Mechanisms and Barriers in Nanomedicine: Progress in the Field and Future Directions.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

The Role of Biological Sex in Pre-Clinical (Mouse) mRNA Vaccine Studies.

In this study, we consider the influence of biological sex-specific immune responses on the assessment of mRNA vaccines in pre-clinical murine studies. Recognising the established disparities in immune function attributed to genetic and hormonal differences between individuals of different biological sexes, we compared the mRNA expression and immune responses in mice of both biological sexes after intramuscular injection with mRNA incorporated within lipid nanoparticles. Regarding mRNA expression, no significant difference in protein (luciferase) expression at the injection site was observed between female and male mice following intramuscular administration; however, we found that female BALB/c mice exhibit significantly greater total IgG responses across the concentration range of mRNA lipid nanoparticles (LNPs) in comparison to their male counterparts. This study not only contributes to the scientific understanding of mRNA vaccine evaluation but also emphasizes the importance of considering biological sex in vaccine study designs during pre-clinical evaluation in murine studies.

Chemical Zymogens and Transmembrane Activation of Transcription in Synthetic Cells.

In this work, synthetic cells equipped with an artificial signaling pathway that connects an extracellular trigger event to the activation of intracellular transcription are engineered. Learning from nature, this is done via an engineering of responsive enzymes, such that activation of enzymatic activity can be triggered by an external biochemical stimulus. Reversibly deactivated creatine kinase to achieve triggered production of adenosine triphosphate, and a reversibly deactivated nucleic acid polymerase for on-demand synthesis of RNA are engineered. An extracellular, enzyme-activated production of a diffusible zymogen activator is also designed. The key achievement of this work is that the importance of cellularity is illustrated whereby the separation of biochemical partners is essential to resolve their incompatibility, to enable transcription within the confines of a synthetic cell. The herein designed biochemical pathway and the engineered synthetic cells are arguably primitive compared to their natural counterpart. Nevertheless, the results present a significant step toward the design of synthetic cells with responsive behavior, en route from abiotic to life-like cell mimics.

Esterase-Responsive Polyglycerol-Based Nanogels for Intracellular Drug Delivery in Rare Gastrointestinal Stromal Tumors.

Rare gastrointestinal stromal tumors (GISTs) are caused by mutations in the KIT and PDGFRA genes. Avapritinib (BLU-285) is a targeted selective inhibitor for mutated KIT and PDGFRA receptors that can be used to treat these tumors. However, there are subtypes of GISTs that exhibit resistance against BLU-285 and thus require other treatment strategies. This can be addressed by employing a drug delivery system that transports a combination of drugs with distinct cell targets. In this work, we present the synthesis of esterase-responsive polyglycerol-based nanogels (NGs) to overcome drug resistance in rare GISTs. Using inverse nanoprecipitation mediated with inverse electron-demand Diels-Alder cyclizations (iEDDA) between dPG-methyl tetrazine and dPG-norbornene, multi-drug-loaded NGs were formed based on a surfactant-free encapsulation protocol. The obtained NGs displayed great stability in the presence of fetal bovine serum (FBS) and did not trigger hemolysis in red blood cells over a period of 24 h. Exposing the NGs to Candida Antarctica Lipase B (CALB) led to the degradation of the NG network, indicating the capability of targeted drug release. The bioactivity of the loaded NGs was tested in vitro on various cell lines of the GIST-T1 family, which exhibit different drug resistances. Cell internalization with comparable uptake kinetics of the NGs could be confirmed by confocal laser scanning microscopy (CLSM) and flow cytometry for all cell lines. Cell viability and live cell imaging studies revealed that the loaded NGs are capable of intracellular drug release by showing similar IC50 values to those of the free drugs. Furthermore, multi-drug-loaded NGs were capable of overcoming BLU-285 resistance in T1-α-D842V + G680R cells, demonstrating the utility of this carrier system.

Brain-Targeted Liposomes Loaded with Monoclonal Antibodies Reduce Alpha-Synuclein Aggregation and Improve Behavioral Symptoms in Parkinson's Disease.

