Preparation and evaluation of N-acetylgalactosamine-modified hydroxypropyl-β-cyclodextrin as a potential therapeutic for MASLD/MASH

Preparation and evaluation of N-acetylgalactosamine-modified hydroxypropyl-β-cyclodextrin as a potential therapeutic for MASLD/MASH | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Preparation and evaluation of N-acetylgalactosamine-modified hydroxypropyl-β-cyclodextrin as a potential therapeutic for MASLD/MASH Rin Onaga, Toru Taharabaru, Yuto Higa, Taishi Higashi, Keiichi Motoyama This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8527688/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Metabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic liver disorder characterized by lipid accumulationthat affects approximately 30% of the global population. MASLD is classified as simple steatosis or metabolic dysfunction-associated steatohepatitis (MASH), which can progress to fibrosis, cirrhosis, and hepatocellular carcinoma. The excessive accumulation of free cholesterol in the liver is partially involved in the pathogenesis of MASH. Recently, 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) has shown therapeutic potential in lipid storage disorders such as Niemann–Pick disease type C, owing to its cholesterol-lowering effect. Therefore, HP-β-CyD is considered a potential therapeutic agent for MASLD/MASH. In this study, to enhance liver accumulation and therapeutic potential of HP-β-CyD, we prepared N -acetylgalactosamine-modified HP-β-CyD (GalNAc-HP-β-CyD) to target the asialoglycoprotein receptor (ASGPR), which is highly expressed on the hepatocellular membrane. GalNAc-HP-β-CyD was taken up by hepatocytes via ASGPR-mediated endocytosis and accumulated in the liver with greater efficiency than HP-β-CyD. Furthermore, GalNAc-HP-β-CyD reduced free cholesterol levels in cholesterol-accumulated hepatocytes. These results suggest that GalNAc-HP-β-CyD has the potential to serve as a cholesterol-lowering agent for MASLD/MASH. Cyclodextrin N-acetylgalactosamine Asialoglycoprotein receptor MASH Cholesterol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Metabolic dysfunction-associated steatotic liver disease (MASLD) is emerging as the leading chronic liver disease following the increase in obesity. MASLD is associated with various chronic diseases, such as cardiovascular disease, type 2 diabetes, and chronic kidney disease. Consequently, approximately 30% of the global population is affected by MASLD [ 1 ]. Its pathogenesis is characterized by excessive lipid accumulation in hepatocytes and is classified into simple steatosis or metabolic dysfunction-associated steatohepatitis (MASH) [ 2 ]. Simple steatosis shows negligible progression, whereas 20–30% of patients with MASLD progress to MASH, which progresses to liver injury, inflammation, and fibrosis, leading to cirrhosis, liver failure, or hepatocellular carcinoma [ 3 ]. Following the increase in obesity, the risk of hepatocellular carcinoma caused by MASH has increased sharply [ 4 ]. Furthermore, the incidence of MASH is rapidly increasing, and it is a leading indication for liver transplantation worldwide [ 5 – 8 ]. In recent years, resmetirom, a selective thyroid hormone receptor agonist, and semaglutide, a glucagon-like peptide-1 receptor agonist, have been approved as treatments for MASH with fibrosis. Both improve steatosis, lobular inflammation, and hepatocyte ballooning, thereby suppressing MASH progression. However, their efficacies in ameliorating liver fibrosis are limited [ 9 ],[ 10 ]. Therefore, the development of MASH therapeutics based on novel approaches is required. Several pieces of evidence suggest that lipid toxicity induced by hepatic free cholesterol contributes significantly to MASH progression [ 11 – 13 ]. Cholesterol accumulation is frequently observed in patients with MASH [ 14 , 15 ], and high-cholesterol diets have been reported to promote MASH progression in mice and nonhuman primates [ 16 – 18 ]. Furthermore, excessive free cholesterol accumulation within hepatocytes promotes hepatocyte death, activates hepatic macrophages and stellate cells, and contributes to MASH progression and liver fibrosis [ 19 – 21 ]. Therefore, reducing free cholesterol levels in hepatocytes may represent a potential therapeutic strategy for MASH and MASH-related liver fibrosis. Cyclodextrins (CyDs) are cyclic oligosaccharides that can incorporate hydrophobic compounds into their hydrophobic cavities to form inclusion complexes [ 22 – 26 ]. In recent years, various derivatives with enhanced functionality and biocompatibility, which are expected to serve as active pharmaceutical ingredients, have been developed [ 27 ]. Notably, 2-hydroxypropyl-β-CyD (HP-β-CyD) has been reported to reduce the lysosomal accumulation of free cholesterol in the hepatocytes of Niemann–Pick type C model mice, which are characterized by excessive cholesterol storage in lysosomes [ 28 ]. In addition, HP-β-CyD reduces excessive cholesterol accumulation in Alzheimer's disease model mice, thereby improving impaired autophagy and amyloid β degradation associated with cholesterol overload [ 29 ]. Therefore, we hypothesized that HP-β-CyD could exert a therapeutic effect on MASLD/MASH via its cholesterol-lowering effect. However, HP-β-CyD has low intracellular uptake efficiency. Furthermore, HP-β-CyD lacks liver selectivity. Therefore, providing the ability to be actively internalized into hepatocytes could be a promising strategy for its application as a therapeutic agent for MASLD/MASH. In this study, we synthesized N -acetylgalactosamine (GalNAc)-modified HP-β-CyDs (GalNAc-HP-β-CyD) to enhance hepatocyte selectivity and permeability of HP-β-CyD. As GalNAc is known to be the targeting ligand for the asialoglycoprotein receptor (ASGPR), which is highly expressed on the hepatocellular membrane [ 30 , 31 ], GalNAc-HP-β-CyD is expected to be recognized by ASGPR and selectively internalized into hepatocytes. To verify this hypothesis, in vitro cellular uptake and in vivo biodistribution of GalNAc-HP-β-CyD were evaluated. Moreover, the cholesterol-lowering effect and cytotoxicity of GalNAc-HP-β-CyD were examined in vitro to explore its feasibility as a therapeutic agent for MASH. Materials and methods Materials HP-β-CyD was donated by Nihon Shokuhin Kako (Tokyo, Japan). Galactosamine pentaacetate (GAPA) was purchased from Combi Blocks, Inc. (San Diego, CA, USA). 