Harnessing biomaterial application strategies for neurorestoration after ischemic stroke injury: An emerging treatment option

Abstract

Background

Ischemic stroke is a medical condition caused by occlusion of blood vessels in brain, resulting in disruption of blood flow to the brain and triggering irreversible damage to the neuronal cells. While stem cells transplantation has been proposed as a potential alternative therapym for ischemic stroke, its effectiveness is limited due to low cell survival rate and potential side effects following transplantation. To overcome these challenges and enhance therapeutics efficacy, researchers have focused on developing various biomaterials to create a sustainable cellular microenvironment or to modify the properties of donor stem cell which could optimize their reparative functions in injured brain tissues.

Objective

This review aims to explore and discuss the different types of biomaterials that have been applied in the treatment of ischemic stroke, shedding light on their potentials as promising therapeutics options for this debilitating condition.

Methods

Literature search was performed to identify publications studying the potential of three biomaterials namely: nanobioparticles, hydrogels and extracellular vesicles for ischemic stroke therapy in vitro, in vivo or in clinical using four databases, namely: PubMed, ScienceDirect, Web of Science and Scopus.

Results and discussion

The major benefits obtained from the application of nanobioparticles for ischemic stroke therapy included as the nanocarrier for drug/cell delivery, cell tracking, real time imaging, promote cell proliferation, while hydrogels provided scaffold support and conferred neuroprotection to stem cells, as well as provided neurotropic effects and controlled drug release for localized treatment. Lastly the extracellular vesicles were identified as a cell-free treatment strategy in promoting angiogenesis, neuronal differentiation and neurogenesis for ischemic stroke treatment.

Conclusion

Biomaterial-based therapies have their own potentials and further clinical investigations are strongly recommended to translate the therapies into more conscientious evidence-based therapy for clinical application.

Keywords

biomaterial ischemic stroke nanobioparticles hydrogel extracellular vesicles

Introduction

Pathophysiology of ischemic stroke

Ischemic stroke is an irreversible cerebrovascular disease causes by permanently injured brain tissue due to the interruption of blood supply to the brain. Ischemic stroke can be divided into three types, which are thrombotic stroke, embolic stroke and lacunar stroke. Thrombotic stroke happens when the brain arteries are occluded by thrombus (blood clot) while the occurrence of embolic stroke is normally due to the formation of embolus somewhere in the body and travels until it reaches the blood vessel in the brain. Lacunar stroke, on the other hand, specifically occurs when the small artery that penetrates deep into the brain is narrowed due to chronic high blood pressure.¹

The progression of hypoxic ischemic injury in all types of ischemic stroke is triggered by the blockage of a blood vessel, leading to localized deprivation of oxygen and glucose supply. Following the deprivation of glucose and oxygen (OGD), there would be a shift in the glucose oxidation from aerobic respiration to anaerobic respiration and the process would lead to the reduction of adenosine triphosphate (ATP) synthesis (Figure 1).^2–4 Following the deprivation of ATP supply, a cascade of events that ultimately lead to cell death in domino-like situation will take place as the brain is a high energy-demanding organ.⁵

Figure 1.

Cellular respiration during (A) normal physiological condition and (B) ischemic stroke condition. (A) In the presence of oxygen, glucose is converted into pyruvate, which produces 2 ATP and NADH. Pyruvate then enters the mitochondria and undergoes further processing to form acetyl CoA, releasing CO₂ and generating additional NADH. Following that, the oxidation of Acetyl CoA in the TCA cycle would occur, leading to the production of 2 ATP, NADH and FADH₂. Through a series of protein complexes in the electron transport chain located in the inner membrane of mitochondria, the high-energy electrons carried by NADH and FADH₂ are transported to another 32 ATP (B). When oxygen is deprived during ischemic stroke, glucose would be processed via anaerobic respiration, causing damaging side effects of acidosis and generating less ATP [5]. ATP: Adenosine triphosphate; ADP: Adenosine diphosphate; acetyl CoA: acetyl coenzyme A; Cyt C: Cytochrome C; CO₂: carbon dioxide; e⁻: Electron; FADH₂: flavin adenine dinucleotide; H⁺: Proton; H₂O: Water; NADH: Nicotinamide adenine dinucleotide (NAD) + hydrogen; O₂: Oxygen; Pi: Phosphate; TCA: Tricarboxylic acid.

