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.

Therapeutic application of circular RNA aptamers in a mouse model of psoriasis.

Efforts to advance RNA aptamers as a new therapeutic modality have been limited by their susceptibility to degradation and immunogenicity. In a previous study, we demonstrated synthesized short double-stranded region-containing circular RNAs (ds-cRNAs) with minimal immunogenicity targeted to dsRNA-activated protein kinase R (PKR). Here we test the therapeutic potential of ds-cRNAs in a mouse model of imiquimod-induced psoriasis. We find that genetic supplementation of ds-cRNAs leads to inhibition of PKR, resulting in alleviation of downstream interferon-α and dsRNA signals and attenuation of psoriasis phenotypes. Delivery of ds-cRNAs by lipid nanoparticles to the spleen attenuates PKR activity in examined splenocytes, resulting in reduced epidermal thickness. These findings suggest that ds-cRNAs represent a promising approach to mitigate excessive PKR activation for therapeutic purposes.

Endosomal escape: A bottleneck for LNP-mediated therapeutics.

Lipid nanoparticles (LNPs) have recently emerged as a powerful and versatile clinically approved platform for nucleic acid delivery, specifically for mRNA vaccines. A major bottleneck in the field is the release of mRNA-LNPs from the endosomal pathways into the cytosol of cells where they can execute their encoded functions. The data regarding the mechanism of these endosomal escape processes are limited and contradicting. Despite extensive research, there is no consensus regarding the compartment of escape, the cause of the inefficient escape and are currently lacking a robust method to detect the escape. Here, we review the currently known mechanisms of endosomal escape and the available methods to study this process. We critically discuss the limitations and challenges of these methods and the possibilities to overcome these challenges. We propose that the development of currently lacking robust, quantitative high-throughput techniques to study endosomal escape is timely and essential. A better understanding of this process will enable better RNA-LNP designs with improved efficiency to unlock new therapeutic modalities.

The immunostimulatory nature of mRNA lipid nanoparticles.

mRNA-Lipid nanoparticles (LNPs) are at the forefront of global medical research. With the development of mRNA-LNP vaccines to combat the COVID-19 pandemic, the clinical potential of this platform was unleashed. Upon administering 16 billion doses that protected billions of people, it became clear that a fraction of them witnessed mild and in some cases even severe adverse effects. Therefore, it is paramount to define the safety along with the therapeutic efficacy of the mRNA-LNP platform for the successful translation of new genetic medicines based on this technology. While mRNA was the effector molecule of this platform, the ionizable lipid component of the LNPs played an indispensable role in its success. However, both of these components possess the ability to induce undesired immunostimulation, which is an area that needs to be addressed systematically. The immune cell agitation caused by this platform is a two-edged sword as it may prove beneficial for vaccination but detrimental to other applications. Therefore, a key challenge in advancing the mRNA-LNP drug delivery platform from bench to bedside is understanding the immunostimulatory behavior of these components. Herein, we provide a detailed overview of the structural modifications and immunogenicity of synthetic mRNA. We discuss the effect of ionizable lipid structure on LNP functionality and offer a mechanistic overview of the ability of LNPs to elicit an immune response. Finally, we shed some light on the current status of this technology in clinical trials and discuss a few challenges to be addressed to advance the field.

Cytotoxicity and reversal effect of sertraline, fluoxetine, and citalopram on MRP1- and MRP7-mediated MDR.

