A Glycolipid-Peptide-Hapten Tricomponent Conjugate Vaccine Generates Durable Antihapten Antibody Responses in Mice.

Eliciting an antihapten antibody response to vaccination typically requires the use of constructs where multiple copies of the hapten are covalently attached to a larger carrier molecule. The carrier is required to elicit T cell help via presentation of peptide epitopes on major histocompatibility complex (MHC) class II molecules; as such, attachment to full-sized proteins, alone or in a complex, is generally used to account for the significant MHC diversity in humans. While such carrier-based vaccines have proven extremely successful, particularly in protecting against bacterial diseases, they can be challenging to manufacture, and repeated use can be compromised by pre-existing immunity against the carrier. One approach to reducing these complications is to recruit help from type I natural killer T (NKT) cells, which exhibit limited diversity in their antigen receptors and respond to glycolipid antigens presented by the highly conserved presenting molecule CD1d. Synthetic vaccines for universal use can, therefore, be prepared by conjugating haptens to an NKT cell agonist such as α-galactosylceramide (αGalCer, KRN7000). An additional advantage is that the quality of NKT cell help is sufficient to overcome the need for an extra immune adjuvant. However, while initial studies with αGalCer-hapten conjugate vaccines report strong and rapid antihapten antibody responses, they can fail to generate lasting memory. Here, we show that antibody responses to the hapten 4-hydoxy-3-nitrophenyl acetyl (NP) can be improved through additional attachment of a fusion peptide containing a promiscuous helper T cell epitope (Pan DR epitope, PADRE) that binds diverse MHC class II molecules. Such αGalCer-hapten-peptide tricomponent vaccines generate strong and sustained anti-NP antibody titers with increased hapten affinity compared to vaccines without the helper epitope. The tricomponent vaccine platform is therefore suitable for further exploration in the pursuit of efficacious antihapten immunotherapies.

Alpha-galactosylceramide improves the potency of mRNA LNP vaccines against cancer and intracellular bacteria.

Although various types of mRNA-based vaccines have been explored, the optimal conditions for induction of both humoral and cellular immunity remain rather unknown. In this study, mRNA vaccines of nucleoside-modified mRNA in lipoplexes (LPXs) or lipid nanoparticles (LNPs) were evaluated after administration in mice through different routes, assessing mRNA delivery, tolerability and immunogenicity. In addition, we investigated whether mRNA vaccines could benefit from the inclusion of the adjuvant alpha-galactosylceramide (αGC), an invariant Natural Killer T (iNKT) cell ligand. Intramuscular (IM) vaccination with ovalbumin (OVA)-encoding mRNA encapsulated in LNPs adjuvanted with αGC showed the highest antibody- and CD8+ T cell responses. Furthermore, we observed that addition of signal peptides and endocytic sorting signals of either LAMP1 or HLA-B7 in the OVA-encoding mRNA sequence further enhanced CD8+ T cell activation although reducing the induction of IgG antibody responses. Moreover, mRNA LNPs with the ionizable lipidoid C12-200 exhibited higher pro-inflammatory- and reactogenic activity compared to mRNA LNPs with SM-102, correlating with increased T cell activation and antitumor potential. We also observed that αGC could further enhance the cellular immunity of clinically relevant mRNA LNP vaccines, thereby promoting therapeutic antitumor potential. Finally, a Listeria monocytogenes mRNA LNP vaccine supplemented with αGC showed synergistic protective effects against listeriosis, highlighting a key advantage of co-activating iNKT cells in antibacterial mRNA vaccines. Taken together, our study offers multiple insights for optimizing the design of mRNA vaccines for disease applications, such as cancer and intracellular bacterial infections.

A natural killer T cell nanoagonist-initiated immune cascade for hepatocellular carcinoma synergistic immunotherapy.