Firstly, the ATP-dependent sodium-potassium (Na⁺/K⁺) pump, which is crucial to maintain the ionic balance across the neuronal cell membranes for proper neuronal membrane potential,⁶ will be shut down due to lack of ATP,⁷ leading to the influx of water and rapid swelling of neurons and glial cells (cytotoxic edema).⁸ Furthermore, the failed ionic pumps can also cause neurons and astrocytes to depolarize, this would trigger an excessive release of neurotransmitter, particularly glutamate, leading to neuronal excitotoxicity.⁹

Excitotoxicity occurs when there is an excessive release of neurotransmitters, primarily glutamate, and an overreaction of their receptors, particularly the N-methy1-D-asapartate receptor (NMDAR).¹⁰ Normally glutamate can be located within the synaptic terminal and it can be cleared from the extracellular space through the GluN2A subunit of NMDAR, which is predominantly located at synaptic sites (Figure 2A).¹¹ Nonetheless, in the occurrence of acute ischemia, it would be difficult for glutamate to be regulated by the GluN2A subunit, leading to its accumulation in the synaptic cleft and extracellular space. This would cause the neuronal membrane to depolarize, resulting in the opening of voltage gated sodium (Na²⁺) and calcium (Ca²⁺) channels and an increase in intracellular Na²⁺ and Ca²⁺ levels.^9,10 The persistent membrane depolarization and overstimulation by glutamate lead to further influx of Na²⁺ and Ca²⁺ ions, and efflux of potassium (K⁺), exacerbating the ionic imbalance and excitotoxicity. Consequently, cell death signaling is activated through the GluN2B subunit at the extrasynaptic sites, causing neuronal cell death (Figure 2B).¹²

Figure 2.

Glutamate expression at synaptic terminal under (A) normal condition and (B) ischemic stroke condition. (A) Under normal condition, through the activation of the GluN2A NMDAR subunit, glutamate would be cleared from the extracellular space. However, it is also dependent on the calcium influx through the receptors. (B) During cerebral ischemia, NMDAR would be reverted to the inactive state due to the failure of ionic pumps, leading to glutamate to be accumulated excessively at the synaptic cleft and extracellular space. As a result, the ionic imbalance and glutamate excitotoxicity would further deteriorate, leading to the activation of GluN2B subunit that recruits death signaling protein which eventually induce neuronal cell death (Figure modified from¹²).

Emerging novel biomaterial-based treatments for ischemic stroke therapy

Restoration or enhancement of blood flow to the ischemic area has emerged as a crucial therapeutic approach for treating strokes, which are primarily caused by thromboembolic occlusion. Currently, intravenous recombinant tissue plasminogen activator (rtPA) is the only United States Food and Drug Administration (FDA)-approved medication in treating ischemic stroke. This drug functions by activating plasminogen, leading to the formation of plasmin, a crucial proteolytic enzyme that breaks down the cross-links between the fibrin, restoring blood flow in the blocked blood vessels. However, the use of this thrombolytic drug heavily associates with several limitations including an increased risk of fatal intracranial hemorrhage (6.3%), short therapeutic window of less than 4.5 h, high rate of re-incidence, and a short half-life of less than 5 min, restricting the widespread application of this medication rather significantly.¹³

The usage of endovascular mechanical thrombectomy is another alternative method in treating ischemic stroke. This minimally invasive surgical procedure is utilized to remove the blood clot in the brain by inserting a catheter into the affected blood vessel, guided by the imaging technology. However, specialized equipment and trained personnel are required for this procedure due to the associated risks such as bleeding, infection and potential damage to the blood vessel, limiting the utilization of this procedure.¹⁴

In the past decades, there have been lack of significant breakthroughs in stroke treatment. Nonetheless, recently, a ray of hope emerges from a promising research that combines a safer, multi-targeted biomaterial-based novel treatment for ischemic stroke therapy. The increasing developments in biomaterials have made them valuable in the medical field, offering unique properties that can be easily modified to achieve desirable pharmaceutical and biomedical characteristics.¹⁵ This advancement has led to the emergence of an extensive selection of biomaterials, including drug delivery carriers, diagnostic tools, biomedical implants and devices, biosensors and tissue engineering in regenerative medicine.¹⁶ Despite these developments, there is a lack of comprehensive reviews that examine the properties of potential biomaterials and their therapeutic applications, particularly for ischemic stroke treatment. Therefore, this review article conducted a thorough search of literature databases to provide an inclusive summary of the latest findings on the properties and applications of three selected state-of-art biomaterials namely: nanobioparticles, hydrogels and extracellular vesicles, used for ischemic stroke therapy.¹⁷

Methodology

Literature review search strategy

In this review, a literature search was performed to identify publications studying the potential of three biomaterials namely: nanobioparticles, hydrogels and extracellular vesicles for ischemic stroke therapy in vitro, in vivo or in clinical. Four specialized databases: PubMed, ScienceDirect, Web of Science and Scopus were used for the literature search using the keywords “Biomaterial for ischemic stroke treatment”, “nanobioparticle”, “hydrogel” and “extracellular vesicle”.

Research article selection and evaluation

Search results were limited to fully documented articles in English following the inclusion and exclusion criteria listed below:

Inclusion criteria:

Full-text articles

In vitro studies related to ischemic stroke therapy using biomaterial such as nanobioparticles, hydrogels or extracellular vesicles.

In vivo studies related to ischemic stroke therapy using biomaterial such as nanobioparticles, hydrogels or extracellular vesicles.

