Blood-brain Barrier Targeting Research Articles

Lactoferrin/CD133 antibody conjugated nanostructured lipid carriers for dual targeting of blood–brain-barrier and glioblastoma stem cells

The main reasons for the difficulty in curing and high recurrence rate of glioblastoma multiforme (GBM) include: 1. The difficulty of chemotherapy drugs in penetrating the blood-brain barrier (BBB) to target tumor cells; 2. The presence of glioma stem cells (GSCs) leading to chemotherapy resistance. Therefore, breaking through the limitations of the BBB and overcoming the drug resistance caused by GSCs are the main strategies to address this problem. This study presents our results on the development of lactoferrin (Lf)/CD133 antibody conjugated nanostructured lipid carriers (Lf/CD133-NLCS) for simultaneously targeting BBB and GSCs. Temozolomide (TMZ) loaded Lf/CD133-NLCS (Lf/CD133-NLCS-TMZ) exhibited high-efficiencyin vitroanti-tumor effects toward malignant glioma cells (U87-MG) and GSCs, while demonstrating no significant toxicity to normal cells at concentrations lower than 200 μg ml-1. The results of thein vitrotargeting GBM study revealed a notably higher cellular uptake of Lf/CD133-NLCS-TMZ in U87-MG cells and GSCs in comparison to Lf/CD133 unconjugated counterpart (NLCS-TMZ). In addition, increased BBB permeability were confirmed for Lf/CD133-NLCS-TMZ compared to NLCS-TMZ bothin vitroandin vivo. Taking together, Lf/CD133-NLCS-TMZ show great potential for dual targeting of BBB and GSCs, as well as GBM therapy based on this strategy.

Chemical engineering of zein with polyethylene glycol and Angiopep-2 to manufacture a brain-targeted docetaxel nanomedicine for glioblastoma treatment.

Glioblastoma (GBM) is the deadliest adult brain cancer. The current standard-of-care chemotherapy using orally administered temozolomide (TMZ) presents poor improvement in patient survival, emphasizing the compelling need for new therapies. A possible chemotherapeutic alternative is docetaxel (DTX), which possesses higher tumoricidal potency against GBM cells. However, its limited blood-brain barrier (BBB) permeability poses a constraint on its application. Nonetheless, nanomedicine offers promising avenues for overcoming this challenge. Angiopep-2 (ANG2) is a peptide that targets the BBB-overexpressed low-density lipoprotein receptor (LDLR). In this work, we managed, for the first time, to employ a pioneering approach of covalently linking zein protein with polyethylene glycol (PEG) and ANG2 prior to its formulation into nanoparticles (ZNPs) with enhanced stability and LDLR-mediated brain targetability, respectively. Carbodiimide and click chemistry approaches were optimized, resulting in functional modification of zein with around 25% PEG, followed by functional modification of PEG with nearly 100% ANG2. DTX-loaded ZNPs presented 100nm average size, indicating high suitability for BBB crossing through receptor-mediated transcytosis. ZNPs maintained the cytotoxic effect of the loaded DTX against GBM cells, while demonstrating a safe matrix against BBB cells. Importantly, these brain-targeted ZNPs showcased up to fourfold enhancement in blood-to-brain permeability in a BBB in vitro model, highlighting the potential of this novel approach of BBB targeting in significantly improving therapeutic outcomes for GBM patients. The versatility of the system and the possibility of significantly increasing drug concentration in the brain open the door to its future application in a wide range of other brain-related diseases.

Lipid Nanoparticles: Versatile Drug Delivery Vehicles for Traversing the Blood Brain Barrier to Treat Brain Cancer

AbstractThe blood‐brain barrier (BBB) poses a significant challenge in delivering therapeutic agents for brain diseases due to its high selectivity against foreign substances. This limitation greatly hampers the effectiveness of conventional chemotherapeutic drugs in treating brain cancers. In response, lipid‐based nanoparticles (LNPs) have emerged as a promising approach, offering opportunities for targeted drug delivery by conjugating targeting ligands onto their surface. This review provides a comprehensive overview of recent advancements in utilizing LNPs to traverse the BBB for enhanced transport of bioactive compounds into the brain, specifically for cancer treatments. Beginning with an exploration of the biological structure and functions of the BBB and the blood‐brain tumor barrier (BBTB), the review highlights the advantages presented by LNPs. Subsequently, it delves into strategies for surface modification of nanoparticles to enhance BBB targeting and improve efficacy in brain cancer treatment. Finally, the review offers insights into future prospects for designing the next generation of LNPs. The review presented herein aims to contribute to the ongoing efforts in overcoming the challenges associated with BBB penetration, ultimately advancing therapeutic strategies for brain cancer and other neurological disorders.

