Selective Replacement of Cholesterol with Cationic Amphiphilic Drugs Enables the Design of Lipid Nanoparticles with Improved RNA Delivery.

The delivery of RNA across biological barriers can be achieved by encapsulation in lipid nanoparticles (LNPs). Cationic amphiphilic drugs (CADs) are pharmacologically diverse compounds with ionizable lipid-like features. In this work, we applied CADs as a fifth component of state-of-the-art LNPs via microfluidic mixing. Improved cytosolic delivery of both siRNA and mRNA was achieved by partly replacing the cholesterol fraction of LNPs with CADs. The LNPs could cross the mucus layer in a mucus-producing air-liquid interface model of human primary bronchial epithelial cells following nebulization. Moreover, CAD-LNPs demonstrated improved epithelial and endothelial targeting following intranasal administration in mice, without a marked pro-inflammatory signature. Importantly, quantification of the CAD-LNP molar composition, as demonstrated for nortriptyline, revealed a gradual leakage of the CAD from the formulation during LNP dialysis. Altogether, these data suggest that the addition of a CAD prior to the rapid mixing process might have an impact on the composition, structure, and performance of LNPs.

mRNA in cancer immunotherapy: beyond a source of antigen.

mRNA therapeutics have become the focus of molecular medicine research. Various mRNA applications have reached major milestones at high speed in the immuno-oncology field. This can be attributed to the knowledge that mRNA is one of nature's core building blocks carrying important information and can be considered as a powerful vector for delivery of therapeutic proteins to the patient.For a long time, the major focus in the use of in vitro transcribed mRNA was on development of cancer vaccines, using mRNA encoding tumor antigens to modify dendritic cells ex vivo. However, the versatility of mRNA and its many advantages have paved the path beyond this application. In addition, due to smart design of both the structural properties of the mRNA molecule as well as pharmaceutical formulations that improve its in vivo stability and selective targeting, the therapeutic potential of mRNA can be considered as endless.As a consequence, many novel immunotherapeutic strategies focus on the use of mRNA beyond its use as the source of tumor antigens. This review aims to summarize the state-of-the-art on these applications and to provide a rationale for their clinical application.

The Role of Ultrasound-Driven Microbubble Dynamics in Drug Delivery: From Microbubble Fundamentals to Clinical Translation.

In the last couple of decades, ultrasound-driven microbubbles have proven excellent candidates for local drug delivery applications. Besides being useful drug carriers, microbubbles have demonstrated the ability to enhance cell and tissue permeability and, as a consequence, drug uptake herein. Notwithstanding the large amount of evidence for their therapeutic efficacy, open issues remain. Because of the vast number of ultrasound- and microbubble-related parameters that can be altered and the variability in different models, the translation from basic research to (pre)clinical studies has been hindered. This review aims at connecting the knowledge gained from fundamental microbubble studies to the therapeutic efficacy seen in in vitro and in vivo studies, with an emphasis on a better understanding of the response of a microbubble upon exposure to ultrasound and its interaction with cells and tissues. More specifically, we address the acoustic settings and microbubble-related parameters (i.e., bubble size and physicochemistry of the bubble shell) that play a key role in microbubble-cell interactions and in the associated therapeutic outcome. Additionally, new techniques that may provide additional control over the treatment, such as monodisperse microbubble formulations, tunable ultrasound scanners, and cavitation detection techniques, are discussed. An in-depth understanding of the aspects presented in this work could eventually lead the way to more efficient and tailored microbubble-assisted ultrasound therapy in the future.

Challenges for labeling and longitudinal tracking of adoptively transferred autoreactive T lymphocytes in an experimental type-1 diabetes model.

OBJECTIVE: Tracking the autoreactive T-cell migration in the pancreatic region after labeling with fluorinated nanoparticles (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate]-perfluoro-15-crown-5-ether nanoparticles, PDP-PFCE NPs) in a diabetic murine model using 19F MRI. MATERIALS AND METHODS: Synthesis of novel PDP-PFCE fluorine tracer was performed for in vitro labeling of T cells. Labeling conditions were optimized using different PDP-PFCE NPs concentrations. For in vivo 19F MRI, mice were longitudinally followed after adoptive transfer of activated, autoreactive, labeled T cells in NOD.SCID mice. RESULTS: Established MR protocols were used for challenging T cell labeling to track inflammation in a model of diabetes after successful labeling of CD4+ and CD8+ T cells with PDP-PFCE NPs. However, T cells were difficult to be detected in vivo after their engraftment in animals. DISCUSSION: We showed successful in vitro labeling of T cells using novel fluorinated liposomal nanoparticles. However, insufficient and slow accumulation of labeled T cells and subsequent T cell proliferation in the pancreatic region remains as limitations of in vivo cell imaging by 19F MRI.

