Seizure-induced neutrophil adhesion in brain capillaries leads to a decrease in postictal cerebral blood flow.

Cerebral hypoperfusion has been proposed as a potential cause of postictal neurological dysfunction in epilepsy, but its underlying mechanism is still unclear. We show that a 30% reduction in postictal cerebral blood flow (CBF) has two contributing factors: the early hypoperfusion up to ∼30 min post-seizure was mainly induced by arteriolar constriction, while the hypoperfusion that persisted for over an hour was due to increased capillary stalling induced by neutrophil adhesion to brain capillaries, decreased red blood cell (RBC) flow accompanied by constriction of capillaries and venules, and elevated intercellular adhesion molecule-1 (ICAM-1) expression. Administration of antibodies against the neutrophil marker Ly6G and against LFA-1, which mediates adhesive interactions with ICAM-1, prevented neutrophil adhesion and recovered the prolonged CBF reductions to control levels. Our findings provide evidence that seizure-induced neutrophil adhesion to cerebral microvessels via ICAM-1 leads to prolonged postictal hypoperfusion, which may underlie neurological dysfunction in epilepsy.

Parvalbumin interneuron activity drives fast inhibition-induced vasoconstriction followed by slow substance P-mediated vasodilation.

The role of parvalbumin (PV) interneurons in vascular control is poorly understood. Here, we investigated the hemodynamic responses elicited by optogenetic stimulation of PV interneurons using electrophysiology, functional magnetic resonance imaging (fMRI), wide-field optical imaging (OIS), and pharmacological applications. As a control, forepaw stimulation was used. Stimulation of PV interneurons in the somatosensory cortex evoked a biphasic fMRI response in the photostimulation site and negative fMRI signals in projection regions. Activation of PV neurons engaged two separable neurovascular mechanisms in the stimulation site. First, an early vasoconstrictive response caused by the PV-driven inhibition is sensitive to the brain state affected by anesthesia or wakefulness. Second, a later ultraslow vasodilation lasting a minute is closely dependent on the sum of interneuron multiunit activities, but is not due to increased metabolism, neural or vascular rebound, or increased glial activity. The ultraslow response is mediated by neuropeptide substance P (SP) released from PV neurons under anesthesia, but disappears during wakefulness, suggesting that SP signaling is important for vascular regulation during sleep. Our findings provide a comprehensive perspective about the role of PV neurons in controlling the vascular response.

Micro-ultrasonic Assessment of Early Stage Clot Formation and Whole Blood Coagulation Using an All-Optical Ultrasound Transducer and Adaptive Signal Processing Algorithm.

Abnormal formation of solid thrombus inside a blood vessel can cause thrombotic morbidity and mortality. This necessitates early stage diagnosis, which requires quantitative assessment with a small volume, for effective therapy with low risk to unwanted development of various diseases. We propose a micro-ultrasonic diagnosis using an all-optical ultrasound-based spectral sensing (AOUSS) technique for sensitive and quantitative characterization of early stage and whole blood coagulation. The AOUSS technique detects and analyzes minute viscoelastic variations of blood at a micro-ultrasonic spot (<100 µm) defined by laser-generated focused ultrasound (LGFU). This utilizes (1) a uniquely designed optical transducer configuration for frequency-spectral matching and wideband operation (6 dB widths: 7-32 MHz and d.c. ∼ 46 MHz, respectively) and (2) an empirical mode decomposition (EMD)-based signal process particularly adapted to nonstationary LGFU signals backscattered from the spot. An EMD-derived spectral analysis enables one to assess viscoelastic variations during the initiation of fibrin formation, which occurs at a very early stage of blood coagulation (1 min) with high sensitivity (frequency transition per storage modulus increment = 8.81 MHz/MPa). Our results exhibit strong agreement with those obtained by conventional rheometry (Pearson's R > 0.95), which are also confirmed by optical microscopy. The micro-ultrasonic and high-sensitivity detection of AOUSS poses a potential clinical significance, serving as a screening modality to diagnose early stage clot formation (e.g., as an indicator for hypercoagulation of blood) and stages of blood-to-clot transition to check a potential risk for development into thrombotic diseases.

