iGEM 2021 — Team GreatBay_SCIE

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# Team:GreatBay SCIE/Notebook

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* [Modeling](https://2021.igem.org/Team%3AGreatBay_SCIE/Model)
  + [Overview](https://2021.igem.org/Team%3AGreatBay_SCIE/Model)
  + [Optimum Liposome-Aptamer Ratio](https://2021.igem.org/Team%3AGreatBay_SCIE/Optimum_Aptamer_Concentration)
  + [Drug Loading](https://2021.igem.org/Team%3AGreatBay_SCIE/Drug_Loading)
  + [Dissociation Constant](https://2021.igem.org/Team%3AGreatBay_SCIE/Dissociation_constant)
  + [3D Models](https://2021.igem.org/Team%3AGreatBay_SCIE/3D_Model)
* [Human Practices](https://2021.igem.org/Team%3AGreatBay_SCIE/Human_Practices)
  + [Integrated Human Practices](https://2021.igem.org/Team%3AGreatBay_SCIE/Human_Practices)
  + [Public Engagement & Education](https://2021.igem.org/Team%3AGreatBay_SCIE/Communication)
  + [GreatBay\_SCIE X Pink Ribbon](https://2021.igem.org/Team%3AGreatBay_SCIE/BreastCancerInChina)
  + [Collaborations](https://2021.igem.org/Team%3AGreatBay_SCIE/Collaborations)
  + [Partnership with SMS\_Shenzhen](https://2021.igem.org/Team%3AGreatBay_SCIE/Partnership)

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* [Aptamer Lab Notebook](#Aptamer)
  [ELONA](#ELONA)
  [Asymmetric PCR](#Asymmetric PCR)* [PLGA Lab Notebook](#PLGA)
  * [Liposome Lab Notebook](#Liposome)
  * [Cell Room Notebook](#Cell)

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Lab Notebook

# Aptamer Lab Note

## ELONA

(Scroll down to see more in the pdf below)

All raw data for aptamer lab are compiled [here](https://2021.igem.org/File%3AT--GreatBay_SCIE--Aptamer_ELONA_Lab.zip)

## Asymmetric PCR

# PLGA Lab Notebook

# Liposome Lab Notebook

# Cell Room Notebook

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:GreatBay SCIE/Engineering

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  + [Protocol](https://2021.igem.org/Team%3AGreatBay_SCIE/Protocol)
  + [Measurement](https://2021.igem.org/Team%3AGreatBay_SCIE/Measurement)
* [Modeling](https://2021.igem.org/Team%3AGreatBay_SCIE/Model)
  + [Overview](https://2021.igem.org/Team%3AGreatBay_SCIE/Model)
  + [Optimum Liposome-Aptamer Ratio](https://2021.igem.org/Team%3AGreatBay_SCIE/Optimum_Aptamer_Concentration)
  + [Drug Loading](https://2021.igem.org/Team%3AGreatBay_SCIE/Drug_Loading)
  + [Dissociation Constant](https://2021.igem.org/Team%3AGreatBay_SCIE/Dissociation_constant)
  + [3D Models](https://2021.igem.org/Team%3AGreatBay_SCIE/3D_Model)
* [Human Practices](https://2021.igem.org/Team%3AGreatBay_SCIE/Human_Practices)
  + [Integrated Human Practices](https://2021.igem.org/Team%3AGreatBay_SCIE/Human_Practices)
  + [Public Engagement & Education](https://2021.igem.org/Team%3AGreatBay_SCIE/Communication)
  + [GreatBay\_SCIE X Pink Ribbon](https://2021.igem.org/Team%3AGreatBay_SCIE/BreastCancerInChina)
  + [Collaborations](https://2021.igem.org/Team%3AGreatBay_SCIE/Collaborations)
  + [Partnership with SMS\_Shenzhen](https://2021.igem.org/Team%3AGreatBay_SCIE/Partnership)

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* [Testing Methods for HER2-Targeting Aptamers](#HR2 Apt)
  [Iteration 1](#I1)
  [Iteration 2](#I2)
  [Iteration 3](#I3)
* [Reference](#Reference)

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Engineering

During the implementation of our project, it is crucial to measure the binding affinity of aptamer against HER2 receptor. At the beginning of the experiment, we ran into some difficulties. However, utilizing the *design*, *test*, and *learn* cycle at experimental design, testing, analyzing and learning steps, We continue to enhance our design, optimize the experimental process and improve detection methods，in order to improve detection sensitivity and accuracy. We tried many variations of ELONA methods. All of our efforts can be found here.

# Testing Methods for HER2-Targeting Aptamers

To test the binding affinity of our aptamer, we tried our many variations of ELONA methods. All of the protocols can be found [here](./Protocol).

## Iteration 1: MB-ELONA

First, we used Magnetic Beads-ELONA.

This assay employs Magnetic Beads, which is modified with -NH2. This method uses MB to localize proteins for the ligand to bind on. HER2 ECD solution is added to activated-MB, and incubated for 1hr. Then it is placed on a magnetic rack, and the supernatant is removed. It is washed several times to remove unbound protein and biotinylated aptamer of different concentrations are added to incubate with protein-coated MB. The MB is then washed again to remove unbound aptamer and HRP-conjugated streptavidin is added for incubation, followed by washing and the addition of TMB solution. At last, stopping solution is added after incubation. MB is removed from the resulting yellow solution(if positive) and can be tested for absorbance at OD450 under the microplate reader.

We employed this method testing the binding affinity of HR2 aptamer. However, our results were not strong enough to prove its specificity (Figure 1).

![](https://static.igem.org/mediawiki/2021/4/42/T--GreatBay_SCIE--MB-ELONA_Qualitative.png)

Figure 1 A qualitative test for the binding affinity of HR2 aptamer using MB-ELONA.

As can be observed in the result, BSA-coated MB showed OD450 values of roughly 0.2 in both aptamer concentrations, proving that our aptamer did not bind to BSA protein; whereas HER2-coated MB showed an increase in OD450 value when aptamer concentration increased by 0.1 when aptamer concentration increased from 0.0 to 1.0 μM. This provided evidence that our aptamer does have some degree of affinity for HER2 protein. However, we were not satisfied with this result as we think the experiment group is not very different from the control group. We also performed MB-ELONA with no protein-coated, this gave us insight into possible reasons for the lack of difference between the control and experiment groups.

