Glucose- and temperature-sensitive nanoparticles for insulin delivery

Figures & data

Table 1 The compositions of the amphiphilic copolymers produced

Sample ID	Monomer:RAFT agent ratio DEGMA:p(AAPBA) (mol/mol)	Yield (wt%)a
PAD-50-1	100:2 (454 mg:9.6 mg)	82.4
PAD-20-1	100:5 (454 mg:24 mg)	79.6
PAD-10-1	100:10 (454 mg:48 mg)	75.5
PAD-5-1	100:20 (454 mg:96 mg)	74.2
PAD-2-1	100:50 (454 mg:242 mg)	72.5

Notes:

a Yield was calculated via the weight method: Yield (wt%) =100× [mass of p(AAPBA-b-DEGMA)]/[mass of DEGMA + mass of p(AAPBA)]. PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y.

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; p(AAPBA), poly(3-acrylamidophenylboronic acid); RAFT, reversible addition–fragmentation chain transfer.

Figure 1 FT-IR spectra of AAPBA, DEGMA, p(AAPBA), and PAD-5-1.

Note: PAD-5-1, p(AAPBA-b-DEGMA) (pAAPBA:DEGMA =1:5).

Abbreviations: AAPBA, 3-acrylamidophenylboronic acid; DEGMA, diethylene glycol methyl ether methacrylate; p(AAPBA), poly(3-acrylamidophenylboronic acid); FTIR, Fourier transform infrared spectroscopy.

Figure 2 ¹H-NMR spectra of (A) DEGMA, (B) AAPBA, (C) p(AAPBA), and (D) PAD-5-1.

Notes: PAD-5-1, p(AAPBA-b-DEGMA) (pAAPBA:DEGMA =1:5). Peaks crossed through correspond to the solvents used for NMR.

Abbreviations: ¹H-NMR, ¹H nuclear magnetic resonance; AAPBA, 3-acrylamidophenylboronic acid; DEGMA, diethylene glycol methyl ether methacrylate; p(AAPBA), poly(3-acrylamidophenylboronic acid).

Figure 3 Thermal analysis of p(AAPBA) and PAD-5-1, showing (A) DTG and (B) TG curves.

Note: PAD-5-1, p(AAPBA-b-DEGMA) (pAAPBA:DEGMA =1:5).

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; DTG, derivative thermogravimetric; p(AAPBA), poly(3-acrylamidophenylboronic acid); TG, thermogravimetric.

Table 2 The molecular weights (Mw and Mn) and molecular weight distributions (PDI) of the copolymers prepared in this work

Sample	DEGMA/p(AAPBA) molar ratio	Mw	Mn	PDI
PAD-50-1	100:2	86,454	74,792	1.16
PAD-20-1	100:5	99,436	86,742	1.15
PAD-10-1	100:10	101,438	88,489	1.15
PAD-5-1	100:20	106,562	94,838	1.12
PAD-2-1	100:50	107,651	92,546	1.16

Note: PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y.

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; p(AAPBA), poly(3-acryl amidophenylboronic acid); PDI, polydispersity index.

Figure 4 Images of the p(AAPBA-b-DEGMA) NP suspensions (1) and TEM micro-graphs of the dried NPs (2).

Notes: (A) PAD-50-1, (B) PAD-20-1, (C) PAD-10-1, (D) PAD-5-1, and (E) PAD-2-1. PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y.

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; NP, nanoparticle; p(AAPBA), poly(3-acrylamidophenylboronic acid); TEM, transmission electron microscopy.

Table 3 The LCSTs, zeta potentials, and polydispersity indices of the copolymer NPs

Samples	DEGMA/p(AAPBA) molar ratio	Zeta potential (mV)	LCST (°C)	PDI
PAD-50-1	100:2	−21.4±2.3	32.0	0.14±0.04
PAD-20-1	100:5	−26.9±1.8	34.5	0.13±0.02
PAD-10-1	100:10	−33.6±1.7	36.5	0.09±0.01
PAD-5-1	100:20	−37.2±0.9	38.5	0.12±0.02
PAD-2-1	100:50	−40.3±1.2	42.5	0.11±0.03

Notes: PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y. Data are presented as mean ± standard deviation.

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; LCST, lower critical solution temperature; NP, nanoparticle; p(AAPBA), poly(3-acrylamidophenylboronic acid); PDI, polydispersity index.

Figure 5 The hydrodynamic diameters of copolymer NPs at different (A) pH and (B) temperatures.

Note: PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y.

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; NP, nanoparticle; p(AAPBA), poly(3-acrylamidophenylboronic acid); T, temperature.

Figure 6 Changes in the size of the p(AAPBA-b-DEGMA) NPs as a function of immersion time (t) in pH 7.4 PBS solutions of glucose.

