A novel intranasal peptide vaccine inhibits non-small cell lung cancer with KRAS mutation.

KRAS mutations occur commonly in the lung and can lead to the development of non-small cell lung cancer (NSCLC). While the mutated KRAS protein is a neoantigen, it usually does not generate an effective anti-tumor immune response on mucosal/epithelial surfaces. Despite this, mutated KRAS remains a potential target for immunotherapy since immune targeting of this protein in animal models has been effective at eliminating tumor cells. We attempted to develop a KRAS vaccine using mutated and wild-type KRAS peptides in combination with a nanoemulsion (NE) adjuvant. The efficacy of this approach was tested in an inducible mutant KRAS-mouse lung tumor model. Animals were immunized intranasally using NE with KRAS peptides. These animals had decreased CD4+FoxP3+ T cells in both lymph nodes and spleen. Immunized animals also showed higher IFN-γ and IL-17a levels to mutated KRAS that were produced by CD8+ T cells and enhancement in KRAS-specific Th1 and Th17 responses that persisted for 3 months after the last vaccination. Importantly, the immunized animals had significantly decreased tumor incidence compared to control animals. In conclusion, a mucosal approach to KRAS vaccination demonstrated the ability to induce local KRAS-specific immune responses in the lung and resulted in reduced tumor incidence.

Delicate Hybrid Laponite-Cyclic Poly(ethylene glycol) Nanoparticles as a Potential Drug Delivery System.

The objective of the study was to explore the feasibility of a new drug delivery system using laponite (LAP) and cyclic poly(ethylene glycol) (cPEG). Variously shaped and flexible hybrid nanocrystals were made by both the covalent and physical attachment of chemically homogeneous cyclized PEG to laponite nanodisc plates. The size of the resulting, nearly spherical particles ranged from 1 to 1.5 µm, while PEGylation with linear methoxy poly (ethylene glycol) (mPEG) resulted in fragile sheets of different shapes and sizes. When infused with 10% doxorubicin (DOX), a drug commonly used in the treatment of various cancers, the LAP-cPEG/DOX formulation was transparent and maintained liquid-like homogeneity without delamination, and the drug loading efficiency of the LAP-cPEG nano system was found to be higher than that of the laponite-poly(ethylene glycol) LAP-mPEG system. Furthermore, the LAP-cPEG/DOX formulation showed relative stability in phosphate-buffered saline (PBS) with only 15% of the drug released. However, in the presence of human plasma, about 90% of the drug was released continuously over a period of 24 h for the LAP-cPEG/DOX, while the LAP-mPEG/DOX formulation released 90% of DOX in a 6 h burst. The results of the cell viability assay indicated that the LAP-cPEG/DOX formulation could effectively inhibit the proliferation of A549 lung carcinoma epithelial cells. With the DOX concentration in the range of 1-2 µM in the LAP-cPEG/DOX formulation, enhanced drug effects in both A549 lung carcinoma epithelial cells and primary lung epithelial cells were observed compared to LAP-mPEG/DOX. The unique properties and effects of cPEG nanoparticles provide a potentially better drug delivery system and generate interest for further targeting studies and applications.

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.

Measuring the Adhesion Forces for the Multivalent Binding of Vancomycin-Conjugated Dendrimer to Bacterial Cell-Wall Peptide.

