Multifunctional PVCL nanogels with redox-responsiveness enable enhanced MR imaging and ultrasound-promoted tumor chemotherapy

Development of versatile nanoplatforms that simultaneously integrate therapeutic and diagnostic features for stimuli-responsive delivery to tumors remains a great challenge. In this work, we report a novel intelligent redox-responsive hybrid nanosystem composed of MnO2 nanoparticles (NPs) and doxorubicin (DOX) co-loaded within poly(N-vinylcaprolactam) nanogels (PVCL NGs) for magnetic resonance (MR) imaging-guided and ultrasound-targeted microbubble destruction (UTMD)-promoted tumor chemotherapy. Methods: PVCL NGs were first synthesized via a precipitation polymerization method, decorated with amines using ethylenediamine, and loaded with MnO2 NPs through oxidation with permanganate and DOX via physical encapsulation and Mn-N coordination bonding. The as-prepared DOX/MnO2@PVCL NGs were well characterized. UTMD-promoted cellular uptake and therapeutic efficacy of the hybrid NGs were assessed in vitro, and a xenografted tumor model was used to test the NGs for MR imaging and UTMD-promoted tumor therapy in vivo. Results: The as-prepared DOX/MnO2@PVCL NGs with a size of 106.8 nm display excellent colloidal stability, favorable biocompatibility, and redox-responsiveness to the reductive intracellular environment and tumor tissues having a relatively high glutathione (GSH) concentration that can trigger the synchronous release of Mn2+ for enhanced T1-weighted MR imaging and DOX for enhanced cancer chemotherapy. Moreover, the DOX/MnO2@PVCL NGs upon the UTMD-promotion exhibit a significantly enhanced tumor growth inhibition effect toward subcutaneous B16 melanoma owing to the UTMD-improved cellular internalization and tumor penetration. Conclusion: Our work thereby proposes a promising theranostic nanoplatform for stimuli-responsive T1-weighted MR imaging-guided tumor chemotherapy.


Preparation of PVCL NGs
PVCL NGs with carboxylic groups were first synthesized using a precipitation polymerization approach referring to the literature [1,2]. In brief, VCL (1.878 g), BAC (98.28 mg) and SDS (20 mg) were co-dissolved in 120 mL of water under stirring at 70 o C for 30 min with N 2 protection to obtain a homogenous solution. Then, ACMA (70 mg, in 5 mL water) was added to the above solution to initiate the polymerization. After 5 to 8 min, AAc (108 mg, in 25 mL water) was dropped into the above mixture. The sequential addition of chemicals was under N 2 protection, and the whole reaction was maintained at 70 o C for 4 h under N 2 protection. Afterwards, the reaction mixture was cooled S-3 down to room temperature (RT) and the obtained white dispersion was dialyzed against water with a dialysis membrane having an MWCO of 12,000-14,000 for 3 days to remove the unreacted monomers. A fraction of the dialysis liquid was subjected to lyophilization to determine the mass concentration and the left was stored at 4 o C for further use.
Primary amine groups were then introduced to the above NGs via an EDC/NHS-mediated coupling reaction between the carboxyl groups of the above NGs and the amine groups of EDA to obtain the aminated PVCL NGs. Briefly, EDC (287.55 mg) and NHS (172.635 mg) in 6 mL of water were added to the above NG dispersion (210 mg, in 30 mL water) under stirring for 2 h to activate the carboxyl groups. Excess EDA (200.4 μL) was quickly injected to the above solution and the reaction was kept at RT for 3 days. Subsequently, the mixture was dialyzed against water using protocols as described above for 3 days to remove the impurity. The purified NGs solution was stored at 4 o C and a small portion was freeze-dried to determine the mass concentration.

Synthesis of MnO 2 @PVCL NGs
MnO 2 NPs were loaded within the PVCL NGs via a redox reaction between the primary amine groups of the PVCL NGs and potassium permanganate [3]. Briefly, different volume of KMnO 4 solution (5 mg/mL) was added to 10 mL of PVCL NGs dispersion (7 mg/mL) using a syringe pump with a flow rate of 0.1 mL/min. The mass ratio of PVCL NGs to KMnO 4 was set as 1 : 0.1, 1 : 0.25, 1 : 0.5, 1 : 0.75, and 1 : 1, respectively in order to optimize the preparation of the NGs. The mixture was stirred overnight, purified through dialysis against water for 3 days. A fraction of purified brown liquid was lyophilized to determine the mass concentration and the left was stored at 4 o C for further use.

Synthesis of DOX/MnO 2 @PVCL NGs
DOX was encapsulated within the MnO 2 @PVCL NGs by physical interaction and Mn-N coordinate bonds. Briefly, an aqueous DOX solution (1.54 mg/mL, 3.25 mL in water) was added into a solution of MnO 2 @PVCL NGs (5.88 mg/mL, 1.7 mL in water), followed by adjusting the S-4 solution pH to 8 with NaOH (1 M). The mixture was stirred at RT in the dark for 24 h. The dispersion was then centrifuged (13 000 rpm, 30 min) to collect the precipitate (the final DOX/MnO 2 @PVCL NGs), and the supernatant containing non-loaded free DOX was also collected for quantification of the DOX loading percentage and efficiency. The DOX loading efficiency and

