The purpose of this article is to describe the design of a unique, bilingual (English and French) Master of Applied Sciences (M.Sc.) in Anatomical Sciences Education (ASE) program at a Canadian postsecondary institution. Anatomy is a core foundational discipline that is essential to many undergraduate, graduate, and professional programs in the health sciences. However, the number of new individuals with the necessary knowledge base and the pedagogical training to teach cadaveric anatomy are in short supply and cannot satisfy the number of openings for trained educators in the field. The M.Sc. in ASE was created to meet the increasingly critical need for instructors trained in human anatomy. The program is designed to prepare students for a career teaching human anatomy to students in the health sciences, emphasizing hands-on cadaveric dissection. Moreover, this program aims to develop educational scholarship skills in trainees by leveraging faculty expertise in medical education research, particularly in the field of anatomy education research. This focus on scholarships will make graduates more competitive for future faculty positions. During their first year of the program, learners will develop clinically relevant anatomy knowledge, teaching skills, and anatomy education scholarship. In the second year, students will benefit from an immediate, hands-on application of this acquired knowledge. In the same year, students will serve as anatomy teachers in the faculty's Medical Program and conduct their education scholarship projects, culminating in a formal research paper. Although similar programs have been developed in recent years, this article provides the first description of the creation of a graduate program in anatomy education. It includes needs assessment, program development, challenges faced, and lessons learned during the approval process. The article serves as a valuable resource for other institutions aspiring to develop similar initiatives. For at least 2 decades, new individuals qualified to teach anatomy  have been in short supply and cannot fill the number of openings for trained educators in the field.Anatomy is a core foundation discipline for many undergraduate, graduate, and professional programs in the health sciences. Despite the ubiquitous nature of anatomy as a required course in university-level life science degrees, the number of people who are trained and prepared to teach anatomy at the postsecondary level has been precipitously declining globally. These programs have typically leveraged the local expertise of professors experienced in delivering human anatomy education to local health care programs  and existing infrastructure  to deliver anatomy instruction to graduate students in ASE. These graduate programs have evolved to complement cadaveric anatomy-related knowledge and teaching expertise with education in related disciplines such as embryology, neuroanatomy, and histology. This will be beneficial as many position descriptions state that some degree of teaching versatility is desirable in potential hires.3 In more recent years, an enhanced focus has been placed on developing educational research and scholarship skills in future anatomists because these skills are highly desired by hiring institutions.To help address the need for individuals capable of teaching human anatomy, several graduate-level anatomical sciences education (ASE) programs have been developed in the last 2 decades, mainly in the United States. Graduates from the first ten years of the program mentioned several strengths of their training, including the development of transferable skills such as pedagogical expertise, public speaking, and collaboration. However, graduates also noted several areas of potential improvement, including the fact that they would have benefitted from (i) a greater depth of education scholarship training, (ii) more opportunities to apply education scholarship training in formal dissemination environments, and (iii) increased training related to best teaching practices. Relatedly, the scholarly output of graduates in master-level ASE programs across North America remains poorly characterized. Finally, current training opportunities in ASE appear to be provided entirely in English. This is a critically important limitation in Canada given the need to develop anatomy educator competencies in French-speaking teachers to address anatomy-related course and program needs at French-speaking or bilingual colleges and universities.Although several ASE programs are already in place, most of them are situated in the United States, and scarce information has been published in terms of program evaluation regarding the successes and lessons learned from these initiatives. A program evaluation study was recently conducted on the 16-month Master of Science in Anatomical Sciences at Queen's University, aimed to prepare trainees as anatomy educators.With these considerations in mind, the purpose of this paper is to characterize the development of a novel Master of Applied Sciences in ASE program planned to begin in September 2023 in the University of Ottawa's Faculty of Medicine. This descriptive article will capture key elements during the program's conception, from the needs assessment stage to program approval, and considerations and lessons learned along the way. The resulting program will offer complete training in either French or English and emphasize knowledge application. Trainees will apply the anatomical knowledge they developed in their first year in the program in year 2 when they serve as anatomy educators in the faculty's Doctor of Medicine (MD) Program. Trainees will then apply their training in education scholarship to their research projects, culminating in a research paper at the end of their second year of study. adapted to this proposal, and invited participants to offer qualitative feedback. Item 1 demonstrated the perceived need for a program dedicated to the anatomical sciences. Participants surveyed expressed that a potential master-level program in ASE would address a gap in local education. Further, such a program would provide immense value to future healthcare professionals in terms of providing foundational knowledge necessary for clinical competence and the safe treatment of patients. Surveyed TMM students also stated that an anatomy-education training program should emphasize hands-on, cadaveric-dissection-based education and training related to evidence-based best practices in teaching. Finally, respondents generally expressed that select courses should be made available to graduate students registered in other faculties’ graduate programs.In January 2019, a needs assessment was conducted involving one of the Faculty of Medicine's bachelor's degree programs, Transitional and Molecular Medicine (TMM). The TMM program was designed for upper-year undergraduate students interested in developing their applied skills in translational research and developing students who would be successful in graduate programs in the biomedical sciences. While this program initially aimed to develop strong biomedical researchers, the main career path for TMM alumni to date has been the healthcare professions , which mirrors a primary outcome of existing graduate programs in anatomy education. With these considerations in mind, the TMM students were queried regarding the need for a Master of Applied Sciences in Anatomical Science Education and what such a program should include and emphasize . Twenty- year 1 course were also designed to provide training in histology, embryology, and pathology in environments that also emphasize hands-on, lab-based activities to provide learners with this foundational knowledge that would be required to successfully teach this content. Finally, a boot camp-like introductory course in point-of-care ultrasound