Layer-by-layer nanoparticle encapsulating all-trans retinoic acid and CpG as a mucosal adjuvant targeting colorectal cancer.

Colorectal cancer (CRC) ranks among the most prevalent cancers globally, demanding innovative therapeutic strategies. Immunotherapy, a promising avenue, employs cancer vaccines to activate the immune system against tumors. However, conventional approaches fall short of eliciting robust responses within the gastrointestinal (GI) tract, where CRC originates. Harnessing the potential of all-trans retinoic acid (ATRA) and cytosine-phosphorothioate-guanine (CpG), we developed layered nanoparticles using a layer-by-layer assembly method to co-deliver these agents. ATRA, crucial for gut immunity, was efficiently encapsulated alongside CpG within these nanoparticles. Administering these ATRA@CpG-NPs, combined with ovalbumin peptide (OVA), effectively inhibited orthotopic CRC growth in mice. Our approach leveraged the inherent benefits of ATRA and CpG, demonstrating superior efficacy in activating dendritic cells, imprinting T cells with gut-homing receptors, and inhibiting tumor growth. This mucosal adjuvant presents a promising strategy for CRC immunotherapy, showcasing the potential for targeting gut-associated immune responses in combating colorectal malignancies.

Non-Modified CpG Oligodeoxynucleotide Forming Guanine-Quadruplex Structure Complexes with ε-Poly-L-Lysine Induce Antibody Production as Vaccine Adjuvants.

Unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) induce inflammatory cytokines and type I interferons (IFNs) to activate the immune system. To apply CpG ODNs as vaccine adjuvants, the cellular uptake and stability of phosphodiester-based, non-modified ODNs require further improvement. Previously developed new CpG ODNs forming guanine-quadruplex (G4) structures showed higher nuclease resistance and cellular uptake than linear CpG ODNs; however, the complex formation of G4-CpG ODNs with antigen proteins is necessary for their application as vaccine adjuvants. In this study, we utilized a cationic polymer, ε-poly-L-lysine (ε-PLL), as a carrier for G4-CpG ODNs and antigen. The ε-PLL/G4-CpG ODN complex exhibited enhanced stability against nucleases. Cellular uptake of the ε-PLL/G4-CpG ODN complex positively correlated with the N/P ratio. In comparison to naked G4-CpG ODNs, the ε-PLL/G4-CpG ODN complex induced extremely high levels of interleukin (IL)-6, IL-12, and IFN-ß. Relative immune cytokine production was successfully tuned by N/P ratio modification. Mice with the ε-PLL/G4-CpG ODN/ovalbumin (OVA) complex showed increased OVA-specific immunoglobulin (Ig)G, IgG1, and IgG2c levels, whereas total IgE levels did not increase and weight gain rates were not affected. Therefore, ε-PLL can serve as a safe and effective phosphodiester-based, non-modified CpG ODN delivery system, and the ε-PLL/G4-CpG ODN/antigen complex is a highly promising candidate for vaccine adjuvants and can be further used in clinical research.

Development of Hydrophobic Interaction-based DNA Supramolecules as Efficient Delivery Carriers of CpG DNA to Immune cells.

Unmethylated cytosine-phosphate-guanine (CpG) DNA stimulates mammalian immune cells through recognition by Toll-like receptor 9 (TLR9). Therefore, CpG DNA is expected to be an effective adjuvant for the treatment of immune and allergic diseases. However, challenges, such as low stability against DNase and low delivery efficiency for immune cells, still need to be resolved for the application of CpG DNA. To overcome these challenges, we developed DNA supramolecules consisting of long single-stranded DNA (lss-DNA) synthesized using rolling circle amplification (RCA) and cholesterol-modified DNA (chol-DNA). Lss-DNAs containing multiple CpG motifs were annealed with complementary chol-DNAs to form DNA supramolecules through hydrophobic interactions. Transmission electron microscopy revealed that lss-DNA mixed with chol-DNA formed micrometer-sized DNA supramolecules. The formation of DNA supramolecules increased their stability against DNase compared to lss DNA, which was evaluated using FBS. Furthermore, DNA supramolecules induced three-times higher TNF-α release from RAW264.7 cells than lss-DNA alone. These results demonstrate that DNA supramolecules are efficient delivery carriers of CpG DNA to immune cells.

