synthesis-cd-free-inpzns-quantum-dots-suitable-for-biomedical

Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, ...

Frederick National Laboratory for Cancer Research

A versatile polycation scaffold that can easily be modified with targeting ligands has been designed, synthesized, and characterized. A series of galactose-containing polymers has been produced to demonstrate the ease of modification of this polynucleotide delivery vehicle motif via the click reaction and to study how various structural modifications affect recognition by ASGPr on hepatocytes. A small library of structures was created where DCS and alkyl spacer length between the targeting group and the polymer backbone was varied. The novel polymer scaffold described proves to be a valuable tool for understanding structure/activity relationships of complexes made with receptor-targeted polymers.

A polycation scaffold presenting tunable "click" sites: conjugation to carbohydrate ligands and examination of hepatocyte-targeted pDNA delivery.

A series of glycopolymers composed of 2-deoxy-2-methacrylamido glucopyranose (MAG) and the primary amine-containing N-(2-aminoethyl) methacrylamide (AEMA) were synthesized via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. The colloidal stability of the polyplexes formed with three diblock glycopolymers and pDNA was assessed using dynamic light scattering, and the polyplexes were found to be stable against aggregation in the presence of salt and serum over the 4 h time period studied. Delivery experiments were performed in vitro to examine the cellular uptake, transfection efficiency, and cytotoxicity of the glycopolymer/pDNA polyplexes in cultured HeLa cells and the diblock copolymer with the shortest AEMA block was found to be the most effective. Additionally, the ability of the diblock glycopolymers to deliver siRNA to U-87 (glioblastoma) cells was screened, and the diblock copolymer with the longest AEMA block was found to have gene knockdown efficacy similar to Lipofectamine 2000.

Diblock glycopolymers promote colloidal stability of polyplexes and effective pDNA and siRNA delivery under physiological salt and serum conditions.

Poly(ethylenimine) (PEI) and PEI-based systems have been widely studied for use as nucleic acid delivery vehicles. However, many of these vehicles display high cytotoxicity, rendering them unfit for therapeutic use. By exploring the mechanisms that cause cytotoxicity, and through understanding structure-function relationships between polymers and intracellular interactions, nucleic acid delivery vehicles with precise intracellular properties can be tailored for specific function. Previous research has shown that PEI is able to depolarize mitochondria, but the exact mechanism as to how depolarization is induced remains elusive and therefore is the focus of the current study. Potential mechanisms for mitochondrial depolarization include direct mitochondrial membrane permeabilization by PEI or PEI polyplexes, activation of the mitochondrial permeability transition pore, and interference with mitochondrial membrane proton pumps, specifically Complex I of the electron transport chain and F(0)F(1)-ATPase. Herein, confocal microscopy and live cell imaging showed that PEI polyplexes do colocalize to some degree with mitochondria early in transfection, and the degree of colocalization increases over time. Cyclosporin a was used to prevent activation of the mitochondrial membrane permeability transition pore, and it was found that early in transfection cyclosporin a was unable to prevent the loss of mitochondrial membrane potential. Further studies done using rotenone and oligomycin to inhibit Complex I of the electron transport chain and F(0)F(1)-ATPase, respectively, indicate that both of these mitochondrial proton pumps are functioning during PEI transfection. Overall, we conclude that direct interaction between polyplexes and mitochondria may be the reason why mitochondrial function is impaired during PEI transfection.

Interaction of poly(ethylenimine)-DNA polyplexes with mitochondria: implications for a mechanism of cytotoxicity.

Progenitor and pluripotent cell types offer promise as regenerative therapies but transfecting these sensitive cells has proven difficult. Herein, a series of linear trehalose-oligoethyleneamine "click" copolymers were synthesized and examined for their ability to deliver plasmid DNA (pDNA) to two progenitor cell types, human dermal fibroblasts (HDFn) and rat mesenchymal stem cells (RMSC). Seven polymer vehicle analogs were synthesized in which three parameters were systematically varied: the number of secondary amines (4-6) within the polymer repeat unit (Tr4(33), Tr5(30), and Tr6(32)), the end group functionalities [PEG (Tr4(128)PEG-a, Tr4(118)PEG-b), triphenyl (Tr4(107)-c), or azido (Tr4(99)-d)], and the molecular weight (degree of polymerization of about 30 or about 100) and the biological efficacy of these vehicles was compared to three controls: Lipofectamine 2000, JetPEI, and Glycofect. The trehalose polymers were all able to bind and compact pDNA polyplexes, and promote pDNA uptake and gene expression [luciferase and enhanced green fluorescent protein (EGFP)] with these primary cell types and the results varied significantly depending on the polymer structure. Interestingly, in both cell types, Tr4(33) and Tr5(30) yielded the highest luciferase gene expression. However, when comparing the number of cells transfected with a reporter plasmid encoding enhanced green fluorescent protein, Tr4(33) and Tr4(107)-c yielded the highest number of HDFn cells positive for EGFP. Interestingly, with RMSCs, all of the higher molecular weight analogs (Tr4(128)PEG-a, Tr4(118)PEG-b, Tr4(107)-c, Tr4(99)-d) yielded high percentages of cells positive for EGFP (30-40%).

Cationic glycopolymers for the delivery of pDNA to human dermal fibroblasts and rat mesenchymal stem cells.

