Effects of chemical and green nano-zinc oxide on histological changes, oxidative stress, and apoptosis in rat kidney associated with cisplatin

Abstract Cisplatin (CP) is used to treat various tumors. A main restriction of cisplatin is nephrotoxicity. This study aimed to evaluate the protective effects of ZnONPs on cisplatin-induced oxidative stress and rat kidney tissue damage. Eighty adult male Wistar rats (250g-270g) were divided into ten groups: Control (CON), Sham (SH), Bulk ZnO (BZnO), Chemical ZnONPs (ChZnONPs), Green ZnONPs (GrZnONPs), Cisplatin (CP), Cisplatin+BulkZnO (CP+BZnO), Cisplatin+Green ZnONPs (CP+GrZnONPs), Cisplatin+Chemical ZnONPs (CP+ChZnONPs), Cisplatin+Explant (CP+EX). CP was i.p administered 5mg/kg/week and BZnO, ChZnONPs and GrZnONPs were i.p administered at a dose of 5mg/kg/day. After 30 days of the treatment, the expression of apoptosis/anti apoptosis related genes oxidant/antioxidant factors and histological changes in the were studied. The CP-treated group showed a decrease in body weight, while the Co-administration of ZGNPs to CP-treated rats showed a significant increase compared to the CP group. The results showed that the increased mRNA level of bax, MDA and the decreased mRNA level of bcl2, SOD and CAT activities in kidney of CP group were improved when animals were treated with ZnO NPs. Our results showed that GrZnONPs, ChZnONPs and BZnO had the potential to protect against oxidative stress and cisplatin-induced neurotoxicity that this protective effect was more evident in GrZnONPs.

Effects of Chitosan Nanoparticles with Long Synthetic siRNAs Targeting VEGF in Triple-Negative Breast Cancer Cells

Abstract Vascular endothelial growth factor (VEGF) is an essential angiogenic factor in breast cancer development and metastasis. Small interfering RNAs (siRNAs) can specifically silence genes via the RNA interference pathway, therefore were investigated as cancer therapeutics. In this study, we investigated the effects of siRNAs longer than 30 base pairs (bp) loaded into chitosan nanoparticles in triple-negative breast cancer cells, compared with conventional siRNAs. 35 bp long synthetic siRNAs inhibited VEGF gene expression by 51.2% and increased apoptosis level by 1.75-fold in MDA-MB-231 cell lines. Furthermore, blank and siRNA-loaded chitosan nanoparticles induced expression of IFN-γ in breast cancer cells. These results suggest that long synthetic siRNAs can be as effective as conventional siRNAs, when introduced into cells with chitosan nanoparticles

Development of novel mucoadhesive gels containing nanoparticle for buccal administration of dexamethasone

Abstract This study aimed to develop promising and innovative mucoadhesive gel systems containing dexamethasone-loaded nanoparticle to increase the effectiveness of treatment for oral precancerous lesions and to reduce side effects. In this respect, a dexamethasone-loaded nanoparticle formulation was prepared by using emulsification/solvent evaporation method. The nanoparticle has high zeta potential (-10.3±0.5 mV), low particle size (218.42±2.1), low polydispersity index (0.070±0.014) and high encapsulation efficiency (95.018±2.982%). To improve the mucosal retention time, the dexamethasone-loaded nanoparticle was dispersed in mucoadhesive gel using gellan gum. The developed gels offered appropriate pH value, high drug content, suitable mechanical and mucoadhesive performance and appropriate viscosity for mucosal administration. All formulations exhibited plastic flow and typical gel-type mechanical spectra after the determined frequency value. The developed formulations exhibited extended drug release as intended for these systems. Cytotoxicity was tested by MTT assay in human epithelioid carcinoma cell (HeLa) in vitro. The MTT assay showed that the blank formulations were non-toxic to cells. It was observed that the bioactivity of the free dexamethasone was potentiated by mucoadhesive gels containing dexamethasone-loaded nanoparticle in HeLa cells. Results from this study indicate that mucoadhesive gels are effective for the local treatment of precancerous lesions. Our findings showed that the developed formulations were worthy of further studies.