Intervention clinical trial related to ischemic stroke therapy using biomaterial such as nanobioparticles, hydrogels or extracellular vesicles.

Exclusion criteria:

Irrelevant titles and abstracts

Duplicated studies

Review articles/meta-analyses

News/editorials/letters

Case reports

Non-English language

Two independent reviewers screened the articles based on the inclusion and exclusion criteria stated above. For the first screening, the related articles were screened based on their titles and abstracts. Next, the remaining papers were checked for duplications. Finally, the selected full-text articles were checked by the third reviewer according to the inclusion criteria for final validation.

Result and discussion

Based on the collected literature review, the in-depth discussion about the application of nanobioparticles, hydrogels and extracellular vesicles for the treatment of ischemic stroke disease were presented. We found that the major benefits obtained from the application of these strategies included roles for drug/cells delivery, cell tracking, real time imaging, promote cell proliferation, conferred neuroprotection and neurotropic effects and controlled drug release. In addition, a side-by-side comparison of the different types of biomaterials, their treatment strategies and the key beneficial outcomes for ischemic stroke therapy were summarized into Table 1. We also highlighted the limitations of current treatment for ischemic stroke and describe how did the novel treatment strategies using the three types of biomaterials able to upgrade current treatment for ischemic stroke (Table 1).

Table 1.

Summary of different types of nanobioparticles, hydrogels or extracellular vesicles biomaterial, treatment strategy and key beneficial outcomes of using these three groups of biomaterials for ischemic stroke therapy.