technology in anatomy education was included given this tool's growing prominence in the healthcare professions and its relation to anatomy education. Early consultations also led to the consensus that content delivery  in these courses would need to be complemented with science communication and presentation opportunities and independent research components wherever possible. These elements would help support program-level learning outcomes relevant to developing educator- and/or education scholar-specific competencies. Overall, all courses in the anatomical sciences were created to emphasize student-centered learning approaches, which have been proven to stimulate engagement, participation, and teamwork in the MD Program.79 Instructors of individual courses would be encouraged to implement flipped classroom pedagogy as much as possible, which would have the added benefit of creating in-class opportunities for the application of knowledge in addition to increased time dedicated to hands-on activities, such as cadaveric dissection.10Building from the needs assessment, this program was designed with an emphasis on hands-on dissection-based courses that mirror the sequence and content of prosection-based anatomy education in the faculty's MD Program. The suggested graduate program would mirror the MD program anatomy education sequence in chronology but not necessarily in content. Early consultations with anatomy faculty led to the realization that the graduate program (with different endpoints to consider) would have to have more comprehensive, detailed anatomy content, in certain areas compared to the MD Program. Head and neck and reproductive/pelvic anatomy were areas where the graduate program needed to emphasize, in far greater detail than what was being delivered to medical students. This organization of content delivery would allow for a comprehensive, full-body anatomy education experience in dissection in the first year of the program and then allow trainees to participate as laboratory teachers in all aspects of the MD Program in their second and final years. Early consultations among relevant Faculty and program organizers advocated for the inclusion of a broad array of anatomical science content, including histology, embryology, and pathology. Furthermore, because many job postings characterize an ideal candidate as being able to teach in related anatomical disciplines, To address this concern, initial drafts of the program emphasized the importance of beginning training in education research as early as possible. At the start of the program (semester 1 of year 1), students will be registered in the immersive and established Introduction to Research in Education course offered through the Faculty of Education. This course will offer a broad, foundational introduction to education research that students can then build on throughout their studies. This course will also include an introduction to common principles and methodologies commonly used in medical education. Additionally, in Semester 5 of the program (during year 2), students will participate in the Faculty of Education's Seminar in Health Professions Education course, allowing for critical examination of applied research in a seminar format, as well as providing students with the opportunity to develop scientific communication skills by presenting a seminar focused on their research projects.Much like comparable existing programs in anatomy education, the proposed program aimed to incorporate pedagogical training that would allow graduates to develop and apply best practices in their teaching activities. As alluded to above, even courses dedicated to knowledge acquisition and training in the anatomical sciences would incorporate presentation components  related to best practices in pedagogy related to the specific course. However, several existing master-level anatomy educator programs have been poorly characterized in terms of providing a substantive education research experience and this characterization may be an area of concern in existing programs.By the beginning of semester 2 of year 1, students will have chosen a research mentor and will begin their research projects, thereby permitting 4 complete semesters to be dedicated to their research paper. To assist with being matched with research mentors, available research projects and research aims of faculty will be collated and disseminated to students during the first semester. There are also networking events to help familiarize students with potential mentors during the program's first semester. To fit with the expertise within the home department, all projects will be focused on education science with faculty mentors who are experts in different fields of education. This extended research period was designed to address potential issues with research outputs derived from programs with shorter research durations4 and would therefore provide trainees with more complete, immersive research experiences. Ideally, at the end of their second year of study and completion of the program, students’ research projects will culminate in research papers and related scholarly products such as international presentations and/or peer-reviewed publications.From the first iterations of this program, it was clear that it would have 2 points of emphasis: i) training in laboratory-based anatomical sciences and ii) pedagogical training and education research. The primary faculty for anatomical-sciences-related courses would be the established professors in the Division of Clinical and Functional Anatomy who have long contributed to the delivery of basic science education in the Faculty's MD program. On the other hand, students would receive pedagogical training by registering in previously established courses offered through the Faculty of Education and led by education professors. While not every Faculty of Education professor involved in course delivery may be an expert in medical education specifically, these professors would have expertise in the teaching of pedagogical principles and education research principles that are commonly employed in medical education. Regarding research mentorship, students would have a wide array of options for potential supervisors from the Faculty of Education and from Medicine's own Department of Innovation in Medical Education .During the External Review Process, 2 external reviewers  reviewed program documents and were provided an online ‘site visit’  in April 2021 that included interviews with relevant students, faculty, program organizers, and administrators. While the external reviewers recognized the need and strengths of the program proposal, including the emphasis on bilingualism, and the immediate application of knowledge in both education scholarship and anatomical science expertise, they also articulated constructive suggestions in the following areas.11 The focus on education technologies in anatomy is particularly useful given the wide array of modern tools that were developed, optimized, and applied to delivering anatomy education during the pandemic.13 The second course that was included at this stage  would emphasize theoretical and evidence-based practical considerations for promoting interprofessional, collaborative competencies in learners. This course will include the study of modern teaching and learning principles, curricular design, and evaluation strategies used as best practices for health profession students in interprofessional contexts. Anatomy laboratory-based education offers a unique environment to promote these valuable skills in future healthcare professionals.The external review process brought forward thoughtful and insightful suggestions to improve the program. In terms of program content, external reviewers agreed that trainees would benefit from additional training in pedagogy. These