Introduction

The tumor microenvironment (TME) is a complex network of cancer-associated cells and non-cellular components [1]. The characteristics of the TME, such as redox state, reactive oxygen species, low pH, tumor-associated receptors, etc. provide promising triggers for the stimulus response feature used in cancer diagnosis and therapy [2, 2(b). In the swollen state (25 ℃, pH = 7.4), the diameter of PNIPAM-VAA shells was 220 nm. However, the diameter is about 150 nm in the state of collapse (50 ℃, pH = 5.8), and the volume of PNIPAM-VAA shells is reduced by about 68.30% after phase transformation. In addition, TEM images show that the single core-shell has a spherical shape and good dispersion. The hydrodynamic diameter and size distribution of AuNR@PNIPAM-VAA nanogel were measured by DLS technology. The results are shown in Fig. 2(c). The length of AuNR is about 130 nm, diameter is about 16 nm, and transverse diameter ratio is about 8, which is consistent with the TEM result. The hydrodynamic radius of AuNR@PNIPAM-VAA nanogel measured at collapse (50 ℃, pH = 5.8) and swelling (25 ℃, pH = 7.4) is about 406 nm and 452 nm, respectively. The changes tendency of hydrated particle size is consistent with TEM images, but the hydrated particle size is significantly larger than that of TEM results. This is because AuNR@PNIPAM-VAA nanogel in the measured hydrated particle size state contain a higher proportion of water. The results showed that the PNIPAM-VAA shell existed in a large swelling state in normal tissue at room temperature, and the swelling-collapse phase transition occurred with the increase of temperature in response to the low pH. Secondly, the variation of AuNR surface potential during the wrap** of PNIPAM-VAA shell was measured as shown in Fig. 2(d). The zeta potential of AuNR is about 39.48 mV, which decreases to 30.91 mV after VAA functionalization and − 21.97 mV after PNIPAM-VAA shell encapsulation, confirming the successful preparation of AuNR@PNIPAM-VAA core-shell structure.

In order to verify that AuNR@PNIPAM-VAA nanogel can only undergo thermal-sensitive phase transition at low pH in TME, the absorption spectra of AuNR@PNIPAM-VAA nanogel at temperature ranging from 34 ℃ to 46 ℃ were measured at pH 5.8 as simulated tumor extracellular matrix and at pH 7.4 as normal physiological environment, as shown in Fig. 2(e). The results show that the absorption spectrum of AuNR@PNIPAM-VAA nanogel remains unchanged before and after temperature change at pH 7.4. On the contrary, at pH 5.8, the longitudinal plasmon resonance absorption peak of AuNR@PNIPAM-VAA nanogel redshifts from 919 nm at 38 ℃ to 949 nm at 46 ℃ with increasing temperature in the range of 34 to 46 ℃. Figure 2(f) shows that the light absorption at 1064 nm of AuNR@PNIPAM-VAA nanogel at pH 5.8 is enhanced with increasing temperature. At the same time, quantitative calculation shows that the light absorption at 1064 nm was enhanced by 88.86% at pH 5.8. Figure 2(g) shows the relationship at pH 5.8 between temperature and light absorption of AuNR@PNIPAM-VAA nanogel at 1064 nm. VPTT is determined to be about 45 ℃ by calculating the first derivative of curve, and the enhancement rate of light absorption of AuNR@PNIPAM-VAA nanogel changes the fastest with temperature. To investigate the reversibility of thermal-sensitive phase transitions triggered by low pH of AuNR@PNIPAM-VAA nanogel, the light absorption changes at 1064 nm were measured at pH 5.8 during five consecutive heat-cooling cycles (from 30 °C to 60 °C) at temperatures above and below the midpoint of the VPTT. As shown in Fig. 2(h), periodic changes in light absorption are observed, demonstrating that AuNR@PNIPAM-VAA nanogel possesses the sustainability and repeatability of the phase transition, and the stability of the thermal-response. These results show that the thermal-sensitive phase transition of AuNR@PNIPAM-VAA nanogel is pH-dependent and highly reversible at low pH as in TME.

