Reversible Assembly of Proteins and Phenolic Polymers for Intracellular Protein Delivery with Serum Stability.

The design of intracellular delivery systems for protein drugs remains a challenge due to limited delivery efficacy and serum stability. Herein, we propose a reversible assembly strategy to assemble cargo proteins and phenolic polymers into stable nanoparticles for this purpose using a heterobifunctional adaptor (2-formylbenzeneboronic acid). The adaptor is easily decorated on cargo proteins via iminoboronate chemistry and further conjugates with catechol-bearing polymers to form nanoparticles via boronate diester linkages. The nanoparticles exhibit excellent serum stability in culture media but rapidly release the cargo proteins triggered by lysosomal acidity and GSH after endocytosis. In a proof-of-concept animal model, the strategy successfully transports superoxide dismutase to retina via intravitreal injection and efficiently ameliorates the oxidative stress and cellular damage in the retina induced by ischemia-reperfusion (I/R) with minimal adverse effects. The reversible assembly strategy represents a robust and efficient method to develop serum-stable systems for the intracellular delivery of biomacromolecules.

Fluorination Promotes the Cytosolic Delivery of Genes, Proteins, and Peptides.

The cytosolic delivery of biomolecules such as genes, proteins, and peptides is of great importance for biotherapy but usually limited by multiple barriers during the process. Cell membrane with high hydrophobic character is one of the representative biological barriers for cytosolic delivery. The introduction of hydrophobic ligands such as aliphatic lipids onto materials or biomolecules could improve their membrane permeability. However, these ligands are lipophilic and tend to interact with the phospholipids in the membrane as well as serum proteins, which may hinder efficient intracellular delivery. To solve this issue, our research group proposed the use of fluorous ligands with both hydrophobicity and lipophobicity as ideal alternatives to aliphatic lipids to promote cytosolic delivery.In our first attempt, fluorous ligands were conjugated onto cationic polymers to increase their gene delivery efficacy. The fluorination dramatically increased the gene delivery performance at low polymer doses. In addition, the strategy greatly improved the serum tolerance of cationic polymers, which is critical for efficient gene delivery in vivo. Besides serum tolerance, mechanism studies revealed that fluorination increases multiple steps such as cellular uptake and endosomal escape. Fluorination also allowed the assembly of low-molecular-weight polymers and achieved highly efficient gene delivery with minimal material toxicity. The method showed robust efficiency for polymers, including linear polymers, branched polymers, dendrimers, bola amphiphilies, and dendronized polymers.Besides gene delivery, fluorinated polymers were also used for intracellular protein delivery via a coassembly strategy. For this purpose, two lead fluoropolymers were screened from a library of amphiphilic materials. The fluoropolymers are greatly superior to their nonfluorinated analogues conjugated with aliphatic lipids. The fluorous lipids are beneficial for polymer assembly and protein encapsulation, reduced protein denaturation, facilitated endocytosis, and decreased polymer toxicity compared to nonfluorinated lipids. The materials exhibited potent efficacy in therapeutic protein and peptide delivery to achieve cancer therapy and were able to fabricate a personalized nanovaccine for cancer immunotherapy. Finally, the fluorous lipids were directly conjugated to peptides via a disulfide bond for cytosolic peptide delivery. Fluorous lipids drive the assembly of cargo peptides into uniform nanoparticles with much improved proteolytic stability and promote their delivery into various types of cells. The delivery efficacy of this strategy is greatly superior to traditional techniques such as cell-penetrating peptides both in vitro and in vivo. Overall, the fluorination techniques provide efficient and promising strategies for the cytosolic delivery of biomolecules.

Polycatechols with Robust Efficiency in Cytosolic Peptide Delivery via Catechol-Boronate Chemistry.

Cytosolic delivery of peptides remains a challenging task because of the limited binding sites on peptides and the existence of multiple intracellular barriers. Here, we proposed the use of polycatechols with a high cell permeability to deliver peptides of different physicochemical properties using the catechol-boronate chemistry. Peptides were decorated with boronate moieties via three strategies, and the introduced boronate groups greatly increased the binding affinity of cargo peptides with polycatechols. The loading peptides could be released under the endolysosomal acidity. When the cargo peptide was modified with boronate moiety via a p-hydroxybenzylcarbamate self-immolative spacer, it could be loaded by polycatechols and released in a traceless manner triggered by reactive oxygen species. The proposed strategies greatly promote the cytosolic delivery efficiency of different peptides into various cell lines and restored their biofunctions after intracellular delivery and release. This study provides a general and robust platform for the intracellular delivery of membrane-impermeable peptides.