Cancer is one of the leading causes of death worldwide, and the development of resistance to chemotherapy drugs is a major challenge in treating malignancies. In recent years, researchers have focused on understanding the mechanisms of multidrug resistance (MDR) in cancer cells and have identified the overexpression of ATP-binding cassette (ABC) transporters, including ABCC1/MRP1 and ABCC10/MRP7, as a key factor in the development of MDR. In this study, we aimed to investigate whether three drugs (sertraline, fluoxetine, and citalopram) from the selective serotonin reuptake inhibitor (SSRI) family, commonly used as antidepressants, could be repurposed as inhibitors of MRP1 and MRP7 transporters and reverse MDR in cancer cells. Using a combination of in silico predictions and in vitro validations, we analyzed the interaction of MRP1 and MRP7 with the drugs and evaluated their ability to hinder cell resistance. We used computational tools to identify and analyze the binding site of these three molecules and determine their binding energy. Subsequently, we conducted experimental assays to assess cell viability when treated with various standard chemotherapies, both with and without the presence of SSRI inhibitors. Our results show that all three SSRI drugs exhibited inhibitory/reversal effects in the presence of chemotherapies on both MRP1-overexpressed cells and MRP7-overexpressed cells, suggesting that these medications have the potential to be repurposed to target MDR in cancer cells. These findings may open the door to using FDA-approved medications in combination therapy protocols to treat highly resistant malignancies and improve the efficacy of chemotherapy treatment. Our research highlights the importance of investigating and repurposing existing drugs to overcome MDR in cancer treatment.

Targeting cancer with mRNA-lipid nanoparticles: key considerations and future prospects.

Harnessing mRNA-lipid nanoparticles (LNPs) to treat patients with cancer has been an ongoing research area that started before these versatile nanoparticles were successfully used as COVID-19 vaccines. Currently, efforts are underway to harness this platform for oncology therapeutics, mainly focusing on cancer vaccines targeting multiple neoantigens or direct intratumoural injections of mRNA-LNPs encoding pro-inflammatory cytokines. In this Review, we describe the opportunities of using mRNA-LNPs in oncology applications and discuss the challenges for successfully translating the findings of preclinical studies of these nanoparticles into the clinic. We critically appraise the potential of various mRNA-LNP targeting and delivery strategies, considering physiological, technological and manufacturing challenges. We explore these approaches in the context of the potential clinical applications best suited to each approach and highlight the obstacles that currently need to be addressed to achieve these applications. Finally, we provide insights from preclinical and clinical studies that are leading to this powerful platform being considered the next frontier in oncology treatment.

Transport by circulating myeloid cells drives liposomal accumulation in inflamed synovium.

The therapeutic potential of liposomes to deliver drugs into inflamed tissue is well documented. Liposomes are believed to largely transport drugs into inflamed joints by selective extravasation through endothelial gaps at the inflammatory sites, known as the enhanced permeation and retention effect. However, the potential of blood-circulating myeloid cells for the uptake and delivery of liposomes has been largely overlooked. Here we show that myeloid cells can transport liposomes to inflammatory sites in a collagen-induced arthritis model. It is shown that the selective depletion of the circulating myeloid cells reduces the accumulation of liposomes up to 50-60%, suggesting that myeloid-cell-mediated transport accounts for more than half of liposomal accumulation in inflamed regions. Although it is widely believed that PEGylation inhibits premature liposome clearance by the mononuclear phagocytic system, our data show that the long blood circulation times of PEGylated liposomes rather favours uptake by myeloid cells. This challenges the prevailing theory that synovial liposomal accumulation is primarily due to the enhanced permeation and retention effect and highlights the potential for other pathways of delivery in inflammatory diseases.

Lipid nanoparticles-loaded with toxin mRNA represents a new strategy for the treatment of solid tumors.

Background and rationale: Cancer therapy have evolved remarkably over the past decade, providing new strategies to inhibit cancer cell growth using immune modulation, with or without gene therapy. Specifically, suicide gene therapies and immunotoxins have been investigated for the treatment of tumors by direct cancer cell cytotoxicity. Recent advances in mRNA delivery also demonstrated the potential of mRNA-based vaccines and immune-modulators for cancer therapeutics by utilizing nanocarriers for mRNA delivery. Methods: We designed a bacterial toxin-encoding modified mRNA, delivered by lipid nanoparticles into a B16-melanoma mouse model. Results: We showed that local administration of LNPs entrapping a modified mRNA that encodes for a bacterial toxin, induced significant anti-tumor effects and improved overall survival of treated mice. Conclusions: We propose mmRNA-loaded LNPs as a new class of anti-tumoral, toxin-based therapy.