Cerebrospinal Fluid-philic and Biocompatibility-Enhanced Soft Cranial Window for Long-Term In Vivo Brain Imaging.

Soft, transparent poly(dimethyl siloxane) (PDMS)-based cranial windows in animal models have created many opportunities to investigate brain functions with multiple in vivo imaging modalities. However, due to the hydrophobic nature of PDMS, the wettability by cerebrospinal fluid (CSF) is poor, which may cause air bubble trapping beneath the window during implantation surgery, and favorable heterogeneous bubble nucleation at the interface between hydrophobic PDMS and CSF. This may result in excessive growth of the entrapped bubble under the soft cranial window. Herein, to yield biocompatibility-enhanced, trapped bubble-minimized, and soft cranial windows, this report introduces a CSF-philic PDMS window coated with hydroxyl-enriched poly(vinyl alcohol) (PVA) for long-term in vivo imaging. The PVA-coated PDMS (PVA/PDMS) film exhibits a low contact angle θACA (33.7 ± 1.9°) with artificial CSF solution and maintains sustained CSF-philicity. The presence of the PVA layer achieves air bubble-free implantation of the soft cranial window, as well as induces the formation of a thin wetting film that shows anti-biofouling performance through abundant water molecules on the surface, leading to long-term optical clarity. In vivo studies on the mice cortex verify that the soft and CSF-philic features of the PVA/PDMS film provide minimal damage to neuronal tissues and attenuate immune response. These advantages of the PVA/PDMS window are strongly correlated with the enhancement of cortical hemodynamic changes and the local field potential recorded through the PVA/PDMS film, respectively. This collection of results demonstrates the potential for future microfluidic platforms for minimally invasive CSF extraction utilizing a CSF-philic fluidic passage.

Deep brain stimulation of the anterior nuclei of the thalamus can alleviate seizure severity and induce hippocampal GABAergic neuronal changes in a pilocarpine-induced epileptic mouse brain.

Deep brain stimulation (DBS) of the anterior nucleus of the thalamus (ANT) has been widely used as an effective treatment for refractory temporal lobe epilepsy. Despite its promising clinical outcome, the exact mechanism of how ANT-DBS alleviates seizure severity has not been fully understood, especially at the cellular level. To assess effects of DBS, the present study examined electroencephalography (EEG) signals and locomotor behavior changes and conducted immunohistochemical analyses to examine changes in neuronal activity, number of neurons, and neurogenesis of inhibitory neurons in different hippocampal subregions. ANT-DBS alleviated seizure activity, abnormal locomotor behaviors, reduced theta-band, increased gamma-band EEG power in the interictal state, and increased the number of neurons in the dentate gyrus (DG). The number of parvalbumin- and somatostatin-expressing inhibitory neurons was recovered to the level in DG and CA1 of naïve mice. Notably, BrdU-positive inhibitory neurons were increased. In conclusion, ANT-DBS not only could reduce the number of seizures, but also could induce neuronal changes in the hippocampus, which is a key region involved in chronic epileptogenesis. Importantly, our results suggest that ANT-DBS may lead to hippocampal subregion-specific cellular recovery of GABAergic inhibitory neurons.

Development of an Electrochemical Dual H2S/Ca2+ Microsensor and Its In Vivo Application to a Rat Seizure Model.

