Strategies for enhanced gene delivery to the central nervous system.

The delivery of genes to the central nervous system (CNS) has been a persistent challenge due to various biological barriers. The blood-brain barrier (BBB), in particular, hampers the access of systemically injected drugs to parenchymal cells, allowing only a minimal percentage (<1%) to pass through. Recent scientific insights highlight the crucial role of the extracellular space (ECS) in governing drug diffusion. Taking into account advancements in vectors, techniques, and knowledge, the discussion will center on the most notable vectors utilized for gene delivery to the CNS. This review will explore the influence of the ECS - a dynamically regulated barrier-on drug diffusion. Furthermore, we will underscore the significance of employing remote-control technologies to facilitate BBB traversal and modulate the ECS. Given the rapid progress in gene editing, our discussion will also encompass the latest advances focused on delivering therapeutic editing in vivo to the CNS tissue. In the end, a brief summary on the impact of Artificial Intelligence (AI)/Machine Learning (ML), ultrasmall, soft endovascular robots, and high-resolution endovascular cameras on improving the gene delivery to the CNS will be provided.

Tarlov Cysts and Premature Ejaculation.

Tarlov cysts adjacent to the spinal cord are usually asymptomatic and found incidentally via magnetic resonance imaging. On rare occasions, they increase in size to produce symptoms resembling disk herniation. We report a rare case of a sacral cyst resulting in premature ejaculation in a 32-year-old man who presented with pelvic pain and acquired premature ejaculation. Spinal nerve root decompression, excision of intraspinal Tarlov cyst, and spinal nerve root adhesion release surgery significantly improved his pain and premature ejaculation at a six-month follow-up.

Clinical gene therapy development for the central nervous system: Candidates and challenges for AAVs.

Many diseases affecting the central nervous system (CNS) are deadly but less understood, leading to impaired mental and motor capabilities and poor patient prospects. Gene therapy is a promising therapeutic modality for correcting many genetic disorders, expanding in breadth and scope with further advances. This review summarizes the candidate CNS disorders for gene therapy, mechanisms of gene therapy, and recent clinical advances and limitations of gene therapy in CNS disorders. We highlight that improving delivery across CNS barriers, safety, monitoring techniques, and multiplexing therapies are predominant factors in advancing long-term outcomes from gene therapy.

Integrated Imaging Methodology Detects Claudin-1 Expression in Premalignant Nonpolypoid and Polypoid Colonic Epithelium in Mice.

OBJECTIVES: Conventional colonoscopy with white light illumination detects colonic adenomas based on structural changes alone and is limited by a high miss rate. We aim to demonstrate an integrated imaging strategy that combines wide-field endoscopy and confocal endomicroscopy in real time to visualize molecular expression patterns in vivo to detect premalignant colonic mucosa. METHODS: A peptide specific for claudin-1 is labeled with Cy5.5 and administrated intravenously in genetically engineered mice that develop adenomas spontaneously in the distal colon. Wide-field endoscopy is used to identify the presence of nonpolypoid and polypoid adenomas. Anatomic landmarks are used to guide placement of a confocal endomicroscope with side-view optics to visualize claudin-1 expression patterns with subcellular resolution. RESULTS: Wide-field fluorescence images show peak uptake in colon adenoma at ∼1 hour after systemic peptide administration, and lesion margins are clearly defined. Further examination of the lesion using a confocal endomicroscope shows dysplastic crypts with large size, elongated shape, distorted architecture, and variable dimension compared with normal. The mean fluorescence intensity is significantly higher for dysplasia than normal. Increased claudin-1 expression in dysplasia vs normal is confirmed ex vivo, and the binding pattern is consistent with the in vivo imaging results. DISCUSSION: Wide-field endoscopy can visualize molecular expression of claudin-1 in vivo to localize premalignant colonic mucosa, and confocal endomicroscopy can identify subcellular feature to distinguish dysplasia from normal.

Dual-modal in vivo fluorescence and photoacoustic imaging using a heterodimeric peptide.

A heterodimeric peptide labeled with IRDye800 is used to perform dual-modal imaging of human esophageal xenograft tumors in vivo. Fluorescence and photoacoustic images provide complementary visualization of tumor dimensions in planar and sagittal views, respectively, demonstrating promise for targeted cancer diagnosis and staging.

Multiplexed Targeting of Barrett's Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft in Vivo and Human Specimens ex Vivo.