Intelligent Tumor Microenvironment-Activated Multifunctional Nanoplatform Coupled with Turn-on and Always-on Fluorescence Probes for Imaging-Guided Cancer Treatment.

Intrinsic tumor microenvironment (TME)-related therapeutic resistance and nontumor-specific imaging have limited the application of imaging-guided cancer therapy. Herein, a TME-responsive MnO2-based nanoplatform coupled with turn-on and always-on fluorescence probes was designed through a facile biomineralization method for imaging-guided photodynamic/chemodynamic/photothermal therapy (PDT/CDT/PTT). After the tumor-targeting delivery of the AuNCs@MnO2-ICG@AS1411 (AMIT) nanoplatform via aptamer AS1411, the TME-responsive dissociation of MnO2 generated sufficient O2 and Mn2+ with the consumption of GSH for improving PDT efficacy and Fenton-like reaction-mediated CDT. Simultaneously, the released small-sized ICG and AuNCs facilitated PDT and PTT efficacy via the deep tumor penetration. Moreover, the turn-on fluorescence of AuNCs revealed the real-time TME-responsive MnO2 degradation process, and the always-on ICG fluorescence enabled the in situ monitoring of the payload distribution in vitro and in vivo. The AMIT NPs also provided magnetic resonance and thermal imaging guidance for the enhanced PDT, CDT, and PTT. Therefore, this all-in-one nanosystem provides a simple and versatile strategy for multiple imaging-guided theranostic applications.

Iron(II) phthalocyanine Loaded and AS1411 Aptamer Targeting Nanoparticles: A Nanocomplex for Dual Modal Imaging and Photothermal Therapy of Breast Cancer.

PURPOSE: A multi-functional nanoplatform with diagnostic imaging and targeted treatment functions has aroused much interest in the nanomedical research field and has been paid more attention in the field of tumor diagnosis and treatment. However, some existing nano-contrast agents have encountered difficulties in different aspects during clinical promotion, such as complicated preparation process and low specificity. Therefore, it is urgent to find a nanocomplex with good targeting effect, high biocompatibility and significant therapeutic effect for the integration of diagnosis and treatment and clinical transformation. MATERIALS AND METHODS: Nanoparticles (NPs) targeting breast cancer were synthesized by phacoemulsification which had liquid fluorocarbon perfluoropentane(PFP) in the core and were loaded with Iron(II) phthalocyanine (FePc) on the shell. The aptamer (APT) AS1411 was outside the shell used as a molecular probe. Basic characterization and targeting abilities of the NPs were tested, and their cytotoxicity and biological safety in vivo were evaluated through CCK-8 assay and blood bio-chemical analysis. The photoacoustic (PA) and ultrasound (US) imaging system were used to assess the effects of AS1411-PLGA@FePc@PFP (A-FP NPs) as dual modal contrast agent in vitro and in vivo. The effects of photothermal therapy (PTT) in vitro and in vivo were evaluated through MCF-7 cells and tumor-bearing nude mouse models. RESULTS: A-FP NPs, with good stability, great biocompatibility and low toxicity, were of 201.87 ± 1.60 nm in diameter, and have an active targeting effect on breast cancer cells and tissues. With the help of PA/US imaging, it was proved to be an excellent dual modal contrast agent for diagnosis and guidance of targeted therapy. Meanwhile, it can heat up under near-infrared (NIR) laser irradiation and has achieved obvious antitumor effect both in vitro and in vivo experiments. CONCLUSION: As a kind of nanomedicine, A-FP NPs can be used in the integration of diagnosis and treatment. The treatment effects and biocompatibility in vivo may provide new thoughts in the clinical transformation of nanomedicine and early diagnosis and treatment of breast cancer.

The CpG molecular structure controls the mineralization of calcium phosphate nanoparticles and their immunostimulation efficacy as vaccine adjuvants.

