Coaxial 3D printing of hierarchical structured hydrogel scaffolds for on-demand repair of spinal cord injury.

After spinal cord injury (SCI), endogenous neural stem cells (NSCs) near the damaged site are activated, but few NSCs migrate to the injury epicenter and differentiate into neurons because of the harsh microenvironment. It has demonstrated that implantation of hydrogel scaffold loaded with multiple cues can enhance the function of endogenous NSCs. However, programming different cues on request remains a great challenge. Herein, a time-programmed linear hierarchical structure scaffold is developed for spinal cord injury recovery. The scaffold is obtained through coaxial 3D printing by encapsulating a dual-network hydrogel (composed of hyaluronic acid derivatives and N-cadherin modified sodium alginate, inner layer) into a temperature responsive gelatin/cellulose nanofiber hydrogel (Gel/CNF, outer layer). The reactive species scavenger, metalloporphyrin, loaded in the outer layer is released rapidly by the degradation of Gel/CNF, inhibiting the initial oxidative stress at lesion site to protect endogenous NSCs; while the inner hydrogel with appropriate mechanical support, linear topology structure and bioactive cues facilitates the migration and neuronal differentiation of NSCs at the later stage of SCI treatment, thereby promoting motor functional restorations in SCI rats. This study offers an innovative strategy for fabrication of multifunctional nerve regeneration scaffold, which has potential for clinical treatment of SCI. STATEMENT OF SIGNIFICANCE: Two major challenges facing the recovery from spinal cord injury (SCI) are the low viability of endogenous neural stem cells (NSCs) within the damaged microenvironment, as well as the difficulty of neuronal regeneration at the injured site. To address these issues, a spinal cord-like coaxial scaffold was fabricated with free radical scavenging agent metalloporphyrin Mn (III) tetrakis (4-benzoic acid) porphyrin and chemokine N-cadherin. The scaffold was constructed by 3D bioprinting for time-programmed protection and modulation of NSCs to effectively repair SCI. This 3D coaxially bioprinted biomimetic construct enables multi-factor on-demand repair and may be a promising therapeutic strategy for SCI.

Ratiometric chemical exchange saturation transfer pH mapping using two iodinated agents with nonequivalent amide protons and a single low saturation power.

Background: As an essential physiological parameter, pH plays a critical role in maintaining cellular and tissue homeostasis. The ratiometric chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) method using clinically approved iodinated agents has emerged as one of the most promising noninvasive techniques for pH assessment. Methods: In this study, we investigated the ability to use the combination of two different nonequivalent amide protons, chosen from five iodinated agents, namely iodixanol, iohexol, iobitridol, iopamidol, and iopromide, for pH measurement. The ratio of two nonequivalent amide CEST signals was calculated and compared for pH measurements in the range of 5.6 to 7.6. To quantify the CEST signals at 4.3 and 5.5 parts per million (ppm), we employed two analytic methods: magnetization transfer ratio asymmetry and Lorentzian fitting analysis. Lastly, the established protocol was used to measure the pH values in healthy rat kidneys (n=5). Results: The combination of iodixanol and iobitridol at a ratio of 1:1 was found to be suitable for pH mapping. The saturation power level (B1) was also investigated, and a low B1 of 1.5 µT was adopted for subsequent pH measurements. Improved precision and an extended pH detection range were achieved using iodixanol and iobitridol (1:1 ratio) and a single low B1 of 1.5 µT in vitro. In vivo renal pH values were measured as 7.23±0.09, 6.55±0.15, and 6.29±0.23 for the cortex, medulla, and calyx, respectively. Conclusions: These results show that the ratiometric CEST method using two iodinated agents with nonequivalent amide protons could be used for in vivo pH mapping of the kidney under a single low B1 saturation power.

FITC characterization of a cathepsin B-responsive nanoprobe for report of differentiation of HL60 cells into macrophages.

