Tumor microenvironment-responsive spherical nucleic acid nanoparticles for enhanced chemo-immunotherapy.

Certain chemotherapeutics can induce tumor cells' immunogenic cell death (ICD), release tumor antigens, and thereby trigger personalized antitumor immune responses. Co-delivery of adjuvants using nanocarriers could amplify the ICD-induced tumor-specific immunity achieving a synergistic chemo-immunotherapeutic effect. However, complicated preparation, low drug loading efficiency, and potential carrier-associated toxicity are the major challenges that limited its clinical applications. Herein, a carrier-free core-shell nanoparticle (MPLA-CpG-sMMP9-DOX, MCMD NPs) was constructed by facile self-assembly of spherical nucleic acids (SNA) with two adjuvants of CpG ODN and monophosphoryl lipid A (MPLA) as a core and doxorubicin (DOX) radially around the dual-adjuvants SNA as a shell. The results demonstrated that MCMD NPs could enhance drugs accumulation in tumors, and release DOX upon enzymatic degradation of matrix metalloproteinase-9 (MMP-9) peptide in the tumor microenvironment (TME), which enhanced the direct-killing effect of DOX on tumor cells. The core of MPLA-CpG SNA efficiently boosted the ICD-induced antitumor immune response to further attack tumor cells. Thus, MCMD NPs achieved a synergistic therapeutic effect of chemo-immunotherapy with reduced off-target toxicity. This study provided an efficient strategy for the development of a carrier-free nano-delivery system for enhanced cancer chemo-immunotherapy.

Urine 2-hydroxyphenanthrene is associated with current asthma: evidence from NHANES 2007-2012.

OBJECTIVE: The current study aims to explore the effects of nine urine monohydroxy PAH metabolites (OHPAH) including 1-hydroxynaphthalene (1-OHNAP), 2-hydroxynaphthalene (2-OHNAP), 3-hydroxyfluorene (3-OHFLU), 9-hydroxyfluorene (9-OHFLU), 1-hydroxyphenanthrene (1-OHPHE), 2-hydroxyphenanthrene (2-OHPHE), 3-hydroxyphenanthrene (3-OHPHE), and 1-hydroxypyrene (1-OHPYR) on current asthma in people in the United States using a variety of statistical techniques. METHODS: A cross-sectional examination of a subsample of 3804 adults aged ≥20 from the National Health and Nutrition Examination Survey (NHANES) was conducted between 2007 and 2012. To investigate the relationship between urine OHPAHs levels and current asthma, multivariate logistic regression, Bayesian kernel machine regression (BKMR), and quantile g-computation (qgcomp) were utilized. RESULTS: In the multivariate logistic regression model, after controlling for confounders, urine 2-OHPHE was associated with current asthma in both male (AOR = 7.17, 95% CI: 1.28-40.08) and female (AOR = 2.91, 95% CI: 1.06-8.01) smokers. In the qgcomp analysis, 2-OHPHE (39.5%), 1-OHNAP (33.1%), and 2-OHNAP (22.5%) were the major positive contributors to the risk of current asthma (OR = 2.29, 95% CI: 0.99, 5.25), and in female smokers, 9-OHFLU (25.8%), 2-OHFLU (21.5%), and 2-OHPHE (15.1%) were the major positive contributors (OR = 2.19, 95% CI: 1.06, 4.47). The results of the BKMR model basically agreed with qgcomp analysis. CONCLUSION: Our results demonstrate a strong association of urine 2-OHPHE with current asthma, and further longitudinal studies are needed to understand the precise relationship between PAH exposure and current asthma risk.

Maximizing TLR9 Activation in Cancer Immunotherapy with Dual-Adjuvanted Spherical Nucleic Acids.