Blood-Brain Barrier-Targeting Nanoparticles: Biomaterial Properties and Biomedical Applications in Translational Neuroscience.

Overcoming the blood-brain barrier (BBB) remains a significant hurdle in effective drug delivery to the brain. While the BBB serves as a crucial protective barrier, it poses challenges in delivering therapeutic agents to their intended targets within the brain parenchyma. To enhance drug delivery for the treatment of neurological diseases, several delivery technologies to circumvent the BBB have been developed in the last few years. Among them, nanoparticles (NPs) are one of the most versatile and promising tools. Here, we summarize the characteristics of NPs that facilitate BBB penetration, including their size, shape, chemical composition, surface charge, and importantly, their conjugation with various biological or synthetic molecules such as glucose, transferrin, insulin, polyethylene glycol, peptides, and aptamers. Additionally, we discuss the coating of NPs with surfactants. A comprehensive overview of the common in vitro and in vivo models of the BBB for NP penetration studies is also provided. The discussion extends to discussing BBB impairment under pathological conditions and leveraging BBB alterations under pathological conditions to enhance drug delivery. Emphasizing the need for future studies to uncover the inherent therapeutic properties of NPs, the review advocates for their role beyond delivery systems and calls for efforts translating NPs to the clinic as therapeutics. Overall, NPs stand out as a highly promising therapeutic strategy for precise BBB targeting and drug delivery in neurological disorders.

Alleviating Recombinant Tissue Plasminogen Activator-induced Hemorrhagic Transformation in Ischemic Stroke via Targeted Delivery of a Ferroptosis Inhibitor.

Intravenous thrombolysis with recombinant tissue plasminogen activator (rtPA) is the primary treatment for ischemic stroke. However, rtPA treatment can substantially increase blood-brain barrier (BBB) permeability and susceptibility to hemorrhagic transformation. Herein, the mechanism underlying the side effects of rtPA treatment is investigated and demonstrated that ferroptosis plays an important role. The ferroptosis inhibitor, liproxstatin-1 (Lip) is proposed to alleviate the side effects. A well-designed macrocyclic carrier, glucose-modified azocalix[4]arene (GluAC4A), is prepared to deliver Lip to the ischemic site. GluAC4A bound tightly to Lip and markedly improved its solubility. Glucose, modified at the upper rim of GluAC4A, imparts BBB targeting to the drug delivery system owing to the presence of glucose transporter 1 on the BBB surface. The responsiveness of GluAC4A to hypoxia due to the presence of azo groups enabled the targeted release of Lip at the ischemic site. GluAC4A successfully improved drug accumulation in the brain, and Lip@GluAC4A significantly reduced ferroptosis, BBB leakage, and neurological deficits induced by rtPA in vivo. These findings deepen the understanding of the side effects of rtPA treatment and provide a novel strategy for their effective mitigation, which is of great significance for the treatment and prognosis of patients with ischemic stroke.

The solute carrier SLC7A1 may act as a protein transporter at the blood-brain barrier

Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood-brain barrier (BBB). Most molecules require either carrier- or receptor-mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium-enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBB-enriched genes according to established selection criteria. As a result, we propose the high-affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1-specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB.

