Chemical Engineering

Drug-containing nanoparticles (NPs) with monodisperse, controlled particle sizes are highly desirable for drug delivery. Accumulating evidence suggests that NPs with sizes less than 50 nm demonstrate superior performance in vitro and in vivo. However, it is difficult to fabricate monodisperse, drug-containing NPs with discrete sizes required for studying and characterizing existing relationships among particle size, biologic processing, and therapeutic functionality. Here, we report a scalable process of fabricating drugÀsilica conjugated nanoparticles, termed drugÀsilica nanoconjugates (drug-NCs), which possess monodisperse size distributions and desirable particle sizes as small as 20 nm. We find that 20 nm NCs are superior to their 50 and 200 nm NC analogues by 2À5-and 10À20-fold, respectively, with regard to tumor accumulation and penetration and cellular internalization. These fundamental findings underscore the importance and necessity of further miniaturizing nanomedicine size for optimized drug delivery applications.

CXCL13/CXCR5 and CCL19/CCR7 play a quite important role in normal physiological conditions, but the functions of both chemokine/receptor pairs in pathophysiological events are not well-investigated. We have investigated expression and functions of CXCL13/CXCR5 and CCL19/CCR7 in CD23 ؉ CD5 ؉ and CD23 ؉ CD5 ؊ B cells from cord blood (CB) and patients with B cell lineage acute or chronic lymphocytic leukemia (B-ALL or B-CLL). CXCR5 and CCR7 are selectively expressed on B-ALL, B-CLL, and CB CD23 ؉ CD5 ؉ B cells at high frequency, but not on CD23 ؉ CD5 ؊ B cells. Although no significant chemotactic responsiveness was observed, CXCL13 and CCL19 cooperatively induce significant resistance to TNF-␣-mediated apoptosis in B-ALL and B-CLL CD23 ؉ CD5 ؉ B cells, but not in the cells from CB. B-ALL and B-CLL CD23 ؉ CD5 ؉ B cells express elevated levels of paternally expressed gene 10 (PEG10). CXCL13 and CCL19 together significantly up-regulate PEG10 expression in the same cells. We have found that CXCL13 and CCL19 together by means of activation of CXCR5 and CCR7 up-regulate PEG10 expression and function, subsequently stabilize caspase-3 and caspase-8 in B-ALL and B-CLL CD23 ؉ CD5 ؉ B cells, and further rescue the cells from TNF-␣-mediated apoptosis. Therefore, we suggest that normal lymphocytes, especially naive B and T cells, use CXCL13/CXCR5 and CCL19/CCR7 for migration, homing, maturation, and cell homeostasis as well as secondary lymphoid tissues organogenesis. In addition, certain malignant cells take advantages of CXCL13/CXCR5 and CCL19/CCR7 for infiltration, resistance to apoptosis, and inappropriate proliferation.

Bioreactors have become a critical step for the testing of new biomaterials and pharmaceuticals. They need to be controllable, ideally high-throughput, and produce a biologically relevant environment. For example, in the brain, it is essential to recreate multiple flow-pressure profiles daily and mimic brain fluid movement for a bioreactor to be more physiologic. In this study, we demonstrate a scalable system that regulates flow rate, pressure, and pulsation amplitude. We also show that with new microcontroller technology, up to 15 chambers running in parallel is theoretically possible. Our system, the Flow Limiting Operator (FLO), achieves these goals by multiplexing a series of valves and pumps to control pressure and volumetric flow rate instead of relying on head gas pressure. With the ability to control multiple parameters and its ease of use, both scientists and clinicians can use FLO to study the effects of pulsation amplitude of the fluid flow, flow rate, and pressure on intercellular interactions for both biomaterials and pharmaceuticals.

Cell-based immunotherapies have tremendous potential to treat many diseases, such as activating immunity in cancer or suppressing it in autoimmune diseases. Most cell-based cancer immunotherapies in the clinic provide adjuvant signals through genetic engineering to enhance T cell functions. However, genetically encoded signals have minimal control over dosing and persist for the life of a cell lineage. These properties make it difficult to balance increasing therapeutic efficacy with reducing toxicities. Here, we demonstrated the potential of phospholipid-coupled ligands as a non-genetic system for immune cell engineering. This system provides simple, controlled, non-genetic adjuvant delivery to immune cells via lipid-mediated insertion into plasma membranes. Lipid-mediated insertion (termed depoting) successfully delivered Toll-like receptor (TLR) ligands intracellularly and onto cell surfaces of diverse immune cells. These ligands depoted into immune cells in a dose-controlled fashion and did not compete during multiplex pairwise loading. Immune cell activation could be enhanced by autocrine and paracrine mechanisms depending on the biology of the TLR ligand tested. Depoted ligands functionally persisted on plasma membranes for up to 4 days in naïve and activated T cells, enhancing their activation, proliferation, and skewing cytokine secretion. Our data showed that depoted ligands provided a persistent yet non-permanent adjuvant signal to immune cells that may minimize the intensity and duration of toxicities compared to permanent genetic delivery. Altogether, these findings demonstrate potential for lipid-mediated depoting as a universal cell engineering approach with unique, complementary advantages to other cell engineering methods.

