Team:ShanghaiTech China/DrugDelivery
Bacteria carriers for drug delivery: biocompatible agarose vesicles to encapsulate GFP+ bacteria.
Key achievements
·Constructed agarose vesicles as bacteria loaders.
·Constructed silk fibroin micrococoons as bacteria loaders.
·Accomplished characterization of constructed vesicles in an intestine-like environment.
Overview
As our engineered bacteria are aimed to settle down in the human gut, it is necessary to ensure the safety of both bacteria and humans. As a result, we constructed agarose vesicles using micro-fluid technology to protect the engineered bacteria from adverse conditions in the gut. The vesicles also serve as a physical barrier to limit bacteria growth within the vesicles. Hopefully, it would minimally affect the intestinal bacterial balances.
Micro-scale and high biocompatibility
The average scale of 50μm-200μm of our agarose vesicles and the biocompatible quality of agarose material makes the vesicles a safe bacterial carrier to shuttle in the intestinal tract.
Transparency
The transparency of agarose allows our near-infrared light to go through smoothly, making our light-controlled module possible.
Good adhesion to the intestinal wall
Referring to related paper[1], we find that our micro-scale vesicles bear good quality to adhere to the intestinal wall. This would contribute to a longer time of treatment per dose.
Suitable for bacteria proliferation
Our engineered bacteria can still proliferate quickly inside the vesicles for over 28 hours.
Good stability
Compared to silk fibroin micrococoons, agarose vesicles can maintain their shape in water for 28h hours.
Our approach
The prevention of the bacterial leakage is one of the component parts of our design of the agarose vesicles. Thus we did some optimization of the concentration of the agarose for vesicles, making the agarose wall a better barrier to limit the proliferation of the engineered bacteria outside the vesicles.
To guarantee the repeatability of our characterization experiments, we considered utilizing the micro-fluid technology to product micro-scale vesicles of similar size. The large volume-surface ratio of the product is also beneficial for the drug diffusion in the drug delivery section.
In the characterization experiment of agarose vesicles, we set the condition of pH ≈ 8.3 LB to imitate the pH condition in the human gut. By comparing the fluorescence intensity/OD600nm of trans5-α-psgkp-km-GFP in pH = 7 and pH ≈ 8.3 condition, vesicles in pH ≈ 8.3 condition and bacteria in pH ≈ 8.3 condition, we could analyze the effect of the alkaline condition on bacterial growth and the feasibility of vesicles to protect the engineered bacteria.
Experimental design
Construction of the agarose vesicles
We generated the vesicles in a micro-fluid chip. There are three tubes to let fluids to enter on one working unit of the chip(figure 1.1). As shown in the video in our experiment, flowing in the sloping two tubes are agarose solution and trans5α-psgkp-km-GFP, respectively. The two-fluid join as one fluid at the Y-cross. The single fluid is then sheared by the oil fluid in the third tube, horizontal in the video. Vesicles are taken shape as a result of shearing and are collected in a new 1.5ml EP microcentrifuge tube.
The emulsion in the tube was then broken using 0.1% Tween20. The agarose vesicles were in the lower layer of water phase.
Initially, we used a 1% (wt) agarose solution as the raw material. Yet, we found severe leakage of trans5-α-psgkp-km-GFP into the solution. We then increased the concentration of agarose to make the wall of vesicles more concrete so that less bacteria can penetrate. After characterizing 1.4wt% and 1.5wt% agarose vesicles, we finally selected 1.5wt% agarose as the optimum concentration.
Also, we tried to add the procedure of soaking the vesicles in oil in RT for 5 hours. This process is to inhibit the growth of the bacteria outside the vesicles by reducing the availability of air and nutrition so that there would be less background fluorescence at the beginning of the characterization experiment（Figure 1.2,1.3）
Figure 1.1 The demonstration of one working unit of our micro-fluid chip. Every component is marked as above.
Characterization of agarose vesicles
As shown in the table below, we set up four groups, Control 1 (Ph = 7, LB media ), Control 2 (Ph = 8.3 media), Agarose Ph = 8.3, Agarose Ph = 7.
We incubated the four groups for 28 hours (37℃, 220 rpm), and used ELISA plate to detect the change of fluorescence and OD600 over time. We also took pictures at 0 h,2 h,3 h,5 h,6.5 h,15 h,and 28 h starting from the incubation.
Figure 1.2（left） Agarose vesicles after 28h incubation with 1wt% agarose (not soaking in oil)
Figure 1.3(right) Agarose vesicles after 28h incubation with 1.5wt% agarose (sat at RT for 5 hours as a emulsion)
Result
We use fluorescence intensity/OD600 to measure the bacterial proliferation. As is displayed in the line chart below (Figure 2.1), in the late stage of the incubation, the curve of fluorescence/OD of Agarose pH=8.3 is close to that of Control 1, indicating a good encapsulation quality of the agarose vesicle; Control 2 is below Agarose pH=8.3, indicating that the agarose vesicles have competent abilities to protect trans5α-psgkp-km-GFP from alkaline environment. The data suggest the agarose vesicle a promising bacterial loader in the gut.
Figure2.1
At the first two hours, we could see particle-shaped trans5α-psgkp-km-GFP in the vesicle. There is also a video about the proliferation of trans5α-psgkp-km-GFP during 1h-2h.
After long time incubation, we could obviously notice that the particle-shaped trans-GFP had grown into green spots, indicative of bacteria proliferation in the vesicles over time. The fluorescence intensity also continued to grow during these times (Figure 2.2).
Figure2.2: Morphology and fluorescence changes of agarose vesicles over 28h
Besides the agarose vesicles, we also tried to encapsulate bacteria with silk fibroin [3], which bears better biocompatibility. We succeeded in collecting some silk micrococoons in water phase (Figure 2.3). However, these micrococoons are easy to crack. It cracked only a few hours after being made, which prevented us to continue.
Our future plan is to encapsulate the agarose vesicles using an enteric-coated capsule to protect the vesicles from gastric acid, also realize the orientation release of agarose vesicles in the gut.
Figure 2.3: silk fibroin micrococoons in the water phase
References
[1] ZHU Lihui, CHEN Aizheng & WANG Shibin. Application and Advances on Polymer Carriers for the Pharmaceutical Microspheres. Chemical Industry and Engineering Progress,Vol.33(07), pp.1832-1838 (2014)
[2] Nitta, S. K. & Numata, K. Biopolymer-based nanoparticles for drug/gene delivery and tissue engineering. Int J Mol Sci 14, 1629-1654, doi:10.3390/ijms14011629 (2013).
[3] Shimanovich, U. et al. Silk micrococoons for protein stabilisation and molecular encapsulation. Nat Commun 8, 15902, doi:10.1038/ncomms15902 (2017).