Universal immunotherapeutic strategy for hepatocellular carcinoma with exosome vaccines that engage adaptive and innate immune responses.

BACKGROUND: Personalized immunotherapy utilizing cancer vaccines tailored to the tumors of individual patients holds promise for tumors with high genetic heterogeneity, potentially enabling eradication of the tumor in its entirety. METHODS: Here, we demonstrate a general strategy for biological nanovaccines that trigger tailored tumor-specific immune responses for hepatocellular carcinoma (HCC). Dendritic cell (DC)-derived exosomes (DEX) are painted with a HCC-targeting peptide (P47-P), an α-fetoprotein epitope (AFP212-A2) and a functional domain of high mobility group nucleosome-binding protein 1 (N1ND-N), an immunoadjuvant for DC recruitment and activation, via an exosomal anchor peptide to form a "trigger" DEX vaccine (DEXP&A2&N). RESULTS: DEXP&A2&N specifically promoted recruitment, accumulation and activation of DCs in mice with orthotopic HCC tumor, resulting in enhanced cross-presentation of tumor neoantigens and de novo T cell response. DEXP&A2&N elicited significant tumor retardation and tumor-specific immune responses in HCC mice with large tumor burdens. Importantly, tumor eradication was achieved in orthotopic HCC mice when antigenic AFP peptide was replaced with the full-length AFP (A) to form DEXP&A&N. Supplementation of Fms-related tyrosine kinase 3 ligand greatly augmented the antitumor immunity of DEXP&A&N by increasing immunological memory against tumor re-challenge in orthotopic HCC mice. Depletion of T cells, cross-presenting DCs and other innate immune cells abrogated the functionality of DEXP&A&N. CONCLUSIONS: These findings demonstrate the capacity of universal DEX vaccines to induce tumor-specific immune responses by triggering an immune response tailored to the tumors of each individual, thus presenting a generalizable approach for personalized immunotherapy of HCC, by extension of other tumors, without the need to identify tumor antigens.

In vitro naphthylquinoxaline thymidine conjugate and UVA treated cancer cells are effective therapeutic vaccines for tumors in vivo with CpG as the adjuvant.

Introduction: Cancer cells induced into immunogenic cell death (ICD) in vitro can be directly used as a whole cell vaccine for tumor immunotherapy with many advantages, especially enacting immediate and intense 'eat me' signals to engage immune system. Unfortunately, there have been few successes with in vitro ICD cancer cells as a treatment vaccine. Objective: To demonstrate that cancer cells treated in vitro with a new class of potent ICD inducer, naphthylquinoxaline thymidine conjugate (NAP) followed by UVA irradiation would be able to act as an effective tumor immunotherapy directly. Methods: The therapeutic potentials of treated cancer cell plus different vaccine adjuvants were assessed by in vivo liver tumor model and in vitro mixed lymphocyte reaction studies. The elicited activated T cells were determined with immunohistochemistry and T cell induced cytotoxicity studies. Results: Treatment of established H22 tumor with in vitro NAP and UVA treated cancer cell vaccine led to significantly improved survival. Further mixed lymphocyte reaction study implied that adjuvants alum and CpG would improve the therapeutic potential whereas poly IC would not be as effective. Subsequent in vivo validation of alum and CpG adjuvants indicated that only CpG in NAP and UVA treated cell vaccine resulted in markedly enhanced survival (median at 71 days and 50% tumor-free) as compared with PBS group (14.5 days, 0%) and CpG alone (36 days, 0%). It was revealed that the enhanced efficacy by CpG was specific to NAP and UVA treated cells. Moreover, the effective tumor immunotherapy was achieved through the infiltration of active CD4 and CD8 T cells in tumors and acquisition of cancer cell-specific cytotoxic CD8 T cells. Conclusion: In vitro NAP and UVA treated cancer cells plus CpG adjuvant are effective tumor therapeutic vaccines per se.

Alarmin-painted exosomes elicit persistent antitumor immunity in large established tumors in mice.

