Aerosol droplet delivery of mesoporous silica nanoparticles: A strategy for respiratory-based therapeutics.

A highly versatile nanoplatform that couples mesoporous silica nanoparticles (MSNs) with an aerosol technology to achieve direct nanoscale delivery to the respiratory tract is described. This novel method can deposit MSN nanoparticles throughout the entire respiratory tract, including nasal, tracheobronchial and pulmonary regions using a water-based aerosol. This delivery method was successfully tested in mice by inhalation. The MSN nanoparticles used have the potential for carrying and delivering therapeutic agents to highly specific target sites of the respiratory tract. The approach provides a critical foundation for developing therapeutic treatment protocols for a wide range of diseases where aerosol delivery to the respiratory system would be desirable. FROM THE CLINICAL EDITOR: Delivery of drugs via the respiratory tract is an attractive route of administration. In this article, the authors described the design of mesoporous silica nanoparticles which could act as carriers for drugs. The underlying efficacy was successfully tested in a mouse model. This drug-carrier inhalation nanotechnology should potentially be useful in human clinical setting in the future.

Study of nebulization delivery of aerosolized fluorescent microspheres to the avian respiratory tract.

This study investigated the delivery of an aerosol of monodisperse microspheres to the respiratory tract of birds following aerosol exposure. Adult domestic pigeons (Columbia livia domestica, n = 5 birds per timed treatment) were exposed to an aerosol of fluorescent 1.0 microm diameter carboxylate microspheres for 0.5, 1, 2, or 4 hr. During the aerosolization period, the birds were free-standing in a plexiglass treatment chamber and the aerosol was delivered using a commercial nebulizer. Immediately following aerosol exposure, the birds were euthanatized and the carcasses were intravenously infused with a modified paraformaldehyde/gluteraldehyde fixative. Evaluation of microsphere distribution was performed using a stereoscopic microscope with an epifluorescent module. The results from this study revealed that the amount of aerosolized particles delivered using a commercial nebulizer was proportional to exposure periods. Aerosol exposure periods of 0.5 hr or 1 hr did not result in a readily observable distribution of 1.0 microm fluorescent microspheres to the cranial thoracic, caudal thoracic, or abdominal air sac membranes. This was partly attributed to the relatively low concentration of the individual monodisperse microspheres in the aerosolized suspension. The 2- and 4-hr exposure periods resulted in readily observable deposition of the 1.0 mirom fluorescent microspheres in the cranial thoracic, caudal thoracic, or abdominal air sac membranes, with the 4-hr exposure period resulting in the greatest number of particles on the membrane surfaces. For each of the exposure periods, there was individual animal variation regarding the distribution and relative number of spheres deposited. This study demonstrates the widespread deposition of particles that had an aerodynamic equivalent diameter of approximately 1 microm and provides a better understanding of particle deposition efficiency within the respiratory system following aerosol exposure in birds.

Effect of ATM heterozygosity on heritable DNA damage in mice following paternal F0 germline irradiation.

The ataxia telangiectasia mutated (ATM) gene product maintains genome integrity and initiates cellular DNA repair pathways following exposures to genotoxic agents. ATM also plays a significant role in meiotic recombination during spermatogenesis. Fertilization with sperm carrying damaged DNA could lead to adverse effects in offspring including developmental defects or increased cancer susceptibility. Currently, there is little information regarding the effect of ATM heterozygosity on germline DNA repair and heritable effects of paternal germline-ionizing irradiation. We used neutral pH comet assays to evaluate spermatozoa 45 days after acute whole-body irradiation of male mice (0.1Gy, attenuated (137)Cs gamma rays) to determine the effect of ATM heterozygosity on delayed DNA damage effects of Type A/B spermatogonial irradiation. Using the neutral pH sperm comet assay, significant irradiation-related differences were found in comet tail length, percent tail DNA and tail extent moment, but there were no observed differences in effect between wild-type and ATM +/- mice. However, evaluation of spermatozoa from third generation descendants of irradiated male mice for heritable chromatin effects revealed significant differences in DNA electrophoretic mobility in the F(3) descendants that were based upon the irradiated F(0) sire's genotype. In this study, radiation-induced chromatin alterations to Type A/B spermatogonia, detected in mature sperm 45 days post-irradiation, led to chromatin effects in mature sperm three generations later. The early cellular response to and repair of DNA damage is critical and appears to be affected by ATM zygosity. Our results indicate that there is potential for heritable genetic or epigenetic changes following Type A/B spermatogonial irradiation and that ATM heterozygosity increases this effect.

