Enhanced mucosal B- and T-cell responses against SARS-CoV-2 after heterologous intramuscular mRNA prime/intranasal protein boost vaccination with a combination adjuvant.

Current COVID-19 mRNA vaccines delivered intramuscularly (IM) induce effective systemic immunity, but with suboptimal immunity at mucosal sites, limiting their ability to impart sterilizing immunity. There is strong interest in rerouting immune responses induced in the periphery by parenteral vaccination to the portal entry site of respiratory viruses, such as SARS-CoV-2, by mucosal vaccination. We previously demonstrated the combination adjuvant, NE/IVT, consisting of a nanoemulsion (NE) and an RNA-based RIG-I agonist (IVT) induces potent systemic and mucosal immune responses in protein-based SARS-CoV-2 vaccines administered intranasally (IN). Herein, we demonstrate priming IM with mRNA followed by heterologous IN boosting with NE/IVT adjuvanted recombinant antigen induces strong mucosal and systemic antibody responses and enhances antigen-specific T cell responses in mucosa-draining lymph nodes compared to IM/IM and IN/IN prime/boost regimens. While all regimens induced cross-neutralizing antibodies against divergent variants and sterilizing immunity in the lungs of challenged mice, mucosal vaccination, either as homologous prime/boost or heterologous IN boost after IM mRNA prime was required to impart sterilizing immunity in the upper respiratory tract. Our data demonstrate the benefit of hybrid regimens whereby strong immune responses primed via IM vaccination are rerouted by IN vaccination to mucosal sites to provide optimal protection to SARS-CoV-2.

Multicomponent intranasal adjuvant for mucosal and durable systemic SARS-CoV-2 immunity in young and aged mice.

Multiple FDA-approved SARS-CoV-2 vaccines currently provide excellent protection against severe disease. Despite this, immunity can wane relatively fast, particularly in the elderly and novel viral variants capable of evading infection- and vaccination-induced immunity continue to emerge. Intranasal (IN) vaccination more effectively induces mucosal immune responses than parenteral vaccines, which would improve protection and reduce viral transmission. Here, we developed a rationally designed IN adjuvant consisting of a combined nanoemulsion (NE)-based adjuvant and an RNA-based RIG-I agonist (IVT DI) to drive more robust, broadly protective antibody and T cell responses. We previously demonstrated this combination adjuvant (NE/IVT) potently induces protective immunity through synergistic activation of an array of innate receptors. We now demonstrate that NE/IVT with the SARS-CoV-2 receptor binding domain (RBD), induces robust and durable humoral, mucosal, and cellular immune responses of equivalent magnitude and quality in young and aged mice. This contrasted with the MF59-like intramuscular adjuvant, Addavax, which showed a decrease in immunogenicity with age. Robust antigen-specific IFN-γ/IL-2/TNF-α was induced in both young and aged NE/IVT-immunized animals, which is significant as their reduced production is associated with suboptimal protective immunity in the elderly. These findings highlight the potential of adjuvanted mucosal vaccines for improving protection against COVID-19.

Wild-type SARS-CoV-2 neutralizing immunity decreases across variants and over time but correlates well with diagnostic testing.

Importance: The degree of immune protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants provided by infection versus vaccination with wild-type virus remains unresolved, which could influence future vaccine strategies. The gold-standard for assessing immune protection is viral neutralization; however, few studies involve a large-scale analysis of viral neutralization against the Omicron variant by sera from individuals infected with wild-type virus. Objectives: 1) To define the degree to which infection versus vaccination with wild-type SARS-CoV-2 induced neutralizing antibodies against Delta and Omicron variants.2) To determine whether clinically available data, such as infection/vaccination timing or antibody status, can predict variant neutralization. Methods: We examined a longitudinal cohort of 653 subjects with sera collected three times at 3-to-6-month intervals from April 2020 to June 2021. Individuals were categorized according to SARS-CoV-2 infection and vaccination status. Spike and nucleocapsid antibodies were detected via ADVIA Centaur® (Siemens) and Elecsys® (Roche) assays, respectively. The Healgen Scientific® lateral flow assay was used to detect IgG and IgM spike antibody responses. Pseudoviral neutralization assays were performed on all samples using human ACE2 receptor-expressing HEK-293T cells infected with SARS-CoV-2 spike protein pseudotyped lentiviral particles for wild-type (WT), B.1.617.2 (Delta), and B.1.1.529 (Omicron) variants. Results: Vaccination after infection led to the highest neutralization titers at all timepoints for all variants. Neutralization was also more durable in the setting of prior infection versus vaccination alone. Spike antibody clinical testing effectively predicted neutralization for wild-type and Delta. However, nucleocapsid antibody presence was the best independent predictor of Omicron neutralization. Neutralization of Omicron was lower than neutralization of either wild-type or Delta virus across all groups and timepoints, with significant activity only present in patients that were first infected and later immunized. Conclusions: Participants having both infection and vaccination with wild-type virus had the highest neutralizing antibody levels against all variants and had persistence of activity. Neutralization of WT and Delta virus correlated with spike antibody levels against wild-type and Delta variants, but Omicron neutralization was better correlated with evidence of prior infection. These data help explain why 'breakthrough' Omicron infections occurred in previously vaccinated individuals and suggest better protection is observed in those with both vaccination and previous infection. This study also supports the concept of future SARS-CoV-2 Omicron-specific vaccine boosters.

