TACE

Introduction

In order to prevent and treat rice sheath blight, 2022 SZU-China designed TACE (Trichoderma's acest carrier ever) to carry and deliver Trichoderma atroviride spores, blocking the transmission of sclerotia at aquatic interfaces or from soils. As the project progressed, we iterated on TACE1.0 to launch TACE2.0. The matrix material of TACE1.0 is starch grafted acrylate, and the matrix material of TACE2.0 is HPMC (Hypromellose). Due to their different chemical nature and modes of action, we designed different experiments to explore the properties of TACE1.0 and TACE2.0, and compared them horizontally. The following are important quantitative experimental results of TACE.

Correlation curve of absorbance and spore concentration in spore solution

During our experiments, we found that it was complicated to count spores number though microscopes, and the concentration of spores in solution cannot be directly converted by measuring OD 550 of spore solution. Therefore, we established a correlation curve of absorbance and spore concentration in Trichoderma atroviride spore solution, which guided our subsequent experiments.

We defined the correlation between the concentration of the spore solution obtained by microscopy and OD 550 of the spore solution. After eluting Trichoderma atroviride spores from PDA culture medium, the spore solution was diluted in gradient, observed and counted using a blood cell counting plate. OD 550 of the corresponding spore solution was measured. We finally obtained the correlation curve shown in figure 1.

Figure 1. Correlation curve of absorbance and spore concentration in spore solution. The equation of this curve is y=0.5715x, `R^2`=0.8667.

In addition, we performed a t-test for the obtained data sets of the spore concentration obtained by microscopy and OD 550 of the spore solution (Fig. 2). The correlation between absorbance and spore concentration in spore solution is proved.

Figure 2. Raw data and t-test calculations.

Thus, we used the equation of this curve (y=0.5715x, `R^2`=0.8667; y, OD 550; x spore concentration) in subsequent experiments on Trichoderma atroviride.

TACE1.0

Imbibition of TACE1.0

In our design, Trichoderma spores suspension was poured into TACE1.0 to fully load the spores. TACE1.0 is known to have a density of 0.60 to 0.73 `g/cm^3` and an extremely high imbibition rate. In order to explore the imbibition capacity of TACE1.0 on different common solutions and explain the underlying mechanism, we conducted experiments and obtained the following results.

Figure 3. Water absorption of TACE1.0 todifferentsolution.

Inside the dried TACE1.0, the long chains of the grafted acrylate starch were interwoven. After TACE1.0 contacts water, the long-chain molecules dissociate and become negatively charged, which leads to electrostatic repulsion between the long-chain molecules. The long chains of the starch, which were originally curly, were stretched and straightened, resulting in an increase in the volume of TACE1.0. On the other hand, due to the confrontation of positive and negative ions inside, polar water molecules are attracted. Based on that, TACE1.0 absorbs water and increases in volume when it meets water.

It can be seen that TACE1.0 has the greatest absorption of deionized water, reaching an astonishing 206.20g/g. However, the absorption of physiological saline was the lowest, which was merely 21.12 g/g. We've learnt that the water absorption of TACE1.0 is closely related to the osmotic pressure of ions in the solution, while the osmotic pressure of uncharged molecules has little effect, such as the difference between the water absorption of deionized water and physiological saline.

The principle behind these phenomena is that the salt ions in the system interfere with the water absorption mechanism of TACE1.0 and seriously reduce the water absorption. This reminds us that the ionic osmotic pressure of spore fluid must be paid great attention to.

Degradation of TACE1.0

Settlement of TACE1.0 occurs at least 30 minutes after TACE1.0 is applied to paddy fields. The settled TACE1.0 will be degraded by microorganisms. In order to investigate the biodegradability of TACE1.0, 0.5g 40 mesh TACE was buried in the paddy fields, and the wet weight was weighed every 5 days.

Figure 4. (A)TACE1.0 was buried in the experimental fields. (B)TACE1.0 one day later. (C)TACE1.0 thirty days later.

