An erythrocyte membrane coated mimetic nano-platform for chemo-phototherapy and multimodal imaging

Abstract

The tumor variability and low efficiency associated with conventional chemical drugs provide an impetus to develop drug-carrying systems with targeted accumulation and controllable release behavior. Herein, DOX-loaded Prussian blue (PB) nano-composites are developed after coating with erythrocyte membrane (EM) and modifying with folic acid (FA). In these nano-composites, PB nanoparticles mixture with different shapes were adopted to improve the photothermal performance, which is highly helpful for cancer photothermal ablation and controllable drug release. In addition, the nano-composites were endowed with high biocompatibility, immune evading capacity, pH-/photo-responsive release behavior, and markedly prolonged blood circulation time, which was reflected by a 99.6% cervical tumor growth inhibition value (TGI) in vivo. Meanwhile, they functioned as multimodal bioimaging agents for photothermal, fluorescence, and photoacoustic imaging of tumors. The reported strategy can be applied for personalized therapy of various tumors by modifying the tumor-targeting molecule on the surface of nanoparticles.

Scheme 1. Illustration of the preparation and anti-tumor application of drug loaded SCPB@DOX@EM@FA NPs.

Fig. 1. (A and B) Scanning electron microscope (SEM) images. (C) The molar extinction coefficient and photothermal conversion efficiency of spheric, spheric & cubic and cubic HMPB NPs. (D) Temperature change during 808 nm laser irradiation (0.8 W cm⁻²) of the spheric and cubic with different ratios for 5 min. (E) DOX loading efficiency and encapsulation efficiency of spheric, spheric & cubic and cubic HMPB NPs when the DOX concentration was 3.0 mg mL⁻¹. (F) Controllable release of different DOX-loading NPs triggered by laser. (G) and (H) TEM of SPB NPs, CPB NPs and SCPB NPs with erythrocyte membrane modification (SCPB@EM NPs). (I) Surface charge of SCPB NPs, SCPB@DOX NPs, EM, SCPB@DOX@EM NPs, and SCPB@DOX@EM@FA NPs. (J) Size distribution and (K) UV-Vis absorption spectra of DOX, SCPB NPs, SCPB@DOX NPs, SCPB@DOX@EM NPs, and SCPB@DOX@EM@FA NPs. (L) SDS-PAGE of protein samples. Samples 1 to 4 is protein marker, sole erythrocyte membrane, SCPB@DOX@EM NPs, and SCPB@DOX@EM@FA NPs, respectively.

Fig. 2. (A) Infrared thermal images of (1) PBS, (2) SCPB NPs, (3) SCPB@DOX NPs, (4) SCPB@DOX@EM NPs and (5) SCPB@DOX@EM@FA NPs (with SCPB concentration at 0.2 mg mL⁻¹) during 808 nm laser irradiation (0.8 W cm⁻²) for 5 min. (B) Photostability of SCPB@EM@FA NPs (with SCPB concentration at 0.2 mg mL⁻¹) under 808 nm laser irradiation (0.8 W cm⁻²) using ICG as control and (C) UV-Vis spectra of ICG and SCPB@EM@FA NPs (with SCPB concentration at 0.2 mg mL⁻¹) before and after 808 nm laser irradiation. Inset: images of SCPB@EM@FA NPs and ICG before and after 808 nm laser irradiation. (D) DOX loading efficiency and encapsulation efficiency of SCPB@DOX NPs when the DOX concentration was 0.125, 0.25, 0.5, 1.0, 2.0 and 3.0 mg mL⁻¹, respectively. (E) DOX release from SCPB@DOX NPs and SCPB@DOX@EM@FA NPs at pH 5.0 and 7.4. (F) DOX release behavior from SCPB@DOX@EM@FA NPs with 4 laser on/off cycles. All release studies were performed in triplicate.

