Multi-Armed Star-Shaped Block Copolymers of Poly(ethylene glycol)-Poly(furfuryl glycidol) as Long Circulating Nanocarriers.

Multi-arm star-shaped block copolymers with precisely tuned nano-architectures are promising candidates for drug delivery. Herein, we developed 4- and 6-arm star-shaped block copolymers consisting of poly(furfuryl glycidol) (PFG) as the core-forming segments and biocompatible poly(ethylene glycol) (PEG) as the shell-forming blocks. The polymerization degree of each block was controlled by adjusting the feeding ratio of a furfuryl glycidyl ether and ethylene oxide. The size of the series of block copolymers was found to be less than 10 nm in DMF. In water, the polymers showed sizes larger than 20 nm, which can be related to the association of the polymers. The star-shaped block copolymers effectively loaded maleimide-bearing model drugs in their core-forming segment with the Diels-Alder reaction. These drugs were rapidly released upon heating via a retro Diels-Alder step. When the star-shaped block copolymers were injected intravenously in mice, they showed prolonged blood circulation, with more than 80% of the injected dose remaining in the bloodstream at 6 h after intravenous injection. These results indicate the potential of the star-shaped PFG-PEG block copolymers as long-circulating nanocarriers.

Preceding Balance Disorders Affect Vestibular Function in Persistent Postural-Perceptual Dizziness.

Persistent postural-perceptual dizziness (PPPD) is induced by preceding conditions that cause balance disorders. To investigate the association between vestibular function and preceding balance disorders in PPPD patients, a retrospective chart review was performed. Vestibular function in 55 PPPD patients was measured using the caloric test, cervical vestibular evoked myogenic potential testing to air-conducted sound (ACS cVEMP), ocular vestibular evoked myogenic potential testing to bone-conducted vibration (BCV oVEMP), and video head impulse testing (vHIT). Patients were classified according to the type of preceding balance disorder. The age-stratified Cochran-Mantel-Haenszel (CMH) test and the exact test for the common odds ratio were conducted to evaluate the association between preceding n ≥ 4 balance disorders and present peripheral vestibular dysfunction. PPPD patients with preceding vestibular neuritis presented a significant positive association with abnormal caloric responses (p = 0.013), while those with preceding benign paroxysmal positional vertigo (BPPV) had significantly lower rates of abnormal BCV oVEMP (p = 0.003). Furthermore, patients with preceding vestibular neuritis showed lateral semicircular canal dysfunction, while those with preceding BPPV presented normal utricular functions. These results present the influence of preceding balance disorders on the vestibular function in PPPD.

An IL-12-Based Nanocytokine Safely Potentiates Anticancer Immunity through Spatiotemporal Control of Inflammation to Eradicate Advanced Cold Tumors.

Treatment of immunologically cold tumors is a major challenge for immune checkpoint inhibitors (ICIs). Interleukin 12 (IL-12) can invigorate ICIs against cold tumors by establishing a robust antitumor immunity. However, its toxicity and systemic induction of counteracting immunosuppressive signals have hindered translation. Here, IL-12 activity is spatiotemporally controlled for safely boosting efficacy without the stimulation of interfering immune responses by generating a nanocytokine that remains inactive at physiological pH, but unleashes its full activity at acidic tumor pH. The IL-12-based nanocytokine (Nano-IL-12) accumulate and release IL-12 in tumor tissues, eliciting localized antitumoral inflammation, while preventing systemic immune response, counteractive immune reactions, and adverse toxicities even after repeated intravenous administration. The Nano-IL-12-mediated spatiotemporal control of inflammation prompt superior anticancer efficacy, and synergize with ICIs to profoundly inflame the tumor microenvironment and completely eradicate ICI-resistant primary and metastatic tumors. The strategy could be a promising approach toward safer and more effective immunotherapies.

Vestibular Imaging and Function in Patients With Inner Ear Malformation Presenting With Profound Hearing Loss.