Nanomedicine to aid immunogenic cell death (ICD)-based anticancer therapy.

Immunogenic cell death (ICD) is emerging as a key component of antitumor therapy that harnesses the immune system of the patient to combat cancer. In recent years, several efforts were made to improve the ICD-based therapies. Here, we discuss how nanomaterial-based strategies increase the efficacy of ICD and highlight their benefits and challenges.

Considerations on the Design of Lipid-based mRNA Vaccines Against Cancer.

Throughout the last decades, mRNA vaccines have been developed as a cancer immunotherapeutic and the technology recently gained momentum during the COVID-19 pandemic. Recent promising results obtained from clinical trials investigating lipid-based mRNA vaccines in cancer therapy further highlighted the potential of this therapy. Interestingly, while the technologies being used in authorized mRNA vaccines for the prevention of COVID-19 are relatively similar, mRNA vaccines in clinical development for cancer vaccination show marked differences in mRNA modification, lipid carrier, and administration route. In this review, we describe findings on how these factors can impact the potency of mRNA vaccines in cancer therapy and provide insights into the complex interplay between them. We discuss how lipid carrier composition can affect passive targeting to immune cells to improve the efficacy and safety of mRNA vaccines. Finally, we summarize strategies that are established or still being explored to improve the efficacy of mRNA cancer vaccines and include next-generation vaccines that are on the horizon in clinical development.

Laser-induced vapor nanobubbles for B16-F10 melanoma cell killing and intracellular delivery of chemotherapeutics.

The most lethal form of skin cancer is cutaneous melanoma, a tumor that develops in the melanocytes, which are found in the epidermis. The treatment strategy of melanoma is dependent on the stage of the disease and often requires combined local and systemic treatment. Over the years, systemic treatment of melanoma has been revolutionized and shifted toward immunotherapeutic approaches. Phototherapies like photothermal therapy (PTT) have gained considerable attention in the field, mainly because of their straightforward applicability in melanoma skin cancer, combined with the fact that these strategies are able to induce immunogenic cell death (ICD), linked with a specific antitumor immune response. However, PTT comes with the risk of uncontrolled heating of the surrounding healthy tissue due to heat dissipation. Here, we used pulsed laser irradiation of endogenous melanin-containing melanosomes to induce cell killing of B16-F10 murine melanoma cells in a non-thermal manner. Pulsed laser irradiation of the B16-F10 cells resulted in the formation of water vapor nanobubbles (VNBs) around endogenous melanin-containing melanosomes, causing mechanical cell damage. We demonstrated that laser-induced VNBs are able to kill B16-F10 cells with high spatial resolution. When looking more deeply into the cell death mechanism, we found that a large part of the B16-F10 cells succumbed rapidly after pulsed laser irradiation, reaching maximum cell death already after 4 h. Practically all necrotic cells demonstrated exposure of phosphatidylserine on the plasma membrane and caspase-3/7 activity, indicative of regulated cell death. Furthermore, calreticulin, adenosine triphosphate (ATP) and high-mobility group box 1 (HMGB1), three key damage-associated molecular patterns (DAMPs) in ICD, were found to be exposed from B16-F10 cells upon pulsed laser irradiation to an extent that exceeded or was comparable to the bona fide ICD-inducer, doxorubicin. Finally, we could demonstrate that VNB formation from melanosomes induced plasma membrane permeabilization. This allowed for enhanced intracellular delivery of bleomycin, an ICD-inducing chemotherapeutic, which further boosted cell death with the potential to improve the systemic antitumor immune response.

Alpha-galactosylceramide improves the potency of mRNA LNP vaccines against cancer and intracellular bacteria.

