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Made available in DSpace on 2019-10-06T16:32:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-09-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Percutaneous medical devices are prone to bacterial contamination that causes dramatic clinical conditions. At the percutaneous level of dental implant systems, microbial pathogens induce biofilm formation that may result in bone resorption and dental implant loss. In view of peri-implantitis caused by bacterial inflammation at the percutaneous abutment region, we here establish a novel drug release system based on layer-by-layer (LbL)-deposited poly(acrylic acid) (PAA) and poly-L-lysine (PLL) coatings on titanium (Ti). Detailed multilayer coating characterization was performed by different microscopy and spectroscopy techniques to probe physical and chemical properties. Our data revealed a significant difference in roughness average between ten double layers coated (141 nm ±30) and uncoated Ti discs (115 nm ±40). Although roughness of the coatings increased significantly after immersion in water for 24 h at 37 °C, this physical property remained below 200 nm. Coating stability was confirmed under neutral and acidic pH, mimicking healthy and diseased/inflammatory environments, respectively. LbL coatings supported in vitro human keratinocytes growth, demonstrating absence of cytotoxic effects. Tetracycline (TC) showed an initial burst release under neutral and acidic conditions, which further demonstrated robust antibacterial efficacy against Porphyromonas gingivalis. However, a convenient pH-dependent 2-folds increase in TC release was observed for coatings incubated at pH = 4.5. Sustained TC release was observed from coatings up till 15 days of incubation in both pH conditions. These results demonstrate the potential application of this simple surface modification to leverage anti-bacterial efficacy at the percutaneous abutment region. Regenerative Biomaterials Radboudumc, Philips van Leydenlaan 25 Department of Dental Materials and Prosthodontics School of Dentistry at Araraquara Sao Paulo State University (Unesp), Humaita, Sao Paulo Department of Tumor Immunology Radboudumc and Radboud Institute for Molecular Life Sciences (RIMLS), Geert Grooteplein Zuid Department of Preventive Dentistry Academic Centre for Dentistry Amsterdam (ACTA) University of Amsterdam and Vrije Universiteit Amsterdam, Gustav Mahlerlaan Department of Organic Chemistry Radboud University, Heyendaalseweg 135 Faculty of Applied Sciences Delft University of Technology, Lorentzweg 1 Department of Dental Materials and Prosthodontics School of Dentistry at Araraquara Sao Paulo State University (Unesp), Humaita, Sao Paulo FAPESP: 2015/03567-7 FAPESP: 2016/19650-3

With the advent of novel immunotherapies, interest in ex vivo autologous cell labeling for in vivo cell tracking has revived. However, current clinically available labeling strategies have several drawbacks, such as release of radiolabel over time and cytotoxicity. Poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) are clinically used biodegradable carriers of contrast agents, with high loading capacity for multimodal imaging agents. Here we show the development of PLGA-based NPs for ex vivo cell labeling and in vivo cell tracking with SPECT. We used primary amine-modified PLGA polymers (PLGA-NH2) to construct NPs similar to unmodified PLGA NPs. PLGA-NH2 NPs were efficiently radiolabeled without chelator and retained the radionuclide for 2 weeks. Monocyte-derived dendritic cells labeled with [111In]In-PLGA-NH2 showed higher specific activity than those labeled with [111In]In-oxine, with no negative effect on cell viability. SPECT/CT imaging showed that radiolabeled THP-1 cells accumulated at the Staphylococcus aureus infection site in mice. In conclusion, PLGA-NH2 NPs are able to retain 111In, independent of chelator presence. Furthermore, [111In]In-PLGA-NH2 allows cell labeling with high specific activity and no loss of activity over prolonged time intervals. Finally, in vivo tracking of ex vivo labeled THP-1 cells was demonstrated in an infection model using SPECT/CT imaging.