2-[2-(2-Chloroethoxy)ethoxy]ethanol (TEG-Cl) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC) and Hoechst 33342 were purchased from Thermo Fisher Scientific K.K. (Tokyo, Japan). Asialofetuin (AF, Type I) and RNase A were purchased from Sigma-Aldrich (St. Louis, MO, USA). Filipin III was purchased from Cayman Chemical (Ann Arbor, MI, USA). The chemicals and solvents used in this study were of analytical reagent grade. Preparation of NH-HP-β-CyD HP-β-CyD (1.0 g) was dissolved in dry dimethyl sulfoxide (DMSO; 2.0 mL), and N,N -carbonyldiimidazole (CDI; 0.98 g) was added to the solution and the mixture was stirred at room temperature for 16 h under nitrogen replacement. The solution was then added dropwise to ethylenediamine (EDA; 4.89 mL) under nitrogen replacement and stirred at room temperature for 24 h. The resulting solution was dialyzed (molecular weight cutoff [MWCO], 0.5–1.0 kDa) against water for 1 day and then lyophilized to obtain NH 2 -HP-β-CyD. Preparation of GAPA-TEG-Cl GAPA-TEG-Cl was prepared from GAPA according to the procedure reported by Pujol et al . [ 32 ] with a slight modification [ 33 ]. GAPA (9.6 g) was dissolved in dry dichloromethane (DCM; 80 mL). Molecular sieves (4 Å) and trimethylsilyl trifluoromethanesulfonate (TMSOTf; 15.12 mL) were added to the stirred solution of GAPA at 50°C, and the mixture was stirred at 50°C for 18 h. Then, triethylamine (TEA; 6.4 mL) was added to the reaction at 0°C for quenching. DCM (400 mL) was added to the solution, and the organic phase was washed with an equivalent volume of saturated aqueous NaHCO 3 solution and water. The resulting organic phase was concentrated by evaporation and dried under reduced pressure to obtain crude GAPA-oxazoline. Crude GAPA-oxazoline was dissolved in dry DCM (136 mL). Molecular sieves (4 Å), TEG-Cl (5.52 mL), and TMSOTf (2.48 mL) were added to the stirred solution of crude GAPA-oxazoline at room temperature. Then, the reaction mixture was stirred for 18 h. TEA (6.4 mL) was added to the reaction at 0°C for quenching. DCM (250 mL) was added to the solution, and the organic phase was washed with an equivalent volume of saturated aqueous NaHCO 3 solution and water. The resulting organic phase was filtered, concentrated by evaporation, and dried under reduced pressure to obtain GAPA-TEG-Cl. Preparation of GalNAc-HP-β-CyD GAPA-TEG-Cl (14.298 g), NH 2 -HP-β-CyD (1.0 g), and TEA (4.0 µL) were mixed in dry DMSO (25 mL), and stirred for 24 h at 60°C. The resulting solution was dialyzed (MWCO, 1.0 kDa) against water for 1 day to remove unreacted GAPA-TEG-Cl and TEA. Then, an aqueous NaOH solution was added to the resulting solution (final concentration: 1 N) and stirred for 2 h at 0°C to deprotect the Ac of the acetoxy (OAc) group of GAPA. Further dialysis (MWCO, 1.0 kDa against water for 1 day) was conducted, and the resulting solution was lyophilized to obtain GalNAc-HP-β-CyD. TRITC modification for fluorescent detection of CyDs NH 2 -HP-β-CyD or GalNAc-HP-β-CyD (50 mg) was dissolved in dry DMSO (2.0 mL). Then, a TRITC solution (100 µL, 5.0 mg/mL in dry DMSO) was added to the CyD solution and stirred in the dark for 1 day at room temperature. The resulting solution was dialyzed (MWCO, 1.0 kDa) against water for 1 day. The resulting solution was lyophilized to obtain TRITC-HP-β-CyD and TRITC-GalNAc-HP-β-CyD. Cell culture Human hepatocellular carcinoma–derived HepG2 cells (1.0 × 10 6 cells) or human cervical cancer–derived HeLa cells (1.0 × 10 6 cells) were suspended in culture medium (high-glucose Dulbecco's modified Eagle medium with 10% (v/v), 10 mL). The cells were cultured at 37°C under humidified 5% CO 2 . Intracellular uptake of TRITC-β-CyDs HepG2 and HeLa cells were seeded at 1.0 × 10 5 cells onto a 35-mm glass-base dish and incubated at 37°C for 24 h. The cells were washed with serum-free medium and treated with TRITC-β-CyDs (10 µM) for 24 h with or without AF (1.0 mg/mL), a competitive inhibitor of ASGPR. The cells were washed twice with serum-free medium and fixed with a 4% paraformaldehyde (PFA) solution for 10 min at room temperature. After washing the cells twice with phosphate-buffered saline (PBS), cell nuclei were stained with Hoechst 33342 (5.0 µg/mL). After washing twice with PBS, fresh PBS (1.0 mL) was added. The cells were observed using a Leica THUNDER Imager DMI8 (Leica Microsystems, Wetzlar, Germany). Flow cytometry HepG2 and HeLa cells (1.0 × 10 5 cells) were seeded in each well (24-well plate) and incubated at 37°C for 24 h. The cells were washed with serum-free medium and treated with TRITC-β-CyDs (10 µM) for 24 h with or without AF (1.0 mg/mL). The cells were washed twice with serum-free medium and detached from the plates. The cells were collected by centrifugation (3,000 rpm) and dispersed in 1.0 mL PBS containing 10% (v/v) fetal bovine serum. After filtering through a nylon mesh, data from 10,000 events were obtained using a BD Accuri C6 Plus flow cytometer (Becton Biosciences, Franklin, NJ, USA). Biodistribution of TRITC-β-CyDs The fluorescence intensities of TRITC-HP-β-CyD and TRITC-GalNAc-HP-β-CyD dissolved in PBS were measured using a Synergy H1 microplate reader (BioTek Instruments, Winooski, VT, USA), and solutions with comparable fluorescence intensities were prepared. TRITC-HP-β-CyD (20 mg/kg) or TRITC-GalNAc-HP-β-CyD (8.7 mg/kg) was subcutaneously administered to 6-week-old C57BL/6J mice (Japan SLC Inc., Shizuoka, Japan). The mice were sacrificed and perfused with PBS and a 4% PFA solution 1 h after administration. The biodistribution of TRITC-derived fluorescence in each administered group was observed using an IVIS Lumina XRMS in Vivo Imaging System (PerkinElmer, Inc., Waltham, MA, USA). Intracellular free cholesterol levels HepG2 cells and HeLa cells (1.0 × 10 5 cells) were seeded on a 35-mm glass-base dish and incubated at 37°C for 24 h. The cells were washed with serum-free medium, and U18666A (7.5 µM) was added for 24 h. Then, the cells were washed with serum-free medium and β-CyDs (5 mM) were added to each well and incubated for 48 h with or without AF (1.0 mg/mL). The cells were then fixed with a 4% PFA solution and stained with Filipin III (50 µg/mL in PBS) for 1 h at room temperature. RNase A (0.1 mg/mL in PBS) was added to each well, and then propidium iodide (5.0 µg/mL in PBS) was added and incubated for 15 min at room temperature. The cells were observed using a Leica THUNDER Imager DMI8 fluorescence microscope. Cytotoxicity HepG2 and HeLa cells (1.0 × 10 5 cells) were seeded in each well (96-well plate) and incubated at 37°C for 24 h. Then, the cells were treated with β-CyDs (0.1, 1, and 5 mM) as described