Minimally invasive nasal infusion (MINI) approach for CNS delivery of protein therapeutics: A case study with ovalbumin.

One of the primary obstacles in treating central nervous system (CNS) disorders lies in the limited ability of disease-modifying drugs to cross the blood-brain barrier (BBB). Our previously described Minimally Invasive Nasal Depot (MIND) technique has proven successful in delivering various drugs to the brain in rat models via a trans-olfactory mucosal approach. In this study, we introduce a novel Minimally Invasive Nasal Infusion (MINI) delivery approach for administering ovalbumin, a model protein, utilizing a programmable infusion pump (iPRECIO SMP-310R) in a mouse model. This research highlights the significant role of olfactory mucosa in nose-to-brain delivery, with an efficacy of nearly 45% compared to intracerebroventricular (ICV) administration. This demonstrates its potential as an alternative procedure for treating CNS diseases, offering a greater safety profile relative to the highly invasive clinical routes traditionally adopted for CNS drug delivery.

RNA therapies for CNS diseases.

Neurological disorders are a diverse group of conditions that pose an increasing health burden worldwide. There is a general lack of effective therapies due to multiple reasons, of which a key obstacle is the presence of the blood-brain barrier, which limits drug delivery to the central nervous system, and generally restricts the pool of candidate drugs to small, lipophilic molecules. However, in many cases, these are unable to target key pathways in the pathogenesis of neurological disorders. As a group, RNA therapies have shown tremendous promise in treating various conditions because they offer unique opportunities for specific targeting by leveraging Watson-Crick base pairing systems, opening up possibilities to modulate pathological mechanisms that previously could not be addressed by small molecules or antibody-protein interactions. This potential paradigm shift in disease management has been enabled by recent advances in synthesizing, purifying, and delivering RNA. This review explores the use of RNA-based therapies specifically for central nervous system disorders, where we highlight the inherent limitations of RNA therapy and present strategies to augment the effectiveness of RNA therapeutics, including physical, chemical, and biological methods. We then describe translational challenges to the widespread use of RNA therapies and close with a consideration of future prospects in this field.

Strategies to reduce the risks of mRNA drug and vaccine toxicity.

mRNA formulated with lipid nanoparticles is a transformative technology that has enabled the rapid development and administration of billions of coronavirus disease 2019 (COVID-19) vaccine doses worldwide. However, avoiding unacceptable toxicity with mRNA drugs and vaccines presents challenges. Lipid nanoparticle structural components, production methods, route of administration and proteins produced from complexed mRNAs all present toxicity concerns. Here, we discuss these concerns, specifically how cell tropism and tissue distribution of mRNA and lipid nanoparticles can lead to toxicity, and their possible reactogenicity. We focus on adverse events from mRNA applications for protein replacement and gene editing therapies as well as vaccines, tracing common biochemical and cellular pathways. The potential and limitations of existing models and tools used to screen for on-target efficacy and de-risk off-target toxicity, including in vivo and next-generation in vitro models, are also discussed.

Mild repetitive TBI reduces brain-derived neurotrophic factor (BDNF) in the substantia nigra and hippocampus: A preclinical model for testing BDNF-targeted therapeutics.

Clinical studies have consistently shown that neurodegenerative diseases (NDs) such as Parkinson's disease, Alzheimer's disease, Amyotrophic Lateral Sclerosis, and Huntington's disease show absent or low levels of brain-derived neurotrophic factor (BDNF). Despite this relationship between BDNF and ND, only a few ND animal models have been able to recapitulate the low BDNF state, thereby hindering research into the therapeutic targeting of this important neurotrophic factor. In order to address this unmet need, we sought to develop a reproducible model of BDNF reduction by inducing traumatic brain injury (TBI) using a closed head momentum exchange injury model in mature 9-month-old male and female rats. Head impacts were repetitive and varied in intensity from mild to severe. BDNF levels, as assessed by ELISA, were significantly reduced in the hippocampus of both males and females as well as in the substantia nigra of males 12 days after mild TBI. However, we observed significant sexual dimorphism in multiple sequelae, including magnetic resonance imaging-determined vasogenic edema, astrogliosis (GFAP-activation), and microgliosis (Iba1 activation). This study provides an opportunity to investigate the mechanism of BDNF reduction in rodent models and provides a reliable paradigm to test BDNF-targeted therapeutics for the treatment of ND.