A dual electrochemical microsensor was fabricated for concurrent monitoring of hydrogen sulfide (H2S) and calcium ions (Ca2+), which are closely linked important signaling species involved in various physiological processes. The dual sensor was prepared using a dual recessed electrode consisting of two platinum (Pt) microdisks (50 µm in diameter). Each electrode was individually optimized for the best sensing ability toward a target analyte. One electrode (WE1, amperometric H2S sensor) was modified with electrodeposition of Au and electropolymerized polyaniline coating. The other electrode (WE2, all-solid-state Ca2+-selective electrode) was composed of Ag/AgCl onto the recessed Pt disk formed via electrodeposition/chloridation, followed by silanization and Ca2+-selective membrane loading. The current of WE1 and the potential of WE2 in a dual sensor responded linearly to H2S concentration and logarithm of Ca2+ concentration, respectively, without a crosstalk between the sensing signals. Both WE1 and WE2 presented excellent sensitivity, selectivity (log⁡KH2S,iAmp≤-3.5, i = CO, NO, O2, NO2-, AP, AA, DA, and GABA; and log⁡KCa2+,jPot≤-3.2, j = Na+, K+, and Mg2+), and fast response time with reasonable stability (during ca. 6 h in vivo experiment). Particularly, WE2 prepared using a mixture of two ionophores (ETH1001 and ETH129) and two plasticizers (2-nitrophenyl octyl ether and bis(2-ethylhexyl) sebacate) showed a very shortened response time (tR to attain the ΔE/Δt slope of 0.6 mV/min = 3.0 ± 0.2 s, n ≥ 10), a critically required factor for real-time analysis. The developed sensor was utilized for simultaneous real-time monitoring of H2S and Ca2+ changes at the brain cortex surface of a living rat during spontaneous epileptic seizures induced by a cortical 4-aminopyridine injection. The dynamic changes of H2S and Ca2+ were clearly observed in an intimate correlation with the electrophysiological recording of seizures, demonstrating the sensor feasibility of in vivo and real-time simultaneous measurements of H2S and Ca2+.

Orthopedic surgery-induced cognitive dysfunction is mediated by CX3CL1/R1 signaling.

BACKGROUND: Postoperative pain is a common phenomenon after surgery and is closely associated with the development of postoperative cognitive dysfunction (POCD). Persistent pain and systemic inflammation caused by surgery have been suggested as key factors for the development of POCD. Fractalkine (CX3CL1) and its receptor, the CX3C chemokine receptor 1 (CX3CR1), are known to play a key role in pain and inflammation signaling pathways. Recent studies have shown that the regulation of CX3CR1/L1 signaling influences the development of various diseases including neuronal diseases. We determined whether CX3CR1/L1 signaling is a putative therapeutic target for POCD in a mouse model. METHODS: Adult (9-11 weeks) male mice were treated with neutralizing antibody to block CX3CR1/L1 signaling both before and after surgery. Inflammatory and behavioral responses including pain were assessed postoperatively. Also, CX3CR1 mRNA level was assessed. Hippocampal astrocyte activation, Mao B expression, and GABA expression were assessed at 2 days after surgery following neutralizing antibody administration. RESULTS: The behavioral response indicated cognitive dysfunction and development of pain in the surgery group compared with the control group. Also, increased levels of pro-inflammatory cytokines and CX3CR1 mRNA were observed in the surgery group. In addition, increased levels of GABA and increased Mao B expression were observed in reactive astrocytes in the surgery group; these responses were attenuated by neutralizing antibody administration. CONCLUSIONS: Increased CX3CR1 after surgery is both necessary and sufficient to induce cognitive dysfunction. CX3CR1 could be an important target for therapeutic strategies to prevent the development of POCD.

Differential contribution of excitatory and inhibitory neurons in shaping neurovascular coupling in different epileptic neural states.

