Docetaxel–trastuzumab stealth immunoliposome: development and in vitro proof of concept studies in breast cancer

Figures & data

Figure 1 Formulation and testing of two different nanoparticles.

Abbreviations: Lipo, liposome; PC, phosphatidylcholine; Chol, cholesterol; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; ANC, antibody nanoconjugate.

Table 1 Summary of the different extrusion conditions tested on Liposome-1a

Size reduction technique	Extrusion conditions (amount of cycles and type of filters used)	Size (nm)	PDI
Extrusion	2×200 nm+4×100 nm	151±23	0.088±0.04
Extrusion	6×100 nm	141±4	0.09±0.03
Extrusion	4×100 nm	169±3	0.13±0.02
Extrusion	2×100 nm + 2×80 nm	133±2	0.12±0.01
Extrusion	2×400 nm + 2×100 nm + 2×80 nm	135±3	0.1±0.01
Sonication	–	141.4±9	0.39±0.01

Note:

a Values are mean of three or more experiments.

Abbreviation: PDI, polydispersity index.

Table 2 Characteristics of ANC-1 and ANC-2a

Phospholipids composition	Molar ratio	Docetaxel:lipid molar ratio (%)	Size reduction technique	Encapsulation rate (%)	Encapsulation efficiency (%)	Size (nm)	PDI
PC:Chol:PG:Mal-PEG	50:19:15:1	1.9	Extrusion	73±6	>90%	140±3.4	0.1±0.01
DOTAP:PC:Mal-PEG	18:17:1	5.0	Extrusion	53±4	>75%	150±5	0.13±0.05

Note:

a Values are mean of three or more experiments.

Abbreviations: ANC, antibody nanoconjugate; PDI, polydispersity index; PC, phosphatidylcholine; Chol, cholesterol; PG, phosphatidylglycerol; Mal-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000]; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane.

Table 3 Characteristics of liposome-1a

Phospholipids composition	Molar ratio	Docetaxel:lipid molar ratio (%)	Encapsulation rate (%)	Size reduction technique	Size (nm)
PC:Chol:Mal-PEG	50:29:1	2.9	17±9	Extrusion	141.7±2.3
PC:Chol:Mal-PEG	50:19:1	3.2	35±19	Extrusion	175±31
PC:Chol:Mal-PEG	50:19:1	3.2	76±11	Sonication	141.4±9
PC:Chol:PG:Mal-PEG	50:19:15:1	1.8	70±19	Extrusion	–
PC:Chol:PG:Mal-PEG	50:19:15:1	1.9	85±2	Extrusion	141±2.4
PC:Chol:PG:Mal-PEG	50:19:15:1	2.0	63±18	Extrusion	158.8

Note:

a Values are mean of three or more experiments.

Abbreviations: PC, phosphatidylcholine; Chol, cholesterol; PG, phosphatidylglycerol; Mal-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000]; PG, phosphatidylglycerol.

Table 4 Characteristics of Liposome-2a

Phospholipids composition	Molar ratio	Docetaxel:lipid molar ratio (%)	Encapsulation rate (%)	Size reduction technique	Size (nm)
DOTAP:Mal-PEG	47:1	10.0	13±2	Extrusion	173±5
DOTAP:Chol:Mal-PEG	34:13:1	2.0	59±8	Extrusion	134±7
DOTAP:Chol:Mal-PEG	34:13:1	4.3	58±3	Extrusion	154±5
DOTAP:Chol:Mal-PEG	49:43:1	5.0	36±9	Extrusion	150
DOTAP:PC:Mal-PEG	13:22:1	3.7	49±12	Extrusion	137±19
DOTAP:PC:Mal-PEG	18:17:1	5.0	72±1	Extrusion	133±2

Note:

a Values are mean of three or more experiments.

Abbreviations: DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; Mal-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000]; PG, phosphatidylglycerol; Chol, cholesterol; PC, phosphatidylcholine.

Figure 2 Monitoring of docetaxel encapsulation (%) over time for ANC-1 (A) and ANC-2 (B) following different storage conditions: diluted at −20°C (■), concentrated at 4°C [img], diluted at 4°C [img] and diluted at 25°C ([img]).^a

Note: ^aValues are mean ± SEM of three or more experiments.

Abbreviation: ANC, antibody nanoconjugate.

Figure 3 Quantification of HER2 using QuantiBRITE kit in standard conditions on MDA-MB-231 and MDA-MB-453 (A) and in lower intensity conditions on SKBR3 (B).

