Development of an ultrasound triggered nanomedicine-microbubble complex for chemo-photodynamic-gene therapy.

Recently, combination therapy has received much attention because of its highly therapeutic effect in various types of cancers. In particular, chemo-photodynamic combination therapy has been considered as an outstanding strategy. However, an abnormal increase in tumor angiogenesis caused by reactive oxygen species (ROS) generated during photodynamic therapy (PDT) has been reported. In this study, the complex of doxorubicin (DOX)-encapsulating anti-angiogenic small interfering RNA (siRNA) nanoparticle and chlorin e6 (Ce6)-encapsulating microbubble has been developed to suppress tumor angiogenesis. The first compartment, doxorubicin-encapsulating siRNA nanoparticle, was electrostatically coated using two biocompatible polymers to prevent the damage of genetic materials. The other part, Ce6-encapsulating microbubble, serves as an ultrasound-triggered local delivery system as well as a drug carrier. Both the in vitro and in vivo experimental results demonstrate successful inhibition of angiogenesis with a minimized damage of siRNAs caused by ROS as well as improved therapeutic effect by chemo-photodynamic-gene triple combination therapy using ultrasound-triggered local delivery.

Systematic identification of a synthetic lethal interaction in brain-metastatic lung adenocarcinoma.

Metastatic lung adenocarcinoma (LuAC) presents a significant clinical challenge due to the short latency and the lack of efficient treatment options. Therefore, identification of molecular vulnerabilities in metastatic LuAC holds great importance in the development of therapeutic drugs against this disease. In this study, we performed a genome-wide siRNA screening using poorly and highly brain-metastatic LuAC cell lines. Using this approach, we discovered that compared to poorly metastatic LuAC (LuAC-Par) cells, brain-metastatic LuAC (LuAC-BrM) cells exhibited a significantly higher vulnerability to c-FLIP (an inhibitor of caspase-8)-depletion-induced apoptosis. Furthermore, in vivo studies demonstrated that c-FLIP knockdown specifically inhibited growth of LuAC-BrM, but not the LuAC-Par, tumors, suggesting the addiction of LuAC-BrM to the function of c-FLIP for their survival. Our in vitro and in vivo analyses also demonstrated that LuAC-BrM is more sensitive to c-FLIP-depletion due to ER stress-induced activation of the c-JUN and subsequent induction of stress genes including ATF4 and DDIT3. Finally, we found that c-JUN not only sensitized LuAC-BrM to c-FLIP-depletion-induced cell death but also promoted brain metastasis in vivo, providing strong evidence for c-JUN's function as a double-edged sword in LuAC-BrM. Collectively, our findings not only reveal a novel link between c-JUN, brain metastasis, and c-FLIP addiction in LuAC-BrM but also present an opportunity for potential therapeutic intervention.

Elimination of Nontargeted Photoacoustic Signals for Combined Photoacoustic and Ultrasound Imaging.

As a molecular imaging modality, photoacoustic (PA) imaging has been in the spotlight because it can provide an optical contrast image of physiological information and a relatively deep imaging depth. However, its sensitivity is limited despite the use of exogenous contrast agents due to the background PA signals generated from nontargeted absorbers, such as blood and boundaries between different biological tissues. In addition, clutter artifacts generated in both in-plane and out-of-plane imaging region degrade the sensitivity of PA imaging. We propose a method to eliminate the nontargeted PA signals. For this study, we used a dual-modal ultrasound (US)-PA contrast agent that is capable of generating both the backscattered US and PA signals in response to the transmitted US and irradiated light, respectively. The US images of the contrast agents are used to construct a masking image that contains the location information about the target site and is applied to the PA image acquired after contrast agent injection. In vitro and in vivo experimental results demonstrated that the masking image constructed using the US images makes it possible to completely remove nontargeted PA signals. The proposed method can be used to enhance the clear visualization of the target area in PA images.

Exosome-based photoacoustic imaging guided photodynamic and immunotherapy for the treatment of pancreatic cancer.

Exosomes, which are released from all cells and take part in cell-to-cell communication, have been utilized as drug delivery vehicles in many recent studies. Immunotherapy is an emerging technology which uses patients' innate immune systems. In immunotherapy, immune cells are stimulated through antibodies, the other immune cells and genetic modifications for the purposes of, for instance, cancer therapy. In this study, tumor-derived re-assembled exosome (R-Exo) was simultaneously utilized as both a drug delivery carrier and an immunostimulatory agent. A chlorin e6 photosensitizer was loaded into tumor-derived exosomes during exosomal re-assembly. After this modification, R-Exo retains its original average size and has the same membrane proteins, which allows for targeting of tumor cells. Chlorin e6-loaded R-Exo (Ce6-R-Exo) can be visualized by photoacoustic imaging and can efficiently generate reactive oxygen species inside tumor cells under laser irradiation. In addition, Ce6-R-Exo increased the release of cytokines from immune cells, which indicates that these modified exosomes can be used as an immunotherapeutic agent. In conclusion, we developed a novel strategy that enables photoacoustic imaging-guided photodynamic and immune-combination therapy for the treatment of cancer with tumor-derived Ce6-R-Exo.

