N.A. Costa^1,2,3,4, C. Monteiro^2,3, A.R. Ribeiro⁵, P.N. Lisboa-Filho¹, M.C.L. Martins^2,3,6

¹ UNESP – Universidade Estadual Paulista, Faculdade de Ciências, Bauru, SP, Brazil.

² i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.

³ INEB – Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal.

⁴ FEUP – Faculdade de Engenharia, Departamento de Engenharia Metalúrgica e de Materiais, Universidade do Porto, Porto, Portugal.

⁵ INL – International Iberian Nanotechnology Laboratory, NanoSafety Group, Braga, Portugal.

⁶ ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal.

INTRODUCTION: Infection of titanium (Ti) implants is still a serious problem in orthopedic applications [1]. Surface conjugation with antimicrobial peptides (AMPs), such as MSI-78 can be a promising strategy to prevent bacterial colonization of the implant [2]. This work intends to investigate the bactericidal activity of MSI-78 conjugated onto porous TiO₂ coatings prepared by micro-arc oxidation (MAO) technique [3] that is commonly used to induce osseointegration.

METHODS: Ti substrates were coated by MAO, using electrolyte containing calcium and phosphorus. Then, the AMP MSI-78 (GIGKFLKKAKKFGKAFVKILKK 22 AA) was conjugated onto the surface through the 1,1′-Carbonyldiimidazole (CDI) chemistry [4]. Coatings were exposed to O₂ plasma to create functional surface OH groups. Afterward, the OH groups were activated with a CDI solution (30 mg/mL in tetrahydrofuran) for 2 h at room temperature. Then, an AMP solution (0.5 mg/mL) in 0.01 M sodium borate (pH 9.15) was prepared to react the amine AMP groups with the surface imidazole group, for 24 h at 40°C. Coatings with only physiosorbed AMP (without CDI as anchor) were also tested. Samples were analyzed by scanning electron microscopy, X-ray photoelectron spectroscopy and water contact angle. To create a surface protein layer (conditioning film), the samples were incubated with 1% v/v human plasma proteins in phosphate-buffered saline (PBS) solution for 30 min at 37°C. Then, their antibacterial potential was evaluated against Methicillin-Resistant Staphylococcus aureus (MRSA; ATCC 33591) in PBS for 5 h at 37°C. The viability of surface adherent bacteria was observed by a confocal high-content screening microscope.

RESULTS: MAO TiO₂ coatings presented a porous structure composed of calcium and phosphorus. The presence of AMPs on the surface was identified by the introduction of nitrogen and induction of hydrophobicity, but no difference was found between physically adsorbed and conjugated AMP. Regardless of the treatment, the AMPs on the surface may attract and kill bacteria (almost 60%) (Fig. 1), even in the presence of human plasma proteins. 

Fig. 1: MRSA viability on the different surfaces.

DISCUSSION & CONCLUSIONS: MSI78-treated MAO surfaces exhibited an excellent antibacterial effect against MRSA even with a conditioning protein film.

REFERENCES: ¹ S. Spriano, S. Yamaguchi, F. Baino, et al (2018) Acta Biomater 79:1–22. ² B. Costa, G. Martínez-de-Tejada, P.A.C. Gomes, et al (2021) Pharmaceutics 13:1918. ³ G. Li, F. Ma, P. Liu, et al (2023) J Alloys Compd 948:169773. ⁴ P. Parreira, C. Monteiro, V. Graça, et al. (2019) Sci Rep 9:1–11.

ACKNOWLEDGEMENTS: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES) – Finance Codes 88887.600413/2021-00 and 88887.802752/2023-00, São Paulo Research Foundation (FAPESP) (grants #2020/10125-9 and #2021/11461-5), and European Union’s H2020 project Sinfonia (N.857253).

H. Teoh ¹, E. Soh ¹, H. Le Ferrand ^{1, 2, 3, 4*}

¹ School of Mechanical and Aerospace Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore.

² School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore.

³ Singapore Center for 3D Printing, Nanyang Technological University, 65 Nanyang Drive, Singapore 639798, Singapore.

⁴ Future Cities Laboratory, Singapore ETH Centre, 1 Create Way, Create Tower #06-01, 138602 Singapore, Singapore

* Corresponding author: hortense@ntu.edu.sg

INTRODUCTION: Engineered living materials (ELMs) are an emerging brand of materials, comprising of a non-living polymer matrix containing living cells that provide additional biological functionalities or transform the structure of the surrounding matrix through their growth¹. By incorporating fungal cells into a suitable matrix, they will form a network of hyphae known as mycelium which will envelop the surface of the material over time. One main challenge of emerging fungal-based ELMs lies in achieving localized multi-material properties in these structures. Although bioprinting can efficiently vary the local composition and properties, it has yet to be demonstrated in fungal-based ELMs.

METHODS: The concept of manipulating fungal foraging behaviour using nutrients in three-dimensional structures fabricated using Direct Ink Writing (DIW) was explored. Using two fungal strains (Pleurotus ostreatus and Ganoderma lucidum), the effect of varying two different nutrient components (malt and peptone) on the formation of mycelium was investigated. DIW was then used to fabricate structures with local variations in nutrient and the growth behaviour of mycelium from both strains in these structures were investigated.

RESULTS: This study showed that the ink formulation used is suitable for both DIW and mycelium growth. Varying the nutrient content allowed for either the inhibition or promotion of exploration (see Fig 1) and bridging of mycelium in different sections. This also allows for the control of mycelium density in three dimensions and the fabrication of patterned surfaces.

Fig 1. Growth of mycelium from G. lucidum and P. ostreatus (right) on structures (left) printed using DIW containing local spatial variations in nutrient content. Scale bars represent 20 mm.

DISCUSSION & CONCLUSIONS: There is potential for the fabrication of patterned fungal-based ELMs and lab-on-a-chip systems to investigate the effects of other substances such as antifungal compounds and other microorganisms on the foraging behaviour of mycelium. Since mycelium is also comprised partly of chitin, which is a natural polymer commonly investigated for its wound healing properties, there is also the potential for this material to be further processed and utilized as wound dressings.

REFERENCES: ¹ Rodrigo-Navarro, A., Sankaran, S., Dalby, M.J., del Campo, A., and Salmeron-Sanchez, M. (2021). Engineered living biomaterials. Nat Rev Mater 6, 1175–1190. 10.1038/s41578-021-00350-8.

ACKNOWLEDGEMENTS: The authors acknowledge funding from the National Research Foundation of Singapore and ETH Zurich, Switzerland with the grant Future Cities Laboratory Global, Module A4: Mycelium digitalization.

V. Monteiro¹, M. Carvalho¹, I. Freitas¹, M. Rocha¹, A. J.R. Amaral¹, F. L. Sousa², V. M. Gaspar¹ and J. F. Mano¹

¹ Department of Chemistry, CICECO – Aveiro Institute of Materials, University of Aveiro, Campus Universitário de Santiago, 3810-193, Aveiro, Portugal.

² Department of Physics, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal.

INTRODUCTION: The establishment of preclinical models that accurately resemble the native tumor microenvironment at an anatomic human scale are highly desirable to level-up in vitro platforms potential for screening candidate therapies. Herein we leveraged on advanced suspension 3D bioprinting for biofabricating pancreatic ductal adenocarcinoma (PDAC) in vitro models combining gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) with human pancreatic cancer cells to generate in vitro platforms capable of emulate native tumor size and composition. The effect of tumor stroma was also revealed after anti-cancer drug exposure by embedding the bioprinted tumor in a matrix composed by cancer-associated fibroblasts (CAFs). Overall, we report for the first time freeform biofabricated PDAC models exhibiting modular anatomic scale biomimetic features, being highly valuable for drug screening and dose escalation studies.

METHODS: 3D Pancreatic cancer models were bioprinted by using a polymeric supporting bath composed by xanthan gum 1.5% (w/v, PBS) (Figure 1A). The ECM-mimetic bioink was obtained by combining Human Pancreatic cancer (PANC-1) cell line with GelMA 5% (w/v) and HAMA 1% (w/v). The computer-aided design (CAD) model was generated in SOLIDWORKS 2021 3D CAD software, in order to recapitulate native tumor size and the different tumor architectures that pancreatic duct suffers during tumor progression. 3D bioprinting was performed in an extrusion bioprinter by using a 22G nozzle, and a printing speed of 10 mm/s at 20◦C. The 3D models embedded in the suspension bath were then photocrosslinked by using a blue light LED curing system (7.5 min, 9.25 mW·cm²). Tumor-stroma platforms were obtained by embedding the bioprinted tumor model in a matrix composed by pancreatic CAFs and GelMA-HAMA. Bioinks shear thinning behavior and mechanical properties were evaluated through rheology. Bioprinted tumors viability were accessed through Live/dead assays, and the susceptibility to Gemcitabine was evaluated by 3D CellTiter Glo assay was well as the impact of stroma in the therapy efficacy. 

RESULTS:  The results showed that post-printing magnetic resonance imaging (MRI) analysis evidenced that in-bath fabricated tumor constructs are seamless architectures (Fig1,B). Cancer cells laden in such tumor-scale models also remained viable up to 14 days and were responsive to gemcitabine chemotherapeutics in a dose-dependent mode (Fig1,C). 3D tumor-stroma models, where cancer cells are surrounded by the stroma compartment (Fig1,D) enabled to emulate native tumor cellular organization and showed increased resistance to chemotherapy, highlighting the key role of stroma components in tumor resistance.

Fig. 1: Analysis of 3D bioprinted PDAC models. (A) 3D tumor models after recovered from the printing bath. (B) MRI images showing 3D models architecture. (C) 3D tumor models response to gemcitabine and (D) Tumor-stroma bioprinted models.

DISCUSSION & CONCLUSIONS: Overall, herein explored for the first-time suspension 3D bioprinting for generating tumor models with a bioinspired design-to-manufacture approach, so as to fabricate tumor-sized PDAC in vitro models that can be useful as platforms for predictive drug screening analysis.

ACKNOWLEDGEMENTS: This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020, financed by national funds through the FCT/MEC (PIDDAC). This work was also supported by the Programa Operacional Competitividade e Internacionalização (POCI), in the component FEDER. The authors acknowledge the financial support by the Portuguese Foundation for Science and Technology (FCT) through a Doctoral Grant (DFA/BD/7692/2020, M.V.M.). The NMR spectrometers are part of the National NMR Network (PTNMR), partially supported by Infrastructure Project No. 022161 (co-financed by FEDER through COMPETE 2020, POCI, and PORL and FCT through PIDDAC). This work was also funded by the European Union’s Horizon Europe research and innovation programme under the scope of project INSPIRE (Grant agreement ID: 101057777).

R. Pinho¹, M. C. Gomes¹, J. F. Mano¹

¹ CICECO, Department of Chemistry, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal.

INTRODUCTION: Bone-like organoids are being explored as a promising alternative to studying bone tissue and related diseases by simulating physically and biochemically bone activity in vitro. However, it is challenging to mimic the intricate bone-building (osteoblasts) and bone-resorbing (osteoclasts) balance, mineralization, and bone tissue organization [1]. Bioengineered bone-like microtissues have recently been developed using gelatine-based microcapsules (mCAPs) as micro-incubators with mineralization capabilities [2 and 3]. These mCAPs, which contain catechol analogues (Hydroxypiridinone-HOPO, and dopamine) were produced from the coordination of HOPO with Fe(III) ions, showing an immense potential to autonomously promote osteogenesis. Here, we showcase a model able to simulate bone regeneration processes, following the co-encapsulation of human-derived bone marrow mesenchymal stem cells (BM-hMSCs) and monocytes cell line (THP-1).

METHODS: The autonomous differentiation of BM-hMSCs and THP-1 cells, the cellular crosstalk, and the mineralization/reabsorption of the formed extracellular matrix were investigated. As the co-culture progressed, it was observed an autonomous differentiation and activation into bone-forming (osteoblasts) and bone-resorbing (osteoclasts) cells (Figure 1), with gene expression profiles exhibiting an active remodeling. 

RESULTS: Results showed that cellular homeostasis is regulated by key factors able to promote (RANK-L) or inhibit (OPG) the resorption of the produced bone tissue. At the same time, the observation of an intense activity of alkaline and acid phosphatases can be associated to both bone formation and resorption, respectively.

CONCLUSION & DISCUSSION: In the end, an autonomous bone tissue incubator unit was engineered, fostering dynamic mineralization and balanced bone regeneration. Through a harmonious osteoblast and osteoclast crosstalk, the bone-like microtissues nurture a conducive microenvironment, advancing our understanding of bone tissue regeneration mechanisms, while offering a feasible alternative to in vivo assays.

Figure 1: Scanning electron microscopy images of active osteoblasts (OB) and active osteoclasts (OC) in protein-based microunits.

REFERENCES: ¹ A. Salhotra, H. N. Shah, B. Levi, M. T. Longaker (2020) Nat. Rev. Mol. Cell. Biol. 21:696–711. ² M. C. Gomes, D. C. S. Costa, C. S. Oliveira, J. F. Mano (2021) Adv. Healthc. Mater 10:2100782. ³ A. R. Pinho, M. C. Gomes, D. C. S. Costa, J. F. Mano (2024) Small 20:2305029.

ACKNOWLEDGEMENTS: This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020, financed by national funds through the FCT/MCTES (PIDDAC). The authors acknowledge funding from the European Research Council (ERC) through the project “Reborn” (ERC-2019-ADG-883370) and the project “TETRISSUE” (PTDC/BTM-MAT/3201/2020) under the program FCT/PTDC/2020. Ana R. Pinho acknowledges FCT for a PhD grant (2021.05888.BD).

S. Parihar¹, H. Jongaprasitkul^1,2, S. Turunen^1,3, M. Kellomäki¹

¹Biomaterials and Tissue Engineering Group, Faculty of Medicine and Health Technology, Tampere University, Finland.
²Chemistry-School of Natural Science and Environmental Sciences, Newcastle University, Newcastle-upon-Tyne, UK.

