Advances in Brain-Targeted Therapies for Neurodegenerative Diseases

Existing treatments for neurodegenerative diseases remain ineffective which is a major problem currently. Lipid nanoparticles offer a delivery system for innovative molecular combinations that can target multiple mechanisms in neurodegenerative diseases. The blood-brain barrier (BBB) presents a significant obstacle to brain drug delivery for neurodegenerative disease treatment because it restricts traditional delivery approaches. The present-day lipid nanoparticle and cell-penetrating peptide (CPP) approaches demonstrate multiple absorption mechanisms which could extend both the duration and bioavailability of encapsulated pharmaceuticals. Bioactive molecules with neurotrophic or neuroprotective properties can be utilized to mediate ND-targeted pathways including neurotrophic factor deficiencies, impaired lipid metabolism, mitochondrial dysfunction, endoplasmic reticulum stress, accumulation of misfolded proteins or peptide fragments, toxic protein aggregates, oxidative stress damage, and neuroinflammation. This review examines new developments in lipid nanoparticles and CPPs to create dual-targeted nanoparticle systems for brain delivery in neurodegenerative disease treatment. 

Introduction to Overcoming BBB in Drug Delivery

Major health challenges in the central nervous system (CNS) include neurodegenerative diseases like Alzheimer's, Parkinson's, and Huntington's disease. Current treatments for CNS-related disorders which affect 1.5 billion people around the world focus mainly on symptom relief instead of disease cure. The BBB constitutes a significant obstacle for ND treatment because it restricts the ability to deliver drugs to the brain. Current advancements in CNS drug delivery involve nanoparticles along with receptor-mediated transport systems Trojan horse-like methods and nasal delivery techniques. These strategies focus on boosting treatment success and preserving BBB integrity by reducing invasive damage. Researchers examine how CPPs combined with nanoparticles can improve drug delivery methods to the brain.

Overview of Blood Brain Barrier Drug Transport

The BBB is a highly dynamic and complex system composed of blood vessels and cells, acting as a protective barrier between the bloodstream and the brain. It plays a critical role in regulating the transport of molecules to the brain. The BBB's structure includes endothelial cells, astrocytes, microglia, and pericytes, all of which collaborate to maintain brain function and protect it from harmful substances. Dysfunction in the neurovascular unit, which includes these cells, is linked to various neurological diseases. The selective permeability of the BBB presents both challenges and opportunities for drug delivery to the brain.

Strategies Using CPPs and Lipid Nanocarriers to Overcome BBB

Overcoming the BBB to deliver therapeutic drugs remains a challenge in treating neurological diseases. Researchers have focused on strategies using CPPs and lipid-based nanocarriers to improve drug delivery. CPPs, such as PACAP, can cross the BBB through specific transport systems and have neuroprotective properties. Lipid-based nanocarriers, widely used in COVID-19 vaccine development, can enhance drug penetration and retention in the brain. These nanocarriers can also be functionalized for active targeting, improving drug delivery by interacting with proteins expressed in the BBB, offering a promising approach for treating central nervous system disorders.

CPPs and BBB Penetrating Peptides in CNS Treatment

Types of CPP

Cell-penetrating peptides are short chains of amino acids that can efficiently cross cell membranes and biological barriers. The first identified CPP, TAT, derived from HIV-1, demonstrated this ability to penetrate cells. CPPs are categorized into three types based on their physicochemical properties: cationic, amphipathic, and hydrophobic. Cationic CPPs, like TAT, are the most common and carry a positive charge that facilitates cellular uptake. Amphipathic CPPs have both hydrophilic and hydrophobic regions, while hydrophobic CPPs, which contain non-polar elements, are less common but show promising low toxicity and efficient membrane interaction. CPPs are explored for drug delivery, particularly in treating neurological diseases.

Table.1 Classification of cell penetrating peptides at Creative Peptides.

CPPs Enhance Cellular Uptake

Cell-penetrating peptides are powerful tools for enhancing the delivery of various cargoes, including nanoparticles, proteins, drugs, and nucleic acids, into cells. These peptides can be combined with their cargo through methods like co-incubation, encapsulation, chemical binding, and self-assembly. One well-known CPP, TAT, is capable of crossing not only cell membranes but also neuronal membranes, making it a promising vehicle for intracellular delivery. Researchers have explored combining TAT with nanoparticles, enhancing the delivery of therapeutic molecules, such as in brain endothelial cells, and overcoming the BBB. CPPs like TAT are also used to improve the delivery of DNA, including vaccines, by stabilizing them into nanoparticles, which efficiently deliver the genetic material into cells. Additionally, CPPs can be coupled with specific targeting peptides, improving their effectiveness in targeting particular cells or tissues, such as in the treatment of brain disorders or cancer.