Author Correction: CRISPR/Cas9-mediated knockout of Mct8 reveals a functional involvement of Mct8 in testis and sperm development in a rat.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CRISRP/Cas9-mediated knockout of Mct8 reveals a functional involvement of Mct8 in testis and sperm development in a rat.

Thyroid hormone (TH) has long been believed to play a minor role in male reproduction. However, evidences from experimental model of thyrotoxicosis or hypothyroidism suggests its role in spermatogenesis. Cellular action of TH requires membrane transport via specific transporters such as monocarboxylate transporter 8 (MCT8). SLC16A2 (encodes for MCT8) inactivating mutation in humans can lead to Allan-Herndon Dudley-syndrome, a X-linked psychomotor and growth retardation. These patients present cryptorchidism which suggests a role of MCT8 during spermatogenesis. In this study, we found that Mct8 is highly expressed during early postnatal development and decreases its expression in the adulthood of testis of wild-type male rats. Histological analysis revealed that spermatogonia largely lacks MCT8 expression while spermatocytes and maturing spermatids highly express MCT8. To further understand the role of Mct8 during spermatogenesis, we generated Slc16a2 (encodes MCT8) knockout rats using CRISPR/Cas9. Serum THs (T3 and T4) level were significantly altered in Slc16a2 knockout rats when compared to wild-type littermates during early to late postnatal development. Unlike Slc16a2 knockout mice, Slc16a2 knockout rats showed growth delay during early to late postnatal development. In adult Slc16a2 knockout rats, we observed reduced sperm motility and viability. Collectively, our data unveil a functional involvement of MCT8 in spermatogenesis, underscoring the importance of TH signaling and action during spermatogenesis.

Combination of chemotherapy and photodynamic therapy for cancer treatment with sonoporation effects.

To overcome the limitations of single therapy, chemotherapy has been studied to be combined with photodynamic therapy. However, nanomedicine combining anticancer drug and photosensitizer still cannot address the insufficiency of drug delivery and the off-targeting effect. To address drug delivery issue, we have developed a doxorubicin encapsulating human serum albumin nanoparticles/chlorin e6 encapsulating microbubbles (DOX-NPs/Ce6-MBs) complex system. Microbubbles enable ultrasound-triggered local delivery via sonoporation for maximizing the drug delivery to a target site. In both in vitro and in vivo experiments, the developed DOX-NPs/Ce6-MBs drug delivery complex could be confirmed to transfer drugs deeply and effectively into cancerous tumors through the following three steps; (1) the local release of nanoparticles due to the cavitation of DOX-NPs/Ce6-MBs; (2) the enhanced extravasation of DOX-NPs and Ce6-liposome/micelle due to the sonoporation phenomenon; (3) the improved penetration of extravasated nanomedicines into the deep tumor region due to the mechanical energy of ultrasound. As a result, the developed DOX-NPs/Ce6-MBs complex with ultrasound irradiation showed increased therapeutic effects compared to the case where no ultrasound irradiation was applied. The DOX-NPs/Ce6-MBs was concluded from this study to be the optimal drug delivery system for external-stimuli local combination (chemotherapy + PDT) therapy.

Development and evaluation of a CEACAM6-targeting theranostic nanomedicine for photoacoustic-based diagnosis and chemotherapy of metastatic cancer.

Metastasis is the leading cause of cancer-related deaths. A number of chemotherapeutic and early diagnosis strategies, including nanomedicine, have been developed to target metastatic tumor cells. However, simultaneous inhibition and imaging of metastasis is yet to be fully achieved. Methods: To overcome this limitation, we have developed human serum albumin-based nanoparticles (tHSA-NPs) with photoacoustic imaging capability, which target carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6). CEACAM6 is highly expressed in metastatic anoikis-resistant tumor cells. Results:In vitro, the CEACAM6-targeting tHSA-NPs efficiently targeted CEACAM6-overexpressing metastatic anoikis-resistant tumor cells. In vivo, CEACAM6-targeting tHSA-NPs administered intravenously to BALB/c nude mice efficiently inhibited lung metastasis in circulating anoikis-resistant tumor cells compared to the controls. In addition, anoikis-resistant tumor cells can be successfully detected by photoacoustic imaging, both in vitro and in vivo, using the intrinsic indocyanine green-binding affinity of albumin. Conclusion: In summary, the CEACAM6-targeting albumin-based nanoparticles allowed the delivery of drugs and photoacoustic imaging to metastatic anoikis-resistant tumor cells in vitro and in vivo. Based on the expression of CEACAM6 in a variety of tumors, CEACAM6-targeting nanomedicine might be used to target various types of metastatic tumor cells.