Natural killer T (NKT) cell-mediated immunotherapy shows great promise in hepatocellular carcinoma featuring an inherent immunosuppressive microenvironment. However, targeted delivery of NKT cell agonists remains challenging. Here, we developed a hyaluronic acid (HA) modified metal organic framework (zeolitic imidazolate framework-8, ZIF-8) to encapsulate α-galactosylceramide (α-Galcer), a classic NKT cell agonist, and doxorubicin (DOX) for eliminating liver cancer, denoted as α-Galcer/DOX@ZIF-8@HA. In the tumor microenvironment (TME), these pH-responsive nano-frameworks can gradually collapse to release α-Galcer for activating NKT cells and further boosting other immune cells in order to initiate an antitumor immune cascade. Along with DOX, the released α-Galcer enabled efficient NKT cell activation in TME for synergistic immunotherapy and tumor elimination, leading to evident tumor suppression and prolonged animal survival in both subcutaneous and orthotopic liver tumor models. Manipulating NKT cell agonists into functional nano-frameworks in TME may be matched with other advanced managements applied in a wider range of cancer therapies.

Synthesis and self-assembly behaviors of α-galactosyl ceramide (1,2)-polysaccharide analogue.

α-Galactosyl ceramide (GalCer) as a glycolipid has been long used as a standard reference for positive control in natural killer T cell studies. The (1,2)-disaccharide analogue of GalCer attracts a special attention in the study of lysosomal glycolipid processing. This paper describes the synthesis and self-assembly behaviors of GalCer 1,2-polysaccharide analogue (PolyGalCer), having considered the 1,2-disaccharide analogue as a structural motif. The synthesis of PolyGalCer is performed via one-pot glycosidation technique of 1,2-linked oligogalactan exploiting chain polymerization of galactose-based cyclic sulfite as a monomer initiated with ceramide-based alcoholic aglycon. Through the concentration dependence of PolyGalCer solutions in water or in MeOH on the turbidity, it is found that PolyGalCer forms associates in both media. From the intersection points, the critical aggregation concentration (CAC) values of PolyGalCer in water and MeOH were estimated. To know the self-assembly and the thermal transition behaviors, we performed dynamic light scattering (DLS) analysis of the associates comprising PolyGalCer in water. The transmission electron microscopy observations of the aqueous sample solution indicate that the solution of PolyGalCer includes large spherical associates. The results clarify that the 1,2-galactan moiety of PolyGalCer skeleton contributes on the kinetic inhibition of large associate formation and the metamorphosis of associates.

Diversification of a Novel α-Galactosyl Ceramide Hotspot Boosts the Adjuvant Properties in Parenteral and Mucosal Vaccines.

The development of potent adjuvants is an important step for improving the performance of subunit vaccines. CD1d agonists, such as the prototypical α-galactosyl ceramide (α-GalCer), are of special interest due to their ability to activate iNKT cells and trigger rapid dendritic cell maturation and B-cell activation. Herein, we introduce a novel derivatization hotspot at the α-GalCer skeleton, namely the N-substituent at the amide bond. The multicomponent diversification of this previously unexplored glycolipid chemotype space permitted the introduction of a variety of extra functionalities that can either potentiate the adjuvant properties or serve as handles for further conjugation to antigens toward the development of self-adjuvanting vaccines. This strategy led to the discovery of compounds eliciting enhanced antigen-specific T cell stimulation and a higher antibody response when delivered by either the parenteral or the mucosal route, as compared to a known potent CD1d agonist. Notably, various functionalized α-GalCer analogues showed a more potent adjuvant effect after intranasal immunization than a PEGylated α-GalCer analogue previously optimized for this purpose. Ultimately, this work could open multiple avenues of opportunity for the use of mucosal vaccines against microbial infections.

HIV and SIV Envelope Glycoproteins Interact with Glycolipids and Lipids.