³New Materials and Processes Research Group, Faculty of Engineering and Business, Turku University of Applied Sciences, 20520, Turku, Finland.

INTRODUCTION: The utilization of bioprinting is expanding across various fields, including basic tissue engineering, regenerative medicine, personalized medicine, and organ-on-chip technology. Bioink, a crucial component, plays a significant role in crafting suitable 3D scaffolds, essential for creating more physiologically relevant in vitro models. An optimal bioink should exhibit desired physicochemical properties, encompassing appropriate mechanical, rheological, chemical, and biological characteristics. The choice of printing technique relies on factors such as the chosen biopolymers, the crosslinking chemistry of the bioink, and the size and complexity of the intended scaffold. The injectable hydrogels are expanding applications from basic tissue engineering to organ-on-chip technology. These hydrogels can temporarily adapt under stress and restore their original properties due to reversible chemistry.¹ The combination of injectability and self-healing properties in hydrogels offers high tunability for precise delivery with a narrow syringe. Hyaluronic acid (HA) is a natural glycosaminoglycan containing free carboxylic and hydroxyl groups for various chemical functionalization. In this work, we demonstrated the time-dependent auto-oxidation controlled shear-thinning that provides injectability for the hydrogels. We utilized a dual crosslinking approach that included oxidative coupling and photocrosslinking for stabilization on longer time scales. In our previous work, we utilised the dual crosslinking approach to improve hydrogels’ structural integrity and tissue adhesion.^2,3

METHODS: The methacrylated HA (HAMA) was functionalized with gallol (GA) using EDC coupling chemistry (Fig. 1). ¹H-NMR spectroscopy confirmed the degree of methacrylation and conjugation of GA to the HA backbone. The temporal oxidation was studied using UV spectroscopy. The flow behaviour of biomaterial inks and viscoelastic properties of hydrogels were determined from rheological measurements.

Fig. 1: Synthesis of gallol functionalized hyaluronic acid methacrylate (HAMA-GA).

RESULTS: Viscosity modulation and oxidative crosslinking were accomplished through pH adjustment, increasing from 3.5 to 5.5. Further increase in pH to 7 and basic (7.5–8) resulted in true hydrogels instantly. All samples at pH from 3.5 to 5.5 behaved as viscoelastic fluids at the zero-time point, designated as a sol-state, displaying G” > G’, and exhibiting Newtonian fluid behaviour as shown in Figures 2A and B.

Fig. 2: Gelation kinetics of oxidized HAMA-GA ink at different pH levels, over time, and under photocrosslinking conditions. (A) Time-dependent moduli of oxidative crosslinking in HAMA-GA at pH 3.5, 5.5, and 7. (B) Viscoelasticity of HAMA-GA at pH 3.5, 5.5, and 7 at zero-time point, varying degrees of oxidation, measured by frequency sweep. (C) UV−vis spectra illustrating pH-induced oxidative crosslinking. (D) Gelation of HAMA-GA via photocrosslinking, demonstrating the successful integration of methacrylate and gallol groups in the same ink.

Moreover, the sample at pH 3.5 did not exhibit a sol-gel phase transition until the end of observation, suggesting that oxidation did not occur due to the acidic pH. In contrast, the ink at pH 5.5 demonstrated high shear-thinning behaviour on a short-time scale but gradually increased G’ over time. In addition, the results revealed that the ink at pH 7 showed immediate gelation into a gel state (G’ > G”) as the ink began to escape from the parallel plate geometry at high velocity, leading to an assumption that the ink at pH 7 was not appropriate for printing. We found that the temporal oxidation of the gallol group occurs at higher pH (Fig. 2C). The degree of oxidation in the gallol groups corresponding to the pH level, ranging from acidic to basic, in Fig. 2C, confirms the o-quinone formation during oxidation. This is indicated by the peak of o-quinone observed within the absorption range of 400–500 nm. The intensity of the peak increased with higher pH levels.

After rheological characterizations, the biomaterial ink, HAMA-GA at pH 5.5, was printed using an extrusion-based bioprinter. The temporal oxidative crosslinking was utilized to gain shear-thinning ability. After establishing a printability window, the ink was printed into multi-layered grid structures (Fig. 3). The printed structures were photocrosslinked upon exposure to blue light to stabilize the printed scaffolds, using lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP) as the photoinitiator. Notably, we observed the photobleaching effect upon photocrosslinking due to the radical quenching phenomena in the presence of a photoinitiator during photocrosslinking. This effect is also presumed to be influenced by the antioxidant properties inherent in gallol-based molecules and the oxygen inhibition effect in free-radical polymerization.⁴

DISCUSSION & CONCLUSIONS: The study investigated the complex interplay between pH adjustment and temporal oxidative crosslinking for developing biomaterial inks. The findings highlight the temporal dynamics of auto-oxidation and its impact on ink properties. The precursor solution was not printable at a lower pH (3.5) due to a lack of hydrogen bonding and auto-oxidation. Increasing the pH to 5.5 achieved a balance and facilitated shear-thinning behaviour crucial for printing as the weak hydrogels were formed due to hydrogen bonding and slow auto-oxidation. However, over time, oxidative covalent crosslinking leads to the formation of true hydrogels, which lose their printability. Further increases in pH to 7 and beyond resulted in instant gelation due to oxidative coupling, making them unsuitable for printing.

In conclusion, the pH-responsive viscosity modulation and auto-oxidation of HAMA-GA ink exhibited temporal properties influencing its printability, with pH 5.5 yielding optimal shear-thinning behaviour. Our study showcased the ability to control the shear-thinning behaviour of hydrogels through time-dependent auto-oxidation. Post-printing photocrosslinking enhances the stability of the scaffolds, and this two-step crosslinking strategy can be readily adopted to develop a diverse library of bioinks.

REFERENCES: ¹ P. Bertsch, M. Diba, D. J. Mooney, S. C. G. Leeuwenburgh; (2023) Chem. Rev. 123: 834−873. et al. (2000) J Biomech, 33:1471-77. ² H. Jongprasitkul, S. Turunen, V. S. Parihar, M. Kellomäki; (2022) Biomacromolecules, 24: 1, 502– 514. ³ H. Jongprasitkul, V. S. Parihar, S. Turunen, M. Kellomäki; (2023) ACS Appl. Mater. Interfaces, 15: 28, 33972-33984. ⁴ K. Elkhourya, J. Zuazolaa, S. Vijayavenkataraman; (2023) SLAS Technology, 28: 142–15.

ACKNOWLEDGEMENTS: The authors are grateful to the Centre of Excellence in Body-on-Chip Research (CoEBoC) by the Academy of Finland for financial support.

Ryzhkov¹, N. Colson^2,3, E. Ahmed^2,3, P. Pobedinskas^2,3, K. Haenen^2,3, A. Braun¹, P. J. Janssen⁴

¹ Empa. Swiss Federal Laboratories for Materials Science and Technology, Laboratory for High Performance Ceramics, CH – 8600 Dübendorf, Switzerland.

² Institute for Materials Research (IMO), Hasselt University, Wetenschapspark 1, B-3590 Diepenbeek, Belgium.  

³ IMOMEC, IMEC vzw, Wetenschapspark 1, B-3590 Diepenbeek, Belgium.

⁴ Institute for Nuclear Medical Applications, Belgian Nuclear Research Centre, B-2400 Mol, Belgium.

INTRODUCTION: Cyanobacteria are crucial to the global carbon and nitrogen cycles through photosynthesis, making them important for studying factors that influence their light utilization efficiency. Photosynthetic microorganisms show potential for sustainable energy conversion in photovoltaic applications. Previous research has demonstrated that applying an external electric field to microbial biofilms or cells improves electron transfer kinetics and thus increases power generation efficiency. Biophotovoltaic devices are considered for integration into regenerative life support systems (closed-loop systems), such as those used in space exploration or other harsh environments like deserts. Our study involves the monitoring of cyanobacterial absorbance and the measurement of photocurrents at varying wavelengths of illumination for switched electric fields i.e. using the bioelectrode either as an anode or as cathode.

METHODS: We explored the relationship between electrical polarization and changes in light absorbance in a biophotoelectrode setup using boron-doped diamond (BDD) as the semiconductor and live Limnospira indica PCC 8005 trichomes embedded in either polysaccharide (agar) or conductive conjugated polymer (PEDOT-PSS) matrices. This involved monitoring cyanobacterial absorbance and measuring photocurrents under different wavelengths of illumination with switched electric fields, using the bioelectrode as either an anode or a cathode. The light utilization efficiency of cyanobacteria was assessed using Pulse Amplitude Modulation (PAM) as a non-invasive, real-time monitoring tool under external polarization.

RESULTS: We observed changes in absorbance characteristics, indicating a direct connection between electrical polarization and the light-absorbing properties of L. indica. This discovery suggests a promising method for enhancing the performance of biophotovoltaic devices through controlled polarization. Additionally, our findings highlight the wavelength-dependent behavior of a biophotovoltaic system that uses live cyanobacteria [1].

An improved light utilization efficiency for L. indica PCC 8005 when immobilized in conductive matrix was demonstrated. Simultaneously, the impact of electrical polarization as an environmental factor influencing the photosynthetic apparatus diminishes as matrix conductivity increases. This research demonstrates the complex interplay among electrical polarization, absorbance changes, and photocurrent generation in cyanobacterial biophotovoltaics.

DISCUSSION & CONCLUSIONS: The observed increase in cyanobacterial light absorbance under negative electrical polarization, along with cathodic and anodic photocurrent generation depending on polarization, underscore the importance of investigating the interplay between electrochemical conditions and molecular biological processes in the design of energy-generating technologies that are based on living microorganisms. The ability of cyanobacteria to harness light energy and enhance their light absorbance in response to negative electrical polarizations opens up interesting perspectives for rational biophotocathode design. This knowledge is crucial for the optimization of conditions in the development of more efficient bioelectrochemical systems comprising cyanobacteria and boron-doped diamond substrates.

REFERENCES: ¹ N. Ryzhkov, N. Colson, E. Ahmed, P. Pobedinskas, K. Haenen, A. Braun, and P. J. Janssen, ACS Omega. DOI: 10.1021/acsomega.4c03925

ACKNOWLEDGEMENTS: This work is financially supported by the Swiss National Science Foundation under project number 200021E-189455 and Research Foundation Flanders (FWO) grant no. G0D4920N in the framework of “Flanders/Swiss Lead Agency Process: Charge and energy transfer between cyanobacteria and semiconductor electrodes under gamma-irradiation”

M.Charnley^1,2 and S.Russell^1,2

¹Swinburne University of Technology, Melbourne, Australia, ²Peter MacCallum Cancer Centre, Melbourne, Australia

INTRODUCTION: T cells develop in the thymus and this process is strictly regulated to ensure an effective immune response. A critical stage of T cell development is β-selection; at this stage the TCRβ chain is generated, and the developing T cell starts to acquire antigenic specificity. Multiple cues from the niche and asymmetric cell division (ACD), where cell fates proteins are polarized during division and consequently differentially inherited, control this progression through β-selection¹. In this project we aim to tease out the influence of individual cues within the niche responsible for this progression. To achieve this, we developed a model using functionalised surfaces to individually present proteins to the developing T cells.

METHODS: Mice DN3a thymocytes generated in vitro and sorted using flow cytometry.

Functional surfaces were produced by immobilising Fc-linked proteins onto surfaces via protein A.

ACD was assessed using immunofluorescence and time lapse microscopy. Flow cytometry was used to monitor progression through β-selection.

RESULTS: Using this platform, we demonstrated that the cell fate protein Notch acts as a polarity cue to control the distribution of Notch1 itself and other proteins in dividing T cells². Functionalised surfaces were further exploited to deconvolve the influence of different cues in the thymic niche.

Fig. 1: Different polarity cues cooperate to regulate ACD. Functionalised surfaces were used to determine that Notch and CXCR4 signalling triggers ACD. Conversely, the inclusion of VCAM-1 reduced the response.

We used this platform to identify a new cue, E-cadherin, that is critical for T cell development³. In developing T cells E-cadherin contributed to the formation of an immunological synapse and the alignment of the mitotic spindle during division, which facilitated subsequent T cell development.

Fig. 2: A model of the role of E-cadherin in controlling spindle orientation in dividing T cells. (A) E-cadherin enables spindle orientation proteins to align the mitotic spindle with the axis of polarity of cell fate proteins. (B) In the absence of endogenous E-cadherin in the developing T cell, alignment of the mitotic spindle is disrupted.

DISCUSSION & CONCLUSIONS: This work revealed that Notch and CXCR4 signalling are the critical drivers for the polarisation of cell fate determinants and E-cadherin plays an important role in coordinating the orientation of the mitotic spindle with the axis of polarity. Thus, multiple external cues coordinate to dictate how the developing T cell divides and develops. Ongoing work is focused on using functionalised surfaces to determine if changes in division can be correlated with changes in cell fate at the single cell level. Collectively, this work highlights the potential of functionalised surfaces as a tool to complement traditional techniques and provides new insights into the role of cues within the niche during T cell development.

REFERENCES: ¹Pham K. et al (2015) J Cell Biol 210: 933. ²Charnley M. et al (2020) J Cell Sci 133: jcs235358. ³Charnley et al (2023) Sci Adv 9, eade5348

ACKNOWLEDGEMENTS: CASS Foundation (CASS ID 7818) and SNSF (PA00P3_142120 and P300P3_154664).