Nucleic-acid-based immune adjuvants have been extensively investigated for the design of cancer vaccines. However, nucleic acids often require the assistance of a carrier system to improve cellular uptake. Yet, such systems are prone to carrier-associated adaptive immunity, leading to difficulties in a multidose treatment regimen. Here, we demonstrate that a spherical nucleic acid (SNA)-based self-adjuvanting system consisting of phosphodiester oligonucleotides and vitamin E can function as a potent anticancer vaccine without a carrier. The two functional modules work synergistically, serving as each other's delivery vector to enhance toll-like receptor 9 activation. The vaccine rapidly enters cells carrying OVA model antigens, which enables efficient activation of adaptive immunity in vitro and in vivo. In OVA-expressing tumor allograft models, both prophylactic and therapeutic vaccinations significantly retard tumor growth and prolong animal survival. Furthermore, the vaccinations were also able to reduce lung metastasis in a B16F10-OVA model.

Doxorubicin and CpG loaded liposomal spherical nucleic acid for enhanced Cancer treatment.

Chemotherapeutics that can trigger immunogenic cell death (ICD) and release tumor-specific antigens are effective on treating a variety of cancers. The codelivery of chemotherapeutics with adjuvants is a promising strategy to achieve synergistic therapeutic effect. However, low drug loading and complicated preparation of current delivery systems lead to carrier-associated toxicity and immunogenicity. Herein, we developed a facile approach to construct liposomal spherical nucleic acids (SNA) by the self-assembly of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)-doxorubicin conjugate and DOPE-matrix metalloproteinases-9 (MMP-9) responsive peptide-CpG conjugate (DOPE-MMP-CpG). Liposomal SNAs efficiently co-delivered DOX and CpG into tumors and released the two drugs upon biological stimuli of MMP-9 enzyme in tumor microenvironment (TME) and high concentration of endogenous glutathione in tumor cells. We demonstrated that liposomal SNA enhanced activation of dendritic cells (DCs), promoted expansion of CD8+ and CD4+ T cells in both tumors and spleen, inhibited tumor growth, and extended animal survival. This work provided a simple strategy of delivering chemotherapeutics and adjuvants to tumors with synergistic therapeutic effect and reduced side effect.

Enhanced Antitumor Immune Responses via a Self-Assembled Carrier-Free Nanovaccine.

Nanovaccines have emerged as promising agents for cancer immunotherapy. However, insufficient antitumor immunity caused by inefficient antigen/adjuvant loading and complicated preparation processes are the major obstacles that limit their clinical application. Herein, two adjuvants, monophosphatidyl A (MPLA) and CpG ODN, with antigens were designed into a nanovaccine to overcome the above obstacles. This nanovaccine was constructed with adjuvants (without additional materials) through facile self-assembly, which not only ensured a high loading efficacy and desirable safety but also facilitated clinical translation for convenient fabrication. More importantly, the selected adjuvants could achieve a notable immune response through synergistic activation of Toll-like receptor 4 (TLR4) and TLR9 signaling pathways, and the resulting nanovaccine remarkably inhibited the tumor growth and prolonged the survival of tumor-implanted mice. This nanovaccine system provides an effective strategy to construct vaccines for cancer immunotherapy.

Revitalizing liver function in mice with liver failure through transplantation of 3D-bioprinted liver with expanded primary hepatocytes.

The utilization of three-dimensional (3D) bioprinting technology to create a transplantable bioartificial liver emerges as a promising remedy for the scarcity of liver donors. This study outlines our strategy for constructing a 3D-bioprinted liver, using in vitro-expanded primary hepatocytes recognized for their safety and enhanced functional robustness as hepatic cell sources for bioartificial liver construction. In addition, we have developed bioink biomaterials with mechanical and rheological properties, as well as printing capabilities, tailored for 3D bioprinting. Upon heterotopic transplantation into the mesentery of tyrosinemia or 90% hepatectomy mice, our 3D-bioprinted liver effectively restored lost liver functions, consequently extending the life span of mice afflicted with liver injuries. Notably, the inclusion of an artificial blood vessel in our 3D-bioprinted liver allowed for biomolecule exchange with host blood vessels, demonstrating, in principle, the rapid integration of the bioartificial liver into the host vascular system. This model underscores the therapeutic potential of transplantation for the treatment of liver failure diseases.