consultations resulted in the incorporation of established Faculty of Education courses that would provide benefits specific to future anatomy educators. The first of these courses  would provide training regarding best practices in utilizing modern teaching technologies in anatomy education, which would include applications of modern imaging  tools and simulation technologies that are increasingly relevant in modern anatomy teaching.As initially described, participants in this program would develop teaching skills and experience by serving as small-group laboratory teachers in the faculty's MD Program. However, external reviewers noted that there would be great benefits to providing experiential teaching opportunities in various settings to various learners. Based on this feedback, efforts were made to provide participants additional teaching opportunities during their second year in the program, including serving as anatomy instructors in other sessions that incorporate lab-based demonstrations, including sessions involving undergraduate, graduate, and high school  learners.Another concern brought forward during the external review process was the number of faculty in place to support the program. There is a substantial amount of research expertise in the Faculty of Education and the Faculty of Medicine's Department of Innovation in Medical Education to support ASE graduate students during the progression of their research papers. However, the external review process did highlight a concern with the small number of core faculty dedicated to the anatomical sciences, especially relative to comparable programs. For the program's initial launch, the core faculty in place will drive the delivery of content and the leadership in courses, with support in laboratory teaching environments provided by instructors who have contributed to similar lab-based teaching in the MD Program. As the program grows and evolves during its formative years, any need for more tenure-track professors will be clarified, and such a need will have to be addressed with future recruitment.The concerns raised during the external review in May 2021 were carefully considered and movement was made to address these concerns from both a faculty and an institutional level, as described above. In the eyes of the same external reviewers, the concerns were deemed to be sufficiently addressed during the summer of 2021, culminating in provincial  approval in December 2021 and successful accreditation from the provincial government to commence in September 2023.To be eligible for the program, candidates must have a Bachelor of Science (B.Sc.) degree with a specialization  in biology, biochemistry, pharmacology, physiology, human kinetics (kinesiology), biopharmaceutical, or biomedical or health sciences, with a minimum average of 75% to 79% (B+). Preference will be given to candidates who have completed an undergraduate course in human anatomy . Candidates who have established a level of anatomy training comparable to the courses offered in the program's first year, as assessed by the program director, can be accepted into a 1-year version of the master's program with a focus on applied teaching and scholarship courses. Candidates are also encouraged to document research or scholarly abilities  in a letter of intent. Applicants must understand and fluently speak the language of instruction (English or French) in the program to which they are applying. Finally, this program will likely attract highly trained international medical graduates (IMGs) to facilitate their career progression in pedagogy. The division currently attracts IMGs in great numbers as teaching assistants in our anatomy laboratories. These IMGs commonly participate in certificate programs in education scholarship  and will assuredly be invested in participating in this graduate program. In its final iteration, the program is described as having 3 goals:For students who are aiming for a career in the health profession, this program will provide an excellent foundation in anatomy education that is necessary to treat future patients competently. At the same time, this anatomical expertise will ensure students are highly competitive in pursuing further training .For students aiming for a career in health profession education and scholarship, this program will provide them with experience, knowledge, and mentorship in education research. Therefore, this program will benefit students aiming for further training  or a position  in a related research field. This program is intended to prepare individuals who can excel in delivering modern human anatomy education to various groups of students in a wide range of settings and address the global shortage of competent anatomy educators.Finally, there is a long-standing and well-established lack of individuals in Canada, the United States, and Europe capable of teaching human anatomy, particularly in a cadaveric laboratory-based setting.These proposed benefits for learners are supported by the notion that, in the previously described comparative program at Queen's University, the 3 most frequent career path outcomes (within 1 year of graduation) included enrollment in a professional health care degree , the pursuit of studies at the PhD level, and careers in curriculum development and teaching at the postsecondary level.After the external review process and adaptations to address reviewer concerns, the governing provincial body approved the final iteration of the program in December 2021. This program's graduates indicated that they would have benefitted from (i) a greater depth of education scholarship training, (ii) more opportunities to apply education scholarship training in formal dissemination opportunities, and (iii) increased training related to best teaching practices. It is worth noting that many of the other master-level programs in ASE in North America do not have robust levels of education scholarship output. As such, our proposed program's increased length (relative to the 16-month approach at Queen's University) helps address these issues by giving candidates a complete scholarship experience, including disseminating education scholarship in the required research paper component. Furthermore, a 20-month program would enable candidates to put their anatomy educator training into practice by allowing them to serve as teachers in all parts of the undergraduate medical program (MD program). The experience of teaching all aspects of anatomy in the context of laboratory dissection would be a strength for graduates who intend to apply for anatomy teaching intensive or pathology assistant positions, proven areas of need across North America.This 20-month program is comparable in length to other programs offered in Canada and the United States with similar content (ASE), training as an educator [M.Ed.], and training in education scholarship (Master of Arts in Education [MA]). This program is typically offered at the master's level with a thesis at comparable international universities. In Ontario, Queen's University offers a 16-month Master of Science in Anatomical Sciences program, characterized in a 2018 publication. consistent with modern advances in education in this field. The active learning principles that form the core of this program are consistent with desired qualifications commonly noted in job descriptions in anatomy education.Finally, besides the hands-on learning and skill development gained from anatomy laboratories and cadaveric dissection, the courses offered in this program emphasize student-centered learning approaches based on the Anatomy Division's experience in innovative methods, such as team-based learning,The Master in Applied Science in Anatomical Science Education is distinct because it focuses on ASE, including hands-on, applied teaching skills in the context of the