Protective effects of psoralen polymer lipid nanoparticles on doxorubicin - induced myocardial toxicity

Abstract Doxorubicin (DOX) induced myocardial toxicity may limit its therapeutic use in clinic. Psoralen (PSO), a major active tricyclic furocoumarin extracted from Psoralea corylifolia, is widely used as an antineoplastic agent in treatment of leukemia and other cancers. This study is aim to find the protective effect of psoralen polymer lipid nanoparticles (PSO-PLN) on doxorubicin-induced myocardial toxicity in mice. The model of myocardial toxicity induced by DOX was established. The experiment was divided into 6 groups: normal saline group, DOX + Sulfotanshinone Sodium, DOX + PSO-PLN (3 mg/kg), DOX + PSO-PLN (6 mg/kg), DOX + PSO-PLN (9 mg/ kg), DOX group. DOX alone treated mice lead to a significant decrease in the body weight, heart weight, and increase in the serum levels of lactate dehydrogenase (LDH), creatine kinase (CK) and malondialdehyde (MDA) markers of cardiotoxicity. However, DOX reduced glutathione (GSH) content and activities of antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GPX), were recovered by PSO-PLN. And PSO-PLN also decreased markers of cardiotoxicity in the serum. Western blotting data showed that the protective effects of PSO-PLN might be mediated via regulation of protein kinase A (PKA) and p38. Our study suggest that PSO-PLN possesses antioxidant activities, inactivating PKA and p38 effect, which in turn protect the heart from the DOX-induced cardiotoxicity.

Formulation and evaluation of the vascular endothelial growth factor loaded polycaprolactone nanoparticles

Abstract In an attempt to increase molecular stability and provide controlled release, vascular endothelial growth factor (VEGF) was encapsulated into polycaprolactone (PCL) nanoparticles. Both VEGF-free and VEGF-loaded PCL nanoparticles were formulated by w/o/w double emulsion of the dichloromethane-water system in the presence of polyvinyl alcohol (PVA) and rat serum albumin. To achieve the optimal formulation concerning particle size and monodispersity, studies were carried out with different formulation parameters, including PVA concentration, homogenization time and rate. Scanning electron microscopy and dynamic light scattering analysis showed respectively that particles had a spherical shape with a smooth surface and particle size varying between 58.68-751.9 nm. All of the formulations were negatively charged according to zeta potential analysis. In vitro release study was performed in pH 7.4 phosphate-buffered saline at 37°C and released VEGF amount was measured by enzyme-linked immunosorbent assay (ELISA) method. At the end of the 35th day, 10% of total encapsulated VEGF was released with a sustained-release profile, which fitted the Korsmeyer-Peppas kinetic model. The bioactivation of the nanoparticles was evaluated using XTT and ELISA methods. As a result, the released VEGF was biologically active and also VEGF loaded PCL nanoparticles enhanced proliferation of the human umbilical vein endothelial cells in cell culture.

Synthesis and characterization of Sophora alopecuroides L. green synthesized of Ag nanoparticles for the antioxidant, antimicrobial and DNA damage prevention activity

Abstract In this study, it was aimed to investigate the amount of antioxidant, protective properties against DNA damage and antibacterial properties against various pathogens after the interaction of Ag metal (Ag NPs/Sa) of Sophora alopecuroides L. (S. alopecuroides L) plant seed, which is grown in Igdir and used in the treatment of many diseases. The DPPH radical quenching activity of Ag NPs/Sa was performed by using Blois method, DNA damage prevention activity by gel electrophoresis and antibacterial property by disk diffusion method. With the green synthesis method, AgNPs obtained as a result of the reaction of the plant and Ag metal are UV visible spectrophotometer (UV-vis), fourier-transformed infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and scanning electron microscope (SEM). DPPH radical quenching activity of Ag NPs/Sa was investigated in the concentration range of 25-250 µg/ml. The radical quenching activity at a concentration of 250 µg/ml was 85,215 ± 0,101%, while this value was 93,018% for the positive control BHA. It has been observed that the protective property of pBR322 plasmid DNA damage against OH radicals originating from H2O2 increases with concentration. It has been observed that Ag NPs/Sa has significant antimicrobial properties against some pathogens (B. subtilis ATCC 6633 E. coli ATCC 25952, B. cereus ATCC 10876, P. aeruginosa ATCC 27853, E. faecalis ATCC 29212, S. aureus ATTC 29213 and C. albicans ATTC 90028) that cause disease and even some pathogens are more effective than antibiotics

Supermeres are functional extracellular nanoparticles replete with disease biomarkers and therapeutic targets.

