Tumor microenvironment dual-responsive core-shell nanoparticles with hyaluronic acid-shield for efficient co-delivery of doxorubicin and plasmid DNA

Abstract

As the tumor microenvironment (TME) develops, it is critical to take the alterations of pH value, reduction and various enzymes of the TME into consideration when constructing the desirable co-delivery systems. Herein, TME pH and enzyme dual-responsive core-shell nanoparticles were prepared for the efficient co-delivery of chemotherapy drug and plasmid DNA (pDNA). A novel pH-responsive, positively charged drug loading material, doxorubicin (DOX)-4-hydrazinobenzoic acid (HBA)-polyethyleneimine (PEI) conjugate (DOX-HBA-PEI, DHP), was synthesized to fabricate positively charged polyion complex inner core DHP/DNA nanoparticles (DDN). Hyaluronic acid (HA) was an enzyme-responsive shell which could protect the core and enhance the co-delivery efficiency through CD44-mediated endocytosis. The HA-shielded pH and enzyme dual-responsive nanoparticles (HDDN) were spherical with narrow distribution. The particle size of HDDN was 148.3±3.88 nm and the zeta potential was changed to negative (-18.1±2.03 mV), which led to decreased cytotoxicity. The cumulative release of DOX from DHP at pH 5.0 (66.4%) was higher than that at pH 7.4 (30.1%), which indicated the pH sensitivity of DHP. The transfection efficiency of HDDN in 10% serum was equal to that in the absence of serum, while the transfection of DDN was significantly decreased in the presence of 10% serum. Furthermore, cellular uptake studies and co-localization assay showed that HDDN were internalized effectively through CD44-mediated endocytosis in the tumor cells. The efficient co-delivery of DOX and pEGFP was confirmed by fluorescent image taken by laser confocal microscope. It can be concluded that TME dual-responsive HA-shielded core-shell nanoparticles could be considered as a promising platform for the co-delivery of chemotherapy drug and pDNA.

Keywords: CD44 targeted; co-delivery; core–shell nanoparticles; hyaluronic acid; pH sensitive.

Figure 1

The structure confirmation of synthesized materials. Notes: (A) ¹H NMR of HBA-PEI. (B) ¹H NMR of DOX-HBA-PEI. (C) FTIR spectra of PEI and HBA-PEI. (D) FTIR spectra of DOX and DOX-HBA-PEI. Abbreviations: DOX, doxorubicin; FTIR, Fourier transform infrared; ¹H NMR, proton nuclear magnetic resonance; HBA, hydrazinobenzoic acid; PEI, polyethyleneimine.

Figure 2

Cytotoxicity of free DOX, PEI, HP and DHP against (A) HepG2 and (B) B16, respectively. (C) In vitro DOX release from DHP at pH 7.4, 6.0 and 5.0. Note: **P<0.01. Abbreviations: DHP, DOX-HBA-PEI; DOX, doxorubicin; HBA, hydrazinobenzoic acid; HP, HBA-PEI; PEI, polyethyleneimine.

Figure 3

The characterization of DDN. Notes: (A) The DNA retardation assay of DDN at various weight ratios of DHP to DNA: 1) naked DNA; 2) 30:512; 3) 30:256; 4) 30:128; 5) 30:64; 6) 30:32; 7) 30:16; 8) 30:8; 9) 30:4; 10) 30:2. (B and C) Size distribution and morphology of DDN. Abbreviations: DDN, DHP/DNA nanoparticles; DHP, doxorubicin-4-hydrazinobenzoic acid-polyethyleneimine conjugate.

Figure 4

Transfection of nanoparticles in vitro. Notes: (A) Fluorescent micrographs of transfection of DDN at different ratios. (B and C) Flow cytometric histogram profiles of fluorescence intensity of DDN in HepG2 cells and B16 cells, respectively. (D) Transfection efficiency of DDN in the two kinds of cells following 48 h of incubation, with PEI as control. (E) Transfection efficiency of HDDN in the presence or absence of 10% FBS following 48 h of incubation. Note: *P<0.05. Abbreviations: DDN, doxorubicin-4-hydrazinobenzoic acid-polyethyleneimine conjugate/DNA nanoparticles; FBS, fetal bovine serum; HDDN, hyaluronic acid-shielded pH and enzyme dual-responsive nanoparticles; PEI, polyethyleneimine.

Figure 5

The characterization of HDDN. Notes: (A) The DNA retardation assay of HDDN at various weight ratios of HA to DNA: 1) DNA; 2) DDN; 3) 20:8; 4) 30:8; 5) 40:8; 6) 50:8; 7) 60:8; 8) 70:8; 9) 80:8. (B and C) Size distribution and morphology of HDDN. Abbreviations: DDN, doxorubicin-4-hydrazinobenzoic acid-polyethyleneimine conjugate/DNA nanoparticles; HA, hyaluronic acid; HDDN, hyaluronic acid-shielded pH and enzyme dual-responsive nanoparticles.

Figure 6

Stability of HDDN in serum, DNase I and Hyals. Notes: (A) The changes in size and zeta potential of HDDN incubated with different concentrations of Hyals (0 and 120 units/mL) for 1 h. The lines and the columns represent the changes in zeta potential and size, respectively. (B) The stability of HDDN in the presence or absence of 20% serum for 24 h. The lines and the columns represent the changes in zeta potential and size, respectively. (C and D) The stability of DDN and HDDN after incubation with DNase I for 12 h. Abbreviations: DDN, doxorubicin-4-hydrazinobenzoic acid-polyethyleneimine conjugate/DNA nanoparticles; HDDN, hyaluronic acid-shielded pH and enzyme dual-responsive nanoparticles; Hyals, hyaluronidases.

Figure 7

In vitro toxicity of DDN and HDDN in (A) HepG2 and (B) B16 cells. Abbreviations: DDN, doxorubicin-4-hydrazinobenzoic acid-polyethyleneimine conjugate/DNA nanoparticles; DOX, doxorubicin; HDDN, hyaluronic acid-shielded pH and enzyme dual-responsive nanoparticles.

Figure 8

Cellular uptake of nanoparticles. Notes: (A) Fluorescent micrographs and (B) quantitative data of cellular uptake in HepG2, B16 and NIH 3T3 cells after 4 h of incubation with DOX, DDN and HDDN, in which the concentration of DOX was 20 μg/mL. (C) Mean fluorescence intensity of HDDN after incubation with different inhibitors of internalization. *P<0.05, **P<0.01. Abbreviations: DDN, doxorubicin-4-hydrazinobenzoic acid-polyethyleneimine conjugate/DNA nanoparticles; DHP, DOX-4-hydrazinobenzoic acid-polyethyleneimine conjugate; DOX, doxorubicin; HDDN, hyaluronic acid-shielded pH and enzyme dual-responsive nanoparticles; MFI, mean fluorescence intensity.

Figure 9

Co-localization of CD44 and HDDN in CD44-positive cells at different time points after incubation with HDDN. Notes: In the figure at 0.5, 1 and 2 h, the white arrows indicated the co-localization of CD44 and HDDN. While in the figure at 4 h, the white arrows indicate the co-localization of DOX and nucleus. Abbreviations: DOX, doxorubicin; HDDN, hyaluronic acid-shielded pH and enzyme dual-responsive nanoparticles.

Figure 10

Co-delivery of DOX and EGFP plasmid by HDDN into B16 (A–D) and HepG2 (a–d) cells. Notes: A and a indicate the uptake of DOX (red), B and b represent EGFP transfection (green), C and c are the nuclear region of cells, D and d are the merge images of A, B and C or a, b and c. The white arrow indicates the co-localization of DOX and EGFP (yellow). Abbreviations: DOX, doxorubicin; EGFP, enhanced green fluorescent protein; HDDN, hyaluronic acid-shielded pH and enzyme dual-responsive nanoparticles.

Scheme 1

The scheme shows the synthesis of drug loading material DHP and the rational design of the pH and enzyme dual-sensitive hyaluronic acid-coated nanoparticles for the co-delivery of DOX and pEGFP. When arriving at the tumor tissue, HDDN is disassembled by Hyal2 in the tumor microenvironment and internalized through CD44-mediated endocytosis. In the lysosome, HDDN are further degraded by Hyal1 and other enzymes to expose the DDN. Because of the acidic condition in the lysosome, hydrazone bonds are broken and DHP releases DOX. With the sponge effect of PEI, DOX and gene escape from the lysosome and carry out the antitumor effect. Abbreviations: DDN, DHP/DNA nanoparticles; DHP, doxorubicin-4-hydrazinobenzoic acid-polyethyleneimine conjugate; DOX, doxorubicin; HA, hyaluronic acid; HBA, hydrazinobenzoic acid; HDDN, hyaluronic acid-shielded nanoparticles; PEI, polyethyleneimine.

Scheme 2

Synthesis of DOX-HBA-PEI. Abbreviations: DOX, doxorubicin; HBA, hydrazinobenzoic acid; PEI, polyethyleneimine.