Enhanced anticancer activity of nanopreparation containing an MMP2-sensitive PEG-drug conjugate and cell-penetrating moiety

Abstract

In response to the challenges of cancer chemotherapeutics, including poor physicochemical properties, low tumor targeting, insufficient tumor cell internalization/bioavailability, and side effects, we developed a unique tumor-targeted micellar drug-delivery platform. Using paclitaxel as a model therapeutic, a nanopreparation composed of a matrix metalloproteinase 2 (MMP2)-sensitive self-assembly PEG 2000-paclitaxel conjugate (as a prodrug and MMP 2-sensitive moiety), transactivating transcriptional activator peptide-PEG1000-phosphoethanolamine (PE) (a cell-penetrating enhancer), and PEG1000-PE (a nanocarrier building block) was prepared. Several major drug delivery strategies, including self-assembly, PEGylation, the enhanced permeability and retention effect, stimulus sensitivity, a cell-penetrating moiety, and the concept of prodrug, were used in design of this nanoparticle in a collaborative manner. The nanopreparation allowed superior cell internalization, cytotoxicity, tumor targeting, and antitumor efficacy in vitro and in vivo over its nonsensitive counterpart, free paclitaxel and conventional micelles. This uniquely engineered nanoparticle has potential for effective intracellular delivery of drug into cancer cells.

Keywords: multifunctional; nanomedicine; non-small cell lung cancer; polymer-drug conjugate; polymeric micelles.

Fig. 1.

Drug delivery strategy and characterization of the MMP2-sensitive nanopreparation. (A) Drug delivery strategy. (B) ¹H-NMR of PEG2000-peptide-PTX. Both CDCl₃ (blue) and D₂O (red) were used to determine the chemical structure and nanostructure of the PTX conjugate. (C) TEM. The particle size and morphology of the nanopreparations were analyzed by TEM using negative staining with 1% phosphotungstic acid (PTA). (D) Enzymatic cleavage. To determine the digestion of PEG2000-peptide-PTX (Left) and its nanopreparation (Right), the samples were treated with 5 ng/µL MMP2 followed by TLC and Dragendorff’s reagent staining.

Fig. 2.

In vitro evaluation of paclitaxel conjugate and its nanopreparation. (A) Cytotoxicity of PEG2000-peptide-PTX in A549 and H9C2 cells. The cytotoxicity of monolayer cells was determined by CellTiter-Blue Cell Viability Assay after 72-h treatments. (B) Apoptosis analysis. The apoptosis of A549 cells was determined by FACS using annexin V/propidium iodide double staining after 72-h treatments. (C) Cellular uptake in A549 cell monolayer. Cells were treated with NBD-PE–labeled formulations (green) for 2 h before measurement. For FACS (Left), cells were trypsinized and collected. For confocal microscopy (Right), cells were fixed and stained with Hoechst 33342. (D) Cytotoxicity of the nanopreparations in A549 cell monolayer. Cells were treated with moderate to low doses of PTX formulations for 72 h before CellTiter-Blue Cell Viability Assay. (E) Penetration of the nanopreparations in A549 spheroids. The spheroids were treated with rhodamine-PE-labeled formulations for 2 h before confocal microscopy (a–h). The sections from c and d were stained by Hoechst 33342 (i and j). (a), PEG1000-PE. (b) TATp-PEG1000-PE. (c and d) TATp-PEG1000-PE/PEG2000-peptide-PTX. (e and f) PEG1000-PE/PEG2000-peptide-PTX. (g and h) PEG2000-peptide-PTX. (i and j) TATp-PEG1000-PE/PEG2000-peptide-PTX. (F) Cytotoxicity of the nanopreparations in A549 spheroids. The spheroids were treated with PTX formulations at the dose of 29.5 ng/mL every other day for 6 d, and the cytotoxicity was estimated by the LDH release.

Fig. 3.

In vivo tumor targeting and antitumor efficacy. (A) In vivo cell internalization. HBSS, the rhodamine-labeled nanopreparation, and its nonsensitive counterpart were injected i.v. in tumor-bearing mice at 5 mg/kg PTX, respectively. At 2 h after injection, tumor and major organs were collected. The cells were dissociated from fresh tissue and analyzed immediately by FACS. (B) Intratumor localization. Tumor sections were stained by Hoechst 33342 and detected by confocal microscopy. (C) Tumor growth inhibition (% of the starting tumor volume). Tumor size was measured every 3 d and calculated as V = lw²/2. *P < 0.05 compared with other groups. (D) Tumor cell apoptosis. Tumor sections were stained by Hoechst 33342, and apoptosis was analyzed by TUNEL assay under confocal microscopy.

Fig. 4.

In vivo side toxicity assessment. (A) Mouse body weight (% of starting body weight). (B) White blood cell counts. At the end of the experiment, white blood cells were counted by a hemocytometer. (C) Activity of ALT and AST. (D) H&E staining.