[Analytical method and comparison for static and dynamic headspace gas chromatography of anisole in water].

OBJECTIVE: To establish and compare the method of static headspace gas chromatography hydrogen flame detector (static headspace method) and purge and trap gas chromatography-mass spectrometry (dynamic headspace method) of anisole in water. METHODS: Nitrogen gas was used as carrier gas in the static headspace method, 5 g NaCl as matrix modifier was added into 10 ml water. The sample was balanced with high speed vibration at 75°C for 30 min, and anisole was detected by gas chromatography and quantified with external standard. Helium was used as carrier gas in dynamic headspace method, 5.0 ml water and 0.004 mg/L internal standard fluorobenzene was purged into the purge and trap apparatus. After purging, trapping and desorption, anisole was detected by the gas chromatography-mass spectrograph, confirmed by the retention time and comparison of mass-spectrogram in spectrum library and quantified with internal standard. The repeatability and sensitivity of assay were evaluated. RESULTS: A good linear range for anisole was observed in static headspace gas chromatography and dynamic headspace gas chromatography-mass spectrometry, within the range of 10 - 500 µg/L and 0.5 - 60.0 µg/L respectively. The linear regression equation was Y = 782.150X + 1.3446 and Y = 0.0358X - 0.0209 respectively, both the correlation coefficient ≥ 0.999. The detection limit (LOD) were 0.002 µg/L and 0.110 µg/L, the lower limit of quantitation (LOQ) were 0.006 µg/L and 0.350 µg/L, the relative standard deviation (RSD) were 1.8% - 2.3% and 2.0% - 3.4%, and the spiking recovery were 93% - 101% and 96% - 101% respectively. CONCLUSION: The methods of static headspace gas chromatography and dynamic headspace gas chromatography-mass spectrometry are simple and can measure anisole in water quickly, sensitively and accurately.

[Ultrastructure of hepatocytes in Gilbert's syndrome patients and chronic hepatitis B patients].

OBJECTIVE: To explore the pathological characteristics of inborn hyperbilirubinemia of patients with Gilbert's syndrome (GS). METHODS: Patients with GS (n = 7) and patients with chronic hepatitis B (CHB; n = 8) were enrolled in the study. GS was diagnosed by peripheral blood analysis results showing glucuronyl transferase gene mutation. The histology and ultrastructure of biopsied liver tissues were evaluated by light microscopy and transmission electron microscopy. RESULTS: The GS group showed normal structure in the hepatic portal area and lobule; however, bile pigment granules with high electron density were noted in the hepatocytes. The CHB group showed abnormal structure of the hepatic lobules, including infiltration of inflammatory cells, necrotic regions, degenerated hepatocytes, bile duct injury, and fibrosis in the portal tracts; a few bile pigment granules were observed. The GS group also showed greater quantity and size of bilirubin deposits than the CHB group. CONCLUSION: The histological and ultrastructural features of GS include normal hepatic lobule and deposition of bile pigment granules in hepatocytes.

[Determination of 41 veterinary drug residues in livestock and poultry meat using a composite purification system coupled with direct analysis in real time-tandem mass spectrometry].