A multicomponent intranasal adjuvant drives durable humoral, cellular, and mucosal immune responses to SARS-CoV-2 in young and aged mice.

Multiple FDA-approved SARS-CoV-2 vaccines provide excellent protection against severe disease. Despite this, immunity can wane relatively fast, particularly in the elderly and novel viral variants capable of evading infection- and vaccination-induced immunity continue to emerge. Intranasal (IN) vaccination more effectively induces mucosal immune responses than parenteral vaccines, which would improve protection and reduce viral transmission. Here, we developed a rationally designed IN adjuvant consisting of a combined nanoemulsion (NE)-based adjuvant and an RNA-based RIG-I agonist (IVT DI) to drive more robust, broadly protective antibody and T cell responses. We previously demonstrated this combination adjuvant (NE/IVT) potently induces protective immunity through synergistic activation of an array of innate receptors. We now demonstrate that NE/IVT with the SARS-CoV-2 receptor binding domain (RBD), induces robust and durable humoral, mucosal, and cellular immune responses of equivalent magnitude and quality in young and aged mice. This contrasted with the MF59-like intramuscular adjuvant, Addavax, which showed a marked decrease in immunogenicity with age. Robust antigen-specific IFNγ/IL-2/TNF-α was induced in both young and aged NE/IVT-immunized animals, which is significant as their reduced production is associated with suboptimal protective immunity in the elderly. These findings highlight the potential of adjuvanted mucosal vaccines for improving protection against COVID-19.

The unfulfilled potential of mucosal immunization.

Recent events involving the global coronavirus pandemic have focused attention on vaccination strategies. Although tremendous advances have been made in subcutaneous and intramuscular vaccines during this time, one area that has lagged in implementation is mucosal immunization. Mucosal immunization provides several potential advantages over subcutaneous and intramuscular routes, including protection from localized infection at the site of entry, clearance of organisms on mucosal surfaces, induction of long-term immunity through establishment of central and tissue-resident memory cells, and the ability to shape regulatory responses. Despite these advantages, significant barriers remain to achieving effective mucosal immunization. The epithelium itself provides many obstacles to immunization, and the activation of immune recognition and effector pathways that leads to mucosal immunity has been difficult to achieve. This review will highlight the potential advantages of mucosal immunity, define the barriers to mucosal immunization, examine the immune mechanisms that need to be activated on mucosal surfaces, and finally address recent developments in methods for mucosal vaccination that have shown promise in generating immunity on mucosal surfaces in human trials.

A Combination Adjuvant for the Induction of Potent Antiviral Immune Responses for a Recombinant SARS-CoV-2 Protein Vaccine.

Several SARS-CoV-2 vaccines have received EUAs, but many issues remain unresolved, including duration of conferred immunity and breadth of cross-protection. Adjuvants that enhance and shape adaptive immune responses that confer broad protection against SARS-CoV-2 variants will be pivotal for long-term protection as drift variants continue to emerge. We developed an intranasal, rationally designed adjuvant integrating a nanoemulsion (NE) that activates TLRs and NLRP3 with an RNA agonist of RIG-I (IVT DI). The combination adjuvant with spike protein antigen elicited robust responses to SARS-CoV-2 in mice, with markedly enhanced TH1-biased cellular responses and high virus-neutralizing antibody titers towards both homologous SARS-CoV-2 and a variant harboring the N501Y mutation shared by B1.1.7, B.1.351 and P.1 variants. Furthermore, passive transfer of vaccination-induced antibodies protected naive mice against heterologous viral challenge. NE/IVT DI enables mucosal vaccination, and has the potential to improve the immune profile of a variety of SARS-CoV-2 vaccine candidates to provide effective cross-protection against future drift variants.

