Results

Overview

Our results mainly consist of 4 parts:

1. Binding affinity of aptamer

2. Preparation of Liposome nanoparticle

3. Preparation of PLGA nanoparticle

4. In vitro cytotoxicity assays

Aptamer

ELONA Experiment

The specificity part of our project relies on the specific binding between the aptamer and HER2 receptors. Obtaining quantitative results through our experiment would, first, allow us to know more information about the aptamer we are using, which helps us to better design later experiments. Secondly, we can use the data in our modeling to improve our design, such as the density of aptamer on the surface of nanoparticles. We tried out 2 types of aptamers (Nickname: H2^[1] & HR2^[2]) and synthesized these aptamers at Genscript. However, only HR2 aptamer yielded result.

We designed our experiment based on a sandwich ELISA kit which is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human HER2 in serum, plasma, and cell culture supernatants. Our assay employs a well-plate with an antibody specific for human HER2 receptors coated on a 48/96-well plate. Recombinant Human HER2 standard is pipetted into the wells and is bound by the immobilized antibody. The wells are washed and a biotinylated HR2 aptamers antibody is added. After washing away unbound biotinylated HR2 aptamer, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of HR2 aptamer bound. The Stopping Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

We evaluated the ELISA kit first, with the standard sample given. The results indicated that, when the concentration of coated HER2 protein was at 8ng/mL, it still has reactivity to the provided HRP-antibody conjugates (provided in the kit) with high effectiveness and sensitivity. Thus, we chose to use add 8ng/mL of Recombinant Human HER2 standard.

Figure 1. A standard sample test of the ELISA. At 8ng/mL, the result shows that the antibody already has a high level of specificity

First, we did a qualitative test for HR2 aptamer.We successfully measured the HR2 aptamer bind to HER2 protein with high specificity and sensitivity.

Figure 2. Qualitative test for aptamer affinity.Some degree of specificity for HER2 receptor is shown on HR2 aptamer

The result gave us insight into the range of concentration we should choose for obtaining the exact Kd of the aptamer in the followed-up quantitative experiment.

Next, we evaluated the HER2 binding affinity of the aptamer quantitively. In order to improve the efficiency of the experiment, we modified the experimental process. Firstly, we further reduced the HER2 protein concentration used in the experiment to 4ng/mL. Secondly, we replaced biotinylated HR2 aptamer with FAM-labeled aptamer for convenience, because FAM can be detected sensitively and we can reduce the process at the same time. HER2 proteins were incubated with increasing concentrations of FAM-labeled aptamer and analyzed by spectrofluorometer. Using non-linear regression analysis, the Kd of the aptamer for binding with the HER2 protein was estimated to be 1.803 μM.

Figure 3. A further quantitative test for aptamer affinity. The curve accounts for non-specific binding of aptamer on the bottom of the well plate, achieved through adding a paramater; the equation: $$Y=\frac{Bmax \times X}{(Kd +X)} + M \times X$$. The R² value for the curve is 0.9801 and Kd = 1.803 μM.

Limitations

Asymmetric PCR

After obtaining the binding affinity of HR2 Aptamer, we went on to design a pH-sensitive HR2 aptamer. Tumor cells up-regulate H⁺/Na⁺ antiporters and have produce excessive amounts of lactate, which results in reducing their environmental pH^[4]. Allowing aptamers to be pH-sensitive, and only binds to HER2 receptors when they are in a low pH environment could minimize the damage dealt to normal tissue cells.

Figure 4^[5]. (left) The pH-sensitive extension is an ssDNA chain that can fold into a d8plex under high or normal pH, the ends of the ssDNA chain consists of aptamer (ATP aptamer is used in literature for demonstration) and a complementary chain that binds with the aptamer and inhibits its action. The duplex shape places the two ends together thus aptamer is inhibited by complementary strand. When the aptamer is placed in low pH solutions, protonation of the bases occurs and stabilizes C·G Hoogsteen base pairing, which folds the ssDNA chain into a triplex. The triplex shape separates the two ends so the aptamer functions normally. (right) only C·G Hoogsteen base pairing is stabilized by protonation, thus increasing the percentage of C·G Hoogsteen base pairing increases the sensitivity of the ssDNA chain to protonation. TAT60 is used in our design.

