Brain-specific Targeting Research Articles

The Glycocalyx: The Importance of Sugar Coating the Blood-Brain Barrier.

The endothelial glycocalyx (GCX), located on the luminal surface of vascular endothelial cells, is composed of glycoproteins, proteoglycans, and glycosaminoglycans. It plays a pivotal role in maintaining blood-brain barrier (BBB) integrity and vascular health within the central nervous system (CNS), influencing critical processes such as blood flow regulation, inflammation modulation, and vascular permeability. While the GCX is ubiquitously expressed on the surface of every cell in the body, the GCX at the BBB is highly specialized, with a distinct composition of glycans, physical structure, and surface charge when compared to GCX elsewhere in the body. There is evidence that the GCX at the BBB is disrupted and partially shed in many diseases that impact the CNS. Despite this, the GCX has yet to be a major focus of therapeutic targeting for CNS diseases. This review examines diverse model systems used in cerebrovascular GCX-related research, emphasizing the importance of selecting appropriate models to ensure clinical relevance and translational potential. This review aims to highlight the importance of the GCX in disease and how targeting the GCX at the BBB specifically may be an effective approach for brain specific targeting for therapeutics.

108 A Translational Approach to Suppress Local Inflammation in the Ischemic Penumbra and Deliver Local Anti-platelet Activity After Stroke

INTRODUCTION: Mechanical thrombectomy is the standard of care for ischemic stroke, but there is a significant mismatch between successful recanalization and clinical outcomes. Inflammation in the ischemic penumbra remains a target for neuroprotective interventions even in recanalized and non-recanalized patients. P-selectin (Psel) is a marker of endothelial cell activation and could be used to deliver therapeutics locally to the site of ischemic injury. METHODS: The fusion protein Pselectin-Crry was constructed using single chain antibody sequence linked to the extracellular region of mouse Crry. In-vivo testing using surface plasmon resonance for binding affinity to Psel, and zymosan assay for complement inhibitory activity. In-vivo studies were performed in C57bl/6 mice subjected to transient middle cerebral artery occlusion for 1 hour followed by administration of Psel-Crry 2 hours after reperfusion. Psel-Crry was labelled using far-red fluorescence and live-animal imaging was used to study brain-specific targeting. The modified Neurological Severity (NS) score was used for functional outcome along with survival, infarct volume and histological outcomes. RESULTS: Psel-Crry demonstrated dose-dependent complement inhibitory activity, and localized specifically to the stroke brain on in-vivo live imaging. Ex-vivo imaging of explanted brain showed Psel-Crry binding specifically to the injured hemisphere without off-target binding (Stroke vs. Sham, p < 0.001). Psel-Crry resulted in 60% reduction in infarct volume at 3 days compare to vehicle (N = 8/group, p < 0.05), reduction in NS scores compared to vehicle (Median 3 vs. 1.5, p < 0.05), and improved 1-week survival (80% vs. 50%, p < 0.05). Histological analyses demonstrated preservation of dendritic arborization in the ischemic penumbra with Psel-Crry treatment with concurrent significant reduction in microglial activation. CONCLUSIONS: Psel-Crry is a translational agent that can achieve local complement inhibitory effect and antiplatelet activity after stroke and has promise for potential use as ajuvant therapy in stroke.

Prevention of Alzheimer pathology by blocking neuregulin signaling on microglia.

