Effects of X-shaped reduction-sensitive amphiphilic block copolymer on drug delivery

Figures & data

Figure 1 Synthesis of X-shaped amphiphilic copolymers.

Abbreviations: PLGA-COOH, poly(lactic-co-glycolic acid); SS, disulfide; PEG, poly(ethylene glycol).

Figure 2 Synthesis scheme for amphiphilic copolymers with different shapes and associated ¹H-NMR spectra (400 MHz, DMSO-d₆ as solvent).

Notes: Polymer characterization: ¹H-NMR: A (δ 4.93, s, (–OC H₂COO–)), B (δ 5.22, q, (–OC H(CH₃)CONH–)), C (δ 1.50, t, (–OCH(C H₃)CONH–)), D (δ 3.19, t, (–NH CH₂CH₂NH–)), G (δ 2.61, s, (–C H₂-SS- CH₂–)), J (δ 3.52, t, (–CH₂C H₂O–)).

Abbreviations: PLGA-COOH, poly(lactic-co-glycolic acid); SS, disulfide; PEG, poly(ethylene glycol); ppm, parts per million; ¹H-NMR, ¹H-nuclear magnetic resonance imaging.

Figure 3 FTIR spectra for (A) PLGA-COOH, (B) PLGA-NH₂, (C) PLGA-SS-COOH, (D) (PLGA)₂-SS-4-arm-PEG₂₀₀₀-NH₂.

Abbreviations: PLGA-COOH, poly(lactic-co-glycolic acid); SS, disulfide; PEG, poly(ethylene glycol); FTIR, Fourier transform infrared spectroscopy.

Table 1 Summary of GPC results for the synthesized block copolymers

Amphiphilic copolymers	^1H-NMR		GPC
	GA:LA:EGa	Mnb	Mnc	Mwc	PDI
PLGA5000-SS-PEG1000	44:31:18	6,005	5,413	8,531	1.57
(PLGA5000)2-SS-4-arm-PEG2000	88:58:34	11,326	9,595	15,762	1.64
(PLGA5000)2-4-arm-PEG2000	88:56:33	11,094	9,275	15,043	1.62

Notes:

a Composition ratio of GA to LA and EG calculated by ¹H-NMR.

b Estimated from ¹H-NMR data (g/mol).

c Determined by GPC (g/mol). GPC images for the synthesized polymers as well as their weight information are shown in the Supplementary material (Figures S1, S2, and Tables S1, S2).

Abbreviations: PLGA, poly(lactic-co-glycolic acid); SS, disulfide; PEG, poly(ethylene glycol); ¹H-NMR, ¹H-nuclear magnetic resonance imaging; GPC, gel permeation chromatography; PDI, polydispersity index; Mn, number-average molecular weight; Mw, weight-average molecular weight; GA, glycolic acid; LA, lactic acid; EG, ethylene glycol.

Figure 4 Determination of Micelle Formation.

Notes: (A) The emission fluorescence intensity of coumarin 6 in water. (B) The emission fluorescence of coumarin 6 (20 µg/mL) in acetone and nanomicelles. (C) Fluorescence quenching process of coumarin 6 in acetone caused by the addition of water. (D) SEM image of the formed particles.

Abbreviations: XNMs, X-shaped (PLGA)₂-SS-4-arm-PEG₂₀₀₀ polymer nanomicelles; SEM, scanning electron microscopy; PLGA, poly(lactic-co-glycolic acid); PEG, poly (ethylene glycol).

Figure 5 Comparison of Micellar Stability.

Notes: (A) Plot of fluorescence intensity ratio against the logarithm of polymer concentration. (Fluorescence emission spectra of pyrene are shown in Figure S2). (B) Size changes of nanomicelles. (The original data are shown in Table S2).

Abbreviations: XNMs, X-shaped (PLGA)₂-SS-4-arm-PEG₂₀₀₀ polymer nanomicelles; LNMs, loaded linear (PLGA)₂-SS-4-arm-PEG₂₀₀ polymer nanomicelles; PDI, polydispersity index; PLGA, poly(lactic-co-glycolic acid); PEG, poly(ethylene glycol).

Figure 6 (A) Typical DLS images of XNMs without DTT. (B) The DLS images of XNMs suspensions with DTT (10 mmol/L) for up to 24 hours.

Abbreviations: DLS, dynamic light scattering; XNMs, X-shaped (PLGA)₂-SS-4-arm-PEG₂₀₀₀ polymer nanomicelles; LNMs, loaded linear (PLGA)₂-SS-4-arm-PEG₂₀₀ polymer nanomicelles; DTT, DL-Dithiothreitol; PDI, polydispersity index; PLGA, poly(lactic-co-glycolic acid); PEG, poly(ethylene glycol).

Table 2 Some characteristics of DTX-loaded nanomicelles

Nanomicelle	Z average size (nm)	PDI	Surface zeta potential (mV)	EE (%)	DL (%)
DTX/LNMs	135.7±3.72	0.118	−6.8	95.4±3.34	5.37±0.54
DTX/NNMs	72.43±4.72	0.120	−5.4	93.3±4.23	5.02±0.34
DTX/XNMs	65.66±2.52	0.092	−7.3	90.5±1.72	4.92±0.24

Abbreviations: DTX, docetaxel; XNMs, X-shaped (PLGA)₂-SS-4-arm-PEG₂₀₀₀ polymer nanomicelles; LNMs, loaded linear (PLGA)₂-SS-4-arm-PEG₂₀₀ polymer nanomicelles; PDI, polydispersity index; EE, encapsulation efficiency; DL, drug loading capacity; PLGA, poly(lactic-co-glycolic acid); PEG, poly(ethylene glycol).

