Genetic Modulation of Biosynthetic Gas Vesicles for Ultrasound Imaging.

Gas vesicles (GVs) from microorganisms are genetically air-filled protein nanostructures, and serve as a new class of nanoscale contrast agents for ultrasound imaging. Recently, the genetically encoded GV gene clusters have been heterologously expressed in Escherichia coli, allowing these genetically engineered bacteria to be visualized in vivo in a real-time manner by ultrasound. However, most of the GV genes remained functionally uncharacterized, which makes it difficult to regulate and modify GVs for broad medical applications. Here, the impact of GV proteins on GV formation is systematically investigated. The results first uncovered that the deletions of GvpR or GvpU resulted in the formation of a larger proportion of small, biconical GVs compared to the full-length construct, and the deletion of GvpT resulted in a larger portion of large GVs. Meanwhile, the combination of gene deletions has resulted in several genotypes of ultrasmall GVs that span from 50 to 20 nm. Furthermore, the results showed that E. coli carrying the ΔGvpCRTU mutant can produce strong ultrasound contrast signals in mouse liver. In conclusion, the study provides new insights into the roles of GV proteins in GV formation and produce ultrasmall GVs with a wide range of in vivo research.

Improving the properties of polylactic acid/polypropylene carbonate blends through cardanol-induced compatibility enhancement.

Cardanol (CD) is used as a reactive compatibilizer, and blended with polylactic acid (PLA) and polypropylene carbonate (PPC) resin (70/30(w/w)) to obtain a series of PLA/PPC/CD blends. The systematic study was conducted on the thermal properties, optical properties, rheological properties, mechanical properties, and microscopic morphology of the blend, by varying amounts of CD added to the blends. A detailed explanation and comprehensive analysis of the reaction mechanism between CD and PLA/PPC have been made. The study found that CD acts as a "bridge" between the PLA and PPC, forming the structure of a block copolymer (PLA-b-CD-b-PPC), and the copolymer can greatly improve the compatibility of PLA and PPC. When the amount of CD reaches 8 wt%, only one Tg is observed in the blend, simultaneously, PLA/PPC has already transitioned from a partially compatible system to a completely compatible system. At the same time, the addition of CD does not have any negative impact on the thermal stability of the PLA/PPC blend under processing temperature conditions, and the thermal stability of the PLA/PPC/CD blends can even be improved under extreme conditions. In addition, the addition of CD allows the PLA/PPC/CD blends to maintain a high light transmittance while reducing the opacity of the blend (the light transmittance remains above 92 %, and the opacity is reduced from 37 % to about 24 %), demonstrating excellent optical properties. Moreover, the elongation at break and impact strength of the PLA/PPC/CD blend both show a trend of first increasing and then decreasing with the increase of CD amount. When the CD amount varies within the range of 6- 8 wt%, the blends undergoes a brittle-ductile transition, and its toughness is greatly improved while the rigidity can also meet practical needs. When the amount of CD in the system increases to 12 wt%, the toughness of the blend reaches its peak, and its elongation at break and impact strength reach 513.24 % and 9211.5 J/m2 respectively (increased to 2442.84 % and 270.73 % of the PLA/PPC blend). Concurrently, the fracture surface of the blend exhibits large-scale plastic flow in the direction of the applied force, with marked shear yield phenomena, showing obvious characteristics of tough fracture.

Ultrasound Molecular Imaging of Bladder Cancer via Extradomain B Fibronectin-Targeted Biosynthetic GVs.

Purpose: Ultrasound molecular imaging (UMI) has proven promising to diagnose the onset and progression of diseases such as angiogenesis, inflammation, and thrombosis. However, microbubble-based acoustic probes are confined to intravascular targets due to their relatively large particle size, greatly reducing the application value of UMI, especially for extravascular targets. Extradomain B fibronectin (ED-B FN) is an important glycoprotein associated with tumor genesis and development and highly expressed in many types of tumors. Here, we developed a gas vesicles (GVs)-based nanoscale acoustic probe (ZD2-GVs) through conjugating ZD2 peptides which can specially target to ED-B FN to the biosynthetic GVs. Materials and Methods: ED-B FN expression was evaluated in normal liver and tumor tissues with immunofluorescence and Western blot. ZD2-GVs were prepared by conjugating ZD2 to the surface of GVs by amide reaction. The inverted microscope was used to analyze the targeted binding capacity of ZD2-GVs to MB49 cells (bladder cancer cell line). The contrast-enhanced imaging features of GVs, non-targeted control GVs (CTR-GVs), and targeted GVs (ZD2-GVs) were compared in three MB49 tumor mice. The penetration ability of ZD2-GVs in tumor tissues was assessed by fluorescence immunohistochemistry. The biosafety of GVs was evaluated by CCK8, blood biochemistry, and HE staining. Results: Strong ED-B FN expression was observed in tumor tissues while little expression in normal liver tissues. The resulting ZD2-GVs had only 267.73 ± 2.86 nm particle size and exhibited excellent binding capability to the MB49 tumor cells. The in vivo UMI experiments showed that ZD2-GVs produced stronger and longer retention in the BC tumors than that of the non-targeted CTR-GVs and GVs. Fluorescence immunohistochemistry confirmed that ZD2-GVs could penetrate the tumor vascular into the interstitial space of the tumors. Biosafety analysis revealed there was no significant cytotoxicity to these tested mice. Conclusion: Thus, ZD2-GVs can function as a potential UMI probe for the early diagnosis of bladder cancer.

