Construtos diméricos de anticorpos de domínio único do tipo VHH humanizados contra toxinas majoritárias do veneno botrópico veiculados em nanopartículas de PLGA peguiladas

Abstract

Snakebite envenoming, classified by the World Health Organization as a neglected tropical disease, primarily affects rural populations in vulnerable conditions. In Brazil, envenomings by Bothrops snakes are the most frequent and treatment with conventional antivenoms has limitations, particularly against local tissue damage. Such damage is mainly caused by toxins like phospholipases A2 (PLA2s) and metalloproteinases, which trigger inflammation, tissue destruction, coagulation disorders, and may lead to necrosis, amputations or death. The search for innovative antivenoms aims to overcome these limitations while ensuring access to safe and effective therapeutics. In this context, single-domain antibodies (VHHs) derived from camelids emerge as a promising approach due to their physicochemical properties, low immunogenicity, and biotechnological versatility. Furthermore, nanostructured formulations can enhance pharmacokinetic profiles and enable controlled delivery of the active pharmaceutical ingredient (API), facilitating penetration into dense tissues and prolonged systemic circulation. This study aimed to develop PEGylated formulations containing humanized VHHs to improve anti-bothropic therapy. Homo- and heterodimeric humanized VHH constructs targeting Bothrops jararacussu venom toxins—PLA2s (BthTX-I and BthTX-II) and metalloproteinase (BjussuMP-II)—were expressed in a microbial system and purified via affinity and size-exclusion chromatography. Purification was monitored by SDS-PAGE and Western blot. In silico assays

determined physicochemical parameters and monoisotopic mass. Binding affinity was assessed by bio- layer interferometry, and polymeric (PLGA-PEG) nanoparticles encapsulating or functionalized with

VHHs were developed and characterized. Mass spectrometry-based approaches were employed for structural characterization, proteoform analysis, purity assessment, heterologous protein profiling, and exact mass determination. The homodimer and its nanoformulations were evaluated for their ability to inhibit B. jararacussu venom cytotoxicity in C2C12 myotubes. A proteomic analysis of venoms from 12 Bothrops species (including B. jararacussu) assessed compositional similarity. In vivo and ex vivo radiolabeling studies of the homodimer and PEGylated constructs evaluated pharmacokinetics and tissue distribution. The purified constructs showed high purity, with host-cell heterologous proteins quantified. Key physicochemical parameters—molar extinction coefficients, theoretical masses, percentages of positively/negatively charged amino acids, predicted proteoforms, and signature peptides—were determined. Real-time kinetics revealed affinities of 3.68 nM (homodimer) and 13.4 nM (heterodimer) for BthTX-I. Nanoformulations were characterized by size, polydispersity index, and zeta potential: homodimer VHH with 152 ± 23.7 nm, 0.614 ± 0.1, −23.6 ± 1.6 mV and heterodimer VHH with 505.6 ± 73.8 nm, 0.606 ± 0.1, −17.2 ± 3.6 mV. Intact mass and peptide mapping confirmed high purity (>92% homodimer, >96% heterodimer) and sequence coverage. Amino acid composition was validated via fragmentation spectra and monoisotopic mass analysis. The encapsulated/functionalized homodimer nanoformulation significantly reduced LDH levels (57%) in response to whole B. jararacussu venom, while the functionalized-only formulation showed no significant effect. Comparative proteomics revealed high interspecies venom similarity, suggesting broad cross-reactivity, though lower protein homology in B. insularis and B. alternatus may explain earlier non-reactivity of constructs. Biodistribution studies indicated higher lung uptake of free radiolabeled homodimer versus PEGylated VHH. Both free and PEGylated forms underwent glomerular filtration, but PEGylation enhanced hepatobiliary circulation. Pharmacokinetic data showed a six-fold increase in VHH half-life for the nanoformulated version. PLGA-PEG drug delivery systems loaded with VHHs targeting major Bothrops toxins demonstrate therapeutic potential, offering promising inhibition of local tissue damage and sustained systemic action.