Xiayan Shao

Intranasal nanoparticles of basic fibroblast growth factor for brain delivery to treat Alzheimer's disease.

Disabilities caused by neurodegeneration have become one of the main causes of mortality in elderly population, with drug distribution to the brain remaining one of the most difficult challenges in the treatment of the central nervous system (CNS) diseases due to the existence of blood-brain barrier. Lectins modified polyethylene glycol-polylactide-polyglycolide (PEG-PLGA) nanoparticles could enhance the drug delivery to the brain following intranasal administration. In this study, basic fibroblast growth factor (bFGF) was entrapped in nanoparticles conjugated with Solanum tuberosum lectin (STL), which selectively binds to N-acetylglucosamine on the nasal epithelial membrane for its brain delivery. The resulting nanoparticles had uniform particle size and negative zeta potential. The brain distribution of the formulations following intranasal administration was assessed using radioisotopic tracing method. The areas under the concentration-time curve of (125)I-bFGF in the olfactory bulb, cerebrum, and cerebellum of rats following nasal application of STL modified nanoparticles (STL-bFGF-NP) were 1.79-5.17 folds of that of rats with intravenous administration, and 0.61-2.21 and 0.19-1.07 folds higher compared with intranasal solution and unmodified nanoparticles, respectively. Neuroprotective effect was evaluated using Mirror water maze task in rats with intracerebroventricular injection of β-amyloid25-35 and ibotenic acid. The spatial learning and memory of Alzheimers disease (AD) rats in STL-bFGF-NP group were significantly improved compared with AD model group, and were also better than other preparations. The results were consistent with the value of choline acetyltransferase activity of rat hippocampus as well as the histological observations of rat hippocampal region. The histopathology assays also confirmed the in vivo safety of STL-bFGF-NP. These results clearly indicated that STL-NP was a promising drug delivery system for peptide and protein drugs such as bFGF to enter the CNS and play the therapeutic role.

PEGylated poly(2-(dimethylamino) ethyl methacrylate)/DNA polyplex micelles decorated with phage-displayed TGN peptide for brain-targeted gene delivery

Phage-displayed TGN peptide-decorated polymeric micelle-like polyplexes based on pegylated poly(2-(dimethylamino) ethyl methacrylate) (PEG-PDMAEMA) were prepared for efficient brain-targeted gene delivery. The diblock copolymers Methoxy-PEG-PDMAEMA and Maleimide-PEG-PDMAEMA were synthesized by the atom transfer radical polymerization method. The TGN ligand, a 12-amino acid peptide that could facilitate blood-brain barrier (BBB) targeting, was conjugated to the PEG terminus of the copolymer via a maleimide-mediated covalent binding procedure. TGN-PEG-PDMAEMA was complexed with plasmid DNA to yield polyplexes. The physiochemical properties of the polyplexes, such as morphology, particle size, zeta potential, cytotoxicity and DNA complex formation ability, were studied prior to the successful in vitro and in vivo transfection. The TGN-PEG-PDMAEMA/DNA polyplexes maintained their stable nano-size, were characterized by good condensation capacity and low toxicity and even provided higher cellular uptake than the unmodified polyplexes (PEG-PDMAEMA/DNA polyplexes). Confocal microscopy studies showed that the DNA of TGN-PEG-PDMAEMA/DNA polyplexes entered into the nuclei through the endosome/lysosome pathway. The transfection efficiency of TGN-modified polyplexes was higher than that of unmodified polyplexes both in vitro and in vivo. The results obtained from frozen sections indicated the widespread expression of an exogenous gene in the mouse brain after intravenous injection. Therefore, the results demonstrate that the TGN-decorated PEG-PDMAEMA developed in this study could be utilized as a potential vehicle for gene delivery to the brain.