We also performed MB-ELONA with no-protein conjugated. This proves the validity of MB-ELONA, as aptamer binds non-specifically to MB, which means that proteins were actually coated onto MB.

We ascribe the lack of difference between control and experiment groups to the high amount of non-specific binding on MB, in other words, the background value is too high. During the experiment, even a group of no protein and aptamer showed a yellow color, meaning HRP-conjugated streptavidin bound to MB none specifically.

![](https://static.igem.org/mediawiki/2021/6/61/T--GreatBay_SCIE--Tested_well-plate_of_MB-ELONA.png)

Figure 2 Tested well-plate of MB-ELONA.

## Iteration 2: Traditional ELONA

To solve the issue of high background values. We returned to the traditional methods of ELONA. The method remains very much the same, but instead of coating protein onto MB, it is coated onto the well-plate.

![](https://static.igem.org/mediawiki/2021/5/58/T--GreatBay_SCIE--Result_of_traditional_ELONA.png)

**Figure 3 Result of traditional ELONA.**

However, the result is not expected. All wells showed extremely low OD450 values, similar to that of blank wells. We speculated that perhaps none of the protein is coated onto the well-plate thus, no aptamer was bound to the plate. The binding ability of MB was too high, and too low for well-plate.

## Iteration 3: ELISA-Kit modifed ELONA

To obtain the surface with the suitable binding ability we need for ELONA, we bought ELISA kits and modified them to suit our needs. 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 this time we used FAM-labeled aptamer to reduce the rounds of incubation needed to increase the efficiency of the experiment.

Before any experiment, we evaluated the sensitivity of the Kit by testing with its standard sample provided (Figure 4).

![](https://static.igem.org/mediawiki/2021/6/6e/T--GreatBay_SCIE--Results_Figure_1_Standard_Test.png)

**Figure 4 Standard test of the ELISA Kit.**

We chose to coat 4 ng/mL of protein onto the provided well-plate as shown in result it showed a significant level of value, meaning it contain enough HER2 to bind with aptamer.

Our design were successful and we were able to obtain a qualitative result to prove the specificity of our aptamer, followed by a full quantitative evalutation of HR2 aptamer. (See more in Result)

![](https://static.igem.org/mediawiki/2021/4/4a/T--GreatBay_SCIE--ELONA_Qualitative.png)![](https://static.igem.org/mediawiki/2021/6/6a/T--GreatBay_SCIE--ELONA_Quantitative.png)

**Figure 5** Qualitative result of ELONA (left). Quantative evaluation of HR2 aptamer (right).

As we moved on to the next stage of our lab, we designed pH-sensitive ssDNA sections to add to the aptamer. But the design of the ssDNA is too long (145 bp) to be synthesized by companies through chemical synthesis. So we researched a method called asymmetric PCR to try and replicates ssDNA strands ourselves. (More can also be found in Results)

![](https://static.igem.org/mediawiki/2021/d/d6/T--GreatBay_SCIE--Result_Gel_Image.jpg)

**Figure 6 The agarose gel for asymmetric PCR.** As shown in the picture, when the F : R primer ratio is above 1 : 1, a blurry new band appears above the original band, which is identified as ssDNA.

All of the parts, we used in our engineering, have been added to the iGEM parts registry. You can get more detailed messages from the following links:
- [HR2 Aptamer; Part:BBa\_K3897000](http://parts.igem.org/Part%3ABBa_K3897000)
- [DNA nanoswitch; Part:BBa\_K3897001](http://parts.igem.org/Part%3ABBa_K3897001)
- [pH-sensitive HR2 Aptamer; Part:BBa\_K3897005](http://parts.igem.org/Part%3ABBa_K3897005)

# Testing Method for Aptamer-conjugated Liposome

When we first tried to test the conjugation of our liposomes with FAM, NH2 pre-modified aptamer, using the method of electrophoresis and microplate reader to test the fluorescence of doxorubicin and FAM, we found that doxorubicin has a quite bright fluorescence under a range of wavelength and that covers the wavelength that FAM would be triggered to give out fluorescence.

![](https://static.igem.org/mediawiki/2021/1/19/T--GreatBay_SCIE--Result29.png)

**Figure 7 Agarose gel-electrophoresis of ED-lip formulation** Samples are loaded onto the agarose gel. UV light visualized the gel. A, B, and C are 3 repeats of aptamer conjugated liposome running on Agarose gel-electrophoresis.

We tried to find another fluorescence tag that can be conjugated on the aptamer and has a different excitation wavelength. Sadly, after a series of researches, we found that the wavelength range of the excitation of doxorubicin is too wide that almost covers all the excitation wavelength of current fluorescence tags. Moreover, through electrophoresis, we found that even the excitation wavelengths of doxorubicin and tags were different, the fluorescence of doxorubicin is too strong and will cover the faint fluorescence of tags. In other words, the background interference is too high. This bad news directed us to find another method for the testing of conjugation of liposomes and aptamer.

In order to eliminate the effect of high fluorescence of doxorubicin, we decided to just avoided using it during testing. This means that we only have to test the fluorescence of FAM and that can be much more clear to distinguish whether the aptamer has been conjugated on our liposomes. We quickly decided to prepare another set of liposomes using the same preparation method - Microfluidic - but without doxorubicin in it. So the doxorubicin-PBS solution we used before is replaced by pure PBS solution.

However, our ultimate goal is to prepare a complex of PEGylated liposome encapsulating doxorubicin conjugated with aptamer. We have to develop another method that can somehow put doxorubicin into our prepared liposome. Meanwhile, we were searching on some protocols of increasing encapsulation rate, and we came up with an idea of ammonium sulphate gradient method, which is usually used to increase the drug encapsulation rate by dragging free drugs into the liposomes and forming precipitate inside them. This method works on the different concentration of ammonium sulphate between solution inside the liposomes and the outside. To us, we only need to make sure that we add ammonium sulphate when preparing liposomes and remove the free ammonium sulphate outside our liposomes as much as we can. We believe this method can work thus we can diffuse doxorubicin after conjugation of liposome and aptamer, and conjugation test.

