X-ray reflectivity study of the heat shock protein Hsp70 interaction with an artificial cell membrane model.

Membrane-bound heat shock protein 70 (Hsp70) apart from its intracellular localization was shown to be specifically expressed on the plasma membrane surface of tumor but not normal cells. Although the association of Hsp70 with lipid membranes is well documented the exact mechanisms for chaperone membrane anchoring have not been fully elucidated. Herein, we addressed the question of how Hsp70 interacts with negatively charged phospholipids in artificial lipid compositions employing the X-ray reflectivity (XRR) studies. In a first step, the interactions between dioleoylphosphatidylcholine (DOPC) in the presence or absence of dioleoylphosphatidylserine (DOPS) and Hsp70 had been assessed using Quartz crystal microbalance measurements, suggesting that Hsp70 adsorbs to the surface of DOPC/DOPS bilayer. Atomic force microscopy (AFM) imaging demonstrated that the presence of DOPS is required for stabilization of the lipid bilayer. The interaction of Hsp70 with DOPC/DOPS lipid compositions was further quantitatively determined by high energy X-ray reflectivity. A systematic characterization of the chaperone-lipid membrane interactions by various techniques revealed that artificial membranes can be stabilized by the electrostatic interaction of anionic DOPS lipids with Hsp70.

Novel Strategy to Drive the Intracellular Uptake of Lipid Nanoparticles for Photodynamic Therapy.

Nanoparticles' uptake by cancer cells upon reaching the tumor microenvironment is often the rate-limiting step in cancer nanomedicine. Herein, we report that the inclusion of aminopolycarboxylic acid conjugated lipids, such as EDTA- or DTPA-hexadecylamide lipids in liposome-like porphyrin nanoparticles (PS) enhanced their intracellular uptake by 25-fold, which was attributed to these lipids' ability to fluidize the cell membrane in a detergent-like manner rather than by metal chelation of EDTA or DTPA. EDTA-lipid-incorporated-PS (ePS) take advantage of its unique active uptake mechanism to achieve >95 % photodynamic therapy (PDT) cell killing compared to <5 % cell killing by PS. In multiple tumor models, ePS demonstrated fast fluorescence-enabled tumor delineation within minutes post-injection and increased PDT potency (100 % survival rate) compared to PS (60 %). This study offers a new nanoparticle cellular uptake strategy to overcome challenges associated with conventional drug delivery.

Self-Organization of Lipid-Porphyrin Conjugates at the Air/Water Interface.

Lipid-porphyrin conjugates are versatile compounds which can self-assemble into liposome-like structures with multifunctional properties. Most of the conjugates that have been described so far, consisted in grafting pyropheophorbide-a (Pyro-a) or other porphyrin derivatives through the esterification of the hydroxyl group in the sn-2 position of a lysophosphatidylcholine. However, despite the versatility of these conjugates, less is known about the impact of the lipid backbone structure on their 2D phase behavior at the air/water interface and more precisely on their fine structures normal to the interface as well as on their in-plane organization. Herein, we synthesized a new lipid-porphyrin conjugate (PyroLSM) based on the amide coupling of Pyro-a to a lysosphingomyelin backbone (LSM) and we compared its interfacial behavior to that of Pyro-a and Pyro-a conjugated lysophosphatidylcholine (PyroLPC) using Langmuir balance combined to a variety of other physical techniques. Our results provided evidence on the significant impact of the lipid backbone on the lateral packing of the conjugates as well as on the shape and size of the formed domains. Compared to Pyro-a and PyroLPC monolayers, PyroLSM exhibited the highest lateral packing which highlights the role of the lipid backbone in controlling their 2D organization which in turn may impact the photophysical properties of their assemblies.

Novel liposome-like assemblies composed of phospholipid-porphyrin conjugates with photothermal and photodynamic activities against bacterial biofilms.