Although various types of mRNA-based vaccines have been explored, the optimal conditions for induction of both humoral and cellular immunity remain rather unknown. In this study, mRNA vaccines of nucleoside-modified mRNA in lipoplexes (LPXs) or lipid nanoparticles (LNPs) were evaluated after administration in mice through different routes, assessing mRNA delivery, tolerability and immunogenicity. In addition, we investigated whether mRNA vaccines could benefit from the inclusion of the adjuvant alpha-galactosylceramide (αGC), an invariant Natural Killer T (iNKT) cell ligand. Intramuscular (IM) vaccination with ovalbumin (OVA)-encoding mRNA encapsulated in LNPs adjuvanted with αGC showed the highest antibody- and CD8+ T cell responses. Furthermore, we observed that addition of signal peptides and endocytic sorting signals of either LAMP1 or HLA-B7 in the OVA-encoding mRNA sequence further enhanced CD8+ T cell activation although reducing the induction of IgG antibody responses. Moreover, mRNA LNPs with the ionizable lipidoid C12-200 exhibited higher pro-inflammatory- and reactogenic activity compared to mRNA LNPs with SM-102, correlating with increased T cell activation and antitumor potential. We also observed that αGC could further enhance the cellular immunity of clinically relevant mRNA LNP vaccines, thereby promoting therapeutic antitumor potential. Finally, a Listeria monocytogenes mRNA LNP vaccine supplemented with αGC showed synergistic protective effects against listeriosis, highlighting a key advantage of co-activating iNKT cells in antibacterial mRNA vaccines. Taken together, our study offers multiple insights for optimizing the design of mRNA vaccines for disease applications, such as cancer and intracellular bacterial infections.

Targeted liposome-loaded microbubbles for cell-specific ultrasound-triggered drug delivery.

One of the main problems in cancer treatment is disease relapse through metastatic colonization, which is caused by circulating tumor cells (CTCs). This work reports on liposome-loaded microbubbles targeted to N-cadherin, a cell-cell adhesion molecule expressed by CTCs. It is shown that such microbubbles can indeed bind to N-cadherin at the surface of HMB2 cells. Interestingly, in a mixture of cells with and without N-cadherin expression, binding of the liposome-loaded microbubbles mainly occurs to the N-cadherin-expressing cells. Importantly, applying ultrasound results in the intracellular delivery of a model drug (loaded in the liposomes) in the N-cadherin-expressing cells only. As described in this paper, such liposome-loaded microbubbles may find application as theranostics and in devices aimed for the specific killing of CTCs in blood.

Continuous freeze-drying of messenger RNA lipid nanoparticles enables storage at higher temperatures.

Messenger RNA (mRNA) lipid nanoparticles (LNPs) have emerged at the forefront during the COVID-19 vaccination campaign. Despite their tremendous success, mRNA vaccines currently require storage at deep freeze temperatures which complicates their storage and distribution, and ultimately leads to lower accessibility to low- and middle-income countries. To elaborate on this challenge, we investigated freeze-drying as a method to enable storage of mRNA LNPs at room- and even higher temperatures. More specifically, we explored a novel continuous freeze-drying technique based on spin-freezing, which has several advantages compared to classical batch freeze-drying including a much shorter drying time and improved process and product quality controlling. Here, we give insight into the variables that play a role during freeze-drying by evaluating the impact of the buffer and mRNA LNP formulation (ionizable lipid to mRNA weight ratio) on properties such as size, morphology and mRNA encapsulation. We found that a sufficiently high ionizable lipid to mRNA weight ratio was necessary to prevent leakage of mRNA during freeze-drying and that phosphate and Tris, but not PBS, were appropriate buffers for lyophilization of mRNA LNPs. We also studied the stability of optimally lyophilized mRNA LNPs at 4 °C, 22 °C, and 37 °C and found that transfection properties of lyophilized mRNA LNPs were maintained during at least 12 weeks. To our knowledge, this is the first study that demonstrates that optimally lyophilized mRNA LNPs can be safely stored at higher temperatures for months without losing their transfection properties.

Dependence of sonoporation efficiency on microbubble size: An in vitro monodisperse microbubble study.

Sonoporation is the process where intracellular drug delivery is facilitated by ultrasound-driven microbubble oscillations. Several mechanisms have been proposed to relate microbubble dynamics to sonoporation including shear and normal stress. The present work aims to gain insight into the role of microbubble size on sonoporation and thereby into the relevant mechanism(s) of sonoporation. To this end, we measured the sonoporation efficiency while varying microbubble size using monodisperse microbubble suspensions. Sonoporation experiments were performed in vitro on cell monolayers using a single ultrasound pulse with a fixed frequency of 1 MHz while the acoustic pressure amplitude and pulse length were varied at 250, 500, and 750 kPa, and 10, 100, and 1000 cycles, respectively. Sonoporation efficiency was quantified using flow cytometry by measuring the FITC-dextran (4 kDa and 2 MDa) fluorescence intensity in 10,000 cells per experiment to average out inherent variations in the bioresponse. Using ultra-high-speed imaging at 10 million frames per second, we demonstrate that the bubble oscillation amplitude is nearly independent of the equilibrium bubble radius at acoustic pressure amplitudes that induce sonoporation (≥ 500 kPa). However, we show that sonoporation efficiency is strongly dependent on the equilibrium bubble size and that under all explored driving conditions most efficiently induced by bubbles with a radius of 4.7 µm. Polydisperse microbubbles with a typical ultrasound contrast agent size distribution perform almost an order of magnitude lower in terms of sonoporation efficiency than the 4.7-µm bubbles. We elucidate that for our system shear stress is highly unlikely the mechanism of action. By contrast, we show that sonoporation efficiency correlates well with an estimate of the bubble-induced normal stress.