Design of experiments in the optimization of nanoparticle-based drug delivery systems.

Design of experiment (DoE) is a powerful statistical technique used for variable screening and optimization. It is based on the simultaneous variation of multiple factors with the objective of finding the configuration of parameters that optimizes one or more outputs of interest, while using the minimal number of experimental runs required for testing, resulting very cost and time-efficient. Despite the high potential offered by this approach for innovation and process optimization, DoE is still only marginally applied in the field of nanomedicine and often its rationale application and analysis result is difficult to grasp by many. In this review, we discuss some of the latest applications of DoE in the formulation of nanovectors used for drug delivery across many different applications. First, we introduce general principles of DoE to the reader, which are indispensable to understand the works we report. Then, we give particular attention to the process variables, the specific designs, and the readouts used for process analysis and optimization for different classes of nanovectors. Finally, we try to delve into the current shortcomings of DoE application and possible future directions that could be employed to further improve the information that can be derived from this approach.

Delivery of Therapeutic RNA to the Bone Marrow in Multiple Myeloma Using CD38-Targeted Lipid Nanoparticles.

Multiple myeloma (MM) is a cancer of differentiated plasma cells that occurs in the bone marrow (BM). Despite the recent advancements in drug development, most patients with MM eventually relapse and the disease remains incurable. RNA therapy delivered via lipid nanoparticles (LNPs) has the potential to be a promising cancer treatment, however, its clinical implementation is limited due to inefficient delivery to non-hepatic tissues. Here, targeted (t)LNPs designed for delivery of RNA payload to MM cells are presented. The tLNPs consist of a novel ionizable lipid and are coated with an anti-CD38 antibody (αCD38-tLNPs). To explore their therapeutic potential, it is demonstrated that LNPs encapsulating small interference RNA (siRNA) against cytoskeleton-associated protein 5 (CKAP5) lead to a ≈90% decrease in cell viability of MM cells in vitro. Next, a new xenograft MM mouse model is employed, which clinically resembles the human disease and demonstrates efficient homing of MM cells to the BM. Specific delivery of αCD38-tLNPs to BM-residing and disseminated MM cells and the improvement in therapeutic outcome of MM-bearing mice treated with αCD38-tLNPs-siRNA-CKAP5 are shown. These results underscore the potential of RNA therapeutics for treatment of MM and the importance of developing effective targeted delivery systems and reliable preclinical models.

A Combinatorial Library of Lipid Nanoparticles for Cell Type-Specific mRNA Delivery.

Ionizable lipid-based nanoparticles (LNPs) are the most advanced non-viral drug delivery systems for RNA therapeutics and vaccines. However, cell type-specific, extrahepatic mRNA delivery is still a major hurdle, hampering the development of novel therapeutic modalities. Herein, a novel ionizable lipid library is synthesized by modifying hydrophobic tail chains and linkers. Combined with other helper lipids and utilizing a microfluidic mixing approach, stable LNPs are formed. Using Luciferase-mRNA, mCherry mRNA, and Cre mRNA together with a TdTomato animal model, superior lipids forming LNPs for potent cell-type specific mRNA delivery are identified. In vitro assays concluded that combining branched ester tail chains with hydroxylamine linker negatively affects mRNA delivery efficiency. In vivo studies identify Lipid 23 as a liver-trophic, superior mRNA delivery lipid and Lipid 16 as a potent cell type-specific ionizable lipid for the CD11bhi macrophage population without an additional targeting moiety. Finally, in vivo mRNA delivery efficiency and toxicity of these LNPs are compared with SM-102-based LNP (Moderna's LNP formulation) and are shown to be cell-specific compared to SM-102-based LNPs. Overall, this study suggests that a structural combination of tail and linker can drive a novel functionality of LNPs in vivo.

Therapeutic gene silencing of CKAP5 leads to lethality in genetically unstable cancer cells.