Phospholipid-Porphyrin (PL-Por) conjugates are unique building blocks that can self assemble into liposome-like structures with improved photophysical properties compared to their monomeric counterparts. The high packing density of porphyrin moieties enables these assemblies to exhibit high photothermal conversion efficiency as well as photodynamic activity. Thus, PL-Por conjugates assemblies can be used for photodynamic therapy (PDT) and photothermal therapy (PTT) applications against resistant bacteria and biofilms. In order to tune the PD/PT properties of such nanosystems, we developed six different supramolecular assemblies composed of newly synthesized PL-Por conjugates bearing either pheophorbide-a (PhxLPC) or pyropheophorbide-a (PyrxLPC) photosensitizers (PSs) for combined PDT/PTT against planktonic bacteria and their biofilms. In this study, the influence of the chemical structure of the phospholipid backbone as well as that of the PS on the photothermal conversion efficiency, the photodynamic activity and the stability of these assemblies in biological medium were determined. Then their antimicrobial efficiency was assessed on S. aureus and P. aeruginosa planktonic cultures and biofilms. The two studied systems show almost the same photothermal effect against planktonic cultures and biofilms of S. aureus and P. aeruginosa. However, PhxLPC vesicles exhibit superior photodynamic activity, making them the best combination for PTT/PDT. Such results highlight the higher potential of the photodynamic activity of PL-Por nanoassemblies compared to their photothermal conversion in combating bacterial infections.

Phospholipid-porphyrin conjugates: deciphering the driving forces behind their supramolecular assemblies.

Phospholipid-porphyrin conjugates (PL-Por) are nowadays considered as a unique class of building blocks that can self-assemble into supramolecular structures that possess multifunctional properties and enhanced optoelectronics characteristics compared to their disassembled counterparts. However, despite their versatile properties, little is known about the impact of the packing parameter of PL-Por conjugates on their assembling mechanism and their molecular organization inside these assemblies. To gain a better understanding on their assembling properties, we synthesized two new series of PL-Por conjugates with different alkyl sn2-chain lengths linked via an amide bond to either pheophorbide-a (PhxLPC) or pyropheophorbide-a (PyrxLPC). By combining a variety of experimental techniques with molecular dynamics (MD) simulations, we investigated both the assembling and optical properties of the PL-Por either self-assembled or when incorporated into lipid bilayers. We demonstrated that independently of the linker length, PhxLPC assembled into closed ovoid structures, whereas PyrxLPC formed rigid open sheets. Interestingly, PyrxLPC assemblies displayed a significant red shift and narrowing of the Q-band indicating the formation of ordered J-aggregates. The MD simulations highlighted the central role of the interaction between porphyrin cores rather than the length difference between the two phospholipid chains in controlling the structure of the lipid bilayer membranes and thus their optical properties. Indeed, while PhxLPC have the tendency to form inter-leaflet π-stacked dimers, PyrxLPC conjugates formed dimers within the same leaflet. Altogether, this work could be used as guidelines for the design of new PL-Por conjugates that self-assemble into bilayer-like supramolecular structures with tunable morphology and optical properties.

Photo-triggerable liposomes based on lipid-porphyrin conjugate and cholesterol combination: Formulation and mechanistic study on monolayers and bilayers.

Lipid-porphyrin conjugates are considered nowadays as promising building blocks for the conception of drug delivery systems with multifunctional properties such as photothermal therapy (PTT), photodynamic therapy (PDT), phototriggerable release, photoacoustic and fluorescence imaging. For this aim, we have recently synthesized a new lipid-porphyrin conjugate named PhLSM. This was obtained by coupling pheophorbide-a (Pheo-a), a photosensitizer derived from chlorophyll-a, to egg lyso-sphingomyelin. The pure PhLSMs were able to self-assemble into vesicle-like structures that were however not stable and formed aggregates with undefined structures due to the mismatch between the length of the alkyl chain in sn-1 position and the adjacent porphyrin. Herein, stable PhLSMs lipid bilayers were achieved by mixing PhLSMs with cholesterol which exhibits a complementary packing parameter. The interfacial behavior as well as the fine structures of their equimolar mixture was studied at the air/buffer interface by the mean of Langmuir balance and x-ray reflectomerty (XRR) respectively. Our XRR analysis unraveled the monolayer thickening and the increase in the lateral ordering of PhLSM molecules. Interestingly, we could prepare stable vesicles with this mixture that encapsulate hydrophilic fluorescent probe. The light-triggered release kinetics and the photothermal conversion were studied. Moreover, the obtained vesicles were photo-triggerable and allowed the release of an encapsulated cargo in an ON-OFF fashion.

Influence of the porphyrin structure and linker length on the interfacial behavior of phospholipid-porphyrin conjugates.