Fig. 3. (A) Hemolysis quantification of EM incubated with SCPB@DOX NPs, SCPB@DOX@EM NPs, and SCPB@DOX@EM@FA NPs. (B) Cytotoxicity of SCPB@EM@FA NPs to different cell lines. (C) The confocal scanning microscope images of macrophages after culturing with SCPB@DOX NPs or SCPB@DOX@EM@FA NPs for 12 h, the control was without treatment (scale bar = 50 μm). (D) The effect of SCPB@DOX NPs or SCPB@DOX@EM@FA NPs on the TNF-α secretion. The TNF-α levels in PBS and lipopolysaccharide (LPS)-treated macrophages were negative control (0%) and positive control (100%), respectively.

Fig. 4. (A) Cell viability (up) and FR expression level assay (down) of NIH-3T3, SMMC-77221, and HeLa cell lines treated with SCPB@DOX@EM@FA NPs or FA + SCPB@DOX@EM@FA NPs. (B) Fluorescence images of HeLa cells after incubating with SCPB@DOX@EM NPs, SCPB@DOX@EM@FA NPs, and SCPB@DOX@EM/SCPB@DOX@EM@FA NPs pretreated with excessive FA. (C) CLSM images of HeLa cells with different treatment (DOX, SCPB@DOX, SCPB@DOX@EM, SCPB@DOX@EM@FA, and SCPB@DOX@EM@FA + laser). The endosomes/lysosomes were stained with LysoTracker green (green). (D) Laser confocal scanning microscope images of internalization and subcellular localization of naked SCPB@DOX NPs and erythrocyte membrane coated SCPB@DOX NPs in the presence of three kinds of small-molecule inhibitors.

Fig. 5. In vitro anti-tumor activity of SCPB@DOX@EM@FA NPs. (A) Live/dead cell staining (scale bar = 200 μm). (B) MTT assay. (C) Flow cytometry analysis of HeLa cells after treatment with PBS, SCPB NPs, DOX, SCPB@DOX NPs, SCPB@DOX@EM NPs and SCPB@DOX@EM@FA NPs subjected to 808 nm laser irradiation. The dashed curves denote the boundary of laser irradiation spots. (D) Protein level detection of IKK, NF-κB, caspase-3, PARP, BAX, and Bcl-2 after different treatment. (E) Illustration of the SCPB@DOX@EM@FA NPs induced apoptosis pathway. Data are shown as mean standard deviation (SD), n = 3. (P value: ***P < 0.001).

Fig. 6. (A) PA mapping of SCPB@DOX@EM@FA NPs at different concentrations. PA imaging (B) and the quantification analysis (C) of the HeLa-tumor-bearing mice pre-injection or post-injection of SCPB@DOX@EM@FA NPs. (D) In vivo pharmacokinetic curves over a span of 48 h after intravenous injection of SCPB NPs or SCPB@DOX@EM@FA NPs. (E) Bio distribution of SCPB NPs, SCPB@DOX NPs, SCPB@DOX@EM NPs or SCPB@DOX@EM@FA NPs after injection for 48 h. In vivo fluorescence images (F) of cervical tumor-bearing nude mice at 12, 24, 36 and 48 h after tail vein injection of SCPB@DOX NPs, SCPB@DOX@EM NPs and SCPB@DOX@EM@FA NPs; ex vivo fluorescence images (G) and quantification analysis (H) of main organs from cervical tumor-bearing nude mice after 48 h tail vein injection of PBS, SCPB@DOX NPs, SCPB@DOX@EM NPs, and SCPB@DOX@EM@FA NPs. Data are shown as mean standard deviation (SD), n = 6. (P value: ***P < 0.001).

Fig. 7. Synergetic therapy of HeLa tumor-bearing mice with SCPB@DOX@EM@FA NPs. (A) Time schedule of treatments and experiments. (B) Infrared thermography of HeLa-bearing mice that were tail vein injected with PBS, SCPB@DOX NPs, SCPB@DOX@EM NPs, and SCPB@DOX@EM@FA NPs. (C) Relative body weight and (D) relative tumor volume of cervical tumor-bearing mice with different treatment. (E) Representative photographs of cervical tumor-bearing mice and tumors for each tested group (with NIR laser: 808 nm, 0.8 W cm⁻²). H&E staining and TUNEL assay of the tumor tissues after different treatment (scale bar = 100 μm). *P < 0.1, **P < 0.01, ***P < 0.001. All data represent means ± SD (n = 3).