3 Characterization of Nanoparticles

3.1 Experimental Instruments and Reagents

3.1.1 Instrument

Table5 Experimental Instruments

3.1.2 Reagents

Table6 Experimental Reagents

ContinuationTable 6 Experimental Reagents

3.2 Experimental Content

3.2.1 Observation of PDA Nanoparticles by Transmission Electron Microscopy

Dilute the PDA nanoparticle dispersion with a concentration of 1 mg/mL to an appropriate concentration. Place the copper grid on filter paper, and carefully drop 10 μL of the diluted solution onto the copper grid using a pipette, and place it in a 37 ℃ oven overnight. Observe the sample morphology under a transmission electron microscope and take photos.

3.2.2Determination of Infrared Absorption Spectrum

Disperse the DA and PDA nanoparticles in a solution at 4 °C and a speed of 15557 g/min for 30 minutes, discard the supernatant, and place the precipitate in a 37 °C oven to dry. Use a Fourier transform infrared spectrometer to analyze the powder obtained after drying, scanning its infrared absorption spectrum in the range of 4000-500 cm^-1, save the results, and perform characteristic peak analysis.

3.2.3Determination of Ultraviolet-Visible Absorption Spectrum

Prepare solutions of PDA, ICG, and PDA-ICG at the corresponding concentrations. Use deionized water to perform baseline correction on the UV spectrophotometer, scan the UV-visible absorption spectra of the above solutions from 400 to 1000 nm, save the results, and conduct peak analysis.

3.2.4Determination of Particle Size and Zeta Potential

Dilute PDA, PDA-ICG, PDA-ICG@PEI/siPKM2 nanoparticles to an appropriate concentration, and measure their hydrodynamic diameter, PDI, and Zeta potential using a Malvern laser particle size analyzer.

3.2.5Evaluation of Solar Thermal Performance

The relationship between the photothermal performance of PDA-ICG nanoparticles and power

Dilute the water dispersion of PDA-ICG nanoparticles to a concentration of 200 μg/mL, with three parallel groups, each group 0.5 mL, and irradiate with 808 nm near-infrared light at powers of 0.5 W/cm², 1 W/cm², and 1.5 W/cm² for 5 minutes, recording the temperature every 10 seconds for the first two minutes and every 20 seconds for the last three minutes, to plot the temperature rise curve of PDA-ICG nanoparticles.

The relationship between the photothermal properties of PDA-ICG nanoparticles and concentration

Prepare 0.5 mL of dispersions of PDA-ICG nanoparticles with concentrations of 200 μg/mL, 150 μg/mL, 100 μg/mL, and 50 μg/mL, using deionized water as a control, in five parallel groups. Irradiate the above solutions with 808 nm near-infrared light at a power of 1 W/cm² for 5 minutes, recording the temperature every 10 seconds for the first two minutes and every 20 seconds for the last three minutes, to plot the heating curve of the PDA-ICG nanoparticles.

Evaluation of the photothermal performance of PDA, ICG, and PDA-ICG in vitro

Take 0.5 mL of PDA (200 μg/mL), ICG (39 μg/mL), PDA-ICG (PDA 200 μg/mL; ICG 39 μg/mL) and deionized water, and irradiate with 808 nm near-infrared light at 1 W/cm² for 5 minutes, recording the temperature every 10 seconds for the first two minutes and every 20 seconds for the last three minutes, while capturing thermal images at 0, 1, 2, 3, 4, and 5 minutes to plot the temperature rise curve.

Determination of the photothermal stability of PDA-ICG nanoparticles

Dilute the water dispersion of PDA-ICG nanoparticles to a concentration of 200 μg/mL, take 0.5 mL of the above liquid and irradiate with 808 nm near-infrared light at 1 W/cm² for 5 minutes, then allow to cool naturally for 5 minutes, and irradiate again with laser to raise the temperature, repeating this 3 times. During the heating and cooling phases, record the temperature every 10 seconds for the first two minutes and every 20 seconds for the last three minutes, and plot the curve.

