Novel Nanotherapeutics for Cancer Immunotherapy by PD-L1-Aptamer-Functionalized and Fexofenadine-Loaded Albumin Nanoparticles.

Immune checkpoint blockade (ICB) is an important strategy for cancer treatment and has achieved remarkable clinical results. Further enhancement of the efficacy of ICB therapy with a new technical approach is of potential medical importance. In this study, we constructed a novel nanotherapeutic agent (PDL1-NP-FEXO) for cancer immunotherapy by attaching PD-L1 aptamers to albumin nanoparticles that were loaded with H1-antihitamine fexofenadine (FEXO). FEXO has been reported to enhance the immunotherapy response by reducing the immunosuppressive M2-like macrophages in the tumor microenvironment. The albumin nanoparticle was fabricated using a self-assembly method. A dynamic light scattering (DLS) study revealed that the average size of PD-L1 aptamer-modified nanoparticle without FEXO (PDL1-NP) was 135.5 nm, while that of PDL1-NP-FEXO was 154.6 nm. Similar to free PD-L1 aptamer, PDL1-NP could also bind with PD-L1-expressing tumor cells (MDA-MB-231). Of note, compared with free PD-L1 aptamer, PDL1-NP significantly boosted tumor inhibition in CT26-bearing mice. Moreover, PDL1-NP-FEXO further enhanced the antitumor efficacy vs. PDL1-NP in an animal model, without raising systemic toxicity. These results indicate that PDL1-NP-FEXO represents a promising strategy to improve ICB efficacy and may have application potential in cancer immunotherapy.

Novel Complex of PD-L1 Aptamer and Albumin Enhances Antitumor Efficacy In Vivo.

The PD-1/PD-L1 pathway blockade can generate a good clinical response by reducing immunosuppression and provoking durable antitumor immunity. In addition to antibodies, aptamers can also block the interaction between PD-1 and PD-L1. For the in vivo application, however, free aptamers are usually too small in size and quickly removed from blood via glomerular filtration. To avoid renal clearance of aptamer, we conjugated the PD-L1 aptamer to albumin to form a larger complex (BSA-Apt) and evaluated whether BSA-Apt would enhance the in vivo antitumor efficacy. The PD-L1 aptamer was thiol-modified and conjugated to the amino group of BSA via a SMCC linker. The average size of BSA-Apt was 11.65 nm, which was above the threshold for renal clearance. Functionally, BSA-Apt retained the capability of the PD-L1 aptamer to bind with PDL1-expressing tumor cells. Moreover, both the free aptamer and BSA-Apt augmented the PBMC-induced antitumor cytotoxicity in vitro. Furthermore, BSA-Apt generated a significantly stronger antitumor efficacy than the free PD-L1 aptamer in vivo without raising systemic toxicity. The results indicate that conjugating the PD-L1 aptamer to albumin may serve as a promising strategy to improve the in vivo functionality of the aptamer and that BSA-Apt may have application potential in cancer immunotherapy.

Novel Complex of PD-L1 Aptamer and Holliday Junction Enhances Antitumor Efficacy in Vivo.

Blocking the PD-1/PD-L1 pathway can diminish immunosuppression and enhance anticancer immunity. PD-1/PD-L1 blockade can be realized by aptamers, which have good biocompatibility and can be synthesized in quantity economically. For in vivo applications, aptamers need to evade renal clearance and nuclease digestion. Here we investigated whether DNA nanostructures could be used to enhance the function of PD-L1 aptamers. Four PD-L1 aptamers (Apt) were built into a Holliday Junction (HJ) to form a tetravalent DNA nanostructure (Apt-HJ). The average size of Apt-HJ was 13.22 nm, which was above the threshold for renal clearance. Apt-HJ also underwent partial phosphorothioate modification and had improved nuclease resistance. Compared with the monovalent PD-L1 aptamer, the tetravalent Apt-HJ had stronger affinity to CT26 colon cancer cells. Moreover, Apt-HJ markedly boosted the antitumor efficacy in vivo vs. free PD-L1 aptamers without raising systemic toxicity. The results indicate that multiple aptamers attached to a DNA nanostructure may significantly improve the function of PD-L1 aptamers in vivo.

Novel Bispecific Aptamer Enhances Immune Cytotoxicity Against MUC1-Positive Tumor Cells by MUC1-CD16 Dual Targeting.