Functionalized lipid nanoparticles modulate the blood-brain barrier and eliminate α-synuclein to repair dopamine neurons

The challenge in the clinical treatment of Parkinson's disease lies in the lack of disease-modifying therapies that can halt or slow down the progression. Peptide drugs, such as exenatide (Exe), with potential disease-modifying efficacy, have difficulty in crossing the blood-brain barrier (BBB) due to their large molecular weight. Herein, we fabricate multi-functionalized lipid nanoparticles (LNP) Lpc-BoSA/CSO with BBB targeting, permeability-increasing and responsive release functions. Borneol is chemically bonded with stearic acid and, as one of the components of Lpc-BoSA/CSO, is used to increase BBB permeability. Immunofluorescence results of brain tissue of 15-month-old C57BL/6 mice show that Lpc-BoSA/CSO disperses across the BBB into brain parenchyma, and the amount is 4.21 times greater than that of conventional LNP. Motor symptoms of mice in Lpc-BoSA/CSO-Exe group are significantly improved, and the content of dopamine is 1.85 times (substantia nigra compacta) and 1.49 times (striatum) that of PD mice. α-Synuclein expression and Lewy bodies deposition are reduced to 51.85% and 44.72% of PD mice, respectively. Immunohistochemical mechanism studies show AKT expression in Lpc-BoSA/CSO-Exe is 4.23 times that of PD mice and GSK-3β expression is reduced to 18.41%. Lpc-BoSA/CSO-Exe could reduce the production of α-synuclein and Lewy bodies through AKT/GSK-3β pathway, and effectively prevent the progressive deterioration of Parkinson's disease. In summary, Lpc-BoSA/CSO-Exe increases the entry of exenatide into brain and promotes its clinical application for Parkinson's disease therapy.

Anti‐mouse CD98hc nanobodies for brain delivery of a therapeutic moiety

AbstractBackgroundThe blood‐brain barrier (BBB) limits the therapeutic perspective for central nervous system (CNS) disorders such as Alzheimer’s disease. Interestingly, monoclonal antibodies generated against receptor‐mediated transcytosis receptors are able to transport therapeutic proteins across the BBB. Nanobodies, which are the variable domain isolated from camelid heavy‐chain only antibodies have also shown their potential to deliver therapeutics into the CNS. Here we aimed to expand the panel of nanobodies targeting other brain endothelial cell receptors.MethodCamelids were immunized with a pool of DNA encoding for several brain endothelial cell receptors. One target was chosen to validate the immunization and discovery approach, CD98hc, previously suggested to allow drug shuttling across the BBB. Nanobody libraries were selected on Chinese Hamster Ovarian (CHO) cells overexpressing the target of interest. Selected nanobodies were screened in vivo based on their potential to deliver into the CNS either a biologically active neuropeptide neurotensin to reduce body temperature, or a BACE1 inhibiting antibody that reduces the accumulation of Aβ1‐40 species.ResultDNA immunization followed by selections on CHO cells overexpressing the target of interest resulted in specific anti‐mouse CD98hc binders with affinities in the low nanomolar range. In vivo screening of mouse CD98hc nanobody binders revealed some discrepancy between the two methods used. Whereas none of the tested nanobodies fused to neurotensin was able to reduce body temperature, nanobodies fused to the anti‐BACE1 antibody 1A11 effectively reduced Aβ1‐40 levels in the brain. We suggest that the neurotensin approach, although having shown robustness with anti‐TfR nanobodies, might lack sensitivity when screening non‐TfR binders. We suggest BACE1 inhibition as a more robust model to validate CD98hc‐mediated transport across the BBB.ConclusionThis is a proof of concept study in which we have optimized our nanobody discovery approach to find nanobodies against BBB targets, and validated CD98hc as a receptor to target in order to shuttle drugs into the brain. In the future we will explore whether nanobodies raised against novel brain endothelial cell receptors could also improve brain penetration of biologics.

Cell membrane-based biomimetic vehicles for effective central nervous system target delivery: Insights and challenges.

Central nervous system (CNS) disorders, including brain tumor, ischemic stroke, Alzheimer's disease, and Parkinson's disease, threaten human health. And the existence of the blood-brain barrier (BBB) hinders the delivery of drugs and the design of drug targeting delivery vehicles. Over the past decades, great interest has been given to cell membrane-based biomimetic vehicles since the rise of targeting drug delivery systems and biomimetic nanotechnology. Cell membranes are regarded as natural multifunction biomaterials, and provide potential for targeting delivery design and modification. Cell membrane-based biomimetic vehicles appear timely with the participation of cell membranes and nanoparticles, and raises new lights for BBB recognition and transport, and effective therapy with its biological multifunction and high biocompatibility. This review summarizes existing challenges in CNS target delivery and recent advances of different kinds of cell membrane-based biomimetic vehicles for effective CNS target delivery, and deliberates the BBB targeting mechanism. It also discusses the challenges and possibility of clinical translation, and presents new insights for development.