A cathepsin B (Cat B)-responsive optical nanoprobe is designed and prepared for report of HL60 differentiation into macrophage. A peptide sequence FRFK is linked to fluorescein (FITC) via the distant amino group of its lysine and N-terminated with acrylic acid (AA) to yield a molecular fluorescent probe AA-FRFK (FITC). The molecular probe is further embedded in poly(lactic-co-glycolic acid) (PLGA) to form a fluorescent nanoprobe AA-FRFK (FITC)@PLGA. The resultant optical nanoprobe is degradable by lysosomal Cat B, which is expressed in macrophages with a level of 5-10 times of that in HL60 cells. As a result, a significant decrease in fluorescence intensity is associated with the differentiation process of HL60 to macrophage and can be used as an indication of the differentiation process. The findings may pave a way toward the development of a universal in vitro labeling strategy of exogenous stem cells for report of in vivo cell differentiation by a dual-mode imaging modality involving optical imaging and magnetic resonance imaging.

Naphthalene-facilitated self-assembly of a Gd-chelate as a novel T2 MRI contrast agent for visualization of stem cell transplants.

Naphthalene is coupled with DOTA via a peptide sequence to yield an amphipathic MRI probe Nap-CFGKTG-DOTA-Gd (Nap-Gd) that can self-assemble into nanofibers. Incubation of NSCs, hMSCs and L929 cells in the presence of Nap-Gd in the µM level can introduce a significant amount of Nap-Gd into the cells as nanoclusters or nanofibers. The resultant intracellular Gd content is 10-60 times that achieved by incubation with Dotarem at the same concentration. The labelled cells exhibit a significant hyperintensive effect under T1-weighted MRI and a significant hypointensive effect under T2-weighted MRI. The hypointensive effect is more persistent than the hyperintensive effect, which allows in vivo tracking of labelled hMSCs for over 12 days under T2-weighted MRI. A comprehensive interpretation of the MRI signal intensity and the associated relaxation times reveals the structure-function relationship between the binding status of Nap-Gd in cells (structure) and the magnetic relaxation processes (function) toward a full understanding of the observed hyperintensive and hypointensive effects.

IQF characterization of a cathepsin B-responsive nanoprobe for report of differentiation of HL60 cells into macrophages.

Tracking of in vivo fates of exogenous cell transplants in terms of viability, migration, directional differentiation and function delivery by a suitable method of medical imaging is of great significance in the development and application of various cell therapies. In this contribution directional differentiation of HL60 cells into macrophages and granulocytes, and a difference in the associated expression level of cathepsin B (Cat B) among the parent and daughter cells is used as a model to guide and evaluate the development of a Cat B-responsive Abz-FRFK-Dnp@PLGA nanoprobe for an optical report of the differentiation process. A well-documented internally quenched fluorescence (IQF) pair coupled with a peptide substrate FRFK of Cat B was synthesized and imbedded in PLGA to form the nanoprobe. The nanoprobe is resistant to leakage when dispersed in water for 10 days. Degradation of the nanoprobe is dominated by Cat B. HL60 cells were then labelled with the Abz-FRFK-Dnp@PLGA nanoprobe to track the differentiation process. Differentiation of labelled HL60 cells into macrophages exhibited a significantly higher fluorescence relative to the granulocytes or the labelled parent cells. The fluorescence difference allows the differentiation process to be followed. The established characterization and assessment procedure is to be used for the development and evaluation of nanoprobes for other imaging modalities.

Cell-assembled nanoclusters of MSC-targeting Gd-DOTA-peptide as a T2 contrast agent for MRI cell tracking.

A cyclic peptide CC9 that targets cell membrane of mesenchymal stem cells (MSCs) is coupled with Gd-DOTA to yield a Gd-DOTA-CC9 complex as MRI contrast agent. It is used to label human MSCs (hMSCs) via electroporation. Electroporation-labeling of hMSCs with Gd-DOTA-CC9 induces cell-assembly of Gd-DOTA-CC9 nanoclusters in the cytoplasm, significantly promotes cell-labeling efficacy and intracellular retention time of the agent. In vitro MRI of labeled hMSCs exhibits significant signal reduction under T2 -weighted MRI, which can allow long-term tracking of labeled cell transplants in in vivo migration. The labeling strategy is safe in cytotoxicity and differentiation potential.