Group	Types of biomaterial	Limitations of current treatment for ischemic stroke	Novel treatment strategy using biomaterial to upgrade current treatment for ischemic stroke	Key beneficial outcomes	Ref.
Nanobio-particles	Human serum albumin (HAS) nanocarriers	Ropinirole hydrochloride (Rp) is potential drug candidate for mitochondrial-mediated neuroprotection in ischemic stroke. However, its effectiveness has been hindered by its low bioavailability and limited diffusion across blood-brain-barrier (BBB)	Encapsulation of Rp within HAS nanocarriers could enhance the Rp biodegradability because HAS is water-soluble, non-toxic, non-immunogenic and biodegradable. HAS also can be coated with gamma-L-Glutamyl-L-Cysteine (γ-L-Glu-L-Cys) to enance the targeted delivery of Rp across BBB towards the brain neuron cells.	Enhanced delivery to ischemic brain regions. Prolonged circulation of Rp with higher bioavailability in vivo. Attenuated brain infarction Decreased neurological deficits Protected mitochondrial integrity and augment ATP production Decreased dopamine and inflammatory cytokine	¹⁸
	Magnetic nanobubbles (MNBs) embedded with superparamagnetic iron oxide nanoparticles (SPIONPs)	Neural stem cells (NSCs) are potential cell source for ischemic stroke therapy. However, inability to track their migration in vivo is the major critical challenge for the clinical application of NSC-based treatment.	NSCs can be labelled with MNBs embedded with SPIONPs for effective in vivo tracking purpose. MNBs are effective contrast agent for ultrasound imaging. SPIONPs are biodegradable contrast agents for magnetic resonance imaging (MRI). SPIONPs can be coated on the shell of MNBs to enhance dual imaging (both ultrasound and MRI) for optimum in vivo tracking of NSCs.	MNB-SPIONPs distributed stably in the intracellular of NSCs. NSCs labeled with MNB-SPIONPs were successfully tracked in vivo. The labeled cells survived at ischemic stroke lesions and differentiate within the stroke boundary 14 days after implantation.	^19,20
	Cerium oxide (CeO₂) nanoparticles loaded with Dl-3-n-butylphthalide (NBP)	Oxidative stress and mitochondrial damage are the main ischemia-reperfusion injury in ischemic stroke.	Dl-3-n-butylphthalide (NBP) is a new class I chemical drug targeting not only anti-thrombosis, but also anti-inflammation and protection of mitochondria. CeO₂ nanoparticles was used as carriers for NBP.	Improved the deficits in sensory, motor function and spatial learning ability of ischemic stroke mice. Inhibited neuroinflammation Maintained mitochondrial morphology. Protected the integrity of BBB.	²¹
	pH-responsive fluorescent liposomal probe (BOD@Lip)	Current computed tomography (CT) scan and MRI scanning tools are not efficient for continuous monitor the disease progression or to detect real time changes occurring inside the ischemic brain.	BOD@Lip is near-infrared (NIR) fluorescence probe sensitive to pH changes. Ischemic tissue has a lower pH than normal tissue due to the accumulation of lactic acid, thus higher fluorescent signal released by BOD@Lip. The fluorescent signal from BOD@Lip is long-lasting, thus can be detected continuously to monitor the disease progression in vivo.	Can efficiently enter the brain parenchyma and accumulate at the lesion site through the impaired BBB. Not cytotoxic to brain cells The correlation between fluorescence signals and the degree of ischemic stroke severity was established and confirmed by Neurological Deficit Score.	²²
	Thrombin-responsive polyphenol-based nanoparticles (TPN)	Tissue-plasminogen activator (PA) thrombolytic agent treatment is limited by its narrow therapeutic windows, low biocompatibility and high risk of intracranial bleeding.	PA can be loaded into TPN before administration to minimize premature release of the PA and prevent nonspecific plasminogen activation, which can cause adverse events such as hemorrhagic complications.	Improved PA loading capacities and stability in vivo. Optimized tPA drug release kinetics. Exhibited lower tPA accumulation on non-targeted organs.	²³
Hydrogel	Hyaluronic acid (HA) hydrogel loaded with 6-bromoindirubin-3′-oxime (BIO) and vascular endothelial growth factor (VEGF)	Overactive inflammation and poor endogenous angiogenesis capacity are the two main challenges in ischemic stroke treatment.	Development of modified HA-based hydrogel loaded with BIO (as anti-inflammatory agent) and VEGF (as angiogenesis agent) can effectively tackle these two challenges. This is because hydrogel delivery system allow drugs or growth factors to be encapsulated and delivered directly into the brain, bypassing the BBB, enabling effective drug accumulation and sustained release at the targeted site for enhanced therapeutic outcomes.	Scavenged ROS and attenuate apoptosis. Decreased inflammatory response. Promoted angiogenesis. Enhanced neovascular maturation. Ameliorated functional deficits.	²⁴
	Layer-by-layer assembly of neural stem cells (NSCs) on HA hydrogel-loaded with VEGF	Transplantation of NSCs is a promising regenerative treatment strategy for ischemic stroke. However, the survival rate of transplanted NSCs is low due to the harsh microenvironment after ischemic stroke, limiting the efficiency of the NSC-based therapy.	NSCs can be coated on HA hydrogel layer-by-layer (LbL) to establish a layered scaffold structure for optimal NSCs adhesion, proliferation and survival. Moreover, the LbL HA hydrogel can be loaded with VEGF to enhance angiogenesis after transplantation.	Increased NSCs survival and differentiation into neurons. Enhanced neurogenesis and facilitate angiogenesis. Potentiated neuroblast migration to penumbra. Promoted BBB repair.	²⁵
	Genipin-conjugated sericin hydrogels (Gen-SH)	Cell-based therapy for ischemic stroke is limited by the low cell survival rate after transplantation in vivo.	The survival of cells can be improved by seeding them on a synthetic biomaterial known as Gen-SH. Gen-SH is made of sericin, a natural silk protein with excellent biocompatibility and biodegradability. This biomaterial could provide a highly porous platform to support cell attachment, cell proliferation and long-term cell survival.	Aided adhesion and long term growth of neuronal cells. Maintained primary neurons survival and cellular integrity. Facilitated neural tissue repair and survival. Promoted axons outgrowth.	²⁶
	Thermos-sensitive hydrogel scaffold	Large scale of NSCs died after transplantation due to harsh microenvironment	NSCs can be seeded on a thermos-sensitive hydrogel scaffold known as PFP [Poly (trimethylene carbonate)₁₅-F127-poly(trimethylene carbonate)₁₅ (PTMC₁₅-F127-PTMC₁₅). PFP is a biodegradable hydrogel to encapsulate NSCs and slow release the cells in vivo for up to 16 days. Moreover, the PFP can be loaded with neurotropic factors such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) to confer protection to NSCs and enhance neurogenesis.	Sustained the levels of neurotrophic factors, set good foundation for survival and differentiation of transplanted NSCs. Promoted differentiation of the transplanted NSCs into mature neurons. Reduced apoptosis of ischemic penumbra. Reduced ischemic infarct size. Improved behavioral performance of ischemic rats.	²⁷
Extracellular vesicles (EVs)	M2 phenotype microglia-derived extracellular vesicles modified with DA7R peptide and SDF-1α cytokine	Endogenous NSCs are multipotent cells located in brain as basic unit of tissue repair after ischemic injury. Nonetheless, the number of existing endogenous NSCs is extremely low, leading to insufficient cell sources for nerve regeneration.	EVs derived from M2 microglia can be modified with DA7R peptide and SDF-1α cytokine to effectively recruit NSCs to lesion sites and induce them to differentiate into neurons for repairing ischemic stroke injury.	Targeted delivery to ischemic site. Increased accumulation within ischemic region Effectively recruit NSCs Stimulate neuronal differentiation Promote neurogenesis Enhance neurological recovery	²⁸
Extracellular vesicles (EVs)	Ultrasmall superparamagnetic iron oxide (USPIO)-labeled extracellular vesicles (EVs)	EVs secreted by human stem cells show significance in treating neurological disorders, such as ischemic stroke. However, the clinical applications of these EVs are limited due to their poor targeting capabilities and low therapeutic efficacies after intravenous administration.	EV isolated from induced forebrain neural progenitor cortical organoids (iNPCo) can be loaded with USPIOs by sonication to enhance targeting ability, tracking and imaging. USPIOs are biocompatible nanoparticles which have been shown to improve the targeting ability of EVs when an external magnet field is applied	Enhanced MRI contrast for cell tracking	²⁹