Understanding the neurovascular coupling (NVC) underlying hemodynamic changes in epilepsy is crucial to properly interpreting functional brain imaging signals associated with epileptic events. However, how excitatory and inhibitory neurons affect vascular responses in different epileptic states remains unknown. We conducted real-time in vivo measurements of cerebral blood flow (CBF), vessel diameter, and excitatory and inhibitory neuronal calcium signals during recurrent focal seizures. During preictal states, decreases in CBF and arteriole diameter were closely related to decreased γ-band local field potential (LFP) power, which was linked to relatively elevated excitatory and reduced inhibitory neuronal activity levels. Notably, this preictal condition was followed by a strengthened ictal event. In particular, the preictal inhibitory activity level was positively correlated with coherent oscillating activity specific to inhibitory neurons. In contrast, ictal states were characterized by elevated synchrony in excitatory neurons. Given these findings, we suggest that excitatory and inhibitory neurons differentially contribute to shaping the ictal and preictal neural states, respectively. Moreover, the preictal vascular activity, alongside with the γ-band, may reflect the relative levels of excitatory and inhibitory neuronal activity, and upcoming ictal activity. Our findings provide useful insights into how perfusion signals of different epileptic states are related in terms of NVC.

Excitation-Inhibition Imbalance Leads to Alteration of Neuronal Coherence and Neurovascular Coupling under Acute Stress.

A single stressful event can cause morphologic and functional changes in neurons and even malfunction of vascular systems, which can lead to acute stress disorder or post-traumatic stress disorder. However, there is a lack of evidence regarding how acute stress impacts neuronal activity, the concurrent vascular response, and the relationship between these two factors, which is defined as neurovascular coupling. Here, using in vivo two-photon imaging, we found that NMDA-evoked calcium transients of excitatory neurons were impaired and that vasodilation of penetrating arterioles was concomitantly disrupted in acutely stressed male mice. Furthermore, acute stress altered the relationship between excitatory neuronal calcium coherence and vascular responses. By measuring NMDA-evoked excitatory and inhibitory neuronal calcium activity in acute brain slices, we confirmed that neuronal coherence both between excitatory neurons and between excitatory and inhibitory neurons was reduced by acute stress but restored by blockade of glucocorticoid receptor signaling. Furthermore, the ratio of sEPSCs to sIPSCs was altered by acute stress, suggesting that the excitation-inhibition balance was disrupted by acute stress. In summary, in vivo, ex vivo, and whole-cell recording studies demonstrate that acute stress modifies excitatory-inhibitory neuronal coherence, disrupts the excitation-inhibition balance, and causes consequent neurovascular coupling changes, providing critical insights into the neural mechanism of stress-induced disorders.SIGNIFICANCE STATEMENT Acute stress can cause pathologic conditions, such as acute stress disorder and post-traumatic stress disorder, by affecting the functions of neurons and blood vessels. However, investigations into the impacts of acute stress on neurovascular coupling, the tight connection between local neural activity and subsequent blood flow changes, are lacking. Through investigations at the in vivo, ex vivo, and whole-cell recording levels, we found that acute stress alters the NMDA-evoked vascular response, impairs the function and coherence of excitatory and inhibitory neurons, and disrupts the excitatory and inhibitory balance. These novel findings provide insights into the relevance of the excitatory-inhibitory balance, neuronal coherence, and neurovascular coupling to stress-induced disorders.

Supramolecular Peptide Hydrogel-Based Soft Neural Interface Augments Brain Signals through a Three-Dimensional Electrical Network.

Recording neural activity from the living brain is of great interest in neuroscience for interpreting cognitive processing or neurological disorders. Despite recent advances in neural technologies, development of a soft neural interface that integrates with neural tissues, increases recording sensitivity, and prevents signal dissipation still remains a major challenge. Here, we introduce a biocompatible, conductive, and biostable neural interface, a supramolecular ß-peptide-based hydrogel that allows signal amplification via tight neural/hydrogel contact without neuroinflammation. The non-biodegradable ß-peptide forms a multihierarchical structure with conductive nanomaterial, creating a three-dimensional electrical network, which can augment brain signal efficiently. By achieving seamless integration in brain tissue with increased contact area and tight neural tissue coupling, the epidural and intracortical neural signals recorded with the hydrogel were augmented, especially in the high frequency range. Overall, our tissuelike chronic neural interface will facilitate a deeper understanding of brain oscillation in broad brain states and further lead to more efficient brain-computer interfaces.

