Microbubbles coupled to methotrexate-loaded liposomes for ultrasound-mediated delivery of methotrexate across the blood–brain barrier

Figures & data

Table 1 Treatment conditions for studying BBB disruption

Group	Ultrasound intensity (W/cm^2)	Duration (minutes)	Duty cycle (%)	MTX-liposome-coupled microbubbles (mL/kg)
1	1.0	2	10	0.2
2	1.0	5	20	0.5
3	1.0	10	50	1.0
4	2.0	2	10	1.0
5	2.0	5	20	0.5
6	2.0	10	50	0.2
7	2.4	2	50	0.5
8	2.4	5	10	1.0
9	2.4	10	20	0.2

Abbreviations: BBB, blood–brain barrier; MTX, methotrexate.

Figure 1 Transmission electron microscopic image of methotrexate-loaded biotinylated liposomes, composed of biotin-DSPE-PEG2000, dipalmitoyl phosphatidyl-glycerol, distearoyl phosphatidylcholine, and PEG4000, showing spherical morphology.

Note: Bar 100 nm.

Abbreviations: DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; PEG, polyethylene glycol.

Figure 2 Size distribution of methotrexate-liposome-coupled microbubbles.

Notes: The size distribution of methotrexate-liposome-coupled microbubbles (A) was different from that of unmodified ZHIFUXIAN microbubbles (B).

Figure 3 Inhibition of proliferation of Walker-256 cells after treatment for 24 hours with or without sonication.

Notes: Data are presented as the mean ± standard deviation (n=9). **P<0.01; ***P<0.001.

Abbreviations: MTX, methotrexate; MNL, MTX-loaded liposomes; MNL-CA, MTX-liposome-coupled microbubbles; US, ultrasound; UCA, unmodified ZHIFUXIAN.

Figure 4 Induction of apoptosis in Walker-256 cells after treatment for 24 hours with or without sonication.

Notes: Data are presented as the mean ± standard deviation (n=9). ***P<0.001.

Abbreviations: Con, control; MTX, methotrexate; MNL, MTX-loaded liposomes; MNL-CA, MTX-liposome-coupled microbubbles; US, ultrasound; UCA, unmodified ZHIFUXIAN.

Figure 5 Microphotographs of Evans blue staining in rat brains to monitor the extent of blood–brain barrier disruption in rats following treatment with methotrexate-liposome-coupled microbubbles and ultrasound.

Notes: (A) Rats receiving Evans blue injection only. (B) Rats treated with methotrexate-methotrexate-liposome-coupled microbubbles and ultrasound under the conditions shown for group 5 in Table 1.

Figure 6 EB extravasation into the rat brain following the treatments shown in Table 1.

Notes: EB was extracted from the brain tissues and determined by absorbance at 620 nm. The rats in the control group received the EB injection only. Data are presented as the mean ± standard deviation (n=10). *P<0.05 versus control; ***P<0.001 versus control; NS, P>0.05 versus control.

Abbreviations: Con, control; EB, Evans blue.

Figure 7 Concentration of MTX in the parietal lobe of the rat brain after treatment with MTX-liposome-coupled microbubbles with or without ultrasound.

Notes: Data are presented as the mean ± standard deviation (n=10). ***P<0.001.

Abbreviations: MTX, methotrexate; US, ultrasound; MNL-CA, MTX-liposome-coupled microbubbles; UCA, unmodified ZHUFIXIAN.

Figure 8 H&E-stained brain tissue after optimal ultrasound treatment.

Notes: Every tenth brain section was stained with H&E. No tissue or capillary damage was noted in rats treated with sonication. An example section is shown. Bar 200 μm.

Abbreviation: H&E, hematoxylin-eosin.

Figure 9 Transmission electron microscopic images of lanthanum nitrate-perfused rat brain.

Notes: (A) Control rats; bar 500 nm. (B) Rats treated with methotrexate-liposome-coupled microbubbles and ultrasound showing lanthanum deposition outside the basement membrane of the vascular wall. The arrow shows lanthanum deposition between the nerve fibers; bar 400 nm.