The co-precipitation of calcium phosphate nanoparticles (CaPs) in the presence of nucleotide chains such as polynucleotides (i.e., plasmid DNA and siRNA) and oligonucleotides has been extensively used for pre-clinical gene or drug delivery and immunotherapy studies. However, the exact role of these molecules in mineralization and tuning the physicochemical characteristics of the synthesized CaPs is still not entirely clear. In this study, we evaluated the effects of three different CpG oligodeoxynucleotides (ODN) and two representative nucleic acids (siRNA and DNA), when used as templates for the formation of CaPs. We examined the influence of CpGs with naturally-occurring phosphodiester or modified phosphorothioate backbones on the homogeneous formation of CaPs from a modified simulated body fluid solution. The hydrodynamic size, size polydispersity, morphology and surface charge of the CaPs were used as the most critical checkpoints to unravel the involved mechanisms. Our results show that the characteristics of CaPs are highly dependent on the composition, backbone, sequence and concentrations of the CpGs. The CpG type and concentration control the size distribution of the mineralized CaPs and their immunostimulation performance as verified by the activation of dendritic cells and secretion of the pro-inflammatory interleukin-6 (IL-6) cytokine, type I interferon-α (IFN-α) and co-stimulatory CD80, CD86 and CD40 markers. This study paves the way for better design of more efficient CaPs loaded with different types of CpGs for immunostimulation applications as vaccine adjuvants.

Enhanced Immunostimulatory Activity of a Cytosine-Phosphate-Guanosine Immunomodulator by the Assembly of Polymer DNA Wires and Spheres.

Unmethylated cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides are immunostimulatory nucleic acids wildly utilized as adjuvants or for vaccines to treat diseases. However, there is a lack of simple and efficient vectors for CpG oligodeoxynucleotide delivery with long-lasting immune stimulation. Herein, self-assembled polymer wires consisting of CpG motifs by hybridization chain reaction were constructed with excellent biocompatibility and immunostimulatory activity. The designed polymer DNA wires acted as programmable multivalent immunoadjuvants and triggered immune response, stimulated pro-inflammatory cytokine secretion, and induced the apoptosis of cancer cells. More strikingly, polymer nanospheres assembled from the polymer DNA wires and cationic poly-l-lysine further improved cellular uptake and continuously stimulate the lysosomal Toll-like receptor 9 of immune cells, thereby remarkably enhancing the activation of immune cells. These results demonstrated that self-assembled polymer DNA nanoassemblies with multivalent CpG could trigger strong immune response and further induce cancer cell death.

A trustworthy CpG nanoplatform for highly safe and efficient cancer photothermal combined immunotherapy.

Palladium nanosheets (Pd NSs) have recently attracted increasing research interest in the biomedical field due to their excellent near-infrared absorption, photothermal conversion capability and biocompatibility. However, the application of Pd NSs in immunotherapy has not been reported. Here, Pd NSs were used as the carriers of immunoadjuvant CpG ODNs for not only efficient delivery of CpG but also for enhancing the immunotherapeutic effects of CpG by the Pd NS-based photothermal therapy (PTT). Pd NSs had no influence on the immune system, and the prepared Pd-CpG nanocomposites, especially Pd(5)-CpG(PS), could significantly increase the uptake of CpG by immune cells and enhance the immunostimulatory activity of CpG in vitro and in vivo. With the combination of Pd(5)-CpG(PS) mediated PTT and immunotherapy, highly efficient tumor inhibition was achieved and the survival rate of the tumor-bearing mice was greatly increased depending on Pd(5)-CpG(PS) with safe near-infrared (NIR) irradiation (808 nm laser, 0.15 W cm-2). Importantly, the combination therapy induced tumor cell death and released tumor-associated antigens, which could be effectively taken up and presented by antigen presenting cells with the assistance of CpG, leading to increased TNF-α and IL-6 production and enhanced cytotoxic T lymphocyte (CTL) activity. This work provides a new paradigm of utilizing photothermal nanomaterials for safe and highly efficient cancer photothermal combined immunotherapy.

Conformational Preferences of DNA Strands from the Promoter Region of the c-MYC Oncogene.