Characterization Techniques
Dynamic light scattering (DLS) and zeta potential measurements were carried out using a Malvern Zetasizer Nano ZS model ZEN3600 (Worcestershire, UK) equipped with a standard 633 nm laser. The morphology of the MnO 2 @PVCL NGs was observed using scanning electron microscope (SEM, S-4800 analytical electron microscope, Tokyo, Japan) at a voltage of 15 kV. The sample was prepared by dropping an NG suspension (1 mg/mL, 5 μL) onto aluminum foil, followed by air drying and sputter coating of a gold film with a thickness of 10 nm. Transmission electron microscopy (TEM) was performed using a JEOL 2010F electron microscope (Tokyo, Japan) at an operating voltage of 200 kV. One drop of the DOX/MnO 2 @PVCL NGs in water (1 mg/mL) was deposited onto a carbon-coated copper grid and air-dried before measurements. The particle size distribution was measured using Image J 1.40 G software (http://rsb.info.nih.gov/ij/download.html).
For each sample, at least 200 MnO 2 @PVCL NGs, DOX/MnO 2 @PVCL NGs or MnO 2 NPs were randomly selected from SEM or TEM images. UV-vis spectroscopy was carried out using a Lambda 25 UV-vis spectrophotometer (PerkinElmer, Waltham, MA). X-ray photoelectron spectroscopy (XPS) data were obtained with an Escalab 250Xi spectrometer (ThermoFisher Scientific, Waltham, MA), equipped with an analyzer mode (pass energy of 50 eV) and an Al Kα X-ray source. Mn

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Cell Culture B16 melanoma cancer cells were regularly cultured and passaged in RPMI-1640 medium supplemented with 10% FBS and 1% P/S in a Thermo Scientific cell incubator (Waltham, MA) at 37 o C and 5% CO 2 .

In Vitro Ultrasound-Targeted Microbubble Destruction (UTMD) Treatment of Cells
UTMD treatment was conducted using a therapeutic ultrasound machine (PHYSIOMED ELEKTROMEDIZIN, Schnaittach, Germany). According to our previous work [4], the procedure was set as follows: B16 cells were suspended with medium containing NGs and SonoVue and subsequently exposed to the ultrasound apparatus with the following optimized parameters: 0.4 W/cm 2 , 1 MHz, 20% microbubbles, PRF 1 kHz, and 30 s. The processed cell suspension was then seeded into 12-well or 6-well plate for 6 h to study the influence of UTMD on the cellular uptake behavior of NGs. Cells treated with phosphate buffered saline (PBS) were used as control. To investigate the UTMD-promoted therapeutic efficacy of the NGs, the seeded cells were treated with medium containing NGs and SonoVue, UTMD treated for 30 s, and further incubated for 24 h before regular cell viability assay (see below). involved as aforementioned. The cytotoxicity was then measured following the same procedure mentioned above.

In Vitro Cellular Uptake
The cellular uptake behavior of DOX/MnO 2 @PVCL NGs in the presence or absence of  intraperitoneal injection of pentobarbital sodium (40 mg/kg) and immobilized in the rodent receiver coil using medical adhesive tape during the imaging process.

In Vivo Antitumor Efficacy
Mice bearing B16 subcutaneous tumors were randomly divided into 5 groups (5 mice for each group) which were separately treated with (1) [5,6]. In the process of therapy, the tumor volume (V = a × b 2 /2, a represents the tumor length and b the tumor width) and mice body weight were recorded every other day for 12 days.
B-mode ultrasound (US) imaging was used to visualize the tumor size using a clinical diagnostic ultrasound scanner (LOGIQ E9, GE, Fairfield, CT). In addition, mice after the treatment course were injected with SonoVue (1.18 mg/mL, 0.1 mL saline), and contrast-enhanced ultrasound (CEUS) imaging was conducted using the same probe (LOGIQ E9, ML 6-15 MHz) for the contrast mode at a low mechanical index (0.12). All CEUS images were collected under the same conditions (20 mm depth and 5 gains). In the end, the mice were anesthetized and sacrificed. The tumor pathological changes were analyzed by hematoxylin-eosin (H&E) and TdT-mediated dUTP Nick-End Labeling staining. To assess the organ toxicity of NGs, the major organs including heart, liver, spleen, lung, and kidney were harvested for H&E staining.

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To explore the in vivo biodistribution of NGs, mice bearing B16 tumors were treated with DOX/MnO 2 @PVCL NGs at a DOX dosage of 5 mg/kg in the presence or absence of UTMD, and were euthanized at different time points post-injection (20 min, 40 min, 90 min, 1 day, and 2 days, respectively). The major organs including the heart, liver, spleen, lungs, kidneys and tumor were extracted, weighed, digested by nitrohydrochloric acid overnight and then analyzed by ICP-OES to quantify Mn element. The tumor-bearing mice injected with PBS were used as control.

Hemolysis Assay and Blood Routine Tests
Hemolysis assay and blood routine tests were performed to evaluate the biosafety of the hybrid NGs. The healthy mice were anaesthetized and the eyeballs were removed to collect blood samples.
Hemolysis assay was performed according to the literature [7]. Briefly, 1.5 mL of blood was diluted

Statistical Analysis
One-way analysis of variance (ANOVA) method was used to evaluate the significance of the experimental data with a significance level (p-value) of 0.05. The data were marked with (*) for p < 0.05, (**) for p < 0.01, (***) for p < 0.001, respectively. All experimental data were displayed as the mean ± standard deviation (n ≥ 3).        The data are shown as mean ± SD (n = 3).