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Focused Library Approach to Discover Discrete Dipeptide Bolaamphiphiles for siRNA Delivery.

ABSTRACT: In this study, we report a new dipeptide functionalization strategy for developing new dendritic bolaamphiphile vectors for efficient siRNA transfection. A focused library of dipeptides was constructed using four amino acids: l-arginine, l-histidine, l-lysine, and l-tryptophan. The dipeptides were coupled to two dendritic bolaamphiphile scaffolds that we developed previously, allowing us to quickly access a focused library of discrete vectors with multivalent dendritic dipeptide functionalities. The resulting discrete bolaamphiphiles were screened for siRNA delivery in vitro in HEK-293 and HeLa cells. Bolaamphiphiles functionalized with dipeptides containing Lys or Arg and either His or Trp were the most effective for in vitro siRNA delivery. Necessary cationic charge to ensure efficient siRNA binding are provided by Arg and Lys residues, whereas endosomal escape is provided through pH responsive buffering of His or membrane interactions of Trp. The most effective vectors (F10 HR/RH) exhibited greater than 75% gene silencing in multiple cell lines and exhibited serum stability.

SUBMITTER: Eldredge AC

PROVIDER: S-EPMC5612378 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Focused Library Approach to Discover Discrete Dipeptide Bolaamphiphiles for siRNA Delivery.

Eldredge Alexander C AC Johnson Mark E ME Oldenhuis Nathan J NJ Guan Zhibin Z

Biomacromolecules 20160907 10

In this study, we report a new dipeptide functionalization strategy for developing new dendritic bolaamphiphile vectors for efficient siRNA transfection. A focused library of dipeptides was constructed using four amino acids: l-arginine, l-histidine, l-lysine, and l-tryptophan. The dipeptides were coupled to two dendritic bolaamphiphile scaffolds that we developed previously, allowing us to quickly access a focused library of discrete vectors with multivalent dendritic dipeptide functionalities. ...[more]

PMID: 27563833

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Project description:The ability to coat scaffolds and wound dressings with therapeutic short interfering RNA (siRNA) holds much potential for applications in wound healing, cancer treatment, and regenerative medicine. Layer-by-layer (LbL) technology is an effective method to formulate polyelectrolyte thin films for local delivery of siRNA; however, the formation and efficacy of LbL coatings as drug delivery systems are highly contingent on the assembly conditions. Here, we investigate the effects of LbL assembly parameters on film composition and consequent siRNA-mediated gene knockdown efficiency in vitro. Films comprising poly(β-amino ester) (PBAE) and siRNA were built on polyglactin 910 (Vicryl) sutures consisting of poly(10% L-lactide, 90% glycolide). A fractional factorial design was employed, varying the following LbL assembly conditions: pH, ionic strength, PBAE concentration, and siRNA concentration. Effects of these parameters on PBAE loading, siRNA loading, their respective weight ratios, and in vitro siRNA-mediated knockdown were elucidated. The parameter effects were leveraged to create a rationally designed set of solution conditions that was predicted to give effective siRNA-mediated knockdown, but not included in any of the original experimental conditions. This level of knockdown with our rationally designed loading conditions (47%) is comparable to previous formulations from our lab while being simpler in construction and requiring fewer film layers, which could save time and cost in manufacturing. This study highlights the importance of LbL solution conditions in the preparation of surface-mediated siRNA delivery systems and presents an adaptable methodology for extending these electrostatically-assembled coatings to the delivery of other therapeutic nucleic acids. STATEMENT OF SIGNIFICANCE: Short interfering RNA (siRNA) therapeutics are powerful tools to silence aberrant gene expression in the diseased state; however, the clinical utility of these therapies relies on effective controlled delivery approaches. Electrostatic self-assembly through the layer-by-layer (LbL) process enables direct siRNA release from surfaces, but this method is highly dependent upon the specific solution conditions used. Here, we use a fractional factorial design to illustrate how these assembly conditions impact composition of siRNA-eluting LbL thin films. We then elucidate how these properties mediate in vitro transfection efficacy. Ultimately, this work presents a significant step towards understanding how optimization of assembly conditions for surface-mediated LbL delivery can promote transfection efficacy while reducing the processing and material required.

Dataset Information

Focused Library Approach to Discover Discrete Dipeptide Bolaamphiphiles for siRNA Delivery.

Publications

Focused Library Approach to Discover Discrete Dipeptide Bolaamphiphiles for siRNA Delivery.

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