Treating large established tumors is challenging for dendritic cell (DC)-based immunotherapy. DC activation with tumor cell-derived exosomes (TEXs) carrying multiple tumor-associated antigen can enhance tumor recognition. Adding a potent adjuvant, high mobility group nucleosome-binding protein 1 (HMGN1), boosts DCs' ability to activate T cells and improves vaccine efficiency. Here, we demonstrate that TEXs painted with the functional domain of HMGN1 (TEX-N1ND) via an exosomal anchor peptide potentiates DC immunogenicity. TEX-N1ND pulsed DCs (DCTEX-N1ND) elicit long-lasting antitumor immunity and tumor suppression in different syngeneic mouse models with large tumor burdens, most notably large, poorly immunogenic orthotopic hepatocellular carcinoma (HCC). DCTEX-N1ND show increased homing to lymphoid tissues and contribute to augmented memory T cells. Importantly, N1ND-painted serum exosomes from cancer patients also promote DC activation. Our study demonstrates the potency of TEX-N1ND to strengthen DC immunogenicity and to suppress large established tumors, and thus provides an avenue to improve DC-based immunotherapy.

Naphthyl quinoxaline thymidine conjugate is a potent anticancer agent post UVA activation and elicits marked inhibition of tumor growth through vaccination.

Anticancer anthracyclines are cytotoxic drugs that can induce antitumor immune response as a secondary effect through immunogenic cell death (ICD) mechanism. However, the immunogenic potency is quite limited, possibly due to that these chemotherapeutic agents are not specifically developed as ICD inducers. Thus, new drug entities through studies focusing on enhanced ICD induction would significantly promote antitumor immune response in the vaccination application. We report here a naphthyl quinoxaline thymidine conjugate as a new class of cytotoxic compounds that effectively induced in vivo antitumor activity through the vaccination application. Synthesized naphthyl quinoxaline conjugates were weak fluorescent thymidine analog yet exhibited a pronounced anticancer activity in the low nanomolar range post UVA activation. The potent activity of naphthyl conjugate was able to induce the marked detection of ICD markers including ATP and HMGB1 extracellular and calreticulin intracellularly at 2 h post UVA activation. Most importantly, mice vaccinated with cells treated with naphthyl conjugate plus UVA exhibited complete tumor growth inhibition in the tumor challenge study, and the induced immunogenic inhibition was much more effective than that of mitoxantrone anthracycline drug. All these results demonstrate the high potential of naphthyl quinoxaline conjugate for the cancer cell vaccine against tumor.

cRGD target liposome delivery system promoted immunogenic cell death through enhanced anticancer potency of a thymidine conjugate under UVA activation as a cancer vaccine.

Conventional chemotherapeutic and photodynamic therapy have recently been shown to also elicit immune response against cancer through the immunogenic cell death mechanism, which can be potentially translated into effective cancer vaccines. However, there are few studies on the potential of nanodelivery system to promote the immunogenic cell death as a cancer vaccine. We reported here that cRGD target liposome delivery system was capable to promote the immunogenic cell death through enhanced potency of a thymidine conjugate post UVA activation as a cancer vaccine. Liposomes and cRGD target liposomes were found to significantly increase the cellular accumulation of the thymidine conjugate and subsequently translated into enhanced cytotoxic potency after UVA activation. More importantly, cRGD target liposomes of the thymidine conjugate markedly promoted the early detection of immunogenic cell death markers including ATP, HMGB1 and calreticulin. Subsequent in vivo vaccination-challenge study confirmed effective tumor growth inhibition by the cRGD liposomal thymidine conjugate and UVA treated cancer cells as the cancer vaccine. In addition, liposomes and cRGD target liposomes alone did not shown any induction of the immunogenic cell death markers, revealing the adjuvant nature of the nanodelivery system.

Evaluation of non-targeting, C- or N-pH (low) insertion peptide modified superparamagnetic iron oxide nanoclusters for selective MRI of liver tumors and their potential toxicity in cirrhosis.

