Introduction

In light of our proof of concept, we have conducted a heat experiment to assess the usability of our produced nanoparticles for PTT. For our experiment we have used a near infrared (NIR) laser that excites nanoparticles with an absorbance around 800 nm (A800 nm). Here we briefly explain what our samples are, we describe the experimental design of our heating experiment, provide the protocol and delineate what was needed to obtain results.

We have provided a clear description of our experiment and analysis enabling future iGEM teams to reproduce this heating experiment investigating photothermal conversion. Furthermore, this can be done using a NIR laser or at different wavelengths depending on what is necessary.

Experiments

Samples

The optimal biologically produced nanoparticles defined in our modeling experiments were used with HAuCl4:AgNO3 ≈ 1.85:1, optimizing the A800 nm. For the controls, Mueller Hinton (MH) broth and silver and gold salts dissolved in ddH2O were used since our previous research findings suggest that medium alone also reduces the salts to some extent. The absorbance spectra were measured using a UV-vis NIR spectrophotometer. Furthermore, transmission electron microscopy (TEM) was used to determine the shape and size of the nanoparticles and a Zetasizer to determine the polydispersity of the nanoparticle population.

Experimental design heating experiment

Heat experiments were performed in which a NIR laser with a power of 1.3 W was shone on nanoparticle dispersion and controls (with volumes of 3 mL) for a total duration of five minutes. Subsequently, the relaxation time was measured, which is the time needed for the sample to cool down to ambient temperature. Temperature change was measured using a thermometer, which was placed above the beam of the laser. The moving average of the temperature was calculated to correct for the sensitivity of the thermometer by calculating the average over five time points. A visual representation of the experimental setup is provided in Figure 1. To be able to assess the heat conversion per concentration of nanoparticles, the mass of the nanoparticle sample was determined by freeze drying. Taking safety into account, the freeze drying experiments were performed by an expert, and the nanoparticles were not taken out of the solution but put into an Eppendorf tube. The mass of the nanoparticles was determined by weighing the tube before and after the experiment.

Fig. 1 | Experimental setup proof of concept

Materials

  • 20 mL Escherichia coli (E. coli) BL21-pET16b overnight culture in Mueller Hinton Broth (+150 µg/mL Ampicillin)
  • Mueller Hinton Broth (+150 µg/mL Ampicillin)
  • Acetate Buffer pH 5
  • Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H20, 99.9%, Acros)
    • 100 mM aqueous HAuCl4 solution
  • Silver nitrate (AgNO3, 99%, Acros)
    • 100 mM aqueous AgNO3 solution
  • ddH2O
  • Sodium acetate
  • Acetic acid
  • 10 M HCl
  • 50 mL Falcon tube
  • 0.2 µm Syringe filter
  • 15 mL Syringe
  • 50 mL Flasks
  • 1 mL plastic cuvettes
  • 3 mL plastic cuvettes
  • 3 mL Quartz cuvettes

Equipment

  • Shaking incubator
  • Tabletop centrifuge
  • Refrigerated micro centrifuge
  • Vortex mixer
  • Rod sonicator (MS-72 probe)
  • Scale
  • UV-vis NIR spectrophotometer
  • Zetasizer
  • Temperature probe
  • 1.3 W NIR Laser

Protocol

  1. For 100 mM acetate buffer pH 5:
    1. In a suitable container, add 800 mL ddH2O.
    2. Add 5.772 grams of sodium acetate to the solution.
    3. Add 1.778 grams of acetic acid to the solution.
    4. Adjust pH with 10M HCl to pH 5.
    5. Add ddH2O to a final volume of 1 L.
  2. Dilute the overnight culture down to an OD600 of 0.2.
  3. Grow diluted cultures in a shaking incubator set to 37°C at 200 rpm until they reach an OD600 of 0.4-0.6 (~1 hour), (Protein synthesis is maximal during log phase growth therefore samples are taken during this phase.)
  4. Once the culture has reached this phase transfer the contents to a 50 mL falcon tube and centrifuge at 4000 rpm for 10 minutes.
  5. Take the supernatant of centrifuged samples and filter sterilize with a 0.2 µm filter into a new falcon tube. This will now be referred to as supernatant. Do not discard the pellet, this will be used in the next step.
  6. Add 1 mL of Mueller Hinton Broth to the pellet and vortex until the pellet is completely resuspended. Add 500 µL of the resuspended pellet into two Eppendorf tubes.
  7. Sonicate the resuspended pellet with a rod sonicator (MS72, 10% amplitude for 30 seconds x2 (1 second on, 1 second off)).
  8. Centrifuge the sonicated lysate samples at max speed (>12,000 RCF) in a chilled (4° C) micro centrifuge for 30 minutes. This will now be referred to as lysate.
  9. Adjust the pH of the supernatant to pH 5 by adding 500 µL of Acetate buffer (pH 5) to 3 mL of the supernatant. This will be pH 5 supernatant.
  10. Add either supernatant or ddH2O into assigned flasks according to the layout below (e.g. 9076 µL).
  11. Add lysate into the assigned flasks according to the layout below.
  12. Add 100 mM aqueous HAuCl4 solution (e.g. 600 µL) into the assigned flasks according to the layout below.
  13. Add 100 mM aqueous AgNO3 solution (e.g. 324 µL) into the assigned flasks according to the layout below.
  14. Place flasks in a shaking incubator set to 49°C at 200 rpm for 20 hours.
  15. For all samples:
    1. Absorbance Scan: Transfer 1 mL into a 1 mL plastic cuvette and scan absorbance from 350 nm to 1000 nm with a UV-vis spectrophotometer.
    2. Zetasizer: Transfer 500 µL into a 50 mL falcon tube, and dilute 100x with ddH2O. Then transfer 1 mL of sample into a 1 mL plastic cuvette and measure the polydispersity index and size on a Zetasizer.
    3. Freeze Drying: Transfer 1 mL into a 1.5 mL Eppendorf tube, and centrifuge at max speed (>12,000 RCF) for 10 minutes. Remove the supernatant. Freeze the pellet and afterward weigh the mass.
    4. Heat Experiment: Transfer 3 mL of sample into a 3 mL quartz cuvette, and place the temperature probe into the cuvette. Aim 1.3 W NIR laser set to 850 nm at the cuvette not directly at the temperature probe, and switch on for 5 minutes.

