Neutron reflectometry as a powerful tool to elucidate membrane interactions of drug delivery systems.

The last couple of decades have seen an explosion of novel colloidal drug delivery systems, which have been demonstrated to increase drug efficacy, reduce side-effects, and provide various other advantages for both small-molecule and biomacromolecular drugs. The interactions of delivery systems with biomembranes are increasingly recognized to play a key role for efficient eradication of pathogens and cancer cells, as well as for intracellular delivery of protein and nucleic acid drugs. In parallel, there has been a broadening of methodologies for investigating such systems. For example, advanced microscopy, mass-spectroscopic "omic"-techniques, as well as small-angle X-ray and neutron scattering techniques, which only a few years ago were largely restricted to rather specialized areas within basic research, are currently seeing increased interest from researchers within wide application fields. In the present discussion, focus is placed on the use of neutron reflectometry to investigate membrane interactions of colloidal drug delivery systems. Although the technique is still less extensively employed for investigations of drug delivery systems than, e.g., X-ray scattering, such studies may provide key mechanistic information regarding membrane binding, re-modelling, translocation, and permeation, of key importance for efficacy and toxicity of antimicrobial, cancer, and other therapeutics. In the following, examples of this are discussed and gaps/opportunities in the research field identified.

Evolution of the structure of lipid nanoparticles for nucleic acid delivery: From in situ studies of formulation to colloidal stability.

The development of lipid nanoparticle (LNP) based therapeutics for delivery of RNA has triggered the advance of new strategies for formulation, such as high throughput microfluidics for precise mixing of components into well-defined particles. In this study, we have characterised the structure of LNPs throughout the formulation process using in situ small angle x-ray scattering in the microfluidic chip, then by sampling in the subsequent dialysis process. The final formulation was investigated with small angle x-ray (SAXS) and neutron (SANS) scattering, dynamic light scattering (DLS) and cryo-TEM. The effect on structure was investigated for LNPs with a benchmark lipid composition and containing different cargos: calf thymus DNA (DNA) and two model mRNAs, polyadenylic acid (polyA) and polyuridylic acid (polyU). The LNP structure evolved during mixing in the microfluidic channel, however was only fully developed during the dialysis. The colloidal stability of the final formulation was affected by the type of incorporated nucleic acids (NAs) and decreased with the degree of base-pairing, as polyU induced extensive particle aggregation. The main NA LNP peak in the SAXS data for the final formulation were similar, with the repeat distance increasing from polyU

On the interactions between RNA and titrateable lipid layers: implications for RNA delivery with lipid nanoparticles.

Characterising the interaction between cationic ionisable lipids (CIL) and nucleic acids (NAs) is key to understanding the process of RNA lipid nanoparticle (LNP) formation and release of NAs from LNPs. Here, we have used different surface techniques to reveal the effect of pH and NA type on the interaction with a model system of DOPC and the CIL DLin-MC3-DMA (MC3). At only 5% MC3, differences in the structure and dynamics of the lipid layer were observed. Both pH and %MC3 were shown to affect the absorption behaviour of erythropoietin mRNA, polyadenylic acid (polyA) and polyuridylic acid (polyU). The adsorbed amount of all studied NAs was found to increase with decreasing pH and increasing %MC3 but with different effects on the lipid layer, which could be linked to the NA secondary structure. For polyA at pH 6, adsorption to the surface of the layer was observed, whereas for other conditions and NAs, penetration of the NA into the layer resulted in the formation of a multilayer structure. By comparison to simulations excluding the secondary structure, differences in adsorption behaviours between polyA and polyU could be observed, indicating that the NA's secondary structure also affected the MC3-NA interactions.

Particle Adsorption Using a Quartz Crystal Microbalance with Dissipation by Applying a Kelvin-Voigt-Based Viscoelastic Model and the Gauss-Newton Method.

