Structural disruption of the blood-brain barrier in repetitive primary blast injury.

BACKGROUND: Blast-induced traumatic brain injury (bTBI) is a growing health concern due to the increased use of low-cost improvised explosive devices in modern warfare. Mild blast exposures are common amongst military personnel; however, these women and men typically do not have adequate recovery time from their injuries due to the transient nature of behavioral symptoms. bTBI has been linked to heterogeneous neuropathology, including brain edema, neuronal degeneration and cognitive abnormalities depending on the intensity of blast overpressure and frequency. Recent studies have reported heterogeneity in blood-brain barrier (BBB) permeability following blast injury. There still remains a limited understanding of the pathologic changes in the BBB following primary blast injuries. In this study, our goal was to elucidate the pathologic pattern of BBB damage through structural analysis following single and repetitive blast injury using a clinically relevant rat model of bTBI. METHODS: A validated, open-ended shock tube model was used to deliver single or repetitive primary blast waves. The pathology of the BBB was assessed using immunofluorescence and immunohistochemistry assays. All data were analyzed using the one-way ANOVA test. RESULTS: We have demonstrated that exposure to repetitive blast injury affects the desmin-positive and CD13-positive subpopulations of pericytes in the BBB. Changes in astrocytes and microglia were also detected. CONCLUSION: This study provides analysis of the BBB components after repetitive blast injury. These results will be critical as preventative and therapeutic strategies are established for veterans recovering from blast-induced traumatic brain injury.

Peptide gels for controlled release of proteins.

Treatment strategies in clinics have been shifting from small molecules to protein drugs due to the promising results of a highly specific mechanism of action and reduced toxicity. Despite their prominent roles in disease treatment, delivery of the protein therapeutics is challenging due to chemical instability, immunogenicity and biological barriers. Peptide hydrogels with spatiotemporally tunable properties have shown an outstanding potential to deliver complex protein therapeutics, maintain drug efficacy and stability over time, mimicking the extracellular matrix, and responding to external stimuli. In this review, we present recent advances in peptide hydrogel design strategies, protein release kinetics and mechanisms for protein drug delivery in cellular engineering, tissue engineering, immunotherapy and disease treatments.

Harnessing Fiber Diameter-Dependent Effects of Myoblasts Toward Biomimetic Scaffold-Based Skeletal Muscle Regeneration.

Regeneration of skeletal muscles is limited in cases of volumetric muscle loss and muscle degenerative diseases. Therefore, there is a critical need for developing strategies that provide cellular and structural support for skeletal muscle regeneration. In the present work, a bioengineered cell niche composed of mechanically competent aligned polyester fiber scaffolds is developed to mimic the oriented muscle fiber microenvironment by electrospinning poly(lactide-co-glycolide) (PLGA) using a custom-designed rotating collector with interspaced parallel blades. Aligned fiber scaffolds with fiber diameters ranging from 335 ± 154 nm to 3013 ± 531 nm are characterized for their bioactivities in supporting growth and differentiation of myoblasts. During in vitro culture, polymeric scaffolds with larger fiber diameter support enhanced alignment, growth, and differentiation of myoblasts associated with phosphorylation of p38 MAPK and upregulated expression of myogenin and myosin heavy chain. In vivo studies using a dystrophin-deficient mdx mouse model show that optimized fiber scaffolds seeded with primary myoblasts result in formation of dystrophin-positive myofibers network in tibialis anterior muscles. Collectively, these experiments provide critical insights on harnessing interactions between muscle cells and engineered fiber matrices to develop effective biomaterials for accelerated muscle regeneration.

Dynamic transition of the blood-brain barrier in the development of non-small cell lung cancer brain metastases.

Invasion of the brain by non-small cell lung cancer (NSCLC) results in a shift of the blood-brain barrier (BBB) to the insufficiently characterized blood-tumor barrier (BTB). Effective drug delivery through the BTB is one of the greatest therapeutic obstacles in treating brain metastases. Using an experimental model, we defined key changes within the BTB and the BBB in the brain around the tumor (BAT) region over time. Brain-seeking NSCLC cells were delivered into the circulation of athymic-nude mice via intracardiac injection and developing brain metastases were evaluated over six-weeks. Components of the BBB and BTB were analyzed using immunofluorescence microscopy and compared using a mixed model of regression. Our results demonstrate a dynamic time-dependent BTB phenotype. Capillaries of the BAT and BTB were dilated with increased CD31 expression compared to controls. Expression of collagen IV, a pan-basement membrane component, was significantly decreased in the BTB compared to the BBB. There was also a significant increase in the desmin-positive pericyte subpopulation in the BTB compared to the BBB. The most striking changes were identified in astrocyte water channels with a 12.18-fold (p < 0.001) decrease in aquaporin-4 in the BTB; the BAT was unchanged. Analysis of NSCLC brain metastases from patient samples similarly demonstrated dilated capillaries and loss of both collagen IV and aquaporin-4. These data provide a comprehensive analysis of the BTB in NSCLC brain metastasis. Astrocytic endfeet, pericytes, and the basement membrane are potential therapeutic targets to improve efficacy of chemotherapeutic delivery into NSCLC brain metastases.

