Currently, scientists are developing functionalized NPs in order to target AD specific pathologies and effectively deliver their drug across the BBB. Figure 3 shows the mechanism of action that many treatments target to decrease physiology associated with AD, along with results of various studies that have shown successful transfection to brain tissue. One study, conducted by Zhou et al., studied the use of an siRNA drug with a polymeric NP, specifically a galactose-modified poly(ethylene glycol)-block-poly[N-(3-methacrylamidopropyl)] guanidium (GalPEG-b-P(Gu)) vehicle, to block the activity of the BACE1 enzyme in the brain that increases the accumulation of Aβ (Zhou Yutong et al., 2020; Cole and Vassar, 2007). This system offers an effective, nonviral, BBB-penetrable way to decrease the activity of this enzyme, which has been impossible previously at a size of 118 nm in a spherical morphology. siRNAs are short pieces of RNA that are able to attack specific targets, including genetic material or proteins, in order to impact the expression of specific genes and therefore expressed proteins. The complex was administered glycosylated, which gave it “triple interaction” stabilization to cross the BBB after going through the blood stream, and fluorinated medicines were also found to be more stable during the administration process. In order to test the delivery and efficacy of this complex, an in vitro using a Transwell model and an in vivo mouse model were utilized. Results of these tests were measured using stability studies of the NP complex itself, the biodistribution of a tagged version of the drug after administration through tissue studies, cytotoxicity, and the qualitative behavior of the mice. Using these measures, the drug was found to be effective in decreasing symptoms and crossing the BBB, indicating a potential future avenue for drug investigation.

A liposome-based therapy for AD was researched by Arora et al.to develop an alternative method to cross the BBB and target another enzyme involved with Aβ, apolipoprotein E (Arora et al., 2021). Apolipoprotein E (ApoE2) is a protein that plays an important role in the removal of Aβ synthesizing proteins and is made by astrocytes located around the BBB (Li et al., 2020). In this study, the gene encoding this protein was introduced to the brain using a liposome NP. The NP was surface functionalized using a GLUT-1 targeting ligand mannose and a cell penetrating peptide (CPP) to increase brain targeting and cellular uptake. These NPs were spherical and were found to be around 172 nm with a zeta potential of 19 mV. This NP was only tested in vitro using a Transwell model, both as a single culture and a coculture. The cells used in these experiments were primary cells from rats, both astrocytes and bEnd-3 cells, which are both integral parts of the rat BBB anatomy. For both models, transendothelial electrical resistance (TEER), which is the most common model validation technique for in vitro BBB experiments, was recorded to measure the barrier integrity, with experiments being conducted once the barrier integrity was optimized (Vigh et al., 2021). The coculture experiment had a peak TEER of around 175 Ω/cm² at which point the liposome was successfully delivered across the barrier. Results showed that the liposome system helped to improve transport of the gene across the BBB by preventing endonuclease digestion and improving cytotoxicity of the drug itself. In mouse models, the pApoE2 genes were shown to be expressed after treatment, indicating therapeutic potential of this delivery method in the treatment of AD.

In order to develop a more targeted approach, researchers have been investigating NP formulations that focus on specific properties of the BBB and other pathological features of an AD brain to create a more effective drug. In a study by Cesur et al., microbubbles were exploded to create a complexed NP with portions of the FDA approved therapy donepezil and BiFeO₃, making the NP electrically sensitive to control release, with a base of polyvinyl alcohol (PVA) (Cesur et al., 2022). It had a size of around 164 nm. This NP was explored to increase the efficacy of donepezil while decreasing side effects, and the frequency of administration typically needed (Knowles, 2006). This complex was biocompatible and found to be effective in creating an electrically active NP that could be delivered across the BBB, creating new avenues to control the specificity of a drug. Another approach, studied in Ribeiro et al., used mesoporous silica to create nanocontainers used to store curcumin, which is a natural drug which has anti-inflammatory and anti-amyloid accumulation activity, that were then dispersed in a thermally active hydrogel (Ribeiro et al., 2022; Tang and Taghibiglou, 2017). The NP was characterized to be spherical with a size of around 158.10 nm and a charge of around −16.73 mV. This NP was tested ex vivo in cells, using a microfluidic model as detailed in Rodero et al., and in vivo in mouse models to determine the cytotoxicity, which was insignificant, and the efficacy in AD expressing mice versus controls (Rodero et al., 2018). Microfluidics allows for testing more closely related to the actual BBB than a Transwell plate because of the dynamic media flowing through the model, especially made possible through tissue chips (Yau et al., 2023). The administration of this treatment was found to reverse the cognitive deficiencies of these mice, which opens another potential drug investigation avenue.