Concerning ionizing radiation-induced cancer from internally deposited radionuclides.

PURPOSE: A comparative evaluation was conducted of ionizing radiation-induced cancer from internally deposited radionuclides. MATERIALS AND METHODS: Data were evaluated for humans for (226)Ra, and for laboratory animal studies for alpha-emitters (228)Ra, (226)Ra, (224)Ra, (238)Pu, (239)Pu, (228)Th, (252)Cf, (249)Cf, and (241)Am, and for beta-emitters (90)Sr, (90)Y, (91)Y, and (144)Ce. Intake routes included ingestion, inhalation, and injection. RESULTS: Cancer risk associated with protracted ionizing radiation exposure is a non-linear function of lifetime average dose rate to the affected tissues. The lifetime effects are best described by three-dimensional average-dose-rate/time/response surfaces that compete with other causes of death during an individual's lifetime. At the higher average dose rates the principal deleterious effects are those associated with radiation-induced injury, while at intermediate average dose rates radiation-induced cancer predominates. CONCLUSIONS: The cumulative radiation dose is neither an accurate nor an appropriate measure of cancer risk associated with protracted ionizing radiation exposure. At low average dose rates the long latency time required for radiation-induced cancer may exceed the natural lifespan yielding a lifespan virtual threshold for radiation-induced cancer for cumulative doses below about 5-10 Sv for bone, bone marrow and lungs.

Instillation versus inhalation of multiwalled carbon nanotubes: exposure-related health effects, clearance, and the role of particle characteristics.

Inhaled multiwalled carbon nanotubes (MWCNTs) may cause adverse pulmonary responses due to their nanoscale, fibrous morphology and/or biopersistance. This study tested multiple factors (dose, time, physicochemical characteristics, and administration method) shown to affect MWCNT toxicity with the hypothesis that these factors will influence significantly different responses upon MWCNT exposure. The study is unique in that (1) multiple administration methods were tested using particles from the same stock; (2) bulk MWCNT formulations had few differences (metal content, surface area/functionalization); and (3) MWCNT retention was quantified using a specialized approach for measuring unlabeled MWCNTs in rodent lungs. Male Sprague-Dawley rats were exposed to original (O), purified (P), and carboxylic acid functionalized (F) MWCNTs via intratracheal instillation and inhalation. Blood, bronchoalveolar lavage fluid (BALF), and lung tissues were collected at postexposure days 1 and 21 for quantifying biological responses and MWCNTs in lung tissues by programmed thermal analysis. At day 1, MWCNT instillation produced significant BALF neutrophilia and MWCNT-positive macrophages. Instilled O- and P-MWCNTs produced significant inflammation in lung tissues, which resolved by day 21 despite MWCNT retention. MWCNT inhalation produced no BALF neutrophilia and no significant histopathology past day 1. However, on days 1 and 21 postinhalation of nebulized MWCNTs, significantly increased numbers of MWCNT-positive macrophages were observed in BALF. Results suggest (1) MWCNTs produce transient inflammation if any despite persistence in the lungs; (2) instilled O-MWCNTs cause more inflammation than P- or F-MWCNTs; and (3) MWCNT suspension media produce strikingly different effects on physicochemical particle characteristics and pulmonary responses.

Toward improved ionizing radiation safety standards.

