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Nanomedicines are increasingly researched and used for the treatment of chronic inflammatory diseases. Herein, the effect of the size of nanoparticles on their distribution and retention in chronic inflammatory sites, as compared to healthy tissues, was studied in a mouse model with chronic inflammation in one of the hind footpads. Using PEGylated gold nanoparticles of 2, 10, 100, and 200 nm, we found that although the smaller nanoparticles of 2 and 10 nm showed greater distribution and slower clearance in the inflamed footpad than the relatively larger nanoparticles of 100 and 200 nm, the larger nanoparticles of 100 and 200 nm were more selectively distributed in the inflamed hind footpad than in the healthy hind footpad in the same mouse. Based on these findings, we prepared protein nanoparticles of 100-200 nm with albumin, IgG antibody, or anti-TNF-α monoclonal antibody (mAb). The nanoparticles can release proteins in response to high redox activity and/or low pH, conditions seen in chronic inflammation sites. We then showed that upon intravenous injection, those stimuli-responsive protein nanoparticles distributed more selectively in the inflamed footpad than free proteins and remained longer in the inflamed footpad than similar protein nanoparticles that are not sensitive to high redox activity or low pH. These findings support the feasibility of increasing the selectivity of nanomedicines and protein therapeutics to chronic inflammation sites and prolonging their retention at the sites by innovative nanoparticle engineering., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Docosahexaenoyl difluorodeoxycytidine (DHA-dFdC) is an amide with potent, broad-spectrum antitumor activity. In the present study, DHA-dFdC's ability to induce immunogenic cell death (ICD) was tested using CT26 mouse colorectal cancer cells, an established cell line commonly used for identifying ICD inducers, as well as Panc-02 mouse pancreatic cancer cells., Methods: The three primary surrogate markers of ICD (i.e., calreticulin (CRT) surface translocation, ATP release, and high mobility group box 1 protein (HMGB1) release) were measured in vitro. To confirm DHA-dFdC's ability to induce ICD in vivo, the gold standard mouse vaccination studies were conducted using both CT26 and Panc-02 models. Additionally, the effect of DHA-dFdC on tumor response to anti-programmed cell death protein 1 monoclonal antibody (anti-PD-1 mAb) were tested in mice with pre-established Panc-02 tumors. RNA sequencing experiments were conducted on PANC-1 human pancreatic cancer cells treated with DHA-dFdC, dFdC, or vehicle control in vitro., Results: DHA-dFdC elicited CRT surface translocation and ATP and HMGB1 release in both cell lines. Immunization of mice with CT26 or Panc-02 cells pretreated with DHA-dFdC prevented or delayed the development of corresponding secondary live challenge tumor. DHA-dFdC enabled Panc-02 tumors to respond to anti-PD-1 mAb. RNA sequencing experiments revealed that DHA-dFdC and dFdC differentially impacted genes related to the KRAS, TP53, and inflammatory pathways, and DHA-dFdC enriched for the unfolded protein response (UPR) compared to control, providing insight into DHA-dFdC's potential mechanism of inducing ICD., Conclusion: DHA-dFdC is a bona fide ICD inducer and can render pancreatic tumors responsive to anti-PD-1 mAb therapy., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Understanding the effects of atrazine exposure on embryo development in oviparous animals may provide important data regarding the impacts of agrochemical use on wildlife and the ecosystem. This study set out to determine the effects of embryonic atrazine exposure on the development of osseous and cartilaginous components of scleral ossicles in Podocnemis expansa . Eggs were collected at the Environmental Protection Area Meandros do Rio Araguaia, Brazil, and artificially incubated in sand treated with solutions containing 2, 20 or 200 µg/L of atrazine. Sixty embryos were collected per treatment throughout the incubation period. Embryos were diaphanized with potassium hydroxide (KOH) and stained with Alizarin Red S and Alcian blue (bone and cartilage tissue respectively). Scleral ossicles were then counted and examined for skeletal abnormalities at different stages of embryonic development. Scleral ossicle counts were significantly reduced in P. expansa embryos treated with 200 μg/L atrazine solution. Rudimentary ossicles and gaps were also noted in embryos exposed to atrazine concentrations of 2 μg/L or 200 μg/L. Findings of this study emphasize the relevance of ecotoxicological investigations in determining the impacts of agrochemicals on native fauna.

