Intracellular delivery mechanism and brain delivery kinetics of biodegradable cationic bovine serum albumin-conjugated polymersomes

Figures & data

Table 1 Particle sizes and zeta potential values of PO and CBSA-PO, loaded or not loaded with coumarin-6 (n = 3)

Vesicle	Mean size, nm	Zeta potential (mV)a
PO	95.2 ± 4.3	−19.2 ± 0.8
CBSA-PO	101.1 ± 7.6	9.3 ± 0.5
Coumarin-6-loaded PO	96.6 ± 5.2	−20.3 ± 0.7
Coumarin-6-loaded CBSA-PO	102.7 ± 5.5	9.5 ± 0.3

Note:

a Measured in NaCl solution (10⁻³ M).

Abbreviations: PO, polymersomes; CBSA, cationic bovine serum albumin.

Figure 1 Transmission electron microscopy images of cationic albumin-conjugated biodegradable polymersomes negatively stained with 1% uranyl acetate solution (bar 100 nm).

Table 2 X-ray photoelectron spectroscopy of maleimide-PEG-PCL:MPEG-PCL mixture (1:10), PO-coupled with or without CBSA

Samples	XPS elemental ratio (%)			XPS C1s envelope ratios (%)				XPS O1s envelope ratios (%)
	C	O	N	C–C/C–H	C–O–C	C–O–C=O	O–C=O	O=C	O–C
				Binding energy (eV)
				285.0	286.6	287.3	289.4	533.5	534.4
Copolymers	75.1	24.9	*	7.5	18.7	56.6	17.2	34.7	65.3
PO	74.2	25.8	*	27.4	30.8	27.3	14.5	44	56
CBSA-PO	74.1	25.5	0.4	13.5	33.6	33.1	19.8	37.7	62.3

Notes:

* Below the detection limit. Copolymers are maleimide-PEG-PCL:MPEG-PCL mixture (1:10).

Abbreviations: XPS, x-ray photoelectron spectroscopy; PO, polymersomes; PEG, poly(ethylene glycol); PCL, poly(ɛ-caprolactone); CBSA, cationic bovine serum albumin; MPEG, methoxy poly(ethylene glycol).

Figure 2 Carbon C1s envelopes from X-ray photoelectron spectroscopy analysis of (A) mixture of maleimide-PEG-PCL:MPEG-PCL (1:10) copolymers, (B) PO without CBSA attached, and (C) CBSA-PO (maleimide-PEG-PCL:MPEG-PCL, 1:10 ratio). (D) Nitrogen N1s envelopes from x-ray photoelectron spectroscopic analysis of CBSA-PO.

Abbreviations: PO, polymersomes; PEG, poly(ethylene glycol); CBSA, cationic bovine serum albumin; PCL, poly(ɛ-caprolactone); MPEG, methoxy poly(ethylene glycol).

Figure 3 Cell uptake of polymersomes at (A) different incubation temperatures and in the presence of various inhibitors of endocytosis for (B) CBSA-PO and (C) PO.

Notes: The bEND.3 cells were incubated with 100 μg/mL PO or CBSA-PO for one hour. Data presented as the mean ± standard deviation (n = 5). **P < 0.01, statistically significant differences by Student’s t-test when compared with the corresponding value of the control.

Abbreviations: PO, polymersomes; CBSA, cationic bovine serum albumin; pheASO, phenylarsine oxide.

Figure 4 In vitro cytotoxicity of PO, CBSA-PO, and CBSA against bEnd.3 cells, in a series of concentrations from 0.25 to 8 mg/mL.

Note: Data presented as the mean ± standard deviation (n = 4).

Abbreviations: PO, polymersomes; CBSA, cationic bovine serum albumin.

Table 3 Steady-state area under the plasma concentration-time curve and plasma clearance of different formulations labeled with coumarin-6

Group	AUCss (% ID*h/mL)	Cl (mL/h/kg)	MRT (hours)	PS (μL/h/g tissue)	% ID/g tissue
PO	2.94 ± 0.52	154 ± 24	1.47 ± 0.34	23.8 ± 2.7	0.052 ± 0.013
CBSA-PO	1.37 ± 0.42**	343 ± 83**	0.82 ± 0.25*	86.3 ± 8.6**	0.111 ± 0.015**
CBSA-PO + CBSA	2.20 ± 0.34*,†	205 ± 30*,†	0.96 ± 0.36	37.5 ± 5.8*,††	0.075 ± 0.019*,†

Notes: Data are the mean ± standard deviation, n = 4. Statistically significant differences by Student’s t-test when compared with the corresponding value of control:

* P < 0.05;

** P < 0.01 versus PO;

† P < 0.05;

†† P < 0.01 versus CBSA-PO.

Abbreviations: AUC_ss, steady-state area under the concentration time curve; Cl, clearance; MRT, mean residence time; PO, polymersomes; PS, permeability surface area product; CBSA, cationic bovine serum albumin.

Figure 5 Blood polymersome concentration (% ID/mL) versus time after intravenous injection of 10 mg/kg of PO, CBSA-PO, and CBSA-PO plus 10 mg/kg of free CBSA.

Note: Data are presented as the mean ± standard deviation of n = 4 rats/point.

Abbreviations: PO, polymersomes; CBSA, cationic bovine serum albumin; ID, the injected dose.

Figure 6 Polymersome concentrations (% ID/g) versus time curves in the rat brain fractions after intravenous injection of 10 mg/kg of PO, CBSA-PO, and CBSA-PO plus 10 mg/kg of free CBSA. Data are presented as the mean ± standard deviation of n = 4 rats/point. (A) Whole homogenate, (B) vascular pellet, (C) supernatant, and (D) ratio of polymersomes concentrations in the supernatant to those in the vascular pellet.

Notes: Statistically significant differences by Student’s t-test when compared with the corresponding value of control: *P < 0.05; **P < 0.01 versus PO; ^†P < 0.05; ^††P < 0.01 versus CBSA-PO.

Abbreviations: PO, polymersomes; CBSA, cationic bovine serum albumin.

Graphic abstract Brain delivery kinetics of cationic bovine serum albumin-conjugated biodegradable polymersomes.

Abbreviation: BBB, blood–brain barrier.