Characterization of the mPEI-CTX-99mTc/DOX complex
The theranostic complexes and the intermediate products obtained during the preparation process were characterized using different techniques. Firstly, 1H nuclear magnetic resonance (NMR) spectroscopy was used to characterize the intermediate products including PEI.NH2-mPEG, PEI.NH2-(PEG-MAL)-mPEG, PEI.NH2-(PEG-CTX)-mPEG, PEI.NH2-DTPA-(PEG-MAL)-mPEG, and PEI.NH2-DTPA-(PEG-CTX)-mPEG. As shown in Fig. S1a and S1b, the peaks at 3.5–3.7 ppm could be assigned to the aromatic protons of PEG, while the peaks at 1.0–1.5 ppm were attributed to the protons of CTX (Fig. 1c). By NMR integration analysis, each PEI was estimated to have 13.3 mPEG, 14.2 PEG-MAL, and 5.4 CTX moieties. Likewise, the average number of DTPA moieties (at 3.8 ppm) attached onto each PEI was measured to be approximately 7.5 and 7.2, as shown in Fig. S1d and S1e, respectively.
Secondly, after acetylation of the PEI.NH2-DTPA-(PEG-CTX)-mPEG and PEI.NH2-DTPA-(PEG-MAL)-mPEG, the mPEG-CTX and mPEG were formed, and then was used for the encapsulation of DOX to synthesize the mPEI-CTX/DOX and mPEI/DOX complexes, which could be easily dissolved in different solvents such as water and cell culture media (Fig. S2a-f). Subsequently, UV-vis spectroscopy was performed to confirm the loading of DOX. As shown in Fig. 2a, the mPEI-CTX/DOX complex showed an enhanced absorption at 490 nm, which was related to the typical absorption peak of DOX in the UV-vis spectra, while no absorption at this wavelength could be observed for the intermediate products PEI.NH2-(PEG-CTX)-mPEG and PEI.NH2-DTPA-(PEG-CTX)-mPEG without DOX. The amount of DOX loaded within mPEI-CTX/DOX complex was calculated to be 20.07 DOX molecules per PEI and the DOX percentage reached 7.02%, which was calculated and analyzed via the standard DOX absorbance/concentration calibration curve (Fig. S2g-i). Similar results were found for the mPEI/DOX complex. Into each PEI, 19.70 DOX molecules were encapsulated, and the DOX percentage was calculated to be 6.99%. Meanwhile, the hydrodynamic size and zeta potential value of mPEI-CTX/DOX complex were measured by dynamic light scattering. As shown in Table S1 and Fig. S3a-c, the hydrodynamic sizes of both mPEI-CTX/DOX and mPEI/DOX complexes had relatively uniform distributions and were larger than that of mPEI-CTX before DOX loading, which reflected the success of DOX loading. As shown in Table S2, the zeta potentials of PEI.NH2-DTPA-(PEG-CTX)-mPEG and mPEI-CTX/DOX showed no significant difference under different pH values, suggesting that the DOX loading did not obviously change the surface potentials of the complexes. Furthermore, the surface potentials of both mPEI/DOX and mPEI-CTX/DOX complexes under a slightly acidic environment (pH = 5.0) were more positive than those under physiological condition (pH = 7.4). This was likely due to the protonation of partial PEI tertiary amines under a slightly acidic environment (pH = 5.0), as observed in previous studies [46-48].
Thirdly, the release kinetics of DOX from the mPEI-CTX/DOX complex were analyzed under two different pH conditions (Fig. 2b). We found that DOX release occurred more rapidly in the initial phase than in the latter, which is in good agreement with that reported previously [45]. At 48 h, the DOX release percentage could be achieved at 26.6% (pH = 5.0) and 22.9% (pH = 7.4), respectively.
Finally, mPEI-CTX/DOX was effectively radiolabeled with 99mTc via DTPA. Instant thin-layer chromatography (ITLC) was used to assess the radiochemical yields (RCYs) and stabilities of the 99mTc-labeled PEI-based NPs. The RCYs of mPEI-CTX-99mTc/DOX and mPEI-99mTc/DOX were found to be 80.3 ± 2.8% and 78.8 ± 0.9% (n = 5), respectively. After PD-10 column purification, the radiochemical purities of both mPEI-CTX-99mTc/DOX and mPEI-99mTc/DOX were over 99% (Fig. S3d-f), and remained above 95% after 12 h in phosphate buffered saline (PBS) at room temperature (Fig. S4a), indicating excellent stabilities in vitro.
In vitro cytotoxicity assays
CCK-8 assay was used to test the cytocompatibility of mPEI-CTX without DOX encapsulation and evaluate the therapeutic efficacy of the mPEI-CTX/DOX complex against C6 cells in vitro. As shown in Fig. S4b, mPEI and mPEI-CTX displayed little cytotoxicity, and the viabilities of C6 cells after treatment remained more than 90% for all the studied polymer concentrations at 24 h and 48 h. On the contrary, the growth of C6 cells was significantly inhibited by the mPEI-CTX/DOX complex and free DOX in a dose-dependent and time-dependent manner (Fig. 2c). After exposure to the mPEI-CTX/DOX complex and free DOX for 48 h at the DOX concentration of 10 µg/mL, 31.9% and 29.5% of C6 cells, respectively, survived. The half maximal inhibitory concentration (IC50) values of mPEI-CTX/DOX and free DOX were calculated to be 9.18 µg/mL and 6.59 µg/mL at 24 h, respectively, and their corresponding IC50 values decreased to 4.87 µg/mL and 4.36 µg/mL, respectively, as the incubation time increased to 48 h.