Multivalent ligand-receptor interaction provides the fundamental basis for the hypothetical notion that high binding avidity relates to the strong force of adhesion. Despite its increasing importance in the design of targeted nanoconjugates, an understanding of the physical forces underlying the multivalent interaction remains a subject of urgent investigation. In this study, we designed three vancomycin (Van)-conjugated dendrimers G5(Van) n ( n = mean valency = 0, 1, 4) for bacterial targeting with generation 5 (G5) poly(amidoamine) dendrimer as a multivalent scaffold and evaluated both their binding avidity and physical force of adhesion to a bacterial model surface by employing surface plasmon resonance (SPR) spectroscopy and atomic force microscopy. The SPR experiment for these conjugates was performed in a biosensor chip surface immobilized with a bacterial cell-wall peptide Lys-d-Ala-d-Ala. Of these, G5(Van)4 bound most tightly with a KD of 0.34 nM, which represents an increase in avidity by 2 or 3 orders of magnitude relative to a monovalent conjugate G5(Van)1 or free vancomycin, respectively. By single-molecule force spectroscopy, we measured the adhesion force between G5(Van) n and the same cell-wall peptide immobilized on the surface. The distribution of adhesion forces increased in proportion to vancomycin valency with the mean force of 134 pN at n = 4 greater than 96 pN at n = 1 at a loading rate of 5200 pN/s. In summary, our results are strongly supportive of the positive correlation between the avidity and adhesion force in the multivalent interaction of vancomycin nanoconjugates.

Oritavancin Retains a High Affinity for a Vancomycin-Resistant Cell-Wall Precursor via Its Bivalent Motifs of Interaction.

Despite its potent antibacterial activities against drug-resistant Gram-positive pathogens, oritavancin remains partially understood with respect to its primary mode of hydrogen bond interaction with a cell-wall peptide regarding the role of its lipophilic 4'-chlorobiphenyl moiety. Here we report a surface plasmon resonance (SPR) study performed in two cell-wall model surfaces, each prepared by immobilization with a vancomycin-susceptible Lys-d-Ala-d-Ala or vancomycin-resistant Lys-d-Ala-d-Lac peptide. Analysis of binding kinetics performed on the peptide surface showed that oritavancin bound ∼100-1000-fold more tightly than vancomycin on each model surface. Ligand competition experiments conducted by SPR and fluorescence spectroscopy provided evidence that such affinity enhancement can be attributed to its 4'-chlorobiphenyl moiety, possibly through a hydrophobic interaction that led to a gain of free energy with a contribution from enthalpy as suggested by a variable-temperature SPR experiment. On the basis of these findings, we propose a model for the bivalent motifs of interaction of oritavancin with cell-wall peptides, by which the drug molecule can retain a strong interaction even with the vancomycin-resistant peptide. In summary, this study advances our understanding of oritavancin and offers new insight into the significance of bivalent motifs in the design of glycopeptide antibiotics.

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.

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.

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.

Force spectroscopy of multivalent binding of riboflavin-conjugated dendrimers to riboflavin binding protein.

Putative riboflavin receptors are considered as biomarkers due to their overexpression in breast and prostate cancers. Hence, these receptors can be potentially exploited for use in targeted drug delivery systems where dendrimer nanoparticles with multivalent ligand attachments can lead to greater specificity in cellular interactions. In this study, the single molecule force spectroscopy technique was used to assess the physical strength of multivalent interactions by employing a riboflavin (RF)-conjugated generation 5 PAMAM dendrimer G5(RF)n nanoparticle. By varying the average RF ligand valency (n = 0, 3, 5), the rupture force was measured between G5(RF)n and the riboflavin binding protein (RFBP). The rupture force increased when the valency of RF increased. We observed at the higher valency (n = 5) three binding events that increased in rupture force with increasing loading rate. Assuming a single energy barrier, the Bell-Evans model was used to determine the kinetic off-rate and barrier width for all binding interactions. The analysis of our results appears to indicate that multivalent interactions are resulting in changes to rupture force and kinetic off-rates.

4-Hydroxytamoxifen probes for light-dependent spatiotemporal control of Cre-ER mediated reporter gene expression.

The tamoxifen inducible Cre-ER/loxP system provides tissue specific temporal control of gene recombination events, and can be used to induce expression of reporter genes (e.g. GFP, LacZ) for lineage tracing studies. Cre enzyme fused with estrogen receptor (Cre-ER) is released upon tamoxifen binding, resulting in permanent activation of reporter genes within cells and their progeny. Tamoxifen and its active metabolite, hydroxytamoxifen (4OHT) diffuses rapidly in vivo, making it difficult to restrict labeling to specific locations. In this study, we developed a photocaged 4OHT molecule by covalently attaching 4OHT to an ortho-nitrobenzyl (ONB1) group, rendering 4OHT inactive. Exposure to UV radiation cleaves the bond between ONB1 and 4OHT, freeing the 4OHT to bind Cre-ER to result in downstream genetic recombination and reporter activation. We show that caged ONB1-4OHT crosses the cell membrane and uncages after short UV exposure, resulting in Cre-driven genetic recombination that can be localized to specific regions or tissues. ONB1-4OHT can provide spatial control of reporter activation and be adapted with any existing Cre-ER/loxP based system.