The use of a quartz crystal microbalance with dissipation (QCM-D) to study the adsorption of particles larger than 100 nm, such as liposomes, viruses, and nano/micro-plastics, remains challenging owing to the lack of appropriate models for data evaluation. This study presents a method for quantifying the adsorption of negatively charged polystyrene latex (100 nm-1 µm) at the solid-liquid interface. The validity of a viscoelastic model based on Kelvin-Voigt theory was assessed, and the model was used to evaluate particle adsorption data obtained from QCM-D measurements. The Gauss-Newton method was used to fit the data; the values obtained were larger than results from atomic force microscopy, indicating that the viscoelastic model combined with the Gauss-Newton method can quantify the adsorption of large polystyrene particles and the surrounding water around them. We suggested that QCM-D, in combination with an appropriate viscoelastic model, is applicable to estimate adsorption at the solid-liquid interface even for soft particles larger than 1 µm, which are out of the range of applications to the hydrodynamics model. Furthermore, we successfully showed that the recorded dissipation reflects the viscoelastic properties of the layer. The viscoelastic model allowed quantification of the rheological properties of the layer. The ratio of the viscous and elastic contributions was characterized by using loss tangent (tan δ) values that were extracted from the experimental data by applying the viscoelastic model. These values were lower for the adsorption of the negatively charged polystyrene particles on a positive surface than on a negative surface. This suggests that tan δ reflects the strength of the contact between the particle and substrate.

Electrostatic interactions control the adsorption of extracellular vesicles onto supported lipid bilayers.

Communication between cells located in different parts of an organism is often mediated by membrane-enveloped nanoparticles, such as extracellular vesicles (EVs). EV binding and cell uptake mechanisms depend on the heterogeneous composition of the EV membrane. From a colloidal perspective, the EV membrane interacts with other biological interfaces via both specific and non-specific interactions, where the latter include long-ranged electrostatic and van der Waals forces, and short-ranged repulsive "steric-hydration" forces. While electrostatic forces are generally exploited in most EV immobilization protocols, the roles played by various colloidal forces in controlling EV adsorption on surfaces have not yet been thoroughly addressed. In the present work, we study the adsorption of EVs onto supported lipid bilayers (SLBs) carrying different surface charge densities using a combination of quartz crystal microbalance with dissipation monitoring (QCM-D) and confocal laser scanning microscopy (CLSM). We demonstrate that EV adsorption onto lipid membranes can be controlled by varying the strength of electrostatic forces and we theoretically describe the observed phenomena within the framework of nonlinear Poisson-Boltzmann theory. Our modelling results confirm the experimental observations and highlight the crucial role played by attractive electrostatics in EV adsorption onto lipid membranes. They furthermore show that simplified theories developed for model lipid systems can be successfully applied to the study of their biological analogues and provide new fundamental insights into EV-membrane interactions with potential use in developing novel EV separation and immobilization strategies.

Structural insights on ionizable Dlin-MC3-DMA lipids in DOPC layers by combining accurate atomistic force fields, molecular dynamics simulations and neutron reflectivity.

Ionizable lipids such as the promising Dlin-MC3-DMA (MC3) are essential for the successful design of lipid nanoparticles (LNPs) as drug delivery agents. Combining molecular dynamics simulations with experimental data, such as neutron reflectivity experiments and other scattering techniques, is essential to provide insights into the internal structure of LNPs, which is not fully understood to date. However, the accuracy of the simulations relies on the choice of force field parameters and high-quality experimental data is indispensable to verify the parametrization. For MC3, different parameterizations in combination with the CHARMM and the Slipids force fields have recently emerged. Here, we complement the existing efforts by providing parameters for cationic and neutral MC3 compatible with the AMBER Lipid17 force field. Subsequently, we carefully assess the accuracy of the different force fields by providing a direct comparison to neutron reflectivity experiments of mixed lipid bilayers consisting of MC3 and DOPC at different pHs. At low pH (cationic MC3) and at high pH (neutral MC3) the newly developed MC3 parameters in combination with AMBER Lipid17 for DOPC give good agreement with the experiments. Overall, the agreement is similar compared to the Park-Im parameters for MC3 in combination with the CHARMM36 force field for DOPC. The Ermilova-Swenson MC3 parameters in combination with the Slipids force field underestimate the bilayer thickness. While the distribution of cationic MC3 is very similar, the different force fields for neutral MC3 reveal distinct differences ranging from strong accumulation in the membrane center (current MC3/AMBER Lipid17 DOPC), over mild accumulation (Park-Im MC3/CHARMM36 DOPC) to surface accumulation (Ermilova-Swenson MC3/Slipids DOPC). These pronounced differences highlight the importance of accurate force field parameters and their experimental validation.

Investigating thermally induced aggregation of Somatropin- new insights using orthogonal techniques.

