LI+LI+Log

Assignment 3 - Molecule of interest: Vinylpyrrolidone
 * Nov12, 2009**

**Density** 1.043g.cm3 [|wiki] 1.144 g/cm3 (predicted, [|chemspider]) 1.045g/cm3 [|MSDS] 1.043 g/cm3 [|Alfa Aesar] Flashing point ** 95 C [|MSDS] 93.9 C [|chemspider] 93C [|Alfa Aesar] 13-14C [|Wiki] 17C [|Journal paper] 13-14C [|Alfa Aesar] 90-92C (1.3kPa) [|Wiki] 92-95 C [|MSDS] 91-93C [|Patent article] 91-93C [|patent article] 90-92C ( [] ) 93-95 ([])
 * Melting point **
 * Boiling point **

** Reflective index ** 1.5117 ( [] ) 1.5113 ( [] ) 1.5110 ( [] ) 1.593 (predicted, [] ) 1.5120 ( [] ) 1470mg/kg (MSDS, [])
 * LD 50 (in rat)**
 * 2000mg/Kg (**Toxicological-hygienic study of vinylpyrrolidinone.** Kvasov, A. R. USSR. Gidrokhim. Issled. (1972), 88-9. From: Ref. Zh., Biol. Khim. 1972, Abstr. No. 21F2061. Journal written in Russian. )
 * 0.9 ml/kg **(Determination of the oral acute toxicity of methacrylates and vinylpyrrolidone in mouse.** Schwach, Gerhard W.; Hofer, Heinz. Inst. Biol., Oesterr. Studienges. Atomenerg. G.m.b.H., Vienna, Austria. Ber. Oesterr. Studienges. Atomenerg. (1978), (SGAE Ber. No. 3004), 15 pp. CODEN: BOAEBM ISSN: 0366-1504. Report written in German. )

**[You shouldn't need ILL to complete this assignment - you can take screenshots from Beilstein without getting the original papers.JCB]**
 * Note: I don’t' have PDF of the articles from on-line. I did request the copies through Merck inter-library. Once I have the copies I will post to my page.
 * [Partial Marks - several properties don't have a correct name, boiling points are not converted to common units at 760 mmHg, use ChemSpider permalinks, you don't have 5 sources for 5 properties JCB]**


 * 29OCT2009**


 * Assignment II

Link to original article: [] [Full Marks JCB]