Effect of high intensity focused ultrasound (HIFU) in conjunction with a nanomedicines-microbubble complex for enhanced drug delivery.

Although nanomedicines have been intensively investigated for cancer therapy in the past, poor accumulation of nanomedicines in tumor sites remains a serious problem. Therefore, a novel drug delivery system is required to enhance accumulation and penetration of nanomedicines at the tumor site. Recently, high-intensity focused ultrasound (HIFU) has been highlighted as a non-invasive therapeutic modality, and showed enhanced therapeutic efficacy in combination with nanomedicines. Cavitation effect induced by the combination of HIFU and microbubbles results in transiently enhanced cell membrane permeability, facilitating improved drug delivery efficiency into tumor sites. Therefore, we introduce the acoustic cavitation and thermal/mechanical effects of HIFU in conjunction with microbubble to overcome the limitation of conventional drug delivery. The cavitation effect maximized by the strong acoustic energy of HIFU induced the preferential accumulation of nanomedicine locally released from the nanomedicines-microbubble complex in the tumor. In addition, the mechanical effect of HIFU allowed the accumulated nanomedicines to penetrate into deeper tumor region. The preferential accumulation and deeper penetration of nanomedicines by HIFU showed enhanced therapeutic efficacy, compared to low frequency ultrasound (US). These overall results demonstrate that the strategy combined nanomedicines-microbubble complex with HIFU is a promising tools for cancer therapy.

Microbubbles used for contrast enhanced ultrasound and theragnosis: a review of principles to applications.

Ultrasound was developed several decades ago as a useful imaging modality, and it became the second most popular diagnostic tool due to its non-invasiveness, real-time capabilities, and safety. Additionally, ultrasound has been used as a therapeutic tool with several therapeutic agents and in nanomedicine. Ultrasound imaging is often used to diagnose many types of cancers, including breast, stomach, and thyroid cancers. In addition, ultrasound-mediated therapy is used in cases of joint inflammation, rheumatoid arthritis, and osteoarthritis. Microbubbles, when used as ultrasound contrast agents, can act as echo-enhancers and therapeutic agents, and they can play an essential role in ultrasound imaging and ultrasound-mediated therapy. Recently, various types of ultrasound contrast agents made of lipid, polymer, and protein shells have been used. Air, nitrogen, and perfluorocarbon are usually included in the core of the microbubbles to enhance ultrasound imaging, and therapeutic drugs are conjugated and loaded onto the surface or into the core of the microbubbles, depending on the purpose and properties of the substance. Many research groups have utilized ultrasound contrast agents to enhance the imaging signal in blood vessels or tissues and to overcome the blood-brain barrier or blood-retina barrier. These agents are also used to help treat diseases in various regions or systems of the body, such as the cardiovascular system, or as a cancer treatment. In addition, with the introduction of targeted moiety and multiple functional groups, ultrasound contrast agents are expected to have a potential future in ultrasound imaging and therapy. In this paper, we briefly review the principles of ultrasound and introduce the underlying theory, applications, limitations, and future perspectives of ultrasound contrast agents.

Near-Infrared Plasmonic Assemblies of Gold Nanoparticles with Multimodal Function for Targeted Cancer Theragnosis.