Monoclonal antibodies (mAbs) hold promise in treating Parkinson's disease (PD), although poor delivery to the brain hinders their therapeutic application. In the current study, it is demonstrated that brain-targeted liposomes (BTL) enhance the delivery of mAbs across the blood-brain-barrier (BBB) and into neurons, thereby allowing the intracellular and extracellular treatment of the PD brain. BTL are decorated with transferrin to improve brain targeting through overexpressed transferrin-receptors on the BBB during PD. BTL are loaded with SynO4, a mAb that inhibits alpha-synuclein (AS) aggregation, a pathological hallmark of PD. It is shown that 100-nm BTL cross human BBB models intact and are taken up by primary neurons. Within neurons, SynO4 is released from the nanoparticles and bound to its target, thereby reducing AS aggregation, and enhancing neuronal viability. In vivo, intravenous BTL administration results in a sevenfold increase in mAbs in brain cells, decreasing AS aggregation and neuroinflammation. Treatment with BTL also improve behavioral motor function and learning ability in mice, with a favorable safety profile. Accordingly, targeted nanotechnologies offer a valuable platform for drug delivery to treat brain neurodegeneration.

NETosis induction reflects COVID-19 severity and long COVID: insights from a 2-center patient cohort study in Israel.

BACKGROUND: COVID-19 severity and its late complications continue to be poorly understood. Neutrophil extracellular traps (NETs) form in acute COVID-19, likely contributing to morbidity and mortality. OBJECTIVES: This study evaluated immunothrombosis markers in a comprehensive cohort of acute and recovered COVID-19 patients, including the association of NETs with long COVID. METHODS: One-hundred-seventy-seven patients were recruited from clinical cohorts at 2 Israeli centers: acute COVID-19 (mild/moderate, severe/critical), convalescent COVID-19 (recovered and long COVID), along with 54 non-COVID controls. Plasma was examined for markers of platelet activation, coagulation, and NETs. Ex vivo NETosis induction capability was evaluated after neutrophil incubation with patient plasma. RESULTS: Soluble P-selectin, factor VIII, von Willebrand factor, and platelet factor 4 were significantly elevated in patients with COVID-19 versus controls. Myeloperoxidase (MPO)-DNA complex levels were increased only in severe COVID-19 and did not differentiate between COVID-19 severities or correlate with thrombotic markers. NETosis induction levels strongly correlated with illness severity/duration, platelet activation markers, and coagulation factors, and were significantly reduced upon dexamethasone treatment and recovery. Patients with long COVID maintained higher NETosis induction, but not NET fragments, compared to recovered convalescent patients. CONCLUSIONS: Increased NETosis induction can be detected in patients with long COVID. NETosis induction appears to be a more sensitive NET measurement than MPO-DNA levels in COVID-19, differentiating between disease severity and patients with long COVID. Ongoing NETosis induction capability in long COVID may provide insights into pathogenesis and serve as a surrogate marker for persistent pathology. This study emphasizes the need to explore neutrophil-targeted therapies in acute and chronic COVID-19.

A mucoadhesive patch loaded with freeze-dried liposomes for the local treatment of oral tumors.

Oral cancers affect millions of people globally, with increasing incidences among adults aged 35 and above. Poor drug uptake by lesions in the oral cavity following systemic administration, as well as limited localized treatment modalities for oral tumors, result in poor patient quality of life and high mortality. Here, we describe a solid, dissolvable, bioadhesive alginate patch containing freeze-dried doxorubicin-loaded liposomes as a local treatment for oral tumors located on the tongue. By varying the alginate-to-liposome ratio in the mucoadhesive patch, we could control the degree of bioadhesion to the tongue and the release profile of the drug-loaded liposomes from the matrix. In vitro, exposing squamous cell carcinoma (SCC) to the alginate mucoadhesive patch or tablet resulted in dose-dependent cancer-cell death. In vivo, the efficacy of the local treatment was demonstrated in mice bearing orthotopic SCC tumors in the tongue. The bioadhesive patch, applied directly above the lesion, significantly reduced the tumor size and treatment-associated side effects compared to implanted patches or systemic drug administration. This study demonstrates that local bioadhesive therapies are effective in treating cancers of the oral cavity.

Implanted synthetic cells trigger tissue angiogenesis through de novo production of recombinant growth factors.