A Soft Housing Needle Ultrasonic Transducer for Focal Stimulation to Small Animal Brain.

Conventional acoustic brain stimulators that transmit low frequency (< 1 MHz) bursts in a pulse repetition frequency with large-sized transducers are barely compatible with small animal models because of broad beam width, possible stimulation of auditory pathways, and blocking of field-of-view for in vivo imaging of brain hemodynamics and neuronal activities. A miniaturized ultrasound stimulator with higher stimulation frequencies will enhance spatial specificity and enable simultaneous eliciting and monitoring brain activities. Moreover, the use of non-periodic pulse sequences may reduce unintended stimulations on auditory cortex, which might be caused by transmitting periodic bursting patterns. A platform for ultrasound brain stimulations for small animal models, including a soft housing 10 MHz needle transducer with a beam size of 680 µm, random transmission sequences, and optical imaging systems, was developed. The platform can deliver focal stimulations to the visual and barrel cortex of mice and monitor subsequent brain activities. The stimulated sites in both the visual and primary somatosensory cortices (S1) showed approximately two to three times higher neuronal calcium signal levels than those in peripheral regions. Activities in the auditory cortex were elicited by periodic sequence stimulation, while it was reduced by 67 and 35% for barrel and visual cortex stimulation with the random sequence, respectively.

Neurovascular Coupling under Chronic Stress Is Modified by Altered GABAergic Interneuron Activity.

Neurovascular coupling (NVC), the interaction between neural activity and vascular response, ensures normal brain function by maintaining brain homeostasis. We previously reported altered cerebrovascular responses during functional hyperemia in chronically stressed animals. However, the underlying neuronal-level changes associated with those hemodynamic changes remained unclear. Here, using in vivo and ex vivo experiments, we investigate the neuronal origins of altered NVC dynamics under chronic stress conditions in adult male mice. Stimulus-evoked hemodynamic and neural responses, especially beta and gamma-band local field potential activity, were significantly lower in chronically stressed animals, and the NVC relationship, itself, had changed. Further, using acute brain slices, we discovered that the underlying cause of this change was dysfunction of neuronal nitric oxide synthase (nNOS)-mediated vascular responses. Using FISH to check the mRNA expression of several GABAergic subtypes, we confirmed that only nNOS mRNA was significantly decreased in chronically stressed mice. Ultimately, chronic stress impairs NVC by diminishing nNOS-mediated vasodilation responses to local neural activity. Overall, these findings provide useful information in understanding NVC dynamics in the healthy brain. More importantly, this study reveals that impaired nNOS-mediated NVC function may be a contributory factor in the progression of stress-related diseases.SIGNIFICANCE STATEMENT The correlation between neuronal activity and cerebral vascular dynamics is defined as neurovascular coupling (NVC), which plays an important role for meeting the metabolic demands of the brain. However, the impact of chronic stress, which is a contributory factor of many cerebrovascular diseases, on NVC is poorly understood. We therefore investigated the effects of chronic stress on impaired neurovascular response to sensory stimulation and their underlying mechanisms. Multimodal approaches, from in vivo hemodynamic imaging and electrophysiology to ex vivo vascular imaging with pharmacological treatment, patch-clamp recording, FISH, and immunohistochemistry revealed that chronic stress-induced dysfunction of nNOS-expressing interneurons contributes to NVC impairment. These findings will provide useful information to understand the role of nNOS interneurons in NVC in normal and pathological conditions.

Lipopeptide-Based Nanosome-Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing.