In a market environment where food safety problems still occur despite repeated prohibitions, food safety problems caused by veterinary drug residues and biological safety problems caused by the transfer of drug resistance have attracted much attention. Herein, a method based on a compound purification system coupled with direct analysis in real time-tandem mass spectrometry (DART-MS/MS) was established to determine 41 different types of veterinary drug residues in livestock and poultry products. First, a single-standard solution sampling method was used to optimize the selection of the best quasi-molecular ion, two daughter ions, and their cone-hole and collision voltages; qualitative and quantitative ion pairs are composed of a quasi-molecular ion and its corresponding daughter ion. The abundance ratios of the drug compounds in standard solutions of the solvent and matrix mixtures were then calculated according to the requirements of the European Union 2002/657 specification. DART-MS/MS was subsequently developed for the accurate characterization and quantitative analysis of the veterinary drugs. Finally, a composite purification pretreatment system was formed by combining the primary secondary amine (PSA) and octadecyl bonded silica gel (C18) of a QuEChERS technology with multiwalled carbon nanotubes (MWCNTs) to achieve the one-step purification of the drug compounds. The influence of the key parameters of the DART ion source on the determination of the drugs was investigated using the peak areas of the quantitative ions as the criterion. The optimum conditions were as follows: ion source temperature of 350 ℃, 12-Dip-it Samplers module, sample injection speed of 0.6 mm/s, and external vacuum pump pressure of -75 kPa. According to the differences in the dissociation constant (pKa) ranges of the 41 types of veterinary drug compounds and the characteristics of the sample matrixes, the extraction solvent, matrix-dispersing solvent, and purification method were optimized based on the recovery. The extraction solvent was 1.0% acetonitrile formate solution, and the pretreatment column included MWCNTs containing 50 mg of PSA and 50 mg of C18. The three chloramphenicol drugs showed a linear relationship in the ranges of 0.5-20 µg/L with correlation coefficients of 0.9995-0.9997,and the detection and quantification limits of three chloramphenicol drugs were 0.1 and 0.5 µg/kg, respectively. The 38 other drugs, including quinolones, sulfonamides, and nitro-imidazoles showed a linear relationship in the ranges of 2-200 µg/L with correlation coefficients of 0.9979-0.9999, and the detection and quantification limits of the 38 other drugs were 0.5 and 2.0 µg/kg, respectively. The recoveries of the 41 veterinary drugs at low, medium, and high spiked levels in chicken, pork, beef, and mutton samples were 80.0%-109.6%, with intra- and inter-day precisions of 0.3%-6.8% and 0.4%-7.0%, respectively. A total of 100 batches of animal meat (pork, chicken, beef, and mutton; 25 batches each) and known positive samples were simultaneously analyzed using the national standard method and the detection method established in this study. Sulfadiazine (89.2, 78.1, and 105.3 µg/kg) was detected in three batches of pork samples, and sarafloxacin (56.3, 102.0 µg/kg) was detected in two batches of chicken samples and no veterinary drugs were detected in the other samples; both methods yielded consistent results for known positive samples. The proposed method is rapid, simple, sensitive, environmentally friendly, and suitable for the simultaneous screening and detection of multiple veterinary drug residues in animal meat.

Tissue factor pathway inhibitor suppresses the growth of human vascular smooth muscle cells through regulating cell cycle.

Tissue factor pathway inhibitor (TFPI) was reported to suppress the proliferation and migration of vascular smooth muscle cells (VSMCs) which play an important role in several vascular proliferative disorders including restenosis. Our preliminary studies demonstrated that TFPI gene induced apoptosis in human vascular smooth muscle cells (hVSMCs). The current study was designed to address the role TFPI gene plays in the cell cycle of hVSMCs. hVSMCs isolated from human umbilical artery were transfected with pIRES-TFPI plasmid which expresses TFPI in eukaryotic cells. As measured by RT-PCR, the expression of TFPI was elevated in the TFPI treated cells, leading to the arrest of the cells at G1 phase as analyzed by flow cytometry. Further study by Western blotting demonstrated that TFPI gene transfer increased the amount of p21 and p53 and decreased the amount of cyclin D and phosphorylated cdk4 and cdk6 in the cells.

A simple self-adjuvanting biomimetic nanovaccine self-assembled with the conjugate of phospholipids and nucleotides can induce a strong cancer immunotherapeutic effect.