Objective: Vestibular impairment has been observed in patients with congenital hearing loss, but little is known about the vestibular anatomy and function of those in this group with inner ear malformations. This study aims to investigate the association between vestibulocochlear anatomy and vestibular function test results in children with inner ear malformations. Study Design: Case series with chart review. Setting: Pediatric patients with inner ear malformations presenting with bilateral profound hearing loss at a tertiary hospital from 1999 to 2017. Methods: Ears were classified into subgroups based on anatomic abnormalities seen on computed tomography imaging. Cervical vestibular evoked myogenic potential (cVEMP), rotatory chair, and caloric test results were obtained and collated. Descriptive and inferential statistics were calculated. Results: Of 82 ears, 29.3% had incomplete partition type II malformation, the most common type. The second-most common type was isolated vestibular organ anomaly (20.7%), which is not included in currently accepted categories. Most ears exhibited abnormal vestibular function. Abnormal vestibule volume was associated with a nonreactive cVEMP (P < .001). Radiologically abnormal lateral semicircular canals were associated with abnormal caloric and rotatory chair results (P < .001). Conclusion: With a relatively large number of cases of isolated vestibular organ anomaly not only in our study but also in previous publications, we suggest that this category be added to the subsets of inner ear malformations. Abnormal vestibule volume was significantly associated with a nonreactive cVEMP finding. The majority of patients with hearing loss secondary to inner ear malformations have abnormal vestibular function test results.

A Hoechst Reporter Enables Visualization of Drug Engagement In Vitro and In Vivo: Toward Safe and Effective Nanodrug Delivery.

Assessment of drug activation and subsequent interaction with targets in living tissues could guide nanomedicine design, but technologies enabling insight into how a drug reaches and binds its target are limited. We show that a Hoechst-based reporter system can monitor drug release and engagement from a nanoparticle delivery system in vitro and in vivo, elucidating differences in target-bound drug distribution related to drug-linker and nanoparticle properties. Drug engagement is defined as chemical detachment of drug or reporter from a nanoparticle and subsequent binding to a subcellular target, which in the case of Hoechst results in a fluorescence signal. Hoechst-based nanoreporters for drug activation contain prodrug elements such as dipeptide linkers, conjugation handles, and nanoparticle modifications such as targeting ligands to determine how nanomedicine design affects distribution of drug engaged with a subcellular target, which is tracked via cellular nuclear fluorescence in situ. Furthermore, the nanoplatform is amenable toward common maleimide-based linkers found in many prodrug-based delivery systems including polymer-, peptide-, and antibody-drug conjugates. Findings from the Hoechst reporter system were applied to develop highly potent, targeted, anticancer micelle nanoparticles delivering a monomethyl auristatin E (MMAE) prodrug comprising the same linkers employed in Hoechst studies. MMAE nanomedicine with the optimal drug-linker resulted in effective tumor growth inhibition in mice without associated acute toxicity, whereas the nonoptimal linker that showed broader drug activation in Hoechst reporter studies resulted in severe toxicity. Our results demonstrate the potential to synergize direct visualization of drug engagement with nanomedicine drug-linker design to optimize safety and efficacy.

In vivo stealthified molecularly imprinted polymer nanogels incorporated with gold nanoparticles for radiation therapy.

Radiation therapy is a representative therapeutic approach for cancer treatment, wherein the development of efficient radiation sensitizers with low side effects is critical. In this study, a novel stealth radiation sensitizer based on Au-embedded molecularly imprinted polymer nanogels (Au MIP-NGs) was developed for low-dose X-ray radiation therapy. Surface plasmon resonance measurements reveal the good affinity and selectivity of the obtained Au MIP-NGs toward the target dysopsonic protein, human serum albumin. The protein recognition capability of the nanogels led to the formation of the albumin-rich protein corona in the plasma. The Au MIP-NGs acquire stealth capability in vivo through protein corona regulation using the intrinsic dysopsonic proteins. The injection of Au MIP-NGs improved the efficiency of the radiation therapy in mouse models of pancreatic cancer. The growth of the pancreatic tumor was inhibited even at low X-ray doses (2 Gy). The novel strategy reported in this study for the synthesis of stealth nanomaterials based on nanomaterial-protein interaction control shows significant potential for application even in other approaches for cancer treatment, diagnostics, and theranostics. This strategy paves a way for the development of a wide range of effective nanomedicines for cancer therapy.

Fc Domain-Imprinted Stealth Nanogels Capable of Orientational Control of Immunoglobulin G Adsorbed In Vivo.

Regulation of nanomaterial-cell interaction is an important requisite for a variety of biomedical applications such as drug delivery systems and theranostics. Here, we demonstrate the regulation of nanomaterial-cell interaction using the oriented adsorption of intrinsic immunoglobulin G (IgG) on molecularly imprinted polymer nanogels (MIP-NGs) capable of recognizing the fragment crystallizable (Fc) domain of IgG. The unique domain recognition property resulted in the suppression of the immune response in Fc domain receptor-possessing macrophages and natural killer cells due to the regulation of protein corona based on the oriented adsorption of IgG. This resulted in the hindrance of the Fc domain, which is the trigger of an immune response. Furthermore, the acquisition of stealth capability was successfully demonstrated in vivo using intravital confocal laser scanning microscopy. The domain imprinting proposed in this study will provide a new strategy for creating nanomaterials capable of domain recognition-based oriented adsorption of intrinsic proteins in situ, thus regulating the protein corona formed on the nanomaterials. Thus, the unique Fc domain-recognition nanomaterial developed in our study can be used for various biomedical applications to target specific cells without triggering an immune response.

Vascular Bursts Act as a Versatile Tumor Vessel Permeation Route for Blood-Borne Particles and Cells.

Dynamic bursting in tumor vasculature has recently sparked interest as a novel particle transportation route for drug delivery. These bursts facilitate the transport of sub-100 nm nanoparticles into tumors, though their contribution on the access of other blood-borne particles remains unknown. To evaluate the versatility of this phenomenon, the in vivo kinetics of a variety of intravenously injected particles and their penetration in tumor xenografts and allografts are compared. Dextran, polymeric micelles, liposomes, and polymeric vesicles with diameters ranging from 32 to 302 nm are found to colocalize in virtually all vascular bursts. By mathematical modeling, the burst vent size is estimated to be 625 nm or larger, indicating the dynamic and stochastic formation of large permeation routes in tumor vasculature. Furthermore, some burst vents are found to be µm-sized, allowing the transport of 1 µm microspheres. Moreover, antibody drugs and platelets are capable of utilizing vascular burst transportation, demonstrating the application of this phenomenon to other types of therapeutics and cellular components. These findings indicate the vast potential of vascular bursts, extending the biological and therapeutic significance of this phenomenon to a wide range of blood-borne particles and cells.

Manipulating dynamic tumor vessel permeability to enhance polymeric micelle accumulation.

Selectively delivering anticancer drugs to solid tumors while avoiding their accumulation in healthy tissues is a major goal in polymeric micelle research. We have recently discovered that the extravasation and permeation of polymeric micelles occur in a dynamic manner characterized by vascular bursts followed by a brief and vigorous outward flow of fluid (called "nano-eruptions"). Nano-eruptions allow delivery of polymeric micelle-associated drugs, though delivery can be heterogeneous both among tumors and within an individual tumor, leading to suboptimal intratumoral distribution. Manipulation of nano-eruptions is expected to improve the efficiency of drug delivery systems (DDSs). By using compounds that affect the intratumoral environment, i.e. a TGF-ß inhibitor and chloroquine, the possibility of manipulating nano-eruptions to improve delivery efficiency was investigated. Both compounds were tested in a mouse xenograft model of GFP-labeled pancreatic tumor cells by tracing nano-eruption events and extravasation of size-modulated polymeric micelles in real-time through intravital confocal laser scanning microscopy. The TGF-ß inhibitor increased the number of dynamic vents, extended duration time, and generated dynamic vents with a wide range of sizes. Chloroquine did not affect the frequency of nano-eruptions, but it increased tumor vessel diameter, maximum nano-eruption area, and maximum radial increase. Both the TGF-ß inhibitor and chloroquine augmented nano-eruptions to diffuse polymeric micelles through tumor stroma, and these medications had a greater effect on the polymeric micelles with larger size, i.e. 70-nm, than on the smaller polymeric micelles having a 30-nm diameter. The results indicate that TGF-ß inhibition and chloroquine refashion the intratumoral distribution of DDSs by different mechanisms.

Structural Control of Boronic Acid Ligands Enhances Intratumoral Targeting of Sialic Acid To Eradicate Cancer Stem-like Cells.

