Mesoporous Silica Nanoparticles for Delivery of Therapeutic Agents in Brain Disorders

Olia Alijanpourtolouti

doi:10.60867/00000107

Parkinson’s and Alzheimer’s diseases are neurodegenerative disorders that progressively impair memory and cognitive function. Early stages of these brain disorders have been linked to congenital genetic mutations and abnormal regulation of gene expression. Gene therapy offers a promising strategy for treating both genetic and acquired brain disorders; however, delivering large therapeutic molecules such as DNA and mRNA remains challenging due to their high molecular weight, negative charge, and susceptibility to enzymatic degradation. The blood-brain barrier (BBB) further limits delivery of therapeutics to target brain cells. Therefore, an effective delivery system capable of carrying, protecting, and transporting these large biomolecules across the BBB is essential. Among various strategies explored, including lipid nanoparticles, viral vectors, and ligand-conjugated RNAs, mesoporous silica nanoparticles (MSNPs) have emerged as a promising platform. This study aimed to develop MSNPs with large pores capable of accommodating sizeable biomolecules while maintaining sub-100 nm dimensions to facilitate BBB penetration, and to evaluate the effects of polymer coating and peptide conjugation on their ability to protect and deliver PARK7 mRNA to brain cells. First, MSNPs were synthesized using two different surfactants—cetyltrimethylammonium bromide (CTAB) and cetyltrimethylammonium chloride (CTAC)—via both one-stage and two-stage synthesis methods to evaluate their morphological and functional properties for drug delivery applications. TEM analysis revealed that MSNPs synthesized with CTAC exhibited larger particle and pore sizes than those produced with CTAB, likely due to the influence of halide ions on micelle formation. Furthermore, MSNPs prepared via the two-stage method demonstrated smaller sizes, reduced aggregation, and higher structural uniformity compared to those synthesized via the one-stage method. Fourier Transform Infrared spectroscopy (FTIR) and X-ray Photoelectron Spectroscopy (XPS) analyses verified successful amine functionalization and Rhodamine B (RhB) incorporation. The ninhydrin assay confirmed more efficient amine grafting in MSNPs synthesised via the two-stage method compared to the one-stage method. UV-Vis, HPLC, and LC/MS results confirmed effective RhB dye encapsulation and stability, with the two-stage method showing higher encapsulation efficiency and lower dye leakage. Overall, MSNPs synthesized via the two-stage method exhibited superior physicochemical properties and greater surface functionalization control. Notably, CTAB-derived MSNPs (<50 nm, ~5 nm pores) were suitable for small-molecule delivery, whereas CTAC-derived MSNPs (<100 nm, ~20 nm pores) offered potential for transporting larger therapeutic agents across the blood brain barrier. The following chapter aimed to develop a biocompatible method for covalently coating MSNPs with polyethylenimine (PEI) to enhance their stability, cellular uptake, and potential for safe and effective biomolecule delivery. Covalent attachment was achieved through CDI-mediated coupling between hydroxyl groups on MSNPs and amine groups of PEI. Various solvents were evaluated to ensure compatibility with sensitive therapeutic agents such as RNA and DNA. FTIR analysis confirmed the activation of hydroxyl groups and successful PEI conjugation in non-aqueous solvents, while TGA results further validated the formation and stability of the PEI coating. Among the solvents tested, a chloroform:acetonitrile (20:80) mixture demonstrated efficiency comparable to DMF but with improved biocompatibility and reduced risk of nucleic acid degradation. DLS, zeta potential, and TEM analyses revealed well-dispersed, pH-dependent nanoparticles with a thin PEI layer, confirming successful surface modification. Overall, this method establishes a stable and biocompatible strategy for covalent PEI functionalization of MSNPs, providing a promising platform for the safe encapsulation and targeted delivery of sensitive biomolecules such as mRNA. The next chapter focused on the covalent conjugation of the TGN peptide to PEI-coated MSNPs (MSNP-PEI) to enhance their brain-targeting potential. TEM analysis revealed that while unmodified MSNP-PEI particles were well-dispersed, peptide conjugation initially caused aggregation due to unwanted side reactions during EDC/NHS coupling in phosphate buffer. To address this, optimization experiments using glycine as a model molecule demonstrated that replacing phosphate buffer with MES buffer and adding 2-mercaptoethanol prior to the coupling reaction effectively prevented nanoparticle crosslinking, yielding well separated products. LC-MS/MS analysis confirmed partial glycine conjugation (approximately 47%). Applying the optimized protocol to TGN peptide conjugation produced well-dispersed MSNP-PEI-peptide nanoparticles, as confirmed by TEM and UV-Vis spectroscopy, which showed characteristic absorbance bands at 260 and 280 nm. Further evidence of successful conjugation was provided by the increased zeta potential (+63.12 mV) and the distinct FITC-associated absorbance and fluorescence emission. RP-HPLC analysis indicated a peptide conjugation efficiency of approximately 27.7%. Overall, this chapter established a reliable and optimized NHS/EDC coupling approach for covalent attachment of TGN peptide to MSNP-PEI, achieving stable, well-dispersed nanoparticles suitable for targeted brain delivery applications. The last chapter focused on evaluating the functional performance of the synthesized MSNPs as gene delivery carriers, particularly their cytocompatibility, cellular uptake, mRNA encapsulation efficiency, and protection against enzymatic degradation. CCK-8 assays demonstrated that both MSNP-CTAC2 and MSNP-CTAB2 exhibited low cytotoxicity toward SH-SY5Y cells, with cell viability remaining above 80% at most concentrations. PEI- and peptide-functionalized MSNPs showed no detectable toxicity and even promoted cell proliferation at certain levels. Fluorescence microscopy and flow cytometry confirmed concentration-dependent internalization of FITC-labelled MSNP–PEI–peptide into SH-SY5Y cells. Gel retardation and RNase A protection assays revealed that MSNP–CTAC2 possessed a higher mRNA loading capacity than MSNP–CTAB2, though both failed to protect mRNA from nuclease degradation. In contrast, covalently PEI-coated MSNPs (MSNP/mRNA–PEI) significantly enhanced mRNA stability, and further conjugation with the TGN peptide (MSNP/mRNA–PEI–peptide) provided superior encapsulation efficiency and protection, preventing RNase-mediated degradation while enabling mRNA release inside cells, as confirmed by FLAG-tag immunofluorescence imaging. Overall, the combination of PEI coating and TGN peptide functionalization produced a biocompatible, stable, and brain targeted nanocarrier with strong potential for safe and effective mRNA delivery in gene therapy applications.

Mesoporous Silica Nanoparticles for Delivery of Therapeutic Agents in Brain Disorders

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