Figure 4 Cell viability (%) of MDA-MB-231 when exposed to free docetaxel [img], free drugs [img], Liposome-1 plus free trastuzumab [img], ANC-1 [img], Liposome-2 plus free trastuzumab [img] and ANC-2 ([img]).^a

Figure 5 Cell viability (%) of MDA-MB-453 when exposed to free docetaxel [img], free drugs [img], Liposome-1 plus free trastuzumab [img], ANC-1 [img], Liposome-2 plus free trastuzumab [img] and ANC-2 ([img]).^a

Figure 6 Cell viability (%) of SKBR3 when exposed to free docetaxel [img], free drugs [img]), Liposome-1 plus free trastuzumab [img], ANC-1 [img], Liposome-2 with free trastuzumab [img] and ANC-2 ([img]).^a

Figure 7 Modulation of docetaxel IC₅₀ of MDA-MB-231, MDA-MB-453 and SKBR3 when treated for 72 hours with free drugs [img], Liposome-1 [img], ANC-1 [img], Liposome-2 [img] and ANC-2 ([img]).^a

Note: ^aValues are mean ± SEM of three or more experiments.

Abbreviation: ANC, antibody nanoconjugate.

Notes: ^aValues are mean ± SD of three or more experiments. (A) Comparison of SKBR3 cell viability when treated with free docetaxel, free drugs, Liposome-1 plus free trastuzumab or ANC-1. (B) Comparison of SKBR3 cell viability when treated with free docetaxel, free drugs, Liposome-2 plus free trastuzumab or ANC-2. (C) Comparison of SKBR3 cell viability when treated with Liposome-I plus free trastuzumab, ANC-1, Liposome-2 plus free trastuzumab or ANC-2.

Abbreviation: ANC, antibody nanoconjugate.

Notes: ^aValues are mean ± SD of three or more experiments. (A) Comparison of MDA-MB-453 cell viability when treated with free docetaxel, free drugs, Liposome-1 plus free trastuzumab or ANC-1. (B) Comparison of MDA-MB-453 cell viability when treated with free docetaxel, free drugs, Liposome-2 plus free trastuzumab or ANC-2. (C) Comparison of MDA-MB-453 cell viability when treated with Liposome-I plus free trastuzumab, ANC-1, Liposome-2 plus free trastuzumab or ANC-2.

Abbreviation: ANC, antibody nanoconjugate.

Notes: ^aValues are mean ± SD of three or more experiments. (A) Comparison of MDA-MB-231 cell viability when treated with free docetaxel, free drugs, Liposome-1 plus free trastuzumab or ANC-1. (B) Comparison of MDA-MB-231 cell viability when treated with free docetaxel, free drugs, Liposome-2 plus free trastuzumab or ANC-2. (C) Comparison of MDA-MB-231 cell viability when treated with Liposome-I plus free trastuzumab, ANC-1, Liposome-2 plus free trastuzumab or ANC-2.

Abbreviation: ANC, antibody nanoconjugate.

Figure 5 Cell viability (%) of MDA-MB-453 when exposed to free docetaxel [img], free drugs [img], Liposome-1 plus free trastuzumab [img], ANC-1 [img], Liposome-2 plus free trastuzumab [img] and ANC-2 ([img]).^a

Figure 6 Cell viability (%) of SKBR3 when exposed to free docetaxel [img], free drugs [img]), Liposome-1 plus free trastuzumab [img], ANC-1 [img], Liposome-2 with free trastuzumab [img] and ANC-2 ([img]).^a

Figure 7 Modulation of docetaxel IC₅₀ of MDA-MB-231, MDA-MB-453 and SKBR3 when treated for 72 hours with free drugs [img], Liposome-1 [img], ANC-1 [img], Liposome-2 [img] and ANC-2 ([img]).^a

Note: ^aValues are mean ± SEM of three or more experiments.

Abbreviation: ANC, antibody nanoconjugate.

Notes: ^aValues are mean ± SD of three or more experiments. (A) Comparison of SKBR3 cell viability when treated with free docetaxel, free drugs, Liposome-1 plus free trastuzumab or ANC-1. (B) Comparison of SKBR3 cell viability when treated with free docetaxel, free drugs, Liposome-2 plus free trastuzumab or ANC-2. (C) Comparison of SKBR3 cell viability when treated with Liposome-I plus free trastuzumab, ANC-1, Liposome-2 plus free trastuzumab or ANC-2.