Fluorous-Tagged Peptide Nanoparticles Ameliorate Acute Lung Injury via Lysosomal Stabilization and Inflammation Inhibition in Pulmonary Macrophages.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a common respiratory critical syndrome that currently has no effective therapeutic interventions. Pulmonary macrophages play a principal role in the initiation and progression of the overwhelming inflammation in ALI/ARDS. Here, a type of fluorous-tagged bioactive peptide nanoparticle termed CFF13F is developed, which can be efficiently internalized by macrophages and suppress the excessive expression of cytokines and the overproduction of reactive oxygen species (ROS) triggered by lipopolysaccharide (LPS). The cytoprotective effect of CFF13F may be attributed to the lysosomal-stabilization property and regulation of the antioxidative system. Moreover, intratracheal pretreatment with CFF13F can effectively reduce local and systematic inflammation, and ameliorate pulmonary damage in an LPS-induced ALI murine model. The therapeutic efficacy of CFF13F is affected by the administration routes, and the local intratracheal injection is found to be the optimal choice for ALI treatment, with preferred biodistribution profiles. The present study provides solid evidence of the potent immunomodulatory bioactivity of the fluorous-tagged peptide nanoparticles CFF13F in vitro and in vivo, and sheds light on the development of novel efficient nanodrugs for ALI/ARDS.

Dendrigraft poly-L-lysines delivery of DNA vaccine effectively enhances the immunogenic responses against H9N2 avian influenza virus infection in chickens.

Biodegradable nanomaterials can protect antigens from degradation, promote cellular absorption, and enhance immune responses. We constructed a eukaryotic plasmid [pCAGGS-opti441-hemagglutinin (HA)] by inserting the optimized HA gene fragment of H9N2 AIV into the pCAGGS vector. The pCAGGS-opti441-HA/DGL was developed through packaging the pCAGGS-opti441-HA with dendrigraft poly-l-lysines (DGLs). DGL not only protected the pCAGGS-opti441-HA from degradation, but also exhibited high transfection efficiency. Strong cellular immune responses were induced in chickens immunized with the pCAGGS-opti441-HA/DGL. The levels of IFN-γ and IL-2, and lymphocyte transformation rate of the vaccinated chickens increased at the third week post the immunization. For the vaccinated chickens, T lymphocytes were activated and proliferated, the numbers of CD3+CD4+ and CD4+/CD8+ increased, and the chickens were protected completely against H9N2 AIV challenge. This study provides a method for the development of novel AIV vaccines, and a theoretical basis for the development of safe and efficient gene delivery carriers.

A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice.

H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs. IMPORTANCE: Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance due to their potential threat to mammals, including humans.

Protective efficacy of an inactivated vaccine against H9N2 avian influenza virus in ducks.

BACKGROUND: Wild ducks play an important role in the evolution of avian influenza viruses (AIVs). Domestic ducks in China are known to carry and spread H9N2 AIVs that are thought to have contributed internal genes for the recent outbreak of zoonotic H7N9 virus. In order to protect animal and public health, an effective vaccine is urgently needed to block and prevent the spread of H9N2 virus in ducks. We developed an inactivated H9N2 vaccine (with adjuvant Montanide ISA 70VG) based on an endemic H9N2 AIV and evaluated this vaccine in ducks. FINDINGS: The results showed that the inactivated H9N2 vaccine was able to induce a strong and fast humoral immune response in vaccinated ducks. The hemagglutination inhibition titer in the sera increased fast, and reached its peak of 12.3 log2 at 5 weeks post-vaccination in immunized birds and remained at a high level for at least 37 weeks post-vaccination. Moreover, viral shedding was completely blocked in vaccinated ducks after challenge with a homologous H9N2 AIV at both 3 and 37 weeks post-vaccination. CONCLUSIONS: The results of this study indicate that the inactivated H9N2 vaccine induces high and prolonged immune response in vaccinated ducks and are efficacious in protecting ducks from H9N2 infection.

