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.

Photoimmuno-antimicrobial therapy for Staphylococcus aureus implant infection.

INTRODUCTION: Implant infections caused by Staphylococcus aureus are responsible for high mortality and morbidity worldwide. Treatment of these infections can be difficult especially when bacterial biofilms are involved. In this study we investigate the potential of infrared photoimmunotherapy to eradicate staphylococcal infection in a mouse model. METHODS: A monoclonal antibody that targets Wall Teichoic Acid surface components of both S. aureus and its biofilm (4497-IgG1) was conjugated to a photosensitizer (IRDye700DX) and used as photoimmunotherapy in vitro and in vivo in mice with a subcutaneous implant pre-colonized with biofilm of Staphylococcus aureus. A dose of 400 µg and 200 µg of antibody-photosensitizer conjugate 4497-IgG-IRDye700DXwas administered intravenously to two groups of 5 mice. In addition, multiple control groups (vancomycin treated, unconjugated IRDye700DX and IRDye700DX conjugated to a non-specific antibody) were used to verify anti-microbial effects. RESULTS: In vitro results of 4497-IgG-IRDye700DX on pre-colonized (biofilm) implants showed significant (p<0.01) colony-forming units (CFU) reduction at a concentration of 5 µg of the antibody-photosensitizer conjugate. In vivo, treatment with 4497-IgG-IRDye700DX showed no significant CFU reduction at the implant infection. However, tissue around the implant did show a significant CFU reduction with 400 µg 4497-IgG-IRDye700DX compared to control groups (p = 0.037). CONCLUSION: This study demonstrated the antimicrobial potential of photoimmunotherapy for selectively eliminating S. aureus in vivo. However, using a solid implant instead of a catheter could result in an increased bactericidal effect of 4497-IgG-IRDye700DX and administration locally around an implant (per operative) could become valuable applications in patients that are difficult to treat with conventional methods. We conclude that photoimmunotherapy could be a potential additional therapy in the treatment of implant related infections, but requires further improvement.

Predator-Prey Interaction Between Xylocoris sordidus (Hemiptera: Anthocoridae) and Enneothrips enigmaticus (Thysanoptera: Thripidae).

The peanut thrips, Enneothrips enigmaticus (Thysanoptera: Thrypidae), is an important pest of the peanut (Arachis hypogaea) in South America. Due to concerns about the environment and human health induced by the extensive use of pesticides in the management control of pests, environmentally and friendlier tactics must be targeted. Thus, this study investigates, for the first time, the behavior of Xylocoris sordidus (Hemiptera: Anthocoridae) as a biological control agent for E. enigmaticus. The methodology included no-choice tests to assess whether the predation rate varies according to the developmental stage of the prey, as well as the predator's developmental stage with the highest predation capacity. Additionally, an analysis of the functional response of adult and 5th instar nymphs of X. sordidus exposed to different densities of E. enigmaticus nymphs (1, 2, 4, 8, 16, and 32) was conducted. The results confirm the predation of peanut thrips by X. sordidus, with a higher predation rate in the nymphal stages of the prey. There was no difference in predation capacity between predator nymphs and adults, and exhibiting a type II functional response. Therefore, the potential of X. sordidus as a biological control agent for E. enigmaticus is confirmed, showing the importance of adopting measures to preserve this predator in peanut crops.

Erratum: Acute cellular and vascular responses to photodynamic therapy using EGFR-targeted nanobody-photosensitizer conjugates studied with intravital optical imaging and magnetic resonance imaging: Erratum.

[This corrects the article DOI: 10.7150/thno.37949.].

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.

Modification of resting metabolism, body composition, and muscle strength after resolution of coronavirus disease 2019.