Superparamagnetic iron oxide nanoclusters (SPIONs) modified with pH (low) insertion peptide (pHLIP) could be advantageous for magnetic resonance imaging (MRI) diagnosis of liver tumors at the early stage due to their unique responsiveness to the tumor acidic microenvironment when tumor markers are unknown. However, many critical aspects including the effectiveness of selective MRI in liver tumors, types of delivery and the potential safety profile in cirrhosis need to be fully evaluated. In this study, we report the evaluation of non-targeting, C- or N-pHLIP modified SPIONs as the contrast agent for selective MRI of liver tumors and their potential toxicity profile in cirrhosis. It was found that N-pHLIP modified SPIONs did not result in the loss of liver tumor in the T2-weight MRI but provided additional dynamic details of tumor structures that would enhance the diagnosis of liver tumors at a small size below 8 mm. In addition, an enhanced safety profile was found for N-pHLIP modified SPIONs with almost fully recoverable impact in cirrhosis. In contrast, the poly-d-lysine assembled SPIONs and C-terminus linked pHLIP SPIONs had non-tumor specific MRI contrast enhancement and potential safety risks in cirrhosis due to the iron overload post injection. All these results implied the promising potential of N-terminus linked pHLIP SPIONs as an MRI contrast agent for the diagnosis of liver tumors.

Anchor peptide captures, targets, and loads exosomes of diverse origins for diagnostics and therapy.

Exosomes are circulating nanovesicular carriers of macromolecules, increasingly used for diagnostics and therapeutics. The ability to load and target patient-derived exosomes without altering exosomal surfaces is key to unlocking their therapeutic potential. We demonstrate that a peptide (CP05) identified by phage display enables targeting, cargo loading, and capture of exosomes from diverse origins, including patient-derived exosomes, through binding to CD63-an exosomal surface protein. Systemic administration of exosomes loaded with CP05-modified, dystrophin splice-correcting phosphorodiamidate morpholino oligomer (EXOPMO) increased dystrophin protein 18-fold in quadriceps of dystrophin-deficient mdx mice compared to CP05-PMO. Loading CP05-muscle-targeting peptide on EXOPMO further increased dystrophin expression in muscle with functional improvement without any detectable toxicity. Our study demonstrates that an exosomal anchor peptide enables direct, effective functionalization and capture of exosomes, thus providing a tool for exosome engineering, probing gene function in vivo, and targeted therapeutic drug delivery.

Fluorescent peptide highlights micronodules in murine hepatocellular carcinoma models and humans in vitro.

Early detection and clear delineation of microscopic lesions during surgery are critical to the prognosis and survival of patients with hepatocellular carcinoma (HCC), a devastating malignancy without effective treatments except for resection. Tools to specifically identify and differentiate micronodules from normal tissue in HCC patients can have a positive impact on survival. Here, we discovered a peptide that preferentially binds to HCC cells through phage display. Significant accumulation of the fluorescence-labeled peptide in tumor from ectopic and orthotopic HCC mice was observed within 2 hours of systemic injection. Contrast between tumor and surrounding liver is up to 6.5-fold, and useful contrast lasts for 30 hours. Micronodules (0.03 cm in diameter) in liver and lung can clearly be distinguished from normal tissue with this fluorescence-labeled peptide in orthotopic HCC mice and HCC patients. Compared to indocyanine green, a Food and Drug Administration-approved imaging contrast agent, an up to 8.7-fold higher differentiation ratio of tumor to fibrosis is achieved with this fluorescence-labeled peptide. Importantly, this peptide enables up to 10-fold differentiation between HCC and peritumoral tissue in human tissues and the complete removal of tumor in HCC mice with surgical navigation. No abnormalities in behavior or activity are observed after systemic treatment, indicating the absence of overt toxicity. The peptide is metabolized with a half-life of approximately 4 hours in serum. CONCLUSION: Our findings demonstrate that micronodules can be specifically differentiated with high sensitivity from surrounding tissue with this molecule, opening clinical possibilities for early detection and precise surgery of HCC. (Hepatology 2018).

