Combination Chemotherapy of Lung Cancer – Co-Delivery of Docetaxel Prodrug and Cisplatin Using Aptamer-Decorated Lipid–Polymer Hybrid Nanoparticles

Figures & data

Figure 1 Synthesis of aptamer-conjugated lipid–polymer ligands (PLA-PEG-APT) and redox-sensitive docetaxel prodrug (DTX-GM-TA).

Notes: Aptamer-conjugated lipid–polymer ligands were synthesized by conjugating the aptamer to PLA-PEG-Mal. Redox-sensitive docetaxel prodrug (DTXp) was synthesized by conjugating the DTX with glyceryl monostearate (GM) using redox-responsive thiodiglycolic anhydride (TA).
Abbreviations PLA, poly(L-lactide); PEG, poly(ethylene glycol); APT, aptamer; DTX, docetaxel; GM, glyceryl monostearate; TA, thiodiglycolic anhydride; NMR, nuclear magnetic resonance; DTXp, docetaxel prodrug; MAL, maleimide.

Table 1 Characterization of LPHNs (Mean ± SD, n=3)

Formulation	Particle Size (nm)	Zeta Potential (mV)	LE (%)		LC (%)
			DTX	DDP	DTX	DDP
APT-DTXp/DDP-LPHNs	213.5 ± 5.3	15.9 ± 1.9	81.2 ± 3.1	82.1 ± 2.2	5.1 ± 0.9	4.9 ± 0.5
DTXp/DDP-LPHNs	208.9 ± 4.9	−13.7 ± 1.6	80.9 ± 2.1	81.3 ± 2.3	6.3 ± 0.6	5.2 ± 0.6
APT-DTXp-LPHNs	211.3 ± 5.7	17.1 ± 2.1	80.3 ± 2.6	/	5.3 ± 0.8	/
APT-DTX-LPHNs	212.4 ± 5.1	16.5 ± 1.5	81.6 ± 2.9	/	5.5 ± 0.6	/
APT-DDP-LPHNs	209.9 ± 4.3	12.7 ± 1.3	/	80.9 ± 2.1	/	5.1 ± 0.7
APT-LPHNs	215.1 ± 3.9	18.7 ± 1.8	/	/	/	/

Abbreviations: LPHNs, lipid–polymer hybrid nanoparticles; APT, aptamer; DTXp, docetaxel prodrug; DTX, docetaxel; DDP, cisplatin; LE, loading efficiency; LC, loading content.

Figure 2 TEM images show the morphologies and size of APT-DTXp/DDP-LPHNs and DTXp/DDP-LPHNs.

Notes: Besides spherical in shape and uniform in size, APT-DTXp/DDP-LPHNs showed coats on the particles’ surface which do not exist in DTXp/DDP-LPHNs.
Abbreviations: TEM, transmission electron microscopy; APT, aptamer; DTXp, docetaxel prodrug; DDP, cisplatin; LPHNs, lipid–polymer hybrid nanoparticles.

Figure 3 The stability of LPHNs in the presence of serum evaluated by testing the particle size (A), zeta potential (B), and LE (C, D) variations.

Notes: The stability of LPHNs in serum was investigated by mixing the LPHNs with FBS (55% in volume). The changes in particle size, zeta potential, and LE LPHNs were analyzed by the same methods. Data presented as mean ±SD, n=3.
Abbreviations: LPHNs, lipid–polymer hybrid nanoparticles; LE, loading efficiency; APT, aptamer; DTXp, docetaxel prodrug; DTX, docetaxel; DDP, cisplatin; FBS, fetal bovine serum.

Figure 4 In vitro DTX (A) and DDP (B) release profiles of APT-DTXp/DDP-LPHNs and other LPHNs.

Notes: Sustained release behaviors were observed for DTXp, DTX and/or DDP-loaded LPHNs. DDP release profiles of APT-DTXp/DDP-LPHNs, DTXp/DDP-LPHNs and APT-DDP-LPHNs were similar in hypoxic and normal condition. However, DTX release manners of APT-DTXp/DDP-LPHNs, DTXp/DDP-LPHNs and APT-DTXp-LPHNs were different, faster and more in hypoxic compared with normal condition. Data presented as mean ±SD, n=3.
Abbreviations: DTX, docetaxel; DDP, cisplatin; APT, aptamer; DTXp, docetaxel prodrug; LPHNs, lipid–polymer hybrid nanoparticles; H, hypoxic condition; N, normal condition.

Figure 5 The cell uptake efficiency of APT-DTXp/DDP-LPHNs and DTXp/DDP-LPHNs.

Notes: FITC was applied to the LPHNs systems and incubated with A549 cells or BEAS2B cells for 2 h. The fluorescence intensity of the cells was determined by flow cytometer. Cell uptake of APT-DTXp/DDP-LPHNs was higher than that of non-modified DTXp/DDP-LPHNs. Data presented as mean ±SD, n=6. * P < 0.05.
Abbreviations: APT, aptamer; DTXp, docetaxel prodrug; DTX, docetaxel; DDP, cisplatin; LPHNs, lipid–polymer hybrid nanoparticles; FITC, fluorescein 5-isothiocyanate.

