Modified Bovine Milk Exosomes for Doxorubicin Delivery to Triple-Negative Breast Cancer Cells.

Biological nanoparticles, such as exosomes, offer an approach to drug delivery because of their innate ability to transport biomolecules. Exosomes are derived from cells and an integral component of cellular communication. However, the cellular cargo of human exosomes could negatively impact their use as a safe drug carrier. Additionally, exosomes have the intrinsic yet enigmatic, targeting characteristics of complex cellular communication. Hence, harnessing the natural transport abilities of exosomes for drug delivery requires predictably targeting these biological nanoparticles. This manuscript describes the use of two chemical modifications, incorporating a neuropilin receptor agonist peptide (iRGD) and a hypoxia-responsive lipid for targeting and release of an encapsulated drug from bovine milk exosomes to triple-negative breast cancer cells. Triple-negative breast cancer is a very aggressive and deadly form of malignancy with limited treatment options. Incorporation of both the iRGD peptide and hypoxia-responsive lipid into the lipid bilayer of bovine milk exosomes and encapsulation of the anticancer drug, doxorubicin, created the peptide targeted, hypoxia-responsive bovine milk exosomes, iDHRX. Initial studies confirmed the presence of iRGD peptide and the exosomes' ability to target the αvß3 integrin, overexpressed on triple-negative breast cancer cells' surface. These modified exosomes were stable under normoxic conditions but fragmented in the reducing microenvironment created by 10 mM glutathione. In vitro cellular internalization studies in monolayer and three-dimensional (3D) spheroids of triple-negative breast cancer cells confirmed the cell-killing ability of iDHRX. Cell viability of 50% was reached at 10 µM iDHRX in the 3D spheroid models using four different triple-negative breast cancer cell lines. Overall, the tumor penetrating, hypoxia-responsive exosomes encapsulating doxorubicin would be effective in reducing triple-negative breast cancer cells' survival.

Estrogen receptors differentially regulate intracellular calcium handling in human nonasthmatic and asthmatic airway smooth muscle cells.

Asthma is defined as chronic inflammation of the airways and is characterized by airway remodeling, hyperresponsiveness, and acute bronchoconstriction of airway smooth muscle (ASM) cells. Clinical findings suggest a higher incidence and severity of asthma in adult women, indicating a concrete role of sex steroids in modulating the airway tone. Estrogen, a major female sex steroid mediates its role through estrogen receptors (ER) ERα and ERß, which are shown to be expressed in human ASM, and their expression is upregulated in lung inflammation and asthma. Previous studies suggested rapid, nongenomic signaling of estrogen via ERs reduces intracellular calcium ([Ca2+]i), thereby promoting relaxation of ASM. However, long-term ER activation on [Ca2+]i regulation in human ASM during inflammation or in asthma is still not known. In Fura-2-loaded nonasthmatic and asthmatic human ASM cells, we found that prolonged (24 h) exposure to ERα agonist (PPT) increased [Ca2+]i response to histamine, whereas ERß activation (WAY) led to decreased [Ca2+] compared with vehicle. This was further confirmed by ER overexpression and knockdown studies using various bronchoconstrictor agents. Interestingly, ERß activation was more effective than 17ß-estradiol in reducing [Ca2+]i responses in the presence of TNF-α or IL-13, while no observable changes were noticed with PPT in the presence of either cytokine. The [Ca2+]i-reducing effects of ERß were mediated partially via L-type calcium channel inhibition and increased Ca2+ sequestration by sarcoplasmic reticulum. Overall, these data highlight the differential signaling of ERα and ERß in ASM during inflammation. Specific ERß activation reduces [Ca2+]i in the inflamed ASM cells and is likely to play a crucial role in regulating ASM contractility, thereby relaxing airways.

Role of Estrogen Receptors α and ß in a Murine Model of Asthma: Exacerbated Airway Hyperresponsiveness and Remodeling in ERß Knockout Mice.

Epidemiological data suggests increased prevalence of asthma in females than males, suggesting a plausible role for sex-steroids, especially estrogen in the lungs. Estrogen primarily acts through estrogen-receptors (ERα and ERß), which play a differential role in asthma. Our previous studies demonstrated increased expression of ERß in asthmatic human airway smooth muscle (ASM) cells and its activation diminished ASM proliferation in vitro and airway hyperresponsiveness (AHR) in vivo in a mouse (wild-type, WT) model of asthma. In this study, we evaluated the receptor specific effect of circulating endogenous estrogen in regulating AHR and remodeling using ERα and ERß knockout (KO) mice. C57BL/6J WT, ERα KO, and ERß KO mice were challenged intranasally with a mixed-allergen (MA) or PBS. Lung function was measured using flexiVent followed by collection of broncho-alveolar lavage fluid for differential leukocyte count (DLC), histology using hematoxylin and eosin (H&E) and Sirius red-fast green (SRFG) and detecting αsmooth muscle actin (α-SMA), fibronectin and vimentin expression using immunofluorescence (IF). Resistance (Rrs), elastance (Ers), tissue-damping (G) and tissue-elasticity (H) were significantly increased, whereas compliance (Crs) was significantly decreased in WT, ERα KO, and ERß KO mice (males and females) challenged with MA compared to PBS. Interestingly, ERß KO mice showed declined lung function compared to ERα KO and WT mice at baseline. MA induced AHR, remodeling and immune-cell infiltration was more prominent in females compared to males across all populations, while ERß KO females showed maximum AHR and DLC, except for neutrophil count. Histology using H&E suggests increased smooth muscle mass in airways with recruitment of inflammatory cells, while SRFG staining showed increased collagen deposition in MA challenged ERß KO mice compared to ERα KO and WT mice (males and females), with pronounced effects in ERß KO females. Furthermore, IF studies showed increased expression of α-SMA, fibronectin and vimentin in MA challenged populations compared to PBS, with prominent changes in ERß KO females. This novel study indicates ERß plays a pivotal role in airway remodeling and AHR and understanding the mechanisms involved might help to surface it out as a potential target to treat asthma.