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.

Nanobody-mediated SPECT/CT imaging reveals the spatiotemporal expression of programmed death-ligand 1 in response to a CD8+ T cell and iNKT cell activating mRNA vaccine.

Rationale: Although promising responses are obtained in patients treated with immune checkpoint inhibitors targeting programmed death ligand 1 (PD-L1) and its receptor programmed death-1 (PD-1), only a fraction of patients benefits from this immunotherapy. Cancer vaccination may be an effective approach to improve the response to immune checkpoint inhibitors anti-PD-L1/PD-1 therapy. However, there is a lack of research on the dynamics of PD-L1 expression in response to cancer vaccination. Methods: We performed non-invasive whole-body imaging to visualize PD-L1 expression at different timepoints after vaccination of melanoma-bearing mice. Mice bearing ovalbumin (OVA) expressing B16 tumors were i.v. injected with the Galsome mRNA vaccine: OVA encoding mRNA lipoplexes co-encapsulating a low or a high dose of the atypical adjuvant α-galactosylceramide (αGC) to activate invariant natural killer T (iNKT) cells. Serial non-invasive whole-body immune imaging was performed using a technetium-99m (99mTc)-labeled anti-PD-L1 nanobody, single-photon emission computerized tomography (SPECT) and X-ray computed tomography (CT) images were quantified. Additionally, cellular expression of PD-L1 was evaluated with flow cytometry. Results: SPECT/CT-imaging showed a rapid and systemic upregulation of PD-L1 after vaccination. PD-L1 expression could not be correlated to the αGC-dose, although we observed a dose-dependent iNKT cell activation. Dynamics of PD-L1 expression were organ-dependent and most pronounced in lungs and liver, organs to which the vaccine was distributed. PD-L1 expression in lungs increased immediately after vaccination and gradually decreased over time, whereas in liver, vaccination-induced PD-L1 upregulation was short-lived. Flow cytometric analysis of these organs further showed myeloid cells as well as non-immune cells with elevated PD-L1 expression in response to vaccination. SPECT/CT imaging of the tumor demonstrated that the expression of PD-L1 remained stable over time and was overall not affected by vaccination although flow cytometric analysis at the cellular level demonstrated changes in PD-L1 expression in various immune cell populations following vaccination. Conclusion: Repeated non-invasive whole-body imaging using 99mTc-labeled anti-PD-L1 nanobodies allows to document the dynamic nature of PD-L1 expression upon vaccination. Galsome vaccination rapidly induced systemic upregulation of PD-L1 expression with the most pronounced upregulation in lungs and liver while flow cytometry analysis showed upregulation of PD-L1 in the tumor microenvironment. This study shows that imaging using nanobodies may be useful for monitoring vaccine-mediated PD-L1 modulation in patients and could provide a rationale for combination therapy. To the best of our knowledge, this is the first report that visualizes PD-L1 expression upon cancer vaccination.

Nanoparticle mediated targeting of toll-like receptors to treat colorectal cancer.

Colorectal cancer (CRC) accounts for approximately 10% of all cancer cases worldwide. Conventional treatment has relied on chemotherapy, radiation therapy and surgery with limited success for patients with metastatic CRC. Toll like receptor (TLR) agonists have garnered attention for their ability to stimulate the innate immune system and consequently stimulate production of proinflammatory cytokines and activate an antitumor T cell response. However, activation of TLRs can also result in tumorigenesis and drug resistance depending on the specific TLR and cell that is targeted. Due to these contradictory effects of TLR stimulation, a key challenge is targeting specific cells, such as the dendritic cells or macrophages, to ensure the most optimal result. Additionally, TLR agonists are small molecules that can be cleared rapidly after local administration and can result in severe systemic side effects. This demonstrates the need to develop appropriate nanoparticle delivery systems for TLR agonists that can specifically target the innate immune system as a tool to treat CRC. In this review, the challenges in designing these nanoparticles will be discussed together with the recent advances of nanoparticle formulations containing TLR agonists.