BACKGROUND & AIMS: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes changes that can influence human metabolism and modify the distribution of body compartments. We aimed to describe the clinical findings of changes in resting metabolism, muscle strength, and body composition in nonhospitalized patients after being diagnosed with coronavirus disease 2019 (COVID-19). METHODS: Physically active patients were evaluated at a nutrition clinic, and indirect calorimetry (IC) and body composition analysis using portable ultrasound were performed. After a routine appointment, all patients were instructed to inform the staff if they tested positive for SARS-CoV-2 infection. Our sample included individuals diagnosed with COVID-19, confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR), within 7 days of the routine appointment. After an average incubation period of 14-21 days, in which there was no proven transmission of disease by RT-PCR, all of the patients were re-evaluated. RESULTS: A total of 38 volunteers (63.2% female) completed the study and were included in the analysis. The mean age of the participants was 37.3 ± 8.8 years. The comparison between pre- and post-COVID-19 stratified by sex demonstrated significant reduction in the RMR and RMR adjusted for weight (p < 0.0001) for both groups. Regarding body composition, there was a significant increase observed in fat mass in men (p < 0.002) and women (p < 0.01), and a significant reduction observed in fat-free mass (men: p < 0.002; women: p < 0.001) and skeletal muscle mass (men: p = 0.003; women: p < 0.0001). There was a significant difference between the change in the RMR measured by IC (p < 0.0001) and that calculated by the predictive equation of Cunningham (1980) (p < 0.0001), whereas the Harris and Benedict (1918) and Mifflin (1990) equations exhibited no difference. However, the mean difference in RMR between the post- and pre-COVID-19 calculated by the Cunningham equation was -40.4 kcal/day (95% confidence interval [CI]: -56.38 to -24.45), whereas the mean difference measured by IC was -362.3 kcal/day (95% CI: -452.7 to -271.9). CONCLUSION: This study describes the trends in the RMR, and body composition in individuals with COVID-19 who were not hospitalized from the pre-COVID-19 period to the post-COVID-19 period. A significant reduction in resting energy expenditure, and loss of fat-free mass and muscle mass in the post-COVID-19 period were observed in both men and women.

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.

Fatal fall-from-height accidents: Statistical treatment using the Human Factors Analysis and Classification System - HFACS.

INTRODUCTION: The civil construction industry (CCI) is one of the most dangerous sectors for occupational accidents. Studies conducted in several countries show that occupational accidents involving falls from height are the main cause of deaths in recent years. METHOD: This article analyzed the combinations of causal factors with the highest likelihood of accidents involving falls from height in construction to assist in decision-making. The methodology was divided into four stages: accident collection and sample definition; accident analysis; probability determination; and obtaining the theoretical curve of an accident probability distribution. The methodology was applied to reports of fatal fall-from-height accidents that occurred in the United States between 1997 and 2020. RESULTS: The results show that among the accidents analyzed, the highest probability of fatality is when a roofer aged between 31 and 44 years performs their activity on a roof between 10:00 and 11:59 am. It is also noted that the three causal factors most present in the accidents were: organizational process (97.7%); poor management of worker resources (96.6%); and organizational climate (95.4%). From the probability distribution curve, 68% of the fatal accidents occurred after reaching between 18 and 34 causal factors present in the HFACS method categories.

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.

Spontaneous fermentation improves the physicochemical characteristics, bioactive compounds, and antioxidant activity of acerola (Malpighia emarginata D.C.) and guava (Psidium guajava L.) fruit processing by-products.

This study aimed to investigate the effects of spontaneous fermentation on physicochemical characteristics, bioactive compounds, and antioxidant activity of acerola and guava fruit industrial by-products. Viable cell counts of lactic acid bacterial (LAB) in acerola and guava by-products were ≥ 5.0 log CFU/mL from 24 h up to 120 h of fermentation. Fermented acerola and guava by-products had increased luminosity and decreased contrast. Contents of total soluble solids and pH decreased, and titrable acidity increased in acerola and guava by-products during fermentation. Ascorbic acid contents decreased in acerola by-product and increased in guava by-product during fermentation. Different phenolic compounds were found in acerola and guava by-products during fermentation. Fermented acerola and guava by-products had increased contents of total flavonoids, total phenolics, and antioxidant activity. The contents of total flavonoids and total phenolics positively correlated with antioxidant activity in fermented acerola and guava by-products. These results indicate that spontaneous fermentation could be a strategy to improve the contents of bioactive compounds and the antioxidant activity of acerola and guava by-products, adding value and functionalities to these agro-industrial residues.