Fig. 2
figure 2

Characterization of AuNR@PNIPAM-VAA nanogel. (a) HAADF and element map** images; (b) TEM image of the thermal-response size and morphology; (c) DLS analysis and (d) Zeta potential of AuNR, AuNR@PNIPAM-VAA ; (e) Absorption spectra and (f) quantitative absorption intensity at 1064 nm; (g) Derivative absorption intensity of AuNR@PNIPAM nanogel with temperature at pH 5.8; (h) Reversibility of light absorption at 1064 nm during 30 °C to 60 °C heating and cooling cycle

Full size image

Photothermal properties of AuNR@PNIPAM-VAA nanogel

The photothermal conversion of AuNR@PNIPAM-VAA nanogel in vitro was investigated due to the presence of AuNR nuclei with light absorption. As shown in Fig. 3(a), the temperature of the two groups AuNR@PNIPAM-VAA increased rapidly, and the temperature tended to equilibrium when the irradiation time reached 10 min. When the irradiation time reaches 30 min, the temperature of the group at pH 5.8 reaches 55.1 ℃, and the group at pH 7.4 is up to 53.6 ℃. Then, the temperature rise characteristics of AuNR@PNIPAM nanogel were measured under 980 nm CW laser irradiation with different optical power densities. Figure 3(b) shows that the higher laser power resulted in faster heating rate and higher final temperature. When the laser power reaches 0.75 W/cm2, the AuNR@PNIPAM-VAA solution temperature of 1 mg/mL AuNR@PNIPAM nanogel rapidly rises to about 52.5 ℃ in a short time (10 min). As shown in Fig. 3(c), the maximum temperature of AuNR@PNIPAM-VAA aqueous solution during six laser irradiation cycles (0.75 W/cm2, 10 min) is always about 53.3 ℃, and the peak shape has no significant change, indicating that these AuNR@PNIPAM-VAA nanogel has good photothermal stability. These results show that AuNR@PNIPAM-VAA nanogel has high efficiency and stable photothermal conversion, which is important for the in vivo application.

The photothermal effect of the AuNR@PNIPAM-VAA nanogel in vivo was monitored using an infrared thermal imager, showing a pH-dependent photothermal behavior. Two groups of AuNR@PNIPAM-VAA nanogel buffer (1 mg/mL) at pH 7.4 and 5.8 were injected into the subcutaneous tissue of normal mice respectively, and then real-time imaging was performed. Figure 3(d) records the photothermal images under irradiation with a laser power density of 0.5 W/cm2. As shown in the figure, the temperature of the simulated normal tissue area (pH = 7.4) in the control group increased rapidly with the extension of laser irradiation time. Meanwhile, the local temperature of the simulated tumor region (pH = 5.8) injected with the nanogel increased more significantly with irradiation time, while no significant temperature rise was observed in other body parts of the mice. Figure 3(e) statistically analyzes the changes of temperature rise in the area injected with AuNR@PNIPAM-VAA nanogel with irradiation time. Compared with the temperature rise in the simulated tumor area (pH = 5.8), the temperature rose rapidly to about 47.7 ℃, and the temperature in the simulated normal tissue area (pH = 7.4) finally stabilized at about 45.7 ℃. All of above results confirmed that AuNR@PNIPAM-VAA nanogel has excellent photothermal properties which can be dynamically modulated by laser irradiation time and power, and shows great potential to be the “switch” for transferring light to heat.