The potential of microtubule-associated protein targets for cancer therapeutics remains largely unexplored due to the lack of target-specific agents. Here, we explored the therapeutic potential of targeting cytoskeleton-associated protein 5 (CKAP5), an important microtubule-associated protein, with CKAP5-targeting siRNAs encapsulated in lipid nanoparticles (LNPs). Our screening of 20 solid cancer cell lines demonstrated selective vulnerability of genetically unstable cancer cell lines in response to CKAP5 silencing. We identified a highly responsive chemo-resistant ovarian cancer cell line, in which CKAP5 silencing led to significant loss in EB1 dynamics during mitosis. Last, we demonstrated the therapeutic potential in an in vivo ovarian cancer model, showing 80% survival rate of siCKAP5 LNPs-treated animals. Together, our results highlight the importance of CKAP5 as a therapeutic target for genetically unstable ovarian cancer and warrants further investigation into its mechanistic aspects.

A single-dose F1-based mRNA-LNP vaccine provides protection against the lethal plague bacterium.

Messenger RNA (mRNA) lipid nanoparticle (LNP) vaccines have emerged as an effective vaccination strategy. Although currently applied toward viral pathogens, data concerning the platform's effectiveness against bacterial pathogens are limited. Here, we developed an effective mRNA-LNP vaccine against a lethal bacterial pathogen by optimizing mRNA payload guanine and cytosine content and antigen design. We designed a nucleoside-modified mRNA-LNP vaccine based on the bacterial F1 capsule antigen, a major protective component of Yersinia pestis, the etiological agent of plague. Plague is a rapidly deteriorating contagious disease that has killed millions of people during the history of humankind. Now, the disease is treated effectively with antibiotics; however, in the case of a multiple-antibiotic-resistant strain outbreak, alternative countermeasures are required. Our mRNA-LNP vaccine elicited humoral and cellular immunological responses in C57BL/6 mice and conferred rapid, full protection against lethal Y. pestis infection after a single dose. These data open avenues for urgently needed effective antibacterial vaccines.

Targeted nanomedicine: Lessons learned and future directions.

Designing a therapeutic modality that will reach a certain organ, tissue, or cell type is crucial for both the therapeutic efficiency and to limit off-target adverse effects. Nanoparticles carrying various drugs, such as nucleic acids, small molecules and proteins, are promoting modalities to this end. Beyond the need to identify a target for a specific indication, an adequate design has to address the multiple biological barriers, such as systemic barriers, dilution and unspecific distribution, tissue penetration and intracellular trafficking. The field of targeted delivery has developed rapidly in recent years, with tremendous progress made in understating the biological barriers, and new technologies to functionalize nanoparticles with targeting moieties for an accurate, specific and highly selective delivery. Implementing new approaches like multi-functionalized nanocarriers and machine learning models will advance the field for designing safe, cell -specific nanoparticle delivery systems. Here, we will critically review the current progress in the field and suggest novel strategies to improve cell specific delivery of therapeutic payloads.

Genome editing in cancer: Challenges and potential opportunities.

Ever since its mechanism was discovered back in 2012, the CRISPR/Cas9 system have revolutionized the field of genome editing. While at first it was seen as a therapeutic tool mostly relevant for curing genetic diseases, it has been recently shown to also hold the potential to become a clinically relevant therapy for cancer. However, there are multiple challenges that must be addressed prior to clinical testing. Predominantly, the safety of the system when used for in-vivo therapies, including off-target activity and the effects of the double strand break induction on genomic stability. Here, we will focus on the inherent challenges in the CRISPR/Cas9 system and discuss various opportunities to overcoming these challenges.

Identification of Cancer Cells in the Human Body by Anti-Telomerase Peptide Antibody: Towards the Isolation of Circulating Tumor Cells.