A transient cytosolic delivery system for accurate Cas9 ribonucleoprotein is a key factor for target specificity of the CRIPSR/Cas9 toolkit. Owing to the large size of the Cas9 protein and a long negative strand RNA, the development of the delivery system is still a major challenge. Here, a size-controlled lipopeptide-based nanosome system is reported, derived from the blood-brain barrier-permeable dNP2 peptide which is capable of delivering a hyperaccurate Cas9 ribonucleoprotein complex (HypaRNP) into human cells for gene editing. Each nanosome is capable of encapsulating and delivering ≈2 HypaRNP molecules into the cytoplasm, followed by nuclear localization at 4 h post-treatment without significant cytotoxicity. The HypaRNP thus efficiently enacts endogenous eGFP silencing and editing in human embryonic kidney cells (up to 27.6%) and glioblastoma (up to 19.7% frequency of modification). The lipopeptide-based nanosome system shows superior delivery efficiency, high controllability, and simplicity, thus providing biocompatibility and versatile platform approach for CRISPR-mediated transient gene editing applications.

Longitudinal study of hemodynamics and dendritic membrane potential changes in the mouse cortex following a soft cranial window installation.

The soft cranial window using polydimethylsiloxane allows direct multiple access to neural tissue during long-term monitoring. However, the chronic effects of soft window installation on the brain have not been fully studied. Here, we investigate the long-term effects of soft window installation on sensory-evoked cerebral hemodynamics and neuronal activity. We monitored the brain tissue immunocytohistology for 6 weeks postinstallation. Heightened reactive astrocytic and microglia levels were found at 2 weeks postinstallation. By 6 weeks postinstallation, mice had expression levels similar to those of normal animals. We recorded sensory-evoked hemodynamics of the barrel cortex and LFP during whisker stimulation at these time points. Animals at 6 weeks postinstallation showed stronger hemodynamic responses and focalized barrel mapping than 2-week postoperative mice. LFP recordings of 6-week postoperative mice also showed higher neural activity at the barrel column corresponding to the stimulated whisker. Furthermore, the expression level of interleukin- 1 ß was highly upregulated at 2 weeks postinstallation. When we treated animals postoperatively with minocycline plus N-acetylcystein, a drug-suppressing inflammatory cytokine, these animals did not show declined hemodynamic responses and neuronal activities. This result suggests that neuroinflammation following soft window installation may alter hemodynamic and neuronal responses upon sensory stimulation.

Chronic and acute stress monitoring by electrophysiological signals from adrenal gland.

We present electrophysiological (EP) signals correlated with cellular cell activities in the adrenal cortex and medulla using an adrenal gland implantable flexible EP probe. With such a probe, we could observe the EP signals from the adrenal cortex and medulla in response to various stress stimuli, such as enhanced hormone activity with adrenocorticotropic hormone, a biomarker for chronic stress response, and an actual stress environment, like a forced swimming test. This technique could be useful to continuously monitor the elevation of cortisol level, a useful indicator of chronic stress that potentially causes various diseases.

Dominance of layer-specific microvessel dilation in contrast-enhanced high-resolution fMRI: Comparison between hemodynamic spread and vascular architecture with CLARITY.

Contrast-enhanced cerebral blood volume-weighted (CBVw) fMRI response peaks are specific to the layer of evoked synaptic activity (Poplawsky et al., 2015), but the spatial resolution limit of CBVw fMRI is unknown. In this study, we measured the laminar spread of the CBVw fMRI evoked response in the external plexiform layer (EPL, 265 ± 65 µm anatomical thickness, mean ± SD, n = 30 locations from 5 rats) of the rat olfactory bulb during electrical stimulation of the lateral olfactory tract and examined its potential vascular source. First, we obtained the evoked CBVw fMRI responses with a 55 × 55 µm2 in-plane resolution and a 500-µm thickness at 9.4 T, and found that the fMRI signal peaked predominantly in the inner half of EPL (136 ± 54 µm anatomical thickness). The mean full-width at half-maximum of these fMRI peaks was 347 ± 102 µm and the functional spread was approximately 100 or 200 µm when the effects of the laminar thicknesses of EPL or inner EPL were removed, respectively. Second, we visualized the vascular architecture of EPL from a different rat using a Clear Lipid-exchanged Anatomically Rigid Imaging/immunostaining-compatible Tissue hYdrogel (CLARITY)-based tissue preparation method and confocal microscopy. Microvascular segments with an outer diameter of <11 µm accounted for 64.3% of the total vascular volume within EPL and had a mean segment length of 55 ± 40 µm (n = 472). Additionally, vessels that crossed the EPL border had a mean segment length outside of EPL equal to 73 ± 61 µm (n = 28), which is comparable to half of the functional spread (50-100 µm). Therefore, we conclude that dilation of these microvessels, including capillaries, likely dominate the CBVw fMRI response and that the biological limit of the fMRI spatial resolution is approximately the average length of 1-2 microvessel segments, which may be sufficient for examining sublaminar circuits.