Extracellular vesicles and exomere nanoparticles are under intense investigation as sources of clinically relevant cargo. Here we report the discovery of a distinct extracellular nanoparticle, termed supermere. Supermeres are morphologically distinct from exomeres and display a markedly greater uptake in vivo compared with small extracellular vesicles and exomeres. The protein and RNA composition of supermeres differs from small extracellular vesicles and exomeres. Supermeres are highly enriched with cargo involved in multiple cancers (glycolytic enzymes, TGFBI, miR-1246, MET, GPC1 and AGO2), Alzheimer's disease (APP) and cardiovascular disease (ACE2, ACE and PCSK9). The majority of extracellular RNA is associated with supermeres rather than small extracellular vesicles and exomeres. Cancer-derived supermeres increase lactate secretion, transfer cetuximab resistance and decrease hepatic lipids and glycogen in vivo. This study identifies a distinct functional nanoparticle replete with potential circulating biomarkers and therapeutic targets for a host of human diseases.

Inhaled nano-based therapeutics for inflammatory lung diseases: Recent advances and future prospects.

Inflammatory lung diseases related morbidity and mortality impose a significant financial burden. Inflammation is a hallmark of many diseases of the respiratory system which is directly or indirectly linked to adverse health conditions, air pollution, rapid lifestyle changes, and regular outbreaks of microbial infections. The unique anatomical and physiological features of the lungs make them an ideal target organ in the treatment of inflammatory respiratory disease and with the help of inhaled therapy lungs can be targeted directly. The principal objective of this review is to present the comprehensive role of inhaled nano-based therapeutics such as liposomes, niosomes, nanoparticles, nanoemulsion, nanosuspension, and exosomes in the treatment and management of inflammatory respiratory diseases. Inhaled nanomedicines provide targeted diagnosis and treatment, improved drug solubility and distribution, prevent first-pass hepatic metabolism, improved patient compliance, and reduced drug side effects. They overcome several biological barriers in the human body and provide immediate, and quick-onset of action. Future research should be focused on improving the therapeutic efficiency of inhaled nanocarriers and to carry out in-depth mechanistic studies to translate current scientific knowledge for the efficient management of inflammatory lung diseases with minimal or no toxicity.

Nanoparticles as a novel and promising antiviral platform in veterinary medicine.

Traditional veterinary virus vaccines, such as inactivated and live-attenuated vaccines, have achieved tremendous success in controlling many viral diseases of livestock and chickens worldwide. However, many recent viral outbreaks caused by different emerging and re-emerging viruses continue to be reported annually worldwide. It is therefore necessary to develop new control regimens. Nanoparticle research has received considerable attention in the last two decades as a promising platform with significant success in veterinary medicine, replacing traditional viral vector vaccines. However, the field of nanoparticle applications is still in its initial phase of growth. Here, we discuss various preparation methods, characteristics, physical properties, antiviral effects, and pharmacokinetics of well-developed nanoparticles and the potential of nanoparticles or nano-vaccines as a promising antiviral platform for veterinary medicine.

In-depth characterization of the aggregation state of cellulose nanocrystals through analysis of transmission electron microscopy images.

Dispersion of cellulose nanocrystals (CNCs) is of utmost importance to guarantee their reliable application. Nevertheless, there is still no consensual method to characterize CNC aggregation. The hypothesis of this paper is that dispersion could be quantified through the classification of aggregates detected in transmission electron microscopy images. k-Means was used to classify image particulate elements of five CNC samples into groups according to their geometric features. Particles were classified into five groups according to their maximum Feret diameter, elongation, circularity and area. Two groups encompassed the most application-critical aggregates: one integrated aggregates of high complexity and low compactness while the other included elongated aggregates. In addition, the characterization of CNC dispersion after different levels of sonication was achieved by assessing the change in the number of elements belonging to each cluster after sonication. This approach could be used as a standard for the characterization of the aggregation state of CNCs.

Tuning cell behavior with nanoparticle shape.

We investigated how the shape of polymeric vesicles, made by the exact same material, impacts the replication activity and metabolic state of both cancer and non-cancer cell types. First, we isolated discrete geometrical structures (spheres and tubes) from a heterogeneous sample using density-gradient centrifugation. Then, we characterized the cellular internalization and the kinetics of uptake of both types of polymersomes in different cell types (either cancer or non-cancer cells). We also investigated the cellular metabolic response as a function of the shape of the structures internalized and discovered that tubular vesicles induce a significant decrease in the replication activity of cancer cells compared to spherical vesicles. We related this effect to the significant up-regulation of the tumor suppressor genes p21 and p53 with a concomitant activation of caspase 3/7. Finally, we demonstrated that combining the intrinsic shape-dependent effects of tubes with the delivery of doxorubicin significantly increases the cytotoxicity of the system. Our results illustrate how the geometrical conformation of nanoparticles could impact cell behavior and how this could be tuned to create novel drug delivery systems tailored to specific biomedical application.

