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.

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.

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.

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.

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.

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.

SPANX Control of Lamin A/C Modulates Nuclear Architecture and Promotes Melanoma Growth.

Mechanisms regulating nuclear organization control fundamental cellular processes, including the cell and chromatin organization. Their disorganization, including aberrant nuclear architecture, has been often implicated in cellular transformation. Here, we identify Lamin A, among proteins essential for nuclear architecture, as SPANX (sperm protein associated with the nucleus on the X chromosome), a cancer testis antigen previously linked to invasive tumor phenotypes, interacting protein in melanoma. SPANX interaction with Lamin A was mapped to the immunoglobulin fold-like domain, a region critical for Lamin A function, which is often mutated in laminopathies. SPANX downregulation in melanoma cell lines perturbed nuclear organization, decreased cell viability, and promoted senescence-associated phenotypes. Moreover, SPANX knockdown (KD) in melanoma cells promoted proliferation arrest, a phenotype mediated in part by IRF3/IL1A signaling. SPANX KD in melanoma cells also prompted the secretion of IL1A, which attenuated the proliferation of naïve melanoma cells. Identification of SPANX as a nuclear architecture complex component provides an unexpected insight into the regulation of Lamin A and its importance in melanoma. IMPLICATIONS: SPANX, a testis protein, interacts with LMNA and controls nuclear architecture and melanoma growth.

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA→RNA→protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

Integrating Artificial Intelligence and Nanotechnology for Precision Cancer Medicine.

Artificial intelligence (AI) and nanotechnology are two fields that are instrumental in realizing the goal of precision medicine-tailoring the best treatment for each cancer patient. Recent conversion between these two fields is enabling better patient data acquisition and improved design of nanomaterials for precision cancer medicine. Diagnostic nanomaterials are used to assemble a patient-specific disease profile, which is then leveraged, through a set of therapeutic nanotechnologies, to improve the treatment outcome. However, high intratumor and interpatient heterogeneities make the rational design of diagnostic and therapeutic platforms, and analysis of their output, extremely difficult. Integration of AI approaches can bridge this gap, using pattern analysis and classification algorithms for improved diagnostic and therapeutic accuracy. Nanomedicine design also benefits from the application of AI, by optimizing material properties according to predicted interactions with the target drug, biological fluids, immune system, vasculature, and cell membranes, all affecting therapeutic efficacy. Here, fundamental concepts in AI are described and the contributions and promise of nanotechnology coupled with AI to the future of precision cancer medicine are reviewed.

Bovine pericardial extracellular matrix niche modulates human aortic endothelial cell phenotype and function.

Xenogeneic biomaterials contain biologically relevant extracellular matrix (ECM) composition and organization, making them potentially ideal surgical grafts and tissue engineering scaffolds. Defining the effect of ECM niche (e.g., basement membrane vs. non-basement membrane) on repopulating cell phenotype and function has important implications for use of xenogeneic biomaterials, particularly in vascular applications. We aim to understand how serous (i.e., basement membrane) versus fibrous (i.e., non-basement membrane) ECM niche of antigen-removed bovine pericardium (AR-BP) scaffolds influence human aortic endothelial cell (hAEC) adhesion, growth, phenotype, inflammatory response and laminin production. At low and moderate seeding densities hAEC proliferation was significantly increased on the serous side. Similarly, ECM niche modulated cellular morphology, with serous side seeding resulting in a more rounded aspect ratio and intact endothelial layer formation. At moderate seeding densities, hAEC production of human laminin was enhanced following serous seeding. Finally, inflammatory marker and pro-inflammatory cytokine expression decreased following long-term cell growth regardless of seeding side. This work demonstrates that at low and moderate seeding densities AR-BP sidedness significantly impacts endothelial cell growth, morphology, human laminin production, and inflammatory state. These findings suggest that ECM niche has a role in modulating response of repopulating recipient cells toward AR-BP scaffolds for vascular applications.

Collagenase Nanoparticles Enhance the Penetration of Drugs into Pancreatic Tumors.