Shaulli¹, A.M.M. Echeverri², M. Andoni ¹, E. Waeber ¹, S. N. Ramakrishna ³, C. Fritsch ⁴, B.R. Rutishauser ², and F. Scheffold ¹

1) Department of Physics, University of Fribourg, Chemin du Musée 3  CH-1700 Fribourg, Switzerland    

2) Adolphe Merkle Institute, Chemin des Verdiers 4, CH-1700 Fribourg, Switzerland

3) Department of Materials, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, CH-8093 Zurich, Switzerland

4) Department of Biology, University of Fribourg, Chemin du Musée 10 CH-1700 Fribourg, Switzerland

INTRODUCTION: The success of gene therapy hinges on the effective encapsulation, protection, and compression of genes. Quantifying the encapsulation efficiency of small molecules of interest like DNA or RNA into delivery carriers remains challenging. Here, we use super-resolution microscopy, specifically direct stochastic optical reconstruction microscopy (dSTORM), to visualize and measure the quantity of DNA entering a single microgel particle. Utilizing pNIPAM/bPEI microgels as model nano-carriers to form polyplexes, we visualize and quantify DNA entry across different charge ratios and monitor polyplex dynamics under temperature variations. In addition, we compare encapsulation efficiency when varying DNA length and shape. Our study shows that dSTORM is a potent tool for fine-tuning and creating polyplex carrier systems with precise size, shape, and loading capacity at the individual particle level.

RESULTS: By employing advanced imaging techniques such as dSTORM, AFM, and CLSM, we were able to characterize individual DNA molecules and monitor the polyplex architecture with unprecedented clarity, on the molecular level (Fig. 1). We show that the number of DNA molecules encapsulated in a polyplex does not change upon temperature increase indicating no leaks in the polyplex system and establishing them as stable carriers at near body temperature. Moreover, our findings reveal that the choice of DNA length and shape significantly impacts polyplexes’ final structure and loading capacity. We observed that shorter linear DNA molecules allowed for the encapsulation of a greater number of DNA strands compared to longer linear DNA molecules. Furthermore, dSTORM reveals the longer DNA chains hanging outside the complex, influencing the polyplex’s final architecture. The plasmid DNA exhibited a higher encapsulation efficiency than linear DNA under identical conditions, highlighting the importance of considering DNA conformation in polyplex design.

Fig. 1 (a) Schematic illustration of DNA length and conformation. (b,c) Polyplexes formed at 25 °C, 37°C (from left to right) schematic illustration, dSTORM images of single polyplex formed with DNA 500 bp, DNA 3527 bp, and pDNA3527 bp. Scale bar 500 nm. (d) The quantification graph shows in column bars the number of encapsulated DNA for each polyplex system. left- 25 °C and right- 37 °C.

DISCUSSION & CONCLUSIONS: Our findings demonstrate dSTORM microscopy as a quantitative tool for designing synthetic gene delivery carriers. We achieved these important results by elucidating, via dSTORM, the key factors influencing polyplex formation, such as N/P mixing ratios, DNA length, and conformation. These results highlight the importance of visualizing the complex formation at relevant conditions to develop efficient gene delivery systems.

ACKNOWLEDGEMENTS: We acknowledge funding from the European Union’s Horizon 2020. Marie Sklodowska-Curie (ITN SUPERCOL, Grant Agreement 860914)

L. Cao¹, B. Lewille², D. Walle², K. Dewettinck², A. Skirtach¹, and B. Parakhonskiy¹

¹ Nano-Biotechnology Laboratory, Department of Biotechnology, Ghent University, Ghent, Belgium. ² Food Structure and Function Laboratory, Department of Food Technology, Safety and Health, Ghent University, Ghent, Belgium.

INTRODUCTION: Biomaterials composed of food polysaccharides and proteins have garnered significant interest due to their exceptional biocompatibility, tunable mechanical properties, and intricate architectural designs. In this study, a stable emulsion-filled gel of alginate and gellan gum was developed by a mild emulsification and gelation approach, with the aim of encapsulating curcumin. Various formulations of the emulsion-filled gel matrices were systematically investigated by varying the emulsion fractions (10, 30, 50, and 70%), allowing for the comprehensive characterization of their microstructural features, mechanical properties, and swelling behavior. Upon encapsulation of curcumin, crucial parameters such as encapsulation efficiency, in vitro simulated digestion kinetics, and antioxidant activity of the encapsulated bioactive ingredients were assessed. The encapsulation of curcumin within these emulsion-filled gels resulted in remarkable stability against heat and ultraviolet radiation and significantly enhanced its bioaccessibility during in vitro digestion. These findings might facilitate the preparation of emulsion-filled gels with excellent stability for bioactive compound delivery in food and pharmaceutical applications.

METHODS: The alginate/gellan gum mixture stocking solution was prepared by dispersing sodium alginate (1.65%, w/v) and gellan gum (1.65%, w/v) in deionized water and stirring with a magnetic stirrer at 60 ^oC for 6 h, and then placed overnight at 4 ^oC to allow complete hydration. Different ratios of emulsion and alginate/gellan gum solution (v/v: 0:10; 1:9; 3:7; 5:5; 7:3; 10:0) were mixed to prepare emulsion-filled gel matrices with different contents of emulsion (0%, 10%, 30%, 50%, 70%, and 100%). The final concentration of alginate/gellan gum solution in each formulation was 1% (w/v). For gelation, firstly, CaCO₃ (0.18 g) was dispersed into the emulsion and alginate/gellan gum mixture solution (10 mL) to form a suspension with stirring at 2000 rpm for 5 min. Then, to induce the crosslinking of Ca²⁺ with alginate and gellan gum, GDL (0.36 g) was added to the above suspension with stirring at 2000 rpm for 30 s. Consequently, the mixed dispersions were quickly poured into a 24-well plate and incubated at room temperature for 24 h. Finally, the formed emulsion-filled gel matrices were washed three times and stored in deionized water at 4 ^oC until a further experiment was performed.

The comprehensive characterizations of emulsion-filled gel matrices, including stability, microstructural features, mechanical properties, encapsulation efficiency, and antioxidant activity, were investigated by a Nikon TI microscope, scanning electronic microscopy, universal testing machine, and UV-visible spectrophotometer.

RESULTS: Within a certain range of emulsion fraction, increasing the content of emulsion improved the gel matrix mechanical properties, as well as the anti-swelling properties. Simulated gastrointestinal fluid digestion indicated that emulsion-filled gel can tolerate the damage imposed by the gastric environment, and sustain the release of curcumin in the intestinal conditions.

Figure 1. Schematic diagram of preparation of emulsion-filled gel loading with curcumin.

DISCUSSION & CONCLUSIONS: We developed a facile method to prepare physically crosslinked alginate and gellan gum-based emulsion gel with different W/O emulsion content. The advantages of the emulsion and hydrogel network characteristics were exploited to encapsulate the hydrophobic functional ingredient curcumin into this emulsion-filled gel matrix. This study has provided some important and useful information regarding the design and development of delivery systems for hydrophobic functional ingredients.

ACKNOWLEDGEMENTS: China Scholarship Council (CSC No. 202106910013), FWO (Research Foundation Flanders): G043322N, I002620N, and EOS (Excellence of Science) program: 40007488.

Isli Cela¹, Trang-Anh Nguyen-Le¹, Christin Neuber¹, Željko Janićijević¹, Daniel Nieder¹, Liliana Rodrigues Loureiro¹, Lydia Hoffmann¹, Anja Feldmann¹, Michael Bachmann¹, Larysa Baraban^1,2

¹ Helmholtz-Zentrum Dresden-Rossendorf e. V. (HZDR), Dresden, Germany.

² Dresden University of Technology, Dresden, Germany

INTRODUCTION:  Universal Chimeric Antigen Receptor (UniCAR) T-cell therapy, has proven to be a successful and safer cancer treatment strategy especially in targeting non-solid tumors. Unlike conventional CAR T-cell, UniCAR T-cells recognize the Tumor Associated Antigen (TAA) indirectly through an adaptor molecule called the Target Module (TM). The administration of the TMs should be carefully monitored, in order to avert undesired effects such as cytokine release syndrome (CRS) or tumor lysis syndrome (TLS), and the dose administration needs to be adjusted according to the patient´s response ¹. Established monitoring techniques such as Enzyme-Linked Immunosorbent Assay (ELISA), Positron Emission Tomography (PET) or Magnetic Resonance Imaging (MRI) face their own limitations such as low-sensitivity, high cost and radiolabeling requirements ². Therefore, in this pilot study we introduce an Extended Gate Field Effect Transistor (EG-FET) based biosensor for label-free, high sensitivity and low-cost monitoring of the TM concentration. Additionally, our sensor is an effective tool for tracking the pharmacokinetics of TMs in mice models, demonstrating results comparable to those achieved by conventional radiolabeling.

METHODS: The EG-FET sensing platform was built in house, combining the readout based on a commercial n-channel enhancement mode MOSFET with a multiplexing module, thus allowing to perform multiple measurements simultaneously. The sensing chip was fabricated via a photolithography process on a glass substrate, followed by plasma-activated bonding of PDMS reservoirs. The sensing surface was biofunctionalized with a small peptide using cysteamine/glutaraldehyde crosslinking chemistry. Using this peptide and the indirect sensing strategy, the same sensing chip could be utilized to detect different TMs, making the sensor more versatile. For the pharmacokinetics study, Cu-64 labelled TMs were injected in mice and blood was withdrawn 1 min post-injection, followed by an end-point withdrawal at a different timepoint for each mouse (t_end= 5 min, 30 min, 60min, 3h, 6h, 24h, 48h). The samples were subjected to radioactivity and EG-FET measurements.

RESULTS: In EG-FET sensing, we measured potential changes caused at the surface of the gate electrode. Due to the employed indirect sensing strategy, greater voltage shifts from the baseline were observed at lower concentrations of TMs. Calibration curves for analytes of different sizes are shown in Fig. 1A. We demonstrate superior sensitivity of the sensor compared to ELISA even in complex media. Based on the measured radioactivity and surface potential changes from samples at different timepoints, we constructed time-series graphs (Fig. 1B) showing that both methods suggest similar decay trends for the TMs in this study.

Fig. 1: (A) Sensor calibration curves in diluted serum for different sized TMs (B) Concept and results of the pharmacokinetics study.

DISCUSSION & CONCLUSIONS: Results of the pilot study showcase that our EG-FET biosensor is a promising, label-free, portable, and cost-efficient tool for monitoring immunotherapeutic drug administration. Further research on the optimization of the sensing system will aim towards achieving improved accuracy suitable for direct, real-time clinical applications.

REFERENCES: ¹ M. Bachmann (2019), The UniCAR system: A modular CAR T cell approach to improve the safety of CAR T cells, Immunology Letters, 211, 13-22 ² B. Beuthien-Baumann, C. Sachpekidis, R. Gnirs, & O. Sedlaczek (2021), Adapting Imaging Protocols for PET-CT and PET-MRI for Immunotherapy Monitoring, Cancers (Basel), 13(23)

ACKNOWLEDGEMENTS: We acknowledge the financial support of the European Research Council (ERC) through the ERC- Consolidator Grant (ImmunoChip, 101045415).

K.S. Park¹

¹ Department of Biological Engineering, Konkuk University, Republic of Korea.

INTRODUCTION: Colorectal cancer (CRC) as the second leading cause of global cancer deaths poses critical challenges in clinical settings. Cancer-derived small extracellular vesicles (sEVs), which are secreted by cancer cells, have been shown to mediate tumor development, invasion, and even metastasis, and have thus received increasing attention for the development of cancer diagnostic or therapeutic platforms. In this work, the sEV-targeted systematic evolution of ligands by exponential enrichment (E-SELEX) is developed to generate a high-quality aptamer (CCE-10F) that recognizes and binds to CRC-derived sEVs.

METHODS: In our E-SELEX procedure (Fig. 1), negative selection was performed to eliminate off-target ssDNA that nonspecifically binds to the components in buffer reagents and the immunoplate itself. The unbound ssDNAs obtained from negative selection were then enriched for further selection comprising three E-SELEX loops, where each loop (indicated as A, B, and C-loops) contained four consecutive positive selections and one counter selection consisting of a total of 15 individual selection steps. Considering their high metastatic potential, SW620 cell-derived sEVs were chosen as the target for positive selection while healthy HS-derived sEVs were selected as the target for counter selection to mimic the clinical setting for potential in vivo applications. Once the final loop was reached, aptamer pools were analyzed by next-generation sequencing (NGS) to obtain sequences of aptamer candidates and their attributes were further evaluated for potential diagnostic and therapeutic applications.

Fig. 1. Workflow of E-SELEX and its evaluation.

RESULTS: This novel aptamer possesses high affinity (K_d = 3.41 nm) for CRC-derived sEVs and exhibits a wide linear range (2.0 × 10⁴ -1.0 × 10⁶ particles µL^-1) with a limit of detection (LOD) of 1.0 × 10³ particles µL^-1. Furthermore, the aptamer discriminates CRC cell-derived sEVs from those derived from normal colon cell, human serum, and other cancer cells, showing high specificity for CRC cell-derived sEVs and significantly suppresses the critical processes of metastasis, including cellular migration, invasion, and angiogenesis, which are originally induced by sEVs themselves (Fig. 2).

Fig. 2. Effect of sEVs and Apt-sEVs on HUVEC angiogenesis.

DISCUSSION & CONCLUSIONS: We expect that this work will serve as fundamental research and have a significant impact on molecular diagnostics and therapeutics by i) providing valuable information on biomarkers that are specifically expressed in CRC cells and their sEVs, ii) contributing to the development of various aptamer-based biosensors to specifically detect CRC sEVs, iii) suggesting a novel therapeutic modality with a combination of chemotherapeutics and siRNA to alleviate sEV-mediated tumor progression, and iv) advancing potential application of targeted drug delivery with the help of DNA nanotechnology.

REFERENCES: B. S. Cha, Y. J. Jang, E. S. Lee, D. Y. Kim, J. S. Woo, J. Son, S. Kim, J. Shin, J. Han, S. Kim, K. S. Park, Adv Healthc Mater 2023, 12, 2300854.

ACKNOWLEDGEMENTS: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government(MSIT)[NRF-2020R1C1C1012275].

Nikoletić^1,2,3, D. Ikkenne², E. Natsaridis¹, C. Palivan^2,3, O. Tagit^1,3

¹ FHNW, Muttenz, Switzerland.

² University of Basel, Basel, Switzerland.

³ Swiss Nanoscience Institute, Basel, Switzerland.

