Ocimum basilicum L. (basil) presents pro-apoptotic activity in an Ehrlich's experimental tumor murine model.

PURPOSE: This study aimed to evaluate the therapeutic effect of an ethanol extract of Ocimum basilicum L. (EEOb) aerial parts against Ehrlich's experimental tumor (EET) in mice. METHODS: Swiss mice were divided into two groups (control and treated; n = 6). On day 21, all mice were inoculated subcutaneously with 2 × 106 (0.05 mL) EET cells in the left paw for solid tumor development. This study lasted 28 days. Treatment began 24 hours after inoculation with EET. Measurements of dorsoplantar thickness were used to assess tumor growth. The paw pad was collected for histopathological analysis and stained using the argyrophilic nucleolar organizing regions (AgNOR) technique and immunohistochemistry for proliferating cell nuclear antigen, Bcl-2 and Bax. RESULTS: The treatment of animals with EEOb at 100 mg/kg intraperitoneally was able to reduce the growth (Control = 3.7 ± 0.1 mm vs. EEOb = 5.7 ± 0.2 mm) and the number of AgNORs of solid Ehrlich tumor. The antitumor effect of EEOb was associated with the induction of apoptosis of tumoral cell, as suggested by the reduction of the content of Bcl-2 induced by extract. CONCLUSIONS: The study demonstrated that daily administration of EEOb is able to reduce the growth of EET by induce apoptosis of tumoral cells.

A multispectral 3D live organoid imaging platform to screen probes for fluorescence guided surgery.

Achieving complete tumor resection is challenging and can be improved by real-time fluorescence-guided surgery with molecular-targeted probes. However, pre-clinical identification and validation of probes presents a lengthy process that is traditionally performed in animal models and further hampered by inter- and intra-tumoral heterogeneity in target expression. To screen multiple probes at patient scale, we developed a multispectral real-time 3D imaging platform that implements organoid technology to effectively model patient tumor heterogeneity and, importantly, healthy human tissue binding.

Nanobody-mediated targeting of zinc phthalocyanine with polymer micelles as nanocarriers.

Photodynamic therapy (PDT) is a suitable alternative to currently employed cancer treatments. However, the hydrophobicity of most photosensitizers (e.g., zinc phthalocyanine (ZnPC)) leads to their aggregation in blood. Moreover, non-specific accumulation in skin and low clearance rate of ZnPC leads to long-lasting skin photosensitization, forcing patients with a short life expectancy to remain indoors. Consequently, the clinical implementation of these photosensitizers is limited. Here, benzyl-poly(ε-caprolactone)-b-poly(ethylene glycol) micelles encapsulating ZnPC (ZnPC-M) were investigated to increase the solubility of ZnPC and its specificity towards cancers cells. Asymmetric flow field-flow fractionation was used to characterize micelles with different ZnPC-to-polymer ratios and their stability in human plasma. The ZnPC-M with the lowest payload (0.2 and 0.4% ZnPC w/w) were the most stable in plasma, exhibiting minimal ZnPC transfer to lipoproteins, and induced the highest phototoxicity in three cancer cell lines. Nanobodies (Nbs) with binding specificity towards hepatocyte growth factor receptor (MET) or epidermal growth factor receptor (EGFR) were conjugated to ZnPC-M to facilitate cell targeting and internalization. MET- and EGFR-targeting micelles enhanced the association and the phototoxicity in cells expressing the target receptor. Altogether, these results indicate that ZnPC-M decorated with Nbs targeting overexpressed proteins on cancer cells may provide a better alternative to currently approved formulations.

Ocimum basilicum L. (basil) presents pro-apoptotic activity in an Ehrlich's experimental tumor murine model