A promising strategy in cancer immunotherapy is the employment of a bispecific agent that can bind with both tumor markers and immunocytes for recruitment of lymphocytes to tumor sites and enhancement of anticancer immune reactions. Mucin1 (MUC1) is a tumor marker overexpressed in almost all adenocarcinomas, making it a potentially important therapeutic target. CD16 is expressed in several types of immunocytes, including NK cells, γδ-T cells, monocytes, and macrophages. In this study, we constructed the first bispecific aptamer (BBiApt) targeting both MUC1 and CD16. This aptamer consisted of two MUC1 aptamers and two CD16 aptamers linked together by three 60 nt DNA spacers. Compared with monovalent MUC1 or CD16 aptamers, BBiApt showed more potent avidity to both MUC1-positive tumor cells and CD16-positive immunocytes. Competition experiments indicated that BBiApt and monovalent aptamers bound to the same sites on the target cells. Moreover, BBiApt recruited more CD16-positive immunocytes around MUC1-positive tumor cells and enhanced the immune cytotoxicity against the tumor cells in vitro. The results suggest that, apart from bispecific antibodies, bispecific aptamers may also potentially serve as a novel strategy for targeted enhancement of antitumor immune reactions against MUC1-expressing malignancies.

An In Vitro Feasibility Study of the Influence of Configurations and Leaflet Thickness on the Hydrodynamics of Deformed Transcatheter Aortic Valve.

Clinically, the percutaneous transcatheter aortic valve (TAV) has been reported to be deformed in a noncircular configuration after its implant. The deformation is universal and various, and it leads to serious leakage and durability problems. Even in the same deformation, the leaflets made in different tissue thicknesses may cause different hydrodynamic performances. Simulating the left heart cardiac conditions by a pulse duplicator system, the present study investigated the effects of the aortic annulus deformation and the leaflet tissue thickness on the hydrodynamics of the TAV. Three 22 mm self-expanding TAV samples were fabricated with three different leaflet thicknesses (0.25, 0.4, 0.55 mm). Every sample was successively deformed to be elliptical, triangular, and undersized circular shapes. The hydrodynamics of the TAV were assessed through a quasi-physiological artery pulsatile flow duplicator system. The transvalvular pressure difference, effective orifice area, and regurgitation flow were determined. High-speed video recordings were taken to investigate the leaflet kinematics. The results showed that the triangular deformation produced the poorest valve function while the elliptical deformation led to the slightest difference from the nominal. With increasing leaflet thickness, the effect of configuration deformation on the regurgitation increased. The thinner leaflets were better than the thicker ones in adapting to the deformation but had a higher risk of deterioration.

Combined use of iterative reconstruction and monochromatic imaging in spinal fusion CT images.

BACKGROUND: Spinal fusion surgery is an important procedure for treating spinal diseases and computed tomography (CT) is a critical tool for postoperative evaluation. However, CT image quality is considerably impaired by metal artifacts and image noise. PURPOSE: To explore whether metal artifacts and image noise can be reduced by combining two technologies, adaptive statistical iterative reconstruction (ASIR) and monochromatic imaging generated by gemstone spectral imaging (GSI) dual-energy CT. MATERIAL AND METHODS: A total of 51 patients with 318 spinal pedicle screws were prospectively scanned by dual-energy CT using fast kV-switching GSI between 80 and 140 kVp. Monochromatic GSI images at 110 keV were reconstructed either without or with various levels of ASIR (30%, 50%, 70%, and 100%). The quality of five sets of images was objectively and subjectively assessed. RESULTS: With objective image quality assessment, metal artifacts decreased when increasing levels of ASIR were applied (P < 0.001). Moreover, adding ASIR to GSI also decreased image noise (P < 0.001) and improved the signal-to-noise ratio (P < 0.001). The subjective image quality analysis showed good inter-reader concordance, with intra-class correlation coefficients between 0.89 and 0.99. The visualization of peri-implant soft tissue was improved at higher ASIR levels (P < 0.001). CONCLUSION: Combined use of ASIR and GSI decreased image noise and improved image quality in post-spinal fusion CT scans. Optimal results were achieved with ASIR levels ≥70%.

Enhancement of Thermal Damage to Adenocarcinoma Cells by Iron Nanoparticles Modified with MUC1 Aptamer.