Progress in bottom-up synthetic biology has stimulated the development of synthetic cells (SCs), autonomous protein-manufacturing particles, as dynamic biomimetics for replacing diseased natural cells and addressing medical needs. Here, we report that SCs genetically encoded to produce proangiogenic factors triggered the physiological process of neovascularization in mice. The SCs were constructed of giant lipid vesicles and were optimized to facilitate enhanced protein production. When introduced with the appropriate genetic code, the SCs synthesized a recombinant human basic fibroblast growth factor (bFGF), reaching expression levels of up to 9⋅106 protein copies per SC. In culture, the SCs induced endothelial cell proliferation, migration, tube formation, and angiogenesis-related intracellular signaling, confirming their proangiogenic activity. Integrating the SCs with bioengineered constructs bearing endothelial cells promoted the remodeling of mature vascular networks, supported by a collagen-IV basement membrane-like matrix. In vivo, prolonged local administration of the SCs in mice triggered the infiltration of blood vessels into implanted Matrigel plugs without recorded systemic immunogenicity. These findings emphasize the potential of SCs as therapeutic platforms for activating physiological processes by autonomously producing biological drugs inside the body.

Artificially Intelligent Nanoarray Detects Various Cancers by Liquid Biopsy of Volatile Markers.

Cancer is usually not symptomatic in its early stages. However, early detection can vastly improve prognosis. Liquid biopsy holds great promise for early detection, although it still suffers from many disadvantages, mainly searching for specific cancer biomarkers. Here, a new approach for liquid biopsies is proposed, based on volatile organic compound (VOC) patterns in the blood headspace. An artificial intelligence nanoarray based on a varied set of chemi-sensitive nano-based structured films is developed and used to detect and stage cancer. As a proof-of-concept, three cancer models are tested showing high incidence and mortality rates in the population: breast cancer, ovarian cancer, and pancreatic cancer. The nanoarray has >84% accuracy, >81% sensitivity, and >80% specificity for early detection and >97% accuracy, 100% sensitivity, and >88% specificity for metastasis detection. Complementary mass spectrometry analysis validates these results. The ability to analyze such a complex biological fluid as blood, while considering data of many VOCs at a time using the artificially intelligent nanoarray, increases the sensitivity of predictive models and leads to a potential efficient early diagnosis and disease-monitoring tool for cancer.

Nanodelivery of nucleic acids.

There is growing need for a safe, efficient, specific and non-pathogenic means for delivery of gene therapy materials. Nanomaterials for nucleic acid delivery offer an unprecedented opportunity to overcome these drawbacks; owing to their tunability with diverse physico-chemical properties, they can readily be functionalized with any type of biomolecules/moieties for selective targeting. Nucleic acid therapeutics such as antisense DNA, mRNA, small interfering RNA (siRNA) or microRNA (miRNA) have been widely explored to modulate DNA or RNA expression Strikingly, gene therapies combined with nanoscale delivery systems have broadened the therapeutic and biomedical applications of these molecules, such as bioanalysis, gene silencing, protein replacement and vaccines. Here, we overview how to design smart nucleic acid delivery methods, which provide functionality and efficacy in the layout of molecular diagnostics and therapeutic systems. It is crucial to outline some of the general design considerations of nucleic acid delivery nanoparticles, their extraordinary properties and the structure-function relationships of these nanomaterials with biological systems and diseased cells and tissues.

Synthetic cells with self-activating optogenetic proteins communicate with natural cells.

Development of regulated cellular processes and signaling methods in synthetic cells is essential for their integration with living materials. Light is an attractive tool to achieve this, but the limited penetration depth into tissue of visible light restricts its usability for in-vivo applications. Here, we describe the design and implementation of bioluminescent intercellular and intracellular signaling mechanisms in synthetic cells, dismissing the need for an external light source. First, we engineer light generating SCs with an optimized lipid membrane and internal composition, to maximize luciferase expression levels and enable high-intensity emission. Next, we show these cells' capacity to trigger bioprocesses in natural cells by initiating asexual sporulation of dark-grown mycelial cells of the fungus Trichoderma atroviride. Finally, we demonstrate regulated transcription and membrane recruitment in synthetic cells using bioluminescent intracellular signaling with self-activating fusion proteins. These functionalities pave the way for deploying synthetic cells as embeddable microscale light sources that are capable of controlling engineered processes inside tissues.

Lung targeted liposomes for treating ARDS.

