Stroke is defined as a neurological impairment lasting for at least 24 h that results from an acute focal injury in the central nervous system (CNS) attributed to a vascular cause (Sacco et al., 2013). This debilitating neurovascular disease occurs when the brain fails to receive an adequate supply of oxygen, which can lead to long-term consequences such as cognitive decline, dementia, and other neurodegenerative disorders (Freitas-Andrade et al., 2020). Cerebrovascular disorders, including stroke, demonstrate high rates of mortality, as the global lifetime risk is approximately 25% (Johnson et al., 2019), with roughly 800,000 individuals experiencing a new or recurring stroke in the United States each year (Kim et al., 2020). Strokes are classified as either ischemic or hemorrhagic depending on the underlying mechanism. According to a 2021 report from the American Heart Association (AHA), 87% of all strokes are ischemic (Virani et al., 2021), which most commonly occur from an occlusion in the cerebral vasculature (Siegel and Sapru, 2019). Due to the high metabolism of the brain, a reduction in blood flow and oxygen to the cerebral tissue distal to the occlusion can lead to irreversible neuronal cell death within a relatively brief timeframe (Fifield and Vanderluit, 2020; Pekny et al., 2019).

Despite stroke being a leading cause of both death and long-term disability worldwide, there are presently no effective therapeutic approaches that can alleviate the functional deficits associated with cerebral ischemia. Currently, the AHA guidelines recommend endovascular techniques, such as mechanical thrombectomy, or the use of pharmacological agents, such as recombinant tissue-type plasminogen activator (rtPA) in the management of acute ischemic stroke (Powers et al., 2019). Although rtPA is commonly administered for the treatment of vascular occlusion, its therapeutic use is limited by several factors, including a narrow time window of 3–4.5 h after symptom onset for proper administration and potential adverse side effects, such as vasogenic edema and cerebral hemorrhage (Liu and Chopp, 2016; Powers et al., 2019). Additionally, patients treated with either endovascular or pharmacological interventions often experience poor outcomes due to the likelihood of increased oxidative stress and neuroinflammation (Altinay et al., 2017). Although reperfusion therapies may be useful in reestablishing arterial flow to the ischemic region, rapid restoration of blood flow is known to trigger a cascade of pathophysiological events which exacerbates tissue damage, referred to as ischemia/reperfusion (I/R) injury (Campbell and Khatri, 2020). Therefore, considerable attention should be devoted to developing therapeutic strategies that attenuate the deleterious effects of reperfusion injury following ischemic stroke.

Cerebral ischemia produces a central core of permanently damaged tissue and surrounding area, which is known as the penumbra (McCabe et al., 2018). The penumbra is a primary target for ischemic stroke therapy, as it represents the hypo-perfused region of non-functional but still viable brain tissue in which the damage can potentially be reversed (González-Nieto et al., 2020; Schaller and Graf, 2004). Ischemic damage continues to evolve over the following hours to days after stroke, with the initial injury expanding into the surrounding penumbra leading to further cell death (Moskowitz et al., 2010). I/R injury is a common consequence of stroke and occurs when cerebral blood flow is rapidly restored to the infarcted tissue (Eghbaliferiz et al., 2020). The spatiotemporal progression of events after I/R injury can be categorized into multiple distinct phases of response to acute focal damage to the CNS, including (1) cell death, (2) neuroinflammation, (3) activation and proliferation of glial cells, and (4) tissue remodeling (Burda and Sofroniew, 2014; Buscemi et al., 2019; Pekny and Nilsson, 2005; Sofroniew, 2015).

The neuroinflammatory cascade following cerebral ischemia results in a multitude of pathophysiological effects that can be attributed to an array of cellular and molecular events (Bao et al., 2012). Following ischemic stroke, cerebral ischemia triggers an immune-mediated inflammatory response resulting in the promotion of pro-inflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, IL-10, and tumor necrosis factor-α (TNF-α; Bavarsad et al., 2019). These pro-inflammatory cytokines amplify the inflammatory response and lead to disruption of the blood-brain barrier (BBB), thus allowing certain molecules to penetrate between the endothelial cells of the cerebral vasculature (Babazadeh et al., 2020). Consequently, a major component of this process relates to the activity of astrocytes, which are glial cells that function to maintain the integrity of the BBB through their support of extensive tight junctions (TJs). These specialized TJs are located between cerebral endothelial cells and limit the passage of various substances across the BBB, whereas the astrocytic end-feet surround the cerebral capillary walls and regulate ion balance (Ballabh et al., 2004). However, the cellular stress and pro-inflammatory signaling caused by cerebral ischemia induces drastic changes in BBB permeability that allows for the infiltration of pro-inflammatory substances that would otherwise not be able to penetrate the BBB (Huang et al., 1999).