above. Cell viability in each well was quantified using a Cell Counting Kit-8 solution. Data analysis Data represent means ± standard error. Scheffé’s test was used to assess statistical significance. Statistical significance was set at p < 0.05. Results and discussion Preparation of GalNAc-HP-β-CyD GalNAc-HP-β-CyD was synthesized by the reaction of GAPA-TEG-Cl with NH 2 -HP-β-CyD, followed by deprotection of the OAc groups using NaOH (Fig. 1 ). The precursor, GAPA-TEG-Cl, was prepared according to the method reported by Pujol et al . [ 32 ]. Briefly, GAPA was dissolved in DCM and activated by TMSOTf, and the crude oxazoline obtained was reacted with TEG-Cl in DCM (Fig. 1 ). The preparation of GAPA-oxazoline was confirmed by the decrease in the integral value of the -NHAc group of GAPA in proton nuclear magnetic resonance ( 1 H-NMR) spectrum and by the observation of the peaks of GAPA-oxazoline in fast atom bombardment mass spectrometry (FAB-MS) spectrum (Fig. 2 ). The fragment ion at m/z 168 was observed, which is a characteristic fragment ion of N -acetylhexosamine derivatives and may be formed by dehydration during MS analysis [ 34 , 35 ]. In addition, 1 H-NMR and FAB-MS spectra of GAPA-TEG-Cl were obtained, and the results suggested that GAPA-TEG-Cl was successfully prepared (Fig. 3 ). The hydroxyl group of HP-β-CyD was activated by CDI and reacted with excess EDA to obtain NH 2 -HP-β-CyD, another precursor of GalNAc-HP-β-CyD. The modification ratio of EDA per HP-β-CyD was determined by the integral values of 1 H-NMR peaks (Fig. 4 ) of the anomeric proton derived from HP-β-CyD (~ 5.0 ppm) and the methylene proton adjacent to the primary amine of EDA (~ 2.6 ppm). As a result, the degree of substitution (DS) of the amino groups in NH 2 -HP-β-CyD was ~ 5.2. NH 2 -HP-β-CyD and GAPA-TEG-Cl were mixed and reacted at 60°C in DMSO. Following purification by dialysis against water, the OAc groups were deprotected using NaOH, and additional dialysis was performed to obtain GalNAc-HP-β-CyD. The preparation of GalNAc-HP-β-CyD was confirmed by 1 H-NMR, and the DS of GalNAc in GalNAc-HP-β-CyD was determined to be 4.5 based on the integral values of the peaks of the anomeric protons of HP-β-CyD (~ 5.0 ppm) and GalNAc (~ 4.4 ppm). These results suggest that GalNAc-HP-β-CyD was successfully prepared. Intracellular uptake of TRITC-GalNAc-HP-β-CyD The intracellular uptake of β-CyDs by HepG2 cells was evaluated to clarify ASGPR-specific internalization of GalNAc-HP-β-CyD (Fig. 6 a, b). Cellular internalization of β-CyDs was detected by the fluorescence of TRITC-labeled β-CyD. TRITC-GalNAc-HP-β-CyD showed a significantly higher intracellular uptake efficiency in HepG2 cells than TRITC-HP-β-CyD. In addition, the intracellular uptake of TRITC-GalNAc-HP-β-CyD decreased with the addition of AF, a competitive inhibitor of ASGPR, whereas that of TRITC-HP-β-CyD remained unchanged. In contrast, TRITC-GalNAc-HP-β-CyD showed negligible cellular internalization in HeLa cells regardless of the presence or absence of AF (Fig. 6 c, d). These results suggest that GalNAc-HP-β-CyD was internalized by hepatocytes via ASGPR-mediated endocytosis. Biodistribution of TRITC-β-CyDs after subcutaneous administration To clarify the role of GalNAc in the liver accumulation of HP-β-CyD, the biodistribution of TRITC-GalNAc-HP-β-CyD and TRITC-HP-β-CyD was compared after subcutaneous administration in mice. One hour after the administration of TRITC-β-CyDs, fluorescent images of the major organs were obtained using IVIS. TRITC-GalNAc-HP-β-CyD showed markedly higher accumulation in the liver than TRITC-HP-β-CyD (Fig. 7 ). TRITC-GalNAc-HP-β-CyD also accumulated in the kidneys, probably due to renal clearance; however, the accumulation level was lower than that of TRITC-HP-β-CyD. These results indicate that GalNAc modification of HP-β-CyD enhances its accumulation in the liver and decreases its accumulation in the kidneys. Cholesterol-lowering effects of GalNAc-HP-β-CyD Free cholesterol promotes the progression of MASH/MASLD [ 11 – 13 ]. Therefore, reducing free cholesterol levels in hepatocytes may be an effective treatment strategy for MASH/MASLD. To investigate whether GalNAc-HP-β-CyD lowers free cholesterol levels in cholesterol-accumulated hepatocytes, free cholesterol in U18666A (an inhibitor of intracellular cholesterol transport)-treated HepG2 cells was stained with Filipin III after treatment with GalNAc-HP-β-CyD (Fig. 8 ). Filipin III fluorescence increased following treatment with U18666A, suggesting that cholesterol-accumulated HepG2 cells were successfully prepared. Both HP-β-CyD and GalNAc-HP-β-CyD showed a cholesterol-lowering effect in U18666A-treated HepG2 cells without cytotoxicity (Fig. 9 ). Most importantly, the cholesterol-lowering effect of GalNAc-HP-β-CyD was attenuated in the presence of AF, whereas that of HP-β-CyD was unaffected. These results suggest that GalNAc-HP-β-CyD, which is internalized by hepatocytes via ASGPR-mediated endocytosis, may lower intracellular free cholesterol. HP-β-CyD showed no such selectivity; however, its cholesterol-lowering effect was comparable to that of GalNAc-HP-β-CyD. These findings suggest that the intracellular and extracellular mechanisms underlying the cholesterol-lowering effects of these CyDs may be fundamentally different, warranting further investigation. In this study, GalNAc-HP-β-CyD was newly synthesized (Figs. 1 – 5 ) to endow HP-β-CyD with liver-targeting ability, and was evaluated as a therapeutic agent for MASLD/MASH. ASGPR is highly expressed on the surface of hepatocytes, rapidly recognizes and internalizes sugar ligands such as galactose, lactose, and GalNAc, and is subsequently re-presented and recycled on the cell membrane. Owing to its rapid internalization and representation, ASGPR is widely used for liver-targeted drug delivery [ 31 , 36 , 37 ]. Notably, GalNAc has a high binding affinity for ASGPR and has been reported to be recognized approximately 50 times more strongly than galactose [ 38 , 39 ]. Therefore, GalNAc-HP-β-CyD showed a significantly higher intracellular uptake efficiency than HP-β-CyD in HepG2 cells, which highly express ASGPR (Fig. 6 ). Furthermore, the intracellular uptake and cholesterol-lowering effect of GalNAc-HP-β-CyD were attenuated in the presence of AF (Figs. 6 , 8 ). These findings suggest that GalNAc-HP-β-CyD is internalized into HepG2 cells and exerts a cholesterol-lowering capacity in HepG2 cells. GalNAc and the triethylene glycol linker of GalNAc-HP-β-CyD are hydrophilic, and