Silver Nanoparticles Enhance Antimicrobial Efficacy of Antibiotics and Restore That Efficacy against the Melioidosis Pathogen.

Melioidosis is an infectious disease caused by Gram-negative bacillus bacteria Burkholderia pseudomallei. Due to the emerging resistance of B. pseudomallei to antibiotics including ceftazidime (CAZ), the development of novel antibiotics and alternative modes of treatment has become an urgent issue. Here, we demonstrated an ability to synergistically increase the efficiency of antibiotics through their combination with silver nanoparticles (AgNPs). Combinations of four conventional antibiotics including CAZ, imipenem (IMI), meropenem (MER), and gentamicin sulfate (GENT) with starch-stabilized AgNPs were tested for their antibacterial effects against three isolates of B. pseudomallei. The combination of each antibiotic with AgNPs featured fractional inhibitory concentration (FIC) index values and fractional bactericidal concentration (FBC) index values ranging from 0.312 to 0.75 µg/mL and 0.252 to 0.625 µg/mL, respectively, against the three isolates of B. pseudomallei. The study clearly showed that most of the combinatorial treatments exhibited synergistic antimicrobial effects against all three isolates of B. pseudomallei. The highest enhancing effect was observed for GENT with AgNPs. These results confirmed the combination of each antibiotic with AgNPs restored their bactericidal potency in the bacterial strains that had previously been shown to be resistant to the antibiotics. In addition, morphological changes examined by SEM confirmed that the bacterial cells were severely damaged by combinations at the FBC level. Although bacteria produce fibers to protect themselves, ultimately the bacteria were killed by the antibiotic-AgNPs combinations. Overall, these results suggest the study of antibiotic-AgNPs combinations as an alternative design strategy for potential therapeutics to more effectively combat the melioidosis pathogen.

A Combination Adjuvant for the Induction of Potent Antiviral Immune Responses for a Recombinant SARS-CoV-2 Protein Vaccine.

Several SARS-CoV-2 vaccines have received EUAs, but many issues remain unresolved, including duration of conferred immunity and breadth of cross-protection. Adjuvants that enhance and shape adaptive immune responses that confer broad protection against SARS-CoV-2 variants will be pivotal for long-term protection. We developed an intranasal, rationally designed adjuvant integrating a nanoemulsion (NE) that activates TLRs and NLRP3 with an RNA agonist of RIG-I (IVT DI). The combination adjuvant with spike protein antigen elicited robust responses to SARS-CoV-2 in mice, with markedly enhanced T H 1-biased cellular responses and high virus-neutralizing antibody titers towards both homologous SARS-CoV-2 and a variant harboring the N501Y mutation shared by B1.1.7, B.1.351 and P.1 variants. Furthermore, passive transfer of vaccination-induced antibodies protected naive mice against heterologous viral challenge. NE/IVT DI enables mucosal vaccination, and has the potential to improve the immune profile of a variety of SARS-CoV-2 vaccine candidates to provide effective cross-protection against future drift variants.

Combined Intranasal Nanoemulsion and RIG-I Activating RNA Adjuvants Enhance Mucosal, Humoral, and Cellular Immunity to Influenza Virus.