Determination of the photothermal conversion efficiency of PDA and PDA-ICG nanoparticles

Prepare PDA (200 μg/mL), PDA-ICG (PDA 200 μg/mL; ICG 39 μg/mL) nano-particle aqueous dispersions, and measure their UV-visible absorbance values at 808 nm. Irradiate the above solutions with 1 W/cm² of 808 nm near-infrared light for 5 minutes, with a volume of 0.5 mL, then allow to cool naturally for 10 minutes to approach room temperature. Record the temperature every 10 seconds for the first two minutes during heating and cooling, and then every 20 seconds. Calculate θ using formula (4) and (5), plot the cooling time of PDA and PDA-ICG against -ln(θ), and obtain τ_s, then calculate the photothermal conversion efficiency of PDA and PDA-ICG according to formula (3) and (6).

h: heat transfer coefficient; S: surface area of the container; T_max: maximum temperature; T_Surr: ambient temperature; I: laser power density; Q_s: the heat emitted by the solvent and container after absorbing light; m_D: mass of the nanoparticles; C_D: specific heat capacity of the solution; A₈₀₈: absorbance of the nanoparticles at 808 nm.

3.2.6ICG's release

Cut the dialysis bag with a molecular weight cutoff of 3500 Da into two suitable and equal lengths, soak in deionized water for 24 hours, then take out and soak in 30% alcohol, and store in a 4 °C refrigerator for later use.

Set up two parallel experiments: the light group and the control group without light exposure. Rinse the two dialysis bags mentioned above with deionized water, use a pipette to take 1 mL of the PBS dispersion of 1 mg/mL PDA-ICG nanoparticles and add it to the dialysis bags, tie both ends tightly with cotton thread, and then place them in a 50 mL centrifuge tube containing 40 mL of PBS solution, wrap the centrifuge tube with aluminum foil, and place it in a 37 °C constant temperature water bath shaker. Remove the dialysis bags at 2 h and 5 h, irradiate the light group with an 808 nm laser (1.5 W/cm²) for 5 min, and do not expose the control group to light, then return them to the centrifuge tube. At 0.5, 1, 2, 3.5, 5, 6.5, 8, and 24 h, use a pipette to take out 1 mL of the external dialysis medium, protect it from light for storage, and add back 1 mL of PBS solution. Measure the UV absorbance value of the extracted solution at 777 nm, calculate its ICG concentration based on the ICG standard curve, and from this calculate the cumulative release rate, plotting the ICG release curve.

3.2.7Evaluation of Photodynamic Performance

The superoxide anion reagent kit was used to evaluate the photodynamic performance of nanoparticles.The yield of superoxide anions is proportional to the increase in absorbance of the samples at 530 nm before and after laser irradiation.

Set up four parallel experiments of PBS, PDA, ICG, and PDA-ICG, with six wells in each group, three of which are illuminated and the other three are not illuminated as a control. Take two 12-well enzyme-labeled strips and break them into six rows and four columns, adding different volumes of 0.01 M PBS solution and 1 mg/mL PDA, ICG, and PDA-ICG solutions to each group according to the table below, so that the concentrations before adding the color development solution correspond to PDA 80 μg/mL, ICG 15 μg/mL. After adding hydroxylamine and mixing evenly, each group of three wells is irradiated with 808 nm laser (1.5 W/cm²) for 5 min, and then placed in a water bath at 25 ℃ for 20 min. Then add the color development solution, and incubate in a water bath at 30 ℃ for 30 min, finally using an enzyme-labeled instrument to detect the absorbance value at 530 nm, calculating the increase in absorbance before and after illumination.

Table7 Evaluation of Photodynamic Performance with Sample Amount

3.3 Results and Discussion

3.3.1 Observation of PDA Nanoparticles by Transmission Electron Microscopy

Figure5 Transmission Electron Microscopy Image of PDA Nanoparticles

Under transmission electron microscopy, PDA nanoparticles appear smooth, round, and spherical, with a uniform size of about 100 nm.

3.3.2Infrared Spectroscopy and UV-Visible Absorption Spectroscopy

Figure6 Infrared absorption spectra of DA and PDA nanoparticles (a) and their photos in aqueous solution (b)

Asshown in Figure 6, the infrared spectroscopy results indicate that both DA and PDA nanoparticles exhibit stretching vibration peaks of hydroxyl and amine groups in the range of 3500-3200 cm^-1, with a typical benzene ring skeletal vibration peak around 1600 cm^-1. Compared to DA, the NH₂ stretching vibration peak at 3334.68 cm^-1 and 3198.92 cm^-1 disappears in PDA nanoparticles. Due to different substitutions on the aromatic ring, DA has C-H bending vibration peaks of 1,2,4-trisubstituted benzene at 887.09 cm^-1 and 813.35 cm^-1, while in PDA nanoparticles, the absorption peak in the range of 900-800 cm^-1 is significantly weakened because all six H atoms of the benzene ring are substituted. This indicates the successful synthesis of PDA nanoparticles. Furthermore, the DA aqueous solution is colorless and transparent, while the PDA nanoparticle aqueous solution is dark brown, further confirming the occurrence of the oxidation self-polymerization reaction of DA.

