disruption-blood-spinal-cord-barrier-using-low-intensity-focused

Disruption of the Blood-Spinal Cord Barrier Using Low-Intensity Focused Ultrasound in a Rat Model

Low-intensity focused ultrasound (LIFU) uses ultrasonic pulsations at lower intensities than ultrasound and is being tested as a reversible and precise ...

Johns Hopkins University School of Medicine

Johns Hopkins University

Translation of biomaterial-based nanoparticle formulations to the clinic faces significant challenges including efficacy, safety, consistency and scale-up of manufacturing, and stability during long-term storage. Continuous microfluidic fabrication of polymeric nanoparticles has the potential to alleviate the challenges associated with manufacture, while offering a scalable solution for clinical level production. Poly(beta-amino esters) (PBAE)s are a class of biodegradable cationic polymers that self-assemble with anionic plasmid DNA to form polyplex nanoparticles that have been shown to be effective for transfecting cancer cells specifically in vitro and in vivo. Here, we demonstrate the use of a microfluidic device for the continuous and scalable production of PBAE/DNA nanoparticles followed by lyophilization and long term storage that results in improved in vitro efficacy in multiple cancer cell lines compared to nanoparticles produced by bulk mixing as well as in comparison to widely used commercially available transfection reagents polyethylenimine and Lipofectamine® 2000. We further characterized the nanoparticles using nanoparticle tracking analysis (NTA) to show that microfluidic mixing resulted in fewer DNA-free polymeric nanoparticles compared to those produced by bulk mixing. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1813-1825, 2017.

Continuous microfluidic assembly of biodegradable poly(beta-amino ester)/DNA nanoparticles for enhanced gene delivery.

There is a need for new tools to better quantify intracellular delivery barriers in high-throughput and high-content ways. Here, we synthesized a triple-fluorophore-labeled nucleic acid pH nanosensor for measuring intracellular pH of exogenous DNA at specific time points in a high-throughput manner by flow cytometry following non-viral transfection. By including two pH-sensitive fluorophores and one pH-insensitive fluorophore in the nanosensor, detection of pH was possible over the full physiological range. We further assessed possible correlation between intracellular pH of delivered DNA, cellular uptake of DNA, and DNA reporter gene expression at 24 hr post-transfection for poly-L-lysine and branched polyethylenimine polyplex nanoparticles. While successful transfection was shown to clearly depend on median cellular pH of delivered DNA at the cell population level, surprisingly, on an individual cell basis, there was no significant correlation between intracellular pH and transfection efficacy. To our knowledge, this is the first reported instance of high-throughput single-cell analysis between cellular uptake of DNA, intracellular pH of delivered DNA, and gene expression of the delivered DNA. Using the nanosensor, we demonstrate that the ability of polymeric nanoparticles to avoid an acidic environment is necessary, but not sufficient, for successful transfection.

A Triple-Fluorophore-Labeled Nucleic Acid pH Nanosensor to Investigate Non-viral Gene Delivery.

Development of highly effective nonviral gene delivery vectors for transfection of diverse cell populations remains a challenge despite utilization of both rational and combinatorial driven approaches to nanoparticle engineering. In this work, multifunctional polyesters are synthesized with well-defined branching structures via A2 + B2/B3 + C1 Michael addition reactions from small molecule acrylate and amine monomers and then end-capped with amine-containing small molecules to assess the influence of polymer branching structure on transfection. These Branched poly(Ester Amine) Quadpolymers (BEAQs) are highly effective for delivery of plasmid DNA to retinal pigment epithelial cells and demonstrate multiple improvements over previously reported leading linear poly(beta-amino ester)s, particularly for volume-limited applications where improved efficiency is required. BEAQs with moderate degrees of branching are demonstrated to be optimal for delivery under high serum conditions and low nanoparticle doses further relevant for therapeutic gene delivery applications. Defined structural properties of each polymer in the series, including tertiary amine content, correlated with cellular transfection efficacy and viability. Trends that can be applied to the rational design of future generations of biodegradable polymers are elucidated.

Differentially Branched Ester Amine Quadpolymers with Amphiphilic and pH-Sensitive Properties for Efficient Plasmid DNA Delivery.

The tumor-selective human adenovirus Delta24-RGD is currently under investigation in phase II clinical trials for patients with recurrent glioblastoma (GBM). To improve treatments for patients with GBM, we explored the potential of combining Delta24-RGD with antibodies targeting immune checkpoints.

