Biocompatible and biodegradable macromolecules have been intensively used in pharmaceutical industries in various formulation preparations to fulfill different requirements. In the last two decades, RNA interference (RNAi) gene regulation has attracted a lot of attention for its high potency in medical applications. However, inadequate delivery of RNA molecules to intracellular target sites has become the bottleneck in both laboratory use and clinical therapy in the future. In general, the small interfering RNA molecules as well as other RNA molecules have poor pharmacokinetic profiles, can easily be hydrolyzed or degraded, cause serious immunogenic response and can be toxic to particular cell types and organs. To fulfill the demands, a variety of polymeric vehicles have been synthesized and investigated, and various chemical reactions have endowed the delivery systems with properties like high membrane permeability, low toxicity and tunable size and surface functionalities for anti-aggregation, etc. In our research, we have explored the major parameters that influenced the efficiency of siRNA delivery based on the experiments using carbohydrate based cationic polymers as successful biocompatible polymer models. Structure-property relationship has been studied from many perspectives, including the carbohydrate units, length of the polymers, dose response, and topology of the polymer-siRNA polyplexes in this study. Herein, two series of carbohydrate-based polycations were synthesized and examined that varied in the degree of polymerization (n)--one containing trehalose [Tr4(n) series: Tr4(23), Tr4(55), Tr4(77)] and the other containing beta-cyclodextrin [CD4(n) series: CD4(10), CD4(26), CD4(39), CD4(143), CD4(239)]. In addition, two monosaccharide models were examined for comparison that contains tartaramidoamine (T4) and galactaramidoamine (G4 or Glycofect) repeats. Delivery profiles for pDNA were compared with those obtained for siRNA delivery and reveal that efficacy differs significantly as a function of carbohydrate type, nucleic acid type and dose, polymer length, and presence of excess polymer in the formulation. The Tr4 polymers yielded higher efficacy for pDNA delivery, yet, the CD4 polymers achieved higher siRNA delivery and gene down regulation. The T4 and Glycofect derivatives, while efficient for pDNA delivery, were completely ineffective for siRNA delivery. A strong polymer length and dose dependence on target gene knockdown was observed for all polymers tested. Also, free polymer in solution (uncomplexed) was demonstrated to be a key factor in promoting siRNA uptake and gene down regulation. The carbohydrate family has been systematically studies in biochemistry. Inspired by the nature, carbohydrates have been a platform that polymer chemists have gained ideas from to improve stability and biocompatibility of nanosystems, for example, by incorporating them as degradable domains or using them as new coating materials. Herein, we have examined nanosystems that have been coated with synthetic polycarbohydrates: poly(glucose) and poly(trehalose), as coating materials aimed to improve the profile of siRNA delivery into brain cancer cells. The poly(trehalose) coated nanocomplexes were demonstrated to be substantially effective for quantitative siRNA delivery in presence of high salt concentrations and serum proteins. The ability of trehalose to lower phase transition energy associated with water freezing and protective properties have shown that poly(trehalose) has great promise to serve as an important component in formulation of effective nanomedicines. In order to fulfill the requirement for tracking the nanoparticles, studying the intracellular unpacking mechanism as well as pharmacokinetic studies in future, we have also developed series of cationic polymers combining lanthanide ion chelating domains. To improve the biocompatibility and the performance of the polymers in facilitating the dissociation of the nanoparticles, we have incorporated the alpha,alpha-(D)-trehalose, a naturally abundant disaccharide into the polymer chains. The trehalose has been known for its potency in protecting biological materials from dehydration and aggregation. The so-formed trehalose containing polymers have shown to be effective material to facilitate siRNA mediated target gene knockdown. The complexation and dissociation with siRNA can also be monitored via FRET (F�rster resonance energy transfer) by chelating the polymers with luminescent lanthanide ions (Eu3+ and Tb3+) and labeling the siRNA with organic dyes (Cy5 and TMR). On the other hand, compared with the currently commercially-available MRI contrast agent Magnevist, the polymers have achieved twice the relaxivity in two different magnetic fields. In summary, by combining the carbohydrate moieties and oligoamines, we have systematically examined the structure-property relationship of various well designed polymers on siRNA delivery profiles using glioblastoma cells as model. On the other hand, we have successfully incorporated the trehalose moieties into the lanthanide chelating polymers to increase the biocompatibility and performance of the polymers for both siRNA delivery and imaging. By incorporating different lanthanide ions, we can potentially use these well structured polymers as theranostic models in fundamental biological research.
University of Minnesota Ph.D. dissertation. July 2014. Major: Chemistry. Advisor: Theresa Reineke. 1 computer file (PDF); xvii, 202 pages.
Synthesis and Evaluation of Carbohydrate Based Cationic Polymers for siRNA Delivery and Tracking.
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