Fluorine MR Imaging Probes Dynamic Migratory Profiles of Perfluorocarbon-Loaded Dendritic Cells After Streptozotocin-Induced Inflammation.

PURPOSE: The pathogenesis of type 1 diabetes (T1D) involves presentation of islet-specific self-antigens by dendritic cells (DCs) to autoreactive T cells, resulting in the destruction of insulin-producing pancreatic beta cells. We aimed to study the dynamic homing of diabetes-prone DCs to the pancreas and nearby organs with and without induction of pancreatic stress in a T1D susceptible model of repeated streptozotocin (STZ) injection. PROCEDURES: In vitro labeling of activated bone marrow-derived DCs (BMDCs) from NOD (Nonobese diabetes) mice was performed using zonyl perfluoro-15-crown-5-ether nanoparticles (ZPFCE-NPs). Internalization of particles was confirmed by confocal microscopy. Two groups of NOD.SCID (nonobese diabetic/severe combined immunodeficiency) mice with (induced by low dose STZ administration) or without pancreatic stress were compared. Diabetogenic BMDCs loaded with BDC2.5 mimotope were pre-labeled with ZPFCE-NPs and adoptively transferred into mice. Longitudinal in vivo fluorine MRI (19F MRI) was performed 24 h, 36 h and 48 h after transfer of BMDCs. For ex vivo quantification of labeled cells, 19F NMR and flow cytometry were performed on dissected tissues to validate in vivo 19F MRI data. RESULTS: In vitro flow cytometry and confocal microscopy confirmed high uptake of nanoparticles in BMDCs during the process of maturation. Migration/homing of activated and ZPFCE-NP- labeled BMDCs to different organs was monitored and quantified longitudinally, showing highest cell density in pancreas at 48-h time-point. Based on 19F MRI, STZ induced mild inflammation in the pancreatic region, as indicated by high accumulation of ZPFCE-NP-labeled BMDCs in the pancreas when compared to the vehicle group. Pancreatic draining lymph nodes showed elevated homing of labeled BMDCs in the vehicle groups in contrast to the STZ group after 72 h. The effect of STZ was confirmed by increased blood glucose levels. CONCLUSION: We showed the potential of 19F MRI for the non-invasive visualization and quantification of migrating immune cells in models for pancreatic inflammation after STZ administration. Without any intrinsic background signal, 19F MRI serves as a highly specific imaging tool to study the migration of diabetic-prone BMDCs in T1D models in vivo. This approach could particularly be of interest for the longitudinal assessment of established or novel anti-inflammatory therapeutic approaches in preclinical models.

Immunopeptidomics-based design of mRNA vaccine formulations against Listeria monocytogenes.

Listeria monocytogenes is a foodborne intracellular bacterial pathogen leading to human listeriosis. Despite a high mortality rate and increasing antibiotic resistance no clinically approved vaccine against Listeria is available. Attenuated Listeria strains offer protection and are tested as antitumor vaccine vectors, but would benefit from a better knowledge on immunodominant vector antigens. To identify novel antigens, we screen for Listeria peptides presented on the surface of infected human cell lines by mass spectrometry-based immunopeptidomics. In between more than 15,000 human self-peptides, we detect 68 Listeria immunopeptides from 42 different bacterial proteins, including several known antigens. Peptides presented on different cell lines are often derived from the same bacterial surface proteins, classifying these antigens as potential vaccine candidates. Encoding these highly presented antigens in lipid nanoparticle mRNA vaccine formulations results in specific CD8+ T-cell responses and induces protection in vaccination challenge experiments in mice. Our results can serve as a starting point for the development of a clinical mRNA vaccine against Listeria and aid to improve attenuated Listeria vaccines and vectors, demonstrating the power of immunopeptidomics for next-generation bacterial vaccine development.

Elucidating the mechanisms behind sonoporation with adeno-associated virus-loaded microbubbles.

Microbubbles are Food and Drug Administration (FDA) approved contrast agents for ultrasound imaging. It has been reported that applying ultrasound on drug-loaded microbubbles facilitates drug uptake by cells, due to so-named sonoporation. However, the biophysics behind sonoporation are not fully understood. It is believed that sonoporation results in a "direct" delivery of drugs in the cytoplasm of cells, though it has been suggested as well that sonoporation facilitates endocytosis which would improve the internalization of drugs by cells. To get a better understanding of sonoporation, this study reports on the ultrasound assisted delivery of adeno-associated virus (AAV) loaded on the surface of microbubbles. AAVs rely on endocytosis for efficient transduction of cells and are, consequently, an elegant tool to evaluate whether endocytosis is involved in ultrasound-induced sonoporation. Applying ultrasound on AAV-loaded microbubbles clearly improved the internalization of AAVs by cells, though transduction of the cells did not occur, indicating that by sonoporation substances become directly delivered in the cytosol of cells.

