Juan Diego Unciti-Broceta

Nanobody conjugated PLGA nanoparticles for active targeting of African Trypanosomiasis

Targeted delivery of therapeutics is an alternative approach for the selective treatment of infectious diseases. The surface of African trypanosomes, the causative agents of African trypanosomiasis, is covered by a surface coat consisting of a single variant surface glycoprotein, termed VSG. This coat is recycled by endocytosis at a very high speed, making the trypanosome surface an excellent target for the delivery of trypanocidal drugs. Here, we report the design of a drug nanocarrier based on poly ethylen glycol (PEG) covalently attached (PEGylated) to poly(D,L-lactide-co-glycolide acid) (PLGA) to generate PEGylated PLGA nanoparticles. This nanocarrier was coupled to a single domain heavy chain antibody fragment (nanobody) that specifically recognizes the surface of the protozoan pathogen Trypanosoma brucei. Nanoparticles were loaded with pentamidine, the first-line drug for T. b. gambiense acute infection. An in vitro effectiveness assay showed a 7-fold decrease in the half-inhibitory concentration (IC50) of the formulation relative to free drug. Furthermore, in vivo therapy using a murine model of African trypanosomiasis demonstrated that the formulation cured all infected mice at a 10-fold lower dose than the minimal full curative dose of free pentamidine and 60% of mice at a 100-fold lower dose. This nanocarrier has been designed with components approved for use in humans and loaded with a drug that is currently in use to treat the disease. Moreover, this flexible nanobody-based system can be adapted to load any compound, opening a range of new potential therapies with application to other diseases.

Novel therapy based on camelid nanobodies

Nanobodies (Nbs) are small antibody fragments derived from camelid heavy chain antibodies through recombinant gene technology. Their exceptional physicochemical properties, possibility of humanization and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components. Several different therapeutic approaches based on the novel camelid Nbs have been developed to treat a wide range of diseases ranging from immune, bone, blood and neurological disorders; infectious diseases and cancer. This review provides a comprehensive overview of the current state of the use of camelid-derived Nbs as novel therapeutic agents against multiple diseases.

Specific Cell Targeting Therapy Bypasses Drug Resistance Mechanisms in African Trypanosomiasis

African trypanosomiasis is a deadly neglected disease caused by the extracellular parasite Trypanosoma brucei. Current therapies are characterized by high drug toxicity and increasing drug resistance mainly associated with loss-of-function mutations in the transporters involved in drug import. The introduction of new antiparasitic drugs into therapeutic use is a slow and expensive process. In contrast, specific targeting of existing drugs could represent a more rapid and cost-effective approach for neglected disease treatment, impacting through reduced systemic toxicity and circumventing resistance acquired through impaired compound uptake. We have generated nanoparticles of chitosan loaded with the trypanocidal drug pentamidine and coated by a single domain nanobody that specifically targets the surface of African trypanosomes. Once loaded into this nanocarrier, pentamidine enters trypanosomes through endocytosis instead of via classical cell surface transporters. The curative dose of pentamidine-loaded nanobody-chitosan nanoparticles was 100-fold lower than pentamidine alone in a murine model of acute African trypanosomiasis. Crucially, this new formulation displayed undiminished in vitro and in vivo activity against a trypanosome cell line resistant to pentamidine as a result of mutations in the surface transporter aquaglyceroporin 2. We conclude that this new drug delivery system increases drug efficacy and has the ability to overcome resistance to some anti-protozoal drugs.

Number of Nanoparticles per Cell through a Spectrophotometric Method - A key parameter to Assess Nanoparticle-based Cellular Assays

Engineered nanoparticles (eNPs) for biological and biomedical applications are produced from functionalised nanoparticles (NPs) after undergoing multiple handling steps, giving rise to an inevitable loss of NPs. Herein we present a practical method to quantify nanoparticles (NPs) number per volume in an aqueous suspension using standard spectrophotometers and minute amounts of the suspensions (up to 1 μL). This method allows, for the first time, to analyse cellular uptake by reporting NPs number added per cell, as opposed to current methods which are related to solid content (w/V) of NPs. In analogy to the parameter used in viral infective assays (multiplicity of infection), we propose to name this novel parameter as multiplicity of nanofection.

New Approaches to Overcome Transport Related Drug Resistance in Trypanosomatid Parasites

Leishmania and Trypanosoma are members of the Trypanosomatidae family that cause severe human infections such as leishmaniasis, Chagas disease, and sleeping sickness affecting millions of people worldwide. Despite efforts to eradicate them, migrations are expanding these infections to developing countries. There are no vaccines available and current treatments depend only on chemotherapy. Drug resistance is a major obstacle for the treatment of these diseases given that existing drugs are old and limited, with some having severe side effects. Most resistance mechanisms developed by these parasites are related with a decreased uptake or increased efflux of the drug due to mutations or altered expression of membrane transporters. Different new approaches have been elaborated that can overcome these mechanisms of resistance including the use of inhibitors of efflux pumps and drug carriers for both active and passive targeting. Here we review new formulations that have been successfully applied to circumvent resistance related to drug transporters, opening alternative ways to solve drug resistance in protozoan parasitic diseases.