Nanobioparticles

Nanobioparticles are small biological particles with a specific range of size between 1–100 nanometres that can be employed in medical fields to enhance the therapeutic potential of various drugs or cells.³⁰ This is mainly because of their ability to overcome the biological barriers along the passages to deliver the useful drugs or cells to the targeted area efficiently.³⁰ In medical fields, different types of nanobioparticle products have been applied such as human serum albumin nanocarriers, superparamagnetic iron oxide nanoparticles (SPIONPs), cerium oxide nanoparticles, liposome-based and polyphenol-based nanoprobe/nanoparticles.

Human serum albumin nanocarriers

The role of mitochondria in energy homeostasis, ROS formation and apoptotic pathway is crucial in the pathophysiology of ischemic stroke.³¹ Therefore, therapeutic interventions that target mitochondria hold promise for the management of this disease. Ropinirole hydrochloride (Rp) has been proposed as a potential drug candidate for mitochondrial-mediated neuroprotection in ischemic stroke. However, its effectiveness has been hindered by its low bioavailability properties.³² To overcome this limitation, a study by Saman et al. developed ligand-linked human serum albumin (HSA) as a nancocarrier of Rp (Rp-NPs).¹⁸ HSA was chosen as a nanocarrier as it is easily accessible, with beneficial properties including water-solubility, non-toxic nature, non-immunogenic, biodegradable, and great amenability for surface decoration with apposite ligand to improve targeting and nanoparticles stability.³³ To improve the delivery of Rp across BBB, molecular docking technique was employed to coat Rp-NPs using gamma-L-glutamyl-L-cysteine (γ-L-Glu-L-Cys), a dipeptide and glutathione-precursor. The γ-L-Glu-L-Cys was selected as is has strong affinity to the N-methyl-D-aspartate receptor (NMDAR) for enhanced brain targeting. The resulting polymer demonstrated enhanced targeting ability towards the ischemic area, prolonged Rp retention in the systematic circulation and attenuated the ischemic injury progression. This was achieved by disrupting the apoptotic mechanism, reducing oxidative stress and suppressing inflammation.¹⁸

Magnetic nanobubbles (MNBs) embedded with superparamagnetic iron oxide nanoparticles (SPIONPs)

The utilization of NSCs in the treatment of various nervous system diseases is due to their potential in multidirectional differentiation and self-renewal ability. However, the clinical application of NSCs faces some challenges due to the lack of ability in tracking their migration in vivo and having poor control over their survival and differentiation efficiency. In 2022, Jin et al. fabricated magnetic nanobubbles (MNBs) through the self-assembly of poly-glucose sorbitol carboxymethyl ether-modified γ-Fe₂ O₃ superparamagnetic iron oxide nanoparticles (SPIONPs) to label the NSCs for in vivo tracking.²⁰ SPIONPs, nanoparticles with unique physical, chemical and biological properties, were integrated into micro/nanobubble shells to enhance multimode imaging and drug delivery function.¹⁹ The SPIONPs assembled MNBs were synthesized by a repeated compression method to enable the nanoparticles to be absorbed on the surfaces of the nanobubbles without any polymer or phospholipid shells. The compression speed of 30 mm/s and 150 compression cycles were the optimum rate for MNBs with a concentration of 11.34 µg/mL. Additionally, the morphological images of MNBs under the transmission electronic microscopy (TEM) clearly showed that the surface formed a regular circle structure that was almost consistent with 150 nm in size. The MNBs embedded with SPIONPs were then added to NSCs and administered into a photothrombotic ischemic stroke model. According to the results, MRI and ultrasound imaging could be used to track the transplanted MNB-NCSs effectively. Following the tracking for 14 days post implantation, the NSCs were found to migrate to stroke lesion areas, survive and differentiate within the stroke boundary, confirming the efficiency of NSC-based therapy for ischemic stroke treatment.

Cerium oxide nanoparticles as carriers for Dl-3-n-butylphthalide

In another study done by Li et al., cerium oxide nanoparticles (CeO₂) was produced by thermally decompose cerium acetonate hydrate in a mixed organic solvent of oleylamine and octadecene prior to coat with polyethylene glycol (PEG) at a high temperature.²¹ The CeO₂ nanoparticles were used as carriers for a chemical drug known as Dl-3-n-butylphthalide (NBP) for the synergistic treatment of ischemic stroke. The NBP-loaded cerium oxide nanoparticles (NBP–CeO₂ NPs) demonstrated a strong capability to neutralize free radicals, effectively reducing ROS in brain microvascular endothelial cells (BMVECs) and hippocampal neurons (HT22) following oxygen-glucose deprivation and reoxygenation (OGD/R). This intervention helped restore mitochondrial integrity. Additionally, damage to the BBB and neuronal apoptosis were alleviated by preserving mitochondrial structure and functionality. In a mouse model of middle cerebral artery occlusion/reperfusion (MCAO/R), NBP–CeO₂ NPs protected mitochondrial health, stabilized the BBB, significantly reduced cerebral infarction and edema, and minimized neuroinflammation and neuronal apoptosis. Long-term studies indicated that the NBP–CeO₂ NPs facilitated angiogenesis and markedly improved neural function recovery post-ischemic stroke in mice. Overall, NBP–CeO₂ NPs offer a promising therapeutic strategy for ischemic stroke by integrating antioxidant and neurovascular repair properties, underscoring their potential for treating ischemia-reperfusion injuries.