Biomimetic nanoparticles have potential applications in many fields due to their favorable properties. Here, we developed a self-adjuvanting biomimetic anti-tumor nanovaccine, which was self-assembled with an amphiphilic conjugate synthesized with the phospholipids of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and hydrophilic Toll-like receptor (TLR9) agonist CpG ODN. The nanovaccine could not only provide effective initial antigen stimulation and sustained long-term antigen supply with a controlled release, but also induce antigen cross-presentation via the MHC-I pathway initiating CD8+ T-cell responses. Moreover, the dense nucleotide shell around the nanovaccine could promote antigen endocytosis via various receptor-mediated pathways into dendritic cells. CpG ODN interacted with TLR9 triggering the cytokine secretion of TNF-α and IL-10, which further boosted the anti-tumor humoral and cellular immune responses, which led to a significant tumor suppressive effect and remarkable survival prolongation. So, this nanovaccine self-assembled with phospholipid-nucleotide amphiphiles can serve as a safe, simple and efficient approach for anti-tumor immunotherapy.

Hydrophilic/lipophilic N-methylene phosphonic chitosan as a promising non-viral vector for gene delivery.

Cationic amphiphilic drugs have recently been shown to inhibit receptor recycling by disrupting the assembly-disassembly of clathrin at the plasma membrane and endosomes. It is therefore proposed that amphiphilic and cationic polysaccharide macromolecule, when used as gene delivery vectors, may have potential ability to direct the disassembly process of cell membrane organization, and penetrate across the cell membrane into cell and nucleus. In the current study, N-methylene phosphonic chitosan (NMPCS), an amphiphilic macromolecule, was synthesized by incorporating the methylene phosphonic group into the amino groups of chitosan (CS) using formaldehyde as the coupling agent, and characterized with a FTIR spectrometer. NMPCS/DNA or CS/DNA complexes were prepared using a complex coacervation method, and characterized by agarose gel electrophoresis retardation assay and dynamic light scattering (DLS). MTT assay was employed to evaluate the cytotoxicity of the polymers and pGL3-control luciferase plasmid was utilized as a reporter gene to assess the transgenic efficacy of the polymers. It was demonstrated that NMPCS was able to fully entrap the DNA at N/P ratio of 2:1, whereas CS entrapped the DNA completely at N/P ratio of 1:1. DLS showed that the NMPCS/DNA or CS/DNA complexes were of mean diameters ranging from 110 to 180 nm. Neither NMPCS nor CS induced significant loss of cell viability at the concentrations ranging from 1 to 50 microg/ml, whereas PEI at 5 microg/ml started to result in significantly decreased cell viability. The expression of transgene mediated by NMPCS was much higher (more than 100-folds) than that mediated by CS, indicating that NMPCS was a more efficacious gene ferrying vector than CS.

[Effect of functionalized multi-walled carbon nanotubes on L02 cells].

OBJECTIVE: To investigate the cytotoxic effect of multi-walled carbon nanotubes (MWCNTs) on human liver L02 cells and its relevant mechanism. METHODS: MWCNTs, carboxyl modification MWCNTs (MWCNTs-COOH), and hydroxyl modification MWCNTs (MWCNTs-OH) were characterized by transmission electron microscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. The carbon nanotubes at concentrations of 12.5, 25, 50, 100, and 200 µg/ml were incubated with human liver L02 cells for 24, 48 and 72 hours, respectively. The cell viability was evaluated by water soluble tetrazolium salts assay and the intercellular reactive oxygen species induced by the carbon nanotubes were detected by 2', 7'-dichlorodihydrofluorescein diacetate method. RESULTS: Transmission electron microscope showed that the average outside diameters (10 to 20 nm) and the average length (10 to 30 µm) of the three MWCNTs were similar. Scanning electron microscope indicated that the three MWCNTs had a similar surface topography. X-ray photoelectron spectroscopy demonstrated that the MWCNTs-COOH and MWCNTs-OH had relatively high peak areas at 289 and 286ev, respectively,indicating that they have been modified by carboxyl and hydroxyl groups,respectively. Water soluble tetrazolium salts assay showed that the MWCNTs-COOH was less cytotoxic when compared to MWCNTs which demonstrated to be slightly more cytotoxic than MWCNTs-OH. The capability to induce increase in intracellular reactive oxygen species was in the following order: MWCNTs > MWCNTs-COOH > MWCNTs-OH. CONCLUSIONS: Modification of MWCNTs with carboxyl group and hydroxyl group improves the biocompatibility of MWCNTs to some extents. MWCNTs-COOH has better compatibility than MWCNTs at the low concentration,and MWCNTs-OH showed better compatibility than MWCNTs after 48 hours. Different mechanisms may be involved in the interaction between cells and the MWCNTs with different chemical surfaces.