Subcellular targeting strategies for protein and peptide delivery.

Cytosolic delivery of proteins and peptides provides opportunities for effective disease treatment, as they can specifically modulate intracellular processes. However, most of protein-based therapeutics only have extracellular targets and are cell-membrane impermeable due to relatively large size and hydrophilicity. The use of organelle-targeting strategy offers great potential to overcome extracellular and cell membrane barriers, and enables localization of protein and peptide therapeutics in the organelles. Although progresses have been made in the recent years, organelle-targeted protein and peptide delivery is still challenging and under exploration. We reviewed recent advances in subcellular targeted delivery of proteins/peptides with a focus on targeting mechanisms and strategies, and highlight recent examples of active and passive organelle-specific protein and peptide delivery systems. This emerging platform could open a new avenue to develop more effective protein and peptide therapeutics.

Membrane Fusion-Mediated Loading of Therapeutic siRNA into Exosome for Tissue-Specific Application.

Tissue-specific delivery of oligonucleotide therapeutics beyond the liver remains a key challenge in nucleic acid drug development. To address this issue, exploiting exosomes as a novel carrier has emerged as a promising approach for efficient nucleic acid drug delivery. However, current exosome-based delivery systems still face multiple hurdles in their clinical applications. Herein, this work presents a strategy for constructing a hybrid exosome vehicle (HEV) through a DNA zipper-mediated membrane fusion approach for tissue-specific siRNA delivery. As a proof-of-concept, this work successfully fuses a liposome encapsulating anti-NFKBIZ siRNAs with corneal epithelium cell (CEC)-derived exosomes to form a HEV construct for the treatment of dry eye disease (DED). With homing characteristics inherited from exosomes, the siRNA-bearing HEV can target its parent cells and efficiently deliver the siRNA payloads to the cornea. Subsequently, the NFKBIZ gene silencing significantly reduces pro-inflammatory cytokine secretions from the ocular surface, reshapes its inflammatory microenvironment, and ultimately achieves an excellent therapeutic outcome in a DED mouse model. As a versatile platform, this hybrid exosome with targeting capability and designed therapeutic siRNAs may hold great potential in various disease treatments.

Fluorous Tagged Peptides for Intracellular Delivery and Biomedical Imaging.

Fluorous tagged peptides have shown promising features for biomedical applications such as drug delivery and multimodal imaging. The bioconjugation of fluoroalkyl ligands onto cargo peptides greatly enhances their proteolytic stability and membrane penetration via a proposed "fluorine effect". The tagged peptides also efficiently deliver other biomolecules such as DNA and siRNA into cells via a co-assembly strategy. The fluoroalkyl chains on peptides with antifouling properties enable efficient gene delivery in the presence of serum proteins. Besides intracellular biomolecule delivery, the amphiphilic peptides can be used to stabilized perfluorocarbon-filled microbubbles for ultrasound imaging. The fluorine nucleus on fluoroalkyls provides intrinsic probes for background-free magnetic resonance imaging. Labeling of fluorous tags with radionuclide 18 F also allows tracing the biodistribution of peptides via positron emission tomography imaging. This mini-review will discuss properties and mechanism of the fluorous tagged peptides in these applications.

Dual-responsive bioconjugates bearing a bifunctional adaptor for robust cytosolic peptide delivery.

Peptide drugs have been successfully used for the treatment of various diseases. However, it is still challenging to develop therapeutic peptides working on intracellular targets due to their poor membrane permeability. Here, we proposed a type of dual-responsive bioconjugates bearing a heterobifunctional adaptor containing both aldehyde and catechol moieties for efficient cytosolic peptide delivery. Hydrazine-terminated cargo peptides were tagged to a boronated dendrimer with the help of the adaptor via dynamic acylhydrazone and catechol­boronate linkages. The bioconjugates efficiently delivered peptides with distinct physicochemical properties into various cells, and could release the cargo peptides triggered by intracellular reactive oxygen species and endolysosomal acidity, restoring the biofunctions of delivered peptides. In addition, the designed complexes efficiently delivered a pro-apoptotic peptide into osteosarcoma cancer cells and successfully inhibited the tumor growth both in vitro and in vivo. This study provides a universal and efficient platform for cytosolic therapeutic peptide delivery to intracellular targets for treating various diseases.

