DCE-MRI quantitative transport mapping for noninvasively detecting hypoxia inducible factor-1α, epidermal growth factor receptor overexpression, and Ki-67 in nasopharyngeal carcinoma patients.

BACKGROUND: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has the potential to noninvasively detect expression of hypoxia inducible factor-1-alpha (HIF-1α), epidermal growth factor receptor (EGFR), and Ki-67 in nasopharyngeal carcinoma (NPC) by quantitatively measuring tumor blood flow, vascularity, and permeability. PURPOSE: We aim to explore the utility of DCE-MRI in detecting HIF-1α, EGFR, and Ki-67 expression levels using traditional Kety's/Tofts' modeling and quantitative transport mapping (QTM). MATERIALS AND METHODS: Eighty-nine NPC patients underwent DCE-MRI before treatment were enrolled. DCE-MRI was processed to generate the following kinetic parameters: |u| and D from the QTM model, tumor blood flow (TBF) from Kety's model, and Ktrans, Ve, and Kep from Tofts' model. Pretreatment levels of HIF-1α, EGFR, and Ki-67 were assessed by immunohistochemistry and classified into low and high expression groups. RESULTS: |u| (p < 0.001) and TBF (p = 0.015) values were significantly higher in the HIF-1α high-expression group compared to low-expression group. Only Ktrans (p = 0.016) was significantly higher in the EGFR high-expression group. Only |u| (p < 0.001) values were significantly higher in the Ki-67 high-expression group compared to low-expression group. Multiple linear regression analyses showed that |u| independently correlated with HIF-1α and Ki-67 expression, and Ktrans independently correlated with EGFR. The areas under the ROC curves of |u| for HIF-1α and Ki-67, and Ktrans for EGFR were 0.83, 0.74, and 0.70, respectively. CONCLUSION: |u| and Ktrans derived from DCE-MRI may be considered as noninvasive imaging markers for detecting hypoxia and proliferation in NPC patients.

Bone-Inspired Tube Filling Decellularized Matrix of Toad Cartilage Provided an Osteoinductive Microenvironment for Mesenchymal Stem Cells to Facilitate the Radius Defect Repair of Rabbit.

Toad bone not only contains the rich cartilage-like matrix but also presents low immunogenicity. It is inferred that decellularized toad bone matrix (dBECM) may provide the more profitable osteoinductive microenvironment for mesenchymal stem cells (MSCs) to promote the repair of bone defects. Herein, a hollow bone-inspired tube is first made from hydroxyapatite (HA) and poly (γ-glutamic acid) (PGA), and then MSCs/dBECM hydrogel is uniformly filled to its central cavity, constructing a biomimetic bone (dBECM + MSCs - PGA + HA). In vitro scratch and transwell experiments show that dBECM hydrogel not only effectively promotes migration and proliferation of MSCs but also induces their osteogenic differentiation. Moreover, the less inflammatory macrophages infiltrate at rat skin after subcutaneously injecting dBECM hydrogel, indicating its low potential for inflammatory attack. After implanting dBECM + MSCs - PGA + HA to critical radius defect of rabbit, X-ray and CT imaging shows that the cortex is effectively regenerated and the medullary cavity recanalization is completed at 20 weeks. Moreover, the expression of Collagen-II and OCN are obviously increased in the defect after implanting dBECM + MSCs - PGA + HA. The therapeutic mechanism of dBECM + MSCs - PGA + HA scaffold are highly associated with the enhanced angiogenesis. Collectively, the biomimetic dBECM + MSCs - PGA + HA scaffold may be a promising strategy to improve radius defect healing efficiency.

Skin-permeable liposome improved stability and permeability of bFGF against skin of mice with deep second degree scald to promote hair follicle neogenesis through inhibition of scar formation.