Notes: (A) PAD-50-1; (B) PAD-20-1; (C) PAD-10-1; (D) PAD-5-1; and (E) PAD-2-1 are shown. The left images show the hydrodynamic diameters, while those on the right give I/I₀ (the light scattering intensity of the glucose-treated NP suspensions divided by the scattering intensity of the particles without glucose treatment). I/I₀ values reflected the extent of swelling. PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y.

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; NP, nanoparticle; p(AAPBA), poly(3-acrylamidophenylboronic acid); PBS, phosphate-buffered saline.

Table 4 Insulin LCs and EEs of PAD-5-1 NPs

NP sample	Group	Insulin concentration (mg·mL^−1)	LC (%)	EE (%)
PAD-5-1	I-L-1	0.25	12.4±3.2	75.1±9.2
	I-L-2	0.50	13.2±2.9	74.2±7.4
	I-L-3	0.75	16.7±3.1	72.4±8.4
	I-L-4	1.00	18.7±2.9	70.1±8.3
	I-L-5	1.25	19.2±3.7	66.7±9.7

Notes: PAD-5-1, p(AAPBA-b-DEGMA) (pAAPBA:DEGMA =1:5). Data are presented as mean ± standard deviation.

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; EE, encapsulation efficiency; I-L, insulin loading; LC, loading capacity; NP, nanoparticle; p(AAPBA), poly(3-acrylamidophenylboronic acid).

Figure 7 In vitro release of the insulin-loaded I-L-4 particles in PBS (pH 7.4) at different (A) temperatures and (B) glucose concentrations.

Note: I-L-4, insulin-loaded NPs prepared from a 1 mg·mL⁻¹ insulin solution.

Abbreviations: NP, nanoparticle; PBS, phosphate-buffered saline.

Figure 8 Cell viability as a function of the concentration of p(AAPBA-b-DEGMA) NPs, as assessed using the MTT assay at 37°C after 24 h exposure.

Notes: The data are expressed as mean ± SD from five independent experiments, with three replicates per experiment. PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y. The concentration refers to the initial concentration of the NP suspension; the final concentration in culture is one-third of this value.

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; NP, nano-particle; p(AAPBA), poly(3-acrylamidophenylboronic acid); SD, standard deviation.

Table 5 Blood biochemical markers measured after injection of the PAD-5-1 NPs into mice for 60 days (n=5, mean ± SD)

Marker (units)	Control group	Observation group (mg·kg^−1·d^−1)
		10	20	40
RBC (×10^6 µL^−1)	2.80±0.26	2.89±0.23	2.84±0.15	2.85±0.13
WBC (×10^3 µL^−1)	9.54±0.31	9.44±0.62	9.38±0.16	9.29±0.43
Hemoglobin (g·dL^−1)	14.12±1.16	14.79±1.23	14.64±1.86	14.83±2.25
Hematocrit (vol%)	35.92±1.17	36.73±1.57	35.85±2.27	36.01±1.85
Platelet (×10^3 µL^−1)	89.07±3.17	90.15±2.15	91.33±2.59	90.36±2.76
Serum protein (g·L^−1)	56.02±3.12	54.63±3.62	56.82±2.21	57.56±2.82
Serum creatinine (mg·dL^−1)	3.47±0.19	3.47±0.21	3.54±0.38	3.52±0.45
Serum glutathione (mg·dL^−1)	0.64±0.11	0.61±0.10	0.60±0.16	0.59±0.12
Total cholesterol (mg·dL^−1)	72.02±4.34	72.15±5.43	72.02±4.84	71.01±5.08
Glucose (mg·dL^−1)	262.31±10.13	253.35±9.12	250.39±8.17	246.58±9.92
Uric acid (mg·dL^−1)	5.21±0.27	5.26±0.38	5.42±0.46	5.38±0.37
AST (U·L^−1)	39.45±3.32	40.32±3.27	40.54±2.21	41.12±3.04
ALT (U·L^−1)	96.62±4.78	97.89±4.78	99.25±4.92	102.06±5.87

Note: PAD-5-1, p(AAPBA-b-DEGMA) (pAAPBA:DEGMA =1:5).

Abbreviations: ALT, alanine aminotransferase; AST, aspartate transaminase; DEGMA, diethylene glycol methyl ether methacrylate; p(AAPBA), poly(3-acrylamidophenylboronic acid); RBC, red blood cell count; SD, standard deviation; WBC, white blood cell count.

Figure 9 Representative HE staining images (200× magnification) from the (A) liver, (B) lung, (C) spleen, (D) kidney, and (E) heart with images marked (1) denoting the negative control group, and the observation groups were given (2) 10, (3) 20, and (4) 40 mg·kg⁻¹·d⁻¹ of PAD-5-1 by IP injection.