Hyperthermia cancer treatment is an adjunctive therapy that aims at killing the tumor cells with excessive heat that is usually generated by metal contrasts exposed to alternating magnetic field. The efficacy of hyperthermia is often limited by the heat damage to normal tissue due to indiscriminate distribution of the metal contrasts within the body. Tumor-targeting metal contrasts may reduce the toxicity of hyperthermia and improve the efficacy of thermotherapy against cancer. MUC1 is a glycoprotein over expressed in most adenocarcinomas, and represents an attractive therapeutic target. In this study, a MUC1 aptamer is conjugated with iron nanoparticles to construct adenocarcinoma-targeting metal contrasts. DNA hybridization studies confirmed that the aptamers were conjugated to the iron nanoparticles. Importantly, more aptamer-modified nanoparticles attached to the MUC1-positive cancer cells compared with the unmodified nanoparticles. Moreover, aptamer-modified nanoparticles significantly enhanced the targeted hyperthermia damage to MUC1-positive cancer cells in vitro (p < 0.05). The results suggest that MUC1 aptamer-modified metal particles may have potential in development of targeted hyperthermia therapy against adenocarcinomas.

Reduction of metal artifacts from alloy hip prostheses in computer tomography.

OBJECTIVE: The objective of this study was to evaluate the feasibility of reducing artifacts from large metal implants with gemstone spectral imaging (GSI) and metal artifact reduction software (MARS). METHODS: Twenty-three in-vivo cobalt-chromium-molybdenum alloy total hip prostheses were prospectively scanned by fast kV-switching GSI between 80 and 140 kVp. The computed tomography images were reconstructed with monochromatic energy and with/without MARS. Both subjective and objective measurements were performed to assess the severity of metal artifacts. RESULTS: Increasing photon energy was associated with reduced metal artifacts in GSI images (P < 0.001). Combination of GSI with MARS further diminished the metal artifacts (P < 0.001). Artifact reduction at 3 anatomical levels (femoral head, neck, and shaft) were evaluated, with data showing that GSI and MARS could reduce metal artifacts at all 3 levels (P = 0.011, P < 0.001, and P = 0.003, respectively). Nevertheless, in certain cases, GSI without MARS produced more realistic images for the clinical situation. CONCLUSIONS: Proper usage of GSI with/without MARS could reduce the computed tomography artifacts of large metal parts and improve the radiological evaluation of postarthroplasty patients.

Bovine pericardium leaflet damage during transcatheter aortic valve crimping: a study of the mechanisms.

Leaflet damage has been documented to occur while deploying a transcatheter aortic valve (TAV) due to mechanical loads during the crimping procedures. In this study, the impact of compressive stress on folded leaflets was measured to investigate the mechanism of traumatic leaflet tissue damage. Numerical simulation of TAV crimping procedure was adapted to calculate stress magnitude and distribution of leaflets. A 20 mm balloon expanding short stent TAV with 0.25 mm thickness leaflets was used in the simulation. Then the calculated stresses were applied on leaflet material (bovine pericardium) samples by loading experiments. Mechanical properties evaluation combined with histological and microscopy observation were used to investigate the tissue damage. The elastic modulus and the tensile strength of the tissue began to decrease significantly at 2 MPa stress and 2.5 MPa stress, respectively. No significant differences were observed at 0-1.5 MPa stress. When the TAV was crimped to 14 Fr and 12 Fr, the 2 MPa greater areas on leaflets increased from 18.17% to 76.96%. 2 MPa compressive stress might be the threshold value for leaflet damage. The TAV crimping size should be paid attention to avoid the compressive stress higher than 2 MPa.

Novel nanotherapeutics for cancer immunotherapy by CTLA-4 aptamer-functionalized albumin nanoparticle loaded with antihistamine.

