In order to be active, many drugs need to pass through one or more cell membranes. Cell membranes are about 10 nm thick and highly impermeable. A primary focus of my research is to use nanoparticles to increase cell permeability. Current research employs peptide-based materials that self-assemble into vesicular nanoparticles. These nano-vesicles have lipid-like properties, including solute encapsulation, fusion, and resizing. They can penetrate cells and deliver therapeutic DNA and mRNA. My laboratory is also investigating new methods for gene silencing in insect models by adding dsRNA associated with peptide nanoparticles to their diet. Gene silencing by feeding dsRNA in insects has excellent potential as a tool for pest management because it can reduce the off-target effect and slow down resistance development to chemical insecticides.

Branched Amphiphilic Peptide Capsules (BAPCs) as a Delivery Systems for mRNA vaccines

In this project, we are testing the ability of BAPCs to deliver therapeutic mRNA in murine models. Similarly to PEG, mRNA shields the BAPC cationic surface and prevents early degradation by the mononuclear phagocytic system (MPS), enhancing with this its circulation time and therapeutics effects. In addition, BAPCs resists disruption by chaotropes, proteases, and temperature, thus displaying significant stability and shelf-life. (Fig. 1).

Figure 1. Branched Amphipathic Peptide Capsule (BAPC) Forming Sequences.

In the presence of DNA, they can act as cationic nucleation centers around which DNA wraps (Fig. 2A-C). We demonstrated that BAPCs were capable of delivering a vaccine DNA encoding E7 oncoproteins of HPV-16 (pgDE7) in mice. Mice immunized with pgDE7-BAPC nanoparticles managed to constrain tumor growth up to one month after transplantation of tumor cells without significant toxic effects.  Furthermore, survival time was enhanced by two-fold in comparison to the control group.  We are currently testing the ability of BAPCs to delivery mRNA vaccines in murine models using the antigen model OVA.

Figure 2. TEM images of the BACP:DNA nanoparticles at N:P = 20.8. (A) Single BAPCs interacting with pDNA. Scale bar = 10 nm. (B) Cluster of BAPCs interacting with DNA. Scale bar = 100 nm. (C) Schematic representation of potential BAPC-DNA interactions.

Oral Delivery of dsRNA to Inhibit Gene Expression in Insects.

Entry of dsRNA into cells is the first step in one of the most powerful tools in contemporary molecular biology: transcript knockdown via RNA interference (RNAi). RNAi-based transcript knockdown has been used in insects of numerous Orders and will, without doubt, continue to be a very important part of the reverse-genetics toolbox in insects. For the most part, dsRNA has been administered to insects by microinjection into hemolymph. While effective, this approach has limitations, not the least of which is the tedium it involves, and the smaller the insect species, the greater the challenge presented by injection. Feeding of dsRNA is a more attractive approach than hemolymph injection because it is non-invasive and also opens the possibility of developing new methods of pest control. However, the stability of dsRNA during or after oral delivery remains a large problem for this approach. To increase the stability of dsRNA and enhance their cellular uptake, polymeric nanoparticles have been used for nucleic acid delivery in RNAi-based gene therapeutics. The Avila-Flores Research Group is combing two threads of research – the use of BAPCs to facilitate the uptake of double-stranded nucleic acids; and the desirability of obtaining efficient transcript knockdown in insects by feeding dsRNA.

We demonstrated the ability of BAPCs to deliver dsRNA orally, and inhibit gene function in insect species from two different Orders and with different feeding mechanisms: Tribolium castaneum, the red flour beetle fed with solid flour diet; and the pea aphid, Acyrthosiphon pisum, fed in artificial liquid diet. The gene transcripts tested (BiP and Armet) are part of the unfolded protein response (UPR) and suppressing their translation resulted in lethality. In Tribolium, we also suppressed the expression of the Vermillion gene, that acts in the developing eye with its  transcript encoding the protein required for the development of  normal eye color. Ingestion of dsVermillion-RNA in complex with BAPCs during larval stages gave rise to adults with white (non-colored) eyes at a rather high frequency (about 50% with n = 20), thus verifying the systemic nature of the RNAi effect created by ingestion of dsRNA/BAPC complexes (Fig. 1). These results show that complexation of dsRNA with BAPCs greatly enhances the oral delivery of dsRNA over dsRNA alone in the diet.

Figure 1. Effects of feeding dsVermillion-RNA -/+ BAPCs in Tribolium. Feeding of BAPCs- dsVermillion complexes (as a “supplement” to the flour diet of Tribolium larvae) resulted in the
absence of Vermillion color in the eye of treated insects, right panel.

We are currently applying this technology to different species such as Popillia japonica (Japanese beetle). The Japanese beetle is an invasive and generalist herbivore. Japanese beetle adults and larvae attack >300 species in >70 plant families including the major field crops in the US (corn, soybeans, and cotton). Our aim is to evaluate if BAPCs/dsRNA can provide effective plant protection against Popillia japonica by simple applying these formulations on plant leaves. We are also studying the bio-distribution of fluorescence-labeled dsRNAs with and without BACPs in different species, in order to shed a light on how dsRNA is transported from cell to cell to induce systemic RNAi (Fig. 2).

Figure 2. Localization of fluorescently labeled Armet-dsRNA in Tribolium larvae 8 hr after the feeding. The fluorescence was shown as magenta on the bright-field background. All pictures were captured in the same condition in a LSM700 confocal microscope. (A) midgut; (B) Fat body (C) Malpighian tubule, (D) to (F) are same tissues in the Tribolium fed with labeled Armet- dsRNA alone. Scale bar: 20 mm.

Laser-assisted Delivery of Molecules into Fungal Cells

Fungal infections are becoming increasingly clinically relevant, but current methods to treat them are limited. Many fungi are resistant to available antifungal drugs, and other molecule-delivery methods, such as electroporation, are only designed for research techniques. Delivering molecules into fungal cells can be quite tricky due to the presence of a rigid carbohydrate cell wall. Femtosecond lasers are more frequently being used as a combinatorial method for delivering small molecules. The femtosecond laser creates ultrafast pulses of photons that interact with nanoparticles in solution, which ultimately results in the formation of pores in the cell wall or membrane, a process called photoporation. The exact mechanisms of photoporation is still being studied, but recent studies have found it is likely a combination of indirect effects such as cavitation, shockwaves, and other photo-chemical, -thermal, or -mechanical phenomena.

Figure 1. Diagram of photoporation phenomena. When the femtosecond laser pulses activate
the nanoparticle, a series of effects are triggered, such as cavitation and shock waves. These
effect concuss the cells, creating pores that allow DNA and other molecules to enter.

Until this point, the process of photoporation has exclusively been studied in mammalian cells. We demonstrated that in Saccharomyces cerevisiae, a single-celled yeast, this combinatorial approach was able to effectively deliver small fluorescent molecules and plasmid DNA through the cell wall. The gold nanoparticles were also synthesized through photoreduction chemistry which removed the need for harsh reducing agents and creation of cytotoxic oxidized products. Nearly 60% of cells were successfully irradiated and alive when analyzed via flow cytometry, and laser conditions where both yeast and mammalian CHO cells maintained viability. This method has potential for further preclinical optimizations for oral, vaginal, and skin mycoses.

Figure 2. Poration of S. cerevisiae and Chinese Hamster Ovary (CHO) cells. (A) Flow cytometry data of fluorescently positive viable cells under either varying laser fluence or irradiation time. (B) Confocal micrographs of photoporated S. cerevisiae and CHO cells irradiated in the presence of calcein.