ABSTRACT Purpose: This study aimed to evaluate the therapeutic effect of an ethanol extract of Ocimum basilicum L. (EEOb) aerial parts against Ehrlich's experimental tumor (EET) in mice. Methods: Swiss mice were divided into two groups (control and treated; n = 6). On day 21, all mice were inoculated subcutaneously with 2 × 106 (0.05 mL) EET cells in the left paw for solid tumor development. This study lasted 28 days. Treatment began 24 hours after inoculation with EET. Measurements of dorsoplantar thickness were used to assess tumor growth. The paw pad was collected for histopathological analysis and stained using the argyrophilic nucleolar organizing regions (AgNOR) technique and immunohistochemistry for proliferating cell nuclear antigen, Bcl-2 and Bax. Results: The treatment of animals with EEOb at 100 mg/kg intraperitoneally was able to reduce the growth (Control = 3.7 ± 0.1 mm vs. EEOb = 5.7 ± 0.2 mm) and the number of AgNORs of solid Ehrlich tumor. The antitumor effect of EEOb was associated with the induction of apoptosis of tumoral cell, as suggested by the reduction of the content of Bcl-2 induced by extract. Conclusions: The study demonstrated that daily administration of EEOb is able to reduce the growth of EET by induce apoptosis of tumoral cells.

Structural characterization of the human DjC20/HscB cochaperone in solution.

J-domain proteins (JDPs) form a very large molecular chaperone family involved in proteostasis processes, such as protein folding, trafficking through membranes and degradation/disaggregation. JDPs are Hsp70 co-chaperones capable of stimulating ATPase activity as well as selecting and presenting client proteins to Hsp70. In mitochondria, human DjC20/HscB (a type III JDP that possesses only the conserved J-domain in some region of the protein) is involved in [FeS] protein biogenesis and assists human mitochondrial Hsp70 (HSPA9). Human DjC20 possesses a zinc-finger domain in its N-terminus, which closely contacts the J-domain and appears to be essential for its function. Here, we investigated the hDjC20 structure in solution as well as the importance of Zn+2 for its stability. The recombinant hDjC20 was pure, folded and capable of stimulating HSPA9 ATPase activity. It behaved as a slightly elongated monomer, as attested by small-angle X-ray scattering and SEC-MALS. The presence of Zn2+ in the hDjC20 samples was verified, a stoichiometry of 1:1 was observed, and its removal by high concentrations of EDTA and DTPA was unfeasible. However, thermal and chemical denaturation in the presence of EDTA led to a reduction in protein stability, suggesting a synergistic action between the chelating agent and denaturators that facilitate protein unfolding depending on metal removal. These data suggest that the affinity of Zn+2 for the protein is very high, evidencing its importance for the hDjC20 structure.

Penetration of Nanobody-Dextran Polymer Conjugates through Tumor Spheroids.

Here we report the generation of nanobody dextran polymer conjugates (dextraknobs) that are loaded with small molecules, i.e., fluorophores or photosensitizers, for potential applications in cancer diagnostics and therapy. To this end, the molecules are conjugated to the dextran polymer which is coupled to the C-terminus of an EGFR-specific nanobody using chemoenzymatic approaches. A monovalent EGFR-targeted nanobody and biparatopic version modified with different dextran average molecular weights (1000, 5000, and 10,000) were probed for their ability to penetrate tumor spheroids. For monovalent Cy5-labeled dextraknobs, the utilization of smaller sized dextran (MW 5000 vs. 10,000) was found to be beneficial for more homogeneous penetration into A431 tumor spheroids over time. For the biparatopic dual nanobody comprising MW 1000, 5000, and 10,000 dextran labeled with photosensitizer IRDye700DX, penetration behavior was comparable to that of a direct nanobody-photosensitizer conjugate lacking a dextran scaffold. Additionally, dextraknobs labeled with IRDye700DX incubated with cells in 2D and 3D showed potent cell killing upon illumination, thus inducing photodynamic therapy (PDT). In line with previous results, monovalent nanobody conjugates displayed deeper and more homogenous penetration through spheroids than the bivalent conjugates. Importantly, the smaller size dextrans did not affect the distribution of the conjugates, thus encouraging further development of dextraknobs.

Influence of sodium hypochlorite on cyclic fatigue resistance of nickel-titanium instruments: A systematic review and meta-analysis of in vitro studies.

