Effect of Polyplex Size on Penetration into Tumor Spheroids

Ovarian cancer is one of the most lethal gynecological cancers in the world. In recent years, nucleic acid (NA)-based formulations have been shown to be promising treatments for ovarian cancer, including tumor nodules. However, gene therapy is not that far advanced in clinical reality due to unfavorable physicochemical properties of the NAs, such as high molecular weight, poor cellular uptake, rapid degradation by nucleases, etc. One of the strategies used to overcome these drawbacks is the complexation of anionic NAs via electrostatic interactions with cationic polymers, resulting in the formation of so-called polyplexes. In this work, the role of the size of pDNA and siRNA polyplexes on their penetration into ovarian-cancer-based tumor spheroids was investigated. For this, a methoxypoly(ethylene glycol) poly(2-(dimethylamino)ethyl methacrylate) (mPEG-pDMAEMA) diblock copolymer was synthesized as a polymeric carrier for NA binding and condensation with either plasmid DNA (pDNA) or short interfering RNA (siRNA). When prepared in HEPES buffer (10 mM, pH 7.4) at a nitrogen/phosphate (N/P) charge ratio of 5 and pDNA polyplexes were formed with a size of 162 +/- 11 nm, while siRNA-based polyplexes displayed a size of 25 +/- 2 nm. The polyplexes had a slightly positive zeta potential of +7-8 mV in the same buffer. SiRNA and pDNA polyplexes were tracked in vitro into tumor spheroids, resembling in vivo avascular ovarian tumor nodules. For this purpose, reproducible spheroids were obtained by coculturing ovarian carcinoma cells with primary mouse embryonic fibroblasts in different ratios (5:2, 1:1, and 2:5). Penetration studies revealed that after 24 h of incubation, siRNA polyplexes were able to penetrate deeper into the homospheroids (composed of only cancer cells) and heterospheroids (cancer cells cocultured with fibroblasts) compared to pDNA polyplexes which were mainly located in the rim. The penetration of the polyplexes was slowed when increasing the fraction of fibroblasts present in the spheroids. Furthermore, in the presence of serum siRNA polyplexes encoding for luciferase showed a high cellular uptake in 2D cells resulting in similar to 50% silencing of luciferase expression. Taken together, these findings show that self-assembled small siRNA polyplexes have good potential as a platform to test ovarian tumor nodulus penetration.

Polysarcosine-based lipid formulations for intracranial delivery of mRNA

Messenger RNA (mRNA) is revolutionizing the future of therapeutics in a variety of diseases, including neurological disorders. Lipid formulations have shown to be an effective platform technology for mRNA delivery and are the basis for the approved mRNA vaccines. In many of these lipid formulations, polyethylene glycol (PEG)-functionalized lipid provides steric stabilization and thus plays a key role in improving the stability both ex vivo and in vivo. However, immune responses towards PEGylated lipids may compromise the use of those lipids in some applications (e.g., induction of antigen specific tolerance), or within sensitive tissues (e.g., central nervous system (CNS)). With respect to this issue, polysarcosine (pSar)-based lipopolymers were investigated as an alternative to PEG-lipid in mRNA lipoplexes for controlled intracerebral protein expression in this study. Four polysarcosine-lipids with defined sarcosine average molecular weight (Mn = 2 k, 5 k) and anchor diacyl chain length (m = 14, 18) were synthesized, and incorporated into cationic liposomes. We found that the content, pSar chain length and carbon tail lengths of pSar-lipids govern the transfection efficiency and biodistribution. Increasing carbon diacyl chain length of pSar-lipid led up to 4- and 6-fold lower protein expression in vitro. When the length of either pSar chain or lipid carbon tail increased, the transfection efficiency decreased while the circulation time was prolonged. mRNA lipoplexes containing 2.5% C14-pSar2k resulted in the highest mRNA translation in the brain of zebrafish embryos through intraventricular injection, while C18-pSar2k-liposomes showed a comparable circulation with DSPE-PEG2k-liposomes after systemic administration. To conclude, pSar-lipid enable efficient mRNA delivery, and can substitute PEG-lipids in lipid formulations for controlled protein expression within the CNS.Drug Delivery Technolog

Together is better: mRNA co-encapsulation in lipoplexes is required to obtain ratiometric co-delivery and protein expression on the single cell level

Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co-deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle-lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre-defined ratio. A mixture of lipoplexes containing only one mRNA type (single-lipoplexes), however, seem to follow the "first come - first serve" principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single-cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle- and single-lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle-lipoplexes outperformed single-lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells.Drug Delivery Technolog

Structure of a Chaperone-Usher Pilus reveals the molecular basis of rod uncoiling

Types 1 and P pili are prototypical bacterial cell-surface appendages playing essential roles in mediating adhesion of bacteria to the urinary tract. These pili, assembled by the chaperone-usher pathway, are polymers of pilus subunits assembling into two parts: a thin, short tip fibrillum at the top, mounted on a long pilus rod. The rod adopts a helical quaternary structure and is thought to play essential roles: its formation may drive pilus extrusion by preventing backsliding of the nascent growing pilus within the secretion pore; the rod also has striking spring-like properties, being able to uncoil and recoil depending on the intensity of shear forces generated by urine flow. Here, we present an atomic model of the P pilus generated from a 3.8 Å resolution cryo-electron microscopy reconstruction. This structure provides the molecular basis for the rod’s remarkable mechanical properties and illuminates its role in pilus secretion

Architectures and biogenesis of non-flagellar protein appendages in Gram-negative bacteria