Elevated expression of C-type like receptors (CLRs) by tumor cells and tumor-associated macrophages (TAMs) present a unique target for the delivery of anticancer agents. Stearoyl gemcitabine (GemC18)-incorporated, acid-sensitive micelles (G-AS-M) prepared with a stearoyl polyethylene glycol (PEG2000) hydrazone were surface-mannosylated in this study for potential targeted killing of tumor cells and TAMs. The surface mannosylated micelles (i.e. G-MAS-M) were significantly more cytotoxic than the G-AS-M micelles to macrophages and tumor cells that express CLRs. Surprisingly, the uptake of GemC18 in the mannosylated G-MAS-M micelles by the macrophages and tumor cells was lower than that of GemC18 in the G-AS-M micelles. The lack of correlation between the cytoxicity and cellular uptake of GemC18 in the micelles was likely caused by a reduction in the sensitivity of the hydrazone bond linking the PEG2000 to the mannosylated G-MAS-M micelles to hydrolysis, resulting in more stable micelles., Competing Interests: Declaration of Interest There is no conflict to declare.

Objective: Cigarette smoking is one of the leading causes of death in the world. The majority of the smokers have tried to quit, but only a few of them were able to achieve long-term abstinence, due to the high addictiveness of nicotine. Nicotine-specific antibodies have the potential to block the euphoric effect of nicotine by forming antibody-antigen complexes in the blood circulation. Since nicotine is taken largely by inhalation, inducing anti-nicotine antibodies in lung and nasal mucosal secretions, in addition to blood circulation, is expected to be beneficial., Significance: The importance of this study is to establish the feasibility of inducing nicotine-neutralizing antibodies not only in the blood, but also in the lung and nasal mucosal secretions, by intranasal administration of a nicotine vaccine candidate., Methods: Nicotine-keyhole limpet hemocyanin conjugate (Nic-KLH) was prepared and mixed with monophosphoryl lipid A (MPL) as an adjuvant. Nic-KLH/MPL was given intranasally or subcutaneously to mice, and the titers, affinity, and specificity of the nicotine-specific antibodies in nasal and lung mucosal secretions and blood samples were determined using (competitive) ELISA., Results: Nasal Nic-KLH/MPL immunization elicited robust nicotine-specific neutralizing IgA in mouse nasal and lung secretions, in additional to anti-nicotine IgG in blood circulation. The nicotine-specific IgG level in mice nasally immunized with Nic-KLH/MPL was lower than in mice subcutaneously immunized with the same Nic-KLH/MPL, but a heterologous prime-boost immunization strategy helped to increase it., Conclusion: Intranasal immunization with a nicotine vaccine candidate can induce systemic and mucosal antibodies that specifically neutralize nicotine.

Previously, we developed a solid lipid nanoparticle (SLN) formulation of 4-(N)-docosahexaenoyl 2', 2'-difluorodeoxycytidine (DHA-dFdC), a compound with promising antitumor activity. Herein, we studied the feasibility of administering the DHA-dFdC by the oral route using the solid lipid nanoparticles (i.e., DHA-dFdC-SLNs). In simulated gastrointestinal fluids, the DHA-dFdC-SLNs did not aggregate. The release of the DHA-dFdC from the solid lipid nanoparticles in simulated gastrointestinal fluid was slow, but was slightly faster in simulated intestinal fluid than in simulated gastric fluid. In mice orally administered with DHA-dFdC-SLNs, plasma DHA-dFdC concentration vs. time curve has a T max of ~ 1.7 h and a C max of 17.01 μg/mL. The absolute oral bioavailability of DHA-dFdC when given as DHA-dFdC-SLNs was ~ 68% (based on AUC 0-24 h values), while the relative oral bioavailability DHA-dFdC (compared with DHA-dFdC in a Tween 80/ethanol-in-water solution) was 126%. Finally, in mice with pre-establish B16-F10 murine melanoma, oral DHA-dFdC-SLNs increased their survival significantly, as compared with oral administration of the DHA-dFdC solution. It is concluded that the solid lipid nanoparticle formulation increased the bioavailability of the DHA-dFdC upon oral administration, as compared with the DHA-dFdC solution.