INTRODUCTION: Immune checkpoint blockade (ICB) is a promising strategy for cancer treatment and has generated remarkable clinical results against multiple malignancies. Exploration of new technical approaches to further boost the therapeutic efficacy of ICB is of potential medical importance. In this study, we designed a novel nanotherapeutics for ICB immunotherapy. METHODS: CTLA-4 aptamers were conjugated to the surface of albumin nanoparticle to construct an aptamer-modified nanostructure (Apt-NP). To improve ICB efficacy, fexofenadine (FEXO), an antihistamine, was encapsulated into Apt-NP to make a drug-loaded nanoparticle (Apt-NP-FEXO). The antitumor efficacies of Apt-NP and Apt-NP-FEXO were evaluated in vitro and in vivo. RESULTS: Apt-NP and Apt-NP-FEXO had average diameters of 149 nm and 159 nm, respectively. Similar to free CTLA-4 aptamers, Apt-modified NPs could selectively bind with CTLA-4 positive cells and improve lymphocyte-mediated antitumor cytotoxicity in vitro. In animal studies, compared with free CTLA-4 aptamer, Apt-NP significantly enhanced antitumor immunity. Moreover, Apt-NP-FEXO further improved antitumor efficacy vs. Apt-NP in vivo. CONCLUSION: The results suggest that Apt-NP-FEXO represents a novel strategy to improve ICB outcome and may have application potential in cancer immunotherapy.

Novel HER2 aptamer selectively delivers cytotoxic drug to HER2-positive breast cancer cells in vitro.

BACKGROUND: Aptamer-based tumor targeted drug delivery system is a promising approach that may increase the efficacy of chemotherapy and reduce the related toxicity. HER2 protein is an attractive target for tumor-specific drug delivery because of its overexpression in multiple malignancies, including breast, gastric, ovarian, and lung cancers. METHODS: In this paper, we developed a new HER2 aptamer (HB5) by using systematic evolution of ligands by exponential enrichment technology (SELEX) and exploited its role as a targeting ligand for delivering doxorubicin (Dox) to breast cancer cells in vitro. RESULTS: The selected aptamer was an 86-nucleotide DNA molecule that bound to an epitope peptide of HER2 with a Kd of 18.9 nM. The aptamer also bound to the extracellular domain (ECD) of HER2 protein with a Kdof 316 nM, and had minimal cross reactivity to albumin or trypsin. In addition, the aptamer was found to preferentially bind to HER2-positive but not HER2-negative breast cancer cells. An aptamer-doxorubicin complex (Apt-Dox) was formulated by intercalating Dox into the DNA structure of HB5. The Apt-Dox complex could selectively deliver Dox to HER2-positive breast cancer cells while reducing the drug intake by HER2-negative cells in vitro. Moreover, Apt-Dox retained the cytotoxicity of Dox against HER2-positive breast cancer cells, but reduced the cytotoxicity to HER2-negative cells. CONCLUSIONS: The results suggest that the selected HER2 aptamer may have application potentials in targeted therapy against HER2-positive breast cancer cells.

The differences between surface degradation and bulk degradation of FEM on the prediction of the degradation time for poly (lactic-co-glycolic acid) stent.

The degradation time is a crucial factor in evaluating the performance of poly (lactic-co-glycolic acid) (PLGA) stents. Bulk degradation mode was commonly used to analyze the stent degradation behavior by finite element approach. However, the PLGA stents may present surface degradation more than bulk degradation under certain conditions, which will greatly affect the degradation time after implantation. In this study, the degradation processes of the poly (lactic-co-glycolic acid) stent were reproduced utilizing finite element analysis. Both bulk degradation and surface degradation modes were considered. The correlation between tensile stress and degradation rate was investigated. The degradation time was analyzed selectively. The stress distribution, fracture, and mass loss were also compared between bulk degradation mode and surface degradation mode. The simulation results showed that, in both evolution modes, the degradation began at the 'peak-valley' region and fracture occurred at the cross of links and rings. Additionally, high levels of Von-Mises stress were observed in these two regions. Compared with bulk degradation, the fracture time of the stent was delayed by 63% in the surface degradation mode. In conclusion, the mass loss rate and scaffolding period showed great differences between surface degradation and bulk degradation. Based on this study, it is suggested that bulk degradation mode is not applicable to the case of inadequate water uptake mode, such as the tracheal stent degradation process. More experimental research should be carried out to accurately predict the scaffolding period after implantation. The mechanical properties of the fracture zone should be strengthened.

The Regulatory Effect of Braided Silk Fiber Skeletons with Differential Porosities on In Vivo Vascular Tissue Regeneration and Long-Term Patency.