INTRODUCTION: Polymers that display either a lower critical solution temperature (LCST) or an upper critical solution temperature (UCST) in an aqueous medium may be the key to developing thermoresponsive nanocarriers for spatial and temporal control over delivery. Polymers with LCST have hydrated polymer chains below the critical temperature (cloud point), but once heated beyond the LCST they undergo entropy-driven hydrophobic collapse and aggregation.¹ Poly(oligo ethylene glycol acrylates) are a class of thermoresponsive polymers that have gained interest in recent years. These polymers offer a variety of LCST and levels of hydrophobicity and are promising building blocks for materials with LCST above physiological temperatures.²

METHODS: Herein, we present the synthesis of a novel thermoresponsive amphiphilic block copolymers based on polydimethylsiloxane (PDMS) and poly(di- and poly(tri(ethylene glycol)  methyl ether acrylate) (PDEGA and PTEGA). We have synthesized a library of block copolymers using RAFT polymerization to investigate the influence of block chain lengths on the thermoresponsive behaviour and architecture of self-assembled structures. Synthesized copolymers were characterized by ¹H NMR spectroscopy and GPC, and polymer self-assemblies were characterized with dynamic light scattering (DLS) and transmission electron microscopy (TEM). To evaluate the thermoresponsive behaviour of copolymers, DLS and turbidimetry (cloud point) experiments were conducted in aqueous solutions.

RESULTS: We synthesized a library of new PDMS-b-PDEGA and PDMS-b-PTEGA polymers with varying block lengths while maintaining precise control over polymer molecular weight and polydispersity. Obtained block copolymers display LCST in aqueous solutions, with critical temperature depending on block lengths. When heated above LCST, the hydrophilic PDEGA and PTEGA blocks undergo hydrophobic transition, leading to reversible aggregation behaviour. Our polymers self-assemble into uniformly sized micelles (Figure 1).

Fig. 1: TEM images of micelles formed by self-assembly of PDMS-b-PDEGA (left) and PDMS-b-PTEGA (right) polymers.

DISCUSSION & CONCLUSIONS: New poly(oligo ethylene glycol acrylates) block copolymers with critical temperatures above physiological are promising materials for advanced drug delivery systems. Thermoresponsive micelles based on our amphiphilic block copolymers can be used to encapsulate different cargoes during self-assembly. Once heated above their LCST, release rate is significantly enhanced due to collapse of nanocarriers.

REFERENCES: ¹ A. Bordat, T. Boissenot, J. Nicolas, et al (2019) Adv. Drug Deliv. Rev. 138: 167-192. ² G. Vancoillie, D. Frank, and R. Hoogenboom (2014) Prog. Polym. Sci. 39: 1074–1095.

ACKNOWLEDGEMENTS: This work was supported by the Swiss Nanoscience Institute. I want to thank Dr. Daniel Messmer and John Coats for helpful discussion regarding polymer synthesis.

H. Kolbe¹, L. Strauss², D. Pappas³, D. B. Salem⁴, T. Schmitt-John⁴

¹ The University of Adelaide, Adelaide, Australia.

² University of the Bundeswehr in Munich, Neubiberg, Germany. 

³ Plasmatreat USA Inc., Hayward, California, USA.

⁴ Plasmatreat GmbH, Steinhagen, Germany

INTRODUCTION: The interface between implants and cartilage or bone tissue is crucial for successful integration within the human body and overall treatment success. In addition to promising mechanical properties, polymeric implant materials like polyetheretherketon (PEEK) often show bioinert surface properties. These properties must be tailored through modifications to reduce the risk of inflammation and to improve biocompatibility and cell adherence. Plasma processes are already well established within the medical field for activation, sterilization and coating applications through plasma-enhanced vapour deposition (PECVD). Generated coatings can enhance the bonding and biocompatibility of polymers and metals and can be used to create customized implant tissue interfaces.

METHODS: Low and atmospheric pressure plasma were used to create various surface modifications and coatings on important implant materials including stainless steel and polymers like Polyetheretherketon (PEEK) as shown in Fig 1. The research investigates the biocompatibility, antimicrobial properties and cytotoxic effects of the generated plasma modifications.

Fig. 1: PECVD coatings for improved biocompatibility of implant surfaces. PECVD coatings and functionalized surfaces SM1 to SM4 and A1-2 using atmospheric pressure plasma (SM1& A1-2) and low-pressure plasma (SM 2 to 4). Adjusted form ¹ with permission.

RESULTS: The generated coatings and surface modifications were assessed and demonstrated improved wettability, at no cytotoxic effects (Fig 2). These treatments also increased the cell attachment and viability of osteoblast cells. The coatings have the potential to enhance the biocompatibility of implant materials while maintaining bulk properties. Other coatings demonstrated antimicrobial properties, reducing the risk of inflammation and infections in the early stages of implantation.

Fig. 2: Proliferation of cultured osteoblast after 7 days in culture on test surfaces SM1 to SM4 in relation to uncoated polymer surface, SM0. Proliferation rates were determined relatively to confluent cells grown in a common multi well plate after 7 days of incubation (positive control = 100 %), * represents statistical significance (p < 0.05).¹

DISCUSSION & CONCLUSIONS: Plasma modification and coatings can improve the surface properties of implant materials to achieve functionalised, non-toxic interfaces for improved bioactivity, osseointegration and antimicrobial properties.

REFERENCES: ¹ L. Strauss, A. Bruyas, R. Gonzalez, D. Pappas, D. Ben Salem, E. Krampe, T. Schmitt‐John, S. Leonhardt, Plasma Process. Polym. 2024;21:e2300082.

Jung¹, A. Moya², P. Pfister^2,3, R. Paillaud², R. Marek¹, I. Martin², A. Scherberich², M. de Wild¹

¹ University of Applied Sciences Northwestern Switzerland, Institute for Medical Engineering and Medical Informatics, Muttenz, CH.

² Department of Biomedicine, University Hospital Basel, University of Basel, Basel, CH.

³ Department of Plastic, Reconstructive, Aesthetic, and Hand Surgery, University Hospital Basel, Basel, CH.

INTRODUCTION: Symbrachydactyly is a rare congenital malformation of upper limb indicated by short or even absent fingers in children¹. Adipose-derived stromal cells (ASC) from adult and paediatric donors are used in this project to engineer osteogenic grafts for phalanx construction. The aim of this work is to investigate their biomechanical properties. Additionally, densitometric testing of volumetric tissue density and tissue dimensions is performed by quantitative contrast-enhanced computed micro-tomography (µCT). Mineralization is then compared to the elastic modulus of the tissue engineered constructs.

METHODS: To engineer the phalanges, ASCs were associated with commercially available collagen sponges and assembled to form in vitro cartilaginous tissue constructs. They were then implanted ectopically in immunocompromised mice for up to 12 weeks to undergo in vivo tissue remodelling via endochondral ossification (ECO), see Fig. 1a. Two key methods were utilised to determine the structural and mechanical properties of the engineered phalanges: The specimens were imaged using contrast-enhanced μCT (SkyScan 1172, NV) with Phosphotungstic Acid staining² to calculate their dimensions and tissue composition (Fig. 1b). Furthermore, 3-point bending tests were conducted using a custom-made test setup (UMT-Tribolab, Bruker, Fig. 1c) for the biomechanical assessment of stiffness and deformation from the elastic part of the load-displacement curve.

RESULTS: The μCT imaging showed consistent sample dimensions throughout the study (length: 6 to 14 mm, width: 2 to 5 mm). After 4 weeks of implantation, early signs of endochondral ossification were evidenced by the mineralization of the implanted cartilage tissues. However, it revealed significant differences between the different donors tested. The biomechanical testing demonstrated a continuous increase in the elastic modulus of the phalange grafts across the different time points, indicating improved mechanical strength (Fig. 2a). Additionally, a positive correlation between the elastic modulus and the percentage of mineralised tissue provided valuable insights into the bone remodelling processes associated with the engineered phalanges (Fig. 2b).

Fig. 1: a) Contrast-enhanced μCT imaging by Phosphotungstic Acid staining of a phalanx graft (7 mm long). b) Analysis of the mineralization. c) Biomechanical 3-point bending test.

Fig. 2: a) Relative Elastic Modulus and b) its correlation to mineralization after in vitro, 4 weeks and 12 weeks of implantation in vivo.

DISCUSSION & CONCLUSIONS: This work contributes valuable knowledge about the biomechanical properties of engineered phalanges. The observed correlation between the observed mechanical properties and tissue composition supports the validation of the functionality of the engineered paediatric phalanges.

REFERENCES: ¹ A. Bartsch et al (2023) Correction of symbrachydactyly: a systematic review of surgical options, Syst Rev 16;12(1):218. ² S. Sutter et al., Contrast-Enhanced Micro-tomographic Characterisation of Vessels in Native Bone and Engineered Vascularised Grafts Using Ink-Gelatin Perfusion and Phosphotungstic Acid (2017) Contrast media & molecular imaging 017, 4035160.

ACKNOWLEDGEMENTS: The ALBUCOL project is supported by the Region Grand Est, Land Baden-Württemberg, Land Rheinland-Pfalz, Cantons Baselstadt, Basellandschaft, Swiss Confederation and by the program INTERREG Upper Rhine from the ERDF (European Regional Development Fund).

Soumya Sethi¹, Andreas Walther²

¹ JGU Mainz.

²JGU Mainz, Max Plank Institute of Polymer Research

INTRODUCTION: Cells transduce mechanical signals into biochemical signals to regulate their fate, these processes are governed by interactions between the cell surface receptors and the cytoskeleton. These cell surface receptors interact with their respective ligands and generate piconewton forces. Various DNA based tension probes have paved the path for understanding the complexities of mechanotransduction.  Despite the many advantages of DNA based probes, they suffer from degradation issues in cell culture medium and thus, long term cellular phenomenon like cell migration, cell division, differentiation etc. are still challenging to map in terms of forces. Here, we introduce L-DNA (stereoisomer of D-DNA) probes that are remarkably resistant to nucleases and sustain cell culture environments for long periods of time.

METHODS: The design of the DNA probes was inspired by previously published articles^[1–3]. Briefly, DNA was immobilised onto glass coverslips using BSA biotin and neutravidin. The DNA was judiciously designed to quench the fluorescence signals and conjugated with an integrin ligand cRGD. Cells used in the study were cultured according to ATCC guidelines.

RESULTS: A direct comparison of L-DNA probes and D-DNA probes in various geometries was carried out, which made it clear that the L-DNA probes were stable in cell culture conditions for several days. Firstly, using L-DNA probes we mapped forces of cells for several days and during important phenomenon like cell migration, division etc. Secondly, L-DNA probes were used to capture the most minute cellular forces during processes like membrane ruffling. Lastly, these probes were used to map forces of collective cell sheets in processes like wound healing. These finding help us delve further into understanding mechanobiology.

Fig. 1: Describes traction force applied by cells as they adhere to surface (left), Depicts the 2 types of DNA – biological and artificial and their degradation potential (right).

DISCUSSION & CONCLUSIONS: We have presented L-DNA – non biological stero-isomer of D-DNA mechanoprobes for imaging cellular traction forces. This new class of probes can not only map cellular forces for significantly long periods of time with utmost detail and clarity but also help us better understand forces involved in in vivo scenarios like wound healing, morphogenesis etc.

Thus, making them highly beneficial in expanding our knowledge of how the cells interact with the extracellular environment and among themselves.

REFERENCES:

[1] Y. Liu, K. Galior, V. P.-Y. Ma, K. Salaita, Acc. Chem. Res. 2017, 50, 2915–2924.

[2] Y. Zhang, C. Ge, C. Zhu, K. Salaita, Nat. Commun. 2014, 5, 5167.

[3] Y. Duan, R. Glazier, A. Bazrafshan, Y. Hu, S. A. Rashid, B. G. Petrich, Y. Ke, K. Salaita, Angew. Chem. Int. Ed. 2021, 60, 19974–19981.

ACKNOWLEDGEMENTS: We express our gratitude to all the Walther lab members and our funding agencies.

Abdallah ALHALABI¹ʻ²; Christine SAINT-PIERRE²; Didier GASPARUTTO²; Xavier LE GUEVEL¹

¹ Univ. Grenoble Alpes, Institut pour l’Avancée des Biosciences, Site Santé – Allée des Alpes, Grenoble, France

² Univ. Grenoble Alpes, CEA, CNRS, SyMMES-UMR 5819, F-38000 Grenoble, France

KEYWORDS: self-assembly, gold nanoclusters, DNA nanostructures, photoluminescence, biosensors, imaging probes.

INTRODUCTION: The development of multidimensional self-assembled nanostructures has gained a lot of attention recently for their biophotonic applications such as biosensors, imaging probes, implant devices, and remotely activated therapies¹.

In this context, we are particularly interested in a promising class of atomically precise ultra-small particles (3nm<) called Gold NanoClusters (AuNCs) which show unique photoluminescence properties tunable from UV to infrared range (370-1300 nm) with high photostability and biocompatibility. In addition, AuNCs are easy to functionalize, and it was shown recently striking modifications their optical properties when they  assemble into different ^2,3.

We aim first to better understand the change of the absorbance and photoluminescence of atomically precise AuNC when they assemble by precisely controlling the distance between AuNCs. The second task is to define their behavior in complex environment (serum, intracellular) and evaluate their potential as optical transductors for biosensing applications. 

RESULTS: To this purpose, we have chosen DNA nanotechnology as it is one of the most successful approaches to design well-controlled nanostructures with sub-nano resolution, to build one-, two- and three-dimensional DNA assemblies with AuNCs emitters. We showed on the first stage of this project the ability to control the stoichiometry of single stranded Oligonucleotides grafting to different sizes of atomically precise Au_nNCs (n=22, 25). The control of the hybridization of such species and therefore controlling precise the distance between clusters were subjected to extensive analytical and optical characterizations (see Fig. 1 ).

Fig. 1: a schematic representation of the fabrication of 1D, 2D & 3D gold nanoclusters superstructures by controlling the biofunctionalization of gold nanoclusters with DNA.