Figure7 UV-visible absorption spectra of ICG, PDA NPs, and PDA-ICG nanoparticles

Asshown in Figure 7, in the wavelength range of 400-1000 nm, PDA nanoparticles did not show any absorption peaks, while ICG exhibited two absorption peaks around 780 nm, with the maximum absorption wavelength at 777 nm. The PDA-ICG nanoparticles also have an absorption peak around 780 nm, similar to ICG, which preliminarily indicates that PDA has successfully loaded ICG.

3.3.3Appearance

Figure8 PDA, ICG, PDA-ICG, PDA-ICG@PEI/siPKM2 aqueous solution (from left to right)

Asshown in Figure 8, the PDA nanoparticles are dark brown, and the ICG aqueous solution is bright green. Due to the loading of ICG onto the PDA nanoparticles, the PDA-ICG nanoparticles and PDA-ICG@PEI/siPKM2 nanoparticles appear dark green.

3.3.4Particle Size and Zeta Potential

Table8 Particle size and Zeta potential of PDA, PDA-ICG, and PDA-ICG@PEI/siPKM2 nanoparticles

Figure9 Hydrated particle size (b) and its distribution (a), Zeta potential (d) and its distribution (c) of PDA, PDA-ICG, and PDA-ICG@PEI/siPKM2 nanoparticles

Asshown in Table 8andFigure 9, the hydrodynamic diameter of PDA nanoparticles is around 150 nm, while the diameter of PDA-ICG@PEI/siPKM2 slightly increases to about 180 nm, indicating that PDA nanoparticles successfully loaded ICG, PEI, and siPKM2. All formulations have a relatively small PDI, meaning the particle size distribution is narrow, which indicates that the size of the nanoparticles is uniform. The Zeta potential of both PDA and PDA-ICG nanoparticles is around -50 mV, indicating a relatively stable system. Due to the high density of positive charges on PEI, the Zeta potential of PDA-ICG@PEI/siPKM2 nanoparticles reverses to positive, around 55 mV, indicating that the nanoparticles successfully encapsulated PEI, and the electrostatic repulsion is significant, resulting in better system stability.

3.3.5Evaluation of Solar Thermal Performance

The relationship between the photothermal performance of PDA-ICG nanoparticles and power

Figure10 Temperature rise of PDA-ICG nanoparticles under laser irradiation of different powers

Asshown in Figure 11, with laser powers of 0.5 W/cm², 1 W/cm², and 1.5 W/cm², after irradiating PDA-ICG nanoparticles for 5 minutes, the temperature of the nanoparticle aqueous dispersion increased by 8.7 ℃, 22.9 ℃, and 36.6 ℃, respectively. This indicates that as the laser power increases, the temperature rise of the nanoparticles also increases, demonstrating that the photothermal performance of the nanoparticles is closely related to the power.

Figure11 Temperature rise of PDA-ICG nanoparticles at different concentrations under laser irradiation

As shown in Figure 12, after irradiating the PDA-ICG nanoparticles with laser at concentrations of 0, 50, 100, 150, and 200 μg/mL for 5 minutes, the temperature of the nanoparticle aqueous dispersion increased by 0.3, 18.8, 20.2, 22.5, and 23.5 ℃. It can be seen that as the concentration of PDA-ICG nanoparticles increases, the temperature rise of the nanoparticles also increases, indicating that the photothermal performance of PDA-ICG is concentration-dependent.

(2) Evaluation of the photothermal performance of PDA, ICG, and PDA-ICG in vitro

Figure12 Temperature increase of ICG, PDA, and PDA-ICG nanoparticles under 808 nm laser irradiation for 5 minutes

Figure13 Representative real-time thermal imaging of ICG, PDA, and PDA-ICG nanoparticles

Record the temperature rise of the samples using a thermal imager (Figure 14), comparing the temperature changes of ICG, PDA, and PDA-ICG nanoparticle aqueous solutions. After 5 minutes of laser irradiation, their temperatures increased by 20.4 ℃、23.4 ℃ and 23.9 ℃ respectively. Among them, the highest temperature of PDA-ICG nanoparticles reached 47.8 ℃, which can induce thermal ablation of tumor cells^[10]. The heating effect of PDA-ICG is slightly higher than that of PDA and ICG, which is due to both PDA and ICG being good photothermal materials, and their combination gives PDA-ICG better photothermal performance^[11].

