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Amphotericin B (AMB) has been a mainstay therapy for fungal infections of the central nervous system, but its use has been limited by its poor penetration into the brain, the mechanism of which remains unclear. In this study, we aimed to investigate the role of P-glycoprotein (P-gp) in AMB crossing the blood-brain barrier (BBB). The uptake of AMB by primary brain capillary endothelial cells in vitro was significantly enhanced after inhibition of P-gp by verapamil. The impact of two model P-gp inhibitors, verapamil and itraconazole, on brain/plasma ratios of AMB was examined in both uninfected CD-1 mice and those intracerebrally infected with Cryptococcus neoformans. In uninfected mice, the brain/plasma ratios of AMB were increased 15 min (3.5 versus 2.0; P < 0.05) and 30 min (5.2 versus 2.8; P < 0.05) after administration of verapamil or 45 min (6.0 versus 3.9; P < 0.05) and 60 min (5.4 versus 3.8; P < 0.05) after itraconazole administration. The increases in brain/plasma ratios were also observed in infected mice treated with AMB and P-gp inhibitors. The brain tissue fungal CFU in infected mice were significantly lower in AMB-plus-itraconazole or verapamil groups than in the untreated group (P < 0.005), but none of the treatments protected the mice from succumbing to the infection. In conclusion, we demonstrated that P-gp inhibitors can enhance the uptake of AMB through the BBB, suggesting that AMB is a P-gp substrate., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The blood-brain barrier (BBB) exerts its central nervous system (CNS) protective function as it hinders the delivery of diagnostic and therapeutic agents to the brain. With the development of nanotechnology during the last thirty years, the nanocarriers for delivering drugs make it possible to transport drugs across the BBB. The brain-targeted drug delivery system usually consists of two parts: nanocarriers and brain-targeted strategies. In this review, several kinds of nanocarriers are introduced for brain-targeted drug delivery. We focus on several possible strategies for brain-targeting and comment on their advantages and disadvantages in application.

Aim: The gene encoding neurotrophic factor was transfected into brain capillary endothelial cells with the aim of delivering the gene product extensively into the brain parenchyma by making use of the secretory function of BCECs., Methods: Plasmid DNA encoding mouse glial cell-derived neurotrophic factor (mGDNF gene) was constructed and prepared. Then, mGDNF gene was transfected into cultured mouse brain capillary endothelial cells (BCECs) in vitro. The amount of mGDNF protein in the transfected cells and secreted from the transfected cells were determined by ELISA. The polarity of the secretion of mGDNF protein from BCECs was investigated in a bicameral culture system., Results: The mGDNF protein was detected out not only from the transfected cells but also the cultured media. And mGDNF protein was mainly found in the brain side of the culture compartment., Conclusion: It has been demonstrated that a secretory protein can be successfully delivered into brain parenchyma by utilizing the secretory pathway of BCECs.

The gene encoding mouse glial cell-derived neurotrophic factor (mGDNF gene) was transfected into brain capillary endothelial cells (BCECs) with the aim of delivering the gene product extensively into the brain parenchyma by making use of the secretory function of BCECs. First, we transfected mGDNF gene into cultured BCECs (MBEC4; mouse brain capillary endothelial cells) in vitro. The amount of mGDNF protein secreted from the transfected cells into the medium was 1500 to 3200 pg/mg of cell protein per day, being about sevenfold higher than that accumulated intracellularly. Furthermore, the basolateral-directed secretion of mGDNF protein from the transfected MBEC4 cells was fivefold higher than the apical-directed secretion. Next, the hemagglutination virus of Japan (HVJ)-liposomes encapsulating mGDNF gene were administered to rats in vivo via the internal carotid artery. The transfected rats showed a marked increase in the brain level of GDNF as assessed by means of enzyme-linked immunosorbent assay (ELISA) and Western blotting on day 3 after the administration, and the level remained significantly elevated for at least 12 days. Furthermore, immunohistochemical staining revealed an increase in GDNF immunoreactivity throughout the transfected forebrain. These results indicate that the gene was successfully transferred in vivo from HVJ-liposomes into BCECs, where it was expressed, and the gene product was secreted into the brain. Then, using this delivery method, we evaluated the protective effect for dopamine neuron against a retrograde 6-hydroxydopamine (6-OHDA) lesion, as assessed by behavioral and neurochemical indices.