Made available in DSpace on 2021-06-25T10:49:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) This study demonstrated a drug-delivery system with anionic beta cyclodextrin (β-CD) complexes to retain tetracycline (TC) and control its release from multilayers of poly(acrylic acid) (PAA) and poly(l-lysine) (PLL) in a ten double layers ([PAA/PLL]10) coating onto titanium. The drug-delivery capacity of the multilayer system was proven by controlled drug release over 15 days and sustained released over 30 days. Qualitative images confirmed TC retention within the layer-by-layer (LbL) over 30 days of incubation. Antibacterial activity of TC/anionic β-CD released from the LbL was established against Staphylococcus aureus species. Remarkably, [PAA/PLL]10/TC/anionic β-CD antibacterial effect was sustained even after 30 days of incubation. The non-cytotoxic effect of the multilayer system revealed normal human gingival fibroblast growth. It is expected that this novel approach and the chemical concept to improve drug incorporation into the multilayer system will open up possibilities to make the drug release system more applicable to implantable percutaneous devices. Department of Dental Materials and Prosthodontics School of Dentistry at Araraquara São Paulo State University (UNESP), Humaita, 1680 Araraquara Regenerative Biomaterials Radboudumc, Philips Van Leydenlaan 25 Department of Physical Chemistry Institute of Chemistry São Paulo State University (UNESP) Department of Prosthodontics and Periodontology Piracicaba Dental School University of Campinas (UNICAMP), Av. Limeira, 901 Department of Tumor Immunology Radboudumc and Radboud Institute for Molecular Life Sciences (RIMLS), Geert Grooteplein Zuid, 28 Nijmegen Department of Physiology and Pathology School of Dentistry at Araraquara São Paulo State University (UNESP) Department of Dental Materials and Prosthodontics School of Dentistry at Araraquara São Paulo State University (UNESP), Humaita, 1680 Araraquara Department of Physical Chemistry Institute of Chemistry São Paulo State University (UNESP) Department of Physiology and Pathology School of Dentistry at Araraquara São Paulo State University (UNESP) FAPESP: 2015/03567-7 FAPESP: 2018/19345-1

With the advent of novel immunotherapies, interest in ex vivo autologous cell labeling for in vivo cell tracking has revived. However, current clinically available labeling strategies have several drawbacks, such as release of radiolabel over time and cytotoxicity. Poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) are clinically used biodegradable carriers of contrast agents, with high loading capacity for multimodal imaging agents. Here we show the development of PLGA-based NPs for ex vivo cell labeling and in vivo cell tracking with SPECT. We used primary amine-modified PLGA polymers (PLGA-NH2) to construct NPs similar to unmodified PLGA NPs. PLGA-NH2 NPs were efficiently radiolabeled without chelator and retained the radionuclide for 2 weeks. Monocyte-derived dendritic cells labeled with [111In]In-PLGA-NH2 showed higher specific activity than those labeled with [111In]In-oxine, with no negative effect on cell viability. SPECT/CT imaging showed that radiolabeled THP-1 cells accumulated at the Staphylococcus aureus infection site in mice. In conclusion, PLGA-NH2 NPs are able to retain 111In, independent of chelator presence. Furthermore, [111In]In-PLGA-NH2 allows cell labeling with high specific activity and no loss of activity over prolonged time intervals. Finally, in vivo tracking of ex vivo labeled THP-1 cells was demonstrated in an infection model using SPECT/CT imaging.

Contains fulltext : 206962.pdf (Publisher’s version ) (Open Access) The knowledge of in vitro and in vivo stability of polymeric nanoparticles is vital for the development of clinical formulations for drug delivery and cell labeling applications. Forster resonance energy transfer (FRET)-based fluorescence labeling approaches are promising tools to study nanoparticle stability under different physiological conditions. Here, we present the FRET-based stability assessment of poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating BODIPY-FL12 and Nile Red as the donor and acceptor, respectively. The stability of PLGA nanoparticles is studied via monitoring the variations of fluorescence emission characteristics along with colloidal characterization. Accordingly, PLGA nanoparticles are colloidally stable for more than 2 weeks when incubated in aqueous buffers in situ, whereas in vitro particle degradation starts in between 24 and 48 h, reaching a complete loss of FRET at 72 h as shown with fluorescence microscopy imaging and flow cytometry analysis. PLGA nanoparticles systemically administered to mice predominantly accumulate in the liver, in which FRET no longer takes place at time points as early as 24 h postadministration as determined by ex vivo organ imaging and flow cytometry analysis. The results of this study expand our knowledge on drug release and degradation behavior of PLGA nanoparticles under different physiological conditions, which will prove useful for the rational design of PLGA-based formulations for various applications that can be translated into clinical practice.

