Introduction

Our nanoparticles are produced with the application of photothermal therapy (PTT) in mind, as mentioned in our proposed Implementation. In this therapy, light from a laser is converted to heat by nanoparticles localized on tumor cells. When the temperature of the tumor cells reaches 42-43 °C, proteins in the cells will denature, leading to both acute and chronic cell injury and eventually cell death1 (Fig. 1).

Visual representation of the concept of photothermal therapy, in which nanoparticles are specifically targeted to the tumor cells and triggered with a near-infrared laser. The nanoparticles will increase in temperature and cause cell death.
Fig. 1 | Visual representation of the concept of photothermal therapy, in which nanoparticles are specifically targeted to the tumor cells and triggered with a near-infrared laser. The nanoparticles will increase in temperature and cause cell death.

The laser shines light from the near-infrared (NIR) spectrum on the tumor since other wavelengths of light will damage vessels and healthy tissue (see the interview with Prof. Dr. Sterenborg on our Human Practices page). Furthermore, NIR light will penetrate deeper into the tissue than visible light, making this wavelength suitable for PTT since it can reach the tumor and activate the nanoparticles. This is due to the minimal absorption coefficient of melanin, hemoglobin, and water molecules in the NIR range2. The NIR region can be divided into two windows functional for PTT. The first window (λ =650-850 nm) is optimal for superficial tumors, with a maximal depth of 2-3 cm under the skin. Laser light in the second window (λ =950-1350 nm) can reach deeply embedded tumors, with a maximal depth of 10 cm under the skin3,4.

We were advised to focus on shallow tumors during our Human Practices by Dick Sterrenborg, Professor of biomedical engineering and physics, and Dimitri Pappaioannou, director of business development at Oncolines. Squamous cell carcinomas make up 90% of all head and neck cancers. These occur on the outermost surface of the skin, or in certain tissues of the head and neck region like the throat, sinuses, nose, and mouth8. Due to the shallow depth of these superficial tumors, a wavelength of 800 nm was chosen. This falls within the first window of the NIR region and can reach these tumors.

In addition to having absorbance in the NIR region, nanoparticles for use in PTT should show a high photothermal conversion efficiency. This property is based on both the shape and the size of the nanoparticles. A higher photothermal conversion efficiency can be reached by reducing the size of the nanoparticles5. Additionally, changing the shape from spherical to a non-spherical (e. g. nanorod, nanostar, or nanocage), can increase the photothermal conversion efficiency9.

During our project, we successfully produced nanoparticles using a cell-free biological system, as described on our Results page. The experiments needed for a valid proof of concept should show that our produced nanoparticles have absorbance in the first window of the NIR region, and have a high photothermal conversion efficiency. These properties would make them applicable in PTT.

Surface plasmon resonance: in-depth explanation

The ability of metallic nanoparticles to convert light into heat is caused by surface plasmon resonance, which is the oscillation of electrons activated by photons of incident light. It occurs on the interface of a conductor and the external medium12. When the size of the conductor is similar to or smaller than the wavelength of the incident light, which is the case for nanoparticles, localized surface plasmon resonance (LSPR) occurs. This means that the electrons start oscillating coherently (Fig. 2)13. The LSPR wavelength is dependent on the electron charge density of the surface of the nanoparticles. Factors influencing this charge density, such as the size and shape of the nanoparticles and dielectric properties of the metal and the medium, can change the LSPR wavelength14.

Visual representation of localized surface plasmon resonance with coherent oscillation of the electrons.
Fig. 2 | Visual representation of localized surface plasmon resonance with coherent oscillation of the electrons.

The absorbance and scattering efficiency of noble metal nanoparticles is strongly enhanced by the LSPR effect. The sum of photon energy obtained from absorbance and scattering is called extinction and represents the total loss of photons. The extinction of small nanoparticles (20 nm) is only caused by absorbance15. For bigger nanoparticles, scattering starts playing a role. The photons that are scattered are no longer available for light conversion into heat. Therefore, smaller nanoparticles are able to generate more heat.