Acute Respiratory Distress Syndrome (ARDS), associated with Covid-19 infections, is characterized by diffuse lung damage, inflammation and alveolar collapse that impairs gas exchange, leading to hypoxemia and patient' mortality rates above 40%. Here, we describe the development and assessment of 100-nm liposomes that are tailored for pulmonary delivery for treating ARDS, as a model for lung diseases. The liposomal lipid composition (primarily DPPC) was optimized to mimic the lung surfactant composition, and the drug loading process of both methylprednisolone (MPS), a steroid, and N-acetyl cysteine (NAC), a mucolytic agent, reached an encapsulation efficiency of 98% and 92%, respectively. In vitro, treating lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages with the liposomes decreased TNFα and nitric oxide (NO) secretion, while NAC increased the penetration of nanoparticles through the mucus. In vivo, we used LPS-induced lung inflammation model to assess the accumulation and therapeutic efficacy of the liposomes in C57BL/6 mice, either by intravenous (IV), endotracheal (ET) or IV plus ET nanoparticles administrations. Using both administration methods, liposomes exhibited an increased accumulation profile in the inflamed lungs over 48 h. Interestingly, while IV-administrated liposomes distributed widely throughout the lung, ET liposomes were present in lungs parenchyma but were not detected at some distal regions of the lungs, possibly due to imperfect airflow regimes. Twenty hours after the different treatments, lungs were assessed for markers of inflammation. We found that the nanoparticle treatment had a superior therapeutic effect compared to free drugs in treating ARDS, reducing inflammation and TNFα, IL-6 and IL-1ß cytokine secretion in bronchoalveolar lavage (BAL), and that the combined treatment, delivering nanoparticles IV and ET simultaneously, had the best outcome of all treatments. Interestingly, also the DPPC lipid component alone played a therapeutic role in reducing inflammatory markers in the lungs. Collectively, we show that therapeutic nanoparticles accumulate in inflamed lungs holding potential for treating lung disorders. SIGNIFICANCE: In this study we compare intravenous versus intratracheal delivery of nanoparticles for treating lung disorders, specifically, acute respiratory distress syndrome (ARDS). By co-loading two medications into lipid nanoparticles, we were able to reduce both inflammation and mucus secretion in the inflamed lungs. Both modes of delivery resulted in high nanoparticle accumulation in the lungs, intravenously administered nanoparticles reached lung endothelial while endotracheal delivery reached lung epithelial. Combining both delivery approaches simultaneously provided the best ARDS treatment outcome.

Nanoparticles Accumulate in the Female Reproductive System during Ovulation Affecting Cancer Treatment and Fertility.

Throughout the female menstrual cycle, physiological changes occur that affect the biodistribution of nanoparticles within the reproductive system. We demonstrate a 2-fold increase in nanoparticle accumulation in murine ovaries and uterus during ovulation, compared to the nonovulatory stage, following intravenous administration. This biodistribution pattern had positive or negative effects when drug-loaded nanoparticles, sized 100 nm or smaller, were used to treat different cancers. For example, treating ovarian cancer with nanomedicines during mouse ovulation resulted in higher drug accumulation in the ovaries, improving therapeutic efficacy. Conversely, treating breast cancer during ovulation, led to reduced therapeutic efficacy, due to enhanced nanoparticle accumulation in the reproductive system rather than at the tumor site. Moreover, chemotherapeutic nanoparticles administered during ovulation increased ovarian toxicity and decreased fertility compared to the free drug. The menstrual cycle should be accounted for when designing and implementing nanomedicines for females.

Foliar Application of dsRNA Targeting Endogenous Potato (Solanum tuberosum) Isoamylase Genes ISA1, ISA2, and ISA3 Confers Transgenic Phenotype.

Isoamylase (ISA) is a debranching enzyme found in many plants, which hydrolyzes (1-6)-α-D glucosidic linkages in starch, amylopectin, and ß-dextrins, and is thought to be responsible for starch granule formation (ISA1 and ISA2) and degradation (ISA3). Lipid-modified PEI (lmPEI) was synthesized as a carrier for long double-stranded RNA (dsRNA, 250-bp), which targets the three isoamylase isoforms. The particles were applied to the plant via the foliar spray and were differentially effective in suppressing the expressions of ISA1 and ISA2 in the potato leaves, and ISA3 in the tubers. Plant growth was not significantly impaired, and starch levels in the tubers were not affected as well. Interestingly, the treated plants had significantly smaller starch granule sizes as well as increased sucrose content, which led to an early sprouting phenotype. We confirm the proposal of previous research that an increased number of small starch granules could be responsible for an accelerated turnover of glucan chains and, thus, the rapid synthesis of sucrose, and we propose a new relationship between ISA3 and the starch granule size. The implications of this study are in achieving a transgenic phenotype for endogenous plant genes using a systemic, novel delivery system, and foliar applications of dsRNA for agriculture.

