*3.3. Delivery of siRNA*

Small interfering RNAs (siRNAs) can suppress the expression of a specific gene and are also used in ischemic stroke treatment [85]. Caspase-3 activation contributes to brain tissue loss and downstream biochemical events, which lead to programmed cell death in many brain injuries including ischemic stroke [86]. Khuloud et al. investigated whether using carbon nanotube-mediated in vivo RNA interference (RNAi) to silence Caspase-3 could offer a therapeutic opportunity against stroke. Peri-lesional stereotactic administration of functionalized carbon nanotubes (f-CNT) carrying Caspase-3 siRNA (siCas 3) decreased the neurodegeneration, improved behaviors, and reduced ischemic lesion in an endothelin-1 induced stroke model [69].

In another study, a gene delivery vector, PAMAM-Arg, consisting of a polyamidoamine (PAMAM) dendrimer amide grafted with basic L-arginine residues (e-PAM-R), was used to deliver the high mobility group box-1 (HGMB1) siRNA [70]. HMGB1 serves as a danger signal that evokes inflammatory responses in various types of cells. After the e-PAM-R/siRNA complex was administered in the rat MCAO model, HMGB1 levels and neuronal cell death were significantly decreased. In addition, the reduction of infarction sizes in the brain was also observed.

#### **4. Stroke Diagnosis using Nanotechnology**

Magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), and ultrasound are employed for the diagnosis of stroke. Brain CT and MRI imaging are important to detect the early stage of stroke in patients [87]. CT angiography can discover the arterial dissection in the brain vessels and changes of collateral blood flow. Diffusion-weighted imaging is a type of MRI methods used to detect the early stage of ischemic stroke because the imaging system can localize and measure the ischemic lesion to confirm the infarct sites [88]. I/R injury is associated with vascular inflammation, so fluorescence imaging can be used to investigate the changes of brain vessels using IV injection of fluorescent dyes such as dextran- or BSA-conjugates [89].

Shen's research groups developed a method to track administered stem cells and investigated cell-based therapies in ischemic stroke. Cationic polymersomes formed polymeric vesicles and they were loaded with superparamagnetic iron oxide nanoparticles (SPIONs) and quantum dots (QDs) as imaging agents. Cationic polymersome vesicles were incubated with neural stem cells (NSCs) to label the NSCs, thus NSCs could be tracked after they were injected to the striatum contralateral of the ischemic hemisphere. The migration and location of NSCs were monitored using MRI imaging in six weeks in a rat MCAO model and the optical imaging tracked the cells for four weeks [90]. Another example is that neural stem cells (NSCs) were labeled with an MRI reporter ferritin heavy chain (FTH) and enhanced green fluorescent protein (EGFP) to monitor the stem cells in the long-term for detecting ischemic stroke [91]. Those approaches are promising to understand how stem cells target the ischemic tissues and to develop the cell-based therapies to treat ischemic stroke.

Andreas et al. conducted a clinical phase II pilot trial using ultra small superparamagnetic iron oxide (USPIO)-enhanced MRI for macrophage imaging since USPIO particles have been introduced as a cell-specific MRI contrast agents taken up by macrophages. USPIO contrast agent was infused in ten patients 5 to 6 days after stroke onset, and MRI was performed within 24–36 h or 48–72 h after the infusion. Results showed that USPIO was much better than gadolinium (a clinically used imaging agent). The study indicates that USPIO-enhanced MRI may provide an in vivo tool to track cells in stroke and other CNS pathologies [92].

Theranostics nano-platform is a new way to combine diagnostics and therapeutics in ischemic stroke. In a study, HSP72 antibodies against HSP72, a specific molecular biomarker of the peri-infarct region, were conjugated to liposomes. Rhodamine/gadolinium labelled- and citicoline (CDP-Choline)-loaded liposomes specifically targeted the peri-infarct tissue in cerebral ischemia. MRI results demonstrated that the nanoparticles were accumulated in the injured brain and significantly reduced lesion volumes [93].

Clinically used gadolinium (Gd) chelates as contrast agents have several issues, such as short circulation time, rapid clearance, and low T1 signal. These issues limit the imaging time and resolution [94]. Upconversion nanoparticles doped with Gd ions can greatly enhance the T1 signals. In addition, upconversion nanoparticles can image the deep tissue and possess the photostability. Jing et al. reported synthetic UCNPs of core/shell structure (NaYF4:Yb/Er@NaGdF4) and coated with PEG (PEG-UCNPs). PEG-UCNPs showed the high diagnostic sensitivity to image acute ischemic stroke at a low dosage (5 mg Gd kg−1) compared to the clinical dosage of 108 mg Gd kg−<sup>1</sup> [95]. This new formulation may be promising in clinic.