The present study demonstrates that, in addition to interacting with galactosylceramide (GalCer), HIV-1, HIV-2, and SIV envelope glycoproteins are able to interact with glucosylceramide (GlcCer), lactosylceramide (LacCer), and ceramide. These interactions were characterized by using three complementary approaches based on molecular binding and physicochemical assays. The binding assays showed that iodinated radiolabeled HIV-1 and HIV-2 glycoproteins (125I-gp) interact physically with GalCer, GlcCer, LacCer, and ceramide previously separated by thin layer chromatography (TLC) or directly coated on a flexible 96-well plate. These interactions are specific as demonstrated, on the one hand, by the dose-dependent inhibition in the presence of various dilutions of immune, but not non-immune, sera, and, on the other hand, by the absence of interaction of these glycolipids/lipids with 125I-IgG used as an unrelated control protein. These interactions were further confirmed in a physicochemical assay, based on the capacity of these glycolipids for insertion in a pre-established monomolecular film, as a model of the cell membrane, with each glycolipid/lipid. The addition of HIV envelope glycoproteins, but not ovomucoid protein used as a negative control, resulted in a rapid increase in surface pressure of the glycolipid/lipid films, thus indirectly confirming their interactions with GalCer, GlcCer, LacCer, and ceramide. In summary, we show that HIV and SIV envelope glycoproteins bind to GalCer, GlcCer, LacCer, and ceramide in a dose-dependent, saturable, and specific manner. These interactions may function as receptors of attachment in order to facilitate infection of CD4 low or negative cells or promote interactions with other receptors leading to the activation of signaling pathways or pathogenesis.

Chemical Synthesis and Molecular Interaction Analysis of α-Galactosyl Ceramide Derivatives as CD1d Ligands.

CD1d is a non-classical major histocompatibility complex (MHC) protein, responsible for lipid antigen presentation, which presents lipids to natural killer T (NKT) cells. Various CD1d lipid ligands have been reported, including microbial and endogenous glycolipids/phospholipids. Among them, an α-galactosylceramide (α-GalCer), a representative CD1d ligand, is one of the most potent ligands and its derivatives have been developed. In this chapter, the chemistry of α-GalCer and its derivatives are described with an emphasis on their chemical syntheses and molecular interaction analysis with CD1d are described.

Unique symbiont-derived sphingolipids: Dietary amino acids source branch formation.

Metabolites derived from symbionts have the potential to regulate host pathophysiological conditions, especially in the gut. In a recent issue of Nature, Oh et al. clarify unique molecular structures of α-galactosylceramides derived from B. fragilis and their immune-modulatory functions against host natural killer T (NKT) cells.

Self-Adjuvanting Lipoprotein Conjugate αGalCer-RBD Induces Potent Immunity against SARS-CoV-2 and its Variants of Concern.

Safe and effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are the best approach to successfully combat the COVID-19 pandemic. The receptor-binding domain (RBD) of the viral spike protein is a major target to develop candidate vaccines. α-Galactosylceramide (αGalCer), a potent invariant natural killer T cell (iNKT) agonist, was site-specifically conjugated to the N-terminus of the RBD to form an adjuvant-protein conjugate, which was anchored on the liposome surface. This is the first time that an iNKT cell agonist was conjugated to the protein antigen. Compared to the unconjugated RBD/αGalCer mixture, the αGalCer-RBD conjugate induced significantly stronger humoral and cellular responses. The conjugate vaccine also showed effective cross-neutralization to all variants of concern (B.1.1.7/alpha, B.1.351/beta, P.1/gamma, B.1.617.2/delta, and B.1.1.529/omicron). These results suggest that the self-adjuvanting αGalCer-RBD has great potential to be an effective COVID-19 vaccine candidate, and this strategy might be useful for designing various subunit vaccines.

Unravelling the structural complexity of glycolipids with cryogenic infrared spectroscopy.

Glycolipids are complex glycoconjugates composed of a glycan headgroup and a lipid moiety. Their modular biosynthesis creates a vast amount of diverse and often isomeric structures, which fulfill highly specific biological functions. To date, no gold-standard analytical technique can provide a comprehensive structural elucidation of complex glycolipids, and insufficient tools for isomer distinction can lead to wrong assignments. Herein we use cryogenic gas-phase infrared spectroscopy to systematically investigate different kinds of isomerism in immunologically relevant glycolipids. We show that all structural features, including isomeric glycan headgroups, anomeric configurations and different lipid moieties, can be unambiguously resolved by diagnostic spectroscopic fingerprints in a narrow spectral range. The results allow for the characterization of isomeric glycolipid mixtures and biological applications.