Then our protocol underwent considerable revision. We substituted the doxorubicin-PBS solution we used before when preparing liposomes into the same amount of 200mM ammonium sulphate dissolving in double distilled water. Then for the empty liposomes we produced, they went through the same conjugation and test process and fortunately we got the results that can answer our expectation.

For the process of doxorubicin diffusion, we purified the liposome solution thoroughly which can result in almost zero concentration of ammonium sulphate outside our liposomes. After that, we added doxorubicin-PBS solution to the outside of our liposomes, heated the solution using metal dry bath, and centrifuged it. We concluded the success of our protocol because of the red precipitate we saw after centrifugation, showing that the doxorubicin had been dragged into our liposomes.

![](https://static.igem.org/mediawiki/2021/6/6d/T--GreatBay_SCIE--Result210.png)

Figure 8 The Doxorubicin sulphate shown as red precipitate after centrifugation

# 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

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# Team:GreatBay SCIE/Experiments

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* [Lab](https://2021.igem.org/Team%3AGreatBay_SCIE/Experiments)
  + [Experimental Design](https://2021.igem.org/Team%3AGreatBay_SCIE/Experiments)
  + [Results](https://2021.igem.org/Team%3AGreatBay_SCIE/Results)
  + [Safety](https://2021.igem.org/Team%3AGreatBay_SCIE/Safety)
  + [Lab Notes](https://2021.igem.org/Team%3AGreatBay_SCIE/Notebook)
  + [Protocol](https://2021.igem.org/Team%3AGreatBay_SCIE/Protocol)
  + [Measurement](https://2021.igem.org/Team%3AGreatBay_SCIE/Measurement)
* [Modeling](https://2021.igem.org/Team%3AGreatBay_SCIE/Model)
  + [Overview](https://2021.igem.org/Team%3AGreatBay_SCIE/Model)
  + [Optimum Liposome-Aptamer Ratio](https://2021.igem.org/Team%3AGreatBay_SCIE/Optimum_Aptamer_Concentration)
  + [Drug Loading](https://2021.igem.org/Team%3AGreatBay_SCIE/Drug_Loading)
  + [Dissociation Constant](https://2021.igem.org/Team%3AGreatBay_SCIE/Dissociation_constant)
  + [3D Models](https://2021.igem.org/Team%3AGreatBay_SCIE/3D_Model)
* [Human Practices](https://2021.igem.org/Team%3AGreatBay_SCIE/Human_Practices)
  + [Integrated Human Practices](https://2021.igem.org/Team%3AGreatBay_SCIE/Human_Practices)
  + [Public Engagement & Education](https://2021.igem.org/Team%3AGreatBay_SCIE/Communication)
  + [GreatBay\_SCIE X Pink Ribbon](https://2021.igem.org/Team%3AGreatBay_SCIE/BreastCancerInChina)
  + [Collaborations](https://2021.igem.org/Team%3AGreatBay_SCIE/Collaborations)
  + [Partnership with SMS\_Shenzhen](https://2021.igem.org/Team%3AGreatBay_SCIE/Partnership)

![](https://static.igem.org/mediawiki/2021/9/9c/T--GreatBay_SCIE--Cat.gif)

* [Aptamer](#Apt)
  [Aptamer-HER2 binding](#Bind)
  [pH sensitive DNA switch](#pH)
  [Asymmetric PCR synthesis](#PCR)
* [Nanoparticle](#Nano)
  [Production of PLGA-CS-PEG](#PLGA)
  [Production of PEGylated liposome](#Lipo)
  [Conjugation between Nanoparticles and Aptamer](#Conj)
* [*In Vitro* Verification of Drug System](#In Vitro)
  [Cell Culturing](#Cell)
  [Cytotoxicity of our Drug system](#Cyto)
  [Plasma stability assay](#Plasma)
* [Reference](#Reference)

![](https://static.igem.org/mediawiki/2021/9/94/T--GreatBay_SCIE--Button.png)

Experiment Design

![](https://static.igem.org/mediawiki/2021/5/5f/T--GreatBay_SCIE--Design_Map.png)

Many experiments have to be done for this sophisticated project, so we organized the experiment into four parts:
1. Aptamer
2. PLGA
3. Liposome
4. *In Vitro* Verification

Below is our experimental design, which is all the experiments we planned to do (including a brief description of their procedure and principle) in order to produce our final drug.

The result of the experiment can be found on our [Results page.](./Results)

The protocol of the experiment can be found on our [Protocol Summary](./Protocol) page.

# Aptamer

## Aptamer-HER2 binding

As a targeted drug, the specificity is crucial for our system and the first step of achieving our goal is to prove the specificity of aptamers to HER2 receptors. The affinity of our aptamer directly affects the specificity of the whole system.
The binding affinity of aptamer against HER2 directly affects the specificity of our drug system. Thus we tested the binding affinity of our aptamer using ELNA. We employed an ELISA Kit to be used for ELONA testing.

ELONA assay[1] imploys immobilized HER2 protein on well-plate. The aptamer is incubated in the well and free aptamers are washed. The aptamer is either tagged with biotin or FAM, which can be amplified by either Streptavidin-HRP or directly measured under a microplate reader. The higher the value measured, means more aptamers bound on HER2.

![](https://static.igem.org/mediawiki/2021/9/95/T--GreatBay_SCIE--ELONA_Experiment.png)

**Figure 1 The general process of ELONA assay.**A) coating of HER2 onto the well-plate. B) Incubation of aptamer into the well and then wash. C) If biotin-labelled aptamer is used, extra steps of incubation with S-HRP and TMB is performed, separately. D) Tested under microplate reader.

## PH sensitive DNA switch

One of the most important designs in our system is the pH-sensitive motif of the aptamer. The pH-sensitive strand displacement aptamer switch system consists of three parts——Aptamer, Intramolecular triplex motif, Displacement strand. This design adopted from previous literature[5][6].
The aptamer is connected to the displacement strand(DS) by an internal triplex domain. Both the target particle and DS can bind to the aptamer. This design allows the aptamer to selectively binding to the target at low pH, without changing the aptamer sequence itself. The spacers are added to reduce the undesired interactions between functional regions.[1]

![](https://static.igem.org/mediawiki/2021/e/e4/T--GreatBay_SCIE--experiment1.jpg)

Figure 2[6] The sequential structure of the pH-sensitive motif.

