Folic acid-conjugated magnetic mesoporous silica nanoparticles loaded with quercetin: a theranostic approach for cancer management

Abstract

The development of drug carriers based on nanomaterials that can selectively carry chemotherapeutic agents to cancer cells has become a major focus in biomedical research. A novel pH-sensitive multifunctional envelope-type mesoporous silica nanoparticle (SBA-15) was fabricated for targeted drug delivery to human colorectal carcinoma cells (HCT-116). SBA-15 was functionalized with folic acid (FA), and the material was loaded with the water-insoluble flavonoid, quercetin (QN). Additionally, acid-labile magnetite Fe₃O₄ nanoparticles were embedded over the FA-functionalized QN-loaded monodisperse SBA-15 to prepare the highly orchestrated material FA-FE-SBA15QN. The in vitro and in vivo anti-carcinogenic efficacy of FA-FE-SBA15QN was carried out to explore the pH-sensitive QN release with putative mechanistic aspects. FA-FE-SBA15QN caused a marked tumor suppression, and triggered mitochondrial-dependent apoptosis through a redox-regulated cellular signaling system. Furthermore, FA-IO-SBA-15-QN initiated the c-Jun N-terminal Kinase (JNK)-guided H2AX phosphorylation, which relayed the downstream apoptotic signal to the phosphorylate tumor suppressor protein, p53. On the other hand, the selective inhibition of heat shock protein-27 (HSP-27) by FA-FE-SBA15QN augmented the apoptotic fate through JNK/H2AX/p53 axis. The in vitro and in vivo magnetic resonance imaging (MRI) studies have indicated the theranostic perspective of the composite. Thus, the result suggested that the newly synthesized FA-FE-SBA15QN could be used as a promising chemo theranostic material for the management of carcinoma.

Scheme 1. Schematic representation of FA-FE-SBA15QN synthesis and graphical representation illustrating the probable mechanism of action of FA-FE-SBA15QN in modulating the cancer microenvironment.

Fig. 1. Schematic representation of the experimental design outlining the phases of the CT-26-challenge, along with the QN and FA-FE-SBA15QN application (15 mg kg⁻¹) in the murine system.

Fig. 2. FTIR spectra (A) and small angle powder XRD patterns (B) of SBA-15 (black), FA-SBA15QN (red), and FA-FE-SBA15QN (blue). N₂ adsorption/desorption isotherms of FA-FE-SBA15QN (C) at 77 K and respective pore size distribution (D). Adsorption points are marked with filled black circles and desorption points by empty red circles. ¹³C CP MAS NMR of folic acid-functionalized mesoporous material FA-SBA15 (E).

Fig. 3. Representative TEM image and FFT pattern of SBA-15 (A and B), TEM (C), HR-TEM (D) and SAED (F) of Fe₃O₄ nanoparticles, and FA-FE-SBA15QN (E).

Fig. 4. (A) FL emission released by QN after the dispersion of FA-FE-SBA15QN in PBS at different pH values (5.5, 7 and 7.4). (B) Line diagram representing the release kinetics of QN from FA-FE-SBA15QN in different pH values (7.4 and 5.5). (C) In vitro T₂ weight MRI images of HCT 116 and HEK-293 cells after the treatment of FA-FE-SBA15QN at different concentrations (2.5, 5, and 10 μg ml⁻¹). Values are represented as the mean ± SEM (n = 5).

Fig. 5. Determination of cell viability, iROS, apoptosis/necrosis, and mitochondrial membrane potential upon the treatment of QN (15 μg ml⁻¹) and FA-FE-SBA15QN (10 μg ml⁻¹), along with/without NAC, SP600125, p53 siRNA, and HSP27 siRNA application in HCT 116 cells. (A) Bar graph showing cell viability. Representative flow cytometric dot plot and gating hierarchy used to define (B) DCF +Ve cells and DCF −Ve cells, (B) viable cells (VB), early apoptotic cells (EA), late apoptotic cells (LA), and necrotic cells (NC), (C) FITC +Ve cells and FITC −Ve cells, (D) high ΔΨ and low ΔΨ. Values are represented as the mean ± SEM (n = 5). p < 0.05 was considered as significant. Statistical comparison: *control vs. QN (15 μg ml⁻¹); **control vs. FA-FE-SBA15QN (10 μg ml⁻¹); ***FA-FE-SBA15QN (10 μg ml⁻¹) vs. FA-FE-SBA15QN (10 μg ml⁻¹) + NAC; ^#FA-FE-SBA15QN (10 μg ml⁻¹) vs. FA-FE-SBA15QN (10 μg ml⁻¹) + SP600125; ^##FA-FE-SBA15QN (10 μg ml⁻¹) vs. FA-FE-SBA15QN (10 μg ml⁻¹) + siRNA (p53); ^###FA-FE-SBA15QN (10 μg ml⁻¹) vs. FA-FE-SBA15QN (10 μg ml⁻¹) + siRNA (HSP27).

