Polyethylenimine-Based Theranostic Nanoplatform for Glioma-Targeting SPECT Imaging and Anticancer Drug Delivery


 Background Glioma is the deadliest brain cancer in adults because the blood-brain-barrier (BBB) prevents the vast majority of therapeutic drugs from entering into the central nervous system. The development of BBB-penetrating drug delivery systems for glioma therapy still remains a great challenge. In this study, we aimed to design and develop a theranostic nanocomplex with enhanced BBB penetrability and tumor-targeting efficiency for glioma (SPECT) imaging and anticancer drug delivery. Results This multifunctional nanocomplex was manufactured using branched polyethylenimine (PEI) as a template to sequentially conjugate with methoxypolyethylene glycol ( m PEG), glioma-targeting peptide chlorotoxin (CTX), and diethylenetriaminepentaacetic acid (DTPA) for 99m Tc radiolabeling on the surface of PEI. After the acetylation of the remaining PEI surface amines using acetic anhydride (Ac 2 O), the CTX-modified PEI ( m PEI-CTX) was utilized as a carrier to load chemotherapeutic drug doxorubicin (DOX) in its interior cavity. The m PEI-CTX/DOX complex had excellent water dispersibility and released DOX in a sustainable and pH-dependent manner; furthermore, it showed targeting specificity and therapeutic effect of DOX toward glioma cells in vitro and in vivo (a subcutaneous tumor mouse model). Owing to the unique biological properties of CTX, the m PEI-CTX/DOX complex was able to cross the BBB and accumulate at the tumor site in an orthotopic rat glioma model. In addition, after efficient radiolabeling of PEI with 99m Tc via DTPA, the 99m Tc-labeled complex could help to visualize the drug accumulation in tumors of glioma-bearing mice and the drug delivery into the brains of rats through SPECT imaging. Conclusions These results indicate the potential of the developed PEI-based nanocomplex in facilitating glioma-targeting SPECT imaging and chemotherapy.


Background
The diffuse invasion and in ltrative overgrowth of glioma cells lead to the development of irregular and indistinct tumor margins, making surgical resection di cult; consequently, patients with malignant gliomas always have a very poor prognosis [1,2]. The special pathological and physiological characteristics of the blood-brain barrier (BBB) allow merely a few chemotherapeutic drugs into the brain in the early stage [3]. This strictly limited selection imposes a great challenge on glioma treatment. To overcome this issue, various nanoparticulate platforms have been widely investigated to develop BBBpenetrating delivery systems because of their great clinical potential in cancer diagnosis and treatment [4][5][6]. These nanoplatforms can be exploited as imaging agents to guide the maximal surgical resection, drug carriers to improve chemotherapy, or theranostic systems to achieve imaging-guided drug delivery and therapy monitoring [7][8][9][10][11].
Although the BBB penetrability of some nanoparticles (NPs) can be improved through speci c modi cation (for example attaching PEG chain or charged/lipophilic groups) [12][13][14], an active strategy has attracted much more attention [15,16]. In this active strategy, NPs are generally modi ed with targeting molecules such as peptides or antibodies, which help NPs bind to endothelial cells of BBB selectively and traverse into the brain via receptor/transporter-mediated transcytosis [17][18][19]. In previous studies, some receptors and transporters, including but not limited to transferrin receptor, low density lipoprotein receptor, insulin receptor, nicotinic acetylcholine receptor, amino acid transporter, choline transporter, hexose transporter, and monocarboxylic acid transporter, have been determined to be involved in receptor/transporter-mediated transcytosis across BBB [19][20][21][22][23][24][25][26]. These ndings have motivated numerous researchers around the world to focus on the development of novel brain targeting systems for glioma imaging and treatment. Moreover, some promising targets, such as the chloride ion channel (CLC), which is overexpressed on glioma cells and involved in multiple malignant features of glioma including proliferation, migration, and apoptosis, also arouse intensive interest [27][28][29][30]. As a ligand of the CLC, chlorotoxin (CTX), a small peptide puri ed from scorpion venom, has been proven to have the ability to penetrate the BBB and show high a nity binding to glioma cells via CLC and matrix metalloproteinase 2 (MMP-2) [31]. Once bound to the receptors on glioma cell surface, the peptide can be internalized into cells. These unique biological properties of CTX make it a potential targeting agent for glioma diagnosis and therapy [32][33][34]. Perceiving this, the feasibility of using radionuclide 131 I and uorescent molecule (indocyanine green) labeled CTX molecules for glioma imaging and treatment has been investigated in clinical trials [35,36]. Notably, a variety of CTX peptide modi ed nanoparticles, for instance, CTXconjugated iron oxides, liposomes, dendrimers, quantum dots, and rare-earth up-conversion NPs, have been studied as potential candidates in this eld [37,38]. These NPs can not only be used as imaging agents for diagnosis or as drug carriers for treatment, but also as multifunctional systems for theranostic applications. Among these NPs, dendritic polymers such as poly(amidoamine) (PAMAM) and polyetherimide (PEI) dendrimers, have been considered as promising templates for the construction of theranostic nanosystems for various kinds of tumors, including brain cancer [39,40].
In our previous study, we reported CTX-modi ed dendrimers for glioma imaging and therapy [41]. The developed multifunctional dendrimers exhibited acceptable imaging performance and targeted radionuclide therapy effect, and they could further entrap gold NPs for glioma SPECT/CT imaging [42].
More importantly, because of the modi cation of CTX peptide, this kind of dendrimer nanoplatform possesses the ability to cross the BBB and target glioma cells. These results together with the attractive features of CTX facilitated our further investigation of dendrimer-based BBB-penetrating NPs as chemotherapeutic drug carriers for glioma therapy. In addition, compared to the 131 I used in our previous studies, 99m Tc is a better radionuclide for SPECT imaging, which is associated with a higher imaging resolution because of its physical properties such as nearly pure electron capture decay and low γ-ray energy (140 keV) [43,44]. Therefore, in the present study, a BBB-penetrating imaging-guided drug delivery nanosystem was designed and synthesized using PEI dendrimer as the template by encapsulation of doxorubicin (DOX) and conjugation of CTX peptide and radionuclide 99m Tc. DOX was encapsulated into the interior cavities by physical interactions, while CTX and 99m Tc were covalently conjugated on the surface of PEI via a PEG chain and bifunctional chelating agent (diethylenetriaminepentaacetic acid, DTPA), respectively. The designed theranostic nanosystem was characterized via different techniques to determine the structure, size, release kinetics, and stability. The targeting speci city and therapeutic e cacy toward glioma cells were evaluated in vitro and in vivo using a subcutaneous glioma tumor model, and the BBB penetrability was investigated via an intracranial rat model, which could be further visualized after the 99m Tc radiolabeling through SPECT imaging. Therefore, a combination of gliomaspeci c agent, chemotherapeutic drug, and radionuclide imaging could be a novel strategy for the imaging-guided drug delivery for brain cancer.