Perfluorocarbons hold great promise both as imaging agents, particularly for

The clinical and commercial development of polymeric sub-micron size formulations based on poly(lactic-co-glycolic acid) (PLGA) particles is hampered by the challenges related to their good manufacturing practice (GMP)-compliant, scale-up production without affecting the formulation specifications. Continuous process technologies enable large-scale production without changing the process or formulation parameters by increasing the operation time. Here, we explore three well-established process technologies regarding continuity for the large-scale production of sub-micron size PLGA particles developed at the lab scale using a batch method. We demonstrate optimization of critical process and formulation parameters for high-shear mixing, high-pressure homogenization and microfluidics technologies to obtain PLGA particles with a mean diameter of 150–250 nm and a small polydispersity index (PDI, ≤0.2). The most influential parameters on the particle size distribution are discussed for each technique with a critical evaluation of their suitability for GMP production. Although each technique can provide particles in the desired size range, high-shear mixing is found to be particularly promising due to the availability of GMP-ready equipment and large throughput of production. Overall, our results will be of great guidance for establishing continuous process technologies for the GMP-compliant, large-scale production of sub-micron size PLGA particles, facilitating their commercial and clinical development.

Contains fulltext : 244666.pdf (Publisher’s version ) (Open Access) Nanomedicines based on poly(lactic-co-glycolic acid) (PLGA) carriers offer tremendous opportunities for biomedical research. Although several PLGA-based systems have already been approved by both the Food and Drug Administration (FDA) and the European Medicine Agency (EMA), and are widely used in the clinics for the treatment or diagnosis of diseases, no PLGA nanomedicine formulation is currently available on the global market. One of the most impeding barriers is the development of a manufacturing technique that allows for the transfer of nanomedicine production from the laboratory to an industrial scale with proper characterization and quality control methods. This review provides a comprehensive overview of the technologies currently available for the manufacturing and analysis of polymeric nanomedicines based on PLGA nanoparticles, the scale-up challenges that hinder their industrial applicability, and the issues associated with their successful translation into clinical practice.

Nanovaccines, co-delivering antigen and invariant natural killer T (iNKT) cell agonists, proved to be very effective in inducing anti-tumor T cell responses due to their exceptional helper function. However, it is known that iNKT cells are not equally present in all lymphoid organs and nanoparticles do not get evenly distributed to all immune compartments. In this study, we evaluated the effect of the vaccination route on iNKT cell help to T and B cell responses for the first time in an antigen and agonist co-delivery setting. Intravenous administration of PLGA nanoparticles was mainly targeting liver and spleen where iNKT1 cells are abundant and induced the highest serum IFN-y levels, T cell cytotoxicity, and Th-1 type antibody responses. In comparison, after subcutaneous or intranodal injections, nanoparticles mostly drained or remained in regional lymph nodes where iNKT17 cells were abundant. After subcutaneous and intranodal injections, antigen-specific IgG2 c production was hampered and IFN-y production, as well as cytotoxic T cell responses, depended on sporadic systemic drainage. Therapeutic anti-tumor experiments also demonstrated a clear advantage of intravenous injection over intranodal or subcutaneous vaccinations. Moreover, tumor control could be further improved by PD-1 immune checkpoint blockade after intravenous vaccination, but not by intranodal vaccination. Anti PD-1 antibody combination mainly exerts its effect by prolonging the cytotoxicity of T cells. Nanovaccines also demonstrated synergism with anti-4-1BB agonistic antibody treatment in controlling tumor growth. We conclude that nanovaccines containing iNKT cell agonists shall be preferentially administered intravenously, to optimally reach cellular partners for inducing effective anti-tumor immune responses.

