Bioengineering CXCR4-overexpressing cell membrane functionalized ROS-responsive nanotherapeutics for targeting cerebral ischemia-reperfusion injury

Rationale: As a potentially life-threatening disorder, cerebral ischemia-reperfusion (I/R) injury is associated with significantly high mortality, especially the irreversible brain tissue damage associated with increased reactive oxygen radical production and excessive inflammation. Currently, the insufficiency of targeted drug delivery and “on-demand” drug release remain the greatest challenges for cerebral I/R injury therapy. Bioengineered cell membrane-based nanotherapeutics mimic and enhance natural membrane functions and represent a potentially promising approach, relying on selective interactions between receptors and chemokines and increase nanomedicine delivery efficiency into the target tissues. Methods: We employed a systematic method to synthesize biomimetic smart nanoparticles. The CXCR4-overexpressing primary mouse thoracic aorta endothelial cell (PMTAEC) membranes and RAPA@HOP were extruded through a 200 nm polycarbonate porous membrane using a mini-extruder to harvest the RAPA@BMHOP. The bioengineered CXCR4-overexpressing cell membrane-functionalized ROS-responsive nanotherapeutics, loaded with rapamycin (RAPA), were fabricated to enhance the targeted delivery to lesions with pathological overexpression of SDF-1. Results: RAPA@BMHOP exhibited a three-fold higher rate of target delivery efficacy via the CXCR4/SDF-1 axis than its non-targeting counterpart in an in vivo model. Additionally, in response to the excessive pathological ROS, nanotherapeutics could be degraded to promote “on-demand” cargo release and balance the ROS level by p-hydroxy-benzyl alcohol degradation, thereby scavenging excessive ROS and suppressing the free radical-induced focal damage and local inflammation. Also, the stealth effect of cell membrane coating functionalization on the surface resulted in extended circulation time and high stability of nanoparticles. Conclusion: The biomimetic smart nanotherapeutics with active targeting, developed in this study, significantly improved the therapeutic efficacy and biosafety profiles. Thus, these nanoparticles could be a candidate for efficient therapy of cerebral I/R injury.


The extraction of cell membrane
Reagents A melt at room temperature and PMSF (Phenylmethylsulfonyl fluoride) was added. A few minutes before use to make the final concentration of PMSF 1 mM.
Cultured about 20-50 million cells, washed them with PBS, and scraped the cells with a cell scraper. Cells were collected by centrifugation, and cell precipitation was left for later use after supernatant was removed. Added 1 mL working solution to 20-50 million cells, gently and fully suspend the cells, and left in an ice bath for 10-15 min.
The samples were successively frozen and thawed three times in liquid nitrogen and room temperature. Removed nuclei and unfragmented cells: centrifuged at 4 ºC, 700g, for 10 min. Carefully collected the supernatant into a new centrifuge tube. When the supernatant was absorbed, did not contact with the precipitation to precipitate cell membrane fragments: centrifuged at 4 ºC for 14000×g for 30 min to precipitate cell membrane fragments.

Preparation of NPs
HBA-OC-PEG 2000 (HOP) was prepared by chemical synthesis. Briefly, HBA, PEG 2000 were dissolved into CH 2 Cl 2 (methylene chloride). Mixed the dissolved HBA with PEG 2000 , and the whole reaction process must be operated at low temperature (in an ice bath), OC (oxaloyl chloride) was rapidly added into the above mixture solution.
It had been reacted at room temperature and stayed overnight, then the HOP was obtained. RAPA@HOP was prepared by nanoprecipitation method. HOP and RAPA were respectively dissolved in the tetrahydrofuran (THF), under the action of full agitation, appropriate amount of RAPA THF solution was dropped into the HOP THF solution which were mixed with H 2 O. The mixture was further dialyzed using dialysis bag (molecular weight cut-off, MWCO: 3500 Da) against water to remove the free RAPA and THF.

The construction of intracellular ROS overexpression
The cells were plated at a density of 5 × 10 5 cells/well in six-well Biocoat plates and grown for 24 h in complete medium that 1640 culture medium supplemented with 10% fetal bovine serum (FBS), at 37 °C in a normoxia with 5% CO 2 atmosphere.   and there was no significant difference between the results (intra-group analysis), but between-group comparisons demonstrated that significant differences exists between the groups ≥ 300 μM and groups < 300 μM; T1: the nanoparticle size after 10 min; T2: the nanoparticle size after 12 h].

1). Mouse thoracic aortic endothelial cells and bioengineering cells were
respectively removed from the culture flashs and resuspended at 0.5×10 6 cells/mL in serum free culture medium.
2). SDF-1(100 ng/mL) was placed in the lower wells. Test cells were then placed in the upper chamber for 60 min.
3). After incubation, the upper surface of the transwell membrane was wiped gently with a cotton swab to remove non-migrating cells. Cells which migrated to the lower surface of the membrane were stained using DAPI. 4). Migrated cells were counted in 3 different fields of a defined size (5×0.25 mm 2 ) using a phase contrast microscope and the mean cellular migration rate was calculated.