Three orthogonal techniques were used to provide new insights into thermally induced aggregation of the therapeutic protein Somatropin at pH 5.8 and 7.0. The techniques were Dynamic Light Scattering (DLS), Asymmetric Flow-Field Flow-Fractionation (AF4), and the TEM-based analysis system MiniTEM™. In addition, Differential Scanning Calorimetry (DSC) was used to study the thermal unfolding and stability. DSC and DLS were used to explain the initial aggregation process and aggregation rate at the two pH values. The results suggest that less electrostatic stabilization seems to be the main reason for the faster initial aggregation at pH 5.8, i.e., closer to the isoelectric point of Somatropin. AF4 and MiniTEM were used to investigate the aggregation pathway further. Combining the results allowed us to demonstrate Somatropin's thermal aggregation pathway at pH 7.0. The growth of the aggregates appears to follow two steps. Smaller elongated aggregates are formed in the first step, possibly initiated by partly unfolded species. In the second step, occurring during longer heating, the smaller aggregates assemble into larger aggregates with more complex structures.

Cation and buffer specific effects on the DNA-lipid interaction.

Knowledge of DNA - lipid layer interactions is key for the development of biosensors, synthetic nanopores, scaffolds, and gene-delivery systems. These interactions are strongly affected by the ionic composition of the solvent. We have combined quartz crystal microbalance (QCM) and ellipsometry measurements to reveal how pH, buffers and alkali metal chloride salts affect the interaction of DNA with lipid bilayers (DOTAP/DOPC 30:70 in moles). We found that the thickness of the DNA layer adsorbed onto the lipid bilayer decreased in the order citrate > phosphate > Tris > HEPES. The effect of cations on the thickness of the DNA layer decreased in the order (K+ > Na+ > Cs+ ∼ Li+). Rationalization of the experimental results requires that adsorption, due to cation specific charge screening, is driven by the simultaneous action of two mechanisms namely, the law of matching water affinities for kosmotropes (Li+) and ion dispersion forces for chaotropes (Cs+). The outcome of these two opposing mechanisms is a "bell-shaped" specific cations sequence. Moreover, a superimposed buffer specificity, which goes beyond the simple effect of pH regulation, further modulated cation specificity. In summary, DNA-lipid bilayer interactions are maximized if citrate buffer (50 mM, pH 7.4) and KCl (100 mM) are used.

Single-Standard Quantification Strategy for Lignin Dimers by Supercritical Fluid Chromatography with Charged Aerosol Detection.

The increased interest in utilizing lignin as a feedstock to produce various aromatic compounds requires advanced chemical analysis methods to provide qualitative and quantitative characterization of lignin samples along different technology streamlines. However, due to the lack of commercially available chemical standards, routine quantification of industrially relevant lignin oligomers in complex lignin samples remains a challenge. This study presents a novel method for universal quantification of lignin dimers based on supercritical fluid chromatography with charged aerosol detection (CAD). A series of lignin-derived dimeric compounds that have been reported from reductive catalytic fractionation (RCF) were synthesized and used as standards. The applicability of using linear regression instead of quadratic calibration curves was evaluated over a concentration range of 15-125 mg/L, demonstrating that the former calibration method is as appropriate as the latter. The response factors of lignin dimeric compounds were compared to assess the uniformity of the CAD signal, revealing that the CAD response for the tested lignin dimers did not differ substantially. It was also found that the response factors were not dependent on the number of methoxy groups or linkage motifs, ultimately enabling the use of only one calibrant for these compounds. The importance of chromatographic peak resolution in CAD was stressed, and the use of a digital peak sharpening technique was adopted and applied to address this challenge. The developed method was verified and used for the quantification of lignin dimers in an oil obtained by a RCF of birch sawdust.

Topological Dynamics of Micelles Formed by Geometrically Varied Surfactants.

The molecular architecture of sugar-based surfactants strongly affects their self-assembled structure, i.e., the type of micelles they form, which in turn controls both the dynamics and rheological properties of the system. Here, we report the segmental and mesoscopic structure and dynamics of a series of C16 maltosides with differences in the anomeric configuration and degree of tail unsaturation. Neutron spin-echo measurements showed that the segmental dynamics can be modeled as a one-dimensional array of segments where the dynamics increase with inefficient monomer packing. The network dynamics as characterized by dynamic light scattering show different relaxation modes that can be associated with the micelle structure. Hindered dynamics are observed for arrested networks of worm-like micelles, connected to their shear-thinning rheology, while nonentangled diffusing rods relate to Newtonian rheological behavior. While the design of novel surfactants with controlled properties poses a challenge for synthetic chemistry, we demonstrate how simple variations in the monomer structure can significantly influence the behavior of surfactants.

