Figure 2. (a) Chemical structure of DOX-hydrazone linker conjugate (DOX-PDPH). Scheme of renal-clearable Au-DOX, which consists of the gold core, surface coating with poly (ethylene glycol) thiol (mPEG-SH, MW: 5000 Da) and acid sensitive linker (PDPH), and loaded DOX molecules. (b) HR-TEM image and size distribution of Au-DOX. The scale bar is 10 nm. (c) UV-Vis absorbance spectra of Au-PEG, DOX, DOX-PDPH and Au-DOX in aqueous solution. (d) Fluorescence emission spectra of Au-DOX and DOX at the same concentrations.
Preparation and characterization of Au-DOX
The Au-PEG nanoparticles (AuNPs) were synthesized following a reported method [25]. DOX was connected with a 3-[2-Pyridyldithio]propionyl hydrazide (PDPH crosslinker) [26] (Figure S1c), which not only can form a pH-responsive hydrazone bond with the carbonyl group of DOX, but also can introduce a thiol functional group to conjugate DOX with AuNPs (Fig. 2a) [27]. The uniform size distribution of Au-DOX nanoparticles was confirmed by high-resolution transmission electron microscopy (HR-TEM) imaging, with a particle size of 1.8 ± 0.2 nm (Fig. 2b). The absorption spectrum of Au-DOX exhibited a characteristic absorbance of DOX at 488 nm, indicating the successful conjugation of DOX onto AuNPs [28]. Notably, the loaded DOX showed an 11 nm redshift (from 488 to 499 nm) compared to free DOX, suggesting the formation of J-aggregates of DOX after conjugation onto AuNPs, which was resulted from dipole-dipole interactions among the loaded DOX molecules (Fig. 2c). Moreover, the fluorescence intensity of DOX decreased about 9 times due to the fluorescence resonance energy transfer (FRET) process between DOX and AuNPs (Fig. 2d) [29]. The successful conjugation of DOX onto AuNPs was also confirmed by agarose gel electrophoresis. The gel mobility of Au-DOX was in between that of pure DOX and Au-PEG (Figure S1a), and the fluorescence imaging of the agarose gel showed a similar mobility behavior (Figure S1b).
Figure 3. (a) The illustration of the acidic pH response of Au-DOX. (b) The fluorescence intensity changes of Au-DOX at pH 5.5, 6.5, and 7.4 in 31 h. (c) Time-dependent DOX release from Au-DOX at different pH values. (d) The illustration of the GSH response of Au-DOX. (e) The change of fluorescence intensity of Au-DOX in 31h after adding 5 mM GSH. (f) Time-dependent DOX release from Au-DOX in 5 mM GSH.
Drug release characteristics triggered by pH and GSH in vitro
Cell cytosol contains high concentrations of GSH (1–10 mM) [30, 31], and the cell endocytic vesicles were acidic with pH as low as 4.5 [32]. To evaluate the pH sensitivity of Au-DOX, the fluorescence intensity of the NPs at different pH values were tested. Three pH values were selected to monitor the pH of blood (pH 7.4), the tumor microenvironment (pH 6.5), and lysosomes (pH 5.5). As expected, the cleavage of hydrazone bond at acidic environment resulted in the release of DOX from Au-DOX, leading to an increase in fluorescence intensity (Fig. 3a). Within 31 h, the fluorescence intensity increased by factors of 3.9, 4.9, and 7 at pH 7.4, 6.5, and 5.5, respectively (Fig. 3b, c, Figure S2a-c). The result indicated that the pH responsive property would facilitate the release of DOX in the tumor acidic environment [33]. Since GSH can displace the ligands of gold nanoparticle through Au-S bond [34, 35], DOX can effectively release from Au nanoparticle surface in the presence of GSH (Fig. 3d). To assess the thiol activation property of Au-DOX, the NPs were incubated in PBS solution containing 5 mM GSH. As the incubation time elapsed, the fluorescence of Au-DOX increased, as revealed by Figure S2d. The fluorescence intensity at the maximum emission wavelength (594 nm) increased by a factor of 8.9 times gradually after 31h incubation with GSH (Fig. 3e, f).
