Abstract
Antibodies and antigen-binding fragments (Fabs) can be used to modify the surface of nanoparticles for enhanced target binding. In our previous work, site-specific conjugation of Fabs to polymeric micelles using conventional methods was limited to approximately 30% efficiency, possibly due to steric hindrance related to macromolecular reactants. Here, we report a new method that enables conjugation of Fabs onto a micelle surface in a controlled manner with up to quantitative conversion of nanoparticle reactive groups. Variation of (i) PEG spacer length in a heterofunctionalized cross-linker and (ii) Fab/polymer feed ratios resulted in production of nanoparticles with a range of Fab densities on the surface up to the theoretical maximum value. The biological impact of variable Fab density was evaluated in vitro with respect to cell uptake and cytotoxicity of a drug-loaded (SN38) targeted polymeric micelle bearing anti-EphA2 Fabs. Fab conjugation increased cell uptake and potency compared with non-targeted micelles, although a Fab density of 60% resulted in decreased uptake and potency of the targeted micelles. Altogether, our findings demonstrate that conjugation strategies can be optimized to allow control of Fab density on the surface of nanoparticles and also that Fab density may need to be optimized for a given cell-surface target to achieve the highest bioactivity.
Highlights
Functionalizing nanoparticles with targeting biomolecules provides biofunctionality, which allows access to natural biological substrates and/or processes that could improve their desired function [1,2,3]
We have previously explored these strategies for installing Fabs onto the poly(ethylene glycol) (PEG) shell of polymeric micelles [15,16], the number of Fabs on the micelles by using these methods was limited to 20–30% modification of the PEG chains
Our results showed that the elongation of the PEG spacer improved the conjugation efficiency on the surface of micelles, while sustaining similar micelle binding ability, cellular uptake and cytotoxicity under the same Fab density
Summary
Functionalizing nanoparticles with targeting biomolecules provides biofunctionality, which allows access to natural biological substrates and/or processes that could improve their desired function [1,2,3]. Covalent attachment of biomolecules to nanoparticles has great potential for generating therapeutics with advanced properties such as cell targeting and penetration through biological barriers [4,5,6] These macromolecular reactions usually present low yield and conjugation efficiencies, as they are more challenging than analogous small-molecule reactions due to factors such as steric hindrance, diffusion limitations and high solvent sensitivity [7,8,9]. We developed polymeric micelles containing the anticancer agent SN38 linked through enzymatically sensitive bonds, which can provide selective drug activation and minimal drug leakage in biological settings These SN38-loaded micelles (SN38/m) were further modified with 1C1 Fabs (directed to the Ephrin receptor EphA2 on cancer cells) by using the heterobifunctional linkers having 4 or 23 unit PEG spacers. This study demonstrates that conjugation of Fabs to nanoparticles can be optimized by linker design and provides insight into the practical formulation of effective ligandinstalled nanomaterials
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