Nanotechnology-enabled topical delivery of therapeutics in chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a chronic inflammatory disease of the paranasal sinuses which represents a significant health burden due to its widespread prevalence and impact on patients' quality of life. As the molecular pathways driving and sustaining inflammation in CRS become better elucidated, the diversity of treatment options is likely to widen significantly. Nanotechnology offers several tools to enhance the effectiveness of topical therapies, which has been limited by factors such as poor drug retention, mucosal permeation and adhesion, removal by epithelial efflux pumps and the inability to effectively penetrate biofilms. In this review, we highlight the successful application of nanomedicine in the field of CRS therapeutics, discuss current limitations and propose opportunities for future work.

Chronic sinusitis is a common inflammatory condition of the sinuses, which affects patients' quality of life and consumes significant healthcare resources. It is primarily treated with corticosteroids, a type of medication that reduces inflammation, as a nasal spray or taken orally. Nasal sprays are preferred, to minimize side effects elsewhere in the body. Recently, another class of drugs ­ 'biologic agents' ­ has been approved for a subtype of chronic sinusitis that causes polyps (grape-like swellings of the sinus lining). However, a lasting cure is elusive, because inflammation frequently returns once these medications are stopped. As our understanding of what causes chronic sinusitis improves, researchers are seeking therapies that more accurately target the cause of inflammation, rather than broadly suppressing all types of inflammation using corticosteroids. The use of nanotechnology allows the design of drugs to overcome various challenges in treating chronic sinusitis, potentially enabling more accurate delivery of drugs into the sinuses, improving drugs' ability to remain on the sinus lining and penetrate it, reducing the amount of drug lost due to the action of outflow pumps and overcoming additional defenses built up by bacteria when they form thick films. Here, we describe how nanomedicine has been used to develop drugs for chronic sinusitis, discuss current limitations and propose opportunities for future work.

Long-acting therapeutic delivery systems for the treatment of gliomas.

Despite the emergence of cutting-edge therapeutic strategies and tremendous progress in research, a complete cure of glioma remains elusive. The heterogenous nature of tumor, immunosuppressive state and presence of blood brain barrier are few of the major obstacles in this regard. Long-acting depot formulations such as injectables and implantables are gaining attention for drug delivery to brain owing to their ease in administration and ability to elute drug locally for extended durations in a controlled manner with minimal toxicity. Hybrid matrices fabricated by incorporating nanoparticulates within such systems help to enhance pharmaceutical advantages. Utilization of long-acting depots as monotherapy or in conjunction with existing strategies rendered significant survival benefits in many preclinical studies and some clinical trials. The discovery of novel targets, immunotherapeutic strategies and alternative drug administration routes are now coupled with several long-acting systems with an ultimate aim to enhance patient survival and prevent glioma recurrences.

Topical Administration of Verapamil in Poly(ethylene glycol)-Modified Liposomes for Enhanced Sinonasal Tissue Residence in Chronic Rhinosinusitis: Ex Vivo and In Vivo Evaluations.