Efficient intracellular and in vivo delivery of toxin proteins by a ROS-responsive polymer for cancer therapy.

Rational design of efficient cytosolic protein delivery carriers holds enormous promise for biotherapeutics development. Several delivery systems have been developed during the past decades, while tailoring the balance between extracellular protein binding and intracellular cargo release is still challenging. In this study, we synthesized a series of oxygen-sensitive reactive polymers, rich in boron, by radical polymerization and post-modification for cytosolic protein delivery in vitro and in vivo. The introduction of boronate building blocks into the polymer scaffold significantly enhanced its protein binding affinity, and the polymer/protein complexes with high stability were obtained by tailoring the molecular ratios between the boronate ligands and the amine groups. The lead material screened from the polymer library exhibited efficient protein delivery efficacy that can release cargo proteins in cytosol in a reactive oxygen species responsive manner, which enables intracellular delivery of proteins with maintained bioactivity. In addition, the polymer-based nanoformulations efficiently delivered saporin, a toxin protein, into osteosarcoma cells and tumor tissues, and exhibited high therapeutic efficacy in an osteosarcoma mouse model. The synthesized polymer in this study can be developed as a promising nanocarrier for cytosolic delivery of protein therapeutics to treat a variety of diseases.

Benzaldehyde-tethered fluorous tags for cytosolic delivery of bioactive peptides.

Development of intracellular delivery systems for bioactive peptides remains challenging. Herein, we report a facile strategy to address this issue by conjugating peptides with benzaldehyde-tethered fluorous tags to generate dynamic peptide amphiphiles via a hydrazone bond for efficient cytosolic delivery. Those dynamic peptide fluoroamphiphiles could self-assemble into nanoparticles that readily cross the cell membrane. Using this strategy, several bioactive peptides were efficiently internalized by cancer cells and released into the cytosol to exert their biological functions, which showed much higher efficacies than non-fluorous lipids and cell penetrating peptide decorated peptides. Moreover, the fluorous tagged proapoptotic peptide was able to efficiently inhibit tumor growth in vivo. This report provides a new family of fluorous tags based on benzaldehyde for efficient cytosolic peptide delivery.

Fluoroalkylation promotes cytosolic peptide delivery.

Cytosolic delivery of peptides remains a challenging task owing to their susceptibility to enzymatic degradation and the existence of multiple intracellular barriers. Here, we report a new strategy to address these issues by decoration of a fluorous tag on the terminal of cargo peptides. The fluorous-tagged peptides were assembled into nanostructures, efficiently internalized by cells via several endocytic pathways and released into the cytosol after endosomal escape. They were relatively stable against enzymatic degradation and showed much higher efficiency than nonfluorinated analogs and cell penetrant peptide-conjugated ones. The proposed strategy also efficiently delivered a proapoptotic peptide into specific sites in the cells and restored the function of cargo peptide after cytosolic delivery. The fluorous-tagged proapoptotic peptide efficiently inhibited tumor growth in vivo. This study provides an efficient fluorination strategy to promote the cytosolic delivery of peptides.

In Vivo Tracking of Fluorinated Polypeptide Gene Carriers by Positron Emission Tomography Imaging.

Fluorinated polymers have attracted increasing attention in gene delivery and cytosolic protein delivery in recent years. In vivo tracking of fluorinated polymers will be of great importance to evaluate their biodistribution, clearance, and safety. However, tracking of polymeric carriers without changing their chemical structures remains a huge challenge. Herein, we reported a series of fluorinated poly-l-(lysine) (F-PLL) with high gene transfection efficiency and excellent biodegradation. Radionuclide 18F was radiolabeled on F-PLL by halogen replacement without chemical modification. The radiolabeling of F-PLL offers positron emission tomography (PET) imaging for in vivo tracking of the polymers. The biodistribution of F-PLL and the DNA complexes revealed by micro-PET imaging illustrated the rapid clearance of fluorinated polymers from liver and intestine after intravenous administration. The results demonstrated that the polymer F-PLL will not be accumulated in the liver and spleen when administrated as a gene carrier. This work presents a new strategy for in vivo tracking fluorinated polymers via PET imaging.

