Visualizing vasculature and its response to therapy in the tumor microenvironment.

Tumor vasculature plays a critical role in the progression and metastasis of tumors, antitumor immunity, drug delivery, and resistance to therapies. The morphological and functional changes of tumor vasculature in response to therapy take place in a spatiotemporal-dependent manner, which can be predictive of treatment outcomes. Dynamic monitoring of intratumor vasculature contributes to an improved understanding of the mechanisms of action of specific therapies or reasons for treatment failure, leading to therapy optimization. There is a rich history of methods used to image the vasculature. This review describes recent advances in imaging technologies to visualize the tumor vasculature, with a focus on enhanced intravital imaging techniques and tumor window models. We summarize new insights on spatial-temporal vascular responses to various therapies, including changes in vascular perfusion and permeability and immune-vascular crosstalk, obtained from intravital imaging. Finally, we briefly discuss the clinical applications of intravital imaging techniques.

Surface biomineralization of uranium onto Shewanella putrefaciens with or without extracellular polymeric substances.

The influence of extracellular polymeric substances (EPS) on the interaction between uranium [U(VI)] and Shewanella putrefaciens (S. putrefaciens), especially the U(VI) biomineralization process occurring on whole cells and cell components of S. putrefaciens was investigated in this study. The removal efficiency of U(VI) by S. putrefaciens was decreased by 22% after extraction of EPS. Proteins were identified as the main components of EPS by EEM analysis and were determined to play a major role in the biosorption of uranium. SEM-EDS results showed that U(VI) was distributed around the whole cell as 500-nanometer schistose structures, which consisted primarily of U and P. However, similar uranium lamellar crystal were wrapped only on the surface of EPS-free S. putrefaciens cells. FTIR and XPS analysis indicated that phosphorus- and nitrogen-containing groups played important roles in complexing U (VI). XRD and U LIII-edge EXAFS analyses demonstrated that the schistose structure consisted of hydrogen uranyl phosphate [H2(UO2)2(PO4)2•8H2O]. Our study provides new insight into the mechanisms of induced uranium crystallization by EPS and cell wall membranes of living bacterial cells under aerobic conditions.

Reactive Oxygen Species Activatable Heterodimeric Prodrug as Tumor-Selective Nanotheranostics.

Nanotheranostics based on tumor-selective small molecular prodrugs could be more advantageous in clinical translation for cancer treatment, given its defined chemical structure, high drug loading efficiency, controlled drug release, and reduced side effects. To this end, we have designed and synthesized a reactive oxygen species (ROS)-activatable heterodimeric prodrug, namely, HRC, and nanoformulated it for tumor-selective imaging and synergistic chemo- and photodynamic therapy. The prodrug consists of the chemodrug camptothecin (CPT), the photosensitizer 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH), and a thioketal linker. Compared to CPT- or HPPH-loaded polymeric nanoparticles (NPs), HRC-loaded NPs possess higher drug loading capacity, better colloidal stability, and less premature drug leakage. Interestingly, HRC NPs were almost nonfluorescent due to the strong π-π stacking and could be effectively activated by endogenous ROS once entering cells. Thanks to the higher ROS levels in cancer cells than normal cells, HRC NPs could selectively light up the cancer cells and exhibit much more potent cytotoxicity to cancer cells. Moreover, HRC NPs demonstrated highly effective tumor accumulation and synergistic tumor inhibition with reduced side effects on mice.

Nanoparticle delivery in vivo: A fresh look from intravital imaging.

Nanomedicine has proven promising in preclinical studies. However, only few formulations have been successfully translated to clinical use. A thorough understanding of how nanoparticles interact with cells in vivo is essential to accelerate the clinical translation of nanomedicine. Intravital imaging is a crucial tool to reveal the mechanisms of nanoparticle transport in vivo, allowing for the development of new strategies for nanomaterial design. Here, we first review the most recent progress in using intravital imaging to answer fundamental questions about nanoparticle delivery in vivo. We then elaborate on how nanoparticles interact with different cell types and how such interactions determine the fate of nanoparticles in vivo. Lastly, we discuss ways in which the use of intravital imaging can be expanded in the future to facilitate the clinical translation of nanomedicine.

Visualizing DC morphology and T cell motility to characterize DC-T cell encounters in mouse lymph nodes under mTOR inhibition.