We characterized the conformational preferences of DNA in an equimolar mixture of complementary G-rich and C-rich strands from the promoter region of the c-MYC oncogene. Our CD-based approach presupposes that the CD spectrum of such a mixture is the spectral sum of the constituent duplex, G-quadruplex, i-motif, and coiled conformations. Spectra were acquired over a range of temperatures at different pHs and concentrations of KCl. Each spectrum was unmixed in terms of the predetermined spectra of the constituent conformational states to obtain the corresponding weighting factors for their fractional contributions to the total population of DNA. The temperature dependences of those contributions then were analyzed in concert according to a model based on a thermodynamic representation of the underlying equilibria. Fitted estimates of the melting enthalpy and temperature obtained for the duplex, G-quadruplex, and i-motif imply that the driving force behind dissociation of the duplex and the concomitant formation of tetrahelical structures is the folding of the G-strand into the G-quadruplex. The liberated C-strand adopts the i-motif conformation at acidic pH and exists in the coiled state at neutral pH. The i-motif alone cannot induce dissociation of the duplex even at pH 5.0, at which it is most stable. Under the physiological conditions of neutral pH, elevated potassium, and room temperature, the duplex and G-quadruplex conformations coexist with the C-strand in the coiled state. Taken together, our results suggest a novel, thermodynamically controlled mechanism for the regulation of gene expression.

Compact quantum dot surface modification to enable emergent behaviors in quantum dot-DNA composites.

Quantum dot (QD) biological imaging and sensing applications often require surface modification with single-stranded deoxyribonucleic acid (ssDNA) oligonucleotides. Furthermore, ssDNA conjugation can be leveraged for precision QD templating via higher-order DNA nanostructures to exploit emergent behaviors in photonic applications. Use of ssDNA-QDs across these platforms requires compact, controlled conjugation that engenders QD stability over a wide pH range and in solutions of high ionic strength. However, current ssDNA-QD conjugation approaches suffer from limitations, such as the requirement for thick coatings, low control over ssDNA labeling density, requirement of large amounts of ssDNA, or low colloidal or photostability, restraining implementation in many applications. Here, we combine thin, multidentate, phytochelatin-3 (PC3) QD passivation techniques with strain-promoted copper-free alkyne-azide click chemistry to yield functional ssDNA-QDs with high stability. This process was broadly applicable across QD sizes (i.e., λem = 540, 560, 600 nm), ssDNA lengths (i.e., 10-16 base pairs, bps), and sequences (poly thymine, mixed bps). The resulting compact ssDNA-QDs displayed a fluorescence quenching efficiency of up to 89% by hybridization with complementary ssDNA-AuNPs. Furthermore, ssDNA-QDs were successfully incorporated with higher-order DNA origami nanostructure templates. Thus, this approach, combining PC3 passivation with click chemistry, generates ssDNA-PC3-QDs that enable emergent QD properties in DNA-based devices and applications.

High affinity of AS1411 toward copper; its application in a sensitive aptasensor for copper detection.

AS1411 is a 26-mer G-rich DNA aptamer which has been broadly employed in the field of targeted drug delivery, due to its capability to bind to nucleolin protein on the surface of cancer cells. In this work, it has been shown for the first time that in addition to nucleolin, AS1411 aptamer could bind to copper ions (Cu2+) with high affinity and selectivity, affecting AS1411 usage in drug delivery systems as a targeting agent. In this study, besides the evaluations of the affinity of AS1411 to different ions and the impact of Cu2+ on targeted drug delivery employed AS1411 aptamer as a targeting agent, a simple and ultra-sensitive fluorescent aptasensor was fabricated for Cu2+ detection through applying AS1411, its complementary strand and magnetic beads coated with streptavidin. Gel Red (GR) was also used as a fluorescent dye. The fabricated aptasensor offered the possibility of condensing samples with different volumes. The detection limit of the sensor was 0.01 µM towards Cu2+ in serum samples. The efficacy of this sensor was further confirmed by comparing Cu2+ levels in serums of healthy people with patients suffering from Wilson's disease, Alzheimer's disease and Diabetes Mellitus using the proposed sensing platform.

