Dataset Information

Legomedicine-A Versatile Chemo-Enzymatic Approach for the Preparation of Targeted Dual-Labeled Llama Antibody-Nanoparticle Conjugates.

ABSTRACT: Conjugation of llama single domain antibody fragments (Variable Heavy chain domains of Heavy chain antibodies, VHHs) to diagnostic or therapeutic nanoparticles, peptides, proteins, or drugs offers many opportunities for optimized targeted cancer treatment. Currently, mostly nonspecific conjugation strategies or genetic fusions are used that may compromise VHH functionality. In this paper we present a versatile modular approach for bioorthogonal VHH modification and conjugation. First, sortase A mediated transPEGylation is used for introduction of a chemical click moiety. The resulting clickable VHHs are then used for conjugation to other groups employing the Cu⁺-independent strain-promoted alkyne-azide cycloadition (SPAAC) reaction. Using this approach, tail-to-tail bispecific VHHs and VHH-targeted nanoparticles are generated without affecting VHH functionality. Furthermore, this approach allows the bioconjugation of multiple moieties to VHHs for simple and convenient production of VHH-based theranostics.

SUBMITTER: van Lith SA

PROVIDER: S-EPMC5330650 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Legomedicine-A Versatile Chemo-Enzymatic Approach for the Preparation of Targeted Dual-Labeled Llama Antibody-Nanoparticle Conjugates.

van Lith Sanne A M SA van Duijnhoven Sander M J SM Navis Anna C AC Leenders William P J WP Dolk Edward E Wennink Jos W H JW van Nostrum Cornelus F CF van Hest Jan C M JC

Bioconjugate chemistry 20170118 2

Conjugation of llama single domain antibody fragments (Variable Heavy chain domains of Heavy chain antibodies, VHHs) to diagnostic or therapeutic nanoparticles, peptides, proteins, or drugs offers many opportunities for optimized targeted cancer treatment. Currently, mostly nonspecific conjugation strategies or genetic fusions are used that may compromise VHH functionality. In this paper we present a versatile modular approach for bioorthogonal VHH modification and conjugation. First, sortase A ...[more]

PMID: 28045502

Similar Datasets

Project description:PurposeTargeted delivery in vivo remains an immense roadblock for the translation of nanomaterials into the clinic. The greatest obstacle is the mononuclear phagocyte system (MPS), which sequesters foreign substances from general circulation and causes accumulation in organs such as the liver and spleen. The purpose of this study was to determine whether attaching an active targeting antibody, 5B1, to the surface of gold nanoparticles and using clodronate liposomes to deplete liver and splenic macrophages could help to minimize uptake by MPS organs, increase targeted delivery to CA19.9-positive pancreatic tumors, and enhance pancreatic tumor delineation.ProceduresTo produce the antibody-gold nanoparticle conjugate (Ab-AuNP), the Ab was conjugated to p-isothiocyanatobenzyl-desferrioxamine (p-SCN-DFO) and subsequently conjugated to NHS-activated gold nanoparticles. The Ab-AuNP was characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Modified Lindmo assay was performed to assess binding affinity and internalization potential in vitro. The Ab-AuNP was radiolabeled with 89Zr and injected into CA19.9-positive BxPc-3 pancreatic orthotopic tumor-bearing mice pretreated with or without clodronate liposomes for PET imaging and biodistribution studies. Inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis was used to confirm delivery of gold nanoparticles to BxPc-3 pancreatic subcutaneous xenografts.ResultsMice pretreated with clodronate liposomes in an orthotopic setting demonstrated decreased liver uptake at early time points (12.2 ± 2.3 % ID/g vs. 22.8 ± 3.8 % ID/g at 24 h) and increased tumor uptake at 120 h (13.8 ± 8.0 % ID/g vs. 6.0 ± 1.2 % ID/g). This allowed for delineation of orthotopic pancreatic xenografts in significantly more mice treated with clodronate (6/6) than in mice not treated with clodronate (2/6) or mice injected with gold nanoparticles labeled with a nonspecific antibody (0/5).ConclusionsThe combination of clodronate liposomes and an active targeting antibody on the surface of gold nanoparticles allowed for PET/CT imaging of subcutaneous and orthotopic pancreatic xenografts in mice.

Project description:RNAs are an important class of cellular regulatory elements, and they are well characterized by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy in their folded or bound states. However, the apo or unfolded states are more difficult to characterize by either method. Particularly, effective NMR spectroscopy studies of RNAs in the past were hampered by chemical shift overlap of resonances and associated rapid signal loss due to line broadening for RNAs larger than the median size found in the PDB (~25 nt); most functional riboswitches are bigger than this median size. Incorporation of selective site-specific (13)C/(15)N-labeled nucleotides into RNAs promises to overcome this NMR size limitation. Unlike previous isotopic enrichment methods such as phosphoramidite, de novo, uniform-labeling, and selective-biomass approaches, this newer chemical-enzymatic selective method presents a number of advantages for producing labeled nucleotides over these other methods. For example, total chemical synthesis of nucleotides, followed by solid-phase synthesis of RNA using phosphoramidite chemistry, while versatile in incorporating isotope labels into RNA at any desired position, faces problems of low yields (<10%) that drop precipitously for oligonucleotides larger than 50 nt. The alternative method of de novo pyrimidine biosynthesis of NTPs is also a robust technique, with modest yields of up to 45%, but it comes at the cost of using 16 enzymes, expensive substrates, and difficulty in making some needed labeling patterns such as selective labeling of the ribose C1' and C5' and the pyrimidine nucleobase C2, C4, C5, or C6. Biomass-produced, uniformly or selectively labeled NTPs offer a third method, but suffer from low overall yield per labeled input metabolite and isotopic scrambling with only modest suppression of (13)C-(13)C couplings. In contrast to these four methods, our current chemo-enzymatic approach overcomes most of these shortcomings and allows for the synthesis of gram quantities of nucleotides with >80% yields while using a limited number of enzymes, six at most. The unavailability of selectively labeled ribose and base precursors had prevented the effective use of this versatile method until now. Recently, we combined an improved organic synthetic approach that selectively places (13)C/(15)N labels in the pyrimidine nucleobase (either (15)N1, (15)N3, (13)C2, (13)C4, (13)C5, or (13)C6 or any combination) with a very efficient enzymatic method to couple ribose with uracil to produce previously unattainable labeling patterns (Alvarado et al., 2014). Herein we provide detailed steps of both our chemo-enzymatic synthesis of custom nucleotides and their incorporation into RNAs with sizes ranging from 29 to 155 nt and showcase the dramatic improvement in spectral quality of reduced crowding and narrow linewidths. Applications of this selective labeling technology should prove valuable in overcoming two major obstacles, chemical shift overlap of resonances and associated rapid signal loss due to line broadening, that have impeded studying the structure and dynamics of large RNAs such as full-length riboswitches larger than the ~25 nt median size of RNA NMR structures found in the PDB.

Dataset Information

Legomedicine-A Versatile Chemo-Enzymatic Approach for the Preparation of Targeted Dual-Labeled Llama Antibody-Nanoparticle Conjugates.

Publications

Legomedicine-A Versatile Chemo-Enzymatic Approach for the Preparation of Targeted Dual-Labeled Llama Antibody-Nanoparticle Conjugates.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets