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

Abstract

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.

Keywords: chemotherapy; magnetic resonance imaging; manganese dioxide nanoparticles; nanogels; ultrasound-targeted microbubble destruction.

Figure 1

(A) Synthetic route for the fabrication of DOX/MnO₂@PVCL NGs. (B) Schematic illustration of the utilization of DOX/MnO₂@PVCL NGs for UTMD-promoted delivery of DOX/MnO₂@PVCL NGs for MR imaging-guided cancer chemotherapy. (C) Hydrodynamic size distribution and relative correlation coefficient (inset) of PVCL, MnO₂@PVCL and DOX/MnO₂@PVCL NGs in water. (D) Zeta potentials of PVCL, MnO₂@PVCL and DOX/MnO₂@PVCL NGs in different aqueous media (n = 3). (E) XPS spectrum of DOX/MnO₂@PVCL NGs. (F) UV-vis spectra of free DOX, MnO₂@PVCL and DOX/MnO₂@PVCL NGs. (G) TEM image and (H) EDX elemental mapping analysis of DOX/MnO₂@PVCL NGs. (I) DOX release profile from DOX/MnO₂@PVCL NGs at pH 7.4/6.5 in the presence or absence of GSH (10 mM). Data are shown as mean ± SD (n = 3). (J) Pseudo-colored T₁-weighted MR images of DOX/MnO₂@PVCL NGs with different Mn concentrations in the presence or absence of GSH (10 mM). The color bar from blue to red indicates the gradual increase of MR signal intensity.

Figure 2

(A) Cell viability of B16 cells treated with free DOX, MnO₂@PVCL, DOX/MnO₂@PVCL and DOX/MnO₂@PVCL NGs + UTMD at different DOX concentrations for 24 h using CCK-8 assays (n = 5). (B) Quantified FACS analysis of DOX mean fluorescence intensity and (C) FACS histograms of B16 cells treated with DOX/MnO₂@PVCL NGs in the presence and absence of UTMD at different DOX concentrations (n = 3). (D) CLSM images of B16 cells incubated with free DOX, DOX/MnO₂@PVCL and DOX/MnO₂@PVCL NGs + UTMD. Scale bar: 20 µm for each panel. (E) In vivo T₁-weighted MR images (grey and pseudo-color) of mice bearing subcutaneous B16 tumors before and at different time points post-injection of DOX/MnO₂@PVCL NGs ([DOX] = 5 mg/kg, in 200 µL of PBS for each mouse). The color bar from blue to red indicates the gradual increase of MR signal intensity. (F) MR signal to noise ratio (SNR) of the tumor region at different time points post-injection of DOX/MnO₂@PVCL NGs (n = 3).

Figure 3

(A) Photograph of tumors on day 12 after different treatments. (B) Tumor growth curves after different treatments (n = 5). Tumor volumes (V) were normalized to their initial values (V₀). (C) B-mode US images and (D) CEUS images of the tumor on day 12 after different treatments. (E) H&E staining and (F) TUNEL staining of tumor slices taken on day 12 after different treatments. Scale bar for (E) and (F) represents 200 µm for each panel.

Figure 4

In vivo biodistribution of Mn in the (A) major organs of the mice (including the heart, liver, spleen, lung, and kidney) and (B) tumor at different time points post single intravenous injection of DOX/MnO₂@PVCL NGs in the absence and presence of UTMD ([DOX] = 5 mg/kg, in 200 µL of PBS for each mouse, n = 3). (C) Mouse body weight changes in different groups over the in vivo therapy process (n = 5).