Nano-sized cytochrome P450 3A4 inhibitors to block hepatic metabolism of docetaxel

Figures & data

Table 1 Summary of physicochemical properties of the nanocarriers

Composition	Size (by DLS)		Surface functionalization	Surface charge		Surfactant, lipid or polymer concentration (g/L)	BM concentration (µM)
	Mean hydrodynamic diameter (nm)	Polydispersity index		Zeta potential (mV)	Zeta deviation (mV)
PS80–EtOH micelle	10.7±0.07	0.06±0.002	–	−9.03±1.04	8.6±1.1	54.5	1,500
DSPE-PEG micelle	16±0.3	0.145±0.03	–	−9.93±0.81	15.4±12	7.1	189
DSPE-PEG (lactosyl) micelle	58±0.4	0.45±0.01	Lactosyl	−5.79±0.94	13.4±4.7	7.2	45
PLGA	67±1.6	0.16±0.04	–	−48.4±1.37	7.9±0.3	4.4	28.7
PLGA-Ga	63±1.1	0.07±0.01	Ga	−39±0.93	6.9±0.8	4.4	28.7
HA	96±0.4	0.15±0.01	–	−42.2±1.46	13.3±0.451	3.0	–

Note: Data presented as mean ± standard deviation, unless otherwise stated.

Abbreviations: BM, bergamottin; DLS, dynamic light scattering; DSPE-PEG, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-(carboxy(polyethylene glycol)-2000); EtOH, ethanol; Ga, galactosamine; HA, hyaluronic acid; PLGA, poly(lactic-co-glycolic) acid; PLGA-Ga, PLGA nanoparticles functionalized with Ga; PS80, polysorbate 80.

Figure 1 FEG-SEM image of PLGA nanoparticles.

Abbreviations: FEG-SEM, field emission gun-scanning electron microscope; PLGA, poly(lactic-co-glycolic) acid.

Table 2 Measured zeta potentials of PLGA nanoparticles, depending on their load and surface functionalization

Composition	PLGA (empty)	PLGA (encapsulating BM)
PLGA nanoparticles
Zeta potential (mV ± SD)	−47.9±1.80	−48.4±1.37
Zeta deviation (mV ± SD)	11.4±0.45	7.91±0.27
PLGA-Ga nanoparticles
Zeta potential (mV ± SD)	−37.4±1.34	−39±0.93
Zeta deviation (mV ± SD)	13.3±0.44	6.92±0.77

Abbreviations: BM, bergamottin; Ga, galactosamine; PLGA, poly(lactic-co-glycolic) acid; PLGA-Ga, PLGA nanoparticles functionalized with Ga.

Table 3 In vitro IC₅₀ of BM in different nanocarriers, measured with the fluorogenic DBOMF probe, after 3 hours of co-incubation on HepaRG cells; results are the mean of at least three independent experiments performed in triplicate, presented with 95% confidence interval

Composition	Unformulated compound	Formulations suitable for injection			Stable and size-appropriate encapsulation	Stable, functionalized and size-appropriate encapsulation
Sample	BM in ethanol	PS80–EtOH BM	DSPE-PEG BM	DSPE-PEG BM lactosyl	PLGA BM	PLGA-Ga BM
IC50 (in µM) with 95% CI	2.1 (1.3–3.4)	1.0 (0.6–1.8)	2.9 (2.1–3.9)	1.4 (1.0–2.1)	0.26 (0.18–0.37)	0.56 (0.40–0.79)

Abbreviations: BM, bergamottin; DBOMF, di(benzyloxymethoxy)fluorescein; DSPE-PEG, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-(carboxy(polyethylene glycol)-2000); EtOH, ethanol; Ga, galactosamine; IC₅₀, half-maximal inhibitory concentration; PLGA, poly(lactic-co-glycolic) acid; PLGA-Ga, PLGA nanoparticles functionalized with Ga; PS80, polysorbate 80.

Figure 2 In vivo imaging of mice injected with (A) micelles of (i) PS80–EtOH DilC₁₈, (ii) DSPE-PEG DilC₁₈, (iii) DSPE-PEG lactosyl DilC₁₈, (B) nanoparticles of (i) PLGA DilC₁₈, (ii) PLGA-Ga DilC₁₈ and (iii) HA DilC₁₈, with solutions on the same scale of fluorescence, 10 minutes after injection. At least three mice per condition were imaged in vivo. Scales: radiant efficiency in (p/second/cm²/sr)/(µW/cm²); min =7.02E7; max =4.14E9. (C) Ex vivo imaging of livers and spleens of mice injected with (i) PLGA DilC₁₈, (ii) PLGA-Ga DilC₁₈ and (iii) HA DilC₁₈ 1 hour after injection. At least two mice per condition were imaged. Scales: radiant efficiency in (p/second/cm²/sr)/(µW/cm²); min =1.11E8; max =6.5E8.