human (cadaveric) anatomy laboratory. This training includes human-anatomy dissection, which is vital for teaching anatomy in many allied health professional programs across North America and numerous undergraduate institutions.https://www.uottawa.ca/faculty-medicine/graduate-postdoctoral/students-hub/research-paper-guidelines-epidemiology. Specifically, evaluation of the final scholarly product will be performed by the student's mentor and 1 other Faculty member involved in student supervision in the program and deemed to be Satisfactory if the product meets expectations of scholarly rigor and is judged to be of comparable quality to published articles in the same field. Graduation will require the completion of all 42 Units as described above. Additionally, students will also be encouraged to present their Research Paper at Meridith Marks Day, our local conference dedicated to medical education research and scholarship, to further apply their scholarly dissemination skills: https://www.uottawa.ca/faculty-medicine/department-innovation/educational-events/medical-education-day/meridith-marks-day.The coherence in the Master in Applied Science in Anatomical Science Education can be viewed in a detailed description of the program in To complement standard knowledge-based assessments, observation-based assessment strategies will need to be developed that are appropriate to monitor the progression of teaching competencies and behaviors and to ensure students are graduating with the necessary educational skill sets. Further, the formative years of program evaluation and individual course evaluation will be critical to ensure that teaching and assessment strategies are optimally employed so that students are successfully meeting course-level expectations and program-level learning outcomes. Evidence-based teaching strategies, including best practices and applications of cognitive learning theories and assessment approaches  that support the evolving independence of learners as teachers, will need to be continually evaluated and purposefully employed. While this paper only focuses on the program development and approval phase, a subsequent report 5 to 10 years after the program launch can characterize how this ASE program fares during the initial accreditation phase and include data (currently scarcely reported in the literature) regarding career path outcomes for the first several cohorts of graduates. Finally, there was a sudden and dramatic shift in anatomy education forced by the coronavirus disease 2019 (COVID-19) pandemic, which created a massive shift to virtual education in anatomy education and generally limited (at least in the short-term) student opportunities for in-person learning opportunities in the anatomy laboratory. Lessons learned from this era will need to be integrated into forward planning and delivery in this program. These lessons include how to best adapt, optimize and maintain hands-on cadaveric-based education in the context of stringent public health restrictions. It also ensures that students are familiar with and can be flexible in transitioning to best practices in virtual anatomy education approaches when needed.In this descriptive article, the authors outline the design of a unique M.Sc. program in anatomical sciences, and the authors hope that this outline is useful for other institutions and individuals who aspire to develop a similar program. The external evaluation of this program suggests that it will be a significantly valuable experience for its graduates in enhancing their anatomy learning and teaching experiences and in subsequent successful employment outcomes.Authors anticipate that this report will prompt further refinement and improvement in this program and beyond, ultimately providing many more students valuable opportunities to extend and deepen their learning and grow as anatomical science scholars. While this paper only focuses on the program development and approval phase, a subsequent report 5 to 10 years after program launch can characterize how this ASE program fares during the initial accreditation phase and include data (currently scarcely reported in the literature) regarding career path outcomes for the first several cohorts of graduates.Click here for additional data file.Supplemental material, sj-docx-1-mde-10.1177_23821205231183866 for An Innovative Master in Anatomy: Combining Anatomy With Educational Scholarship by Alireza Jalali and Christopher J. Ramnanan in Journal of Medical Education and Curricular DevelopmentClick here for additional data file.Supplemental material, sj-docx-2-mde-10.1177_23821205231183866 for An Innovative Master in Anatomy: Combining Anatomy With Educational Scholarship by Alireza Jalali and Christopher J. Ramnanan in Journal of Medical Education and Curricular Development


This demonstratesa game-changing impact of multivalent presentation on the inhibitoryeffect of a ligand as simple as lactose. Moreover, using DLS hydrodynamicdiameter measurements, we correlated the increased affinity of theglycodendrimer ligands to Gal-3 and Gal-8 but especially to Gal-9with the formation of relatively uniform and stable galectin/Lac-CS-DDMaggregates.Galectins, the glycanbinding proteins, and their respective carbohydrateligands represent a unique fundamental regulatory network modulatinga plethora of biological processes. The advances in galectin-targetedtherapy must be based on a deep understanding of the mechanism ofligand–protein recognition. Carbosilane dendrimers, the well-definedand finely tunable nanoscaffolds with low toxicity, are promisingfor multivalent carbohydrate ligand presentation to target galectinreceptors. The study discloses a synthetic method for two types oflactose-functionalized carbosilane glycodendrimers (Lac-CS-DDMs).Furthermore, we report their outstanding, dendritic effect-drivenaffinity to tandem-type galectins, especially Gal-9. In the enzyme-linkedimmunosorbent assay, the affinity of the third-generation multivalentdendritic ligand bearing 32 lactose units to Gal-9 reached nanomolarvalues (IC Generally, tandem-repeatgalectins are challenging to study due to their complex structureencompassing two distinct carbohydrate recognition domains (CRD) interconnectedwith a peptide linker.18 Though basicallyhighly conserved, the particular features in the structure of CRDof each galectin are responsible for the selectivity of their carbohydrateligands. Numerous factors determine the extraordinary selectivityof the glycan–galectin recognition,13 even if not all of them are fully understood. A structural comparisonin the binding between Gal-1, Gal-3, and Gal-9 CRDs and poly-LacNAcoligosaccharides is contained in a study by Nagae and Yamaguchi.19 It showed similarities between the interactionsof Gal-3 and Gal-9N. In Gal-8N, a specific binding-site residue isArg59 for recognition of sialylated and sulfated oligosaccharides,which is absent in Gal-9.20 Gal-1 comparedto Gal-3 also contains a specific residue, His52, interacting withligands in subsites C and D; in contrast to Gal-3, Gal-1 is well knownto be acting as an exo-type lectin for longer oligosaccharide ligands,21 recognizing only the terminal disaccharide.The systematic development of small-molecule glycomimetic ligandsrevealed the influence of configuration and substitution at individualpositions of the carbohydrate, e.g., an aromatic substitution at theC-3 position, on the potency and selectivity of the ligand.24 Substantial differences in the binding modes are also found in theinteractions with the less defined subsite E, localized at the reducingend of carbohydrate ligands.26 The multivalent presentationof glycan ligands in macromolecules represents another natural strategyto ensure effective ligand–protein interaction. In multivalentligand molecules, the overall binding potency can exceed the solesum of monovalent