Hybrid stem cell-derived bioresponsive vesicles for effective inflamed blood-brain barrier targeting delivery

Blood-brain barrier (BBB) dis-integrity, a characteristic of extensive inflammatory encephalopathy, provides the access for brain-targeted drug delivery. Inspired from the bioresponsiveness of stem cell, we conceived of new nano-scale delivery strategy via neural stem cell membrane-derived bioresponsive vesicles (NSC-Lipo) for inflamed BBB target delivery. After recombination of neural stem cell membranes with simple liposomes, the functional ligands, especially very late antigen-4 (VLA-4), were entirely engrafted into NSC-Lipo and equipped the NSC-Lipo with “pass order” to the “selective gate”. It bestowed the bioresponsive vesicles with selective target the injured brain microvessel endothelial cells (BMECs) and penetrate into the lesion through the vascular cell adhesion molecule-1 (VCAM-1)/VLA-4 interaction in the post-ischemic mice model. The released anti-inflammatory agent, metformin, could reverse the inflammatory response of BMECs and rapidly recovers BBB’s integrity. With the repair of BBB’s integrity and down regulation of VCAM-1 expression, the targeting ability of bioresponsive vesicles cease to be in force as well. The bioresponsive vesicles led to enhanced survival rate of ischemic stroke mice (from 30 % to 90 %) after just a single injection compared to metformin loaded bare liposomes.

Transferrin Aptamers Increase the In Vivo Blood-Brain Barrier Targeting of Protein Spherical Nucleic Acids.

The systemic delivery of exogenous proteins to cells within the brain and central nervous system (CNS) is challenging due to the selective impermeability of the blood-brain barrier (BBB). Herein, we hypothesized that protein delivery to the brain could be improved via functionalization with DNA aptamers designed to bind transferrin (TfR) receptors present on the endothelial cells that line the BBB. Using β-galactosidase (β-Gal) as a model protein, we synthesized protein spherical nucleic acids (ProSNAs) comprised of β-Gal decorated with TfR aptamers (Transferrin-ProSNAs). The TfR aptamer motif significantly increases the accumulation of β-Gal in brain tissue in vivo following intravenous injection over both the native protein and ProSNAs containing nontargeting DNA sequences. Furthermore, the widespread distribution of β-Gal throughout the brain is only observed for Transferrin-ProSNAs. Together, this work shows that the SNA architecture can be used to selectively deliver protein cargo to the brain and CNS if the appropriate aptamer sequence is employed as the DNA shell. Moreover, this highlights the importance of DNA sequence design and provides a potential new avenue for designing highly targeted protein delivery systems by combining the power of DNA aptamers together with the SNA platform.

TfR Aptamer Enhanced Blood-Brain Barrier Penetration of Biomimetic Nanocomplexes for Intracellular Transglutaminase 2 Imaging and Silencing in Glioma.

Engineering a versatile nanocomplex integrating effective penetration of the blood-brain barrier (BBB), accurate diagnosis, and boosting therapy has always been an intractable challenge in glioblastoma multiforme (GBM). Herein, biomimetic nanocomplexes (TMPsM) for single intracellular transglutaminase 2 (TG2)-triggered self-assembly imaging and RNAi therapy for GBM are subtly developed. To prove the concept, transferrin receptor (TfR) aptamer-modified brain metastatic tumor cell membrane is prepared as the shell for dual BBB targeting capability and prolonged blood retention time. Upon targeting entering into GBM, hollow MnO2 is decomposed to release KKGKGQQ-tetraphenylethene (Pep-TPE) and siRNA. Owing to TG2 dependence, the non-emissive Pep-TPE would be self-aggregated to induce the emission turn-on in GBM that contain overexpressed TG2. The resulting aggregation-induced emission fluorescence imaging with a high signal-to-noise ratio can achieve the precise localization of the tumor and dynamic detection of TG2 activity, thereby allowing the GBM accurate diagnosis. Notably, the TG2 can be silenced by the released siRNA to cause cell apoptosis and increase chemotherapeutic sensitivity, ultimately realizing excellent antitumor efficacy. In vitro and in vivo results demonstrate that the as-prepared TMPsM indeed possess superior BBB penetration, precise diagnosis, and effective therapy of GBM. The proposed strategy may pioneer a new path for the theranostics of brain tumors.