pH-responsive fluorescent liposomal probe (BOD@Lip)

A nanoparticle probe known as pH-responsive fluorescent liposomal probe (BOD@Lip) was first developed by Yao et al. to visualize the degree of ischemic stroke in vivo real-time.²² The work described in the study provided a novel method to overcome the limitation of current ischemic stroke assessment methods which are mainly carried out by computed tomography (CT) and magnetic resonance imaging (MRI) scanning. CT scan is not able to identify early ischemic stroke while the MRI scan is limited by lengthy scanning time and it is also not suitable for patients with metal implantation.²² In the study, the BOD@Lip was prepared by loading a hydrophobic pH responsive near-infrared (NIR) fluorescent probe into amphipathic liposomes with a molar ratio of 7/3 (phosphatidylethanolamine/oleic acid). The purpose of amphipathic liposomes which is also known as lipidic supramolecular is to encapsulate and carry molecules of both hydrophilic and hydrophobic particles.³⁴ The size of the synthesized BOD@Lip was 191.0 nm in diameter with a low polydispersity index and it was consistently in regular spherical structure and size, which indicated excellent stability in different storage temperatures. BOD@Lip was found to accumulate at the ischemic lesion site in the brain once injected into the animal model with ischemic stroke. Furthermore, they are sensitive to pH changes, resulting to higher fluorescent signal responses with lower pH Values. Due to the accumulation of lactic acid because of high rate of glycolysis, ischemic tissue has a lower pH than normal tissue. Prolonged ischemic condition would result in higher accumulation of lactic acid and lower pH value at the ischemic brain area, leading to higher BOD@Lip fluorescence signal. Based on this principle, the degree of ischemic stroke can be assessed by real-time fluorescence imaging with BOD@Lip. The study also established the correlation between Neurological Deficit Score and the detected BOD@Lip fluorescence signals, which could be applied by other clinicians to determine the ischemic stroke based on the BOD@Lip fluorescence signal rapidly and comprehensively. Through real-time and accurate monitoring of ischemic stroke severity, clinicians can objectively evaluate and adopt appropriate treatment methods for individual patients.

Thrombin-responsive polyphenol-based nanoparticles (TPN) as carrier for tissue plasminogen activators (tPAs)

The first-line treatments against acute thrombosis and ischemic stroke are thrombolytic (clot busting) therapies with tissue plasminogen activators (PAs). Due to several factors, the systemic delivery of PAs is clinically limited, leading to diminished therapeutic outcomes and compromised risk−benefit profiles. These include rapid neutralization by endogenous inhibitors and high risk of severe side effects. In 2022, Yu et. al. developed a thrombin-responsive nanocarrier which aimed to deliver PAs specifically to the thrombus-site in the ischemic stroke brain.²³ The nanocarrier was synthesized using a mesoporous silica nanoparticles (MS NP) loaded with urokinase-type thrombolytic PA (uPA). Then, the uPA-loaded MS NP was incubated with thrombin-cleavable low-fouling peptide (Pep) and tannic acid (TA) to produce MS@PA/Pep/TA NPs. As a naturally occurring polyphenol, TA is known for its broad adherence to diverse substances, non-covalent interactions and responsive properties.^35,36 The synthesized MS@PA/Pep/TA NPs was found to significantly improve the PA loading capacity, delay PA blood clearance as well as reduce nonspecific cell association, providing a desirable thrombolysis effects for the ischemic stroke patients.

Hydrogels

Hydrogel delivery systems offer promising advancements in stroke treatment by allowing drugs or growth factors to be encapsulated and delivered directly into the brain through local injection or implantation. This approach bypasses the BBB, enabling effective drug accumulation and sustained release at the targeted site, which enhances therapeutic outcomes while minimizing side effects. Additionally, hydrogels form three-dimensional polymer networks that mimic the native extracellular matrix (ECM), providing structural scaffolds within the stroke-affected area to promote regeneration and recovery after an ischemic stroke. In addition, hydrogel could be loaded with various cytokines or drugs to create a microenvironment favourable for neurogenesis.

