ZrMOF nanoparticles as quenchers to conjugate DNA aptamers for target-induced bioimaging and photodynamic therapy

Aptamer conjugated porphyrinic metal–organic framework (MOF) achieved target-induced bioimaging and photodynamic therapy.


Introduction
As emerging materials, nanoscale metal-organic frameworks (MOFs) are attracting intense interest in different areas such as energy storage, 1-3 catalysis, 4-7 and especially biochemical applications [8][9][10][11] in sensing, nanomedicine and bioimaging, due to their well-dened structure with unique physical and chemical properties. 12 Photodynamic therapy for cancer treatment involves generation of reactive oxygen species (ROS) by irradiation of photosensitizers at the tumor site. 13,14 As a key to this method, porphyrin and its derivatives as photosensitizers have frequently been a limiting factor due to poor solubility, selfquenching and aggregation issues. 15 These problems can be overcome using metal-organic framework (MOF) nanoparticles with precise spatial control and monomeric form, in particular porphyrin-based MOFs, 16 which are promising candidates for imaging contrast, drug delivery and photodynamic therapy.
Despite the success of nanoscale MOFs in biomedical science, 8,17,18 conventional bioimaging with these materials usually involves loading dyes into porous MOF nanoparticles or preparing intrinsic uorescent MOF nanoparticles for further imaging study. 19,20 However, leakage of dye from porous MOF nanoparticles is a potential problem. Meanwhile, non-specic accumulation of dye-loaded MOF and intrinsically uorescent MOF nanoparticles can cause strong background signals and fake imaging information. 21 Target-induced bioimaging can signicantly decrease the background and fake imaging information. Current cancer therapy with nanoscale MOFs mainly relies on passive targeting (enhanced permeability and retention (EPR) effect) to improve the specic accumulation of drug at tumor sites. 22,23 However, the EPR effect is complex and strongly depends on the size, surface properties and circulation time of nanoparticles, and large nanoparticles may have limited extracellular diffusion. In addition, some well-designed nanoparticles with a good EPR effect can penetrate throughout large tumor tissues following systemic administration, possibly causing side effects. 24 By conjugating nanoparticles with targeting ligands, such as small molecules, peptides, antibodies or aptamers, the nanoparticles can bind with cell-surface receptors and enter cells by receptor-mediated endocytosis, thus enhancing cellular uptake into cancer cells rather than increasing accumulation in the tumor. 13,[25][26][27][28][29] Although nanoscale MOF-DNA conjugates have been studied, 21,30 their applications have been limited by complex organic synthesis before and post MOF construction, as well as as-synthesized organic linkers. We here developed general facile one-step aptamer conjugation to nanoscale MOFs for targetinduced bioimaging and photodynamic therapy. Aptamers, selected from a large library by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), 31-34 are single-stranded oligonucleotides that can specically bind to the target by folding into distinct secondary or tertiary structures. Phosphate, a functional group which has a strong coordination interaction with zirconium, 35,36 was coupled to the 5 0 end of an aptamer through solid-phase DNA synthesis for direct conjugation to zirconium-based MOF nanoparticles as shown in Scheme 1.