Esophageal adenocarcinoma (EAC) is a molecularly heterogeneous disease that is rising rapidly in incidence and has poor prognosis. We developed a heterobivalent peptide to target detection of early Barrett's neoplasia by combining monomer heptapeptides specific for either EGFR or ErbB2 in a heterodimer configuration. The structure of a triethylene glycol linker was optimized to maximize binding interactions to the surface receptors on cells. The Cy5.5-labeled heterodimer QRH*-KSP*-E3-Cy5.5 demonstrated specific binding to each target and showed 3-fold greater fluorescence intensity and 2-fold higher affinity compared with those of either monomer alone. Peak uptake in xenograft tumors was observed at 2 h postinjection with systemic clearance by ∼24 h in vivo. Furthermore, ligand binding was evaluated on human esophageal specimens ex vivo, and 88% sensitivity and 87% specificity were found for the detection of either high-grade dysplasia (HGD) or EAC. This peptide heterodimer shows promise for targeted detection of early Barrett's neoplasia in clinical study.

Ultrashort Carbon Nanotubes That Fluoresce Brightly in the Near-Infrared.

The intrinsic near-infrared photoluminescence observed in long single-walled carbon nanotubes is known to be quenched in ultrashort nanotubes due to their tiny size as compared to the exciton diffusion length in these materials (>100 nm). Here, we show that intense photoluminescence can be created in ultrashort nanotubes (∼40 nm length) upon incorporation of exciton-trapping sp3 defect sites. Using super-resolution photoluminescence imaging at <25 nm resolution, we directly show the preferential localization of excitons at the nanotube ends, which separate by less than 40 nm and behave as independent emitters. This unexpected observation opens the possibility to synthesize fluorescent ultrashort nanotubes-a goal that has been long thought impossible-for bioimaging applications, where bright near-infrared photoluminescence and small size are highly desirable, and for quantum information science, where high quality and well-controlled near-infrared single photon emitters are needed.

In vivo near-infrared imaging of ErbB2 expressing breast tumors with dual-axes confocal endomicroscopy using a targeted peptide.

ErbB2 expression in early breast cancer can predict tumor aggressiveness and clinical outcomes in large patient populations. Accurate assessment with physical biopsy and conventional pathology can be limited by tumor heterogeneity. We aim to demonstrate real-time optical sectioning using a near-infrared labeled ErbB2 peptide that generates tumor-specific contrast in human xenograft breast tumors in vivo. We used IRDye800CW as the fluorophore, validated performance characteristics for specific peptide binding to cells in vitro, and investigated peak peptide uptake in tumors using photoacoustic tomography. We performed real-time optical imaging using a handheld dual-axes confocal fluorescence endomicroscope that collects light off-axis to reduce tissue scattering for greater imaging depths. Optical sections in either the vertical or horizontal plane were collected with sub-cellular resolution. Also, we found significantly greater peptide binding to pre-clinical xenograft breast cancer in vivo and to human specimens of invasive ductal carcinoma that express ErbB2 ex vivo. We used a scrambled peptide for control. Peptide biodistribution showed high tumor uptake by comparison with other organs to support safety. This novel integrated imaging strategy is promising for visualizing ErbB2 expression in breast tumors and serve as an adjunct during surgery to improve diagnostic accuracy, identify tumor margins, and stage early cancers.

Evaluation of Different Single-Walled Carbon Nanotube Surface Coatings for Single-Particle Tracking Applications in Biological Environments.

Fluorescence imaging of biological systems down to the single-molecule level has generated many advances in cellular biology. For applications within intact tissue, single-walled carbon nanotubes (SWCNTs) are emerging as distinctive single-molecule nanoprobes, due to their near-infrared photoluminescence properties. For this, SWCNT surfaces must be coated using adequate molecular moieties. Yet, the choice of the suspension agent is critical since it influences both the chemical and emission properties of the SWCNTs within their environment. Here, we compare the most commonly used surface coatings for encapsulating photoluminescent SWCNTs in the context of bio-imaging applications. To be applied as single-molecule nanoprobes, encapsulated nanotubes should display low cytotoxicity, and minimal unspecific interactions with cells while still being highly luminescent so as to be imaged and tracked down to the single nanotube level for long periods of time. We tested the cell proliferation and cellular viability of each surface coating and evaluated the impact of the biocompatible surface coatings on nanotube photoluminescence brightness. Our study establishes that phospholipid-polyethylene glycol-coated carbon nanotube is the best current choice for single nanotube tracking experiments in live biological samples.

Single-nanotube tracking reveals the nanoscale organization of the extracellular space in the live brain.