Preclinical Evaluation of Invariant Natural Killer T Cells Modified with CD38 or BCMA Chimeric Antigen Receptors for Multiple Myeloma.

Due to the CD1d restricted recognition of altered glycolipids, Vα24-invariant natural killer T (iNKT) cells are excellent tools for cancer immunotherapy with a significantly reduced risk for graft-versus-host disease when applied as off-the shelf-therapeutics across Human Leukocyte Antigen (HLA) barriers. To maximally harness their therapeutic potential for multiple myeloma (MM) treatment, we here armed iNKT cells with chimeric antigen receptors (CAR) directed against the MM-associated antigen CD38 and the plasma cell specific B cell maturation antigen (BCMA). We demonstrate that both CD38- and BCMA-CAR iNKT cells effectively eliminated MM cells in a CAR-dependent manner, without losing their T cell receptor (TCR)-mediated cytotoxic activity. Importantly, iNKT cells expressing either BCMA-CARs or affinity-optimized CD38-CARs spared normal hematopoietic cells and displayed a Th1-like cytokine profile, indicating their therapeutic utility. While the costimulatory domain of CD38-CARs had no influence on the cytotoxic functions of iNKT cells, CARs containing the 4-1BB domain showed a better expansion capacity. Interestingly, when stimulated only via CD1d+ dendritic cells (DCs) loaded with α-galactosylceramide (α-GalCer), both CD38- and BCMA-CAR iNKT cells expanded well, without losing their CAR- or TCR-dependent cytotoxic activities. This suggests the possibility of developing an off-the-shelf therapy with CAR iNKT cells, which might even be boostable in vivo by administration α-GalCer pulsed DCs.

The Adjuvant of α-Galactosylceramide Presented by Gold Nanoparticles Enhances Antitumor Immune Responses of MUC1 Antigen-Based Tumor Vaccines.

BACKGROUND: Therapeutic tumor vaccines are one of the most promising strategies and have attracted great attention in cancer treatment. However, most of them have shown unsatisfactory immunogenicity, there are still few available vaccines for clinical use. Therefore, there is an urgent demand to develop novel strategies to improve the immune efficacy of antitumor vaccines. PURPOSE: This study aimed to develop novel adjuvants and carriers to enhance the immune effect of MUC1 glycopeptide antigen-based antitumor vaccines. METHODS: An antitumor vaccine was developed, in which MUC1 glycopeptide was used as tumor-associated antigen, α-GalCer served as an immune adjuvant and AuNPs was a multivalent carrier. RESULTS: Immunological evaluation results indicated that the constructed vaccines enabled a significant antibody response. FACS analysis and immunofluorescence assay showed that the induced antisera exhibited a specific binding with MUC1 positive MCF-7 cells. Moreover, the induced antibody can mediate CDC to kill MCF-7 cells. Besides stimulating B cells to produce MUC1-specific antibodies, the prepared vaccines also induced MUC1-specific CTLs in vitro. Furthermore, the vaccines significantly delayed tumor development in tumor-bearing mice model. CONCLUSION: These results showed that the construction of vaccines by presenting α-GalCer adjuvant and an antigen on gold nanoparticles offers a potential strategy to improve the antitumor response in cancer immunotherapy.

Serial Stimulation of Invariant Natural Killer T Cells with Covalently Stabilized Bispecific T-cell Engagers Generates Antitumor Immunity While Avoiding Anergy.

CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong antitumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T-cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogues of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell-specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8+ cytolytic T-cell responses. The antitumor effects of iNKT-cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell-specific BiTEs activated iNKT cells without causing iNKT cell anergy or exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens. SIGNIFICANCE: Covalently stabilized conjugates that engage the antigen receptors of iNKT cells and target a tumor antigen activate potent antitumor immunity without induction of anergy or depletion of the responding iNKT cells.

Amide-Linked C4″-Saccharide Modification of KRN7000 Provides Potent Stimulation of Human Invariant NKT Cells and Anti-Tumor Immunity in a Humanized Mouse Model.