HYPOTHESIS: Phospholipid-porphyrin (Pl-Por) conjugates consist of porphyrin derivatives grafted to a lysophosphatidylcholine backbone. Owing to their structural similarities with phospholipids, Pl-Por conjugates can self-assemble into liposome-like assemblies. However, there is a significant lack of information concerning the impact of the porphyrin type and the length of the alkyl chain bearing the porphyrin on the interfacial behavior of the Pl-Por conjugates. We hypothesized that changing the chain length and the porphyrin type could impact their two-dimensional phase behavior and modulate the alignment between the two chains. EXPERIMENTS: 6 Pl-Por conjugates with different alkyl chain lengths in the sn2 position of C16 lysophosphatidylcholine and coupled to either pheophorbide-a or pyropheophorbide-a were synthesized. Their interfacial behavior at the air/water interface was assessed using Langmuir balance combined to a variety of other physical techniques including Brewster angle microscopy, atomic force microscopy and X-ray reflectometry. FINDINGS: Our results showed that all 6 Pl-Por form stable monolayers with the porphyrin moiety at the air/water interface. We also showed that changing the porphyrin moiety controlled the packing of the monolayer and thus the formation of organized domains. The chain length dictated the structure of the formed domains with no evidence of the alignment between the two chains.

Design and Synthesis of New PEGylated Polydopamine-Based Nanoconstructs Bearing ROS-Responsive Linkers and a Photosensitizer for Bimodal Photothermal and Photodynamic Therapies against Cancer.

Polydopamine (PDA) nanoparticles (NPs) have recently acquired considerable attention for the development of nanoplatforms with multifunctional properties including photothermal (PTT) and photodynamic (PDT) activities. In addition to their high PTT performance, they can be easily conjugated to different types of photosensitizers (PSs) to acquire PDT activity. However, because of PDA free-radical scavenging properties, grafting the PSs directly to PDA surfaces may lead to an inefficient PDT outcome. Thus, the present work aims at synthesizing and characterizing a new PEGylated PDA-based nanoplatform with bifunctional PTT and PDT properties, which allows bimodal cancer therapy with the possibility to release the PS on demand in a spatiotemporal fashion. To do so, PDA NPs with a well-defined size and shape were prepared by the auto-oxidative self-polymerization process of dopamine hydrochloride in mild alkaline solution. The impact of the size on the PTT conversion efficiency was then determined. This allowed us to choose the optimal PDA NP size for PTT applications. Next, PDA NPs were decorated with SH-PEG polymers that bear at their extremity a thioketal reactive oxygen species-cleavable linker coupled to trisulfonated-tetraphenylporphyrin (TPPS3) chosen as a hydrophilic PS. The grafting efficiency of PS-conjugated PEG on PDA was demonstrated in situ using a quartz crystal microbalance with dissipation monitoring. In addition, the photoinduced release of the PS was demonstrated by 1H NMR. Finally, PTT/PDT bimodal therapy was assessed in vitro on human squamous esophageal cells by illuminating the PDA NPs at two different wavelengths, which showed the strong synergistic effect of combining PTT and PDT within this nanoplatform.

Atomic Force Microscopy Imaging and Nanomechanical Properties of Six Tau Isoform Assemblies.

The amyloid fibrillar form of the protein Tau is involved in a number of neurodegenerative diseases, also known as tauopathies. In this work, six different fibrillar Tau isoforms were assembled in vitro. The morphological and nanomechanical properties of these isoforms were studied using atomic force microscopy at high resolution in air and buffer. Our results demonstrate that all Tau isoform fibrils exhibit paired-helical-filament-like structures consisting of two protofibrils separated by a shallow groove. Interestingly, whereas the N-terminal inserts do not contribute to any morphological or mechanical difference between the isoforms with the same carboxyl-terminal microtubule-binding domain repeats, isoforms with four microtubule repeats (4R) exhibited a persistence length ranging from 2.0 to 2.8 µm, almost twofold higher than those with three repeats (3R). In addition, the axial Young's modulus values derived from the persistence lengths, as well as their radial ones determined via nanoindentation experiments, were very low compared to amyloid fibrils made of other proteins. This sheds light on the weak intermolecular interaction acting between the paired ß-sheets within Tau fibrils. This may play an important role in their association into high molecular weight assemblies, their dynamics, their persistence, their clearance in cells, and their propagation.

Bioinspired polydopamine nanoparticles: synthesis, nanomechanical properties, and efficient PEGylation strategy.