Intelligent lesion blood–brain barrier targeting nano-missiles for Alzheimer's disease treatment by anti-neuroinflammation and neuroprotection

The treatment of Alzheimer's disease (AD) is one of the most difficult challenges in neurodegenerative diseases due to the insufficient blood‒brain barrier (BBB) permeability and unsatisfactory intra-brain distribution of drugs. Therefore, we established an ibuprofen and FK506 encapsulated drug co-delivery system (Ibu&FK@RNPs), which can target the receptor of advanced glycation endproducts (RAGE) and response to the high level of reactive oxygen species (ROS) in AD. RAGE is highly and specifically expressed on the lesion neurovascular unit of AD, this property helps to improve targeting specificity of the system and reduce unselective distribution in normal brain. Meanwhile, these two drugs can be specifically released in astrocytes of AD lesion in response to high levels of ROS. As a result, the cognition of AD mice was significantly improved and the quantity of Aβ plaques was decreased. Neurotoxicity was also alleviated with structural regeneration and functional recovery of neurons. Besides, the neuroinflammation dominated by NF-κB pathway was significantly inhibited with decreased NF-κB and IL-1β in the brain. Overall, Ibu&FK@RNPs can efficiently and successively target diseased BBB and astrocytes in AD lesion. Thus it significantly enhances intracephalic accumulation of drugs and efficiently treats AD by anti-neuroinflammation and neuroprotection.

Highly Specific Blood-Brain Barrier Transmigrating Single-Domain Antibodies Selected by an In Vivo Phage Display Screening.

A major bottleneck in the successful development of central nervous system (CNS) drugs is the discovery and design of molecules that can cross the blood-brain barrier (BBB). Nano-delivery strategies are a promising approach that take advantage of natural portals of entry into the brain such as monoclonal antibodies (mAbs) targeting endogenous BBB receptors. However, the main selected mAbs rely on targeting broadly expressed receptors, such as the transferrin and insulin receptors, and in selection processes that do not fully mimic the native receptor conformation, leading to mistargeting and a low fraction of the administered dose effectively reaching the brain. Thus, there is an urgent need to identify new BBB receptors and explore novel antibody selection approaches that can allow a more selective delivery into the brain. Considering that in vitro models fail to completely mimic brain structure complexity, we explored an in vivo cell immunization approach to construct a rabbit derived single-domain antibody (sdAb) library towards BBB endothelial cell receptors. The sdAb antibody library was used in an in vivo phage display screening as a functional selection of novel BBB targeting antibodies. Following three rounds of selections, next generation sequencing analysis, in vitro brain endothelial barrier (BEB) model screenings and in vivo biodistribution studies, five potential sdAbs were identified, three of which reaching >0.6% ID/g in the brain. To validate the brain drug delivery proof-of-concept, the most promising sdAb, namely RG3, was conjugated at the surface of liposomes encapsulated with a model drug, the pan-histone deacetylase inhibitor panobinostat (PAN). The translocation efficiency and activity of the conjugate liposome was determined in a dual functional in vitro BEB-glioblastoma model. The RG3 conjugated PAN liposomes enabled an efficient BEB translocation and presented a potent antitumoral activity against LN229 glioblastoma cells without influencing BEB integrity. In conclusion, our in vivo screening approach allowed the selection of highly specific nano-antibody scaffolds with promising properties for brain targeting and drug delivery.

