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Here we report the generation of nanobody dextran polymer conjugates (dextraknobs) that are loaded with small molecules, i.e., fluorophores or photosensitizers, for potential applications in cancer diagnostics and therapy. To this end, the molecules are conjugated to the dextran polymer which is coupled to the C-terminus of an EGFR-specific nanobody using chemoenzymatic approaches. A monovalent EGFR-targeted nanobody and biparatopic version modified with different dextran average molecular weights (1000, 5000, and 10,000) were probed for their ability to penetrate tumor spheroids. For monovalent Cy5-labeled dextraknobs, the utilization of smaller sized dextran (MW 5000 vs. 10,000) was found to be beneficial for more homogeneous penetration into A431 tumor spheroids over time. For the biparatopic dual nanobody comprising MW 1000, 5000, and 10,000 dextran labeled with photosensitizer IRDye700DX, penetration behavior was comparable to that of a direct nanobody-photosensitizer conjugate lacking a dextran scaffold. Additionally, dextraknobs labeled with IRDye700DX incubated with cells in 2D and 3D showed potent cell killing upon illumination, thus inducing photodynamic therapy (PDT). In line with previous results, monovalent nanobody conjugates displayed deeper and more homogenous penetration through spheroids than the bivalent conjugates. Importantly, the smaller size dextrans did not affect the distribution of the conjugates, thus encouraging further development of dextraknobs.

Here we report the generation of nanobody dextran polymer conjugates (dextraknobs) that are loaded with small molecules, i.e., fluorophores or photosensitizers, for potential applications in cancer diagnostics and therapy. To this end, the molecules are conjugated to the dextran polymer which is coupled to the C-terminus of an EGFR-specific nanobody using chemoenzymatic approaches. A monovalent EGFR-targeted nanobody and biparatopic version modified with different dextran average molecular weights (1000, 5000, and 10,000) were probed for their ability to penetrate tumor spheroids. For monovalent Cy5-labeled dextraknobs, the utilization of smaller sized dextran (MW 5000 vs. 10,000) was found to be beneficial for more homogeneous penetration into A431 tumor spheroids over time. For the biparatopic dual nanobody comprising MW 1000, 5000, and 10,000 dextran labeled with photosensitizer IRDye700DX, penetration behavior was comparable to that of a direct nanobody-photosensitizer conjugate lacking a dextran scaffold. Additionally, dextraknobs labeled with IRDye700DX incubated with cells in 2D and 3D showed potent cell killing upon illumination, thus inducing photodynamic therapy (PDT). In line with previous results, monovalent nanobody conjugates displayed deeper and more homogenous penetration through spheroids than the bivalent conjugates. Importantly, the smaller size dextrans did not affect the distribution of the conjugates, thus encouraging further development of dextraknobs.

Since the pandemic outbreak of Covid-19 in December 2019, several lateral flow assay (LFA) devices were developed to enable the constant monitoring of regional and global infection processes. Additionally, innumerable lateral flow test devices are frequently used for determination of different clinical parameters, food safety, and environmental factors. Since common LFAs rely on non-biodegradable nitrocellulose membranes, we focused on their replacement by cellulose-composed, biodegradable papers. We report the development of cellulose paper-based lateral flow immunoassays using a carbohydrate-binding module-fused to detection antibodies. Studies regarding the protein binding capacity and potential protein wash-off effects on cellulose paper demonstrated a 2.7-fold protein binding capacity of CBM-fused antibody fragments compared to the sole antibody fragment. Furthermore, this strategy improved the spatial retention of CBM-fused detection antibodies to the test area, which resulted in an enhanced sensitivity and improved overall LFA-performance compared to the naked detection antibody. CBM-assisted antibodies were validated by implementation into two model lateral flow test devices (pregnancy detection and the detection of SARS-CoV-2 specific antibodies). The CBM-assisted pregnancy LFA demonstrated sensitive detection of human gonadotropin (hCG) in synthetic urine and the CBM-assisted Covid-19 antibody LFA was able to detect SARS-CoV-2 specific antibodies present in serum. Our findings pave the way to the more frequent use of cellulose-based papers instead of nitrocellulose in LFA devices and thus potentially improve the sustainability in the field of POC diagnostics.

Jan Zaloga,1 Christina Janko,1 Johannes Nowak,2 Jasmin Matuszak,1 Sabine Knaup,1 Dietmar Eberbeck,3 Rainer Tietze,1 Harald Unterweger,1 Ralf P Friedrich,1 Stephan Duerr,1 Ralph Heimke-Brinck,4 Eva Baum,4 Iwona Cicha,1 Frank Dörje,4 Stefan Odenbach,2 Stefan Lyer,1 Geoffrey Lee,5 Christoph Alexiou1 1Department of Otorhinolaryngology, Head and Neck Surgery, Section for Experimental Oncology and Nanomedicine (SEON), Else Kröner-Fresenius-Stiftung-Professorship, University Hospital Erlangen, Erlangen, Germany; 2Measuring and Automation Technology, Technical University Dresden, Dresden, Germany; 3Physikalisch-Technische-Bundesanstalt, Berlin, Germany; 4Pharmacy Department, University Hospital Erlangen, Erlangen, Germany; 5Division of Pharmaceutics, Friedrich Alexander University Erlangen-Nuremberg, Erlangen, Germany Abstract: The promising potential of superparamagnetic iron oxide nanoparticles (SPIONs) in various nanomedical applications has been frequently reported. However, although many different synthesis methods, coatings, and functionalization techniques have been described, not many core-shell SPION drug delivery systems are available for clinicians at the moment. Here, bovine serum albumin was adsorbed onto lauric acid-stabilized SPIONs. The agglomeration behavior, zeta potential, and their dependence on the synthesis conditions were characterized with dynamic light scattering. The existence and composition of the core-shell-matrix structure was investigated by transmission electron microscopy, Fourier transform infrared spectroscopy, and zeta potential measurements. We showed that the iron oxide cores form agglomerates in the range of 80 nm. Moreover, despite their remarkably low tendency to aggregate even in a complex media like whole blood, the SPIONs still maintained their magnetic properties and were well attractable with a magnet. The magnetic properties were quantified by vibrating sample magnetometry and a superconducting quantu