Figure 7 (A) Typical SEM images of DTX/XNMs (insert: typical appearance of DTX/XNMs suspension). (B) Typical TEM images of DTX/XNMs. (C) In vitro drug release profile of DTX/XNMs. (D) Changes of particle size, PDI, and drug remaining percentage of DTX/XNMs in process of time.

Abbreviations: DTX, docetaxel; XNMs, X-shaped (PLGA)₂-SS-4-arm-PEG₂₀₀₀ polymer nanomicelles; TEM, transmission electron microscopy; PDI, polydispersity index; PLGA, poly(lactic-co-glycolic acid); PEG, poly(ethylene glycol).

Figure 8 (A) The column graph showing cellular coumarin 6 fluorescence in A2780 cells following certain time incubation. Indicated values were mean ± SD (n=6). *P<0.05, **P<0.01, ***P<0.001. (B) Cellular uptake of coumarin 6, coumarin 6/LNMs, and coumarin 6/XNMs analyzed by flow cytometry.

Abbreviations: XNMs, X-shaped (PLGA)₂-SS-4-arm-PEG₂₀₀₀ polymer nanomicelles; LNMs, loaded linear (PLGA)₂-SS-4-arm-PEG₂₀₀ polymer nanomicelles; FITC, fluorescein isothiocyanate; SD, standard deviation; PLGA, poly(lactic-co-glycolic acid); PEG, poly(ethylene glycol).

Figure 9 The in vitro cytotoxicity assessment of XNMs and drug loaded formulations using MTT assay.

Notes: (A) Biocompatibility assessment of XNMs against A2780 cells following incubation for 48 hours. (B) Cytotoxicity effect of DTX and DTX loaded formulations on A2780 after treatment for 24 hours. (C) Cytotoxicity effect of DTX and DTX loaded formulations on A2780 after 48 hours incubation. Indicated values were mean ± SD (n=6). *P<0.05, **P<0.01, ***P<0.001.

Abbreviations: DTX, docetaxel; XNMs, X-shaped (PLGA)₂-SS-4-arm-PEG₂₀₀₀ polymer nanomicelles; PLGA, poly(lactic-co-glycolic acid); PEG, poly(ethylene glycol); LNMs, loaded linear (PLGA)₂-SS-4-arm-PEG₂₀₀ polymer nanomicelles.

Figure 10 (A) Immunofluorescence images for nuclear staining (Hoechst33342: blue) and β-tubulin (FITC: green) following the treatment with different DTX formulations for 24 hours. (B) Fluorescence imaging analysis of apoptosis in A2780 cells.

Abbreviations: DTX, docetaxel; XNMs, X-shaped (PLGA)₂-SS-4-arm-PEG₂₀₀₀ polymer nanomicelles; LNMs, loaded linear (PLGA)₂-SS-4-arm-PEG₂₀₀ polymer nanomicelles; FITC, fluorescein isothiocyanate; PLGA, poly(lactic-co-glycolic acid); PEG, poly(ethylene glycol).

Figure S1 Typical GPC chromatograms of polymers (A) PLGA-NH₂ (B) PLGA-SS-COOH.

Abbreviations: GPC, gel permeation chromatography; PLGA, poly(lactic-co-glycolic acid); SS, disulfide; MV, micro voltage.

Figure S2 Typical GPC chromatograms of three synthesized amphiphilic block copolymers.

Note: –SS– represents the disulphide bonds.

Abbreviations: GPC, gel permeation chromatography; PLGA, poly(lactic-co-glycolic acid); MV, micro voltage.

Figure S3 Fluorescence emission spectra of pyrene.

Abbreviation: con, concentration.

Table S1 Summary of GPC results

Amphiphilic copolymers	GPC
	Mn	Mw	PDI
PLGA-NH2	4,371	6,206	1.42
PLGA-SS-COOH	4,520	6,915	1.53

Abbreviations: PLGA, poly(lactic-co-glycolic acid); SS, disulfide; GPC, gel permeation chromatography; PDI, polydispersity index; Mn, number-average molecular weight; Mw, weight-average molecular weight.

Table S2 Changes of size, PDI, and zeta potential of XNMs and LNMs

Nanomicelles		Day 1	Day 3	Day 5	Day 7	Day 14
Size (nm)	XNMs	64.77±2.89	65.10±2.85	73.60±2.42	77.97±2.98	92.77±2.86
	LNMs	135.70±2.54	137.14±1.73	145.34±3.72	152.34±3.52	227.10±4.87
PDI	XNMs	0.118±0.030	0.103±0.015	0.112±0.007	0.167±0.023	0.130±0.028
	LNMs	0.117±0.047	0.223±0.038	0.307±0.054	0.558±0.044	0.663±0.072
Zeta potential (mV)	XNMs	11.20±0.03	10.50±0.134	10.70±0.21	9.80±0.12	9.50±0.28
	LNMs	10.80±0.02	10.12±0.13	9.86±0.21	8.76±0.12	8.54±0.28

Abbreviations: PDI, polydispersity index; XNMs, X-shaped (PLGA)₂-SS-4-arm-PEG₂₀₀₀ polymer nanomicelles; LNMs, loaded linear (PLGA)₂-SS-4-arm-PEG₂₀₀ polymer nanomicelles.