Contains fulltext : 229164.pdf (Publisher’s version ) (Open Access) Perfluorocarbons hold great promise both as imaging agents, particularly for (19)F MRI, and in therapy, such as oxygen delivery. (19)F MRI is unique in its ability to unambiguously track and quantify a tracer while maintaining anatomic context, and without the use of ionizing radiation. This is particularly well-suited for inflammation imaging and quantitative cell tracking. However, perfluorocarbons, which are best suited for imaging - like perfluoro-15-crown-5 ether (PFCE) - tend to have extremely long biological retention. Here, we showed that the use of a multi-core PLGA nanoparticle entrapping PFCE allows for a 15-fold reduction of half-life in vivo compared to what is reported in literature. This unexpected rapid decrease in (19)F signal was observed in liver, spleen and within the infarcted region after myocardial infarction and was confirmed by whole body NMR spectroscopy. We demonstrate that the fast clearance is due to disassembly of the ~200 nm nanoparticle into ~30 nm domains that remain soluble and are cleared quickly. We show here that the nanoparticle ultrastructure has a direct impact on in vivo clearance of its cargo i.e. allowing fast release of PFCE, and therefore also bringing the possibility of multifunctional nanoparticle-based imaging to translational imaging, therapy and diagnostics.

Polymeric particles made up of biodegradable and biocompatible polymers such as poly(lactic-co-glycolic acid) (PLGA) are promising tools for several biomedical applications including drug delivery. Particular emphasis is placed on the size and surface functionality of these systems as they are regarded as the main protagonists in dictating the particle behavior in vitro and in vivo. Current methods of manufacturing polymeric drug carriers offer a wide range of achievable particle sizes, however, they are unlikely to accurately control the size while maintaining the same production method and particle uniformity, as well as final production yield. Microfluidics technology has emerged as an efficient tool to manufacture particles in a highly controllable manner. Here, we report on tuning the size of PLGA particles at diameters ranging from sub-micron to microns using a single microfluidics device, and demonstrate how particle size influences the release characteristics, cellular uptake and in vivo clearance of these particles. Highly controlled production of PLGA particles with ~100 nm, ~200 nm, and &gt, 1000 nm diameter is achieved through modification of flow and formulation parameters. Efficiency of particle uptake by dendritic cells and myeloid-derived suppressor cells isolated from mice is strongly correlated with particle size and is most efficient for ~100 nm particles. Particles systemically administered to mice mainly accumulate in liver and ~100 nm particles are cleared slower. Our study shows the direct relation between particle size varied through microfluidics and the pharmacokinetics behavior of particles, which provides a further step towards the establishment of a customizable production process to generate tailor-made nanomedicines.

Unraveling the mechanisms that govern the formation and function of invadopodia is essential towards the prevention of cancer spread. Here, we characterize the ultrastructural organization, dynamics and mechanical properties of collagenotytic invadopodia forming at the interface between breast cancer cells and a physiologic fibrillary type I collagen matrix. Our study highlights an uncovered role for MT1-MMP in directing invadopodia assembly independent of its proteolytic activity. Electron microscopy analysis reveals a polymerized Arp2/3 actin network at the concave side of the curved invadopodia in association with the collagen fibers. Actin polymerization is shown to produce pushing forces that repel the confining matrix fibers, and requires MT1-MMP matrix-degradative activity to widen the matrix pores and generate the invasive pathway. A theoretical model is proposed whereby pushing forces result from actin assembly and frictional forces in the actin meshwork due to the curved geometry of the matrix fibers that counterbalance resisting forces by the collagen fibers., The mechanism of force production by invadopodia is unclear. Here, the authors show that cell surface MT1-MMP when in contact with collagen, induces Arp2/3 branched actin polymerisation on the concave side of invadopodia, which generates a pushing force along with collagen cleavage by MT1-MMP to invade.

Item does not contain fulltext The use of nanotechnologies for the development of highly sensitive and affordable diagnostic assays has significantly improved the ability to detect and characterize multiple types of biomarkers. Semiconductor and metal nanoparticles with unique optical properties have been successfully integrated within biomarker detection schemes for the generation and enhancement of optical signals in label-based and label-free assays. Highly sensitive label-based diagnostics has been realized particularly via using quantum dots (QDs) as labeling probes. Similarly, many label-free techniques that are emerging as potential complements to label-based approaches benefit from signal enhancement strategies using e.g., metal nanoparticles. This review presents a concise overview of recent advances in diagnostic assays that utilize nanoparticles for the generation and enhancement of optical signals in fluorescence- and surface plasmon resonance-based techniques. Advanced diagnostic assays that utilize nanoparticles provide major improvements in detection sensitivity, which can potentially meet the challenging requirements of clinical diagnostics.