The regulation of genes utilizing the RNA interference (RNAi) mechanism via the delivery of synthetic siRNA has great potential in the treatment of a variety of lung diseases. However, the delivery of siRNA to the lungs is challenging due to the poor bioavailability siRNA when delivered intraveneously, and difficulty in formulating and maintaining the activity of free siRNA when delivered directly to the lungs using inhalation devices. The use of non-viral vectors such as cationic dendrimers can help enhance the stability of siRNA and its delivery to the cell cytosol. Therefore, in this work, we investigate the ability of a triphenylphosphonium (TPP) modified generation 4 poly(amidoamine) (PAMAM) dendrimer (G4NH2-TPP) to enhance the in vitro transfection efficiency of siRNA in a model of the pulmonary epithelium and their aerosol formulations in pressurized metered dose inhalers (pMDIs) and dry powder inhalers (DPIs). Complexes of siRNA and G4NH2-TPP were prepared with varying TPP d...

Pulmonary administration of polymer drug conjugates is of great potential clinical significance for treating lung cancer as such regimen significantly increases local drug concentrations while decreases systemic and local side effects. In this work, we demonstrate that nanoparticles prepared with methoxypoly(ethylene glycol) (mPEG)-doxorubicin (DOX) conjugates (mPEG-DOX) that have a pH-sensitive imine bond (Schiff base) can at the same time work as efficient carriers for DOX to kill cancer cells and also as a strategy to directly formulate nanoparticles in propellant-based inhalers. Nanoparticles prepared by precipitation in water had a diameter in the range between 100 and 120 nm. We investigated the effects of molecular weight (MW) of mPEG (1K, 2K, and 5K Da) on the in vitro release kinetics, cellular internalization, and cytotoxicity on in vitro model of lung adenocarcinoma and aerosol characters. It is observed that the DOX released from mPEG-DOX nanoparticles was significantly ...

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The lungs are major sites of metastases for several cancer types, including breast cancer (BC). Prognosis and quality of life of BC patients that develop pulmonary metastases are negatively impacted. The development of strategies to slow the growth and relieve the symptoms of BC lung metastases (BCLM) is thus an important goal in the management of BC. However, systemically administered first line small molecule chemotherapeutics have poor pharmacokinetic profiles and biodistribution to the lungs, as well as significant off-target toxicity, severely compromising their effectiveness. In this work we propose the local delivery of an add-on immunotherapy to the lungs to support first line chemotherapy treatment of advanced BC. In a syngeneic murine model of BCLM we show that local pulmonary administration (p.a.) of PLX-3397 (PLX), a colony stimulating factor 1 receptor inhibitor (CSF-1Ri), is capable of overcoming physiological barriers of the lung epithelium, penetrating the tumor microenvironment (TME), and decreasing phosphorylation of CSF-1 receptors, as shown by western blot of lung tumor nodules. That inhibition is accompanied by an overall decrease in the abundance of pro-tumorigenic (M2-like) macrophages in the TME, with a concomitant increase in the amount of anti-tumor (M1-like) macrophages when compared to vehicle treated control. These effects with PLX p.a. were

Demonstrating bioequivalence (BE) of nasal suspension sprays is a challenging task. Analytical tools are required to determine the particle size of the active pharmaceutical ingredient (API) and the structure of a relatively complex formulation. This study investigated the utility of the morphologically-directed Raman spectroscopy (MDRS) method to investigate the particle size distribution (PSD) of nasal suspensions. Dissolution was also investigated as an orthogonal technique. Nasal suspension formulations containing different PSD of mometasone furoate monohydrate (MFM) were manufactured. The PSD of the MFM batches was characterized before formulation manufacture using laser diffraction and automated imaging. Upon formulation manufacture, the droplet size, single actuation content, spray pattern, plume geometry, the API dissolution rate, and the API PSD by MDRS were determined. A systematic approach was utilized to develop a robust method for the analysis of the PSD of MFM in Nason...

Dry powder inhalers (DPIs) are drug–device combination products where the complexity of the formulation, its interaction with the device, and input from users play important roles in the drug delivery. As the landscape of DPI products advances with new powder formulations and novel device designs, understanding how these advancements impact performance can aid in developing generics that are therapeutically equivalent to the reference listed drug (RLD) products. This review details the current understanding of the formulation and device related principles driving DPI performance, past and present research efforts to characterize these performance factors, and the implications that advances in formulation and device design may present for evaluating bioequivalence (BE) for generic development.