There is evidence that encapsulating glucocorticoids into nucleic acid-containing nanoparticles reduces the inflammatory toxicities of the nanoparticles. Herein, using betamethasone acetate (BA), a glucocorticoid, and a solid lipid nanoparticle formulation of siRNA, we confirmed that coencapsulating BA into the siRNA solid lipid nanoparticles significantly reduced the proinflammatory activity of the siRNA nanoparticles in a mouse model. Using TNF-α siRNA, we then showed that the BA and TNF-α siRNA coencapsulated into the solid lipid nanoparticles acted as a dual anti-inflammatory and synergistically reduced TNF-α release by mouse macrophages in culture following stimulation with lipopolysaccharide, as compared to solid lipid nanoparticles encapsulated with TNF-α siRNA or BA alone. Importantly, upon studying the effect of the ratio of BA and TNF-α siRNA on the proinflammatory activity of the resultant nanoparticles, we identified that BA and TNF-α siRNA coencapsulated solid lipid nanoparticles prepared with a BA to TNF-α siRNA weight ratio of 2:1 induced the lowest proinflammatory cytokine production by macrophages in culture. This result was in comparison to nanoparticles prepared with BA to TNF-α siRNA ratios both higher and lower than 2:1 (i.e., 4:1, 1:1, and 0.5:1) and is likely due to differences in molecular interactions among the various components in the BA and TNF-α-siRNA coencapsulated solid lipid nanoparticles at these ratios. Encapsulating glucocorticoids into siRNA-nanoparticles represents a viable strategy to reduce the proinflammatory activity of the nanoparticles; however, the ratio of the glucocorticoid to siRNA in the nanoparticles requires optimization.

Previously, we synthesized 4-(N)-docosahexaenoyl 2', 2'-difluorodeoxycytidine (DHA-dFdC), a novel lipophilic compound with a potent, broad-spectrum antitumor activity. Herein, we report a solid lipid nanoparticle (SLN) formulation of DHA-dFdC with improved apparent aqueous solubility, chemical stability, as well as efficacy in a mouse model. The SLNs were prepared from lecithin/glycerol monostearate-in-water emulsions emulsified with D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) and Tween 20. The resultant DHA-dFdC-SLNs were 102.2 ± 7.3 nm in diameter and increased the apparent solubility of DHA-dFdC in water to at least 5.2 mg/mL, more than 200-fold higher than its intrinsic water solubility. DHA-dFdC in a lyophilized powder of DHA-dFdC-SLNs was significantly more stable than the waxy solid of pure DHA-dFdC. DHA-dFdC-SLNs also showed an increased cytotoxicity against certain tumor cells than DHA-dFdC. The plasma concentration of DHA-dFdC in mice intravenously injected with DHA-dFdC-SLNs in dispersion followed a bi-exponential model, with a half-life of ~44 h. In mice bearing B16-F10 murine melanoma, DHA-dFdC-SLNs were significantly more effective than DHA-dFdC in controlling the tumor growth. In addition, histology evaluation revealed a high level of apoptosis and tumor encapsulation in tumors in mice treated with DHA-dFdC-SLNs. DHA-dFdC-SLNs represents a new DHA-dFdC formulation with improved antitumor activity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bisphosphonates are generally used to treat bone diseases, such as bone metastasis from cancer. There is evidence that, through the modification of the pharmacokinetics and biodistribution of bisphosphonates by formulating them into nanoparticles, they may be able to treat extraskeletal tumors. However, many previously reported bisphosphonate nanoparticle formulations show extensive premature release of bisphosphonates. Herein, using zoledronate (Zol), a third-generation bisphosphonate, we developed a new Zol nanoparticle formulation (denoted as Zol-NPs) by encapsulating anionic lipid-coated Zol-calcium nanocomplexes into poly(lactic- co-glycolic) acid nanoparticles emulsified with octadecanoic acid-hydrazone-polyethylene glycol (2000), an acid-sensitive cleavable emulsifying agent. The resultant Zol-NPs, about 180 nm in hydrodynamic diameter, show very limited premature release of Zol (i.e., <5% in 48 h in a simulated physiological condition) and enhanced cytotoxicity to both murine cancer cells and macrophages. In a mouse model with orthotopically transplanted mammary tumors, Zol-NPs significantly reduced the distribution of Zol in bones, but increased its distribution in tumors. Importantly, Zol-NPs also significantly inhibited tumor growth, whereas the equivalent dose of free Zol did not. This platform technology may be exploited to treat extraskeletal tumors with bisphosphonates.