Title: Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes**

1. Gene-silencing siRNAs are promising therapeutic agents with broad applications for treating a wide array of diseases. Developing siRNA-based medicinal drug requires a safe and effective delivery of siRNA to target cells through viral or non-viral based delivery platforms. 2. Hepatocytes are desired target cells for siRNA owning to its easy access through a simple intravenous (IV) injection. The delivery methods include local delivery of naked siRNAs or a systemic delivery of formulated siRNAs using viral or non-viral based platforms. Two non-viral based delivery platforms, including si-RNA cholesterol conjugates or lipid-nano particles, showed some early promises for effective target delivery to liver cells. However, these delivery approaches often suffer from a narrow therapeutic index, where the efficacious dose is close to the margin of toxic dose. 3. Key considerations for designing siRNA delivery vehicles include the selection of shielding ligand to prevent non-specific interactions as well as the cell-targeting ligands (e.g Galactose) to promote a specific hepatocyte uptake, avoiding a non-specific uptake by the resident immuno-cell, the Kupffer cells. 4. The siRNA polyconjugates enter the target cell through a receptor-mediated endocytosis, followed by a selective activation of maleamate bonds in the acidic environment of endosome to form the un-masked amine groups. The free amine of the endosomolytic agent can further activate the disruption endocytosis membrane, enabling the escape of siRNA. 5. The dynamic siRNA polymer conjugate was constructed through a reversible attachment of siRNA through a disulfide bond, a shielding agent (PEG group) and a receptor-targeting NAG bond, respectively. The endosomolytic properties of the polyconjugate can be modulated by the chemical environment. 6. The assembling of a negatively charged, non-aggregating siRNA polyconjugate involves a reversible linkage of three main structural motifs onto the polymer backbone (PBAVE). The key functional moieties include siRNA, the shielding group (PEG) and the cell-targeting group (NAG). The disulfide bond links siRNA to the polymer backbone and PEG and NAG groups are attached to the polymer through reversible modification with maleic anhydride derivatives. Particle size measurement suggested the polyconjugate is the 10 nm range size domain. 7. Polyconjugate enters the hepatocytes through ASGPr receptor-mediated endocytosis. The low pH environment of endosome cleaves the acid labile maleamate bond, releasing PEG and NAG groups. The de-protected free amine groups can disrupt the endosome membrane, releasing polymer-siRNA conjugate into cytoplasm. The disulfide bond linking siRNA and polymer will be subsequently oxidized in cytoplasma, resulting in the release of the free siRNA in the cell. 8. The apolipoprotein B gene is responsible for cholesterol transport. The efficiency of apoB gene knock-down in cell culture was evaluated in mouse primary hepatocyte. All siRNAs tested showed ~ 80% gene knockdown, which is comparable to the benchmark using a commercial siQUEST. 9. The Dissociation of PEG group from polyconjugate in endosome is critical for an effective gene knock-down. When PED group was permanently linked to polymer backbone, the polyconjugate become completely inactive. 10. NAG functional group is attached to polymer backbone through a CDB linkage. The impact of NAG group on hepatocyte cell was evaluated by IV injection of Cy3-labeled siRNA polyconjugate into mice. Histo analysis of the liver cross-section under confocal micoscope suggested preferential and uniform distribution of siRNA in hepatocyte cells. The hepatocyte uptake of siRNA was significantly reduced when NAG was replaced with glucose in the polyconjugate assembly, confirming NAG as cell targeting ligand for the liver. 11. The necessity of covalent bond between siRNA and polymer backbone was evaluated by injecting the mice with a polymer conjugate formulation, where the siRNS was attached to polymer through electrostatic interaction. The non-covelent bonded polyconjugate showed a marked decrease in siRNA accumulation in liver, suggesting the importance of covalent bonding between siRNA and polymer backbone. 12. The in vivo gene knockdown was evaluated by quantitative assessing the apoB mRNA level post IV injection of apoB-1 siRNA polycomjugate. Mice treating with active siRNA showed ~ 76% reduction in gene expression when compared to the control group. 13. The polyconjugate-induced gene knock-down is specific with respect to target issue. The apoB gene expression in other organs was not effected for mice post treatment. 14. PPARA gene is an important gene in controlling fatty acid metabolism. The siRNA polyconjugate designed for silencing ppara gene was injected to mice and the assay of PPARA mRNA showed ~ 62% reduction in gene expression when compared to the control. 15. The toxicity-indicating biomarkers (e.g the liver enzyme, cytokines level, and TNF and IL-6 level in serum) were evaluated for mice treated with active polyconjugates. Results suggested the safety profiles are satisfactory with respect to key toxicity indicators. 