these moieties could inhibit the hydrophobic interaction between cholesterol and the cavities of HP-β-CyDs. Optimization of the linker in future studies may help improve the interaction between GalNAc-HP-β-CyDs and cholesterol. GalNAc-HP-β-CyD accumulated in the liver to a greater extent than HP-β-CyD after subcutaneous injection (Fig. 7 ). However, GalNAc-HP-β-CyD also accumulated in the kidneys, probably due to renal clearance. To avoid renal clearance and improve blood retention, polyethylene glycol modification or polymerization of GalNAc-HP-β-CyD may be useful. Furthermore, in vivo safety should be evaluated in addition to in vitro studies (Fig. 9 ). Conclusions In this study, the newly prepared GalNAc-HP-β-CyD was efficiently internalized into hepatocytes via ASGPR and accumulated in the liver to a greater extent than HP-β-CyD. Furthermore, GalNAc-HP-β-CyD reduced cholesterol accumulation in cholesterol-accumulated hepatocytes, a factor that contributes to MASH progression. These results suggest that GalNAc-HP-β-CyD may have the potential to serve as a novel therapeutic agent for MASH. Declarations Competing interests The authors have no competing interests to disclose. Funding This work was partially supported by JST SPRING (Grant Number JPMJSP2127). Ethics approval All animal experiments in this study were approved by the Ethics Committee for Animal Care and Use of Kumamoto University (Approval ID: A2024-034). Data availability No datasets were generated or analyzed during the current study. Author contributions Rin Onaga: Data curation, formal analysis, investigation, methodology, and writing of the original draft. Toru Taharabaru: Investigation, methodology, supervision, writing–original draft, and writing–review and editing. Yuto Higa: Investigation and methodology. Taishi Higashi: Methodology and supervision. Keiichi Motoyama: Conceptualization, funding acquisition, investigation, methodology, supervision, and writing–review and editing. Acknowledgements The authors thank Nihon Shokuhin Kako Co., Ltd. for providing HP-β-CyD. References Stefan, N., Yki-Järvinen, H., Neuschwander-Tetri, B.A.: Metabolic dysfunction-associated steatotic liver disease: heterogeneous pathomechanisms and effectiveness of metabolism-based treatment. 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Chem. 256 , 4929–4933 (1981) Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":135176,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation pathway of GalNAc-HP-β-CyD\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8527688/v1/9ce9f4c6bfb5ac6b6a2dfc77.png"},{"id":99884998,"identity":"9c4dc871-68f4-4bb6-92e8-7b5030bb6383","added_by":"auto","created_at":"2026-01-09 12:18:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":128831,"visible":true,"origin":"","legend":"\u003cp\u003e(a) \u003csup\u003e1\u003c/sup\u003eH-NMR and (b) FAB-MS spectra of GAPA-oxazoline. 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Solvent: D\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8527688/v1/7f1e11e8dea1e0d55b5f45c2.png"},{"id":99885006,"identity":"e4f32786-7cef-429b-a250-92e327805438","added_by":"auto","created_at":"2026-01-09 12:18:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":148281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH-NMR spectra of GalNAc-HP-β-CyD. Solvent: D\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8527688/v1/c94943a7082978f507b214c6.png"},{"id":100359084,"identity":"be061df1-f419-4376-bfa3-1a16e994271b","added_by":"auto","created_at":"2026-01-16 07:21:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":291606,"visible":true,"origin":"","legend":"\u003cp\u003eIntracellular uptake of TRITC-GalNAc-HP-β-CyD in (a, b) HepG2 and (c, d) HeLa cells. (a, c) Fluorescent microscopic images. A representative image of three independent experiments is shown. Scale bars = 50 µm. (b, d) Flow cytometric analysis (n = 3). *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e TRITC-HP-β-CyD. †\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e TRITC-GalNAc-HP-β-CyD.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8527688/v1/6ccec7d293e647dbffae9796.png"},{"id":100358742,"identity":"20b18ffc-f420-4aca-8cb2-4a66207a655d","added_by":"auto","created_at":"2026-01-16 07:21:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":187306,"visible":true,"origin":"","legend":"\u003cp\u003eBiodistribution of TRITC-HP-β-CyDsafter subcutaneous administrationin C57BL/6J mice. A representative image from three mice is shown.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8527688/v1/6fc538d7f34f035683f02885.png"},{"id":100357791,"identity":"097b4b34-3749-42da-94a2-9c7ad06242d8","added_by":"auto","created_at":"2026-01-16 07:20:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":364420,"visible":true,"origin":"","legend":"\u003cp\u003eFree cholesterol levels in U18666A-treated HepG2 cells after treatment with GalNAc-HP-β-CyD or HP-β-CyD with or without AF. A representative image from three independent experiments is shown. Scale bars = 50 µm.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8527688/v1/5e0af31e8fd916fb4a14e4d2.png"},{"id":100358856,"identity":"329f420b-c99b-46a7-b8d9-3a0f82d9d2ff","added_by":"auto","created_at":"2026-01-16 07:21:30","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":31886,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of HepG2 cells after treatment with GalNAc-HP-β-CyD or HP-β-CyD (n = 4–5). *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e Control.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8527688/v1/35637fd0bd75e5f4fcebd7b4.png"},{"id":109505556,"identity":"e0dfe24f-d8da-4d06-9797-405bf4fc7bfc","added_by":"auto","created_at":"2026-05-19 01:55:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1489123,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8527688/v1/4451271b-5ed4-4dfc-b11b-2ede60a9ca4f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preparation and evaluation of N-acetylgalactosamine-modified hydroxypropyl-β-cyclodextrin as a potential therapeutic for MASLD/MASH","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMetabolic dysfunction-associated steatotic liver disease (MASLD) is emerging as the leading chronic liver disease following the increase in obesity. MASLD is associated with various chronic diseases, such as cardiovascular disease, type 2 diabetes, and chronic kidney disease. Consequently, approximately 30% of the global population is affected by MASLD [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Its