Electrostatic interactions between polyglutamic acid and polylysine yields stable polyion complex micelles for deoxypodophyllotoxin delivery

Figures & data

Figure 1 Synthesis steps of different polymers.

Notes: (A) d,l-Lactide; (B) mPEG-PDLLA; (C) mPEG-PDLLA-Phe-^NBOC; (D) mPEG-PDLLA-NH₂; (E) mPEG-PDLLA-PBLG and mPEG-PDLLA-PBLL; (F) mPEG-PDLLA-PLG and mPEG-PDLLA-PLL.

Abbreviations: mPEG, methoxy PEG; PDLLA, poly(d,l-lactide); PEG, poly(ethylene glycol); Phe-NBOC, N-(tert-butoxycarbonyl)-l-phenylalanine; PLG, polyglutamate; PLL, poly(l-lysine); PBLG, poly(γ-benzyl-l-glutamate); PBLL, poly(γ-benzyl-l-lysine); TFA, trifluoroacetic acid.

Figure 2 Structural representations of mPEG-PDLLA-PBLG and mPEG-PDLLA-PBLL.

Abbreviations: PEG, poly(ethylene glycol); PDLLA, poly(d,l-lactic acid); mPEG, methoxy PEG; PBLG, poly(γ-benzyl-l-glutamate); PBLL, poly(γ-benzyl-l-lysine).

Table 1 Composition and molecular characteristics of polymers

Polymer	Composition of copolymer			Molecular weight (Mw)
	x	y	z
mPEG2000-PDLLA2000-PBLG1095	45.50	26.70	4.83	4,973.6
mPEG2000-PDLLA2000-PBLL1230	45.50	26.70	4.91	5,127.8

Abbreviations: mPEG, methoxy poly(ethylene glycol); PDLLA, poly(d,l-lactic acid); PBLG, poly(γ-benzyl-l-glutamate); PBLL, poly(γ-benzyl-l-lysine).

Table 2 Molecular weight calculated from ¹H-NMR and GPC

Polymer	Mn × 10^−3 (^1H-NMR)	Mn × 10^−3 (GPC)	Mw × 10^−3 (GPC)	Mw/Mn
mPEG2000-PDLLA2000-PBLG1095	5.0	4.3	5.6	1.29
mPEG2000-PDLLA2000-PBLL1230	5.1	4.8	5.8	1.21
mPEG2000-PDLLA2000-PLG735	4.6	4.1	5.1	1.24
mPEG2000-PDLLA2000-PLL730	4.6	4.3	5.7	1.33

Notes: M_n indicates number-average molecular weight; M_w indicates weight-average molecular weight, M_w/M_n indicates the polydispersity of molecular weight distribution.

Abbreviations: GPC, gel permeation chromatography; mPEG, methoxy polyethylene glycol; NMR, nuclear magnetic resonance; PLG, polyglutamate; PLL, poly(l-lysine); PDLLA, poly(d,l-lactic acid); PBLG, poly(γ-benzyl-l-glutamate); PBLL, poly(γ-benzyl-l-lysine).

Table 3 Drug loading and entrapment efficiency of DPT micelle solutions

Sample	LE (%)	EE (%)
DPT-PCM solution	16.41±0.10	98.46±0.70
DPT-PM solution	16.35±0.14	98.11±0.99

Note: Data are shown as mean value ± SD (n=3).

Abbreviations: DPT, deoxypodophyllotoxin; EE, entrapment efficiency; LE, loading efficiency; PCM, polyion complex micelle; PM, polymeric micelle.

Table 4 Particle size distribution of DPT micelles after reconstitution

Sample	Particle size, nm	Polydispersity	Zeta potential, mV
DPT-PCM	36.3±0.8***	0.218±0.009**	−3.31±0.17
DPT-PM	27.3±0.4	0.130±0.026	−4.15±0.29

Notes: Data are shown as mean value ± SD (n=3).

** P<0.01 and

*** P<0.001 as compared to DPT-PM.

Abbreviations: DPT, deoxypodophyllotoxin; PCM, polyion complex micelle; PM, polymeric micelle.

Figure 3 TEM images and DSC thermograms of DPT formulations.

Notes: (A) TEM images of (a) DPT-PM and (b) DPT-PCM. (B) DSC thermograms of (a) DPT; (b) PICs; (c) physical mixture composed of DPT and PICs (weight ratio of DPT/PICs =1/5); and (d) DPT-PCM (weight ratio of DPT/PICs =1/5).