The 3D structure of the modified aptamer can change between a triplex and a duplex under different pH. This is due to two types of base-pairing: Watson-Crick interaction and Hoogsteen interaction.
Conventional base-pairing present in DNA between A-T and C-G are known as Watson-Crick base pairing denoted as "-", also known as complementary base pairing. However, another type of pairing exists as well, known as Hoogsteen base pairing. One of the bases of A-T or C-G is flipped, and hydrogen bonds hold them together as well. However, C·G hoogsteen bonds (denoted as "·")require protonation of Cytosine to form one essential hydrogen bond.
A DNA switch can be designed to be pH-sensitive. As shown in Figure 2, the DNA can fold into a triplex under low pH, and unfold into a duplex at high pH. Due to the protonation of cytosine to form C·G bonds, regulating the amount of C·G and A·T present in the switch could regulate its threshold value of folding.

![](https://static.igem.org/mediawiki/2021/7/7b/T--GreatBay_SCIE--experiment2.jpg)

Figure 3[5] A DNA nanoswitch that folds under different pH ranges depending on its proportion of C·G and A·T pairs.

At higher pH, within normal tissues, the mostly unprotonated linker becomes a duplex by Watson-Crick base pairing. The arm of DS region is long enough for it to competitively bind to the aptamer, thus decreasing the affinity for targets due to the present of competitors.

![](https://static.igem.org/mediawiki/2021/7/7d/T--GreatBay_SCIE--pH_Fold.png)

Figure 4[6] The structure of pH-sensitive aptamer under low pH. The linker is a triplex due to Hoogsteen base pairing.

At lower pH, within cancerous tissues, the protonated cytosine bases turn to Hoogsteen base pairing, making the linker a triplex. The arm of DS region is so short that it cannot bind to the aptamer, thus increasing the affinity for targets due to the absence of competitors.
We attached the pH-sensitive motif to HR2 aptamer, a full sequence can be found [here](http://parts.igem.org/Part%3ABBa_K3897005).
To verify the effectiveness of pHs-HR2 aptamer on targeting HER2 and simulate *in vivo* environment, we plan to perform ELONA under different pH for a single concentration of aptamer (A qualitative test).

## Asymmetric PCR synthesis

After designing the pH-sensitive sequence of the aptamer, we noticed that it is too long (145 bp) to be synthesized by chemical methods. Thus, we plan to split the aptamer sequence into 2 partially complementary 'pre-aptamer' sequences, which can be synthesized chemically.

After linking the 2 'pre-aptamer' sequences using PCR, we plan to utilize asymmetric PCR (asPCR), an analog of normal PCR but with different concentrations of forward and reverse primer, to generate ssDNA from the dsDNA template. As, previous literature has shown that with a forward: reverse primer ratio of 20: 1, the asPCR could generate a maximum amount of more than 50% of ssDNA[7], we plan to a similar primer ratio. (The capability of asPCR to generate ssDNA can be rationalized that as the PCR cycle proceeds, the less concentrated primer will be eventually used up, and by then the system can only generate ssDNA from the only available primer). The graphic illustration of the PCR strategy is shown in Figure.

![](https://static.igem.org/mediawiki/2021/0/07/T--GreatBay_SCIE--Design_ASP_L.png)
![](https://static.igem.org/mediawiki/2021/1/18/T--GreatBay_SCIE--Design_ASP_R.png)

**Figure 5 PCR strategy for ssDNA synthesis.** Left: normal PCR that links the two pre-aptamer together. Right: asymmetric PCR that generates ssDNA and dsDNA(inevitably).

After PCR, we plan to verify the presence of ssDNA with agarose gel electrophoresis, for the speed by which ssDNA travels will deviate from that of the dsDNA generated, and eventually the single strands will be separated and can be extracted for use.

# Nanoparticle

## Production of PLGA-CS-PEG encapsulating doxorubicin

The coating of CS-PEG layer on PLGA to form PLGA-CS-PEG nanoparticle is a double-step procedure.

Firstly, PLGA molecules containing the negatively charged carboxyl groups can strongly attract the chitosan molecules containing the positively charged amino groups.

Secondly, the PEG chains are attached to the PLGA-CS complex due to the hydrogen bonds forming between amino groups on the chitosan and the electrons of oxygen atoms of the PEG molecules.[3]

![](https://static.igem.org/mediawiki/2021/8/89/T--GreatBay_SCIE--Design_PLGA_Production.png)

**Figure 6[2]Illustration of the manufacturing procedures of PLGA nanoparticles.** The nanoparticles are produced form oil-in-water single emulsion solvent evaporation method.

Through FTIR(Fourier Transform Infrared Spectrometry) and TEM (Transmission Electron Microscopy), we were able to prove the existence of suitable sized nanoparticles in our resulting product.

To prove the successful encapsulation of doxorubicin in PLGA nanoparticles, we plan to apply HPLC (High-Performance Liquid Chromatography). Both drug-containing nanoparticles and drug-free nanoparticles were tested for comparative analysis[2].

## Production of PEGylated liposome encapsulating doxorubicin

![](https://static.igem.org/mediawiki/2021/6/6d/T--GreatBay_SCIE--Lipo_Design.png)

**Figure 7 Method used for the production of PEGylated liposome nanoparticles.**

For liposome, since it possesses the same issue as PLGA( the rapid opsonization by phagocytes in the blood circulation ), we will further stabilize it by applying a surface modification using PEG. This can significantly prolong the half-lives of the liposome nanoparticles. The PEG chains attach to liposomes due to the hydrogen bonds, similar to the binding between PEG and PLGA.

We plan to manufacture our PEGylated liposomes via hydration. After the production of the nanoparticles, we will apply DLS(dynamic light scattering) for testing the diameter of the nanoparticle[4].

Then, we will calculate the encapsulation efficiency of doxorubicin in the nanoparticles by the formula below.

$$\text{Encapsulation Rate} = \frac{(\text{Amount of initial Dox} - \text{Free Dox})}{\text{Amount of initial Dox} \times \text{100%}}$$

The final drug amount is obtained by measuring the amount of drug recovered from destroyed liposomes by Triton X-100, a surfactant breaking down the spheres of liposomes.