Ionizing radiation safety standards developed by the International Commission on Radiological Protection (ICRP) during the past 50-plus years have provided guidance for effective protection of workers and the public from the potentially harmful effects of exposure to ionizing radiation, including cancer. Earlier standards were based primarily on radiation dose rate to organs of the body. More recent recommendations have calculated cancer risk as a function of cumulative dose using a linear no-threshold cancer risk model based on the acute high dose rate exposures received by the Japanese atomic bomb survivors. The underlying assumption in these current recommendations is that risk of radiation-induced cancer is proportional to cumulative dose without threshold. In conflict with this position are the studies of protracted exposures from internally-deposited radionuclides in people and laboratory animals that have demonstrated that cancer induction risk is a function of average dose rate for protracted exposures to ionizing radiation. At lower average dose rates, cancer latency can exceed natural lifespan leading to a virtual threshold. This forum statement proposes that the conflict of these two cancer risk models is explained by the fact that the increased risk of cancer observed in the atomic bomb survivor studies was primarily the result of acute high dose rate promotion of ongoing biological processes that lead to cancer rather than cancer induction. In addition, ionizing radiation-induced cancer is not the result of a simple stochastic event in a single living cell but rather a complex deterministic systemic effect in living tissues. It is recommended that the ICRP consider revising its position in light of this important distinction between cancer promotion and cancer induction.

Concerning the health effects of internally deposited radionuclides.

The ionizing radiation dose-response relationships for internally deposited radionuclides are examined using data from humans involving Ra and laboratory animal studies involving alpha-emitters Ra, Ra, Ra, Th, Pu, Pu, and Am and beta-emitters Y, Sr, Y, and Ce. Intake routes included ingestion, inhalation, and injection. The lifetime effects are best described by three-dimensional average-dose-rate/time/response surfaces that compete with other causes of death during an individual's lifetime. Using maximum likelihood survival regression methods, the characteristic logarithmic slope for cancer induction was found to be about negative one-third for alpha-emitters or about negative two-thirds for beta-emitters. At the higher average dose-rates the principal deleterious effects were those associated with radiation-induced injury while at intermediate average dose-rates radiation-induced cancer predominates. The relative biological effectiveness for cancer induction of high linear energy transfer alpha radiation with respect to low linear energy transfer beta radiation is a strong function of dose-rate. As average dose-rate decreases, the effectiveness of the beta irradiation drops off more rapidly than that of the alpha irradiation. The cumulative dose yielding a specific level of induced cancer risk is less at lower dose-rates than at higher dose-rates showing an apparent inverse dose-rate effect (up to a factor of about 10 for high linear energy transfer alpha radiation and a factor of about 2 for low linear energy transfer beta radiation). The cumulative radiation dose is neither an accurate nor an appropriate measure of cancer risk associated with protracted ionizing radiation exposure. Cancer risk associated with protracted ionizing radiation exposure is a non-linear function of lifetime average dose-rate to the affected tissues. At low average dose-rates the long latency time required for radiation-induced cancer may exceed the natural lifespan. This long latency results in a lifespan virtual threshold (cancer risk p < 0.001) for each internally deposited radionuclide. For young adult beagles, bone sarcoma induction from alpha-emitting radionuclides was unlikely for cumulative doses below about 1 Gy (20 Sv) delivered specifically to the sensitive tissues at bone surfaces in a manner associated with radionuclide relative potency from highest for Th, Pu, and Pu to lowest for Ra. Bone sarcoma induction from ingested Sr was unlikely for cumulative beta radiation doses below about 20 Gy (20 Sv), but beta irradiation of tissues adjacent to bone also induced leukemia and soft tissue carcinomas above 10 Gy (10 Sv). Inhaled radionuclides tended to be most potent in producing lung carcinoma when the radiation dose was most uniformly distributed in the lung. In young adult beagles lung carcinoma from inhaled alpha-emitting Pu in the dispersible nitrate form was unlikely for cumulative doses below about 0.5 Gy (10 Sv) and below higher cumulative doses for other forms of Pu and Pu depending on relative potency. Lung carcinoma from inhaled beta-emitting Y in relatively insoluble fused aluminosilicate particles was unlikely for cumulative doses below about 5 Gy (5 Sv) and below higher doses for inhaled particles with Y, Ce, or Sr in order of decreasing potency.

Gamma irradiation of Type B spermatogonia leads to heritable genomic instability in four generations of mice.