Plaque formation, microglial activation, and synaptic loss are pathological hallmarks of Alzheimer's disease, however, removing plaques have had little clinical benefit. Here, we show that neuregulin-1, a glial growth factor, induces inflammatory cytokines and promotes phagocytic activity in vitro and augments microglial activation and plaque formation in 5XFAD Alzheimer's mice. Brain-specific targeting of neuregulin-1 by intraventricular delivery of a novel neuregulin-1 fusion protein antagonist GlyB4 significantly alters microglial morphology and function to a non-pathogenic morphology in early-stage 5XFAD mice and prevents plaques from forming. Once plaques have already formed, GlyB4 reduces new plaque formation and prevents synaptic loss. Selective, targeted disruption of neuregulin-1 signaling on brain microglia with GlyB4 could be a novel 'upstream' approach to slow or stop disease progression in Alzheimer's disease.Significance StatementMicroglia-associated neuroinflammation is a major hallmark of Alzheimer's disease. Here, we show that neuregulin1 (NRG1), an important neuron-glial signaling factor in development, induces microglial activation and promotes pathological changes in the 5XFAD mouse model of Alzheimer's disease. Inhibition of NRG1 using a targeted NRG1 antagonist called GlyB4 blocks NRG1 receptor activation in microglia, reduces Amyloid β plaque formation, and prevents synaptic loss. While NRG1 clearly has important functions at other sites in the nervous system, excessive NRG1 signaling in microglia leads to pathological neuroinflammation and neurodegeneration. Selective disruption of NRG1 signaling in microglia using GlyB4 could be a novel approach to block disease progression in patients with Alzheimer's disease.

Nose-to-Brain Delivery of Dexamethasone: Biodistribution Studies in Mice.

Neuroinflammation (NI) is an important physiologic process which promotes the tissue repair and homeostatic maintenance in the central nervous system after different types of insults. However, when it is exacerbated and sustained in time, NI plays a critical role in the pathogenesis of different neurologic diseases. The high systemic doses required for brain-specific targeting lead to severe undesirable effects. The intranasal (IN) route has been proposed as an alternative drug administration route for a better NI control. Herein, the brain biodistribution of intranasally administered dexamethasone versus intravenously administered one is reported. A higher amount of dexamethasone was found in every analyzed region of those brains of intranasally administered mice. HPLC analysis also revealed that IN administration allows Dex to arrive faster and in a greater concentration to the brain in comparison with intravenous administration, data confirmed by immunofluorescence and HPLC analysis. These data support the proposal of the IN administration of Dex as an alternative for a more efficient control of NI. SIGNIFICANCE STATEMENT: This work highlights the biodistribution of dexamethasone after its intranasal administration. Intranasal administration allows for a faster arrival, better distribution, and a higher concentration of the drug within the brain compared to its intravenous administration. These results explain some of the evidence shown in a previous work in which dexamethasone controls neuroinflammation in a murine stroke model and can be used to propose alternative treatments for neuroinflammatory diseases.

Formulation and cognitive evaluation of self-assembled phosphatidylserine-chitosan nanoparticles of lycopene, an innovative technique to lessen STZ-induced oxidative stress: A vital persuader of major neurological diseases

The present study deals with the encapsulation and brain-specific targeting of Lycopene (LYC) as Polysorbate-80 (P-80) coated Phosphatidylserine-chitosan self-assembled nanoparticles (P-80-LYC-PSCNP), with an approach to reduce oxidative stress which is rationalized as a major cause of neurological disease. P-80-LYC-PSCNP was formulated by using the injection technique for self-assembly of Phosphatidylserine and chitosan, applying P-80 coating for surface modification and were assessed for their morphology and physicochemical attributes. The efficiency of LYC liberated from P-80-LYC-PSCNP was determined in Streptozotocin-induced Oxidative stress Model (SOSM); cognitively by behavioral despair-test and biochemically by enzymatic assays at 5 mg/kg LYC-equivalent dose. P-80-LYC-PSCNP were formulated as stable (ζ = 3.89 mV) spherical, regular (≈220 nm), and homogenous entities. It demonstrated a total 76.89% LYC in-vitro release during 28 hrs and shown efficacy for brain-specific delivery with a relative bioavailability of 20.4, Cmax 4.6 times, and 2.5 times swifter than normal LYC. After 28 days, it shortened immobility time by 72.57 ± 8.12 s (P < 0.01) against STZ-control (134.58 ± 13.85 s, P < 0.001). It also improved the antioxidant enzymatic functioning of CAT, SOD, and GPx by 7.90 ± 0.45 Δ240/min/mgP (P < 0.05), 32.50 ± 1.70 U/mgP (P < 0.01), and 59.40 ± 3.70 U/mgP (P < 0.01) respectively against STZ-control which showed 3.05 ± 0.80 Δ240/min/mgP (P < 0.05), 15.20 ± 3.10 U/mgP (P < 0.05), and 25.15 ± 3.05 U/mgP at 5 mg/kg LYC-equivalent dose respectively. P-80-LYC-PSCNP was successfully formulated, analyzed and had delivered Lycopene (5 mg/kg) by trespassing the BBB with improved pharmacokinetics. It alleviated the experimental Streptozotocin-induced Oxidative stress in turn improving behavioral and cognitive anomalies, while enhancing the antioxidant-related activity of enzymes.