Hyaluronic acid (ha) hydrogel-loaded with 6-bromoindirubin-3′-oxime (BIO) and vascular endothelial growth factor (VEGF)

Study by Liu et al. implemented hydrogel made from modified hyaluronic acid (HA) to deliver a combination of 6-bromoindirubin-3′-oxime (BIO) and vascular endothelial growth factor (VEGF) as anti-inflammatory and pro-angiogenesis agents, respectively, to treat photothrombotic stroke animal models.²⁴ BIO is a semisynthetic cell-permeable indirubin derivative that had been reported to alleviate the early inflammatory response of ischemic stroke due to its ability to inhibit glycogen synthase kinase 3 beta (GSK3β) which plays role in the release of inflammatory cytokines from lipopolysaccharides (LPS).³⁷ Nonetheless, BIO has poor aqueous solubility and adverse side effects, therefore, a triblock amphiphilic copolymer known as PF127 was used to form drug-encapsulated nanoparticles, enabling slow release of BIO. On the other hand, VEGF is a protein with vascular permeability activity that could enhance vascular endothelial cells proliferation and angiogenesis. In the study, VEGF was encapsulated in poly lactic-co-glycolic acid (PLGA) porous microspheres to sustain and control the release of VEGF through pore size modulation. The combination of PF127/BIO and PLG/VEGF was incorporated in HA-based hydrogels to allow effective BBB penetration. Due to smaller particle size, PF127/BIO were released from the hydrogel faster, reduced the inflammatory response and cell apoptosis. Meanwhile, due to its relatively large particle size, the PLGA/VEGF porous microspheres enabled slower and more sustained release, promoting angiogenesis in the area affected by the stroke.²⁴ Overall, PF127/BIO nanoparticles and PLGA/VEGF porous microspheres loaded onto the HA hydrogel system resulted in decreased inflammation, enhanced vascular regeneration and improved long-term neurological recovery after ischemic stroke.

Layer-by-layer hyaluronic acid (Ha) hydrogel-loaded with vascular endothelial growth factor (VEGF) and neural stem cells (NSCs)

In another study conducted by Ge et al., HA hydrogel loaded with VEGF (HA-VEGF) also was utilized as the main component for constructing a hydrogel.²⁵ In addition, Ge et al. also added NSCs to the HA-VEGF layer-by-layer (LbL) to construct a layered platform for optimal NSCs adhesion, proliferation and survival. The LbL HA hydrogel can stabilize the microenvironment of NSCs and confer neuroprotective effects on NSCs. Moreover, the LbL HA hydrogel was loaded with VEGF to enhance angiogenesis after transplantation. The results showed that the biomaterial structure enhanced survival and differentiation of implanted NSCs into neurons in mice with distal middle cerebral artery occlusion (dMCAO). Moreover, the engraftment of LbL (VEGF)-NSCs promoted the functional recovery by reducing the volume of infarct core in ischemic stroke mice as it enhanced neurogenesis, angiogenesis, survival of host neurons, and repaired the BBB.²⁵

Genipin-conjugated sericin hydrogels (Gen-Sh)

Cell transplantation has long been considered as a promising approach for neural repair. However, challenges such as low cell survival rates in vivo has hindered the successful implementation of this strategy. To overcome these obstacles, Zhao et al. designed a genipin-conjugated sericin hydrogels (Gen-SH) utilizing sericin, a natural silk protein spun by the domestic silkworm Bombyx mori.²⁶ Sericin was chosen as the natural polymer material for this study due to its excellent biocompatibility, mild immunogenicity, biodegradability, and the ability to promote cell adhesion and proliferation.³⁸ However, its amorphous structure poses a challenge in biomedical applications due to its poor mechanical performance. To address this issue, the researchers conjugated the sericin hydrogels with a biocompatible cross-linker known as genipin, resulting in genipin-conjugated sericin hydrogels (Gen-SH) that exhibited a highly porous morphology and a moderate swelling rate. In vitro experiments demonstrated that Gen-SH facilitated the attachment and long term growth of neurons. Furthermore, the study revealed that sericin inherently exhibited neuroprotective and neurotropic effects, promoting branching and extensions of axons while preventing hypoxia-induced cell death in primary neurons. Not only that, the breakdown products of Gen-SH inherited these neuroprotective and neurotropic capabilities, eliminating the need for cytokines. Additionally, the study also highlighted the Gen-SH hydrogels promoted cell proliferation and demonstrated a high percentage of cell survival when they were transplanted in vivo.²⁶