Polydopamine (PDA) is a bioinspired fascinating polymer which is considered nowadays as a material of choice for designing drug delivery nanosystems. Indeed, PDA exhibits multiple interesting features including simple preparation protocols, biocompatibility, simple functionalization procedures, free radicals scavenging and photothermal/photoacoustic properties. However, because of its heterogeneous structure, clear procedures about PDA nanoparticles synthesis and PEGylation with well-defined and reproducible physicochemical properties such as size, shape and nanomechanics are still needed. In this work, we established tightly controlled experimental conditions to synthesize PDA nanoparticles with well-defined size and yield. This allowed us to identify the factors that affect the most these two parameters and to construct surface response plots with accurate predictive values of size and yield. The nanomechanical properties of PDA NPs exhibiting different sizes have been studied with AFM nanoindentation experiments. Our results demonstrated for the first time that the elasticity of PDA NPs was decreasing with their size. This could be explained by the higher geometric packing order of the stacked oligomeric fractions inside the core of the biggest PDA NPs. Next, in order to determine the best PEGylation experimental conditions of PDA NPs using thiol-terminated PEG that allow grafting the highest polymer density with proteins repelling properties, we have first optimized the PEGylation strategy on PDA films. By using a combination of QCM-D and AFM experiments, we could demonstrate that efficient PEGylation of PDA films could be done even at low PEG concentration but in the presence of NaCl which exerts a salting out effect on PEG chains improving thus the grafting density. Finally, we transposed these experimental conditions to PDA NPs and we could synthesize PEGylated PDA NPs exhibiting high stability in physiological conditions as revealed by FTIR and DLS experiments respectively.

Two new polymorphic structures of human full-length alpha-synuclein fibrils solved by cryo-electron microscopy.

Intracellular inclusions rich in alpha-synuclein are a hallmark of several neuropathological diseases including Parkinson's disease (PD). Previously, we reported the structure of alpha-synuclein fibrils (residues 1-121), composed of two protofibrils that are connected via a densely-packed interface formed by residues 50-57 (Guerrero-Ferreira, eLife 218;7:e36402). We here report two new polymorphic atomic structures of alpha-synuclein fibrils termed polymorphs 2a and 2b, at 3.0 Å and 3.4 Å resolution, respectively. These polymorphs show a radically different structure compared to previously reported polymorphs. The new structures have a 10 nm fibril diameter and are composed of two protofilaments which interact via intermolecular salt-bridges between amino acids K45, E57 (polymorph 2a) or E46 (polymorph 2b). The non-amyloid component (NAC) region of alpha-synuclein is fully buried by previously non-described interactions with the N-terminus. A hydrophobic cleft, the location of familial PD mutation sites, and the nature of the protofilament interface now invite to formulate hypotheses about fibril formation, growth and stability.

Capillary zone electrophoresis-native mass spectrometry for the quality control of intact therapeutic monoclonal antibodies.

Therapeutic monoclonal antibodies (mAbs) are complex glycoproteins and ensuring their safety, efficacy and quality is still challenging. Indeed, during their manufacturing process, they are exposed to several stresses that can lead to their denaturation, misfolding or dimerization. We report here a new method based on capillary electrophoresis coupled to native mass spectrometry (MS) with a sheath liquid interface to analyze an intact therapeutic mAb, Infliximab, under non-denaturing conditions that preserve its conformational heterogeneity as well as self-association without inducing further unfolding / denaturation. For capillary zone electrophoresis (CZE) separation, a triple layer coating using polybrene-dextran sulfate-polybrene was employed. A sheath liquid composed of isopropanol - water - acetic acid with a flow rate of 10 µL min-1 and mild MS conditions allowed optimal signal intensities. A specific mass spectrum was obtained for each Infliximab conformation in a "stressed" formulated preparation. This is the first time that within a single analysis different conformational states, i.e. native and unfolded monomers as well as dimers are simultaneously detected. The results and the lack of analytical bias arising from the CZE-MS conditions were confirmed by using atomic force microscopy (AFM) as an orthogonal technique. A middle-up approach combined to CZE-MS analysis of the stressed samples suggested that the dimer formation involved mostly Fab-Fab interactions.

Photo-triggerable liposomal drug delivery systems: from simple porphyrin insertion in the lipid bilayer towards supramolecular assemblies of lipid-porphyrin conjugates.

