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:

• matrix metalloproteinases
• stimuli responsive
• nanocarriers
• TPGS
• FRET

Supplementary materials

Figure S1 Chemical structure of TPGS.

Abbreviations: TPGS, d-α-tocopheryl polyethylene glycol 1000 succinate; PEG1000, methoxy-polyethylene glycol (MW 1,000 Da); α-TOS, d-α-tocopheryl succinate; MW, molecular weight.

Figure S2 ¹H NMR spectrum of α-TOS (A), mPEG2K-NH₂ (B), and T2K (C) in CDCl₃.

Note: (a, b, c) are the key protons on the molecule that were labeled for ¹H-NMR analysis.

Abbreviations: ¹H-NMR, ¹H-nuclear magnetic resonance spectroscopy; α-TOS, d-α-tocopheryl succinate; T2K, mPEG2K-α-TOS conjugate; α-TOS, α-tocopherol succinate.

Figure S3 RP-HPLC of TPGS, T2K, and TGK.

Notes: Mobile phase, methanol with 3.2% acetic acid:water =95:5; wavelength: 285 nm.

Abbreviations: RP-HPLC, reverse-phase high-performance liquid chromatography; TPGS, d-α-tocopheryl polyethylene glycol 1000 succinate; TGK, mPEG2K-GK8-α-TOS conjugate; T2K, mPEG2K-α-TOS conjugate; α-TOS, α-tocopherol succinate; Min, minutes; GK8, GPVGLIGK-NH2 peptide.

Figure S4 Cellular uptake of DTX, T2K, and TGK micelles in the presence of NaN₃ (ATP depletion agent) and GM6001 (MMPs inhibitor) in HT1080 cells.

Notes: Values are expressed as mean ± SD (n=3). HT1080 is the human fibrosarcoma cell line.

Abbreviations: T2K micelles, micelles composed of TPGS/T2K (n:n =40:60) loaded with DTX; TGK micelles, micelles composed of TPGS/TGK (n:n =40:60) loaded with DTX; DTX, docetaxel; SD, standard deviation; TPGS, d-α-tocopheryl polyethylene glycol 1000 succinate; MMP, matrix metalloproteinase; ATP, adenosine triphosphate; ns, no significance.

Figure S5 Cytotoxicity of blank T2K and TGK micelles in HUVEC cells at 48 hours with Tween 80 used as control.

Note: Values are expressed as mean ± SD (n=3).

Abbreviations: HUVEC, human umbilical vein endothelial cells; 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.

Figure S6 Cytotoxicity of DTX, TGK, and T2K micelles in the presence of 10 nM GM6001 in HT1080 cells at 48 hours.

Notes: Values are expressed as mean ± SD (n=3). HT1080 is the human fibrosarcoma cell line.

Abbreviations: T2K micelles, micelles composed of TPGS/T2K (n:n =40:60) loaded with DTX; TGK micelles, micelles composed of TPGS/TGK (n:n =40:60) loaded with DTX; DTX, docetaxel; SD, standard deviation; TPGS, d-α-tocopheryl polyethylene glycol 1000 succinate.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81373354).

Disclosure

The authors report no conflicts of interest in this work.