Flasks

Flask Supernatant Lysate 100 mM HAuCl4 100 mM AgNO3 ddH2O
1

8576 µL

500 µL

600 µL

324 µL

0 µL
2

8576 µL

500 µL

600 µL

324 µL

0 µL
3

0 µL

0 µL

600 µL

324 µL

9076 µL
4

0 µL

0 µL

600 µL

324 µL

9076 µL

Results

Absorbance

The absorbance spectra showed an overall higher absorbance for biological samples compared to controls (Fig. 2A). High absorbance was measured for the range of 400-1000 nm. This could indicate the presence of a diverse population of nanoparticles since mono- and bimetallic nanoparticles, also depending on being silver or gold in their composition, have absorbance at different wavelengths. This heterodisperse population was confirmed using the zetasizer which showed a polydispersity index of 1.0, indicating nanoparticles of different sizes. The presence of nanoparticles was confirmed using TEM (Fig. 3), showing a diverse population of nanoparticles with different shapes and sizes. The nanoparticle population did not show an urchin-like shape but absorbed 800 nm light (Fig. 2B) and was therefore used for the heat experiments.

Fig. 2 | Absorbance spectrum of biological nanoparticles produced using a microbial factory. Nanoparticles were produced using wildtype Escherichia coli BL21 supernatant with HAuCl4:AgNO3 ≈ 1.85:1. A.) Absorbance spectra (400-1000 nm) of the biological nanoparticles and a solution of ddH2O with gold and silver salts, representing the mean and the standard error of the mean. B.) Barplots of the average absorbance at 800 nm with standard error for the biological nanoparticles and the solution of ddH2O with gold and silver salts.

Fig. 3 | Transmission electron microscopy image of biologically produced nanoparticles with HAuCl4:AgNO3 ≈ 1.85:1 produced using wildtype Escherichia coli BL21 supernatant.

Heat experiments

Heat experiments were performed to assess if our nanoparticles are able to convert the light of a NIR laser into heat. Shining the laser on the nanoparticles resulted almost directly in an increase in temperature, in which the slope was higher initially and flattened over time (Fig. 4). The flattening of the curve is likely caused by increased heat transfer to the environment at higher temperatures of the solution. The nanoparticle sample reached a maximal temperature of 34.91±0.49°C and thereby showed a ΔTemp of 7.1°C. Half of this temperature increase was already reached after 100 s, which is at a third of the total laser exposure time. After turning the laser off, the biological sample quickly cooled down to room temperature. The controls showed minimal temperature change over time, with a ΔTemp of ~1.5°C for ddH2O with salts and a ΔTemp of ~0.44°C for medium alone. Thus, our research findings indicate that the increase in temperature is caused by the nanoparticles converting the light of the laser into heat.

Fig. 4 | Temperature changes over time after shining a near-infrared (NIR) laser on biologically produced nanoparticles. Nanoparticles were produced using a wildtype Escherichia coli BL21 supernatant with HAuCl4:AgNO3 ≈ 1.85:1. For controls, Mueller Hinton broth and ddH2O with silver and gold salts were used. The samples were exposed to the laser for five minutes after which the relaxation time was measured.

