Treatment of Alzheimer's disease with framework nucleic acids

Abstract Objectives To provide a new research direction for nerve regeneration and strategy for Alzheimer's disease treatment, tetrahedral DNA nanostructures (TDNs)—novel tetrahedral framework nucleic acid molecule nanoparticles (tFNA) that can inhibit the apoptosis of nerve cells are employed in the experiment. Materials and methods To verify the successful preparation of TDNs, the morphology of TDNs was observed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The expression of apoptosis‐related genes and proteins was investigated by confocal microscope, flow cytometry, PCR and Western blot to detect the impact of TDNs on the Alzheimer's model. And finally, Morris water maze experiment was used to test behavioural changes and Nissl stain was detected to observe the morphology and quantity of neurons in the hippocampus. Immunofluorescence stain was used to observe the Aβ stain, and TUNEL dyeing was utilized to observe neuronal apoptosis. Results In vitro and in vivo experiments confirm that TDNs, in a specific concentration range, have no toxic or side effects on nerve cells, can effectively inhibit apoptosis in an Alzheimer's disease cell model and effectively improve memory and learning ability in a rat model of Alzheimer's disease. Conclusions These findings suggest that TDNs may be a promising drug for the treatment of Alzheimer's disease.

changes, which ultimately induces cytotoxicity and cell death, including mitochondria-dependent apoptosis. 4,5 Studies have shown that some drugs for Alzheimer's disease treatment may cause cytotoxicity (eg, tacrine can cause significant hepatotoxicity); therefore, we aimed to find a new drug that can inhibit nerve cell apoptosis and thus become a safer option for the treatment of Alzheimer's disease. [6][7][8] Tetrahedral DNA nanostructures (TDNs) are novel three-dimensional framework nucleic acids (tFNA) which currently have broad application prospects in the biomedical field. The advantages of TDNs include simple synthesis, high yield, good biocompatibility and good in nuclease resistance. [9][10][11][12] Fortunately, according to previous literature reports, TDNs can partially pass the blood-brain barrier (BBB), 13 a special structure that separates the central nervous system (CNS) from the blood circulation and prevents substances in the plasma that are toxic to brain tissue from entering the brain, thereby maintaining a good living environment for nerve cells. At present, there is a lack of reports on the role of TDNs in the treatment of Alzheimer's disease.

| Materials
TDNs were fabricated on the basis of a previous method. 14,15 The capillary electrophoresis technique (Qsep 100TM; Bioptic) and nondenaturing polypropylene gel electrophoresis were used to verify successful TDN synthesis, by measuring the molecular weight of DNA single strand and TDN. Subsequently, we measured TDN particle size by dynamic light scattering (DLS).

| Real-time cell analysis
PC12 cells were seeded into Real-time cell analysis (RTCA)-specific culture plates at a concentration of 5000 cells/well and cultured in an incubator at a temperature of 37°C and a carbon dioxide concentration of 5% for 48 hours. Next, Aβ was added to PC12 cells at a concentration of 25 μmol/L for 24 hours to generate an Alzheimer's disease cell model, as previously described. 16 After Alzheimer's disease was successfully modelled, the well plate was rinsed 3 times with 0.02 mol/L phosphate-buffered saline (PBS), and TDN was added at a concentration of 250 nmol/L. At the corresponding time points, the corresponding values on the RTCA apparatus were used for analysis.

| Detection of apoptosis by flow cytometry
After 24 hours of TDN or serum-free medium treatment, cells were digested with trypsin containing no EDTA and collected in a 15 mL centrifuge tube (300 g, 5 minutes). The supernatant was discarded, washed with PBS and centrifuged (300 g, 5 minutes). The cell pellet was suspended in 400 μL of Annexin V binding solution at a concentration of approximately 10 6 cells/mL. Then, 5 μL of Annexin V-FITC staining solution was added to the cell suspension and gently mixed at 4°C for 15 minutes in the dark, followed by addition of 5 μL propidium iodide staining solution, gentle mixing and incubation at 4°C for 5 minutes in the dark. Subsequently, cells were transferred to a flow cytometry tube and analysed using a flow cytometer (FC500 Beckman).

| Immunofluorescence analysis
Cells were fixed with 4% paraformaldehyde at 37°C for 20 minutes, incubated with 0.5% Triton X-100 for 15 minutes and blocked with 5% skimmed milk powder at 37°C for 50 minutes before adding the primary antibodies against caspase-3 (1:100; Abcam) and Bax (1:100; Abcam). After incubation at 4°C overnight, cells were incubated with a secondary antibody (1:2000, Beyotime) for 1 hour at room temperature. Finally, cells were stained with phalloidin and DAPI to observe the cell skeleton and nucleus and photographed using a fluorescence or a laser confocal microscope (N-SIM, Nikon).

