Microrheological Phenomenon and Mechanical Properties of High-Aspect-Ratio Microgroove Injection Moulding of Kaolin/PP Composites.

The microrheological phenomenon of kaolin-filled polypropylene (kaolin/PP) composites was investigated for the first time. The microviscosity of kaolin/PP composites was studied by changing the melt temperature and shear rate. Then, injection moulding experiments of rectangular microgrooves with different aspect ratios using kaolin/PP composites and mechanical property tests of the samples were carried out. The results showed that with increasing kaolin content, the microviscosity of the kaolin/PP composites gradually increases. The shear rate had the greatest influence on the microviscosity, and the kaolin content had the least influence. When the aspect ratio of rectangular microgrooves is small, with an increasing kaolin content, the microgroove filling rate increases, and the microstructured sample geometric shape replication effect is good; however, when the aspect ratio reaches 10:1, the microgroove filling rate decreases with an increasing kaolin content. The microstructured sample geometric shape replication effect is also poor, and size effects appear. Different factors control the microrheological morphology of composites with different aspect ratios, including the shear deformation and viscous flow of composites. The increase in kaolin content leads to a decrease in the friction coefficient and an increase in the wear resistance of the composites. We concluded that the best composite formulation for kaolin/PP composites in microinjection is the 7KL/PP composite with 7% kaolin. When the aspect ratio is 5:1, the reproduction of the microstructured sample geometry is the best, and the comprehensive mechanical properties of the sample are the best.

Oxygen nanocarrier broke the hypoxia trap of solid tumors and rescued transfection efficiency for gene therapy.

BACKGROUND: Gene therapy shows great promise for a broad array of diseases. However, we found that hypoxic tumor microenvironment (TME) exerted significant inhibitory effects on transfection efficiency of a variety of gene vectors (such as Lipo 2000 and PEI) in an oxygen-dependent manner. Solid tumors inevitably resulted in acute hypoxic areas due to the rapid proliferation of tumor cells and the aberrant structure of blood vessels. Thus, the hypoxic TME severely limited the efficiency and application of gene therapy. METHODS: In our previous study, we constructed endoplasmic reticulum-targeted cationic liposomes, PAR-Lipo, which could effectively deliver genes and ensure high transfection efficiency under normoxia. Unsatisfactorily, the transfection efficiency of PAR-Lipo was rather poor under hypoxia. We believed that reoxygenation was the most direct and effective means to rescue the low transfection under hypoxia. Hence, we fabricated liposomes modified with perfluorooctyl bromide (PFOB@Lipo) to load oxygen and deliver it to tumor sites, which effectively alleviated the hypoxic nature of tumor. Then PAR-Lipo were applied to mediate high-efficiency delivery of tumor suppressor gene pTP53 to inhibit tumor progression. RESULTS: The results showed that such staged strategy augmented the expression of P53 protein in tumors and extremely suppressed tumor growth. CONCLUSION: This work was the first attempt to utilize an oxygen nanocarrier to assist the therapeutic effect of gene therapy under hypoxia, providing a new reference for gene therapy in malignant tumors. GRAPHICAL ABSTARCT.

Epirubicin-loaded superparamagnetic iron-oxide nanoparticles for transdermal delivery: cancer therapy by circumventing the skin barrier.

The transdermal administration of chemotherapeutic agents is a persistent challenge for tumor treatments. A model anticancer agent, epirubicin (EPI), is attached to functionalized superparamagnetic iron-oxide nanoparticles (SPION). The covalent modification of the SPION results in EPI-SPION, a potential drug delivery vector that uses magnetism for the targeted transdermal chemotherapy of skin tumors. The spherical EPI-SPION composite exhibits excellent magnetic responsiveness with a saturation magnetization intensity of 77.8 emu g(-1) . They feature specific pH-sensitive drug release, targeting the acidic microenvironment typical in common tumor tissues or endosomes/lysosomes. Cellular uptake studies using human keratinocyte HaCaT cells and melanoma WM266 cells demonstrate that SPION have good biocompatibility. After conjugation with EPI, the nanoparticles can inhibit WM266 cell proliferation; its inhibitory effect on tumor proliferation is determined to be dose-dependent. In vitro transdermal studies demonstrate that the EPI-SPION composites can penetrate deep inside the skin driven by an external magnetic field. The magnetic-field-assisted SPION transdermal vector can circumvent the stratum corneum via follicular pathways. The study indicates the potential of a SPION-based vector for feasible transdermal therapy of skin cancer.