The development of small-diameter vascular grafts that can meet the long-term patency required for implementation in clinical practice presents a key challenge to the research field. Although techniques such as the braiding of scaffolds can offer a tunable platform for fabricating vascular grafts, the effects of braided silk fiber skeletons on the porosity, remodeling, and patency in vivo have not been thoroughly investigated. Here, we used finite element analysis of simulated deformation and compliance to design vascular grafts comprised of braided silk fiber skeletons with three different degrees of porosity. Following the synthesis of low-, medium-, and high-porosity silk fiber skeletons, we coated them with hemocompatible sulfated silk fibroin sponges and then evaluated the mechanical and biological functions of the resultant silk tubes with different porosities. Our data showed that high-porosity grafts exhibited higher elastic moduli and compliance but lower suture retention strength, which contrasted with low-porosity grafts. Medium-porosity grafts offered a favorable balance of mechanical properties. Short-term in vivo implantation in rats indicated that porosity served as an effective means to regulate blood leakage, cell infiltration, and neointima formation. High-porosity grafts were susceptible to blood leakage, while low-porosity grafts hindered graft cellularization and tended to induce intimal hyperplasia. Medium-porosity grafts closely mimicked the biomechanical behaviors of native blood vessels and facilitated vascular smooth muscle layer regeneration and polarization of infiltrated macrophages to the M2 phenotype. Due to their superior performance and lack of occlusion, the medium-porosity vascular grafts were evaluated in long-term (24-months) in vivo implantation. The medium-porosity grafts regenerated the vascular smooth muscle cell layers and collagen extracellular matrix, which were circumferentially aligned and resembled the native artery. Furthermore, the formed neoarteries pulsed synchronously with the adjacent native artery and demonstrated contractile function. Overall, our study underscores the importance of braided silk fiber skeleton porosity on long-term vascular graft performance and will help to guide the design of next-generation vascular grafts.

Investigation of failure modes of explanted porcine valves in the mitral position.

BACKGROUND: Porcine valves are used for mitral valve replacement, but the limited long-term durability has restricted the application in younger patients. Degenerated porcine mitral valves were explanted to analyze the failure modes and damage characteristics. METHODS: Twelve porcine valves were explanted via secondary mitral valve replacement surgery. Microcomputed tomography scanning, morphological and pathological examinations were performed to classify the cusp tears, calcification, and pannus formation. The causes of valve deterioration were subsequently analyzed. RESULTS: The mean age at first implantation was 45.42±19.58 years (range, 11-64 years). The mean duration of implantation was 9.39±4.14 years (range, 4.25-18.75 years). The indications for first surgery were rheumatic heart disease in 8 patients (66.67%), infective endocarditis in 2 patients (16.67%), degenerative valvular disease in one patient (8.33%), and congenital heart disease in one patient (8.33%). Type I cusp tears and commissural dehiscence that occurred near the stent post position were found in 6 (50%) and 5 (41.67%) valves, respectively. Calcification was detected in 6 (50%) cases, and pannus was found in most valves (91.67%). CONCLUSIONS: Leaflet damage occurred near the stent posts area was the main failure mode of porcine mitral valves in this study. Patients who undergo the first surgery at younger age, the higher prevalence rate of rheumatic heart disease, the structure of bioprosthetic porcine valve, and left ventricular stresses could be considered as the main factors causing valve deterioration.

Targeted Treatment of Colon Cancer with Aptamer-Guided Albumin Nanoparticles Loaded with Docetaxel.

PURPOSE: Chemotherapy of colon cancer needs improvement to mitigate the severe adverse effects (AEs) associated with the cytotoxic drugs. The aim of this study is to develop a novel targeted drug delivery system (TDDS) with practical application potential for colon cancer treatment. METHODS: The TDDS was built by loading docetaxel (DTX) in albumin nanoparticles (NPs) that were functionalized with nucleolin-targeted aptamers (AS1411). RESULTS: The TDDS (Apt-NPs-DTX) had an average size of 62 nm and was negatively charged with a zeta potential of -31.2 mV. DTX was released from the albumin NP with a typical sustained release profile. Aptamer-guided NPs were preferentially ingested by nucleolin-expressing CT26 colon cancer cells vs the control cells. In vitro cytotoxicity study showed that Apt-NPs-DTX significantly enhanced the killing of CT26 colon cancer cells. Importantly, compared with non-targeted drug delivery, Apt-NPs-DTX treatment significantly improved antitumor efficacy and prolonged the survival of CT26-bearing mice, without raising systemic toxicity. CONCLUSION: The results suggest that Apt-NPs-DTX has potential in the targeted treatment of colon cancer.

