Physicochemical Properties of A New PEGylated Polybenzofulvene Brush for Drug Encapsulation

Abstract

A new polymer brush was synthesized by spontaneous polymerization of benzofulvene macromonomer 6-MOEG-9-T-BF3k bearing a nona(ethylene glycol) side chain linked to the 3-phenylindene scaffold by means of a triazole heterocycle. The polymer structure was studied by SEC-MALS, NMR spectroscopy, and MALDI-TOF MS techniques, and the results supported the role of oligomeric initiatory species in the spontaneous polymerization of polybenzofulvene derivatives. The aggregation features of high molecular weight poly-6-MOEG-9-T-BF3k-FE were investigated by pyrene fluorescence analysis, dynamic light scattering studies, and transmission electron microscopy, which suggested a tendency towards the formation of spherical objects showing dimensions in the range of 20-200 nm. Moreover, poly-6-MOEG-9-T-BF3k-FE showed an interesting cytocompatibility in the whole concentration range tested that, besides its aggregation features, makes this polybenzofulvene brush a good polymer candidate for nanoencapsulation and delivery of drug molecules. Finally, the photo-physical features of poly-6-MOEG-9-T-BF3k-FE could allow the biodistribution of the resulting drug delivery systems to be monitored by fluorescence microscopy techniques.

Keywords: PEGylation; affinity polymerization; drug delivery systems; grafting through; nanocarrier; polybenzofulvene; spontaneous polymerization.

Figure 1

The previously proposed aggregation mechanism in PEGylated polybenzofulvene brush (PPBFB) derivatives.

Figure 2

Design of PPBFB poly-6-MOEG-9-T-BF3k from poly-6-MOEG-9-TM-BF3k.

Scheme 1

“Grafting through” approach to PPBFB derivative poly-6-MOEG-9-T-BF3k. Reagents: (i) ethynyltrimethylsilane, CuI, Pd(PPh₃)₂Cl₂, TBAI, TEA, DMF; (ii) C₂H₅ONa, C₂H₅OH; (iii) CH₃(OCH₂CH₂)₉N₃, CuBr, DIPEA, THF; (iv) Al(CH₃)₃, CH₂Cl₂; (v) Procedure A: PTSA, TFA, CDCl₃; Procedure B: PTSA, CD₃CN; (vi) solvent elimination (for the details see the Materials and Methods section).

Figure 3

Structure of indenone derivative 3 found by crystallography. Ellipsoids enclose 50% probability.

Figure 4

Comparison of the ¹H NMR spectra (500 MHz) of monomer 6-MOEG-9-T-BF3k acquired respectively in CDCl₃ (bottom trace) and in CD₃CN (top trace).

Figure 5

Comparison of the ¹³C NMR spectra (125 MHz) of monomer 6-MOEG-9-T-BF3k acquired respectively in CDCl₃ (bottom trace) and in CD₃CN (top trace).

Figure 6

¹H NMR spectra performed during the transferring of the benzofulvene monomer 6-MOEG-9-T-BF3k in the water environment. Comparison of ¹H NMR spectrum of the benzofulvene monomer in CD₃CN (bottom trace) with that of the same monomer after diluting (1:1) with D₂O (middle trace) the initial monomer solution and after the partial evaporation of the organic solvent (top trace).

Figure 7

Comparison of the ¹H NMR spectrum of monomer 6-MOEG-9-T-BF3k (500 MHz, CDCl₃, top trace) with those of the corresponding polymers: poly-6-MOEG-9-T-BF3k-WA, poly-6-MOEG-9-T-BF3k-FE, and poly-6-MOEG-9-T-BF3k-SE.

Figure 8

Comparison of the ¹³C NMR spectrum of monomer 6-MOEG-9-T-BF3k (125 MHz, CDCl₃, top trace) with those of the corresponding polymers: poly-6-MOEG-9-T-BF3k-WA, poly-6-MOEG-9-T-BF3k-FE, and poly-6-MOEG-9-T-BF3k-SE.

Figure 9

Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum of poly-6-MOEG-9-T-BF3k-FE (i.e., the sample obtained by the standard procedure consisting of the fast solvent evaporation at reduced pressure into a rotary evaporator apparatus). The inset displays an enlarged section of the mass spectrum recorded in reflectron mode using DCTB as matrix and CF₃COOLi salt as doping agent.

Figure 10

I₃₇₃/I₃₈₄ intensity ratio obtained from pyrene emission spectra in the presence of poly-6-MOEG-9-T-BF3k-FE (blue curve) and Z-potential values (orange curve) as a function of polymer concentration.

Figure 11

DLS size distribution histograms of poly-6-MOEG-9-T-BF3k-FE dispersions in ultrapure water.

Figure 12

Structure of macromolecular aggregates found by TEM analysis of poly-6-MOEG-9-T-BF3k-FE solutions in water. The scale bar corresponds to 1 μm in the left panel and 200 nm in the right panel.

Figure 13

Normalized absorption and emission spectra of poly-6-MOEG-9-T-BF3k-FE in dichloromethane solution (top) and in the solid state (bottom). Optical absorption (black solid line), emission excitation profiles (λ_em = 470 nm, dotted blue lines) and emission spectra (λ_ex = 330 nm, blue solid line; λ_ex = 390 nm, black solid line; λ_ex = 420 nm, green solid line; λ_ex = 490 nm, red solid line).

Figure 14

Cell viability % (tetrazolium salt (MTS) assay) of poly-6-MOEG-9-T-BF3k-FE on human colon cancer cells HCT116 (panel A) and normal human bronchial epithelial cells 16HBE (panel B) cells, after 24 h and 48 h of incubation at concentrations of 0.005, 0.01, 0.03, 0.1 and 0.5 mg/mL.