Fig. 6. QN and FA-FE-SBA15QN-induced modulation of HSP60, HSP27, p-JNK, and Bax expression in HCT 116 cells with/without application of NAC, SP600125, and siRNA (HSP27). Representative flow cytometric dot plot and gating hierarchy used to define (A) Q1: HSP60-AF647 −Ve/HSP27-AF488 −Ve cells; Q2: HSP60-AF647 −Ve/HSP27-AF488 +Ve cells; Q3: HSP60-AF647 +Ve/HSP27-AF488 +Ve cells; Q4: HSP60-AF647 +Ve/HSP27-AF488 −Ve cells, (B) p-JNK-FITC −Ve and p-JNK-FITC +Ve cells. (C) Immunofluorescence images showing the expression of p-JNK and Bax. DAPI was used for nuclear staining. Slides were viewed using a confocal microscope (magnification 20×). Respective fluorescence intensities (p-JNK-AF555, Bax-AF647, and DAPI) were analyzed using ImageJ software through an RGB calculator.

Fig. 7. Assessment of mitochondrial-dependant apoptosis upon QN and FA-FE-SBA15QN treatment, along with/without application of NAC, SP600125, and siRNA (p53). Representative flow cytometric dot plot and gating hierarchy used to define (A) p53-AF647 +Ve and p53-AF647 −Ve cells, (B) Bax-FITC +Ve and Bax-FITC −Ve cells, (C) CytC-FITC +Ve and CytC-FITC −Ve cells. Representative flow cytometric histogram showing (D) Bcl2 and (F) Apaf1. Bar graph shows relative fluorescence intensities of (E) Bcl2-FITC and (G) Apaf1-FITC. Bar graph shows (H) caspase 3 and (I) 9 activity in the different experimental conditions in HCT-116 cells. Values are represented as the mean ± SEM (n = 6). p < 0.05 was considered as significant. Statistical comparison: *control vs. QN (15 μg ml⁻¹); **control vs. FA-FE-SBA15QN (10 μg ml⁻¹); ***FA-FE-SBA15QN (10 μg ml⁻¹) vs. FA-IO-SBA-15-QN (10 μg ml⁻¹) + NAC; ^#FA-FE-SBA15QN (10 μg ml⁻¹) vs. FA-FE-SBA15QN (10 μg ml⁻¹) + SP600125; ^##FA-FE-SBA15QN (10 μg ml⁻¹) vs. FA-FE-SBA15QN (10 μg ml⁻¹) + siRNA (p53); NS = not significant.

Fig. 8. In vivo assessment of chemo theranostic potential of QN and FA-FE-SBA15QN. (A) Experimental animals showing tumor growth inhibitory activity of QN and FA-FE-SBA15QN. (B) Representative images of tumor harvested after the completion of the experiment. (C) Kaplan–Meier analysis of 30 days survival of CT-26 tumor-bearing mice post-administrated with QN and FA-FE-SBA15QN (15 mg kg⁻¹). (D) Representative line diagram showing body weight change throughout the experimental period. (E) Bar graph showing tumor weight. (F) In vivo T₂ weight MRI images of CT-26 tumor-bearing mice. Rectangular zone indicating the tumor area. Values are represented as mean ± SEM (n = 5). p < 0.05 was considered as significant. Statistical comparison: *CT-26 vs. CT-26 + QN (15 mg kg⁻¹); **CT-26 vs. CT-26 + FA-FE-SBA15QN (15 mg kg⁻¹).

Fig. 9. (A) Histopathological assessment of tumor microenvironment. The tumor tissue architectural assessment was done using hematoxylin & eosin staining (H&E) (nagnification: 20× and 40×). (B) Immunofluorescence images showing the expression of Ki-67 and Bax. DAPI was used for nuclear staining. Slides were viewed using a confocal microscope (magnification 20×). Respective fluorescence intensities (Ki-67-AF546, Bax-AF647, and DAPI) were analyzed using ImageJ software through an RGB calculator. (C) Representative immunoblots of cleaved PARP, PCNA, BAX, Bcl2, Bid, cytochrome C, cleaved caspase 3 and cleaved caspase 9. (D) Densitometric analysis of the relative protein expression of cleaved PARP, PCNA, BAX, Bcl2, Bid, cytochrome C, cleaved caspase 3 and cleaved caspase 9. β-Actin served as the internal control. Values are presented as mean ± SEM (n = 6). p < 0.05 was considered as significant. Statistical comparison: *CT-26 vs. CT-26 + QN (15 mg kg⁻¹); ^#CT-26 vs. CT-26 + FA-FE-SBA15QN (15 mg kg⁻¹); @ = not significant.