Transferrin-Pep63-liposomes accelerate the clearance of A\u03b2 and rescue impaired synaptic plasticity in early Alzheimer\u2019s disease models

Alzheimer’s disease (AD) is characterized by aberrant accumulation of extracellular β-amyloid (Aβ) peptides in the brain. Soluble Aβ oligomers are thought to be the most neurotoxic species and are correlated with cognitive dysfunction in early AD. However, there is still no effective treatment so far. We determined that Pep63, a small peptide, had a neuroprotective effect on synaptic plasticity and memory in our previous study. Here, we developed novel and multifunctional liposomes targeting both Aβ oligomers and fibrils based on a liposome delivery system. Transferrin-Pep63-liposomes (Tf-Pep63-Lip), possessing the ability for blood-brain barrier targeting, were also incorporated with phosphatidic acid (PA) and loaded with neuroprotective Pep63. We discovered that administration of Tf-Pep63-Lip could significantly reduce the Aβ burden in the hippocampus, and improve cognitive deficits in 6-month-old APP/PS1 mice in the Morris-Water maze task and fear-conditioning test with the combined effects of PA and Pep63. Tf-Pep63-Lip could capture Aβ oligomers or fibrils and then facilitated microglial chemotaxis nearby for clearance. Simultaneously, Tf-Pep63-Lip hindered Aβ1-42 aggregation and disaggregated Aβ1-42 assembly due to multivalent PA-Aβ. Pep63 effectively inhibited the binding between EphB2 and Aβ oligomers after release from liposomes and rescued NMDA receptors trafficking, the basis of synaptic plasticity. No side effects were observed in either APP/PS1 or wild-type mice, indicating that Tf-Pep63-Lip might be safe under the dosing regimen used in our experiment. Taken together, our results suggested that Tf-Pep63-Lip may serve as a safe and efficient agent for AD combination therapy.

Discovery of a Highly Conserved Peptide in the Iron Transporter Melanotransferrin that Traverses an Intact Blood Brain Barrier and Localizes in Neural Cells.

The blood-brain barrier (BBB) hinders the distribution of therapeutics intended for treatment of diseases of the brain. Our previous studies demonstrated that that a soluble form of melanotransferrin (MTf; Uniprot P08582; also known as p97, MFI2, and CD228), a mammalian iron-transport protein, is an effective carrier for delivery of drug conjugates across the BBB into the brain and was the first BBB targeting delivery system to demonstrate therapeutic efficacy within the brain. Here, we performed a screen to identify peptides from MTf capable of traversing the BBB. We identified a highly conserved 12-amino acid peptide, termed MTfp, that retains the ability to cross the intact BBB undigested, distribute throughout the parenchyma, and enter endosomes and lysosomes within neurons, astrocytes and microglia in the brain. This peptide may provide a platform for the transport of therapeutics to the CNS, and thereby offers new avenues for potential treatments of neuropathologies that are currently refractory to existing therapies.

Multifunctional nanorods based on self-assembly of biomimetic apolipoprotein E peptide for the treatment of Alzheimer's disease

Targeting a single molecule or a single pathway and poor drug delivery to the brain hamper the therapy of Alzheimer's disease (AD) based on abnormal metabolism of amyloid-β (Aβ). To solve these problems, we designed and synthesized a multi - strategy peptide (MOP), an ingenious apolipoprotein E mimetic peptide, which could reduce Aβ deposition via inhibiting Aβ aggregation and at the same time accelerate Aβ clearance. Meanwhile, MOP could be self-assembled into different nanostructure, thus we constructed a multifunctional delivery system (APND-3) based on MOP self-assembled nanorods (aspect ratios of 3) that was a favorable morphology to enhance the permeation across the blood brain barrier (BBB) to address the poor delivery to brain issues. Besides, the drug delivery system introduces polydopamine (PDA) and COG1410 ligand as a shell to keep the favorable morphology of core and enhance the BBB targeting efficiency. As a result, the delivery system significantly enhances the delivery of MOP to the brain, thus reducing Aβ deposition, mitigating the memory deficits, and ameliorating neurologic damage in AD model mice. Our findings suggest that our drug and carrier integrated multifunctional delivery system has the potential for AD treatment.

Two peptides targeting endothelial receptors are internalized into murine brain endothelial cells.