Excessive deposition of extracellular matrix (ECM) usually resulted in scar formation during wound healing, which caused skin dysfunction, such as hair loss. Basic fibroblast growth factor (bFGF) was very helpful for promoting hair follicle neogenesis and regulating the remodeling of ECM during wound healing. Because of its poor stability in wound fluids and low permeability against the dense wound scar, the repairing quality of bFGF on wound was hindered largely in clinical practice. To overcome these drawbacks, herein, a novel liposome with silk fibroin hydrogel core (bFGF-SF-LIP) was firstly prepared to stabilize bFGF, followed by insertion of laurocapam, a permeation enhancer, into the liposomal membrane to construct a skin-permeable liposome (SP-bFGF-SF-LIP). The encapsulated efficiency of bFGF was reaching to nearly 90% when ratio of drug/lipids above 1:300, and it activity was not compromised by laurocapam. SP-bFGF-SF-LIP exhibited a hydrodynamic diameter of 103.3 nm and Zeta potential of -2.31 mV. The stability of the encapsulated bFGF in wound fluid was obviously enhanced. After 24 h of incubation with wound fluid containing MMP-9, the remaining bFGF was as high as 65.4 ± 0.5% for SP-bFGF-SF-LIP, while only 2.1 ± 0.2% of free bFGF was remained. The skin-permeability of bFGF was significantly enhanced by SP-bFGF-SF-LIP and most of the encapsulated bFGF penetrated into the dermis. After treatment with SP-bFGF-SF-LIP, the morphology of hair follicle at wound zone was obviously improved and the hair regrew on the deep second scald mice model. The therapeutic mechanism was highly associated with inhibiting scar formation and promoting vascular growth in dermis. Conclusively, SP-bFGF-SF-LIP may a potential option to improve wound healing with high-quality.

Sustained-release of FGF-2 from a hybrid hydrogel of heparin-poloxamer and decellular matrix promotes the neuroprotective effects of proteins after spinal injury.

INTRODUCTION: The short lifetime of protein-based therapies has largely limited their therapeutic efficacy in injured nervous post-spinal cord injury (post-SCI). METHODS: In this study, an affinity-based hydrogel delivery system provided sustained-release of proteins, thereby extending the efficacy of such therapies. The affinity-based hydrogel was constructed using a novel polymer, heparin-poloxamer (HP), as a temperature-sensitive bulk matrix and decellular spinal cord extracellular matrix (dscECM) as an affinity depot of drug. By tuning the concentration of HP in formulation, the cold ternary fibroblast growth factor-2 (FGF2)-dscECM-HP solution could rapidly gelatinize into a hydrogel at body temperature. Due to the strong affinity for FGF2, hybrid FGF2-dscECM-HP hydrogel enabled sustained-release of encapsulated FGF2 over an extended period in vitro. RESULTS: Compared to free FGF2, it was observed that both neuron functions and tissue morphology after SCI were clearly recovered in rats treated with FGF2-dscECM-HP hydrogel. Moreover, the expression of neurofilament protein and the density of axons were increased after treatment with hybrid FGF2-dscECM-HP. In addition, the neuroprotective effects of FGF2-dscECM-HP were related to inhibition of chronic endoplasmic reticulum stress-induced apoptosis. CONCLUSION: The results revealed that a hybrid hydrogel system may be a potential carrier to deliver macromolecular proteins to the injured site and enhance the therapeutic effects of proteins.

Silk fibroin nanoparticles dyeing indocyanine green for imaging-guided photo-thermal therapy of glioblastoma.

Silk was easily dyed in traditional textile industry because of its strong affinity to many colorants. Herein, the biocompatible silk fibroin was firstly extracted from Bombyx mori silkworm cocoons. And SF nanoparticles (SFNPs) were prepared for dyeing indocyanine green (ICG) and construct a therapeutic nano-platform (ICG-SFNPs) for photo-thermal therapy of glioblastoma. ICG was easily encapsulated into SFNPs with a very high encapsulation efficiency reaching to 97.7 ± 1.1%. ICG-SFNPs exhibited a spherical morphology with a mean particle size of 209.4 ± 1.4 nm and a negative zeta potential of -31.9 mV, exhibiting a good stability in physiological medium. Moreover, ICG-SFNPs showed a slow release profile of ICG in vitro, and only 24.51 ± 2.27% of the encapsulated ICG was released even at 72 h. Meanwhile, ICG-SFNPs exhibited a more stable photo-thermal effect than free ICG after exposure to near-infrared irradiation. The temperature of ICG-SFNPs rapidly increased by 33.9 °C within 10 min and maintained for a longer time. ICG-SFNPs were also easily internalized with C6 tumor cells in vitro, and a strong red fluorescence of ICG was observed in cytoplasm for cellular imaging. In vivo imaging showed that ICG-SFNPs were effectively accumulated inside tumor site of C6 glioma-bearing Xenograft nude mice through vein injection. Moreover, the temperature of tumor site was rapidly rising up to kill tumor cells after local NIR irradiation. After treatment, its growth was completely suppressed with the relative tumor volume of 0.55 ± 033 while free ICG of 33.72 ± 1.90. Overall, ICG-SFNPs may be an effective therapeutic means for intraoperative phototherapy and imaging.