Maintaining pH-dependent conformational flexibility of M1 is critical for efficient influenza A virus replication.

The M gene segment of influenza A virus has been shown to be a contributing factor to the high growth phenotype. However, it remains largely unknown why matrix protein 1 (M1), the major structural protein encoded by M gene, exhibits pH-dependent conformational changes during virus replication. Understanding the mechanisms underlying efficient virus replication can help to develop strategies not only to combat influenza infections but also to improve vaccine supplies. M(NLS-88R) and M(NLS-88E) are two M1 mutants differing by only a single amino acid: G88R vs G88E. G88R but not G88E was the compensatory mutation naturally selected by the virus after its nuclear localization signal was disrupted. Our study shows that, compared with M(NLS-88E) M1, M(NLS-88R) M1 dissociated quickly from viral ribonucleoproteins (vRNPs) at higher pH and took less time to dissemble in vitro, despite forming thicker matrix layer and having stronger association with vRNP in assembled virions. Correspondingly, M(NLS-88R) replicated more efficiently and was genetically more stable than M(NLS-88E). Crystallographic analysis indicated that M(NLS-88R) M1, like wild-type M1, is able to switch from a face-to-back-oriented conformation to a face-to-face-oriented conformation when pH drops from neutral to acidic, whereas G88E mutation causes M(NLS-88E) M1 to be trapped in a face-to-face-arranged conformation regardless of environmental pH. Our results suggest that maintaining M1 pH-dependent conformational flexibility is critical for efficient virus replication, and position 88 is a key residue controlling M1 pH-dependent conformational changes. Our findings provide insights into developing M1-based antiviral agents.

pH-responsive pHLIP (pH low insertion peptide) nanoclusters of superparamagnetic iron oxide nanoparticles as a tumor-selective MRI contrast agent.

Superparamagnetic iron oxide nanoparticles (SPION) are contrast agents used for noninvasive tumor magnetic resonance imaging (MRI). SPION with active targeting by tumor-specific ligands can effectively enhance the MRI sensitivity and specificity of tumors. However, the challenge remains when the tumor specific markers are yet to be determined, especially in the case of early tumor detection. In this study, the effectiveness of pH-responsive SPION via a pH low insertion peptide (pHLIP) to target tumor acidic microenvironments was investigated. Polylysine polymers were first successfully modified with pHLIP to have the pH-responsive capability. SPION pHLIP nanoclusters of 64, 82, 103, and 121nm size were then assembled by the pH-responsive polymers in a size-controlled manner. The pH-responsive SPION nanoclusters of the 64nm size exhibited the most effective pH-responsive retention in cells and tumor selective imaging in MRI. More importantly, the unique contrast enhancement of tumor inner core by the pH-responsive SPION in three different tumor models demonstrated the clinical potential to target tumor acidic microenvironment through pHLIP for tumor early detection and diagnosis by MRI. STATEMENT OF SIGNIFICANCE: Detection and diagnosis of tumors at early stage are critical for the improvement of the survival rate of cancer patients. However, the challenge remains when the tumor specific markers are yet to be determined, especially in early tumor detection. pH low insertion peptide (pHLIP) has been used as a specific ligand to target the tumor acidic microenvironment for tumors at early and metastatic stages. Superparamagnetic iron nanoparticles (SPION) are contrast enhancing agents used in the noninvasive magnetic resonance imaging for tumors. This research has demonstrated that pH-responsive pHLIP nanoclusters of SPION were able to target different tumors and facilitate the noninvasive diagnosis of tumors by MRI.

For Better or Worse, Iron Overload by Superparamagnetic Iron Oxide Nanoparticles as a MRI Contrast Agent for Chronic Liver Diseases.