Nanoparticles based therapeutic efficacy against Acanthamoeba: Updates and future prospect.

Acanthamoeba sp. is a free living amoeba that causes severe, painful and fatal infections, viz. Acanthamoeba keratitis and granulomatous amoebic encephalitis among humans. Antimicrobial chemotherapy used against Acanthamoeba is toxic to human cells and show side effects as well. Infections due to Acanthamoeba also pose challenges towards currently used antimicrobial treatment including resistance and transformation of trophozoites to resistant cyst forms that can lead to recurrence of infection. Therapeutic agents targeting central nervous system infections caused by Acanthamoeba should be able to cross blood-brain barrier. Nanoparticles based drug delivery put forth an effective therapeutic method to overcome the limitations of currently used antimicrobial chemotherapy. In recent years, various researchers investigated the effectiveness of nanoparticles conjugated drug and/or naturally occurring plant compounds against both trophozoites and cyst form of Acanthamoeba. In the current review, a reasonable effort has been made to provide a comprehensive overview of various nanoparticles tested for their efficacy against Acanthamoeba. This review summarizes the noteworthy details of research performed to elucidate the effect of nanoparticles conjugated drugs against Acanthamoeba.

Paclitaxel and spirulina co-loaded polymeric nanoparticles: in-vitro and in-vivo anticancer study

Paclitaxel spirulina nanoparticles were said to have promising anticancer activity against gastric cancer. Nanoparticles of paclitaxel-spirulina were prepared for treating gastric cancer using precipitation technique. The synergistic anticancer efficiency againstMKN45 cells retains when the paclitaxel and spirulina were encapsulated into nanoparticles. To increase the site specific delivery, intra-tumoral administration was carried in the in vivo evaluation. There was an increase in overall survival in an MKN45-transplanted mice model and notable improvement in anti-tumour efficacy when paclitaxel-spirulina nanoparticles were delivered through intra-tumoral administration. The further investigation of overall anticancer mechanism of these nanoparticles is made as a major part in this research. Hence, the conjecture of this research is that, the paclitaxel-spirulina encapsulated nanoparticles could be an effective chemotherapeutic formulation for gastric cancer.

Organic Nanoparticles as Drug Delivery Systems and Their Potential Role in the Treatment of Chronic Myeloid Leukemia.

Chronic myeloid leukemia is a myeloproliferative neoplasm that occurs more prominently in the older population, with a peak incidence at ages 45 to 85 years and a median age at diagnosis of 65 years. This disease comprises roughly 15% of all leukemias in adults. It is a clonal stem cell disorder of myeloid cells characterized by the presence of t(9;22) chromosomal translocation, also known as the Philadelphia chromosome, or its byproducts BCR-ABL fusion protein/messenger RNA, leading to the expression of a protein with enhanced tyrosine kinase activity. This fusion protein has become the main therapeutic target in chronic myeloid leukemia therapy, with imatinib displaying superior antileukemic effects, placing it at the forefront of current treatment protocols and displaying great efficacy. Alternatively, nanomedicine and employing nanoparticles as drug delivery systems may represent new approaches in future anticancer therapy. This review focuses primarily on the use of organic nanoparticles aimed at chronic myeloid leukemia therapy in both in vitro and in vivo settings, by going through a thorough survey of published literature. After a brief introduction on the pathogenesis of chronic myeloid leukemia, a description of conventional, first- and second-line, treatment modalities of chronic myeloid leukemia is presented. Finally, some of the general applications of nanostrategies in medicine are presented, with a detailed focus on organic nanocarriers and their constituents used in chronic myeloid leukemia treatment from the literature.

Application of Nanoparticles for Targeting G Protein-Coupled Receptors.

