In vivo senescence imaging nanoprobe targets the associated reactive oxygen species.

Cellular senescence, a cell-cycle arrest state upon stress or damage, can adversely impact aging and cancers. We have designed a novel near infrared fluorogenic nanoprobe, named D3, which can only be turned on by highly elevated levels of reactive oxygen species (ROS), critical players for the induction and maintenance of senescence, for real-time senescence sensing and imaging. In contrast to glowing senescent cells, non-senescent cells whose ROS levels are too low to activate the D3 signal remain optically silent. Upon systemic injection into senescent tumor-bearing mice, the D3 nanoprobe quickly accumulates in tumors, and its fluorescence signal is turned on specifically by senescence-associated ROS in the senescent tumors. The fluorescence signal at senescent tumors was 3-fold higher than that of non-senescent tumors. This groundbreaking design introduces a novel activation mechanism and a powerful imaging nanoprobe to identify and assess cellular senescence in living organisms.

Aptamer-Gemcitabine Conjugates with Enzymatically Cleavable Linker for Targeted Delivery and Intracellular Drug Release in Cancer Cells.

Gemcitabine is a chemotherapeutic used clinically to treat a variety of cancers. However, because it lacks tumor cell specificity, gemcitabine may cause off-target cytotoxicity and adversely impact patients. To impart cancer cell specificity to gemcitabine and improve its therapeutic efficacy, we synthesized a unique aptamer-drug conjugate that carries a high gemcitabine payload (three molecules) via a dendrimer structure and enzymatically cleavable linkers for controlled intracellular drug release. First, linker-gemcitabinedendrimer-linker-gemcitabine products were produced, which had significantly lower cytotoxicity than an equimolar amount of free drug. Biochemical analysis revealed that lysosomal cathepsin B protease rapidly cleaved the dendritic linkers and released the conjugated gemcitabine as a free drug. Subsequently, the dendrimer-linker-gemcitabine was coupled with a cell-specific aptamer to form aptamer-gemcitabine conjugates. Functional assays confirmed that, under aptamer guidance, aptamer-gemcitabine conjugates were selectively bound to and then internalized by triple-negative breast cancer cells. Cellular therapy studies indicated that the aptamer-gemcitabine conjugates potentiated cytotoxic activity to targeted cancer cells but did not affect off-target control cells. Our study demonstrates a novel approach to aptamer-mediated targeted drug delivery that combines a high drug payload and an enzymatically controlled drug release switch to achieve higher therapeutic efficacy and fewer off-target effects relative to free-drug chemotherapy.

Developing a far-red fluorogenic beta-galactosidase probe for senescent cell imaging and photoablation.

A methylene blue (MB)-based beta-galactosidase (ß-gal) activatable molecule, Gal-MB, was developed for senescence imaging and light-triggered senolysis. When in contact with LacZ ß-gal or senescence-associated ß-gal (SA-ß-gal), the photoinsensitive Gal-MB becomes fluorescent. Gal-MB also offered selective phototoxicity toward LacZ ß-gal expressing cells and drug-induced senescent cells, which express SA-ß-gal, after light illumination at 665 nm.

RHAMMB-mediated bifunctional nanotherapy targeting Bcl-xL and mitochondria for pancreatic neuroendocrine tumor treatment.

The incidence of pancreatic neuroendocrine tumor (PNET) has continued to rise. Due to their indolent feature, PNET patients often present with incurable, metastatic diseases. Novel therapies are urgently needed. We have previously shown that Receptor for Hyaluronic Acid-Mediated Motility isoform B (RHAMMB) and Bcl-xL are upregulated in PNETs and both of them promote PNET metastasis. Because RHAMM protein is undetectable in most adult tissues, we hypothesized that RHAMMB could be a gateway for nanomedicine delivery into PNETs. To test this, we developed a RHAMMB-targeting nanoparticle (NP). Inside this NP, we assembled small interfering RNA (siRNA) against Bcl-xL (siBcl-xL) and mitochondria-fusing peptide KLA. We demonstrated that RHAMMB-positive PNETs picked up the RHAMMB-targeting NPs. siBcl-xL or KLA alone killed only 30% of PNET cells. In contrast, a synergistic killing effect was achieved with the co-delivery of siBcl-xL and KLA peptide in vitro. Unexpectedly, siBcl-xL induced cell death before reducing Bcl-xL protein levels. The systemically injected RHAMMB-targeting NPs carrying siBcl-xL and KLA peptide significantly reduced tumor burden in mice bearing RHAMMB-positive PNETs. Together, these findings indicate that the RHAMMB-targeting nanotherapy serves as a promising drug delivery system for PNET and possibly other malignancies with upregulated RHAMMB. The combination of siBcl-xL and KLA peptide can be a therapy for PNET treatment.