REFERENCES:

1. Pang, Z. et al. Multifunctional Gold Nanoclusters for Effective Targeting, Near-Infrared Fluorescence Imaging, Diagnosis, and Treatment of Cancer Lymphatic Metastasis. ACS Nano 16, 16019–16037 (2022).
2. Musnier, B. et al. High photoluminescence of shortwave infrared-emitting anisotropic surface charged gold nanoclusters. Nanoscale 11, 12092–12096 (2019).
3. Yahia-Ammar, A., Sierra, D., Mérola, F., Hildebrandt, N. & Le Guével, X. Self-Assembled Gold Nanoclusters for Bright Fluorescence Imaging and Enhanced Drug Delivery. ACS Nano 10, 2591–2599 (2016).

ACKNOWLEDGEMENTS: This research project is financially supported by the Grenoble Alpes University (ANR-17-EURE-0003).

Hidaka, M. Kojima, I. Horiguchi, S. Sakai

Division of Chemical Engineering, Graduate School of Osaka University

INTRODUCTION: 3D bioprinting is a technology that constructs three-dimensional (3D) structures using cells and biomaterials (bioink) with a 3D printer. To create functional 3D structures, it is essential to control the stiffness and cell density of the bioink during the printing process. Therefore, nozzles with micromixers have been explored to meet this demand. However, current passive mixers have limitations in mixing viscous bioinks and require a long time to achieve homogeneous mixing¹. Additionally, active mixers can cause cell damage due to high shear stress during mixing². Given these challenges, we focused on a micromixer driven by acoustic stimulation. This mixer uses microstreaming induced by bubbles trapped in the nozzle path under acoustic stimulation, enabling gentle yet rapid mixing in a smaller mixing chamber³. Our aim was to develop a novel type of nozzle with an acoustic micromixer for 3D bioprinting and evaluate its performance with a typical bioink, sodium alginate (SA).

METHODS: The micromixer has two inlets and one outlet with a diameter of 700 µm. Holes to trap bubbles were placed along the path of the mixer. Microstreaming was generated by acoustic stimulation from a piezo transducer installed beside the nozzle. The effects of bubble size, frequency of acoustic wave, and voltage applied to the piezo on microstreaming strength were evaluated using a sodium alginate (SA) aqueous solution containing a pigment. Next, the effect of bubble arrangement was investigated using three types of nozzles: one with a single bubble, one with two parallel bubbles, and one with two bubbles arranged in a zigzag pattern. SA solutions with and without pigment were flowed through the nozzles simultaneously at a flow rate of 0.25 mm/s under acoustic stimulation. Mixing efficiency (MI) was calculated from the gray-scale images obtained at the outlet area.

where Ii  denotes the white-point intensity of the mixing chamber shown as a red square in Fig 1(b),  represents its average intensity, and N indicates the number of sampling points. Based on the results, the nozzle with high MI was proposed and its mixing performance was evaluated. Additionally, SA ink containing 10T1/2 cells were mixed inside the nozzle under the acoustic stimulation, and the cell viability was evaluated.

Fig. (a) Schematic of the acoustic micromixer and photo of bubble design (b) Effect of flow rate on mixing (c) Printed line structures with and without acoustic stimulation (d) Effect of acoustic stimulation on cell viability in acoustic micromixer.

RESULTS & DISCUSSION: From the observation results of microstreaming, the bubble design shown in Fig. (a) exhibited the most prominent stream at 3.0 kHz and 45 V. Additionally, the zigzag bubble arrangement demonstrated efficient mixing. Based on these results, a nozzle design with six bubbles arranged in a zigzag pattern was proposed (Fig. (a)). High mixing efficiency (over 80%) was achieved at flow rates of 0.45-1.8 mm/s (Fig. (b)) when mixing 0.5 wt% SA solutions with and without pigment inside the nozzle. Finally, a line structure of 0.5 wt% SA solutions with different colors was successfully printed using the nozzle under these conditions (Fig. (c)). Moreover, there was no significant difference in cell viability between conditions with and without acoustic stimulation, indicating the effectiveness of the acoustic micromixer in 3D bioprinting (Fig. (d)).

CONCLUSIONS: We developed the acoustic nozzle for mixing bioinks, and showed its effectiveness for bioprinting application.

REFERENCES: ¹Lavrentieva et al, (2020), Macromol Biosci, 20: 200107. ²Blaeser et al, (2016), Adv Healthc Mater, 5(3): 326-333.³Rasouli et al, (2019), Lab on a chip, 19: 3316-3325

ACKNOWLEDGEMENTS: This study was supported by JST SPRING (JPMJSP2138) and JSPS KAKENHI (24KJ1594).

Selina Camenisch¹, Natalia Velez Char², Marian Neidert², Anna Zeitlberger², Thomas Hundsberger², Wolfram Jochum², Peter Wick¹, Vanesa Ayala-Nunez¹

¹Swiss Federal Laboratories for Materials and Science and Technology (Empa), St. Gallen, CH

² Kantonsspital St. Gallen, St. Gallen, CH

INTRODUCTION: Glioblastoma is the most frequent and malignant primary brain tumor. It carries a poor prognosis despite advances in surgery, radio- and chemotherapy. Human organoids are an excellent alternative to recapitulate a human organ-specific architecture and microenvironment in vitro. In this project, we propose an ex vivo glioblastoma organoid model embedded in an organ-on-chip platform that is fully patient-derived, with tissue taken from different tumor sub-regions, and vascularized. This patient-derived system can be used for predicting the response to treatment with different chemotherapeutics and/or radiotherapy, aiding in the decision making process of clinicians.

METHODS: To generate the patient-derived organoids (PDO), we used patient navigated biopsies from three different sub-regions of glioblastoma (hypoxic/necrotic core, contrast-enhancing, and peritumoral infiltration zone). The procedure was based on an established protocol by Jacob et al.¹ The patient material was provided by the Kantonsspital St. Gallen (KSSG) in Switzerland.

Afterwards, we investigated the cell composition, viability and drug response of the organoids. The composition and viability were characterized through immunohistochemistry stainings (H&E, GFAP, MAP2, CD45, CD3, CD68, and Ki-67). The evaluation was done with the assistance of a neuropathologist.

To characterize the drug response of the organoids, we treated them with Temozolamide (TMZ) under a similar therapeutic scheme used in patients (Stupp regimen). We evaluated organoid’s size, NGS mutational profiling, apoptotic/necrotic cell fraction, cell metabolic activity (XTT assay), and toxicity (LDH assay). The apoptotic cell fraction was quantified by analyzing the nuclear morphology in H&E stained tissue slices with a pre-trained AI-driven image readout software.

RESULTS: It was possible to establish glioblastoma organoids from the three sub-regions of the tumor. The morphology, growth rate and cell composition were patient- dependent. The histophathology analysis showed that the organoids preserved features from the original tumor tissue: abundant pleomorphic nuclei (like high-grade gliomas) and the presence of macrophages, leukocytes (including T lymphocytes) and proliferating Ki-67⁺ cells.

Using glioblastoma spheroids and patient-derived organoids, we established a multidimensional drug assessment tool to characterize the response to drugs with anti-tumor activity. With this tool, it was possible to quantify the effects of TMZ on tumor size reduction, metabolic activity, cytotoxicity, appearance of new drug-induced mutations, and extent of apoptosis and necrosis. The in vitro data will be compared to clinical data to define the predictivity of our model.

DISCUSSION & CONCLUSIONS: This project sets the basis for a patient-relevant experimental system that can be used to determine effective drug doses and combinations in a patient-specific manner, but also to screen for novel anti-cancer therapeutics.

REFERENCES: ^1.       F. Jacob et al. Nat Protocols 2020, Vol 15, 4000-4033

Sivakumar¹, J. Pinheiro Marques¹, A. Roux¹

¹ Tissue Engineering Group, HEPIA HES-SO Genève, Geneva, Switzerland

INTRODUCTION: Cell barrier integrity is crucial for proper regulation of molecule and ion movement between different tissues. Understanding mechanisms leading to barrier dysfunction in disease, such as gastrointestinal and neurological disorders, is essential for the development of new therapies^1,2. This study introduces an advanced, cost-effective biochip designed to measure Transendothelial/epithelial Electrical Resistance (TEER) in an Barrier-on-Chip model.

METHODS: The electrodes were microfabricated using photolithography, polyimide deposition, Pt sputtering, lift-off, and etching techniques. The designs were created in CAO and converted for cleanroom fabrication, followed by anodic dissolution to release the electrodes. The fluidic device was constructed using fast prototyping techniques, including laser-cut stacked cast PMMA for precision. It was bonded with double-sided tape and incorporated a semi-permeable PET membrane. Fluid perfusion was facilitated with polycarbonate female Luers and clear optical epoxy for waterproofing. Caco2 cells were seeded into the fluidic channels and cultured under standard conditions to validate the device’s performance. Impedance spectrometry and TEER were measured using an in-house measuring device (TEEROC)³.

Fig. 1. On the left, CAD exploded view of the designed device, comprising the two Pt over PI electrodes, the PCB with a mounted horizontal header and the fluidic PMMA layers with the semi-permeable  PET membrane and the female luerlock. On the right, a produced device picture after cell seeding. Male luer plugs are closing the fluidic channels.

RESULTS: The electrodes, PCB, and OOC designs were changed to improve production efficiency and performance. Experiments with Caco2 cells confirmed reliable TEER measurements, validating the device’s application for in vitro cell barrier studies.

Fig. 2. Measured average TEER of Caco2 cells over four days using two Barrier-on-Chip devices. Measurements were done in triplicate; error bars represent standard deviation.

Fig. 3. Confocal image of Caco2 layer after 7 days of culture. the cells nuclei are represented in blue, using DAPI, and cells tight junctions are expressed in red using ZO-1.

DISCUSSION & CONCLUSIONS: The advanced electrode design and compact PCB integration of this Barrier-on-Chip device significantly enhance TEER measurement accuracy. Its versatile PMMA structure and electrode designs allow rapid customisation for different biological barriers, paving the way for broader tissue engineering and biomedical research applications. Future work will focus on optimising the device for diverse cell types and drug testing scenarios.

REFERENCES: ¹Choublier, N. et al. Blood–Brain Barrier Dynamic Device with Uniform Shear Stress Distribution for Microscopy and Permeability Measurements. Appl. Sci. 11, 5584 (2021). ²Wei, W., et al. 3D In Vitro Blood‐Brain‐Barrier Model for Investigating Barrier Insults. Adv. Sci. 10, 2205752 (2023). ³Roux, A. et al. Assessment of the in vitro TEER measurement for toxicology screening. Toxicol. Lett. 238S, S56-S383 (2015)

ACKNOWLEDGEMENTS: Foundation Campus Biotech Geneva and Neural Microfabrication Platform for cleanroom facilities and technical support, HEPIA and HES-SO for funding.

Prannoy Seth¹, Jens Friedrichs¹, Yanuar Dwi Putra Limasale¹, Nicole Fertala¹, Uwe Freudenberg¹, Yixin Zhang^2,4, Ayala Lampel³, Carsten Werner^1,4

¹ Leibniz Institute of Polymer Research Dresden, Dresden, Germany.

² B CUBE – Center for Molecular Bioengineering, Dresden, Germany.

³ Shmunis School of Biomedicine and Cancer Research, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

⁴ Technical University Dresden, Dresden, Germany.

INTRODUCTION: The complexity of the extracellular matrix (ECM) goes beyond simple cell-matrix interaction and involves a multitude of responses, including dynamic matrix remodeling via force dissipation and cell-mediated matrix cleavage and the incorporation of glycosaminoglycans (GAGs), notably heparin, which contributes to the structural functionality and presentation of numerous signaling molecules¹. In this study, we design biohybrid sulfated GAG (sGAG)-based interpenetrating polymer networks formed by covalent² and non-covalent³ crosslinking schemes to tunable the viscoelasticity and proteolytic cleavage within the hydrogels (Fig. 1 A) and investigate their influence on human induced pluripotent stem cells (hiPSCs; Fig. 1 B) morphology, pluripotency, ECM secretion, and Yes-associated protein (YAP) transcriptional activity.

METHODS: Pre-formed hiPSCs were embedded in hydrogels and cultured for five days. Afterward, the cysts were fixed and stained for various features for immunohistochemical imaging. The resulting images were analysed with Imaris image analysis software. Differences between conditions were then assessed using ANOVA and Tukey’s post hoc test in GraphPad Prism.

RESULTS: The viscoelasticity and cleavability of the hydrogel significantly influenced hiPSC cyst morphology. In non-cleavable, medium-viscoelastic hydrogels, cysts maintained a spherical shape, whereas those in high-viscoelastic hydrogels were slightly less spherical. For cysts in cleavable viscoelastic hydrogels, increasing the viscoelastic fraction led to a decrease in sphericity, with those in high-viscoelastic gels exhibiting an elongated morphology. Importantly, hiPSC cells retained pluripotency across all hydrogel types, regardless of the matrix’s stress relaxation or cleavage properties. Additionally, in viscoelastic hydrogels with cleavable covalent crosslinks, there was an increase in ECM protein secretion and YAP transcriptional activity with increasing viscoelasticity of the hydrogels. Selective inhibition of actin contraction, network formation, and proteolytic cleavage resulted in morphological alterations of hiPSC cysts, depending on the dominant mode of matrix remodeling within the hydrogels.

DISCUSSION & CONCLUSIONS: Slow-relaxing gels likely provide resistance that decelerates cell invasion, leading to more stable and gradual morphological development. Conversely, cells within fast-relaxing gels may encounter less resistance due to a higher proportion of non-covalent crosslinks. The inclusion of sGAG heparin potentially allows for the sequestration of growth factors, thereby enabling the retention of pluripotency in the embedded cells. In conclusion, the presented interpenetrating sGAG-based biohybrid hydrogels underscore the importance of combining viscoelasticity and cell-mediated matrix cleavage in creating cell-instructive matrices to support stem cell culture and morphogenesis.

