POROUS SILICON FUNCTIONALIZATION FOR DRUG DELIVERY AND BIOSENSING BY IN SITU PEPTIDE NUCLEIC ACID SYNTHESIS

POROUS SILICON FUNCTIONALIZATION FOR DRUG DELIVERY AND BIOSENSING BY IN SITU PEPTIDE NUCLEIC ACID SYNTHESIS K. R. BEAVERS 1 , J. W. MARES 2 , C. M. SWARTZ 3 , Y. ZHAO 1 , S. M. WEISS 1,2* , C. L. DUVALL 1,4* 1 Interdisciplinary Graduate Program in Materials Science, Vanderbilt University, Nashville, Tennessee, USA E-mail:  kelsey.r.beavers@vanderbilt.edu;   Tel: (706) 621-3696 2 Department of Electrical Engineering and Computer Science, Vanderbilt University, Nashville, Tennessee, USA 3 LaVergne High School, LaVergne, Tennessee 37086, USA 4 Department of Biomedical Engineering, Vanderbilt University, Nashville, Tennessee, USA SUMMARY We report the automated synthesis of peptide nucleic acids (PNA) directly from porous silicon (PSi) films. Base-by-base nucleotide additions were monitored by non-destructive optical reflectance measurements, and synthesis of the completed PNA sequence was verified by mass spectrometry.  The versatility of the in situ synthesis technique for loading PSi with PNA molecules is demonstrated for both drug delivery and biosensor devices. 1. INTRODUCTION PNA are synthetic nucleic acids in which nucleobases (A, C, T, and G) are spaced along a neutrally-charged peptide backbone. 1 PNA bind to complementary DNA and RNA in a highly stable, sequence-specific manner 1 . Importantly, the lack of electrostatic repulsion between PNA and oligonucleotide targets improves hybrid stability relative to natural nucleic acid hybrids (e.g., DNA-DNA). Due to their stability and specificity, PNA show promise as both therapeutic regulators of gene expression 2 and biosensor probe molecules. 3 The clinical translation of PNA therapeutics is currently limited by the poor cellular uptake and bioavailability of PNA. 2 Moreover, in order for PNA probes to be effective for biosensing applications, they must be stably attached to biosensor surfaces in a sufficiently high density to ensure hybridization response reproducibility. 3 The central hypothesis of this work is that PNA efficacy in gene therapy and biosensing applications can be improved by coupling to PSi, which is known to be biocompatible and biodegradable material that possesses a large internal surface area. In this study, we demonstrate a novel strategy for the automated, base-by-base synthesis of PNA directly from PSi. In situ PNA synthesis is then used to load PSi nanoparticles for therapeutic PNA delivery and to functionalize PSi biosensors for sequence-specific DNA detection. 2. EXPERIMENTAL RESULTS AND DISCUSSIONS In situ synthesis of PNA from PSi. PSi films were etched from p-type Si (0.01Ω-cm) using 15% hydrofluoric acid in ethanol. PSi films were either fabricated as 10 µm thick single layers (70% porosity, 30nm average pore diameter), or, for nanoparticle generation, as multilayers stacks of ~200nm thick particle layers (70% porosity, 30nm pores) and thin, mechanically fragile separation layers (>85% porosity).  PSi films were oxidized at 800°C for 30 min, and silanized with 3-aminopropyltriethoxy silane (APTES). PNA was synthesized by standard solid-phase Fmoc-peptide chemistry using silanized PSi as the solid phase support. 4 Reflectometry was used to monitor PNA synthesis progression by tracking total change in optical thickness of the PSi film relative to APTES-functionalized PSi ( Figure 1 ). Coupling of PNA monomers to growing oligos inside the PSi films leads to a change in the effective refractive index of the films, which is measured by tracking changes in the reflectance spectra. Successful synthesis of the full 23mer PNA was verified by mass spectrometry ( Figure 1 inset ), which also provided insights into the PNA coupling efficiency inside the PSi. Functionalization of PSi Nanoparticles for Delivery of PNA Therapeutics. Naked, unmodified PNA has poor bioactivity as a result of its limited intracellular uptake.  To improve intracellular PNA delivery, a 23mer therapeutic PNA (5'-ACA AAC ACC ATT GTC ACA CTC CA-3') was synthesized from the surface of a multilayer PSi film. Following synthesis, the multilayer film was ultrasonically fractured and filtered to produced PNA-functionalized nanoparticles with an average size of ~240nm. To compare the efficiency of intracellular PNA delivery with or without the aid of a PSi nanocarrier, Huh7 human liver cancer cells were incubated with fluorescently-labeled, naked (free) PNA or in situ PNA-functionalized PSi nanoparticles. The resultant uptake was evaluated by confocal microscopy ( Figure 2 ). PNA delivery with PSi nanoparticles resulted in significantly more PNA uptake relative to free PNA at all treatments times investigated. The fluorescence signal of PNA delivered with PSNPs was more intense, distributed and less punctate than that introduced without a carrier. This preliminary study indicates that PSi nanoparticles can be used improve the intracellular delivery of therapeutic PNA without the need for fusion with cell-penetrating peptides or other modifications. Furthermore, it demonstrates the utility of in situ synthesis as a PNA loading strategy for PSi nanoparticles. Functionalization of a PSi Biosensor. A 16-mer probe PNA (5'-TAG CTA TGG TCC TCG T-3') was synthesized in situ from a 10 µm thick PSi film to test for the selective detection of a target DNA (5'-G GTT TCT GAT GCT GAC-3'). PNA-functionalized PSi films were incubated with buffer alone, 10 µM 100% mismatch “scrambled” DNA, or 10 µM target DNA ( Figure 3 ). No significant changes in optical thickness (signified by shifts in interference fringes) were observed for sensors incubated with either the scrambled DNA sequence or buffer alone, indicating negligible non-specific binding events. In contrast, a 30 nm red shift was observed following incubation with the complementary DNA target, confirming selective hybridization with PNA probe molecules. Comparison experiments (not shown) with PNA probes attached using traditional covalent chemistry methods showed a more than five times reduced optical response to target binding. This initial study demonstrates the successful fabrication of a highly-selective biosensor by in situ synthesis from a single layer PSi film.  Future studies focused on translating this functionalization strategy to resonant photonic PSi structures are expected to enable higher detection sensitivity and compatibility with planar lab-on-chip devices. 5. CONCLUSIONS We have demonstrated that PSi can be used as a solid phase support for the automated synthesis of PNA. In addition, we have demonstrated the utility of this strategy for the functionalization of both PSi drug delivery vehicles and PSi biosensors. REFERENCES (1) Ratilainen, T.; Holmen, A.; Tuite, E.; Nielsen, P. E.; Norden, B., Thermodynamics of sequence-specific binding of PNA to DNA. Biochemistry-Us 2000, 39 (26), 7781-7791. (2) Torres, A. G.; Fabani, M. M.; Vigorito, E.; Williams, D.; Al-Obaidi, N.; Wojciechowski, F.; Hudson, R. H. E.; Seitz, O.; Gait, M. J., Chemical structure requirements and cellular targeting of microRNA-122 by peptide nucleic acids anti-miRs. Nucleic Acids Res 2012, 40 (5), 2152-2167. (3) Briones, C.; Moreno, M., Applications of peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) in biosensor development. Analytical and Bioanalytical Chemistry 2012, 402 (10), 3071-3089. (4) Braasch, D.; Corey, D., Synthesis, analysis, purification, and intracellular delivery of peptide nucleic acids. Methods 2001, 23 (2), 97-107.