16. The apoB gene expression was evaluated as function of polyconjugate dose level. Different potencies of siRNAs were prepared through serial dilution from a high dose siRNA polyconjugate. Alternatively, siRNAs of different potencies were also prepared by conjugating a varying level of siRNA with polymer at a fix level. For both sample sets, similar dose-response curves were obtained, where the top and mid dose afforded 84% and 50% reduction in gene-expression, respectively. 17. Cholesterol level in liver is directly linked to the level of the expressed apoB protein, An apoB gene knockdown in theory would elicit a reduced cholesterol level in liver. Mice treated with aopB gene-silencing polyconjugate indeed showed a significant reduction in cholesterol level in liver, 18. The apoB gene knockdown reduces the cholesterol level in serum through the impairment of VLDL ( **Very-low-density lipoprotein)** assembly and cholesterol transport from the liver. This pathological pathway also promotes a significant reduction in triglyceride transport from liver, as indicated by marked increase in triglyceride accumulation in liver. 19. Following a single dose injection of 50 ug of apoB siRNA polyconjugate, a sustained reduction was achieved through day 10 and day 4 for apoB gene expression and serum cholesterol level in mice, respectively. 20. Dynamic polyconjugates deliver siRNA effectively to liver cell for both in-vitro and in-vivo models. Significant gene knockdown was demonstrated for apoB and pparp genes with maximum efficiency of 80 to 90% at 2.5 mg/kg dose. The time duration of gene knock down is satisfactory. The dose level tested is well tolerated and no toxicity was indicated based on several safety biomarkers. 21. The apoB gene silencing with siRNA polyconjugates effectively elicit a reduction in apoB gene expression and cholesterol level in serum as well as an impairment of triglyceride transport from the liver. However, the fatty liver induced by the gene-knock and its potential acute effect in human should be further investigated prior to advancing apoB siRNA into a viable therapeutic agent. 22. The ppara siRNA polyconjugates promotes a significant increase for triglyceride serum level. However, this pharmacological effect is less robust than that with apoB gene knockdown. 23. The siRNA polymerconjugate facilitates a specific cell delivery through ligand-mediated receptor interaction on helpatocyte cell membrane. The key advantage over the nanoparticle-based delivery platforms (such as SNALP and iNOP) includes minimal accumulation in non-heptocyte cells. Particle size reduction of polyconjygate compared to SNALP and iNOP further eliminates the potential activation of immuno cells, resulting in negligible toxicity effects. 24. The siRNA polyconjugates offer a promising delivery platform for gene-based therapeutically applications with high potency and low toxicity. The reversible covalent bonds used in linking siRNA, cell targeting ligand, and shielding ligand provide added flexibility for targeting variety cell types. 25. The synthesis of SATA-modified siRNA involves reacting 5` amine siRNA with SATA and sodium bicarbonate at 1:1 :0.36 mass ratio in water at 4C fro 16 hrs. The product was isolated through precipitation with ethanol, followed by incubation at -80C fro 2 hrs. 26. PBAVE polymer was prepared per the procedure reported in the literature. The polymer was then further modified with SMPT, followed by a 16-hour coupling reaction with SATA-siRNA in pH 9 buffer. The assembly of NAG and PEG groups were achieved by mixing siRNA-polymer conjugate with CDM-NAG and CDM-PEG in Hepes free base for 1 hour at room temperature. 27. CDM-PEG and CDM-NAG were prepared by mixing equal molar of acid chloride and respective PEG or NAG derivatives, followed by purification with HPLC.
 * Introduction **
 * Results **
 * Formulation of siRNA polyconjugates and cellular delivery **
 * Activity of the siRNA polyconjugate in tissue culture **
 * Targeting of the siRNA polyconjugate to hepatocyte in vivo **
 * Knockdown of target gene in liver of mice by using siRNA polyconjugate **
 * Dose-response and phenotypic analysis of mice receiving apoB siRNA polyconjugates **
 * Longevity and phenotypic effect of apoB knockdown **
 * Discussion **
 * Methods: polyconjugate synthesis and formulation **

1. Conducted serch in Scifinder using "Polymer conjugate for siRNA delivery" as the entry under "explore literature" and "research topic search".
 * 22OCT2009

Found 22 hits with all key words closely associated. Out of the 22 hits, the refine search yielded 4 review articles. I

I am still not clear on how to add DOI link to my wiki page on the review articles I found?? [For articles found via SciFinder do a search for doi on the page and you should find the link - right click on that link and copy it then paste on the wiki JCB]

15OCT2009** 1. About Wiki - explain how to use "notify me" function. 2. How to add link to use DOI. No PDF files can be added to Wiki. 3. Discussed assignment. 4. Discussed Chemspider
 * I Class note:**

Search results: Found ~ 48 paper.
 * II: Search Scifinder (use Research Topic function) using the title "siRNA polymer conjugates for therapeutic applications" as entry.**

01OCT2009 I am thinking about working on a review article on "siRNA polymer conjugate for therapeutic application".