Table 2 CI Values Were Evaluated According to the IC₅₀ Values (Mean ± SD, n=3)

Formulations	DTXp/DDP Ratio (w/w)	DTX IC50 (mg/mL)	DDP IC50 (mg/mL)	CI Value
APT-DTXp-LPHNs	/	3.12 ± 0.12	/	/
APT-DDP-LPHNs	/	/	1.79 ± 0.09	/
APT-DTXp/DDP-LPHNs	5/1	2.17 ± 0.13	0.43 ± 0.04	0.94
APT-DTXp/DDP-LPHNs	2/1	1.52 ± 0.10	0.76 ± 0.08	0.91
APT-DTXp/DDP-LPHNs	1/1	0.71 ± 0.09	0.71 ± 0.09	0.62
APT-DTXp/DDP-LPHNs	1/2	0.57 ± 0.06	1.14 ± 0.12	0.82
APT-DTXp/DDP-LPHNs	1/5	0.31 ± 0.02	1.55 ± 0.11	0.97

Abbreviations: CI, combination index; IC₅₀, the half maximal inhibitory concentration; LPHNs, lipid–polymer hybrid nanoparticles; APT, aptamer; DTXp, docetaxel prodrug; DTX, docetaxel; DDP, cisplatin.

Figure 6 Cell viability of LPHNs evaluated at various drug concentrations using MTT assay.

Notes: Blank APT-LPHNs was nontoxic to A549 cells, while drug-loaded LPHNs and free drugs exhibited dose-dependent cytotoxicity. Drug-loaded LPHNs showed significantly higher cytotoxicity than free drugs, especially in lower concentrations. Aptamer-decorated APT-DTXp/DDP-LPHNs exhibited better cell inhibition ability than that of APT-DTXp-LPHNs, APT-DTX-LPHNs, APT-DDP-LPHNs, and non-decorated DTXp/DDP-LPHNs. Data presented as mean ±SD, n=6. * P < 0.05.
Abbreviations: LPHNs, lipid–polymer hybrid nanoparticles; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; APT, aptamer; DTXp, docetaxel prodrug; DTX, docetaxel; DDP, cisplatin.

Table 3 BUN, AST, and ALT Levels (Mean ± SD, n=3)

Parameters (U/L)	BUN	AST	ALT
APT-DTXp-LPHNs	33.5 ± 2.9	22.3 ± 1.9	18.3 ± 1.3
APT-DDP-LPHNs	35.1 ± 2.5	20.6 ± 1.5	17.8 ± 1.9
APT-DTXp/DDP-LPHNs	32.7 ± 2.7	21.4 ± 1.6	16.9 ± 1.5
APT-DTXp/DDP-LPHNs	30.9 ± 3.1	22.7 ± 1.8	15.9 ± 1.4
APT-DTXp/DDP-LPHNs	34.8 ± 2.6	20.5 ± 1.7	18.1 ± 1.5
APT-DTXp/DDP-LPHNs	36.1 ± 2.7	21.7 ± 2.1	16.7 ± 1.6
APT-DTXp/DDP-LPHNs	35.4 ± 3.0	22.4 ± 1.8	17.4 ± 1.3
Free DTX/DDP	51.6 ± 3.7	38.9 ± 2.3	28.6 ± 2.2
Normal saline	30.4 ± 2.4	21.3 ± 1.4	15.5 ± 1.2

Abbreviations: BUN, blood urea nitrogen; AST, serum aspartate aminotransferase; ALT, alanine aminotransferase; LPHNs, lipid–polymer hybrid nanoparticles; APT, aptamer; DTXp, docetaxel prodrug; DTX, docetaxel; DDP, cisplatin.

Figure 7 In vivo DTX (A) and DDP (B) biodistribution of drug-loaded LPHNs and free drugs. Note: Free drugs mainly accumulated in kidney, and heart compared with tumor tissue. On the contrary, LPHNs distribution in the tumor was significantly higher than free drugs. APT-DTXp/DDP-LPHNs showed higher tumor distribution than non-decorated DTXp/DDP-LPHNs. Data presented as mean ±SD, n=6. * P < 0.05. DTX: docetaxel; DDP: cisplatin; LPHNs; lipid–polymer hybrid nanoparticles; APT: aptamer; DTXp: docetaxel prodrug.

Figure 8 In vivo anti-tumor activity of drug-loaded LPHNs and free drugs.

Notes: APT-DTXp/DDP-LPHNs showed higher tumor distribution than non-decorated DTXp/DDP-LPHNs. Single drug contained APT-DTXp-LPHNs and APT-DDP-LPHNs exhibited weaker tumor inhibition efficiency than APT-DTXp/DDP-LPHNs. Prodrug-based APT-DTXp-LPHNs gained smaller tumor size than that of APT-DTX-LPHNs at the end of the study. Data presented as mean ±SD, n=6. * P < 0.05.
Abbreviations: LPHNs, lipid–polymer hybrid nanoparticles; DTX, docetaxel; DDP, cisplatin; APT, aptamer; DTXp, docetaxel prodrug.