Fig. 3
figure 3

Photothermal conversion performance of AuNR@PNIPAM-VAA nanogel. (a) Temperature rise curve as a function of time; (b) Temperature changes under different power density laser irradiation; (c) Photothermal stability under cyclic irradiation; (d) Photothermal imaging and (e) real-time temperature rise curve of mouse subcutaneous tissues under irradiation after injection of AuNR@PNIPAM-VAA nanogel

Full size image

Photoacoustic properties of AuNR@PNIPAM-VAA nanogel

Based on the above pH-triggered thermal property of AuNR@PNIPAM-VAA nanogel, Fig. 4(a) records the relationship between the photoacoustic signal amplitude at different pH and temperature. The results show that under neutral conditions (pH = 7.4), photoacoustic signal has hardly change with increasing temperature. Under acidic conditions (pH < 6), photoacoustic signal increases with increasing temperature; and with decreasing pH, the enhancement trend of photoacoustic signal becomes more and more significant. Figure 4(b) and 4(c) describe the statistical photoacoustic signal intensity of AuNR@PNIPAM-VAA nanogel with different pH value at 45 ℃ and 37.5 ℃, respectively. The insert were the corresponding images acquired by PAM at 1064 nm. The photoacoustic signal amplitude is inert to pH near the physiological temperature, and increases significantly with the decrease of pH near the hyperthermia temperature (45 ℃). In order to illustrate the synergistic effect of thermal and pH on PAI, AuNR@PNIPAM-VAA solution at different temperature and pH levels were imaged by a 1064 nm by PAM system. Figure 4(d) is the corresponding sample photo, and the white dotted line box indicates the imaging area. Point-by-point scanning in the x-y plane (C-scan) was performed, and the maximum value of the photoacoustic signal at each position is projected to obtain the photoacoustic image in the x-y plane as shown in Fig. 4(e). It can be clearly seen that the photoacoustic signal intensity of AuNR@PNIPAM-VAA nanogel increases with the increase of temperature under acidic conditions, but remains basically unchanged at pH 7.4. Figure 4(f) is the 45 °C and 37.5 °C differential image of Fig. 4(e), where AuNR@PNIPAM-VAA nanogel under acidic conditions are clearly identified owing to the enhancement of AuNR@PNIPAM-VAA photoacoustic signal under the synergistic effect of thermal and pH. The signal intensity of the sample in Fig. 4(e) was quantified and statistically analyzed to get Fig. 4(g). The results show that with the decrease of pH, the thermal induction enhancement effect of photoacoustic signal amplitude becomes more obvious. As shown in Fig. 4(h), when the sample is heated from 37.5 ℃ to 45 ℃, the photoacoustic signal amplitude is enhanced by 60% at pH 5.8 and slightly by 19% at pH 7.4. In summary, the photoacoustic signal of AuNR@PNIPAM-VAA nanogel shows a significant pH-dependent thermal-trigged enhancement in the pH range of 5.8 to 7.4, indicating that the nanogel can target tumor stroma at low pH.

To further verify that NIR light-switching stimulation (980 nm CW laser) can be used to regulate the thermal switch of the thermal-sensitive phase transition in the TME, PAI of simulated tissue samples on AuNR@PNIPAM-VAA nanogel were performed as shown in Fig. 4(i). Three polyethylene tubes were injected with 1 mg/mL AuNR@PNIPAM-VAA nanogel solution with different pH values. Two tubes with pH levels of 7.4 and 5.8 were irradiated with 980 nm CW laser (0.5 W/cm2) for 0, 5, and 10 min. Meanwhile, and the third tube containing AuNR@PNIPAM-VAA nanogel solution at pH 5.8 without light irradiation was used as a control. Point-by-point scanning in the x-axis direction (B-scan) and the photoacoustic signals in the z-axis direction at each position are projected to obtain x-z plane photoacoustic images at different exposure times were obtained as shown in Fig. 4(j). The photoacoustic signal of the nanogel solution with pH = 5.8 increased significantly with the increase of irradiation time under irradiation, however, the photoacoustic signal of the AuNR@PNIPAM-VAA nanogel buffer solution with pH 7.4 increased slightly. The photoacoustic signal intensity of the sample in Fig. 4(j) was quantified and statistically analyzed to obtain Fig. 4(k). The photoacoustic signal of AuNR@PNIPAM-VAA nanogel was enhanced by 37% in the simulated tumor region (pH = 5.8) after 10 min of laser irradiation, and the enhancement amplitude was only 5% in the simulated normal tissue region (pH = 7.4). The above results verified that the AuNR@PNIPAM-VAA nanogel has a pH-responsive photothermal conversion ability, and NIR light (980 nm) can be used to modulate the thermal field to trigger the thermal-sensitive phase transition.