Hybrid spherical nucleotide nanoparticles can enhance the synergistic anti-tumor effect of CTLA-4 and PD-1 blockades.

Combined blockades of CTLA-4 and PD-1 can yield better overall complementary clinical outcomes than individual blockades, but the response rates are still relatively low. To investigate the anti-tumor effects of various combined strategies, we designed various spherical nucleotide nanoparticles (SNPs) loaded with CTLA-4 aptamer (cSNPs), PD-1 siRNA (pSNPs) or both (hybrid SNPs, or hSNPs). The results demonstrated that hSNPs could promote significantly stronger anti-tumor immune responses in a nonredundant fashion than the mixture of pSNPs and cSNPs (pSNPs & cSNPs). We reasoned that this is because all individual immune cells could receive both CTLA-4 and PD-1 blockades when they engulfed hSNPs, but it is much less likely that individual immune cells could receive both CTLA-4 and PD-1 blockades as many of them may not take both pSNPs and cSNPS from pSNPs & cSNPs. Further results revealed that the synergistic immune stimulatory effects of CTLA-4 and PD-1 blockades in the form of hSNPs were at least partly through regulating the immune suppressive function of both Tregs and TIM3+ exhausted-like CD8 T cells and allowing effector T cells to expand. This mechanism is not identical to earlier reported mechanisms of CTLA-4 and PD-1 blockades.

A cell-penetrating peptide-assisted nanovaccine promotes antigen cross-presentation and anti-tumor immune response.

Exogenous antigens processed in the cytosol and subsequently cross-presented on major histocompatibility complex class I (MHC-I) molecules activate cytotoxic CD8+ lymphocytes (CTL), which are crucial in cancer immunotherapy. Here, we reported a nanovaccine, which was produced by encapsulating OVA (ovalbumin, a model antigen) chemically modified with MPGΔNLS (MPGΔNLS-OVA conjugate) into poly(lactide-co-glycolide) acid (PLGA) nanoparticles. We hypothesized that after the uptake of the nanovaccine into immune cells, MPGΔNLS, a cell-penetrating peptide (CPP), would assist the escape of the antigens from lysosomes into the cytosol, increase the amount of antigens processed in the cytosol and subsequently enhance antigen cross-presentation via MHC-I molecules to elicit cytotoxic CD8+ T cell responses. The results of the in vitro experiments demonstrated that the MPGΔNLS-OVA-loaded PLGA NPs not only elevated the release of OVA into the cytosol of dendritic cells (BMDCs), but also promoted the maturation and activation of BMDCs. It was also observed in mice vaccinated with MPGΔNLS-OVA-loaded PLGA NPs that the MPGΔNLS modification could stimulate the expansion of OVA-specific T-cells, generation of OVA-specific IgG antibodies and proliferation of OVA-specific memory T cells. Moreover, the treatment of E·G7-OVA tumor-bearing mice with MPGΔNLS-OVA-loaded PLGA NPs resulted in significantly suppressed tumor growth and prolonged survival periods of the mice compared to the treatment with unmodified OVA-PLGA NPs or free OVA. In summary, cell-penetrating peptides linked with antigens encapsulated in nanovaccines can spatiotemporally affect the intracellular localization of antigens, promote antigen cross-presentation and stimulate antigen-specific immune responses, especially CTL responses. Therefore, the CPP modification on antigens is an innovative approach to enhance the efficacy of nanovaccines for cancer immunotherapy.