Solanum tuberosum lectin-conjugated PLGA nanoparticles for nose-to-brain delivery: in vivo and in vitro evaluations

Solanum tuberosum lectin (STL) conjugated poly (DL-lactic-co- glycolic acid) (PLGA) nanoparticle (STL-NP) was constructed in this paper as a novel biodegradable nose-to-brain drug delivery system. The in vitro uptake study showed markedly enhanced endocytosis of STL-NP compared to unmodified PLGA nanoparticles (NP) in Calu-3 cells and significant inhibition of uptake in the presence of inhibitor sugar (chitin hydrolysate). Following intranasal administration, coumarin-6 carried by STL-NP was rapidly absorbed into blood and brain. The AUC(0→12 h) of coumarin-6 in blood, olfactory bulb, cerebrum and cerebellum were about 0.77-, 1.48-, 1.89- and 1.45-fold of those of NP, respectively (p < 0.05). STL-NP demonstrated 1.89–2.45 times (p < 0.01) higher brain targeting efficiency in different brain tissues than unmodified NP. Enhanced accumulation of STL-NP in the brain was also observed by near infrared fluorescence probe image following intranasal administration. The fluorescence signal of STL-NP appeared in olfactory bulb, cerebrum and brainstem early at 0.25 h. The signal in olfactory bulb decreased gradually after 2 h, while the obvious signal in brainstem, cerebrum and cerebellum lasted for more than 8 h. The STL-NP safety experiments showed mild cytotoxicity and negligible cilia irritation. These intriguing in vitro and in vivo results suggest that STL-NP might serve as a promising brain drug delivery system.

In vivo toxicity and immunogenicity of wheat germ agglutinin conjugated poly (ethylene glycol)-poly(lactic acid) nanoparticles for intranasal delivery to the brain

Biodegradable polymer-based nanoparticles have been widely studied to deliver therapeutic agents to the brain after intranasal administration. However, knowledge as to the side effects of nanoparticle delivery system to the brain is limited. The aim of this study was to investigate the in vivo toxicity and immunogenicity of wheat germ agglutinin (WGA) conjugated poly(ethylene glycol)-poly(lactic acid) nanoparticles (WGA-NP) after intranasal instillation. Sprague-Dawley rats were intranasally given WGA-NP for 7 continuous days. Amino acid neurotransmitters, lactate dehydrogenase (LDH) activity, reduced glutathione (GSH), acetylcholine, acetylcholinesterase activity, tumor necrosis factor α (TNF-α) and interleukin-8 (IL-8) in rat olfactory bulb (OB) and brain were measured to estimate the in vivo toxicity of WGA-NP. Balb/C mice were intranasally immunized by WGA-NP and then WGA-specific antibodies in serum and nasal wash were detected by indirect ELISA. WGA-NP showed slight toxicity to brain tissue, as evidenced by increased glutamate level in rat brain and enhanced LDH activity in rat OB. No significant changes in acetylcholine level, acetylcholinesterase activity, GSH level, TNF-α level and IL-8 level were observed in rat OB and brain for the WGA-NP group. WGA-specific antibodies in mice serum and nasal wash were not increased after two intranasal immunizations of WGA-NP. These results demonstrate that WGA-NP is a safe carrier system for intranasal delivery of therapeutic agents to the brain.

Enhancement of nose-to-brain delivery of basic fibroblast growth factor for improving rat memory impairments induced by co-injection of β-amyloid and ibotenic acid into the bilateral hippocampus.

Basic fibroblast growth factor (bFGF) delivery to the brain of animals appears to be an emerging potential therapeutic approach to neurodegenerative diseases, such as Alzheimers disease (AD). The intranasal route of administration could provide an alternative to intracerebroventricular infusion. A nasal spray of bFGF had been developed previously and the objective of the present study was to investigate whether bFGF nasal spray could enhance brain uptake of bFGF and ameliorate memory impairment induced by co-injection of β-amyloid(25-35) and ibotenic acid into bilateral hippocampus of rats. The results of brain uptake study showed that the AUC(0-12h) of bFGF nasal spray in olfactory bulb, cerebrum, cerebellum and hippocampus was respectively 2.47, 2.38, 2.56 and 2.19 times that of intravenous bFGF solution, and 1.11, 1.95, 1.40 and 1.93 times that of intranasal bFGF solution, indicating that intranasal administration of bFGF nasal spray was an effective means of delivering bFGF to the brain, especially to cerebrum and hippocampus. In Morris water maze tasks, intravenous administration of bFGF solution at high dose (40 μg/kg) showed little improvement on spatial memory impairment. In contrast, bFGF solution of the same dose following intranasal administration could significantly ameliorate spatial memory impairment. bFGF nasal spray obviously improved spatial memory impairment even at a dose half (20 μg/kg) of bFGF solution, recovered their acetylcholinesterase and choline acetyltransferase activity to the sham control level, and alleviated neuronal degeneration in rat hippocampus, indicating neuroprotective effects on the central nerve system. In a word, bFGF nasal spray may be a new formulation of great potential for treating AD.