The targeted antitumor efficacy of the mPEI-CTX/DOX complex was also evaluated using CCK-8 assay in vitro. Compared with the mPEI/DOX complex without CTX modification, the targeted mPEI-CTX/DOX complex displayed a stronger inhibitory effect on C6 cells proliferation (Fig. 2d). The viabilities of C6 cells incubated with the mPEI-CTX/DOX complex were much weaker than that of the cells treated with mPEI/DOX at the same DOX concentrations and time points. The cell survival rate after the mPEI-CTX/DOX complex treatment at the DOX concentration of 10 µg/mL for 48 h (31.9%) was much smaller than that after mPEI/DOX complex (49.2%) treatment under the same condition.
Furthermore, we checked the cytoskeleton and nucleus of the cells after treatment (Fig. 3). Obviously, in the absence of DOX such as in the PBS and mPEI-CTX groups, the cytoskeleton and nucleus of the treated cells maintained a normal state, and no cytoskeletal injury or cellular membrane dysfunction could be observed. In contrast, severe cytoskeleton damage occurred in C6 cells after incubation with the mPEI-CTX/DOX complex and free DOX, and the cytoskeleton of cells treated with the mPEI-CTX/DOX complex was almost completely destroyed.
In vitro targeting specificity
To investigate the targeting specificity of the mPEI-CTX/DOX complex in vitro, the fluorescence intensities of DOX in C6 cells were qualitatively tested using confocal laser scanning microscopy (CLSM) and quantitatively analyzed using flow cytometry. As shown in Fig. 4, CLSM revealed that the C6 cells incubated with the mPEI-CTX/DOX complex had more intense red DOX fluorescence signals both inside the cytosol and on the surface of the cells than those incubated with the mPEI/DOX complex. Similarly, because of the presence of DOX, the targeting specificity of the mPEI-CTX/DOX complex could be evaluated by flow cytometry. As shown in Fig. 5a and 5b, the C6 cells incubated with the mPEI-CTX/DOX complex for 4 h showed significantly higher DOX fluorescence signals compared with those treated with free DOX at the same DOX concentration.
The cellular uptake of mPEI-CTX/DOX by C6 cells after 99mTc radiolabeling was also validated in vitro. After incubation with mPEI-CTX-99mTc/DOX or mPEI-99mTc/DOX for 4 h, SPECT images of these cells were acquired (Fig. 5c and 5d). It could be clearly seen that the cells treated with mPEI-CTX-99mTc/DOX displayed higher signal intensities than those treated with mPEI-99mTc/DOX at different radioactive concentrations. The SPECT signal intensity of mPEI-CTX-99mTc/DOX was much higher than that of mPEI-99mTc/DOX at the highest 99mTc concentration.
In vivo SPECT imaging and antitumor efficacy in a subcutaneous glioma tumor model
To evaluate the performance of mPEI-CTX-99mTc/DOX in vivo, SPECT imaging was performed using a xenografted nude mouse model. Unsurprisingly, the 99mTc-radiolabeled CTX-modified PEI complex exhibited acceptable SPECT imaging results (Fig. 6a and 6b). The tumor accumulation of mPEI-CTX-99mTc/DOX could be observable at 2 h post-injection, which increased with the progression of time. Higher signal intensities could be found in tumor regions at 4 h, and 6 h post-injection, and the highest seemed to be at 8 h post-injection followed by attenuated tumor accumulation at 12 h post-injection. Conversely, inconspicuous SPECT signal intensity changes could be found in the tumors for 12 h following the injection of mPEI-99mTc/DOX, suggesting the key role of CTX peptide in the process of glioma-targeting. This could be further confirmed by the SPECT image of ex vivo tumors at 12 h post-injection (Fig. 6c), and much higher tumor SPECT signal intensity was observed in the mice treated with mPEI-CTX-99mTc/DOX. In addition, biodistribution studies were performed at 12 h post-injection to analyze the accumulation of mPEI-CTX-99mTc/DOX and mPEI-99mTc/DOX in major organs (Fig. S5). Similar to the high radioactive intensities in SPECT images of the abdomen of mice, the biodistribution data showed that both the mPEI-CTX-99mTc/DOX and mPEI-99mTc/DOX were mainly accumulated in the liver, kidneys, and spleen with mild accumulation in the lung, heart, and intestines, which resulted in low radioactivity uptake in other orangs such as the stomach and muscle. Notably, the mice treated with mPEI-CTX-99mTc/DOX exhibited a higher tumor uptake than those treated with mPEI-99mTc/DOX (4.72 󠅕± 0.19 ID%/g vs 1.61 ± 0.18 ID%/g), further corroborating the targeting specificity of mPEI-CTX-99mTc/DOX in vivo.