Superparamagnetic iron oxide nanoparticles (SPIONs) have recently been used as an effective magnetic resonance imaging (MRI) contrast agent for the noninvasive diagnosis of chronic liver diseases including nonalcohol fatty liver diseases, nonalcohol steatohepatitis, and cirrhosis as well as liver tumors. However, the potential risk of the iron overload by SPIONs has been highly underestimated in chronic liver diseases. While most of SPIONs have been shown safe in the healthy group, significant toxicity potential by the iron overload has been revealed through immunotoxicity, lipid peroxidation, and fatty acid and cholesterol metabolism in cirrhosis as a high risk factor. As a result, the systems toxicology assessments of SPIONs are crucial in both healthy ones and chronic liver disease models to determine the margin of safety. In addition, the challenge of the iron overload by SPIONs requires better designed SPIONs as MRI contrast agents for chronic liver diseases such as the biodegradable nanocluster assembly with urine clearance.

Broad Spectrum Anti-Influenza Agents by Inhibiting Self-Association of Matrix Protein 1.

The matrix protein 1 (M1) of influenza A virus (IAV) exists as a three-dimensional oligomeric structure in mature virions with high sequence conservation across different IAV subtypes, which makes it a potential broad spectrum antiviral target. We hypothesized that impairing self-association of M1 through a small molecule 'wedge', which avidly binds to an M1-M1 interface, would result in a completely new class of anti-influenza agents. To establish this proof-of-principle, we performed virtual screening on a library of >70,000 commercially available small molecules that resulted in several plausible 'wedges'. Biophysical studies showed that the best molecule bound the M1 protein potently and weakened M1-M1 self-association. Most importantly, the agent reduced the thickness of the M1 layer in mature virions and inhibited in ovo propagation of multiple IAV strains including H1N1, pandemic H1N1, H3N2 and H5N1, which supports the "wedge" hypothesis. These results demonstrate that M1 is a promising druggable target for the discovery of a completely new line of broad spectrum anti-IAV agents.

Iron overload by Superparamagnetic Iron Oxide Nanoparticles is a High Risk Factor in Cirrhosis by a Systems Toxicology Assessment.

Superparamagnetic iron oxide nanoparticles (SPIONs) as a contrast agent have been widely used in magnetic resonance imaging for tumor diagnosis and theranostics. However, there has been safety concern of SPIONs with cirrhosis related to excess iron-induced oxidative stress. In this study, the impact of iron overload by SPIONs was assessed on a mouse cirrhosis model. A single dose of SPION injection at 0.5 or 5 mg Fe/kg in the cirrhosis group induced a septic shock response at 24 h with elevated serum levels of liver and kidney function markers and extended impacts over 14 days including high levels of serum cholesterols and persistent low serum iron level. In contrast, full restoration of liver functions was found in the normal group with the same dosages over time. Analysis with PCR array of the toxicity pathways revealed the high dose of SPIONs induced significant expression changes of a distinct subset of genes in the cirrhosis liver. All these results suggested that excess iron of the high dose of SPIONs might be a risk factor for cirrhosis because of the marked impacts of elevated lipid metabolism, disruption of iron homeostasis and possibly, aggravated loss of liver functions.

Structure-activity relationship study of anticancer thymidine-quinoxaline conjugates under the low radiance of long wavelength ultraviolet light for photodynamic therapy.

Thymidine quinoxaline conjugate (dT-QX) is a thymidine analog with selective cytotoxicity against different cancer cells. In this study, the structure activity relationship study of dT-QX analogs was carried out under the low radiance of black fluorescent (UVA-1) light. Significantly enhanced cytotoxicity was observed under UVA-1 activation among analogs containing both thymidine and quinoxaline moieties with different length of the linker, stereochemical configuration and halogenated substituents. Among these analogs, the thymidine dichloroquinoxaline conjugate exhibited potent activity under UVA-1 activation as the best candidate with EC50 at 0.67 µM and 1.3 µM against liver and pancreatic cancer cells, respectively. In contrast, the replacement of thymidine moiety with a galactosyl residue or the replacement of quinoxaline moiety with a fluorescent pyrenyl residue or a simplified diketone structure resulted in the full loss of activity. Furthermore, it was revealed that the low radiance of UVA-1 at 3 mW/cm(2) for 20 min was sufficient enough to induce the full cytotoxicity of thymidine dichloroquinoxaline conjugate and that the cytotoxic mechanism was achieved through a rapid and steady production of reactive oxygen species.