Ultrasound Molecular Imaging of Epithelial Mesenchymal Transition for Evaluating Tumor Metastatic Potential via Targeted Biosynthetic Gas Vesicles.

Epithelial mesenchymal transition (EMT) of tumor cells is recognized as the main driver to promote metastasis. Extensive researches suggest that gradually decreased E-cadherin (E-cad) and increased N-cadherin (N-cad) exist in the tumor cells during the EMT process. However, there still lacks suitable imaging methods to monitor the status of EMT for evaluating tumor metastatic potentials. Herein, the E-cad-targeted and N-cad-targeted gas vesicles (GVs) are developed as the acoustic probes to monitor the EMT status in tumor. The resulting probes have ≈200 nm particle size and good tumor cell targeting performance. Upon systemic administration, E-cad-GVs and N-cad-GVs can traverse through blood vessels and bind to the tumor cells, producing strong contrast imaging signals in comparison with the nontargeted GVs. The contrast imaging signals correlate well with the expression levels of E-cad and N-cad and tumor metastatic ability. This study provides a new strategy to noninvasively monitor the EMT status and help to evaluate tumor metastatic potential in vivo.

Ultrasound molecular imaging of p32 protein translocation for evaluation of tumor metastasis.

Protein translocation is an essential process for living cells to respond to different physiological, pathological or environmental stimuli. However, its abnormal occurrence usually results in undesirable outcomes such as tumors. To date, there is still a lack of appropriate methods to detect this event in live animals in a real-time manner. Here, we identified the gradually increased cell-surface translocation of p32 protein from mitochondria during tumor progression. LyP-1-modified gas vesicles (LyP-1-GVs) were developed through conjugating LyP-1 (p32-targeting peptide) to the biosynthetic GVs to monitor the cell-surface level of p32 translocation. The resulting LyP-1-GVs have about 200 nm particle size and good tumor cell targeting performance. Upon systemic administration, LyP-1-GVs can traverse through blood vessels and bind to the tumor cells, producing strong contrast imaging signals in comparison with the non-targeted GVs. The contrast imaging signals correlate well with the cell-surface translocation level of p32 protein and tumor metastatic ability. To our knowledge, this is the first report about the in vivo detection of protein translocation to cell membrane from mitochondria by ultrasound molecular imaging. Our study provides a new strategy to explore the molecular events of protein membrane translocations for evaluation of tumor metastasis at the live animal level.

Effect of Different Comonomers Added to Graft Copolymers on the Properties of PLA/PPC/PLA-g-GMA Blends.

The melt-free radical grafting of glycidyl methacrylate (GMA) onto poly (lactic acid) (PLA) with styrene (St), α-methylstyrene (AMS), and epoxy resin (EP) as comonomers in a twin-screw extruder was used to prepare PLA-g-GMA graft copolymers. The prepared graft copolymers were then used as compatibilizers to prepare PLA/PPC/PLA-g-GMA blends by melt blending with PLA and polypropylene carbonate (PPC), respectively. The effects of different comonomers in the PLA-g-GMA graft copolymers on the thermal, rheological, optical, and mechanical properties and microstructure of the blends were studied. It was found that the grafting degree of PLA-g-GMA graft copolymers was increased to varying degrees after the introduction of comonomers in the PLA-g-GMA grafting reaction system. When St was used as the comonomer, the grafting degree of the PLA-g-GMA graft copolymer increased most significantly, from 0.8 to 1.6 phr. St as a comonomer also most improved the compatibility between PLA and PPC, and the haze of the blends was reduced while maintaining high transmittance. In addition, the PLA-g-GMA graft copolymer with the introduction of St as a comonomer significantly improved the impact toughness of the blends, while the thermal stability and tensile strength of the blends remained largely unchanged.

Ultrasound-mediated blood-brain barrier opening: An effective drug delivery system for theranostics of brain diseases.

