Design of pH-sensitive methotrexate prodrug-targeted curcumin nanoparticles for efficient dual-drug delivery and combination cancer therapy

Abstract

Aim: We designed acid-labile methotrexate (MTX) targeting prodrug self-assembling nanoparticles loaded with curcumin (CUR) drug for simultaneous delivery of multi-chemotherapeutic drugs and combination cancer therapy.

Methods: A dual-acting MTX, acting as both an anticancer drug and as a tumor-targeting ligand, was coupled to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[aldehyde(polyethylene glycol)-2000] via Schiff's base reaction. The synthesized prodrug conjugate (DSPE-PEG-Imine-MTX) could be self-assembled into micellar nanoparticles (MTX-Imine-M) in aqueous solution, which encapsulated CUR into their core by hydrophobic interactions (MTX-Imine-M-CUR).

Results: The prepared MTX-Imine-M-CUR nanoparticles were composed of an inner hydrophobic DSPE/CUR core and an outside hydrophilic bishydroxyl poly (ethyleneglycol) (PEG) shell with a self-targeting MTX prodrug corona. The imine linker between 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[aldehyde(polyethyleneglycol)-2000] and MTX, as a dynamic covalent bond, was strong enough to remain intact in physiological pH, even though it is rapidly cleaved in acidic pH. The MTX-Imine-M-CUR could codeliver MTX and CUR selectively and efficiently into the cancer cells via folate receptor-mediated endocytosis followed by the rapid intracellular release of CUR and the active form of MTX via the acidity of endosomes/lysosomes. Moreover, the MTX-Imine-M-CUR resulted in significantly higher in vitro and in vivo anticancer activity than pH-insensitive DSPE-PEGAmide-MTX assembling nanoparticles loaded with CUR (MTX-Amide-M-CUR), MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR (M-CUR), combination of both free drugs, and individual free drugs.

Conclusion: The smart system provided a simple, yet feasible, drug delivery strategy for targeted combination chemotherapy.

Keywords: combination therapy; nanoparticles; pH-sensitive prodrug; self-assembly; targeting.

Figure 1

(A) Synthetic route of DSPE-PEG-Imine-MTX conjugate via imine reaction between the aldehyde group of DSPE-PEG-CHO and the aromatic amino group of MTX. (B) Schematic representation of preparation of MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR (M-CUR), DSPE-PEG-Amide-MTX nanoparticles (MTX-Amide-M-CUR), and DSPE-PEG-Imine-MTX nanoparticles (MTX-Imine-M-CUR). (C) Schematic representation of active selective cellular uptake via folate receptor-mediated endocytosis, pH-controlled intracellular dual-drug release, and combination therapy of MTX-Imine-M-CUR nanoparticles after passive tumor accumulation by EPR effect. Abbreviations: AcOH, acetic acid; CHO, aldehyde group; CUR, curcumin; DHFR, dihydrofolate reductase; DMSO, dimethyl sulfoxide; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; EPR, enhanced permeability and retention; MTX, methotrexate.

Figure 2

Hydrodynamic size distribution, zeta potential, and TEM images of (A) M-CUR, (B) MTX-Amide-M-CUR, and (C) MTX-Imine-M-CUR. (D) XRD spectra of CUR, MTX-Amide-M nanocarriers, MTX-Amide-M nanocarriers/CUR mixture, MTX-Amide-M-CUR nanosystems, MTX-Imine-M nanocarriers, MTX-Imine-M nanocarriers/CUR mixture, and MTX-Imine-M-CUR nanosystems. (E) In vitro release profiles of CUR from M-CUR, MTX-Amide-M-CUR, and MTX-Imine-M-CUR in PBS buffer (pH 7.4 and 5.0) at 37°C. (F) In vitro release profiles of MTX from MTX-Amide-M-CUR and MTX-Imine-M-CUR in PBS buffer (pH 7.4 and 5.0) at 37°C. Abbreviations: CUR, curcumin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate; PBS, phosphate buffer saline; TEM, transmission electron microscope; XRD, X-ray diffractometer.

Figure 3

CLSM images of folate receptor-overexpressing (A) HeLa and (B) MCF-7 cells incubated with free CUR, M-CUR, and MTX-Imine-M-CUR for 0.5 and 2 h. Notes: DAPI (false-color blue) was used to identify the nucleus. Scale bars are 15 µm. Abbreviations: CLSM, confocal laser scanning microscopy; CUR, curcumin; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate.

Figure 4

(A) CLSM images, (B) flow cytometry profiles, and (C) mean fluorescence intensity of HeLa cells incubated with free CUR, M-CUR, MTX-Amide-M-CUR (without/with FA pretreatment), or MTX-Imine-M-CUR (without/with FA pretreatment) for 2 h. Error bars indicate SD (n=4). *P<0.05. (D) Subcellular location of MTX-Imine-M-CUR in HeLa cells after incubation for 1 h. Notes: DAPI (false-color blue) and LysoTracker Red (false-color red) were used to identify the nucleus and lysosomes, respectively. Scale bars are 20 µm. Abbreviations: CLSM, confocal laser scanning microscopy; CUR, curcumin; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; FA, folic acid; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate; NA, no answer.

Figure 5

(A) Intracellular drug/nanocarriers distribution of HeLa cells treated with MTX-Imine-M-CUR for 1 and 6 h. DSPE-PEG-Cy5.5 (false-color red) was used to label nanocarriers. (B, C) In vitro cell viability of (B) HeLa cells and (C) MCF-7 cells incubated with free CUR, free CUR/MTX mixture, M-CUR nanosystems, MTX-Amide-M-CUR nanosystems, or MTX-Imine-M-CUR nanosystems for 24 h. (D, E) In vitro cell viability of (D) HeLa and (E) MCF-7 cells incubated with MTX-Imine-M-CUR nanosystems without/with FA pretreatment for 24 h. Note: Error bars indicate SD (n=4). Abbreviations: C, cytoplasm; CUR, curcumin; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; FA, folic acid; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate; N, nucleus.

Figure 6

In vivo antitumor efficacy of HeLa tumor-bearing nude mice after intravenous injection of 0.9% NaCl, free CUR, free CUR/MTX, M-CUR, MTX-Amide-M-CUR, or MTX-Imine-M-CUR at an equivalent dose of CUR (8 mg/kg). Notes: (A) Tumor volumes changes, (B) body weight changes, (C) isolated tumor weight, and (D) representative H&E staining histologic images from the tumor tissues. Error bars indicate SD (n=5). *P<0.05. Abbreviations: CUR, curcumin; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000]; M-CUR, MTX unconjugated DSPE-PEG assembling micellar nanoparticles loaded with CUR; MTX, methotrexate.