Abbreviations: DilC₁₈, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine; DSPE-PEG, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-(carboxy(polyethylene glycol)-2000); EtOH, ethanol; Ga, galactosamine; PLGA, poly(lactic-co-glycolic) acid; PLGA-Ga, PLGA nanoparticles functionalized with Ga; HA, hyaluronic acid; PS80, polysorbate 80.

Figure 3 FACS analysis of hepatocytes (A) 1 hour after mice injected with (i) PS80–EtOH DilC₁₈, (ii) PLGA DilC₁₈, (iii) HA DilC₁₈ or (iv) PLGA-Ga DilC₁₈ and (B) 20 hours after mice injected with (i) HA DilC₁₈ or (ii) PLGA-Ga DilC₁₈.

Notes: FITC-positive population (FITC+) represents hepatocytes, due to their autofluorescence at this wavelength. The population in the upper right corner is the APC-positive hepatocytes (APC+/FITC+): hepatocytes after DilC₁₈ uptake.

Abbreviations: DilC₁₈, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine; FACS, fluorescence-activated cell sorting (flow cytometry); EtOH, ethanol; FITC, fluorescein isothiocyanate; Ga, galactosamine; HA, hyaluronic acid; PLGA, poly(lactic-co-glycolic) acid; PLGA-Ga, PLGA nanoparticles coated with Ga; PS80, polysorbate 80.

Figure 4 (A) Median tumor volume (mm³) (n=4 for groups 1–2, n=6 for groups 3–5), (B) individual tumor volume (mm³) of groups 1–2 and 4–5, (C) individual tumor volume (mm³) of groups 1–2, 3 and 5, (D) Kaplan–Meier diagram of groups 1–2 and 4–5, and (E) Kaplan–Meier diagram of groups 1–2, 3 and 5. Difference between the PLGA-Ga BM and docetaxel alone groups was evaluated with a two-way ANOVA analysis and a Bonferroni post test, ***P-value <0.001. Arrows: injections (gray arrow PS80–EtOH BM or PLGA-Ga BM injections and black arrow docetaxel injections).

Abbreviations: BM, bergamottin; EtOH, ethanol; Ga, galactosamine; PLGA, poly(lactic-co-glycolic) acid; PLGA-Ga, PLGA nanoparticles functionalized with Ga; PS80, polysorbate 80.

Figure S1 (A) Schematic representation of the nanocarriers and synthesis process of (B) PS80 micelles, (C) DSPE-PEG micelles, (D) PLGA nanoparticles and (E) HA nanoparticles.

Abbreviations: DSPE-PEG, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-(carboxy(polyethylene glycol)-2000); EDC, 1-ethyl-3-(3-dimethlyaminopropyl) carbodiimide; HA, hyaluronic acid; lactosyl-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl; NHS, N-hydroxysulfosuccinimide; PLGA, poly(lactic-co-glycolic) acid; PS80, polysorbate 80.

Figure S2 Schematic representation of the functionalization of PLGA nanoparticles with galactosamine.

Abbreviations: BM, bergamottin; EDC, 1-ethyl-3-(3-dimethlyaminopropyl) carbodiimide; NHS, N-hydroxysulfosuccinimide; PLGA, poly(lactic-co-glycolic) acid.

Figure S3 Favored intake of coated nanoparticles by HepaRG cells seen with Zeiss LSM710 confocal microscope with fixed optical parameters after 20 minutes (A, B), 3 hours (C, D) or 24 hours (E, F) of in vitro incubation of coated PLGA-Ga nanoparticles (A, C, E) or uncoated PLGA nanoparticles (B, D, F).

Notes: Fixed cells were imaged on a Zeiss confocal LSM710 microscope. Images were acquired using the 63× objective in oil, as 12-bit images. To image DilC₁₈, DsRed filters were used and the Zeiss confocal LSM710 microscope was set as follows: masgain =600; digital gain =1.0; laser line (561 nm): 15%. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Master gain =600; digital gain =1.0; laser line (561 nm): 15%.

Abbreviations: PLGA, poly(lactic-co-glycolic) acid; PLGA-Ga, PLGA nanoparticles functionalized with galactosamine.

Figure S4 In vitro uptake of nanoparticles by HepaRG cells seen with confocal microscope (Zeiss LSM710) using the 63× objective in oil.

Notes: Uptake of (A) coated DSPE-PEG micelles with lactosyl containing 27 mg/L of DilC₁₈ and (B) uncoated DSPE-PEG micelles containing 27 mg/L of DilC₁₈ after 3 hours of in vitro incubation on HepaRG cells. Images were acquired with identical optical parameters (master gain =600; digital gain =1.0; laser line [561 nm]: 15%) and after DAPI staining of the nuclei and fixation of HepaRG cells.

Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; DilC₁₈, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine; DSPE-PEG, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-(carboxy(polyethylene glycol)-2000).

Figure S5 In vivo imaging of a mouse injected with DilC₁₈ in H₂O-EtOH (1:1 v/v).