Monocultures of primary porcine brain capillary endothelial cells: Still a functional in vitro model for the blood-brain-barrier

The main obstacle for the treatment of brain diseases is the restriction of the passage of pharmaceuticals across the blood-brain barrier. Endothelial cells line up the cerebral micro vessels and prevent the uncontrolled transfer of polar substances by intercellular tight junctions. In addition to this physical barrier, active transporters of the multi-drug-resistance prevent the passage of hydrophobic substances by refluxing them back to the blood stream. This paper reviews the development and selected applications of an in vitro porcine brain derived primary cell culture system established in the authors lab that closely resembles the BBB in vivo and could thus be used to study beyond other applications drug delivery to the brain. An essential technique to control the intactness or destruction of the barrier, the impedance spectroscopy, will be introduced. It will be shown that nanoparticles can cross the blood brain barrier by two mechanisms: opening the tight junctions and thus allowing parallel import of substances into the brain as well as receptor mediated endocytosis using brain specific target molecules. However cytotoxic effects have to be considered as well which beside standard cytotoxicity assays could be also determined by impedance technology. Moreover it will be shown that enzymes e.g. for enzyme replacement therapy could be transferred across the barrier by proper tuning or chemical modification of the enzyme. Since this review is based on a conference presentation it will mainly focus on applications of the monoculture system developed in the authors lab which under given culture conditions is useful due to its easy availability, robustness, good reproducibility and also due to its simplicity. Improvements of this model made by other groups will be acknowledged but not discussed here in detail.

Intranasal delivery of dexamethasone efficiently controls LPS-induced murine neuroinflammation.

Neuroinflammation is the hallmark of several infectious and neurodegenerative diseases. Synthetic glucocorticoids (GCs) are the first-line immunosuppressive drugs used for controlling neuroinflammation. A delayed diffusion of GCs molecules and the high systemic doses required for brain-specific targeting lead to severe undesirable effects, particularly when lifelong treatment is required. Therefore, there is an urgent need for improving this current therapeutic approach. The intranasal (i.n.) route is being employed increasingly for drug delivery to the brain via the olfactory system. In this study, the i.n. route is compared to the intravenous (i.v.) administration of GCs with respect to their effectiveness in controlling neuroinflammation induced experimentally by systemic lipopolysaccharide (LPS) injection. A statistically significant reduction in interleukin (IL)-6 levels in the central nervous system (CNS) in the percentage of CD45+ /CD11b+ /lymphocyte antigen 6 complex locus G6D [Ly6G+ and in glial fibrillary acidic protein (GFAP) immunostaining was observed in mice from the i.n.-dexamethasone (DX] group compared to control and i.v.-DX-treated animals. DX treatment did not modify the percentage of microglia and perivascular macrophages as determined by ionized calcium binding adaptor molecule 1 (Iba1) immunostaining of the cortex and hippocampus. The increased accumulation of DX in brain microvasculature in DX-i.n.-treated mice compared with controls and DX-IV-treated animals may underlie the higher effectiveness in controlling neuroinflammation. Altogether, these results indicate that IN-DX administration may offer a more efficient alternative than systemic administration to control neuroinflammation in different neuropathologies.