Methods
Synthesis of PEI.NHAc-DOX-(PEG-CTX)-mPEG-DTPA-99m Tc In this study, the PEI-based theranostic nanosystem was prepared according to protocols reported in the literature [42]. Brie y, reaction of PEI.NH 2 with mPEG-COOH led to the formation of PEGylated PEI (PEI.NH 2 -mPEG). MAL-PEG-SVA was attached to the PEI.NH 2 -mPEG to modify with CTX peptide in the next step. To achieve the CTX modi cation in a convenient way, an extra cysteine residue was added at its C-terminal, which could react with the maleimide group on the PEI surface to form PEI.NH 2 -(PEG-CTX)-mPEG. After that, the PEI.NH 2 -(PEG-CTX)-mPEG was decorated with DTPA, which is one of the most frequently used bifunctional chelating agent for 99m Tc radiolabeling, and the remaining PEI terminal amines were acetylated using excess Ac 2 O. Finally, the PEI.NHAc-DTPA-(PEG-CTX)-mPEG (mPEI-CTX) was used to encapsulate the anticancer drug DOX, and the synthesized PEI.NHAc-DTPA-(PEG-CTX)-mPEG/DOX (mPEI-CTX/DOX) complex could be further labeled with 99m Tc using SnCl 2 as the reductant.
The schematic illustration of the synthetic process is shown in Fig. 1, and the details are provided in supplementary information.
Cytocompatibility and cytotoxicity analysis CCK-8 assay was employed to assess the cytocompatibility of mPEI-CTX without DOX and the cell cytotoxicity after DOX encapsulation. For this assay, C6 cells were plated in 96-well plates at a density of 8,000 cells per well and incubated for 24 h. These cells were then treated with mPEI-CTX/DOX, mPEI/DOX, and free DOX at different DOX concentrations (0-10 μg/mL) or mPEI-CTX and mPEI at different polymer concentrations (0-100 μg/mL) for 24 h and 48 h. After washing the cells with PBS 3 times, CCK-8 solution (100 μL) was added to each well, and the cells were cultured for 2 h. The absorbance at 450 nm was measured using a Varioskan Flash multimode microplate reader (Thermo Fisher Scienti c, Waltham, MA, USA).
To test the in uence of the complex on the cytoskeleton, C6 cells were seeded in a 12-well plate at a density of 2 × 10 5 cells per well. Subsequently, these cells were incubated with mPEI-CTX/DOX, mPEI-CTX, and free DOX at the DOX concentration of 5 µg/mL. PBS was set as a negative control. After 24 h, the cytoskeleton was visualized by confocal microscopic imaging using the standard protocols [42].

