Aptamer-Aptamer Chimera for Targeted Delivery and ATP-Responsive Release of Doxorubicin into Cancer Cells

Aptamers offer a great opportunity to develop innovative drug delivery systems that can deliver cargos specifically into targeted cells. In this study, a chimera consisting of two aptamers was developed to deliver doxorubicin into cancer cells and release the drug in cytoplasm in response to adenosine-5′-triphosphate (ATP) binding. The chimera was composed of the AS1411 anti-nucleolin aptamer for cancer cell targeting and the ATP aptamer for loading and triggering the release of doxorubicin in cells. The chimera was first produced by hybridizing the ATP aptamer with its complementary DNA sequence, which is linked with the AS1411 aptamer via a poly-thymine linker. Doxorubicin was then loaded inside the hybridized DNA region of the chimera. Our results show that the AS1411–ATP aptamer chimera was able to release loaded doxorubicin in cells in response to ATP. In addition, selective uptake of the chimera into cancer cells was demonstrated using flow cytometry. Furthermore, confocal laser scanning microscopy showed the successful delivery of the doxorubicin loaded in chimeras to the nuclei of targeted cells. Moreover, the doxorubicin-loaded chimeras effectively inhibited the growth of cancer cell lines and reduced the cytotoxic effect on the normal cells. Overall, the results of this study show that the AS1411–ATP aptamer chimera could be used as an innovative approach for the selective delivery of doxorubicin to cancer cells, which may improve the therapeutic potency and decrease the off-target cytotoxicity of doxorubicin

Synthesis of Mono-Amino Substituted γ-CD: Host–Guest Complexation and In Vitro Cytotoxicity Investigation

Cyclodextrins (CDs) are cyclic oligosaccharides which can trap hydrophobic molecules and improve their chemical, physical, and biological properties. γ-CD showed the highest aqueous solubility with the largest cavity diameter among other CD types. The current study describes a direct and easy method for nucleophilic mono-aminos to be substituted with γ-CD and tested for their ability to host the guest curcumin (CUR) as a hydrophobic drug model. The mass spectrometry and NMR analyses showed the successful synthesis of three amino-modified γ-CDs: mono-6-amino-6-deoxy-cyclodextrine (γ-CD-NH2), mono-6-deoxy-6-ethanolamine-γ-cyclodextrine (γ-CD-NHCH2CH2OH), and mono-6-deoxy-6-aminoethylamino)-γ-cyclodextrin (γ-CD-NHCH2CH2NH2). These three amino-modified γ-CDs were proven to be able to host CUR as native γ-CDs with formation constants equal to 6.70 ± 1.02, 5.85 ± 0.80, and 8.98 ± 0.90 mM−1, respectively. Moreover, these amino-modified γ-CDs showed no significant toxicity against human dermal fibroblast cells. In conclusion, the current work describes a mono-substitution of amino-modified γ-CDs that can still host guests and showed low toxicity in human dermal fibroblasts cells. Therefore, the amino-modified γ-CDs can be used as a carrier host and be conjugated with a wide range of molecules for different biomedical applications, especially for active loading methods

Fig 4 -

A) Induced apoptosis of MCF-7_WT cells treated with Dis and Hyd alone and combined B) Induced apoptosis of MCF-7_DoxR cells treated with Dis and Hyd alone and combined.</p

Diagnostic graphics produced for synergistic effect quantification of DOX/Dis/Hyd combination against MCF-7_DoxR cells.

(A) Dose -Fa curve for DOX alone and for DOX with (DOX/Dis/Hyd combination) at different concentration points (B) Table of IC50 of DOX, Dis, Hyd alone and combination (C) The fraction affected (Fa) versus combination index (CI) plot after treatment with DOX/Dis/Hyd combination, that most of CI values are < 1 for the range of 0.25–0.8 (D) The Fa-DRI plot for the non-constant ratios of DOX/Dis/Hyd combination.</p

Biological Performance of Primary Dental Pulp Stem Cells Treated with Gold Nanoparticles

Gold nanoparticles (AuNPs) are one of the most stable nanoparticles that have been prevalently used as examples for biological and biomedical applications. Herein, we evaluate the effect of AuNPs on the biological processes of dental pulp stem cells derived from exfoliated deciduous teeth (SHED). Two different shapes of PEGylated AuNPs, rods (AuNR-PEG) and spheres (AuNS-PEG), were prepared and characterized. SHED cells were treated with different concentrations of AuNR-PEG and AuNS-PEG to determine their effect on the stemness profile of stem cells (SCs), proliferation, cytotoxicity, cellular uptake, and reactive oxygen species (ROS), for cells cultured in media containing-fetal bovine serum (FBS) and serum-free media (SFM). Our results showed that both nanoparticle shapes maintained the expression profile of MSC surface markers. Moreover, AuNS-PEG showed a stimulatory effect on the proliferation rate and lower toxicity on SHED, compared to AuNR-PEG. Higher concentrations of 0.5–0.125 nM of AuNR-PEG have been demonstrated to cause more toxicity in cells. Additionally, cells treated with AuNPs and cultured in FBS showed a higher proliferative rate and lower toxicity when compared to the SFM. For cellular uptake, both AuNS-PEG and AuNR-PEG were uptaken by treated cells efficiently. However, cells cultured in SFM media showed a higher percentage of cellular uptake. For ROS, AuNR-PEG showed a significant reduction in ROS at lower concentrations (<0.03 nM), while AuNS-PEG did not show any significant difference compared to the control untreated cells. Thus, our results give evidence about the optimum concentration and shape of AuNPs that can be used for the differentiation of stem cells into specific cell lineages in tissue engineering and regenerative medicine

STRING analysis of protein–protein interactions.

(A) Interaction between proteins involved in drug resistance STAT3, NOTCH, DNMT, (B) Illustration of combination of HDACi and DNMTi pathway, (C) combination of HDACi and DNMTi and the effect of cell apoptosis proteins, (D) interactions of DNMTi and proteins ivoved in cell cycle, (E) Effect of ALDH enzymes on P53 proteins, (F) Interactions between ALDH and glutathione (GSH), (G) Interactions between ALDH, ABCC1 and p53 proteins, (H) Signalling pathway showed interaction of DNMT, ALDH, P53, TOP2A, ABCB1. STAT3, NOCH1. (thickness of edges indicate confidence).</p

Diagnostic graphics produced for synergistic affect quantification.

(A) Isobologram for Dis/Hyd combination at different concentration points, (B) The Fa-DRI plot (Chou-Martin plot) for the non-constant ratios of Dis/Hyd combination, (C) The fraction affected (Fa) versus combination index (CI) plot after treatment with Dis/Hyd combination, that most of CI values are 50 of Dis, Hyd and Dis/Hyd combination.</p

Apoptosis Induced by DOX/Dis/Hyd single treatment and combinations.

A) and B) MCF-7_WT cells, C) and D) MCF-7_DoxR cells.</p