Ultrasmall graphene oxide based T1 MRI contrast agent for in vitro and in vivo labeling of human mesenchymal stem cells.

Herein, we report on development of a two-dimensional nanomaterial graphene oxide (GO)-based T1 magnetic resonance imaging (MRI) contrast agent (CA) for in vitro and in vivo labeling of human mesenchymal stem cells (hMSCs). The CA was synthesized by PEGylation of ultrasmall GO, followed by conjugation with a chelating agent DOTA and then gadolinium(III) to form GO-DOTA-Gd complexes. Thus-prepared GO-DOTA-Gd complexes exhibited significantly improved T1 relaxivity, and the r1 value was 14.2 mM-1s-1 at 11.7 T, approximately three times higher than Magnevist, a commercially available CA. hMSCs can be effectively labeled by GO-DOTA-Gd, leading to remarkably enhanced cellular MRI effect without obvious adverse effects on proliferation and differentiation of hMSCs. More importantly, in vivo experiment revealed that intracranial detection of 5×105 hMSCs labeled with GO-DOTA-Gd is achieved. The current work demonstrates the feasibility of the GO-based T1 MRI CA for stem cell labeling, which may find potential applications in regenerative medicine.

MRI reveals slow clearance of dead cell transplants in mouse forelimb muscles.

A small molecule tetraazacyclododecane-1,4,7,10-tetraacetic acid (Gd­DOTA)4­TPP agent is used to label human mesenchymal stem cells (hMSCs) via electroporation (EP). The present study assessed the cytotoxicity of cell labeling, in addition to its effect on cell differentiation potential. There were no significant adverse effects on cell viability or differentiation induced by either EP or cellular uptake of (Gd­DOTA)4­TPP. Labeled live and dead hMSCs were transplanted into mouse forelimb muscles. T2­weighted magnetic resonance imaging (MRI) was used to track the in vivo fate of the cell transplants. The labeling and imaging strategy allowed long term tracking of the cell transplants and unambiguous distinguishing of the cell transplants from their surrounding tissues. Cell migration was observed for live hMSCs injected into subcutaneous tissues, however not for either live or dead hMSCS injected into limb muscles. A slow clearance process occurred of the dead cell transplants in the limb muscular tissue. The MRI results therefore reveal that the fate and physiological activities of cell transplants depend on the nature of their host tissue.

Magnetic Resonance Imaging Revealed Splenic Targeting of Canine Parvovirus Capsid Protein VP2.

Canine parvovirus (CPV) is a highly contagious infectious virus, whose infectious mechanism remains unclear because of acute gastroenteritis and the lack of an efficient tool to visualize the virus in real time during virology research. In this study, we developed an iron oxide nanoparticle supported by graphene quantum dots (GQD), namely, FeGQD. In this composite material, GQD acts as a stabilizer; thus, vacancies are retained on the surface for further physical adsorption of the CPV VP2 protein. The FeGQD@VP2 nanocomposite product showed largely enhanced colloidal stability in comparison with bare FeGQD, as well as negligible toxicity both in vitro and in vivo. The composite displayed high uptake into transferrin receptor (TfR) positive cells, which are distinguishable from FeGQD or TfR negative cells. In addition, the composite developed a significant accumulation in spleen rather than in liver, where bare FeGQD or most iron oxide nanoparticles gather. As these evident targeting abilities of FeGQD@VP2 strongly suggested, the biological activity of CPV VP2 was retained in our study, and its biological functions might correspond to CPV when the rare splenic targeting ability is considered. This approach can be applied to numerous other biomedical studies that require a simple yet efficient approach to track proteins in vivo while retaining biological function and may facilitate virus-related research.

Improving the solubility of dexlansoprazole by cocrystallization with isonicotinamide.