ligand affinities by orders of magnitude.28 Moreover, unlike monovalent systems, the multivalent glycoconjugatescan form unique two- and three-dimensional lattices with galectinreceptors. There are clear differences between Gal-1 and Gal-3 inthe recognition of multivalent ligands.30 For tandem-repeat galectins, the available systematicaffinity data are rather limited.31 Therefore,despite potential therapeutical promises, the design of selectiveligands/inhibitors of Gal-8 and Gal-9 remains to a large extent “terra incognita”. Recent studies have associatedsuch multi-dimensional structures with significant biological activity,32 including induction of apoptosis in activatedhuman T cells34 or negative regulation of neuroblastomacell growth.35 Bertozzi et al. provideddirect evidence of galectin ability to cross-link glycoligands onthe cell surface.36 In the last few years,various multivalent carbohydrate-based or carbohydrate-decorated materialshave been designed.38 Many types of scaffolds havebeen used for the multivalent presentation, such as polymers,39 calixarenes,40 serum albumins,42 peptides,44 or dendrimers.47To date, 12 members ofthe human galectin family have been described:Gal-1, Gal-2, Gal-3, Gal-4, Gal-7, Gal-8, Gal-9, Gal-10, Gal-12, Gal-13,Gal-14, and Gal-16.49 As such, dendritic structures provide control over the physicochemicalproperties and valency of the system. Unlike some other syntheticplatforms for multivalent presentation,50 dendriticstructures allow fine-tuning of the attributes of the multivalentsystem by controlling the size, branching level, peripheral derivatization,spatial distribution, and density of the ligands. Thanks to theirversatility, glycodendrimers (glyco-DDMs) are extensively exploitedto study ligand–galectin interactions. In their pioneer work,André et al. demonstrated the different inhibitory potencyof poly(amidoamine) (PAMAM) DDMs bearing aromatic p-isothiocyanatophenyl β-d-lactoside ligands (up togeneration five) for Gal-1 and Gal-3.51 Cousin and Cloninger used lactose (Lac)-functionalized PAMAMs toinvestigate the multivalent Gal-1-mediated interactions. Remarkablyhomogeneous clusters were formed in the case of a significant excessof Gal-1 over glyco-DDMs. Another study reported that the glyco-DDMsinhibited Gal-1-moderated cellular aggregation of prostate carcinomacells by providing a competitive binding site.52Dendrimers(DDMs) are regular, highly branched spherical macromoleculesprepared by repetition of individual synthetic steps.N-Acetyllactosamine (LacNAc)-functionalizedPAMAMs synthesized by a chemoenzymatic method either inhibited orenhanced Gal-3-mediated cancer cell aggregation depending on the DDMstructure and generation.46 An optimaltopological distribution and density of surface glycans were studiedon fully programmable supramolecular assemblies of Lac-decorated amphiphilicJanus DDMs known as glycodendrimersomes (GDSs). Agglutination assaysof Gal-153 and Gal-854 with Lac-presenting GDSs suggested the optimal ligand topologyand density, the increase in which did not lead to higher galectinreactivity.A different density and spatial distribution of multivalentlypresentedligands can be responsible for the differences in the behavior ofsupramolecular assemblies of DDM-galectin complexes. Thus, the structuralparameter variations may serve for modulating the inter- and intra-cellularprocesses. 55 Still, we expect that new findings concerningother less common galectins can contribute to a deeper understandingof the related processes in living cells. To our best knowledge, nostudy has been published to date on the interactions between Gal-9and DDMs or, importantly, any other synthetic multivalent systems.Gal-1 and Gal-3 are the most studied representativesof the galectinfamily due to their biological significance and high abundance. Onthe other hand, only limited data are available for other galectins.56 the activityof functionalized dendrimers depends not only on peripheral derivatizationbut also on the structure of the dendritic scaffold. Carbosilane (CS)based CS-DDMs represent a well-established material with many benefitsin the biomedical field. Yet, CS-DDMs have not been investigated asmultivalent platforms to display galectin-binding ligands. Therefore,we present the very first study revealing the potential of glyco-CS-DDMsfor galectin-targeted research. Two series of Lac-decorated carbosilanedendrimers (Lac-CS-DDMs) of the first to third generation have beenprepared for this study. Lactose ligands were grafted to the peripheryof the alkyne-terminated CS-DDMs using copper-catalyzed azide–alkynecycloaddition (CuAAC). The two Lac-CS-DDMs series differ in the triazolering position, which is either separated from the carbohydrate ligandby a triethylene glycol (TEG) spacer (series A) or directly linkedto the C-1 position of lactose via a triazole moiety (series B). Wepreviously found that a triazole next to the carbohydrate unit mayhave a positive effect on the affinity, especially to Gal-3.39 The interactions of Lac-CS-DDMs with human galectins  were thoroughly studied using two complementarymethods: (i) competitive enzyme-linked immunosorbent assay (ELISA),which assessed the ligand inhibitory potency, and (ii) dynamic lightscattering (DLS), which correlated the Lac-CS-DDM-galectin aggregationbehavior with an increased affinity.As recently demonstrated,22.11I–G3I) and propargyl-terminated (G1A–G3A)CS-DDMs and 5-(OMOM)isophtalic acid were prepared according to ourpreviously reported method.57 The acetylatedlactose ligands N3-TEG-AcLac and N3-AcLac wereprepared adopting the reported procedure.58 Unless otherwise stated, d-lactose, 2-{2-[2-(2-propynyloxy)ethoxy]ethoxy}ethanamine,hydroxybenzotriazole (HOBt), N,N′-dicyclohexylcarbodiimide (DCC), N-methylmorpholine(NMM), dimethylformamide (DMF), potassium carbonate, CuI, N,N-diisopropylethylamine (DIPEA), triethylamine,Dowex 50 WX8, Amberlite IRA402, and Chelex 100 (sodium form) werepurchased from commercial sources and used without further purification.The 3-iodopropyl-terminated at 79.5 MHz) at 25 °C. 1H and 13C NMR signals of the prepared compounds were assignedto corresponding atoms utilizing gHSQC, gCOSY, gHMBC and HSQC TOCSY2D NMR correlation spectra. 1H and 13C chemicalshifts (δ/ppm) are given relative to residual solvent signals; 29Si spectra werereferenced to external standard hexamethyldisilane (−19.87ppm). MALDI-TOF spectra were measured on an UltrafleXtreme MALDI-TOF/TOFmass spectrometer  with a 1 kHz smartbeamII laser. The measurements of G1-DDMs were done in thepositive reflectron mode technique, with the mass range of 2–6kDa. The measurements of G2-DDMs were done in the positive/negativelinear mode technique, with the mass range of 2–20 kDa. Theaccelerating voltage was set at 25 kV. Typically, spectra were obtainedby accumulating 3000 shots. Dihydroxybenzoic acid was used as thematrix (10 mg/mL in acetonitrile/0.1% TFA 1:1). Gel permeation chromatography(GPC) was performed on a Dionex UltiMate 3000 HPLC system equippedwith a Phenogel 10E3Å column (Phenomenex), with 100% methanolasthe mobile phase and diode array detector. Chromatograms were acquiredat λ = 225 nm. Fourier transform infrared spectroscopy (FTIR)was carried out using a Nicolet 6700 with a mid-IR DTGS detector.The spectra were recorded in the range of 650–4000 cm–1 at a resolution of 4 cm–1 with the ATR technique.NMR spectra were measured on a Bruker Avance 400  and m is the number of isophthalic moieties in the DDM outer layer, whichimplies that the number of peripheral Lac units in Lac-CS-DDMs is2m.Herein below, G2.1.12 (1.1 equiv) and dry K2CO3 (1.3 equiv) weresuspended in dry DMF. The iodopropyl-terminated DDM  was dissolved in petroleum ether (35–60°C fraction) and added dropwise to the suspension. The reactionmixture was stirred overnight at 80 °C. Then, after evaporatingDMF under vacuum, the