Association between Periodontitis and Aortic Calcification: A Cohort Study.

The present study investigated the association between the presence of periodontitis and aortic calcification (AC) risk among Chinese adults. A total of 6059 individuals who underwent regular health check-ups and received a diagnosis of periodontitis between 2009 and 2016 were included. The outcome was AC, assessed by a chest low-dose spiral CT scan. Cox proportional hazards regression analysis was used to assess the association between periodontitis and AC risk after adjusting for several confounders. After a median follow-up period of 2.3 years (interquartile range: 1.03-4.97 years), 843 cases of AC were identified, with 532 (12.13%) and 311 (18.59%) patients in the non-periodontitis group and periodontitis group, respectively. Multivariate analyses demonstrated that, compared with those without periodontitis, the hazard ratio and 95% confidence interval for AC risk in participants with periodontitis was 1.18 (1.02-1.36) (P = .025) in the fully adjusted model. Stratified analyses showed that the positive relationship between periodontitis and AC was more evident in males and participants <65 years of age (pinteraction = .005 and .004, respectively). Our results show that the presence of periodontitis was positively associated with AC among Chinese adults, especially among males and younger participants.

Quantifying the interactions between biomimetic biomaterials - collagen I, collagen IV, laminin 521 and cellulose nanofibrils - by colloidal probe microscopy.

Biomaterials of different nature have been and are widely studied for various biomedical applications. In many cases, biomaterial assemblies are designed to mimic biological systems. Although biomaterials have been thoroughly characterized in many aspects, not much quantitative information on the molecular level interactions between different biomaterials is available. That information is very important, on the one hand, to understand the properties of biological systems and, on the other hand, to develop new composite biomaterials for special applications. This work presents a systematic, quantitative analysis of self- and cross-interactions between films of collagen I (Col I), collagen IV (Col IV), laminin (LN-521), and cellulose nanofibrils (CNF), that is, biomaterials of different nature and structure that either exist in biological systems (e.g., extracellular matrices) or have shown potential for 3D cell culture and tissue engineering. Direct surface forces and adhesion between biomaterials-coated spherical microparticles and flat substrates were measured in phosphate-buffered saline using an atomic force microscope and the colloidal probe technique. Different methods (Langmuir-Schaefer deposition, spin-coating, or adsorption) were applied to completely coat the flat substrates and the spherical microparticles with homogeneous biomaterial films. The adhesion between biomaterials films increased with the time that the films were kept in contact. The strongest adhesion was observed between Col IV films, and between Col IV and LN-521 films after 30 s contact time. In contrast, low adhesion was measured between CNF films, as well as between CNF and LN-521 films. Nevertheless, a good adhesion between CNF and collagen films (especially Col I) was observed. These results increase our understanding of the structure of biological systems and can support the design of new matrices or scaffolds where different biomaterials are combined for diverse biological or medical applications.

Quantified forces between HepG2 hepatocarcinoma and WA07 pluripotent stem cells with natural biomaterials correlate with in vitro cell behavior.

In vitro cell culture or tissue models that mimic in vivo cellular response have potential in tissue engineering and regenerative medicine, and are a more economical and accurate option for drug toxicity tests than animal experimentation. The design of in vivo-like cell culture models should take into account how the cells interact with the surrounding materials and how these interactions affect the cell behavior. Cell-material interactions are furthermore important in cancer metastasis and tumor progression, so deeper understanding of them can support the development of new cancer treatments. Herein, the colloidal probe microscopy technique was used to quantify the interactions of two cell lines (human pluripotent stem cell line WA07 and human hepatocellular carcinoma cell line HepG2) with natural, xeno-free biomaterials of different chemistry, morphology, and origin. Key components of extracellular matrices -human collagens I and IV, and human recombinant laminin-521-, as well as wood-derived, cellulose nanofibrils -with evidenced potential for 3D cell culture and tissue engineering- were analysed. Both strength of adhesion and force curve profiles depended on biomaterial nature and cell characteristics. The successful growth of the cells on a particular biomaterial required cell-biomaterial adhesion energies above 0.23 nJ/m. The information obtained in this work supports the development of new materials or hybrid scaffolds with tuned cell adhesion properties for tissue engineering, and provides a better understanding of the interactions of normal and cancerous cells with biomaterials in the human body.