## Conjugation between Nanoparticles and Aptamer:

Conjugation between nanoparticles and aptamers is an important section of our project, as it combines the two important functions—killing & targeting.

The conjugations of aptamer-liposome and aptamer-PLGA are similar, and thus they are demonstrated together. By modifying aptamers with an amine group at the 3' end, we linked the aptamer to the exterior carboxyl ends of the nanoparticle with cross-linking agents N-hydroxy-sulfo-succinimide (NHS) and 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC), which assist the amide formation between the aptamer and the nanoparticle. The mechanism of the reaction was given in the Figure below.

![](https://static.igem.org/mediawiki/2021/8/84/T--GreatBay_SCIE--Design_PLGA_Mechanism.png)

**Figure 8 Mechanism scheme for the conjugation between aptamer and nanoparticle.** The nanoparticle is represented by 'NP' in brown; the aptamer is shown by 'Apt' in red. The O-Acylisourea intermediate produced form EDC and the carboxilic acid, which is not very stable, is immediately attacked by NHS forming an NHS ester, which is more stable than O-Acylisourea and maintains adequate leaving group ability that allows for efficient conjugation to primary amines.[8]

The verifications of successful conjugation are done by AGE(agarose gel electrophoresis). Since nanoparticles attached by aptamers are more heavy than those without aptamers, we plan to prove the effectiveness of conjugation by comparing the distance traveled by nanoparticles with and without aptamers.

# *In Vitro* Verification of Drug System

In order to prove the effectiveness of our drug delivery system. We set up several cell experiments, including cell culturing, cytotoxicity of the system, and the plasma stability assay verify different properties of our system.

## Cell Culturing

Cell culturing is the first step of testing the cytotoxicity of our system. It is crucial to cultivate cancer cells for the following experiments.

The two cell lines we plan to cultivate for our in vitro cytotoxicity assay are SK-BR-3(human breast cancer cell, HER2 positive), and MDA-MB-231(human breast cancer cell, HER2 negative).

## Cytotoxicity of our Drug system

By incubating our drug with cells, we were able to verify the cytotoxicity of our drug system as well as the effectiveness of our aptamer.

We will incubate our drug with the cancer cells and test the cell viability at regular intervals using CCK-8 (Cell Counting Kit 8). CCK-8 test will allow us to use a microplate reader to quantify the number of live cells. We will cultivate the cancer cells with the addition of our two drug delivery systems——PLGA-based and liposome-based system, and identify the cell viability. Cell Counting Kit-8(CCK-8) is a widely used test kit for cytotoxicity and cell proliferation. It contains a chemical called WST-8, which will react with 1-Methoxy PMS. The slightly yellow WST-8 will turn to WST-8 Formazan orange dye as the dehydrogenase in mitochondria is reduced. The faster and more the cells proliferate, the darker the color; the higher cytotoxicity, the lighter the color. The depth of the color is directly proportional to the number of living cells, which can be used to analyze cell proliferation and cytotoxicity directly.

![](https://static.igem.org/mediawiki/2021/9/95/T--GreatBay_SCIE--Design_CCK8.png)

Figure 9 An Overview of CCK-8[11].

## Plasma stability assay

To verify the stability of our system, we plan to carry our plasma stability assay to find out its half-life within the body. The drug will be incubated in blood plasma and then the resulting solution will be separated using HPLC.[9]

Improving stability is an important aspect of our system. A lack of stability will directly result in serious side effects since toxins will be released in the bloodstream or taken in by various cells such as phagocytes.

To verify the stability of our system, we plan to apply a method called plasma stability assay to find out the half-life of our drug delivery system within the body. The drug will be incubated in blood plasma to mimic the in vivo environment and then the resulting solution will be separated using a machine called HPLC(High-Performance Liquid Chromatography). We will then calculate the amount of drug maintaining, compare the data with the original amount of drug, and thus deduce the half-life of the system.

HPLC can separate the matters dissolving in a solvent due to the different flow rates of various particles on an absorbent(column of the machine), and give the quantity of each component. In this case, we separate doxorubicin, the toxin we apply, and other parts of our system to calculate the amount of drug remaining.[9]

![](https://static.igem.org/mediawiki/2021/f/f2/T--GreatBay_SCIE--Design_HPLC.png)

Figure 10 Overview to HPLC[10].

Proceed to [Results Page](./Results).

# Reference

1. Drolet, D. W., Moon-McDermott, L., & Romig, T. S. (1996). An enzyme-linked oligonucleotide assay. *Nature biotechnology*, *14*(8), 1021–1025. <https://doi.org/10.1038/nbt0896-1021>
2. Parveen S, Sahoo SK. Long circulating chitosan/PEG blended PLGA nanoparticle for tumor drug delivery. Eur J Pharmacol. 2011 Nov 30;670(2-3):372-83. doi: 10.1016/j.ejphar.2011.09.023. Epub 2011 Sep 21. Erratum in: Eur J Pharmacol. 2014 Mar 15;727:186. PMID: 21951969.
3. Zhang, M., Li, X., Gong, Y., Zhao, N., & Zhang, X. (2002b). Properties and biocompatibility of chitosan films modified by blending with PEG. *Biomaterials*, *23*(13), 2641–2648. [https://doi.org/10.1016/s0142-9612(01)00403-3](https://doi.org/10.1016/s0142-9612%2801%2900403-3)
4. Cadinoiu, A. N., Rata, D. M., Atanase, L. I., Daraba, O. M., Gherghel, D., Vochita, G., & Popa, M. (2019). Aptamer-Functionalized Liposomes as a Potential Treatment for Basal Cell Carcinoma. *Polymers*, *11*(9), 1515. <https://doi.org/10.3390/polym11091515>
5. Idili, A., Vallée-Bélisle, A., & Ricci, F. (2014). Programmable pH-Triggered DNA Nanoswitches. *Journal of the American Chemical Society*, *136*(16), 5836–5839. <https://doi.org/10.1021/ja500619w>
6. Thompson, I. A. P., Zheng, L., Eisenstein, M., & Soh, H. T. (2020). Rational design of aptamer switches with programmable pH response. *Nature Communications*, *11*(1). <https://doi.org/10.1038/s41467-020-16808-2>
7. Marimuthu C, Thean T, Soo T, Chee H, Rajan S, Junji T, Subash G. Asymmetric PCR for good quality ssDNA generation towards DNA aptamer production. Songklanakarin J. Sci. Technol. 34 (2), 125-131, Mar. - Apr. 2012
8. *Crosslinking Applications | Thermo Fisher Scientific - NL*. (n.d.). Thermo Fisher Scientific. <https://www.thermofisher.com/nl/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/crosslinking-applications.html>
9. Di, L., Kerns, E. H., Hong, Y., & Chen, H. (2005). Development and application of high throughput plasma stability assay for drug discovery. *International journal of pharmaceutics*, *297*(1-2), 110–119. <https://doi.org/10.1016/j.ijpharm.2005.03.022>
10. What is HPLC (High Performance Liquid Chromatography) ？. (n.d.). SHIMADZU. <https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/basic/what_is_hplc.html>
11. Têtu, J., PhD. (2016, May 20). Still using MTT or WST-1 for cell counting? | tebu-bio’s blog. Tebu-Bio’s Blog | Acting and Reacting in Life Sciences and Biotechnologies. <https://www.tebu-bio.com/blog/still-using-mtt-or-swt-1-for-cell-counting/>

