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.

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.

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.

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.

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.

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.

Synthetic Cells Synthesize Therapeutic Proteins inside Tumors.

Synthetic cells, artificial cell-like particles, capable of autonomously synthesizing RNA and proteins based on a DNA template, are emerging platforms for studying cellular functions and for revealing the origins-of-life. Here, it is shown for the first time that artificial lipid-based vesicles, containing the molecular machinery necessary for transcription and translation, can be used to synthesize anticancer proteins inside tumors. The synthetic cells are engineered as stand-alone systems, sourcing nutrients from their biological microenvironment to trigger protein synthesis. When pre-loaded with template DNA, amino acids and energy-supplying molecules, up to 2 × 107 copies of green fluorescent protein are synthesized in each synthetic cell. A variety of proteins, having molecular weights reaching 66 kDa and with diagnostic and therapeutic activities, are synthesized inside the particles. Incubating synthetic cells, encoded to secrete Pseudomonas exotoxin A (PE) with 4T1 breast cancer cells in culture, resulted in killing of most of the malignant cells. In mice bearing 4T1 tumors, histological evaluation of the tumor tissue after a local injection of PE-producing particles indicates robust apoptosis. Synthetic cells are new platforms for synthesizing therapeutic proteins on-demand in diseased tissues.

A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.

Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40-150 µg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.