Nanoparticles (NPs) have attracted unequivocal attention in recent years due to their potential applications in therapeutics, bio-imaging and material sciences. For drug delivery, NP-based carrier systems offer several advantages over conventional methods. When conjugated with ligands and drugs (or other therapeutic molecules), administrated NPs are able to deliver cargo to targeted sites through ligand-receptor recognition. Such targeted delivery is especially important in cancer therapy. Through this targeted cancer nanotherapy, cancer cells are killed with higher specificity, while the healthy cells are spared. Furthermore, NP drug delivery leads to improved drug load, enhanced drug solubility and stability, and controlled drug release. G protein-coupled receptors (GPCRs) are a superfamily of cell transmembrane receptors. They regulate a plethora of physiological processes through ligand-receptor-binding-induced signaling transduction. With recent evidence unveiling their roles in cancer, GPCR agonists and antagonists have quickly become new targets in cancer therapy. This review focuses on the application of some notable nanomaterials, such as dendrimers, quantum dots, gold nanoparticles, and magnetic nanoparticles, in GPCR-related cancers.

Vetorização de compostos 5-nitro-heterocí­clicos com atividade frente a microrganismos multirresistentes. Obtenção de nanopartí­culas carregadas e avaliação da atividade antimicrobiana / Vectorization of 5-nitro-heterocyclic compounds with activity against multiresistant microorganisms. Obtaining of loaded nanoparticles and evaluation of antimicrobial activity

Pacientes com queimaduras e outras lesões cutâneas de grande extensão apresentam alta propensão a infecções multirresistentes. Neste contexto, o objetivo deste trabalho é a obtenção de nanopartículas de quitosana carregadas com composto derivado de 5-nitro-heterocíclico com estrutura análoga à nifuroxazida, N'-((5-nitrofurano-2-il)metileno)-2-benzidrazida (C-H), que apresentou importante atividade frente a diversas cepas de bactérias multirresistentes. Por sua vez, a quitosana é um biopolímero com atividade antimicrobiana, analgésica, regeneradora tecidual e que, mediante contato com exsudato de lesões cutâneas forma filmes hidrogéis protetores no local de aplicação, sendo estas atividades importantes para a prevenção e tratamento de infecções e da exsudação excessiva de lesões de grande extensão. As nanopartículas de quitosana carregadas com o composto (Nps-H) foram obtidas pelo método de gelificação iônica com tripolifosfato de sódio como agente reticulante, variando a concentração de NaCl e polissorbato 80 do sistema, orientada por análise fatorial. As Nps-H obtidas foram caracterizadas por Dynamic Light Scattering (DLS) para determinação do tamanho, índice de polidispersão (IPD) e potencial zeta (ζ). A eficiência de encapsulação (EE%) foi determinada por espectrofotometria UV/VIS a 370 nm por método indireto. Entre os experimentos desenvolvidos, aquele que apresentou os melhores resultados resultou em partículas de tamanho médio de 321 d.nm, IPD 0,18, Pζ +37 mV e EE% de 48%. A morfologia e superfície das Nps-H foram analisadas por Microscopia Eletrônica de Varredura (MEV) e mostraram que as Nps-H são esféricas e de superfície irregular. A partir da obtenção e caracterização das Nps-H determinou-se a concentração inibitória mínima (CIM) das Nps-H, do composto livre (C-H) e das nanopartículas de quitosana vazias (Nps-Cs) por método colorimétrico e microdiluição frente às cepas de Staphylococcus aureus ATCC 29213, hVISA e ORSA. As Nps-H apresentaram atividade bastante superior comparando-se ao C-H e as Nps-Cs frente às cepas de S. aureus estudadas. Com vista à preparação de uma formulação farmacêutica estável, partiu-se para a liofilização das Nps-H e com esse objetivo realizou-se análises térmicas das Nps-H por Differential Scanning Calorimetric (DSC) para determinação das temperaturas de transição vítrea e eutética (Tg' e Teut) e análise por criomicroscopia para determinação da temperatura de colapso (Tcol). Amostras de Nps-H com lio/crioprotetores glicina, lactose e sacarose a diferentes concentrações foram liofilizadas a -40 ºC a 100 mTorr. As amostras de Nps-H com lactose e sacarose ambas a 2,5% e 5% demonstraram preservar as características originais das Nps-H após o processo de liofilização. Observou-se, com os resultados obtidos neste trabalho que as Nps-H representam uma promissora alternativa na prevenção e tratamento de pacientes com lesões cutâneas infeccionadas por bactérias multirresistentes e no controle da exsudação excessiva, principalmente por suas atividades terapêuticas em conjunto, diminuindo a mortalidade e morbidade desses quadros