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# Team:GreatBay SCIE/Results

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* [Overview](#Overview)
* [Aptamer](#Aptamer)
  [ELONA Experiment](#ELONA)
  [Asymmetric PCR](#PCR)
* [Liposome](#Liposome)
  [Manufacturing of Dox-encapsulated Liposome](#DoxLip)
  [Encapsulation rate testing of Dox-Liposome](#Rate)
  [Modification of Doxorubicin Liposome with Aptamer](#Conjugation)
  [Ammonium sulphate diffusion](#Ammo)
* [PLGA](#PLGA)
* [*In Vitro* Verification](#Cell)
  [Cell Culturing](#Culture)
  [Evaluation of Cytotoxicity of Dox](#Dox Cyto)
  [Evaluation of Cytotoxicity of Apt-Dox-Liposome](#Lipo Cyto)
* [Attributions](#Atr)
* [Summary](#Summary)
* [Outlook](#Outlook)
* [Reference](#Refe)

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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 results. The result for H2 is presented in [Supplement](./Results/Supplement), as it did not aid out project.

HR2 Aptamer sequence:
5' -
AACCGCCCAAATCCCTAAGAGTCTGCACTTGTCATTTTGTATA
TGTATTTGGTTTTTGGCTCTCACAGACACACTACACACGCACA
- 3'

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.

![](https://static.igem.org/mediawiki/2021/6/6e/T--GreatBay_SCIE--Results_Figure_1_Standard_Test.png)

**Figure 1.1 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.

![](https://static.igem.org/mediawiki/2021/4/4a/T--GreatBay_SCIE--ELONA_Qualitative.png)

**Figure 1.2 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.

![](https://static.igem.org/mediawiki/2021/6/6a/T--GreatBay_SCIE--ELONA_Quantitative.png)

**Figure 1.3 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: ![](https://static.igem.org/mediawiki/2021/a/ac/T--GreatBay_SCIE--Kd_equation.svg). The R2 value for the curve is 0.9801 and Kd = 1.803 μM.

## 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.

![](https://static.igem.org/mediawiki/2021/a/ac/T--GreatBay_SCIE--Results_Figure_4._pHs.png)

**Figure 1.4[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 the 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.

Asymmetric PCR differs from normal PCR in the system used. In asymmetric PCR, forward and reverse primers are added with different ratios. So, ssDNA strands will be produced as there will be more forward (or backward) strands than the other.
pH-sensitive HR2 sequence, composed of HR2 Aptamer, pH-sensitive DNA switch, and complementary strand:

5' -
AACCGCCCAAATCCCTAAGAGTCTGCACTTGTCATTTTGTATA
TGTATTTGGTTTTTGGCTCTCACAGACACACTACACACGCACA

TTTTGAGGGAAAGAATCATTTCTT
TCCCTATGTTTCCCTTTCTTTTTAA

TTGGGCGGTT
- 3'

**Table 1 Primers and sequence.** The PCR programme can be found in the protocol summary [here](https://2021.igem.org/Team%3AGreatBay_SCIE/Protocol)

| Primer | 5‘ to 3' Sequence |
| --- | --- |
| Forward Template | AACCGCCCAAATCCCTAAGAGTCTGCACTTGTCATTTTGTA TATGTATTTGGTTTTTGGCTCTCACAGACACACTACACACGC |
| Forward Primer | AACCGCCCAAATCCCTAAGAG |
| Reverse Template | AACCGCCCAATTAAAAAGAAAGGGAAACATAGGGAAAGAAA TGATTCTTTCCCTCAAAATGTGCGTGTGTAGTGTGTCTGTGAG |
| Reverse Primer | AACCGCCCAATTAAAAAGAAAGGG |

![](https://static.igem.org/mediawiki/2021/d/d6/T--GreatBay_SCIE--Result_Gel_Image.jpg)

**Figure 1.5 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 1.5 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 were able to prove the validity of our experiment by repeating with ssDNA strands synthesized by Genscript, which was used previously in ELONA (Figure 1.6). 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.

![](https://static.igem.org/mediawiki/2021/2/20/T--GreatBay_SCIE--Results_Figure6%2C_asPCR_Validity.png)

**Figure 1.6 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](./Protocol).

**Table 2.1 Specific amounts of our used materials**

![](https://static.igem.org/mediawiki/2021/d/da/T--GreatBay_SCIE--Result21.png)

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.

**Table 2.2 Liposome's diameters**

![](https://static.igem.org/mediawiki/2021/a/a8/T--GreatBay_SCIE--Result22.png)

*Molar ratio of the samples LA is 10 : 6 : 1 ; for LC is 15 : 6 : 1.5. The liposomes were made on Jul 30th 2021.*

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.

