(628b) pH-Responsive, Core-Shell Magnetite-Silver Nanoparticles for the Guided Transport and Delivery of Nucleotide Cargoes: An Avenue for Highly-Targeted Gene Therapies

Over the past few years, gene therapies have attracted much attention for the development of therapies for various conditions including cancer, neurodegenerative diseases, protein deficiencies and autoimmune disorders. Despite the benefits of this approach, several challenges are yet to be solved to eventually reach clinical implementation. Some of these challenges include low transfection rates, limited stability under physiological conditions and poor specificity towards the target cells. An avenue to overcome such issues is to deliver the therapies with the aid of potent cell-penetrating vectors, which include viral and non-viral vehicles. The use of viral vectors has been criticized due to issues concerning genome integration, a potential for oncogenicity and induced mutagenesis in the host cell1. Alternatively, non-viral vectors, such as nanostructured materials, have been successfully tested in drug and gene delivery without the concerns of the viral ones 2. This has been attributed to their high surface area to volume ratio, ease of synthesis, and the possibility for surface modification to incorporate various surface chemistries and thereby a tunable multifunctional response. This has led to an increasing number of applications where the delivered molecules have shown a considerable increase in bioavailability in both in vitro and in vivo applications3. Among the most successful nanostructured vehicles, magnetite nanoparticles stand out due to their superior biocompatibility and the possibility for manipulation with the aid of magnetic fields.

Here, we propose to develop a nanostructured core-shell cell-penetrating vehicle composed of magnetite at the core and surrounded by a silver shell. A subsequent superficial conjugation of a pH-responsive polymer was proposed to assure that the vehicle  is capable of carrying and releasing circular DNA. Accordingly, the magnetite core was synthesized by co-precipitation of 500mM ferric chloride and 250mM ferrous chloride (molar ratio 2:1) in the presence of a 5M NaOH aqueous solution at 90°C under mechanical agitation at 300 rpm. This was followed by deposition of the silver from a 1 mM silver nitrate solution in a reducing honey solution 20% (w/v). During the process, pH was maintained above 4 to prevent undesirable degradation reactions. Silver on the surface was subsequently chlorinated with the aid of a 1M HCl solution. The prepared core-shell system exhibited colloidal stability such that agglomerate formation and thereby precipitation was avoided. A Hofmann elimination reaction was further conducted on the system to conjugate the pH-responsive polymer poly(2-dimethylamino) ethyl methacrylate (pDMAEMA) to the chlorine atoms on the surface. The conjugation reaction proceeded at 50°C under continuous mechanical agitation at 500 rpm. Besides the role as DNA carrier, the pDMAEMA was conjugated to attempt to avoid endosome formation, which is a cell internalization mechanism that traps exogenous material and consequently might lead to a significant reduction in transfection rate4. Finally, a 10kb circular DNA vector was loaded on the modified nanoparticles at pH values in the range of 6 to 8.5 to estimate the loading efficiency, which was defined as the DNA trapped by the system divided by total loaded DNA. The designed vector contained information for a Cas9 enzyme, a CRISPR gRNA and an mCherry reporter. This in an attempt to explore the suitability of the vehicle in the delivery of gene editing systems. The release of DNA was also tested in vitro, by exposing the loaded systems to a medium with pH values in the range of 8.5 to 6. In this case, the efficiency was calculated as the total DNA released divided by the loaded DNA. The obtained nanoconjugates were characterized by UV-Vis spectrophotometer, dynamic light scattering (DLS), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscope equipped with energy dispersive spectroscopy (SEM+EDS). Furthermore, particle size was determined by DLS.

Figure 1.b shows the FTIR spectra of the core-shell nanoparticles, the core-shell+pDMAEMA conjugate and the free pDMAEMA. The distinctive peaks of pDMAEMA were observed on the conjugate, thereby confirming successful immobilization. Additionally, EDS analysis confirmed the presence of silver and the polymer as evidenced by the presence of silver and nitrogen, respectively (Figure 1.c). DLS results the magnetite core had a diameter around 124 nm, with an increase in particle diameter to 342 nm after coating with silver.

Figure 1. (a) Scanning electron microscopy (SEM) image of a core-shell nanoparticle with pDMAEMA in the surface. (b) FTIR results green is core-shell nanoparticles, red is core-shell nanoparticles with the polymer and blue is the polymer. polymer shows characteristic peaks at 1453, 1639 and 3400 [1/cm]. (c) EDS spectrum of core-shell nanoparticle + pDMAEMA shows the presence of atoms as spotted in (a)  .  

The study presented here provides a route for the development of gene delivery systems based on core-shell magnetic nanoparticles, and pH-responsive polymers. Also, it highlights a methodology for the synthesis, functionalization, and loading of a circular DNA vector with the size typical of those for applications in CRISPR/Cas9 editing systems. Finally, the developed system is likely to provide opportunities to overcome issues regarding low stability and endosome formation, which are commonly present when delivering with the aid of nanostructured materials.

Keywords: gene delivery, core-shell, magnetite, pDMAEMA, nanoparticles, surface modifications

References:

1.            Ho, B. X., Loh, S. J. H., Chan, W. K. & Soh, B. S. In vivo genome editing as a therapeutic approach. International Journal of Molecular Sciences 19, (2018).

2.            Jin, S. & Ye, K. Nanoparticle-mediated drug delivery and gene therapy. in Biotechnology Progress 23, 32–41 (2007).

3.            Varadan, V. K. Nanomedicine : design and applications of magnetic nanomaterials, nanosensors, and nanosystems LK  - https://univdelosandes.on.worldcat.org/oclc/916159742.  TA  - TT  - (Wiley, 2008).

4.            Cuellar, M. et al. Novel BUF2-magnetite nanobioconjugates with cell-penetrating abilities. Int. J. Nanomedicine Volume 13, 8087–8094 (2018).