These therapies show that depending on the drug target, NP material, and surface functionalization can be modified to develop an effective delivery and treatment option for AD. As the knowledge of AD progression increases, numerous potential avenues for the use of nanotherapeutics will expand. NPs can be customized to complex with many different drugs and adapted for different targets, which shows that they are applicable to future drug development.

Currently in the United States, there is only one AD diagnostic based on nanomedicine that is in clinical trials that is registered with the government, which is a diagnostic known as ADx-001. This trial is being run by the biopharmaceutical company Alzeca Biosciences, Inc., which is based in Houston, Texas and is investigating a proof-of-concept in humans of their contrast agent ADx-001, which is a lipid-PEG conjugated styryl-pyrimidine. This drug is used to visualize Aβ plaques that form in the brain, providing an avenue to early diagnosis for AD and dementia because it has been shown that Aβ plaques predate symptoms and cause the formation of neurofibrillary tangles. Patients are monitored using MRI scans that are sensitive to specific anatomical changes that happen with AD and can show its progression in patients while they are still alive. The NP is made from the imaging molecule Gd(III)-DSPE-DOTA sodium salt complexed with ET3-73 which targets amyloid plaque specifically. Since this trial is in Phase 1 with the FDA, researchers are measuring immediate adverse reactions to the drug that may raise concern for its safety and are looking longitudinally over 2 years to observe any differences in the anatomical structure of patients’ brains (Andrew et al., 2020).

Scientists have been centering glioblastoma research around the use of NPs that are able to change conformation in response to a variety of different pathogenic factors of tumors in order to achieve more targeted drug delivery. Tumor microenvironments often have altered pHs or other conditions that can be exploited to target specific areas of the brain and avoid having the systemic effects that current treatments, such as chemotherapy and radiation, have (Barar and Omidi, 2013). These environmental changes often affect how drugs and other delivery systems function, so designing a vehicle that can adapt to the change in environment is important to effective therapy (Jayasuriya et al., 2016). This is done to deliver a variety of drugs thought to help reduce tumor size, or to deliver chemotherapy drugs to a more targeted location. Another common experimental drug to be delivered are proteins, which are being highly investigated because they have high specificity and specific function in cells (Yau et al., 2021a).

A study by Guo et al. showed that a thiolated paclitaxel-oligo with an angiopep-2 unit, or a modified protein NP, can be used to complex with a variety of different drugs and use its ability of size changing to be able to cross the BBB effectively and target only the tumor tissue (Guo et al., 2020). The NPs only self-assemble into the active drug form when they are in the acidic tumor microenvironment, thus allowing targeted delivery of the drug and avoiding systemic effects based on pH. This method was tested both in vitro using fluorescence and in vivo in mouse models and found to be effective. Using the angiopep-2 peptide side chain here shows the efficacy of side chain manipulation to functionalize an NP for its intended therapeutic goal.

This strategy of modifying NPs for the delivery of drugs specifically to tumors is also being used in other research. Wang et al. developed a pH sensitive polymeric NP that delivers a antiprogrammed death-ligand 1 (a cancer antibody) across the BBB as tested in a Transwell tissue model (Wang Hairong et al., 2022). The NP is made from p-2-methacryloyloxyethyl phosphorylcholine-co-p-poly(ethylene glycol) methacrylate (pMPC-co-pPEGMA) and exhibits spherical morphology with a diameter around 45 nm. This experiment transfected pMPC-co-pPEGMA, which disassembled at high pHs to release immune checkpoint blockade (ICB) antibodies across the barrier successfully by interacting with endothelial cell surfaces for transfection and triggering an antitumor immune response. The system was then tested in a mouse model, which also showed successful delivery using red tagged mRNA as measured in a confocal microscope, which is the standard for visualization. mRNA can also be detected through other methods, including chemically through rtPCR or through its interactions with other molecules, which has been utilized previously in other nanoparticle studies and has the potential to be used in future CNS studies (Li et al., 2022). Another study by Tan et al. investigated the use of an NP made from oleic manganese monoxide contained in micelles made from polyethylene glycol-poly(2-diisopropylamino)ethyl methacrylate (PEG-PDPA) containing temozolomide (TMZ) and the guiding peptide internalizing arginine-glycine-aspartic acid to cross the BBB and reach the brain (Tan et al., 2020). The NP had a dodecahedron crystal shape and had a size of around 71 nm with a zeta potential of +4.3 mV. This NP was successful at passing through both the in vitro Transwell model of the BBB and the in vivo mouse model as measured using MRI. The complex was able to successfully enter the glioma and respond to the tumor microenvironment, releasing manganese, O₂ and TMZ by inducing apoptosis in cancer cells and causing oxidative stress. TMZ is a drug that was derived from various common chemotherapies to combat gliomas and complex with this NP (Fan et al., 2014). Figure 4 shows the testing model of the vehicle, along with the transfection results. Both vehicles used existing biological structures and proven molecules that can cross the BBB to complex with their drugs through modifications and deliver to the affected area successfully.