Intranasal vaccination using dry powder vaccine formulation represents an attractive, non-invasive vaccination modality with better storage stability and added protection at the mucosal surfaces. Herein we report that it is feasible to induce specific mucosal and systemic antibody responses by intranasal immunization with a dry powder vaccine adjuvanted with an insoluble aluminum salt. The dry powder vaccine was prepared by thin-film freeze-drying of a model antigen, ovalbumin, adsorbed on aluminum (oxy)hydroxide as an adjuvant. Special emphasis was placed on the characterization of the dry powder vaccine formulation that can be realistically used in humans by a nasal dry powder delivery device. The vaccine powder was found to have "passable" to "good" flow properties, and the vaccine was uniformly distributed in the dry powder. An in vitro nasal deposition study using nasal casts of adult humans showed that around 90% of the powder was deposited in the nasal cavity. Intranasal immunization of rats with the dry powder vaccine elicited a specific serum antibody response as well as specific IgA responses in the nose and lung secretions of the rats. This study demonstrates the generation of systemic and mucosal immune responses by intranasal immunization using a dry powder vaccine adjuvanted with an aluminum salt., (Copyright © 2018 Elsevier B.V. All rights reserved.)

TNF-α siRNA has shown promising therapeutic benefits in animal models of rheumatoid arthritis. However, there continues to be a need for siRNA delivery systems that have high siRNA encapsulation efficiency and minimum burst release of TNF-α siRNA, and can target inflamed tissues after intravenous administration. Herein we report a novel acid-sensitive sheddable PEGylated solid-lipid nanoparticle formulation of TNF-α-siRNA, AS-TNF-α-siRNA-SLNs, prepared by incorporating lipophilized TNF-α-siRNA into solid-lipid nanoparticles composed of biocompatible lipids such as lecithin and cholesterol. The nanoparticles are approximately 120 nm in diameter, have a high siRNA encapsulation efficiency (>90%) and a minimum burst release of siRNA (<5%), and increase the deilvery of the siRNA in chronic inflammation sites in mouse models, including in a mouse model with collagen-induced arthritis. Importantly, in a mouse model of collagen antibody-induced arthritis that does not respond to methotrexate therapy, intravenous injection of the AS-TNF-α-siRNA-SLNs significantly reduced paw thickness, bone loss, and histopathological scores. These findings highlight the potential of using this novel siRNA nanoparticle formulation to effectively treat arthritis, potentially in patients who do not respond adequately to methotrexate., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In spite of recent advances in targeted tumor therapy, systemic chemotherapy with cytotoxic agents remains a vital cancer treatment modality. Gemcitabine is a nucleoside analog commonly used in the treatment of various solid tumors, but an oral gemcitabine dosage form remain unavailable. Previously, we developed the 4-( N )-stearoyl gemcitabine solid lipid nanoparticles (GemC18-SLNs) by incorporating 4-( N )-stearoyl gemcitabine (GemC18), an amide prodrug of gemcitabine, into solid lipid nanoparticles. GemC18-SLNs, when administered intravenously, showed strong antitumor activity against various human and mouse tumors in mouse models. In the present study, we defined the plasma pharmacokinetics of gemcitabine when GemC18-SLNs were given orally to healthy mice and evaluated the antitumor activity of GemC18-SLNs when given orally in mouse models of lung cancer. In mice orally gavaged with GemC18-SLNs, plasma gemcitabine concentration followed an absorption phase and then clearance phase, with a T max of ~2 h. The absolute oral bioavailability of gemcitabine in the GemC18-SLNs was ~70% (based on AUC 0-24 h values). In mice with pre-established tumors (i.e. mouse TC-1 or LLC lung cancer cells), oral GemC18-SLNs significantly inhibited the tumor growth and increased mouse survival time, as compared to the molar equivalent dose of gemcitabine hydrochloride or GemC18 in vegetable oil or in Tween 20. Immunohistostaining revealed that oral GemC18-SLNs also have significant antiproliferative, antiangiogenic, and proapoptotic activity in LLC tumors. Formulating a lipophilic amide prodrug of gemcitabine into solid lipid nanoparticles may represent a viable approach toward developing a safe and efficacious gemcitabine oral dosage form., Competing Interests: CONFLICTS OF INTEREST None.