pathogenesis is characterized by excessive lipid accumulation in hepatocytes and is classified into simple steatosis or metabolic dysfunction-associated steatohepatitis (MASH) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Simple steatosis shows negligible progression, whereas 20\u0026ndash;30% of patients with MASLD progress to MASH, which progresses to liver injury, inflammation, and fibrosis, leading to cirrhosis, liver failure, or hepatocellular carcinoma [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Following the increase in obesity, the risk of hepatocellular carcinoma caused by MASH has increased sharply [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, the incidence of MASH is rapidly increasing, and it is a leading indication for liver transplantation worldwide [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent years, resmetirom, a selective thyroid hormone receptor agonist, and semaglutide, a glucagon-like peptide-1 receptor agonist, have been approved as treatments for MASH with fibrosis. Both improve steatosis, lobular inflammation, and hepatocyte ballooning, thereby suppressing MASH progression. However, their efficacies in ameliorating liver fibrosis are limited [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e],[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, the development of MASH therapeutics based on novel approaches is required.\u003c/p\u003e \u003cp\u003eSeveral pieces of evidence suggest that lipid toxicity induced by hepatic free cholesterol contributes significantly to MASH progression [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Cholesterol accumulation is frequently observed in patients with MASH [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and high-cholesterol diets have been reported to promote MASH progression in mice and nonhuman primates [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, excessive free cholesterol accumulation within hepatocytes promotes hepatocyte death, activates hepatic macrophages and stellate cells, and contributes to MASH progression and liver fibrosis [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Therefore, reducing free cholesterol levels in hepatocytes may represent a potential therapeutic strategy for MASH and MASH-related liver fibrosis.\u003c/p\u003e \u003cp\u003eCyclodextrins (CyDs) are cyclic oligosaccharides that can incorporate hydrophobic compounds into their hydrophobic cavities to form inclusion complexes [\u003cspan additionalcitationids=\"CR23 CR24 CR25\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In recent years, various derivatives with enhanced functionality and biocompatibility, which are expected to serve as active pharmaceutical ingredients, have been developed [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Notably, 2-hydroxypropyl-β-CyD (HP-β-CyD) has been reported to reduce the lysosomal accumulation of free cholesterol in the hepatocytes of Niemann\u0026ndash;Pick type C model mice, which are characterized by excessive cholesterol storage in lysosomes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In addition, HP-β-CyD reduces excessive cholesterol accumulation in Alzheimer's disease model mice, thereby improving impaired autophagy and amyloid β degradation associated with cholesterol overload [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, we hypothesized that HP-β-CyD could exert a therapeutic effect on MASLD/MASH via its cholesterol-lowering effect. However, HP-β-CyD has low intracellular uptake efficiency. Furthermore, HP-β-CyD lacks liver selectivity. Therefore, providing the ability to be actively internalized into hepatocytes could be a promising strategy for its application as a therapeutic agent for MASLD/MASH.\u003c/p\u003e \u003cp\u003eIn this study, we synthesized \u003cem\u003eN\u003c/em\u003e-acetylgalactosamine (GalNAc)-modified HP-β-CyDs (GalNAc-HP-β-CyD) to enhance hepatocyte selectivity and permeability of HP-β-CyD. As GalNAc is known to be the targeting ligand for the asialoglycoprotein receptor (ASGPR), which is highly expressed on the hepatocellular membrane [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], GalNAc-HP-β-CyD is expected to be recognized by ASGPR and selectively internalized into hepatocytes. To verify this hypothesis, \u003cem\u003ein vitro\u003c/em\u003e cellular uptake and \u003cem\u003ein vivo\u003c/em\u003e biodistribution of GalNAc-HP-β-CyD were evaluated. Moreover, the cholesterol-lowering effect and cytotoxicity of GalNAc-HP-β-CyD were examined \u003cem\u003ein vitro\u003c/em\u003e to explore its feasibility as a therapeutic agent for MASH.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eHP-β-CyD was donated by Nihon Shokuhin Kako (Tokyo, Japan). Galactosamine pentaacetate (GAPA) was purchased from Combi Blocks, Inc. (San Diego, CA, USA). 2-[2-(2-Chloroethoxy)ethoxy]ethanol (TEG-Cl) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC) and Hoechst 33342 were purchased from Thermo Fisher Scientific K.K. (Tokyo, Japan). Asialofetuin (AF, Type I) and RNase A were purchased from Sigma-Aldrich (St. Louis, MO, USA). Filipin III was purchased from Cayman Chemical (Ann Arbor, MI, USA). The chemicals and solvents used in this study were of analytical reagent grade.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of NH-HP-β-CyD\u003c/h3\u003e\n\u003cp\u003eHP-β-CyD (1.0 g) was dissolved in dry dimethyl sulfoxide (DMSO; 2.0 mL), and \u003cem\u003eN,N\u003c/em\u003e-carbonyldiimidazole (CDI; 0.98 g) was added to the solution and the mixture was stirred at room temperature for 16 h under nitrogen replacement. The solution was then added dropwise to ethylenediamine (EDA; 4.89 mL) under nitrogen replacement and stirred at room temperature for 24 h. The resulting solution was dialyzed (molecular weight cutoff [MWCO], 0.5\u0026ndash;1.0 kDa) against water for 1 day and then lyophilized to obtain NH\u003csub\u003e2\u003c/sub\u003e-HP-β-CyD.\u003c/p\u003e\n\u003ch3\u003ePreparation of GAPA-TEG-Cl\u003c/h3\u003e\n\u003cp\u003eGAPA-TEG-Cl was prepared from GAPA according to the procedure reported by Pujol \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] with a slight modification [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. GAPA (9.6 g) was dissolved in dry dichloromethane (DCM; 80 mL). Molecular sieves (4 \u0026Aring;) and trimethylsilyl trifluoromethanesulfonate (TMSOTf; 15.12 mL) were added to the stirred solution of GAPA at 50\u0026deg;C, and the mixture was stirred at 50\u0026deg;C for 18 h. Then, triethylamine (TEA; 6.4 mL) was added to the reaction at 0\u0026deg;C for quenching. DCM (400 mL) was added to the solution, and the organic phase was washed with an equivalent volume of saturated aqueous NaHCO\u003csub\u003e3\u003c/sub\u003e solution and water. The resulting organic phase was concentrated by evaporation and dried under reduced pressure to obtain crude GAPA-oxazoline.