In a study by Janowicz et al., PEGylated hyperbranched polymers to deliver doxorubicin targeted to an ephrin receptor (EphA2) to reduce tumor growth were used to test at what stage of brain cancer nanomedicine was most effective as a treatment (Janowicz et al., 2022). This study was conducted using mouse models exploring how changes in brain anatomy and the BBB that occur during the progression of brain cancer affect delivery of nanomedicines to affected areas. As cancer progresses, the BBB becomes leaky, which can also cause medication delivery to become more complicated (Arvanitis et al., 2020). The study found that delivering nanomedicines at the early stages of leakiness lead to the most effective release of the drug and treatment of the disease.

Other avenues for glioblastoma therapeutics investigate the exploitation of native processes in the body, including specific materials and receptors to treat glioblastoma. Lu et al. studied the use of a self-assembled complex of hemoglobin, lactate oxidase, and bis[2,4,5-trichloro-6-(pentyloxycarbonyl)phenyl] oxalate – photosensitizer chlorin e6 (CPPO-Ce6) encapsulated with U251 glioma cell membranes to increase the metabolism of lactate in cancer cells through the delivery of synergistic lactic acid metabolic therapy and chemiexcited photodynamic therapy (PDT) (Lu et al., 2022). This biomimetic complex, called M@HPLC, was studied in both Transwell and mouse models which both showed uptake specifically in U251 Glioma cells, which are rat-derived glioma cells. The NP system had a diameter of around 118.7 nm and had a zeta potential of −29.1 mV. In the Transwell model, there was successful delivery to both endothelial cells and lower glioma cells using the M@HPLC complex. In mice, the drug was found to cross the BBB and deliver the synergistic system successfully. In Ramalho et al., Asiatic acid, a natural compound with anti-tumor properties and low toxicity but poor bioavailability, was delivered using a Transferrin coupled poly(lactic-co-glycolic acid) (PLGA) NP (Ramalho et al., 2022; Nagoor Meeran et al., 2018). The NP showed a mean size less than 200 nm and a negative zeta potential, which indicated that it could interact with the BBB. The transferrin allowed for interaction with specifically BBB cells, which carry the transferrin receptor to allow passage of the compound to the glioblastoma. This use of transferrin to allow a small drug complex to cross the BBB effectively is a technique that can be extended to many CNS targeting drugs (Wiley et al., 2013). Lu et al. describes the use of a graphene oxide based vehicle conjugated with irinotecan (CPT-11) and cetuximab (CET) that allows for pH sensitive and body temperature dependent release of shRNA and CPT-11 into a tumor to induce apoptosis (Lu et al., 2020). The NP exhibited a 279 nm size with a −11.7 mV zeta potential. The vehicle was studied found to show a controlled release in the formed hydrogel and was able to be targeted to specific areas of the cell using antibody conjugation to the CET, as shown in mouse models. These techniques use the natural anatomy of the BBB to deliver drugs to their targeted location in the treatment of glioblastoma.

There are currently three active clinical trials in progress testing different nanomedicine techniques in the treatment of glioblastomas. One trial is currently in Phase I with the FDA and being run by the University of California, San Francisco. The drug uses a nanoliposome to encase CPT-11 for individuals genetically predisposed to develop one of the various types of glioblastoma multiforme and have previously been diagnosed with it (ClinicalTrials.gov, 2022a). This drug is administered intravenously every 3 weeks until improvement is seen or until there are signs of toxicity. This study is measuring the safety of the drug as well as monitoring for any signs of efficacy in human subjects. The second study, currently in Phase II with the FDA and sponsored by the University of Regensburg, is a PEGylated liposome containing Doxorubicin that is used in conjunction with radiation therapy and TMZ to treat glioblastoma. In vivo models, the liposome was shown to improve the drug’s penetration through the BBB, which makes it beneficial in the delivery of the effective drug doxorubicin across the BBB to help treat the condition. This drug is administered twice a month on the 1st and 15th days and is being monitored longitudinally based on data from completed toxicity studies (ClinicalTrials.gov, 2009). The third study is currently in Phase II as well and uses a PEGylated topoisomerase I inhibitor polymer etirinotecan pegol (NKTR-102) conjugated with CPT-11 to improve the BBB penetration of CPT-11 and increase the inhibition caused by CPT-11. Previous in vivo models have found that this polymer ensures that the cancer is exposed to higher doses of the drug and has fewer systemic side effects due to the localization of treatment (ClinicalTrials.gov, 1663).