The blood-brain barrier (BBB) is a special structure in the central nervous system (CNS), whose delicate properties can protect the brain from the attack by harmful large molecules and organisms within blood circulation. However, the very barrier also prevents the brain-targeted drugs from taking effects, posing great challenge to the treatment of neurodegenerative diseases, glioma and other brain diseases. With the development of nanotechnology, more and more nanoparticles (NPs) have been developed as drug carriers, which can facilitate the drug with various mechanisms to get across the BBB. This article starts with introduction of the BBB, explaining the complexity of BBB structure and its unique features to form barriers, so that we can understand the difficulty in transporting drug into the BBB and the potential methods of targeting and permeate the barrier. Next, we focus on three main types of NPs that have been applied in the brain-targeted drug delivery system: polymeric-based NPs; biomimetic-based NPs and inorganic-based NPs. Furthermore, we deal with the specific NPs drug delivery strategies, which include absorption-mediated, carrier-mediated and receptor-mediated transcytosis. At the end of the article, we talk about the leading trend and future development of the brain-targeted nanoparticle drug delivery systems.

As a neurological disorder in the brain, epilepsy is not only associated with abnormal synchronized discharging of neurons, but also inseparable from non-neuronal elements in the altered microenvironment. Anti-epileptic drugs (AEDs) merely focusing on neuronal circuits frequently turn out deficient, which is necessitating comprehensive strategies of medications to cover over-exciting neurons, activated glial cells, oxidative stress and chronic inflammation synchronously. Therefore, we would report the design of a polymeric micelle drug delivery system that was functioned with brain targeting and cerebral microenvironment modulation. In brief, reactive oxygen species (ROS)-sensitive phenylboronic ester was conjugated with poly-ethylene glycol (PEG) to form amphiphilic copolymers. Additionally, dehydroascorbic acid (DHAA), an analogue of glucose, was applied to target glucose transporter 1 (GLUT1) and facilitate micelle penetration across the blood‒brain barrier (BBB). A classic hydrophobic AED, lamotrigine (LTG), was encapsulated in the micelles via self-assembly. When administrated and transferred across the BBB, ROS-scavenging polymers were expected to integrate anti-oxidation, anti-inflammation and neuro-electric modulation into one strategy. Moreover, micelles would alter LTG distribution in vivo with improved efficacy. Overall, the combined anti-epileptic therapy might provide effective opinions on how to maximize neuroprotection during early epileptogenesis. The micelles manage to cross the blood–brain barrier and then degrade under reactive oxygen species stimulation to release lamotrigine for anti-epileptic neuroprotection and glial stabilization. [Display omitted] [ABSTRACT FROM AUTHOR]

Brain diseases, including neurodegenerative diseases, acute ischemic stroke and brain tumors, have become a major health problem and a huge burden on society with high morbidity and mortality. However, most of the current therapeutic drugs can only relieve the symptoms of brain diseases, and it is difficult to achieve satisfactory therapeutic effects fundamentally. Extensive studies have shown that the therapeutic effects of brain diseases are mainly affected by two factors: the conservation of the blood–brain barrier (BBB) and the complexity of the brain micro‐environment. Brain‐targeting drug delivery systems provide new possibilities for overcoming these barriers with versatility. In this review, it provides an overview of BBB alteration and discusses targeting delivery strategies for brain diseases therapy. Furthermore, delivery systems which are designed to modulate the brain micro‐environment with synergistic effects were also highlighted. This article is categorized under:Therapeutic Approaches and Drug Discovery > Emerging Technologies [ABSTRACT FROM AUTHOR]

Blood–brain barrier (BBB) strictly controls matter exchange between blood and brain, and severely limits brain penetration of systemically administered drugs, resulting in ineffective drug therapy of brain diseases. However, during the onset and progression of brain diseases, BBB alterations evolve inevitably. In this review, we focus on nanoscale brain-targeting drug delivery strategies designed based on BBB evolutions and related applications in various brain diseases including Alzheimer's disease, Parkinson's disease, epilepsy, stroke, traumatic brain injury and brain tumor. The advances on optimization of small molecules for BBB crossing and non-systemic administration routes (e. g., intranasal treatment) for BBB bypassing are not included in this review. Blood–brain barrier (BBB) is evolving during the onset and progression of brain diseases. BBB evolution-based nanoscale brain-targeting drug delivery strategies are summarized in this review for various brain diseases. [Display omitted] [ABSTRACT FROM AUTHOR]