OBJECTIVE: This systematic review/meta-analysis investigated the influence of NaOCl on cyclic fatigue resistance of endodontic NiTi instruments. MATERIALS AND METHODS: A systematic search until July 2022 in PubMed/MEDLINE, Embase, Scopus, Web of Science, SciELO, Cochrane Library, and grey literature was conducted. According to the PECOS strategy, only in vitro studies evaluating the effects of NaOCl on the cyclic fatigue resistance of NiTi instruments were eligible. Cyclic fatigue resistance was the primary outcome. A modified Joanna Briggs Institute's Checklist was used for risk of bias assessment. RESULTS: Of the 2,445 records screened, 37 studies were included. Most studies used simulated canals made of stainless-steel block with severe to moderate curvatures. NaOCl concentration varied from 1-6%, mainly at 37 °C. Regarding fatigue resistance, 23 studies using 1.2% to 6% NaOCl showed a reduction in the resistance compared to the control groups, especially when pre-heated. Four meta-analyses were performed according to the tested NiTi systems. The meta-analyses indicated that the PTU F2 files had higher reduction of fatigue resistance after exposure to 5.25% NaOCl; no differences between NaOCl and no immersion were observed for Reciproc R25, WaveOne 25.08, and WaveOne Gold Primary files. Included studies had low risk of bias. CONCLUSION: NaOCl appears to reduce cyclic fatigue resistance of certain NiTi files, especially when they are pre-heated, particularly in conventional NiTi files compared to some heat-treated instruments. It is possible that the temperature of the solution may have a greater influence on resistance than NaOCl itself. Important to note that an overall tendency toward no significant influence was observed among various systems. CLINICAL RELEVANCE: Precautions are necessary when a pre-heated high-concentration NaOCl is used to enhance its properties during root canal preparation, mainly using conventional wire.

A Correlational Study Between Executive Functions and Conditional Discriminations in Children with Autism / Um Estudo Correlacional Entre Funções Executivas e Discriminações Condicionais em Crianças com Autismo

Abstract Neuropsychology is a science that allows tracing the profile of cognitive impairments and preserved skills to design appropriate treatments and educational practices aiming at a better quality of life for the individual. This is basic correlational research, which objective was to verify if the results found in the executive functions in children with autism spectrum disorder (ASD) are predictive of or have some correlation with the performance in conditional discrimination through choice tasks according to the identity matching-to-sample (MTS) model. Correlations revealed significant associations between neuropsychological tests and MTS tasks. Future research may further explore MTS tasks for the assessment and intervention of individuals with ASD.

Resumo A Neuropsicologia é uma ciência que permite traçar o perfil dos comprometimentos cognitivos e habilidades preservadas a fim de delinear tratamentos e práticas educativas adequadas, almejando melhor qualidade de vida do indivíduo. Trata-se de uma pesquisa básica correlacional, cujo objetivo foi verificar se os resultados encontrados nas funções executivas em crianças com Transtorno do Espectro do Autismo (TEA) são preditivas de ou tem alguma correlação com o desempenho em discriminação condicional por meio de tarefas de escolha de acordo com o modelo MTS de identidade. As correlações revelaram associações significativas entre os testes neuropsicológicos e as tarefas de MTS. Pesquisas futuras poderão explorar melhor as tarefas de MTS para avaliação e intervenção de indivíduos com TEA.

The Impact of Nanobody Density on the Targeting Efficiency of PEGylated Liposomes.

Nanoparticles (NPs) are commonly modified with tumor-targeting moieties that recognize proteins overexpressed on the extracellular membrane to increase their specific interaction with target cells. Nanobodies (Nbs), the variable domain of heavy chain-only antibodies, are a robust targeting ligand due to their small size, superior stability, and strong binding affinity. For the clinical translation of targeted Nb-NPs, it is essential to understand how the number of Nbs per NP impacts the receptor recognition on cells. To study this, Nbs targeting the hepatocyte growth factor receptor (MET-Nbs) were conjugated to PEGylated liposomes at a density from 20 to 800 per liposome and their targeting efficiency was evaluated in vitro. MET-targeted liposomes (MET-TLs) associated more profoundly with MET-expressing cells than non-targeted liposomes (NTLs). MET-TLs with approximately 150-300 Nbs per liposome exhibited the highest association and specificity towards MET-expressing cells and retained their targeting capacity when pre-incubated with proteins from different sources. Furthermore, a MET-Nb density above 300 Nbs per liposome increased the interaction of MET-TLs with phagocytic cells by 2-fold in ex vivo human blood compared to NTLs. Overall, this study demonstrates that adjusting the MET-Nb density can increase the specificity of NPs towards their intended cellular target and reduce NP interaction with phagocytic cells.

Conjugation of IRDye Photosensitizers or Fluorophores to Nanobodies.