Specific heat conversion of nanoparticles

The mass of the nanoparticles was determined to assess the heat conversion per concentration of nanoparticles. Freeze drying the samples showed a concentration of 2.5±0.095 mg/mL while 1.53 mg/mL silver and gold salts were added to the solution. This could suggest that other salts present in the medium were also involved in the formation of nanoparticles. Another explanation would be that after filtering still cell debris, medium components or other proteins were present in the sample. This heat conversion per concentration of nanoparticles was used to estimate the amount of nanoparticles necessary to actually kill a tumor cell. As mentioned before, cell death will occur at a temperature of 42-43°C.1 Therefore, starting from body temperature, a ΔTemp of 5°C is required to kill a tumor cell. Previous heating experiments were performed at room temperature. For the simplicity of our experiments, we assumed that the starting temperature would not affect the absorbance of our nanoparticles at 800 nm and thereby the ΔTemp. According to literature, the average size of a laryngeal tumor is 2.74 cm,2,3 giving a volume of 10.77 cm3 assuming that the tumor is a perfect sphere. Our results revealed that 2.5 mg nanoparticles could cause a ΔTemp of 7°C for 3 mL solution after five minutes of irradiation with a NIR laser. Assuming a linear correlation, 19.23 mg of nanoparticles would be required to kill the tumor cells.

Assumptions:

  • The starting temperature will not affect the absorbance of the nanoparticles at 800 nm.
  • The tumor is a perfect sphere.
  • Linear correlation between mass of nanoparticles and corresponding ΔTemp.
  • The tumor has the same specific heat capacity as the medium.

Photothermal conversion efficiency

Heating experiments can be used to calculate the photothermal conversion efficiency of our nanoparticles.4 In this experiment, the temperature of the nanoparticles in solution was measured while it was irradiated with an infrared laser. The temperature increased to an equilibrium and cooled to ambient temperature. An energy balance equation can be used to describe the temperature change of the system.

Eq. 1)

$$\sum_i m_iC_{p,i}\frac{dT}{dt} = Q_1 + Q_{dis} + Q_{ext}$$

m and Cp are the mass and the heat capacity of the components of the system (i). T is the temperature of the system and t is the time. Q1, Qdis, and Qext describe the energy produced by nanoparticles, the baseline energy in the system (the temperature rise of the sample due to direct heating of the laser), and the outgoing energy, respectively.

This formula can be rewritten as described in5 to calculate the photothermal conversion efficiency (η).

Eq. 2)

$$\eta = \displaystyle \frac{hs(T_{max} - T_{Surr})- Q_{dis}}{I(1- 10^{-A_{800~nm}})}$$

The heat transfer coefficient (h) and the surface area of the container (s) are obtained by using eq. 3. The maximum temperature Tmax is 35.61 °C, and the temperature of the surroundings Tsurr is 28.28 °C. Qdis is calculated with eq. 6. The power of the laser (I) is 1300 mW and the absorbance at 800 nm (A800 nm) is 5.7.

Eq. 3)

$$h s=\frac{m C_p}{\tau}$$

The mass of the sample (m) is 3 g. The specific heat (Cp) is 4.2 J/g K. To calculate the time constant τ, eq. 4 is used.

Eq. 4)

$$t=-\tau \ln (\theta)$$

The time constant τ was determined by making a plot of -ln(θ) over time (Fig. 5). θ is a dimensionless parameter, described in eq. 5. It can be calculated by using the heating or the cooling data of the experiment. In this calculation the cooling data was used because the graph showed a higher R2. The slope of this plot represents the time constant τ, which is 154 s. The time constant τ can then be used to determine hs, with eq. 3. This gives 81.8 mW/K.

Eq. 5)

$$\theta=\frac{T-T_{\text {Surr }}}{T_{\text {max }}-T_{\text {Surr }}}$$

Fig. 5 | A.) Temperature change over time of biologically produced nanoparticles using a microbial factory. Nanoparticles were irradiated with a near-infrared laser for five minutes after which the relaxation time was measured. B.) Cooling data was used to compute the θ, in which the slope represents the time constant. This information was required to calculate the photothermal conversion efficiency of the nanoparticles.

The dissipated heat or Qdis is calculated using equation 6. The temperature change of the medium after irradiation with the laser is used, this is 0.54 K. This gives a Qdis of 6.804 J. Over a time of 300 seconds, this is 22.68 mW.

Eq. 6)

$$\text Q_{dis}=m * C_p * \Delta T$$

Substituting the according values of all parameters to eq. 2, the photothermal conversion efficiency is found to be 44.3%. This is in agreement with values found in literature, where nanoparticles show a photothermal conversion efficiency of 20-60%.4,5,6

Assumptions:

  • The heat dissipated to the surrounding is negligible.
  • The heat is evenly distributed through the solution.
  • Heat capacity of the nanoparticles is negligible compared to the surrounding liquid.

Conclusion

In our heating experiment, it was revealed that the nanoparticles were able to convert light of a NIR laser into heat with a ΔTemp of 7.1°C and a PTT conversion efficiency of 44.3%. We have provided the experimental design including a description of our samples and controls, as well as the protocol and a detailed analysis to obtain the results of a simple but novel experiment. This page described a reproducible experiment that enables personalization by other iGEM teams.

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