Electrostatic interactions between cationic dendrimers and anionic model biomembrane.

The electrostatic interactions between cationic poly(amidoamine) (PAMAM) dendrimers of different generations, G3, G4, and G6, with net anionic model biomembranes have been predicted by adopting an analytical model based on two dissimilar soft spheres. The influence of bilayer surface charge density, ionic strength, pH, temperature, membrane softness (modeled as changes in bilayer thickness), and dendrimer generation on the attractive interaction was investigated. The attraction was found to decrease with increasing salt concentration, dendrimer charge, and thickness (or softness) of the membrane. On the other hand, the attraction increased with the surface charge density of the membrane, and the size of dendrimer generation. In fact, the attraction was found to be much larger for large generations, like G6 dendrimer that have a higher charge, than it is with small ones like G3 and G4 dendrimers. These results have implications for the use of PAMAM dendrimers as potential gene transfection vectors.

Effect of encapsulated protein on the dynamics of lipid sponge phase: a neutron spin echo and molecular dynamics simulation study.

Lipid membranes are highly mobile systems with hierarchical, time and length scale dependent, collective motions including thickness fluctuations, undulations, and topological membrane changes, which play an important role in membrane interactions. In this work we have characterised the effect of encapsulating two industrially important enzymes, ß-galactosidase and aspartic protease, in lipid sponge phase nanoparticles on the dynamics of the lipid membrane using neutron spin echo (NSE) spectroscopy and molecular dynamics (MD) simulations. From NSE, reduced membrane dynamics were observed upon enzyme encapsulation, which were dependent on the enzyme concentration and type. By fitting the intermediate scattering functions (ISFs) with a modified Zilman and Granek model including nanoparticle diffusion, an increase in membrane bending rigidity was observed, with a larger effect for ß-galactosidase than aspartic protease at the same concentration. MD simulations for the system with and without aspartic protease showed that the lipids relax more slowly in the system with protein due to the replacement of the lipid carbonyl-water hydrogen bonds with lipid-protein hydrogen bonds. This indicates that the most likely cause of the increase in membrane rigidity observed in the NSE measurements was dehydration of the lipid head groups. The dynamics of the protein itself were also studied, which showed a stable secondary structure of protein over the simulation, indicating no unfolding events occurred.

Self-Diffusive Properties of the Intrinsically Disordered Protein Histatin 5 and the Impact of Crowding Thereon: A Combined Neutron Spectroscopy and Molecular Dynamics Simulation Study.

Intrinsically disordered proteins (IDPs) are proteins that, in comparison with globular/structured proteins, lack a distinct tertiary structure. Here, we use the model IDP, Histatin 5, for studying its dynamical properties under self-crowding conditions with quasi-elastic neutron scattering in combination with full atomistic molecular dynamics (MD) simulations. The aim is to determine the effects of crowding on the center-of-mass diffusion as well as the internal diffusive behavior. The diffusion was found to decrease significantly, which we hypothesize can be attributed to some degree of aggregation at higher protein concentrations, (≥100 mg/mL), as indicated by recent small-angle X-ray scattering studies. Temperature effects are also considered and found to, largely, follow Stokes-Einstein behavior. Simple geometric considerations fail to accurately predict the rates of diffusion, while simulations show semiquantitative agreement with experiments, dependent on assumptions of the ratio between translational and rotational diffusion. A scaling law that previously was found to successfully describe the behavior of globular proteins was found to be inadequate for the IDP, Histatin 5. Analysis of the MD simulations show that the width of the distribution with respect to diffusion is not a simplistic mirroring of the distribution of radius of gyration, hence, displaying the particular features of IDPs that need to be accounted for.

Nanoscale structural and mechanical characterization of thin bicontinuous cubic phase lipid films.