The brain is a dynamic structure with the extracellular space (ECS) taking up almost a quarter of its volume. Signalling molecules, neurotransmitters and nutrients transit via the ECS, which constitutes a key microenvironment for cellular communication and the clearance of toxic metabolites. The spatial organization of the ECS varies during sleep, development and aging and is probably altered in neuropsychiatric and degenerative diseases, as inferred from electron microscopy and macroscopic biophysical investigations. Here we show an approach to directly observe the local ECS structures and rheology in brain tissue using super-resolution imaging. We inject single-walled carbon nanotubes into rat cerebroventricles and follow the near-infrared emission of individual nanotubes as they diffuse inside the ECS for tens of minutes in acute slices. Because of the interplay between the nanotube geometry and the ECS local environment, we can extract information about the dimensions and local viscosity of the ECS. We find a striking diversity of ECS dimensions down to 40 nm, and as well as of local viscosity values. Moreover, by chemically altering the extracellular matrix of the brains of live animals before nanotube injection, we reveal that the rheological properties of the ECS are affected, but these alterations are local and inhomogeneous at the nanoscale.

Detection of Sessile Serrated Adenomas in the Proximal Colon Using Wide-Field Fluorescence Endoscopy.

BACKGROUND & AIMS: Many cancers in the proximal colon develop via from sessile serrated adenomas (SSAs), which have flat, subtle features that are difficult to detect with conventional white-light colonoscopy. Many SSA cells have the V600E mutation in BRAF. We investigated whether this feature could be used with imaging methods to detect SSAs in patients. METHODS: We used phage display to identify a peptide that binds specifically to SSAs, using subtractive hybridization with HT29 colorectal cancer cells containing the V600E mutation in BRAF and Hs738.St/Int cells as a control. Binding of fluorescently labeled peptide to colorectal cancer cells was evaluated with confocal fluorescence microscopy. Rats received intra-colonic 0.0086 mg/kg, 0.026 mg/kg, or 0.86 mg/kg peptide or vehicle and morbidity, mortality, and injury were monitored twice daily to assess toxicity. In the clinical safety study, fluorescently labeled peptide was topically administered, using a spray catheter, to the proximal colon of 25 subjects undergoing routine outpatient colonoscopies (3 subjects were given 2.25 µmol/L and 22 patients were given 76.4 µmol/L). We performed blood cell count, chemistry, liver function, and urine analyses approximately 24 hours after peptide administration. In the clinical imaging study, 38 subjects undergoing routine outpatient colonoscopies, at high risk for colorectal cancer, or with a suspected unresected proximal colonic polyp, were first evaluated by white-light endoscopy to identify suspicious regions. The fluorescently labeled peptide (76.4 µmol/L) was administered topically to proximal colon, unbound peptide was washed away, and white-light, reflectance, and fluorescence videos were recorded digitally. Fluorescence intensities of SSAs were compared with those of normal colonic mucosa. Endoscopists resected identified lesions, which were analyzed histologically by gastrointestinal pathologists (reference standard). We also analyzed the ability of the peptide to identify SSAs vs adenomas, hyperplastic polyps, and normal colonic mucosa in specimens obtained from the tissue bank at the University of Michigan. RESULTS: We identified the peptide sequence KCCFPAQ and measured an apparent dissociation constant of Kd = 72 nM and an apparent association time constant of K = 0.174 min-1 (5.76 minutes). During fluorescence imaging of patients during endoscopy, regions of SSA had 2.43-fold higher mean fluorescence intensity than that for normal colonic mucosa. Fluorescence labeling distinguished SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity. The peptide had no observed toxic effects in animals or patients. In the analysis of ex vivo specimens, peptide bound to SSAs had significantly higher mean fluorescence intensity than to hyperplastic polyps. CONCLUSIONS: We have identified a fluorescently labeled peptide that has no observed toxic effects in animals or humans and can be used for wide-field imaging of lesions in the proximal colon. It distinguishes SSAs from normal colonic mucosa with 89% sensitivity and 92% specificity. This targeted imaging method might be used in early detection of premalignant serrated lesions during routine colonoscopies. ClinicalTrials.gov ID: NCT02156557.

Toward the suppression of cellular toxicity from single-walled carbon nanotubes.

In the multidisciplinary fields of nanobiology and nanomedicine, single-walled carbon nanotubes (SWCNTs) have shown great promise due to their unique morphological, physical and chemical properties. However, understanding and suppressing their cellular toxicity is a mandatory step before promoting their biomedical applications. In light of the flourishing recent literature, we provide here an extensive review on SWCNT cellular toxicity and an attempt to identify the key parameters to be considered in order to obtain SWCNT samples with minimal or no cellular toxicity.

Optical detection of individual ultra-short carbon nanotubes enables their length characterization down to 10 nm.

Ultrashort single-walled carbon nanotubes, i.e. with length below ~30 nm, display length-dependent physical, chemical and biological properties that are attractive for the development of novel nanodevices and nanomaterials. Whether fundamental or applicative, such developments require that ultrashort nanotube lengths can be routinely and reliably characterized with high statistical data for high-quality sample production. However, no methods currently fulfill these requirements. Here, we demonstrate that photothermal microscopy achieves fast and reliable optical single nanotube analysis down to ~10 nm lengths. Compared to atomic force microscopy, this method provides ultrashort nanotubes length distribution with high statistics, and neither requires specific sample preparation nor tip-dependent image analysis.