Kangfuxin promotes apoptosis of gastric cancer cells through activating ER­stress and autophagy.

Gastric cancer is a leading cause of cancer­associated mortality worldwide. In studies on the mechanisms of antigastric cancer drugs, autophagy and endoplasmic reticulum (ER) stress have been demonstrated to serve an active role in gastric cancer. The organic extract of Periplaneta americana (also termed American Cockroach), which is named Kangfuxin (KFX) in China, has been used clinically as a traditional Chinese medicine against disorders, including stomach bleeding, gastric ulcers, tuberculosis, burns and trauma. However, the role of KFX and its mechanism in gastric cancer remains to be elucidated. The present study aimed to determine the effects of KFX in vitro against cultured the human carcinoma SGC­7901 cell line, and to explore the potential mechanism of the anticancer effects of KFX in gastric cancer. SGC­7901 cells were treated with different concentrations of KFX for varying amounts of time. As a result, KFX treatment decreased the ratio of apoptosis regulators Bcl­2/Bax, activated ER stress and induced significant apoptosis in SGC­7901 cells. Furthermore, KFX was able to restore the ER stress activation blocked by 4­phenylbutyrate. In addition, KFX activated autophagy in SGC­7901 cells. These results demonstrated that ER stress, autophagy and the apoptosis­inducing effects of KFX in SGC­7901 cells may achieve promising anticancer effects in numerous other types of cancer. In particular, ER stress may serve an essential role in KFX­induced anticancer effects on gastric carcinoma and a secondary role in autophagy.

Thermo-sensitive hydrogels combined with decellularised matrix deliver bFGF for the functional recovery of rats after a spinal cord injury.

Because of the short half-life, either systemic or local administration of bFGF shows significant drawbacks to spinal injury. In this study, an acellular spinal cord scaffold (ASC) was encapsulated in a thermo-sensitive hydrogel to overcome these limitations. The ASC was firstly prepared from the spinal cord of healthy rats and characterized by scanning electronic microscopy and immunohistochemical staining. bFGF could specifically complex with the ASC scaffold via electrostatic or receptor-mediated interactions. The bFGF-ASC complex was further encapsulated into a heparin modified poloxamer (HP) solution to prepare atemperature-sensitive hydrogel (bFGF-ASC-HP). bFGF release from the ASC-HP hydrogel was more slower than that from the bFGF-ASC complex alone. An in vitro cell survival study showed that the bFGF-ASC-HP hydrogel could more effectively promote the proliferation of PC12 cells than a bFGF solution, with an approximate 50% increase in the cell survival rate within 24 h (P < 0.05). Compared with the bFGF solution, bFGF-ASC-HP hydrogel displayed enhanced inhibition of glial scars and obviously improved the functional recovery of the SCI model rat through regeneration of nerve axons and the differentiation of the neural stem cells. In summary, an ASC-HP hydrogel might be a promising carrier to deliver bFGF to an injured spinal cord.

An injectable acellular matrix scaffold with absorbable permeable nanoparticles improves the therapeutic effects of docetaxel on glioblastoma.

Intratumoral drug delivery (IT) is an inherently appealing approach for concentrating toxic chemotherapies at the site of action. However, for most chemotherapies, poor tumor penetration and short retention at the administration site limit their anti-tumor effects. In this work, we describe permeable nanoparticles (NPs) prepared with a novel amphiphilic polymer, RRR-α-tocopheryl succinate-grafted-ε-polylysine conjugate (VES-g-ε-PLL). The nanoparticles (NPs) of VES-g-ε-PLL exhibited an ultra-small hydrodynamic diameter (20.8 nm) and positive zeta potential (20.6 mV), which facilitate strong glioma spheroid penetration ability in vitro. Additionally, the hydrophobic model drug docetaxel (DTX) could be effectively encapsulated in the nanoparticles with 3.99% drug loading and 73.37% encapsulation efficiency. To prolong the retention time of DTX-loaded nanoparticles (DTX-NPs) in the tumor, intact decellularized brain extracellular matrix (dBECM) derived from healthy rats was used as a drug depot to adsorb the ultra-small DTX-NPs. The intact DTX-NPs-adsorbing dBECM scaffold was further homogenized into an injectable DTX-NPs-dBECM suspension for intratumoral administration. The DTX-NPs-dBECM suspension exhibited slower DTX release than naked DTX-NPs without compromising the tumor penetration ability of DTX-NPs. An antitumor study showed that the DTX-NPs-dBECM suspension exhibited more powerful in vitro inhibition of tumor spheroid growth than free DTX solution or DTX-NPs. Due to strong tumor penetration ability and prolonged retention, DTX-NPs-dBECM led to complete suppression of glioma growth in vivo at 28 days after treatment. The therapeutic mechanism was due to enhanced proliferation inhibition and apoptosis of tumor cells and angiogenesis inhibition of glioma after treatment with DTX-NPs-dBECM. Finally, the safety of DTX-NPs-dBECM at the therapeutic dose was demonstrated via pathological HE assay from heart, liver, spleen, lung and kidney tissues. In conclusion, permeable nanoparticle-absorbing dBECM is a potential carrier for intratumoral delivery of common chemotherapeutics.