Subsequently, the in vivo antitumor effect of the mPEI-CTX/DOX complex was investigated using the xenografted tumor model. The mPEI/DOX, mPEI-CTX, mPEI, free DOX, and saline were used as the control groups. The ability to inhibit tumor growth was in the followed the order: mPEI-CTX/DOX > free DOX > mPEI/DOX > mPEI-CTX ≈ mPEI ≈ saline (Fig. 7a). Inhibition of tumor growth in the mPEI-CTX/DOX complex group was higher than that in the control groups, and the relative tumor volumes after the 21-day treatment in each group had increased 5.77 ± 0.68 (mPEI-CTX/DOX), 9.26 ± 1.51 (free DOX), 15.1 ± 1.67 (mPEI/DOX), 21.8 ± 2.58 (mPEI-CTX), 23.42 ± 2.09 (mPEI), and 25.47 ± 2.19 (saline) times, respectively. The antitumor effect of the designed mPEI-CTX/DOX complex could also be confirmed by the survival rate data (Fig. 7b). In the studied time period, the survival rate followed the same order of the ability to inhibit tumor growth, and the mice in the mPEI-CTX/DOX complex group displayed the longest survival time. The overall survival time was 51 days in the mPEI-CTX/DOX complex group and 40 days in the mPEI/DOX complex group, which was longer than that in the other control groups. This further demonstrated that the CTX modification enhanced the anti-glioma effect and prolonged survival time by specific targeting. Furthermore, the toxicity and side effects of the drug delivery systems were evaluated according to body weights of the mice during the entire treatment period. As shown in Fig. S6, a slighter body weight loss was observed in the mice of the free DOX group than those of the other groups, indicating certain toxicity of free DOX to the mice. However, the mice of the mPEI-CTX/DOX complex group and those of the other control groups showed no significant differences in weights. This seems to be explained by the inhibited toxicity of DOX after being encapsulated into the PEGylated PEI.
Subsequently, we performed HE and TUNEL staining to check the biosafety and therapeutic effect of the developed mPEI-CTX/DOX complex. As shown in Fig. 7c, the H&E staining showed that the tumor sections exhibited well-shaped cells. No obvious necrotic areas could be observed in the mPEI-CTX, mPEI, and saline groups, while the tumor necrosis was apparent in the other groups. The mPEI-CTX/DOX group showed a much larger necrotic area than the mPEI/DOX and free DOX groups, indicating that the CTX-modified complex had the strongest anticancer efficiency among the studied groups. Similarly, as shown in Fig. 7d, TUNEL assay revealed no apoptotic cells in the saline, mPEI, and mPEI-CTX groups. Unlike the mPEI/DOX and free DOX groups, which showed a small number of apoptotic cells, the mPEI-CTX/DOX group displayed obvious positive staining of apoptotic cells, confirming the antitumor performance in vivo. In addition, the biosafety in the complex in vivo system was checked by observing the H&E stained morphology of the major organs of tumor-bearing mice after treatment (Fig. S7). Myocardial damage could be found in the free DOX group because of the compound’s cardiotoxicity; however, no obvious damages to the hearts were observed after the encapsulation of DOX into the carriers. As for other major organs, no obvious tissue damage, necrotic areas, or abnormalities could be found in the six groups after treatment. These results revealed the good organ compatibility and low systemic toxicity of the synthesized mPEI-CTX/DOX complex to the mice.
Targeted SPECT imaging of glioma in an orthotopic rat glioma model
In view of the unique biological properties of CTX, we evaluated the BBB penetrability and targeting ability of the mPEI-CTX/DOX complex using Sprague Dawley (SD) rats bearing intracranial glioma in vivo. The mPEI-CTX/DOX and mPEI/DOX complexes after 99mTc radiolabeling were injected via tail vein, and their SPECT images were acquired at different time points. As shown in Fig. 8a, the mPEI-CTX-99mTc/DOX crossed the BBB, and the tumor uptake in the brains was observable after its accumulation for 2 h, followed by increased signal intensities at tumor sites at 4 h post-injection. The tumor SPECT signal intensities seemed to be stronger at 6 and 8 h post-injection, and they could still be detectable at 12 h post-injection. On the contrary, during the studied period, the rats injected with the mPEI-99mTc/DOX complex without CTX modification exhibited insignificant radioactivity accumulation in the glioma regions. These data indicated that CTX peptide could promote the BBB penetrability and glioma-targeting efficiency in PEI-based drug delivery systems. Furthermore, unlike the stable tumor-to-background ratio (TBR) in the rats treated with mPEI-99mTc/DOX (Fig. 8b), a rising trend of TBR values was observed in the rats injected with mPEI-CTX-99mTc/DOX, which revealed the efficient BBB penetrability and targeting effect. This could also be confirmed by the obvious difference of the signal intensities in the brains resected from the rats after SPECT imaging (Fig. 8c).