Extremely Effective Chemoradiotherapy by Inducing Immunogenic Cell Death and Radio-Triggered Drug Release under Hypoxia Alleviation.

Local hypoxia in solid malignancies often results in resistance to radiotherapy (RT) and chemotherapy (CT), which may be one of the main reasons for their failure in clinical application. Especially, oxygen is an essential element for enhancing DNA damage caused by ionizing radiation in radiotherapy. Here, two biomimetic oxygen delivery systems were designed by encapsulating hemoglobin (Hb) alone into a liposome (Hb-Lipo) or co-encapsulating Hb and doxorubicin (DOX) into a liposome (DOX-Hb-Lipo). Our data indicated that both Hb-Lipo and DOX-Hb-Lipo could effectively alleviate hypoxia in tumors. We demonstrated that RT plus tumor-targeting delivery of oxygen mediated by Hb-Lipo could significantly overcome the tolerance of hypoxic cancer cells to RT, showing significantly enhanced cancer-cell killing and tumor growth inhibition ability, mainly attributing to hypoxia alleviation and increased reactive oxygen species production under RT in cancer cells. Furthermore, a melanoma model that was quite insensitive to both RT and CT was used to test the efficacy of chemoradiotherapy combined with hypoxia alleviation. RT plus Hb-Lipo only caused a limited increase in antitumor activity. However, extremely strong tumor inhibition could be obtained by RT combined with DOX-Hb-Lipo-mediated CT, attributed to radio-triggered DOX release and enhanced immunogenic cell death induced by RT under an oxygen supplement. Our study provided a valuable reference for overcoming hypoxia-induced radioresistance and a useful therapeutic strategy for cancers that are extremely insensitive to chemo- or radiotherapy.

Synchronous delivery of oxygen and photosensitizer for alleviation of hypoxia tumor microenvironment and dramatically enhanced photodynamic therapy.

Photosensitizer, proper laser irradiation, and oxygen are essential components for effective photodynamic therapy (PDT) in clinical cancer therapy. However, native hypoxic tumoral microenvironment is a major barrier hindering photodynamic reactions in vivo. Thus, we have prepared biocompatible liposomes by loading complexes of oxygen-carrier (hemoglobin, Hb) and photosensitizer (indocyanine green, ICG) for enhanced PDT against hypoxic tumor. Ideal oxygen donor Hb, which is an oxygen-carried protein in red blood cells, makes such liposome which provide stable oxygen supply. ICG, as a photosensitizer, could transfer energy from lasers to oxygen to generate cytotoxic reactive oxygen species (ROS) for treatment. The liposomes loading ICG and Hb (LIH) exhibited efficient tumor homing upon intravenous injection. As revealed by T2-weighted magnetic resonance imaging and immunohistochemical analysis, the intratumoral hypoxia was greatly alleviated, and the level of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in tumor was obviously down-regulated. A weak PDT efficiency was found in cells incubated in simulated hypoxia condition in vitro, while PDT effect was dramatically enhanced in LIH treated hypoxia cells under near-infrared (NIR) laser, which was mainly attributed to massive generation of ROS with sufficient oxygen supply. ROS trigger oxidative damage of tumors and induce complete suppression of tumor growth and 100% survival rate of mice, which were also in good health condition. Our work highlights a liposome-based nanomedicine that could effectively deliver oxygen to tumor and alleviate tumor hypoxia state, inducing greatly improved efficacy compared to conventional cancer PDT and demonstrates the promise of modulating unfavorable tumor microenvironment with nanotechnology to overcome limitations of cancer therapies.

Nanofibrillar cellulose-alginate hydrogel coated surgical sutures as cell-carrier systems.