Notes: Presented images were taken 1 minute, 5 minutes, 30 minutes and 60 minutes after intravenous injection in the tail vein. Scale: min =7.00E7; max =2.00E8 radiant efficiency in (p/second/cm²/sr)/(µW/cm²).

Abbreviations: DilC₁₈, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine; EtOH, ethanol.

Figure S6 In vivo imaging of mice injected with (A) PS80–EtOH DilC₁₈, (B) DSPE-PEG DilC₁₈, (C) DSPE-PEG lactosyl DilC₁₈, (D) HA DilC₁₈, (E) PLGA DilC₁₈ and (F) PLGA-Ga DilC₁₈.

Notes: Presented images were taken approximately every 2 minutes until 15 minutes, every 4 minutes from 15 minutes to 30 minutes and every 7 minutes from 30 minutes to 1 hour. At least three mice per condition were imaged. Scale: min =7.02E7; max =4.14E9 radiant efficiency in (p/second/cm²/sr)/(µW/cm²).

Abbreviations: DilC₁₈, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine; DSPE-PEG, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-(carboxy(polyethylene glycol)-2000); EtOH, ethanol; HA, hyaluronic acid; PLGA, poly(lactic-co-glycolic) acid; PLGA-Ga, PLGA nanoparticles coated with galactosamine; PS80, polysorbate 80.

Figure S7 In vivo imaging of mice injected with (A) HA DilC₁₈, (B) PLGA DilC₁₈ and (C) PLGA-Ga DilC₁₈, at lowered concentration of nanoparticles (0.25 g/L of polymer).

Notes: Presented images were taken approximately every 2 minutes until 15 minutes, every 4 minutes from 15 minutes to 30 minutes and every 7 minutes from 30 minutes to 1 hour. At least three mice per condition were imaged. Scale: min =7.02E7; max =4.14E9 radiant efficiency in (p/second/cm²/sr)/(µW/cm²).

Abbreviations: DilC₁₈, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine; HA, hyaluronic acid; PLGA, poly(lactic-co-glycolic) acid; PLGA-Ga, PLGA nanoparticles coated with galactosamine.

Figure S8 In vitro control of MDA-MB-231 cells sensitization to docetaxel by BM.

Notes: Cell sensitization to docetaxel diluted in NaCl 1% (1:9 v/v) prior to injection was measured using a WST-1 assay according to the manufacturer’s instructions, after 48 hours of co-incubation of BM (0–5 µM) and docetaxel (0–25 nM) in cell culture medium. A statistical analysis was performed by a two-way analysis of variance (ANOVA) analysis (Prism software). The concentration in BM did not cause significant differin the response of MDA-MB-231 cells to docetaxel. As measured by WST-1, MDA-MB-231 cells were not sensitized to docetaxel in vitro, by the co-incubation with polysorbate 80 (PS80)-EtOH BM micelles for 48 hours. In vitro viability of MDA-MB-231 cells after a 48-hour co-incubation of PS80–EtOH BM (0 µM, 0.2 µM, 1 µM and 5 µM) and docetaxel, evaluated by WST-1 assay. Results are the mean of triplicates ± SD, presented with a statistical analysis (two-way ANOVA, Prism software).

Abbreviations: BM, bergamottin; EtOH, ethanol; PS80, polysorbate 80.

Figure S9 Relative mean weight evolution per treatment group, relative to the mean weight per group at the first day of injection, ±SD.

Notes: No difference in weight evolution was noted between treated groups. No significant weight loss was observed.

Abbreviations: BM, bergamottin; EtOH, ethanol; PLGA, poly(lactic-co-glycolic) acid; PLGA-Ga, PLGA nanoparticles functionalized with galactosamine; PS80, polysorbate 80.

Table S1 Treatment groups for in vivo study: antitumor efficacy

Group number	Groups	Products injected	Days of treatment	Number of mice
1	Control	Glucose: 5%	0, 4, 8	4
2	PLGA-Ga BM micelles	PLGA-Ga BM micelles: 0.097 mg/kg	0, 4, 8	4
3	Docetaxel micelles	Glucose: 5%	0, 4, 8	6
		Docetaxel micelles: 20 mg/kg	1, 5, 9
4	PS80–EtOH BM micelles + docetaxel micelles	PS80–EtOH BM micelles: 5 mg/kg	0, 4, 8	6
		Docetaxel micelles: 20 mg/kg	1, 5, 9
5	PLGA-Ga BM micelles + docetaxel micelles	PLGA-Ga BM micelles: 0.097 mg/kg	0, 4, 8	6
		Docetaxel micelles: 20 mg/kg	1, 5, 9

Note: All solutions were injected at 10 mL/kg.

Abbreviations: PS80–EtOH BM, polysorbate 80–ethanol micelles encapsulating bergamottin; PLGA-Ga BM, poly(lactic-co-glycolic) acid nanoparticles functionalized with galactosamine, encapsulating bergamottin.