Ischemic stroke is a cerebrovascular disease normally caused by interrupted blood supply to the brain. Ischemia would initiate the cascade reaction consisted of multiple biochemical events in the damaged areas of the brain, where the ischemic cascade eventually leads to cell death and brain infarction. Extensive researches focusing on different stages of the cascade reaction have been conducted with the aim of curing ischemic stroke. However, traditional treatment methods based on antithrombotic therapy and neuroprotective therapy are greatly limited for their poor safety and treatment efficacy. Nanomedicine provides new possibilities for treating stroke as they could improve the pharmacokinetic behavior of drugs in vivo , achieve effective drug accumulation at the target site, enhance the therapeutic effect and meanwhile reduce the side effect. In this review, we comprehensively describe the pathophysiology of stroke, traditional treatment strategies and emerging nanomedicines, summarize the barriers and methods for transporting nanomedicine to the lesions, and illustrate the latest progress of nanomedicine in treating ischemic stroke, with a view to providing a new feasible path for the treatment of cerebral ischemia. This review summarizes various nanomedicines, including liposomes, micelles, dendrimer, nanoparticles, and exosomes, which have been applied for the treatment of ischemic stroke. It especially focuses on their abilities to normalize various abnormalities at different phases of ischemic cascade, such as dissolving the clot and salvaging the penumbra. [Display omitted] [ABSTRACT FROM AUTHOR]

The blood–brain barrier (BBB) and the poor ability of many drugs to cross that barrier greatly limits the efficacy of chemotherapies for glioblastoma multiforme (GBM). The present study exploits albumin as drug delivery vehicle to promote the chemotherapeutic efficacy of paclitaxel (PTX) by improving the stability and targeting efficiency of PTX/albumin nanoparticles (NPs). Here we characterize PTX-loaded human serum albumin (HSA) NPs stabilized with intramolecular disulfide bonds and modified with substance P (SP) peptide as the targeting ligand. The fabricated SP-HSA-PTX NPs exhibited satisfactory drug-loading content (7.89%) and entrapment efficiency (85.7%) with a spherical structure (about 150 nm) and zeta potential of −12.0 mV. The in vitro drug release from SP-HSA-PTX NPs occurred in a redox-responsive manner. Due to the targeting effect of the SP peptide, cellular uptake of SP-HSA-PTX NPs into brain capillary endothelial cells (BCECs) and U87 cells was greatly improved. The low IC 50 , prolonged survival period and the obvious pro-apoptotic effect shown by TUNEL analysis all demonstrated that the fabricated SP-HSA-PTX NPs showed a satisfactory anti-tumor effect and could serve as a novel strategy for GBM treatment. [ABSTRACT FROM AUTHOR]

Metabolic microenvironment dysfunction remains hallmarks of acute ischemic stroke (AIS) throughout the ischemia, reperfusion, and recovery stages. Existing therapeutic strategies focus only on a single pathological process, while cascading metabolic changes are neglected. For the sake of better long-term prognosis, interventions in each stage are certainly worthwhile if achievable. Herein, inspired by the natural hemoglobin (Hb) carrying oxygen mechanism, a polymer-based nanoerythrocyte is designed to remodel the metabolic microenvironment in the whole stages of AIS. Briefly, Hb and methoxatin are incorporated into polydopamine nanocage which is further coated with the polymer layer formed by in-situ free radical polymerization. Modified with microthrombus-targeting peptide, this nanoerythrocyte can actively accumulate in the ischemic core and respond to the metabolic microenvironment. In vivo results demonstrate that cellular metabolic homeostasis and responsiveness are restored by oxygen balance regulation, microglia polarization, glucose metabolism activation, and neovascularization in permanent and transient middle cerebral artery occlusion model mice. The behavioral experiment results also confirm the effects of nanoerythrocyte for long-term prognosis. This strategy gives a proof of concept that metabolic microenvironment modulation is a promising perspective for AIS treatment with nanoerythrocytes. [Display omitted] • Microthrombus-targeted nanoerythrocyte penetrates the damaged blood-brain barrier and reaches the ischemic core. • Nanoerythrocyte responds to the level of oxygen and reactive oxygen species during different stages of acute ischemic stroke. • A multi-cellular metabolic modulation strategy is beneficial for the long-term prognosis of acute ischemic stroke. [ABSTRACT FROM AUTHOR]

