Chemoselective Methionine Labelling of Recombinant Trastuzumab Shows High In Vitro and In Vivo Tumour Targeting.

A highly effective 2-step system for site-specific antibody modification and conjugation of the monoclonal antibody Herceptin (commercially available under Trastuzumab) in a cysteine-independent manner was used to generate labelled antibodies for in vivo imaging. The first step contains redox-activated chemical tagging (ReACT) of thioethers via engineered methionine residues to introduce specific alkyne moieties, thereby offering a novel easy way to fundamentally change the process of antibody bioconjugation. The second step involves modification of the introduced alkyne via azide-alkyne cycloaddition 'click' conjugation. The versatility of this 2-step approach is demonstrated here by the selective incorporation of a fluorescent dye but can also be applied to a wide variety of different conjugation partners depending on the desired application in a facile manner. Methionine-modified antibodies were characterised in vitro, and the diagnostic potential of the most promising variant was further analysed in an in vivo xenograft animal model using a fluorescence imaging modality. This study demonstrates how methionine-mediated antibody conjugation offers an orthogonal and versatile route to the generation of tailored antibody conjugates with in vivo applicability.

Chemoenzymatic surface decoration of Nisin-shelled nanoemulsions: Novel targeted drug-nanocarriers for cancer applications.

Nisin, a peptide used as a natural food preservative, is employed in this work for the development of a novel nanocarrier system. Stable and uniform nisin-shelled nanoemulsions (NSNE) with a diameter of 100 ± 20 nm were successfully prepared using 20 kHz flow-through ultrasonication technique. The NSNE showed limited toxicity, high bactericidal activity and high drug loading capacity (EE 65 % w/w). In addition, the nisin shell was exploited for the site-specific attachment of a recombinantly produced cancer targeting ligand (αHER2LPETG IgG). Employing a unique two phases (bio-click) approach which involved both Sortase A mediated Azide Bioconjugation (SMAB) and Strain Promoted Azide Alkyne Cycloaddition (SPAAC) reactions, targeted NSNE (NSNEDOX-αHER2 IgG) were successfully assembled and loaded with the chemotherapeutic drug Doxorubicin (DOX). Finally, NSNEDOX-αHER2 IgG showed cancer-specific binding and augmented cytotoxicity to HER2 expressing tumour cells.

Self-Assembly of Oriented Antibody-Decorated Metal-Organic Framework Nanocrystals for Active-Targeting Applications.

Antibody (Ab)-targeted nanoparticles are becoming increasingly important for precision medicine. By controlling the Ab orientation, targeting properties can be enhanced; however, to afford such an ordered configuration, cumbersome chemical functionalization protocols are usually required. This aspect limits the progress of Abs-nanoparticles toward nanomedicine translation. Herein, a novel one-step synthesis of oriented monoclonal Ab-decorated metal-organic framework (MOF) nanocrystals is presented. The crystallization of a zinc-based MOF, Zn2 (mIM)2 (CO3 ), from a solution of Zn2+ and 2-methylimidazole (mIM), is triggered by the fragment crystallizable (Fc) region of the Ab. This selective growth yields biocomposites with oriented Abs on the MOF nanocrystals (MOF*Ab): the Fc regions are partially inserted within the MOF surface and the antibody-binding regions protrude from the MOF surface toward the target. This ordered configuration imparts antibody-antigen recognition properties to the biocomposite and shows preserved target binding when compared to the parental antibodies. Next, the biosensing performance of the system is tested by loading MOF*Ab with luminescent quantum dots (QD). The targeting efficiency of the QD-containing MOF*Ab is again, fully preserved. The present work represents a simple self-assembly approach for the fabrication of antibody-decorated MOF nanocrystals with broad potential for sensing, diagnostic imaging, and targeted drug delivery.

Engineering Antibodies with C-Terminal Sortase-Mediated Modification for Targeted Nanomedicine.