Advances and Potential Applications of Chitosan Nanoparticles as a Delivery Carrier for the Mucosal Immunity of Vaccine.

BACKGROUND: Drug research and development has entered into the new epoch of innovation formulation, and the drug delivery system has been in the forefront of pharmaceutical innovation. Chitosan, a natural polysaccharide derived from chitin, due to its well-known biocompatibility and biodegradability, it has been widely used in drug delivery, immunostimulation, tissue regeneration, blood coagulation, wound healing, drug delivery and tissue engineering. Chitosan has become a valuable vaccine adjuvant and delivery carrier, which have attracted increasing attention for its applications. In this paper, we reviewed chitosan nanoparticles, which is a promising biomaterial as vaccine adjuvant and delivery carrier, including characteristics, preparation methods and applications, or even its limitations. We also investigated the mucosal immune delivery route for drug loaded chitosan nanoparticles, such as the routes of oral and nasal. Due to the low toxicity, better biodegradability and adhesivity of chitosan nanoparticles, it can be used as the delivery carrier of vaccine antigens and drugs. These promising studies laid a foundation for the applications of chitosan nanoparticles as a delivery carrier in the vaccine or drug. METHODS: We undertook a structured research of biodegradable polymeric nanoparticles of chitosan used as a delivery carrier for the mucosal immunity of vaccine. We have searched the bibliographic databases for peer-reviewed research literature. The outstanding characteristics of the screened papers were described respectively, and a systematic content analysis methodology was used to analyse the findings. RESULTS: Sixty-three papers were included in the review, the majority defined leadership and governance approaches that had impacted upon the polymeric nanoparticles as the delivery carrier for the mucosal immunity of vaccine in therapeutic applications and developments. Thirty-five papers outlined the superiority characteristics of chitosan nanoparticles that applied in the field of vaccine. Twenty-eight papers overviewed the application prospects of chitosan derivatives used as drug delivery systerm. These included current advances in research and clinical applications of chitosan derivatives. This review identified the drug delivery systerm of chitosan or its derivatives, and we described the synthesis methods, applications and challenges of chitosan. CONCLUSION: The findings of this review identified that the chitosan derivatives were used as delivery carrier for vaccines. It also indicates that the chitosan or its derivatives play a vital role in the drug and vaccine delivery systerm.

Biological evaluation of N-2-hydroxypropyl trimethyl ammonium chloride chitosan as a carrier for the delivery of live Newcastle disease vaccine.

Mucosal immune system plays a very important role in antiviral immune response. We prepared Newcastle disease viruses (NDV) encapsulated in N-2-hydroxypropyl trimethyl ammonium chloride chitosan (N-2-HACC) nanoparticles (NDV/La Sota-N-2-HACC-NPs) by an ionic cross linking method, and assessed the potential of N-2-HACC-NPs as a mucosal immune delivery carrier. The properties of the nanoparticles were determined by transmission electron microscopy, Zeta potential and particle size analysis, encapsulation efficiency and loading capacity. NDV/La Sota-N-2-HACC-NPs have regular spherical morphologies and high stability; with 303.88±49.8nm mean diameter, 45.77±0.75mV Zeta potential, 94.26±0.42% encapsulation efficiency and 54.06±0.21% loading capacity. In vitro release assay indicated that the release of NDV from NDV/La Sota-N-2-HACC-NPs is slow. The NDV/La Sota-N-2-HACC-NPs have good biological characteristics, very low toxicity and high level of safety. Additionally, specific pathogen-free chickens immunized with NDV/La Sota-N-2-HACC-NPs showed much stronger cellular, humoral and mucosal immune responses than commercial attenuated live Newcastle disease vaccine, and NDV/La Sota-N-2-HACC-NPs reached the sustainable release effect. Our study here provides a foundation for the further development of mucosal vaccines and drugs, and the N-2-HACC-NPs should be a potential drug delivery carrier with immense potential in medical applications.

IgA response and protection following nasal vaccination of chickens with Newcastle disease virus DNA vaccine nanoencapsulated with Ag@SiO2 hollow nanoparticles.