Low-dose oncolytic adenovirus therapy overcomes tumor-induced immune suppression and sensitizes intracranial gliomas to anti-PD-1 therapy.

Cerebral vasospasm is a major source of morbidity and mortality following aneurysm rupture and has limited treatment options.

PD-1+ Monocytes Mediate Cerebral Vasospasm Following Subarachnoid Hemorrhage.

The mechanism by which cationic polymers containing titratable amines mediate effective endosomal escape and cytosolic delivery of nucleic acids is not well understood despite the decades of research devoted to these materials. Here, we utilize multiple assays investigating the endosomal escape step associated with plasmid delivery by polyethylenimine (PEI) and poly(β-amino esters) (PBAEs) to improve the understanding of how these cationic polymers enable gene delivery. To probe the role of these materials in facilitating endosomal escape, we utilized vesicle membrane leakage and extracellular pH modulation assays to demonstrate the influence of polymer buffering capacity and effective p on the delivery of the plasmid DNA. Our results demonstrate that transfection with PBAEs is highly sensitive to the effective p of the overall polymer, which has broad implications for transfection. In more acidic environments, PBAE-mediated transfection was inhibited, while PEI was relatively unaffected. In neutral to basic environments, PBAEs have high buffering capacities that led to dramatically improved transfection efficacy. The cellular uptake of polymeric nanoparticles overall was unchanged as a function of pH, indicating that microenvironmental acidity was important for downstream intracellular delivery efficiency. Overall, this study motivates the use of polymer chemical characteristics, such as effective p values, to more efficiently evaluate new polymeric materials for enhanced intracellular delivery characteristics.

Efficiency of Cytosolic Delivery with Poly(β-amino ester) Nanoparticles is Dependent on the Effective p of the Polymer.

Immune checkpoint inhibitors such as anti-programmed cell death protein 1 (anti-PD-1) have shown promise for the treatment of cancers such as melanoma, but results for glioblastoma (GBM) have been disappointing thus far. It has been suggested that GBM has multiple mechanisms of immunosuppression, indicating a need for combinatorial treatment strategies. It is well understood that GBM increases glutamate in the tumor microenvironment (TME); however, the significance of this is not well understood. The authors posit that glutamate upregulation in the GBM TME is immunosuppressive. The authors utilized a novel glutamate modulator, BHV-4157, to determine synergy between glutamate modulation and the well-established anti-PD-1 immunotherapy for GBM.

Synergy between glutamate modulation and anti-programmed cell death protein 1 immunotherapy for glioblastoma.

Elastography is an imaging technology capable of measuring tissue stiffness and consistency. The technology has achieved widespread use in the workup and management of diseases of the liver, breast, thyroid, and prostate. Although elastography is increasingly being applied in neurosurgery, it has not yet achieved widespread adoption and many clinicians remain unfamiliar with the technology. Therefore, we sought to summarize the range of applications and elastography modalities available for neurosurgery, report its effectiveness in comparison with conventional imaging methods, and offer recommendations. All full-text English-language manuscripts on the use of elastography for neurosurgical procedures were screened using the PubMed/MEDLINE, Embase, Cochrane Library, Scopus, and Web of Science databases. Thirty-two studies were included with 990 patients, including 21 studies on intracranial tumors, 5 on hydrocephalus, 4 on epilepsy, 1 on spinal cord compression, and 1 on adolescent scoliosis. Twenty studies used ultrasound elastography (USE) whereas 12 used magnetic resonance elastography (MRE). MRE studies were mostly used in the preoperative setting for assessment of lesion stiffness, tumor-brain adherence, diagnostic workup, and operative planning. USE studies were performed intraoperatively to guide resection of lesions, determine residual microscopic abnormalities, assess the tumor-brain interface, and study mechanical properties of tumors. Elastography can assist with resection of brain tissue, detection of microscopic lesions, and workup of hydrocephalus, among other applications under investigation. Its sensitivity often exceeds that of conventional MRI and ultrasound for identifying abnormal tissue and lesion margins.

Applications of elastography in operative neurosurgery: A systematic review.