The internal network structure of TACE1.0 has a large surface area. The long starch chains of TACE1.0 were first broken by microorganisms, followed by the release of free acrylic acid. Acrylic acid can be actively transported to the cytoplasm by microorganisms and degraded by related enzymes.

Figure 5. Degradation of TACE 1.0.

Experimental results show that the wet weight of TACE1.0 can be reduced by 50% in about 5 days after it is disseminated in paddy fields, and the wet weight is only 3.4% after 15 days. However, during the period of 20 to 30 days, TACE1.0 still left about 0.2% to 0.3% of refractory substance, with a mass of about 0.2g. Generally speaking, TACE1.0 is environmentally friendly, but it can not be ignored that there remains refractory substance after TACE’s usage, which limits its large-scale application in farmland.

TACE2.0

2022 SZU-China launched TACE2.0 in response to the shortcomings of TACE1.0. We experimented TACE2.0 with essential properties.

Measurement of basic parameters of TACE2.0

According to the recommended prescription (15% spore powder, 15% glucose, 60% HPMC4000, 10% HPMC15000), we prepared TACE2.0 powder and then carried out tablet compressing. We used a 10mm tablet mold with pressure of 9.11MPa and a 6mm tablet mold with pressures of 1.58 MPa and 6.33MPa. After tableting, we used a quarter-cut method to obtain 3mm TACE2.0.

Among them, the 6mm TACE 2.0 tablets pressed under the pressure of 1.58MPa were not hard enough and easily fell apart under external force. After due consideration, we used a 10mm tablet mold with pressure of 9.11MPa and a 6mm tablet mold with tablet pressures of 6.33MPa for tabletting. The following are the measurements of the physical parameters of TACE2.0.

Table1. Physical parameters of TACE2.0 tableted by different methods.

We measured an average weight of 257.23mg for 10mm TACE2.0 and 59.10mg for 6mm TACE2.0, with a density of 0.97g/cm³ for 10 mm TACE and 0.87g/cm³ for 6mm and 3mm TACE2.0, which means that TACE2.0 is able to float on water. TACE2.0's good floatage capacity is a huge improvement over TACE1.0.

TACE1.0 vs. TACE2.0

Spore release rate of TACE1.0

We set up a series of experiments to characterize the release of Trichoderma spores loaded by TACE1.0 of different particle sizes (40 mesh and 80 mesh). First, we eluted Trichoderma spores in PDA culture medium to a concentration of order of `10^8`. We used a certain mass of TACE1.0 to load Trichoderma spore suspension, based on the mass ratio we had previously explored to achieve good encapsulation and loading rates simultaneously at the liquid-said rate of 50:1. Then, the spores absorbed TACE1.0 were contained in a filter bag which allows spores free entry and exit, and the filter bag was completely immersed in a known volume of deionized water. OD550 was measured within 200 minutes to obtain the short-time release curve of Trichoderma spores loaded in TACE1.0.

Figure 6. Short-time release curve of Trichoderma spores loaded in TACE1.0 (left: 40 mesh, right: 80 mesh). Fitted by the first order kinetic equation.

Generally, first-order kinetic is a pharmacokinetic concept that states the transport rate of drug in a compartment is proportional to the first power of the drug concentration (C) in that compartment. First-order kinetic equation （1）is used to fit the data of TACE1.0:

$$C = C_0e^{-kt} \ (1)$$

Where C is the spore concentration at time t; t is the release time; K is the first-order kinetic reaction rate constant; and `C_0` is the initial concentration of the chemical.

We observed that the release characteristics of TACE1.0 are basically the same whether it is 40 mesh or 80 mesh. In the early stage, the spores were released rapidly, and half of them were released in about 30 minutes. In the late stage, the release rate of spores greatly slowed down in 80-90 minutes, and spores are almost completely released in 100 minutes.