Cocrystallization of an active pharmaceutical ingredient (API) with a cocrystal former (co-former) is widely used to tailor the physicochemical properties of parent APIs. For proton-pump inhibitors (PPIs), the isolation of cocrystals has not been widely investigated. Here, a 1:1 cocrystal of a PPI molecule, dexlansoprazole (DLS), was obtained by solvent crystallization with isonicotinamide (INM). The product was characterized by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), solid-state and liquid NMR, as well as Fourier transform infrared spectroscopy (FTIR) techniques. A two-point R2(2)(9) hetero-synthon was proposed to exist in the cocrystal, where intermolecular hydrogen bonding occurs between NH, SO groups of DLS and amide of INM. The dissolution profiles of DLS and DLS-INM in water were also collected, and the results demonstrate the cocrystal exhibits superior apparent maximum solubility relative to the pure drug.

Modified VEGF targets the ischemic myocardium and promotes functional recovery after myocardial infarction.

Vascular endothelial growth factor (VEGF) promotes angiogenesis and improves cardiac function after myocardial infarction (MI). However, the non-targeted delivery of VEGF decreases its therapeutic efficacy due to an insufficient local concentration in the ischemic myocardium. In this study, we used a specific peptide to modify VEGF and determined that this modified VEGF (IMT-VEGF) localized to the ischemic myocardium through intravenous injection by interacting with cardiac troponin I (cTnI). When IMT-VEGF was used to mediate cardiac repair in a rat model of ischemia-reperfusion (I-R) injury, we observed a decreased scar size, enhanced angiogenesis and improved cardiac function. Moreover, an alternative treatment using the repeated administration of a low-dose IMT-VEGF also promoted angiogenesis and functional recovery. The therapeutic effects of IMT-VEGF were further confirmed in a pig model of MI as the result of the conserved properties of its interacting protein, cTnI. These results suggest a promising therapeutic strategy for MI based on the targeted delivery of IMT-VEGF.

Rational Design and Synthesis of γFe2 O3 @Au Magnetic Gold Nanoflowers for Efficient Cancer Theranostics.

An γFe2 O3 @Au core/shell-type magnetic gold nanoflower-based theranostic nano-platform is developed. It is integrated with ultrasensitive surface-enhanced Raman scattering imaging, high-resolution photo-acoustics imaging, real-time magnetic resonance imaging, and photothermal therapy capabilities.

A Carbon-Neutral Photosynthetic Microbial Fuel Cell Powered by Microcystis aeruginosa.

A photosynthetic microbial fuel cell (m-PMFC) is developed for generating electricity by harnessing solar energy using Microcystis aeruginosa. In this m-PMFC, commensal bacteria can consume the nutrients that Microcystis aeruginosa produces to generate electricity so that no net CO2production occurs. A b-MFC is constructed to confirm the role of commensal bacteria in electric generation. An s-PMFC is constructed to confirm the contribution of Microcystis aeruginosa as substrates. The power outputs of m-PMFCs exhibit no significant difference in terms of different inoculation amount of Microcystis aeruginosa or light/dark cycles. The power density of m-PMFC exhibits similar response to bubbling of N2and O2as that of b-MFC, as confirmed by cyclic voltammetry analysis of m-PMFC and b-MFC. Scanning electron microscope images demonstrate that the biofilm of m-PMFC consists mainly of commensal bacteria. These results suggest that commensal bacteria act as the main biocatalysts and Microcystis aeruginosa as the anode substrates in the m-PMFC.

Preoperative Detection and Intraoperative Visualization of Brain Tumors for More Precise Surgery: A New Dual-Modality MRI and NIR Nanoprobe.