Confocal laser scanning microscopy (CLSM) and ow cytometry analysis
Confocal microscopy and ow cytometry analysis were applied to determine the targeting speci city of mPEI-CTX/DOX in vitro. For confocal microscopy, C6 cells were seeded into each well of 24-well plates at a density of 5 × 10 4 and cultured for 24 h. The medium was replaced with 1 mL of fresh medium containing mPEI-CTX/DOX or mPEI/DOX at the DOX concentration of 8 µg/mL. PBS was set as a negative control. After 4 h, the cells were rinsed, xed, counterstained, and observed. For ow cytometry analysis, C6 cells were seeded in a 12-well plate (2 × 10 5 cells per well) and incubated for 24 h. The C6 cells were treated with mPEI-CTX/DOX and mPEI/DOX at a different DOX concentrations (0-8 µg/mL). After 4 h, the cells were harvested and washed 3 times with PBS, and the uorescence intensities per 10,000 cells were recorded in the FL1-uorescence channel using a BD AccuriTM C6 Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

In vitro SPECT imaging
In vitro SPECT imaging was used to con rm the cellular uptake of mPEI-CTX/DOX by glioma cells after 99m Tc radiolabeling. In brief, C6 cells were seeded in 12-well plates at a density of 2 × 10 5 cells/well. After incubation for 24 h, the culture medium was replaced with 2 mL of fresh medium containing 99m Tc-mPEI-CTX/DOX and 99m Tc-mPEI/DOX at different radioactivity concentrations (50, 100, 200, and 400 mCi/mL).
After 4 h incubation, the cells were harvested and washed 3 times with PBS, centrifuged in 1.5 mL tubes, and imaged by a SPECT imaging system equipped with Xeleris 2.0 Workstation and low-energy generalpurpose collimators (In nia, Denver, CO).

Targeted SPECT imaging of glioma in tumor model
Before SPECT imaging in vivo, the mice were divided into targeted and non-targeted groups (6 mice per group) at random. The targeted group was intravenously administrated saline solution containing 99m Tc-mPEI-CTX/DOX (600 mCi, 100 mL), and a same dose of 99m Tc-mPEI/DOX was injected in the nontargeted group. SPECT images were then captured at 0.5, 2, 4, 6, 8, and 12 h post-injection. At 12 h postinjection, one mouse from each group was euthanized, and the tumors and major organs (heart, lung, liver, stomach, spleen, kidneys, soft tissue, and intestines) were collected for analysis of the relative signal intensities.
We subsequently evaluated the therapeutic e cacy of mPEI-CTX/DOX in vivo in the subcutaneous tumor model. Ten days after tumor inoculation, the mice were split randomly into 6 groups (6 mice per group).
The mice in each group were sequentially treated with mPEI-CTX/DOX, mPEI/DOX, mPEI-CTX, mPEI, free DOX, and saline via intravenous injection at a DOX concentration of 1 mg/mL in 100 mL saline solution. The treatments were then performed every 3 days, accounting for a total of 7 times, and the tumor size and body weight of each mouse were recorded after each treatment. Their relative tumor volumes, body weights, and survival rates were calculated as described in our previous work [45]. After a three-week treatment, the representative mice from these groups were sacri ced to harvest the tumors and major organs including the heart, liver, spleen, lung, and kidneys. The hematoxylin and eosin (HE) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were performed according to the standard protocols to investigate the potential toxicity of mPEI-CTX/DOX in vivo [45].
Targeted SPECT imaging of glioma in an orthotopic glioma rat model To verify the BBB penetrability and tumor-targeting e ciency of mPEI-CTX/DOX, an orthotopic glioma rat model was established according to the method described previously [42]. Fourteen days later, twelve rats were equally divided into two groups and injected with 99m Tc-mPEI-CTX/DOX and 99m Tc-mPEI/DOX according to the assigned group. SPECT imaging was carried out at 0.5, 2, 4, 6, 8, and 12 h post-injection. All the rats were euthanized after SPECT imaging to separate the brains and measure their relative radioactivity intensities.

Statistical analysis
Data are presented as mean ± standard deviation, and one-way analysis of variance was performed to evaluate the signi cance of the data. A p value of 0.05 was selected as the threshold of signi cance, and the data were denoted with (**) for p < 0.01 and (***) for p < 0.001.