Current influenza virus vaccines are focused on humoral immunity and are limited by the short duration of protection, narrow cross-strain efficacy, and suboptimal immunogenicity. Here, we combined two chemically and biologically distinct adjuvants, an oil-in-water nanoemulsion (NE) and RNA-based agonists of RIG-I, to determine whether the diverse mechanisms of these adjuvants could lead to improved immunogenicity and breadth of protection against the influenza virus. NE activates TLRs, stimulates immunogenic apoptosis, and enhances cellular antigen uptake, leading to a balanced TH1/TH2/TH17 response when administered intranasally. RIG-I agonists included RNAs derived from Sendai and influenza viral defective interfering RNAs (IVT DI, 3php, respectively) and RIG-I/TLR3 agonist, poly(I:C) (pIC), which induce IFN-Is and TH1-polarized responses. NE/RNA combined adjuvants potentially allow for costimulation of multiple innate immune receptor pathways, more closely mimicking patterns of activation occurring during natural viral infection. Mice intranasally immunized with inactivated A/Puerto Rico/8/1934 (H1N1) (PR/8) adjuvanted with NE/IVT DI or NE/3php (but not NE/pIC) showed synergistic enhancement of systemic PR/8-specific IgG with significantly greater avidity and virus neutralization activity than the individual adjuvants. Notably, NE/IVT DI induced protective neutralizing titers after a single immunization. Hemagglutinin stem-specific antibodies were also improved, allowing recognition of heterologous and heterosubtypic hemagglutinins. All NE/RNAs elicited substantial PR/8-specific sIgA. Finally, a unique cellular response with enhanced TH1/TH17 immunity was induced with the NE/RNAs. These results demonstrate that the enhanced immunogenicity of the adjuvant combinations was synergistic and not simply additive, highlighting the potential value of a combined adjuvant approach for improving the efficacy of vaccination against the influenza virus.

Shielded α-Nucleophile Nanoreactor for Topical Decontamination of Reactive Organophosphate.

Here, we describe a nanoscale reactor strategy with a topical application in the therapeutic decontamination of reactive organophosphates (OPs) as chemical threat agents. It involves functionalization of poly(amidoamine) dendrimer through a combination of its partial PEG shielding and exhaustive conjugation with an OP-reactive α-nucleophile moiety at its peripheral branches. We prepared a 16-member library composed of two α-nucleophile classes (oxime, hydroxamic acid), each varying in its reactor valency (43-176 reactive units per nanoparticle), and linker framework for α-nucleophile tethering. Their mechanism for OP inactivation occurred via nucleophilic catalysis as verified against P-O and P-S bonded OPs including paraoxon-ethyl (POX), malaoxon, and omethoate by 1H NMR spectroscopy. Screening their reactivity for POX inactivation was performed under pH- and temperature-controlled conditions, which resulted in identifying 13 conjugates, each showing shorter POX half-life up to 2 times as compared to a reference Dekon 139 at pH 10.5, 37 °C. Of these, 10 conjugates were further confirmed for greater efficacy in POX decontamination experiments performed in two skin models, porcine skin and an artificial human microtissue. Finally, a few lead conjugates were selected and demonstrated for their biocompatibility in vitro as evident with lack of skin absorption, no inhibition of acetylcholinesterase (AChE), and no cytotoxicity in human neuroblastoma cells. In summary, this study presents a novel nanoreactor library, its screening methods, and identification of potent lead conjugates with potential for therapeutic OP decontamination.

Reactivity and mechanism of α-nucleophile scaffolds as catalytic organophosphate scavengers.

Despite their unique benefits imparted by their structure and reactivity, certain α-nucleophile molecules remain underexplored as chemical inactivators for the topical decontamination of reactive organophosphates (OPs). Here, we present a library of thirty α-nucleophile scaffolds, each designed with either a pyridinium aldoxime (PAM) or hydroxamic acid (HA) α-nucleophile core tethered to a polar or charged scaffold for optimized physicochemical properties and reactivity. These library compounds were screened for their abilities to catalyze the hydrolysis of a model OP, paraoxon (POX), in kinetic assays. These screening experiments led to the identification of multiple lead compounds with the ability to inactivate POX two- to four-times more rapidly than Dekon 139-the active ingredient currently used for skin decontamination of OPs. Our mechanistic studies, performed under variable pH and temperature conditions suggested that the differences in the reactivity and activation energy of these compounds are fundamentally attributable to the core nucleophilicity and pKa. Following their screening and mechanistic studies, select lead compounds were further evaluated and demonstrated greater efficacy than Dekon 139 in the topical decontamination of POX in an ex vivo porcine skin model. In addition to OP reactivity, several compounds in the PAM class displayed a dual mode of activity, as they retained the ability to reactivate POX-inhibited acetylcholine esterase (AChE). In summary, this report describes a rationale for the hydrophilic scaffold design of α-nucleophiles, and it offers advanced insights into their chemical reactivity, mechanism, and practical utility as OP decontaminants.

