Aim 3: Assess the activity of PNA-loaded PSNPs targeted against miR-122 in vivo
A) Non-destructive optical characterization of in situ synthesis of 23-mer anti-miR122 PNA using reflectometry and Fourier transform analysis to determine the optical thickness of the loaded PSi
4.4 Conclusion
This is, to our knowledge, only the fourth report of in vivo miRNA silencing with a PNA therapeutic [66, 68, 69], and the first report of an endosomolytic PNA delivery system. All other reports have focused on inhibition of miR-155 for treatment of lymphoma. Initial work by Fabani et al. modified PNA with positively-charged lysine residues and saw effective miR-155 inhibition at a 50 mg kg-1 PNA dose. In the study by Babar et al., PNA encapsulation into PLGA nanoparticles coated with a cell-penetrating peptide (0.35 wt% PNA loading) reduced the effective PNA dose to 1.5 mg kg-1. Cheng et. al. conjugated PNA to a pH-responsive peptide which enables targeted delivery of anti-miRNA PNA to the acidic tumor microenvironment. Additionally, the peptide utilizes an environmentally-activated non-endocytic cell uptake/membrane transduction mechanism. This enabled miR-155 inhibition at a 1 mg kg-1 dose in a mouse model of lymphoma.
Our non-targeted PNA delivery system beat or approached the level of miRNA inhibitory potency of these different targeted delivery approaches, and we hypothesize that this new long-circulating nanocomposite can be made more potent by adding targeting ligands, such as GalNAc for hepatocyte targeting [123].
In summary, this chapter showcases a new nanocomposite PNA delivery vehicle proven to overcome both systemic and intracellular delivery barriers. Porous silicon is leveraged as a highly porous scaffold amenable to high drug cargo loading; this substrate enables much higher and simpler drug loading relative to water in oil in water (W/O/W) emulsion methods commonly utilized to load hydrophilic cargo into hydrophobic polymer-based nano- and micro- carriers.
Furthermore, simple electrostatic assembly was utilized for PSNP surface functionalization with a multifunctional polymer that enhances resultant particle colloidal stability, in vivo circulation, and intracellular bioavailability. This fabrication process is both facile and rapid, requiring only
sequential centrifugal wash steps for composite purification. This approach would also be potentially amenable to adaptation to more controlled microfluidic synthesis techniques, such as those developed by Santos et. al. [68, 124]. Furthermore, it is well-established that porous silicon can host a wide range of cargos [85, 125]. We anticipate that this composite system can facilitate the intracellular activity of a broad range of therapeutic and diagnostic payloads. Finally, based on its high bioavailability and long blood circulation half-life, this system can also potentially be functionalized with active targeting ligands and further tuned to facilitate preferential delivery to defined cells and tissues. Thus, PSNP-polymer nanocomposites represent a promising material platform with potential high impact in miRNA inhibitory and other biologic nanomedicines.
CHAPTER 5
CONCLUSIONS 5.1 Summary
Unraveling the secrets of the human genome, researchers have learned more about the role of non- coding RNAs in disease. The existence of functional non-coding RNAs (like miRNA) now makes sense. MiRNAs are well adapted to silence genes because they share the same nucleic acid language as their targets. Similarly, it seems that nucleic acid-based drugs are ideal for therapeutic silencing of pathological miRNAs. Peptide nucleic acids are excellent miRNA inhibitors, yet they have no innate ability to reach miRNA targets in the body. The challenge in developing clinical anti-miRNA therapies is therefore a challenge of delivery. This works’ central hypothesis was that PNA anti-miRNA activity can be improved by engineering PSNPs to increase PNA blood circulation half-life, cellular uptake, and delivery to the cell cytoplasm.
In Aim 1, strategies for loading PNA into PSi were developed and assessed. Covalent attachment of pre-synthesized PNA was found to be the least efficient method of drug loading.
Automated in situ synthesis of PNA from PSi was developed as an alternative, highly efficient covalent drug-loading strategy. Physical adsorption of PNA was an equally efficient drug loading strategy (due to PSi’s high internal surface area), but in situ synthesis enabled more control over PNA release kinetics. Ultrasonic fracture of perforated PSi multilayers was then show to be an effective method for producing PSNPs loaded with PNA. These PSNPs are biocompatible and effective at increasing the uptake and activity of anti-miR122 PNA therapeutic in human liver cancer cells.
Aim 2 expanded PNA-loaded PSNP functionality by developing a method to coat these particles with PEGDB polymer. Polymer/PSi nanocomposites, formed by simple electrostatic self-
assembly, possess the pH-responsive endosomal escape properties of PEGDB without sacrificing the high PNA loading efficiency of PSi. As a result, these nanocomposites show increased cytosolic PNA delivery and improved anti-miRNA activity relative to uncoated PSNPs. Increased anti-miRNA activity was seen in spite of the fact that PEGDB decreased PSNP cellular uptake.
This result highlights the therapeutic importance of PEGDB endosomal escape functionality.
Finally, Aim 3 assessed the activity of PNA-loaded PSNPs targeted against miR-122 in vivo. PEGDB functionalization was shown to enhance particle colloidal stability, and the PEG block of the polymer made particle surfaces more inert. Consequently, nanocomposites extended the blood circulation half-life of intravenously-delivered PNA from <1 min to 70min, increasing its bioavailability by 73x. Finally, these nanocomposites were shown to be non-toxic and enable PNA-mediated miR-122 inhibition in the liver in vivo, decreasing plasma cholesterol by 20%. The culmination of Aims 1 through 3 is a composite nanoparticle proven to overcome both systemic and intracellular barriers to the delivery of PNA therapeutics.