Evaluation of Gallium as a Tracer of Exogenous Hemoglobin–Haptoglobin Complexes for Targeted Drug Delivery Applications

Abstract

Abstract. Haptoglobin (Hp) is a plasma glycoprotein that generates significant interest in the drug delivery community because of its potential for delivery of antiretroviral medicines with high selectivity to macrophages and monocytes, the latent reservoirs of human immunodeficiency virus. As is the case with other therapies that exploit transport networks for targeted drug delivery, the success of the design and optimization of Hp-based therapies will critically depend on the ability to accurately localize and quantitate Hp-drug conjugates on the varying and unpredictable background of endogenous proteins having identical structure. In this work, we introduce a new strategy for detecting and quantitating exogenous Hp and Hp-based drugs with high sensitivity in complex biological samples using gallium as a tracer of this protein and inductively coupled plasma mass spectrometry (ICP MS) as a method of detection. Metal label is introduced by reconstituting hemoglobin (Hb) with gallium(III)-protoporphyrin IX followed by its complexation with Hp. Formation of the Hp/Hb assembly and its stability are evaluated with native electrospray ionization mass spectrometry. Both stable isotopes of Ga give rise to an abundant signal in ICP MS of a human plasma sample spiked with the metallabeled Hp/Hb complex. The metal label signal exceeds the spectral interferences' contributions by more than an order of magnitude even with the concentration of the exogenous protein below 10 nM, the level that is more than adequate for the planned pharmacokinetic studies of Hp-based therapeutics.

In Vitro Functional Quality Characterization of NOTA-Modified Somatropins

“…An overview of the results is given in Figure 4 and Table 1. 46 , the characterization of proteinmetal complexes destined for either diagnostic or therapeutic purposes 47 , and, during the investigation of interactions between protein drugs with their therapeutic targets/physiological partners, including unmodified proteins and their modified versions [48][49][50][51] . In this study, the different substitution-degree species within the protein product were functionally evaluated for hGHBp binding, thereby complementing the information provided by SEC/HPRBC.…”

Exploiting His-Tags for Absolute Quantitation of Exogenous Recombinant Proteins in Biological Matrices: Ruthenium as a Protein Tracer

“…Thus, substituting Fe 3+ with Ga 3+ in met-hemoglobin may allow detection of exogenous hemoglobin/haptoglobin complexes with high sensitivity. 23 Likewise, substituting Fe 3+ with In 3+ in transferrin not only enables sensitive detection of transferrin-based therapies in blood and cerebrospinal fluid, but also allows their biodistribution patterns to be determined within various organs of animal models. 24 Unfortunately, this strategy is restricted to metalloproteins and its success hinges on identification of exogenous metals that can be used to replace the cognate ones without compromising the binding affinity and changing the properties of the protein.…”

Antimicrobial Photodynamic Activity of Gallium-Substituted Haemoglobin on Silver Nanoparticles

“…GaHb was prepared by removing the hemin cofactors from bovine haemoglobin to produce the apoprotein (apoHb) and substituting with GaPpIX, using literature methods. 28,29 Stoichiometric replacement was established by titration and Job plot analysis (see ESI). GaHb-AgNPs were prepared by combining GaHb (1 mg/mL) in phosphate buffered saline (PBS, pH 7.4) with 10-or 40-nm citrate-stabilized AgNPs (20 μg/mL) at 4 C for 12 h, followed by centrifugation and redispersion in 15 mM borate buffer (pH 8.5).…”