Verapamil is a calcium channel blocker that holds promise for the therapy of chronic rhinosinusitis (CRS) with and without nasal polyps. The verapamil-induced side effects limit its tolerated dose via the oral route, underscoring the usefulness of localized intranasal administration. However, the challenge to intranasal administration is mucociliary clearance, which diminishes localized dose availability. To overcome this challenge, verapamil was loaded into a mucoadhesive cationic poly(ethylene glycol)-modified (PEGylated) liposomal carrier. Organotypic nasal explants were exposed to verapamil liposomes under flow conditions to mimic mucociliary clearance. The liposomes resulted in significantly higher tissue residence compared with the free verapamil control. These findings were further confirmed in vivo in C57BL/6 mice following intranasal administration. Liposomes significantly increased the accumulation of verapamil in nasal tissues compared with the control group. The developed tissue-retentive verapamil liposomal formulation is considered a promising intranasal delivery system for CRS therapy.

A Self-Emulsified Adjuvant System Containing the Immune Potentiator Alpha Tocopherol Induces Higher Neutralizing Antibody Responses than a Squalene-Only Emulsion When Evaluated with a Recombinant Cytomegalovirus (CMV) Pentamer Antigen in Mice.

The development of new vaccine adjuvants represents a key approach to improvingi the immune responses to recombinant vaccine antigens. Emulsion adjuvants, such as AS03 and MF59, in combination with influenza vaccines, have allowed antigen dose sparing, greater breadth of responses and fewer immunizations. It has been demonstrated previously that emulsion adjuvants can be prepared using a simple, low-shear process of self-emulsification (SE). The role of alpha tocopherol as an immune potentiator in emulsion adjuvants is clear from the success of AS03 in pandemic responses, both to influenza and COVID-19. Although it was a significant formulation challenge to include alpha tocopherol in an emulsion prepared by a low-shear process, the resultant self-emulsifying adjuvant system (SE-AS) showed a comparable effect to the established AS03 when used with a quadrivalent influenza vaccine (QIV). In this paper, we first optimized the SE-AS with alpha tocopherol to create SE-AS44, which allowed the emulsion to be sterile-filtered. Then, we compared the in vitro cell activation cytokine profile of SE-AS44 with the self-emulsifying adjuvant 160 (SEA160), a squalene-only adjuvant. In addition, we evaluated SE-AS44 and SEA160 competitively, in combination with a recombinant cytomegalovirus (CMV) pentamer antigen mouse.

CNS Delivery of Nucleic Acid Therapeutics: Beyond the Blood-Brain Barrier and Towards Specific Cellular Targeting.

Nucleic acid-based therapeutic molecules including small interfering RNA (siRNA), microRNA(miRNA), antisense oligonucleotides (ASOs), messenger RNA (mRNA), and DNA-based gene therapy have tremendous potential for treating diseases in the central nervous system (CNS). However, achieving clinically meaningful delivery to the brain and particularly to target cells and sub-cellular compartments is typically very challenging. Mediating cell-specific delivery in the CNS would be a crucial advance that mitigates off-target effects and toxicities. In this review, we describe these challenges and provide contemporary evidence of advances in cellular and sub-cellular delivery using a variety of delivery mechanisms and alternative routes of administration, including the nose-to-brain approach. Strategies to achieve subcellular localization, endosomal escape, cytosolic bioavailability, and nuclear transfer are also discussed. Ultimately, there are still many challenges to translating these experimental strategies into effective and clinically viable approaches for treating patients.

Cold exposure impairs extracellular vesicle swarm-mediated nasal antiviral immunity.