Light-responsive liposomes are considered nowadays as one of the most promising nanoparticulate systems for the delivery and release of an active pharmaceutical ingredient (API) in a spatio-temporal manner. Several strategies can be used to design photo-triggered liposomes. One of them consists in the incorporation of a photosensitizer (PS) in the lipid matrix of a liposomal bilayer that induces the release of the cargo either via a photochemical or a photophysical process. Among the described photosensitizers, porphyrin derivatives have appeared as the most potent ones. This review describes the state-of-the-art of photo-triggerable liposomes based on the combination of lipids and porphyrin derivatives either free or conjugated. It focuses on the different light-triggered release mechanisms and the requirements for the development of such systems. It also details the different strategies for the synthesis of lipid-porphyrin conjugates, their self-assembling properties and their biomedical applications.

Newly Synthesized Lipid-Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro.

Lipid-porphyrin conjugates are considered nowadays as promising building blocks for the conception of supramolecular structures with multifunctional properties, required for efficient cancer therapy by photodynamic therapy (PDT). The synthesis of two new lipid-porphyrin conjugates coupling pheophorbide-a (Pheo-a), a photosensitizer derived from chlorophyll-a, to either chemically modified lyso-phosphatidylcholine (PhLPC) or egg lyso-sphingomyelin (PhLSM) is reported. The impact of the lipid backbone of these conjugates on their self-assembling properties, as well as on their physicochemical properties, including interfacial behavior at the air/buffer interface, fluorescence and absorption properties, thermotropic behavior, and incorporation rate in the membrane of liposomes were studied. Finally, their photodynamic activity was evaluated on esophageal squamous cell carcinoma (ESCC) and normal esophageal squamous epithelium cell lines. The liposome-like vesicles resulting from self-assembly of the pure conjugates were unstable and turned into aggregates with undefined structure within few days. However, both lipid-porphyrin conjugates could be efficiently incorporated in lipid vesicles, with higher loading rates than unconjugated Pheo-a. Interestingly, phototoxicity tests of free and liposome-incorporated lipid-porphyrin conjugates demonstrated a better selectivity in vitro to esophageal squamous cell carcinoma relative to normal cells.

Molecular interactions governing the incorporation of cholecalciferol and retinyl-palmitate in mixed taurocholate-lipid micelles.

Cholecalciferol (D3) and retinyl palmitate (RP) are the two main fat-soluble vitamins found in foods from animal origin. It is assumed that they are solubilized in mixed micelles prior to their uptake by intestinal cells, but only scarce data are available on the relative efficiency of this process and the molecular interactions that govern it. The extent of solubilization of D3 and RP in micelles composed of lipids and sodium taurocholate (NaTC) was determined. Then, the molecular interactions between components were analyzed by surface tension and surface pressure measurements. The mixture of lipids and NaTC allowed formation of micelles with higher molecular order, and at lower concentrations than pure NaTC molecules. D3 solubilization in the aqueous phase rich in mixed micelles was several times higher than that of RP. This was explained by interactions between NaTC or lipids and D3 thermodynamically more favorable than with RP, and by D3 self-association.

Ib-AMP4 insertion causes surface rearrangement in the phospholipid bilayer of biomembranes: Implications from quartz-crystal microbalance with dissipation.

Most antimicrobial peptides exert their rapid bactericidal activity through a unique mechanism of bacterial membrane disruption. However, the molecular events that underlie this mechanism remain partly unresolved. In this study, the frequency shift (ΔF) obtained through quartz-crystal microbalance with dissipation (QCM-D) indicated that the initial binding of Ib-AMP4 within the lipid membrane started at a critical Ib-AMP4 concentration that exceeded 100µg/ml. Circular dichroism measurements provided evidence that Ib-AMP4 occurs in a ß-sheet configuration which is adapted for insertion into the lipid membrane. Monolayer experiments and the value of dissipation alteration (ΔD) obtained through QCM-D showed that the pressure increased within the phospholipid bilayer upon peptide insertion, and the increase in pressure subsequently forced the bilayer to wrinkle and form pores. However, D continued to increase, indicating that the membrane surface underwent a dramatic morphological transition: the membrane surface likely became porous and uneven as Ib-AMP4 projected from the external surface of the lipid bilayer. Intensive peptide insertion, however, soon plateaued 1min after the addition of Ib-AMP4. This behaviour corresponded with the results of bactericidal kinetics and liposome leakage assays. A sudden decrease in D accompanied by a negligible decrease in F occurred after replacing the Ib-AMP4 solution with HEPES buffer. This result implied that the bilayer surface rearranged and that poration and wrinkling decreased without further peptide insertion. Transmission electron microscopy results indicated that pore formation occurred during Ib-AMP4 insertion but eventually subsided. Therefore, the mode of action of AMP in bacterial membranes could be elucidated through QCM-D.