Spore release rate of TACE2.0

There are many factors that affect the spore release rate of TACE2.0, such as the properties of matrix materials and the content of excipient in the formulation, the size of tablets and its tabletting process. Here we focus on the effect of TACE's particle size on spore release.

Figure 7. TACE2.0 of different particle size.

After successfully preparing TACE2.0 with different particle sizes, we need to determine the effect of particle size on the release rate, and then decide which particle size for TACE2.0 to use as our final product. 0.05g of powdery TACE2.0, 3 grains of 3mm, 1 grain of 6mm, and 1 grain of 10mm were added to 10ml of deionized water, and the absorbance of the solution was detected at intervals to explore the mechanism of TACE2.0 release. It must be noted that the spore concentration does not represent the actual release of TACE2.0, but the shape of the curve can qualitatively illustrate the releasing process of TACE.

First, we observed the complete dissolution time of TACE2.0 with different particle sizes. Powdery TACE2.0 is completely dissoluted in about 5 hours, 3mm TACE2.0 in about 12 hours and 20 minutes, 6mm TACE2.0 in about 20 hours, and 10mm TACE 2.0 in about 28 hours. As expected, the larger the particle size of TACE2.0, the slower the dissolution. It is worth noting that powdery TACE2.0 should be completely dissolved immediately in theory, but because of the overlap during dissemination, it forms a gel layer, which lengthens the complete dissolution time to 5h. In addition, a vortex oscillator used in the measurement may accelerate the dissolution to some extent.

To learn more about the release principle of sustained-release tablets, we consulted the literature and decided to use Higuchi model (2) to fit our data.

`Q/A=2C_0(Dt/\pi)^{\frac{1}{2}} \ (2)`

Wherein `\frac{Q}{A}` refers to the amount of the spore diffused into the receiving cell per unit diffusion area (`mg/cm^2`), wherein `C_0` is the initial concentration of the spore in the gel `(mg\cdot ml^{-1})`, D is the apparent diffusion coefficient of the drug (`cm^2/min`), and t is the time of drug diffusion (min).

The release model should have three typical release phases: slow release in the early stage, because only the shallow surface gelation began to release spores; rapid release in the middle stage, a considerable part of the tablet had been gelated and released at the same time; slow release in the later stage, when it had been completely gelated, only a small amount of spores were left to diffuse. The Higuchi model fits our expectations of how TACE2.0 works. However, we observed that the Higuchi model is not suitable for TACE2.0, while the first-order kinetic equation has a good fitting.

Figure 8. Fitted by the first order kinetic equation (A).release rate of powdered TACE2.0. (B)3 mm release rate of TACE2.0. (C)6mmTACE 2.0 release rate, (D)10mm TACE 2.0 release rate.

See more about modeling process in models.

From the fitted curve based on the first order kinetic equation, we found that TACE2.0 did not have an initial slow release period, but directly entered a rapid release phase. For example, TACE2.0 of 3mm and 6mm almost reached the maximum release rate when just exposed to water, and then decreased slowly. We believe that this is mainly due to the limitation of our tabletting process, resulting in the surface of TACE2.0 is not smooth enough. When TACE2.0 encounters water, the outer surface gelatinizes rapidly and begins to release spores, causing it to enter the intermediate rapid release phase.

Determination of adsorption radius

The floatage and adsorption of TACE is a complex and dynamic process. We built a simple model that TACE first floated randomly, and then adsorbed by rice plant if it entered the position of the adsorption radius.

We collected gramineous plants in paddy fields and set up a simulated environment in the laboratory. TACE1.0 pre-loaded with acid-base pairs, 3mm and 6mm TACE2.0 were put into the simulated field, and TACE floated. After stirring, we observed the floating situation of TACE in a certain area. When the TACE particles were adsorbed at some certain points, we measured the distance from this point to the plant and calculated the adsorption radius.

Figure 9. (A)TACE1.0 is adsorbed to the rice stem, (B)TACE 2.0 is adhered to the rice stem.