Family Refusal of Heart Valve Donation.

BACKGROUND: Impairment of the heart valves can occur due to many diseases that cause deterioration of the contractile function and harm the body, making it necessary for the heart valves to be transplanted. This study's objective was to analyze families' refusal to donate heart valves between 2001 and 2020. METHODS: This was a cross-sectional study conducted in accordance with the Terms of Family Authorization for Donation of Organs and Tissues from patients diagnosed with brain death by an Organ Procurement Organization in the state of São Paulo. The variables analyzed were sex, age, cause of death, hospital type (private or public), and refusal to donate heart valves. Data analysis was performed using Stata software version 15.0  (StataCorp, LLC, College Station, Tex, United States) in a descriptive and inferential way. RESULTS: A total of 236 people (9.65%) refused to specifically donate heart valves of their relatives, the majority of whom were between 41 and 59 years old. Most potential donors had suffered a stroke and had been in a private hospital. From 2001 to 2009, there was a decreasing trend in males and the age group from 0 to 11 years old, whereas there was an increasing trend in those aged 60 years or older and in the general population. Between 2010 and 2020, there was a downward trend in the 41- to 59-year-old age group and the general population. CONCLUSIONS: The specific refusal to donate heart valves was associated with age, diagnosis, and whether the institution was public or private.

RuBisCO as a protein source for potential food applications: A review.

RuBisCO is a complete protein, widely abundant and recognized as ideal for human consumption. Further, its biochemical composition, organoleptic and physical features mean RuBisCO has potential as a nutritionally beneficial food additive. Nonetheless, despite growing plant-based market trends, there is a lack of information about the applications of this protein. Here, we explored the biochemical features of RuBisCO as a potential food additive and compared it with other plant protein sources currently available. We describe potential advantages, including nutritional content, digestibility, non-allergenicity and, potential bioactivities. Despite the lack of industrial procedures for RuBisCO purification, a growing number of novel methods are emerging, justifying discussion of their feasibilities. Overall, this information can help both researchers and industry to review the use RuBisCO as a sustainable source of protein for plant-based food products or formulation of novel functional foods.

Influence of ethylenediaminetetraacetic acid irrigation on the regenerative endodontic procedure in an immature rat molar model.

AIM: To analyse the influence of ethylenediaminetetraacetic acid (EDTA) on the repair process in immature rat molars after a regenerative endodontic procedure (REP). METHODOLOGY: The lower first molars of 12 4-week-old Wistar rats underwent pulpectomy in the mesial root and were divided into the following groups: sodium hypochlorite (NaOCl; n = 6) - the mesial canals were irrigated with 2.5% NaOCl for 5 min, and NaOCl-EDTA (n = 6) - the canals were irrigated with 2.5% NaOCl, followed by 17% EDTA for 5 min each. After evoking bleeding using a size 10 K-file, the cavities were sealed. Three molars on the untreated side were randomly used as control (control-15 d; n = 3), and three molars from the other three rats untreated were used as immediate control (n = 3). After 15 days (NaOCl, NaOCl-EDTA and control-15 d groups) or immediately (control-immediate), the animals were euthanized, and the teeth were subjected to histologic evaluation of tissue regeneration and presence of collagen fibres. Mann-Whitney U-test was used (p < .05). RESULTS: The experimental groups had newly formed cementum-like tissue and increased root length and thickness. Half of the specimens in NaOCl-EDTA group showed apical foramen closure, whilst the NaOCl group had partial apical closure. The experimental groups showed inflammatory infiltrate extending mainly to the medium third of the root canal. These parameters were similar between experimental groups (p > .05). Newly formed connective tissue in the pulp space was significantly higher in the NaOCl-EDTA group than in NaOCl group (p < .05). Regarding the collagen fibres, the NaOCl-EDTA group had more collagen fibres in the root tip, but there was no significant difference compared to NaOCl group, and both groups showed greater amount of immature fibres in this area; in the centre of the apical third of root canal, there was equivalence between mature and immature fibres from both groups (p > .05). CONCLUSIONS: Ethylenediaminetetraacetic acid irrigation improved newly formed intracanal connective tissue after REP in immature molars of rats; however, EDTA did not influence cementum-like tissue formation, apical closure, inflammatory infiltrate and maturation of collagen fibres.