Preparation, characterization, in vivo and biochemical evaluation of brain targeted Piperine solid lipid nanoparticles in an experimentally induced Alzheimer’s disease model

Background: Alzheimer is a fast growing disease with imprecise chemical treatments. Increased oxidative stress, decrease in acetylcholine concentration, and appearance of amyloidal proteins are reported in pathology of Alzheimer. Chemical drugs are effective but on the cost of detrimental side effects.Purpose: Present research is based on Preparation, characterization, behavioral and biochemical evaluation of brain targeted Piperine solid lipid nanoparticles in an experimentally induced Alzheimer’s model at a low dose of 2 mg/kg.Methods: Piperine solid lipid nanoparticles were prepared by Emulsification-Solvent Diffusion technique with polysorbate-80 coating to impart Brain specific targeting. Experimental Ibotenic acid induced Alzheimer’s, Force swimming test, superoxide dismutase, acetylcholenesterase enzymatic assays and also Histopathology of brain cortex was conducted to evaluate the Piperine therapeutic effects in Alzheimer’s Disease.Results: Piperine in solid lipid nanoformulation (2 mg/kg equivalent) reduced the SOD values by 504 ± 44.24 m units, p < 0.05, increased the acetylcholenesterase values by 29.24 ± 4.29 µg/mg, p < 0.01 and reduced immobility to 41.36 ± 3.53 s, p < 0.001 and has shown superior results than Donepezil (5 mg/kg). Histopathology studies revealed the reduced plaques and tangles.Conclusions: P-80-PIP-SLN has shown therapeutic effects in Alzheimer via reducing the oxidative stress and reducing the cholinergic degradation at 2 mg/kg dose equivalent.

A Comparative Study of Transfection Efficiency between Liposomes, Immunoliposomes and Brain-specific Immunoliposomes

This study investigated the transfection ability and efficiency of liposomes and immunoliposomes for exogenous gene delivery into the brain via the venous system. Four groups of rats underwent tail vein injection with one of the following: liposomes encapsulating pCMV (human cytomegalovirus promoter)-LacZ plasmid 80 microg (low dose) or 300 microg (high dose); general immunoliposomes encapsulating 80 microg transferrin receptor antibodies (OX26)-pCMV-LacZ plasmid; or brain-specific immunoliposomes encapsulating 80 microg OX26-pGFAP (glial fibrillary acidic protein promoter)-LacZ plasmid. A control group received no injected agent. The LacZ mRNA levels (1 h post-injection) and beta-galactosidase activity (48 h post-injection) in the brain and peripheral organs were assayed using real-time reverse transcription-polymerase chain reaction and histochemical staining, respectively. Both immunoliposomes delivered exogenous DNA containing the LacZ gene into the brain after venous injection, resulting in extensive LacZ expression in the brain. Furthermore, the brain-specific OX26-pGFAP-LacZ immunoliposome decreased the non-specific expression of LacZ in peripheral organs without affecting transfection efficiency in the brain. Thus, brain-specific immunoliposomes are an efficient and brain-specific targeting vector.

Short biodegradable polyamines for gene delivery and transfection of brain capillary endothelial cells

With the goal of rational design of systemic gene delivery system and efficient transfection of capillary endothelial cells forming the blood–brain barrier (BBB), we synthesized several short polyamines with reducible disulfide backbones for pDNA packaging, internalization and consequent release from endosomal compartments. The synthetic cationic polymers prepared from short linear PEI (pLPEI), triethylenetetramine (pTETA), and spermine (pSPE), demonstrated very low toxicity, good condensation capacity, and high levels of pDNA protection, producing small particulate nanoformulations. Mild reduction of the disulfide backbone allowed complete release of pDNA from these polyplexes. In vitro transfection of murine brain capillary endothelial bEnd.3 cells with pSPE, pTETA, and pLPEI polyplexes was 2.3–4.9 times more effective compared with the non-degradable LPEI 22 kDa reagent (ExGen500) in the presence of serum. Their transfection ability was noticeably decreased following inhibition of the cellular reduced glutathione (GSH). After cellular uptake of biodegradable polyplexes, a disperse distribution of labeled pDNA in the cytoplasm of transfected cells was observed in contrast to ExGen500. Based on these polyamines, novel multifunctional polyplexes have been developed for efficient nuclear delivery of pDNA by co-application of NLS peptide and PEG-modified intercalating conjugates. Significant increase of nuclear accumulation was observed, and the transfection of bEnd.3 cells was additionally enhanced nearly 2-fold, demonstrating 8.5-, 6.3- and 3.7-fold better levels for pLPEI, pTETA, and pSPE, respectively, compared to ExGen500. Following brain-specific targeting, these safe and effective polyplexes may be converted into systemic nanocarriers for gene delivery and transfection of the BBB.