Aberrant sialylation of cancer cells is emerging as an attractive method for generating effective antitumor strategies. However, as sialic acid (SA) is also present in healthy tissues, systems targeting SA in tumors must be strategically designed to be specifically activated in an intratumoral environment while avoiding systemic interaction. Phenylboronic acid (PBA) and its derivatives have shown potential for developing such smart ligands based on its triggered binding to SA at intratumoral pH. Because the affinity of PBAs against SA can be structurally controlled, the approach may further offer the possibility to enhance tumor targeting by molecularly engineering PBAs. Thus, to demonstrate that the modification of the chemical structure of PBAs can promote tumor targeting, we compared nanomedicines installed with the standard PBA or 5-boronopicolinic acid (5-BPA), which shows an exceptionally high binding affinity to SA in acidic pH. Platinum anticancer drugs were loaded into these nanomedicines and evaluated against orthotopic head and neck tumors, featuring a large fraction of SA-rich cancer stem-like cells (CSCs) that are resistant to platinum drugs. The 5-BPA ligands increased intracellular drug delivery of nanomedicines at intratumoral pH (pH 6.5) and enhanced the accumulation of nanomedicines in tumors to efficaciously eliminate the malignant CSCs, suppress tumor growth, and prolong mice survival. These findings indicate the potential of engineered PBA ligands for developing effective strategies targeting SA in tumors.

Polymeric Micelles Loading Proteins through Concurrent Ion Complexation and pH-Cleavable Covalent Bonding for In Vivo Delivery.

Protein drugs have great potential as targeted therapies, yet their application suffers from several drawbacks, such as instability, short half-life, and adverse immune responses. Thus, protein delivery approaches based on stimuli-responsive nanocarriers can provide effective strategies for selectively enhancing the availability and activation of proteins in targeted tissues. Herein, polymeric micelles with the ability of encapsulating proteins are developed via concurrent ion complexation and pH-cleavable covalent bonding between proteins and block copolymers directed to pH-triggered release of the protein payload. Carboxydimethylmaleic anhydride (CDM) is selected as the pH-sensitive moiety, since the CDMamide bond is stable at physiological pH (pH 7.4), while it cleaves at pH 6.5, that is, the pathophysiological pH of tumors and inflammatory tissues. By using poly(ethylene glycol)-poly(l-lysine) block copolymers having 45% CDM addition, different proteins with various sizes and isoelectric points are loaded successfully. By using myoglobin-loaded micelles (myo/m) as a model, the stability of the micelles in physiological conditions and the dissociation and release of functional myoglobin at pH 6.5 are successfully confirmed. Moreover, myo/m shows extended half-life in blood compared to free myoglobin and micelles assembled solely by polyion complex, indicating the potential of this system for in vivo delivery of proteins.

PEG-OligoRNA Hybridization of mRNA for Developing Sterically Stable Lipid Nanoparticles toward In Vivo Administration.

Lipid nanoparticles (LNPs) exhibit high potential as carriers of messenger RNA (mRNA). However, the arduous preparation process of mRNA-loaded LNPs remains a huge obstacle for their widespread clinical application. Herein, we tackled this issue by mRNA PEGylation through hybridization with polyethylene glycol (PEG)-conjugated RNA oligonucleotides (PEG-OligoRNAs). Importantly, mRNA translational activity was preserved even after hybridization of 20 PEG-OligoRNAs per mRNA. The straightforward mixing of the PEGylated mRNA with lipofectamine LTX, a commercial lipid-based carrier, just by pipetting in aqueous solution, allowed the successful preparation of mRNA-loaded LNPs with a diameter below 100 nm, whereas the use of non-PEGylated mRNA provided large aggregates above 100- and 1000-nm. In vivo, LNPs prepared from PEG-OligoRNA-hybridized mRNA exhibited high structural stability in biological milieu, without forming detectable aggregates in mouse blood after intravenous injection. In contrast, LNPs from non-PEGylated mRNA formed several micrometer-sized aggregates in blood, leading to rapid clearance from blood circulation and deposition of the aggregates in lung capillaries. Our strategy of mRNA PEGylation was also versatile to prevent aggregation of another type of mRNA-loaded LNP, DOTAP/Chol liposomes. Together, our approach provides a simple and robust preparation method to LNPs for in vivo application.

