Abdelkader A. Metwally

Measurement of polyamine pKa values.

The extent of ionization of the polyamines is an important factor in their interactions with cellular components. The pK(a) is the pH at which a functional group is 50% ionized. For compounds such as polyamines with more than one ionizable center (atom or functional group), there is a pK(a) value for each center of ionization. This chapter describes the pK(a) values for each amine group in many important polyamines, the factors influencing these values and methods for their determination using potentiometric titration and nuclear magnetic resonance spectroscopy.

Asymmetrical N4,N9-Diacyl Spermines: SAR Studies of Nonviral Lipopolyamine Vectors for Efficient siRNA Delivery with Silencing of EGFP Reporter Gene

Our aim is to study the effects of varying the two acyl moieties in synthesized N(4),N(9)-diacyl spermines on siRNA formulations and their delivery efficiency in cell lines. Six novel asymmetrical lipopolyamines, [N(4)-cholesteryloxy-3-carbonyl-N(9)-oleoyl-, N(4)-decanoyl-N(9)-oleoyl-, N(4)-decanoyl-N(9)-stearoyl-, N(4)-lithocholoyl-N(9)-oleoyl-, N(4)-myristoleoyl-N(9)-myristoyl-, and N(4)-oleoyl-N(9)-stearoyl]-1,12-diamino-4,9-diazadodecane, were assessed for their abilities to bind to siRNA, studied using a RiboGreen intercalation assay, and to form nanoparticles. Their siRNA delivery efficiencies were quantified in FEK4 primary skin cells and in an immortalized cancer cell line (HtTA) using a fluorescein-tagged siRNA, and compared with formulations of N(4),N(9)-dioleoyl-1,12-diamino-4,9-diazadodecane and of a leading transfecting agent, TransIT-TKO. Transfection was measured in terms of siRNA delivery and silencing of EGFP reporter gene in HeLa cells. By incorporating two different acyl moieties, changing their length and oxidation level in a controlled manner, we show efficient fluorescein-tagged siRNA formulation, delivery, and knock-down of EGFP reporter gene. N(4)-Oleoyl-N(9)-stearoyl spermine and N(4)-myristoleoyl-N(9)-myristoyl spermine are effective siRNA delivery vectors typically resulting in 89% cell delivery and gene silencing to 34% in the presence of serum, comparable with the results obtained with TransIT-TKO; adding a second lipid chain is better than incorporating a steroid moiety.

Efficient silencing of EGFP reporter gene with siRNA delivered by asymmetrical N4,N9-diacyl spermines.

It is important to obtain structure-activity relationship (SAR) data across cationic lipids for the self-assembly and nonviral intracellular delivery of siRNA. The aims of this work are to carry out a SAR study on the efficiency of asymmetrical N(4),N(9)-diacyl spermines in siRNA delivery and EGFP reporter gene silencing, with comparisons to selected mixtures composed of symmetrical N(4),N(9)-diacyl spermines. Another important aim of these studies is to quantify the changes in cell viability, assayed with alamarBlue, as a function of lipid structure. Therefore, we have designed, synthesized, purified, and assayed novel cationic lipids that are asymmetrical lipopolyamines based on spermine. Flow cytometry and fluorescence microscopy in an EGFP stably transfected HeLa cell line, measuring both delivery of fluorescently tagged siRNAs and silencing the EGFP signal, allowed quantitation of the differences between asymmetrical cationic lipids, mixtures of their symmetrical counterparts, and comparison with commercial nonviral delivery agents. Intracellular delivery of siRNA and gene silencing by siRNA differ with different hydrophobic domains. In these asymmetrical N(4),N(9)-diacyl spermines, lipids that enhance siRNA uptake do not necessarily enhance siRNA-induced inhibition of gene expression: C18 and longer saturated chains promote uptake, while more unsaturated C18 chains promote gene silencing. These properties are efficiently demonstrated in a new nontoxic cationic lipid siRNA vector, N(4)-linoleoyl-N(9)-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), which is also shown to be comparable with or superior to TransIT-TKO and Lipofectamine 2000.