Targeted Therapy of Colon Cancer by Aptamer-Guided Holliday Junctions Loaded with Doxorubicin.

PURPOSE: Chemotherapy is the primary treatment for advanced colon cancer, but its efficacy is often limited by severe toxicities. Targeted therapy in the form of selectively drug delivery system (SDDS) is an important strategy to reduce adverse effects. Here, we aim to design a novel SDDS with potential for practical application using biocompatible components and scalable production process, for targeted delivery of doxorubicin (Dox) to colon cancer cells. METHODS: The SDDS was made of a self-assembled DNA nano-cross (Holliday junction, or HJ) functionalized by four AS1411 aptamers (Apt-HJ) and loaded with Dox. RESULTS: Apt-HJ had an average size of 12.45 nm and a zeta potential of -11.6 mV. Compared with the monovalent AS1411 aptamer, the quadrivalent Apt-HJ showed stronger binding to target cancer cells (CT26). A complex of Apt-HJ and doxorubicin (Apt-HJ-Dox) was formed by intercalating Dox into the DNA structure of Apt-HJ, with each complex carrying approximately 17 Dox molecules. Confocal microscopy revealed that Apt-HJ-Dox selectively delivered Dox into CT26 colon cancer cells but not the control cells. Moreover, Apt-HJ-Dox achieved targeted killing of CT26 cancer cells in vitro and reduced the damage to control cells. Importantly, compared with free Dox, Apt-HJ-Dox significantly enhanced the antitumor efficacy in vivo without boosting the adverse effects. CONCLUSION: These results suggest that Apt-HJ-Dox has application potential in targeted treatment of colon cancer.

Influence of Configuration on Stress Distribution of Pulmonary Monocusp Leaflet.

PURPOSE: For the relief of right ventricular outflow tract obstruction in operative treatment of tetralogy of Fallot and other complex congenital heart diseases, transannular monocusp patch operations are often necessary to prevent right ventricular pressure overload and reduce pulmonary regurgitation. However, long-term durability of a monocusp leaflet is unsatisfactory, its failure is believed to be related to mechanical stress, whose distribution is primarily affected by geometric configurations. Therefore, the influence of several geometrical parameters on stress distribution of leaflet is investigated. METHODS: Five parameters affecting leaflet configuration were established: angle between free edge of the leaflet and vessel wall, angle formed by the two end points of free edge, length of the free edge of the leaflet, height of the leaflet, and shape of elliptic conical surface constituting the leaflet surface. The first four parameters were fixed, and two factors were defined to describe the last parameter. Seven models with different values of these factors were analyzed using finite element method at the pressure of the pulmonary artery loaded on the leaflet. RESULTS: The peak stresses of all models occurred at end points of the free edge of the leaflet (tear high-risk regions). The middle of leaflet had the greatest stress gradient and produced tissue wrinkling; this area could be the risk region of calcification. Both factors were noted to influence the stress distribution, and one of the factors could also relieve the wrinkling. CONCLUSIONS: The leaflet of model (1.2_min) had the most even stress distribution and lowest peak principal stress, which was the optimal choice among all the models.

Excretion of SARS-CoV-2 through faecal specimens.

Coronavirus disease 2019 (COVID-19) has become a pandemic with increasing numbers of cases worldwide. SARS-CoV-2, the causative virus of COVID-19, is mainly transmitted through respiratory droplets or through direct and indirect contact with an infected person. The possibility of potential faecal-oral transmission was investigated in this study. We collected 258 faecal specimens from nine provinces in China and detected the nucleic acid of SARS-CoV-2 using real-time RT-PCR. Vero cells were used to isolate the virus from SARS-CoV-2 nucleic acid positive samples, after which sequencing of Spike gene in eight samples was performed. In all, 93 of 258 (36%) stool samples were positive for SARS-CoV-2 RNA. The positive rates of critical, severe, moderate, and mild patients were 54.4%, 56.1%, 30.8%, and 33.3%, respectively. The content of nucleic acid increased within 2 weeks after the onset of the disease. From the perspective of clinical typing, the nucleic acid can be detected in the faeces of critical patients within two weeks and until four to five weeks in the faeces of severe and mild patients. SARS-CoV-2 was isolated from stool specimens of two severe patients. Four non-synonymous mutations in Spike gene were newly detected in three stool samples. A small number of patients had strong faecal detoxification ability. The live virus in faeces could be an important source of contamination, which may lead to infection and further spread in areas with poor sanitary conditions. The findings of this study have public health significance and they should be considered when formulating disease control strategies.