BACKGROUND: The human upper respiratory tract is the first site of contact for inhaled respiratory viruses and elaborates an array of innate immune responses. Seasonal variation in respiratory viral infections and the importance of ambient temperature in modulating immune responses to infections have been well recognized; however, the underlying biological mechanisms remain understudied. OBJECTIVE: We investigated the role of nasal epithelium-derived extracellular vesicles (EVs) in innate Toll-like receptor 3 (TLR3)-dependent antiviral immunity. METHODS: We evaluated the secretion and composition of nasal epithelial EVs after TLR3 stimulation in human autologous cells and fresh human nasal mucosal surgical specimens. We also explored the antiviral activity and mechanisms of TLR3-stimulated EVs against respiratory viruses as well as the effect of cool ambient temperature on TLR3-dependent antiviral immunity. RESULTS: We found that polyinosinic:polycytidylic acid, aka poly(I:C), exposure induced a swarm-like increase in the secretion of nasal epithelial EVs via the TLR3 signaling. EVs participated in TLR3-dependent antiviral immunity, protecting the host from viral infections through both EV-mediated functional delivery of miR-17 and direct virion neutralization after binding to virus ligands via surface receptors, including LDLR and ICAM-1. These potent antiviral immune defense functions mediated by TLR3-stimulated EVs were impaired by cold exposure via a decrease in total EV secretion as well as diminished microRNA packaging and antiviral binding affinity of individual EV. CONCLUSION: TLR3-dependent nasal epithelial EVs exhibit multiple innate antiviral mechanisms to suppress respiratory viral infections. Furthermore, our study provides a direct quantitative mechanistic explanation for seasonal variation in upper respiratory tract infection prevalence.

Tumor-targeted miRNA nanomedicine for overcoming challenges in immunity and therapeutic resistance.

miRNA are critical messengers in the tumor microenvironment (TME) that influence various processes leading to immune suppression, tumor progression, metastasis and resistance. Strategies to modulate miRNAs in the TME have important implications in overcoming these challenges. However, miR delivery to specific cells in the TME has been challenging. This review discusses nanomedicine strategies to achieve cell-specific delivery of miRNAs. The key goal of delivery is to activate the tumor immune landscape as well as to prevent chemotherapy resistance. Specifically, the use of hyaluronic acid-based nanoparticle miRNA delivery to the TME is discussed. The discussion is focused on miRNA-125b for reprogramming tumor-associated macrophages to overcome immunosuppression and miRNA-let-7b to overcome resistance to anticancer chemotherapeutics because both these miRNAs have been extensively evaluated for delivery with hyaluronic acid-based delivery systems.

miRNAs are the messenger molecules with the tumor that have significant influence on the cancer growth and progression. Many strategies have been evaluated to modulate these messengers artificially to obstruct cancer growth and destroy cancer cells. This review discusses one such strategy to deliver these messenger miRNAs using hyaluronic acid-based nanoparticles that harness the body's own immune system to fight cancer. The two miRNAs that this review discusses are miRNA-125b and miRNA-let7b.

Nucleic acid therapies for CNS diseases: Pathophysiology, targets, barriers, and delivery strategies.

Nucleic acid therapeutics have emerged as one of the very advanced and efficacious treatment approaches for debilitating health conditions, including those diseases affecting the central nervous system (CNS). Precise targeting with an optimal control over gene regulation confers long-lasting benefits through the administration of nucleic acid payloads via viral, non-viral, and engineered vectors. The current review majorly focuses on the development and clinical translational potential of non-viral vectors for treating CNS diseases with a focus on their specific design and targeting approaches. These carriers must be able to surmount the various intracellular and extracellular barriers, to ensure successful neuronal transfection and ultimately attain higher therapeutic efficacies. Additionally, the specific challenges associated with CNS administration also include the presence of blood-brain barrier (BBB), the complex pathophysiological and biochemical changes associated with different disease conditions and the existence of non-dividing cells. The advantages offered by lipid-based or polymeric systems, engineered proteins, particle-based systems coupled with various approaches of neuronal targeting have been discussed in the context of a variety of CNS diseases. The possibilities of rapid yet highly efficient gene modifications rendered by the breakthrough methodologies for gene editing and gene manipulation have also opened vast avenues of research in neuroscience and CNS disease therapy. The current review also underscores the extensive scientific efforts to optimize specialized, efficacious yet non-invasive and safer administration approaches to overcome the therapeutic delivery challenges specifically posed by the CNS transport barriers and the overall obstacles to clinical translation.

Mathematical Modeling and Experimental Validation of Extracellular Vesicle-Mediated Tumor Suppressor MicroRNA Delivery and Propagation in Ovarian Cancer Cells.

