Synthesis and characterization of ionic block copolymer templated calcium phosphate nanocomposites

Self-assembling thermo-reversibly gelling anionic and zwitterionic pentablock copolymers were used as templates for precipitation of calcium phosphate nanostructures, controlling their size and ordered structural arrangement. Calcium and phosphate ions were dissolved in a block-copolymer micellar dispersion at low temperatures. Aging at ambient temperature produced inorganic nanoparticles, presumably nucleated by ionic interactions. The self-assembled nanocomposites were characterized by small-angle X-ray and neutron scattering (SAXS/SANS), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and transmission electron microscopy (TEM). 1H-31P NMR with 1H spin diffusion from polymer to phosphate proved the formation of nanocomposites, with inorganic particle sizes from ∼2 nm, characterized by 1H-31P dipolar couplings, to \u3e 100 nm. TEM analysis showed polymer micelles surrounded by calcium phosphate. SAXS attested that a significant fraction of the calcium phosphate was templated by the polymer micelles. SANS data indicated that the order of the polymer was enhanced by the inorganic phase. The nanocomposite gels exhibited higher moduli than the neat polymer gels. The calcium phosphate was characterized by TGA, X-ray diffraction, high-resolution TEM, and various NMR techniques. An unusual crystalline phase with \u3e2 chemically and \u3e3 magnetically inequivalent HPO4 2- ions was observed with the zwitterionic copolymer, highlighting the influence of the polymer on the calcium phosphate crystallization. The inorganic fraction of the nanocomposite was around 30 wt % of the dried hydrogel. Thus, a significant fraction of calcium phosphate has been templated by the tailored self-assembling ionic block copolymers, providing a bottom-up approach to nanocomposite synthesis

The Possible "Proton Sponge " Effect of Polyethylenimine (PEI) Does Not Include Change in Lysosomal pH.

Polycations such as polyethylenimine (PEI) are used in many novel nonviral vector designs and there are continuous efforts to increase our mechanistic understanding of their interactions with cells. Even so, the mechanism of polyplex escape from the endosomal/lysosomal pathway after internalization is still elusive. The “proton sponge ” hypothesis remains the most generally accepted mechanism, although it is heavily debated. This hypothesis is associated with the large buffering capacity of PEI and other polycations, which has been interpreted to cause an increase in lysosomal pH even though no conclusive proof has been provided. In the present study, we have used a nanoparticle pH sensor that was developed for pH measurements in the endosomal/lysosomal pathway. We have carried out quantitative measurements of lysosomal pH as a function of PEI content and correlate the results to the “proton sponge ” hypothesis. Our measurements show that PEI does not induce change in lysosomal pH as previously suggested and quantification of PEI concentrations in lysosomes makes it uncertain that the “proton sponge ” effect is the dominant mechanism of polyplex escape

Pyruvate kinase M2-specific siRNA induces apoptosis and tumor regression

Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20111487/DC1.The development of cancer-specific therapeutics has been limited because most healthy cells and cancer cells depend on common pathways. Pyruvate kinase (PK) exists in M1 (PKM1) and M2 (PKM2) isoforms. PKM2, whose expression in cancer cells results in aerobic glycolysis and is suggested to bestow a selective growth advantage, is a promising target. Because many oncogenes impart a common alteration in cell metabolism, inhibition of the M2 isoform might be of broad applicability. We show that several small interfering (si) RNAs designed to target mismatches between the M2 and M1 isoforms confer specific knockdown of the former, resulting in decreased viability and increased apoptosis in multiple cancer cell lines but less so in normal fibroblasts or endothelial cells. In vivo delivery of siPKM2 additionally causes substantial tumor regression of established xenografts. Our results suggest that the inherent nucleotide-level specificity of siRNA can be harnessed to develop therapeutics that target isoform-specific exons in genes exhibiting differential splicing patterns in various cell types.MIT-Harvard Center for Cancer Nanotechnology ExcellenceNational Cancer Institute (U.S.) (Grant U54 CA151884)Marie D. and Pierre Casimir-Lambert FundNational Cancer Institute (U.S.) (Cancer Center Support (core) grant P30-CA14051

MICU2, a Paralog of MICU1, Resides within the Mitochondrial Uniporter Complex to Regulate Calcium Handling