Demonstrating bioequivalence (BE) of nasal suspension sprays is a challenging task. Analytical tools are required to determine the particle size of the active pharmaceutical ingredient (API) and the structure of a relatively complex formulation. This study investigated the utility of the morphologically-directed Raman spectroscopy (MDRS) method to investigate the particle size distribution (PSD) of nasal suspensions. Dissolution was also investigated as an orthogonal technique. Nasal suspension formulations containing different PSD of mometasone furoate monohydrate (MFM) were manufactured. The PSD of the MFM batches was characterized before formulation manufacture using laser diffraction and automated imaging. Upon formulation manufacture, the droplet size, single actuation content, spray pattern, plume geometry, the API dissolution rate, and the API PSD by MDRS were determined. A systematic approach was utilized to develop a robust method for the analysis of the PSD of MFM in Nasonex® and four test formulations containing the MFM API with different particle size specifications. Although the PSD between distinct techniques cannot be directly compared due to inherent differences between these methodologies, the same trend is observed for three out of the four batches. Dissolution analysis confirmed the trend observed by MDRS in terms of PSD. For suspension-based nasal products, MDRS allows the measurement of API PSD which is critical for BE assessment. This approach has been approved for use in lieu of a comparative clinical endpoint BE study [1]. The correlation observed between PSD and dissolution rate extends the use of dissolution as a critical analytical tool demonstrating BE between test and reference products.

Pulmonary administration of polymer drug conjugates is of great potential clinical significance for treating lung cancer as such regimen significantly increases local drug concentrations while decreases systemic and local side effects. In this work, we demonstrate that nanoparticles prepared with methoxypoly(ethylene glycol) (mPEG)-doxorubicin (DOX) conjugates (mPEG-DOX) that have a pH-sensitive imine bond (Schiff base) can at the same time work as efficient carriers for DOX to kill cancer cells, and also as a strategy to directly formulate nanoparticles in propellant-based inhalers. Nanoparticles prepared by precipitation in water had a diameter in the range between 100-120 nm. We investigated the effects of molecular weight (MW) of mPEG (1K, 2K, and 5K Da) on the in vitro release kinetics, cellular internalization and cytotoxicity on in vitro model of lung adenocarcinoma and aerosol characters. It is observed that the DOX released from mPEG-DOX nanoparticles was significantly accelerated in acidic environment-pH 5.5 (endosomal/lysosomal pH) in comparison with pH 7.4 (physiological pH), as designed. Release of DOX from mPEG1K-DOX nanoparticles was significantly greater than those from mPEG2K and mPEG5K counterparts. In vitro cytotoxicity of nanoparticles, followed the sequence of mPEG1K-DOX > free DOX > mPEG2K-DOX >> mPEG5K-DOX, a trend closely following their rate and extent of cellular internalization. mPEG-DOX nanoparticles with mPEG1K and mPEG2K were directly dispersed in hydrofluoroalkane (HFA), while a trace of ethanol was required to disperse mPEG5K-DOX nanoparticles in HFA. These pMDI formulations with high physical stability in HFAs display superior aerosol characteristics conducive to deep lung deposition. The fine particle fractions of these formulations ranged from 40% to 60%, higher than those of commercial products. Such formulations prepared from nanoparticles of pH-sensitive PEG-drug conjugates may also be envisioned to be extended to

The lungs are major sites of metastases for several cancer types, including breast cancer (BC). Prognosis and quality of life of BC patients that develop pulmonary metastases are negatively impacted. The development of strategies to slow the growth and relieve the symptoms of BC lung metastases (BCLM) is thus an important goal in the management of BC. However, systemically administered first line small molecule chemotherapeutics have poor pharmacokinetic profiles and biodistribution to the lungs, as well as significant off-target toxicity, severely compromising their effectiveness. In this work we propose the local delivery of an add-on immunotherapy to the lungs to support first line chemotherapy treatment of advanced BC. In a syngeneic murine model of BCLM we show that local pulmonary administration (p.a.) of PLX-3397 (PLX), a colony stimulating factor 1 receptor inhibitor (CSF-1Ri), is capable of overcoming physiological barriers of the lung epithelium, penetrating the tumor microenvironment (TME), and decreasing phosphorylation of CSF-1 receptors, as shown by western blot of lung tumor nodules. That inhibition is accompanied by an overall decrease in the abundance of pro-tumorigenic (M2-like) macrophages in the TME, with a concomitant increase in the amount of anti-tumor (M1-like) macrophages when compared to vehicle treated control. These effects with PLX p.a. were achieved using a much smaller dose (1 mg/kg, every other day) compared to the systemic doses typically used in preclinical studies (40–800 mg/kg/day). As an additive in combination with intravenous (i.v.) administration of paclitaxel (PTX), p.a. PLX leads to a decrease in tumor burden without additional toxicity. These results suggested that the proposed immunochemotherapy, with regional pulmonary delivery of PLX along with i.v. standard of care chemotherapy, may lead to new opportunities to improve treatment, quality of life, and survival of patients with BCLM.