Current research investigating nanotherapies for PD centers around the delivery of controlling enzymes and activities known to be associated with PD, as current treatments revolve around dopamine replacement or brain stimulation to compensate for neuron death. One study by Zhao et al. investigates the use of Ginkgolide B, a plant drug thought to be effective against PD, which has poor bioavailability, paired with a poly(ethylene glycol)-co-poly(ε-caprolactone) (PEG-PCL) vehicle to help it cross the BBB. Ginkgolide B is a drug that has shown neuroprotective effects in stroke patients, and is a research target for treatment of both PD and AD patients (Mohammad Nabavi et al., 2015). The NP complex had a size of 91.26 nm with a zeta potential of −12.09 mV. In zebrafish models, it was shown that the vehicle helped the drug to have an extended release, which would allow more periodic injections of the drug into the brain during treatment. In mouse models, the drug was successful in crossing the BBB, which was discovered through tissue studies (Zhao et al., 2020). Figure 6 shows the cellular uptake of the vehicle into zebrafish cells which shows the model’s potential use. Another study by Jennings et al. investigated the use of a nanoparticle-based system to inhibit leucine-rich repeat kinase 2 (LRRK2) activity, which would increase lysosome activity that is thought to alleviate symptoms of PD (Jeong and Lee, 2020). The NP used is DNL2101, a selective, ATP-competitive, CNS penetrable LRRK2 inhibitor with formula 2-methyl-2-(3-methyl-4-((4-(methylamino)-5-(trifluoromethyl)pyrimidin-2-yl)amino)-1H-pyrazol-1-yl)propanenitrile. In this case, however, the NP itself is the drug that works to do this and can pass through the BBB, but also targets other tissues such as the kidneys and liver. This drug was found to be somewhat harsh, with some mouse models experiencing kidney and lung issues that were reversible and some toxicity after a prolonged period. However, when tested in humans, it was found that the drug was not toxic in lower dosages and was effective in inhibiting the gene LRRK2 (Jennings et al., 2022).

Joy et al. describe the use of a transdermal delivery system to deliver a complex of Brahmi, a herbal plant thought to have significant nervous system therapeutic effects, and a lipid nanoparticle (Joy et al., 2022). This patch system, made from a combination of polyvinyl alcohol (PVA) and the Brahmi-solid lipid nanoparticle complex, is a dissolvable and painless method of distribution of the NP, which makes patient compliance and treatment more successful, and are able to be manufactured without a cleanroom leading to greater ability to create them at a greater scale to treat more people (Ghanbariamin et al., 2023). The formulation had a zeta potential of −19.21 mV and the NP was in a size range between 120 and 200 nm. Patches in general are very useful for situations that call for targeted drug delivery, such as treatment of osteoarthritis, and therefore may be applied to targeted delivery to the brain (Cao et al., 2021). The lipid NP made the structure of the drug itself more optimal for delivery, which allowed for uptake to the brain as seen in mice. The patch itself was also tested on rat skin and found to have no irritating effect, which means that the patch system would be effective in delivering the medication. This method of delivering an effective lipid NP treatment is important because it indicates a new method of drug delivery to offer to patients.

Wang et al. studied the use of poly(ethylene glycol)-b-poly(trimethylene carbonate) (PEG-PTMC) NPs to deliver Ginkgolide B (Wang Q. et al., 2022). PEG-PTMC is an FDA-approved material that can be used for drug delivery, so using this complex is a method to ensure adequate complexing of the drug to the vehicle. In zebrafish, the NP was found to be highly stable and noncytotoxic. In a mouse model, the drug was successful in delivering across the BBB, which offers another avenue for investigating the use of FDA approved NPs with PD treating medications. In Gao et al., the use of MgOp@PPLP, which is a complex of MgO nanoparticles and a polydopamine shell with polyethylene glycol (PEG), lactoferrin, and puerarin on the surface to improve hydrophilicity and tissue targeting of the drug, with an anti-SNCA plasmid inside, which combats alpha synuclein creation (Gao et al., 2022). These NPs were guided to their target tissue using near-infrared radiation and crossed the BBB, allowing the delivery of gene therapy and antioxidant therapy to the disease source. These treatments offer potential avenues of investigation for various complexes that drugs can be paired with to effectively deliver across the BBB.