To enable pH-sensitive property of HR2 aptamer, a DNA strand has to be added to the original aptamer which would fold into different shapes, which inhibits HR2 aptamer under high pH and allows high affinity for HER2 receptor under low pH. However, the pH-sensitive extension would extend the aptamer to 145 bp, which exceeds the limit for chemical synthesis, and even if we managed to reduce the length, the price of synthesis would remain expensive. Thus, we performed asymmetric PCR^[3] to synthesize ssDNA strand.

First, we designed primers to synthesize the template; PCR has been carried out to replicate the template using the Forward Template and Reverse Template(shown below), where they would overlap and allow the replication of 145bp long template. Then, asymmetric PCR(asPCR) is performed, forward and reverse primer ratio of 1:1 up to 100:1 is added respectively in different groups to synthesize ssDNA strand.

Figure 5. The agarose gel for assymmetic PCR. As shown in the picture, when the F : R primer ratio is above 1 : 1, a blurry new band appears above the orginal band, which is identified as ssDNA.

Figure 5 is our result for asymmetric, as can be seen, an extra band of DNA appeared above the dsDNA band, which can be identified as ssDNA. We noticed that this is not concordant with our referenced literature^[3], as in the literature ssDNA ends up below the dsDNA bands. However, we are able to prove the validity of our experiment by repeating with ssDNA strands synthesized by Genscript, which was used previously in ELONA (Figure 6). The ssDNA strand is 86bp long, however, it lies above the 100bp long ladder band. Through this, we can be sure of our result that we have synthesized the ssDNA strand of the desired length, though, due to unknown reasons, its band appeared above dsDNA strands.

Figure 6. The agarose gel for company-synthesized ssDNA strands of 86bp As shown in the picture, ssDNA of 86bp lies about the 100bp ladder, due to unknown reasons.

Through asPCR, we were able to synthesize ssDNA strands which are our aptamers. Our next step is to buy FAM-modified primers to synthesize aptamers for the ELONA test of the pHs-HR2 aptamer. However, due to a lack of time and effort, we were unable to finish future experiments. Our original plan includes a qualitative test for the affinity of our pH sensitive aptamer at 5 μM, under pH 6.5, 7.1 and 8.0 to verify our theory. Our expected result would be an decrease in affinity when changing from pH 6.5 to 8.0.

Reference

1. Niazi, J. H., Verma, S. K., Niazi, S., & Qureshi, A. (2015). In vitro HER2 protein-induced affinity dissociation of carbon nanotube-wrapped anti-HER2 aptamers for HER2 protein detection. The Analyst, 140(1), 243–249. https://doi.org/10.1039/c4an01665c
2. Liu, Z., Duan, J. H., Song, Y. M., Ma, J., Wang, F. D., Lu, X., & Yang, X. D. (2012). Novel HER2 aptamer selectively delivers cytotoxic drug to HER2-positive breast cancer cells in vitro. Journal of translational medicine, 10, 148. https://doi.org/10.1186/1479-5876-10-148
3. Marimuthu C, Thean-Hock Tang, Soo-Choon Tan, Chee-Hock Hoe, Rajan Saini, Junji Tominaga and Subash C.B. Gopinath songklanakarin J. Sci. Technol. 34 (2), 125-131, Mar. - Apr. 2012
4. Tannock, I. F., & Rotin, D. (1989). Acid pH in tumors and its potential for therapeutic exploitation. Cancer research, 49(16), 4373–4384.
5. Thompson, I., Zheng, L., Eisenstein, M., & Soh, H. T. (2020). Rational design of aptamer switches with programmable pH response. Nature communications, 11(1), 2946. https://doi.org/10.1038/s41467-020-16808-2