Hydrogel nanomaterials, especially those that are of non-human and non-animal origins, have great potential in biomedical and pharmaceutical sciences due to their versatility and inherent soft-tissue like properties. With the ability to simulate native tissue function, hydrogels are potentially well suited for cellular therapy applications. In this study, we have fabricated nanofibrillar cellulose-alginate (NFCA) suture coatings as biomedical devices to help overcome some of the limitations related to cellular therapy, such as low cell survivability and distribution out of target tissue. The addition of sodium alginate 8% (w/v) increased the NFCA hydrogel viscosity, storage and loss moduli by slightly under one order of magnitude, thus contributing significantly to coating strength. Confocal microscopy showed nearly 100% cell viability throughout the 2-week incubation period within and on the surface of the coating. Additionally, typical morphologies in the dual cell culture of spheroid forming HepG2 and monolayer type SK-HEP-1 were observed. Twelve out of 14 NFCA coated surgical sutures remained intact during the suturing operation with various mice and rat tissue; however, partial peeling off was observed in 2 of the coated sutures. We conclude that NFCA suture coatings could perform as cell-carrier systems for cellular based therapy and post-surgical treatment.

Preparation of artificial red cell and its application on alleviation of tumor hypoxia.

Hemoglobin-based oxygen carriers were developed as an alternative for blood transfusion. However, the research progress for their further clinic applications was slow in recent several years. Hypoxia is found in most solid tumors, which is responsible for the tumor formation, increased metastasis, drug resistance during therapeutic process as well as poor patient survival. In this work, novel hemoglobin (Hb) loaded nanoliposomes, as artificial red cells for oxygen delivery, were optimized by screening various types of phospholipids and analyzing different mole ratio of phospholipid to cholesterol. The nanoliposomes presented a high encapsulating efficiency to hemoglobin and also significantly enhanced its stability. The obtained hemoglobin loaded nanoliposome (HLL) could be lyophilized for long term storage. HLL did not cause significant cell death in the concentration range of 0-100µg equivalent Hb/mL under normoxia and hypoxia incubation conditions, suggesting the low cytotoxicity and high biocompatibility of HLL. Importantly, HLL could efficiently accumulate into subcutaneous and deep orthotopic tumors, inducing a significant decrease of hypoxia-inducible factors 1α subunits (HIF-1α) in the tumors and remarkably reduced expression of vascular endothelial growth factor (VEGF). The study of acute and chronic toxicity indicated that HLL did not induce obvious damage to main organs of mice after intravenous injections with total Hb dose of 120mg/kg. We presented a promising method for relieving the hypoxia degree in solid tumors and down-regulating HIF-1α protein by directly delivering oxygen into tumors, which will be very helpful for subsequent cancer therapy.

Feasibility Study of the Permeability and Uptake of Mesoporous Silica Nanoparticles across the Blood-Brain Barrier.

Drug delivery into the brain is impeded by the blood-brain-barrier (BBB) that filters out the vast majority of drugs after systemic administration. In this work, we assessed the transport, uptake and cytotoxicity of promising drug nanocarriers, mesoporous silica nanoparticles (MSNs), in in vitro models of the BBB. RBE4 rat brain endothelial cells and Madin-Darby canine kidney epithelial cells, strain II, were used as BBB models. We studied spherical and rod-shaped MSNs with the following modifications: bare MSNs and MSNs coated with a poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) block copolymer. In transport studies, MSNs showed low permeability, whereas the results of the cellular uptake studies suggest robust uptake of PEG-PEI-coated MSNs. None of the MSNs showed significant toxic effects in the cell viability studies. While the shape effect was detectable but small, especially in the real-time surface plasmon resonance measurements, coating with PEG-PEI copolymers clearly facilitated the uptake of MSNs. Finally, we evaluated the in vivo detectability of one of the best candidates, i.e. the copolymer-coated rod-shaped MSNs, by two-photon in vivo imaging in the brain vasculature. The particles were clearly detectable after intravenous injection and caused no damage to the BBB. Thus, when properly designed, the uptake of MSNs could potentially be utilized for the delivery of drugs into the brain via transcellular transport.

Technetium-99m-labeled nanofibrillar cellulose hydrogel for in vivo drug release.

Nanoscale celluloses have recently gained an increasing interest in modern medicine. In this study, we investigated the properties of plant derived nanofibrillar cellulose (NFC) as an injectable drug releasing hydrogel in vivo. We demonstrated a reliable and efficient method of technetium-99m-NFC labeling, which enables us to trace the in vivo localization of the hydrogel. The release and distribution of study compounds from the NFC hydrogel after subcutaneous injection in the pelvic region of BALB/c mice were examined with a multimodality imaging device SPECT/CT. The drug release profiles were simulated by 1-compartmental models of Phoenix® WinNonlin®. The NFC hydrogel remained intact at the injection site during the study. The study compounds are more concentrated at the injection site when administered with the NFC hydrogel compared with saline solutions. In addition, the NFC hydrogel reduced the elimination rate of a large compound, technetium-99m-labeled human serum albumin by 2 folds, but did not alter the release rate of a small compound (123)I-ß-CIT (a cocaine analogue). In conclusion, the NFC hydrogels is easily prepared and readily injected, and it has potential use as a matrix for controlled release or local delivery of large compounds. The interactions between NFC and specific therapeutic compounds are possible and should be investigated further.