Real-time in vivo two-photon imaging study reveals decreased cerebro-vascular volume and increased blood-brain barrier permeability in chronically stressed mice.

Chronic stress disrupts brain homeostasis and adversely affects the cerebro-vascular system. Even though the effects of chronic stress on brain system have been extensively studied, there are few in vivo dynamic studies on the effects of chronic stress on the cerebro-vascular system. In this study, the effects of chronic stress on cerebral vasculature and BBB permeability were studied using in vivo two-photon (2p) microscopic imaging with an injection of fluorescence-conjugated dextran. Our real-time 2p imaging results showed that chronic stress reduced the vessel diameter and reconstructed vascular volume, regardless of vessel type and branching order. BBB permeability was investigated with two different size of tracers. Stressed animals exhibited a greater BBB permeability to 40-kDa dextran, but not to 70-kDa dextran, which is suggestive of weakened vascular integrity following stress. Molecular analysis revealed significantly higher VEGFa mRNA expression and a reduction in claudin-5. In summary, chronic stress decreases the size of cerebral vessels and increases BBB permeability. These results may suggest that the sustained decrease in cerebro-vascular volume due to chronic stress leads to a hypoxic condition that causes molecular changes such as VEGF and claudin-5, which eventually impairs the function of BBB.

Ultrasmall gold nanosatellite-bearing transformable hybrid nanoparticles for deep tumor penetration.

Since delivering drugs to an entire tumoral region leads to high therapeutic efficacy and good prognosis, achieving deep tumoral penetration of drugs is a major issue in cancer treatment. In this regard, conventional nanomedicines (>50 nm) have shown limitations in cancer therapy, primarily attributed to the heterogeneous distribution of drugs because of the physiological barrier of the tumor interstitial space. To address this issue, we prepared transformable hybrid nanoparticles (TNPs) consisting of a pH-responsive nanocarrier (PEG-PBAE) and doxorubicin (DOX)-conjugated ultrasmall (<3 nm) gold nanoparticles (nanosatellites). It has been shown that PEG-PBAE can serve as a reservoir for nanosatellites and release them in mildly acidic conditions (pH 6.5), mimicking the tumor microenvironment. When DOX-loaded TNPs (DOX-TNPs) were intravenously injected into tumor-bearing mice, they successfully accumulated and dissociated at the extracellular level of the tumor, leading to the disclosure of nanosatellites and free DOX. While the free DOX accumulated in tumor tissue near blood vessels, the deeply diffused nanosatellites were taken up by the tumor cell, followed by the release of DOX via cleavage of pH-responsive ester linkages in the nanosatellites at the intracellular level. Consequently, the DOX-TNPs effectively suppressed tumor growth through improved tumor penetration of DOX, suggesting their promising potential as a cancer nanomedicine. STATEMENT OF SIGNIFICANCE: Deep tumor penetration of anticancer drug is an important issue for high therapeutic efficacy. If the drugs cannot reach cancer cells in a sufficient concentration, their effectiveness will be limited. In this regard, conventional nanomedicine showed only modest therapeutic efficacy since they cannot deliver their payloads to the deep site of tumor tissue. This heterogeneous distribution of the drug is primarily attributed to the physiological barriers of the tumor microenvironment, including a dense extracellular matrix. To surmount this challenge, we developed tumor acidity-triggered transformable nanoparticles. By encapsulating doxorubicin-conjugated ultrasmall gold nanosatellites into the nanoparticles, the drug was not significantly bound to genetic materials, resulting in its minimal sequestration near the vasculature and deep tumor penetration. Our strategy could resolve not only the poor penetration issue of the drug but also its restricted tumor accumulation, suggesting the potential as an effective nanotherapeutics.