Experiments

Experimental design

Heat experiments were performed to assess the usability of our produced nanoparticles for PTT. A visual representation of the experimental setup is provided in Figure 3. For the nanoparticles to be excitable by the NIR laser, it is crucial to have absorbance at 800 nm (A800 nm). Therefore, the optimal biologically produced nanoparticles defined in our modeling experiments were used with HAuCl4:AgNO3 ≈ 1.85:1. For the controls, Mueller Hinton broth (MH broth) and ddH2O with silver and gold salts were used since our previous research findings suggested that medium alone also reduces the salts to some extent. The absorption 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 the Zetasizer to determine the polydispersity of the nanoparticle population.

Heat experiments were performed in which a NIR laser with a power of 1.3 W was emitted on the nanoparticle sample and controls (with a volume 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 room temperature. The 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. Finally, using freeze drying, the mass of the nanoparticle sample was determined to assess the specific heat conversion per concentration of nanoparticles. Taking safety into account, the freeze drying experiments were performed by a professional, 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.

Visual representation of the experimental setup of the heating experiments with the near-infrared laser.
Fig. 3 | Visual representation of the experimental setup of the heating experiments with the near-infrared laser.

Results

Absorbance

The absorption spectra showed an overall higher absorbance for the biological sample than for the control (Fig. 4A). High absorbance was measured for the range of 400-1000 nm. This could indicate the presence of a diverse population of nanoparticles since monometallic and bimetallic nanoparticles, also depending on being silver or gold and their composition, have absorbance at different wavelengths. This heterodisperse population was confirmed using the zetasizer which showed a polydispersity index of 1000, indicating nanoparticles of different sizes. The presence of nanoparticles was confirmed using the TEM (Fig. 5), showing a diverse population of nanoparticles with different shapes and sizes. The nanoparticle population did not show an urchin-like shape, however, showed A800 nm (Fig. 4B) and was therefore used for the heat experiments.

Absorbance spectrum of biological nanoparticles produced using a microbial factory.
Fig. 4 | Absorption 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.) Absorption 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.
Transmission electron microscopy image of biologically produced nanoparticles.
Fig. 5 | 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 steeper at the beginning and flattened over time (Fig. 6). 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±0.487°C and thereby showed an 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. This short relaxation time indicates that the nanoparticles are unable to retain the heat, which is desirable since otherwise too much tissue will get damaged. The controls showed minimal temperature change over time, ΔTemp of ~1.5°C for the ddH2O with salts and ΔTemp of ~0.44°C for the medium. Thereby our research findings suggest that the increase in heat is caused by the nanoparticles converting the light of the laser into heat.

Temperature change over time after shining a near-infrared (NIR) laser on biologically produced nanoparticles.
Fig. 6 | Temperature change 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 (MH) 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.

Heat conversion per concentration 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°C1. 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,10,11 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 1 mL solution after five minutes of irradiation with a NIR laser. Assuming a linear correlation, 19.23 mg nanoparticles would be required to kill the tumor cells.

  • 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 nanoparticles6 . 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 in Cole at al.7 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 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. 7).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)

$$\theta=\frac{T-T_{\text {Surr }}}{T_{\text {max }}-T_{\text {Surr }}}$$
Two graphs displaying temperature change over time of biologically produced nanoparticles using a microbial factory.
Fig. 7 | 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%6,7,16.

  • 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 this project, we aimed to biologically produce the optimal nanoparticles for PTT using a microbial factory. Our produced nanoparticles did not satisfy all the desired properties for PTT, since the nanoparticles were not urchin-like and aggregates were formed. However, the heat experiment 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%.

In the future, heat experiments on cancer cell lines should be performed to confirm their ability to kill tumor cells. Also, as a starting point, body temperature should be used to verify that this will not affect the functionality of the nanoparticles. Finally, a monodisperse nanoparticle population is required for applicability in the clinic. This could be obtained by adding surfactants, however, the effect of surfactants on the heat conversion of the nanoparticles should be tested. For more detailed information see future experiments.

All in all, this work revealed the ability of our produced nanoparticles to convert light of a NIR laser into heat and thereby opens avenues for an application in PTT. This work could contribute to new insights into the production of the optimal nanoparticles for PTT and thereby pave the way for a new treatment strategy for patients with head and neck cancer.

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