Activation of invariant natural killer T (iNKT) cells by α-galactosylceramides (α-GalCers) stimulates strong immune responses and potent anti-tumor immunity. Numerous modifications of the glycolipid structure have been assessed to derive activating ligands for these T cells with altered and potentially advantageous properties in the induction of immune responses. Here, we synthesized variants of the prototypical α-GalCer, KRN7000, with amide-linked phenyl alkane substitutions on the C4″-position of the galactose ring. We show that these variants have weak iNKT cell stimulating activity in mouse models but substantially greater activity for human iNKT cells. The most active of the C4″-amides in our study showed strong anti-tumor effects in a partially humanized mouse model for iNKT cell responses. In silico analysis suggested that the tether length and degree of flexibility of the amide substituent affected the recognition by iNKT cell antigen receptors of the C4″-amide substituted glycolipids in complex with their antigen presenting molecule CD1d. Our findings establish the use of stable C4″-amide linked additions to the sugar moiety for further exploration of the immunological effects of structural modifications of iNKT cell activating glycolipids and highlight the critical need for more accurate animal models to assess these compounds for immunotherapeutic potential in humans.

Supramolecular organization of α-galactosylceramide in pure dispersions and in cationic DODAB bilayers.

α-galactosylceramide (α-GalCer; KRN7000) strongly stimulates NKT cells. The structures of α-GalCer assemblies and of cationic DODAB bilayers containing α-GalCer were investigated by differential scanning calorimetry (DSC) and electron spin resonance (ESR) spectroscopy. Assemblies of α-GalCer have a very tightly packed gel phase, causing spin labels to cluster and display spin exchange interactions. An endothermic phase transition is observed by DSC, leading to a fluid phase. This phase transition peak disappears upon mixing with DODAB, showing that up to 9 mol% α-GalCer is miscible with the cationic lipid. ESR spectra show that α-GalCer decreases DODAB gel phase packing, resulting in a decrease of gel-fluid transition temperature and cooperativity in DSC thermograms of mixed bilayers. In contrast, α-GalCer increases the rigidity of the fluid phase. These effects are probably due to the conformation of the rigid amide bond that connects the phytosphingosine base of α-GalCer to its long and saturated acyl chain. Possibly, α-GalCer adopts a V-shaped conformation because of the perpendicular orientation of the amide bond towards the axes of the hydrocarbon chains. Apparently, the effect of the amide bond configuration is a key structural feature for the interaction between ceramide-based glycolipids and DODAB molecules, since we have previously reported a similar decrease of gel phase packing and increase in fluid phase rigidity for DODAB bilayers containing C24:1ß-glucosylceramide. Since the structure of delivery systems is critical to the biological activity of α-GalCer, this work certainly contributes to the planning and development of novel immunotherapeutic tools.

Polar functional group-containing glycolipid CD1d ligands modulate cytokine-biasing responses and prevent experimental colitis.

The MHC class I-like molecule CD1d is a nonpolymorphic antigen-presenting glycoprotein, and its ligands include glycolipids, such as α-GalCer. The complexes between CD1d and ligands activate natural killer T cells by T cell receptor recognition, leading to the secretion of various cytokines (IFN-γ, IL-4, IL-17A, etc.). Herein, we report structure-activity relationship studies of α-GalCer derivatives containing various functional groups in their lipid acyl chains. Several derivatives have been identified as potent CD1d ligands displaying higher cytokine induction levels and/or unique cytokine polarization. The studies also indicated that flexibility of the lipid moiety can affect the binding affinity, the total cytokine production level and/or cytokine biasing. Based on our immunological evaluation and investigation of physicochemical properties, we chose bisamide- and Bz amide-containing derivatives 2 and 3, and evaluated their in vivo efficacy in a DSS-induced model of ulcerative colitis. The derivative 3 that exhibits Th2- and Th17-biasing responses, demonstrated significant protective effects against intestinal inflammation in the DSS-induced model, after a single intraperitoneal injection.

Wrapping axons in mammals and Drosophila: Different lipids, same principle.