The experimental results show that the adsorption radius of TACE1.0 is 0.824±0.209 cm. The adsorption capacity of TACE1.0 is not ideal and is greatly affected by external forces. Generally speaking, if TACE1.0 falls near the plant, it can form a good encirclement. However, if the surrounding water surface is stirred vigorously, it will cause TACE1.0 to be blown away and difficult to re-adsorb to the plant.

Figure 10. Adsorption radius of TACE 2.0 with different particle sizes.

After our determination, the adsorption radius of 3mm TACE2.0 is 1.5±0.267 cm, and the adsorption radius of 6mm TACE2.0 is 2.54±0.38 cm. The adsorption radius of TACE2.0 is significantly larger than that of TACE1.0. Because of the gelation of the outer layer of TACE2.0, it can effectively adhere to the stem of rice. Slight external force did not lead to the detachment of TACE2.0, which formed a stable spore release source at the rice stem and helped the colonization of Trichoderma atroviride.

Encapsulation rate and loading rate

To put it simply, the encapsulation rate refers to the effective utilization degree of the added spore suspension and the effective utilization rate of the spore suspension (in the imbibition step for TACE1.0); the loading rate refers to the proportion of the effective ingredients of a single TACE particle or the proportion of the absorbed spore suspension to the total weight.

TACE1.0 loads spores by absorbing spore suspension, while TACE2.0 contains its own spore components. In order to understand the carry capacity of the two different generations of TACE, we conducted the following experiment, calculating the encapsulation rate (ER%) according to formula (3), and the loading rate (LR%) according to formula (4):

$$（ER\%）=M1/M2\times 100% \ （3）$$

$$（LR\%）=M1/M3\times 100% \ （4）$$

Where M1 is the mass of solution encapsulated in a single particle；M2 is the total weight of spore suspension; M3 is the weight of TACE 1.0 used.

Figure 11. Carry capacity of TACE 1.0 to Trichoderma spores at different liquid-said ratio.

For TACE 1.0, when the dosage of spore suspension tends to infinity, the encapsulation rate tends to 0 and the loading rate tends to 100%. From the point of view of maximum utilization, the liquid-said ratio should be controlled at 45 ~ 50 g/g. However, we found that even at the optimal ratio of 1:45, the encapsulation rate of TACE1.0 was only 17%, and the loading rate was only 13%. Therefore, the absolute values of TACE 1.0 loading rate and efficiency are still very low.

Table 2. Carry capacity of TACE1.0 to Trichoderma spores at different liquid-said ratio.

In contrast, TACE2.0 is a hydrophilic matrix sustained-release tablet pressed from powder with clear composition, its theoretical encapsulation rate reaches 100%, and the loading rate is the proportion of spore powder in the prescription, which reaches 15%. In fact, TACE2.0 is loaded with dry Trichoderma spore powder, the absolute quantity of spores loaded will be much larger than that of TACE1.0. To sum up, TACE2.0 has unique advantages as a commodity.

Experimental evidence shows that SZU-China has a successful iteration of TACE. In addition, we also verified the functionality of TACE. See Proof-of-concept for details.

Trichoderma atroviride

Spores' concentration test

We tested the inhibition effect of different concentrations of Trichoderma atroviride(T.a) spores on R.solani as a way to help us better determine the amount of T.a to be applied in product implementation so as to achieve the desired effect of killing R.solani at a smaller cost.

Process

The T.a spores incubated for 7 days from PDA and were washed off with sterile water and their concentrations were determined by OD550-spores curve.

$$y=0.5715X, \ R^2=0.8667$$

`y` represents the absorption value of OD550,
`X` represents the concentration of T.a spore(`10^7` per ml)

Then 100ul of different spore concentrations of `1.2 \ \times 10^7`, `1.2 \ \times 10^6`, `1.2 \ \times 10^5`, `1.2 \ \times 10^4`, `1.2 \ \times 10^3`, `1.2 \ \times 10^2`per ml of T.a solution were taken in 20ml PDA,

After mixing, plates were poured and solid.
Then a 5 mm R.solani piece is attached to the centre of the plate.
PDA with 100 ul sterile water was used as a control. Each treatment was repeated 3 times.
We calculated the area of R.solani of different groups through algorithm and the following formula:

`S_0`represents the area of control R.solani.
`S_1`represents the area of R.solani which is inhibited by T.a.