A Hybrid Nanogel to Preserve Lysosome Integrity for Fluorescence Imaging.

Fluorescence imaging of lysosomes provides a powerful tool to probe the lysosome physiology in living cells, yet the continuous light exposure inevitably causes lysosome damage and phototoxicity, which remains a formidable challenge. Here the long-term lysosome tracking with minimized photodamage was realized using a multifunctional nanoprobe, a platinum nanoparticle, and a quinacrine co-loaded nanogel. To construct the hybrid nanogel, cisplatin first functioned as cross-linker to withhold all components and then was reduced to a platinum nanoparticle in situ by ethanol. The platinum nanoparticle enabled a long-term quinacrine fluorescence imaging of lysosome by scavenging the light induced reactive oxygen species which could damage lysosomal membranes.

Near-Infrared Fluorogenic Spray for Rapid Tumor Sensing.

Surgical resection of cancerous tissues is a critical procedure for solid tumor treatment. During the operation, the surgeon mostly identifies the cancerous tissues by naked-eye visualization under white light without aid, therefore, the outcome heavily relies on the surgeon's experience. A near-infrared pH-responsive fluorogenic dye, CypH-11, was designed to be used as a sensitive cancer spray to highlight cancerous tissues during surgical operations, minimizing the surgeon's subjective judgment. CypH-11, pKa 6.0, emits almost no fluorescence at neutral pH but fluoresces brightly in an acidic environment, a ubiquitous consequence of cancer cell proliferation. After topical application, CypH-11 was absorbed quickly, and its fluorescence signal in the cancerous tissue was developed within a minute. The signal-to-background ratio was 1.3 and 1.5 at 1 and 10 min, respectively. The fluorogenic property and near-instant signal development capability enable the "spray-and-see" concept. This fast-acting CypH-11 spray could be a handy and effective tool for fluorescence-guided surgery, identifying small cancerous lesions in real time for optimal resection without systemic toxicity.

Selective photo-ablation of glioma cells using an enzyme activatable photosensitizer.

An activatable photosensitizer based on methylene blue was developed and validated for its efficacy in the selective killing of γ-glutamyl transpeptidase expressing cells. The 1O2 deactivation via bond breakage, rapid in situ enzymatic photosensitivity conversion, long lysosomal retention, and nucleus relocation collectively contribute to its strong and selective photodynamic effects.

A combined approach of convection-enhanced delivery of peptide nanofiber reservoir to prolong local DM1 retention for diffuse intrinsic pontine glioma treatment.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is a highly lethal malignancy that occurs predominantly in children. DIPG is inoperable and post-diagnosis survival is less than 1 year, as conventional chemotherapy is ineffective. The intact blood-brain barrier (BBB) blocks drugs from entering the brain. Convection-enhanced delivery (CED) is a direct infusion technique delivering drugs to the brain, but it suffers from rapid drug clearance. Our goal is to overcome the delivery barrier via CED and maintain a therapeutic concentration at the glioma site with a payload-adjustable peptide nanofiber precursor (NFP) that displays a prolonged retention property as a drug carrier. METHODS: The post-CED retention of 89Zr-NFP was determined in real time using PET/CT imaging. Emtansine (DM1), a microtubule inhibitor, was conjugated to NFP. The cytotoxicity of the resulting DM1-NFP was tested against patient-derived DIPG cell lines. The therapeutic efficacy was evaluated in animals bearing orthotopic DIPG, according to glioma growth (measured using bioluminescence imaging) and the long-term survival. RESULTS: DM1-NFP demonstrated potency against multiple glioma cell lines. The half-maximal inhibitory concentration values were in the nanomolar range. NFP remained at the infusion site (pons) for weeks, with a clearance half-life of 60 days. DM1-NFP inhibited glioma progression in animals, and offered a survival benefit (median survival of 62 days) compared with the untreated controls (28 days) and DM1-treated animal group (26 days). CONCLUSIONS: CED, in combination with DM1-NFP, complementarily functions to bypass the BBB, prolong drug retention at the fusion site, and maintain an effective therapeutic effect against DIPG to improve treatment outcome.