Biocompatible Low-Retention Superparamagnetic Iron Oxide Nanoclusters as Contrast Agents for Magnetic Resonance Imaging of Liver Tumor.

Although superparamagnetic iron oxide (SPIO) nanoparticles have been developed as a contrast agent for magnetic resonance imaging (MRI), acute iron overload due to the persistently high retention of SPIOs in the liver and spleen that are slowly converted to ferroproteins is a serious safety concern. Here, we report that the addition of poly-L-lysine polymers to an SPIO hydroxyethyl starch solution produced tightly controlled, monodispersed nanoparticles in a size-dependent manner as effective contrast agents for the MRI of liver tumors. High MRI contrast was demonstrated with an orthotopic liver tumor model at a low injection dose. Simultaneously, rapid bioclearance of excess iron in the lung and spleen and in blood serum was observed within 24 h post-injection. The full excretion of excess iron was confirmed in urine post-intravenous injection, suggesting that the effective clearance of SPIOs could be achieved with our SPIO nanoclusters as a liver imaging contrast agent to resolve acute iron overload in the clinical usage of SPIOs as a contrast agent.

Anticancer activity of a thymidine quinoxaline conjugate is modulated by cytosolic thymidine pathways.

BACKGROUND: High levels of thymidine kinase 1 (TK1) and thymidine phosphorylase (TYMP) are key molecular targets by thymidine therapeutics in cancer treatment. The dual roles of TYMP as a tumor growth factor and a key activation enzyme of anticancer metabolites resulted in a mixed outcome in cancer patients. In this study, we investigated the roles of TK1 and TYMP on a thymidine quinoxaline conjugate to evaluate an alternative to circumvent the contradictive role of TYMP. METHODS: TK1 and TYMP levels in multiple liver cell lines were assessed along with the cytotoxicity of the thymidine conjugate. Cellular accumulation of the thymidine conjugate was determined with organelle-specific dyes. The impacts of TK1 and TYMP were evaluated with siRNA/shRNA suppression and pseudoviral overexpression. Immunohistochemical analysis was performed on both normal and tumor tissues. In vivo study was carried out with a subcutaneous liver tumor model. RESULTS: We found that the thymidine conjugate had varied activities in liver cancer cells with different levels of TK1 and TYMP. The conjugate mainly accumulated at endothelial reticulum and was consistent with cytosolic pathways. TK1 was responsible for the cytotoxicity yet high levels of TYMP counteracted such activities. Levels of TYMP and TK1 in the liver tumor tissues were significantly higher than those of normal liver tissues. Induced TK1 overexpression decreased the selectivity of dT-QX due to the concurring cytotoxicity in normal cells. In contrast, shRNA suppression of TYMP significantly enhanced the selective of the conjugate in vitro and reduced the tumor growth in vivo. CONCLUSIONS: TK1 was responsible for anticancer activity of dT-QX while levels of TYMP counteracted such an activity. The counteraction by TYMP could be overcome with RNA silencing to significantly enhance the dT-QX selectivity in cancer cells.

6-Hydroxyflavone and derivatives exhibit potent anti-inflammatory activity among mono-, di- and polyhydroxylated flavones in kidney mesangial cells.

Inflammatory responses by kidney mesangial cells play a critical role in the glomerulonephritis. The anti-inflammatory potential of nineteen mono-, di- and polyhydroxylated flavones including fisetin, quercetin, morin, tricetin, gossypetin, apigenin and myricetin were investigated on rat mesangial cells with lipopolysaccharide (LPS) as the inflammatory stimuli. 6-Hydroxyflavone and 4',6-dihydroxyflavone exhibited high activity with IC50 in the range of 2.0 µM, a much better inhibition potential in comparison to the well-studied polyhydroxylated flavones. Interestingly, the anti-inflammatory activity was not due to direct quenching of NO radicals. Investigation on derivatives with methylation, acetylation or sulfation of 6-hydroxyl group revealed that 6-methoxyflavone was the most potent with an IC50 of 192 nM. Mechanistic study indicated that the anti-inflammatory activity of 6-methoxyflavone arose via the inhibition of LPS-induced downstream inducible NO synthase in mesangial cells. The identification of 6-hydroxyflavone and 6-methoxyflavone with potent anti-inflammatory activity in kidney mesangial cells provides a new flavone scaffold and direction to develop naturally derived products for potential nephritis prevention and treatment.