Fig. 4
figure 4

Photoacoustic properties of AuNR@PNIPAM-VAA nanogel. (a) Photoacoustic signal amplitudes; (b) and (c) Statistics signal amplitudes of AuNR@PNIPAM-VAA nanogel at different pH values at 45 ℃ and 37.5 ℃, respectively; (d) Photograph and (e) photoacoustic images in x-y plane of AuNR@PNIPAM-VAA nanogel and AuNR samples; (f) Differential photoacoustic images basing on the results at 45 °C and 37.5 °C in Fig. 4(e); (g) Statistic photoacoustic signal amplitude varied with temperature in Fig. 4(e); (h) Quantitative enhancement of photoacoustic signal; (i) Schematic diagram of AuNR@PNIPAM-VAA nanogel at pH 7.4 and pH 5.8; (j) Photoacoustic image in x-z plane at different exposure time; (k) Quantitative enhancement of photoacoustic signal after the irradiation of 980 nm CW laser (0.5 W/cm2)

Full size image

In vivo dynamic enhanced photoacoustic imaging of tumor

To verify the possibility of AuNR@PNIPAM-VAA nanogel to distinguish the normal and tumor tissue, a mouse model simulating the TME was constructed and imaged, and the specific protocol is shown in Fig. 5(a). 1 mg/mL of AuNR@PNIPAM-VAA nanogel was prepared in PBS buffer that mimics TME (pH = 5.8) and normal physiology (pH = 7.4), respectively. The nanogel was injected into the subcutaneous tissue of the normal mice back, and irradiated by a 980 nm CW laser (0.5 W/cm2). The x-z plane point scan was performed on the area of interest to obtain the photoacoustic image in Fig. 5(b). The photoacoustic image intensity of the AuNR@PNIPAM-VAA nanogel in the simulated tumor region (pH = 5.8) gradually increased with the increase of irradiation time, while the photoacoustic image intensity in the simulated normal tissue region (pH = 7.4) remained almost constant. In order to quantitatively compare the differences, we depicted the maximum projection curve of photoacoustic signal under different 980 nm laser irradiation time as shown in Fig. 5(c). The results showed that the photoacoustic signal in the simulated tumor area was enhanced by 35.5% after 15 min of exposure. The above results demonstrated the possibility that the prepared AuNR@PNIPAM-VAA nanogel can achieve tumor-specific high-contrast imaging by utilizing a thermal-sensitive phase transition triggered in response to the low pH in TME.

Photoacoustic + ultrasound image of normal tissue and tumor was presented in Fig. 5(d). Figure 5(e) shows the statistical analysis of the photoacoustic signal amplitude after different laser irradiation time. Results show that photoacoustic signal in the tumor region increased after laser irradiation, while that in the normal tissue presents limited differences. All of these can be ascribed to the pH dependence of AuNR@PNIPAM-VAA nanogel. Point-by-point photoacoustic scanning along the dashed line was shown in Fig. 5(f). Fig.  5(g) shows the superposition of ultrasound image and differential photoacoustic image of normal tissues and tumors after injection of AuNR@PNIPAM-VAA nanogel. The results showed that the enhanced photoacoustic intensity in the tumor region increased significantly with the increase of irradiation time, while that of the normal tissue remained almost unchanged. Figure 5(h) shows the quantified statistical analysis of the enhanced photoacoustic signal. After exposed to 980 nm CW laser with 15 min (0.5 W/cm2), the enhanced photoacoustic amplitude increased by 42% compared to that with 7 min laser irradiation in the tumor region. However, the photoacoustic amplitude of the subcutaneously injected AuNR@PNIPAM-VAA nanogel in the control group has slightly change. Thus, AuNR@PNIPAM-VAA exhibits strong enhanced photoacoustic properties in acidic TME based on its thermal-triggered NIR-II light absorption enhancement. These results indicate that the prepared AuNR@PNIPAM-VAA nanogel can achieve tumor-specific high-contrast PAI by dynamically adjusting the laser irradiation.