Patients with burns and others extensive skin lesions show high propension to multiresistant infections. In this context, the aim of this project is to obtain chitosan nanoparticles carried with 5-nitro-heterocyclic derivate with structure analogue to nifuroxazide N'-((5-nitrofuran-2-yl) methylene)-2-benzhydrazide, that showed important activity against multiresistant bacteria strains. In its turn, the chitosan is a biopolymer with antimicrobial, analgesic, tecidual regenerator activities and by contact with excessive burns and extensive skin lesions exsudate process, form protective hydrogel films on place of application, being these activities important to infection and excessive exsudate treatment and prevention of extensive burns and lesions. The chitosan nanoparticles carried with compound (Nps-H) were obtained by ionic gelation method with sodium tripolyphosphate as crosslinker agent, varing the NaCl and polysorbate 80 concentrations in the system, oriented by factorial analyze. The Nps-H obtained were characterized by Dynamic Light Scattering (DLS) to size determination, polydispersion index (PDI) and zeta potential (ζ-P). The encapsulation efficiency (EE%) were determined by spectrophotometry UV/VIS at 370 nm by indirect method. Bepolissorbato the experiments developed, the one who showed the best results, resulted in particles with size of 321 d.nm, PDI of 0,18, ζ-P of +37 mV and EE% of 48%. The Nps-H morphology and surface were analyzed by Scanning Electronic Microscopy (SEM) and showed that Nps-H are spherical and with irregular surface. Starting of Nps-H obtain and characterization were determined the Nps-H, free compound (C-H) and empty chitosan nanoparticles (Nps-Cs) Minimal Inhibitory Concentrations (MIC) by colorimetric method and microdilution against Staphylococcus aureus ATCC 29213, ORSA and hVISA strains. The Nps-H showed the superior activity comparing to C-H and Nps-H against all strains tested. With a view to preparation of stable pharmaceutic formulation, started to Nps-H freeze-drying and with this aim, were realized Nps-H thermal analyzes by Differential Scanning Calorimetric (DSC) to determine glass transition and euthetic temperatures (Tg' and Teut) and the cryomicroscopy analyze to determine collapse temperature (Tcol). The Nps-H samples with lyo/cryoprotectants as glycine, lactose and sucrose at different concentrations were lyophilized at -40 ºC at 100 mTorr. The Nps-H samples with lactose and sucrose both at 2,5% and 5% demonstrated to preserve the original Nps-H characteristics after freeze-drying process. Were observed, with the results obtained in this project that Nps-H represent the promising alternative to prevention and treatment of patients with infected skin lesions by multiresitant bacteria and to control of excessive exudate process, mainly by their therapeutic activities combined, decreasing the mortality and morbidity of these cases

Chitosan and carboxymethylated derivative nanoparticles as delivery systems for biological products: preparation, characterization, stability and in vitro/in vivo evaluation / Nanopartículas de quitosana e derivado carboximetilado como sistemas de fornecimento (delivery) de produtos biológicos: preparo, caracterização, estabilidade e avaliação in vitro/in vivo