Blood Brain Barrier Penetrating Peptides  

BBB penetrating peptides are a special type of cell-penetrating peptides that help various substances, such as small molecules, proteins, nanoparticles, and genetic material, cross the blood-brain barrier. Discovered in 1986, certain small peptides were found to cross the BBB, prompting researchers to explore them as potential brain-targeting delivery systems. For example, Angiopep-2 has gained attention for its ability to transport therapeutic cargo, such as siRNA, enzymes, and drugs, across the BBB by interacting with a specific receptor. Despite their potential, challenges remain, such as targeting the right brain regions without affecting other areas and ensuring safe, effective transport. Advances in peptide design, such as modifications to enhance stability and reduce toxicity, are helping improve the delivery of drugs to the brain and may pave the way for new treatments.

Peptide-Mediated BBB Transcytosis and Internalization Mechanisms

The exact mechanism of CPP internalization remains complex, influenced by factors such as peptide sequence, concentration, cargo type, and the lipid composition of cell membranes. Two main transport pathways are widely accepted: direct translocation and endocytosis. Direct translocation is an energy-independent process, while endocytosis is an energy-dependent active transport. These pathways enable the efficient delivery of peptides and nanoparticles into cells. Some CPPs, like PACAP, can enter cells via both receptor-mediated and receptor-independent mechanisms. Studies also show that CPPs, such as PepNeg, can cross the blood-brain barrier through these pathways, enabling drug delivery to the brain.

Nanoparticle-Mediated Drug Delivery

Types of Lipid Nanoparticle

Nanotechnology-based delivery systems are becoming one of the most common methods for efficiently transporting therapeutic agents. Nanoparticles are being widely explored for various disease treatments and diagnostics, offering significant potential for drug delivery. Among these, lipid nanoparticles are well-known for their safety and effectiveness as carriers. These particles can be classified into several types, including liposomes, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), emulsions, and vesicle nanoparticles.

Liposomes, commonly used in brain-targeted delivery systems, consist of a spherical aqueous core surrounded by a lipid bilayer. However, they typically need targeting ligands to cross the blood-brain barrier effectively. Functionalizing liposomes with cell-penetrating peptides can improve their ability to deliver drugs into the brain.

Solid lipid nanoparticles, containing solid lipid cores, protect therapeutic molecules from being cleared by the body and reduce their toxicity. Nano-structured lipid carriers, made from a mixture of lipids, offer enhanced stability and controlled release, crucial for maintaining drug levels in the brain.

Nan emulsions are stable dispersions of oil and water, providing faster absorption and high surface area, making them suitable for a variety of administration routes.

The choice of nanoparticle type significantly impacts drug delivery to the brain and spinal cord, influencing the effectiveness of treatments for central nervous system disorders.

Targeting and Internalization Capabilities of Nanocarriers

To effectively deliver drugs across the BBB using nanoparticles (NPs), several challenges need to be addressed. First, it is crucial to determine whether NPs can successfully cross the BBB and understand the mechanisms involved. NPs can penetrate the BBB through various mechanisms. Small lipophilic molecules (<400 Da) typically diffuse passively across the endothelial cell layer, while larger molecules, like biological ligands, cross through receptor-mediated transcytosis. Specific surface-exposed ligands bind to endothelial cell receptors, promoting NP internalization and barrier penetration. Additionally, some nanoparticles may interact with the cell membrane components due to their charge or surface properties, facilitating adsorption-mediated transcytosis. Certain NP systems can also induce temporary BBB disruption to enhance penetration. Factors such as size, shape, ligand density, binding affinity, and surface charge influence the ability of NPs to cross the BBB.

To improve targeting, magnetic nanoparticles can be modified with polymers and CPPs, creating third-generation "targeted nanoparticles." These particles, combined with ligands like transferrin, enhance BBB penetration. For instance, nanoparticles loaded with drugs like doxorubicin have been shown to accumulate significantly in brain tumors, increasing survival in animal models. Targeted nanoparticles can offer neuroprotective effects while reducing systemic side effects by reducing the required drug dose.

Lipid nanoparticles (LNPs) are another promising option for brain drug delivery, especially for lipophilic drugs. LNPs are biocompatible, biodegradable, and able to encapsulate both hydrophilic and hydrophobic drugs, making them versatile carriers for treating neurological diseases. However, challenges such as immune responses and difficulties targeting specific brain regions remain. Despite these obstacles, LNPs show great promise for enhancing drug delivery across the BBB and treating neurological disorders.