Here we report a novel assembly structure of near-infrared plasmonic gold nanoparticles (AuNPs), possessing both photoacoustic (PA) and photothermal (PT) properties. The template for the plasmonic AuNP assembly is a bioconjugate between short double-strand DNA (sh-dsDNA) and human methyl binding domain protein 1 (MBD1). MBD1 binds to methylated cytosine-guanine dinucleotides (mCGs) within the sequence of sh-dsDNA. Hexahistidine peptides on the engineered MBD1 function as a nucleation site for AuNP synthesis, allowing the construction of hybrid conjugates, sh-dsDNA-MBD1-AuNPs (named DMAs). By varying the length of sh-dsDNA backbone and the spacer between two adjacent mCGs, we synthesized three different DMAs (DMA_5mCG, DMA_9mCG, and DMA_21mCG), among which DMA_21mCG exhibited a comparable photothermal and surprisingly a higher photoacoustic signals, compared to a plasmonic gold nanorod. Further, epidermal growth factor receptor I (EGFR)-binding peptides are genetically attached to the MBD1 of DMA_21mCG, enabling its efficient endocytosis into EGFR-overexpressing cancer cells. Notably, the denaturation of MBD1 disassembled the DMA and accordingly released the individual small AuNPs (<5 nm) that can be easily cleared from the body through renal excretion without causing accumulation/toxicity problems. This DMA-based novel approach offers a promising platform for targeted cancer theragnosis based on simultaneous PA imaging and PT therapy.

Nanocomposites of Molybdenum Disulfide/Methoxy Polyethylene Glycol-co-Polypyrrole for Amplified Photoacoustic Signal.

Photoacoustic activity is the generation of an ultrasonic signal via thermal expansion or bubble formation, stimulated by laser irradiation. Photoacoustic nanoplatforms have recently gained focus for application in bioelectric interfaces. Various photoacoustic material types have been evaluated, including gold nanoparticles, semiconductive π-conjugating polymers (SP), etc. In this study, surfactant-free methoxy-polyethylene glycol-co-polypyrrole copolymer (mPEG-co-PPyr) nanoparticles (NPs) and mPEG-co-PPyr NP/molybdenum disulfide (mPEG-co-PPyr/MoS2) nanocomposites (NCs) were prepared and their photoacoustic activity was demonstrated. The mPEG-co-PPyr NPs and mPEG-co-PPyr/MoS2 NCs both showed photoacoustic signal activity. The mPEG-co-PPyr/MoS2 NCs presented a higher photoacoustic signal amplitude at 700 nm than the mPEG-co-PPyr NPs. The enhanced photoacoustic activity of the mPEG-co-PPyr/MoS2 NCs might be attributed to heterogeneous interfacial contact between mPEG-co-PPyr and the MoS2 nanosheets due to complex formation. Laser ablation of MoS2 might elevate the local temperature and facilitate the thermal conductive transfer in the mPEG-co-PPyr/MoS2 NCs, amplifying PA signal. Our study, for the first time, demonstrates enhanced PA activity in SP/transition metal disulfide (TMD) composites as photoacoustic nanoplatforms.

Reducible Polyethylenimine Nanoparticles for Efficient siRNA Delivery in Corneal Neovascularization Therapy.

The aim of this study is to establish the safe and effective ocular delivery system of therapeutic small interfering RNA (siRNA) in corneal neovascularization therapy. The major hurdle present in siRNA-based corneal neovascularization (CNV) therapy is severe cytotoxicity caused by repetitive drug treatment. A reducible branched polyethylenimine (rBPEI)-based nanoparticle (NP) system is utilized as a new siRNA carrier as a hope for CNV therapy. The thiolated BPEI is readily self-crosslinked in mild conditions to make high molecular weight rBPEI thus allowing the creation of stable siRNA/rBPEI nanoparticles (siRNA-rBPEI-NPs). In the therapeutic region, the rBPEI polymeric matrix is effectively degraded into nontoxic LMW BPEI inside the reductive cytosol causing the rapid release of the encapsulated siRNA into the cytosol to carry out its function. The fluorescent-labeled siRNA-rBPEI-NPs can release siRNA into the entire corneal region after subconjuctival injection into the eye of Sprague Dawley rats thus confirming the proof of concept of this system.

Multifunctional theranostic contrast agent for photoacoustics- and ultrasound-based tumor diagnosis and ultrasound-stimulated local tumor therapy.

Biomedical imaging-guided cancer therapy should have capabilities of both accurate tumor diagnosis and high therapeutic efficacy for the personalized treatment. Various biomedical imaging-guided cancer therapies are currently being investigated to overcome current limitations that include low sensitivity of diagnosis and poor drug delivery to the tumor site. Here, we report the development of a multifunctional theranostic contrast agent demonstrating high sensitive photoacoustic and ultrasound imaging and effective local delivery of anticancer drug to a tumor site. A microbubble (porphyrin-MB) was developed using phospholipid-porphyrin conjugates to enhance ultrasound and photoacoustic signal intensities simultaneously. Paclitaxel-loaded human serum albumin nanoparticles (PTX-HSA-NPs) were then conjugated onto the surface of the microbubble. The developed PTX-HSA-NPs conjugated porphyrin-MB (porphyrin-MB-NPs) provided sensitive, dual modal images of a tumor at 700 nm optimal laser wavelength for photoacoustic imaging and 5-14 MHz operating frequency for the ultrasound imaging. In addition, porphyrin-MB-NPs efficiently suppressed tumor growth by ultrasound exposure. Exposure to the focused ultrasound triggered the collapse of porphyrin-MB-NPs, resulting in the local release of PTX-HSA-NPs and enhanced penetration into the tumor site. The increased preferential accumulation and penetration of PTX-HSA-NPs suppressed tumor growth 10-fold more than without exposure to ultrasound. In conclusion, the developed porphyrin-MB-NPs establish a new paradigm in simultaneous bi-functional ultrasound/photoacoustic imaging diagnosis and locally triggered release of nanomedicine and enhanced chemotherapy efficiency.