The mechanical response of lipid membranes to nanoscale deformations is of fundamental importance for understanding how these interfaces behave in multiple biological processes; in particular, the nanoscale mechanics of non-lamellar membranes represents a largely unexplored research field. Among these mesophases, inverse bicontinuous cubic phase QII membranes have been found to spontaneously occur in stressed or virally infected cells and to play a role in fundamental processes, such as cell fusion and food digestion. We herein report on the fabrication of thin ( Ì´150 nm) supported QII cubic phase lipid films (SQIIFs) and on their characterization via multiple techniques including Small Angle X-Ray Scattering (SAXS), Ellipsometry and Atomic Force Microscopy (AFM). Moreover, we present the first nanomechanical characterization of a cubic phase lipid membrane, through AFM-based Force Spectroscopy (AFM-FS). Our analysis reveals that the mechanical response of these architectures is strictly related to their topology and structure. The observed properties are strikingly similar to those of macroscopic 3D printed cubic structures when subjected to compression tests in material science; suggesting that this behaviour depends on the 3D organisation, rather than on the length-scale of the architecture. We also show for the first time that AFM-FS can be used for characterizing the structure of non-lamellar mesophases, obtaining lattice parameters in agreement with SAXS data. In contrast to classical rheological studies, which can only probe bulk cubic phase solutions, our AFM-FS analysis allows probing the response of cubic membranes to deformations occurring at length and force scales similar to those found in biological interactions.

Interaction of nanoparticles with lipid films: the role of symmetry and shape anisotropy.

The bioactivity, biological fate and cytotoxicity of nanomaterials when they come into contact with living organisms are determined by their interaction with biomacromolecules and biological barriers. In this context, the role of symmetry/shape anisotropy of both the nanomaterials and biological interfaces in their mutual interaction, is a relatively unaddressed issue. Here, we study the interaction of gold nanoparticles (NPs) of different shapes (nanospheres and nanorods) with biomimetic membranes of different morphology, i.e. flat membranes (2D symmetry, representative of the most common plasma membrane geometry), and cubic membranes (3D symmetry, representative of non-lamellar membranes, found in Nature under certain biological conditions). For this purpose we used an ensemble of complementary structural techniques, including Neutron Reflectometry, Grazing Incidence Small-Angle Neutron Scattering, on a nanometer lengthscale and Confocal Laser Scanning Microscopy on a micrometer length scale. We found that the structural stability of the membrane towards NPs is dependent on the topological characteristic of the lipid assembly and of the NPs, where a higher symmetry gave higher stability. In addition, Confocal Laser Scanning Microscopy analyses highlighted that NPs interact with cubic and lamellar phases according to two distinct mechanisms, related to the different structures of the lipid assemblies. This study for the first time systematically addresses the role of NPs shape in the interaction with lipid assemblies with different symmetry. The results will contribute to improve the fundamental knowledge on lipid interfaces and will provide new insights on the biological function of phase transitions as a response strategy to the exposure of NPs.

Shear-induced nanostructural changes in micelles formed by sugar-based surfactants with varied anomeric configuration.

HYPOTHESIS: The self-assembly of long tail sugar-based surfactants into worm-like micelles has recently been demonstrated, and the rheological properties of such systems have been shown to be tuneable through subtle modifications of the molecular characteristics of the surfactant monomer. In particular, the anomeric configuration of the hexadecylmaltoside headgroup was shown to induce profound changes in the nanostructure and rheology of the system. The origin of such changes is hypothesised to arise from differences in the structure and relaxation of the micellar networks in the semi-dilute regime. EXPERIMENTS: Here we explore the molecular background to the flow properties of the two anomers of hexadecylmaltoside (α- and ß-C16G2) by directly connecting their rheological behaviour to the micelle morphology. For this purpose, 1-3 plane rheo-small-angle neutron scattering measurements, using a Couette cell geometry, probed the structural changes in the micellar phase under shear. The effect of surfactant anomeric configuration, surfactant concentration, temperature and mixing ratio of the two anomers were investigated. The static micelle structure in the semi-dilute regime was determined using the polymer reference interaction site model. FINDINGS: The segmental alignment of the micellar phase was studied under several flow conditions, showing that the shear-thinning behaviour relates to the re-arrangement of ß-C16G2 worm-like micelles, whilst shorter α-C16G2 micelles are considerably less affected by the flow. The results are rationalised in terms of micelle alignment and disruption of the entangled network, providing a detailed mechanism by which sugar-based surfactants control the rheology of the fluid. To further enable future studies, we provide the complete code for modelling micelle structure in the semi-dilute regime using the polymer reference interaction site model.

Poly(styrene)-block-Maltoheptaose Films for Sub-10 nm Pattern Transfer: Implications for Transistor Fabrication.