Newcastle disease caused by ND virus (NDV) is a highly contagious disease of birds. Vaccine for effective protection of poultry animals from NDV infection is urgently needed. Mucosal immunity plays a very important role in the antiviral immune response. In this study, a NDV F gene-containing DNA vaccine encapsulated in Ag@SiO2 hollow nanoparticles (pFDNA-Ag@SiO2-NPs) with an average diameter of 500 nm were prepared to assess the mucosal immune response. These nanoparticles exhibited low cytotoxicity and did not destroy the bioactivity of plasmid DNA, which could be expressed in vitro. The plasmid DNA was sustainably released after an initial burst release. In vivo immunization showed that the intranasal immunization of chickens with pFDNA-Ag@SiO2-NPs induced high titers of serum antibody, significantly promoted lymphocyte proliferation and induced higher expression levels of IL-2 and IFN-γ in a dose-dependent manner. These results indicated that the Ag@SiO2 hollow nanoparticles could serve as an efficient and safe delivery carrier for NDV DNA vaccine to induce mucosal immunity. This study has provided promising results for the further development of mucosal vaccines encapsulated in inorganic nanoparticles.

Development of blocking ELISA for detection of antibodies against H9N2 avian influenza viruses.

A blocking enzyme-linked immunosorbent assay (bELISA) was developed for detection of antibodies against H9N2 avian influenza viruses (AIVs) based on a monoclonal antibody specific to the hemagglutinin (HA) protein of H9N2 AIV. The specificity of the bELISA was tested using antisera against H3, H4, H5, H7, and H10 AIVs and other avian viruses. The average percent inhibition (PI) value of 116 non-immune serum samples from ducks and specific pathogen-free (SPF) chickens was 2.80%. The selected cut-off PI values for negative and positive sera were 17.6% and 25.0%, respectively. A high correlation of >96% was identified between the bELISA and hemagglutinin inhibition (HI) assay, according to the detection results of sera from infected chickens (n=30) and from chickens vaccinated with an inactivated H9N2 vaccine (n=40). Sera collected from vaccinated chickens and ducks (n=660) at different weeks post vaccination that were positive by the HI assay were confirmed with the bELISA. The results revealed a time-dependent increase in antibody levels. Therefore, the bELISA offers the potential advantage of a high throughput, rapid, sensitive, and specific method for detection of specific antibodies against H9N2.

Synthesis, characterization, and immune efficacy of layered double hydroxide@SiO2 nanoparticles with shell-core structure as a delivery carrier for Newcastle disease virus DNA vaccine.

Layered double hydroxide (LDH)@SiO2 nanoparticles were developed as a delivery carrier for the plasmid DNA expressing the Newcastle disease virus F gene. The LDH was hydrotalcite-like materials. The plasmid DNA encapsulated in the LDH@SiO2 nanoparticles (pFDNA-LDH@SiO2-NPs) was prepared by the coprecipitation method, and the properties of pFDNA-LDH@SiO2-NPs were characterized by transmission electron microscopy, zeta potential analyzer, Fourier transform infrared spectroscopy, and X-ray diffraction analysis. The results demonstrated that the pFDNA-LDH@SiO2-NPs had a regular morphology and high stability with a mean diameter of 371.93 nm, loading capacity of 39.66%±0.45%, and a zeta potential of +31.63 mV. A release assay in vitro showed that up to 91.36% of the total plasmid DNA could be sustainably released from the pFDNA-LDH@SiO2-NPs within 288 hours. The LDH@SiO2 nanoparticles had very low toxicity. Additionally, their high transfection efficiency in vitro was detected by fluorescent microscopy. Intranasal immunization of specific pathogen-free chickens with pFDNA-LDH@SiO2-NPs induced stronger cellular, humoral, and mucosal immune responses and achieved a greater sustained release effect than intramuscular naked plasmid DNA, and the protective efficacy after challenge with the strain F48E9 with highly virulent (mean death time of chicken embryos ≤60 hours, intracerebral pathogenicity index in 1 -day-old chickens >1.6) was 100%. Based on the results, LDH@SiO2 nanoparticles can be used as a delivery carrier for mucosal immunity of Newcastle disease DNA vaccine, and have great application potential in the future.