There are two nanoparticle-based treatments for Parkinson’s disease that are currently in the clinical trial phase of testing. One drug is developed by InnoMedica Schweiz AG and tests the replacement of ganglioside GM1 in PD patients with a liposome system for successful delivery (Maria et al., 2022). The drug is administered via dose escalation leading up to the maximum acceptable dose. GM1 has previously been found to be a neuroprotective agent that helps fundamental functional processes in neurons, especially in PD patients, so this study aims to explore how this drug can be safely administered, effectively delivered, and how effective it is compared to Levodopa, which is the current treatment (ClinicalTrials.gov, 2022b). Another drug currently in clinical trials for use in treating PD is a LRRK2 inhibitor drug DNL2101 produced by Biogen, Inc. in collaboration with Denali therapeutics. It uses a small molecule inhibitor of LRRK2 to increase lysosome activity in PD patients, relieving symptoms. This drug is administered by mouth four times daily and the study is being conducted longitudinally to explore its long term effects (Jennings et al., 2022).

Current research in the use of nanotherapeutics for the treatment of MS focuses on methods to effectively deliver already FDA approved treatments, such as dimethyl fumarate (DMF) (Bomprezzi, 2015). A study by Mehdi et al. looked at the use of modified platelets to deliver DMF through the BBB, as DMF currently has relatively low bioavailability. The zeta potential of this complex was +7 mV, and the size of 118.33 nm was ideal for successful transfection. This study found that the positive charge of the complex was key to successful interaction with endothelial cells which led to successful transcytosis (Mehdi-almadarlou et al., 2022). Another study by Sinha et al. used chitosan-alginate core-shell-corona shaped NPs to deliver DMF for long-term release, which had negative zeta potential of −27.2 mV and a bigger size of 561 nm, which lended it a larger surface area that was ideal for the matrix delivery system being investigated. This study, however, did not focus on the BBB and instead conducted various dissolution tests of the NP system to determine the best way to create an effective slow-release drug (Sinha et al., 2021). These methods both show that existing therapies can also be improved using NP systems to deliver across the BBB more effectively.

Other therapies for MS focus on blocking the body’s response to the disease, which includes the activation of the immune system against native cells, causing the breakdown of myelin sheaths (Fabrizio and Mario, 2005). In Tredicine et al., the use of liposomes was explored to block the function of T cells that attack myelin in the nervous system by downregulating Th17 activation using a peptide from myelin basic protein to reduce inflammation, promoting T regulatory lymphocytes, downregulating Th1 and Th17, and interfering with the pro/anti inflammatory macrophage balance (Tredicine et al., 2023). This was done using a liposome system, which was made from a double layer of phosphatidylserine, which was able to interact with the markers to achieve downregulation as tested in mice. The NP was a spherical bilayer with a size of 298.51 nm and a zeta potential of −38.87 mV, which showed transfection in monocytes and T cells from MS patients and in mouse models. Another study by Triantafyllakou et al., studied using a PLGA NP encapsulating a myelin peptide from the myelin oligodendrocyte glycoprotein epitope (rMOG) complexed with glucosamine to target antigen presenting immune cells and prevent self-attack (Triantafyllakou et al., 2022). Specifically, this NP is able to bind to dendritic cells and prevent them from inducing additional autoimmune activity. The NP had a spherical morphology with a size of 378.4 nm and a zeta potential of −34.8 mV. This system was tested in both in vitro and in vivo models on mice with experimental autoimmune encephalitis (EAE) which is thought to bear many similarities to MS in the autoimmune triggers. Results showed that there was significant suppression of disease symptoms in treated mice and that the glycosylation played a role in the efficacy of the complex. A study by Titus et al. studied the use of digoxin, which is a common medication used to treat heart issues by inhibiting the sodium-potassium pump and thought to have pro-myelinating properties, in combination with a poly(lactide-co-glycolide) (PLG) NP complexed with autoantigens to both prevent autoimmune attack and stimulate the regeneration of myelin-producing cells, oligodendrocytes, in the CNS (Titus et al., 2022). The NP was of spherical morphology and had a mean size of 483.7 nm with a charge of −84.7 mV. This regimen was tested in vivo in mouse models and was studied both for efficacy in delivery, measured with flow cytometry, and for any alterations made to the makeup of the BBB due to its targeting. The targeting was found to be sufficient to the brain with no specific impact of the makeup of the BBB, which makes this a viable treatment. The NPs investigated in these studies provide viable treatment plans for MS and are potential avenues for future drugs that target the pathophysiology of the disease.