Purpose: To prepare an angiopep-conjugated dendrigraft poly-L-lysine (DGL)-based gene delivery system and evaluate the neuroprotective effects in the rotenone-induced chronic model of Parkinson's disease (PD). Methods: Angiopep was applied as a ligand specifically binding to low-density lipoprotein receptor-related protein (LRP) which is overexpressed on blood-brain barrier (BBB), and conjugated to biodegradable DGL via hydrophilic polyethyleneglycol (PEG), yielding DGL-PEG-angiopep (DPA). In vitro characterization was carried out. The neuroprotective effects were evaluated in a chronic parkinsonian model induced by rotenone using a regimen of multiple dosing intravenous administrations. Results: The successful synthesis of DPA was demonstrated via H-NMR. After encapsulating the therapeutic gene encoding human glial cell line-derived neurotrophic factor ( hGDNF), DPA/ hGDNF NPs showed a sphere-like shape with the size of 119 ± 12 nm and zeta potential of 8.2 ± 0.7 mV. Angiopep-conjugated NPs exhibited higher cellular uptake and gene expression in brain cells compared to unmodified counterpart. The pharmacodynamic results showed that rats in the group with five injections of DPA/ hGDNF NPs obtained best improved locomotor activity and apparent recovery of dopaminergic neurons compared to those in other groups. Conclusion: This work provides a practical non-viral gene vector for long-term gene therapy of chronic neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Tumor metastasis is a significant contributor to the mortality of cancer patients. Specifically, current conventional treatments are unable to achieve complete remission of brain metastasis. This is due to the unique pathological environment of brain metastasis, which differs significantly from peripheral metastasis. Brain metastasis is characterized by high tumor mutation rates and a complex microenvironment with immunosuppression. Additionally, the presence of blood-brain barrier (BBB)/blood tumor barrier (BTB) restricts drug leakage into the brain. Therefore, it is crucial to take account of the specific characteristics of brain metastasis when developing new therapeutic strategies. Nanomaterials offer promising opportunities for targeted therapies in treating brain metastasis. They can be tailored and customized based on specific pathological features and incorporate various treatment approaches, which makes them advantageous in advancing therapeutic strategies for brain metastasis. This review provides an overview of current clinical treatment options for patients with brain metastasis. It also explores the roles and changes that different cells within the complex microenvironment play during tumor spread. Furthermore, it highlights the use of nanomaterials in current brain treatment approaches. [Display omitted] • The unique pathological environment of brain metastases limits clinical outcomes. • Different cell types play different roles and change during the progression of BM. • NMs can be customized to specific pathological features and thus offer significant advantages in BM treatment. [ABSTRACT FROM AUTHOR]

Immunotherapy is regarded as one of the most promising approaches for treating tumors, with a multitude of immunotherapeutic thoughts currently under consideration for the lethal glioblastoma (GBM). However, issues with immunotherapeutic agents, such as limited in vivo stability, poor blood-brain barrier (BBB) penetration, insufficient GBM targeting, and represented monotherapy, have hindered the success of immunotherapeutic interventions. Moreover, even with the aid of conventional drug delivery systems, outcomes remain suboptimal. Biomimetic strategies seek to overcome these formidable drug delivery challenges by emulating nature's intelligent structures and functions. Leveraging the variety of biological structures and functions, biomimetic drug delivery systems afford a versatile platform with enhanced biocompatibility for the co-delivery of diverse immunotherapeutic agents. Moreover, their inherent capacity to traverse the BBB and home in on GBM holds promise for augmenting the efficacy of GBM immunotherapy. Thus, this review begins by revisiting the various thoughts and agents on immunotherapy for GBM. Then, the barriers to successful GBM immunotherapy are analyzed, and the corresponding biomimetic strategies are explored from the perspective of function and structure. Finally, the clinical translation's current state and prospects of biomimetic strategy are addressed. This review aspires to provide fresh perspectives on the advancement of immunotherapy for GBM. [Display omitted] • Immunotherapeutic thoughts and agents for glioblastoma are revisited comprehensively. • Obstacles encountered during the process of achieving immunotherapy for GBM are analyzed. • Biomimetic strategies to overcome all barriers are summarized from the perspective of function and structure. • The future direction of biomimetic strategies in the immunotherapy of glioblastoma are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Combination of gene therapy and chemotherapy is a promising approach for glioma therapy. In this study, a co-delivery system of plasmid encoding human tumor necrosis factor-related apoptosis-inducing ligand (pORF-hTRAIL, Trail) and doxorubicin (DOX) has been simply constructed in two steps. Firstly, DOX was intercalated into Trail to form a stable complex. Secondly, DOX-Trail complex was condensed by Dendrigraft poly-l-lysine (DGL) to form a nanoscaled co-delivery system. Choline transporters are both expressed on blood–brain barrier (BBB) and glioma, Herein, a choline derivate with high choline transporter affinity was chosen as BBB and glioma dual targeting ligand. Choline-derivate modified co-delivery system showed higher cellular uptake efficiency and cytotoxicity than unmodified co-delivery system in U87 MG cells. In comparison with single medication or unmodified delivery system, Choline-derivate modified co-delivery system induced more apoptosis both in vitro and in vivo. The therapeutic efficacy on U87 MG bearing xenografts further confirmed the predominance of this dual targeting and co-delivery system. [Copyright &y& Elsevier]