Chitosan is a biocompatible and biodegradable mucoadhesive polymer with unique advantages, such as the distinct trait of opening the junctions to allow paracellular transport of antigen and good tolerability. However, the poor solubility of chitosan in neutral or alkalinized media has restricted its applications in the pharmaceutical field. Chitosan can be easily carboxymethylated to improve its solubility in aqueous media, while its biodegradability and biocompatibility are preserved. Apart from this, carboxymethyl chitosan (CMCS) can be easily processed into nanoparticles which highlight its suitability and extensive usage for preparing different drug delivery formulations. The present study deals with the development and characterization of a delivery system based on CMCS nanoparticles using ovalbumin as model protein. We demonstrated that ovalbumin loaded nanoparticles were successfully synthetized using calcium chloride as a cross-linker by ionic gelation. The nanoparticles exhibited an average size of approximately 169 nm and presented a pseudo-spherical shape. The nanoparticles size increased according to the addition of CaCl2 due to the strong electrostatic attraction. During storage the nanoparticles size increased was attributed to swelling and aggregation. The loading efficiency of ovalbumin was found to be 17%. Confocal microscopy clearly showed the association between ovalbumin and CMCS chains into nanoparticles. Therefore, we suggest these nanoparticles can be considered as an attractive and promising carrier candidate for proteins and antigens. The major challenge that limits the use of such carriers is their instability in an aqueous medium. Thus, the next step of this work was to determine the robustness of several formulations using distinct freeze-drying protocols. This study demonstrated that mannitol in concentration of 10% (w/v) is well suited to preserve ovalbumin loaded CMCS nanocapsules from aggregation during lyophilization and subsequent reconstitution. Importantly, the results showed that an annealing step has a huge impact on porosity of freeze-dried cake by nearly complete crystallization of mannitol, once the crystalline matrix prevents the partial collapse and the formation of larger pores observed without annealing. Therefore, the usual observation that annealing increases the pore size due to growth of ice crystal size does not always apply, at least when crystallization of solute is involved. Since all characterizations and stability studies had been performed, the main purpose of this study was to develop a stable antigen delivery system for oral immunization using CMCS and inactivated rabies virus (RV) as the antigen. RV loaded nanoparticles was found to enhance both systemic (IgG) and local (IgA) immune responses against RV after oral delivery in mice. The effective doses 50% were 50-times higher than the negative controls, indicating that the immune response started only after the third boosting dose. Furthermore, enough neutralizing antibodies was produced to be protected against the harmful effects of the rabies virus. It is therefore concluded, that the CMCS nanoparticles formulated in this study, are suitable for oral vaccine delivery, and can be suggested as a promising delivery system for a diverse range of antigens as well as a gene/protein delivery system, especially for those positively charged. Since several approaches show that effective intervention in airway allergic inflammation can be achieved with allergen-activated interleukin-10-secreting cells, the final part of this work was dedicated to assessing whether IL-10 loaded chitosan nanoparticles (IL10-CSNPs) could be used as a possible inhalable therapeutic tool for preventing exacerbations in asthmatic patients. As positive controls, we also assess whether interleukin 17A and interleukin 9 have the ability to stimulate human airway smooth muscle (HASM) cell contractility using magnetic twisting cytometry (MTC). Significant decreased baseline cell stiffness was observed in HASM cells pre-treated with IL-10, but not with IL10-CSNPs, whereas treatment with IL-17A significantly enhanced baseline cell stiffening. Our findings reveal a previously unknown mechanism underlying immunotherapy for prevention and treatment of asthma

A quitosana é um polímero mucoadesivo biocompatível e biodegradável, com vantagens únicas, tais como a característica distinta de abrir as junções que permitim o transporte paracelular de antígenos e boa tolerabilidade. No entanto, sua baixa solubilidade em meios neutros ou alcalinizados tem restringido suas aplicações no campo farmacêutico. A quitosana pode ser facilmente carboximetilada para melhorar de sua solubilidade em meios aquosos, enquanto sua biodegradabilidade e biocompatibilidade são preservadas. Além disso, a carboximetilquitosana (CMCS) pode ser facilmente processada na forma de nanopartículas, o que destaca sua adequabilidade para uso extensivo no preparo de sistemas de delivery de medicamentos. O presente estudo trata do desenvolvimento e caracterização de um sistema de delivery baseado em nanopartículas de CMCS utilizando ovalbumina como proteína modelo. Nós demonstramos que as nanopartículas carregadas com ovalbumina foram sintetizadas com sucesso utilizando cloreto de cálcio como agente de reticulação por gelificação iônica. As nanopartículas exibiram um tamanho médio de aproximadamente 169 nm e apresentaram uma forma pseudo-esférica. O tamanho das nanopartículas aumentou de acordo com a adição de CaCl2 devido à forte atração eletrostática. Durante o armazenamento, o tamanho aumentado das nanopartículas foi atribuído a incorporação de água e agregação. A eficiência de encapsulamento da ovalbumina foi de aproximadamente 17%. A microscopia confocal mostrou claramente a associação entre ovalbumina e a cadeias de CMCS nas nanopartículas. Sugerimos, portanto, que tal sistema pode ser considerado como candidato atraente e promissor para o carreamento de proteínas e antígenos. O principal desafio que limita o uso desses carreadores consiste na instabilidade em meio aquoso. Assim, o próximo passo deste trabalho foi determinar a robustez de várias formulações utilizandose diferentes protocolos de liofilização. Este estudo demonstrou que o manitol em uma concentração de 10% (p/v) é adequado para preservar da agregação as nanocápsulas de CMCS carregadas com ovalbumina durante a liofilização e subsequente reconstituição. Mais importante, os resultados mostraram que uma etapa de annealing tem um enorme impacto sobre a porosidade da amostra liofilizada devido a quase completa cristalização do manitol, uma vez que a matriz cristalina evita o colapso parcial e a formação de poros maiores observados na ausência do annealing. Portanto, a observação comum de que o annealing aumenta o tamanho doporos devido ao crescimento dos cristais de gelo nem sempre se aplica, pelo menos quando a cristalização de um soluto está envolvida. Uma vez que todas as caracterizações e estudos de estabilidade foram realizados, o principal objetivo deste estudo foi desenvolver um sistema estável de delivery de antígeno para imunização oral utilizando CMCS e vírus rábico inativado (RV) como antígeno. Verificou-se que as nanopartículas carregadas com RV aumentam as respostas imune sistêmica (IgG) e local (IgA) contra o RV após administração oral em camundongos. As doses efetivas 50% foram 50 vezes maiores que os controles negativos, indicando que a resposta imune foi iniciada apenas após a terceira dose da vacina. Além disso, foram produzidos anticorpos neutralizantes suficientes para proteção contra os efeitos nocivos do vírus rábico. Conclui-se, portanto, que as nanopartículas de CMCS formuladas neste estudo, são adequadas para o delivery oral de vacinas, e podem ser sugeridas como um sistema promissor de delivery para uma gama diversa de antígenos, bem como para o delivery de genes/proteínas, especialmente para aqueles carregados positivamente. Uma vez que diversas abordagens mostram que uma intervenção efetiva em casos de inflamação alérgica de vias aéreas pode ser conseguida por meio de células secretoras de interleucina 10 (IL-10) mediante ativação por alergenos, a parte final deste trabalho esteve dedicada a avaliação de nanopartículas de quitosana carregadas com IL-10 (IL10-CSNPs) como possível ferramenta terapêutica inalável para prevenção de exacerbações em pacientes asmáticos. Como controles positivos, avaliou-se adicionalmente se as interleucinas 17A (IL-17A) e 9 (IL-9) possuem a capacidade de estimular a contratilidade de células humanas de músculo liso de vias aéreas humanas (HASM) por meio de citometria de torção magnética (MTC). Uma diminuição significativa da rigidez celular basal foi observada em células HASM pré-tratadas com IL-10, mas não com IL10-CSNPs, enquanto que o tratamento com IL-17A aumentou significativamente a magnitude rigidez celular basal. Nossos resultados revelam um mecanismo previamente desconhecido subjacente à imunoterapia para prevenção e tratamento da asma