Hydrophilic scaffolds of oxime as the potent catalytic inactivator of reactive organophosphate.

Despite its efficacy as a skin decontaminant of reactive organophosphates (OP), Dekon 139-a potassium salt of 2,3-butanedione monooxime (DAM)-is associated with adverse events related to percutaneous absorption largely due to its small size and lipophilicity. In order to address this physicochemical issue, we synthesized and evaluated the activity of a focused library of 14 hydrophilic oxime compounds, each designed with either a DAM or monoisonitrosoacetone (MINA) oxime tethered to a polar or charged scaffold in order to optimize the size, hydrophilicity, and oxime acidity. High-throughput colorimetric assays were performed with paraoxon (POX) as a model OP to determine the kinetics of POX inactivation by these compounds under various pH and temperature conditions. This primary screening led to the identification of 6 lead compounds, predominantly in the MINA series, which displayed superb catalytic activity by reducing the POX half-life (t1/2) by 2-3 fold relative to Dekon 139. Our mechanistic studies show that POX inactivation by the oxime compounds occurred faster at a higher temperature and in a pH-dependent manner in which the negatively charged oximate species is ≥ 10-fold more effective than the neutral oxime species. Lastly, using one of the lead compounds, we demonstrated its promising efficacy for POX decontamination in porcine skin ex vivo, and showed its potent ability to protect acetylcholine esterase (AChE) through POX inactivation. In summary, we report the rational design and chemical biological validation of novel hydrophilic oximes which address an unmet need in therapeutic OP decontamination.

Nanoemulsion adjuvant-driven redirection of TH2 immunity inhibits allergic reactions in murine models of peanut allergy.

BACKGROUND: Immunotherapy for food allergies involves progressive increased exposures to food that result in desensitization to food allergens in some subjects but not tolerance to the food. Therefore new approaches to suppress allergic immunity to food are necessary. Previously, we demonstrated that intranasal immunization with a nanoemulsion (NE) adjuvant induces robust mucosal antibody and TH17-polarized immunity, as well as systemic TH1-biased cellular immunity with suppression of pre-existing TH2-biased immunity. OBJECTIVE: We hypothesized that immunization with food in conjunction with the nanoemulsion adjuvant could lead to modulation of allergic reactions in food allergy by altering pre-existing allergic immunity and enhancing mucosal immunity. METHODS: Mice were sensitized to peanut with aluminum hydroxide or cholera toxin. The animals were then administered 3 monthly intranasal immunizations with peanut in the nanoemulsion adjuvant or saline. Mice were then challenged with peanut to examine allergen reactivity. RESULTS: The NE intranasal immunizations resulted in marked decreases in TH2 cytokine, IgG1, and IgE levels, whereas TH1 and mucosal TH17 immune responses were increased. After allergen challenge, these mice showed significant reductions in allergic hypersensitivity. Additionally, the NE immunizations significantly increased antigen-specific IL-10 production and regulatory T-cell counts, and the protection induced by NE was dependent in part on IL-10. Control animals immunized with intranasal peanut in saline had no modulation of their allergic response. CONCLUSIONS: NE adjuvant-mediated induction of mucosal TH17 and systemic TH1-biased immunity can suppress TH2-mediated allergy through multiple mechanisms and protect against anaphylaxis. These results suggest the potential therapeutic utility of this approach in the setting of food allergy.

Photocontrolled Release of Doxorubicin Conjugated through a Thioacetal Photocage in Folate-Targeted Nanodelivery Systems.

