Abstract:

Multi-drug delivery focuses on different signaling pathways in cancer cells and has synergistic antiproliferative effects. In this manuscript, we developed folic acid (FA)-conjugated polymeric multi-drug nanoparticles (FA-PMDNPs) consisting of poly-L-lysine (PLL) and poly glutamic-conjugated PTX/GEM (PGA-PTX and PGA-GEM) for FA receptor-targeted synergistic breast cancer therapy. The carboxyl-rich structure of PGA provided plenty reaction sites and negative charge for drug loading. Transmission electron microscopy (TEM) results showed that FA-PMDNPs had uniform particle size and spherical morphology. The hemolysis study proved that FA-PMDNPs had good biocompatibility. In vitro cell viability and in vivo studies showed that FA-PMDNPs more effectively inhibited the proliferation of FA receptor (FR)-overexpressing breast cancer cells (4T1) than the pure drugs.Consequently, these results demonstrated that FA-PMDNPs could be effectively targeted at cancer cells compared with free drugs, indicating their strong potential as efficient multi-drug-carrying nano-platforms for cancer treatment. 

Key words: FA-receptor targeted, Polymeric nanoparticles, Combined chemotherapy, Breast cancer, Drug targeted delivery

CLC Number: 

• R944

Supporting:

Supporting Information

1. Synthesis of poly (L-lysine) (PLL)

N6-Cbz-L-Lysine (5.0 g, 17.8 mmol) in THF (65 mL) was reacted with triphosgene (2.1 g, 7.2 mmol) and the reaction was allowed at 50 ºC for 3 h under nitrogen^[1]. Lys (Z)-NCA was precipitated with the addition of cold n-hexane in the reaction and the product was filtered and dried at room temperature. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.11 (1H，α-NH), 7.34 (5H, Ph), 5.00 (2H, CH₂Ph), 4.42 (1H, -CH), 2.98 (2H, -CH₂), 1.69 (2H, -CH₂), 1.4 (2H, -CH₂), 1.28 (2H, -CH₂). FT-IR (cm-1): 943 (O=C-O-C=O in the NCA), 1688 (C=O in the Cbz group), 1774, 1812 (C=O in the NCA), 1250 (C-O in the Cbz group), 740 cm⁻¹, 697 cm⁻¹ (the deformation vibration of benzene ring).

Lys (Z)-NCA (5.1 g, 16.6 mmol) was dissolved in DMF (25 mL) under nitrogen. Hexylamine (55.6 mg, 0.6 mmol) dissolved in DMF (1 mL) was injected into the Lys (Z)-NCA solution and the reaction was allowed at 35 ºC for 72 h under nitrogen. Then PLL (Z) was precipitated with the addition of ice-cold methyl tert-butyl ether and the product was filtered and dried at room temperature. ¹H NMR (400 MHz, DMSO-d₆) δ: 7.32 (ArH in the Cbz group), 4.99 (-CH₂ in the Cbz group) and 0.82 (-CH₃ in hexylamine). FT-IR (cm⁻¹): 1692, 1627, 1537, 1250, 740, 697.

PLL (Z) (3.6 g) was dissolved in HBr (13.2 g, 33 wt% in acetic acid) and the reaction was stirred at room temperature for 2 h. Then, PLL was precipitated with the addition of ice-cold methyl tert-butyl ether in the reaction and the product was filtered and dried at room temperature. ¹H NMR (400 MHz, D₂O) δ: 4.27 (-CH), 1.13-1.84 (-CH₂ in PLL and hexylamine) 0.82 (-CH₃ in hexylamine). FT-IR (cm^−-1): 1632, 1541.

2. Synthesis of poly (L-glutamic) acid (PGA)

L-Glutamic acid-5-(phenylmethyl) ester (5.0 g, 21.1 mmol) in THF (65 mL) was reacted with triphosgene (2.5 g, 8.4 mmol) and the reaction was allowed at 50 ºC for 3 h under nitrogen^[2]. GluB-NCA was precipitated with the addition of ice-cold n-hexane in the reaction and the product was filtered and dried at room temperature. ¹H NMR (400 MHz, DMSO-d₆) δ: 9.12 (1H, α-NH), 7.48–7.23 (5H, Ph), 5.10 (2H, -CH₂Ph), 4.45 (1H, -CH), 2.53 (2H, -CH₂), 1.99 (2H, -CH₂). FT-IR (cm⁻¹): 927 (O=C-O-C=O in the NCA), 1703 (C=O in the Cbz group), 1780 (C=O in the NCA), 744 cm⁻¹, 696 cm⁻¹ (the deformation vibration of benzene ring).

GluB-NCA (4.5 g, 17.2 mmol) was dissolved in DMF (25 mL) under nitrogen. Hexylamine (31. 6 mg, 0.3 mmol) dissolved in DMF (1 mL) was injected into the GluB-NCA solution and the reaction was allowed at 35 ^oC for 72 h under nitrogen. Then PBLG was precipitated with the addition of ice-cold methyl tert-butyl ether in the reaction and the product was filtered and dried at room temperature. ¹H NMR (400 MHz, DMSO-d₆) δ: 7.24 (Ph in the Cbz), 5.03 (-CH₂ in the Cbz), 3.93 (-CH), 0.81 (-CH₃ in hexylamine). FT-IR (cm⁻¹): 3292, 1652, 1550, 740, 690.

PBLG (3.4 g) dissolved in methanol (20 mL) was added to sodium hydroxide solution (3 mL, 10 M). The reaction was allowed at room temperature for 3 h. Then, the reaction solution was filtered and dried at room temperature to get a white solid. The solid was stirred in 5 mL of EA/HCl for 5 min and solvent was removed in vacuo to give the title compound. ¹H NMR (400 MHz, D₂O) δ: 12.13 (-COOH), 4.24 (-CH), 0.84 (-CH₃ in hexylamine). FT-IR (cm⁻¹): 3292, 1718, 1635, 1539.

 

 

Scheme S1. Synthesis of the PLL.

 

 

Scheme S2. Synthesis of the PGA.

 

 

Figure S1. Characterization of the compounds. (A) FT-IR spectra of Lys-NCA, PLL(Z) and PLL. (B) ¹H NMR spectra of Lys-NCA, PLL(Z) and PLL. (C) FT-IR spectra of BLG-NCA, PBLG and PGA. (D) ¹H NMR spectra of BLG-NCA, PBLG and PGA.

 

 

Scheme S3. Synthesis of the FA-OH.

 

 

Scheme S4. Synthesis of PGA-PTX, PGA-GEM and PGA-FA.

 

 

Figure S2. FT-IR spectra and ¹H NMR spectra of PGA-PTX, PGA-GEM and PGA-FA.

 

 

Figure S3. Hydration particle size (A), and zeta potential (B) of different mass ratios in FA-PMDNPs.

References

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[2] Lee D, Choe K, Jeong Y, et al. Establishment of a controlled insulin delivery system using a glucose-responsive double-layered nanogel. RSC Advances, 2015, 5(19): 14482-14491.