Abbreviation: ANC, antibody nanoconjugate.

Notes: ^aValues are mean ± SD of three or more experiments. (A) Comparison of MDA-MB-453 cell viability when treated with free docetaxel, free drugs, Liposome-1 plus free trastuzumab or ANC-1. (B) Comparison of MDA-MB-453 cell viability when treated with free docetaxel, free drugs, Liposome-2 plus free trastuzumab or ANC-2. (C) Comparison of MDA-MB-453 cell viability when treated with Liposome-I plus free trastuzumab, ANC-1, Liposome-2 plus free trastuzumab or ANC-2.

Abbreviation: ANC, antibody nanoconjugate.

Figure 6 Cell viability (%) of SKBR3 when exposed to free docetaxel [img], free drugs [img]), Liposome-1 plus free trastuzumab [img], ANC-1 [img], Liposome-2 with free trastuzumab [img] and ANC-2 ([img]).^a

Figure 7 Modulation of docetaxel IC₅₀ of MDA-MB-231, MDA-MB-453 and SKBR3 when treated for 72 hours with free drugs [img], Liposome-1 [img], ANC-1 [img], Liposome-2 [img] and ANC-2 ([img]).^a

Note: ^aValues are mean ± SEM of three or more experiments.

Abbreviation: ANC, antibody nanoconjugate.

Notes: ^aValues are mean ± SD of three or more experiments. (A) Comparison of SKBR3 cell viability when treated with free docetaxel, free drugs, Liposome-1 plus free trastuzumab or ANC-1. (B) Comparison of SKBR3 cell viability when treated with free docetaxel, free drugs, Liposome-2 plus free trastuzumab or ANC-2. (C) Comparison of SKBR3 cell viability when treated with Liposome-I plus free trastuzumab, ANC-1, Liposome-2 plus free trastuzumab or ANC-2.

Abbreviation: ANC, antibody nanoconjugate.

Figure 7 Modulation of docetaxel IC₅₀ of MDA-MB-231, MDA-MB-453 and SKBR3 when treated for 72 hours with free drugs [img], Liposome-1 [img], ANC-1 [img], Liposome-2 [img] and ANC-2 ([img]).^a

Note: ^aValues are mean ± SEM of three or more experiments.

Abbreviation: ANC, antibody nanoconjugate.

Figure 8 Confocal microscopy observations of ANC-1 uptake on SKBR3 (A), MDA-MB-453 (B) and MDA-MB-231 (C) cells, after 6 hour exposition.

Note: Magnification ×40.

Abbreviation: ANC, antibody nanoconjugate.

Figure S1 Representative SDS-PAGE gel, showing trastuzumab engraftment on liposome surface. Protein markers (A1), trastuzumab (A2), thiolated trastuzumab (A3), Liposome-1 (A4), ANC-1 engrafted with strategy B (A5), Liposome-1 engrafted with strategy B without maleimide function (A6), ANC-1 engrafted following strategy A (A7), Liposome-1 engrafted following strategy A without maleimide function (A8), protein markers (B1), trastuzumab (B2), ANC-1 engrafted following strategy B (B3), Liposome-1 engrafted following strategy B without maleimide function (B4), ANC-2 engrafted with strategy B (B5), Liposome-2 engrafted with strategy B without maleimide function (B6), ANC-1 engrafted with strategy B after 45 days (B7).

Abbreviations: SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; ANC, antibody nanoconjugate.

Figure S2 TEM observations of ANC-1 treated with osmium tetroxide (A and B) and uranyl acetate (C).

Abbreviations: TEM, transmission electron microscopy; ANC-1, antibody nanoconjugate-1.

Figure S3 TEM observations of ANC-2 treated with osmium tetroxide.

Abbreviations: TEM, transmission electron microscopy; ANC-2, antibody nanoconjugate-2.

Figure S4 TEM observations of liposome-1 before extrusion.

Abbreviation: TEM, transmission electron microscopy.

Figure S5 Monitoring size (nm) over time for ANC-1 following different storage conditions: diluted at −20°C [img], concentrated at 4°C [img], diluted at 4°C [img] and diluted at 25°C ([img]).^a

Note: ^aValues are mean ± SEM of three or more experiments.

Abbreviation: ANC-1, antibody nanoconjugate-1.