Results and discussion
Zr-based porphyrinic MOF (ZrMOF) nanoparticles were synthesized using ZrOCl 2 , tetra(4-carboxyphenyl)porphine (TCPP) and benzoic acid according to a literature report. 13 Xray diffraction conrmed the well-dened crystal structure of the as-synthesized ZrMOF nanoparticles (Fig. S1 †). TEM indicated that the size of a ZrMOF nanoparticle is around 110 nm (Fig. 1a), and dynamic light scattering demonstrated a uniform size distribution of ZrMOF nanoparticles (Fig. S2 †). To conjugate the aptamer, a phosphate-terminal aptamer was added to ZrMOF nanoparticles and incubated for 5 hours. The free aptamer was removed by washing with water and centrifugation. Before aptamer conjugation, ZrMOF nanoparticles showed a positively charged zeta-potential. Aer aptamer conjugation, a negatively charged zeta-potential was observed, because of the negatively charged DNA aptamer (Fig. S3 †). DLS indicated a slight increase in size for ZrMOF nanoparticles aer aptamer conjugation (Fig. S2 †).
The stability of ZrMOF, as one of the most important features for biochemical study, was studied before and aer DNA aptamer conjugation. Aptamer-conjugated ZrMOF nanoparticles showed much better stability aer 24 hours than ZrMOF nanoparticles without any surface modication (Fig. 1c). Thus, phosphate-terminal DNA aptamer conjugation can signicantly enhance the biostability of ZrMOF nanoparticles in buffers and increase their potential for biomedical applications.  Having demonstrated that aptamer-conjugated ZrMOF nanoparticles are stable in different buffers, we next studied their utility in biomedical applications. In previous studies, nanoscale MOF nanoparticles were used as carriers for bioimaging by loading dyes into their well-dened porous structures. We here found that our ZrMOF nanoparticles can be used as quenchers for uorescent dyes, such as RhB and TAMRA. As shown in Fig. 2a, the uorescence of RhB was quenched upon adding to ZrMOF nanoparticles. This can be attributed to the conjugated p-p stacking effect between the TCPP linker and RhB via uorescence resonance energy transfer (FRET). Similarly, the uorescence of TAMRA was quenched as well when the TAMRA-modied aptamer was conjugated on the surface of ZrMOF nanoparticles, as shown in Fig. 2b. The uorescence of TAMRA was recovered when the target complementary DNA (c-DNA) was added to hybridize with the TAMRA-modied aptamer to form a double-stranded DNA (Fig. 2b), thus detaching the TAMRA from the surface of ZrMOF nanoparticles, resulting in the recovery of uorescence. This feature enabled us to construct a target-induced bioimaging system.
To validate the feasibility of target-induced bioimaging, we used ZrMOF nanoparticles conjugated with the TAMRA-modied DNA aptamer. As illustrated in Fig. 3a, the uorescence of TAMRA was quenched aer conjugating the aptamer to the surface of ZrMOF nanoparticles. But the uorescence of TAMRA was recovered aer binding with the target receptor on the cell membrane. Here, we selected the Sgc8 aptamer (Table S1 †), which binds the target membrane protein PTK7 expressed on HeLa cells. Before conducting target-induced imaging, the uorescence-quenching stability in Dulbecco's modied Eagle's medium (DMEM) culture medium was studied. No obvious uorescence recovery was observed from ZrMOF-aptamer-TAMRA and ZrMOF-Library-TAMRA aer four hours in HeLa cell culture medium DMEM (Fig. 2c and d), indicating a stable FRET under physiological conditions. The uorescence of TAMRA was recovered when treated with 10Â PBS buffer overnight, because the high concentration of free phosphate ions decomposed the ZrMOF nanoparticles and released the TAMRA modied DNA from the surface. A 2 hour incubation time of ZrMOF-aptamer-TAMRA and HeLa cells was used to study the target-induced imaging with ZrMOF-Library-TAMRA as a negative control. As shown in Fig. 3b, ZrMOF-aptamer-TAMRA exhibited an excellent target-induced imaging ability when incubated with HeLa cells, while ZrMOF-Library-TAMRA, which has a random DNA sequence and no target binding ability, showed negligible uorescence in HeLa cells, as shown in Fig. 3c.