Fluorophores have been conjugated to nanobodies for approximately a decade, for several applications in molecular biology. More recently, photosensitizers have been conjugated to nanobodies for targeted photodynamic therapy (PDT). The most common chemistry is the random conjugation in which commercial fluorophores or photosensitizers contain a N-hydroxysuccinimide ester (NHS ester) group that reacts specifically and efficiently with lysines in the amino acid sequence of the nanobody and with the N-terminal amino groups to form a stable amide bond. Alternatively, maleimide-containing fluorophores or photosensitizers can be used for conjugation to thiols, in a site-directed manner through a cysteine incorporated at the C-terminal of the nanobody. This chapter addresses both conjugation strategies, providing details on the reaction conditions, purification, and characterization of the conjugates obtained.

Orthotopic Breast Cancer Model to Investigate the Therapeutic Efficacy of Nanobody-Targeted Photodynamic Therapy.

Photodynamic therapy (PDT) is characterized by the local application of laser light, which activates a photosensitizer to lead to the formation of singlet oxygen and other toxic reactive oxygen species, to finally kill cells. Recently, photosensitizers have been conjugated to nanobodies to render PDT more selective to cancer cells. Nanobodies are the smallest naturally derived antibody fragments from heavy-chain antibodies that exist in animals of the Camelidae family. Indeed, we have shown that nanobody-targeted PDT can lead to extensive and selective tumor damage, and thus the subsequent step is to assess whether this damage can delay or even inhibit tumor growth in vivo. To evaluate the therapeutic efficacy of PDT, mouse models are mostly employed in which human tumors are grown subcutaneously in the flank of the animals. Although very useful, it has been suggested that these tumors are further away from their natural environment and that tumors developed in the organ or tissue of origin would be closer to the natural situation. Thus, this chapter describes the development of an orthotopic model of breast cancer and the application of nanobody-targeted PDT, for the assessment of the therapeutic efficacy.

Investigation of the Therapeutic Potential of Nanobody-Targeted Photodynamic Therapy in an Orthotopic Head and Neck Cancer Model.

Photodynamic therapy (PDT) has a great therapeutic potential because it induces local cellular cytotoxicity upon application of a laser light that excites a photosensitizer, leading to toxic reactive oxygen species. Nevertheless, PDT still is underutilized in the clinic, mostly because of damage induced to normal surrounding tissues. Efforts have been made to improve the specificity. Nanobody-targeted PDT is one of such approaches, in which the variable domain of heavy-chain antibodies, i.e., nanobodies, are used to target photosensitizers selectively to cancer cells. In vitro studies are certainly very valuable to evaluate the therapeutic potential of PDT approaches, but many aspects such as bio-distribution of the photosensitizers, penetration through tissues, and clearance are not taken into account. In vivo studies are therefore essential to assess the influence of such factors, in order to gain more insights into the therapeutic potential of a treatment under development. This chapter describes the development of an orthotopic model of head and neck cancer, to which nanobody-targeted PDT is applied, and the therapeutic potential is assessed by immunohistochemistry one day after PDT.

Nanobody-Targeted Photodynamic Therapy: Nanobody Production and Purification.

Nanobodies have recently been introduced to the field of photodynamic therapy (PDT) as a very promising strategy to target photosensitizers selectively to cancer cells. Nanobodies are known for their characteristic small size (15 kDa), high specificity, and high binding affinities. These features allow rapid accumulation of nanobody-photosensitizer conjugates at the tumor site and rapid clearance of unbound fractions, and thus illumination for activation is possible 1 or 2 h postinjection. Preclinical studies have shown extensive tumor damage after nanobody-targeted PDT . This chapter addresses the first steps toward preparing nanobody-photosensitizer conjugates, which are the nanobody production and purification. The protocol for nanobody production addresses either medium- or large-scale bacterial expression, while the nanobody purification is described for two main strategies: affinity chromatography and ion-exchange chromatography. For the first strategy, protocols are described for different affinity tags and purification from either medium-scale or large-scale productions. For the second strategy, the protocol given is for purification from a large-scale production.

In Vitro Assessment of Binding Affinity, Selectivity, Uptake, Intracellular Degradation, and Toxicity of Nanobody-Photosensitizer Conjugates.