In clinical practice, it is difficult to identify tumor margins during brain surgery due to its inherent infiltrative character. Herein, a unique dual-modality nanoprobe (Gd-DOTA-Ag2S QDs, referred as Gd-Ag2S nanoprobe) is reported, which integrates advantages of the deep tissue penetration of enhanced magnetic resonance (MR) imaging of Gd and the high signal-to-noise ratio and high spatiotemporal resolution of fluorescence imaging in the second near-infrared window (NIR-II) of Ag2S quantum dots (QDs). Due to the abundant tumor angiogenesis and the enhanced permeability and retention effect in the tumor, a brain tumor (U87MG) in a mouse model is clearly delineated in situ with the help of the Gd assisted T1 MR imaging and the intraoperative resection of the tumor is precisely accomplished under the guidance of NIR-II fluorescence imaging of Ag2S QDs after intravenous injection of Gd-Ag2S nanoprobe. Additionally, no histologic changes are observed in the main organs of the mouse after administration of Gd-Ag2S nanoprobe for 1 month, indicating the high biocompatibility of the nanoprobe. We expect that such a novel "Detection and Operation" strategy based on Gd-Ag2S nanoprobe is promising in future clinical applications.

Fates of Fe3O4 and Fe3O4@SiO2 nanoparticles in human mesenchymal stem cells assessed by synchrotron radiation-based techniques.

Superparamagnetic iron oxide nanoparticles (SPIOs) have been widely used as the magnetic resonance imaging (MRI) contrast agent in biomedical studies and clinical applications, with special interest recently in in vivo stem cell tracking. However, a full understanding of the fate of SPIOs in cells has not been achieved yet, which is particularly important for stem cells since any change of the microenvironment may disturb their propagation and differentiation behaviors. Herein, synchrotron radiation-based X-ray fluorescence (XRF) in combination with X-ray absorption spectroscopy (XAS) were used to in situ reveal the fate of Fe3O4 and Fe3O4@SiO2 NPs in human mesenchymal stem cells (hMSCs), in which the dynamic changes of their distribution and chemical speciation were precisely determined. The XAS analysis evidences that Fe3O4 NPs cultured with hMSCs are quite stable and almost keep their initial chemical form up to 14 days, which is contradictory to the previous report that Fe3O4 NPs were unstable in cell labeling assessed by using a simplified lysosomal model system. Coating with a SiO2 shell, Fe3O4@SiO2 NPs present higher stability in hMSCs without detectable changes of their chemical form. In addition, XRF analysis demonstrates that Fe3O4@SiO2 NPs can label hMSCs in a high efficiency manner and are solely distributed in cytoplasm during cell proliferation, making it an ideal probe for in vivo stem cell tracking. These findings with the help of synchrotron radiation-based XAS and XRF improve our understanding of the fate of SPIOs administered to hMSCs and will help the future design of SPIOs for safe and efficient stem cells tracking.

The interplay of T1- and T2-relaxation on T1-weighted MRI of hMSCs induced by Gd-DOTA-peptides.

Three Gd-DOTA-peptide complexes with different peptide sequence are synthesized and used as T1 contrast agent to label human mesenchymal stem cells (hMSCs) for magnetic resonance imaging study. The peptides include a universal cell penetrating peptide TAT, a linear MSC-specific peptide EM7, and a cyclic MSC-specific peptide CC9. A significant difference in labeling efficacy is observed between the Gd-DOTA-peptides as well as a control Dotarem. All Gd-DOTA-peptides as well as Dotarem induce significant increase in T1 relaxation rate which is in favor of T1-weighted MR imaging. Gd-DOTA-CC9 yields the maximum labeling efficacy but poor T1 contrast enhancement. Gd-DOTA-EM7 yields the minimum labeling efficacy but better T1 contrast enhancement. Gd-DOTA-TAT yields a similar labeling efficacy as Gd-DOTA-CC9 and similar T1 contrast enhancement as Gd-DOTA-EM7. The underlying mechanism that governs T1 contrast enhancement effect is discussed. Our results suggest that T1 contrast enhancement induced by Gd-DOTA-peptides depends not only on the introduced cellular Gd content, but more importantly on the effect that Gd-DOTA-peptides exert on the T1-relaxation and T2-relaxation processes/rates. Both T1 and particularly T2 relaxation rate have to be taken into account to interpret T1 contrast enhancement. In addition, the interpretation has to be based on cellular instead of aqueous longitudinal and transverse relaxivities of Gd-DOTA-peptides.