The potential use of H102 peptide-loaded dual-functional nanoparticles in the treatment of Alzheimer's disease

Alzheimers disease (AD) is a complex neurodegenerative disease with few effective treatments. The non-targeted distribution of drugs decreases drug efficiency and cause side effects. The cascade targeting strategy has been suggested for precise drug delivery. We developed a dual-functional nanoparticle drug delivery system loaded with β-sheet breaker peptide H102 (TQNP/H102). Two targeting peptides, TGN and QSH, were conjugated to the surface of the nanoparticles for blood-brain barrier transport and Aβ42 targeting, respectively. The prepared nanoparticles were spherical and uniform. The brain distribution study of H102 was conducted with the HPLC-mass spectrometry method to evaluate whether this nano-carrier could achieve increased AD-lesion delivery. The highest uptake of H102 was observed in the hippocampi of the TQNP/H102 group mice 1h after administration, which was 2.62 and 1.86 times the level of non-modified nanoparticles (NP/H102) and TGN modified nanoparticles (TNP/H102), respectively. The neuroprotective effects of H102 preparations were evaluated using Morris water maze experiment, biochemical indexes assay and tissue histology. The spatial learning and memory of the AD model mice in the TQNP/H102 group were significantly improved compared with the AD control group, and were also better than other preparations at the same dosage, even the TNP/H102 group. These results were consistent with the values of biochemical indexes in mouse hippocampi as well as the histological observations. The results demonstrate that TQNP is a promising carrier for peptide or protein drugs, such as H102, for entry into the central nervous system (CNS) and subsequent location of brain AD lesions, thus offering a highly-specific method for AD therapy.

Intranasal H102 Peptide-Loaded Liposomes for Brain Delivery to Treat Alzheimer’s Disease

PurposeH102, a novel β-sheet breaker peptide, was encapsulated into liposomes to reduce its degradation and increase its brain penetration through intranasal administration for the treatment of Alzheimer’s disease (AD).MethodsThe H102 liposomes were prepared using a modified thin film hydration method, and their transport characteristics were tested on Calu-3 cell monolayers. The pharmacokinetics in rats’ blood and brains were also investigated. Behavioral experiments were performed to evaluate the improvements on AD rats’ spatial memory impairment. The neuroprotective effects were tested by detecting acetylcholinesterase (AchE), choline acetyltransferase (ChAT) and insulin degrading enzyme (IDE) activity and conducting histological assays. The safety was evaluated on rats’ nasal mucosa and cilia.ResultsThe liposomes prepared could penetrate Calu-3 cell monolayers consistently. After intranasal administration, H102 could be effectively delivered to the brain, and the AUC of H102 liposomes in the hippocampus was 2.92-fold larger than that of solution group. H102 liposomes could excellently ameliorate spatial memory impairment of AD model rats, increase the activities of ChAT and IDE and inhibit plaque deposition, even in a lower dosage compared with H102 intranasal solution. H102 nasal formulations showed no toxicity on nasal mucosa.ConclusionsThe H102-loaded liposome prepared in this study for nasal administration is stable, effective and safe, which has great potential for AD treatment.

Conjugating influenza a (H1N1) antigen to n‐trimethylaminoethylmethacrylate chitosan nanoparticles improves the immunogenicity of the antigen after nasal administration