Plasma membranes of axon-wrapping glial cells develop specific cylindrical bilayer membranes that surround thin individual axons or axon bundles. Axons are wrapped with single layered glial cells in lower organisms whereas in the mammalian nervous system, axons are surrounded with a characteristic complex multilamellar myelin structure. The high content of lipids in myelin suggests that lipids play crucial roles in the structure and function of myelin. The most striking feature of myelin lipids is the high content of galactosylceramide (GalCer). Serological and genetic studies indicate that GalCer plays a key role in the formation and function of the myelin sheath in mammals. In contrast to mammals, Drosophila lacks GalCer. Instead of GalCer, ceramide phosphoethanolamine (CPE) has an important role to ensheath axons with glial cells in Drosophila. GalCer and CPE share similar physical properties: both lipids have a high phase transition temperature and high packing, are immiscible with cholesterol and form helical liposomes. These properties are caused by both the strong headgroup interactions and the tight packing resulting from the small size of the headgroup and the hydrogen bonds between lipid molecules. These results suggest that mammals and Drosophila wrap axons using different lipids but the same conserved principle.

Immunological Evaluation of Co-Assembling a Lipidated Peptide Antigen and Lipophilic Adjuvants: Self-Adjuvanting Anti-Breast-Cancer Vaccine Candidates.

Co-assembling vaccines composed of a lipidated HER2-derived antigenic CH401 peptide and either a lipophilic adjuvant, Pam3 CSK4 , α-GalCer, or lipid A 506, were evaluated as breast cancer vaccine candidates. This vaccine design was aimed to inherit both antigen multivalency and antigen-specific immunostimulation properties, observed in reported self-adjuvanting vaccine candidates, by using self-assembly and adjuvant-conjugated antigens. Under vaccination concentrations, respective lipophilic adjuvants underwent co-assembly with lipidated CH401, which boosted the anti-CH401 IgG and IgM production. In particular, α-GalCer was responsible for the most significant immune activation. Therefore, the newly developed vaccine design enabled the optimization of adjuvants against the antigenic CH401 peptide in a simple preparatory manner. Overall, the co-assembling vaccine design opens the door for efficient and practical self-adjuvanting vaccine development.

Vaccines adjuvanted with an NKT cell agonist induce effective T-cell responses in models of CNS lymphoma.

Aim: The efficacy of anti-lymphoma vaccines that exploit the cellular adjuvant properties of activated natural killer T (NKT) cells were examined in mouse models of CNS lymphoma. Materials & methods: Vaccines were prepared by either loading the NKT cell agonist, α-galactosylceramide onto irradiated and heat-shocked B- and T-lymphoma cells, or chemically conjugating α-galactosylceramide to MHC-binding peptides from a lymphoma-associated antigen. Vaccine efficacy was analyzed in mice bearing intracranial tumors. Results: Both forms of vaccine proved to be effective in preventing lymphoma engraftment through activity of T cells that accessed the CNS. Established lymphoma was harder to treat with responses constrained by Tregs, but this could be overcome by depleting Tregs prior to vaccination. Conclusion: Simply designed NKT cell-activating vaccines enhance T-cell responses and have the potential to protect against CNS lymphoma development or prevent CNS relapse. To be effective against established CNS lymphoma, vaccines need to be combined with Treg suppression.

Thermodynamic and Structural Behavior of α-Galactosylceramide and C6-Functionalized α-GalCer in 2D Layers at the Air-Liquid Interface.

α-Galactosylceramide (α-GalCer; KRN7000) is a ligand for the glycoprotein CD1d that presents lipid antigens to natural killer T cells. Therefore, KRN7000 as well as some modified versions thereof have been widely investigated as part of novel immunotherapies. To examine the impact of structural modification, we investigated KRN7000 and C6-modified KRN7000 at the air-liquid interface using monolayer isotherms, BAM, IRRAS, GIXD, and TRXF. The amino group has no influence on the highly ordered sub-gel structures found at lateral pressures relevant for biological membranes. Neither lateral compression nor the protonation state of the amino group has a measurable effect on the lattice structure, which is defined by strong and rigid intermolecular hydrogen bonds. However, the first-order phase transition found for the C6-functionalized α-GalCer is connected with an extraordinary surface-inhibited nucleation. Our study demonstrates that KRN7000 can be functionalized at C6 without significantly changing the structural properties.