Dual functional theranostic nanoparticles for rhabdomyosarcoma treatment

Rhabdomyosarcoma (RMS) is one of the most common soft tissue sarcomas and is extremely aggressive and highly malignant. The majority of cases occur in children, and most commonly appear in the arms or legs. Currently, RMS is treated with aggressive chemotherapy, as well as surgery and/or radiation in many cases. The aggressive treatments often cause long term aftereffects, and can even cause permanent life-altering disabilities. Alternative treatment modalities are urgently needed.
The aim of this work is to build a dual-functional therapy/near-infrared (NIR) imaging nanoparticle, which could perform gene therapy with specific RMS gene knock-down regulated by pH-activated-release, and monitor the effects on tumour size with delivered near-infrared (NIR) dye. The nanoparticle will be surface functionalized with novel aptamers for improved targeting. The design can therefore be separated into four parts from outside to inside: aptamerbased targeting system, a pH-responsive charge reversible coating layer, nanoparticle-based NIR non-invasive imaging, and nanoparticle transfected siRNA for alveolar RMS (aRMS) specific gene knockdown.
Aptamers were chosen as the targeting ligand for the nanosystem since compared to antibodies, they are much smaller, have higher affinity and specificity, and lower immunogenicity. Furthermore, they are easier and cheaper to manufacture. The binding target for aptamer generation was Neural EGF-Like 1 (NELL1) chosen based on validation results which suggested overexpression on the surface of the target aRMS cells. Oligos were isolated, and their binding affinity was quantified using Surface Plasmon Resonance (SPR). The selected NELL1 aptamer could locate and firmly bind to the NELL1 on the surface of aRMS cells, which could therefore potentially minimize off-target effects.
The aptamer was attached to a synthesized pH-activated charge reversible PMPC-PDPA@PEG- 2 b-PAMA/DMMA copolymer layer. The PEG-b-PAMA/DMMA within the copolymer was able to efficiently convert the charge from negative (under physiological pH) to positive (under acidic pH), and the PMPC-PDPA could revert from a hydrophilic to a hydrophobic state after being activated by the acidic environment of tumours and subcellular organelles (endosomes & lysosomes). The enhanced tissue accumulation and cellular uptake could be achieved with increased adsorption onto negatively charged cellular membranes.
For NIR imaging, IR820 NIR dye was loaded into the mesoporous silica nanoparticles core to enable non-invasive monitoring of the uptake and accumulation of the nanosystem during treatment. Compared with free IR820 dye which could only rely on passive diffusion, the IR820- loaded MSNPs were found to have significant improvements from the perspectives of cellular uptake, accumulation amount and penetration depth within both 2D and 3D cell models. Two siRNA targeting options were selected and validated for aRMS gene therapy: (i) Human αB-crystallin (CRYAB) & Heat Shock Protein Family B (Small) Member 2 (HSPB2) double knockout, and (ii) Keratin 17 (KRT17) knockdown.
Through linking the validated siRNAs onto the MSNPs surface with the disulfide bound which could only be cleavaged by the high concentration of GSH within RMS cells, it has been found to have a high degree of RNase I degradation prevention, and a significant enhanced transfection efficiency. The total GSH levels within RD and RH30 cells were found to be 5.08 and 9.28 times higher than that of control fibroblast cells. Compared with the commercially available DharmaFect transfection reagent, the nanoparticles transfected siRNA has shown a similar or better level of gene knockdown; with a significant suppression of aRMS cells proliferation and mobility.
Overall, the constructed nanosystem could provide another route for tackling aRMS in a more effective way, with fewer side-effects.

Keywords: NIR imaging
nanoparticles
gene therapy
aptamer
sarcoma
Advisor: Townley, H
Coward, K