Extracellular vesicle (EV)-mediated microRNA transfer and propagation from the donor cell to the recipient cell in the tumor microenvironment have significant implications, including the development of multidrug resistance (MDR). Although miRNA-encapsulated EV have been shown to have functional effects on recipient cells, the quantitative aspects of transfer kinetics and functional effects remain poorly understood. Intracellular events such as degradation of miRNA, loading of miRNA into EVs, cellular release of EVs, and their uptake by recipient cells govern the transfer and functional effect of encapsulated miRNA. Based on these rate-limiting steps, we developed a mathematical model using ordinary differential equations (model 1). We performed coculture experiments using ID8-VEGF ovarian cancer cells to demonstrate EV-mediated propagation of tumor suppressor miRNA Let7b administered with hyaluronic acid-poly(ethyleneimine) (HA-PEI) nanoparticles. Using the experimental data and model fitting, we determined the rate constants for the kinetic events involved in the transfer from the donor cells to the recipient cells. In model 2, we performed Let7b transfection experiments in ID8-VEGF cells with HA-PEI nanoparticles to determine the concentration-effect relationship on HMGA2 mRNA levels. Lastly, in model 3, we combined model 1 and model 2 parameters to describe the kinetics and effect relationship of EV-Let7b in recipient cells to predict the minimum number of miRNA copies needed to show functional effects.

Role of 3D Printing in the Development of Biodegradable Implants for Central Nervous System Drug Delivery.

Increased life expectancy has led to a rise in age-related disorders including neurological diseases such as Alzheimer's disease and Parkinson's disease. Limited progress has been made in the development of clinically translatable therapies for these central nervous system (CNS) diseases. Challenges including the blood-brain barrier, brain complexity, and comorbidities in the elderly population are some of the contributing factors toward lower success rates. Various invasive and noninvasive ways are being employed to deliver small and large molecules across the brain. Biodegradable, implantable drug-delivery systems have gained lot of interest due to advantages such as sustained and targeted delivery, lower side effects, and higher patient compliance. 3D printing is a novel additive manufacturing technique where various materials and printing techniques can be used to fabricate implants with the desired complexity in terms of mechanical properties, shapes, or release profiles. This review discusses an overview of various types of 3D-printing techniques and illustrative examples of the existing literature on 3D-printed systems for CNS drug delivery. Currently, there are various technical and regulatory impediments that need to be addressed for successful translation from the bench to the clinical stage. Overall, 3D printing is a transformative technology with great potential in advancing customizable drug treatment in a high-throughput manner.

Cystatin SN is a potent upstream initiator of epithelial-derived type 2 inflammation in chronic rhinosinusitis.

BACKGROUND: Cystatin SN (CST1) and cystatin SA (CST2) are cysteine protease inhibitors that protect against allergen, viral, and bacterial proteases. Cystatins are overexpressed in the setting of allergic rhinitis and chronic rhinosinusitis with nasal polyps (CRSwNP); however, their role in promoting type 2 inflammation remains poorly characterized. OBJECTIVE: The purpose of this study was to use integrated poly-omics and a murine exposure model to explore the link between cystatin overexpression in CRSwNP and type 2 inflammation. METHODS: In this institutional review board- and institutional animal care and use committee-approved study, we compared tissue, exosome, and mucus CST1 and CST2 between CRSwNP and controls (n = 10 per group) by using matched whole exome sequencing, transcriptomic, proteomic, posttranslational modification, histologic, functional, and bioinformatic analyses. C57/BL6 mice were dosed with 3.9 µg/mL of CST1 or PBS intranasally for 5 to 18 days in the presence or absence of epithelial ABCB1a knockdown. Inflammatory cytokines were quantified by using Quansys multiplex assays or ELISAs. RESULTS: Of the 1305 proteins quantified, CST1 and CST2 were among the most overexpressed protease inhibitors in tissue, exosome, and mucus samples; they were localized to the epithelial layer. Multiple posttranslational modifications were identified in the polyp tissue. Exosomal CST1 and CST2 were strongly and significantly correlated with eosinophils and Lund-Mackay scores. Murine type 2 cytokine secretion and TH2 cell infiltration increased in a time-dependent manner following CST1 exposure and was abrogated by epithelial knockdown of ABCB1a, a regulator of epithelial cytokine secretion. CONCLUSION: CST1 is a potent upstream initiator of epithelial-derived type 2 inflammation in CRSwNP. Therapeutic strategies targeting CST activity and its associated posttranslational modifications deserve further interrogation.