Targeted distribution of bacterial magnetosomes isolated from Magnetospirillum gryphiswaldense MSR-1 in healthy Sprague-Dawley rats.

Bacterial magnetosomes (BMs) have drawn great interest as novel magnetic targeted carriers and have been widely used as carriers for enzymes, nucleic acids and antibodies experimentally. However, BMs have rarely been employed as drug carriers in vivo mainly due to the unclear biocompatibility and pharmacokinetics of BMs. In this study, we provided a unique effective method for purification and sterilization of BMs. The BMs obtained exhibited sterility, high purity, narrowed size-distribution, uniformity in morphology, intact membrane and abundant amino groups in BMs membrane. To elucidate the in vivo body distribution of BMs and if BMs displayed any rejection by animals when they are delivered into the vascular system, the BMs were injected into the sublingual vena of Sprague-Dawley (SD) rats and the tissue distribution of BMs in dejecta, urine, serum and main organs was examined. A target distribution of BMs in SD rats was observed. After injected into the sublingual vena, BMs were only found in livers and there was no obvious evidence to indicate the existence of BMs in the dejecta and urine within 72 h following the intravenous administration.

[Inhibitory effect of low molecular weight heparin on the secretion of vascular endothelial growth factor by tumor cells in vitro].

OBJECTIVE: To investigate whether low molecular weight heparin (LMWH) may suppress the expression and secretion of vascular endothelial growth factor (VEGF) from tumor cells in vitro and inhibit the VEGF-induced proliferation of human tumor vascular endothelial cells. METHODS: Human lung cancer cell line A549, human liver cancer cell line HepG2, human colon carcinoma cell lines HCT116 and HCT8 were used in this study. The expression levels of VEGF and TNF-alpha (tumor necrosis factor-alpha) in the tumor cells with or without pretreatment of LMWH/heparin were measured by standard sandwich ELISA technique. The VEGF mRNA level of HepG2 cells cultured with or without LMWH/heparin was determined by RT-PCR and real time PCR. Human umbilical vein endothelial cells (HUVEC) were cultured in tissue culture medium (TCM) with or without LMWH/heparin for 3 days. Then non-radioactive cell proliferation assay (MTS) kit and cell cycle assay by flow cytometry were performed to measure the proliferation of HUVEC. RESULTS: The VEGF levels in the control, LMWH, and heparin groups of the pulmonary adenocarcinoma cell line A549 were (1045.89 +/- 165.30) pg/ml, (782.45 +/- 67.17) pg/ml and (916.54 +/- 71.25) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups of the colon adenocarcinoma cell line HCT116 were (955.76 +/- 51.14) pg/ml, (822.89 +/- 142.39) pg/ml and (951.77 +/- 188.22) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups in the colon adenocarcinoma cell line HCT8 were (1290.62 +/- 41.23) pg/ml, (1063.34 +/- 63.82) pg/ml and (1257.14 +/- 11.40) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups in the liver cancer cell line HepG2 were (1083.00 +/- 134.35) pg/ml, (758.00 +/- 84.85) pg/ml and (874.00 +/- 22.62) pg/ml, respectively. The VEGF expression levels in the above mentioned cell lines cultured in TCM were significantly reduced in the LMWH-treated groups compared with that of the control group (P < 0.05). But the level of TNF-alpha in TCM-cultured cells was unaffected by LMWH. The VEGF mRNA was reduced in the LMWH-treated HepG2 cell line. Moreover, TCM exhibited stimulating effect on proliferation of HUVEC and the effect was significantly impaired by LMWH treatment. Flow cytometric analysis revealed that LMWH treatment arrested HUVECs at the G1 phase of cell cycle. CONCLUSION: LMWH can suppress the expression and secretion of VEGF by tumor cell lines and therefore have a potential inhibiting effect on angiogenesis induced by VEGF.