Photosensitizers have recently been conjugated to nanobodies for targeted photodynamic therapy (PDT) to selectively kill cancer cells. The success of this approach relies on nanobody-photosensitizer conjugates that bind specifically to their targets with very high affinities (kD in low nM range). Subsequently, upon illumination, these conjugates are very toxic and selective to cells overexpressing the target of interest (EC50 in low nM range). In this chapter, protocols are described to determine the binding affinity of the nanobody-photosensitizer conjugates and assess the toxicity and selectivity of the conjugates when performing in vitro PDT studies. In addition, and because the efficacy of PDT also depends on the (subcellular) localization of the conjugates at the time of illumination, assays are described to investigate the uptake and the intracellular degradation of the nanobody-photosensitizer conjugates.

Assessment of the In Vivo Response to Nanobody-Targeted PDT Through Intravital Microscopy.

Methods that allow real-time, longitudinal, intravital detection of the fluorescence distribution and the cellular and vascular responses within tumor and normal tissue are important tools to obtain valuable information when investigating new photosensitizers and photodynamic therapy (PDT) responses. Intravital confocal microscopy using the dorsal skinfold chamber model gives the opportunity to visualize and determine the distribution of photosensitizers within tumor and normal tissue. Next to that, it also allows the visualization of the effect of treatment with respect to changes in vascular diameter and blood flow, vascular leakage, and tissue necrosis, in the first days post-illumination. Here, we describe the preparation of the skinfold chamber model and the intravital microscopy techniques involved, for a strategy we recently introduced, that is, the nanobody-targeted PDT. In this particular approach, photosensitizers are conjugated to nanobodies to target these specifically to cancer cells.

Influence of ethylenediaminetetraacetic acid on regenerative endodontics: A systematic review.

BACKGROUND: The effects of ethylenediaminetetraacetic acid (EDTA) on regenerative endodontic procedures (REPs) are controversial, because, despite releasing growth factors from dentine, some studies show negative effects on cell behaviour. OBJECTIVES: The aim of the study was to investigate the influence of the use of EDTA in REP on the growth factors' release, cell behaviour and tissue regeneration. METHODS: A systematic search was conducted (PubMed/Medline, Scopus, Cochrane Library, Web of Science, Embase, OpenGrey and reference lists) up to February 2021. Only in vivo and in vitro studies evaluating the effects of EDTA on the biological factors of dentine, pulp/periapical tissues and cell behaviour were eligible. Studies without a control group or available full text were excluded. The growth factors' release was the primary outcome. Risk of bias in the in vitro and in vivo studies was performed according to Joanna Briggs Institute's Checklist and SYRCLE's RoB tool, respectively. RESULTS: Of the 1848 articles retrieved, 36 were selected. Amongst these, 32 were in vitro, three animal studies and one with both models. The EDTA concentrations ranged from 3% to 15%, at different times. Regarding growth factors' release (17 studies), 15 studies found significant transforming growth factor (TGF)-ß release after dentine conditioning with EDTA, and most found no influence on vascular endothelial growth factor release. Regarding cell behaviour (26 studies), eight studies showed no influence of EDTA-treated dentine on cell viability, whereas, five, nine and six studies showed higher cell migration, adhesion and differentiation respectively. No influence of EDTA conditioning was observed in animal studies. In vitro studies had a low risk of bias, whereas animal studies had high risk of bias. Meta-analysis was unfeasible. DISCUSSION: This review found that EDTA increased TGF-ß release and improved cell activity. However, well-designed histological analyses using immature teeth models are needed. CONCLUSIONS: High-quality in vitro evidence suggests that EDTA-treated dentine positively influences TGF-ß release, cell migration, attachment and differentiation; further research to evaluate its influence on tissue regeneration is necessary due to low methodological quality of the animal studies.

The Effect of Microbubble-Assisted Ultrasound on Molecular Permeability across Cell Barriers.