Then we got Figure 13: R.solani inhibition rate of different concentrations of T.a spores in `2_{nd}`day.

Figure 12. Variation of R.solani inhibition degree within 7 days by different concentration T.a spores. These figures show the inhibition degree of every day. Under different concentrations of T.a spores, the inhibition effects are different. In 6 days, T.a can completely inhibit R.solani.

It was observed that by the second day of growth there was a significant difference in the inhibition of R.soalni by T.a. At a spore concentration of `1.2*10^5` spores/ml, the mycelial area of R.solani reduced, as it shown in figure 12. And the inhibition rate increased as the spore concentration increased, as it shown in figure 13. The inhibitory effect of `1.2*10^2-1.2*10^4`spores/ml were still weak and no inhibition effect of the growth of R.solani was seen. By day 6, `1.2*10^5`spores/ml of T.a had completely inhibited the growth of R.solani. So we primarily consider that the amount of `10^5`spores/ml may be appropriate.

Figure 13. R.solani inhibition rate of different concentrations of T.a spores in `2_{nd}`day. After 2 days, concentrations higher than `10^5`spores/ml have significant inhibition effect.

Enzyme activity test of Prb 1

Prb 1 protein is a kind of serine protease, so we test the enzyme activity of wild-type T.a(WT) and engineered T.a.

Process

1 ml `10^4`spores/ml Prb 1 transformed T.a are taken in 100ml Mini medium at 28°C, 180rpm for 2 days.

Then centrifuge the culture at 5000rpm for 10min. Take supernatant for further experiments.
The supernatant is first used for protein concentration determination using BCA protein concentration determination kit in 96 well plates. The standard curve is:

$$Y = 0.7660*X + 0.1363$$

Y represents the absorption values of A 562nm.
X represents the protein concentration.

Figure 14. BSA standard curve.

Through this curve we can figure out the protein concentration of the supernate. For wild-type supernate it is 1.694125326 mg/ml and it is 1.356005222 mg/ml for Prb 1 transformant.

Then test the enzyme activity of Prb 1 by AKP Activity Assay Kit. One unit of enzyme activity(1 U) is defined as catalyze to produce 1 umol of Tyrosine per minute per mg of Prb 1 at 40 °C. The enzyme activity of wild-type(control) T.a in supernate is 5.3319 U and for Prb 1 transformant the enzyme activity has been enhanced to 9.2195U.

Figure 15. Enzyme activity of Prb 1.Wild -type represents the Prb 1 enzyme activity of wild-type T.a and it is the control group. Prb 1+ represents the Prb 1 enzyme activity of Prb 1 integrated T.a. Prb 1 activity of wild-type is 5.3319U and 9.2195U for transformed T.a. After T test, the significance between wild-type T.a and Prb 1+ is confirmed.

RNAi

1. Inhibition of R. solani on PDA medium

For the effect of RNAi product spraying on the growth of R.solani on PDA medium, we evaluated its effect by measuring the radius of Rhizoctonia solani colony on PDA medium.

Figure 16. Effect of spraying shRNA on the growth of R.solani.

Figure 17. Colony radius of R.solani after shRNA treatment.

Similarly, we also sprayed CNT-shRNA and measured the colony radius.

Figure 18. Effect of spraying CNT-shRNA on the growth of R.solani.

Figure 19. Colony radius of R.solani after CNT-shRNA treatment.