Uptake of apolipoprotein E fragment coupled liposomes by cultured brain microvessel endothelial cells and intact brain capillaries

The suitability of surface modified liposomes as drug carriers for brain-specific targeting was investigated using apolipoprotein E fragments as brain-directed vectors. Liposomes coated with polyethylene glycol-2000 (sterically stabilized, PEGylated liposomes) were prepared from hydrogenated egg phosphatidylcholine, cholesterol, and a PEG-derivatized phospholipid. Liposomes were covalently coupled to a peptide of 26 amino acids length, derived from the binding site of human apolipoprotein E4 (ApoE4) and a peptide of random amino acid sequence, respectively. Rhodamine-labeled dipalmitoylphosphatidylethanolamine was incorporated into the lipid bilayer in order to visualize the liposomal interaction with brain capillary endothelial cell monolayers. The interaction of the liposomes with monolayers of porcine brain capillary endothelial cells (BCEC), the rodent cell line RBE4, and freshly isolated porcine brain capillaries was studied by means of confocal laser scanning fluorescence microscopy. In contrast to random peptide coupled liposomes, the ApoE4-fragment coupled liposomes were rapidly taken up by cultured BCECs and RBE4 cells. Uptake could be inhibited by ApoE4, free peptide, and antibodies against the LDL receptor in a concentration-dependent manner. The results indicate that the liposomes are internalized via the LDL receptor, which is expressed at the blood−brain barrier. In conclusion, liposomes coupled to ApoE4 fragments are taken up into brain endothelium via an endocytotic pathway and may therefore be a suitable carrier for drug delivery to the brain.

Uptake of Cationized Albumin Coupled Liposomes by Cultured Porcine Brain Microvessel Endothelial Cells and Intact Brain Capillaries

The suitability of protein-coupled liposomes as drug carriers for brain specific targeting was investigated using albumin (BSA) and cationized albumin (CBSA), respectively, as model proteins. Liposomes coated with polyethylene glycol (sterically stabilized, PEG-liposomes) were prepared from phosphatidylcholine, cholesterol, and a PEG-derivatized phospholipid and covalently coupled to thiolated BSA or CBSA. Liposomes were loaded with carboxy-fluorescein and rhodamine-labeled dipalmitoyl-phosphatidylethanolamine as hydrophilic and lipophilic marker compounds, respectively. The interaction of these constructs with monolayers of porcine brain capillary endothelial cells (BCEC) and freshly isolated porcine brain capillaries was studied by means of fluorescence assays and confocal laser scanning fluorescence microscopy (CLSFM). In contrast to BSA, CBSA was rapidly taken up by cultured BCECs. BSA-coupled liposomes did not interact with endothelial cells, whereas CBSA-coupled liposomes bound to cellular surfaces and exhibited time dependently a high intracellular accumulation. CBSA-conjugated liposomes were also taken up by intact brain capillaries. Cellular uptake could be inhibited by free cationized albumin, phenylarsineoxide, nocodazole, and filipin, but not by dansylcadaverine, suggesting a caveolae-mediated incorporation process. Immunostaining demonstrated a high expression of caveolin in the capillary endothelium. In conclusion, liposomes coupled to CBSA are taken up into brain endothelium via an endocytotic pathway and may therefore be a suitable carrier for drug delivery to the brain.