Despite their proven ability for precise and targeted release, nanoplatform systems for photocontrolled delivery often face formidable synthetic challenges, in part due to the paucity of advanced linker strategies. Here, we report on a novel linker strategy using a thioacetal ortho-nitrobenzaldehyde (TNB) cage, demonstrating its application for delivery of doxorubicin (Dox) in two nanoscale systems. This photocleavable linker, TNB(OH), which presents two identical arms, each terminated with a hydroxyl functionality, was prepared in a single step from 6-nitroveratraldehyde. TNB(OH) was used to cross-link Dox to a folate receptor (FAR)-targeting poly(amidoamine) dendrimer conjugate G5(FA)n=5.4(Dox)m=5.1, and also used to prepare an upconversion nanocrystal (UCN) conjugate, UCN-PPIX@(Dox)(G5FA), a larger core/shell nanostructure. In this core/shell nanostructure, the UCN core emits UV and visible light luminescence upon near-infrared (NIR) excitation, allowing for the photocleavage of the TNB linker as well as the photostimulation of protoporphyrin IX (PPIX) coupled as a cytotoxic photosensitizer. Drug-release experiments performed in aqueous solutions with long-wavelength ultraviolet A (UVA) light showed that Dox release occurred rapidly from its TNB linked form or from its dendrimer conjugated form with comparable decay kinetics. Cellular toxicity studies in FAR-overexpressing KB carcinoma cells demonstrated that each nanoconjugate lacked intrinsic cytotoxicity until exposed to UVA or NIR (980 nm) (for the UCN nanoconjugate), which resulted in induction of potent cytotoxicity. In summary, this new TNB strategy offers synthetic convenience in drug conjugation chemistry with the ability for the temporal control of drug activation at the delivery site.

Control of an Unusual Photo-Claisen Rearrangement in Coumarin Caged Tamoxifen through an Extended Spacer.

The use of coumarin caged molecules has been well documented in numerous photocaging applications including for the spatiotemporal control of Cre-estrogen receptor (Cre-ERT2) recombinase activity. In this article, we report that 4-hydroxytamoxifen (4OHT) caged with coumarin via a conventional ether linkage led to an unexpected photo-Claisen rearrangement which significantly competed with the release of free 4OHT. The basis for this unwanted reaction appears to be related to the coumarin structure and its radical-based mechanism of uncaging, as it did not occur in ortho-nitrobenzyl (ONB) caged 4OHT that was otherwise linked in the same manner. In an effort to perform design optimization, we introduced a self-immolative linker longer than the ether linkage and identified an optimal linker which allowed rapid 4OHT release by both single-photon and two-photon absorption mechanisms. The ability of this construct to actively control Cre-ERT2 mediated gene modifications was investigated in mouse embryonic fibroblasts (MEFs) in which the expression of a green fluorescent protein (GFP) reporter dependent gene recombination was controlled by 4OHT release and measured by confocal fluorescence microscopy and flow cytometry. In summary, we report the implications of this photo-Claisen rearrangement in coumarin caged compounds and demonstrate a rational linker strategy for addressing this unwanted side reaction.

A Thioacetal Photocage Designed for Dual Release: Application in the Quantitation of Therapeutic Release by Synchronous Reporter Decaging.

Despite the immense potential of existing photocaging technology, its application is limited by the paucity of advanced caging tools. Here, we report on the design of a novel thioacetal ortho-nitrobenzaldehyde (TNB) dual arm photocage that enabled control of the simultaneous release of two payloads linked to a single TNB unit. By using this cage, which was prepared in a single step from commercial 6-nitroverataldehyde, three drug-fluorophore conjugates were synthesized: Taxol-TNB-fluorescein, Taxol-TNB-coumarin, and doxorubicin-TNB-coumarin, and long-wavelength UVA light-triggered release experiments demonstrated that dual payload release occurred with rapid decay kinetics for each conjugate. In cell-based assays performed in vitro, dual release could also be controlled by UV exposure, resulting in increased cellular fluorescence and cytotoxicity with potency equal to that of unmodified drug towards the KB carcinoma cell line. The extent of such dual release was quantifiable by reporter fluorescence measured in situ and was found to correlate with the extent of cytotoxicity. Thus, this novel dual arm cage strategy provides a valuable tool that enables both active control and real-time monitoring of drug activation at the delivery site.

Light-controlled active release of photocaged ciprofloxacin for lipopolysaccharide-targeted drug delivery using dendrimer conjugates.

We report an active delivery mechanism targeted specifically to Gram(-) bacteria based on the photochemical release of photocaged ciprofloxacin carried by a cell wall-targeted dendrimer nanoconjugate.

Modular Integration of Upconverting Nanocrystal-Dendrimer Composites for Folate Receptor-Specific NIR Imaging and Light-Triggered Drug Release.