Photoacoustic-based nanomedicine for cancer diagnosis and therapy.

Photoacoustic imaging is the latest promising diagnostic modality that has various advantages such as high spatial resolution, deep penetration depth, and use of non-ionizing radiation. It also employs a non-invasive imaging technique and optically functionalized imaging. The goal of this study was to develop a nanomedicine for simultaneous cancer therapy and diagnosis based on photoacoustic imaging. Human serum albumin nanoparticles loaded with melanin and paclitaxel (HMP-NPs) were developed using the desolvation technique. The photoacoustic-based diagnostic and chemotherapeutic properties of HMP-NPs were evaluated through in vitro and in vivo experiments. The size and zeta potential of the HMP-NPs were found to be 192.8±21.11nm and -22.2±4.39mV, respectively. In in vitro experiments, HMP-NPs produced increased photoacoustic signal intensity because of the loaded melanin and decreased cellular viability because of the encapsulated paclitaxel, compared to the free human serum albumin nanoparticles (the control). In vivo experiments showed that the HMP-NPs efficiently accumulated inside the tumor, resulting in the enhanced photoacoustic signal intensity in the tumor site, compared to the normal tissues. The in vivo chemotherapy study demonstrated that HMP-NPs had the capability to treat cancer for an extended period. In conclusion, HMP-NPs were simultaneously capable of photoacoustic diagnostic and chemotherapy against cancer.

Combination effect of paclitaxel and hyaluronic acid on cancer stem-like side population cells.

Cancer recurrence is the main cause of chemotherapeutic treatment failure. The mechanisms driving cancer recurrence may be due to very rare subpopulation cells, cancer stem-like cells (CSCs). Therefore, the early detection and better treatment of cancer stem-like cells are of great interest. In this study, we investigated how to eliminate the side population cells (SP), which have the characteristics of cancer stem-like cells, and also show chemotherapy resistance. Fluorescence-activated cell sorting (FACS) were used to sort SP and non-SP cells from human liver cancers, Huh-7 Hyaluronic acid (HA), which is an abundant component in the extracellular matrix, is known to involve in proliferation of normal and cancer cells. Herein, we investigated the effect of HA component on chemotherapy against SP cells. Cell growth inhibitory effects of the paclitaxel (PTX) chemotherapy combined with the HA component on SP cells of Huh-7 was determined using the trypan blue dye exclusion test. PTX combined with HA was found to show more increased inhibition of cell growth in both SP and non-SP cells, compared to free PTX treatment. In conclusion, SP cells of Huh-7 shows chemotherapeutic drug resistance due to the over-expressed efflux pumps. HA proposed one of possibilities to overcome the limitation of chemotherapy against cancer stem-like cells.

Entrapped doxorubicin nanoparticles for the treatment of metastatic anoikis-resistant cancer cells.

Metastasized and chemoresistant secondary breast cancer treatment commonly shows very low efficacy. A new efficient treatment method is required to overcome the limitation against the secondary breast cancer. In this study, anoikis-resistant breast cancer cells, MDA-MB-231 and MCF-7 were developed as models of chemoresistant and metastatic breast cancer. Doxorubicin encapsulating human serum albumin nanoparticles (HSA+DOX NPs) were fabricated to confirm the benefits of nanoparticles at the treatment of anoikis-resistant breast cancer cells. The side population (SP) fraction in the anoikis-resistant cancer cells was higher than the parental cells. HSA+DOX NPs were more cytotoxic to anoikis-resistant cancer cells than free doxorubicin. The confocal microscope images demonstrated HSA+DOX NPs to deliver more doxorubicin into cells compared to the free doxorubicin by bypassing the drug efflux pump systems of anoikis-resistant cancer cells. In this study, a nanomedicine-based drug delivery carrier shows a potential in treating a metastasized and chemoresistant breast cancer.