The current advances in nanoengineered materials coupled with the precise targeting capability of recombinant antibodies can create nanoscale diagnostics and therapeutics which show enhanced accumulation and extended retention at a target tissue. Smaller antibodies such as single-chain variable fragments (scFv) preserve the selective and strong binding of their parent antibody to their antigen with the benefits of low immunogenicity, more efficient tissue penetration and easy introduction of functional residues suitable for site-specific conjugation. This is of high importance as nonspecific antibody modification often involves attachment to free cysteine or lysine amino acids which may reside in the active site, leading to reduced antigen binding.In this chapter, we outline a facile and versatile chemoenzymatic approach for production of targeted nanocarrier scFv conjugates using the bacterial trans-peptidase Sortase A (Srt A). Srt A efficiently mediates sequence-specific peptide ligation under mild conditions and has few undesirable side reactions. We first describe the production, purification and characterization of Srt A enzyme and a scFv construct which targets activated platelets, called scFvanti-GPIIb/IIIa. Following this, our protocol illustrates the chemoenzymatic modification of the antibody at the C-terminus with an orthogonal click chemistry linker. This avoids any random attachment to the biologically active antigen binding site of the antibody. Finally, we describe the modification of a nanoparticle surface with scFv attachment via two methods: (1) direct Sortase-mediated conjugation; or (2) a two-step system which consists of scFv Sortase-mediated conjugation followed by strain promoted azide-alkyne cycloaddition. Finally, methodology is described to assess the successful assembly of targeted particles.

Surface functionalization of methotrexate-loaded chitosan nanoparticles with hyaluronic acid/human serum albumin: Comparative characterization and in vitro cytotoxicity.

The active tumor targeting ligands, hyaluronic acid (HA) and human serum albumin (HSA), are considered promising targeting moieties of drug carriers for cancer therapy. The chitosan nanoparticles loaded with methotrexate (MTX-CsNPs) were employed as the core for subsequent coating process. HA and HSA coating solutions were used at different concentrations. The effect of different HA Mw (1000, 360, 10kDa) was also investigated. The coated MTX-CsNPs was characterized proving the success of surface functionalization. The antitumor activity of the prepared MTX-CsNPs was evaluated on MCF-7 breast cancer cell lines. Results showed that both 360 and 10kDa HA allowed for successful HA adsorption, while its Mw and concentration determined negative charge density. HSA coating was accompanied by a slight increase in nanoparticles (NPs) size and a final positive surface charge. The in vitro cytotoxicity proved that HA and HSA coated MTX-CsNPs improved the antitumor activity compared to uncoated NPs and free drug.

Methotrexate loading in chitosan nanoparticles at a novel pH: Response surface modeling, optimization and characterization.

The aim of this study was to assess the feasibility of employing a novel but critical formulation pH (6.2) to encapsulate an anionic model drug (methotrexate, MTX) into chitosan(Cs)-tripolyphosphate nanoparticles(NPs). A response surface methodology using a three-level full factorial design was applied studying the effects of two independent variables namely; Cs concentration and MTX concentration. The responses investigated were the entrapment efficiency (EE%), mean hydrodynamic particle size (PS), polydispersity index (PDI) and zeta potential (ZP). In order to simultaneously optimize the series of models obtained, the desirability function approach was applied with a goal to produce high percent of MTX encapsulated into highly charged Cs-TPP NPs of homogenous optimum PS. MTX-loaded CsNPs were successfully prepared at the novel pH applied. The suggested significant models were found quadratic for EE, PS and ZP responses, while 2-factor interaction model for PDI. The optimization overlay graph showed that the maximum global desirability, D=0.856, was reached when the conditions were set at high Cs and MTX concentration. Thus, the use of such optimized conditions, at this novel pH, achieved a maximum drug EE% (73.38%) into NPs characterized by optimum PS (232.6nm), small PDI value (0.195) and highly surface charged (+18.4mV).

Chitosan-tripolyphosphate nanoparticles: Optimization of formulation parameters for improving process yield at a novel pH using artificial neural networks.

At a novel pH value of the polymeric solution (6.2), variable chitosan (Cs) and sodium tripolyphosphate (TPP) concentrations and mass ratios were optimized to improve the process yield without undesirable particle flocculation. Prepared formulations were characterized in terms of particle size (PS), zeta potential (ZP) and percentage yield (% yield). Artificial neural networks (ANN) were built up and used to identify the parameters that control nanoparticle (NP) size and yield, in addition to being tested for their ability to predict these two experimental outputs. Using these networks, it was found that TPP concentration has the greatest effect on PS and% yield. The most optimum formulation was characterized by a notable process yield reaching 91.5%, a mean hydrodynamic PS 227 nm, ZP+24.13 mv and spherical compact morphology. Successful Cs-TPP interaction in NP formation was confirmed by both Fourier transform-infrared spectroscopy (FT-IR) and differential scanning calorimetry (DSC). This study demonstrated the ability of ANN to predict not only PS of the formed particles but also NP% yield. This may have a great impact on Cs-TPP NPs preparation and can be used to customize the required target formulations.