Upconversion nanocrystals (UCNs) display near-infrared (NIR)-responsive photoluminescent properties for NIR imaging and drug delivery. The development of effective strategies for UCN integration with other complementary nanostructures for targeting and drug conjugation is highly desirable. This study reports on a core/shell-based theranostic system designed by UCN integration with a folate (FA)-conjugated dendrimer for tumor targeting and with photocaged doxorubicin as a cytotoxic agent. Two types of UCNs (NaYF4:Yb/Er (or Yb/Tm); diameter = ≈50 to 54 nm) are described, each displaying distinct emission properties upon NIR (980 nm) excitation. The UCNs are surface modified through covalent attachment of photocaged doxorubicin (ONB-Dox) and a multivalent FA-conjugated polyamidoamine (PAMAM) dendrimer G5(FA)6 to prepare UCN@(ONB-Dox)(G5FA). Surface plasmon resonance experiments performed with G5(FA)6 dendrimer alone show nanomolar binding avidity (KD = 5.9 × 10(-9) M) to the folate binding protein. This dendrimer binding corresponds with selective binding and uptake of UCN@(ONB-Dox)(G5FA) by FAR-positive KB carcinoma cells in vitro. Furthermore, UCN@(ONB-Dox)(G5FA) treatment of FAR(+) KB cells inhibits cell growth in a light dependent manner. These results validate the utility of modularly integrated UCN-dendrimer nanocomposites for cell type specific NIR imaging and light-controlled drug release, thus serving as a new theranostic system.

Mechanism of Cooperativity and Nonlinear Release Kinetics in Multivalent Dendrimer-Atropine Complexes.

Despite extensive studies on drug delivery using multivalent complexation systems, the biophysical basis for release kinetics remains poorly defined. The present study addresses this aspect involved in the complexation of a fifth generation poly(amidoamine) (PAMAM) dendrimer with atropine, an essential antidote used for treating organophosphate poisoning. First, we designed (1)H NMR titration studies for determining the molecular basis of the drug complexation with a glutarate-modified anionic dendrimer. These provide evidence pointing to a combination of electrostatic and hydrophobic interactions as the driving forces for dendrimer complexation with the alkaloid drug molecule. Second, using LC-MS/MS spectrometry, we determined the dissociation constants (KD) at steady state and also measured the drug release kinetics of atropine complexes with four negatively charged dendrimer types. Each of these dendrimers has a high payload capacity for up to ∼ 100 atropine molecules. However, the affinity of the atropine to the carrier was highly dependent on the drug to dendrimer ratio. Thus, a complex made at a lower loading ratio (≤ 0.1) displayed greater atropine affinity (KD ≈ µM) than other complexes prepared at higher ratios (>10), which showed only mM affinity. This negative cooperative variation in affinity is tightly associated with the nonlinear release kinetics observed for each complex in which drug release occurs more slowly at the later time phase at a lower loading ratio. In summary, the present study provides novel insights on the cooperativity as the mechanistic basis for nonlinear release kinetics observed in multivalent carrier systems.

Formulation, high throughput in vitro screening and in vivo functional characterization of nanoemulsion-based intranasal vaccine adjuvants.

Vaccine adjuvants have been reported to induce both mucosal and systemic immunity when applied to mucosal surfaces and this dual response appears important for protection against certain pathogens. Despite the potential advantages, however, no mucosal adjuvants are currently approved for human use. Evaluating compounds as mucosal adjuvants is a slow and costly process due to the need for lengthy animal immunogenicity studies. We have constructed a library of 112 intranasal adjuvant candidate formulations consisting of oil-in-water nanoemulsions that contain various cationic and nonionic surfactants. To facilitate adjuvant development we first evaluated this library in a series of high-throughput, in vitro assays for activities associated with innate and adaptive immune activation in vivo. These in vitro assays screened for the ability of the adjuvant to bind to mucin, induce cytotoxicity, facilitate antigen uptake in epithelial and dendritic cells, and activate cellular pathways. We then sought to determine how these parameters related to adjuvant activity in vivo. While the in vitro assays alone were not enough to predict the in vivo adjuvant activity completely, several interesting relationships were found with immune responses in mice. Furthermore, by varying the physicochemical properties of the surfactant components (charge, surfactant polar head size and hydrophobicity) and the surfactant blend ratio of the formulations, the strength and type of the immune response generated (TH1, TH2, TH17) could be modulated. These findings suggest the possibility of using high-throughput screens to aid in the design of custom adjuvants with unique immunological profiles to match specific mucosal vaccine applications.