TCPP, the organic linker used in the synthesis of ZrMOF nanoparticles, and its derivatives have been used as photosensitizers for photodynamic therapy. However, their photodynamic therapy effect has been limited due to their hydrophobicity, aggregation tendency and insufficient  selectivity to malignant tissues. 15 To overcome these limitations, we took advantage of the aptamer-conjugated porphyrinic ZrMOF nanoparticles for targeted photodynamic cancer therapy. Porphyrinic ZrMOF nanoparticles absorb light at both the Soret band and Q band wavelengths (Fig. S4 †). Irradiation at 650 nm, which can penetrate tissues, was selected to generate reactive oxygen species to kill the cancer cells. Singlet oxygen sensor green, which can be specically oxidized by reactive oxygen species to produce enhanced uorescence, was used as the ROS detector. Upon continuous laser excitation at 650 nm, porphyrinic ZrMOF-aptamer nanoparticles generated increasing amounts of singlet oxygen from 1 to 30 min (Fig. 4a). Moreover, the generation of singlet oxygen was conrmed with confocal imaging (Fig. 3d and e) of HeLa cells with ZrMOFaptamer as the positive control and ZrMOF-Library as the negative control. A signicant signal increase of singlet oxygen was observed when HeLa cells were treated with ZrMOFaptamer (Fig. 3d). Photodynamic therapy of aptamerconjugated porphyrinic ZrMOF nanoparticles was investigated by measuring cell viability using the MTS assay, as shown in Fig. 4b. HeLa cells were treated with ZrMOF-aptamer, ZrMOF-Library-650, and ZrMOF-aptamer-650 (irradiated with a 650 nm laser), respectively. ZrMOF-aptamer only without laser irradiation showed negligible cell death even at concentrations up to 200 mg mL À1 (red column). Aer laser irradiation (650 nm, 200 mW cm À2 ) for 5 min, ZrMOF-Library with a random DNA sequence and no target binding ability exhibited slight cell toxicity when the concentration was increased to 200 mg mL À1 (black column). However, signicantly reduced cell viability was observed for HeLa cells when incubated with ZrMOF-aptamer nanoparticles under the same conditions as ZrMOF-Library nanoparticles (blue column). The cell viability was 85% for HeLa cells when treated with ZrMOF-Library at a concentration of 200 mg mL À1 , while the cell viabilities were 48% and 17% for HeLa cells when treated with ZrMOF-aptamer at a concentration of 100 mg mL À1 and 200 mg mL À1 , respectively. In addition, cell apoptosis analysis-based ow cytometry and live/dead cell staining also indicated that aptamer conjugation signicantly increased the cell apoptosis efficiency (Fig. S8 and S9 †). These results demonstrated that a higher photodynamic therapy effect can be achieved through targeting aptamer conjugation to ZrMOF nanoparticles.
The phosphate-terminal aptamer provided a facile strategy for DNA aptamer conjugation to ZrMOF nanoparticles. This method can be generalized to other types of MOF nanoparticles, such as UiO-66 and HfMOF nanoparticles (Fig. 5), which have potential applications in bioimaging and radiation therapy. As shown in Fig. S5, † UiO-66 nanoparticles were pink aer conjugating with the phosphate-terminal aptamer modied with TAMRA. XRD results indicated that the crystal structure of UiO-66 did not change aer conjugation with the phosphateterminal aptamer modied with TAMRA (Fig. 5d). For HfMOF nanoparticles, their stability in water was signicantly increased aer conjugating with the aptamer through phosphate zirconium coordination (Fig. S6 †).

Conclusion
In conclusion, ZrMOF nanoparticles as quenchers to conjugate DNA aptamers for target-induced imaging and photodynamic  therapy were developed and generalized to other types of MOF nanoparticles, such as UiO-66 and HfMOF. Target-induced imaging and targeted photodynamic therapy were achieved using ZrMOF nanoparticles as quenchers and photosensitizers, and an aptamer as a targeting ligand on the surface of ZrMOF nanoparticles. This facile aptamer conjugation to ZrMOF nanoparticles offers opportunities to develop MOF-based targetdirected biosensors. On the basis of these superior features, we believe that future work can benet from the rational design of engineering DNA aptamers and MOF nanomaterials for biomedical studies.

Conflicts of interest
There are no conicts to declare.