Abstract: The activation of caspase-3 mediated neuron death is a common process in neurodegenerative diseases. Efficient activated caspase-3 imaging in brain would be helpful to monitor the potential lesion and intervene promptly. However, the blood–brain barrier (BBB) is the major obstacle that hinders the delivery of diagnostic agents into the brain. Herein, the fluorescence resonance energy transfer (FRET) was successfully applied to detect in vivo activated caspase-3 in apoptotic neuron with a brain-targeted nano-device, which was based on dendrigraft poly-l-lysines (DGLs) and linked with a brain-targeted peptide RVG29 as well as the caspase-3 cleavable peptide linker (DEVD). This nano-device could detect the level of caspase-3 activation in accordance with the degree of apoptosis in rat brain; meanwhile normal rat showed no fluorescence signal. The location of fluorescence signal was confirmed to accumulate more in caspase-3 activated neurons. Taken together, our nano-device would help to image activated caspase-3 in vivo and hold great promise in early diagnosis of neurodegenerative diseases. [Copyright &y& Elsevier]

Abstract: One of the most common life-threatening infections in immunosuppressive patients, like AIDs patients, is cryptococcal meningitis or meningoencephalitis. Current therapeutic options are mostly ineffective and mortality rates remain high. Hydrophobic antifungal drug Amphotericin B (AmB), has become a golden standard in severe systemic fungal infection therapy. However, most AmB commercial formulations, including deoxycholate AmB and lipid formulations of AmB, show poor penetration into the CNS and difficulty to reach the therapeutic levels. To improve the CNS permeability of AmB, we have successfully developed an effective brain-targeting polymeric micellar system with angiopep-2 modified, named Angiopep-PEG-PE/AmB polymeric micelles. An immunosuppressive murine model with Cryptococcus neoformans meningoencephalitis (CNME) was established to evaluate the CNS penetration efficiency and antifungal treatment efficacy of the AmB-incorporated brain-vectored polymeric micellar formulation, compared with the AmB commercial formulations. After three consecutive days of i.v. administration, the results showed that the group treated with Angiopep-PEG-PE/AmB achieved the greatest treatment efficacy, which reached the highest AmB level in brain, reduced the brain fungal burden significantly, decreased histopathological severity and prolonged the median survival time. The increased treatment efficacy could be attributed to the brain-targeting delivery system promoted AmB crossing the BBB and penetrating into the brain to reach the therapeutic concentration. The underlying mechanism was also explored in this work. Therefore, the brain-targeting delivery system could have potential and promising implications for treatment of intracerebral fungal infection. [Copyright &y& Elsevier]