Efficient Gene Silencing by Self-Assembled Complexes of siRNA and Symmetrical Fatty Acid Amides of Spermine

Gene silencing by siRNA (synthetic dsRNA of 21-25 nucleotides) is a well established biological tool in gene expression studies and has a promising therapeutic potential for difficult-to-treat diseases. Five fatty acids of various chain length and oxidation state (C12:0, C18:0, C18:1, C18:2, C22:1) were conjugated to the naturally occurring polyamine, spermine, and evaluated for siRNA delivery and gene knock-down. siRNA delivery could not be related directly to gene silencing efficiency as N4,N9-dierucoyl spermine resulted in higher siRNA delivery compared to N4,N9-dioleoyl spermine. GFP silencing in HeLa cells showed that the unsaturated fatty acid amides are more efficient than saturated fatty acid amides, with N4,N9-dioleoyl spermine resulting in the most efficient gene silencing in the presence of serum. The alamarBlue cell viability assay showed that fatty acid amides of spermine have good viability (75%–85% compared to control) except N4,N9-dilauroyl spermine which resulted in low cell viability. These results prove that unsaturated fatty acid amides of spermine are efficient, non-toxic, non-viral vectors for siRNA mediated gene silencing.

Self-Assembled Lipoplexes of Short Interfering RNA (siRNA) Using Spermine-Based Fatty Acid Amide Guanidines: Effect on Gene Silencing Efficiency

Four guanidine derivatives of N4, N9-diacylated spermine have been designed, synthesized, and characterized. These guanidine-containing cationic lipids bound siRNA and formed nanoparticles. Two cationic lipids with C18 unsaturated chains, N1,N12-diamidino-N4, N9-dioleoylspermine and N1,N12-diamidino-N4-linoleoyl-N9-oleoylspermine, were more efficient in terms of GFP expression reduction compared to the other cationic lipids with shorter C12 (12:0) and very long C22 (22:1) chains. N1,N12-Diamidino-N4-linoleoyl-N9-oleoylspermine siRNA lipoplexes resulted in GFP reduction (26%) in the presence of serum, and cell viability (64%). These data are comparable to those obtained with TransIT TKO. Thus, cationic lipid guanidines based on N4, N9-diacylated spermines are good candidates for non-viral delivery of siRNA to HeLa cells using self-assembled lipoplexes.

Quantitative Silencing of EGFP Reporter Gene by Self-Assembled siRNA Lipoplexes of LinOS and Cholesterol

Nonviral siRNA vectors prepared by the direct mixing of siRNA and mixtures of an asymmetric N4,N9-diacyl spermine conjugate, N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), with either cholesterol or DOPE, at various molar ratios of the neutral lipids, are reported. The effects of varying the lipid formulation and changing the N/P charge ratio on the intracellular delivery of siRNA to HeLa cells and on the siRNA-mediated gene silencing of a stably expressed reporter gene (EGFP) were evaluated. The presence of either cholesterol or DOPE in the mixture resulted in a marked increase in the delivery of the siRNA as well as enhanced EGFP silencing as evaluated by FACS. A LinOS/Chol 1:2 mixture resulted in the highest siRNA delivery and the most efficient EGFP silencing (reduced to 20%) at N/P = 3.0. Lowering the amount of siRNA from 15 pmol to 3.75 pmol, thus increasing the N/P charge ratio to 11.9, resulted in decreasing the amount of delivered siRNA, while the efficiency of gene silencing was comparable to that obtained with 15 pmol (N/P = 3.0) of siRNA. Mixtures of symmetrical N4,N9-dioleoyl spermine (DOS) with cholesterol at 1:2 molar ratio showed less siRNA delivery than with LinOS/Chol at N/P = 3.0 (15 pmol of siRNA), and comparable delivery at N/P = 11.9 (3.75 pmol of siRNA). The EGFP silencing was comparable with LinOS and with DOS when mixed with cholesterol 1:2 (lipoplexes prepared with 15 pmol of siRNA), but LinOS mixtures showed better EGFP silencing when the siRNA was reduced to 3.75 pmol. Lipoplex particle size determination by DLS of cholesterol mixtures was 106–118 nm, compared to 194–356 nm for lipoplexes prepared with the spermine conjugates only, and to 685 nm for the LinOS/DOPE 1:1 mixture. Confocal microscopy showed successful siRNA delivery of red tagged siRNA and quantitative EGFP knockdown in HeLa EGFP cells; Z-stack photomicrographs showed that the delivered siRNA is distributed intracellularly. Cryo-TEM of siRNA LinOS/Chol 1:2 lipoplexes shows the formation of multilamellar spheres with a size of ∼100 nm, in good agreement with the particle size measured by DLS. The constant distance between lamellar repeats is ∼6 nm, with the electron-dense layers fitting a monolayer of siRNA. AlamarBlue cell viability assay showed that the lipoplexes resulted in cell viability ≥81%, with LinOS/Chol 1:2 mixtures resulting in cell viabilities of 89% and 94% at siRNA 15 nM and 3.75 nM respectively. These results show that lipoplexes of siRNA and LinOS/Chol mixtures prepared by the direct mixing of the lipid mixture and siRNA, without any preceding preformulation steps, result in enhanced siRNA delivery and EGFP knockdown, with excellent cell viability. Thus, LinOS/Chol 1:2 mixture is a promising candidate as a nontoxic nonviral siRNA vector.