Epithelial-mesenchymal Transition Phenotypes in Vertebral Metastases of Lung Cancer.

Vertebral metastases of non-small cell lung cancer (NSCLC) are frequently diagnosed in the metastatic setting and are commonly identified in the thoracic vertebrae in patients. Treatment of NSCLC bone metastases, which are often multiple, is palliative, and the median survival times are 3 to 6 months. We have characterized spontaneous vertebral metastases in a brain metastases model of NSCLC and correlated these findings with epithelial-mesenchymal transition (EMT). Brain metastases were established in athymic nude mice following intracardiac injection of brain-seeking adenocarcinoma NSCLC cells. Thirty-nine percent of mice (14/36) developed spontaneous vertebral metastases, spinal cord compression, and hind-limb paralysis. Vertebral metastases consisted of an adenocarcinoma phenotype with neoplastic epithelial cells arranged in cords or acini and a mesenchymal phenotype with spindloid neoplastic cells arranged in bundles and streams. Quantitative and qualitative immunohistochemical and immunofluorescence assays demonstrated an increase in vimentin expression compared to cytokeratin expression in vertebral metastases. A correlation with EMT was supported by an increase in CD44 in vertebral metastases and parenchymal metastases. These data demonstrate a translational lung cancer metastasis model with spontaneous vertebral metastasis. The mesenchymal and epithelial phenotype of these spontaneous metastases coupled with EMT provide a conduit to improve drug delivery and overall patient survival.

Surface Modification of Polymeric Nanoparticles with M2pep Peptide for Drug Delivery to Tumor-Associated Macrophages.

PURPOSE: Tumor-associated macrophages (TAMs) with immune-suppressive M2-like phenotype constitute a significant part of tumor and support its growth, thus making an attractive therapeutic target for cancer therapy. To improve the delivery of drugs that control the survival and/or functions of TAMs, we developed nanoparticulate drug carriers with high affinity for TAMs. METHODS: Poly(lactic-co-glycolic acid) nanoparticles were coated with M2pep, a peptide ligand selectively binding to M2-polarized macrophages, via a simple surface modification method based on tannic acid-iron complex. The interactions of M2pep-coated nanoparticles with macrophages of different phenotypes were tested in vitro and in vivo. PLX3397, an inhibitor of the colony stimulating factor-1 (CSF-1)/CSF-1 receptor (CSF-1R) pathway and macrophage survival, was delivered to B16F10 tumors via M2pep-modified PLGA nanoparticles. RESULTS: In bone marrow-derived macrophages polarized to M2 phenotype, M2pep-coated nanoparticles showed greater cellular uptake than those without M2pep. Consistently, M2pep-coated nanoparticles showed relatively high localization of CD206+ macrophages in B16F10 tumors. PLX3397 encapsulated in M2pep-coated nanoparticles attenuated tumor growth better than the free drug counterpart. CONCLUSION: These results support that M2pep-coating can help nanoparticles to interact with M2-like TAMs and facilitate the delivery of drugs that control the tumor-supportive functions of TAMs.

Laminin mimetic peptide nanofibers regenerate acute muscle defect.

Skeletal muscle cells are terminally differentiated and require the activation of muscle progenitor (satellite) cells for their regeneration. There is a clinical need for faster and more efficient treatment methods for acute muscle injuries, and the stimulation of satellite cell proliferation is promising in this context. In this study, we designed and synthesized a laminin-mimetic bioactive peptide (LM/E-PA) system that is capable of accelerating satellite cell activation by emulating the structure and function of laminin, a major protein of the basal membrane of the skeletal muscle. The LM/E-PA nanofibers enhance myogenic differentiation in vitro and the clinical relevance of the laminin-mimetic bioactive scaffold system was demonstrated further by assessing its effect on the regeneration of acute muscle injury in a rat model. Laminin mimetic peptide nanofibers significantly promoted satellite cell activation in skeletal muscle and accelerated myofibrillar regeneration following acute muscle injury. In addition, the LM/E-PA scaffold treatment significantly reduced the time required for the structural and functional repair of skeletal muscle. This study represents one of the first examples of molecular- and tissue-level regeneration of skeletal muscle facilitated by bioactive peptide nanofibers following acute muscle injury. SIGNIFICANCE STATEMENT: Sports, heavy lifting and other strength-intensive tasks are ubiquitous in modern life and likely to cause acute skeletal muscle injury. Speeding up regeneration of skeletal muscle injuries would not only shorten the duration of recovery for the patient, but also support the general health and functionality of the repaired muscle tissue. In this work, we designed and synthesized a laminin-mimetic nanosystem to enhance muscle regeneration. We tested its activity in a rat tibialis anterior muscle by injecting the bioactive nanosystem. The evaluation of the regeneration and differentiation capacity of skeletal muscle suggested that the laminin-mimetic nanosystem enhances skeletal muscle regeneration and provides a suitable platform that is highly promising for the regeneration of acute muscle injuries. This work demonstrates for the first time that laminin-mimetic self-assembled peptide nanosystems facilitate myogenic differentiation in vivo without the need for additional treatment.