Aptamer-Equipped Protamine Nanomedicine for Precision Lymphoma Therapy.

Anaplastic large cell lymphoma (ALCL) is the most common T-cell lymphoma in children. ALCL cells characteristically express surface CD30 molecules and carry the pathogenic ALK oncogene, both of which are diagnostic biomarkers and are also potential therapeutic targets. For precision therapy, we report herein a protamine nanomedicine incorporated with oligonucleotide aptamers to selectively target lymphoma cells, a dsDNA/drug payload to efficiently kill targeted cells, and an siRNA to specifically silence ALK oncogenes. The aptamer-equipped protamine nanomedicine was simply fabricated through a non-covalent charge-force reaction. The products had uniform structure morphology under an electron microscope and a peak diameter of 103 nm by dynamic light scattering measurement. Additionally, flow cytometry analysis demonstrated that under CD30 aptamer guidance, the protamine nanomedicine specifically bound to lymphoma cells, but did not react to off-target cells in control experiments. Moreover, specific cell targeting and intracellular delivery of the nanomedicine were also validated by electron and confocal microscopy. Finally, functional studies demonstrated that, through combined cell-selective chemotherapy using a drug payload and oncogene-specific gene therapy using an siRNA, the protamine nanomedicine effectively killed lymphoma cells with little toxicity to off-target cells, indicating its potential for precision therapy.

Multifunctional Nanodelivery Platform for Maximizing Nucleic Acids Combination Therapy.

The silencing of an oncogene with a small interfering RNA (siRNA) is a promising way for cancer therapy. Its efficacy can be further enhanced by integrating with other therapeutics; however, transporting siRNA and other active ingredients to the same location at the same time is challenging. Here, we report a novel multifunctional nanodelivery platform by sequentially layering several functional ingredients, such as siRNAs, microRNAs, peptides, and targeting ligands, onto a core through charge-charge interaction. The prepared nanovectors effectively and programmably delivered multiple active components to maximize therapeutic combination with minimal off-targeting effects.

Enzyme-Assisted Photodynamic Therapy Based on Nanomaterials.

Photodynamic therapy (PDT) is a noninvasive cancer treatment that requires the copresence of a photosensitizer (PS), oxygen, and light. The efficacy of conventional PDT is usually limited by two factors: delivery of the PS to the tumor and the hypoxic solid tumor environment. To improve the efficacy of PDT, nanomaterial-based, enzyme-assisted PDT (nano-ezPDT), which integrates enzyme-responsive components into nanomedicines, was developed for enhanced PS delivery and oxygen generation. Nano-ezPDT is designed to take full advantage of the catalytic function of locally activated tumor-associated enzymes or smuggled exogeneous enzymes. The enhancement of PS release and accumulation is often controlled by endogenous enzymes upregulated at the tumor sites. Oxygen generation, however, relies mostly on catalase-loaded nanomedicines. In this review, we discuss the associated enzymes, constructs, and types of nanocarriers and highlight the principle and utility of nano-ezPDT for cancer therapy.

Multilayered Activatable Nanoprobe for Ultra-Bright Tumor Imaging.