Compromised antitumor efficacy of the drug delivery system
The antitumor efficacy of Au-DOX was evaluated by Female Balb/c mice with 4T1 tumors. Following six treatments of DOX and Au-DOX, respectively, the organs (heart, spleen, lungs, kidneys, liver) and tumors were collected from the Balb/c mice. The fluorescence images showed that (Fig. 4a) the liver fluorescence was much stronger than the other organs for the mice injected with DOX, indicating that DOX were accumulated in liver after intravenous injection. While for the mice injected with Au-DOX, the fluorescence of the tumor area was strong, indicating that the drug accumulated in the tumor through EPR effect after intravenous injection. Quantitative analysis of the fluorescence intensity of the organs and tumors (Fig. 4b) showed that the liver fluorescence intensity of the mice injected with DOX was 5 times higher than the mice injected with Au-DOX, and lung was 1.5 times higher, while the tumor fluorescence intensity of the mice injected with Au-DOX was 2 times higher than the mice injected with DOX, indicating the renal clearable DDSs can enhance the clearance of DOX from the reticuloendothelial system (RES). H&E images of tumor tissue (Figure S3) revealed extensive necrosis with unstructured eosinophilic material and numerous necrotic cell debris (red arrows) surrounding the necrotic area in both the DOX and Au-DOX groups, confirming the effective therapeutic efficacy of Au-DOX. Interestingly, the fluorescence intensity of the liver collected from the mice 6 times treatment of Au-DOX was 12.26 times lower than the liver collected 24 h after treatment of Au-DOX (Figure S4), further confirmed the efficient clearance ability of the renal clearable DDSs [36, 37]. Analysis of the tumor growth curve (Fig. 4c and S5) revealed a significant inhibition of tumor growth in the Au-DOX treatment group compared to the PBS group when administered once every three days, with the tumor volume and weight approximately half that of the PBS treatment group. However, even though Au-DOX showed higher accumulation at the tumor site than free DOX, it did not exhibit superior antitumor effects, the therapeutic effect of the Au-DOX was not as good as free DOX at the same dosage (5 mg/kg body weight), and the tumor volume was nearly twice that of the DOX group. Interestingly, when the Au-DOX injection frequency was increased to once every two days, a similar therapeutic effect was achieved as with free DOX group administered once every three days, the size and weight of these two groups were comparable (Figs. 4d and 4e). To evaluate the possible influence of the antitumor effect of Au-PEG, the Au-PEG and PBS (comparable AuNPs dosage to Au-DOX treatment) were continuously injected into 4T1 tumor-bearing mice for 22 days. As shown in Figure S6a, S6b, and S6c, no significant differences were found in tumor volume growth rate, size, and weight between these two groups, and the Au-PEG treatment group did not exhibit significant tumor injury compared to the PBS group. These findings indicated that the vector Au-PEG did not interfere with the tumor suppression study.
Figure 5. (a) Schematic diagram of lysosome escape. (b) Cellular uptake of DOX and Au-DOX within 2 hours. (c) Fluorescence intensity of DOX and Au-DOX in nucleus and lysosomes. (d) Fluorescence images of Hela cells for intracellular tracking of Au-DOX. scale bar = 5 µm. (e) The cell viability of Au-DOX and DOX towards Hela cells for 24 h. Data points represent mean ± SD (n = 3).
Lysosomal barrier to the drug delivery system
To unveil the origin of the reduced therapeutic efficiency of Au-DOX, intracellular tracking and drug release studies were conducted. After 2h incubation, the small molecule DOX diffused rapidly within the cell, primarily accumulated in the nucleus due to the concentration gradient driving force (Fig. 5b and Figure S7). While for the cells incubated with Au-DOX, a significant fluorescence overlap was observed between the red fluorescence of DOX and the green fluorescence of LysoTracker, indicating the endocytosis and lysosomal localization of the NPs. The fluorescence intensity of Au-DOX and DOX in the lysosome and nucleus were further analysed. As demonstrated in Fig. 5c, the fluorescence intensity of DOX group in the nucleus was 1.6 times higher than that in the lysosome, whereas the fluorescence intensity of Au-DOX group in the lysosome was 3.4 times higher than that in the nucleus. To study whether Au-DOX can evade lysosomal entrapment, the incubation period was extended to 48 hours (Fig. 5d, figure S7) with Hela cells and 4T1 cell, respectively. The result showed that Au-DOX was still remained in the lysosomes. Since Au-DOX being trapped in the lysosomes, the cytotoxicity of Au-DOX within 24 hours was slightly lower compared to an equivalent amount of free DOX (Fig. 5e) [38]. These results indicated that although renal-clearable gold nanoparticles can effectively release drugs in the presence of acid and GSH, it was worth noting that AuNPs without DOX loading exhibited minimal toxicity to Hela cancer cells (Figure S8), but they face challenges in evading lysosomal entrapment in vivo, as illustrated in diagram 5a, thereby limiting their therapeutic efficacy [39].
Reduced toxicity of the drug delivery system
One advantage of Renal clearable DDSs was reduced toxicity [40]. After six injections over a 15 days period (administered every three days) (Fig. 6a), the mice in the DOX group exhibited a weight reduction of 10%, whereas no significant weight loss was observed in the Au-DOX group, no matter whether the drug was administered every two or three days. This observation highlights the biocompatibility and low toxicity of Au-DOX (Figure S6d). Further study showed that Au-DOX exhibited improved renal clearance efficiency compared to DOX (Figure S9a, b, c, d). As depicted in Fig. 6b, the renal clearance rate of Au-DOX within 24 hours was 47%, whereas that of DOX was only 11%. The utilization of renal clearable AuNPs considerably enhanced the renal clearance efficiency while concurrently reducing kidney injury [41]. Au-DOX treatment blood chemistry tests showed that the blood urea nitrogen (BUN) and creatinine (CREA) levels were all within the normal range (Fig. 6c and Figure S9e), indicating the favourable biosafety profile of the Au-DOX. H&E images revealed that the free DOX group induced severe kidney damage, whereas no obvious damage was observed in the Au-DOX group (Fig. 6d). It was worth noting that the primary elimination pathway for free DOX was the liver, which usually leads to liver injury in approximately 40% of patients. Fluorescence imaging of organs demonstrated a higher accumulation of free DOX in the liver compared to Au-DOX. Further examination via H&E staining revealed that the DOX group caused substantial liver injury, as well as damage to other reticuloendothelial system organs such as the spleen and lungs (Fig. 6d, Figure S3). Conversely, no evident damage was observed in the Au-DOX group.