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Investigating the molecular conformations of monoclonal antibodies (mAbs) adsorbed at the solid/liquid interface is crucial for understanding mAb solution stability and advancing the development of mAb-based biosensors. This study examines the pH-dependent conformational plasticity of a human IgG1k mAb, COE-3, at the SiO 2 /water interface under varying pH conditions (pH 5.5 and 9). By integrating neutron reflectivity (NR) and molecular dynamics (MD) simulations, we reveal that the mAb irreversibly deposits onto the interface at pH 5.5, with surface density saturation reached at 20 ppm bulk concentration. At pH 5.5, the adsorbed mAb adopts a stable "flat-on" orientation, while at pH 9, it assumes a more flexible conformation and a "tilted" orientation. This pH-dependent orientation shift is reversible and influenced by the distinct surface charge properties of the Fab and Fc fragments, with the Fc fragment more prone to desorption at higher pH. The root-mean-square deviation (RMSD) analysis further shows that COE-3 maintains structural stability upon adsorption across both pH levels, showing minimal unfolding or denaturation. These findings highlight how pH-dependent electrostatic interactions between mAb fragments and the SiO 2 interface drive conformational adjustments in the intact mAb, offering insights into adsorption-induced aggregation and suggesting pH modulation as a mechanism for controlling biosensor efficiency.

The physical stability of a monoclonal antibody (mAb) solution for injection in a prefilled syringe may in part depend on its behavior at the silicone oil/water interface. Here, the adsorption of a mAb (termed COE-3) and its fragment antigen-binding (Fab) and crystallizable (Fc) at the oil/water interface was measured using neutron reflection. A 1.4 ± 0.1 μm hexadecane oil film was formed on a sapphire block by a spin-freeze-thaw process, retaining its integrity upon contact with the protein solutions. Measurements revealed that adsorbed COE-3 and its Fab and Fc fragments retained their globular structure, forming layers that did not penetrate substantially into the oil phase. COE-3 and Fc were found to adsorb flat-on to the interface, with denser 45 and 42 Å inner layers, respectively, in contact with the oil and a more diffuse 17-21 Å outer layer caused by fragments adsorbing in a tilted manner. In contrast, Fab fragments formed a uniform 60 Å monolayer. Monolayers were formed under all conditions studied (10-200 ppm, using three isotopic contrasts), although changes in packing density across the COE-3 and Fc layers were observed. COE-3 had a higher affinity to the interface than either of its constituent fragments, while Fab had a lower interfacial affinity consistent with its higher net surface charge. This study extends the application of high-resolution neutron reflection measurements to the study of protein adsorption at the oil/water interface using an experimental setup mimicking the protein drug product in a siliconized prefilled syringe.

During the formulation of therapeutic monoclonal antibodies (mAbs), nonionic surfactants are commonly added to attenuate structural rearrangement caused by adsorption/desorption at interfaces during processing, shipping, and storage. We examined the adsorption of a mAb (COE-3) at the SiO 2 /water interface in the presence of pentaethylene glycol monododecyl ether (C 12 E 5 ), polysorbate 80 (PS80-20EO), and a polysorbate 80 analogue with seven ethoxylates (PS80-7EO). Spectroscopic ellipsometry was used to follow COE-3 dynamic adsorption, and neutron reflection was used to determine interfacial structure and composition. Neither PS80-20EO nor C 12 E 5 had a notable affinity for COE-3 or the interface under the conditions studied and thus did not prevent COE-3 adsorption. In contrast, PS80-7EO did coadsorb but did not influence the dynamic process or the equilibrated amount of absorbed COE-3. Near equilibration, COE-3 underwent structural rearrangement and PS80-7EO started to bind the COE-3 interfacial layer and subsequently formed a well-defined surfactant bilayer via self-assembly. The resultant interfacial layer comprised an inner mAb layer of about 70 Å thickness and an outer surfactant layer of a further 70 Å, with distinct transitional regions across the mAb-surfactant and surfactant-bulk water boundaries. Once formed, such interfacial layers were very robust and worked to prevent further mAb adsorption, desorption, and structural rearrangement. Such robust interfacial layers could be anticipated to exist for formulated mAbs stored in type II glass vials; further research is required to understand the behavior of these layers for siliconized glass syringes.