Mammalian target of rapamycin (mTOR), a serine/threonine kinase orchestrating cellular metabolism, is a crucial immune system regulator. However, it remains unclear how mTOR regulates dendritic cell (DC) function in vivo, especially DC-T cell encounters, a critical step for initiating adaptive immune responses. We dynamically visualized DC-T contacts in mouse lymph node using confocal microscopy and established an encounter model to characterize the effect of mTOR inhibition on DC-T cell encounters using DC morphology. Quantitative data showed mTOR inhibition via rapamycin altered DC shape, with an increased form factor (30.17%) and decreased cellular surface area (20.36%) and perimeter (22.43%), which were associated with Cdc42 protein downregulation (52.71%). Additionally, DCs adopted a similar morphological change with Cdc42 inhibition via ZCL278 as that observed with mTOR inhibition. These morphologically altered DCs displayed low encounter rates with T cells. Time-lapse imaging data of T cell motility supported the simulated result of the encounter model, where antigen-specific T cells appeared to reduce arrest in the lymph nodes of rapamycin-pretreated mice relative to the untreated group. Therefore, mTOR inhibition altered DC morphology in vivo and decreased the DC-T cell encounter rate, as well as Cdc42 inhibition. By establishing an encounter model, our study provides an intuitive approach for the early prediction of DC function through morphological quantification of form factor and area.

Self-Assembled "Off/On" Nanopomegranate for In Vivo Photoacoustic and Fluorescence Imaging: Strategic Arrangement of Kupffer Cells in Mouse Hepatic Lobules.

Kupffer cells (KCs), potent scavenger cells located in hepatic sinusoids, constantly phagocytize and degrade foreign materials to maintain metabolism and clearance. Understanding the strategic KC arrangement which links to their spatial location and function in hepatic lobules, the basic functional unit in the liver, is highly valuable for characterizing liver function. However, selectively labeling KCs and characterizing their function in vivo remains challenging. Herein, a fast self-assembled pomegranate structure-like nanoparticle with "nanopomegranate seeds" of dye aggregates has been developed, which has dual-modality "off/on" capability. This nanopomegranate shows good photostability, a high extinction coefficient, a high KC labeling efficiency (98.8%), and better visualization of KC morphology than commercial FluoSpheres. In vivo photoacoustic (PA) and fluorescence imaging consistently visualize that KCs are strategically distributed along the central vein (CV)-portal triad (PT) axis in each liver lobule: more and larger KCs exist in areas closer to the PTs. The high-resolution PA quantitative data further revealed that the density of KCs was linearly dependent on the r n/ rmax ratio (their relative location along the CV-PT axis) ( R2 = 0.7513), and the KC density at the outermost layer is almost 246-fold that at the innermost layer (each layer is 8 µm). Notably, the phagocytic ability of KCs located in layers with r n/ rmax ratios of 0.167-0.3 varies in a zigzag pattern, as evidenced by their different PA intensities. Additionally, the fluorescence imaging quantitation suggests similar fluorescence activation of nanopomegranate in KCs. Nanopomegranates combined with dual-modality imaging reveal the strategic arrangement of KCs in vivo, greatly extending our understanding of liver physiology.

Using Fluorescence Imaging to Track Drug Delivery and Guide Treatment Planning In Vivo.

Imaging has become an indispensable tool in both clinical medicine and preclinical sciences. It enables doctors to locate sites of cancer/disease, track drug delivery, and guide operative planning, thus enhancing the treatment efficacy. Recently, we developed a multimodal theranostic lipid nanoparticles, named HPPS(NIR)-chol-siRNA with its built-in near-infrared (NIR) fluorescent probe core as a useful surrogate for tracking small interfering RNA (siRNA) delivery. By using the image co-registration of computed tomography (CT) and fluorescence molecular tomography (FMT), we achieved noninvasive assessment and treatment planning of siRNA delivery into the orthotopic tumor, thus enabling efficacious RNA interference (RNAi) therapy. In this chapter, we introduce this method to illustrate the use of CT-FMT co-registration for tracking drug delivery and guiding treatment planning in vivo.

A PEGylation-Free Biomimetic Porphyrin Nanoplatform for Personalized Cancer Theranostics.