crude product was dissolved in methanol andfiltered through a short silica-gel column. The organic phase wasevaporated (to ca. 3 mL volume) and purified using organic solventnanofiltration (OSN). After evaporation to dryness, the products Gn-Bm2 (6–8) were obtained as brownish waxy solids .Compound 2.1.2n-Am2 (3–5) or Gn-Bm2 (6–8); 1 equiv; reactions were carried out in a 0.1–0.3mM scale) and azide-functionalized carbohydrates  were dissolved in dryDMF. Then, CuI (0.01 equiv per branch) and DIPEA (5 drops) were added.The suspension was transferred to a microwave reaction vial (10 mL),sealed with a septum, placed into the microwave reactor cavity, andirradiated up to 80 °C with stirring (600 rpm) for 3 h. Aftercooling to ambient temperature, the vial content was concentratedunder reduced pressure. The solid was dissolved in methanol. Then,Chelex 100 was added to remove the Cu residues. After OSN, the productsGn-A-AcLacm2 (12a–14a) and Gn-B-AcLacm2 (15a–17a) were obtained as off-white powders (85–90% yield).The DDMs with peracetylated glycounits Gn-A-AcLacm2 (12a–14a) or Gn-B-AcLacm2 (15a–17a)  were deacetylated under microwave irradiation (55 °C)according to the previously reported procedure.57 The completion of the deacetylation was checked using 1H NMR, and the deacetylation was repeated when necessary.The deacetylated glyco-DDMs were dissolved in distilled water andfreeze-dried overnight to obtain products Gn-A-Lac2m (12b–14b) and Gn-B-Lacm2 (15b–17b) as puffy off-whitepowders . The deacetylatedglyco-DDMs were checked for the presence of Cu ions by inductivelycoupled plasma–optical emission spectroscopy (ICP-OES). Productsof insufficient purity were subsequently dissolved in water and treatedwith Chelex 100 (sodium form) until the complete removal of copperwas achieved according to ICP-OES (detection limit 1–5 ppmCu).The alkyne-terminatedDDMs (G2.1.359 was carried out using solvent-resistant stirredcell Millipore (for 47 mm membranes) equipped with 1 or 3 kDa MWCOregenerated cellulose ultrafiltration discs Ultracel (Millipore) andPTFE encapsulated O-rings Teflex , with nitrogenas a driving gas (transmembrane pressure 5 bar). Crude dendritic productswere dissolved in 50 mL of an appropriate solvent , and the solution was filteredthrough the membrane until a residual volume of 5 mL of the retentatewas reached. The retentate was then diluted with 45 mL of the samesolvent mixture and filtered. 1H NMR was used to monitorthe purification progress. The procedure was repeated as necessary;three to four cycles were typically sufficient to obtain analyticallypure products.60Nanofiltration2.2NcoI/AscI for Gal-1 and Gal-3 and AscI/NotI for Gal-8 and Gal-9). The geneconstruct of Gal-1 contains a mutation C2S, which increases its stabilityand renders it resistant to oxidation.62 The plasmidscarrying the gene constructs of Gal-1 and Gal-3 were prepared as describedpreviously.22 The plasmid containing thegene construct of Gal-8 was a kind gift from Prof. L. Elling, RWTHAachen, Germany; it contains a full-length peptide linker of 34 aa(3717 Da). The gene construct of Gal-9 was designed according to thestudy by Itoh et al.63 where they foundthe most stable and soluble form of Gal-9 . The gene was preparedcommercially . Galectins wereproduced and purified as described previously.64 Briefly, the transformed Escherichia coli Rosetta 2(DE3)pLysS competent cells were inoculated into a Luria-Bertanimedium and cultivated at 37 °C and 220 rpm overnight. The precultureswere inoculated into a Terrific Broth medium  and cultivatedat 37 °C and 150 rpm. The LB and TB media contained ampicillin(100 μg/mL) and chloramphenicol (34 μg/mL). The proteinexpression was induced by adding 0.5 mM isopropyl 1-thio-β-d-galactoside (IPTG) when the culture grew to an optical density(OD600) of 0.6–0.8. Then, the cells were cultivatedat 25 °C for 24 h and harvested by centrifugation .Recombinanthuman galectins Gal-1, Gal-3, Gal-8, and Gal-9 were produced as N-terminalHis-tagged constructs cloned in a pET-Duet1 vector , thecell-free extract was loaded on an equilibrated Ni-NTA column . First, the column was washedwith equilibration buffer, then with equilibration buffer containing0.5% Triton X-100 (10- to 20-fold column volume) to remove tracesof lipopolysaccharide.64 Then, the columnwas rewashed with pure equilibration buffer. Bound galectins wereeluted with an elution buffer containing 500 mM of imidazole. In thecase of Gal-9, a pre-elution step of washing with 50 mL of 50 mM imidazolewas included to reduce non-specific protein adsorption to the column.Then, elution with a gradient of imidazole (100–500 mM) followed.Fractions were analyzed for protein content using the Bradford assay65 calibrated for immunoglobulin G (IgG), pooled,and dialyzed overnight in PBS buffer pH 7.5 (7 L) containing 2 mMEDTA followed by 4 h of dialysis in PBS buffer (7 L). Gal-1, Gal-3,Gal-8, and Gal-9 proteins were stable at 4 °C for approximately2 months. The purity of prepared galectins was confirmed on SDS-PAGE.For the purification of galectins,harvested cells were suspended in an equilibration buffer . Phenylmethylsulfonyl fluoride  was added to prevent cleavage by proteases. The suspension wassonicated using an UltraSonic Processor UP50 H  for six cycles . Aftercentrifugation was coated in microtiter plates  and incubated overnight. Then, the wells were washed with 3 ×250 μL of PBS buffer containing 0.05% Tween; this washing stepfollowed each incubation step. The wells were then blocked with 2mg/mL BSA in PBS buffer (250 μL/well) and incubated for 1 h.After washing, serial dilutions of the ligands (Lac-CS-DDMs or lactosestandard) in EPBS (2 mM EDTA in PBS) buffer with the addition of DMSOfor better solubility (10% v/v)were incubated with Gal-1, Gal-3, Gal-8, or Gal-9 . For the direct binding ELISA assay, the incubation step comprisedserial dilution of the respective galectin in EPBS (50 μL/well).After a washing step, the anti-His-antibody conjugated to horseradishperoxidase  was used for labelingof residual bound galectin and subsequent colorimetric detection.After another washing step, the substrate for the peroxidase reactionTMB One  was added and incubatedthe reactions until visible blue staining appeared (3–20 min).The reaction was stopped by adding 3 M HCl (50 μL per well),accompanied by a color shift to yellow as determined spectrophotometricallyby an absorbance microplate reader .The intensity of the signal at 450 nm corresponded to the amount ofgalectin bound to the wells. A background value, as a mean of thenegative control wells containing buffer instead of galectin/ligand,with an absorbance of ca. 0.05–0.075 was subtracted from themeasured absorbances. The values of half maximal inhibitory constants(IC50) were calculated from the non-linear regression (dose–responseinhibition-variable slope) of the sigmoidal curves using GraphPadPrism 7  from at least three independent experimentswith at least two different galectin batches. Using the standard inhibitorlactose, it was verified that the impact of the DMSO co-solvent concentration(0–10% v/v) on the galectinaffinity was not significant.The inhibitorypotential of prepared Lac-CS-DDMs toward Gal-1, Gal-3, Gal-8, andGal-9 was determined using a competitive ELISA assay with immobilizedasialofetuin .2.4n-A-Lacm2 and galectin/lactose) to obtain a finalgalectin concentration of 10 μM. Mixtures of components wereincubated for 90 min at room temperature. The samples were vortexedprior to measurements. The hydrodynamic diameters were determinedfrom five independent repetitions (each 10–50 runs). Multimodalintensity-weighted particle size distribution was used for data analysis.The hydrodynamicdiameter of the nanoparticles of galectins, Lac-CS-DDMs, and theiraggregates was measured by dynamic light scattering (DLS) using aZetasizer Nano ZS  at 25 °C inMalvern disposable plastic