Abbreviations: DPT, deoxypodophyllotoxin; DSC, differential scanning calorimetry; PCM, polyion complex micelle; PEG, poly(ethylene glycol); PDLLA, poly(d,l-lactide); PIC, polyion complex; mPEG, methoxy PEG; PLG, polyglutamate; PLL, poly(l-lysine); PM, polymeric micelle; TEM, transmission electron microscopy.

Figure 4 Stability tests of DPT-PCM and DPT-PM.

Notes: DPT-loaded PMs and size changes over time at (A) 4°C, (B) 25°C, and (C) 37°C. The effect of temperature on the physical stabilities of drug-loaded micelles at (D) 4°C, (E) 25°C, and (F) 37°C. Data are shown as mean ± SD (n=3). ***P<0.001 as compared to DPT-PM, respectively.

Abbreviations: DPT, deoxypodophyllotoxin; PCM, polyion complex micelle; PM, polymeric micelle.

Figure 5 The in vitro cytotoxicity of blank PM, blank PCM, DPT, DPT-PM, and DPT-PCM against A549 cells after (A) 24 h and (B) 48 h of incubation.

Notes: Data are shown as mean ± SD (n=6). ***P<0.001 as compared to DPT, blank-PM, and blank-PCM at 24 h and 48 h. **P<0.01 as compared to DPT-PM at 48 h.

Abbreviations: DPT, deoxypodophyllotoxin; PCM, polyion complex micelle; PM, polymeric micelle.

Figure 6 Investigation on drug release, pharmacokinetics, and histopathological changes.

Notes: (A) (a) Cumulative release profiles of DPT from DPT-PCM at pH 7.4, pH 6.8, and pH 5.0 (mean ± SD, n=3). *P<0.05 and **P<0.01 as compared to DPT-PM at the same pH. ***P<0.001 as compared to pH 7.4. (b) The profile of the plasma DPT concentration vs time after intravenous administration of 15 mg/kg dose of DPT-PCM and 15 mg/kg dose of DPT-PM (mean ± SD, n=6). (B) Histopathological photograph of an ear vein (scale bar =100 µm).

Abbreviations: DPT, deoxypodophyllotoxin; PCM, polyion complex micelle; PM, polymeric micelle.

Table 5 Pharmacokinetic parameters of DPT-PCM and DPT-PM

Sample	Dose, mg/kg	C0, μg/mL	t1/2, min	AUC0–inf, μg/mL⋅min	CL, mL/min/kg	MRT, min	Vss, μg/(μg/mL)
DPT-PCM	15.00	25.43±1.48***	35.33±4.11	588.62±119.59	26.28±5.92	37.41±6.36	958.03±35.92**
DPT-PM	15.00	33.83±1.00	32.71±4.57	660.66±33.47	22.76±1.15	31.89±2.47	728.69±93.08

Notes: Data are shown as mean ± SD (n=6).

** P<0.01 and

*** P<0.001 as compared to DPT-PM.

Abbreviations: AUC_0–inf, total area under the plasma concentration–time curve; CL, clearance rate; DPT, deoxypodophyllotoxin; MRT, mean residence time; PCM, polyion complex micelle; PM, polymeric micelle; t_1/2, half-time; Vss, volume of distribution at steady state.

Figure S1 ¹H-NMR spectra at 300 M of (A) mPEG-PDLLA; (B) mPEG-PDLLA-Phe-^NBOC; (C) mPEG-PDLLA–NH₂; (D) mPEG-PDLLA-PBLG; (E) mPEG-PDLLA-PBLL; (F) mPEG-PDLLA-PLG; and (G) mPEG-PDLLA-PLL.

Abbreviations: DPT, deoxypodophyllotoxin; mPEG, methoxy polyethylene glycol; NMR, nuclear magnetic resonance; PDLLA, poly(d,l-lactide); PLG, polyglutamate; PLL, poly(l-lysine); Phe-^NBOC, N-(tert-butoxycarbonyl)-l-phenylalanine; PBLG, poly(γ-benzyl-l-glutamate); PBLL, poly(γ-benzyl-l-lysine).

Figure S2 FT-NIR spectra of block copolymer (A) mPEG-PDLLA; (B) mPEG-PDLLA-PBLG; (C) mPEG-PDLLA-PBLL; (D) mPEG-PDLLA-PLG; and (E) mPEG-PDLLA-PLL.

Note: Characteristic peaks are circled in red.

Abbreviations: FT-NIR, Fourier transform near-infrared; mPEG, methoxy polyethylene glycol; PDLLA, poly(d,l-lactide); PLG, polyglutamate; PLL, poly(l-lysine); PBLG, poly(γ-benzyl-l-glutamate); PBLL, poly(γ-benzyl-l-lysine).