siRNA and Gene Formulation for Efficient Gene Therapy

Whilst small interfering RNA (siRNA, also known as short interfering RNA) has a somewhat chequered history with regard to its discovery and initial usage, the “mammalian” research community singularly neither reading nor citing the output from the “plant” research community, it is now recognised in terms of

Chapter 9:Polyamine-Based Agents for Gene and siRNA Transfer

bn being invested and spent that RNA interfer‐ ence (RNAi), sequence specific post-transcriptional gene silencing (PTGS) by siRNA, has many potential therapeutic applications [1] as well as being an important tool in the study of functional genomics. The site and mechanism of action of siRNA requires that these short double-stranded nucleic acids are delivered to the cytosol of target cells. Therefore, formula‐ tion is required in a strategy similar to that for gene therapy, although not requiring access to the nucleus. Efficient medicines design should come with an understanding of the problem at the molecular level. Our contributions are aimed at the use of non-viral gene therapy and this Chapter therefore has such a focus.

Efficient siRNA delivery and gene silencing by unsymmetrical fatty acid amides of spermine

In this chapter, we delineate how one might design, synthesize and formulate an efficient, non-toxic, non-viral lipopolyamine vector for possible future in vivo application. This can be achieved by the ability of novel polyamine conjugates, e.g. spermine, to condense DNA, leading to the self-assembly of nanoparticles that are suitable for gene or siRNA delivery. Our focus is on the lipopolyamines synthesized by conjugation to the naturally occurring tetra-amine spermine. As the first step in gene delivery is the condensation of DNA and formation of nanoparticles, this step must be studied in detail for any understanding of rational design. The ability of lipopolyamines to condense DNA is compared and contrasted with well-known literature DNA condensing agents and with commercially available transfection agents. The transfection efficiency of these novel lipopolyamines has been investigated in cell lines and in animals. There is a tension between the cytotoxicity of these compounds and their delivery efficiency. The self-assembly of nanoparticles of genes and siRNA (lipoplexes) makes polyamine-based agents both attractive and efficient for polynucleotide transfer.

Computer-Assisted Drug Formulation Design: Novel Approach in Drug Delivery

Purpose: To model and interpret drug distribution in the dermis and underlying tissues after topical application which is relevant to the treatment of local conditions. Methods: We created a new physiological pharmacokinetic model to describe the effect of blood flow, blood protein binding and dermal binding on the rate and depth of penetration of topical drugs into the underlying skin. We used this model to interpret literature in vivo human biopsy data on dermal drug concentration at various depths in the dermis after topical application of 6 substances. This interpretation was facilitated by our in vitro human dermal penetration studies in which dermal diffusion coefficient and binding were estimated. Results: The model shows that dermal diffusion alone cannot explain the in vivo data and blood and/or lymphatic transport to deep tissues must be present for almost all of the drugs tested. Conclusion: Topical drug delivery systems for deeper tissue delivery should recognise that blood/ lymphatic transport may dominate over dermal diffusion for certain compounds.