Overexpressed extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDAC) limits drug penetration into the tumor and is associated with poor prognosis. Here, we demonstrate that a pretreatment based on a proteolytic-enzyme nanoparticle system disassembles the dense PDAC collagen stroma and increases drug penetration into the pancreatic tumor. More specifically, the collagozome, a 100 nm liposome encapsulating collagenase, was rationally designed to protect the collagenase from premature deactivation and prolonged its release rate at the target site. Collagen is the main component of the PDAC stroma, reaching 12.8 ± 2.3% vol in diseased mice pancreases, compared to 1.4 ± 0.4% in healthy mice. Upon intravenous injection of the collagozome, ∼1% of the injected dose reached the pancreas over 8 h, reducing the level of fibrotic tissue to 5.6 ± 0.8%. The collagozome pretreatment allowed increased drug penetration into the pancreas and improved PDAC treatment. PDAC tumors, pretreated with the collagozome followed by paclitaxel micelles, were 87% smaller than tumors pretreated with empty liposomes followed by paclitaxel micelles. Interestingly, degrading the ECM did not increase the number of circulating tumor cells or metastasis. This strategy holds promise for degrading the extracellular stroma in other diseases as well, such as liver fibrosis, enhancing tissue permeability before drug administration.

Tailoring the lipid composition of nanoparticles modulates their cellular uptake and affects the viability of triple negative breast cancer cells.

Lipid nanoparticles are used widely as anticancer drug and gene delivery systems. Internalizing into the target cell is a prerequisite for the proper activity of many nanoparticulate drugs. We show here, that the lipid composition of a nanoparticle affects its ability to internalize into triple-negative breast cancer cells. The lipid headgroup had the greatest effect on enhancing cellular uptake compared to other segments of the molecule. Having a receptor-targeted headgroup induced the greatest increase in cellular uptake, followed by cationic amine headgroups, both being superior to neutral (zwitterion) phosphatidylcholine or to negatively-charged headgroups. The lipid tails also affected the magnitude of cellular uptake. Longer acyl chains facilitated greater liposomal cellular uptake compared to shorter tails, 18:0 > 16:0 > 14:0. When having the same lipid tail length, unsaturated lipids were superior to saturated ones, 18:1 > 18:0. Interestingly, liposomes composed of phospholipids having 14:0 or 12:0-carbon-long-tails, such as DMPC and DLPC, decreased cell viability in a concertation dependent manner, due to a destabilizing effect these lipids had on the cancer cell membrane. Contrarily, liposomes composed of phospholipids having longer carbon tails (16:0 and 18:0), such as DPPC and HSPC, enhanced cancer cell proliferation. This effect is attributed to the integration of the exogenous liposomal lipids into the cancer-cell membrane, supporting the proliferation process. Cholesterol is a common lipid additive in nanoscale formulations, rigidifying the membrane and stabilizing its structure. Liposomes composed of DMPC (14:0) showed increased cellular uptake when enriched with cholesterol, both by endocytosis and by fusion. Contrarily, the effect of cholesterol on HSPC (18:0) liposomal uptake was minimal. Furthermore, the concentration of nanoparticles in solution affected their cellular uptake. The higher the concentration of nanoparticles the greater the absolute number of nanoparticles taken up per cell. However, the efficiency of nanoparticle uptake, i.e. the percent of nanoparticles taken up by cells, decreased as the concentration of nanoparticles increased. This study demonstrates that tuning the lipid composition and concentration of nanoscale drug delivery systems can be leveraged to modulate their cellular uptake.

Graft-specific immune tolerance is determined by residual antigenicity of xenogeneic extracellular matrix scaffolds.