The blood-brain barrier (BBB) is one of the main obstacles for therapies targeting brain diseases. Most macromolecules fail to pass the tight BBB, formed by brain endothelial cells interlinked by tight junctions. A wide range of small, lipid-soluble molecules can enter the brain parenchyma via diffusion, whereas macromolecules have to transcytose via vesicular transport. Vesicular transport can thus be utilized as a strategy to deliver brain therapies. By conjugating BBB targeting antibodies and peptides to therapeutic molecules or nanoparticles, it is possible to increase uptake into the brain. Previously, the synthetic peptide GYR and a peptide derived from melanotransferrin (MTfp) have been suggested as candidates for mediating transcytosis in brain endothelial cells (BECs). Here we study uptake, intracellular trafficking, and translocation of these two peptides in BECs. The peptides were synthesized, and binding studies to purified endocytic receptors were performed using surface plasmon resonance. Furthermore, the peptides were conjugated to a fluorophore allowing for live-cell imaging studies of their uptake into murine brain endothelial cells. Both peptides bound to low-density lipoprotein receptor-related protein 1 (LRP-1) and the human transferrin receptor, while lower affinity was observed against the murine transferrin receptor. The MTfp showed a higher binding affinity to all receptors when compared to the GYR peptide. The peptides were internalized by the bEnd.3 mouse endothelial cells within 30 min of incubation and frequently co-localized with endo-lysosomal vesicles. Moreover, our in vitro Transwell translocation experiments confirmed that GYR was able to cross the murine barrier and indicated the successful translocation of MTfp. Thus, despite binding to endocytic receptors with different affinities, both peptides are able to transcytose across the murine BECs.

Examination of a blood-brain barrier targeting β-galactosidase-monoclonal antibody fusion protein in a murine model of GM1-gangliosidosis

GM1-gangliosidosis is a lysosomal disease resulting from a deficiency in the hydrolase β-galactosidase (β-gal) and subsequent accumulation of gangliosides, primarily in neuronal tissue, leading to progressive neurological deterioration and eventually early death. Lysosomal diseases with neurological involvement have limited non-invasive therapies due to the inability of lysosomal enzymes to cross the blood-brain barrier (BBB). A novel fusion enzyme, labeled mTfR-GLB1, was designed to act as a ferry across the BBB by fusing β-gal to the mouse monoclonal antibody against the mouse transferrin receptor and tested in a murine model of GM1-gangliosidosis (β-gal−/−). Twelve hours following a single intravenous dose of mTfR-GLB1 (5.0 mg/kg) into adult β-gal−/− mice showed clearance of enzyme activity in the plasma and an increase in β-gal enzyme activity in the liver and spleen. Long-term efficacy of mTfR-GLB1 was assessed by treating β-gal−/− mice intravenously twice a week with a low (2.5 mg/kg) or high (5.0 mg/kg) dose of mTfR-GLB1 for 17 weeks. Long-term studies showed high dose mice gained weight normally compared to vehicle-treated β-gal−/− mice, which are significantly heavier than heterozygous controls. Behavioral assessment at six months of age using the pole test showed β-gal−/− mice treated with mTfR-GLB1 had improved motor function. Biochemical analysis showed an increase in β-gal enzyme activity in the high dose group from negligible levels to 20% and 11% of heterozygous levels in the liver and spleen, respectively. Together, these data show that mTfR-GLB1 is a catalytically active β-gal fusion enzyme in vivo that is readily taken up into tissues.Despite these indications of bioactivity, behavior tests other than the pole test, including the Barnes maze, inverted screen, and accelerating rotarod, showed limited or no improvement of treated mice compared to β-gal−/− mice receiving vehicle only. Further, administration of mTfR-GLB1 was insufficient to create measurable increases in β-gal enzyme activity in the brain or reduce ganglioside content (biochemically and morphologically).

Examination of a blood-brain barrier targeting β-galactosidase-monoclonal antibody fusion protein in a murine model of GM1-gangliosidosis

Examination of a blood-brain barrier targeting β-galactosidase-monoclonal antibody fusion protein in a murine model of GM1-gangliosidosis