A simple, general approach of allosteric coagulation enzyme inhibition through monosulfated hydrophobic scaffolds.

Allosteric inhibition of coagulation enzymes offers the advantage of controlled inhibition. In this study, a small library of mono sulfated indole and benzothiazole based molecules was synthesized and screened against the panel of coagulation proteases. The results reveal that selected molecules inhibit the thrombin, factor Xa and factor XIa with moderate potency. Compound 6a was found to have an allosteric mode of inhibition against thrombin. Plasma clotting assays suggest that selected inhibitors 14b, 14c and 14d prolong both prothrombin and activated partial thromboplastin time. Overall, this work presents the newer class of allosteric inhibitors of thrombin and factor XIa with improved aqueous solubility profile.

Crystal structures of influenza A virus matrix protein M1: variations on a theme.

Matrix protein 1 (M1) of the influenza A virus plays multiple roles in virion assembly and infection. Interest in the pH dependence of M1's multiple functions led us to study the effect of subtle pH changes on M1 structure, resulting in the elucidation of a unique low-pH crystal structure of the N(1-165)-domain of A/WSN/33 (H1N1) M1 that has never been reported. Although the 2.2 Å crystal structure of M1 N-terminus shows a dimer with the two monomers interacting in a face-to-face fashion at low pH as observed earlier, a 44° rotation of the second monomer has led to a significantly different dimer interface that possibly affects dimer stability. More importantly, while one of the monomers is fully defined, the N-terminal half of the second monomer shows considerable disorder that appears inherent in the protein and is potentially physiologically relevant. Such disorder has not been observed in any other previously reported structure at either low or high pH conditions, despite similar crystallization pH conditions. By comparing our novel N(1-165)-domain structure with other low-pH or neutral-pH M1 structures, it appears that M1 can energetically access different monomer and dimer conformations, as well as oligomeric states, with varying degree of similarities. The study reported here provides further insights into M1 oligomerization that may be essential for viral propagation and infectivity.

Cyanine 5.5 conjugated nanobubbles as a tumor selective contrast agent for dual ultrasound-fluorescence imaging in a mouse model.

Nanobubbles and microbubbles are non-invasive ultrasound imaging contrast agents that may potentially enhance diagnosis of tumors. However, to date, both nanobubbles and microbubbles display poor in vivo tumor-selectivity over non-targeted organs such as liver. We report here cyanine 5.5 conjugated nanobubbles (cy5.5-nanobubbles) of a biocompatible chitosan-vitamin C lipid system as a dual ultrasound-fluorescence contrast agent that achieved tumor-selective imaging in a mouse tumor model. Cy5.5-nanobubble suspension contained single bubble spheres and clusters of bubble spheres with the size ranging between 400-800 nm. In the in vivo mouse study, enhancement of ultrasound signals at tumor site was found to persist over 2 h while tumor-selective fluorescence emission was persistently observed over 24 h with intravenous injection of cy5.5-nanobubbles. In vitro cell study indicated that cy5.5-flurescence dye was able to accumulate in cancer cells due to the unique conjugated nanobubble structure. Further in vivo fluorescence study suggested that cy5.5-nanobubbles were mainly located at tumor site and in the bladder of mice. Subsequent analysis confirmed that accumulation of high fluorescence was present at the intact subcutaneous tumor site and in isolated tumor tissue but not in liver tissue post intravenous injection of cy5.5-nanobubbles. All these results led to the conclusion that cy5.5-nanobubbles with unique crosslinked chitosan-vitamin C lipid system have achieved tumor-selective imaging in vivo.