Determination of the photothermal stability of PDA-ICG nanoparticles

Figure14 Temperature change curve of PDA-ICG nanoparticles after continuous three times of light exposure

Asshown in Figure 15, the trend of the curve's three heating and cooling cycles is almost consistent. That is, under three consecutive laser irradiations, the heating and cooling behavior of the PDA-ICG nanoparticles is similar, and the highest temperature reached is close. After three heating and cooling cycles, the PDA-ICG nanoparticles still maintain good photothermal performance, indicating that they have good photothermal stability.

(4) Measurement of the photothermal conversion efficiency of PDA and PDA-ICG nanoparticles

Figure15 Temperature changes of PDA and PDA-ICG nanoparticles after laser irradiation for 5 min and cooling for 10 min

Figure16 (a) PDA nanoparticles and (b) PDA-ICG nanoparticles' cooling time and the linear correlation curve of -ln(θ) after 5 minutes of laser irradiation

The environmental temperature during the test was 19.8 ℃. According to the previous formula, the photothermal conversion efficiencies of PDA and PDA-ICG nanoparticles were calculated through the time against-ln(θ) linear fitting (Figure 17), resulting in the curves Y = 262.2X - 20.1 (R²=0.9916) and Y = 242X - 48.1 (R²=0.9902), indicating a good linear relationship, and the values of τ_s were 262.2 and 242, respectively. The photothermal conversion efficiency of PDA was calculated to be 17.85%, and that of PDA-ICG was 21.95%, indicating that the photothermal performance of PDA was enhanced after loading ICG.

3.3.6Release of ICG

Figure17 Release curve of ICG from PDA-ICG nanoparticles

Asshown in Figure 10,in a PBS solution (pH 7.4) at 37 °C,ICG is released slowly, with a cumulative release rate of 6.53% after 24 hours.Under the same conditions, when PDA-ICG nanoparticles are irradiated with 808 nm laser (1.5 W/cm²) for 5 minutes at 2 hours and 5 hours, it can be observed that the release rate of ICG rapidly increases after laser irradiation, and after stopping the light, the release rate gradually slows down, with the cumulative release rate increasing to 10.84% after 24 hours, indicating that the release of ICG is light-responsive.

3.2.7Evaluation of Photodynamic Performance

Figure18 Generation of superoxide anions from different samples after 5 minutes of 808 nm laser irradiation (ns representsP＞0.05, **** representsP＜0.0001)

AsFigure 18, after 5 minutes of laser irradiation, the UV-visible absorbance values of PBS, PDA, ICG, and PDA-ICG at 530 nm increased by 0.0097±0.0004, 0.0277±0.029, 0.2677±0.033, and 0.192±0.014, respectively. The small amount of superoxide anions produced in the PDA and PBS groups may be due to the direct absorption of laser energy by oxygen in the air. The production of superoxide anions in the PDA-ICG and ICG groups was significantly higher than that in the PDA and PBS groups, approximately 7-27 times greater, indicating that PDA-ICG and ICG have good photodynamic performance.

3.4Conclusion

In this chapter, we used various methods such as UV spectroscopy, infrared spectroscopy, particle size and Zeta potential measurement, infrared thermal imaging, and superoxide anion reagent kits to characterize the nanoparticles. With the step-by-step preparation of the nanoparticles, changes in the color of their aqueous solution were observed. The characteristic peaks from UV and infrared spectroscopy, the change of the nanoparticles' potential from negative to positive, and the different colors of the nanoparticles' aqueous solution all demonstrate the successful preparation of PDA, PDA-ICG, and PDA-ICG@PEI/siPKM2 nanoparticles. The results of particle size and Zeta potential show that the particle size of the nanoparticles is around 150-180 nm, which is suitable and uniform, and the absolute value of the Zeta potential is about 50 mV, indicating a stable system. The light-responsive release of ICG was detected. In the evaluation of photothermal performance, the photothermal conversion efficiency of PDA-ICG nanoparticles was 21.95%. Since the subsequently loaded PEI and siPKM2 do not have photothermal effects, they do not affect the photothermal performance of the nanoparticles, thus both the obtained PDA-ICG and PDA-ICG@PEI/siPKM2 nanoparticles exhibit good photothermal performance. The detection results of superoxide anion generation indicate that ICG, PDA-ICG, and the subsequently prepared PDA-ICG@PEI/siPKM2 nanoparticles all possess good photodynamic performance.