REFERENCES: ¹U. Freudenberg et al., “Glycosaminoglycan-Based Biohybrid Hydrogels: A Sweet and Smart Choice for Multifunctional Biomaterials,” Adv. Mater., 28(40), 8861–8891. ²R. Wieduwild et al., “Minimal Peptide Motif for Non-covalent Peptide–Heparin Hydrogels,” J. Am. Chem. Soc., 135(8), 2919–2922. ³M. V. Tsurkan et al., “Defined Polymer–Peptide Conjugates to Form Cell-Instructive starPEG–Heparin Matrices In Situ,” Adv. Mater., 25(18), 2606–2610.

ACKNOWLEDGEMENTS: The authors thank the medical faculty at the University Hospital Dresden for providing hMSCs, and the CRTD Stem Cell Engineering Facility for providing hiPSCs. The authors thank Dr. Valentina Magno for scientific discussions. The authors also thank Lisa Ferdinand, Milauscha Grimmer, and Isabell Jeglinski for their technical support. This project was supported by the Deutsche Forschungsgemeinschaft (DFG) Grant IA 33 02 00. 

Das¹, M. Waser¹, K. Choi², C. Jablonski¹, T. Bühler¹, J. Kwon¹, S. Wendeborn¹, R. Raso¹

¹ ICB FHNW Muttenz, Muttenz, Switzerland.

² HeiQ Materials AG, Schlieren, Switzerland.

INTRODUCTION: Since the first demonstration of isolated graphene via a simple scotch tape method [1], graphene has attracted substantial interest due to its exceptional properties (electrical [1], mechanical [2], etc.). Many efforts have been focused on utilizing this “wonder material” in various applications, one of them being membrane applications [3,4]. For membrane applications, graphene has often been perforated either by top-down [3,4] or bottom-up [5] approaches. In both cases, the performance of the graphene-based membrane depends highly on the sizes of the pores [3]. Hence, defects during their processing can tremendously reduce their performance [6]. The presence of defects during the processing of these layers has motivated many researchers to explore innovative ways to modify perforated graphene surfaces. Hence, in recent years, the functionalization of graphene surfaces has gained considerable interest [7,8]. In this framework, this work focuses on finding simple routes to modify these perforated graphene surfaces with selective functionality.

METHODS: This work will show two routes for modifying perforated graphene surfaces. Each route is simple, easy to use, and easy to scale up. We have used two different kinds of polymerization methods to modify the graphene surfaces, and our results will demonstrate the possibility of both spatial specificity and selective functionality.

RESULTS: The work presented herein emphasizes two main objectives: on the one hand, to specifically modify the pores of the graphene surface with the chosen polymer and, on the other hand, to tune the wettability of the graphene surface. Utilizing techniques such as atomic force microscopy and scanning electron microscopy, we identified the influence of each method on the graphene morphology. Furthermore, contact angle measurements demonstrated the phenomenological implication of the second type of modification, where we could show that the contact angle of water can be increased dramatically with the choice of modification. The results would demonstrate the ease of application and the possibility of modifying perforated graphene surfaces to enhance their performance.

Fig. 1: Examples of two routes of graphene modification one focusing on spatial specificity and the other focusing on controlling the wettability of the surface.

DISCUSSION & CONCLUSIONS: In conclusion, we have identified two simple routes to modify perforated graphene surfaces that can open the pathway to myriads of membrane applications. One route of graphene modification demonstrated special specificity, while the other route allowed for tuneable wettability.

REFERENCES: ¹ K. S. Novoselov, A. K. Geim, et al (2004) Science 306: 666–669. ²D. G. Papageorgiou, I. A. Kinloch, R. J. Young (2017) Prog. Mater. Sci. 90: 75–127. ³K. Celebi, J. Buchheim, R. M. Wyss, et al (2014) Science  344: 289–292. ⁴T. Ashirov, A. O. Yazaydin, A. Coskun (2022) Adv. Mater. 34:2106785. ⁵K. Choi, A. Droudian, R. M. Wyss, K.-P. Schlichting, H. G. Park (2018) Sci. Adv. 4: eaau0476. ⁶S. C. O’Hern, C. A. Stewart, M. S. H. Boutilier, et al (2012) ACS Nano 6:10130–10138. ⁷A. Criado, M. Melchionna, S. Marchesan, M. Prato, (2015) Angew. Chemie Int. Ed. 54:10734–10750. ⁸M. Steenackers, A. M. Gigler, N. Zhang, F. Deubel, et al (2011) J. Am. Chem. Soc. 133:10490–10498.

ACKNOWLEDGEMENTS: This work was supported by Innosuisse via the project Swiss HIPOGRAPH (project number: 45921.1 IP-ENG).

Schnapka¹, L Aulich¹, N. Hubert¹, G. Dürre¹, A. Brehmer¹, C. Hein¹

¹ Fraunhofer Institute for Production Systems and Design Technology, Germany.

INTRODUCTION: Lipid nanoparticles (LNPs) have become a useful vehicle for mRNA-based vaccines. Through this time, storage conditions and sensitivity to mechanical stress were found to be problematic factors for subsequent use of the formu-lation. In the course of this study, LNPs were formu-lated using an innovative new microfluidic device. The particles produced were analysed under storage conditions from 4°C, -20 C to -80 °C for their sensi-tivity to mechanical stress, simulated by vortex treatment.

METHODS: LNPs were freshly formulated using the microfluidic device FDmix and dialyzed in PBS with 0.15 M sucrose as cryoprotectant. LNPs were frozen at 4 °C, ‑20 °C or -80 °C. Each batch was thawn after at least 24 h and vortexed at 2500 rpm for 3 s, 5 s and 10 s respectively. Samples were then analysed by DLS measurement. Kolmogorov-Smirnov test and Wilkoxon signed rank test were used to analyse the data.

RESULTS: Before treatment, the microfluidically formulated LNPs had an average particle size of 73.9 nm. Dif-ferences of particle size before and after freezing without mechanical stress (fig. 1) revealed a signify-cantly higher particle growth for samples frozen at ‑20 °C than at 4 °C or -80 °C, whereas samples stored at 4 °C remained unchanged. For all freezing temperatures, a continuous increase in particle size with arising vortex duration was observed (fig. 2), which proved to be significant in statistical analysis. PDI remained unchanged in all conditions (i.e. ΔPDI < 0.06), except after freezing at -20 °C (ΔPDI: 0.12) and at 4 °C with 10 s vortexing (ΔPDI: 0.17).

Fig. 1: Particle growth of thawed LNPs after freezing without vortexing, ** 0.001 ***<0.0001

Fig. 2: Particle growth of thawed LNPs after freezing at different temperatures and vortexing for 3 s, 5 s or 10 s, * <0.05, ** 0.001 ***<0.0001

DISCUSSION & CONCLUSIONS: The effect of the negative freezing temperatures on particle size of LNPs can be explained by ice formation during freezing process, that cause a reduced distance between particles and support their agglomeration [1]. This could also be a reason for an increased PDI after freezing at -20°C. In addition, the electrostatic repulsive force can be removed by increasing the ionic strength during freezing via a cryoprotectant. [1]. Kamiya et al. (2022) showed corresponding results. Thus, the used cryoprotectant concentration might have to be adapted for different storing tem-peratures. In the same study, Kamiya et al. showed less influence of mechanical stress from vortexing, resulting in no effect at shorter times (i.e. 30 s), but particle growth at longer times (i.e. 5 min). LNPs used for Kamiyas experiment showed a larger parti-cle size (114.5 nm) after formulation than LNPs in the present study (73.9 nm), so the effect of vortex duration on final particle size and PDI could be pro-portional to the starting particle size of vortexed LNPs. In conclusion, alternative methods of mixing (e.g. pipetting or inverting the tube) should be used to avoid mechanical stress and a suitable freezing matrix should be carefully selected if LNPs are stored at negative temperatures.

REFERENCES: ¹ M. Kamiya (2022) Stability study of mRNA-Lipid nanoparticles exposed to various conditions based on the evaluation between physicochemical properties and their relation with protein expression ability, Pharmaceutics.

Katharina Hast^1,2,3,4, Markus Rottmar², Marija Buljan^1,5, Katharina Maniura^2,4, Kongchang Wei^2,3

¹ Laboratory for Particles-Biology Interactions

² Laboratory for Biointerfaces

³ Laboratory for Biomimetic Membranes and Textiles

Empa, Swiss Federal Laboratories for Materials Science and Technology , Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland

⁴ Department of Health Sciences and Technology, ETH Zurich, Zurich, Switzerland

⁵ SIB, Swiss Institute of Bioinformatics, Lausanne, Switzerland

INTRODUCTION: 3D in vitro systems are becoming increasingly popular for their ability to mimic tissue structures and bridging the gap between simplistic 2D cell culture models and animal models¹. Hydrogels are essential tools for 3D disease modeling and drug testing due to their tunable properties and ability mimic complex cell interactions². Commonly used hydrogels include Matrigel, collagen, or alginate, which are limited in terms of me-chanical properties and xenogenicity³. Decellularized ECM (dECM) hydrogels replicate native ECM biochemistry of the tissue more accurately but often rely on cytotoxic crosslinkers such as glutaraldehyde, which restricts their applicability. Dynamic hydrogels, crosslinked with non-covalent interactions, support degradation-independent cell activities, including 3D cell spreading and migration^4-5. However, dynamic crosslinking strategies for dECM-based hydrogels are still lacking

METHODS: Our experiments employed porcine skin as an ECM source, being decellularized using freeze-thaw cycles and subsequent Tween and SDS  treatment. The resultant dECM was cryogenically ground, solubilized, and mixed with Ac-β-CD to form a dynamic host-guest complex. UV-initiated polymerization of acrylate groups in the precursor solution resulted in hydrogel formation.

RESULTS: We show that Ac-β-CD-clustering is able to crosslink dECM into host-guest hydrogels, achieving tunable mechanical properties. We demonstrate the modularity and tunability of the hydrogel properties in regards to its composition and Ac-β-CD concentration, achieving increasing stiffness with increasing Ac-β-CD concentration. The dynamic nature of the formed host-guest hydrogel becomes clear when comparing the negligible damping factor of a static equivalent hydrogel (GelMa) to the increased damping factor values in the host-guest hydrogels, indicating a viscoelastic solid-like behavior with dominant elastic properties but non-negligible viscous dissipation.

Figure 1: (A) Ac-β-CD-assisted dECM crosslinking mechanism. (B) UV-Curing of gelatin- and dECM-based dynamic and static hydrogels in the presence of I2959 as photoinitiator (C) Rheological properties of static and dynamic hydrogels with varying ratios of Ac-β-CD.

DISCUSSION & CONCLUSIONS: Our findings support the notion that host-guest-crosslinked dECM hydrogels present a viable platform for creating dynamic, physiologically relevant 3D tissue models.

REFERENCES: ¹Chaicharoenaudomrung, et al. (2019). Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling. World journal of stem cells, ²Kyriakopoulou, et al. (2023) Recreating the extracellular matrix: novel 3D cell culture platforms in cancer research. The FEBS Journal., ³Vitale, et al. (2022) Tumor Microenvironment and Hydrogel-Based 3D Cancer Models for In Vitro Testing Immunotherapies. Cancers., ⁴Feng, et al. (2019) Dynamic and Cell-Infiltratable Hydrogels as Injectable Carrier of Thera-peutic Cells and Drugs for Treating Challenging Bone Defects. ACS Central Science; ⁵Feng, et al. (2016) Mechanically resilient, injectable, and bioadhesive supramolecular gelatin hydrogels crosslinked by weak host-guest interactions assist cell infiltration and in situ tissue regeneration. Biomaterials.

ACKNOWLEDGEMENTS: This project was supported by the Uniscientia foundation.

Khaliq¹, C.D. Franco², H. Hölscher³, E. Macchia⁴, L. Torsi⁵ and  G. Scamarcio^1,2

¹ Dipartimento Interateneo di Fisica, UNIBA, Bari, Italy,

 ² CNR, Istituto di Fotonica e Nanotecnologie Bari, Italy, ³IMT, KIT, Karlsruhe, Germany,

⁴Dipartimento di Farmacia, UNIBA, Bari, Italy, ⁵Dipartimento di chimica, UNIBA, Bari, Italy

aniqa.khaliq@uniba.it

INTRODUCTION: Biofunctionalized interfaces play a critical role in the development of sensing devices, enabling accurate detection and quantification of various biological biomarkers even at the physical limit of single molecule detection[1]. A fundamental aspect in the design and optimization of these interfaces is the comprehension of the mechanical properties exhibited by physiosorbed protein layers. The accurate quantification of tip-sample interaction forces in amplitude modulation atomic force microscopy (AM-AFM) has been a challenging yet crucial objective for nanoscale imaging to measure the mechanical properties of biological samples.

METHODS: Experimental analysis will involve the fabrication of biofunctionalized interfaces with controlled anti-IgM protein layers physiosorbed on gold substrate. Atomic Force Microscopy (AFM) and Force Spectroscopy (FS) will be employed to achieve the quantitative nanomechanical mapping of protein layer to estimate its mechanical properties, including Young’s modulus and adhesion. AFM measurements were performed in air, at room temperature, in tapping mode using an NTEGRA (NT-MDT, Russia) microscope equipped with a platinum-iridium coated tip FMG01/Pt (Tips Nano) with an apex size of 35 nm, a resonance frequency f = 77.7 kHz, spring constant= 2.5 Nm^-1 and a quality factor Q = 242. After acquiring the proteins morphology and identifying the single proteins on the gold surface, based on amplitude modulation force spectroscopy, amplitude–distance, and phase–distance-curves were collected.

