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Multifunctional miRNA-targeting nanodevices for pluripotent cancer theranostics

Periodic Reporting for period 2 - MIRNANO (Multifunctional miRNA-targeting nanodevices for pluripotent cancer theranostics)

Reporting period: 2019-01-15 to 2020-01-14

“MIRNANO” is a multidisciplinary program at the forefront of nanotechnology and nanomedicine. The foundation of the project is the combination of materials chemistry and biomolecular (DNA/RNA) engineering, aiming to develop innovative technologies that can all contribute to advancing the field of bionanotechnology and personalized medicine.

• Nanotechnology has revolutionized modern research and has led to unparalleled achievements in a plethora of fields, including biomedicine and bioengineering.
• MiRNAs are short endogenous noncoding RNAs that regulate gene expression at the post-transcriptional level. There is established evidence that aberrant expression of certain miRNAs (oncomiRs) is associated with cancer. Therefore, miRNAs have emerged as promising targets for silencing-based anticancer therapies (anti-miR) and as biomarkers for early diagnosis.
• Nucleic acid engineering can improve in vivo stability of molecular drugs by using new classes of nucleic acid structures or artificial oligonucleotide mimics and provide intelligent systems by designing programmable DNA nanodevices.
• Delivery systems are needed to provide efficient release of nucleic acids, improve bioavailability, biocompatibility and reduce off-target effects. Porous silicon nanoparticles (pSiNPs) present tunable pore sizes, allow for control of their physicochemical properties and show a biocompatible degradation pathway in vivo.

Objectives:
1) develop novel cancer therapeutics for precision nanomedicine leveraging silencing of oncogenic microRNAs; 2) fabricate innovative hybrid technologies allowing for detection and imaging of target microRNAs in real time; 3) design advanced DNA nanodevices responsive to biomolecular inputs for theranostics.

The research work has generated the following outputs:
1. The development of novel therapeutics for ovarian cancer based on tumor-targeting, microRNA-silencing porous silicon nanoparticles (pSiNPs).This was achieved by using biodegradable porous silicon nanoparticles loaded with specific anti-miR artificial oligonucleotides and decorated with tumor-targeting ligands (ACS Appl. Mater. Interfaces 2019, 11, 27, 23926-23937).
2. The fabrication of a nano-in-nano platform housing miRNA-responsive dynamic DNA nanodevices incorporated into a hybrid polymer/porous silicon scaffold for sensing of miRNA markers in situ and in real time (Nanoscale, 2020,12, 2333-2339).
3. The manufacturing of tissue engineering scaffolds made from biocompatible polymers aimed to provide physical cues to direct the extension of neurites and to encourage repair of damaged nerves. This has been accomplished by including neurotrophic payloads in the scaffold to substantially enhance regrowth and repair processes. The nanofiber hybrids were demonstrated to increase neurite extension relative to drug-free control nanofibers in a dorsal root ganglion explant assay.
4. The design of programmable DNA-based transducers responding to biomolecular inputs for the actuation of synthetic theranostic molecular networks.This has been carried out by designing, developing and testing nucleic acid based-networks, including DNA/RNA strand displacement reactions and activation of functional RNAs, controlled by oncogenic transcription factors through rationally designed DNA actuators.
The research work has focused on innovative hybrid multiscale devices for theranostics and the engineering of functional DNA systems with potential in oncology. This includes:
1. The development of novel cancer therapeutics based on tumor-targeting, microRNA-silencing porous silicon nanoparticles (pSiNPs). We focused on ovarian cancer, which is the most lethal gynecologic malignancy and one of the leading causes of cancer mortality among women, and set out to target miR-21, which is associated with cell proliferation, multidrug resistance, and tumor invasion. We engineered tumor-targeting, anti-miR nanotherapeutics that provided anticancer activity in a mouse model of ovarian cancer. Porous silicon nanoparticles are loaded with an artificial oligonucleotide, a locked nucleic acid (LNA), targeting miR-21, and decorated with a tumor-homing peptide that allows for enhanced accumulation in the tumor microenvironment. After testing in a xenograft mouse model of ovarian cancer developed from human COV-318 cells, we were able to silence miR-21 and achieved complete inhibition of tumor growth with no side effects. We observed no increase of tumor volume over the course of a 10-day treatment. The work has been published in ACS Appl. Mater. Interfaces 2019, 11, 27, 23926-23937.
2. The fabrication of a nano-in-nano platform housing miRNA-responsive dynamic DNA nanodevices incorporated into a hybrid polymer/porous silicon scaffold for sensing of miRNA markers in situ and in real time. . We demonstrated long-term release of the engineered DNA payload with retention of specificity and functionality over 20 days. This suggests that extracellular miRNA markers may be detected in cell culture over several weeks, providing a new means for real-time monitoring of disease conditions. The research has recently led to a publication in Nanoscale, 2020,12, 2333-2339.
3. The manufacturing of tissue engineering scaffolds made from biocompatible polymers aimed to provide physical cues to direct the extension of neurites and to encourage repair of damaged nerves. This has been accomplished by including neurotrophic payloads in the scaffold to substantially enhance regrowth and repair processes. We have tested three different therapeutic payloads, a protein, a small molecule and and an RNA aptamer. Each therapeutic was loaded using a tailor loading chemistry that is optimized to slow the rate of release of these water-soluble payloads.The nanofiber hybrids were demonstrated to increase neurite extension in a dorsal root ganglion explant assay. A manuscript based on this work is currently under review.
4. The design of programmable DNA-based transducers responding to biomolecular inputs for the actuation of synthetic theranostic molecular networks. This has been carried out by designing, developing and testing nucleic acid based-networks controlled by oncogenic transcription factors through specific DNA actuators. The main goal is to craft novel oncogenic TF-controlled molecular technologies with potential in precision oncology.
The anti-miR approach demonstrated for the treatment of ovarian cancer suggests that porous silicon nanoparticles might serve as an effective platform for delivery of microRNA-silencing therapeutics in other diseases.
We showed that nucleic acid-loaded pSiNPs can be incorporated in multiscale technologies that allow for sensing of miRNA markers in situ and in real time by combining miRNA-responsive DNA nanodevices with hybrid tissue engineering scaffolds. This suggests that extracellular miRNA markers may be detected in cell culture over several weeks, providing a new means for monitoring of disease conditions. The strategies developed throughout MIRNANO hold great potential in supporting further development of precision medicine therapeutics. On account of the high multidisciplinarity of MIRNANO, both the published results and the anticipated future outputs are expected to leave a mark on different domains and reach out to a broad scientific community, including supramolecular chemistry, tissue engineering, biomolecular engineering and nanomedicine.
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