The development of tumor targeted probes with strong signal and high contrast is always challenging in cancer imaging. Here, a unique multilayered activatable nanoprobe (MAN) is prepared to fulfill this long-standing goal. MAN adopts a versatile layer-by-layer fabrication technique that sequentially assembles multifunctional polyelectrolytes onto nanoparticles via charge-charge interaction. Unlike the common one-probe-one-fluorochrome construct, MAN offers a dramatic fluorescence enhancement by transporting a large quantity of quenched fluorochromes for maximal signal and contrast. Excellent signal amplification and retention with negligible cytotoxicity is observed in cell study. Upon systemic injection into mice, MAN quickly accumulates in tumor and its fluorescent signal is turned on by proteases overexpressed in tumors, resulting in >700% tumor-to-normal-tissue contrast. This multilayered fabrication provides a simple and powerful universal platform to design sensitive tumor imaging probes.

A Multiresponsive Nanohybrid to Enhance the Lysosomal Delivery of Oxygen and Photosensitizers.

Photodynamic therapy (PDT) is a promising cancer ablation method, but its efficiency is easily affected by several factors, such as the insufficient delivery of photosensitizers, low oxygen levels as well as long distance between singlet oxygen and intended organelles. A multifunctional nanohybrid, named MGAB, consisting of gelatin-coated manganese dioxide and albumin-coated gold nanoclusters, was designed to overcome these issues by improving chlorin e6 (Ce6) delivery and stimulating oxygen production in lysosomes. MGAB were quickly degraded in a high hydrogen peroxide, high protease activity, and low pH microenvironment, which is closely associated with tumor growth. The Ce6-loaded MGAB were picked up by tumor cells through endocytosis, degraded within the lysosomes, and released oxygen and photosensitizers. Upon near-infrared light irradiation, the close proximity of oxygen with photosensitizer within lysosomes enabled the production of cytotoxic singlet oxygen, resulting in more effective PDT.

Layer-by-layer construction of an oxygen-generating photo-responsive nanomedicine for enhanced photothermal and photodynamic combination therapy.

A multifunctional photosensitive and photothermal nanomedicine, AMP-Ce6-HA, was prepared by sequentially layering hard and soft functional molecules. AMP-Ce6-HA was efficiently picked up by MDA-MB-231 breast cancer cells, and then released oxygen in lysosomes. The photothermal and oxygen-enhanced photodynamic combination therapy was much more effective than the single one.

Nanoparticle Delivery of miR-708 Mimetic Impairs Breast Cancer Metastasis.

Triple-negative breast cancer (TNBC) patients exhibit the worst clinical outcome due to its aggressive clinical course, higher rate of recurrence, and a conspicuous lack of FDA-approved targeted therapies. Here, we show that multilayered nanoparticles (NPs) carrying the metastasis suppressor microRNA miR-708 (miR708-NP) localize to orthotopic primary TNBC, and efficiently deliver the miR-708 cargo to reduce lung metastasis. Using a SOX2/OCT4 promoter reporter, we identified a population of miR-708low cancer cells with tumor-initiating properties, enhanced metastatic potential, and marked sensitivity to miR-708 treatment. In vivo, miR708-NP directly targeted the SOX2/OCT4-mCherry+ miR-708low tumor cells to impair metastasis. Together, our preclinical findings provide a mechanism-based antimetastatic therapeutic approach for TNBC, with a marked potential to generate miR-708 replacement therapy for high-risk TNBC patients in the clinic. To our knowledge, this gold nanoparticle-based delivery of microRNA mimetic is the first oligonucleotide-based targeted therapy for TNBC.

Redox-responsive cisplatin nanogels for anticancer drug delivery.

Inspired by cisplatin's deactivation by glutathione (GSH) in cancer, a GSH responsive nanogel loaded with doxorubicin (Dox) was prepared using hyaluronan as a matrix and cisplatin as a crosslinker. The elevated GSH depletes the cisplatin crosslinker in the nanogel, enhances Dox release and boosts cytotoxicity, thus providing a new GSH responsive platform to reverse cisplatin resistance.

Facile metabolic glycan labeling strategy for exosome tracking.