Insoluble aluminum salts such as aluminum (oxy)hydroxide are commonly used as vaccine adjuvants. Recently, there is evidence suggesting that the adjuvant activity of aluminum salt-based materials is tightly related to their physicochemical properties, including nanometer-scale size, shape with long aspect ratio, and low degree of crystallinity. Herein, for the first time, the bicontinuous reverse microemulsion (RM) technique was utilized to synthesize stick-like monodisperse aluminum (oxy)hydroxide nanoparticles with a long aspect ratio of ∼10, length of ∼80 nm, and low degree of crystallinity (denoted as Al-nanosticks). Moreover, the relationship between the physicochemical properties of Al-nanosticks and the bicontinuous RM was discussed. Compared to the commercial Alhydrogel, which contains micrometer-scale aluminum oxyhydroxide particular aggregates with moderate degree of crystallinity, the Al-nanosticks are more effective in adsorbing and delivering antigens (e.g., ovalbumin, OVA) into antigen-presenting cells, activating inflammasomes, and potentiating OVA-specific antibody responses in a mouse model. It is concluded that the aluminum (oxy)hydroxide nanosticks synthesized in the bicontinuous RM are promising new aluminum salt-based vaccine adjuvants.

Inflammation is implicated in a host of chronic illnesses. Within these inflamed tissues, the pH of the microenvironment is decreased and immune cells, particularly macrophages, infiltrate the area. Additionally, the vascular integrity of these sites is altered with increased fenestrations between endothelial cells. These distinctive properties may be exploited to enhance targeted delivery of anti-inflammatory therapies. Using a mouse model of chronic inflammation, we previously showed that acid-sensitive sheddable PEGylation increases the distribution and retention of nanoparticles in chronic inflammation sites. Here we demonstrated that surface modification of the acid-sensitive sheddable PEGylated nanoparticles with mannose, a ligand to mannose receptors present in chronic inflammation sites, significantly increases the targeted delivery of the nanoparticles to these areas. Furthermore, we showed that the acid-sensitive sheddable PEGylated, mannose-modified nanoparticles are able to significantly increase the delivery of betamethasone-21-acetate (BA), a model anti-inflammatory compound, to chronic inflammation sites as compared to free BA. These results highlight the ability to engineer formulations to target chronic inflammation sites by exploiting the microenvironment of these regions.

The use of pesticides is a widely spread practice in Brazilian agriculture, and dispersion of these substances is an important factor for the fauna and flora. Atrazine is an endocrine disruptor in the xenoestrogen class that is used worldwide in agricultural practices. In Brazil, its use is permitted in several crops. Podocnemis expansa is a representative of the Testudines order that is the largest freshwater reptile of South America. Its distribution enables it to get in contact with molecules that are commonly used as pesticides, which may cause deleterious effects in target populations. In order to evaluate the possible effects of the exposure to atrazine on bone ontogeny of this species, eggs were artificially incubated in sand moistened with water contaminated with atrazine at concentrations equal to 0, 2, 20 or 200 μg/L. Embryos were collected throughout incubation and submitted to diaphanization of soft tissues with potassium hydroxide (KOH); bones were stained with Alizarin red S and cartilages by Alcian blue. Embryos were evaluated for the presence of abnormalities during the different stages of pre-natal development of skeletal elements. No effect of atrazine was observed on bone development during the embryonic phase in P. expansa individuals, in the conditions of this study.

Eggshell evaluation may serve as an indicator of the effect of substances released in the environment, which may change eggshell shape, size, structure, and/or chemical composition. Additionally, exposure may interfere with hatching rates in contaminated eggs. The objective of this study was to better understand how exposure to the insecticide methyl parathion interferes with chemical changes in eggshells of Podocnemis expansa throughout their artificial incubation, as well as with egg hatchability. A total of 343 P. expansa eggs were collected in a natural reproduction area for the species. These eggs were transferred to and artificially incubated in the Wild Animal Teaching and Research Laboratory at Universidade Federal de Uberlândia. On the first day of artificial incubation, 0, 35, 350, and 3500 ppb of methyl parathion were incorporated to the substrate. Eggs were collected every three days for chemical analysis of eggshells. Hatchability was evaluated as the number of hatchlings in each treatment, for the eggs that were not used in the chemical analysis. Student's T-test was used for data on eggshell chemical composition, and the Binomial Test for Two Proportions was used in the hatchability analysis, at a 5% significance level. It was observed that the incorporation of methyl parathion to the substrate on the first day of artificial incubation of P. expansa eggs reduced the levels of total fat in the shells throughout their incubation, besides reducing egg hatchability.