Multivalent dendrimer vectors with DNA intercalation motifs for gene delivery.

Poly(amido amine) (PAMAM) dendrimers constitute an important class of nonviral, cationic vectors in gene delivery. Here we report on a new concept for dendrimer vector design based on the incorporation of dual binding motifs: DNA intercalation, and receptor recognition for targeted delivery. We prepared a series of dendrimer conjugates derived from a fifth generation (G5) PAMAM dendrimer, each conjugated with multiple folate (FA) or riboflavin (RF) ligands for cell receptor targeting, and with 3,8-diamino-6-phenylphenanthridinium ("DAPP")-derived ligands for anchoring a DNA payload. Polyplexes of each dendrimer with calf thymus dsDNA were made and characterized by surface plasmon resonance (SPR) spectroscopy, dynamic light scattering (DLS) and zeta potential measurement. These studies provided evidence supporting polyplex formation based on the observation of tight DNA-dendrimer adhesion, and changes in particle size and surface charge upon coincubation. Further SPR studies to investigate the adhesion of the polyplex to a model surface immobilized with folate binding protein (FBP), demonstrated that the DNA payload has only a minimal effect on the receptor binding activity of the polyplex: KD = 0.22 nM for G5(FA)(DAPP) versus 0.98 nM for its polyplex. Finally, we performed in vitro transfection assays to determine the efficiency of conjugate mediated delivery of a luciferase-encoding plasmid into the KB cancer cell line and showed that RF-conjugated dendrimers were 1 to 2 orders of magnitude more effective in enhancing luciferase gene transfection than a plasmid only control. In summary, this study serves as a proof of concept for DNA-ligand intercalation as a motif in the design of multivalent dendrimer vectors for targeted gene delivery.

Design considerations for PAMAM dendrimer therapeutics.

We have previously shown that methotrexate (MTX) conjugated to a cancer-specific poly amido amine (PAMAM) dendrimer has a higher therapeutic index than MTX alone. Unfortunately, these therapeutics have been difficult to advance because of the complicated syntheses and an incomplete understanding of the dendrimer properties. We wished to address these obstacles by using copper-free click chemistry to functionalize the dendrimer scaffolds and to exploring the effects of two dendrimer properties (the targeting ligand and drug linkage) on cytotoxicity. We conjugated either ester or amide-linker modified MTX to dendrimer scaffolds with or without folic acid (FA). Because of multivalency, the FA and MTX functionalized dendrimers had similar capacities to target the folate receptor on cancer cells. Additionally, we found that the ester- and amide-linker modified MTX compounds had similar cytotoxicity but the dendrimer-ester MTX conjugates were much more cytotoxic than the dendrimer-amide MTX conjugates. These results clarify the impact of these properties on therapeutic efficacy and will allow us to design more effective polymer therapeutics.

The synthesis of a c(RGDyK) targeted SN38 prodrug with an indolequinone structure for bioreductive drug release.

Preparation of a novel c(RGDyK) targeted SN38 prodrug incorporating an indolequinone structure for bioreductively triggered drug release is described. This design yields a prodrug that targets surface molecules on tumor cells (alpha(v)beta(3) integrins) and releases drug under bioreductive conditions. There are three moieties in the prodrug design, namely a therapeutic drug SN38, an indolequinone structure serving as a drug releasing trigger, and an alpha(v)beta(3) integrin targeting peptide c(RGDyK). Preliminary studies showed that SN38 is released in the presence of a bioreductive enzyme (DT-diaphorase).