Spinal cord injury (SCI) is a devastating disease with limited effective treatment options. Animal paradigms are vital for understanding the pathogenesis of SCI and testing potential therapeutics. The porcine model of SCI is increasingly favored because of its greater similarity to humans. However, its adoption is limited by the complexities of care and range of testing parameters. Researchers need to consider swine selection, injury method, post-operative care, rehabilitation, behavioral outcomes, and histology metrics. Therefore, we systematically reviewed full-text English-language articles to evaluate study characteristics used in developing a porcine model and summarize the interventions that have been tested using this paradigm. A total of 63 studies were included, with 33 examining SCI pathogenesis and 30 testing interventions. Studies had an average sample size of 15 pigs with an average weight of 26 kg, and most used female swine with injury to the thoracic cord. Injury was most commonly induced by weight drop with compression. The porcine model is amenable to testing various interventions, including mean arterial pressure augmentation ( = 7), electrical stimulation ( = 6), stem cell therapy ( = 5), hypothermia ( = 2), biomaterials ( = 2), gene therapy ( = 2), steroids ( = 1), and nanoparticles ( = 1). It is also notable for its clinical translatability and is emerging as a valuable pre-clinical study tool. This systematic review can serve as a guideline for researchers implementing and testing the porcine SCI model.

Porcine Model of Spinal Cord Injury: A Systematic Review.

Cardiac arrest (CA) remains the leading cause of coma, and early arousal recovery indicators are needed to allocate critical care resources properly. High-frequency oscillations (HFOs) of somatosensory evoked potentials (SSEPs) have been shown to indicate responsive wakefulness days following CA. Nonetheless, their potential in the acute recovery phase, where the injury is reversible, has not been tested. We hypothesize that time-frequency (TF) analysis of HFOs can determine arousal recovery in the acute recovery phase. To test our hypothesis, eleven adult male Wistar rats were subjected to asphyxial CA (five with 3-min mild and six with 7-min moderate to severe CA) and SSEPs were recorded for 60 min post-resuscitation. Arousal level was quantified by the neurological deficit scale (NDS) at 4 h. Our results demonstrated that continuous wavelet transform (CWT) of SSEPs localizes HFOs in the TF domain under baseline conditions. The energy dispersed immediately after injury and gradually recovered. We proposed a novel TF-domain measure of HFO: the total power in the normal time-frequency space (NTFS) of HFO. We found that the NTFS power significantly separated the favorable and unfavorable outcome groups. We conclude that the NTFS power of HFOs provides earlier and objective determination of arousal recovery after CA.

Time-Frequency Analysis of Somatosensory Evoked High-Frequency (600 Hz) Oscillations as an Early Indicator of Arousal Recovery after Hypoxic-Ischemic Brain Injury.

Article	Year
Continuous microfluidic assembly of biodegradable poly(beta-amino ester)/DNA nanoparticles for enhanced gene delivery. Journal of biomedical materials research. Part A\| PubMed ID: 28177587	2017
A Triple-Fluorophore-Labeled Nucleic Acid pH Nanosensor to Investigate Non-viral Gene Delivery. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 28479046	2017
Differentially Branched Ester Amine Quadpolymers with Amphiphilic and pH-Sensitive Properties for Efficient Plasmid DNA Delivery. Molecular pharmaceutics\| PubMed ID: 30615464	2019
Low-dose oncolytic adenovirus therapy overcomes tumor-induced immune suppression and sensitizes intracranial gliomas to anti-PD-1 therapy. Neuro-oncology advances\| PubMed ID: 32642679	2020
PD-1+ Monocytes Mediate Cerebral Vasospasm Following Subarachnoid Hemorrhage. Neurosurgery\| PubMed ID: 33370819	2021
Efficiency of Cytosolic Delivery with Poly(β-amino ester) Nanoparticles is Dependent on the Effective p of the Polymer. ACS biomaterials science & engineering\| PubMed ID: 33463158	2020
Synergy between glutamate modulation and anti-programmed cell death protein 1 immunotherapy for glioblastoma. Journal of neurosurgery\| PubMed ID: 34388730	2022
Applications of elastography in operative neurosurgery: A systematic review. Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia\| PubMed ID: 35933785
Porcine Model of Spinal Cord Injury: A Systematic Review. Neurotrauma reports\| PubMed ID: 36204385	2022
Time-Frequency Analysis of Somatosensory Evoked High-Frequency (600 Hz) Oscillations as an Early Indicator of Arousal Recovery after Hypoxic-Ischemic Brain Injury. Brain sciences\| PubMed ID: 36671984	2022

Denis Routkevitch

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