As the most abundant glial cell of the brain, astrocytes are involved in a wide variety of processes relating to CNS homeostasis other than maintenance of the BBB, including providing structural and metabolic support to surrounding neurons (Jing et al., 2013). Among these effects include the support of synaptic connections and the secretion of various trophic factors (Liu and Chopp, 2016). Furthermore, following cerebral ischemia, astrocytes undergo a process known as astrogliosis, in which astrocytes react to parenchymal injury to surround the infarcted area, forming a glial scar (Sofroniew, 2009). Although the glial scar serves to limit the spread of damage, this process is also thought to prevent axon regeneration (Sofroniew, 2015). Consequently, the complex roles of reactive astrocytes in ischemic damage are not entirely understood and therefore represent a topic of significant research interest. During astrogliosis, reactive astrocytes express cell body hypertrophy, morphologic changes to dendrite length and number, and an upregulated expression of glial fibrillary acidic protein (GFAP), which is the major cytoplasmic intermediate filament (Liddelow and Barres, 2017). Moreover, previous research has indicated that peak levels of neuroinflammation following CNS injury correlates with maximal astrocyte reactivity and thus, GFAP expression (Cekanaviciute and Buckwalter, 2016). Therefore, treatments targeting the activity of reactive astrocytes and their effects on functional recovery are promising approaches for the management of neuroinflammatory conditions.

Although I/R injury is often an unavoidable consequence of ischemic stroke, the surge of blood during reperfusion results in a multitude of cellular and molecular events that occur in a non-linear fashion (Liu and Chopp, 2016). This vicious cycle of inflammation and heightened oxidative stress leads to the production of various pro-inflammatory cytokines and chemokines that cause additional damage and neuronal cell death (Aggarwal & Harikumar, 2009; Del Prado-Audelo et al., 2019). Therefore, the irreversible tissue damage that occurs after stroke is caused not only by the initial ischemic event, but also by the subsequent reperfusion period. Consequently, research efforts focused on developing strategies that reduce the harmful effects associated with I/R injury are of great interest. Despite the global prevalence of ischemic stroke, therapeutic options remain relatively limited. In the search for alternative therapeutic treatments, natural anti-inflammatory and antioxidant compounds, such as curcumin found in turmeric, have been thoroughly investigated.

Curcumin is a natural polyphenol and is the most bioactive compound that can be isolated from the rhizome of turmeric (Schmitt et al., 2020). Curcumin has shown therapeutic promise in treating the adverse effects associated with ischemic stroke and other neurodegenerative diseases via its ability to mitigate excessive autophagy (Huang et al., 2018; Zhang et al., 2018), reduce neuroinflammation (Liu et al., 2013; Miao et al., 2016; Zhang et al., 2017), protect against mitochondrial dysfunction (Bagheri et al., 2020; Daverey and Agrawal, 2016; Liu et al., 2014), decrease oxidative stress (Caillaud et al., 2020; Duan et al., 2022; Lan et al., 2018), limit cellular apoptosis (Altinay et al., 2017; Laorodphun et al., 2022; Xie et al., 2018), and improve functional outcomes (Wicha et al., 2017; Wu et al., 2021). Furthermore, curcumin has an excellent safety profile and has been shown to alleviate the adverse effects of neuroinflammation, often targeting key immunological cascades and intracellular signaling pathways without the worry of added contraindications (Patel et al., 2020).

Curcumin anti-inflammatory properties result from its ability to inhibit nuclear factor-kappa B (NF-κB), a transcription factor that plays a significant role in the production of pro-inflammatory cytokines, chemokines, and other mediators of neuroinflammation (Del Prado-Audelo et al., 2019; Karthikeyan et al., 2020). In particular, endothelial cells’ NF-κB activation during reperfusion leads to oxidative stress and BBB opening (Schaller and Graf, 2004), making NF-κB a promising target for stroke therapy. In addition, curcumin possesses bifunctional dose-dependent antioxidant properties, scavenging reactive oxygen species (ROS) while also stimulating an antioxidant response (Daverey and Agrawal, 2016; Lan et al., 2018), shown to decrease the activity of superoxide dismutase and glutathione peroxidase (Gupta et al., 2013) in the stroke brain. These ROS enzymes are largely responsible for post-ischemic stroke damage, most notably during the reperfusion period (Bavarsad et al., 2019).

Despite its proven immunomodulatory capabilities, the clinical potential of curcumin is severely limited by its poor bioavailability and insolubility (Ghaffari et al., 2020). Studies have shown that following systemic injections of curcumin, only trace amounts are detected in the brain (Pan et al., 1999). This is likely due to the restrictive nature of the BBB, which physiologically blocks most substances in systemic circulation from reaching the brain parenchyma, preventing compounds like curcumin from entering the CNS (Henrich-Noack et al., 2019). To overcome this limitation, nanomolecule-based delivery systems may potentially enhance curcumin’s therapeutic effects.