Sequential infiltration synthesis (SIS) into poly(styrene)-block-maltoheptaose (PS-b-MH) block copolymer using vapors of trimethyl aluminum and water was used to prepare nanostructured surface layers. Prior to the infiltration, the PS-b-MH had been self-assembled into 12 nm pattern periodicity. Scanning electron microscopy indicated that horizontal alumina-like cylinders of 4.9 nm diameter were formed after eight infiltration cycles, while vertical cylinders were 1.3 nm larger. Using homopolymer hydroxyl-terminated poly(styrene) (PS-OH) and MH films, specular neutron reflectometry revealed a preferential reaction of precursors in the MH compared to PS-OH. The infiltration depth into the maltoheptaose homopolymer film was found to be 2.0 nm after the first couple of cycles. It reached 2.5 nm after eight infiltration cycles, and the alumina incorporation within this infiltrated layer corresponded to 23 vol % Al2O3. The alumina-like material, resulting from PS-b-MH infiltration, was used as an etch mask to transfer the sub-10 nm pattern into the underlying silicon substrate, to an aspect ratio of approximately 2:1. These results demonstrate the potential of exploiting SIS into carbohydrate-based polymers for nanofabrication and high pattern density applications, such as transistor devices.

Morphologies and Structure of Brain Lipid Membrane Dispersions.

This study aims to explore the variety of previously unknown morphologies that brain lipids form in aqueous solutions. We study how these structures are dependent on cholesterol content, salt solution composition, and temperature. For this purpose, dispersions of porcine sphingomyelin with varying amounts of cholesterol as well as dispersions of porcine brain lipid extracts were investigated. We used cryo-TEM to investigate the dispersions at high-salt solution content together with small-angle (SAXD) and wide-angle X-ray diffraction (WAXD) and differential scanning calorimetry (DSC) for dispersions in the corresponding salt solution at high lipid content. Sphingomyelin forms multilamellar vesicles in large excess of aqueous salt solution. These vesicles appear as double rippled bilayers in the images and as split Bragg peaks in SAXD together with a very distinct lamellar phase pattern. These features disappear with increasing temperature, and addition of cholesterol as the WAXD data shows that the peak corresponding to the chain crystallinity disappears. The dispersions of sphingomyelin at high cholesterol content form large vesicular type of structures with smooth bilayers. The repeat distance of the lamellar phase depends on temperature, salt solution composition, and slightly with cholesterol content. The brain lipid extracts form large multilamellar vesicles often attached to assemblies of higher electron density. We think that this is probably an example of supra self-assembly with a multiple-layered vesicle surrounding an interior cubic microphase. This is challenging to resolve. DSC shows the presence of different kinds of water bound to the lipid aggregates as a function of the lipid content. Comparison with the effect of lithium, sodium, and calcium salts on the structural parameters of the sphingomyelin and the morphologies of brain lipid extract morphologies demonstrate that lithium has remarkable effects also at low content.

Thermoresponsive Glycopolymers Based on Enzymatically Synthesized Oligo-ß-Mannosyl Ethyl Methacrylates and N-Isopropylacrylamide.

We present here a series of thermoresponsive glycopolymers in the form of poly(N-isopropylacrylamide)-co-(2-[ß-manno[oligo]syloxy] ethyl methacrylate)s. These copolymers were prepared from oligo-ß-mannosyl ethyl methacrylates that were synthesized through enzymatic catalysis, and were subsequently investigated with respect to their aggregation and phase behavior in aqueous solution using a combination of 1H NMR spectroscopy, dynamic light scattering, cryogenic transmission electron microscopy (TEM), and small-angle X-ray scattering (SAXS). The thermoresponsive glycopolymers were prepared by conventional free radical copolymerization of different mixtures of 2-(ß-manno[oligo]syloxy)ethyl methacrylates (with either one or two saccharide units) and N-isopropylacrylamide (NIPAm). The results showed that below the lower critical solution temperature (LCST) of poly(NIPAm), the glycopolymers readily aggregate into nanoscale structures, partly due to the presence of the saccharide moieties. Above the LCST of poly(NIPAm), the glycopolymers rearrange into a heterogeneous mixture of fractal and disc/globular aggregates. Cryo-TEM and SAXS data demonstrated that the presence of the pendant ß-mannosyl moieties in the glycopolymers induces a gradual conformational change over a wide temperature range. Even though the onset of this transition is not different from the LCST of poly(NIPAm), the gradual conformational change offers a variation of the temperature-dependent properties in comparison to poly(NIPAm), which displays a sharp coil-to-globule transition. Importantly, the compacted form of the glycopolymers shows a larger colloidal stability compared to the unmodified poly(NIPAm). In addition, the thermoresponsiveness can be conveniently tuned by varying the sugar unit-length and the oligo-ß-mannosyl ethyl methacrylate content.