waveguide resonance (PWR) sensors exhibit narrow resonances at the two orthogonal polarizations, transverse electric (TE) and transverse magnetic (TM), which are narrower by almost an order of a magnitude than the standard surface plasmon resonance (SPR), and thus the figure of merit is enhanced. This fact is useful for measuring optical anisotropy of materials on the surface and determining the orientation of molecules with high resolution. Using the diverging beam approach and a liquid crystal retarder, we present experimental results by simultaneous detection of TE and TM polarized resonances as well as using fast higher contrast serial detection with a variable liquid crystal retarder. While simultaneous detection makes the system simpler, a serial one has the advantage of obtaining a larger contrast of the resonances and thus an improved signal-to-noise ratio. Although the sensitivity of the PWR resonances is smaller than the standard SPR, the angular width is much smaller, and thus the figure of merit is improved. When the measurement methodology has a high enough angular resolution, as is the one presented here, the PWR becomes advantageous over other SPR modes. The possibility of carrying out exact numerical simulations for anisotropic molecules using the 4 × 4 matrix approach brings another advantage of the PWR over SPR on the possibility of extracting the orientation of molecules adsorbed to the surface. High sensitivity of the TE and TM signals to the anisotropic molecules orientation is found here, and comparison to the experimental data allowed detection of the orientation of lipids on the sensor surface. The molecular orientations cannot be fully determined from the TM polarization alone as in standard SPR, which underlines the additional advantage of the PWR technique.

Thickness determination in anisotropic media with plasmon waveguide resonance imaging.

This paper describes a simple procedure to determine the local thickness of a thin anisotropic layer. It also discriminates between isotropic and anisotropic regions, provided a smoothness hypothesis on the refractive index distribution is satisfied. The procedure is based on the analysis of surface plasmon resonance (SPR) data acquired in an imaging mode. The general arrangement of the setup is the Kretschmann configuration. We show, on an azobenzene modified polymer layer, good agreement between atomic force microscopy and optical measurements of thickness variation.