Microscopic Differentiation of Plasmonic Nanoparticles for the Ratiometric Read-out of Target DNA.

The development of highly sensitive and rapid methods for detecting DNA is of critical importance. Here, we describe a strategy for the digital detection of target DNA at the femto-molar level. Individual DNA molecules were encoded with a single gold nanorod (AuNR), separated and enriched by magnetic immune-separation. The coding gold nanorods were then de-hybridized and dispersed into a gold nanosphere (AuNS) solution at a certain concentration, and both gold nanoparticles were immobilized on glass slides for dark-field microscopic imaging. Using an in-house Matlab program, the concentration of the target DNA was calculated based on the ratio of the coding gold nanorods to gold nanospheres. By combining the coding of individual biomolecules with a single gold nanorod and the use of gold nanospheres as an internal standard, a method for the rapid and accurate digital detection of target DNA to the femto-molar level was developed.

Nanoparticles as Theranostic Vehicles in Experimental and Clinical Applications-Focus on Prostate and Breast Cancer.

Prostate and breast cancer are the second most and most commonly diagnosed cancer in men and women worldwide, respectively. The American Cancer Society estimates that during 2016 in the USA around 430,000 individuals were diagnosed with one of these two types of cancers, and approximately 15% of them will die from the disease. In Europe, the rate of incidences and deaths are similar to those in the USA. Several different more or less successful diagnostic and therapeutic approaches have been developed and evaluated in order to tackle this issue and thereby decrease the death rates. By using nanoparticles as vehicles carrying both diagnostic and therapeutic molecular entities, individualized targeted theranostic nanomedicine has emerged as a promising option to increase the sensitivity and the specificity during diagnosis, as well as the likelihood of survival or prolonged survival after therapy. This article presents and discusses important and promising different kinds of nanoparticles, as well as imaging and therapy options, suitable for theranostic applications. The presentation of different nanoparticles and theranostic applications is quite general, but there is a special focus on prostate cancer. Some references and aspects regarding breast cancer are however also presented and discussed. Finally, the prostate cancer case is presented in more detail regarding diagnosis, staging, recurrence, metastases, and treatment options available today, followed by possible ways to move forward applying theranostics for both prostate and breast cancer based on promising experiments performed until today.