\u003c/p\u003e \u003cp\u003eCrude GAPA-oxazoline was dissolved in dry DCM (136 mL). Molecular sieves (4 \u0026Aring;), TEG-Cl (5.52 mL), and TMSOTf (2.48 mL) were added to the stirred solution of crude GAPA-oxazoline at room temperature. Then, the reaction mixture was stirred for 18 h. TEA (6.4 mL) was added to the reaction at 0\u0026deg;C for quenching. DCM (250 mL) was added to the solution, and the organic phase was washed with an equivalent volume of saturated aqueous NaHCO\u003csub\u003e3\u003c/sub\u003e solution and water. The resulting organic phase was filtered, concentrated by evaporation, and dried under reduced pressure to obtain GAPA-TEG-Cl.\u003c/p\u003e\n\u003ch3\u003ePreparation of GalNAc-HP-β-CyD\u003c/h3\u003e\n\u003cp\u003eGAPA-TEG-Cl (14.298 g), NH\u003csub\u003e2\u003c/sub\u003e-HP-β-CyD (1.0 g), and TEA (4.0 \u0026micro;L) were mixed in dry DMSO (25 mL), and stirred for 24 h at 60\u0026deg;C. The resulting solution was dialyzed (MWCO, 1.0 kDa) against water for 1 day to remove unreacted GAPA-TEG-Cl and TEA. Then, an aqueous NaOH solution was added to the resulting solution (final concentration: 1 N) and stirred for 2 h at 0\u0026deg;C to deprotect the Ac of the acetoxy (OAc) group of GAPA. Further dialysis (MWCO, 1.0 kDa against water for 1 day) was conducted, and the resulting solution was lyophilized to obtain GalNAc-HP-β-CyD.\u003c/p\u003e\n\u003ch3\u003eTRITC modification for fluorescent detection of CyDs\u003c/h3\u003e\n\u003cp\u003eNH\u003csub\u003e2\u003c/sub\u003e-HP-β-CyD or GalNAc-HP-β-CyD (50 mg) was dissolved in dry DMSO (2.0 mL). Then, a TRITC solution (100 \u0026micro;L, 5.0 mg/mL in dry DMSO) was added to the CyD solution and stirred in the dark for 1 day at room temperature. The resulting solution was dialyzed (MWCO, 1.0 kDa) against water for 1 day. The resulting solution was lyophilized to obtain TRITC-HP-β-CyD and TRITC-GalNAc-HP-β-CyD.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHuman hepatocellular carcinoma\u0026ndash;derived HepG2 cells (1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) or human cervical cancer\u0026ndash;derived HeLa cells (1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) were suspended in culture medium (high-glucose Dulbecco's modified Eagle medium with 10% (v/v), 10 mL). The cells were cultured at 37\u0026deg;C under humidified 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIntracellular uptake of TRITC-β-CyDs\u003c/h3\u003e\n\u003cp\u003eHepG2 and HeLa cells were seeded at 1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells onto a 35-mm glass-base dish and incubated at 37\u0026deg;C for 24 h. The cells were washed with serum-free medium and treated with TRITC-β-CyDs (10 \u0026micro;M) for 24 h with or without AF (1.0 mg/mL), a competitive inhibitor of ASGPR. The cells were washed twice with serum-free medium and fixed with a 4% paraformaldehyde (PFA) solution for 10 min at room temperature. After washing the cells twice with phosphate-buffered saline (PBS), cell nuclei were stained with Hoechst 33342 (5.0 \u0026micro;g/mL). After washing twice with PBS, fresh PBS (1.0 mL) was added. The cells were observed using a Leica THUNDER Imager DMI8 (Leica Microsystems, Wetzlar, Germany).\u003c/p\u003e\n\u003ch3\u003eFlow cytometry\u003c/h3\u003e\n\u003cp\u003eHepG2 and HeLa cells (1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) were seeded in each well (24-well plate) and incubated at 37\u0026deg;C for 24 h. The cells were washed with serum-free medium and treated with TRITC-β-CyDs (10 \u0026micro;M) for 24 h with or without AF (1.0 mg/mL). The cells were washed twice with serum-free medium and detached from the plates. The cells were collected by centrifugation (3,000 rpm) and dispersed in 1.0 mL PBS containing 10% (v/v) fetal bovine serum. After filtering through a nylon mesh, data from 10,000 events were obtained using a BD Accuri C6 Plus flow cytometer (Becton Biosciences, Franklin, NJ, USA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBiodistribution of TRITC-β-CyDs\u003c/h2\u003e \u003cp\u003eThe fluorescence intensities of TRITC-HP-β-CyD and TRITC-GalNAc-HP-β-CyD dissolved in PBS were measured using a Synergy H1 microplate reader (BioTek Instruments, Winooski, VT, USA), and solutions with comparable fluorescence intensities were prepared. TRITC-HP-β-CyD (20 mg/kg) or TRITC-GalNAc-HP-β-CyD (8.7 mg/kg) was subcutaneously administered to 6-week-old C57BL/6J mice (Japan SLC Inc., Shizuoka, Japan). The mice were sacrificed and perfused with PBS and a 4% PFA solution 1 h after administration. The biodistribution of TRITC-derived fluorescence in each administered group was observed using an IVIS Lumina XRMS \u003cem\u003ein Vivo\u003c/em\u003e Imaging System (PerkinElmer, Inc., Waltham, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular free cholesterol levels\u003c/h2\u003e \u003cp\u003eHepG2 cells and HeLa cells (1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) were seeded on a 35-mm glass-base dish and incubated at 37\u0026deg;C for 24 h. The cells were washed with serum-free medium, and U18666A (7.5 \u0026micro;M) was added for 24 h. Then, the cells were washed with serum-free medium and β-CyDs (5 mM) were added to each well and incubated for 48 h with or without AF (1.0 mg/mL). The cells were then fixed with a 4% PFA solution and stained with Filipin III (50 \u0026micro;g/mL in PBS) for 1 h at room temperature. RNase A (0.1 mg/mL in PBS) was added to each well, and then propidium iodide (5.0 \u0026micro;g/mL in PBS) was added and incubated for 15 min at room temperature. The cells were observed using a Leica THUNDER Imager DMI8 fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity\u003c/h2\u003e \u003cp\u003eHepG2 and HeLa cells (1.0 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells) were seeded in each well (96-well plate) and incubated at 37\u0026deg;C for 24 h. Then, the cells were treated with β-CyDs (0.1, 1, and 5 mM) as described above. Cell viability in each well was quantified using a Cell Counting Kit-8 solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eData represent means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Scheff\u0026eacute;\u0026rsquo;s test was used to assess statistical significance. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of GalNAc-HP-β-CyD\u003c/h2\u003e \u003cp\u003eGalNAc-HP-β-CyD was synthesized by the reaction of GAPA-TEG-Cl with NH\u003csub\u003e2\u003c/sub\u003e-HP-β-CyD, followed by deprotection of the OAc groups using NaOH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The precursor, GAPA-TEG-Cl, was prepared according to the method reported by Pujol \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Briefly, GAPA was dissolved in DCM and activated by TMSOTf, and the crude oxazoline obtained was reacted with TEG-Cl in DCM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The preparation of GAPA-oxazoline was confirmed by the decrease in the integral value of the -NHAc group of GAPA in proton nuclear magnetic resonance (\u003csup\u003e1\u003c/sup\u003eH-NMR) spectrum and by the observation of the peaks of GAPA-oxazoline in fast atom bombardment mass spectrometry (FAB-MS) spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The fragment ion at \u003cem\u003em/z\u003c/em\u003e 168 was observed, which is a characteristic fragment ion of \u003cem\u003eN\u003c/em\u003e-acetylhexosamine derivatives and may be formed by dehydration during MS analysis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In addition, \u003csup\u003e1\u003c/sup\u003eH-NMR and FAB-MS spectra of GAPA-TEG-Cl were obtained, and the results suggested that GAPA-TEG-Cl was successfully prepared (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe hydroxyl group of HP-β-CyD was activated by CDI and reacted with excess EDA to obtain NH\u003csub\u003e2\u003c/sub\u003e-HP-β-CyD, another precursor of GalNAc-HP-β-CyD. The modification ratio of EDA per HP-β-CyD was determined by the integral values of \u003csup\u003e1\u003c/sup\u003eH-NMR peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) of the anomeric proton derived from HP-β-CyD (~\u0026thinsp;5.0 ppm) and the methylene proton adjacent to the primary amine of EDA (~\u0026thinsp;2.6 ppm). As a result, the degree of substitution (DS) of the amino groups in NH\u003csub\u003e2\u003c/sub\u003e-HP-β-CyD was ~\u0026thinsp;5.2.\u003c/p\u003e \u003cp\u003eNH\u003csub\u003e2\u003c/sub\u003e-HP-β-CyD and GAPA-TEG-Cl were mixed and reacted at 60\u0026deg;C in DMSO. Following purification by dialysis against water, the OAc groups were deprotected using NaOH, and additional dialysis was performed to obtain GalNAc-HP-β-CyD. The preparation of GalNAc-HP-β-CyD was confirmed by \u003csup\u003e1\u003c/sup\u003eH-NMR, and the DS of GalNAc in GalNAc-HP-β-CyD was determined to be 4.5 based on the integral values of the peaks of the anomeric protons of HP-β-CyD (~\u0026thinsp;5.0 ppm) and GalNAc (~\u0026thinsp;4.4 ppm). These results suggest that GalNAc-HP-β-CyD was successfully prepared.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular uptake of TRITC-GalNAc-HP-β-CyD\u003c/h2\u003e \u003cp\u003eThe intracellular uptake of β-CyDs by HepG2 cells was evaluated to clarify ASGPR-specific internalization of GalNAc-HP-β-CyD (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). Cellular internalization of β-CyDs was detected by the fluorescence of TRITC-labeled β-CyD.\u003c/p\u003e \u003cp\u003eTRITC-GalNAc-HP-β-CyD showed a significantly higher intracellular uptake efficiency in HepG2 cells than TRITC-HP-β-CyD. In addition, the intracellular uptake of TRITC-GalNAc-HP-β-CyD decreased with the addition of AF, a competitive inhibitor of ASGPR, whereas that of TRITC-HP-β-CyD remained unchanged.\u003c/p\u003e \u003cp\u003eIn contrast, TRITC-GalNAc-HP-β-CyD showed negligible cellular internalization in HeLa cells regardless of the presence or absence of AF (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d). These results suggest that GalNAc-HP-β-CyD was internalized by hepatocytes via ASGPR-mediated endocytosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBiodistribution of TRITC-β-CyDs after subcutaneous administration\u003c/h2\u003e \u003cp\u003eTo clarify the role of GalNAc in the liver accumulation of HP-β-CyD, the biodistribution of TRITC-GalNAc-HP-β-CyD and TRITC-HP-β-CyD was compared after subcutaneous administration in mice.\u003c/p\u003e \u003cp\u003eOne hour after the administration of TRITC-β-CyDs, fluorescent images of the major organs were obtained using IVIS. TRITC-GalNAc-HP-β-CyD showed markedly higher accumulation in the liver than TRITC-HP-β-CyD (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). TRITC-GalNAc-HP-β-CyD also accumulated in the kidneys, probably due to renal clearance; however, the accumulation level was lower than that of TRITC-HP-β-CyD. These results indicate that GalNAc modification of HP-β-CyD enhances its accumulation in the liver and decreases its accumulation in the kidneys.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCholesterol-lowering effects of GalNAc-HP-β-CyD\u003c/h2\u003e \u003cp\u003eFree cholesterol promotes the progression of MASH/MASLD [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, reducing free cholesterol levels in hepatocytes may be an effective treatment strategy for MASH/MASLD. To investigate whether GalNAc-HP-β-CyD lowers free cholesterol levels in cholesterol-accumulated hepatocytes, free cholesterol in U18666A (an inhibitor of intracellular cholesterol transport)-treated HepG2 cells was stained with Filipin III after treatment with GalNAc-HP-β-CyD (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFilipin III fluorescence increased following treatment with U18666A, suggesting that cholesterol-accumulated HepG2 cells were successfully prepared. Both HP-β-CyD and GalNAc-HP-β-CyD showed a cholesterol-lowering effect in U18666A-treated HepG2 cells without cytotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Most importantly, the cholesterol-lowering effect of GalNAc-HP-β-CyD was attenuated in the presence of AF, whereas that of HP-β-CyD was unaffected. These results suggest that GalNAc-HP-β-CyD, which is internalized by hepatocytes via ASGPR-mediated endocytosis, may lower intracellular free cholesterol. HP-β-CyD showed no such selectivity; however, its cholesterol-lowering effect was comparable to that of GalNAc-HP-β-CyD. These findings suggest that the intracellular and extracellular mechanisms underlying the cholesterol-lowering effects of these CyDs may be fundamentally different, warranting further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this study, GalNAc-HP-β-CyD was newly