Results

Overview

Our results mainly consist of 4 parts:

1. Binding affinity of aptamer
  · ELONA lab for testing the binding affinity of the aptamer
  · Asymmetric PCR for the synthesis of ssDNA

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. The figure is obtained from previous literature: https://doi.org/10.1038/s41467-020-16808-2.

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 strands.

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.

Liposome

Manufacturing of Dox-encapsulated Liposome

We used microfluidics technology, which is on a specific automatic machinery, to manufacture liposomes using 2 different molar ratio of DSPC : cholesterol : PEG (DCP ratio), which is DSPC : cholesterol : PEG = 10 : 6 : 1 or 15 : 6 : 1 of materials at the first time, and also trying different rotate speeds (Fluid Velocity / mL*min^-1): 6, 12, 18. Then we used Malvern Particle Sizer to measure the diameters of liposomes. A detailed protocol can be found in here.

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.

Table 2.2 demonstrated that both DCP ratio and rotate speeds are important factors of liposome's diameter. Generally, liposomes with DCP ratio of 15 : 6 : 1 have larger diameters than 10 : 6 : 1 liposomes, while liposomes with rotate speed of 6 mL/min and 12 mL/min have smaller diameters. The Pdi of most sets are smaller than 0.3, which indicated that the sizes of liposomes are uniform. To meet our requirements of "150--200nm" to best coordinate with EPR effect, we decided only to use the speed 6mL/min the next time.

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.

To better improve the size and stability of liposomes, we set another 2 DCP ratios (LA, 10 : 6 : 1.5 and LC, 15 : 6 : 1.5) for manufacturing. Due to the need of blank liposomes for conjugation testing which will be described in the latter part, we also made blank liposomes with four different ratios the second time. However, due to various reasons, the liposomes' sizes were bigger than the last time, though the results verified that a DCP ration of 10 : 6 : 1 is the best ratio for making suitable liposomes, which is also shown in our first experiment. Possible reasons can be the misoperation of staff, incoherence of experiments, or precipitate's presence during transportation process which caused a change of materials' concentration etc. In addition, we also made blank liposomes, which have relatively small diameters than drug-loaded liposomes. We later used LA-Dox made on 13th and 16th; LA blank liposomes made on 16th, which have relative small diameters and Pdi.

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.

We also got some graphs using specific software to show the distribution of particle size.

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.

Encapsulation rate testing of Dox-Liposome

To measure encapsulation rate of Dox-Liposome, we need to know the amount or concentration of initial doxorubicin and free doxorubicin after purifying. Therefore, we made a doxorubicin gradient which showed the relation between doxorubicin concentration and its absorbance under 485nm waves. All the experiments were repeated at least three times, and error bars represent SEM. a.u. arbitrary units to guarantee the accuracy of the gradient and Figure 2.2 shows one of the gradient (the orange line) with the R² value (0.9983)>0.99, symbolizing its desirable imitative effect.

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.

After that, we took 200 μL of initial solution and supernates from centrifugation of the original solution each to test their absorbance using microplate reader under 485 nm waves, and used the equation we got to calculate their concentrations thus their amounts. By utilizing the formula: $$\text{Encapsulation Rate} = \frac{(\text{Amount of initial Dox} - \text{Free Dox})}{\text{Amount of initial Dox} \times \text{100%}}$$ we obtained the encapsulation results, presented in Table 2.4.

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.

The results showed us that we had successfully wrapped the drugs inside with different degrees (from 27% to 67%). The rates aren't high enough compared to results we found in thesis or other researches, but considering that dox is a highly toxic drug and our aim is to make more numbers of nanoparticles with less drugs each to prevent side effects, the results are acceptable.

Modification of doxorubicin liposome with aptamer

To connect aptamers onto liposomes, we utilized EDC/HNS method for coupling the carboxylic group on PEG and amine group on aptamers. Specifically, this included activation of carboxylic group on PEGylated liposomes first under EDC/NHS environment and then incubation in certain temperature with NH2-modified aptamers.

The first time we used Dox-liposomes for aptamer modification, and got our first result shown in Figure 2.3A. From the picture we can see a free aptamer running to about 100bp place. We expected that, for aptamer-dox liposome, there were fluorescences at the original place as the conjugated aptamer could no longer run to 100bp place like free aptamer. There were also light bands in aptamer-dox liposomes' lanes, which indicated that we hadn't removed all the free aptamer the first time. Nevertheless, the biggest problem we found was that doxorubicin itself has strong fluorescences that can cover the fluorescences of aptamer.

To solve these issues, we decided to use blank liposome to conjugate aptamer first, and then diffuse Dox into the liposome later.

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 3. B showed the results of electrophoresis of aptamer-blank liposomes. Without the disruption of dox fluorescences, we clearly found there are pale fluorescences bands at the original places, showing our success in conjugation.

We also use some blank liposomes as control groups. From figure 3. C, there was not fluorescences in lane 7 but fluorescences in lane 4 and 5, showing blank liposomes hardly affect the fluorescences. However, there was fluorescences in lane 6, which is also blank liposome. This could be caused by contamination of nearby samples, or because we used liposome's precipitate for electrophoresis that time to increase the concentration, as the precipitate was macroscopic.

Ammonium sulphate diffusion

After verifying that blank liposomes had been successfully modified by aptamer, we used ammonium sulphate gradient to help diffuse doxorubicin into liposomes. The nature of this method was let inside ammonium sulphate "drags" doxorubicin outside into liposomes by forming Doxorubicin-sulphate precipitates. Therefore, it was very important to wash away all ammonium sulphate outside the liposomes. The method succeeded when we found red precipitate after diffusion process, showing that red doxorubicin went into liposomes.

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.

The encapsulation rate of the Aptamer-Dox-Liposome is measured with the same method.

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