RESULTS: The analysis is based on the equation of motion for the cantilever[2]. Amplitude/phase versus piezo displacement curves  of IgM proteins on gold substrate serve as vital components in reconstructing force-distance curves. We obtained forces and separation points from FD curve (points are marked in Fig. 2(b) and calculate the Young’s modulus with measured force and distance values. The young’s modulus of the IgM proteins is in the range of 10-20 MPa. F_adh is the adhesion force equal to the pull-off force (i.e., the minimum of the force-versus-distance curve). The adhesion force is directly accessible from the minimum of the graph in Fig. 2(b). The adhesion force for the proteins is 0.8 nN.  The Hamaker constant is indicative of van der Waals forces which contribute to the interactions of proteins with other molecules or with surfaces in contact . Moreover, the van der Waals force is a crucial factor that contributes to the formation of protein–ligand complexes. The Hamaker constant of proteins is in range of  to 10^-19 J.

Fig. 1: Schematic setup of an amplitude modulation atomic force microscope.

Fig. 2:  a) AFM semi-contact 2 x 2 μm² micrograph of a typical anti-IgM layer physiosorbed on gold substrate. b) Reconstructed tip-sample interaction force based on the data points of the amplitude and phase versus distance curves of IgM proteins on gold substrate

DISCUSSION & CONCLUSIONS: The AFM study of IgM protein thin films on gold substrates reveals key mechanical properties crucial for bio-sensing applications. The low Young’s modulus, significant adhesion force, and quantified Hamaker constant provide insights into the proteins’ flexibility, surface interactions, and van der Waals forces, respectively. These findings enhance our understanding of IgM antibody behavior and open new possibilities for their use in medical diagnostics.

REFERENCES: ¹M. Hölscher (2006) Applied Physics Letters, 89.12. ²E. Macchia et al. (2018)  Nat. Commun. 9.

M. Veloso¹, S. Pozzo², M. do Nascimento Tomaz², G. Rocca¹, A. Ferrari¹, G. Frassinella¹, G. Arrigoni¹, T. Neuper³, J. Horejs-Höck³, F. Mancin², E. Papini¹

¹ Department of Biomedical Sciences, University of Padova, Italy. ² Department of Chemical Sciences, University of Padova, Italy. ³ Department of Biosciences and Medical Biology, Paris Lodron University of Salzburg, Austria.

INTRODUCTION: Poly(2-oxazolines) (POx) were suggested as an alternative polymeric coating to poly(ethylene glycol) (PEG) due to their anti-fouling properties and high circulation times in mice models. However, it has been shown that these particles trigger the complement system and are highly uptaken by human macrophages and monocytes after protein corona formation¹. With this in mind, the interaction of POx-coated particles with dendritic cells was analysed, exploring their immunomodulatory properties.

METHODS: Organically-modified silica nanoparticles coated with poly(2-methyl-2-oxazoline) (PMOXA) and poly(2-ethyl-2-oxazoline) (PEtOXA) were pre-incubated with human serum to form the protein corona, characterized by dynamic light scattering, western blot and proteomics. The uptake of the particles by dendritic cells was measured by flow cytometry and transmission electron microscopy. The maturation of dendritic cells was then evaluated by the upregulation of surface maturation markers and release of cytokines and chemokines, all in the presence of serum.

RESULTS: By characterizing the protein corona of POx-coated nanoparticles, we observed that apolipoproteins constitute the majority of proteins bound to the surface, but the relative amounts decrease compared to uncoated particles. In turn, there is an increase in coagulation-, complement- and immunoglobulin-related proteins. As far as the uptake goes, PEtOXA-coated particles are the most uptaken by dendritic cells in the presence of human serum, and located in endosomal compartments. Interestingly, this uptake pattern is only observed in dendritic cells: in macrophages and HEK293T cells, PEG particles are the most favored. We then tested the ability of these particles to trigger dendritic cell maturation and observed that POx-coated particles induced upregulation of maturation markers, and cytokine and chemokine release.  

Fig. 1: Nanoparticle uptake by different cell types, represented by the percentage of cells positive for nanoparticles.

Fig. 2: Electron micrographs of PEtOXA-coated nanoparticles (indicated with arrows) inside dendritic cells.

DISCUSSION & CONCLUSIONS: Results suggest that PEtOXA-coated particles are selectively uptaken by dendritic cells in the presence of human serum, while simultaneously being able to modulate the activation of monocyte-derived dendritic cells. While the mechanism of uptake is still unknown, we believe receptor-mediated phagocytosis is the main trigger. The specific receptor(s) responsible for it are being investigated.

REFERENCES: ¹Tavano, R., Gabrielli, L., Lubian, E., Fedeli, C., et al. C1q-Mediated Complement Activation and C3 Opsonization Trigger Recognition of Stealth Poly(2-methyl-2-oxazoline)-Coated Silica Nanoparticles by Human Phagocytes. ACS Nano 12, 5834–5847 (2018).

ACKNOWLEDGEMENTS: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 956544.

Natsaridis¹, B. Olcay², J.L. Reymond², O. Tagit^1,3

¹ Group of Biointerfaces, Institute for Chemistry and Bioanalytics, FHNW University of Applied Sciences and Arts Northwestern Switzerland, Muttenz, Switzerland.

² Department of Chemistry, Biochemistry and Pharmaceutical Sciences, Universität Bern, Bern, Switzerland,

³ Swiss Nanoscience Institute, University of Basel, Basel, Switzerland.

INTRODUCTION: Antimicrobial resistance is a global health concern. The emergence of pan-resistant bacteria has rendered commonly available antibiotics ineffective [1]. With the development of new antimicrobials stagnating, effective alternative treatment strategies are urgently needed. Among the alternatives, antimicrobial peptides (AMPs) are promising due to their high efficacy and low resistance rates. However, the antimicrobial activity of both synthetic and natural AMPs is compromised in vivo due to rapid degradation and poor tissue penetration [2]. Therefore, more efficient ways to protect and deliver AMPs to infection sites are needed. This project aims to develop a novel strategy for AMP-mediated treatment of antibiotic-resistant infections using nanoparticle technology to encapsulate AMPs and release them directly at the site of infection, improving their in vivo stability and bioavailability.

METHODS: We utilized a synthetic AMP, ln69, and tested its encapsulation in liposomes and PLGA-lipid hybrid nanoparticles (LPNPs) using Thin Lipid Film Hydration methos (TLF) and nanoprecipitation method (NP), respectively. Liposome and LPNPs were studied for their physicochemical properties (DLS), stability under different storage conditions, and antimicrobial efficacy towards an E.coli reference strain, DSM 1103.

RESULTS: In previous studies, the peptide (ln69) has been shown to efficiently destroy various Gram-negative and Gram-positive strains including Methicillin-resistant Staphylococcus aureus [3]. The liposomal (~100 nm in diameter) and LPNP (~200 nm in diameter) (Table 1) nanoformulations of ln69 displayed improved bactericidal activity with reduced doses (1.7 mg/mL and 2.5 mg/mL, respectively) in comparison to ‘free’ peptide (3.3 mg/mL) towards E. coli (Fig.1). The growth curves recorded over time have shown that liposomal and LPNP nanoformulations resulted in significantly higher growth inhibition when compared to free ln69 in the same concentration. Empty liposomes and LPNPs that were used as controls had no antimicrobial effect.

Table 1. Physicochemical characteristics of ln69 encapsulated nanoparticles.

Fig. 1: Antimicrobial efficacy of liposomal and LPNPs ln69 formulations. Comparison with free ln69 and empty formulations.

DISCUSSION & CONCLUSIONS: Encapsulation improves the stability and efficacy of AMPs. We aim to further develop AMP  nanoformulations towards the eradication of resistant infections.

REFERENCES: ¹ Zhang, Q. Y., Yan, Z. B., Meng, Y. M., Hong, X. Y., Shao, G., Ma, J. J., … & Fu, C. Y. (2021). Antimicrobial peptides: mechanism of action, activity and clinical potential. Military Medical Research, 8, 1-25. ² Li, G., Lai, Z., & Shan, A. (2023). Advances of antimicrobial peptide‐based biomaterials for the treatment of bacterial infections. Advanced Science, 10(11), 2206602. ³ A.R Poole, M. Alini, and A Hollander (1995) Chondrocytes and cartilage destruction in Mechanisms and Models in Rheumatoid Arthritis (eds B. Henderson, J. Edwards, and R. Pettipher) Academic Press, pp 163-204.

Yaqi Feng¹²³, B. Mi Li¹²

¹ State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016, China.

²University of Chinese Academy of Sciences, Beijing 100049, China.

³Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH Zürich, 8092 Zürich, Switzerland

INTRODUCTION: In this work, we present the complementary integration of AFM and micropipette micromanipulation, which allows precise 3D manipulations and nanomechanical measurements of single living cells. The experiments on living animal suspended/adherent cells showed the dramatic changes in cell mechanics in different states and revealed the dynamics of single cells grown on micropillar arrays.

METHODS: The established micropipette micromanipulation system (Figure 1) consists of an inverted optical microscope, a micropipette, a plastic tube, a syringe, a syringe pump, a motorized 3D micro- manipulator and a cell incubator. The syringe was fixed on the syringe pump. The micropipette was attached to the end-effector of the micromanipulator. The plastic tube connects the micropipette to the syringe, which creates a closed system. The micropillar array substrates were fabricated with the use of photolithography and polydimethylsiloxane (PDMS) molding.

Figure 1.  Experimental platform of micropipette-assisted AFM for single-cell 3D manipulations and nanomechanical measurements. (A) Schematic illustration of micropipette-assisted AFM. An example of a living adherent cell is shown. (B) Optical image of moving the AFM probe to detect a single cell which is immobilized in the micropillar array. (C) SEM image of a prepared micropipette. The inset shows the detailed aperture of the micropipette’s tip. (D) SEM image of the PDMS micropillar array.

RESULTS: We used the established system to investigate the mechanical changes of single cancer cells in their different states (adherent state and suspended state) during tumor metastasis. During the process of tumor metastasis, cancer cells dramatically change their states.^[1,2] Fig. 2(A–D) show the selection and manipulation process of a living MCF-7 cell with the use of a micropipette. Micropillars with a height of 10 μm were used for immobilizing the detached MCF-7 cells. The AFM probe was then controlled to detect the mechanical properties of MCF-7 cells. For control, the mechanical properties of living MCF-7 cells in their adherent states were also measured. The statistical results clearly show that the Young’s modulus (Fig. 2E), relaxation time (Fig. 2F) and viscosity (Fig. 2G) of MCF-7 cells in the suspended states were all significantly larger than those in the adherent states.

Figure 2.  Micropipette-assisted AFM of single living MCF-7 cells. (A–D) Digesting and moving the targeted single MCF-7 cell onto the micropillar array by micropipette-based micromanipulations. (E–G) Statistical results of the Young’s modulus (E), relaxation time (F) and viscosity (G) of MCF-7 cells in their adherent states (N = 7) and suspended states (N = 6).

DISCUSSION & CONCLUSIONS: In the future, the established method can be used to unveil the detailed dynamics of the same single cells in different states during physiological and pathological processes.

REFERENCES: ¹ L. Keller and K. Pantel, Nat. Rev. Cancer (2019) DOI 10.1038/s41568-019-0180-2. 2 D. Wirtz , K. Konstantopoulos and P. C. Searson, Nat. Rev. Cancer (2011) DOI 10.1038/nrc3080.

Junnan Song, Bogdan V Parakhonskiy, Andre G. Skirtach

Faculty of Bioscience Engineering, Ghent University, 9000 Ghent, Belgium

INTRODUCTION: In the realm of material science, artificial intelligence (AI) and machine learning (ML) are heralding a new age in image analysis, with significant advancements across various microscopy techniques and quality control processes. When it comes to micro-nano particles, size and shape are the most fundamental morphology parameters determining particles’ potential applications, especially in the area of biomedicine, as it has a high relationship to particles’ biodistribution[1]. CaCO₃ – one of the most prominent worldwide inorganic minerals in biological and geological systems – mainly has four forms, namely calcite, aragonite, vaterite, and amorphous and each phase has its own representative shapes. Therefore, it is practical to build up a model to predict the particles phase based on its surface characteristic and the machine learning would be the bridge to connect the both sides.

METHODS: Four methods covering magnetic stirrer, microwave and magnetic stirrer, ultrasound agitation, as well as ultrasound agitation coupled with magnetic stirrer were taken into consideration, given the reaction time was 90s. The size and shape of synthesize particles were extracted from SEM images. The semi-supervised machine learning like Segment Anything Model was utilized to accelerate particles’ characteristic like area, ratio, surface ratio, volume, mass and so on, with the hand-analysed result as a control.

RESULTS: Ultrasound agitation generates uniformly distributed bubbles, resulting in smaller particles than solely magnetic stirring process, while microwave processing yields larger bubbles, leading to bigger particles up to 7.8 ± 3.1 µm. The combination of ultrasound agitation and magnetic stirring produces the most ion distribution, yielding pure spherical vaterite particles with the size of 0.7 ± 0.2 µm. Vaterite particles quickly transform into calcite when exposed to 0.9% NaCl and water, with transformation suppressed in PBS and cell media. Briefly, larger vaterites exhibit greater stability than smaller ones. Both big and small particles demonstrated loading efficiency higher than 95 % for macromolecular drugs verified by FITC-dextran (MW 2,000,000). Cells co-cultured with small( 0.7 ± 0.2 μm ) vaterite exhibit high viability than big ( 2.5 ± 0.6 μm) , suggesting small vaterites potential as drug carriers within cellular environments.

Fig. 1: Effect of processing methods on particles’ morphology.

DISCUSSION & CONCLUSIONS: Ultrasound agitation coupled with magnetic stirrer is suitable for synthesis smaller vaterites with high uniformity. The prediction model based on the SEM images would boost the micro-nano nanoparticle morphology analysis.

REFERENCES ¹J. Song, A. S. Vikulina, B. V. Parakhonskiy and A. G. Skirtach (2023)  Part-II: The place of organics-on-inorganics in it, their composition and applications. Front Chem, 11: p. 1078840.

ACKNOWLEDGEMENTS: We thank the Special Research Fund (BOF) of Ghent University (01IO3618), FWO- Vlaanderen (G043322N; I002620N), and EOS of FWO-F.N.R.S. (40007488) for support. JS acknowledges the support of the China Research Council (CSC, No. 202006150025).