Combination microRNA-based cellular reprogramming with paclitaxel enhances therapeutic efficacy in a relapsed and multidrug-resistant model of epithelial ovarian cancer.

Most advanced-stage ovarian cancer patients, including those with epithelial ovarian cancer (EOC), develop recurrent disease and acquisition of resistance to chemotherapy, leading to limited treatment options. Decrease in Let7b miRNA levels in clinical ovarian cancer has been associated with chemoresistance, increased proliferation, invasion, and relapse in EOC. We have established a murine EOC relapsed model by administering paclitaxel (PTX) and stopping therapy to allow for tumor regrowth. Global microRNA profiling in the relapsed tumor showed significant downregulation of Let7b relative to untreated tumors. Here, we report the use of hyaluronic acid (HA)-based nanoparticle formulation that can deliver Let7b miRNA mimic to tumor cells and achieve cellular programming both in vitro and in vivo. We demonstrate that a therapeutic combination of Let7b miRNA and PTX leads to significant improvement in anti-tumor efficacy in the relapsed model of EOC. We further demonstrate that the combination therapy is safe for repeated administration. This novel approach of cellular reprogramming of tumor cells using clinically relevant miRNA mimic in combination with chemotherapy could enable more effective therapeutic outcomes for patients with advanced-stage relapsed EOC.

Traumatic brain injury and the development of parkinsonism: Understanding pathophysiology, animal models, and therapeutic targets.

The clinical translation of therapeutic approaches to combat debilitating neurodegenerative conditions, such as Parkinson's disease (PD), remains as an urgent unmet challenge. The strong molecular association between the pathogenesis of traumatic brain injury (TBI) and the development of parkinsonism in humans has been well established. Therefore, a lot of ongoing research aims to investigate this pathology overlap in-depth, to exploit the common targets of TBI and PD for development of more effective and long-term treatment strategies. This review article intends to provide a detailed background on TBI pathophysiology and its established overlap with PD with an additional emphasis on the recent findings about their effect on perivascular clearance. Although, the traditional animal models of TBI and PD are still being considered, there is a huge focus on the development of combinatory hybrid animal models coupling concussion with the pre-established PD models for a better recapitulation of the human context of PD pathogenesis. Lastly, the therapeutic targets for TBI and PD, and the contemporary research involving exosomes, DNA vaccines, miRNA, gene therapy and gene editing for the development of potential candidates are discussed, along with the recent development of lesser invasive and promising central nervous system (CNS) drug delivery strategies.

Application of nanotechnology in medical diagnosis and imaging.

Nanotechnology has tremendously impacted the advancement in imaging, early detection, diagnosis, and prognosis of diseases by improving upon existing clinically relevant technologies. The unique biophysical properties of the nanoparticles enable contrast enhancement to improve biomedical imaging while our ability to manipulate nanoparticles for molecular level specificity enables tissue-specific diagnosis. Importantly, subtle variation in size or composition of the nanoparticles can result in great changes in their optical, magnetic or electrical properties that allows unique possibility of multiplexing. This concise but focused review summarizes the important classes of nanoparticles that have been actively used in improving our ability to image diseased tissues and have contributed to develop technologies that has led to early detection and diagnosis of diseases.