Single-molecule imaging in live cell using gold nanoparticles.

Optimal single particle tracking experiments in live cells requires small and photostable probes, which do not modify the behavior of the molecule of interest. Current fluorescence-based microscopy of single molecules and nanoparticles is often limited by bleaching and blinking or by the probe size. As an alternative, we present in this chapter the synthesis of a small and highly specific gold nanoprobe whose detection is based on its absorption properties. We first present a protocol to synthesize 5-nm-diameter gold nanoparticles and functionalize them with a nanobody, a single-domain antibody from camelid, targeting the widespread green fluorescent protein (GFP)-tagged proteins with a high affinity. Then we describe how to detect and track these individual gold nanoparticles in live cell using photothermal imaging microscopy. The combination of a probe with small size, perfect photostability, high specificity, and versatility through the vast existing library of GFP-proteins, with a highly sensitive detection technique enables long-term tracking of proteins with minimal hindrance in confined and crowded environments such as intracellular space.

Nonlinear photoluminescence spectroscopy of carbon nanotubes with localized exciton states.

We report distinctive nonlinear behavior of photoluminescence (PL) intensities from localized exciton states embedded in single-walled carbon nanotubes (SWNTs) at room temperature. We found that PL from the local states exhibits strong nonlinear behavior with increasing continuous-wave excitation power density, whereas free exciton PL shows only weak sublinear behavior. The strong nonlinear behavior was observed regardless of the origin of the local states and found to be nearly independent of the local state density. These results indicate that the strong PL nonlinearity arises from a universal mechanism to SWNTs with sparse local states. The significant nonlinear PL is attributed to rapid ground-state depletion of the local states caused by an efficient accumulation of photogenerated free excitons into the sparse local states through one-dimensional diffusional migration of excitons along the nanotube axis; this mechanism is verified by Monte Carlo simulations of exciton diffusion dynamics.

Noncovalent Functionalization of Boron Nitride Nanotubes in Aqueous Media Opens Application Roads in Nanobiomedicine.

Boron nitride nanotubes (BNNTs) are of intense scientific interest due to their unique physiochemical properties and prospective applications in various nanotechnologies, particularly nanobiomedicine. A critical problem hampering the application processing of BNNTs is the outer sidewall functionalization, which is primarily acquired to lead BNNTs dispersible in various solvents. Furthermore, the surface of BNNTs should be intelligently designed and precisely controlled to satisfy the specific demands of different applications. For these purposes, covalent and noncovalent approaches have been factually developed to help to extend the full potential of applications. Importantly, wrapping the outermost sidewall of BNNTs with either water-soluble polymers or biomolecules through weak noncovalent interactions has been proved to be efficient for giving BNNTs considerable dispersity in aqueous media, and endowing novel chemical functions to BNNTs with almost no change in their pristine physiochemical properties. This article summarizes recent progress in this field and addresses future perspectives on the noncovalent functionalization of BNNTs for promoting their application processing in various bio-related nanotechnologies.

Noncovalent functionalization of disentangled boron nitride nanotubes with flavin mononucleotides for strong and stable visible-light emission in aqueous solution.

Strong and stable visible-light-emitting boron nitride nanotube (BNNT)/biomolecule nanohybrids were successfully fabricated via noncovalent functionalization of BNNTs with flavin mononucleotides (FMN). Atomic force microscopy showed excellent dispersion of the nanohybrids in aqueous solution. Infrared absorption spectroscopy revealed strong π-π stacking interactions between FMN and BNNT sidewalls. Importantly, the fluorescence spectra revealed that the nanohybrids were highly fluorescent in the visible-light spectral range. Moreover, this fluorescence had unique pH-dependent and thermally stable properties. These nanohybrids might be used to construct novel fluorescence imaging probes that function over a wide pH and temperature range.

Isolation of individual boron nitride nanotubes via peptide wrapping.

The isolation of individual boron nitride nanotubes (BNNTs) in aqueous phases has been achieved for the first time from raw materials based on the combination of peptide wrapping with a sonication procedure. Atomic force microscopic observations revealed the representative height and length of individual BNNTs. Fluorescence and infrared absorption spectra suggested the strong pi-pi interactions between BNNTs and the peptide. The absorption maxima of BNNTs were significantly blue-shifted from 200 nm for the original BNNTs to 193 nm. The modulation of the BNNT band gap with peptide wrapping promises potential applications of the peptide/BNNT complexes to various nanotechnologies.