Foliar Delivery of siRNA Particles for Treating Viral Infections in Agricultural Grapevines.

Grapevine leafroll disease (GLD) is a globally spreading viral infection that causes major economic losses by reducing crop yield, plant longevity and berry quality, with no effective treatment. Grapevine leafroll associated virus-3 (GLRaV-3) is the most severe and prevalent GLD strain. Here, we evaluated the ability of RNA interference (RNAi), a non-GMO gene-silencing pathway, to treat GLRaV-3 in infected Cabernet Sauvignon grapevines. We synthesized lipid-modified polyethylenimine (lmPEI) as a carrier for long double-stranded RNA (dsRNA, 250-bp-long) that targets RNA polymerase and coat protein genes that are conserved in the GLRaV-3 genome. Self-assembled dsRNA-lmPEI particles, 220 nm in diameter, displayed inner ordered domains spaced 7.3±2 nm from one another, correlating to lmPEI wrapping spirally around the dsRNA. The particles effectively protected RNA from degradation by ribonucleases, and Europium-loaded particles applied to grapevine leaves were detected as far as 60-cm from the foliar application point. In three field experiments, a single dose of foliar administration knocked down GLRaV-3 titer, and multiple doses of the treatment kept the viral titer at baseline and triggered recovery of the vine and berries. This study demonstrates RNAi as a promising platform for treating viral diseases in agriculture.

Targeting neurons in the tumor microenvironment with bupivacaine nanoparticles reduces breast cancer progression and metastases.

Neurons within the tumor microenvironment promote cancer progression; thus, their local targeting has potential clinical benefits. We designed PEGylated lipid nanoparticles loaded with a non-opioid analgesic, bupivacaine, to target neurons within breast cancer tumors and suppress nerve-to-cancer cross-talk. In vitro, 100-nm nanoparticles were taken up readily by primary neurons, trafficking from the neuronal body and along the axons. We demonstrate that signaling between triple-negative breast cancer cells (4T1) and neurons involves secretion of cytokines stimulating neurite outgrowth. Reciprocally, neurons stimulated 4T1 proliferation, migration, and survival through secretion of neurotransmitters. Bupivacaine curbs neurite growth and signaling with cancer cells, inhibiting cancer cell viability. In vivo, bupivacaine-loaded nanoparticles intravenously administered suppressed neurons in orthotopic triple-negative breast cancer tumors, inhibiting tumor growth and metastatic dissemination. Overall, our findings suggest that reducing nerve involvement in tumors is important for treating cancer.

Advanced human-relevant in vitro pulmonary platforms for respiratory therapeutics.

Over the past years, advanced in vitro pulmonary platforms have witnessed exciting developments that are pushing beyond traditional preclinical cell culture methods. Here, we discuss ongoing efforts in bridging the gap between in vivo and in vitro interfaces and identify some of the bioengineering challenges that lie ahead in delivering new generations of human-relevant in vitro pulmonary platforms. Notably, in vitro strategies using foremost lung-on-chips and biocompatible "soft" membranes have focused on platforms that emphasize phenotypical endpoints recapitulating key physiological and cellular functions. We review some of the most recent in vitro studies underlining seminal therapeutic screens and translational applications and open our discussion to promising avenues of pulmonary therapeutic exploration focusing on liposomes. Undeniably, there still remains a recognized trade-off between the physiological and biological complexity of these in vitro lung models and their ability to deliver assays with throughput capabilities. The upcoming years are thus anticipated to see further developments in broadening the applicability of such in vitro systems and accelerating therapeutic exploration for drug discovery and translational medicine in treating respiratory disorders.

Robotics, microfluidics, nanotechnology and AI in the synthesis and evaluation of liposomes and polymeric drug delivery systems.

The field of nanotechnology and personalised medicine is undergoing drastic changes in the approach and efficiency of experimentation. The COVID-19 pandemic has spiralled into mass stagnation of major laboratories around the globe and led to increased investment into remote systems for nanoparticle experiments. A significant number of laboratories now operate using automated systems; however, the extension to nanoparticle preparation and artificial intelligence-dependent databases holds great translational promise. The strive to combine automation with artificial intelligence (AI) grants the ability to optimise targeted therapeutic nanoparticles for unique cell types and patients. In this perspective, the current and future trends of automated approaches to nanomedicine synthesis are discussed and compared with traditional methods.