Nanoparticle Delivery of STAT3 Alleviates Pulmonary Hypertension in a Mouse Model of Alveolar Capillary Dysplasia.

BACKGROUND: Pulmonary hypertension (PH) is a common complication in patients with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with mutations in the FOXF1 gene. Although the loss of alveolar microvasculature causes PH in patients with ACDMPV, it is unknown whether increasing neonatal lung angiogenesis could prevent PH and right ventricular (RV) hypertrophy. METHODS: We used echocardiography, RV catheterization, immunostaining, and biochemical methods to examine lung and heart remodeling and RV output in Foxf1WT/S52F mice carrying the S52F Foxf1 mutation (identified in patients with ACDMPV). The ability of Foxf1WT/S52F mutant embryonic stem cells to differentiate into respiratory cell lineages in vivo was examined using blastocyst complementation. Intravascular delivery of nanoparticles with a nonintegrating Stat3 expression vector was used to improve neonatal pulmonary angiogenesis in Foxf1WT/S52F mice and determine its effects on PH and RV hypertrophy. RESULTS: Foxf1WT/S52F mice developed PH and RV hypertrophy after birth. The severity of PH in Foxf1WT/S52F mice directly correlated with mortality, low body weight, pulmonary artery muscularization, and increased collagen deposition in the lung tissue. Increased fibrotic remodeling was found in human ACDMPV lungs. Mouse embryonic stem cells carrying the S52F Foxf1 mutation were used to produce chimeras through blastocyst complementation and to demonstrate that Foxf1WT/S52F embryonic stem cells have a propensity to differentiate into pulmonary myofibroblasts. Intravascular delivery of nanoparticles carrying Stat3 cDNA protected Foxf1WT/S52F mice from RV hypertrophy and PH, improved survival, and decreased fibrotic lung remodeling. CONCLUSIONS: Nanoparticle therapies increasing neonatal pulmonary angiogenesis may be considered to prevent PH in ACDMPV.

Nanoparticle Delivery of Proangiogenic Transcription Factors into the Neonatal Circulation Inhibits Alveolar Simplification Caused by Hyperoxia.

Rationale: Advances in neonatal critical care have greatly improved the survival of preterm infants, but the long-term complications of prematurity, including bronchopulmonary dysplasia (BPD), cause mortality and morbidity later in life. Although VEGF (vascular endothelial growth factor) improves lung structure and function in rodent BPD models, severe side effects of VEGF therapy prevent its use in patients with BPD.Objectives: To test whether nanoparticle delivery of proangiogenic transcription factor FOXM1 (forkhead box M1) or FOXF1 (forkhead box F1), both downstream targets of VEGF, can improve lung structure and function after neonatal hyperoxic injury.Methods: Newborn mice were exposed to 75% O2 for the first 7 days of life before being returned to a room air environment. On Postnatal Day 2, polyethylenimine-(5) myristic acid/polyethylene glycol-oleic acid/cholesterol nanoparticles containing nonintegrating expression plasmids with Foxm1 or Foxf1 cDNAs were injected intravenously. The effects of the nanoparticles on lung structure and function were evaluated using confocal microscopy, flow cytometry, and the flexiVent small-animal ventilator.Measurements and Main Results: The nanoparticles efficiently targeted endothelial cells and myofibroblasts in the alveolar region. Nanoparticle delivery of either FOXM1 or FOXF1 did not protect endothelial cells from apoptosis caused by hyperoxia but increased endothelial proliferation and lung angiogenesis after the injury. FOXM1 and FOXF1 improved elastin fiber organization, decreased alveolar simplification, and preserved lung function in mice reaching adulthood.Conclusions: Nanoparticle delivery of FOXM1 or FOXF1 stimulates lung angiogenesis and alveolarization during recovery from neonatal hyperoxic injury. Delivery of proangiogenic transcription factors has promise as a therapy for BPD in preterm infants.

Synthesis and application of POLYseq for profiling human liver organoids.

Detailed herein is the protocol for synthesis, characterization, and application of POLYseq for cell pooling in single-cell sequencing runs. POLYseq is synthesized through commercially available reagents and is highly tailorable. Synthesis is easily performed in a two-step protocol, utilizing Michael addition only requiring a temperature-stable hot bath capable of holding 90°C. However, care must be taken when mixing reagents for synthesis, as the final product is sensitive to initial mixing ratios of POLYseq reagents. For complete details on the use and execution of this protocol, please refer to Dunn et al. (2021).

Highly Efficient In Vivo Targeting of the Pulmonary Endothelium Using Novel Modifications of Polyethylenimine: An Importance of Charge.