PEGylation (PEG) is the most commonly adopted strategy to prolong nanoparticles' vascular circulation by mitigating the reticuloendothelial system uptake. However, there remain many concerns in regards to its immunogenicity, targeting efficiency, etc., which inspires pursuit of alternate, non-PEGylated systems. We introduced here a PEG-free, porphyrin-based ultrasmall nanostructure mimicking nature lipoproteins, termed PLP, that integrates multiple imaging and therapeutic functionalities, including positron emission tomography (PET) imaging, near-infrared (NIR) fluorescence imaging and photodynamic therapy (PDT). With an engineered lipoprotein-mimicking structure, PLP is highly stable in the blood circulation, resulting in favorable pharmacokinetics and biodistribution without the need of PEG. The prompt tumor intracellular trafficking of PLP allows for rapid nanostructure dissociation upon tumor accumulation to release monomeric porphyrins to efficiently generate fluorescence and photodynamic reactivity, which are highly silenced in intact PLP, thus providing an activatable mechanism for low-background NIR fluorescence imaging and tumor-selective PDT. Its intrinsic copper-64 labeling feature allows for noninvasive PET imaging of PLP delivery and quantitative assessment of drug distribution. Using a clinically relevant glioblastoma multiforme model, we demonstrated that PLP enabled accurate delineation of tumor from surrounding healthy brain at size less than 1 mm, exhibiting the potential for intraoperative fluorescence-guided surgery and tumor-selective PDT. Furthermore, we demonstrated the general applicability of PLP for sensitive and accurate detection of primary and metastatic tumors in other clinically relevant animal models. Therefore, PLP offers a biomimetic theranostic nanoplatform for pretreatment stratification using PET and NIR fluorescence imaging and for further customized cancer management via imaging-guided surgery, PDT, or/and potential chemotherapy.

Nanoparticle-enabled, image-guided treatment planning of target specific RNAi therapeutics in an orthotopic prostate cancer model.

The abilities to deliver siRNA to its intended action site and assess the delivery efficiency are challenges for current RNAi therapy, where effective siRNA delivery will join force with patient genetic profiling to achieve optimal treatment outcome. Imaging could become a critical enabler to maximize RNAi efficacy in the context of tracking siRNA delivery, rational dosimetry and treatment planning. Several imaging modalities have been used to visualize nanoparticle-based siRNA delivery but rarely did they guide treatment planning. We report a multimodal theranostic lipid-nanoparticle, HPPS(NIR)-chol-siRNA, which has a near-infrared (NIR) fluorescent core, enveloped by phospholipid monolayer, intercalated with siRNA payloads, and constrained by apoA-I mimetic peptides to give ultra-small particle size (<30 nm). Using fluorescence imaging, we demonstrated its cytosolic delivery capability for both NIR-core and dye-labeled siRNAs and its structural integrity in mice through intravenous administration, validating the usefulness of NIR-core as imaging surrogate for non-labeled therapeutic siRNAs. Next, we validated the targeting specificity of HPPS(NIR)-chol-siRNA to orthotopic tumor using sequential four-steps (in vivo, in situ, ex vivo and frozen-tissue) fluorescence imaging. The image co-registration of computed tomography and fluorescence molecular tomography enabled non-invasive assessment and treatment planning of siRNA delivery into the orthotopic tumor, achieving efficacious RNAi therapy.

Imaging the cytosolic drug delivery mechanism of HDL-like nanoparticles.

PURPOSE: Molecular therapeutics often require an effective nanoparticle-based delivery strategy to transport them to cytosolic organelles to be functional. Recently, a cytosolic delivery strategy based on the scavenger receptor class B type I (SR-BI) mediated pathway has shown great potential for the effective delivery of theranostics agents into the cytoplasm of cells without detrimental endosomal entrapment. This study elucidates this unique delivery mechanism for improving cytosolic drug delivery. METHODS: Multifluorophore-labeled HDL-mimicking peptide phospholipid scaffold (HPPS) nanoparticles were developed. Fluorescence imaging was utilized to examine HPPS transporting payloads into cells step by step through sequential inhibition studies. RESULTS: HPPS specifically recognizes and binds to SR-BI, then interacts with SR-BI, which results in direct transport of payload molecules into the cell cytoplasm without entire particles internalization. The cytosolic transport of payloads occurred through a temperature- and energy-independent pathway, and was also different from actin- and clathrin-mediated endocytosis. Furthermore, this transport was significantly inhibited by disruption of lipid rafts using filipin or methyl-ß-cyclodextrin. CONCLUSIONS: The cytosolic delivery of payloads by HPPS via SR-BI targeting is predominately mediated through a lipid rafts/caveolae-like pathway. This cytosolic delivery strategy can be utilized for transporting molecular therapeutics that require their action sites to be within cytosolic organelles to enhance therapeutic effect.

Laser-assisted subepithelial keratectomy versus epipolis laser in situ keratomileusis for myopia: a meta-analysis of clinical outcomes.