In vitro assessment of the dual-targeting behavior of a peptide-based magnetic resonance imaging contrast agent.

In this study, a peptide-based dual-targeting magnetic resonance imaging (MRI) contrast agent (S8) was designed and synthesized. Arg-Gly-Asp (RGD) and Asn-Gly-Arg (NGR) were combined in the targeting vector so as to allow binding, on the surface of tumor cells, to integrin αvß3 and aminopeptidase N (CD13), respectively. The longitudinal relaxivity (r1) value of S8 was 8.297 mM-1sec-1 at a magnetic field of 11.7 T, which is approximately double the r1 value (4.25 mM-1sec-1) of Magnevist, a commercially available contrast agent. MDA-MB-231 human breast cancer cells (which overexpress αvß3) and human prostate cancer cells PC-3 (which overexpress CD13) were used to investigate the tumor­targeting behavior of S8. The results from the present study indicate that the designed contrast agent, S8, targets both MDA-MB­231 and PC-3 cells.

Graphene oxide based theranostic platform for T1-weighted magnetic resonance imaging and drug delivery.

Magnetic resonance imaging (MRI) is a powerful and widely used clinical technique in cancer diagnosis. MRI contrast agents (CAs) are often used to improve the quality of MRI-based diagnosis. In this work, we developed a positive T1 MRI CA based on graphene oxide (GO)-gadolinium (Gd) complexes. In our strategy, diethylenetriaminepentaacetic acid (DTPA) is chemically conjugated to GO, followed by Gd(III) complexation, to form a T1 MRI CA (GO-DTPA-Gd). We have demonstrated that the GO-DTPA-Gd system significantly improves MRI T1 relaxivity and leads to a better cellular MRI contrast effect than Magnevist, a commercially used CA. Next, an anticancer drug, doxorubicin (DOX), was loaded on the surface of GO sheets via physisorption. Thus-prepared GO-DTPA-Gd/DOX shows significant cytotoxicity to the cancer cells (HepG2). This work provides a novel strategy to build a GO-based theranostic nanoplatform with T1-weighted MRI, fluorescence imaging, and drug delivery functionalities.

Dye sublimation as a measure of accumulated heat exposure.

Heat history monitor: Combination of the sublimation and adsorption processes of specific dyes can be used as a measure of accumulated heat exposure. Mass transfer from the sublimation layer to the adsorption layer strongly depends on temperature and results in a gradual color change in the adsorption layer. The total color change reflects the accumulated heat exposure.

Physicochemical characterization of felodipine-kollidon VA64 amorphous solid dispersions prepared by hot-melt extrusion.

To improve the dissolution and hence the oral bioavailability, amorphous felodipine (FEL) solid dispersions (SDs) with Kollidon® VA 64 (PVP/VA) were prepared. Hot-melt extrusion was employed with an extruding temperature below the melting point (Tm ) of FEL. X-ray powder diffraction (XRPD) and (13) C CP/MAS nuclear magnetic resonance (NMR) measurements show that the extrudates are amorphous. The intermolecular interaction between FEL and PVP/VA in SDs was investigated by Fourier transform infrared spectroscopy, (15) N CP/MAS NMR, and (1) H high-resolution MAS NMR. Furthermore, a single glass transition temperature (Tg ) was detected by differential scanning calorimetry in addition to a single (1) H T1 or T1rho relaxation time detected by (13) C NMR signals. These results confirm that the extrudates contain FEL dispersed into the polymer matrix at a molecular level with no detectable phase separation. This molecular-scale mixing results in a significantly faster dissolution rate compared with the pure crystalline FEL. Additionally, the molecular-scale mixing prevents the amorphous drug from recrystallizing even after being stored at 40°C/75% Relative Humidity for 2 months.