Prussian blue nanoparticle-based antigenicity and adjuvanticity trigger robust antitumor immune responses against neuroblastoma.

We describe the synthesis of CpG oligodeoxynucleotide-coated Prussian blue nanoparticles (CpG-PBNPs) that function as a nanoimmunotherapy for neuroblastoma, a common childhood cancer. These CpG-PBNPs increase the antigenicity and adjuvanticity of the treated tumors, ultimately driving robust antitumor immunity through a multi-pronged mechanism. CpG-PBNPs are synthesized using a facile layer-by-layer coating scheme resulting in nanoparticles that exhibit monodisperse size distributions and multiday stability without cytotoxicity. The strong intrinsic absorption of PBNPs in the CpG-PBNPs enables ablative photothermal therapy (CpG-PBNP-PTT) that triggers tumor cell death, as well as the release of tumor antigens to increase antigenicity. Simultaneously, the CpG coating functions as an exogenous molecular adjuvant that complements the endogenous adjuvants released by the CpG-PBNP-PTT (e.g. ATP, calreticulin, and HMGB1). In cell culture, coating NPs with CpG increases immunogenicity while maintaining the photothermal activity of PBNPs. When administered in a syngeneic, Neuro2a-based, murine model of neuroblastoma, CpG-PBNP-PTT results in complete tumor regression in a significantly higher proportion (70% at 60 days) of treated animals relative to controls. Furthermore, the long-term surviving, CpG-PBNP-PTT-treated animals reject Neuro2a rechallenge, suggesting that this therapy generates immunological memory. Our findings point to the importance of simultaneous cytotoxicity, antigenicity, and adjuvanticity to generate robust and persistent antitumor immune responses against neuroblastoma.

Tunable DNA Hybridization Enables Spatially and Temporally Controlled Surface-Anchoring of Biomolecular Cargo.

The controlled immobilization of biomolecules onto surfaces is relevant in biosensing and cell biological research. Spatial control is achieved by surface-tethering molecules in micro- or nanoscale patterns. Yet, there is an increasing demand for temporal control over how long biomolecular cargo stays immobilized until released into the medium. Here, we present a DNA hybridization-based approach to reversibly anchor biomolecular cargo onto micropatterned surfaces. Cargo is linked to a DNA oligonucleotide that hybridizes to a sequence-complementary, surface-tethered strand. The cargo is released from the substrate by the addition of an oligonucleotide that disrupts the duplex interaction via toehold-mediated strand displacement. The unbound tether strand can be reloaded. The generic strategy is implemented with small-molecule or protein cargo, varying DNA sequences, and multiple surface patterning routes. The approach may be used as a tool in biological research to switch membrane proteins from a locally fixed to a free state, or in biosensing to shed biomolecular receptors to regenerate the sensor surface.

CpG-PEG Conjugates and their Immune Modulating Effects after Systemic Administration.

PURPOSE: Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs were found to be able to target cells that express Toll-like receptor 9 to modulate innate and adaptive immune reactions. But their in vivo application in immunotherapy against cancer has not been successful. We attempted in this study to examine polyethylene-glycol (PEG) conjugated CpG ODNs and investigated their mechanism of immune modulation in anti-cancer therapy. METHODS: CpG-PEG conjugates with different PEG lengths were synthesized. In vitro activity as well as in vivo pharmacokinetics and pharmacodynamics properties were evaluated. RESULTS: CpG-PEG20Ks were found to be able to persist longer in circulation and activate various downstream effector cells. After intravenous injection, they resulted in higher levels of IL-12p70 in the circulation and lower M-MDSC infiltrates in the tumor microenvironment. Such activities were different from those of CpG ODNs without PEGylation, suggesting different PK-PD profiles systemically and locally. CONCLUSIONS: Our data support the development of CpG-PEGs as a new therapeutic agent that can be systemically administered to modulate immune responses and the microenvironment in tumor tissues.

Double-stranded phosphodiester cytosine-guanine oligodeoxynucleotide complexed with calcium phosphate as a potent vaccine adjuvant for activating cellular and Th1-type humoral immunities.