As one of the most serious infectious respiratory diseases, influenza A (H1N1) is a great threat to human health, and it has created an urgent demand for effective vaccines. Nasal immunization can induce both systemic and mucosal immune responses against viruses, and it can serve as an ideal route for vaccination. However, the low immunogenicity of antigens on nasal mucosa is a high barrier for the development of nasal vaccines. In this study, we covalently conjugated an influenza A (H1N1) antigen to the surface of N‐trimethylaminoethylmethacrylate chitosan (TMC) nanoparticles (H1N1‐TMC/NP) through thioester bonds to increase the immunogenicity of the antigen after nasal administration. SDS‐PAGE revealed that most of the antigen was conjugated on TMC nanoparticles, and an in vitro biological activity assay confirmed the stability of the antigen after conjugation. After three nasal immunizations, the H1N1‐TMC/NP induced significantly higher levels of serum IgG and mucosal sIgA compared with free antigen. A hemagglutination inhibition assay showed that H1N1‐TMC/NP induced much more protective antibodies than antigen‐encapsulated nanoparticles or alum‐precipitated antigen (I.M.). In the mechanistic study, H1N1‐TMC/NP was shown to stimulate macrophages to produce IL‐1β and IL‐6 and to stimulate spleen lymphocytes to produce IL‐2 and IFN‐γ. These results indicated that H1N1‐TMC/NP may be an effective vaccine against influenza A (H1N1) viruses for use in nasal immunization. J. Med. Virol. 87:1807–1815, 2015.

Preparation and evaluation of antigen/N-trimethylaminoethylmethacrylate chitosan conjugates for nasal immunization.

The frequent outbreak of respiratory infectious diseases such as influenza and pulmonary tuberculosis calls for new immunization strategies with high effectiveness. Nasal immunization is one of the most potential methods to prevent the diseases infected through the respiratory tract. In this study, we designed a water-soluble system based on antigen/N-trimethylaminoethylmethacrylate chitosan conjugates for nasal immunization. N-trimethylaminoethylmethacrylate chitosan (TMC) was synthesized by free radical polymerization of chitosan and N-trimethylaminoethylmethacrylate chloride and identified by (1)H NMR and FT-IR. Thiolated ovalbumin (OVA) was covalently conjugated to maleimide modified TMC with high conjugation efficiency. OVA conjugated TMC (OVA-TMC) significantly increased uptake of OVA by Raw 264.7 cells, which was 2.38 times higher than that of OVA/TMC physical mixture (OVA+TMC) at 4h. After nasal administration, OVA-TMC showed higher transport efficiency to superficial and deep cervical lymph nodes than OVA+TMC or OVA alone. Balb/C mice were intranasally given with OVA-TMC three times at 2-week internals to evaluate the immunological effect. The serum IgG, IgG1 and IgG2a levels of the OVA-TMC group were 17.9-87.9 times higher than that of the OVA+TMC group and comparable to that of the intramuscular group. The secretory IgA levels in nasal wash and saliva of the OVA-TMC group were 5.2-7.1 times higher than that of the OVA+TMC group while the secretory IgA levels of the intramuscular alum-precipitated OVA group were not increased. After immunofluorescence staining of nasal cavity, IgA antibody secreting cells were mainly observed in the lamina propria regions and glands of nasal mucosa. OVA-TMC showed little toxicity to the nasal epithelia or cilia of rats after nasal administration for three consecutive days. These results demonstrated that antigen conjugated TMC can induce both systemic and mucosal immune responses after nasal administration and may serve as a convenient, safe and effective vaccine for preventing respiratory infectious diseases.

Concanavalin A-conjugated poly(ethylene glycol)–poly(lactic acid) nanoparticles for intranasal drug delivery to the cervical lymph nodes

Abstract Concanavalin A (ConA)-conjugated poly(ethylene glycol)–poly(lactic acid) nanoparticles (ConA-NPs) were prepared for targeted drug delivery to the cervical lymph nodes after intranasal administration. ConA, a lectin specifically binding to α-mannose and α-glucose, was covalently conjugated on NPs without loss of its carbohydrates binding bioactivity. In vitro cellular uptake experiment demonstrated that NPs could be uptaken by Calu-3 cells in a time- and concentration-dependent manner, and conjugation of ConA on NPs could significantly increase the rate and amount of cellular uptake. ConA-NP showed no obvious toxicity to Calu-3 cells in vitro or to the nasal cilia of rats in vivo. Compared with NPs without ConA, ConA-NP is more effective in targeting drugs to the deep cervical lymph nodes, as evidenced by 1.36–2.52 times increase of targeting efficiency, demonstrating that ConA-NP is a potential carrier for targeted drug delivery to the cervical lymph nodes via nasal route.