Examples of Nanodrug Development for Neurodegenerative Diseases

Bioactive Peptides for Neuroprotection and Repair

Bioactive peptides, obtained through chemical synthesis or enzymatic hydrolysis of proteins (such as protein hydrolysates from food sources), are amino acid sequences with therapeutic potential for various human diseases. Many studies suggest that some bioactive peptides, such as PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide), have neuroprotective effects and could play a role in preventing and treating neurodegenerative diseases. PACAP, initially isolated from sheep hypothalamus extracts for its ability to stimulate cyclic AMP (cAMP) formation in rat pituitary cells, has since been shown to exert neurotrophic and neuroprotective effects in various in vitro and in vivo models of neurodegenerative diseases.

PACAP activates CREB (cAMP Response Element-Binding Protein), a key transcription factor involved in many cellular processes. The binding of PACAP to its receptor triggers signaling cascades (e.g., ERK, AKT, or PLC pathways), leading to CREB activation. In Alzheimer's disease (AD) pathology, lower PACAP levels are observed in human cerebrospinal fluid (CSF) and brain tissue, correlating with cognitive decline in mild cognitive impairment and dementia stages. These findings highlight the importance of PACAP in maintaining neuronal function. PACAP has been shown to protect neurons from the toxicity of Aβ-42 oligomers, possibly by enhancing mitochondrial function, thus slowing AD progression.

Further studies have shown that PACAP treatment reduces the expression of genes upregulated after middle cerebral artery occlusion (MCAO), linking PACAP's protective effects with improvements in neurological outcomes after stroke. In Parkinson's disease (PD) rat models, PACAP administration prevents the degeneration of dopaminergic neurons in the substantia nigra, alleviates cognitive decline, and improves behavioral defects by regulating dopamine levels and PD-associated proteins.

However, while PACAP shows promise as a neuroprotective agent, its delivery to the CNS remains challenging. The presence of the BBB and certain efflux mechanisms complicate the transport of peptide or protein-based therapies.

Lipids, particularly lipid nanoparticles, have been explored as carriers for peptides and bioactive molecules due to their biocompatibility, biodegradability, and ability to protect encapsulated bioactive molecules from degradation. LNPs can enhance the stability, circulation time, and targeted delivery of therapeutic agents to the brain. For instance, PEGylated liposomes have been shown to accumulate in ischemic regions of the brain due to the enhanced permeability and retention (EPR) effect. Additionally, LNPs have been successfully used for brain-targeted drug delivery, as evidenced by the ability of PEGylated liposomes to improve the brain accumulation of therapeutic agents, as well as the use of liposomes modified with CPPs and transferrin (Tf) to cross the BBB.

In Alzheimer's disease, peptide inhibitors targeting amyloid-β (Aβ) aggregation have been a focus of research. These inhibitors aim to interfere with the aggregation of Aβ peptides, which form toxic oligomers and fibrils that contribute to amyloid plaque formation, a hallmark of AD pathology. Over the past two decades, amyloid-β peptide inhibitors have been developed to target various stages of Aβ aggregation, offering significant therapeutic potential for AD treatment.

For neuroprotection and repair, LNPs have demonstrated the ability to encapsulate hydrophobic drugs and improve the bioavailability of treatments delivered to target tissues. For example, the herbal compound Andrographolide (AG), known for its neuroprotective effects but with low solubility and bioavailability, can be delivered to the brain using solid lipid nanoparticles (SLNs). These SLNs enhance AG's release characteristics and transport to the BBB, showing promise in animal models.

Overall, while many peptides and proteins have demonstrated neuroprotective properties in cell and animal models, translating these findings into clinical applications remains a major challenge. Lipid nanoparticles hold significant promise for improving the delivery of neuroprotective agents across the BBB, potentially transforming the treatment of neurodegenerative diseases.

Table.2 Active Pharmaceutical Ingredients at Creative Peptides.

Neurotherapeutic Drug Delivery for Alzheimer's Disease

The search for effective treatments for Alzheimer's disease is ongoing. AD, the most common progressive neurodegenerative disease, has been one of the most significant healthcare challenges for many years. Symptoms such as memory loss and cognitive decline profoundly impact patients' daily lives. The number of AD cases continues to rise annually, particularly in developed countries. According to the World Health Organization (WHO), the number of global patients could reach 152 million by 2050.