Results And Discussion
Synthesis and characterization of the mPEI-CTX/DOX complex PEGylated PEI have been developed as multifunctional templates to entrap gold NPs, to be labeled with radionuclides, or to load drugs for tumor imaging and treatment in our previous studies [45][46][47]. We further modi ed these PEI-based NPs with targeting ligands for different tumors, for example, folic acid for cervical carcinoma, arginine-glycine-aspartic acid peptide for hepatic carcinoma, and CTX for glioma [42,45,48]. In the present study, following a similar strategy, PEI was consecutively connected with mPEG, CTX, and DTPA. After acetylation of the remaining PEI surface amines using AcO 2 , mPEI-CTX was loaded with DOX to synthesize the mPEI-CTX/DOX complex that could be further conveniently radiolabeled with 99m Tc through DTPA. The theranostic complexes and the intermediate products obtained during the preparation process were characterized using different techniques.
Secondly, after acetylation of the PEI.NH 2 -DTPA-(PEG-CTX)-mPEG and PEI.NH 2 -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 con rm 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.NH 2 -(PEG-CTX)-mPEG and PEI.NH 2 -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 (DLS). 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 re ected the success of DOX loading. As shown in Table S2, the zeta potentials of PEI.NH 2 -DTPA-(PEG-CTX)-mPEG and mPEI-CTX/DOX showed no signi cant 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 [49][50][51].
Thirdly, the release kinetics of DOX from the mPEI-CTX/DOX complexes 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]. It was clear that DOX could sustainably be released from the complexes in a pH-dependent manner with a faster release rate under the acidic condition. At 48 h, the DOX release percentage could be achieved at 26.6% (pH = 5.0) and 22.9% (pH = 7.4), respectively. The faster DOX release rate under the acidic condition was likely because of high water solubility of a majority of protonated DOX molecules, which leads to the repulsion of the protonated positively charged PEI backbone and enhanced DOX release from the complexes.
In vitro cytotoxicity assays CCK-8 assay was used to test the cytocompatibility of mPEI-CTX without DOX encapsulation and evaluate the therapeutic e cacy 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 and 48 h. On the contrary, the growth of C6 cells was signi cantly inhibited by the mPEI-CTX/DOX complex and free DOX in a dosedependent 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. Obviously, the proliferation inhibition effect of the PEI-based NPs was only relevant to the antitumor drug DOX. The half maximal inhibitory concentration (IC 50 ) 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 IC 50 values decreased to 4.87 µg/mL and 4.36 µg/mL, respectively, as the incubation time increased to 48 h. Notably, the mPEI-CTX/DOX complex showed a higher IC 50 value than free DOX, which might be ascribed to the gradual release of antitumor drug from the mPEI-CTX/DOX complex, that is, the concentration of the released DOX form the complex was lower than that of free DOX at a given time point. The targeted antitumor e cacy of the mPEI-CTX/DOX complex was also evaluated using CCK-8 assay in vitro. Compared with mPEI/DOX complex without CTX modi cation, 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. This result suggests that the targeting ability of CTX made the mPEI-CTX/DOX complex suitable for speci c cellular uptake, thus enhancing cytotoxicity.
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. These data further con rm the cytocompatibility of the synthesized mPEI-CTX and the equivalence of the therapeutic effects of the mPEI-CTX/DOX complex and free DOX in vitro.

In vitro targeting speci city
To investigate the targeting speci city of the mPEI-CTX/DOX complex in vitro, the uorescence intensities of DOX in C6 cells were qualitatively tested using confocal laser scanning microscopy (CLSM) and quantitatively analyzed using ow cytometry. As shown in Fig. 4, CLSM revealed that the C6 cells incubated with the mPEI-CTX/DOX complex had more intense red DOX uorescence 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 speci city of the mPEI-CTX/DOX complex could be evaluated by ow cytometry. As shown in Fig. 5a and 5b, the C6 cells incubated with the mPEI-CTX/DOX complex for 4 h showed signi cantly higher DOX uorescence signals compared with those treated with free DOX at the same DOX concentration, which could be attributed to the targeting ability of CTX and internalization of the complex into the cytoplasm of C6 cells. The CLSM data were in agreement with the ow cytometry results, con rming the targeting speci city and drug delivery property of the mPEI-CTX/DOX complex to glioma cells.
The cellular uptake of mPEI-CTX/DOX by C6 cells after 99m Tc radiolabeling was also validated in vitro.
After incubation with mPEI-CTX-99m Tc/DOX or mPEI-99m Tc/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-99m Tc/DOX displayed higher signal intensities than those treated with mPEI-99m Tc/DOX at different radioactive concentrations. The SPECT signal intensity of mPEI-CTX-99m Tc/DOX was much higher than that of mPEI-99m Tc/DOX at the highest 99m Tc concentration. The imaging performance of mPEI-CTX-99m Tc/DOX in vitro further supported the ow cytometry and CLSM data, which led to the assessment of the applicability of the synthesized complex for targeted SPECT imaging and chemotherapy of glioma models of mice.