Mice conceived 6 weeks after paternal exposure to ionizing radiation were fathered by sperm that were Type B spermatogonia at the time of irradiation. Previous studies of these offspring showed that this paternal F0 germ cell irradiation led to decreased embryonic cell proliferation rates, altered enzyme activities, protein levels and whole-body weights. In the present study, we examined four generations of CD1 mice following paternal F0 irradiation of the Type B spermatogonia (1.0 Gy, (137)Cs gamma rays) to determine the stability of the heritable effects. Offspring were evaluated for changes in protein kinase C and mitogen-activated protein kinase enzyme activities and Trp53 and p21(waf1) protein levels. Two or more endpoints were significantly altered in all four generations of offspring from the irradiated F0 sire (P

Chemoprevention of tobacco smoke-induced lung tumors by inhalation of an epigallocatechin gallate (EGCG) aerosol: a pilot study.

We investigated whether inhalation of aerosolized epigallocatechin gallate (EGCG) would prevent the development of lung tumors produced by tobacco smoke (TS). Male strain A/J mice were exposed for 5 mo, 6 h/day, 5 days/wk, to a mixture of tobacco sidestream and mainstream smoke. At the end of this exposure, 3 groups were formed: (a) mice exposed to TS and left undisturbed in air; (b) animals exposed to TS and given EGCG aerosol by nose-only inhalation for 30 min per session; and (c) animals exposed to TS and then exposed by nose-only inhalation to water aerosol without any EGCG (sham-exposed group). Three similar groups were formed from animals that previously had been kept in filtered air. In experiment 1, the EGCG concentration in the aerosol was 80 microg/L and administered 3 times a week and in experiment 2 it was 191 microg/L administered twice a week. Inhalation of EGCG did not modulate TS-induced tumorigenesis. In two accompanying positive control experiments, animals treated with the tobacco-specific carcinogen NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] were given the same EGCG or water aerosol treatment. In both experiments, EGCG aerosol significantly reduced lung tumor multiplicity by 20% to 30% However, exposure of NNK-treated animals to water solvent alone (sham exposure) produced an even greater reduction in tumor multiplicities (40%). A reduction of lung tumor multiplicities was also observed in animals exposed nose-only once or five times a week to either water aerosols or to filtered air. It is concluded that water-soluble chemopreventive agents that need to be ingested in comparatively high doses are not the most suitable candidates for administration by inhalation.

Germline drift in chimeric male mice possessing an F2 component with a paternal F0 radiation history.

In earlier work using mouse chimeric embryos we have shown that pre-implantation embryonic cells with a history of paternal gamma-irradiation (1 Gy) 6 weeks prior to conception demonstrated a competitive cell proliferation disadvantage when challenged by direct cell-cell contact with control pre-implantation embryos in chimeras even two generations after paternal irradiation. This effect on embryonic cell proliferation was associated with paternal irradiation of sensitive type A/B spermatogonia 6 weeks prior to conception but not from irradiation 5 weeks prior to conception. The purpose of this new study is to test the hypothesis that germ cell lines with a history of acute irradiation may also exhibit a selection disadvantage or germline chimeric drift over time in adult male mice two generations after paternal irradiation in chimeric mice. F(0) male CD1 mice received a 1.0 Gy whole body absorbed dose of attenuated (137)Cs gamma-rays and were mated at post-irradiation weeks 5 and 6. Chimeric XY<---->XY male CD1 mice were constructed with F(2) embryos from the F(1) offspring and with control embryos. To distinguish the control germline cells, the control embryos were heterozygous for the neo transgene, which served as a cell lineage marker. To test for germline chimeric drift, each XY<---->XY chimera was mated periodically from age 7 to 21 weeks with a different CD1 female. The fraction of offspring from each liter with the neo marker was used to quantify the relative contribution of the control germ cell lineage to the fertilizing sperm population. The results showed that there was a significant selection against germ cells with the paternal F(0) post-irradiation week 6 history. In contrast, there was a very small but significant selection in favor of the germ cells with the paternal F(0) post-irradiation week 5 history. These results indicate that the effects of a relatively non-toxic dose of radiation (1 Gy) on cell proliferation transmitted by ancestral type B spermatogonia to the embryo are manifested in the germline of the adult male mice even after two generations.