Antigenicity remains the primary barrier towards expanding the use of unfixed xenogeneic biomaterials in clinical applications. An unfixed xenogeneic biomaterial devoid of antigenicity, with maintained structural and mechanical integrity, has potential to overcome the limitations of current clinically utilized glutaraldehyde-fixed xenogeneic biomaterials, such as heart valve bioprostheses. Unfortunately, the threshold level of residual antigenicity necessary to overcome graft-specific immune responses in unfixed xenogeneic tissue has yet to be determined. Furthermore, little information is known regarding the extent to which in vitro disruption of native ECM properties, resulting from decellularization or antigen removal procedures, are tolerated following in vivo implantation. This manuscript demonstrates that humoral adaptive immune responses are more sensitive to residual xenogeneic biomaterial antigen content than are cell-mediated adaptive responses. Critically, the threshold for tolerable residual antigenicity is identified, with removal of ≥92% of lipophilic antigens required to reduce adaptive immune responses to levels equivalent to glutaraldehyde fixed tissue. Finally, the results demonstrated that the innate immune system tolerates minor changes in protein organization provided that molecular structure is maintained. Antigen removed xenogeneic biomaterials achieving these in vitro success criteria induce in vivo adaptive and innate tolerance, while modulating pro-regenerative constructive remodeling. STATEMENT OF SIGNIFICANCE: Removal of antigenic components from candidate xenogeneic biomaterials is the primary success criteria for development of extracellular matrix (ECM) scaffolds in tissue engineering applications. Currently, the threshold level of residual biomaterial antigenicity required to overcome recipient graft-specific adaptive immune responses is unknown. Additionally, the extent to which the innate immune response tolerates changes to the native ECM, resulting from the ECM scaffold production process, has yet to be determined. This manuscript not only establishes the threshold for tolerable residual antigenicity, but also demonstrates that deviations in protein organization are tolerated by the innate immune system, provided macromolecular structure remains intact. In doing so, we provide the foundation of an immunologically-acceptable unfixed xenogeneic biomaterial for use in clinical applications.

Label-Free Assessment of Collagenase Digestion on Bovine Pericardium Properties by Fluorescence Lifetime Imaging.

The extracellular matrix architecture of bovine pericardium (BP) has distinct biochemical and biomechanical properties that make it a useful biomaterial in the field of regenerative medicine. Collagen represents the dominant structural protein of BP and is therefore intimately associated with the properties of this biomaterial. Enzymatic degradation of collagen molecules is critical for extracellular matrix turnover, remodeling and ultimately tissue regeneration. We present a quantitative, label-free and non-destructive method for monitoring changes in biochemical and biomechanical properties of BP during tissue degradation, based on multi-spectral fluorescence lifetime imaging (ms-FLIm). Strong correlations of fluorescence intensity ratio and average fluorescence lifetime were identified with collagen content, Young's Modulus and Ultimate tensile strength during collagenase degradation, indicating the potential of optically monitoring collagen degradation using ms-FLIm. The obtained results demonstrate the value of ms-FLIm to assess the quality of biomaterials in situ for applications in regenerative medicine.

Fiber-based fluorescence lifetime imaging of recellularization processes on vascular tissue constructs.

New techniques able to monitor the maturation of tissue engineered constructs over time are needed for a more efficient control of developmental parameters. Here, a label-free fluorescence lifetime imaging (FLIm) approach implemented through a single fiber-optic interface is reported for nondestructive in situ assessment of vascular biomaterials. Recellularization processes of antigen removed bovine pericardium scaffolds with endothelial cells and mesenchymal stem cells were evaluated on the serous and the fibrous sides of the scaffolds, 2 distinct extracellular matrix niches, over the course of a 7 day culture period. Results indicated that fluorescence lifetime successfully report cell presence resolved from extracellular matrix fluorescence. The recellularization process was more rapid on the serous side than on the fibrous side for both cell types, and endothelial cells expanded faster than mesenchymal stem cells on antigen-removed bovine pericardium. Fiber-based FLIm has the potential to become a nondestructive tool for the assessment of tissue maturation by allowing in situ imaging of intraluminal vascular biomaterials.

Apoptotic Cell Clearance in Development.

Programmed cell death and its specific form apoptosis play an important role during development of multicellular organisms. They are crucial for morphogenesis and organ sculpting as well as for adjusting cell number in different systems. Removal of apoptotic cells is the last critical step of apoptosis. Apoptotic cells are properly and efficiently recognized and eliminated through phagocytosis, which is performed by professional and nonprofessional phagocytes. Phagocytosis of apoptotic cells or apoptotic cell clearance is a dynamic multistep process, involving interactions between phagocytic receptors and ligands on apoptotic cells, which are highly conserved in evolution. However, this process is extremely redundant in mammals, containing multiple factors playing similar roles in the process. Using model organisms such as Caenorhabditis elegans, Drosophila melanogaster, zebrafish, and mouse permits addressing fundamental questions in developmental cell clearance by a comprehensive approach including powerful genetics and cell biological tools enriched by live imaging. Recent studies in model organisms have enhanced significantly our understanding of the molecular and cellular basis of apoptotic cell clearance during development. Here, we review the current knowledge and illuminate the great potential of the research performed in genetic models, which opens new directions in developmental biology.