Nanoscale Reduced Graphene Oxide-Mediated Photothermal Therapy Together with IDO Inhibition and PD-L1 Blockade Synergistically Promote Antitumor Immunity.

Despite the potential efficacy of immune checkpoint blockade for effective treatment of cancer, this therapeutic modality is not generally curative, and only a fraction of patients respond. Combination approaches provide strategies to target multiple antitumor immune pathways to induce synergistic antitumor immunity. Here, a multi-combination immunotherapy, including photothermal therapy (PTT), indoleamine-2,3-dioxygenase (IDO) inhibition, and programmed cell death-ligand 1 (PD-L1) blockade, is introduced for inducing synergistic antitumor immunity. We designed a multifunctional IDO inhibitor (IDOi)-loaded reduced graphene oxide (rGO)-based nanosheets (IDOi/rGO nanosheets) with the properties to directly kill tumor cells under laser irradiation and in situ trigger antitumor immune response. In vivo experiments further revealed that the triggered immune response can be synergistically promoted by IDO inhibition and PD-L1 blockade; the responses included the enhancement of tumor-infiltrating lymphocytes, including CD45+ leukocytes, CD4+ T cells, CD8+ T cells, and NK cells; the inhibition of the immune suppression activity of regulator T cells (Tregs); and the production of INF-γ. We also demonstrate that the three combinations of PTT, IDO inhibition, and PD-L1 blockade can effectively inhibit the growth of both irradiated tumors and tumors in distant sites without PTT treatment. This work can be thought of as an important proof of concept to target multiple antitumor immune pathways to induce synergistic antitumor immunity.

Immobilization of plasmid DNA on an anti-DNA antibody modified coronary stent for intravascular site-specific gene therapy.

BACKGROUND: The aim of the present study was to investigate the incorporation of plasmid DNA (pDNA) onto a coronary stent by chemo-immunoconjugation for achieving site-specific gene delivery. METHODS: Anti-DNA immunoglobulin M antibody was chemically linked onto collagen-coated stent by using N-succinimidyl-3-(2-pyridyldithiol)-propionate as cross-linker. pDNA was tethered on the antibody-immobilized stent by highly specific antigen-antibody affinity interaction. Radioactive-labeled antibody and pDNA were used to evaluate binding capacity and stability. A reporter plasmid pEGFP was tethered on the antibody-immobilized stents that was assessed in cell culture and in rabbit carotid model. RESULTS: The amount of antibody chemically linked on the stents was 15-fold higher than that of the control and its retention time was also significantly longer. The pEGFP-tethered stents had no detrimental effects on cell growth. In cell culture studies, numerous green fluorescent protein (GFP)-transfected cells were only found on the stent, which demonstrated high localization and efficiency of gene delivery. The overall GFP transfection efficiency in treated rabbit carotid arteries was 2.8 +/- 0.7% of the total cells. However, the rate of neointima transfection was 7.0 +/- 0.8% of total cells in this region. Importantly, no distal spreading of the vector was detected by polymerase chain reaction, either in distal organs or in the downstream segments of the stented arteries. CONCLUSIONS: For the first time, our group reports the successful use of anti-DNA antibody-immobilized metal stent as plasmid gene delivery system that possess high efficiency and site-specificity in vitro and in vivo.

[Effect of chitosan gene nanoparticles on L02 cells].

