Nanobody-functionalized PEG-b-PCL polymersomes and their targeting study

Zou, Tao; Dembele, Fatimata; Beugnet, Anne; Sengmanivong, Lucie; Trépout, Sylvain; Marco, Sergio; Marco, Ario de; Li, Min-Hui

doi:10.1016/j.jbiotec.2015.09.034

Cited by 50 publications

(39 citation statements)

References 29 publications

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“…The copolymers having varied f Hyrdophilic values such as 0.25–0.45 for PEG‐PLA, 0.12–0.32 for PEG‐poly(caprolactone), 0.31–0.34 for poly(butadiene)‐poly(ethylene oxide), 0.42 for hyaluronan‐poly(g‐benzyl‐ L ‐glutamate), 0.25 for polystyrene‐poly(acrylic acid), 0.36 for poly[oligo(ethylene glycol) methyl methacrylate]‐poly(2‐(diisopropylamino)ethyl methacrylate), have been reported to be self‐assembled to form polymersomes.…”

Section: Resultsmentioning

confidence: 99%

Design of Colloidally Stable and Non‐Toxic Petox‐Based Polymersomes for Cargo Molecule Encapsulation

et al. 2019

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Polymersomes are vesicular constructs formed by the self‐assembly of polymer amphiphiles. Here we report the fabrication of PEtOx‐b‐PLA polymersomes at physiological pH/salt concentration and the determination of a fPEtOx range, which is one of the most recognized factors that governs the self‐assembly of amphiphilic copolymers, for the first time. The findings indicate that PEtOx‐b‐PLA copolymers, containing PEtOx fractions of varied lengths, self‐assemble to form polymersomes in the range of 98.35 to 119.50 nm. The encapsulation potential of PEtOx‐b‐PLA polymersomes was demonstrated by loading of docetaxel (hydrophobic) with 17.90% encapsulation efficiency and by loading of pDNA (hydrophilic) with 65.03% encapsulation efficiency. In addition, size distribution profiles showed that polymersomes are colloidally stable for 24 h at 37 °C and for 3 months at +4 °C. The cell viability results showed that PEtOx‐b‐PLA copolymer and polymersomes are not cytotoxic to HUVEC, HEK293 and hMSC cell lines.

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Section: Resultsmentioning

confidence: 99%

Design of Colloidally Stable and Non‐Toxic Petox‐Based Polymersomes for Cargo Molecule Encapsulation

et al. 2019

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“…When the sequence of a nanobody is available after panning, its expression should be planned considering the final application of the resulting construct. We developed and validated a large array of modular vectors based on pET vector scaffolds that share: i) a conserved cloning site for the nanobody sequence (NcoI/NotI); ii) a further cloning cassette for inserting a "functional tag" such as fluorescent proteins, Avitag, SNAP, free cysteine, SpyTag …; iii) a poly-His tag for affinity purification [13,44,47,62,[126][127][128][129][130][131]. This concept enables to exchange the modules and adapt existing constructs to design new vector versions.…”

Section: Practical Advice For Nanobody Expression and Purificationmentioning

confidence: 99%

“…With respect to conventional IgGs (150 kDa), the tiny dimension of nanobody confers the exclusive capacity to bind to cryptic epitopes of viruses [2], a characteristic that at the present could be particularly useful to produce reagents suitable for coronavirus studies. Over the years, it emerged that VHHs are effective crystallography chaperones and molecular tools for protein structural characterization [3][4][5], convenient carriers for radioisotopes with extremely short half-life to use for in vivo PET/ SPECT imaging [6][7][8][9][10], valid immunoreagents for the controlled and oriented functionalization of nanoparticles, nanogels and biosensors [11][12][13][14] and that they can be even internalized by mammalian cells when provided with a suitable leader peptide [15]. More recently, their minimal dimension has been particularly appreciated by scientists looking for binders suitable to optimize the performance of super resolution microscopy [16][17][18] and to create tandem chimeric antigen receptors (CARs) that show higher target specificity due to the possibility of binding simultaneously multiple antigens [19].…”

Section: Introductionmentioning

confidence: 99%

Recombinant expression of nanobodies and nanobody-derived immunoreagents

Marco

2020

Protein Expression and Purification

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A B S T R A C TAntibody fragments for which the sequence is available are suitable for straightforward engineering and expression in both eukaryotic and prokaryotic systems. When produced as fusions with convenient tags, they become reagents which pair their selective binding capacity to an orthogonal function. Several kinds of immunoreagents composed by nanobodies and either large proteins or short sequences have been designed for providing inexpensive ready-to-use biological tools. The possibility to choose among alternative expression strategies is critical because the fusion moieties might require specific conditions for correct folding or posttranslational modifications. In the case of nanobody production, the trend is towards simpler but reliable (bacterial) methods that can substitute for more cumbersome processes requiring the use of eukaryotic systems. The use of these will not disappear, but will be restricted to those cases in which the final immunoconstructs must have features that cannot be obtained in prokaryotic cells. At the same time, bacterial expression has evolved from the conventional procedure which considered exclusively the nanobody and nanobody-fusion accumulation in the periplasm. Several reports show the advantage of cytoplasmic expression, surface-display and secretion for at least some applications. Finally, there is an increasing interest to use as a model the short nanobody sequence for the development of in silico methodologies aimed at optimizing the yields, stability and affinity of recombinant antibodies.

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“…The approach to deliver drugs or toxins specifically to tumors mediated by nanoparticles decorated with nanobodies has been demonstrated in many in vitro examples and is now beginning to be tested in vivo . Polymersomes and polyplexes are relatively new artificial vesicles that can be modified simply by modifying the polymer‐building blocks . It might be very attractive to encapsule recombinant cytosolic sdAb or sdAb expression plasmids into nanoparticles decorated with an specific anti‐receptor nanobody (for example EGFR 122 to deliver cytosolic anti‐cancer single domain antibodies to tumor cells.…”

Section: Protein Transfection With Cytosolic/nuclear Sdabsmentioning

confidence: 99%

“…88 Polymersomes and polyplexes are relatively new artificial vesicles that can be modified simply by modifying the polymer-building blocks. 120,121 It might be very attractive to encapsule recombinant cytosolic sdAb or sdAb expression plasmids into nanoparticles decorated with an specific antireceptor nanobody (for example EGFR 122 to deliver cytosolic anti-cancer single domain antibodies to tumor cells. Furthermore, nanoparticles/nanotubes coated with transferrin or Angiopep-2, a ligand for the low-density lipoprotein receptor-related protein-1 (LRP1), demonstrated translocation across the blood-brain barrier.…”

Section: Protein Transfection With Cytosolic/nuclear Sdabsmentioning

confidence: 99%

Single domain antibodies for the knockdown of cytosolic and nuclear proteins

Böldicke

2017

Protein Science

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Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic.

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Nanobody-functionalized PEG-b-PCL polymersomes and their targeting study

Cited by 50 publications

References 29 publications

Design of Colloidally Stable and Non‐Toxic Petox‐Based Polymersomes for Cargo Molecule Encapsulation

Design of Colloidally Stable and Non‐Toxic Petox‐Based Polymersomes for Cargo Molecule Encapsulation

Recombinant expression of nanobodies and nanobody-derived immunoreagents

Single domain antibodies for the knockdown of cytosolic and nuclear proteins

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