| Alzheimer's disease rat model generation
Sprague-Dawley rats were anesthetized with an intraperitoneal injection of 1% sodium pentobarbital at an injection rate of 40 μg/g and then fixed on a brain stereotaxic instrument. The hippocampal CA1 area was slowly and uniformly injected, bilaterally, with 10 μg (1 μL) of Aβ1-40 or the same amount of normal saline (1 μL; sham operation). The needle was left for 5 minutes, and the wound was sutured after withdrawing the needle. Three days after the operation, rats were randomly divided into two groups: the first received a daily tail-vein injection of 100 μL normal saline for 21 days, and the sec-

| Western blotting
After different treatments, PC12 cell and brain tissue lysate were prepared, and protein samples were separated by SDS-PAGE electropho- [ab32503]; Abcam). The next day, membranes were rewarmed at room temperature for 1 hour and incubated with the appropriate secondary antibody (A0208, A0216, Beyotime) for 1 hour at 37°C. Finally, membranes were incubated with exposure liquid (1705060, Bio-Rad) and developed using enhanced chemiluminescence detection system (Bio-Rad). The data were processed using Quantity One software.

| Quantitative real-time PCR
Total RNA was isolated from PC12 cells or collected brain tissues with the RNeasy Mini Kit (Qiagen). Then, cDNA was prepared by reverse polymerase chain transcription using the Qiagen One-step RT-PCR Kit (Qiagen). All operations were performed according to previously described methods, 17 and the expression of caspase 3, Bcl-2 and Bax was determined by real-time quantitative real-time PCR (qPCR; ABI 7300).
The primer sequences for each gene are displayed in Table 1.

| Detection of the ability to penetrate the blood-brain barrier
We used a method of co-culture of human micro vascular endothelial

| Behavioural testing
For all groups, the Morris water maze test was performed after 21 days saline administration or TDN administration. 18 The test consisted of two phases: a positioning navigation phase and a space exploration phase. In the first, rats received 5 consecutive days of training in the water maze, 4 times a day, 30 minutes each, and the time required to enter the water from four water inlet points and find the platform. The average score of the four trials in each day was used for the final statistical analysis as the final result of the day. In the space exploration phase, on the 6th day of the experiment, the platform was removed, and after entering the water from the farthest end of the platform, the rats were placed in water, the swimming trajectory of the rats within 30 seconds was recorded, and the rats were observed and stayed in the target quadrant. Time, the number of times it traversed the platform and the latency it first found on the platform.

| Nissl staining
After the behavioural testing, rats in each group were sacrificed, and the whole brain was removed, and the cerebellum was cut off on ice. We first made paraffin sections based on the steps in the literature report. 19 Subsequently, we performed Nissl staining on the sections. Sections were microscopically examined using upright optical microscope (CK31; Olympus), and images were captured for analysis.

| TUNEL staining
The frozen sections were fixed with 4% paraformaldehyde for 20 minutes and incubated on ice for 2 minutes in a permeabilization solution. Subsequently, 50 μL of a freshly prepared TUNEL (TdT-mediated dUTP Nick-End Labelling) solution was added onto each slide. Slides were finally examined under a fluorescence or laser confocal microscope. The apoptotic rate was calculated by analysing the relevant hippocampus parts of the sample by microscopy.

| Statistical analysis
Data analysis was performed using SPSS 16.0 statistical software.
All data are expressed as mean ± standard deviation. All groups were compared using multivariate analysis of variance. One-way analysis of variance was used to compare the effect of TDNs at different time points in the same group. P values <.05 were considered as statistically significant. Figure 1A shows a schematic diagram of TDN synthesis, with each DNA single strand forming one side of the structure. We verified

TA B L E 1 The primers sequences of relevant genes designed for qPCR
Primer name Direction Primer sequence the synthesis of the tetrahedrons by non-denaturing polypropylene gel electrophoresis. Figure Figure 1C). Based on these tests, we confirmed the successful production of TDNs.