BACKGROUND: Exosomes are nano-sized vesicles derived from the fusion of multivesicular bodies with the surrounding plasma membrane. Exosomes have various diagnostic and therapeutic potentials in cancer and other diseases, thus tracking exosomes is an important issue. METHODS: Here, we report a facile exosome labeling strategy using a natural metabolic incorporation of an azido-sugar into the glycan, and a strain-promoted azide-alkyne click reaction. In culture, tetra-acetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz) was spontaneously incorporated into glycans within the cells and later redistributed onto their exosomes. These azido-containing exosomes were then labeled with azadibenzylcyclooctyne (ADIBO)-fluorescent dyes by a bioorthogonal click reaction. RESULTS: Cellular uptake and the in vivo tracking of fluorescent labeled exosomes were evaluated in various cells and tumor bearing mice. Highly metastatic cancer-derived exosomes showed an increased self-homing in vitro and selective organ distribution in vivo. CONCLUSION: Our metabolic exosome labeling strategy could be a promising tool in studying the biology and distribution of exosomes, and optimizing exosome based therapeutic approaches. GENERAL SIGNIFICANT: A facile and effective exosome labeling strategy was introduced by presenting azido moiety on the surface of exosome through metabolic glycan synthesis, and then conjugating a strain-promoted fluorescent dye.

Lysosome Enlargement Enhanced Photochemotherapy Using a Multifunctional Nanogel.

Large lysosomes are susceptible toward rupture because of an increased membrane tension. Here we report a strategy to first enlarge and weaken the lysosome and then destroy it to boost the efficiency of photochemotherapy using a hyaluronan nanogel, carrying chloroquine as a lysosomal expander, rhodamine B as a photosensitive lysosomal destroyer, and cisplatin as a chemotherapeutic. This all-in-one nanogel provides a facile approach and new insight into improve the photochemotherapy, by making use of lysosome's size, as a risk factor in lysosomal destabilization.

Real-Time Visualization of Lysosome Destruction Using a Photosensitive Toluidine Blue Nanogel.

Breaking the lysosome helps its sequestered payloads access their molecular targets in cells and thus enhances the intracellular drug delivery. Current strategies for lysosomal escape involve direct physical interactions with the lipid membrane. These interactions pose a systemic toxicity and uncontrolled membrane rupture risk. Here, we report a light-detonated lysosome disruption using a hyaluronan (HA) nanogel packed with toludine blue (TB). The HA/TB nanogel is concentrated within the lysosomes. The applied light assists TB in generating reactive oxygen species and destroying the lysosome in situ, both in cells and isolated lysosomes. Real time fluorescent tracking reveals that quenched TB fluorescence recovers along with lysosome explosion, relocates to the nucleus, and is presented as a fluorescent sparkling in cells. This HA/TB, composed of all clinically approved materials, represents a biocompatible and facile strategy to "bomb" lysosomes in a spatiotemporally controlled fashion.

A cell surface clicked navigation system to direct specific bone targeting.

Cell therapies are promising up-and-coming therapeutic strategies for many diseases. For maximal therapeutic benefits, injected cells have to navigate their way to a designated area, including organ and tissue; unfortunately, the majority of therapeutic cells are currently administered without a guide or homing device. To improve this serious shortcoming, a functionalization method was developed to equip cells with a homing signal. Its application was demonstrated by applying an Azadibenzocyclooctyne-bisphosphonate (ADIBO-BP) and azide paired bioorthogonal chemistry on cells for bone specific homing. Jurkat T cells and bone marrow derived stromal cells (BMSCs) were cultured with tetraacetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) to place unnatural azido groups onto the cell's surface; these azido groups were then reacted with ADIBO-BP. The tethered bisphosphonates were able to bring Jurkat cells to hydroxyapatite, the major component of bone, and mineralized SAOS-2 cells. The incorporated BP groups also enhanced the specific affinity of BMSCs to mouse femur bone fragments in vitro. The introduced navigation strategy is expected to have a broad application in cell therapy, because through the biocompatible ADIBO and azide reactive pair, various homing signals could be efficiently anchored onto therapeutic cells.