From the results, the inhibition effect of spraying shRNA on colony growth is not obvious, which may be due to insufficient spraying amount of shRNA and the too fast growth rate of R.solani, shRNA can not play a very good inhibition effect. However, after binding with CNT, the inhibitory effect of shRNA was significantly improved, which showed that CNT shRNA was more absorbed by R.solani than shRNA, thus playing a better role.

2.Mycelium growth on leaves

To prove that our RNAi product can inhibit the infection of Rhizoctonia solani, we observed the growth of hyphae on leaves after spraying shRNA under a microscope (Fig. 20). At the same time, we also observed the leaves treated with shRNA after CNT binding (Fig. 21).

Figure 20. Effect of spraying different shRNA on mycelial elongation without binding CNT.

Figure 21. Effect of spraying different shRNA after binding CNT on mycelial elongation.

After measuring the mycelium extension length in leaves under different treatments, the following statistical results are obtained (Fig. 22).

Figure 22. Extension length of mycelium under different treatments.(a) The effect of spraying shRNA targeting key genes in infection process on mycelial extension without binding CNT. (b) Effect of spraying shRNA targeting key survival genes of Rhizoctonia solani on mycelial extension without CNT binding. (c) Effect of spraying shRNA targeting key genes in infection process after binding CNT on mycelial extension. (d) Effect of spraying shRNA targeting the key survival genes of Rhizoctonia solani after binding CNT on mycelial extension.

For spraying shRNA without CNT binding, the growth of mycelia on leaves was inhibited to some extent, but the inhibition effect was not ideal. For shRNA that inhibits the key genes in the infection process of R.solani, only the PG gene is inhibited; However, shRNA of key genes inhibiting the growth of Rhizoctonia solani had no significant effect.

After the shRNA was bound with CNT, it was sprayed. Compared with the control group's 21.5 mm mycelium length, the shRNA targeting the key genes in the infection process of Rhizoctonia solani could inhibit the extension of the mycelium to about 12.1 mm. For shRNA targeting the key genes of R.solani growth, the highest inhibition rate of hyphal extension was 40%.

3.Leaves spot area

As one of the important indicators of leaf disease, the area of disease spots on leaves can also be used to evaluate the effect of RNAi products. On the fifth day of spraying, we observed the difference of leaf spot under different treatments(Fig. 23), and quantified the area of the spot through image analysis(Fig. 24).

Figure 23. Distribution of disease spots on infected rice.

Figure 24. Leaves spot area under different treatments.

We can find that compared with the control, spraying siRNA will reduce the area of the diseased spot to 45.53%, and shRNA will reduce the area of the diseased spot to 25.91%. The spraying effect of shRNA-CNT was the best, and the area of disease spot was only 8.53% of the control. It can be concluded that shRNA CNT has obvious advantages over siRNA and shRNA.

4.Target gene expression

The essence of RNAi treatment is that sRNA molecules specifically recognize target genes in R.solani and then conduct post transcriptional silencing. Therefore, in order to verify the therapeutic effect of RNAi, we must measure the changes in the expression level of target genes in R.solani after spraying sRNA. We sprayed siRNA, shRNA and CNT shRNA respectively, and tested the effect of RNAi products through qRT-PCR for 5 consecutive days.

Figure 25. Changes in the expression level of target genes. Blue represents the control group, red represents siRNA treatment, green represents shRNA treatment, and purple represents CNT shRNA treatment.

For siRNA and shRNA treatment, the relative expression of target genes in Rhizoctonia solani decreased to the lowest at the third day, 0.144 and 0.103, respectively. But after that, the expression of target genes in both groups increased rapidly. Under the treatment of CNT shRNA, the expression of target gene decreased to 0.019 on the fourth day, and the effect lasted longer.

To sum up, CNT shRNA has more obvious advantages in comparison with the size of the colony of Rhizoctonia solani growing on PDA medium, the growth degree of the mycelia on the leaves, the size of the diseased spot, the expression inhibition level of the target gene and the duration of the inhibition effect. Therefore, we look forward to its outstanding performance as our RNAi product.