Note: PAD-5-1, p(AAPBA-b-DEGMA) (pAAPBA:DEGMA =1:5).

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; HE, hematoxylin–eosin; IP, intraperitoneal; p(AAPBA), poly(3-acrylamidophenylboronic acid).

Figure 10 Blood glucose concentrations after the treatment of diabetes mellitus mice with insulin formulations.

Notes: The control group comprised normal mice, and the NPs group animals treated with insulin-loaded PAD-5-1 [I-L-4]. I-L-4 nanoparticles were prepared using a 1 mg·mL⁻¹ insulin solution; PAD-5-1, p(AAPBA-b-DEGMA) (pAAPBA:DEGMA =1:5).

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; NP, nanoparticle; p(AAPBA), poly(3-acrylamidophenylboronic acid).

Figure S1 IR spectra of PAD-50-1, PAD-20-1, PAD-10-1, and PAD-2-1, recorded in the form of a KBr tablet (sample:KBr mass ratio 1:100).

Note: PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y.

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; IR, infrared; p(AAPBA), poly(3-acrylamidophenylboronic acid).

Figure S2 ¹H-NMR spectra of PAD-50-1 (NaOD/D₂O, pH 9.5), PAD-20-1 (NaOD/D₂O, pH 9.5), PAD-10-1 (NaOD/D₂O, pH 9.5), and PAD-2-1 (NaOD/D₂O, pH 9.5).

Notes: Peaks crossed through correspond to the solvents used for NMR. PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y.

Abbreviations: ¹H-NMR, ¹H nuclear magnetic resonance; DEGMA, diethylene glycol methyl ether methacrylate; p(AAPBA), poly(3-acrylamidophenylboronic acid).

Figure S3 DTG (left) and TG (right) data for PAD-50-1, PAD-20-1, PAD-10-1, and PAD-2-1.

Note: PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y.

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; DTG, derivative thermogravimetric; p(AAPBA), poly(3-acrylamidophenylboronic acid); TG, thermogravimetric.

Figure S4 (A) The stability of blank p(AAPBA-b-DEGMA) NPs in pH 7.4 PBS and (B) their reversible glucose sensitivity.

Note: PAD-X-Y, p(AAPBA-b-DEGMA) with DEGMA:pAAPBA molar ratios of X:Y.

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; NP, nanoparticle; p(AAPBA), poly(3-acrylamidophenylboronic acid); PBS, phosphate-buffered saline.

Figure S5 In vitro release of insulin into pH 7.4 PBS at different temperatures for (A) I-L-1, (B) I-L-2, (C) I-L-3, and (D) I-L-5 insulin-loaded NPs.

Abbreviations: I-L, insulin loading; NP, nanoparticle; PBS, phosphate-buffered saline.

Figure S6 In vitro release of insulin from I-L-4 into PBS (pH 7.4) at various glucose concentrations at (A) 25, (B) 38.5, and (C) 42°C.

Abbreviations: I-L, insulin loading; PBS, phosphate-buffered saline.

Figure S7 Digital photographs (400× magnification) of NIH 3T3 cells after treatment with PAD-5-1 NPs.

Notes: (A) Untreated cells and cells treated with (B) 8.33, (C) 16.7, (D) 25, (E) 33.3, and (F) 41.7 µg·mL⁻¹ of PAD-5-1. PAD-5-1, p(AAPBA-b-DEGMA) (pAAPBA:DEGMA =1:5).

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; NP, nanoparticle; p(AAPBA), poly(3-acrylamidophenylboronic acid).

Scheme S1 The reaction between PBA and glucose.

Abbreviation: PBA, phenylboronic acid.

Scheme S2 The synthesis of p(AAPBA) and p(AAPBA-b-DEGMA) by RAFT polymerization.

Notes: (A) Synthesis of p(AAPBA); (B) synthesis of p(AAPBA-b-DEGMA).

Abbreviations: AIBN, 2,2-azo-bis-isobutyronitrile; DDATC, S-1-dodecyl-S′-(α,α′,-dimethyl-α″-acetic acid) trithiocarbonate; DEGMA, diethylene glycol methyl ether methacrylate; DMF, dimethyl formamide; p(AAPBA), poly(3-acrylamidophenylboronic acid); RAFT, reversible addition–fragmentation chain transfer.

Scheme S3 A schematic illustrating self-assembly of the block copolymers into nanoparticles in aqueous solution, with and without insulin and glucose.

Abbreviations: DEGMA, diethylene glycol methyl ether methacrylate; p(AAPBA), poly(3-acrylamidophenylboronic acid).