Nanomolecules have been extensively investigated in recent years for their ability to improve the pharmacokinetic properties of therapeutic compounds, provide targeted delivery to sites of injury, and systematically control the release of the drug cargo (Karthikeyan et al., 2020). Polyamidoamine (PAMAM) dendrimers are among the most popular class of nanomolecules used in biomedical applications. These unique polymeric nanostructures are comprised of an inner core surrounding concentric branched layers referred to as generations (G) and terminal end units (Kannan et al., 2014; Yousefi et al., 2020). Their architectural design can be precisely controlled, allowing for modification of their size, shape, and surface functionality (Svenson and Tomalia, 2012). Based on these parameters, PAMAM dendrimer generations are referred to as generation 1 (G1), generation 2 (G2), generation 3 (G3), generation 4 (G4), etc. Importantly, dendrimers of G4 and above possess a three-dimensional structure that allows the encapsulation of small hydrophobic molecules such as curcumin (Esfand and Tomalia, 2001; Kambhampati et al., 2015). The branching, dimensions, and cargo-carrying capabilities of G4 PAMAM dendrimers have been previously described to be suitable for the encapsulation and delivery of curcumin. They can also effectively cross the BBB to reach the brain from the systemic circulation (Florendo et al., 2018; Srinageshwar et al., 2017; 2019). Due to these properties and their modifiable chemistry, PAMAM dendrimers serve as an adequate platform for drug delivery to the brain.

While 100% amine (–NH2) PAMAM dendrimer surfaces have been shown to be highly toxic (Naha et al., 2018), recent studies have shown that hydroxyl (–OH) terminated PAMAM dendrimers have comparatively reduced toxicity (Fana et al., 2020) and have stable complex formation with curcumin, allowing for its release (Gallien et al., 2021). Using a previously described method for the synthesis of G4 PAMAM dendrimers (Srinageshwar et al., 2017), our lab has successfully produced, de novo, mixed-surface G4-70/30-Cys PAMAM dendrimers (70% hydroxyls and 30% amines, with a cystamine core), in which surface amines were changed to net neutral hydroxyls to create a reduced cytotoxicity. Our previous studies have shown that these modified G4-70/30 PAMAM dendrimers possess the ability to encapsulate biomolecules within their internal cavities, are relatively non-toxic, and can safely cross the BBB following systemic administration (Srinageshwar et al., 2017; 2019), making them a promising vehicle to deliver various therapeutic compounds to the brain.

Although several nano-curcumin formulations have been explored in the context of neurodegenerative diseases, to our knowledge, this is the first study to investigate the therapeutic efficacy of curcumin encapsulated in surface-modified G4-70/30 PAMAM dendrimers for the treatment of ischemic stroke in a rat model. The primary aims of this study are to evaluate the therapeutic potential of curcumin encapsulated in PAMAM dendrimers as a method of delivery and a treatment option for ischemic stroke by assessing the effects on (1) functional outcomes, (2) lesion size, and (3) relative expression of astrocyte reactivity.

The current study was designed to determine the efficacy of curcumin encapsulated in G4-70/30-Cys PAMAM dendrimers to treat the behavioral and histopathological outcomes following ischemic stroke in rats. Our results show that MCAo animals treated with either DenCur or Den resulted in a significant reduction in post-stroke neuroinflammation, both in the cortex and striatum. This was demonstrated through decreased levels of GFAP optical densities in comparison with either Cur or HBSS treatments. These results suggest that empty PAMAM dendrimers may possess intrinsic anti-inflammatory properties, and that they likely able to cross the BBB to elicit these effects in the stroke brain. Although the exact mechanisms by which dendrimers alone may offer neuroprotection and repair following I/R injury remain unclear, our findings align with previous research suggesting that dendrimers possess intrinsic anti-inflammatory properties (Chauhan et al., 2009). Most notably, the results of the cylinder testing (paw laterality index) indicates that DenCur-treated animals were the only group that did not show any reliance on one paw when averaged across the duration of the study, whereas the other three treatment groups demonstrated reliance on the unaffected paw, revealing consistent impairment of the contralateral affected paw. This impairment did not normalize in any of these treatment groups for the duration of the study, which further supports the hypothesis that the unique combination of dendrimer and curcumin may have a synergistic effect that is superior to the delivery of dendrimers or curcumin alone on the protection of contralateral paw function following an ischemic stroke. Furthermore, the significant benefits seen in the paw function of MCAo animals receiving DenCur treatment, but not within their overall neurobehavioral scores, suggests the mechanism underlying the effects of DenCur on the multifactorial and deeply complex pathophysiology of ischemic stroke requires further elucidation.