Pulmonary vascular disease encompasses a wide range of serious afflictions with important clinical implications. There is critical need for the development of efficient, nonviral gene therapy delivery systems. Here, a promising avenue to overcome critical issues in efficient cell targeting within the lung via a uniquely designed nanosystem is reported. Polyplexes are created by functionalizing hyperbranched polyethylenimine (PEI) with biological fatty acids and carboxylate-terminated poly(ethylene glycol) (PEG) through a one-pot 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysuccinimide reaction. Following intravenous injection, polyplexes show an exceptionally high specificity to the pulmonary microvascular endothelium, allowing for the successful delivery of stabilized enhanced green fluorescent protein (eGFP) expressing messenger ribonucleic acid (mRNA). It is further shown, quantitatively, that positive surface charge is the main mechanism behind such high targeting efficiency for these polyplexes. Live in vivo imaging, flow cytometry of single cell suspensions, and confocal microscopy are used to demonstrate that positive polyplexes are enriched in the lung tissue and disseminated in 85-90% of the alveolar capillary endothelium, whilst being sparse in large vessels. Charge modification, achieved through poly(acrylic acid) or heparin coating, drives a highly significant reduction in both targeting percentage and targeting strength, highlighting the importance of specific surface charge, derived from chemical formulation, for efficient targeting of the pulmonary microvascular endothelium.

In-vitro depth-dependent hyperthermia of human mammary gland adenocarcinoma.

Nanoparticle mediated photothermal ablation of cancerous tissue shows promising results and applicability as a highly efficacious treatment method. As a majority of the photothermal work has been conducted with minimal attenuation of the laser before reaching the nanoparticles within surface seeded tumors in-vivo or through buffered media in-vitro, it is important to understand the effects of greater laser attenuation on photothermal efficacy mediated by changes in the scattering and absorption of the laser. Photothermal efficacy using a near infrared (NIR) 785nm laser irradiating polystyrene (PS) stabilized magnetite (Fe3O4) nanoparticles (PS-Fe3O4) is examined on MDA-MB-231 human mammary gland adenocarcinoma in-vitro. Agarose gel columns of various heights were created to simulate soft tissue and subsequently used for NIR laser attenuation. Polystyrene was found to significantly improve magnetite nanoparticle stability in serum containing media and modified Hank's Balanced Salt Solution and was able to induce significant hyperthermic ablation at mass concentrations which also did not elicit significant innate toxicity. Furthermore it was found that the polystyrene coating significantly reduced innate toxicity over 48h compared to uncoated magnetite. Agar gel layers provided similar optical attenuation in the NIR region to skin and prostate.

Photo-fluorescent and magnetic properties of iron oxide nanoparticles for biomedical applications.

Iron oxide exhibits fascinating physical properties especially in the nanometer range, not only from the standpoint of basic science, but also for a variety of engineering, particularly biomedical applications. For instance, Fe3O4 behaves as superparamagnetic as the particle size is reduced to a few nanometers in the single-domain region depending on the type of the material. The superparamagnetism is an important property for biomedical applications such as magnetic hyperthermia therapy of cancer. In this review article, we report on some of the most recent experimental and theoretical studies on magnetic heating mechanisms under an alternating (AC) magnetic field. The heating mechanisms are interpreted based on Néel and Brownian relaxations, and hysteresis loss. We also report on the recently discovered photoluminescence of Fe3O4 and explain the emission mechanisms in terms of the electronic band structures. Both optical and magnetic properties are correlated to the materials parameters of particle size, distribution, and physical confinement. By adjusting these parameters, both optical and magnetic properties are optimized. An important motivation to study iron oxide is due to its high potential in biomedical applications. Iron oxide nanoparticles can be used for MRI/optical multimodal imaging as well as the therapeutic mediator in cancer treatment. Both magnetic hyperthermia and photothermal effect has been utilized to kill cancer cells and inhibit tumor growth. Once the iron oxide nanoparticles are up taken by the tumor with sufficient concentration, greater localization provides enhanced effects over disseminated delivery while simultaneously requiring less therapeutic mass to elicit an equal response. Multi-modality provides highly beneficial co-localization. For magnetite (Fe3O4) nanoparticles the co-localization of diagnostics and therapeutics is achieved through magnetic based imaging and local hyperthermia generation through magnetic field or photon application. Here, Fe3O4 nanoparticles are shown to provide excellent conjugation bases for entrapment of therapeutic molecules, fluorescent agents, and targeting ligands; enhancement of solid tumor treatment is achieved through co-application of local hyperthermia with chemotherapeutic agents.

Photothermal effects and toxicity of Fe3O4 nanoparticles via near infrared laser irradiation for cancer therapy.

The photothermal effect of magnetite (Fe3O4) nanoparticles was characterized by photonic absorption in the near-infrared (NIR) region. Upon laser irradiation at 785 nm, the Fe3O4 nanoparticles generate localized hyperthermia in tumorous lesions, which is an effective strategy for cancer therapy; however, uncoated magnetite possesses an innate toxicity which can lead to drawbacks in the clinical setting. To reduce innate toxicity, a poly(acrylic acid) (PAA) coating on the nanoparticles was investigated in order to determine the alterations to stability and the degree of toxicity in an attempt to create a higher utility vector. It was found that the PAA coating significantly reduced the innate toxicity of the uncoated magnetite. Furthermore, the efficacy of PAA-coated magnetite nanoparticles (PAA-Fe3O4) was investigated for treating MDA-MB-231 (human mammary gland adenocarcinoma) cultures in viable concentration ranges (0.1-0.5mg/ml). An appropriate PAA-Fe3O4 concentration range was then established for inducing significant cell death by hyperthermic ablation, but not through innate toxicity.