Brain-targeting, acid-responsive antioxidant nanoparticles for stroke treatment and drug delivery

Abstract

Stroke is the leading cause of death and disability. Currently, there is no effective pharmacological treatment for this disease, which can be partially attributed to the inability to efficiently deliver therapeutics to the brain. Here we report the development of natural compound-derived nanoparticles (NPs), which function both as a potent therapeutic agent for stroke treatment and as an efficient carrier for drug delivery to the ischemic brain. First, we screened a collection of natural nanomaterials and identified betulinic acid (BA) as one of the most potent antioxidants for stroke treatment. Next, we engineered BA NPs for preferential drug release in acidic ischemic tissue through chemically converting BA to betulinic amine (BAM) and for targeted drug delivery through surface conjugation of AMD3100, a CXCR4 antagonist. The resulting AMD3100-conjugated BAM NPs, or A-BAM NPs, were then assessed as a therapeutic agent for stroke treatment and as a carrier for delivery of NA1, a neuroprotective peptide. We show that intravenous administration of A-BAM NPs effectively improved recovery from stroke and its efficacy was further enhanced when NA1 was encapsulated. Due to their multifunctionality and significant efficacy, we anticipate that A-BAM NPs have the potential to be translated both as a therapeutic agent and as a drug carrier to improve the treatment of stroke.

Keywords: A-BAM NPs, A-BAM NPs; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Acid-triggered release; Antioxidant nanoparticles; BA, betulinic acid; BAM, betulinic amine; BBB, blood brain barrier; BIRDS, biosensor imaging of redundant deviation in shifts; BT, ß-sitosterol; DLS, dynamic light scattering; DTA, dehydrotrametenolic acid; DYDA, diketohydrindylidene diketohydrindamine; Drug delivery; GA, glycyrrhetic acid; Ischemic stroke; LCMS, liquid chromatography mass spectrometry; LP, lupeol; MCAO, middle cerebral artery occlusion; NA1; NMR, nuclear magnetic resonance; NP, nanoparticle; OA, oleanolic acid; PAA, poricoic acid; PEG, polyethylene glycol; SA, sumaresinolic acid; SEM, scanning electron microscopy; ST, stigmasterol; TEM, transmission electron microscope; TTC, triphenyltetrazolium chloride; UA, ursolic acid; tPA, tissue-type plasminogen activator.

Graphical abstract

Scheme 1

Schematic diagram of NA1-A-BAM NP synthesis.

Scheme 2

Schematic diagram of application of NA1-A-BAM NPs for stroke treatment.

Fig. 1

Screen of natural nanomaterials for stroke treatment. a) Molecular structures of the selected natural nanomaterials and their derived NPs. Scale bar: 500 nm. b) Characteristics of NPs derived from the indicated nanomaterials. c) Quantification of the percentage of infarct area in the brain of stroke mice receiving treatment of the indicated NPs 3 days after MCAO surgery. Data are presented as mean ± SD (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. t-test).

Fig. 2

Development of BAM NPs for acid-triggered drug release. a) Scheme of BAM synthesis. b) Distribution of pH in the brain at day 0 and day 2 after ischemic insult as determined by BIRDS. c) Release of IR780, a model payload, from BA or BAM NPs with time at pH 6.5 or pH 7.4. d) SEM analysis of morphological changes of BA or BAM NPs after incubation at pH 6.5 or pH 7.4 for the indicated time. Scale bar: 500 nm. Data are presented as mean ± SD (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001. t-test).

Fig. 3

Synthesis and characterization of A-BAM NPs for targeted drug delivery to stroke. a) Western blot analysis of the expression of CXCR4 in ischemic brain tissue at the indicated time. Control: normal brain tissue. b) Representative images of CXCR4 expression in the region normal or ischemic brain tissue. Scale bar: 30 μm. c) Morphology of BAM NPs with and without AMD3100-conjugation as determined by SEM. d-e) Representative images (d) and semi-quantification (e) of the indicated NPs in the brain of stroke mice after intravenous administration. f) Representative images of brain slices with TTC staining of ischemia (left) and fluorescence imaging of IR780 (right). Data are presented as mean ± SD (n = 3; *P < 0.05, **P < 0.01. t-test).

Fig. 4

Evaluation of A-BAM NPs as a therapeutic agent for stroke treatment. a-d) Representative images a) and quantification b) of cerebral infarction (n = 3), c) Kaplan−Meier survival analysis (n = 7), and d) neurological scores (day 3 after surgery, n = 5) of stroke mice receiving the indicated treatments. Infarct area and neurological scores were determined on day 3 after surgery. Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. t-test).

Fig. 5

Characterization of A-BAM NPs for targeted delivery of NA1 for stroke treatment. a) Representative images of NA1-A-BAM NPs by SEM (Scare bar: 200 nm) and TEM (inset, scare bar: 100 nm). b) Release of NA1 from NA1-A-BAM NPs with time at the indicated pH. c) Kaplan−Meier survival analysis (n = 7), d) infarct area (n = 5), and e) neurological scores (day 3 after surgery, n = 5) of stroke mice receiving the indicated treatments. Infarct area and neurological scores were determined on day 3 after surgery. Data are presented as mean ± SD (***P < 0.001, ****P < 0.0001. t-test).