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.

The Role of Phosphate Fertilization on Physiological Responses of the Young Bertholletia excelsa Plants Grown in a P-Deficient Amazon Ferralsol.

Phosphorus (P) reacts with soil minerals, which makes it less available to plants. Considering that Amazonian soils have a low pH and nutrient availability, both of these properties contribute to an increase in P limitation. Here, we investigate how the addition of P to the substrate affects morpho-physiological traits of Brazil nut trees (Bertholletia excelsa Bonpl.). The experiment was carried out in a greenhouse with 24-month-old saplings, and the P treatments consisted of a control (Ferrasol without P addition) and 100, 200, 400, and 500 mg P kg-1 of added to the soil. When B. excelsa saplings were fertilized with phosphate, the N:P leaf ratio reduced from 50 to 26. Addition of P favored the photochemical efficiency of PSII (FV/FM), and the application of 200 mg kg-1 increased photosynthesis (PN) by 50%. Furthermore, phosphorus enhanced light and nutrient use efficiency. An increase in B. excelsa dry biomass was observed when 200 mg P kg-1 was added, with maximum yield occurring at 306.2 mg P kg-1. Physiological parameters suggest robust responses by B. excelsa to P fertilization. In addition, our findings reveal the critical role of P on B. excelsa growth in Ferralsol, as well as the potential of P fertilization to improve functional traits of this important Amazonian tree.

Sleep Quality Predicts Functional Disability in Older Adults with Low Back Pain: A Longitudinal Study.

Low back pain (LBP) and sleep quality are two very prevalent complaints in the older population. However, little is known about the prognostic relationship between sleep quality and disability in older adults with LBP. The aim of this study was to examine the association between sleep quality and disability in older adults with LBP. This was a longitudinal study over a 6-month period. Older adults with LBP in the last 12 weeks and who had preserved cognitive functions were recruited. The questionnaires used were the Pittsburgh Sleep Quality Index and the Roland Morris Disability Questionnaire. At baseline, we collected information on demographic/anthropometric variables, cognitive status, depression, daytime sleepiness, and comorbidities. Linear regression adjusted for potential covariates were performed. Poor sleep at baseline predicted higher disability after 6 months [ß = 0.30 (CI95%:0.07 to 0.55)]. Our results support the existence of an important relationship between sleep and disability in older adults with LBP.

Effects of diabetes mellitus on dental pulp: A systematic review of in vivo and in vitro studies.

OBJECTIVES: This systematic review (PROSPERO CRD42021227711) evaluated the influence of diabetes mellitus (DM) on the response of the pulp tissue and in the pulp cells behaviour. MATERIALS AND METHODS: Searches in PubMed/MEDLINE, Embase, Web of Science and OpenGrey were performed until March 2022. Studies evaluating the effects of DM in the pulp tissue inflammation and in the cell behaviour were included, followed by risk of bias assessment (Methodological Index for Non-Randomized Studies and SYRCLE's RoB tools). The meta-analysis was unfeasible, and a narrative synthesis for each outcome was provided. RESULTS: Of the 615 studies, 21 were eligible, mainly with in vivo analysis (16 studies). The pulp inflammation (10 studies) was analysed mainly by haematoxylin-eosin stain; DM increased pulp inflammation/degeneration in 9 studies, especially after dental procedures. The cell viability (5 studies) was analysed mostly using MTT assay; DM and glycating agents decreased cellular viability in 3 studies. DM reduced collagen in all of three studies. There were controversial results regarding mineralization; however, increased alkaline phosphatase was reported in three of four studies. CONCLUSIONS: DM seems to increase inflammation/degeneration and mineralization in the pulp tissue while reducing cell proliferation. Further analyses in human pulp are important to provide stronger evidence.

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.