microcuvettes (100 μL sample volume).The light scattered at 173° from the incident light was fittedto an autocorrelation function using the method of cumulants . Commercial sterile PBS was used for samplepreparation. Components were (i) dissolved in PBS to obtain a 10 μMfinal concentration  or (ii) mixed in 150:1,6:1, and 2:1 ratios  were prepared accordingto a slightly modified procedure from polyalkyne substrates Gn-Am2, which wepreviously developed for the preparation of glucose- and galactose-decoratedCS-DDMs.57 Analogical series B was synthesizedto investigate the effect of the triazole ring position on the overallavidity of the multivalent dendritic system. First, the phenolic alkynederivative with propargyl-terminated TEG chains 2 wasprepared in two steps. A new type of alkyne-terminated CS-DDMs (Gn-Bm2) was synthesizedfrom dendritic precursors G1I–G3I bynucleophilic substitution of iodine by a phenolic group of the substrate 2 in the presence of a mild base (2I) to 3.85–3.98 ppm (−CH2O−) in 1H NMR spectra indicated the completionof the reaction. The two series of alkyne-terminated CS-DDMs wereused for the attachment of sugar moieties, combining alkyne-terminatedseries (Gn-Am2) with azide-TEG-functionalized lactose and TEG-alkyne-terminatedseries (Gn-Bm2) with azide-functionalized lactose.Lactose-functionalizedLac-CS-DDMs of series A -catalyzedazide–alkyne cycloaddition (CuAAC) (4 and sodium ascorbate as a reducing agent)66 by a procedure facilitating triazole ring formationunder microwave irradiation and elevated temperature .67 We have found that thismethod provides quantitative conversion in a shorter reaction time.Moreover, the yields of the acetylated Lac-CS-DDMs (Gn-A-AcLacm2 and Gn-B-AcLacm2) weregenerally higher due to the absence of aqueous work-up. The microwave-assistedreaction setup is known to accelerate the reaction progress69 and to be generally beneficial for synthesizing glyco-CS-DDMs.Acetylated Lac-decoratedCS-DDMs  (CuAAC) 1. Nevertn-A-AcLacm2 and Gn-B-AcLacm2 by OSN.60 For the final O-deacetylation step, we used triethylamine-catalyzeddeacetylation under microwave irradiation70 as this method accelerates the deprotection process. The disappearanceof seven acetyl group singlets around 2.0 ppm in 1H NMRindicated efficient deacetylation. Finally, the formation of the Lac-CS-DDMsdiffering in the triazole ring position Gn-A-Lacm2 and Gn-B-Lacm2 was confirmed by one-and two-dimensional NMR  and for lower generations of Lac-CS-DDMs also by MALDI-TOF MS analyses.Then, we took advantage of a substantial size difference betweenthe reaction components and purified the G13C signals of the skeletal C-1 and C-1′ carbons aretypically positioned at 104–102 ppm as both C-1 and C-1′carbons form O-glycosidic bonds. Accordingly, therespective signals of H-1 and H-1′ are located at 4.22–4.20ppm. On the contrary, in series B, the N-glycosidicbond to the triazole ring formed as a result of the CuAAC shifts theC-1 carbon upfield (87–86 ppm) compared to the correspondingC-1′ signal (104–99 ppm). Similarly, respective H-1signals are positioned upfield (4.8–4.3 ppm) compared to H-1′signals (5.6–5.1 ppm).Even thoughseries A and B differ only in the triazole ring position,we observed subtle differences in NMR spectra demonstrating the structuralfeatures of both series. In Lac-CS-DDM series A, the characteristic 2 group of the TEG chain and the skeletal H-1 proton of Lac and (ii)CH group of the triazole ring, CH2 group of TEG, and amidicNH protons. On the contrary, in series B, the CH triazole signal interactswith the H-1 skeletal proton of Lac and both carbons of the triazolering interact with CH2 protons of TEG .In addition, the triazole ringposition was identified by two-dimensionalNMR experiments. The H-C correlated gHMBC spectra of the Lac-CS-DDMsof series A showed an interaction between (i) the end CH3.270 in E. coli Rosetta 2(DE3)pLysS. We purified them by Ni-NTA affinity chromatographywith elution by imidazole. The binding affinity of prepared galectinsto ASF was determined by the direct ELISA assay; respective KD values: KD = 4.4μM for Gal-1, KD = 3.2 μMfor Gal-3, KD = 0.37 μM for Gal-8,and KD = 0.30 μM for Gal-9 .Todetermine the affinity of prepared Lac-CS-DDMs to a representativeselection of galectins, we produced prototype Gal-1, chimera-typeGal-3, and tandem-repeat Gal-8 and Gal-9 as His-tagged constructs41 Varying concentrationsof glycodendrimer inhibitors Gn-A-Lacm2 and Gn-B-Lacm2 competed for binding galectins in solutionwith immobilized competitor ASF, which interacts with galectins. Theamount of residual galectin bound to immobilized ASF was determinedspectrophotometrically using the anti-His-tag antibody conjugatedto horseradish peroxidase. The inhibitory potency of prepared Lac-CS-DDMswas compared to monovalent lactose as a standard, and a relative potency(rp) for each galectin was calculated (rp/lac) was also determined . This value representsthe positive cooperativity and, hence, the affinity increase for lactosebound in a multivalent system (rp/lac > 1) compared to the free lactose. Since some compounds werepoorlywater-soluble, the dimethyl sulfoxide (DMSO) co-solvent (2–10% v/v) was applied to reach the saturatingconcentration in the dose–response inhibition curves.To determinethe inhibitory potency of prepared Lac-CS-DDMs toward galectins, weemployed competitive ELISA.lculated 1. To furN-triazole linker exhibited the best resultwith Gal-3 compared to the O-ethyltriazole and thiourealinker in a series of glycopolymers39 andpartially also in the study with glycoclusters, where, however, thiseffect was not apparent with Gal-1.29 Inour present study, the positive impact of the N-triazolelinker adjacent to the carbohydrate moiety is evident for all galectintypes, including tandem-repeat galectins. The N-triazolepart interacts with the less conserved subsite E in the binding grove,which has so far been only marginally studied, especially in connectionwith glycomimetic thiodigalactoside inhibitors.26 There, arginine-π interactions have been deemed responsiblefor the affinity increase, particularly with Gal-3 (Arg186). Sinceconserved arginine residues are found in the subsite E of galectins, we hypothesize that this might be a particular conservedfeature of the galectin CRD domain influencing linker binding to subsiteE; however, this can only be reliably confirmed by a crystallographystudy. The best inhibitor of all galectins was G3-B-Lac32. Especially for Gal-9, it showed IC50 in nanomolarrange (970 nM), 1400 times better than free lactose. Notably, onlyin the case of Gal-9, a strong positive cooperativity was observedbetween multivalent lactosyls (rp/lac was up to 44), in contrast to other tested galectins Gal-1, Gal-3,and Gal-8 . A partial explanation of the binding differences might alreadybe found in the interaction of the binding sites of respective galectinswith lactose . However, the relative potencies presented in Table S1 clearly show that the major contributionof these high avidities lies in the multivalency effect, especiallyfor Gal-9. Tandem-repeat galectins , due to the presenceof two close covalently bound CRDs, have much higher prospects oftaking advantage of, e.g., the statistical rebinding effect, thanother galectin types.Overall, the more glycans on thedendrimer, the better the affinityto the galectins was obtained. The series A of Lac-CS-DDMs featuringa directly bound lactose showed a lower overall inhibitory potencythan the series B with the triazole group adjacent to the glycan.This is in line with our previous studies where we found a considerableeffect of the carbohydrate linker on the affinity to galectins—thedirect 3.33-B-AcLac32 were performed to provide insightinto the complexity ofmultivalent galectin/Lac-CS-DDM interactions  or oligomers 75 via the non-lectin domain (hydrophobic)interactions. Other interactions may also