Adsorption of galloyl catechin aggregates significantly modulates membrane mechanics in the absence of biochemical cues.

Physical interactions of four major green tea catechin derivatives with cell membrane models were systemically investigated. Catechins with the galloyl moiety caused the aggregation of small unilamellar vesicles and an increase in the surface pressure of lipid monolayers, while those without did not. Differential scanning calorimetry revealed that, in a low concentration regime (≤10 µM), catechin molecules are not significantly incorporated into the hydrophobic core of lipid membranes as substitutional impurities. Partition coefficient measurements revealed that the galloyl moiety of catechin and the cationic quaternary amine of lipids dominate the catechin-membrane interaction, which can be attributed to the combination of electrostatic and cation-π interactions. Finally, we shed light on the mechanical consequence of catechin-membrane interactions using the Fourier-transformation of the membrane fluctuation. Surprisingly, the incubation of cell-sized vesicles with 1 µM galloyl catechins, which is comparable to the level in human blood plasma after green tea consumption, significantly increased the bending stiffness of the membranes by a factor of more than 60, while those without the galloyl moiety had no detectable influence. Atomic force microscopy and circular dichroism spectroscopy suggest that the membrane stiffening is mainly attributed to the adsorption of galloyl catechin aggregates to the membrane surfaces. These results contribute to our understanding of the physical and thus the generic functions of green tea catechins in therapeutics, such as cancer prevention.

Impact of lipid composition and photosensitizer hydrophobicity on the efficiency of light-triggered liposomal release.

Photo-triggerable liposomes are considered nowadays as promising drug delivery devices due to their potential to release encapsulated drugs in a spatial and temporal manner. In this work, we have investigated the photopermeation efficiency of three photosensitizers (PSs), namely verteporfin, pheophorbide a and m-THPP when incorporated into liposomes with well-defined lipid compositions (SOPC, DOPC or SLPC). By changing the nature of phospholipids and PSs, the illumination of the studied systems was shown to significantly alter their lipid bilayer properties via the formation of lipid peroxides. The system efficiency depends on the PS/phospholipid association, and the ability of the PS to peroxidize acyl chains. Our results demonstrated the possible use of these three clinically approved (or under investigation) PSs as potential candidates for photo-triggerable liposome conception.

Accumulation of phosphatidylcholine on gut mucosal surface is not dominated by electrostatic interactions.

The accumulation of phosphatidylcholine (PC) in the intestinal mucus layer is crucial for the protection of colon epithelia from the bacterial attack. It has been reported that the depletion of PC is a distinct feature of ulcerative colitis. Here we addressed the question how PC interacts with its binding proteins, the mucins, which may establish the hydrophobic barrier against colonic microbiota. In the first step, the interactions of dioleoylphosphatidylcholine (DOPC) with two mucin preparations from porcine stomach, have been studied using dynamic light scattering, zeta potential measurement, and Langmuir isotherms, suggesting that mucin binds to the surface of DOPC vesicles. The enthalpy of mucin-PC interaction could be determined by isothermal titration calorimetry. The high affinity to PC found for both mucin types seems reasonable, as they mainly consist of mucin 2, a major constituent of the flowing mucus. Moreover, by the systematic variation of net charges, we concluded that the zwitterionic DOPC has the strongest binding affinity that cannot be explained within the electrostatic interactions between charged molecules.

Nanomechanical properties of distinct fibrillar polymorphs of the protein α-synuclein.

Alpha-synuclein (α-Syn) is a small presynaptic protein of 140 amino acids. Its pathologic intracellular aggregation within the central nervous system yields protein fibrillar inclusions named Lewy bodies that are the hallmarks of Parkinson's disease (PD). In solution, pure α-Syn adopts an intrinsically disordered structure and assembles into fibrils that exhibit considerable morphological heterogeneity depending on their assembly conditions. We recently established tightly controlled experimental conditions allowing the assembly of α-Syn into highly homogeneous and pure polymorphs. The latter exhibited differences in their shape, their structure but also in their functional properties. We have conducted an AFM study at high resolution and performed a statistical analysis of fibrillar α-Syn shape and thermal fluctuations to calculate the persistence length to further assess the nanomechanical properties of α-Syn polymorphs. Herein, we demonstrated quantitatively that distinct polymorphs made of the same protein (wild-type α-Syn) show significant differences in their morphology (height, width and periodicity) and physical properties (persistence length, bending rigidity and axial Young's modulus).