Abstract: Clinical diagnosis of cancers using magnetic resonance imaging (MRI) is highly dependent on contrast agents, especially for brain tumors which contain blood–brain barrier (BBB) at the early stage. However, currently mostly used low molecular weight contrast agents such as Gd-DTPA suffer from rapid renal clearance, non-specificity, and low contrast efficiency. The aim of this paper is to investigate the potential of a macromolecular MRI contrast agent based on dendrigraft poly-l-lysines (DGLs), using chlorotoxin (CTX) as a tumor-specific ligand. The contrast agent using CTX-modified conjugate as the main scaffold and Gd-DTPA as the payload was successfully synthesized. The results of fluorescent microscopy showed that the modification of CTX could markedly enhance the cellular uptake in C6 glioma and liver tumor cell lines, but not in normal cell line. Significantly increased accumulation of CTX-modified conjugate within glioma and liver tumor was further demonstrated in tumor-bearing nude mice using in vivo imaging system. The MRI results showed that the signal enhancement of mice treated with CTX-modified contrast reached peak level at 5 min for both glioma and liver tumor, 144.97% ± 19.54% and 158.69% ± 12.41%, respectively, significantly higher than that of unmodified counterpart and commercial control. And most importantly, the signal enhancement of CTX-modified contrast agent maintained much longer compared to that of controls, which might be useful for more exact diagnosis for tumors. CTX-modified dendrimer-based conjugate might be applied as an efficient MRI contrast agent for targeted and accurate tumor diagnosis. This finding is especially important for tumors such as brain glioma which is known hard to be diagnosed due to the presence of BBB. [Copyright &y& Elsevier]

Abstract: The blood–brain barrier is the major obstacle that prevents diagnostic and therapeutic drugs being delivered to the central nervous systems in order to exert their effects. Specific ligand-receptor binding mediated endocytosis is one of the possible strategies to cross this barrier. A 30-amino-acid peptide (leptin30) derived from an endogenic hormone—leptin is exploited as brain-targeting ligand as it is reported to possess the same brain accumulation efficiency after intravenous injection. Dendrigraft poly-l-lysine (DGL) is used as non-viral gene vector in this study. DGL–PEG–Leptin30 was complexed with plasmid DNA yielding nanoparticles (NPs). The cellular uptake characteristic and mechanism were explored in brain capillary endothelial cells (BCECs) which express leptin receptors. Furthermore, brain parenchyma microglia cells such as BV-2 cells expressing leptin receptors could promote ligand-receptor mediated endocytosis leading to enhanced gene transfection ability of DGL–PEG–Leptin30/DNA NPs. The targeted NPs were proved to be transported across in vitro BBB model effectively and accumulate more in brains after i.v. resulting in a relatively high gene transfection efficiency both in vitro and in vivo. Besides, the NPs showed low cytotoxicity after in vitro transfection. Thus, DGL–PEG–Leptin30 provides a safe and noninvasive approach for the delivery of gene across the blood–brain barrier. [Copyright &y& Elsevier]

Abstract: Lactoferrin (Lf)-modified nanoparticles (NPs) have been demonstrated to mediate efficient expression of exogenous genes in the brain via intravenous administration. The brain-targeting properties of Lf-modified NPs were investigated in this study. In vivo imaging results showed that the accumulation of Lf-modified NPs was higher in the brain but lower in the other organs than that of unmodified counterparts. The results of analytical transmission electron microscopy showed that some Lf-modified NPs crossed the blood–brain barrier (BBB) and reached the neural tissues, while some remained within the BBB. Similar results were observed in the distribution of exogenous gene products. All the results demonstrated the successful delivery of Lf-modified NPs into the brain. Lf-modified NPs could be exploited as potential brain-targeting delivery systems for exogenous genes, especially for those encoding secretive proteins. [Copyright &y& Elsevier]

Abstract: Background: Gene therapy is considered one of the most promising approaches to develop an effective treatment for Parkinson''s disease (PD). The existence of blood-brain barrier (BBB) significantly limits its development. In this study, lactoferrin (Lf)-modified nanoparticles (NPs) were used as a potential non-viral gene vector due to its brain-targeting and BBB-crossing ability. Methods and results: The neuroprotective effects were examined in a rotenone-induced chronic rat model of PD after treatment with NPs encapsulating human glial cell line-derived neurotrophic factor gene (hGDNF) via a regimen of multiple dosing intravenous administration. The results showed that multiple injections of Lf-modified NPs obtained higher GDNF expression and this gene expression was maintained for a longer time than the one with a single injection. Multiple dosing intravenous administration of Lf-modified NPs could significantly improve locomotor activity, reduce dopaminergic neuronal loss, and enhance monoamine neurotransmitter levels on rotenone-induced PD rats, which indicates its powerful neuroprotective effects. Conclusion: The findings may have implications for long-term non-invasive gene therapy for neurodegenerative diseases in general. [Copyright &y& Elsevier]