synthesized (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) to endow HP-β-CyD with liver-targeting ability, and was evaluated as a therapeutic agent for MASLD/MASH. ASGPR is highly expressed on the surface of hepatocytes, rapidly recognizes and internalizes sugar ligands such as galactose, lactose, and GalNAc, and is subsequently re-presented and recycled on the cell membrane. Owing to its rapid internalization and representation, ASGPR is widely used for liver-targeted drug delivery [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Notably, GalNAc has a high binding affinity for ASGPR and has been reported to be recognized approximately 50 times more strongly than galactose [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, GalNAc-HP-β-CyD showed a significantly higher intracellular uptake efficiency than HP-β-CyD in HepG2 cells, which highly express ASGPR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Furthermore, the intracellular uptake and cholesterol-lowering effect of GalNAc-HP-β-CyD were attenuated in the presence of AF (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These findings suggest that GalNAc-HP-β-CyD is internalized into HepG2 cells and exerts a cholesterol-lowering capacity in HepG2 cells. GalNAc and the triethylene glycol linker of GalNAc-HP-β-CyD are hydrophilic, and these moieties could inhibit the hydrophobic interaction between cholesterol and the cavities of HP-β-CyDs. Optimization of the linker in future studies may help improve the interaction between GalNAc-HP-β-CyDs and cholesterol.\u003c/p\u003e \u003cp\u003eGalNAc-HP-β-CyD accumulated in the liver to a greater extent than HP-β-CyD after subcutaneous injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, GalNAc-HP-β-CyD also accumulated in the kidneys, probably due to renal clearance. To avoid renal clearance and improve blood retention, polyethylene glycol modification or polymerization of GalNAc-HP-β-CyD may be useful. Furthermore, \u003cem\u003ein vivo\u003c/em\u003e safety should be evaluated in addition to \u003cem\u003ein vitro\u003c/em\u003e studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, the newly prepared GalNAc-HP-β-CyD was efficiently internalized into hepatocytes via ASGPR and accumulated in the liver to a greater extent than HP-β-CyD. Furthermore, GalNAc-HP-β-CyD reduced cholesterol accumulation in cholesterol-accumulated hepatocytes, a factor that contributes to MASH progression. These results suggest that GalNAc-HP-β-CyD may have the potential to serve as a novel therapeutic agent for MASH.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially supported by JST SPRING (Grant Number JPMJSP2127).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments in this study were approved by the Ethics Committee for Animal Care and Use of Kumamoto University (Approval ID: A2024-034).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRin Onaga: Data curation, formal analysis, investigation, methodology, and writing of the original draft. Toru Taharabaru: Investigation, methodology, supervision, writing–original draft, and writing–review and editing. Yuto Higa: Investigation and methodology. Taishi Higashi: Methodology and supervision. Keiichi Motoyama: Conceptualization, funding acquisition, investigation, methodology, supervision, and writing–review and editing.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors thank Nihon Shokuhin Kako Co., Ltd. for providing HP-β-CyD.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStefan, N., Yki-J\u0026auml;rvinen, H., Neuschwander-Tetri, B.A.: Metabolic dysfunction-associated steatotic liver disease: heterogeneous pathomechanisms and effectiveness of metabolism-based treatment. Lancet Diabetes Endocrinol. \u003cb\u003e13\u003c/b\u003e, 134\u0026ndash;148 (2025)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePowell, E.E., Wong, V.W., Rinella, M.: Non-alcoholic fatty liver disease. 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Chem. \u003cb\u003e256\u003c/b\u003e, 4929\u0026ndash;4933 (1981)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cyclodextrin, N-acetylgalactosamine, Asialoglycoprotein receptor, MASH, Cholesterol ","lastPublishedDoi":"10.21203/rs.3.rs-8527688/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8527688/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMetabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic liver disorder characterized by lipid accumulationthat affects approximately 30% of the global population. MASLD is classified as simple steatosis or metabolic dysfunction-associated steatohepatitis (MASH), which can progress to fibrosis, cirrhosis, and hepatocellular carcinoma. The excessive accumulation of free cholesterol in the liver is partially involved in the pathogenesis of MASH. Recently, 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) has shown therapeutic potential in lipid storage disorders such as Niemann–Pick disease type C, owing to its cholesterol-lowering effect. Therefore, HP-β-CyD is considered a potential therapeutic agent for MASLD/MASH. In this study, to enhance liver accumulation and therapeutic potential of HP-β-CyD, we prepared \u003cem\u003eN\u003c/em\u003e-acetylgalactosamine-modified HP-β-CyD (GalNAc-HP-β-CyD) to target the asialoglycoprotein receptor (ASGPR), which is highly expressed on the hepatocellular membrane. GalNAc-HP-β-CyD was taken up by hepatocytes via ASGPR-mediated endocytosis and accumulated in the liver with greater efficiency than HP-β-CyD. Furthermore, GalNAc-HP-β-CyD reduced free cholesterol levels in cholesterol-accumulated hepatocytes. These results suggest that GalNAc-HP-β-CyD has the potential to serve as a cholesterol-lowering agent for MASLD/MASH.\u003c/p\u003e","manuscriptTitle":"Preparation and evaluation of N-acetylgalactosamine-modified hydroxypropyl-β-cyclodextrin as a potential therapeutic for MASLD/MASH","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-09 12:18:21","doi":"10.21203/rs.3.rs-8527688/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b2f99934-2786-4146-95f2-a60f662d373f","owner":[],"postedDate":"January 9th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Withdrawn","date":"2026-05-19T01:44:11+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T01:55:41+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-09 12:18:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8527688","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8527688","identity":"rs-8527688","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}