| TDN has a therapeutic effect on an in vitro Alzheimer's disease model
According to reports in the literature, 9 compared to traditional DNA single strands, TDNs enter living cells through caveolin-mediated endocytosis and maintain their structural integrity for a period of time inside the cell. 9 We first confirmed that TDNs, rather than DNA single strands, successfully entered PC12 cells, by testing their cellular uptake by confocal microscopy and flow cytometry

| TDN treatment reduces Alzheimer's diseaseinduced apoptosis in vitro
To explore the mechanism of TDNs action, we assessed apoptosis by flow cytometry, as well as the expression of proteins and genes related to the apoptotic signalling pathway by Western blot, caspase-3 Activity Assay Kit (Beyotime) and real-time qPCR. Addition of TDNs significantly reduced the proportion of apoptotic cells in the Alzheimer's disease cell model, indicating that TDNs can inhibit apoptosis to a certain extent ( Figure 1H). Moreover, the caspase-3 activity was decreased after TDN addition (Figure 2A,B).
Bax is one of the major apoptotic genes in mitochondria-depen-

| TDNs can cross the BBB
The blood-brain barrier (BBB) separates the central nervous system (CNS) from the blood circulation and prevents entry of substances in the plasma that are toxic to the brain, thereby maintaining a good living environment for nerve cells. However, the BBB does not prevent all substances from entering the brain tissue. For example, essential nutrients (such as glucose, amino acids.) may enter the CNS faster than with simple diffusion. 24,25 Before conducting in vivo experiments, we assessed whether TDNs can cross the BBB. First, we constructed an in vitro BBB model by co-culturing the brain microvascular endothelial cell line Bend.3 with PC 12 cells and tested the ability of TDNs to cross the BBB by flow cytometry. As shown in Figure 3A,B, compared to single-stranded DNA, TDNs could indeed cross the BBB.
Subsequently, we injected the TDN solution into the tail vein of rats, and using small animal in vivo imaging technology, we observed the distribution of TDNs in vivo at different time points.
As shown in Figure 3C, TDNs accumulated in the brain in the first 10 minutes after injection and then gradually decreased. The above experimental results confirm that TDNs can partially cross the BBB, providing an experimental basis for the subsequent in vivo experiments.

| TDNs improve learning and memory in an Alzheimer's disease rat model
To detect behavioural changes, we subjected rats of each group to the Morris water maze test. Figure 3D,E show the experimental results and trajectories of the directional navigation experiments for the five consecutive days. Compared with the first day, the escape latency of each group was reduced over time; however, there was a significant difference among control, sham-operated and the TDNinjected rats on the 5th day (P < .01). On the second day, the escape latency of Alzheimer's disease rats was significantly higher (P < .05), while that of TDN-injected rats was significantly lower (P < .05) than that of control rats. For the days 3, 4 and 5, this difference was more pronounced. On the fifth day, the escape latency was significantly lower in the control, sham and TDN groups than in the Alzheimer's disease group (P < .05). The difference was most significant for the TDN injection group (P < .01).  Figure 3F). Moreover, the number of crossings was greater in these groups than in the Alzheimer's disease group, with a statistically significant difference found between the TDN and Alzheimer's disease groups (P < .05; Figure 3G). Lastly, the latency to find the platform was lower in all groups compared to the Alzheimer's disease group, which was statistically significant between the TDN and Alzheimer's disease groups (P < .05; Figure 3H).
Taken together, the above results suggest that TDNs can improve the learning and memory ability of Alzheimer's disease rats, which will provide new research ideas for the treatment of Alzheimer's disease.

| TDNs partially restore Alzheimer's diseaserelated morphological anomalies in the hippocampus
In order to detect changes in the morphology and number of neurons in the hippocampus after TDN injection in the tail vein, we

| TDN treatment inhibits Alzheimer's diseaseinduced apoptosis in the hippocampus
Next, in order to further explore the effect of TDNs on cell apoptosis in vivo, we performed TUNEL staining of the rat hippocampus.  Nucleic acid nanotechnology is a hot topic in recent years. 33,34 Due to the special molecular properties of DNA, it has great potential for development in many research directions such as biomedi- Our findings suggest that TDNs may be used as a new drug for the treatment of Alzheimer's disease ( Figure 5E). This novel and noteworthy discovery may pave the way for the potential use of TDNs in preventing neuronal cells death in the process of Alzheimer's disease.

ACK N OWLED G EM ENT
This study was supported by National Key R&D Programme of China (2019YFA0110600) and National Natural Science Foundation of China (81970916, 81671031).

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

AUTH O R CO NTR I B UTI O N
All authors contributed to the study concept and design. Xiaoru

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.