In vivo SPECT imaging and antitumor e cacy in a subcutaneous glioma tumor model
To evaluate the performance of mPEI-CTX-99m Tc/DOX in vivo, SPECT imaging was performed using a xenografted nude mouse model. Unsurprisingly, the 99m Tc-radiolabeled CTX-modi ed PEI complex exhibited acceptable SPECT imaging results ( Fig. 6a and 6b). The tumor accumulation of mPEI-CTX-99m Tc/DOX could be observable at 1 h post-injection, which increased with the progression of time.
Higher signal intensities could be found in tumor regions at 2 h, 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-99m Tc/DOX, suggesting the key role of CTX peptide in the process of glioma-targeting. This could be further con rmed by the SPECT image of ex vivo tumors at 12 h postinjection (Fig. 6c), and much higher tumor SPECT signal intensity was observed in the mice treated with mPEI-CTX-99m Tc/DOX. In addition, biodistribution studies were performed at 12 h post-injection to analyze the accumulation of mPEI-CTX-99m Tc/DOX and mPEI-99m Tc/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-99m Tc/DOX and mPEI-99m Tc/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-99m Tc/DOX exhibited a higher tumor uptake than those treated with mPEI-99m Tc/DOX (4.72 ± 0.19 ID%/g vs 1.61 ± 0.18 ID%/g), further corroborating the targeting speci city of mPEI-CTX-99m Tc/DOX in vivo.
Subsequently, the in vivo antitumor effect of the mPEI-CTX/DOX complex was investigated using the xenografted tumor model, and 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 21day 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 enhanced antitumor effect of the mPEI-CTX/DOX complex could be mainly attributed to the synergistic effect of PEGylation and CTX modi cation of the drug delivery system. The PEGylation modi cation prolonged the blood circulation time of the complex, and CTX endowed the complex with target speci city toward glioma cells in vivo, leading to an improved retention time and therapeutic e cacy in tumor region. The antitumor effect of the designed mPEI-CTX/DOX complex could also be con rmed 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 modi cation enhanced the anti-glioma effect and prolonged survival time by speci c 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 signi cant 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 CTXmodi ed complex had the strongest anticancer e ciency 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, con rming 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 99m Tc radiolabeling were injected via tail vein, and their SPECT images were acquired at different time points. As shown in Fig. 8a, the mPEI-CTX-99m Tc-/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-99m Tc-/DOX complex without CTX modi cation exhibited insigni cant radioactivity accumulation in the glioma regions. These data indicated that CTX peptide could promote the BBB penetrability and glioma-targeting e ciency in PEI-based drug delivery systems. Furthermore, unlike the stable tumor-to-background ratio (TBR) in the rats treated with mPEI-99m Tc-/DOX (Fig. 8b), a rising trend of TBR values was observed in the rats injected with mPEI-CTX-99m Tc-/DOX, which reveals the e cient BBB penetrability and targeting effect. This could also be con rmed by the obvious difference of the signal intensities in the brains resected from the rats after SPECT imaging (Fig. 8c).

Conclusions
In this study, we designed and synthesized a PEI-based drug delivery system for targeted glioma therapy. The dendritic PEI could be readily modi ed with CTX peptide on the surface, and effectively encapsulate the anticancer drug DOX into the interior cavities. This CTX-functionalized PEI-based drug delivery system could release DOX in a pH-sensitive manner and displayed good targeting ability and therapeutic e cacy toward glioma cells in vitro and in vivo (a subcutaneous tumor model). More importantly, owing to the unique biological characters of CTX, the developed drug delivery system was able to cross BBB and accumulate in the brain tumor region, and the tumor accumulation could be visualized after 99m Tc radiolabeling. This PEI-based drug carrier not only exhibited a potential strategy to overcome the challenges posed by the BBB barrier through peptide modi cation, but also provided a promising approach to fabricate imaging-guided drug delivery systems for different types of cancers.   Confocal microscopic images of C6 cells treated with mPEI-CTX/DOX, mPEI-CTX, free DOX, and PBS for 24 h. The DOX concentration used was 5 μg/mL.

Supplementary Files
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