Conventional class B cytosine-guanine (CpG) (CpG-B) oligodeoxynucleotide (ODNs) consisting of a single-stranded (ss) phosphorothioate (PT) backbone (ss CpG-B-PT) is converted from a proinflammatory cytokine inducer to a type-I interferon (IFN) inducer when complexed with cationic materials. In this study, we designed ss CpG-B and double-stranded (ds) CpG-B ODNs with a phosphodiester (PD) backbone (ss CpG-B-PD and ds CpG-B-PD, respectively) that became type-I IFN inducers upon complexation with Lipofectamine 2000 (Lipo), a cationic liposome. The ds CpG-B-PD complex induced higher IFN-ß expression in mouse macrophage-like RAW264 cells than ss CpG-B-PD and ss CpG-B-PT complexes. The fold induction of IFN-ß increased with the number of CpG motifs in ds CpG-B-PD, and a complex of ds CpG-B-PD consisting of 72 base pairs with nine CpG motifs (ds CpG-B72-PD) and Lipo showed the highest capacity to induce IFN-ß. The materials and method used for complexation influenced the degree of IFN-ß induction: ds CpG-B72-PD entrapped by calcium phosphate (CaP) (ds CpG-B72-PD/CaP) showed a higher induction capacity than ds CpG-B72-PD adsorbed onto the CaP surface. Entrapment of ds CpG-B72-PD by CaP also enhanced the induction of the proinflammatory cytokine interleukin-12. Vaccinating mice with ds CpG-B72-PD/CaP in conjunction with ovalbumin (OVA) increased the ratios of OVA-specific CD8+ T cells to total CD8+ T cells in peripheral blood and of OVA-specific IgG2a associated with helper T (Th)1 cells to OVA-specific IgG1 associated with Th2 cells. These results indicate that ds CpG-B72-PD/CaP is an effective vaccine adjuvant that can activate both cellular and Th1-type humoral immune responses.

Preclinical Development of a Prophylactic Neuroprotective Therapy for the Preventive Treatment of Anticipated Ischemia-Reperfusion Injury.

Ischemia-reperfusion brain injury can be iatrogenically induced secondary to life-saving procedures. Prophylactic treatment of these patients offers a promising prevention for lifelong complications. We postulate that a cytosine-guanine (CpG) oligodeoxynucleotide (ODN) can provide robust antecedent protection against cerebral ischemic injury with minimal release of pro-inflammatory cytokines, making it an ideal candidate for further clinical development. Mouse and nonhuman primate (NHP) models of cerebral ischemic injury were used to test whether an A-type CpG ODN, which induces minimal systemic inflammatory cytokine responses, can provide prophylactic protection. Extent of injury in the mouse was measured by histological staining of live tissue. In the NHP, injury was assessed 2 and 7 days post-occlusion from T2-weighted magnetic resonance images and neurological and motor deficits were cataloged daily. Plasma cytokine levels were measured using species-specific Luminex assays. Prophylactic administration of an A-type CpG ODN provided robust protection against cerebral ischemic injury in the mouse with minimal systemic inflammation. Rhesus macaques treated with D192935, a mixture of human optimized A-type CpG ODNs, had smaller infarcts and demonstrated significantly less neurological and motor deficits following ischemic injury. Our findings demonstrate the translational potential of D192935 as a prophylactic treatment for patients at risk of cerebral ischemic injury.

A Series of ß-Carboline Alkaloids from the Seeds of Peganum harmala Show G-Quadruplex Interactions.

In this study, we screened 17 medicinal plants for binding activity to G-quadruplex d(TTGGGTT)4 by (1)H NMR spectroscopy and found that the crude extract of Peganum harmala L. seeds showed the most potential binding activity. Subsequently, (1)H NMR- and bioassay-guided isolation of the extract of P. harmala L. was performed to obtain four pairs of partially racemized ß-carboline alkaloids, pegaharmines A-D (1-4). Their structures and absolute configurations were determined by extensive NMR analyses, X-ray crystallography, ECD calculations, and CD exciton chirality approaches. Interestingly, pegaharmine D (4), which showed the strongest G-quadruplex interaction, exhibited significant cytotoxic activity against three cancer cell lines. This work contributed a practical strategy for the discovery of novel G-quadruplex ligands from natural products and provided potential insights for using ß-carboline alkaloids as anticancer lead compounds specifically targeting G-quadruplexes.