Glioma-targeted superparamagnetic iron oxide nanoparticles as drug-carrying vehicles for theranostic effects.

Multifunctional nanoparticles capable of the specific delivery of therapeutics to diseased cells and the real-time imaging of these sites have the potential to improve cancer treatment through personalized therapy. In this study, we have proposed a multifunctional nanoparticle that integrate magnetic targeting, drug-carrier functionality and real-time MRI imaging capabilities in one platform for the theranostic treatment of tumors. The multifunctional nanoparticle was designed with a superparamagnetic iron oxide core and a multifunctional shell composed of PEG/PEI/polysorbate 80 (Ps 80) and was used to encapsulate DOX. DOX-loaded multifunctional nanoparticles (DOX@Ps 80-SPIONs) with a Dh of 58.0 nm, a zeta potential of 28.0 mV, and a drug loading content of 29.3% presented superior superparamagnetic properties with a saturation magnetization (Ms) of 24.1 emu g(-1). The cellular uptake of DOX@Ps 80-SPIONs by C6 cells under a magnetic field was significantly enhanced over that of free DOX in solution, resulting in stronger in vitro cytotoxicity. The real-time therapeutic outcome of DOX@Ps 80-SPIONs was easily monitored by MRI. Furthermore, the negative contrast enhancement effect of the nanoparticles was confirmed in glioma-bearing rats. Prussian blue staining and ex vivo DOX fluorescence assays showed that the magnetic Ps 80-SPIONs and encapsulated DOX were delivered to gliomas by imposing external magnetic fields, indicating effective magnetic targeting. Due to magnetic targeting and Ps 80-mediated endocytosis, DOX@Ps 80-SPIONs in the presence of a magnetic field led to the complete suppression of glioma growth in vivo at 28 days after treatment. The therapeutic mechanism of DOX@Ps 80-SPIONs acted by inducing apoptosis through the caspase-3 pathway. Finally, DOX@Ps 80-SPIONs' safety at therapeutic dosage was verified using pathological HE assays of the heart, liver, spleen, lung and kidney. Multifunctional SPIONs could be used as potential carriers for the theranostic treatment of CNS diseases.

Advanced Interfere Treatment of Diabetic Cardiomyopathy Rats by aFGF-Loaded Heparin-Modified Microbubbles and UTMD Technique.

This study aims to investigate the preclinical performance and mechanism of a novel strategy of aFGF-loaded heparin-modified microbubbles (aFGF-HMB) combined with ultrasound-targeted microbubble destruction (UTMD) technique for diabetic cardiomyopathy (DCM) prevention. Type 1 diabetic rats were induced by streptozotocin. Twelve weeks after intervention, indexes from transthoracic echocardiography and cardiac catheterization showed that the left ventricular function in the aFGF-HMB/UTMD group was significantly improved compared with diabetes control (DM). From Picrosirius Red staining and TUNEL staining, the aFGF-HMB/UTMD group showed significant difference from the other groups. The cardiac collagen volume fraction (CVF) and myocardial cell apoptosis index (AI) in aFGF-HMB/UTMD group decreased to 7.2 % and 7.11 % respectively, compared with the DM group (CVF = 24.5 % and AI =20.3 % respectively). The results of myocardial microvascular density (MCD) also proved the strongest inhibition of aFGF-HMB/UTMD group on DCM progress. CD31 staining of aFGF-HMB/UTMD group reached 22 n/hrp, much higher than that of DM group (9 n/hrp). These results confirmed that the abnormalities including left ventricular dysfunction, myocardial fibrosis, cardiomyocytes apoptosis and microvascular rarefaction could be suppressed by twice weekly aFGF treatments for 12 consecutive weeks (free aFGF or aFGF-HMB+/-UTMD), with the strongest improvements observed in the aFGF-HMB/UTMD group (P < 0.05 vs free aFGF or aFGF-HMB). Western blot analyses of heart tissue further revealed the highest aFGF, anti-apoptosis protein (Bcl-2), VEGF-C, pAkt, pFoxo-3a levels and strongest reduction in pro-apoptosis proteins (Bax) level in aFGF-HMB/UTMD group. Overall, aFGF-HMB combined with UTMD technique might be developed as an effective strategy to prevent DCM in future clinical therapy.