Fig. 5
figure 5

In vivo dynamic enhanced photoacoustic effect of the AuNR@PNIPAM-VAA nanogel for tumor imaging. (a) Schematic PAI of subcutaneous tissue after injection of AuNR@PNIPAM-VAA nanogel; (b) Superposition of photoacoustic and ultrasound images of subcutaneous tissues of normal mice injected with AuNR@PNIPAM-VAA nanogel; (c) Peak-to-peak projection curve of photoacoustic signal amplitude of subcutaneous tissue of normal mice in Fig. 5(b); (d) photoacoustic + ultrasound image of normal tissue and tumor; (e) Photoacoustic signal amplitude and (f) Schematic PAI of mice after injection of AuNR@PNIPAM-VAA nanogel; (g) Superposition of ultrasound image and differential photoacoustic image and (h) enhanced photoacoustic signals of normal tissue and tumor

Full size image

In vivo toxicity assessment

The pathological structure of the major organs of mice injected with PBS and AuNR@PNIPAM-VAA nanogel for 7 and 14 days was evaluated as shown in Fig. 6(a). The AuNR@PNIPAM-VAA nanogel did not damage these major organs, such as heart, liver, spleen, lung and kidney. This was followed by intravenous injection of AuNR@PNIPAM-VAA nanogel in three mice, and then serum biochemical and hematological parameters were systematically studied at 7 and 14 days. The results in Fig. 6(b) showed that there were no obvious abnormal changes in the test group compared with the PBS group, indicating that these AuNR@PNIPAM-VAA nanogels did not cause obvious infection and inflammatory response in the mouse model. The above results indicated that the toxic side effects of AuNR@PNIPAM-VAA nanogel were negligible.

Fig. 6
figure 6

(a) H&E staining of different organs, (b) Serum biochemical and hematological analysis of healthy mice after injection of PBS and AuNR@PNIPAM-VAA nanogel for 7 days and 14 days

Full size image

Conclusions

In conclusion, a volume phase-transition photoacoustic nanogel AuNR@PNIPAM-VAA has been successfully fabricated for tumor-specific high-contrast imaging. VAA monomer was introduced into PNIPAM chain to make the nanogel with pH responsive function. By adjusting the proportion of VAA monomers, PNIPAM-VAA shell obtained the pH-dependence required for thermal-sensitive volume phase transitions to target acid TME. As the temperature exceeded VPTT, the developed AuNR@PNIPAM-VAA nanogel underwent a solgel phase transition in TME and further resulted in enhanced NIR-II light absorption due to the change of nanogel’ refractive index. Based on the pH-dependence of PNIPAM-VAA thermal-sensitive phase transition, AuNR@PNIPAM-VAA nanogel can target TME at low pH and exhibit strong and switchable NIR-II light absorption under NIR light stimulation (980 nm), thereof AuNR@PNIPAM-VAA nanogel in the tumor region exhibited enhanced photoacoustic signal in the NIR-II window. In addition, the nanogel showed good biocompatibility, and no side effects were observed both in vitro and in vivo tests. However, it should be noted that the imaging wavelength is not the absorption peak of AuNR@PNIPAM-VAA, in which the nanoprobe produces the maximum photoacoustic efficiency. In further studies, we commit to develop new materials with strong absorption in the NIR-II region to achieve deeper and more sensitive PAI. In a word, a method of tumor-specific PAI using NIR light to regulate the thermal field and target the low pH TME is proposed, which is expected to realize accurate and dynamic monitoring of tumor diagnosis and treatment.