Particle-by-Particle Charge Analysis of DNA-Modified Nanoparticles Using Tunable Resistive Pulse Sensing.

Resistive pulse sensors, RPS, are allowing the transport mechanism of molecules, proteins and even nanoparticles to be characterized as they traverse pores. Previous work using RPS has shown that the size, concentration and zeta potential of the analyte can be measured. Here we use tunable resistive pulse sensing (TRPS) which utilizes a tunable pore to monitor the translocation times of nanoparticles with DNA modified surfaces. We start by demonstrating that the translocation times of particles can be used to infer the zeta potential of known standards and then apply the method to measure the change in zeta potential of DNA modified particles. By measuring the translocation times of DNA modified nanoparticles as a function of packing density, length, structure, and hybridization time, we observe a clear difference in zeta potential using both mean values and population distributions as a function of the DNA structure. We demonstrate the ability to resolve the signals for ssDNA, dsDNA, small changes in base length for nucleotides between 15 and 40 bases long, and even the discrimination between partial and fully complementary target sequences. Such a method has potential and applications in sensors for the monitoring of nanoparticles in both medical and environmental samples.

Polyethyleneimine-functionalized boron nitride nanospheres as efficient carriers for enhancing the immunostimulatory effect of CpG oligodeoxynucleotides.

CpG oligodeoxynucleotides (ODNs) stimulate innate and adaptive immune responses. Thus, these molecules are promising therapeutic agents and vaccine adjuvants against various diseases. In this study, we developed a novel CpG ODNs delivery system based on polyethyleneimine (PEI)-functionalized boron nitride nanospheres (BNNS). PEI was coated on the surface of BNNS via electrostatic interactions. The prepared BNNS-PEI complexes had positive zeta potential and exhibited enhanced dispersity and stability in aqueous solution. In vitro cytotoxicity assays revealed that the BNNS-PEI complexes with concentrations up to 100 µg/mL exhibited no obvious cytotoxicity. Furthermore, the positively charged surface of the BNNS-PEI complexes greatly improved the loading capacity and cellular uptake efficiency of CpG ODNs. Class B CpG ODNs loaded on the BNNS-PEI complexes enhanced the production of interleukin-6 and tumor necrosis factor-α from peripheral blood mononuclear cells compared with CpG ODNs directly loaded on BNNS. Contrary to the free CpG ODNs or CpG ODNs directly loaded on BNNS, class B CpG ODNs loaded on the BNNS-PEI complexes induced interferon-α simultaneously. PEI coating may have changed the physical form of class B CpG ODNs on BNNS, which further affected their interaction with Toll-like receptor 9 and induced interferon-α. Therefore, BNNS-PEI complexes can be used to enhance the immunostimulatory effect and therapeutic activity of CpG ODNs and the treatment of diseases requiring interleukin-6, tumor necrosis factor-α, and interferon-α.

Gold nanocluster-based vaccines for dual-delivery of antigens and immunostimulatory oligonucleotides.

We here report a facile one-pot synthesis of fluorescent gold nanoclusters (AuNCs) via the peptide biomineralization method, which can elicit specific immunological responses. The as-prepared peptide-protected AuNCs (peptide-AuNCs) display strong red fluorescence, and more importantly, as compared to the peptide alone, the immune stimulatory ability of the resulting peptide-AuNCs can not only be retained, but can also be efficaciously enhanced. Moreover, through a dual-delivery of antigen peptides and cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs), the as-prepared peptide-AuNC-CpG conjugates can also act as smart self-vaccines to assist in the generation of high immunostimulatory activity, and be applied as a probe for intracellular imaging. Both in vitro and in vivo studies provide strong evidence that the AuNC-based vaccines may be utilized as safe and efficient immunostimulatory agents that are able to prevent and/or treat a variety of ailments.