International audience; Quantum dots (QDs) are not only advantageous for color-tuning, improved brightness, and high stability, but their nanoparticle surfaces also allow for the attachment of many biomolecules. Because IgG antibodies (AB) are in the same size range of biocompatible QDs and the AB orientation after conjugation to the QD is often random, it is difficult to predict if few or many AB per QD will lead to an efficient AB-QD conjugate. This is particularly true for homogeneous Förster resonance energy transfer (FRET) sandwich immunoassays, for which the AB on the QD must bind a biomarker that needs to bind a second AB-FRET-conjugate. Here, we investigate the performance of Tb-to-QD FRET immunoassays against total prostate specific antigen (TPSA) by changing the number of AB per QD while leaving all the other assay components unchanged. We first characterize the AB-QD conjugation by various spectroscopic, microscopic, and chromatographic techniques and then quantify the TPSA immunoassay performance regarding sensitivity, limit of detection, and dynamic range. Our results show that an increasing conjugation ratio leads to significantly enhanced FRET immunoassays. These findings will be highly important for developing QD-based immunoassays in which the concentrations of both AB and QDs can significantly influence the assay performance.

A simple method for the encapsulation of quantum dots (QDs) in virus-like particle (VLP) nanoassemblies with tuneable structural properties and enhanced biocompatibility is presented. Cowpea chlorotic mottle virus-based capsid proteins assemble around the carboxylated QDs to form QD/VLP nanoassemblies of different capsid size as a function of pH and ionic strength. Detailed structural characterizations verify that nanoassemblies with probably native capsid icosahedral symmetry (T = 3) are obtained at low pH and high ionic strength (pH 5.0, 1.0 M NaCl), whereas high pH and low ionic strength conditions (pH 7.5, 0.3 M NaCl) result in the formation of smaller assembly sizes similar to T = 1 symmetry. In vitro studies reveal that QD/VLP nanoassemblies are efficiently internalized by RAW 264.7 macrophages and HeLa cells with no signs of toxicity at QD concentrations exceeding the potentially-toxic levels. The presented route holds great promise for preparation of size-tuneable, robust, non-toxic luminescent probes for long term cellular imaging applications. Furthermore, thanks to the possibility of chemical and genetic manipulation of the viral protein shell encaging the QDs, the nanoassemblies have potential for in vivo targeting applications.

The interplay of photonics, nanotechnology, and biochemistry has significantly improved the identification and characterization of multiple types of biomarkers by optical biosensors. Great achievements in fluorescence-based technologies have been realized, for example, by the advancement of multiplexing techniques or the introduction of nanoparticles to biochemical and clinical research. This review presents a concise overview of recent advances in fluorescence sensing techniques for the detection of circulating disease biomarkers. Detection principles of representative approaches, including fluorescence detection using molecular fluorophores, quantum dots, and metallic and silica nanoparticles, are explained and illustrated by pertinent examples from the recent literature. Advanced detection technologies and material development play a major role in modern biosensing and consistently provide significant improvements toward robust, sensitive, and versatile platforms for early detection of circulating diagnostic biomarkers.

We report on the proof-of-principle of using semiconductor nanorods (quantum rods; QRs) in homogeneous Forster resonance energy transfer (FRET) bioassays. Terbium complexes (Tb) with long photoluminescence lifetimes were used as FRET donors, and biological recognition was accomplished by biotin-QR to Tb-streptavidin binding. Time-resolved and steady-state spectroscopy were used to investigate varying relative donor/acceptor concentrations and different lengths of polyethylene glycol (PEG)-based surface coatings. Homogeneous bioassays displayed low picomolar detection limits in 150 μL samples, independent of whether 1 and 10 kDa biotin-PEG-SH surface ligands were used. The results suggest that the combination of Tb-to-QR FRET with time-gated detection may become a powerful tool for homogeneous biosensing.

Drug delivery to the brain is one of the major challenges in the treatment of cerebral diseases and implies extensive understanding of nanomedicine transcytosis pathways across the blood–brain barr...