AD is a complex, multifactorial disease, and the excessive expression and accumulation of amyloid-beta (Aβ) aggregates are widely considered to be key factors in its pathogenesis. As a result, most clinical trials and research efforts have focused on developing drugs and interventions targeting Aβ to slow the progression of AD. Currently, several drug development programs are in Phase 2 and Phase 3. However, since many trials have been discontinued in their early stages, it remains unclear whether long-term treatments will have beneficial effects.

Peptide-Based Strategies

Peptide-based strategies for treating Alzheimer's disease primarily focus on reducing the accumulation of amyloid-beta (Aβ) plaques. One such peptide formulation, CH-3, is derived from casein hydrolysate produced by three enzymes. Akio et al. evaluated its ACE inhibitory activity and antihypertensive effects, showing significant antihypertensive activity compared to other hydrolysates. Min et al. demonstrated that oral CH-3 peptide can improve cognitive function in an AD mouse model. In their study, the AD animal model was created by intracerebroventricular injection of Aβ 1-42, leading to cognitive impairment, which was confirmed through the Morris water maze test. After oral administration of CH-3, cognitive function was restored to a level comparable to the control group. Additionally, another tripeptide, MKP, which is different from CH-3, was also tested in the AD mouse model. MKP was shown to penetrate the BBB and effectively alleviate cognitive impairment caused by Aβ 1-42.

In addition to these neuroprotective peptides, another peptide-based strategy for AD involves Aβ or Tau peptide vaccines, composed of various fragments of Aβ or Tau peptides. These vaccines work by training the immune system to recognize and eliminate harmful Aβ deposits (or Tau protein tangles) present in the brains of AD patients. ACI-35 is one such candidate vaccine, designed as a liposome-based vaccine containing copies of 16 synthetic Tau fragments (Tau393–408), phosphorylated at residues S396 and S404. These Tau phosphopeptides are modified with two palmitic acid chains at both ends, allowing them to assemble into liposomes. Currently, these promising vaccines are undergoing preclinical and clinical studies to assess their safety and efficacy.

Protein-Based Strategies

Proteins play a central role in maintaining the normal function of the CNS. Neurotrophic factors (NTFs) are a class of proteins with neuroprotective effects and potential therapeutic roles in treating Alzheimer's disease, Parkinson's disease, and Huntington's disease. NTFs prevent or reduce neuronal degeneration by providing neurotrophic support to specific neuronal populations. The mammalian neurotrophic factor family includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). Glial cell-derived neurotrophic factor (GDNF) is an important protein that supports the survival of midbrain dopaminergic neurons. Dysfunction of GDNF has been linked to several neurodegenerative diseases. One study reported that GDNF levels were decreased in the plasma of AD patients. Therefore, the administration of GDNF could be a potential therapeutic approach for certain CNS-related diseases. However, GDNF does not cross the BBB.

Dietz et al. demonstrated the effectiveness of using the TAT peptide to deliver GDNF across the BBB in a PD mouse model. Immunohistochemical analysis confirmed that, after systemic administration, Tat-GDNF could reach brain regions. These protein-based strategies have shown low toxicity in preclinical studies, but further formulation and safety studies are needed before clinical trials can proceed.

CPP and Nanoparticle Combination for Neurodegeneration

Neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, present a major challenge in delivering treatment directly to the brain. A promising solution lies in combining CPPs with NPs. This combination enhances cellular uptake and targets specific disease areas, improving the effectiveness of treatments while minimizing side effects.

Recent studies have shown that CPP-NP conjugates can effectively transport therapeutic molecules to the brain and target specific proteins linked to Alzheimer's disease. Additionally, these conjugates can deliver RNA-based treatments to reduce harmful protein formations. Despite their potential, CPP-functionalized nanoparticles still face challenges, including stability, potential toxicity, and poor selectivity in different biological environments. New strategies, like "nose-to-brain" delivery, are emerging to provide faster, more efficient treatments for neurodegenerative diseases.

Summary

In recent years, there has been growing interest in using lipid nanoparticles (LNPs) and cell-penetrating peptides (CPPs) for more efficient targeted drug delivery to treat central nervous system (CNS) diseases. This review summarizes the latest advancements and innovations in this field, focusing on topics such as the delivery of LNP formulations to the brain, the selective targeting abilities of CPPs, and the synergistic effects of combining CPPs with nanoparticles. In the treatment of neurodegenerative diseases, LNP-based drugs show advantages in enhancing the solubility, stability, and bioavailability of therapeutic molecules. On the other hand, CPP-based drug delivery systems hold promise in improving cellular uptake and promoting selective targeting. Developing a dual delivery system combining the strengths of nanoparticles and CPPs holds great potential for the future of targeted drug delivery systems.

References

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