One-Pot Synthesis of PEG-Poly(amino acid) Block Copolymers Assembling Polymeric Micelles with PEG-Detachable Functionality.

Block copolymers composed of poly(ethylene glycol) (PEG) and poly(amino acids) (PAA) segments have shown exceptional features for developing biocompatible nanostructures. While the conventional methods for synthesizing PEG-PAA provide excellent control of the degree of polymerization and polydispersity, these protocols involve several steps, which increase the time and costs, and reduce the number of possible block copolymer designs. In this study, we developed a one-pot synthetic method for PEG-PAA block copolymers by doing sequential ring-opening polymerizations (ROP) of ethylene oxide (EO) and the N-carboxyanhydrides (NCAs) of amino acids promoted by the organic base 1,1,3,3-tetramethylguanidine (TMG) as the catalyst. The procedure was effectively used to synthesize PEG-poly(benzyl-l-glutamate) (PEG-PBLG) and PEG-poly(l-Lysine) (PEG-PLL) with narrow molecular weight distribution, comparable to that of block copolymers synthesized by the conventional method initiating the ROP of NCA by amine-terminated PEG. The resulting block copolymers present an ester bond between the PEG and the PAA segments, which can be gradually hydrolyzed in physiological conditions, being advantageous for improving the biocompatibility. Besides, we confirmed that the one-pot PEG-PBLG self-assembled into micelles in aqueous conditions, which showed comparable blood circulation and biodistribution to the micelles prepared from conventional PEG-PBLG. These results indicate the high potential of our one-pot synthetic approach for preparing hydrolyzable PEG-PAAs for constructing supramolecular assemblies.

Acupressure Bead in the Eustachian Tube.

In this article, we aim to enlighten practitioners and patients involved with acupressure beads and to contribute to their safer use by reporting a unique case of insidious intrusion of an acupressure bead into the eustachian tube. A metallic object was found in the eustachian tube of a patient while conducting a magnetic resonance imaging (MRI) examination. The object was later confirmed to be an auricular acupressure bead, and was successfully removed by performing a tympanoplasty and a canal wall down mastoidectomy. The bead was assumed to have passed through an existing perforation of the tympanic membrane. According to previously published literature, tympanic membrane perforations exist in ∼1% of the population. Therefore, middle-ear foreign bodies are relatively common occurrences for otolaryngologists. However, metallic objects such as acupressure beads are especially important in the sense that they can cause severe burns during MRI. To avoid potential complications, acupressure-bead practitioners should be aware of the possibility that intrusions through the tympanic membrane could go unnoticed.

Biofilm formation ability of Listeria monocytogenes isolates from raw ready-to-eat seafood.

Listeria monocytogenes is of great concern as a foodborne pathogen. Many ready-to-eat foods are widely contaminated with this organism and have caused listeriosis outbreaks and sporadic cases in many countries. In Japan, there is a high incidence of L. monocytogenes contamination, specifically in raw ready-to-eat seafood. Identical L. monocytogenes subtypes have been isolated repeatedly from samples of food manufactured at a given store or processing plant, and researchers suspected that certain L. monocytogenes isolates have formed biofilms at these sites. A microtiter plate biofilm formation assay was conducted, and all raw ready-to-eat seafood isolates tested were able to form biofilms to various degrees. Biofilm formation by L. monocytogenes isolates of lineage I was significantly greater (P = 0.000) than that by isolates of lineage II. However, isolates of clonal lineages formed different levels of biofilms, indicating that the ability to form a biofilm is affected positively or negatively by environmental factors.

Nonsense-mutated inlA and prfA not widely distributed in Listeria monocytogenes isolates from ready-to-eat seafood products in Japan.

InlA is a surface protein participating in the entry of Listeria monocytogenes into mammalian non-phagocytic cells. PrfA is a positive regulatory factor that regulates the expression of a set of virulence genes. Recent studies revealed that some L. monocytogenes strains have a truncated form of these proteins because of nonsense mutations in their sequences, and these truncations contribute to the significant reduction in virulence of this pathogen. In this study, sequence analyses of inlA and prfA among L. monocytogenes isolated from ready-to-eat seafood revealed that only one out of 59 isolates had a nonsense-mutated inlA and all had non-mutated prfA. This indicated that these strains could be fully virulent based on the sizes of these proteins.