Matrix metalloproteinases-2/9-sensitive peptide-conjugated polymer micelles for site-specific release of drugs and enhancing tumor accumulation: preparation and in vitro and in vivo evaluation

Abstract

Since elevated expression of matrix metalloproteinase (MMP)-2 and MMP-9 is commonly observed in several malignant tumors, MMPs have been widely reported as key factors in the design of drug delivery systems. Several strategies have been proposed to develop MMPs-responsive nanoparticles to deliver chemotherapeutics to malignant solid tumors. A stimuli-responsive drug delivery system, which could be cleaved by MMPs, was proposed in this study. By inserting an MMP-2/9 cleavable oligopeptide GPVGLIGK-NH2 (GK8) as spacer between α-tocopherol succinate (α-TOS) and methoxy-polyethylene glycol molecular weight (MW 2000 Da) activated by N-hydroxysuccinimide (mPEG2K-NHS), mPEG2K-GK8-α-TOS (TGK) was synthesized as the primary ingredient for MMP-2/9-sensitive micelles composed of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) and TGK (n:n =40:60, TGK micelles). mPEG2K-α-TOS (T2K) was similarly synthesized as nonsensitive control. The TGK micelles showed better stability than nonsensitive micelles composed of TPGS and T2K (n:n =40:60, T2K micelles) owing to the inserted peptide. Fluorescence resonance energy transfer results indicated that TGK micelles could be successfully cleaved by MMP-2/9. Effective drug release was demonstrated in the presence of collagenase type IV, a mixture of MMP-2 and MMP-9. Compared with nonsensitive micelles, docetaxel (DTX)-loaded TGK micelles showed a fold higher cellular uptake in HT1080 cells. While the half-maximal inhibitory concentration (IC50) of TGK and T2K micelles were similar (P>0.05) in MCF-7 cells (MMP-2/9 underexpression), the IC50 values of the aforementioned micelles were 0.064±0.006 and 0.122±0.009 μg/mL, respectively, in HT1080 cells (MMP-2/9 overexpression). The MMP-2/9-sensitive micelles also demonstrated desired tumor targeting and accumulation ability in vivo. The results of in vivo antitumor effect evaluation indicate that TGK micelles are potent against solid tumors while maintaining minimum systemic toxicity compared with T2K micelles and DTX.

Keywords: FRET; TPGS; matrix metalloproteinases; nanocarriers; stimuli responsive.

Figure 1

Synthetic routes of TGK (A) and T2K (B). Abbreviations: mPEG2K-NHS, methoxy-polyethylene glycol (MW 2,000 Da) activated by N-hydroxysuccinimide; GK8, GPVGLIGK-NH₂ peptide; α-TOS, d-α-tocopheryl succinate; TCTU, O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′,-tetramethyluronium tetrafluoroborate; DMF, dimethylformamide; TEA, triethylamine; TGK, mPEG2K-GK8-α-TOS conjugate; T2K, mPEG2K-α-TOS conjugate; MW, molecular weight.

Figure 2

¹H NMR spectra of mPEG2K-NHS (A), GK-8 (B), mPEG2K-GK8 (C), α-TOS (D) and TGK (E) in CDCl₃. Note: (a, b, c, d, e, f, g, j, h) are the key proton and their signals on the molecule labeled as reference for ¹H-NMR analysis. Abbreviations: ¹H-NMR, ¹H-nuclear magnetic resonance spectroscopy; GK8, GPVGLIGK-NH₂ peptide; mPEG2K-NHS, methoxy-polyethylene glycol (MW 2,000 Da) activated by N-hydroxysuccinimide; TGK, mPEG2K-GK8-α-TOS conjugate; α-TOS, α-tocopherol succinate; MW, molecular weight.

Figure 3

Characteristics of TGK micelles and T2K micelles. Notes: Size distribution of DTX-loaded TGK micelles (A) and T2K micelles (B). (C) Plot of UV absorption of I₂ versus concentration of TPGS/T2K (n:n =40:60) and TPGS/TGK (n:n =40:60). CMC value was calculated by corresponding polymer concentration at which a sharp increase in absorbance was observed. (D) Amount of DTX retained in T2K micelles and TGK micelles at corresponding time, in 48 hours. Stability was indicated by the percentage of DTX remaining in the micelles. Values are expressed as mean ± SD (n=3). Abbreviations: DTX, docetaxel; T2K micelles, micelles composed of TPGS/T2K (n:n =40:60); TGK micelles, micelles composed of TPGS/TGK (n:n =40:60); UV, ultraviolet; TPGS, d-α-tocopheryl polyethylene glycol 1000 succinate; TGK, mPEG2K-GK8-α-TOS conjugate; T2K, mPEG2K-α-TOS conjugate; α-TOS, α-tocopherol succinate; CMC, critical micelle concentration; SD, standard deviation; h, hour.

Figure 4

FRET analysis of DiO/DiI-loaded T2K micelles and TGK micelles. Notes: Emission spectra of DiO/DiI-loaded T2K micelles, TGK micelles, DiO/DiI-loaded T2K micelles and TGK micelles (A), and the ratio of IR/(IR+IG) incubated at 37°C for 2 hours with or without MMP-2/9 (B). Values are expressed as mean ± SD (n=3). **P<0.01. Abbreviations: T2K micelles, micelles composed of TPGS/T2K (n:n =40:60); TGK micelles, micelles composed of TPGS/TGK (n:n =40:60); DiO, 3,3′-dioctadecyloxacarbocyanine perchlorate; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; IG, fluorescence intensity at 510 nm; IR, fluorescence intensity at 565 nm; MMP, matrix metalloproteinase; TPGS, d-α-tocopheryl polyethylene glycol 1000 succinate; SD, standard deviation.