**Table 2.3.1 Dox liposomes's mean diameter**

![](https://static.igem.org/mediawiki/2021/c/ca/T--GreatBay_SCIE--Result23.png)

**Table 2.3.2 Blank liposome's diameters**

![](https://static.igem.org/mediawiki/2021/c/cc/T--GreatBay_SCIE--Result24.png)

*DCP ratio of the samples LA is 10 : 6 : 1 ; for LB is 10 : 6 : 1.5; for LC is 15 : 6 : 1.5; for LD is 15 : 6 : 1. Dox liposomes were made on Sep 13th and Sep 16th, the top table is a representation of the mean diameter of the two results. Blank Liposomes were made on Sep 16th.*

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

![](https://static.igem.org/mediawiki/2021/e/ee/T--GreatBay_SCIE--Result25.png)
![](https://static.igem.org/mediawiki/2021/7/7c/T--GreatBay_SCIE--Result26.png)

**Figure 2.1 The distribution graph** The left one is for LA-Dox-13 while the right one is for LA-16. The peaks in the graphs demonstrated high distribution of liposomes' diameters in these values.

## 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.

![](https://static.igem.org/mediawiki/2021/0/08/T--GreatBay_SCIE--Result27.png )

**Figure 2.2 A standard test of absorbance of Dox** The absorbance of Dox liposome and tested and its concentration is inferred.

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:

![](https://static.igem.org/mediawiki/2021/d/df/T--GreatBay_SCIE--Encap.svg)

we obtained the encapsulation results, presented in Table 2.4.

**Table 2.4 The encapsulation rates of different Dox liposome samples (made on Sep 13th & 16th)**

![](https://static.igem.org/mediawiki/2021/a/ab/T--GreatBay_SCIE--Result28.png)

*There was no data for LB-Dox-13 as it was accidentally lost during transportation.*

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

\*The aptamer we used is HR2 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. We conjugated HR2 Aptamers onto LA-Dox 13, 16 and LA-16.

The first time we used Dox-liposomes for aptamer modification and got our first result shown in Figure 2.3A. From the figure, 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 the aptamer.

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

![](https://static.igem.org/mediawiki/2021/1/19/T--GreatBay_SCIE--Result29.png)

**Figure 2.3 Agarose gel-electrophoresis of ED-lip formulation** Samples are loaded onto the agarose gel. UV light visualized the gel. A, B, and C are 3 repeats of aptamer conjugated liposome running on Agarose gel-electrophoresis.

Figure 2.3B showed the results of electrophoresis of aptamer-blank liposomes. Without the disruption of dox fluorescences, we 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 2.3C, there were no fluorescences in lane 7 but fluorescences in lanes 4 and 5, showing blank liposomes hardly affect the fluorescences. However, there were fluorescences in lane 6, which is also blank liposomes. 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 the diffusion process, showing that red doxorubicin went into liposomes.

![](https://static.igem.org/mediawiki/2021/6/6d/T--GreatBay_SCIE--Result210.png)

**Figure 2.4 The Doxorubicin sulphate shown as red precipitate after centrifugation**

The Dox-Liposomes made in this method were not used in the cell culturing experiment, however, it provides evidence of possible methods to produce liposomes as well as proof of successful aptamer conjugation.

# PLGA nanoparticle

We hope to synthesis PLGA nanoparticle entrapping doxorubicin. To prepare the nanoparticle, we used the single emulsion-solvent evaporation method, which synthesizes particles entrapping hydrophobic or amphiphilic contents. Briefly, the polymer and the drug were dissolved in an organic solvent, which was then mixed and sonicated with an aqueous phase containing surfactants, forming an oil-in-water emulsion. The organic solvent was allowed to evaporate, which hardens particles and formed a nanoparticle suspension. Please go to our [Protocol Summary](./Protocol) for detailed procedures.

Initially, we attempted to produce nanoparticles with an output power of 500W. The diameter of the particle produced is shown in Figure 3.1.

![](https://static.igem.org/mediawiki/2021/8/86/T--GreatBay_SCIE--Results_PLGA_Diameter.png)

**Figure 3.1 Measurement of particle diameter.** Output power 500W. Data was obtained with Laser Diffraction Particle Size Analyzer from Shenzhen University.

Our result indicates that the average diameter is 2.5um, which is too large for a delivery platform for anticancer drugs, whose diameter is usually between 70-200nm[7]. Therefore, after consulting Dr. Wei, we realized that the output power was inadequate for the production of smaller particles, and a new batch of particles was produced with a sonication output power of 800W.

![](https://static.igem.org/mediawiki/2021/c/c2/T--GreatBay_SCIE--Results_PLGA_2nd_Diameter.png)

**Figure 3.2 Mesurement of 2nd batch of particle diameter.** Output power: 800W, measured by Malvern Particle Sizer.

With enhanced output power, nanoparticles of diameter of 251nm were obtained. The morphology of the particle was characterised with transmission electron microscope Figure 3.3.

![](https://static.igem.org/mediawiki/2021/7/72/T--GreatBay_SCIE--POC_F3L.png)

**Figure 3.3 TEM image for nanoparticle morphology.** The PLGA particle was treated with centrifugation, 7000rpm, for 3min, to remove large aggregation formed during transportation.

# *In Vitro*I Verification

## Cell Culturing

Cell lines of **SK-BR-3 (human breast, HER2 positive)**, **MDA-MB-231 (human breast, HER2 negative)**, obtained form National Collection of Authenticated Cell Cultures (Shanghai，China), was cultivated using DMEM medium supplemented with 10% fetal bovine serum in 5% CO2 at 37°C. The morphology of the cell was shown in Figure 4.1. For details of cell thawing and subsulturing, please visit our [Protocol Summary](./Protocol).

![](https://static.igem.org/mediawiki/2021/7/72/T--GreatBay_SCIE--Results_Cell_SK.png)![](https://static.igem.org/mediawiki/2021/7/74/T--GreatBay_SCIE--Results_Cell_MB.png)

**Figure 4.1. Cell morphology of SK-BR-3 (Left) and MDA-MB-231 (Right) under microscope.**

## Evaluation of Cytotoxicity of Dox

To evaluate the cell cytotoxicity of Doxorubicin, both cell lines were cultivated in 96 well plate for 24 hour, with approximately then the DMEM growth medium was removed and the cells were incubated with Dox containing DMEM at various concentration. For previous study has shown that doxorubicin caused a decrease of more than 80% of cell viability in B16F10 murine melanoma cell lines at 24 hours, 1 μg/ml[6], the experiment was designed to tested the cytotoxicity on both cell lines imposed by Dox at a similar time interval and concentration. After co-incubation with doxorubicin, the cell viability was tested with CCK-8 kits. The result of our experiment was shown in Figure 4.2.

