Esterase-Activated, pH-Responsive, and Genetically Targetable Nano-Prodrug for Cancer Cell Photo-Ablation.

Activatable prodrugs have drawn considerable attention for cancer cell ablation owing to their high specificity in drug delivery systems. However, phototheranostic prodrugs with dual organelle-targeting and synergistic effects are still rare due to low intelligence of their structures. Besides, the cell membrane, exocytosis, and diffusional hindrance by the extracellular matrix reduce drug uptake. Moreover, the up-regulation of heat shock protein and short singlet-oxygen lifetime in cancer cells hamper photo-ablation efficacy, especially in the mono-therapeutic model. To overcome those obstacles, we prepare an esterase-activated DM nano-prodrug, which is conjugated by diiodine-substituted fluorogenic malachite green derivative (MG-2I) and phototherapeutic agent DPP-OH via hydrolyzable ester linkage, having pH-responsiveness and genetically targetable activity for dual organelles-targeting to optimize photo-ablation efficacy. The DM nanoparticles (NPs) present improved pH-responsive photothermal/photodynamic property by the protonation of diethylaminophenyl units in acidic environment. More importantly, the MG-2I and DPP-OH moieties can be released from DM nano-prodrug through overexpressed esterase; then specifically target lysosomes and mitochondria in CT-26 Mito-FAP cells. Hence, near-infrared DM NPs can trigger parallel damage in dual-organelles with strong fluorescence and effective phototoxicity, thus inducing serious mitochondrial dysfunction and apoptotic death, showing excellent photo-ablation effect based on esterase-activated, pH-responsive, and genetically targetable activities.

Permeation of macromolecules into the renal glomerular basement membrane and capture by the tubules.

How the kidney prevents urinary excretion of plasma proteins continues to be debated. Here, using unfixed whole-mount mouse kidneys, we show that fluorescent-tagged proteins and neutral dextrans permeate into the glomerular basement membrane (GBM), in general agreement with Ogston's 1958 equation describing how permeation into gels is related to molecular size. Electron-microscopic analyses of kidneys fixed seconds to hours after injecting gold-tagged albumin, negatively charged gold nanoparticles, and stable oligoclusters of gold nanoparticles show that permeation into the lamina densa of the GBM is size-sensitive. Nanoparticles comparable in size with IgG dimers do not permeate into it. IgG monomer-sized particles permeate to some extent. Albumin-sized particles permeate extensively into the lamina densa. Particles traversing the lamina densa tend to accumulate upstream of the podocyte glycocalyx that spans the slit, but none are observed upstream of the slit diaphragm. At low concentrations, ovalbumin-sized nanoparticles reach the primary filtrate, are captured by proximal tubule cells, and are endocytosed. At higher concentrations, tubular capture is saturated, and they reach the urine. In mouse models of Pierson's or Alport's proteinuric syndromes resulting from defects in GBM structural proteins (laminin ß2 or collagen α3 IV), the GBM is irregularly swollen, the lamina densa is absent, and permeation is increased. Our observations indicate that size-dependent permeation into the lamina densa of the GBM and the podocyte glycocalyx, together with saturable tubular capture, determines which macromolecules reach the urine without the need to invoke direct size selection by the slit diaphragm.

Nanocrystal Formulation Improves Vaginal Delivery of CSIC for HIV Prevention.

5-Chloro-3-phenylsulfonylindole-2-carboxamide (CSIC) is a highly potent non-nucleoside reverse transcriptase inhibitor (NNRTI) with potential for use in topical prophylaxis against HIV transmission. However, the hydrophobic nature of CSIC limits its administration through vaginal route. In this study, we developed nanocrystals of CSIC to potentially improve the aqueous solubility and intracellular uptake of CSIC in vitro and in vivo. CSIC nanocrystals were manufactured and stabilized with Pluronic F98 and hydroxypropyl methylcellulose E5. Transmission electron microscopy showed CSIC nanocrystals to be needle-like. Dynamic light scattering measurements showed a hydrodynamic size of 243 nm (polydispersity index < 0.3) and near neutral surface charge (- 7.8 mV). Particle size was maintained for at least 7 days in the liquid state and for at least 5 months after lyophilization. Drug content in the CSIC nanocrystal formulation (nanosuspension) was 0.8 mg/mL, which is 1000 times higher than the aqueous solubility of CSIC. In vitro release study showed that over 90% of CSIC was released from the nanocrystal formulation in a linear fashion over a period of 4 days. Importantly, CSIC nanocrystals showed equivalent cell-based anti-HIV activity (EC50 ~ 1 nM) as that of non-formulated drug. In vitro studies demonstrated rapid macrophage uptake of CSIC nanocrystals via both energy-dependent (endocytosis) and independent processes. In vivo studies in Swiss Webster female mice showed that the nanocrystal formulation significantly improved CSIC delivery to mouse cervicovaginal tissues following intravaginal instillation. In summary, nanocrystals are a promising formulation approach for topical delivery of CSIC for protection against HIV sexual transmission.