Figure 5

In vitro release profile of DTX-loaded micelles. Notes: In vitro release profile of DTX-loaded T2K micelles (A) and TGK micelles (B) in pH 7.4 PBS. Values are expressed as mean ± SD (n=3). Abbreviations: DTX, docetaxel; T2K micelles, micelles composed of TPGS/T2K (n:n =40:60); TGK micelles, micelles composed of TPGS/TGK (n:n =40:60); PBS, phosphate buffered saline; BSA, bovine serum albumin; MMP, matrix metalloproteinase; SD, standard deviation; TPGS, d-α-tocopheryl polyethylene glycol 1000 succinate; h, hour.

Figure 6

The cellular uptake of DTX and DTX-loaded T2K and TGK micelles. Notes: The cellular uptake of DTX and DTX-loaded T2K and TGK micelles in the presence of GM6001 and MMP-2/9 in MCF-7 (A) and HT1080 (B) cells. Values are expressed as mean ± SD (n=3); *P<0.05; **P<0.01. HT1080 is the human fibrosarcoma cell line, MCF-7 is the human breast adenocarcinoma cell line. Abbreviations: T2K micelles, micelles composed of TPGS/T2K (n:n =40:60); TGK micelles, micelles composed of TPGS/TGK (n:n =40:60); SD, standard deviation; TPGS, d-α-tocopheryl polyethylene glycol 1000 succinate; MMP, matrix metalloproteinase.

Figure 7

Cytotoxicity of micelles to cell lines expressing different level of MMPs. Notes: Cytotoxicity of blank T2K and TGK micelles in MCF-7 (A) and HT1080 (B) cells at 48 hours. Cytotoxicity of DTX-loaded T2K micelles and TGK micelles in MCF-7 cells (C) and HT1080 cells (D) at 48 hours. Tween 80 was used as control. Values are expressed as mean ± SD (n=3). HT1080 is the human fibrosarcoma cell line, MCF-7 is the human breast adenocarcinoma cell line. Abbreviations: DTX, docetaxel; T2K micelles, micelles composed of TPGS/T2K (n:n =40:60); TGK micelles, micelles composed of TPGS/TGK (n:n =40:60); SD, standard deviation; TPGS, d-α-tocopheryl polyethylene glycol 1000 succinate; MMPs, matrix metalloproteinases.

Figure 8

Subcellular location of coumarin-6-labeled T2K micelles and TGK micelles. Notes: Images were taken after incubation with micelles for 30 minutes. Blue, Hoechst 33342; green, coumarin-6-labeled micelles or released coumarin-6; red, LysoTracker Red. Original magnification, ×400. HT1080 is the human fibrosarcoma cell line, MCF-7 is the human breast adenocarcinoma cell line. Abbreviations: T2K micelles, coumarin-6-labeled micelles composed of TPGS/T2K (n:n =40:60); TGK micelles, coumarin-6-labeled micelles composed of TPGS/TGK (n:n =40:60); TPGS, d-α-tocopheryl polyethylene glycol 1000 succinate.

Figure 9

In vivo imaging studies for tumor targeting ability of MMP-2/9 sensitive micelles. Notes: (A) In vivo imaging of subcutaneous tumor-bearing nude mice after intravenous injection of DiR-labeled T2K micelles and TGK micelles at 2, 4, 8, 10, 12, and 24 hours postinjection, respectively. Tumor-bearing nude mice injected with saline (left of each group) were used as control. (B) Images of dissected organs of subcutaneous tumor-bearing nude mice executed at 24 hours after intravenous injection of DiR-labeled T2K micelles and TGK micelles. (C) Fluorescence intensity normalized with weights of DiR-labeled T2K micelles and TGK micelles in various organs. Values are expressed as mean ± SD (n=3); *P<0.05; **P<0.01. Abbreviations: DiR, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine; DiR T2K micelles, DiR-labeled micelles composed of TPGS/T2K (n:n =40:60); DiR TGK micelles, DiR-labeled micelles composed of TPGS/TGK (n:n =40:60); SD, standard deviation; TPGS, d-α-tocopheryl polyethylene glycol 1000 succinate.

Figure 10

The antitumor efficacy against subcutaneous tumor-bearing nude mice; 10 mg/kg DTX or equivalent T2K or TGK micelles was administrated at day 1, day 4, and day 7. Notes: (A) The relative tumor volume–time curve. (B) The body weight change after administration of DTX, TGK micelles, or T2K micelles. (C) Photographs of tumors from each group. (D) The weights of excised tumors from subcutaneous tumor-bearing nude mice at the time of execution. Values are expressed as mean ± SD (n=5). *P<0.05. Abbreviations: DTX, docetaxel; T2K micelles, micelles composed of TPGS/T2K (n:n =40:60); TGK micelles, micelles composed of TPGS/TGK (n:n =40:60); SD, standard deviation; TPGS, d-α-tocopheryl polyethylene glycol 1000 succinate.