![](https://static.igem.org/mediawiki/2021/2/2d/T--GreatBay_SCIE--Results_Dox_SK.png)![](https://static.igem.org/mediawiki/2021/b/b7/T--GreatBay_SCIE--Results_Dox_MB.png)

**Figure 4.2 Doxorubicin cytotoxic effect on SK-BR-3(Left) and MDA-MB-231(Right) cell lines.** After co-incubation with growth medium containing Dox for 24 hours, the medium was removed and replaced with CCK-8 test solution and the cells were incubated for another 1.5 hour.

The data suggests that similar to that of the previous literature, no significant cytotoxic effect was exhibited by doxorubicin after 3 hours of co-incubation, possibly because the time interval was inadequate Dox to exert its cytotoxic effects. To our relief, at 24 hours a general trend was shown that the cell viability decreases as the concentration of Dox in the growth medium increases, though the exhibited cytotoxicity on both SK-BR-3 and MDA- MB-231 cell lines was smaller than that on B16F10. We could use this result to decide on the Dox concentration that should be encapsulated into the nanoparticles to produce enough effects to kill the cancer cell.

## Evaluation of Cytotoxicity of Apt-Dox-Liposome

To prove that our system could work as a whole, we tested the cytotoxic efficacies of Apt-Dox-Liposome, on HER2-positive and HER2 negative cell lines using the same method as above. The Liposome drugs we used are LA-Dox-13 and LA-16, which can be found in the previous part above.

![](https://static.igem.org/mediawiki/2021/6/64/T--GreatBay_SCIE--Results_Lipo_Cyto.png)

**Figure 4.3 Cytotoxicity assays of the produced liposome drugs** LA-Dox 13&16 is used in Dox-Liposome and Apt-Dox-Liposome group, LA-16 were used in Blank Liposome group. Dox concentration in all groups were diluted into 20 μg/ml. We also included 20 μg/ml and 40 μg/ml as a comparison for drug cytotoxicity.

From Figure 4.3, the data showed that while sole Dox entrapping liposome exhibits similar cytotoxicity against both HER2-positive and HER2-negative cells, the aptamer conjugated liposome shows an increased cytotoxic effect against the HER2 positive cell, and a reduced cytotoxic effect on the HER2 negative cell, which indicates that, presumably due to the successful binding to the HER2 extracellular domain by the aptamer, a greater amount of Dox is delivered to the targeted cancer cell.

However, the cell viability of Dox-Liposome is even lower than that of the 20 μg/ml Dox group even though it contains the same concentration of Dox. Presumably, this is due to the cytotoxicity of liposomes, during liposome production, the chemical remains unremoved, thus damaging the cell. This is a deficiency of the system we may consider in the future.

The data suggests, preliminarily, that our HER2 aptamer-liposome delivery system could selectively deliver Doxorubicin *in vitro*, and could possibly exhibit decreased cytotoxic effects on normal cells. However, the experimental data now is not sufficient enough to prove the effectiveness as repeating data is missing.

# Attributions

Our experiments are completed with the help of experts:

**ELONA experiments** are completed on our own.

**Liposome experiments** are carried out with the help of SUSTech. They provided laboratory, specific machines and professional guidance. We manufactured lots of liposomes for later experiments and measure their diameters using Malvern Particle Sizer there.

The **production of PLGA nanoparticles** was carried out with the help of Dr. Wei from Northwest University, China. He provided us with numerous constructive pieces of advice on the production process, including oil/water ratio, sonication output, and storage method. With his guidance, we have successfully decreased the size of the particle from 10um to 2um by ourselves. However, we realize that the maximum output of the sonicator in our lab was not sufficient to produce nanoscale particles, so we send our material to Dr. Wei’s lab where he helped us to produce the particle, with a sonication output of 800W.

# Summary

Our experimental results have provided preliminary evidence for the possibility of such a targeted drug. We have successfully tested HR2 Aptamer's binding affinity against HER2 through ELONA and utilized it on synthesized liposome nanoparticles. Finally, we were able to perform in vitro cytotoxicity tests to show the overall effectiveness of the drugs.

However, due to lack of time, there are still things we have not yet finished, which we present below.

**Table 5.1 A lab progress chart of GreatBay\_SCIE 2021.**

![](https://static.igem.org/mediawiki/2021/4/41/T--GreatBay_SCIE--Results_Lab_Progress.png)

The experiment is divided into 4 parts. The completed sections are highlighted in colors, while the incomplete experiments are highlighted in grey.

# Outlook

If OncoKiller can be refined and optimized it could become a universal model for many cancer drugs. Due to the accessibility of aptamers and their versatility, drugs for diverse cancers can be produced using our system, but with different aptamers. For example, targeted drugs for liver cancer can apply aptamers specifically binding to liver cancer cells instead of HER2 aptamers; Triple Negative breast cancer can be targeted with aptamer even though there are no receptors for antibodies to target. We believe that using aptamers in cancer-curing drugs is very prosperous, as it not only explores more opportunities but also allowing more families to be able to afford them.

In the past few months, we've read through literature performed experiments; we overcame many difficulties, tried out different solutions, and eventually led to our preliminary design for our project. Fighting cancer is hard. We know our targeting drug has many issues yet to be solved, but we wish to do our bit in this long-stretched fight.

# Reference

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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>
6. Gaumet, M., Vargas, A., Gurny, R., & Delie, F. (2008). Nanoparticles for drug delivery: the need for precision in reporting particle size parameters. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V, 69(1), 1–9. <https://doi.org/10.1016/j.ejpb.2007.08.001>
7. Eliaz, R. E., Nir, S., Marty, C., & Szoka, F. C., Jr (2004). Determination and modeling of kinetics of cancer cell killing by doxorubicin and doxorubicin encapsulated in targeted liposomes. Cancer research, 64(2), 711–718. <https://doi.org/10.1158/0008-5472.can-03-0654>