Brain tumor-targeted delivery and therapy by focused ultrasound introduced doxorubicin-loaded cationic liposomes.

Brain tumor lacks effective delivery system for treatment. Focused ultrasound (FUS) can reversibly open BBB without impacts on normal tissues. As a potential drug carrier, cationic liposomes (CLs) have the ability to passively accumulate in tumor tissues for their positive charge. In this study, FUS introduced doxorubicin-loaded cationic liposomes (DOX-CLs) were applied to improve the efficiency of glioma-targeted delivery. Doxorubicin-loaded CLs (DOX-CLs) and quantum dot-loaded cationic liposomes (QD-CLs) were prepared using extrusion technology, and their characterizations were evaluated. With the advantage of QDs in tracing images, the glioma-targeted accumulation of FUS + CLs was evaluated by fluorescence imaging and flow cytometer. Cell survival rate, tumor volume, animal survival time, and brain histology in C6 glioma model were investigated to evaluate the glioma-targeted delivery of FUS + DOX-CLs. DOX-CLs and QD-CLs had suitable nanoscale sizes and high entrapment efficiency. The combined strategy of FUS introduced CLs significantly increased the glioma-targeted accumulation for load drugs. FUS + DOX-CLs showed the strongest inhibition on glioma based on glioma cell in vitro and glioma model in vivo experiments. From MRI and histological analysis, FUS + DOX-CLs group strongly suppressed the glioma progression and extended the animal survival time to 81.2 days. Among all the DOX treatment groups, FUS + DOX-CLs group showed the best cell viability and highest level of tumor apoptosis and necrosis. Combining the advantages of BBB reversible opening by FUS and glioma-targeted binding by CLs, ultrasound introduced cationic liposomes could achieve glioma-targeted delivery, which might be developed as a potential strategy for future brain tumor therapy.

Prevent diabetic cardiomyopathy in diabetic rats by combined therapy of aFGF-loaded nanoparticles and ultrasound-targeted microbubble destruction technique.

Acidic fibroblast growth factor (aFGF) has shown the great potential to prevent the structural and functional injuries caused by diabetic cardiomyopathy (DCM). The present study sought to investigate the preclinical performance and mechanism of the combination therapy of aFGF-nanoparticles (aFGF-NP) and ultrasound-targeted microbubble destruction (UTMD) technique for DCM prevention. From Mason staining and TUNEL staining, aFGF-NP+UTMD group showed significant differences from the diabetes group and other groups treated with aFGF or aFGF-NP. The cardiac collagen volume fraction (CVF) and cardiac myocyte apoptosis index in aFGF-NP+UTMD group reduced to 4.15% and 2.31% respectively, compared with those in the diabetes group (20.5% and 11.3% respectively). Myocardial microvascular density (MCD) in aFGF-NP+UTMD group was up to 35n/hpf, much higher than that in the diabetes group (14n/hpf). The diabetes group showed similar results (MCD, CVF and cardiac myocyte apoptosis index) to other aFGF treatment groups (free aFGF±UTMD or aFGF-NP). Indexes from transthoracic echocardiography and hemodynamic evaluation also proved the same conclusion. These results confirmed that the abnormalities including diastolic dysfunctions, myocardial fibrosis and metabolic could be suppressed by the different extents of twice weekly aFGF treatments for 12 consecutive weeks (free aFGF or aFGF-NP±UTMD), with the strongest improvements observed in the aFGF-NP+UTMD group. Western blot and immunohistochemical analyses of heart tissue samples further revealed the high efficiency of heart-targeted delivery and effective cardioprotection with this combination approach. Overall, this study has generated supportive data that are critical for the translation of a promising DCM prevention strategy.