OBJECTIVE: To investigate the effect of the gene nanoparticles using chitosan (CNP), arginine modified chitosan (ANP), or hexadecylated chitosan (HNP) as carriers on the human normal liver cell line L02. METHODS: CNPs, ANPs, and HNPs were prepared using complex coacervation method. The size and zeta potential of the gene nanoparticles were measured using Zetasizer nanoZS. The nanoparticles at concentrations of 5, 10, 30, and 50 microg/ml (based on the content of DNA) were incubated with L02 cells, respectively. The cell viability was evaluated by MTT assay, and the effect of the gene nanoparticles on the cell apoptosis was analyzed by flow cytometry. RESULTS: The zeta potential of the gene nanoparticles ranged from 12.10 to 14.63 mV, and their diameters ranged from 148.07 to 179.47 nm. MTT assay showed that the viability of L02 cells began to decrease when the concentration of CNPs reached 30 microg/ml and higher. Furthermore, the CNPs could induce cell apoptosis as the concentration of CNPs reached 30 microg/ml and higher. CONCLUSION: CNPs can induce L02 cell apoptosis at relatively higher concentrations.

Photothermally Controlled MHC Class I Restricted CD8+ T-Cell Responses Elicited by Hyaluronic Acid Decorated Gold Nanoparticles as a Vaccine for Cancer Immunotherapy.

Cancer vaccines aim to induce a strong major histocompatibility complex class I (MHC-I)-restricted CD8+ cytotoxic T-cell response, which is an important prerequisite for successful cancer immunotherapy. Herein, a hyaluronic acid (HA) and antigen (ovalbumin, OVA)-decorated gold nanoparticle (AuNPs)-based (HA-OVA-AuNPs) vaccine is developed for photothermally controlled cytosolic antigen delivery using near-infrared (NIR) irradiation and is found to induce antigen-specific CD8+ T-cell responses. Chemical binding of thiolated HA and OVA to AuNPs facilitates antigen uptake of dendritic cells via receptor-mediated endocytosis. HA-OVA-AuNPs exhibit enhanced NIR absorption and thermal energy translation. Cytosolic antigen delivery is then permitted through the photothermally controlled process of local heat-mediated endo/lysosome disruption by laser irradiation along with reactive oxygen species generation, which helps to augment proteasome activity and downstream MHC I antigen presentation. Consequently, the HA-OVA-AuNPs nanovaccine can effectively evoke a potent anticancer immune response in mice under laser irradiation. This NIR-responsive nanovaccine is promising as a potent vaccination method for improving cancer vaccine efficacy.

Nanovaccine Incorporated with Hydroxychloroquine Enhances Antigen Cross-Presentation and Promotes Antitumor Immune Responses.

Induction of effective antigen-specific CD8+ T-cell responses is critical for cancer immunotherapy success. Hydroxychloroquine (HCQ) is a widely used classical antimalarial and antirheumatic drug. HCQ is also an endosomal membrane disrupting agent that can lead to vesicular swelling and membrane permeabilization, which likely facilitates the release of therapeutic agents from lysosomes into the cytoplasm. Here, we develop a minimalistic nanovaccine, which is composed of poly(lactide- co-glycolide)acid (PLGA) nanoparticles (NPs) encapsulating a physical mixture of ovalbumin (OVA, a model antigen) and HCQ (HCQ-OVA-PLGA NPs). We tested whether HCQ could spatiotemporally control the cytosolic delivery of antigens, enhance antigen processing and presentation via the major histocompatibility complex (MHC)-I pathway, and thus generate a sufficient antitumor cytotoxic T-cell response. The results of in vitro experiments showed that HCQ-OVA-PLGA NPs significantly enhanced OVA escape from lysosomes into the cytoplasm within bone-marrow-derived dendritic cells. We also observed that HCQ-OVA-PLGA NPs enhanced the expression level of MHC-I on dendritic cells and improved cross-presentation of antigen, compared to free OVA or OVA-PLGA NPs. Results of in vivo experiments confirmed that HCQ initiated Th1-type responses and strong CD8+ T-cell responses that induced tumor cell apoptosis. Moreover, vaccination of mice with HCQ-OVA-PLGA NPs effectively generated memory immune responses in vivo and prevented tumor progression. We conclude that co-encapsulation of HCQ with antigens in nanovaccines can boost antigen-specific antitumor immune responses, particularly through CD8+ T-cells, serving as a simple and effective platform for the treatment of tumors and infectious diseases.