The use of nanofibrillar cellulose hydrogel as a flexible three-dimensional model to culture human pluripotent stem cells.

Human embryonic stem cells and induced pluripotent stem cells have great potential in research and therapies. The current in vitro culture systems for human pluripotent stem cells (hPSCs) do not mimic the three-dimensional (3D) in vivo stem cell niche that transiently supports stem cell proliferation and is subject to changes which facilitate subsequent differentiation during development. Here, we demonstrate, for the first time, that a novel plant-derived nanofibrillar cellulose (NFC) hydrogel creates a flexible 3D environment for hPSC culture. The pluripotency of hPSCs cultured in the NFC hydrogel was maintained for 26 days as evidenced by the expression of OCT4, NANOG, and SSEA-4, in vitro embryoid body formation and in vivo teratoma formation. The use of a cellulose enzyme, cellulase, enables easy cell propagation in 3D culture as well as a shift between 3D and two-dimensional cultures. More importantly, the removal of the NFC hydrogel facilitates differentiation while retaining 3D cell organization. Thus, the NFC hydrogel represents a flexible, xeno-free 3D culture system that supports pluripotency and will be useful in hPSC-based drug research and regenerative medicine.

Differentiation of liver progenitor cell line to functional organotypic cultures in 3D nanofibrillar cellulose and hyaluronan-gelatin hydrogels.

Physiologically relevant hepatic cell culture models must be based on three-dimensional (3D) culture of human cells. However, liver cells are generally cultured in two-dimensional (2D) format that deviates from the normal in vivo morphology. We generated 3D culture environment for HepaRG liver progenitor cells using wood-derived nanofibrillar cellulose (NFC) and hyaluronan-gelatin (HG) hydrogels. Culture of undifferentiated HepaRG cells in NFC and HG hydrogels induced formation of 3D multicellular spheroids with apicobasal polarity and functional bile canaliculi-like structures, structural hallmarks of the liver tissue. Furthermore, hepatobiliary drug transporters, MRP2 and MDR1, were localized on the canalicular membranes of the spheroids and vectorial transport of fluorescent probes towards the biliary compartment was demonstrated. Cell culture in 3D hydrogel supported the mRNA expression of hepatocyte markers (albumin and CYP3A4), and metabolic activity of CYP3A4 in the HepaRG cell cultures. On the contrary, the 3D hydrogel cultures with pre-differentiated HepaRG cells showed decreasing expression of albumin and CYP3A4 transcripts as well as CYP3A4 activity. It is concluded that NFC and HG hydrogels expedite the hepatic differentiation of HepaRG liver progenitor cells better than the standard 2D culture environment. This was shown as improved cell morphology, expression and localization of hepatic markers, metabolic activity and vectorial transport. The NFC and HG hydrogels are promising materials for hepatic cell culture and tissue engineering.

Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture.

Over the recent years, various materials have been introduced as potential 3D cell culture scaffolds. These include protein extracts, peptide amphiphiles, and synthetic polymers. Hydrogel scaffolds without human or animal borne components or added bioactive components are preferred from the immunological point of view. Here we demonstrate that native nanofibrillar cellulose (NFC) hydrogels derived from the abundant plant sources provide the desired functionalities. We show 1) rheological properties that allow formation of a 3D scaffold in-situ after facile injection, 2) cellular biocompatibility without added growth factors, 3) cellular polarization, and 4) differentiation of human hepatic cell lines HepaRG and HepG2. At high shear stress, the aqueous NFC has small viscosity that supports injectability, whereas at low shear stress conditions the material is converted to an elastic gel. Due to the inherent biocompatibility without any additives, we conclude that NFC generates a feasible and sustained microenvironment for 3D cell culture for potential applications, such as drug and chemical testing, tissue engineering, and cell therapy.