Progress Towards New Treatments for Human African Trypanosomiasis

The treatment of African trypanosomiasis has essentially remained unchanged for decades. A mountain of excellent work has been produced on many aspects of trypanosome biochemistry, biology, genetics, etc., but this has not translated into new therapies, although the disease burden has steadily increased through the latter half of the twentieth century. The only new drug to be introduced in the last 50 years or so is eflornithine, in the late 1970s, for the treatment of late-stage gambiense sleeping sickness only. However, this was in many ways unsatisfactory and melarsoprol remained the first-line treatment for late-stage sleeping sickness until an alarming increase in treatment failures necessitated change. Since the emerging sleeping sickness epidemic became widely recognised, around the year 2000, needs-driven development of new drugs, and the preservation of the production of old drugs, has been the result of dedicated work by organisations such as the World Health Organisation, the Drugs for Neglected Diseases initiative (DNDi), the Access to Essential Medicines campaign, and the Consortium for Parasitic Drug Development (CPDD) among others, much of it in partnership with academia and the pharmaceutical industry. This has already resulted in milestones such as the donations of free treatments by producers; improved drug distribution, case finding and clinical care; an improved 10-day melarsoprol treatment; the first clinical trial for an oral sleeping sickness drug—pafuramidine and the introduction of eflornithine–nifurtimox combination therapy to begin replacing melarsoprol. While these efforts have undoubtedly contributed to reducing the disease burden in central Africa, newer treatments are still very necessary, especially as most current treatments are threatened by drug resistance. Here, we review recent advances in understanding drug resistance mechanisms, progress towards new drugs, and new delivery systems to improve efficacy.

Amide-controlled, one-pot synthesis of tri-substituted purines generates structural diversity and analogues with trypanocidal activity

A novel one-pot synthesis of tri-substituted purines and the discovery of purine analogues with trypanocidal activity are reported. The reaction is initiated by a metal-free oxidative coupling of primary alkoxides and diaminopyrimidines with Schiff base formation and subsequent annulation in the presence of large N,N-dimethylamides (e.g. N,N-dimethylpropanamide or larger). This synthetic route is in competition with a reaction previously-reported by our group1, allowing the generation of a combinatorial library of tri-substituted purines by the simple modification of the amide and the alkoxide employed. Among the variety of structures generated, two purine analogues displayed trypanocidal activity against the protozoan parasite Trypanosoma brucei with IC50 < 5 μM, being each of those compounds obtained through each of the synthetic pathways.

Identification and characterization of a bacterial hyaluronidase and its production in recombinant form.

Hyaluronidases (Hyals) are broadly used in medical applications to facilitate the dispersion and/or absorption of fluids or medications. This study reports the isolation, cloning, and industrial‐scale recombinant production, purification and full characterization, including X‐ray structure determination at 1.45 Å, of an extracellular Hyal from the nonpathogenic bacterium Streptomyces koganeiensis. The recombinant S. koganeiensis Hyal (rHyal_Sk) has a novel bacterial catalytic domain with high enzymatic activity, compared with commercially available Hyals, and is more thermostable and presents higher proteolytic resistance, with activity over a broad pH range. Moreover, rHyal_Sk exhibits remarkable substrate specificity for hyaluronic acid (HA) and poses no risk of animal cross‐infection.

Drug ‘clicking’ on cell-penetrating fluorescent nanoparticles for in cellulo chemical proteomics

Chemical proteomics approaches are widely used to identify molecular targets of existing or novel drugs. This manuscript describes the development of a straightforward approach to conjugate azide-labeled drugs via click chemistry to alkyne-tagged cell-penetrating fluorescent nanoparticles as a novel tool to study target engagement and/or identification inside living cells. A modification of the Baeyer test for alkynes allows monitoring the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, guaranteeing the presence of the drug on the solid support. As a proof of concept, the conjugation of the promiscuous kinase inhibitor dasatinib to Cy5-labeled nanoparticles is presented. Dasatinib-decorated fluorescent nanoparticles efficiently inhibited its protein target SRC in vitro, entered cancer cells, and colocalized with SRC in cellulo.

Tracking cell proliferation using a nanotechnology-based approach

AIM To develop an efficient nanotechnology fluorescence-based method to track cell proliferation to avoid the limitations of current cell-labeling dyes. MATERIAL & METHODS Synthesis, PEGylation, bifunctionalization and labeling with a fluorophore (Cy5) of 200 nm polystyrene nanoparticles (NPs) were performed. These NPs were characterized and assessed for in vitro long-term monitoring of cell proliferation. RESULTS The optimization and validation of this method to track long-term cell proliferation assays have been achieved with high reproducibility, without cell cycle disruption. This method has been successfully applied in several adherent and suspension cells including hard-to-transfect cells and isolated human primary lymphocytes. CONCLUSION A novel approach to track efficiently cellular proliferation by flow cytometry using fluorescence labeled NPs has been successfully developed. [Formula: see text].