Cancer treatment using siRNA based therapies pose various limitations such as off-target effects and degradation due to lack of specific delivery in desired cells. The aim of the present study was to develop multifunctional targeted nanoconstructs, which can efficiently and precisely deliver siRNA and silence the desired gene of interest in various LHRH overexpressing cancer cells. Herein, we report the development of triblock, PAMAM-histidine-PEG dendritic nanoconstructs functionalized with triptorelin (an LHRH analog) for targeted siRNA delivery to LHRH overexpressing breast (MCF-7) and prostate (LNCaP) cancer cells. The nanoconstructs were characterized using H NMR and DLS and displayed a very low cationic charge to avoid off-target interactions. The developed nanoconstructs showed negligible cytotoxicity and hemolytic activity with efficient siRNA loading, excellent serum stability, and strongly protected siRNA from degradation. Further, confocal microscopy results confirmed extremely significant (p< 0.001) higher cellular uptake of cy5.5 conjugated targeted nanoparticles (NPs) in both cancer cell lines than nontargeted NPs. Also, targeted NPs specifically delivered cy3-tagged siRNA to MCF-7 cells. Co-localization studies in MCF-7 and LNCaP cells further established that targeted NPs traveled through the endolysosomal pathway and escaped endosomes within 6 h of incubation. Gene silencing studies in luciferase expressing MCF-7 and LNCaP cell lines demonstrated that the targeted NPs exhibited extremely significant (p < 0.001) silencing of luciferase gene. Additionally, receptor blockade studies further confirmed the specificity of targeted NPs and suggested that targeted NPs entered cancer cells via LHRH receptor mediated endocytosis, which was evident through insignificant gene silencing in receptor blocked cells. Thus, the results indicated that PAMAM-histidine-PEG-triptorelin could be a promising approach for siRNA delivery, gene silencing, and tumor therapy in all LHRH overexpressing cancer cells.
BackgroundConsidering the increase in cancer cases and number of deaths per year worldwide, development of potential therapeutics is imperative. Mesoporous silica nanoparticles (MSNPs) are among the potential nanocarriers having unique properties for drug delivery. Doxorubicin (DOX), being the most commonly used drug, can be efficiently delivered to gonadotropin-releasing hormone (GnRH)-overexpressing cancer cells using functionalized MSNPs.AimWe report the development of decapeptide-conjugated MSNPs loaded with DOX for the targeted drug delivery in breast and prostate cancer cells.Materials and methodsMSNPs were synthesized and subsequently functionalized with an analog of GnRH by using a heterobifunctional polyethylene glycol as a linker. These targeted MSNPs were then characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, and Raman spectroscopy. An anticancer drug DOX was loaded and then characterized for drug loading. DOX-loaded nanocarriers were then studied for their cellular uptake using confocal microscopy. The cytotoxicity of DOX-loaded targeted MSNPs and DOX-loaded bare MSNPs was studied by performing MTT assay on MCF-7 (breast cancer) and LNCaP (prostate cancer) cells. Further, acridine orange/ethidium bromide staining, as well as flow cytometry, was performed to confirm the apoptotic mode of cancer cell death.ResultsMSNPs were conjugated with polyethylene glycol as well as an agonist of GnRH and subsequently loaded with DOX. These targeted and bare MSNPs showed excellent porous structure and loading of DOX. Further, higher uptake of DOX-loaded targeted MSNPs was observed as compared to DOX-loaded bare MSNPs in GnRH-overexpressing breast (MCF-7) and prostate (LNCaP) cancer cells. The targeted MSNPs also showed significantly higher (P<0.001) cytotoxicity than DOX-loaded bare MSNPs at different time points. After 48 hours of treatment, the IC50 value for DOX-loaded targeted MSNPs was found to be 0.44 and 0.43 µM in MCF-7 and LNCaP cells, respectively. Acridine orange/ethidium bromide staining and flow cytometry analysis further confirmed the pathway of cell death through apoptosis.ConclusionThis study suggests GnRH analog-conjugated targeted MSNPs can be the suitable and promising approach for targeted drug delivery in all hormone-dependent cancer cells.
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