#### **5. New Opportunities and Perspectives**

Stroke is an acute disease, therefore treating stroke requires the early diagnosis and immediate therapies. This review has highlighted the recent advances in design and engineering of new nanomaterials to target ischemic stroke tissues and new technologies used to image and monitor the disease progression in vivo.

Specifically delivering therapeutics to the injured brain is essential in treating ischemic stroke. While many nanoparticle-based formulations or cell-based platforms have been developed, the fundamental question of how they target ischemic stroke lesion has not been clearly addressed. For example, BBB is the blood vessel barrier to prevent therapeutics across the blood vessels. Most studies showed the dramatic therapeutic effects of using nanoparticle-based formulations compared to free drugs. The enhanced outcomes claimed that nanoparticles transported therapeutics across BBB, but the direct in vivo experimental data were vague to support this conclusion. Developing advanced in vivo imaging systems [96,97] is needed to visualize the intact brain [68] and to address whether and how nanoparticles cross the BBB.

Brain vasculature during stroke shows the temporal opening, and this disruption of BBB may be a target to guide small molecules across the BBB. Further investigation is needed on the time course of BBB opening to design ideal drug delivery platforms. Although ischemic stroke increases brain vascular permeability, the endothelial gaps are unlikely to allow the efficient transport of nanoparticles because their size is usually larger than endothelial gaps. Therefore, developing new and novel concepts is needed to solve the drug delivery across BBB. Ischemic stroke and reperfusion cause acute inflammatory responses including neutrophil infiltration across blood vessels. Recent studies have shown that rational design of nanoparticles (gold nanoparticles, polymeric nanoparticles, or protein nanoparticles) enables neutrophils to transport nanoparticles across the blood vessel barrier in infection and cancer mouse models [6,98–101]. It is expected that this technology of hijacking neutrophils in vivo may transport nanotherapeutics across BBB for therapies of ischemic stroke.

Ischemic stroke is strongly correlated to inflammatory responses. Inflammatory responses include endothelial activation and neutrophil infiltration, which damages the brain tissues. Targeting activated endothelium using cell membrane-derived nanovesicles has demonstrated the value in delivering therapeutics to treat ischemic stroke [68]. To translate cell-derived nanovesicles, developing new technologies is needed to scale up their production. Recent studies show that nitrogen cavitation methods [77] and other approaches [102] could have the potential to scale up cell-derived nanovesicles for clinical applications. In addition, Dong [68] et al. reported an interesting study to deliver lipid mediators [84] (such as Resolvin D2) to treat ischemic stroke. This study is different from current therapies that mainly deliver anti-inflammatory agents. Anti-inflammatory therapies can cause side effects [103], but Resolvin D2 is a new drug to increase the host immune defense via increased neutrophil apoptosis and macrophage phagocytosis [104]. In the future, it is needed to investigate how to efficiently load lipid mediators in nanovesicles [105] for improved treatment of ischemic stroke. Another direction is the design of new nanoparticles in response to inflammatory environments (such as pH or enzymes) [8] to improve the treatment of ischemic stroke.

Targeting inflammatory neutrophils in situ to block brain neutrophil infiltration is a new opportunity to treat ischemic stroke. A recent study [7] shows that albumin protein-formed nanoparticles loaded with doxorubicin could induce neutrophil apoptosis, thus inhibiting neutrophil infiltration to prevent brain damage in a mouse ischemic stroke model. This is an exciting and new research area to develop novel therapies to solve the lacking pharmacological therapies for ischemic stroke in clinic.

In addition, developing new drugs that can target inflammatory pathways for management of the host injury during ischemic stroke is needed. The pathogenesis of ischemic stroke is complicated, and it is involved with multiple signaling pathways. The molecular mechanisms of ischemic stroke-induced brain injury are needed to be further determined. The timing of administering drugs or nanoparticle-based therapeutics is also very critical. Optimizing therapeutic windows in the future is needed.

Theranostics formulations are interesting and promising in treating ischemic stroke since they combine diagnosis and therapies. Nanoparticle-based platforms are novel constructs because they can contain imaging agents and drugs in single nanoparticle platforms. For instance, formulations with both neuroprotectants and Fe2O3 magnetic nanoparticles can achieve the therapy and imaging. In the future, developing more similar drug delivery systems is needed to treat ischemic stroke. However, considering that many inorganic materials do not naturally exist in the body (although iron oxide

nanoparticles (IONPs) have been approved by the US food and drug administration (FDA) to treat anemia), fully evaluating the biodistribution and toxicity after systemic administration is required.