Design and evaluation of doxorubicin-containing microbubbles for ultrasound-triggered doxorubicin delivery: cytotoxicity and mechanisms involved.

Drug delivery with microbubbles and ultrasound is gaining more and more attention in the drug delivery field due to its noninvasiveness, local applicability, and proven safety in ultrasonic imaging techniques. In this article, we tried to improve the cytotoxicity of doxorubicin (DOX)-containing liposomes by preparing DOX-liposome-containing microbubbles for drug delivery with therapeutic ultrasound. In this way, the DOX release and uptake can be restricted to ultrasound-treated areas. Compared to DOX-liposomes, DOX-loaded microbubbles killed at least two times more melanoma cells after exposure to ultrasound. After treatment of the melanoma cells with DOX-liposome-loaded microbubbles and ultrasound, DOX was mainly present in the nuclei of the cancer cells, whereas it was mainly detected in the cytoplasm of cells treated with DOX-liposomes. Exposure of cells to DOX-liposome-loaded microbubbles and ultrasound caused an almost instantaneous cellular entry of the DOX. At least two mechanisms were identified that explain the fast uptake of DOX and the superior cell killing of DOX-liposome-loaded microbubbles and ultrasound. First, exposure of DOX-liposome-loaded microbubbles to ultrasound results in the release of free DOX that is more cytotoxic than DOX-liposomes. Second, the cellular entry of the released DOX is facilitated due to sonoporation of the cell membranes. The in vitro results shown in this article indicate that DOX-liposome-loaded microbubbles could be a very interesting tool to obtain an efficient ultrasound-controlled DOX delivery in vivo.

Physical transfection technologies for macrophages and dendritic cells in immunotherapy.

INTRODUCTION: Dendritic cells (DCs) and macrophages, two important antigen presenting cells (APCs) of the innate immune system, are being explored for the use in cell-based cancer immunotherapy. For this application, the therapeutic potential of patient-derived APCs is increased by delivering different types of functional macromolecules, such as mRNA and pDNA, into their cytosol. Compared to the use of viral and non-viral delivery vectors, physical intracellular delivery techniques are known to be more straightforward, more controllable, faster and generate high delivery efficiencies. AREAS COVERED: This review starts with electroporation as the most traditional physical transfection method, before continuing with the more recent technologies such as sonoporation, nanowires and microfluidic cell squeezing. A description is provided of each of those intracellular delivery technologies with their strengths and weaknesses, especially paying attention to delivery efficiency and safety profile. EXPERT OPINION: Given the common use of electroporation for the production of therapeutic APCs, it is recommended that more detailed studies are performed on the effect of electroporation on APC fitness, even down to the genetic level. Newer intracellular delivery technologies seem to have less impact on APC functionality but further work is needed to fully uncover their suitability to transfect APCs with different types of macromolecules.

The dawn of mRNA vaccines: The COVID-19 case.

In less than one year since the outbreak of the COVID-19 pandemic, two mRNA-based vaccines, BNT162b2 and mRNA-1273, were granted the first historic authorization for emergency use, while another mRNA vaccine, CVnCoV, progressed to phase 3 clinical testing. The COVID-19 mRNA vaccines represent a new class of vaccine products, which consist of synthetic mRNA strands encoding the SARS-CoV-2 Spike glycoprotein, packaged in lipid nanoparticles to deliver mRNA to cells. This review digs deeper into the scientific breakthroughs of the last decades that laid the foundations for the rapid rise of mRNA vaccines during the COVID-19 pandemic. As well as providing momentum for mRNA vaccines, SARS-CoV-2 represents an ideal case study allowing to compare design-activity differences between the different mRNA vaccine candidates. Therefore, a detailed overview of the composition and (pre)clinical performance of the three most advanced mRNA vaccines is provided and the influence of choices in their structural design on to their immunogenicity and reactogenicity profile is discussed in depth. In addition to the new fundamental insights in the mRNA vaccines' mode of action highlighted here, we also point out which unknowns remain that require further investigation and possibly, optimization in future mRNA vaccine development.