Monotherapy and Combination Therapy Using Anti-Angiogenic Nanoagents to Fight Cancer.

Anti-angiogenic therapy, targeting vascular endothelial cells (ECs) to prevent tumor growth, has been attracting increasing attention in recent years, beginning with bevacizumab (Avastin) through its Phase II/III clinical trials on solid tumors. However, these trials showed only modest clinical efficiency; moreover, anti-angiogenic therapy may induce acquired resistance to the drugs employed. Combining advanced drug delivery techniques (e.g., nanotechnology) or other therapeutic strategies (e.g., chemotherapy, radiotherapy, phototherapy, and immunotherapy) with anti-angiogenic therapy results in significantly synergistic effects and has opened a new horizon in fighting cancer. Herein, clinical difficulties in using traditional anti-angiogenic therapy are discussed. Then, several promising applications of anti-angiogenic nanoagents in monotherapies and combination therapies are highlighted. Finally, the challenges and perspectives of anti-angiogenic cancer therapy are summarized. A useful introduction to anti-angiogenic strategies, which may significantly improve therapeutic outcomes, is thus provided.

Long-term persistence and spectral blue shifting of quantum dots in vivo.

Quantum dots are a powerful fluorophore family with desirable attributes for fluorescence imaging. They have been used in several animal models with direct clinical relevance, including sentinel lymph node mapping, tracing vasculature and lymphatics, and targeting specific lesions for diagnosis and removal. (1-12) Despite significant interest for use in translational applications, little is known about the persistence and long-term fate of quantum dots in vivo. We have observed fluorescence of quantum dots injected into Balb/c and nude mice for up to two-years post injection using both whole-body and microscopic fluorescence techniques. Two-photon spectral microscopy was used to verify the existence of quantum dots within two-year tissues, but also revealed a range of significantly blue-shifted emission peaks with increased bandwidths. Systemically administered quantum dots persist and retain fluorescence for up to two-years in vivo, but with significantly blue-shifted emission.

Subcellular Singlet Oxygen and Cell Death: Location Matters.

We developed a tool for targeted generation of singlet oxygen using light activation of a genetically encoded fluorogen-activating protein complexed with a unique dye molecule that becomes a potent photosensitizer upon interaction with the protein. By targeting the protein receptor to activate this dye in distinct subcellular locations at consistent per-cell concentrations, we investigated the impact of localized production of singlet oxygen on induction of cell death. We analyzed light dose-dependent cytotoxic response and characterized the apoptotic vs. necrotic cell death as a function of subcellular location, including the nucleus, the cytosol, the endoplasmic reticulum, the mitochondria, and the membrane. We find that different subcellular origins of singlet oxygen have different potencies in cytotoxic response and the pathways of cell death, and we observed that CT26 and HEK293 cell lines are differentially sensitive to mitochondrially localized singlet oxygen stresses. This work provides new insight into the function of type II reactive oxygen generating photosensitizing processes in inducing targeted cell death and raises interesting mechanistic questions about tolerance and survival mechanisms in studies of oxidative stress in clonal cell populations.

Affibody-targeted fluorogen activating protein for in vivo tumor imaging.

Molecular imaging using near-infrared (NIR) fluorescence is useful for intraoperative imaging and real-time margin identification. Directly conjugated IR dyes possess useful properties for in vivo imaging, but conjugation often substantially alters the circulation dynamics of targeting moieties. We developed and characterized a new tumor-targeting probe, affiFAP, which consists of a protein that specifically binds EGFR (affibody) and a fluorogen activating protein (FAP). This compact molecular recognition reagent can reversibly bind and activate fluorescence of otherwise nonfluorescent dyes and allows tumor visualization with low nonspecific tissue staining. We demonstrate molecular pre-targeting of affiFAPs and subsequent systemic or topical application of fluorogenic dye to achieve high contrast, fast clearance, and good tissue penetration that may be used in clinical settings to molecularly define tumor margins.

Fluorescence imaging of tumors in vivo.

We review recent progress in tumor imaging in vivo using fluorescent tags, highlight the problems of fluorescence imaging in small animals, discuss recent advances in near-infrared fluorochromes and quantum dots, and point to some future possibilities. GFP-based fluorescence imaging is briefly discussed. The authors believe that improvements in near-infrared fluorochromes are required to enable practical imaging in tissues at centimeter depths.

Simultaneous multi-species tracking in live cells with quantum dot conjugates.