The unique structure of dendrimers may provide two major benefits for the treatment of neuroinflammatory conditions. First, dendrimers often exhibit innate anti-inflammatory properties. Second, dendrimers efficiently cross the BBB to deliver anti-inflammatory substances to specific regions in the brain. These advantages highlight the potential of dendrimers as a promising therapeutic option for managing inflammatory disorders. Even in their unmodified form, dendrimers have been found to exhibit anti-inflammatory properties independently of any carried material (Chauhan et al., 2009). One possible mechanism for this effect is the functional inhibition of inflammatory mediators such as cyclooxygenase (COX) enzymes, which has been attributed to the dendrimer surface functionality (Chauhan et al., 2009; Fruchon and Poupot, 2017). The electrochemical properties of dendrimers may enable them to modify key enzymes, particularly through interaction with dendrimeric hydroxyl terminal groups, thus leading to their anti-inflammatory effects (Neibert et al., 2013).

Additionally, studies have shown that G4 PAMAM dendrimers can selectively target activated glial cells in areas of neuroinflammation, thus localizing the dendrimers to sites of damage (Sharma et al., 2018). Research conducted by Dai et al. (2010) in a rabbit model of cerebral palsy revealed that dendrimers were primarily taken up by activated microglia and astrocytes, even in regions far from the site of injection (Dai et al., 2010). Remarkably, oligodendrocytes and neurons, cell types typically not associated with inflammation, showed minimal uptake of the dendrimer. Furthermore, the brain parenchyma of control animals displayed only trace amounts of dendrimers, underscoring their capacity to selectively target regions of neuroinflammation while avoiding noninflamed tissue. These findings were supported by Kannan et al. (2012), who found that systemically administered G4 PAMAM dendrimers accumulated in activated glial cells within the brains of rabbits with cerebral palsy, but not in healthy control subjects (Kannan et al., 2012).

Dendrimer uptake, however, depends on several factors, including disease severity, the amount of astrocyte activation, and the extent of BBB disruption (Nance et al., 2016). Therapeutics delivered by dendrimers, therefore, demonstrate improved efficacy by resulting in greater accumulation at sites of injury. In the setting of ischemic stroke, the targeting ability of dendrimers is extremely important, granting the potential for therapeutics to only elicit their effects on the tissue requiring it while limiting unwanted effects on surrounding regions. Moreover, quantitative studies have shown that G4 PAMAM dendrimers had the greatest uptake volume in the penumbra of the ischemic brain when compared to smaller and larger sized dendrimers (Zheng et al., 2014). This finding is remarkable, as the penumbra comprises the region of potentially salvageable tissue and therefore serves as a primary target of acute stroke therapy.

Dendrimers also possess a profound capacity to target specific areas of acute inflammation, which not only allows them to exert their intrinsic anti-inflammatory effects but also enables them to deliver relevant therapeutic cargo to these sites. These nanomolecules are considered highly effective drug-delivery vehicles due to their small size, biodegradability, lack of immunogenicity, and sustained drug-releasing ability in biological systems (Menjoge et al., 2010; Mukherjee et al., 2019; Naha et al., 2018). The encapsulation of naturally derived anti-inflammatory compounds, such as curcumin, within dendrimers has been shown to not only enhance solubility and bioavailability, but also increase the stability of the cargo by protecting it from the external environment (Del Prado-Audelo et al., 2019). Therefore, combining the intrinsic anti-inflammatory properties of dendrimers with the modulation of key inflammatory pathways by curcumin results in a synergistic therapeutic strategy that can target damaged and vulnerable tissue in the ischemic brain, providing a potent and multifaceted approach to alleviate the detrimental neurodegenerative cascade following ischemic stroke.

Conclusively, our results indicated that intraperitoneal injections of PAMAM dendrimers, whether loaded with curcumin or not, mitigates the astrocyte reactivity seen following ischemic stroke and reperfusion injury in rats and confers accelerated functional recovery as compared to soluble curcumin. Moreover, improved normalization of contralateral paw use seen in rats receiving PAMAM dendrimer-encapsulated curcumin implies a synergistic effect of these compounds, which may indicate successful delivery of curcumin to the brain. Further investigation of these effects, including the potential intrinsic anti-inflammatory effects of PAMAM dendrimers and the degree to which they can deliver their drug cargo, remains strongly warranted.

Treatments for stroke remain limited, so new drugs are necessary to advance brain recovery. Curcumin encapsulated in PAMAM dendrimers shows great promise in treating CNS diseases such as ischemic stroke by overcoming the limitations of free curcumin delivery, which include poor bioavailability and limited penetration into the CNS, thereby enhancing its therapeutic efficacy. In addition to their drug delivery capabilities, PAMAM dendrimers may exhibit anti-inflammatory properties that could be beneficial in stroke treatment. The therapeutic potential of curcumin encapsulated in PAMAM dendrimers were evaluated in a rat model of stroke.