Intracellularly Activatable Nanovasodilators To Enhance Passive Cancer Targeting Regime.

Conventional cancer targeting with nanoparticles has been based on the assumed enhanced permeability and retention (EPR) effect. The data obtained in clinical trials to date, however, have rarely supported the presence of such an effect. To address this challenge, we formulated intracellular nitric oxide-generating nanoparticles (NO-NPs) for the tumor site-specific delivery of NO, a well-known vasodilator, with the intention of boosting EPR. These nanoparticles are self-assembled under aqueous conditions from amphiphilic copolymers of poly(ethylene glycol) and nitrated dextran, which possesses inherent NO release properties in the reductive environment of cancer cells. After systemic administration of the NO-NPs, we quantitatively assessed and visualized increased tumor blood flow as well as enhanced vascular permeability than could be achieved without NO. Additionally, we prepared doxorubicin (DOX)-encapsulated NO-NPs and demonstrated consequential improvement in therapeutic efficacy over the control groups with considerably improved DOX intratumoral accumulation. Overall, this proof of concept study implies a high potency of the NO-NPs as an EPR enhancer to achieve better clinical outcomes.

Protein tyrosine phosphatase conjugated with a novel transdermal delivery peptide, astrotactin 1-derived peptide recombinant protein tyrosine phosphatase (AP-rPTP), alleviates both atopic dermatitis-like and psoriasis-like dermatitis.

BACKGROUND: Atopic dermatitis (AD) and psoriasis are the 2 most common chronic inflammatory skin diseases. There is an unmet medical need to overcome limitations for transcutaneous drug development posed by the skin barrier. OBJECTIVE: We aimed to identify a novel transdermal delivery peptide and to develop a transcutaneously applicable immunomodulatory protein for treating AD and psoriasis. METHODS: We identified and generated reporter proteins conjugated to astrotactin 1-derived peptide (AP), a novel transdermal delivery peptide of human origin, and analyzed the intracellular delivery efficiency of these proteins in mouse and human skin cells and tissues using multiphoton confocal microscopy. We also generated a recombinant therapeutic protein, AP-recombinant protein tyrosine phosphatase (rPTP), consisting of the phosphatase domain of the T-cell protein tyrosine phosphatase conjugated to AP. The immunomodulatory function of AP-rPTP was confirmed in splenocytes on cytokine stimulation and T-cell receptor stimulation. Finally, we confirmed the in vivo efficacy of AP-rPTP transdermal delivery in patients with oxazolone-induced contact hypersensitivity, ovalbumin-induced AD-like, and imiquimod-induced psoriasis-like skin inflammation models. RESULTS: AP-conjugated reporter proteins exhibited significant intracellular transduction efficacy in keratinocytes, fibroblasts, and immune cells. In addition, transcutaneous administration of AP-dTomato resulted in significant localization into the dermis and epidermis in both mouse and human skin. AP-rPTP inhibited phosphorylated signal transducer and activator of transcription (STAT) 1, STAT3, and STAT6 in splenocytes and also regulated T-cell activation and proliferation. Transcutaneous administration of AP-rPTP through the paper-patch technique significantly ameliorated skin tissue thickening, inflammation, and cytokine expression in both AD-like and psoriasis-like dermatitis models. CONCLUSION: We identified a 9-amino-acid novel transdermal delivery peptide, AP, and demonstrated its feasibility for transcutaneous biologic drug development. Moreover, AP-rPTP is a novel immunomodulatory drug candidate for human dermatitis.