Bacteria commonly expose non-flagellar proteinaceous appendages on their outer surfaces. These extracellular structures, called pili or fimbriae, are employed in attachment and invasion, biofilm formation, cell motility or protein and DNA transport across membranes. Over the past 15 years, the power of molecular and structural techniques has revolutionalized our understanding of the biogenesis, structure, function and mode of action of these bacterial organelles. Here, we review the five known classes of Gram-negative non-flagellar appendages from a biosynthetic and structural point of view

In vitro and in vivo mRNA delivery using lipid-enveloped pHresponsive polymer nanoparticles

Biodegradable core−shell structured nanoparticles with a poly(β-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell were developed for in vivo mRNA delivery with a view toward delivery of mRNA-based vaccines. The pH-responsive PBAE component was chosen to promote endosome disruption, while the lipid surface layer was selected to minimize toxicity of the polycation core. Messenger RNA was efficiently adsorbed via electrostatic interactions onto the surface of these net positively charged nanoparticles. In vitro, mRNA-loaded particle uptake by dendritic cells led to mRNA delivery into the cytosol with low cytotoxicity, followed by translation of the encoded protein in these difficult-to-transfect cells at a frequency of 30%. Particles loaded with mRNA administered intranasally (i.n.) in mice led to the expression of the reporter protein luciferase in vivo as soon as 6 h after administration, a time point when naked mRNA given i.n. showed no expression. At later time points, luciferase expression was detected in naked mRNA-treated mice, but this group showed a wide variation in levels of transfection, compared to particle-treated mice. This system may thus be promising for noninvasive delivery of mRNA-based vaccines.United States. Dept. of Defense (Institute for Soldier Nanotechnology, contract W911NF-07-D-0004)Ragon Institute of MGH, MIT and HarvardSingapore. Agency for Science, Technology and ResearchHoward Hughes Medical Institute (Investigator

Preparation and Characterization of Cationic PLA-PEG Nanoparticles for Delivery of Plasmid DNA

The purpose of the present work was to formulate and evaluate cationic poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles as novel non-viral gene delivery nano-device. Cationic PLA-PEG nanoparticles were prepared by nanoprecipitation method. The gene loaded nanoparticles were obtained by incubating the report gene pEGFP with cationic PLA-PEG nanoparticles. The physicochemical properties (e.g., morphology, particle size, surface charge, DNA binding efficiency) and biological properties (e.g., integrity of the released DNA, protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in Hela cells) of the gene loaded PLA-PEG nanoparticles were evaluated, respectively. The obtained cationic PLA-PEG nanoparticles and gene loaded nanoparticles were both spherical in shape with average particle size of 89.7 and 128.9 nm, polydispersity index of 0.185 and 0.161, zeta potentials of +28.9 and +16.8 mV, respectively. The obtained cationic PLA-PEG nanoparticles with high binding efficiency (>95%) could protect the loaded DNA from the degradation by nuclease and plasma. The nanoparticles displayed sustained-release properties in vitro and the released DNA maintained its structural and functional integrity. It also showed lower cytotoxicity than Lipofectamine 2000 and could successfully transfect gene into Hela cells even in presence of serum. It could be concluded that the established gene loaded cationic PLA-PEG nanoparticles with excellent properties were promising non-viral nano-device, which had potential to make cancer gene therapy achievable

Efficient long-range conduction in cable bacteria through nickel protein wires

Filamentous cable bacteria display long-range electron transport, generating electrical currents over centimeter distances through a highly ordered network of fibers embedded in their cell envelope. The conductivity of these periplasmic wires is exceptionally high for a biological material, but their chemical structure and underlying electron transport mechanism remain unresolved. Here, we combine high-resolution microscopy, spectroscopy, and chemical imaging on individual cable bacterium filaments to demonstrate that the periplasmic wires consist of a conductive protein core surrounded by an insulating protein shell layer. The core proteins contain a sulfur-ligated nickel cofactor, and conductivity decreases when nickel is oxidized or selectively removed. The involvement of nickel as the active metal in biological conduction is remarkable, and suggests a hitherto unknown form of electron transport that enables efficient conduction in centimeter-long protein structures

Structural Differences between the Streptococcus agalactiae Housekeeping and Pilus-Specific Sortases: SrtA and SrtC1

The assembly of pili on the cell wall of Gram-positive bacteria requires transpeptidase enzymes called sortases. In Streptococcus agalactiae, the PI-1 pilus island of strain 2603V/R encodes two pilus-specific sortases (SrtC1 and SrtC2) and three pilins (GBS80, GBS52 and GBS104). Although either pilus-specific sortase is sufficient for the polymerization of the major pilin, GBS80, incorporation of the minor pilins GBS52 and GBS104 into the pilus structure requires SrtC1 and SrtC2, respectively. The S. agalactiae housekeeping sortase, SrtA, whose gene is present at a different location and does not catalyze pilus polymerization, was shown to be involved in cell wall anchoring of pilus polymers. To understand the structural basis of sortases involved in such diverse functions, we determined the crystal structures of S. agalactiae SrtC1 and SrtA. Both enzymes are made of an eight-stranded beta-barrel core with variations in their active site architecture. SrtA exhibits a catalytic triad arrangement similar to that in Streptococcus pyogenes SrtA but different from that in Staphylococcus aureus SrtA. In contrast, the SrtC1 enzyme contains an N-terminal helical domain and a ‘lid’ in its putative active site, which is similar to that seen in Streptococcus pneumoniae pilus-specific sortases, although with subtle differences in positioning and composition. To understand the effect of such differences on substrate recognition, we have also determined the crystal structure of a SrtC1 mutant, in which the conserved DP(W/F/Y) motif was replaced with the sorting signal motif of GBS80, IPNTG. By comparing the structures of WT wild type SrtA and SrtC1 and the ‘lid’ mutant of SrtC1, we propose that structural elements within the active site and the lid may be important for defining the role of specific sortase in pili biogenesis