The aim of this study is to compare the cytotoxicity mechanisms of linear PEI to two analogous polymers synthesized by our group: a hydroxyl-containing poly(l-tartaramidoamine) (T4) and a version containing an alkyl chain spacer poly(adipamidopentaethylenetetramine) (A4) by studying the cellular responses to polymer transfection. We have also synthesized analogues of T4 with different molecular weights (degrees of polymerization of 6, 12, and 43) to examine the role of molecular weight on the cytotoxicity mechanisms. Several mechanisms of polymer-induced cytotoxicity are investigated, including plasma membrane permeabilization, the formation of potentially harmful polymer degradation products during transfection including reactive oxygen species, and nuclear membrane permeabilization. We hypothesized that since cationic polymers are capable of disrupting the plasma membrane, they may also be capable of disrupting the nuclear envelope, which could be a potential mechanism of how the pDNA is delivered into the nucleus (other than nuclear envelope breakdown during mitosis). Using flow cytometry and confocal microscopy, we show that the polycations with the highest amount of protein expression and toxicity, PEI and T4(43), are capable of inducing nuclear membrane permeability. This finding is important for the field of nucleic acid delivery in that direct nucleus permeabilization could be not only a mechanism for pDNA nuclear import but also a potential mechanism of cytotoxicity and cell death. We also show that the production of reactive oxygen species is not a main mechanism of cytotoxicity, and that the presence or absence of hydroxyl groups and polymer length play a role in polyplex size and charge in addition to protein expression efficiency and toxicity.

Membrane and nuclear permeabilization by polymeric pDNA vehicles: efficient method for gene delivery or mechanism of cytotoxicity?

Cationic polymers are commonly used to transfect mammalian cells, but their mechanisms of DNA delivery are unknown. This study seeks to decipher the mechanism by which plasmid DNA delivered by a class of cationic polymers traffics to and enters the nucleus. While studies have been performed to elucidate the mechanism of naked plasmid DNA (pDNA) import into the nuclei of mammalian cells, our objectives were to determine the effects of polymer complexation on pDNA nuclear import and the impact of polymer structure on that import. We have performed studies in whole cells and in isolated nuclei using flow cytometry and confocal microscopy to characterize how polymer-DNA complexes (polyplexes) are able to deliver their pDNA cargo to the nuclei of their target cells. The polymers tested herein include (i) linear poly(ethylenimine) (JetPEI), a polyamine, and (ii) two poly(glycoamidoamine)s (PGAAs), polyamines that contain carbohydrate moieties (meso-galactarate, Glycofect (G4), and L-tartarate, T4) within their repeat units. Our results indicate that, when complexed with the PGAAs, pDNA association with the nuclei was severely hampered in isolated nuclei compared to whole cells. When the pDNA was complexed with JetPEI, there was slight inhibition of pDNA-nuclear interaction in isolated nuclei compared to whole cells. However, even in the case of PEI, the amount of pDNA imported into the nucleus increases in the presence of cytosolic extract, thus indicating that intracellular components also play a role in pDNA nuclear import for all polymers tested. Interestingly, PEI and G4 exhibit the highest reporter gene expression as well as inducing higher envelope permeability compared to T4, suggesting that the ability to directly permeabilize the nuclear envelope may play a role in increasing expression efficiency. In addition, both free T4 and G4 polymers are able to cross the nuclear membrane without their pDNA cargo in isolated nuclei, indicating the possibility of different modes of nuclear association for free polymers vs polyplexes. These results yield insight to how the incorporation of carbohydrate moieties influences intracellular mechanisms and will prove useful in the rational design of safe and effective polymer-based gene delivery vehicles for clinical use.

Exploring the mechanism of plasmid DNA nuclear internalization with polymer-based vehicles.

HIV transmission efficiency is greatly increased when viruses are transmitted at virological synapses formed between infected and uninfected cells. We have previously shown that virological synapses formed between HIV-pulsed mature dendritic cells (DCs) and uninfected T cells contain interdigitated membrane surfaces, with T cell filopodia extending toward virions sequestered deep inside invaginations formed on the DC membrane. To explore membrane structural changes relevant to HIV transmission across other types of intercellular conjugates, we used a combination of light and focused ion beam scanning electron microscopy (FIB-SEM) to determine the three-dimensional (3D) architectures of contact regions between HIV-1-infected CD4(+) T cells and either uninfected human CD4(+) T cells or human fetal astrocytes. We present evidence that in each case, membrane extensions that originate from the uninfected cells, either as membrane sheets or filopodial bridges, are present and may be involved in HIV transmission from infected to uninfected cells. We show that individual virions are distributed along the length of astrocyte filopodia, suggesting that virus transfer to the astrocytes is mediated, at least in part, by processes originating from the astrocyte itself. Mechanisms that selectively disrupt the polarization and formation of such membrane extensions could thus represent a possible target for reducing viral spread.

Article	Year
A polycation scaffold presenting tunable "click" sites: conjugation to carbohydrate ligands and examination of hepatocyte-targeted pDNA delivery. Macromolecular bioscience\| PubMed ID: 20480507	2010
Diblock glycopolymers promote colloidal stability of polyplexes and effective pDNA and siRNA delivery under physiological salt and serum conditions. Biomacromolecules\| PubMed ID: 21657209	2011
Interaction of poly(ethylenimine)-DNA polyplexes with mitochondria: implications for a mechanism of cytotoxicity. Molecular pharmaceutics\| PubMed ID: 21699201	2011
Cationic glycopolymers for the delivery of pDNA to human dermal fibroblasts and rat mesenchymal stem cells. Biomaterials\| PubMed ID: 22138032	2012
Membrane and nuclear permeabilization by polymeric pDNA vehicles: efficient method for gene delivery or mechanism of cytotoxicity? Molecular pharmaceutics\| PubMed ID: 22175236	2012
Exploring the mechanism of plasmid DNA nuclear internalization with polymer-based vehicles. Molecular pharmaceutics\| PubMed ID: 22715912	2012
Three-dimensional imaging of HIV-1 virological synapses reveals membrane architectures involved in virus transmission. Journal of virology\| PubMed ID: 24965444	2014

Giovanna Grandinetti

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