Dibenzazepine-Loaded Nanoparticles Induce Local Browning of White Adipose Tissue to Counteract Obesity.

Inhibition of Notch signaling via systemic drug administration triggers conversion of white adipocytes into beige adipocytes (browning) and reduces adiposity. However, translation of this discovery into clinical practice is challenged by potential off-target side effects and lack of control over the location and temporal extent of beige adipocyte biogenesis. Here, we demonstrate an alternative approach to stimulate browning using nanoparticles (NPs) composed of FDA-approved poly(lactide-co-glycolide) that enable sustained local release of a Notch inhibitor (dibenzazepine, DBZ). These DBZ-loaded NPs support rapid cellular internalization and inhibit Notch signaling in adipocytes. Importantly, focal injection of these NPs into the inguinal white adipose tissue depots of diet-induced obese mice results in localized NP retention and browning of adipocytes, consequently improving the glucose homeostasis and attenuating body-weight gain of the treated mice. These findings offer new avenues to develop a potential therapeutic strategy for clinical treatment of obesity and its associated metabolic syndrome.

Angiogenic Heparin-Mimetic Peptide Nanofiber Gel Improves Regenerative Healing of Acute Wounds.

Wound repair in adult mammals typically ends with the formation of a scar, which prevents full restoration of the function of the healthy tissue, although most of the wounded skin heals. Rapid and functional recovery of major wound injuries requires therapeutic approaches that can enhance the healing process via overcoming mechanical and biochemical problems. In this study, we showed that self-assembled heparin-mimetic peptide nanofiber gel was an effective bioactive wound dressing for the rapid and functional repair of full-thickness excisional wounds in the rat model. The bioactive gel-treated wounds exhibited increased angiogenesis (p < 0.05), re-epithelization (p < 0.05), skin appendage formation, and granulation tissue organization (p < 0.05) compared to sucrose-treated samples. Increased blood vessel numbers in the gel-treated wounds on day 7 suggest that angiogenesis played a key role in improvement of tissue healing in bioactive gel-treated wounds. Overall, the angiogenic heparin-mimetic peptide nanofiber gel is a promising platform for enhancing the scar-free recovery of acute wounds.

Improving pancreatic islet in vitro functionality and transplantation efficiency by using heparin mimetic peptide nanofiber gels.

Pancreatic islet transplantation is a promising treatment for type 1 diabetes. However, viability and functionality of the islets after transplantation are limited due to loss of integrity and destruction of blood vessel networks. Thus, it is important to provide a proper mechanically and biologically supportive environment for enhancing both in vitro islet culture and transplantation efficiency. Here, we demonstrate that heparin mimetic peptide amphiphile (HM-PA) nanofibrous network is a promising platform for these purposes. The islets cultured with peptide nanofiber gel containing growth factors exhibited a similar glucose stimulation index as that of the freshly isolated islets even after 7 days. After transplantation of islets to STZ-induced diabetic rats, 28 day-long monitoring displayed that islets that were transplanted in HM-PA nanofiber gels maintained better blood glucose levels at normal levels compared to the only islet transplantation group. In addition, intraperitoneal glucose tolerance test revealed that animals that were transplanted with islets within peptide gels showed a similar pattern with the healthy control group. Histological assessment showed that islets transplanted within peptide nanofiber gels demonstrated better islet integrity due to increased blood vessel density. This work demonstrates that using the HM-PA nanofiber gel platform enhances the islets function and islet transplantation efficiency both in vitro and in vivo.

Effects of flavonoid quercetin on survival of motor neuron gene expression.

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease that results in muscle weakness and atrophy. To attenuate disease severity, drug development studies have been applied mainly to target the Survival of Motor Neuron 2 (SMN2) gene, which is an important modifier of SMA. Although several compounds have been tested, there is still no cure for SMA. In this study, SMN2-inducing effects of quercetin, an abundant flavonoid polyphenol in human diet, was investigated in the fibroblast cell lines of two SMA type I patients. Gene expression studies showed that quercetin upregulates SMN2 mRNA up to fourfold, but not the SMN protein level.