Quantum dots are available in a range of spectrally separated emission colors and with a range of water-stabilizing surface coatings that offers great flexibility for enabling bio-specificity. In this study, we have taken advantage of this flexibility to demonstrate that it is possible to perform a simultaneous investigation of the lateral dynamics in the plasma membrane of i) the transmembrane epidermal growth factor receptor, ii) the glucosylphospatidylinositol-anchored protein CD59, and iii) ganglioside GM1-cholera toxin subunit B clusters in a single cell. We show that a large number of the trajectories are longer than 50 steps, which we by simulations show to be sufficient for robust single trajectory analysis. This analysis shows that the populations of the diffusion coefficients are heterogeneously distributed for all three species, but differ between the different species. We further show that the heterogeneity is decreased upon treating the cells with methyl-ß-cyclodextrin.

Nanoparticle transport from mouse vagina to adjacent lymph nodes.

To test the feasibility of localized intravaginal therapy directed to neighboring lymph nodes, the transport of quantum dots across the vaginal wall was investigated. Quantum dots instilled into the mouse vagina were transported across the vaginal mucosa into draining lymph nodes, but not into distant nodes. Most of the particles were transported to the lumbar nodes; far fewer were transported to the inguinal nodes. A low level of transport was evident at 4 hr after intravaginal instillation, and transport peaked at about 36 hr after instillation. Transport was greatly enhanced by prior vaginal instillation of Nonoxynol-9. Hundreds of micrograms of nanoparticles/kg tissue (ppb) were found in the lumbar lymph nodes at 36 hr post-instillation. Our results imply that targeted transport of microbicides or immunogens from the vagina to local lymph organs is feasible. They also offer an in vivo model for assessing the toxicity of compounds intended for intravaginal use.

Imaging vasculature and lymphatic flow in mice using quantum dots.

Quantum dots are ideal probes for fluorescent imaging of vascular and lymphatic tissues. On injection into appropriate sites, red- and near-infrared-emitting quantum dots provide excellent definition of vasculature, lymphoid organs, and lymph nodes draining both normal tissues and tumors. We detail methods for use with commercially available quantum dots and discuss common difficulties.

Fluorescent DNA nanotags: supramolecular fluorescent labels based on intercalating dye arrays assembled on nanostructured DNA templates.

Fluorescence detection and imaging are vital technologies in the life sciences and clinical diagnostics. The key to obtaining high-resolution images and sensitive detection is to use fluorescent molecules or particles that absorb and emit visible light with high efficiency. We have synthesized supramolecular complexes consisting of a branched DNA template and fluorogenic intercalating dyes. Because dyes can intercalate up to every other base pair, high densities of fluorophores are assembled yet the DNA template keeps them far enough away from each other to prevent self-quenching. The efficiency with which these noncovalent assemblies absorb light is more than 10-fold greater than that of the individual dye molecules. Förster resonance energy transfer from the intercalated dyes to covalently attached acceptor dyes is very efficient, allowing for wavelength shifting of the emission spectrum. Simple biotinylation of the DNA template allows for labeling of streptavidin-coated synthetic microspheres and mouse T-cells.

Cholera toxin B conjugated quantum dots for live cell labeling.

Cholera toxin subunit B (CTB)--quantum dot conjugates were developed for labeling mammalian cells. The conjugates were internalized by all tested cell lines into small vesicles dispersed throughout the cytoplasm, while commercially available polyarginine conjugates rapidly accumulated in large perinuclear endosomes. Although a large proportion of CTB conjugates eventually also accumulated in perinuclear endosomes, this accumulation required several days, and even then many CTB conjugated quantum dots remained in small vesicles dispersed throughout the cytoplasm. Thus CTB conjugates are a practical alternative to polyarginine conjugates for the general labeling of mammalian cells.

Sentinel lymph node imaging using quantum dots in mouse tumor models.

We demonstrate that quantum dots injected into two model tumors rapidly migrate to sentinel lymph nodes. PEG-coated quantum dots having terminal carboxyl, amino, or methoxyl groups all migrated from the tumor to surrounding lymph nodes similarly. Passage from the tumor through lymphatics to adjacent nodes could be visualized dynamically through the skin; at least two nodes could usually be defined. Imaging during necropsy confirmed confinement of the quantum dots to the lymphatic system and demonstrated easy tagging of sentinel lymph nodes for pathology. Examination of the sentinel nodes identified by quantum dot localization showed that at least some contained metastatic tumor foci.

Noninvasive imaging of quantum dots in mice.

Quantum dots having four different surface coatings were tested for use in in vivo imaging. Localization was successfully monitored by fluorescence imaging of living animals, by necropsy, by frozen tissue sections for optical microscopy, and by electron microscopy, on scales ranging from centimeters to nanometers, using only quantum dots for detection. Circulating half-lives were found to be less than 12 min for amphiphilic poly(acrylic acid), short-chain (750 Da) methoxy-PEG or long-chain (3400 Da) carboxy-PEG quantum dots, but approximately 70 min for long-chain (5000 Da) methoxy-PEG quantum dots. Surface coatings also determined the in vivo localization of the quantum dots. Long-term experiments demonstrated that these quantum dots remain fluorescent after at least four months in vivo.