The interaction of nonionic surfactant hexaethylene glycol monododecyl ether (C 12 E 6 ) with a reconstituted cuticular wheat wax film has been investigated by spectroscopic ellipsometry and neutron reflection (NR) to help understand the role of the leaf wax barrier during pesticide uptake, focusing on the mimicry of the actions adjuvants impose on the physical integrity and transport of the cuticular wax films against surfactant concentration. As the C 12 E 6 concentration was increased up to the critical micelle concentration (CMC = 0.067 mM), an increasing amount of surfactant mass was deposited onto the wax film. Alongside surface adsorption, C 12 E 6 was also observed to penetrate the wax film, which is evident from the NR measurements using fully protonated and chain-deuterated surfactants. Furthermore, surfactant action upon the model wax film was found to be physically reversible below the CMC, as water rinsing could readily remove the adsorbed surfactant, leaving the wax film in its original state. Above the CMC, the detergency action of the surfactant became dominant, and a significant proportion of the wax film was removed, causing structural damage. The results thus reveal that both water and C 12 E 6 could easily penetrate the wax film throughout the concentration range measured, indicating a clear pathway for the transport of active ingredients while the removal of the wax components above the CMC must have enhanced the transport process. As the partial removal of the wax film could also expose the underlying cutaneous substrate to the environment and undermine the plant's health, this study has a broad implication to the roles of surfactants in crop care.

Chemical vapor deposition (CVD) is now a well-established method for creating monolayer graphene films. In this method, poly(methyl methacrylate) (PMMA) films are often coated onto monolayer graphene films to make them mechanically robust enough for transfer and further handling. However, it is found that PMMA is hard to remove entirely, and any residual polymers remaining can affect graphene's properties. We demonstrate here a method to determine the amount of PMMA remaining on the graphene sheet fabricated from CVD by a combined study of Raman scattering, atomic force microscopy, and neutron reflection. Neutron reflectivity is a powerful technique which is particularly sensitive to any interfacial structure, so it is able to investigate the density profile of the residual PMMA in the direction perpendicular to the graphene film surface. After the standard process of PMMA removal by acetone-IPA cleaning, we found that the remaining PMMA film could be represented as a two-layer model: an inner layer with a thickness of 17 Å and a roughness of 1 Å mixed with graphene and an outer diffuse layer with an average thickness of 31 Å and a roughness of 4 Å well mixed with water. On the basis of this model analysis, it was demonstrated that the remaining PMMA still occupied a significant fraction of the graphene film surface.

Spectroscopic ellipsometry (SE) and neutron reflection (NR) data for the adsorption of a monoclonal antibody (mAb, termed COE-3, pI 8.44) at the bare SiO 2 /water interface are compared here to the simulations based on Derjaguin-Landau-Verwey-Overbeek theory. COE-3 adsorption was characterized by an initial rapid increase in the surface-adsorbed amount (Γ) followed by a plateau. Only the initial rate of the increase in Γ was strongly correlated with the bulk concentration (0.002-0.2 mg/mL), with Γ at the plateau being about 2.2 mg/m 2 (pH 5.5). Simulations captured COE-3 adsorption at equilibrium most accurately, the point at which the outgoing flux of molecules within the adsorbed plane matched the adsorption flux. Increasing the buffer pH from 5.5 to 9 increased Γ at equilibrium to ∼3 mg/m 2 (0.02 mg/mL COE-3), revealing a dominant role for lateral repulsion between adsorbed mAb molecules. In contrast, increasing the buffer ionic strength (pH 6) reduced Γ, which was captured by simulations accounting for electrostatic screening by ions, in addition to mAb/SiO 2 attractive forces and lateral repulsion. NR data at the same bulk concentrations corroborated the SE data, albeit with slightly higher Γ due to longer adsorption times for data acquisition; for example, at pH 9, Γ was 3.6 mg/m 2 (0.02 mg/mL COE-3), equivalent to a relatively high volume fraction of 0.5. An adsorbed monolayer with a thickness of 50-52 Å was consistently determined by NR, corresponding to the short axial lengths of fragment antigen-binding and fragment crystallization and implying minimal structural perturbation. Thus, the simulations enabled a mechanistic interpretation of the experimental data of mAb adsorption at the SiO 2 /water interface.

Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

Interfaces between materials and cells play a critical role in cell biomedical applications. Here, a simple, robust, and cost-effective method is developed to fabricate patterned thermoresponsive poly(N-isopropylacrylamide-co-styrene) microgel strips on a polyethyleneimine-precoated, non-thermoresponsive cell-adherent glass coverslip. The aim is to investigate whether cell sheets could be harvested from these cell-adherent surfaces patterned with thermoresponsive strips comprised of the microgels. We hypothesize that if the cell-to-cell interaction is strong enough to retain the whole cell sheet from disintegration, the cell segments growing on the thermoresponsive strips may drag the cell segments growing on the cell-adherent gaps to detach, ending with a whole freestanding and transferable cell sheet. Critical value concerning the width of the thermoresponsive strip and its ratio to the non-thermoresponsive gap may exist for cell sheet recovery from this type of surface pattern. To obtain this critical value, a series of strip patterns with various widths of thermoresponsive strip and non-thermoresponsive gap were prepared using negative microcontact printing technology, with COS7 fibroblast cells being used to test the growth and detachment. The results unraveled that COS7 cells preferentially attached and proliferated on the cell-adherent, non-thermoresponsive gaps to form patterned cell layers and that they subsequently proliferated to cover the microgel strips to form a confluent cell layer. Intact COS7 cell sheets could be recovered when the width of the thermoresponsive strip is no smaller than that of the non-thermoresponsive gap. Other cells such as HeLa, NIH3T3, 293E, and L929 could grow similarly; that is, they showed initial preference to the non-thermoresponsive gaps and then migrated to cover the entire patterned surface. However, it was difficult to detach them as cell sheets due to the weak interactions within the cell layers formed. In contrast, when COS7 and HeLa cells were cultured successively, they formed the cocultured cell layer that could be detached together. These freestanding patterned cell sheets could lead to the development of more elaborate tumor models for drug targeting and interrogation.

G(IIKK) 3 I-NH 2 has been recently shown to be highly effective at killing bacteria and inhibiting cancer cell growth while remaining benign to normal host mammalian cells. The aim of this work is to evaluate how residue substitutions of Ala (A), Val (V), Glu (E), and Lys (K) for the N-terminal Gly (G) or C-terminal Ile (I) of G(IIKK) 3 I-NH 2 affect the physiochemical properties and bioactivity of the variants. All substitutions caused the reduction of peptide hydrophobicity, while N-terminal substitutions had a less noticeable effect on the surface activity and helix-forming ability than C-terminal substitutions. N-terminal variants held potent anticancer activity but exhibited reduced hemolytic activity; these actions were related to the maintenance of their moderate surface pressures (12-16 mN m -1 ), while their hydrophobicity was reduced. Thus, N-terminal substitutions enhanced the cell selectivity of the mutants relative to the control peptide G(IIKK) 3 I-NH 2 . In contrast, C-terminal variants exhibited lower anticancer activity and much lower hemolytic activity except for G(IIKK) 3 V-NH 2 . These features were correlated well with their lower surface pressures (≤10 mN m -1 ) and decreased hydrophobicity. In spite of its very low helical content, the C-terminal variant G(IIKK) 3 V-NH 2 still displayed potent anticancer activity while retaining high hemolytic activity as well, again correlating well with its relatively high surface pressure and hydrophobicity. These results together indicated that surface activity governs the anticancer activity of the peptides, but hydrophobicity influences their hemolytic activity. In contrast, helicity appears to be poorly correlated to their bioactivity. This work has demonstrated that N-terminal modifications provide a useful strategy to optimize the anticancer activity of helical anticancer peptides (ACPs) against its potential toxicity to mammalian host cells.