Methylene blue encapsulated mesoporous silica nanoparticles for anticancer and antibacterial photodynamic therapy

Haritha Kirla

Methylene blue (MB) is the first fully synthetic drug used in medicine. It is an organic dye with a rich history ranging from biological staining to antimicrobial applications. It has recently garnered attention for its potential as an anticancer agent, owing to its photosensitizing properties. MB is a widely available and cost-effective photosensitizer, absorbs light at a wavelength of 630 nm to 680 nm and generates a cytotoxic singlet oxygen reactive oxygen species (ROS) that induce oxidative stress inside the cells which eventually leads to the targeted cell death. Furthermore, MB exhibits a propensity to accumulate in cancerous cells. Despite these advantageous characteristics, the utilization of MB in anticancer photodynamic therapy (PDT) remains relatively limited compared to other photosensitizer dyes, primarily due to unwanted biological interactions and low in vivo PDT efficacy. Recent studies have highlighted the enhanced PDT activity of MB when incorporated into nanocarriers. Among the array of available nanocarriers, mesoporous silica nanoparticles (MSNs) present as promising candidates due to their structural advantages, safety profile, and clinical translatability. However, while some research has explored the use of MSNs as nanocarriers for MB, many have reported a reduction in singlet oxygen quantum yield compared to free MB dye. This decline is primarily attributed to the encapsulation of the dye within MSN nanocarriers through physical adsorption methods, which often result in dye leakage, thereby diminishing its efficacy as a PDT agent. This research work primarily focuses on the methods and development of MSNs covalently encapsulating MB for enhanced dye loading, minimized dye leakage, and improved ROS generation. The constraints mentioned can be addressed through the covalent encapsulation of MB within MSNs. To achieve this, two distinct MB silane derivatives and an Azure B (AB) dye silane derivative were synthesized. These new derivatives, namely MBS1, MBS2, and ABS respectively, were characterized for their chemical structures, spectral properties, and ROS generation capabilities. Their chemical structures and silane group functionalization were confirmed through NMR, FT-IR, and mass spectral analysis. UV-Vis spectral studies indicated comparable light absorption properties between the three silane derivatives and their parent dyes. Among the synthesized silane derivatives, MBS1 exhibited the highest singlet oxygen quantum yield (ɸΔ(MBS1) = 0.4), followed by ABS (ɸΔ(ABS) = 0.34), while MBS2 showed theleast (ɸΔ(MBS2) = 0.27) in methanol. However, both MBS1 and MBS2 demonstrated similar singlet oxygen generation abilities in aqueous conditions. Subsequently, MBS1 and MBS2 were utilized for loading onto amine-modified MSNs (AMSNs) via a chemical conjugation method. The resulting MSN nanocomposites, obtained after covalently encapsulating MBS1 and MBS2, were denoted as MBS1-AMSN and MBS2-AMSN. The dye retaining stability, fluorescence imaging capability, and singlet oxygen generation ability of MBS1-AMSN and MBS2-AMSN were evaluated alongside non-covalently encapsulated MSNs (MB@AMSN). All nanocomposites exhibited spherical structures and ultra-small sizes (~ 35 nm). MBS1-AMSN and MBS2-AMSN demonstrated higher dye loading, dye retaining stability, and fluorescence quantum yield compared to MB@AMSN. MBS1-AMSN showed a 70-fold increase, while MBS2-AMSN showed a 33-fold improvement in fluorescence quantum yields compared to free MB dye. Covalent conjugation resulted in a 2-fold enhancement in the singlet oxygen quantum yield of the dye in MBS1-AMSN and a 1.2-fold improvement in MBS2-AMSN, compared to non-covalent encapsulation. Evaluation on RAW 264.7 macrophages revealed superior fluorescence imaging capacity and PDT efficacy for MBS1-AMSN, establishing it as a more efficient PDT agent compared to MBS2-AMSN and MB@AMSN. Building on these promising results, MBS1-AMSN was further assessed for its anticancer PDT efficacy in 4T1 triple-negative breast cancer (TNBC) cells both in vitro and in animal models. Polyethylene glycol (PEG) surface-modified MBS1-AMSN (PEG-MBS1-MSN) demonstrated excellent PDT efficacy in 4T1 cell culture in vitro, showing significant reductions in cell viabilities and high ROS generation. PDT using PEG-MBS1-MSN on BALB/c female mice bearing 4T1 tumours exhibited high rates of tumour apoptosis and immune response at the tumour site, even at very low dosages (2 mg/kg of PEG-MBS1-MSN equivalent to 40 μg/kg MB) of nanocomposites and light exposure (fluence of 60 J/cm²). These results warrant further studies to evaluate systemic immune responses following PDT treatment, which could be valuable in controlling TNBC tumour metastasis. Healthcare-associated infections arising from resistance to traditional antimicrobial strategies are a significant concern. MB has a rich history of use as an antimicrobial compound and can be employed in antimicrobial photodynamic therapy (aPDT) as an alternative approach for treating microorganisms resistant to traditional antibiotics. Therefore, in this study as a proof-of-concept solution, dual-mode antimicrobial surface coatings on glass substrates were developed by incorporating the light-active MBS1 and the well-known antimicrobial disinfectant compound dimethyloctadecyl [3-(trimethoxysilyl)propyl] ammonium chloride (QAS) into silica nanoparticles (QA-MBS1-SNP). The surface coatings were characterized for their surface morphology by SEM and light absorption properties by UV-Visible spectroscopic techniques. The surface coatings demonstrated excellent aPDT bactericidal activity (> 99.999%, > 5-log reduction) and antibiofouling activity against gram-negative microorganism Escherichia. coli, suggesting their potential application in creating light-active surface coatings for various healthcare applications. Further investigations with various microorganisms are anticipated to unveil the full potential of these surface coatings for microbial resistance. In the concluding chapter, the discussion delves into recommendations for future research and potential extensions of this work.

Methylene blue encapsulated mesoporous silica nanoparticles for anticancer and antibacterial photodynamic therapy

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