Porteiro Figueiras¹, P. Taladriz-Blanco¹, B. Rothen-Rutishauser¹ and A. Petri-Fink^1,2

¹ Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland.

² Department of Chemistry, University of Fribourg, Fribourg, Switzerland

INTRODUCTION: Dynamic biomaterials that regulate cellular responses in situ are crucial yet challenging in tissue engineering. This study addresses the underexplored potential of mechano-sensitive cellular behavior by developing light-responsive substrates with plasmonic nanoparticles in a thermoresponsive film.

METHODS: Gold nanoparticles (AuNPs) of various shapes (AuSpheres, AuStars, AuRods) were synthesized using seeded-growth methods. Hydrogels were produced via radical polymerization, incorporating different AuNPs concentrations (0.25, 0.50, 0.75 mM) into a mix of N-isopropylacrylamide, N,N’-Methylenebis(acrylamide), ammonium persulfate, and N,N,N,N’-tetramethylethylenediamine in Milli-Q water under inert conditions. The mixture was placed between glass plates. After polymerization, gels were washed and stored in Milli-Q water.

RESULTS: The transmission electron micrographs (fig. 1a, 1b, 1c) show the different morphology of the AuNPs. The spectra (fig. 1d) indicated localized surface plasmon resonance at 851 nm for AuStars and AuRods, and 525 nm for AuSpheres.  The light-to-heat conversion was studied by lock-in thermography at 850 nm (fig. 1e), being higher upon increasing AuNPs concentration. Among the shapes, AuStars generated the most heat, followed by AuRods and AuSpheres. Rheological analysis showed that the storage modulus (G’) of the hydrogels was consistent at ~3.2 kPa, regardless of AuNPs shape or concentration.

Fig. 1: TEM images of AuSpheres (A), AuStars (B), and AuRods (C). UV-vis-NIR spectra (D) of the AuNPs and the heating power at 850 nm within the hydrogels (E).

DISCUSSION & CONCLUSIONS: The study underscores the significance of nanoparticle shape in modulating the mechanical properties of thermoresponsive films and their potential influence on cellular behavior. The ability to dynamically control substrate stiffness through localized heating presents a novel approach to investigating mechanobiological cues in tissue engineering applications.

REFERENCES: ¹ H. De Belly et al (2022) Nat Rev Mol Cell Biol 23(7):465-480. ² D. Septiadi et al (2020) Adv. Funct. Mater. 30: 2002630. ³ A. Lee et al (2021) Adv. Healthcare Mater. 10: 2001667.

ACKNOWLEDGEMENTS: The authors acknowledge the support of the Swiss National Science Foundation through the National Center of Competence in Research “Bio-Inspired Materials” and the Adolphe Merkle Foundation.

J Chen¹, M Sobecki¹, E Kryzwinska¹, Z Fan¹, C Stockmann¹

¹. Institute of Anatomy, University of Zurich, Zurich, Switzerland

INTRODUCTION: Fibrosis is the final path of various chronic diseases with different causes, and tumor desmoplasia is characterized by an expansion of ⍺-smooth muscle actin-positive cancer-associated fibroblasts (⍺-SMA⁺ CAFs) and a massive increase in extracellular matrix (ECM) components, such as collagen, fibronectin, elastin, etc. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor with the development of tumor desmoplasia, hampering the treatment efficiency of the PDAC tumor¹. During fibrogenesis, distinct genes that are active during embryonic development but silent afterward become reactivated in fibrogenic fibroblasts, e.g., ADAM12². We designed ADAM12 vaccine to stimulate T cell immune response, especially cytotoxic T cell response, to target and kill ADAM12⁺ fibrogenic cells in fibrotic diseases which were tested in liver fibrosis and lung fibrosis³. We further tested the efficacy of ADAM12 vaccination on subcutaneous KPPC-derived KP2 PDAC tumor model, which showed delayed tumor growth in v-A12 tumor-bearing mice, along with decreased ⍺-SMA, ADAM12 expression and collagen deposition. Besides, we identify ADAM12 vaccine primarily induces CD8⁺ T cell stimulation in tumor-bearing mice. In summary, ADAM12 vaccination hampers PDAC tumor growth in mice.

METHODS: 8-week-old C57BL/6JRj female were randomly allocated to two different groups and immunized subcutaneously under the neck with 2×10⁷ TU of either ADAM12 (v-A12), or control (v-CTRL) vector formulated in 200 µL of 50% IFA, 50 µg ODN in LAL water. To generate isografts, 5×10⁴ KPPC-derived KP2 cells were injected subcutaneously into mice in a volume of 100 µL PBS. Mice were immunized on day 9 (prime) and day 2 (boost) before the tumor inoculation (Fig 1A). Tumor size was measured every 2 days when tumors were measurable, and tissue samples and spleens were collected for further analysis at the endpoint.

RESULTS: As shown in Fig. 1B, v-A12 vaccination resulted in delayed PDAC outgrowth as well as a reduction of the tumor volume by 50% at endpoint (Fig. 1C). Tumors from v-A12 vaccinated mice were characterized by a decrease in the area covered by ADAM12-expressing cells (Fig. 1D). The reduction of intratumoral ADAM12⁺ cells upon immunization was associated with a lower tumor collagen content (Fig. 1E) as well as a more tubular morphology of the tumors, and hence more reminiscent of benign pancreatic tissue (Fig. 1F).

Fig. 1: Prophylactic ADAM12 vaccination hampered KP2 PDAC tumor growth.

DISCUSSION & CONCLUSIONS: In summary, our results provide proof-of-concept for the feasibility to reduce tumor desmoplasia in murine PDAC with vaccine-based immunotherapy to target fibroblast-specific transcripts. Of note, we do not target the function of ADAM12 but exploit it as “tag”, that allows immunotherapeutic depletion of CAFs.

REFERENCES:

¹ Lafaro, K. J., Melstrom, L. G. The Paradoxical Web of Pancreatic Cancer Tumor Microenviron-ment. Am J Pathol 189, 44–57 (2019). ² Dulauroy, S., Di Carlo, S. E., Langa, F., Eberl, G. & Peduto, L. Lineage tracing and genetic ablation of ADAM12⁺ perivascular cells identify a major source of profibrotic cells during acute tissue injury. Nat Med 18, 1262–1270 (2012). ³ Sobecki, M. et al. Vaccination-based immunotherapy to target profibrotic cells in liver and lung. Cell Stem Cell 29, 1459-1474.e9 (2022).

C.-V. H. Bui¹, H. Ulusan¹, A. Hierlemann¹, F. Cardes¹

¹ Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.

INTRODUCTION: Complementary metal-oxide-semiconductor microelectrode arrays (CMOS-MEAs) are important tools for large-scale cellular electrophysiology. Owing to on-chip active electronics, CMOS-MEAs allow for simultaneous operation of a large number of electrodes at high spatial density, which is essential for neural-network recordings. Electrode designs of these devices are typically confined to a 2D microscale form factor. Extending to 3D nanoscale geometries could unlock new functionalities. However, 3D nanostructures for neural interfaces have mostly been demonstrated on passive MEAs featuring few electrodes. Current scaling difficulties could be due to CMOS-incompatible processing, low-throughput nanofabrication techniques or the inability to decouple the traditional CMOS fabrication steps from the realization of cell-interfacing 3D nano-structures. Here we experimentally demonstrate direct post-fabrication of 3D nano-electrodes on existing large-area high-density CMOS-MEAs. By selecting post-CMOS-compatible process steps that only rely on high-throughput optical lithography, investigators can decouple CMOS electronics from 3D nano-electrode realization. Specifically, we scaled nano-volcanoes [1], to form 26’400 electrodes on existing CMOS-MEAs [2] using ion beam etching redeposition technique. We also showed that electrode geometries could be varied. Cross-sectional scanning electron microscopy (SEM) showed that neurons tightly engulfed the fabricated nanostructures.

METHODS: For the etch parameter study (Fig.1a), optical lithography was used to pattern openings of 11 mm, 7 mm and 5 mm diameter in negative photoresist of 1.2 mm film thickness on a silicon substrate. Ion beam etching was performed at 0° (surface-orthogonal), 10° and 20° beam angles. Wall heights of the silicon volcano structures were measured via SEM. For nano-volcano electrodes, ion beam etching was performed on a Si_xN_y-TiW-Pt-SiO₂ film, deposited on a silicon substrate (Fig.1b and Fig.2a), or a SiO₂ film, deposited on CMOS-MEA dies (Fig. 2b). Rat cortical neurons were cultured on volcano arrays, fixed and cross-sectioned by a focused ion beam (FIB).

RESULTS: Nano-volcano wall heights showed a dependency on etching angles and photoresist openings, allowing for structural aspect ratio tuning (Fig.1a). By etching multilayer films, volcano-shaped electrodes featuring SiO₂ passivation on the outer surfaces and Pt-coated inner surfaces could be made (Fig.1b). Cross-sectioning SEM showed that in vitro rat cortical neurons tightly engulfed these structures (Fig.2a). The fabrication process was applied to modify planar electrodes on CMOS-MEAs into 3D nano-electrodes at scale (Fig.2b).  Nanostructures were fabricated with high yield and good uniformity. Packaged CMOS-MEAs were operational after post-processing.

Fig. 1: (a) Effect of etch angle and photoresist opening on volcano wall height. (b) SEM image (with partial FIB cut) of a nano-volcano electrode with SiO­2 outer passivation and Pt inner lining.

Fig. 2: (a) Cross-sectional SEM imaging of rat cortical neurons engulfing nano-volcano electrodes. (b) Nano-volcano electrodes post-processed on CMOS-MEAs at scale.

DISCUSSION & CONCLUSIONS: Fabrication of CMOS-MEAs featuring 3D nanostructures was demonstrated. The structures were tunable, enabling customization of bio-interfaces. Neuron engulfment of nano-volcanoes suggests strong coupling, opening a path towards a large-scale intracellular readout of the rich subthreshold signaling repertoire of a neural network.

REFERENCES: ¹ B. X. E. Desbiolles, E. de Coulon, A. Bertsch, S. Rohr, P. Renaud, Nano Lett. 19, 6173–6181 (2019). ² J. Müller et al., Lab Chip. 15, 2767–2780 (2015).

ACKNOWLEDGEMENTS: This work was supported by the Swiss National Science Foundation project 205320_188910/1 and the European Research Council Advanced Grant 694829 ‘neuroXscales’.

Seyed Amirabbas Nazemi¹, Patrick Shahgaldian¹

¹ University of Applied Sciences Northwestern Switzerland, ICB, Muttenz, Switzerland.

INTRODUCTION: In contrast to genetic engineering’s focus on sequence manipulation, supramolecular engineering emphasizes the design of sophisticated systems via non-covalent interactions.

Our research group has developed a method to stabilize a variety of enzymes onto silica supports through immobilization. Here, we present a novel approach for stabilizing glycosylated enzymes with enhanced temporal and thermal stability, using boronate bioconjugation and encapsulation within an organosilica nanoenvironment. Moreover, by tailoring the nanoenvironment to facilitate cyclodextrin-enzyme complexes and leveraging cyclodextrin’s chaperone properties, we achieved a remarkable enhancement in the resistance of a lipase enzyme to chemical and physical stressors.

METHODS: Enzyme immobilization strategies were conducted using silica nanoparticles (SNP) as solid support following various coupling techniques fitting the chemistry of the enzymes, and also introducing a protective layer of organosilica¹ for further stabilization.

In the first example reported, two glycosylated enzymes were immobilized using the boronate affinity towards the glycans of the enzyme and stabilized within an organosilica layer providing them with higher thermal and temporal stability.² In another example we increased a lipase enzyme activity and resistance by engineering the immobilized enzyme nano-environment using a chaperone-mimicking molecule (cyclodextrin) derivative as the building block of the protective layer.³

RESULTS: Figure 1 represents the chemical scheme of glycosylated enzymes’ immobilization and stabilization.

Fig. 1: Chemical scheme of immobilization and stabilization of a glycosylated enzyme on SNPs through boronate ester bioconjugation and organosilica layer protection.

Figure 2 shows thermal and chemical stability of the soluble and shielded lipase enzyme supported by cyclodextrin as molecular chaperone within its organosilica nano-environment.

Fig. 2: A) Thermostability of soluble (red), and shielded enzyme without (blue) and with (black) CD at 50 °C. B) Refolding capacity of aforementioned samples after 20 min treatment with SDS (1%). C) Temperature profile of free and CD-supported shielded enzyme.

DISCUSSION & CONCLUSIONS: Through the process of supramolecular engineering, several enzymes were successfully immobilized and stabilized within their nano-environment. This approach yielded enzymes with significantly enhanced resistance to various physical and chemical stressors, providing them with remarkable stability. Such improvements in stability are particularly advantageous in maintaining enzyme function under harsh conditions that would typically lead to denaturation or loss of activity. The stabilization of these enzymes not only extends their temporal durability but also reveals new possibilities for their repeated use in biocatalytic cycles. This increased reusability could lead to more sustainable and cost-effective biotechnological applications by reducing the need for frequent enzyme replenishment. Consequently, the findings from this study contribute to the growing field of enzyme engineering, offering promising insights for industrial biocatalysis and other related fields.

REFERENCES:

1. M. R. Correro, N. Moridi, H. Schutzinger, S. Sykora, E. M. Ammann, E. H. Peters, Y. Dudal, F. X. Corvini and P. Shahgaldian, Angew. Chem. Int. Edit., 2016, 55, 6285-6289.
2. S. A. Nazemi, M. Olesinska, C. Pezzella, S. Varriale, C. W. Lin, P. F. X. Corvini and P. Shahgaldian, Chem. Commun., 2021, 57, 11960-11963.
3. A. F. Kalourazi, S. A. Nazemi, A. R. U. Parambil, R. Muñoz-Tafalla, P. Vidal, S. S. Shahangian, V. Guallar, M. Ferrer and P. Shahgaldian, Nanoscale, 2024, 16, 5123-5129.