govern the formation ofmore or less irregular aggregates depending on the conditions. Forexample, Miyanishi et al. discovered that human Gal-9 interacts withitself and other galectin family members via CRDs.76Initial experimentsat a 10 μM concentration revealed self-assemblingtendencies of both Lac-CS-DDMs and galectins as individual components.The investigated galectins are known to self-associate into homodimers is in good accordancewith the reported value. Interestingly, most mixed Gal-1/DDM samplesalso display small particles around 1 nm, especially at higher Gal-1/Lac-CS-DDMratios. Gal-3 is known to be monomeric in diluted solutions under100 μM.75 The size of observed particles is in good accordance with the data reportedfor a full-length Gal-3 by Halimi et al.78 Surprisingly, size-related data for tandem galectins are lacking;however, if we consider the rather conserved CRD domain, then we canexpect that the size of tandem galectins will be close to CRD domaindimers observed by Birdsall et al. (6 nm)79 or Gal-1 homodimers .80 Gal-8 and Gal-9 (ca. 36 kDa) showed higher tendencyto form large aggregates during our measurements than Gal-1 or Gal-3.There, the diameter values of minor-intensity fractions in Gal-8 (5.4± 0.5 nm) and Gal-9 (5.8 ± 0.1 nm) samples indicate thepresence of monomers and dimers. In the free Lac-CS-DDMs, the tendencyto form large aggregates increased with increasing DDM generation(entries 5–8). However, we always observed particles around10 nm, representing a dominant fraction corresponding to small clusterscontaining from several units to high tens of DDM molecules, dependingon the generation. The formation of small clusters was also previouslyreported for other carbosilane glycodendrimers.57Although all studied galectins showed some degreeof aggregation,the population corresponding to monomers or dimers was observablein all cases. The size of pure Gal-1 is smaller than observed by Scott et al.;81 Compared to singlecomponents, particles of a different size range were observed in mixedsolutions , Gal-8 (0.6–0.9 μm), and Gal-9 (0.7–1.0μm); Gal-1 formed aggregates in a wider size range dependingon the Lac concentration.Having an insight into the aggregation behavior offree galectinsand glycodendrimers, we conducted a series of tests to evaluate thesize distribution of Gal/Lac-CS-DDM assemblies following the studyby Cloninger et al.olutions 2,indicaolutions 2. Thus, 52 On the other hand,chimera- and tandem-type galectins readily aggregated with Lac-CS-DDMsunder the same experimental conditions. Gal-3 formed rather non-uniformassociates with glycodendrimers (entries 33–44), and fractionsof small particles corresponding either to free Gal-3 or to smallGal-3/Lac-CS-DDM clusters were apparent in most samples. On the otherhand, tandem-type galectins formed rather uniform aggregates in thesub-micron size range for Gal-8 (entries 45–56) and in therange of 1–2 μm for Gal-9 (entries 57–68). Inthe case of Gal-8, the size of particles observed upon mixing withLac-CS-DDMs is not fully indicative of the mutual association as alsothe free Gal-8 aggregates fall into similar size range (entry 3).Nevertheless, fractions of free galectin were apparent only underthe conditions of a vast galectin excess.Next, we focused on the aggregation behavior of Gal/Lac-CS-DDMmixtures. Concerning Gal-1, the observed size diversity points toa very dynamic aggregation process. Particles in the size range offree Gal-1 together with small clusters were predominant in Gal-1/Lac-CS-DDMsolutions, which indicates a poor aggregation tendency even at highDDM concentrations (entries 21–26). A certain level of aggregationwas observed only with the third-generation dendrimers . This is in accordancewith the findings by Cousin and Cloninger, who observed aggregationof Gal-1 with polyamidoamine-based glycodendrimers for the generations3–6 having 20 or more Lac units at the periphery, but not withthe second generation.Gal-9/Lac-CS-DDM solutionsexhibited specific association featurescompared to other tested galectins: (i) very large aggregates of arelatively narrow size range were formed, and (ii) no fractions ofsmall particles corresponding to either free Gal-9 or small Gal/Lac-CS-DDMclusters were detected. The presence of small particles cannot beruled out, but compared to other studied galectin types, Gal-9 showsby far the highest degree of aggregation.3-A-Lac32 than free lactose), whichin the case of Gal-9/G3-B-Lac32 attacks thenanomolar affinity benchmark (1390-fold affinity enhancement overfree lactose).From the comparisonof the results obtained from ELISA and DLS,we may relate the aggregation behavior with the Lac-CS-DDM affinityto galectins. In the case of Gal-1, the poor mutual association correlateswith a low affinity increase, which was only 2–6 times higherin the multivalent system than for monovalent lactose 1. In con41-G3 CS-DDMs wereprepared using CuAAC click reaction from alkyne-terminated dendriticprecursors. The Lac-CS-DDM series differed in the triazole ring position,being distanced from the Lac unit by a TEG linker (series A) or linkeddirectly to the C-1 position of the carbohydrate moiety (series B).The inhibitory activity of the multivalent Lac-CS-DDM ligands wasdetermined in the ELISA assay and compared to the free lactose. Theinhibitory potency was generally higher in series B. Hence, we showedthat positioning the triazole ring in the carbohydrate moiety neighborhoodfurther enhances the affinity of the multivalent ligands. The participationof the triazole ring on the glycan-receptor binding will be furtherstudied.Thestudy provides the very first multivalent galectin inhibitorsbased on CS scaffolds with a high inhibitory potency, especially toGal-9. Lactose-decorated Gn-B-Lacm2, the affinity of G3 was about 20 times higher than G1 . AllLac-CS-DDMs showed higher affinity to the tested galectins than freelactose. The most potent ligand was G3-B-Lac32, in which the affinity raised 13 times to Gal-1, 47 times to Gal-3,and almost 2 orders of magnitude (77 times) to Gal-8 compared to freelactose. Moreover, G3-B-Lac32 showed a solid3 orders of magnitude (1400 times) higher affinity to Gal-9, reachingthe nanomolar IC50 benchmark (970 nM). These findings fullysupport the dendritic effect principle: even simple ligands can achieveoutstanding affinity to target receptors if presented in a multivalentmanner.Generally, the affinity to the tested galectins increasedwiththe generation. In GIn addition, for the first time, we demonstrated thatthe aggregationbehavior is related to the inhibitory potency of the multivalent ligands.We studied the self- and mutual aggregation behavior of the testedgalectins, glycodendrimers, and their mixtures by DLS. Both free galectinsand glycodendrimers formed distinct and relatively stable self-associates.Lac-CS-DDMs formed large, stable, and uniform aggregates with Gal-3(0.3–1.4 μm), Gal-8 (0.5–1.2 μm), and particularlywith Gal-9 (1–2 μm). The mutual Gal-Lac-CS-DDM associatesdiffered from the sole components in size, indicating that the interactionbetween the galectins and DDMs stabilizes the system by reassemblingto mutual aggregates. This was particularly prominent in Gal-9/Lac-CS-DDMmixtures, in which the mutual aggregates (i) were formed even in thesignificant excess of Gal-9 (150:1) and (ii) their diameter was 2–3times larger compared to the aggregates of the sole components. Incontrast, indistinct, polydisperse, and dynamically reassembling aggregateswere detected in Gal-1/Lac-CS-DDM mixtures. In conclusion, besidesproviding highly potent Gal-9 inhibitors, we showed that the increasedaffinity of the multivalent system is associated with the formationof stable, uniform aggregates. Therefore, the investigation of theaggregation behavior of multivalent ligands can serve as an indicativetool to estimate the inhibitory potency toward galectins.