Early detection of tumor cells by identifying universal Tumor Associated Antigens (TAA) can drastically change our diagnostic, theranostic and therapeutic possibilities to cure cancer. Human Telomerase Reverse Transcriptase (hTERT), a hallmark of cancer, could act as an optimal TAA candidate. Here we report about the development of a monoclonal antibody against hTERT peptide (α-hTERT mAb) presented on the surface of cancer cells and its possible applications as a pan-cancer marker. Liquid biopsies, an innovative tool in precision oncology, comprising the noninvasive analysis of circulating tumor-derived material to counteract limitations associated with tissue biopsies. Within the tumor circulome, the US Food and Drug Administration already approved the use of circulating tumor cells (CTCs) as valid liquid biopsies. However, currently CTCs are being trapped using antibodies against specific cancer types, with anti EpCAM as the most common antibody, directed mainly against solid tumors. Moreover, the precision medicine approach is based on specific cancer type directed antibodies. Our novel mAb against the hTERT 16-mer peptide, corresponding to amino acids 611-626, is capable of detecting various types of cancer cells both in vitro and ex vivo from tumors of patients with either hematological or solid tumors. This antibody does not bind to normal lymphocytes cells. Cleavage of our antibody to F(ab')2 fragments increased its binding specificity to the tested cancer cells. Future studies may point to the use of this antibody in the procedure of capturing CTCs.

Nanotechnology-based strategies against SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has already infected more than 500 million people globally (as of May 2022), creating the coronavirus disease 2019 (COVID-19) pandemic. Nanotechnology has played a pivotal role in the fight against SARS-CoV-2 in various aspects, with the successful development of the two highly effective nanotechnology-based messenger RNA vaccines being the most profound. Despite the remarkable efficacy of mRNA vaccines against the original SARS-CoV-2 strain, hopes for quickly ending this pandemic have been dampened by the emerging SARS-CoV-2 variants, which have brought several new pandemic waves. Thus, novel strategies should be proposed to tackle the crisis presented by existing and emerging SARS-CoV-2 variants. Here, we discuss the SARS-CoV-2 variants from biological and immunological perspectives, and the rational design and development of novel and potential nanotechnology-based strategies to combat existing and possible future SARS-CoV-2 variants. The lessons learnt and design strategies developed from this battle against SARS-CoV-2 variants could also inspire innovation in the development of nanotechnology-based strategies for tackling other global infectious diseases and their future variants.

A lipid nanoparticle platform for mRNA delivery through repurposing of cationic amphiphilic drugs.

Since the recent clinical approval of siRNA-based drugs and COVID-19 mRNA vaccines, the potential of RNA therapeutics for patient healthcare has become widely accepted. Lipid nanoparticles (LNPs) are currently the most advanced nanocarriers for RNA packaging and delivery. Nevertheless, the intracellular delivery efficiency of state-of-the-art LNPs remains relatively low and safety and immunogenicity concerns with synthetic lipid components persist, altogether rationalizing the exploration of alternative LNP compositions. In addition, there is an interest in exploiting LNP technology for simultaneous encapsulation of small molecule drugs and RNA in a single nanocarrier. Here, we describe how well-known tricyclic cationic amphiphilic drugs (CADs) can be repurposed as both structural and functional components of lipid-based NPs for mRNA formulation, further referred to as CADosomes. We demonstrate that selected CADs, such as tricyclic antidepressants and antihistamines, self-assemble with the widely-used helper lipid DOPE to form cationic lipid vesicles for subsequent mRNA complexation and delivery, without the need for prior lipophilic derivatization. Selected CADosomes enabled efficient mRNA delivery in various in vitro cell models, including easy-to-transfect cancer cells (e.g. human cervical carcinoma HeLa cell line) as well as hard-to-transfect primary cells (e.g. primary bovine corneal epithelial cells), outperforming commercially available cationic liposomes and state-of-the-art LNPs. In addition, using the antidepressant nortriptyline as a model compound, we show that CADs can maintain their pharmacological activity upon CADosome incorporation. Furthermore, in vivo proof-of-concept was obtained, demonstrating CADosome-mediated mRNA delivery in the corneal epithelial cells of rabbit eyes, which could pave the way for future applications in ophthalmology. Based on our results, the co-formulation of CADs, helper lipids and mRNA into lipid-based nanocarriers is proposed as a versatile and straightforward approach for the rational development of drug combination therapies.