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.

Biocompatibility, biodegradation and excretion of polylactic acid (PLA) in medical implants and theranostic systems.

Polylactic acid (PLA) is the most commonly used biodegradable polymer in clinical applications today. Examples range from drug delivery systems, tissue engineering, temporary and long-term implantable devices; constantly expanding to new fields. This is owed greatly to the polymer's favorable biocompatibility and to its safe degradation products. Once coming in contact with biological media, the polymer begins breaking down, usually by hydrolysis, into lactic acid (LA) or to carbon dioxide and water. These products are metabolized intracellularly or excreted in the urine and breath. Bacterial infection and foreign-body inflammation enhance the breakdown of PLA, through the secretion of enzymes that degrade the polymeric matrix. The biodegradation occurs both on the surface of the polymeric device and inside the polymer body, by diffusion of water between the polymer chains. The median half-life of the polymer is 30 weeks; however, this can be lengthened or shortened to address the clinical needs. Degradation kinetics can be tuned by determining the molecular composition and the physical architecture of the device. Using L- or D- chirality of the LA will greatly slow or lengthen the degradation rates, respectively. Despite the fact that this polymer is more than 150 years old, PLA remains a fertile platform for biomedical innovation and fundamental understanding of how artificial polymers can safely coexist with biological systems.

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.

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.

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.

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.

Three-dimensional localization microscopy in live flowing cells.

Capturing the dynamics of live cell populations with nanoscale resolution poses a significant challenge, primarily owing to the speed-resolution trade-off of existing microscopy techniques. Flow cytometry would offer sufficient throughput, but lacks subsample detail. Here we show that imaging flow cytometry, in which the point detectors of flow cytometry are replaced with a camera to record 2D images, is compatible with 3D localization microscopy through point-spread-function engineering, which encodes the depth of the emitter into the emission pattern captured by the camera. The extraction of 3D positions from sub-cellular objects of interest is achieved by calibrating the depth-dependent response of the imaging system using fluorescent beads mixed with the sample buffer. This approach enables 4D imaging of up to tens of thousands of objects per minute and can be applied to characterize chromatin dynamics and the uptake and spatial distribution of nanoparticles in live cancer cells.

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.

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.

Proteolytic Nanoparticles Replace a Surgical Blade by Controllably Remodeling the Oral Connective Tissue.

Surgical blades are common medical tools. However, blades cannot distinguish between healthy and diseased tissue, thereby creating unnecessary damage, lengthening recovery, and increasing pain. We propose that surgical procedures can rely on natural tissue remodeling tools-enzymes, which are the same tools our body uses to repair itself. Through a combination of nanotechnology and a controllably activated proteolytic enzyme, we performed a targeted surgical task in the oral cavity. More specifically, we engineered nanoparticles that contain collagenase in a deactivated form. Once placed at the surgical site, collagenase was released at a therapeutic concentration and activated by calcium, its biological cofactor that is naturally present in the tissue. Enhanced periodontal remodeling was recorded due to enzymatic cleavage of the supracrestal collagen fibers that connect the teeth to the underlying bone. When positioned in their new orientation, natural tissue repair mechanisms supported soft and hard tissue recovery and reduced tooth relapse. Through the combination of nanotechnology and proteolytic enzymes, localized surgical procedures can now be less invasive.