Blood-brain barrier (BBB) remains a significant obstacle to drug therapy for brain diseases. Focused ultrasound (FUS) combined with microbubbles (MBs) can locally and transiently open the BBB, providing a potential strategy for drug delivery across the BBB into the brain. Nowadays, taking advantage of this technology, many therapeutic agents, such as antibodies, growth factors, and nanomedicine formulations, are intensively investigated across the BBB into specific brain regions for the treatment of various brain diseases. Several preliminary clinical trials also have demonstrated its safety and good tolerance in patients. This review gives an overview of the basic mechanisms, ultrasound contrast agents, evaluation or monitoring methods, and medical applications of FUS-mediated BBB opening in glioblastoma, Alzheimer's disease, and Parkinson's disease.

Ultrasound Molecular Imaging for Multiple Biomarkers by Serial Collapse of Targeting Microbubbles with Distinct Acoustic Pressures.

Ultrasound molecular imaging (UMI) has shown promise for assessing the expression levels of biomarkers for the early detection of various diseases. However, it remains difficult to simultaneously image multiple biomarkers in a single systemic administration, which is important for the accurate diagnosis of diseases and for understanding the dynamic intermolecular mechanisms that drive their malignant progression. The authors develop an ultrasound molecular imaging method by serial collapse of targeting microbubbles with distinct acoustic pressures for the simultaneous detection of two biomarkers. To test this, αv ß3 -targeting lipid microbubbles (L-MBα ) and VEGFR2-targeting lipid-PLGA microbubbles (LP-MBv ) are fabricated and simultaneously injected into tumor-bearing mice at 7 and 14 days, followed by the low-intensity acoustic collapse of L-MBα and high-intensity acoustic collapse of LP-MBv . The UMI signals of L-MBα and LP-MBv are obtained by subtracting the first post-burst signals from the first pre-burst signals, and subtracting the second post-burst signals from the first post-burst signals, respectively. Interestingly, the signal intensities from UMI agree with the immunohistochemical staining results for αv ß3 and VEGFR2. Importantly, they find a better fit for the invasive behavior of MDA-MB-231 breast tumors by analyzing the ratio of αv ß3 integrin to VEGFR2, but not the single αv ß3 or VEGFR2 levels.

Autogenous Fat Transplantation and Botulinum Toxin Injection Into the Masseter Muscle to Create an Ideal Oval Face.

BACKGROUND: East Asian faces vary in shape but only oval faces seem to be considered attractive. Many patients with wide faces seek removal of part of the mandibular angle and/or zygoma to achieve an ideal facial contour, but the procedure is high risk and the recovery period is relatively protracted. OBJECTIVES: We sought to achieve ideal facial contours through the use of autologous fat grafting (AFG) combined with masseter botulinum toxin (BTX) injection for patients with wide faces and masseter hypertrophy. METHODS: Fourteen patients with wide faces underwent AFG of the forehead, temporal region, cheeks, zygomatic body, nose, nasolabial fold, tear trough, and chin; and BTX injection into the masseter muscles. Each patient was photographed more than 6 months after the operation. The pre- and postoperative ratios pertaining to the facial aesthetics of the face were calculated. The Hollowness Severity Rating Scale (HSRS) and Ricketts's E-line were used to evaluate the photographs. Patient satisfaction was also investigated. RESULTS: All patients received AFG and 1 to 3 BTX injections. The face length:bizygomatic breadth, bigonial breadth:bizygomatic breadth, and lower-face height:middle-face height ratios improved greatly after treatment. The mean HSRS score decreased from 2.214 preoperatively to 1.071 postoperatively. The chin and nose became more prominent than before. Facial swelling persisted for an average of 11.929 days. All patients were satisfied with the treatment outcome. CONCLUSIONS: A combination of AFG and BTX injection was able to achieve an ideal oval face in East Asian patients with wide faces and masseter hypertrophy, with very few complications. Recovery was rapid and patient satisfaction was high.

[Check ligament suspension for correction of congenital severe blepharoptosis].

OBJECTIVE: To evaluate the clinical result of check ligament suspension for correction of congenital severe blepharoptosis. METHODS: Since Jan. 2010 to Nov. 2010, 15 eyes in 15 cases with congenital severe blepharoptosis were treated with the check ligament suspension. Palpebral aponeurosis was exposure by opening fascia palpebral during blepharoplasty. Palpebral aponeurosis was cut off about 5 mm above the tarsus. The check ligament was seen in the intermuscular space between the segment of levator and the anterior one third of superior rectus attached to the conjunctival fornix. Congenital blepharoptosis could be corrected by suturing the check ligament and levator palpebrae superior to the upper margin of tarsal plate with 3-0 silk thread. Double eyelid plasty was carried out in the end. RESULTS: The follow-up period was 3-11 months with good cosmetic result. All the cases could close their eyes in 15 to 30 days with no complication. CONCLUSIONS: In conclusion, this technique is quite successful in raising the level of the upper eyelid in severe congenital blepharoptosis. The check ligament moves in a similar direction as the natural movement of levator muscle, so both the postoperative static and dynamic appearance of the upper lid is more natural.