Mitochondrial calcium uptake is present in nearly all vertebrate tissues and is believed to be critical in shaping calcium signaling, regulating ATP synthesis and controlling cell death. Calcium uptake occurs through a channel called the uniporter that resides in the inner mitochondrial membrane. Recently, we used comparative genomics to identify MICU1 and MCU as the key regulatory and putative pore-forming subunits of this channel, respectively. Using bioinformatics, we now report that the human genome encodes two additional paralogs of MICU1, which we call MICU2 and MICU3, each of which likely arose by gene duplication and exhibits distinct patterns of organ expression. We demonstrate that MICU1 and MICU2 are expressed in HeLa and HEK293T cells, and provide multiple lines of biochemical evidence that MCU, MICU1 and MICU2 reside within a complex and cross-stabilize each other's protein expression in a cell-type dependent manner. Using in vivo RNAi technology to silence MICU1, MICU2 or both proteins in mouse liver, we observe an additive impairment in calcium handling without adversely impacting mitochondrial respiration or membrane potential. The results identify MICU2 as a new component of the uniporter complex that may contribute to the tissue-specific regulation of this channel.National Institutes of Health (U.S.) (GM0077465)National Institutes of Health (U.S.) (DK080261

Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system

DNA-based gene therapy has considerable therapeutic potential, but the challenges associated with delivery continue to limit progress. Messenger RNA (mRNA) has the potential to provide for transient production of therapeutic proteins, without the need for nuclear delivery and without the risk of insertional mutagenesis. Here we describe the sustained delivery of therapeutic proteins in vivo in both rodents and non-human primates via nanoparticle-formulated mRNA. Nanoparticles formulated with lipids and lipid-like materials were developed for delivery of two separate mRNA transcripts encoding either human erythropoietin (hEPO) or factor IX (hFIX) protein. Dose-dependent protein production was observed for each mRNA construct. Upon delivery of hEPO mRNA in mice, serum EPO protein levels reached several orders of magnitude (>125 000-fold) over normal physiological values. Further, an increase in hematocrit (Hct) was established, demonstrating that the exogenous mRNA-derived protein maintained normal activity. The capacity of producing EPO in non-human primates via delivery of formulated mRNA was also demonstrated as elevated EPO protein levels were observed over a 72-h time course. Exemplifying the possible broad utility of mRNA drugs, therapeutically relevant amounts of human FIX (hFIX) protein were achieved upon a single intravenous dose of hFIX mRNA-loaded lipid nanoparticles in mice. In addition, therapeutic value was established within a hemophilia B (FIX knockout (KO)) mouse model by demonstrating a marked reduction in Hct loss following injury (incision) to FIX KO mice

The role of kinetic context in apparent biased agonism at GPCRs

Biased agonism describes the ability of ligands to stabilize different conformations of a GPCR linked to distinct functional outcomes and offers the prospect of designing pathway-specific drugs that avoid on-target side effects. This mechanism is usually inferred from pharmacological data with the assumption that the confounding influences of observational (that is, assay dependent) and system (that is, cell background dependent) bias are excluded by experimental design and analysis. Here we reveal that ‘kinetic context’, as determined by ligand-binding kinetics and the temporal pattern of receptor-signalling processes, can have a profound influence on the apparent bias of a series of agonists for the dopamine D2 receptor and can even lead to reversals in the direction of bias. We propose that kinetic context must be acknowledged in the design and interpretation of studies of biased agonism

Layer-by-Layer Nanoparticles for Systemic Codelivery of an Anticancer Drug and siRNA for Potential Triple-Negative Breast Cancer Treatment

A single nanoparticle platform has been developed through the modular and controlled layer-by-layer process to codeliver siRNA that knocks down a drug-resistance pathway in tumor cells and a chemotherapy drug to challenge a highly aggressive form of triple-negative breast cancer. Layer-by-layer films were formed on nanoparticles by alternately depositing siRNA and poly-l-arginine; a single bilayer on the nanoparticle surface could effectively load up to 3500 siRNA molecules, and the resulting LbL nanoparticles exhibit an extended serum half-life of 28 h. In animal models, one dose via intravenous administration significantly reduced the target gene expression in the tumors by almost 80%. By generating the siRNA-loaded film atop a doxorubicin-loaded liposome, we identified an effective combination therapy with siRNA targeting multidrug resistance protein 1, which significantly enhanced doxorubicin efficacy by 4 fold in vitro and led to up to an 8-fold decrease in tumor volume compared to the control treatments with no observed toxicity. The results indicate that the use of layer-by-layer films to modify a simple liposomal doxorubicin delivery construct with a synergistic siRNA can lead to significant tumor reduction in the cancers that are otherwise nonresponsive to treatment with Doxil or other common chemotherapy drugs. This approach provides a potential strategy to treat aggressive and resistant cancers, and a modular platform for a broad range of controlled multidrug therapies customizable to the cancer type in a singular nanoparticle delivery system.Janssen Pharmaceutical Ltd. (TRANSCEND Grant)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)National Health and Medical Research Council (Australia) (CJ Martin Fellowship)National Science Foundation (U.S.). Graduate Research FellowshipNatural Sciences and Engineering Research Council of Canada (Postdoctoral Fellowship

Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling

Despite substantial efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain largely unclear. Here we examined cellular uptake of siRNA delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy as well as defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR, and cathepsins. SiRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes/lysosomes and increased gene silencing of the target gene. Our data suggests that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways

Biomimetic self-assembling copolymer-hydroxyapatite nanocomposites with the nanocrystal size controlled by citrate

Citrate binds strongly to the surface of calcium phosphate (apatite) nanocrystals in bone and is thought to prevent crystal thickening. In this work, citrate added as a regulatory element enabled molecular control of the size and stability of hydroxyapatite (HAp) nanocrystals in synthetic nanocomposites, fabricated with self-assembling block copolymer templates. The decrease of the HAp crystal size within the polymer matrix with increasing citrate concentration was documented by solid-state nuclear magnetic resonance (NMR) techniques and wide-angle X-ray diffraction (XRD), while the shapes of HAp nanocrystals were determined by transmission electron microscopy (TEM). Advanced NMR techniques were used to characterize the interfacial species and reveal enhanced interactions between mineral and organic matrix, concomitant with the size effects. The surface-to-volume ratios determined by NMR spectroscopy and long-range 31P{1H} dipolar dephasing show that 2, 10, and 40 mM citrate changes the thicknesses of the HAp crystals from 4 nm without citrate to 2.9, 2.8, and 2.3 nm, respectively. With citrate concentrations comparable to those in body fluids, HAp nanocrystals of sizes and morphologies similar to those in avian and bovine bones have been produced

Dioctadecyldimethylammonium:monoolein nanocarriers for efficient in vitro gene silencing

This study describes a novel liposomal formulation for siRNA delivery, based on the mixture of the neutral lipid monoolein (MO) and cationic lipids of the dioctadecyldimethylammonium (DODA) family. The cationic lipids dioctadecyldimethylammonium bromide (DODAB) and chloride (DODAC) were compared in order to identify which one will most efficiently induce gene silencing. MO has a fluidizing effect on DODAC and DODAB liposomes, although it was more homogeneously distributed in DODAC bilayers. All MO-based liposomal formulations were able to efficiently encapsulate siRNA. Stable lipoplexes of small size (100-160 nm) with a positive surface charge (>+45 mV) were formed. A more uniform MO incorporation in DODAC:MO may explain an increase of the fusogenic potential of these liposomes. The siRNA-lipoplexes were readily internalized by human nonsmall cell lung carcinoma (H1299) cells, in an energy dependent process. DODAB:MO nanocarriers showed a higher internalization efficiency in comparison to DODAC:MO lipoplexes, and were also more efficient in promoting gene silencing. MO had a similar gene silencing ability as the commonly used helper lipid 1,2-dioleyl-3-phosphatidylethanolamine (DOPE), but with much lower cytotoxicity. Taking in consideration all the results presented, DODAB:MO liposomes are the most promising tested formulation for systemic siRNA delivery.This work was supported by FEDER through POFC - COMPETE and by national funds from FCT through the projects PEst-C/BIA/UI4050/2011 (CBM.A), PEst-C/FIS/UI0607/2011 (CFUM), and PTDC/QUI/69795/2006, while Ana Oliveira holds scholarship SFRH/BD/68588/2010. Eloi Feitosa thanks FAPESP (2011/03566-0) and CNPq (303030/2012-7), and Renata D. Adati thanks FAPESP for scholarship (2011/07414-0). K. Raemdonck is a postdoctoral fellow of the Research Foundation - Flanders (FWO-Vlaanderen). We acknowledge NanoDelivery-I&D em Bionanotecnologia, Lda. for access to their equipment