BACKGROUND: To identify possible differences between laser-assisted subepithelial keratectomy and epipolis laser in situ keratomileusis for myopia. DESIGN: Meta-analysis. PARTICIPANTS: Patients from previously reported comparative studies treated by laser-assisted subepithelial keratectomy versus epipolis laser in situ keratomileusis. METHODS: A systematic literature retrieval was conducted in the MEDLINE, EMBASE and Cochrane Library, up to January 2013. The included studies were subject to a meta-analysis using a RevMan 5.1 version software. MAIN OUTCOME MEASURES: The differences in efficacy, predictability, safety, epithelial healing time, pain perception and corneal haze formation. RESULTS: A total of six studies involving 517 eyes were included. There were no statistically significant differences in the final proportion of eyes with uncorrected visual acuity of 6/6 or better (P = 0.43), mean postoperative uncorrected visual acuity (P = 0.53), final proportion of eyes with refraction within ± 0.50 D (P = 0.62) and ± 1.00 D (P = 0.16) of target, final proportion of eyes losing two or more lines of best spectacle-corrected visual acuity (P = 1.00), healing time of corneal epithelium (P = 0.58), final proportion of eyes with corneal haze grade 0.5 or higher (P = 0.26), and corneal haze levels (P = 0.36). CONCLUSIONS: There were no significant differences in efficacy, predictability, safety, epithelial healing time and corneal haze formation between laser-assisted subepithelial keratectomy and epipolis laser in situ keratomileusis, but the result was limited. Future more data are required to detect the potential differences between the two procedures.

Lipid-based nanoparticles in the systemic delivery of siRNA.

RNAi therapeutics are believed to be the future of personalized medicine and have shown promise in early clinical trials. However, many physiological barriers exist in the systemic delivery of siRNAs to the cytoplasm of targeted cells to perform their function. To overcome these barriers, many siRNA delivery systems have been developed. Among these, lipid-based nanoparticles have great potential owing to their biocompatibility and low toxicity in comparison with inorganic nanoparticles and viral systems. This review discusses the hurdles of systemic siRNA delivery and highlights the recent progress made in lipid-based nanoparticles, which are categorized based on their key lipid components, including cationic lipid, lipoprotein, lipidoid, neutral lipid and anionic lipid-based nanoparticles. It is expected that these lipid nanoparticle-based siRNA delivery systems will have an enabling role for personalized cancer medicine, where siRNA delivery will join forces with genetic profiling of individual patients to achieve the best treatment outcome.

Efficient systemic delivery of siRNA by using high-density lipoprotein-mimicking peptide lipid nanoparticles.

UNLABELLED: The main challenge for RNAi therapeutics lies in systemic delivery of siRNA to the correct tissues and transporting them into the cytoplasm of targeted cells, at safe, therapeutic levels. Recently, we developed a high-density lipoprotein-mimicking peptide-phospholipid scaffold (HPPS) and demonstrated its direct cytosolic delivery of siRNA in vitro, thereby bypassing endosomal trapping. AIM: We investigate the in vivo implementation of HPPS for siRNA delivery. METHOD & RESULTS: After systemic administration in KB tumor-bearing mice, HPPS prolonged the blood circulation time of cholesterol-modified siRNA (chol-siRNA) by a factor of four, improved its biodistribution and facilitated its uptake in scavenger receptor class B type I overexpressed tumors. For therapeutic targeting to the bcl-2 gene, the HPPS-chol-si-bcl-2 nanoparticles downregulated Bcl-2 protein, induced enhanced apoptosis (2.5-fold) in tumors when compared with controls (saline, HPPS, HPPS-chol-si-scramble and chol-si-bcl-2) and significantly inhibited tumor growth with no adverse effect. CONCLUSION: HPPS is a safe, efficient nanocarrier for RNAi therapeutics in vivo.

Mechanistic insights into LDL nanoparticle-mediated siRNA delivery.

Although small interfering RNA (siRNA) can silence the expression of disease-related genes, delivery of these highly charged molecules is challenging. Delivery approaches for siRNAs are actively being pursued, and improved strategies are required for nontoxic and efficient delivery for gene knockdown. Low density lipoprotein (LDL) is a natural and endogenous nanoparticle that has a rich history as a delivery vehicle. Here, we examine purified LDL nanoparticles as carriers for siRNAs. When siRNA was covalently conjugated to cholesterol, over 25 chol-siRNA could be incorporated onto each LDL without changing nanoparticle morphology. The resulting LDL-chol-siRNA nanoparticles were selectively taken up into cells via LDL receptor mediated endocytosis, resulting in enhanced gene silencing compared to free chol-siRNA (38% gene knock down versus 0% knock down at 100 nM). However, silencing efficiency was limited by the receptor-mediated entrapment of the LDL-chol-siRNA nanoparticles in endolysosomes. Photochemical internalization demonstrated that endolysosome disruption strategies significantly enhance LDL-mediated gene silencing (78% at 100 nM).