The combination of ultrasound and microbubbles (USMB) has been applied to enhance drug permeability across tissue barriers. Most studies focused on only one physicochemical aspect (i.e., molecular weight of the delivered molecule). Using an in vitro epithelial (MDCK II) cell barrier, we examined the effects of USMB on the permeability of five molecules varying in molecular weight (182 Da to 20 kDa) and hydrophilicity (LogD at pH 7.4 from 1.5 to highly hydrophilic). Treatment of cells with USMB at increasing ultrasound pressures did not have a significant effect on the permeability of small molecules (molecular weight 259 to 376 Da), despite their differences in hydrophilicity (LogD at pH 7.4 from -3.2 to 1.5). The largest molecules (molecular weight 4 and 20 kDa) showed the highest increase in the epithelial permeability (3-7-fold). Simultaneously, USMB enhanced intracellular accumulation of the same molecules. In the case of the clinically relevant anti- C-X-C Chemokine Receptor Type 4 (CXCR4) nanobody (molecular weight 15 kDa), USMB enhanced paracellular permeability by two-fold and increased binding to retinoblastoma cells by five-fold. Consequently, USMB is a potential tool to improve the efficacy and safety of the delivery of drugs to organs protected by tissue barriers, such as the eye and the brain.

Gold Nanoclusters: Imaging, Therapy, and Theranostic Roles in Biomedical Applications.

For the past two decades, atomic gold nanoclusters (AuNCs, ultrasmall clusters of several to 100 gold atoms, having a total diameter of <2 nm) have emerged as promising agents in the diagnosis and treatment of cancer. Owing to their small size, significant quantization occurs to their conduction band, which leads to emergent photonic properties and the disappearance of the plasmonic responses observed in larger gold nanoparticles. For example, AuNCs exhibit native luminescent properties, which have been well-explored in the literature. Using proteins, peptides, or other biomolecules as structural scaffolds or capping ligands, required for the stabilization of AuNCs, improves their biocompatibility, while retaining their distinct optical properties. This paved the way for the use of AuNCs in fluorescent bioimaging, which later developed into multimodal imaging combined with computer tomography and magnetic resonance imaging as examples. The development of AuNC-based systems for diagnostic applications in cancer treatment was then made possible by employing active or passive tumor targeting strategies. Finally, the potential therapeutic applications of AuNCs are extensive, having been used as light-activated and radiotherapy agents, as well as nanocarriers for chemotherapeutic drugs, which can be bound to the capping ligand or directly to the AuNCs via different mechanisms. In this review, we present an overview of the diverse biomedical applications of AuNCs in terms of cancer imaging, therapy, and combinations thereof, as well as highlighting some additional applications relevant to biomedical research.

Targeting of promising transmembrane proteins for diagnosis and treatment of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal types of cancer due to the relatively late diagnosis and the limited therapeutic options. Current treatment regimens mainly comprise the cytotoxic agents gemcitabine and FOLFIRINOX. These compounds have shown limited efficacy and severe side effects, highlighting the necessity for earlier detection and the development of more effective, and better-tolerated treatments. Although targeted therapies are promising for the treatment of several types of cancer, identification of suitable targets for early diagnosis and targeted therapy of PDAC is challenging. Interestingly, several transmembrane proteins are overexpressed in PDAC cells that show low expression in healthy pancreas and may therefore serve as potential targets for treatment and/or diagnostic purposes. In this review we describe the 11 most promising transmembrane proteins, carefully selected after a thorough literature search. Favorable features and potential applications of each target, as well as the results of the preclinical and clinical studies conducted in the past ten years, are discussed in detail.

Single Domain Antibodies as Carriers for Intracellular Drug Delivery: A Proof of Principle Study.

Antibody-drug conjugates (ADCs) are currently used for the targeted delivery of drugs to diseased cells, but intracellular drug delivery and therefore efficacy may be suboptimal because of the large size, slow internalization and ineffective intracellular trafficking of the antibody. Using a phage display method selecting internalizing phages only, we developed internalizing single domain antibodies (sdAbs) with high binding affinity to rat PDGFRß, a receptor involved in different types of diseases. We demonstrate that these constructs have different characteristics with respect to internalization rates but all traffic to lysosomes. To compare their efficacy in targeted drug delivery, we conjugated the sdAbs to a cytotoxic drug. The conjugates showed improved cytotoxicity correlating to their internalization speed. The efficacy of the conjugates was inhibited in the presence of vacuolin-1, an inhibitor of lysosomal maturation, suggesting lysosomal trafficking is needed for efficient drug release. In conclusion, sdAb constructs with different internalization rates can be designed against the same target, and sdAbs with a high internalization rate induce more cell killing than sdAbs with a lower internalization rate in vitro. Even though the overall efficacy should also be tested in vivo, sdAbs are particularly interesting formats to be explored to obtain different internalization rates.