Thermos-sensitive hydrogel scaffold

According to the study by Qi et al., a biodegradable thermos-sensitive hydrogel scaffold known as poly (trimethylene carbonate)₁₅₋F127-poly (trimethylene carbonate)₁₅ (PTMC₁₅-F127- PTMC₁₅, or in brief PFP) can be synthesized to provide permissive substrates for donor and control drug release as well as increase the survival of NSCs.²⁷ The PFP was prepared through the ring-opening polymerization (ROP) of trimethylene carbonate (TMC) in tetrahydrofuran (THF) using Pluronic (F127) as an initiator and tetramethylethylenediamine (TMEDA) as the catalyst. The PFP is biodegradable biomaterial, it is also non-immunogenic and can be easily transform from liquid to gel at 37°C. In the study, PFP was loaded with NSCs, along with several important neurotropic factors which included with brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and Neurotrophin-3 (NT-3). Taken together, the PFP scaffold became a promising neurotrophic factors release system to support NSCs survival and differentiation. According the study, transplantation of PFP loaded with NSCs and neurotrophic factors (BDNF, NGF and NT-3) effectively reduced the apoptosis of ischemic penumbra in model rats. Furthermore, they also found that neurotropic factors released by PFP increased mature neuron numbers that were differentiated from donor NSCs. The result suggests that this biomaterial can effectively increase the therapeutic potential of NSCs for ischemic stroke by effective controlled release of BDNF, NGF, and NT-3 to support survival of grafted NSCs.

Extracellular vesicles

Membrane-bound vesicles released by cells or commonly known as extracellular vesicles possess active molecules such as RNA and proteins that regulate various cell functions and biological processes.³⁹ These extracellular vesicles have gained significant research interest as a promising form of cell-free therapy due to their ability to imitate the physiological functions of the parent cells and low immunogenicity.⁴⁰

M2 phenotype microglia-derived extracellular vesicles modified with DA7R peptide and SDF-1α cytokine

M2 phenotype microglia is an important type of immune cell that could inhibit neuroinflammation and accelerate neurogenesis.⁴¹ The EV secreted by M2 phenotype microglia has been reported to promote the differentiation of NSCs into neurons.⁴² Thus, Ruan et al. collected the EVs derived from M2 phenotype microglia and further modified them to achieve rapid and effective recruitment and differentiation transformation of NSCs.²⁸ The modification involved adding injured vascular targeting peptide (also known as DA7R peptide; ^DR^DP^DP^DL^DW^DT^DA) and the stromal cell-derived factor-1α (SDF-1α) on the surface of the EV to produce DA7R-SDF-1-EV. DA7R peptide is highly specific and high affinity for neuropilin-1 (NRP-1) and vascular endothelial growth factor receptor 2 (VEGFR2),⁴³ while SDF-1α is as a class of chemokine that could mediate cell migration, effectively inducing NSCs to migrate to the lesion site during nerve injury. Taken together, DA7R-SDF-1-EV can target injured vascular specifically to increase their accumulation at the ischemic region to achieve neurogenesis after stroke. The efficacy of this EV-based system was validated in ischemic stroke mouse model in which the DA7R-SDF-1-EV was able to increase NSCs recruitment, stimulate their neuronal differentiation and enhance neurogenesis.²⁸

Ultrasmall superparamagnetic iron oxide (USPIO)-labeled extracellular vesicles

Through the incorporation of ultra-small superparamagnetic iron oxide (USPIOs), Liu et al. carried out a research to improve the targeting capabilities of extracellular vesicles.²⁹ The extracellular vesicles were derived from the media of induced forebrain neural progenitor cortical organoids (iNPCo)and loaded with USPIOs by sonication. Notably, this study demonstrated that the USPIO-labeled extracellular vesicles exhibited significantly higher contrast in MRI imaging, compared to the unlabelled extracellular vesicles.²⁹ The successful USPIO labelling of extracellular vesicles was deemed as a promising approach for in vitro tracking of extracellular vesicles derived from brain organoids, thus paving the way for future in vivo investigations. This study complements the previous study done by Ruan et al. by displaying a different strategy to enhance extracellular vesicles targeting. While Ruan et al. focused on surface modification for enhanced targeted delivery of extracellular vesicles, Liu et al. employed USPIO labelling to improve the tracking and imaging capabilities of extracellular vesicles.

Conclusion and recommendations

In enhancing the therapeutic approach for ischemic stroke, this review article focuses on the new ideas of biomaterial applications. The biomaterials such as nanobioparticles, hydrogen and extracellular vesicles could be applied in the wide variety of functions due to their special properties, which can easily be modified and adapted further to achieve desirable microenvironment and biomedical characteristics. In conclusion, the biomaterials have high potential to improve current treatments for ischemic stroke from different aspects and future studies should aim on overcoming the remaining obstacles in transforming these potential biomaterials into next-generation ischemic stroke therapy.

Footnotes

Acknowledgments

The authors extend their gratitude to the Universiti Sains Malaysia (USM) libraries, particularly Perpustakaan Hamdan Tahir and Perpustakaan Hamzah Sendut, for providing invaluable resources that have been instrumental in crafting this systematic review. Additionally, the authors would like to acknowledge and express their appreciation to all the scientists whose previous work contributed to this review articles.

Author contributions

A.M.S and F.A.O.: literature review search and write original draft; S.C.T.: literature review search, review and edit original draft. All authors have read and agreed to the final draft.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This article was funded by the Ministry of Higher Education Malaysia for Fundamental Research Grant Scheme with Project Code: FRGS/1/2023/SKK10/USM/03/2.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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