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Treatment of adhesions after Achilles tendon injury using focused ultrasound with targeted bFGF plasmid-loaded cationic microbubbles

  • Yuzhou Shen , Jiancheng Ma , Junsheng Jiang , Zhilin Chen , Wenzhu Yan , Yue Wang , Feng Wang EMAIL logo and Li Liu EMAIL logo
Published/Copyright: June 13, 2020
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Open Chemistry
From the journal Open Chemistry Volume 18 Issue 1

Abstract

Nonviral targeting technology has become promising as a form of gene therapy for diseases and injuries, such as Achilles tendon injuries. In this study, we used avidin–biotin bridge and positive–negative charge attractions to load the intercellular adhesion molecule-1 (ICAM-1) antibody and the basic fibroblast growth factor (bFGF) plasmid onto the surface of the microbubbles. The saturated loading capacity for 1.1 × 108 microbubble was 6.55 ± 0.53 µg. We established the ICAM-1 antigen microenvironment using tumor necrosis factor-alpha-stimulated human umbilical vein endothelial cells and found the targeting ability of the prepared microbubbles in vitro. In vivo, we also found that the injected targeted bFGF gene microbubbles expressed the bFGF gene better when compared with that of the control group. Furthermore, we evaluated adhesions after Achilles tendon injuries in rabbits using hematoxylin and eosin and immunohistochemical (IHC) staining methods. The collagen fibers were properly arranged in the tendon, and there was greater cellularity inside the tendon sheath and a clearer boundary between the internal and external tendon sheath than that of the control group. IHC staining showed greater ICAM-1 expression inside the tendon sheath when compared with outside the tendon sheath. In conclusion, targeted microbubbles can be a useful carrier of genes to provide gene therapy for the prevention of adhesions after tendon injury.

Keywords: microbubbles; ICAM-1; bFGF; tendon adhesion; gene therapy

1 Introduction

Significant progress has been made in the surgical repair of tendons; adhesions frequently occur during the healing process in Achilles tendon injuries, which seriously affect joint function recovery. Therefore, reducing adhesions during tendon healing with a quick restoration of tendon function has become an intense area of research [1].

Currently, the methods to prevent adhesions include drug therapy, physical barriers, bionic sheath membranes, physical rehabilitation training, and gene therapies. Gene therapy has become an intense area of research because of its ability to be expressed in high quantities and to induce high protein activity while having minimal effects in surrounding tissues. Many researchers have found that cytokines, such as IGF-1 (insulin-like growth factor 1), TGF-β1 (transforming growth factor-beta 1), and bFGF (basic fibroblast growth factor), have significantly improved Achilles tendon injury repair in animal models. bFGF is a heparin-binding growth factor, which is a peptide composed of 146 amino acids. Exogenous bFGF accelerated wound healing in various injuries [2]. Nakanishi et al. found that the tendon mechanical strength significantly improved in the group that received a composite bFGF coating in the suture line when compared with the control group in a rabbit model of synovial tendon repair [3,4,5]. Since exogenous healing is the primary cause of tendon adhesions, the question now is how to direct bFGF to promote endogenous healing within the tendon sheath, while avoid it works outside the tendon sheath.

Intercellular adhesion molecule-1 (ICAM-1) is a member of the immunoglobulin family, which is mainly expressed on activated endothelial cells and other antigen transmitter cells, mediates the adhesion reaction between endothelial cells and leukocytes, and is almost not expressed in normal tissues. Research showed that the expression of ICAM-1 on the surface of Achilles tendon cells increased significantly in the model of Achilles tendon inflammation. Therefore, in this study, the Achilles tendon cells expressing ICAM-1 surface antigen after injury were used as target receptors to prepare targeted ultrasound microbubbles carrying the ICAM-1 antibody.

Acoustic microbubbles could be used as a gene carrier to treat diseases [6]. Targeted ultrasound microbubble destruction technology uses microbubble contrast agents as carriers to make an adherent surface or to encapsulate target genes. Targeted acoustic microbubbles containing target genes are injected intravenously to reach the target tissues, where the bubbles are broken to release the gene using ultrasonic irradiation.

In this study, we prepared targeted acoustic microbubbles carrying the bFGF gene and detected the gene carrying efficiency and targeting ability of this conjugated substance in vitro and in vivo. The rabbit model of Achilles tendon injuries has postoperative inflammation that is established by local incision and suture. We used this model to explore the efficiency by which the prepared microbubbles could prevent postoperative adhesions after Achilles tendon injury.

2 Materials and methods

2.1 Preparation of the targeted microbubble contrast agent with the bFGF gene

1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC; 561.9 µL; Avanti Polar Lipids Inc., USA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biothinyl(polyethylene glycol) 2000] (DSPE–PEG 2000–Biotin; 93.5 µL; Avanti Polar Lipids Inc., USA), and polyethyleneimine-600 (94.7 µL; Avanti Polar Lipids Inc., USA) were used to prepare biotinylated cationic microbubbles (BCMs). The rabbit ICAM-1 primary antibody (20 µL; Biosynthesis Biotechnology Co, Ltd, Beijing, China), the bFGF plasmid (bFGFp; 7 µL; ACRO Biosystems, Beijing, China), and the prepared BCMs (500 µL) were mixed using a biotin–avidin bridge to prepare the targeted bFGF gene microbubbles. Unbound antibodies and bFGFp were removed by washing three times with phosphate-buffered saline (PBS; Hua Maike Biotechnology Co, Ltd, Beijing, China).

The average particle size and the concentration of the prepared microbubbles were detected using an automatic dilution particle calculator (Particle Sizing System, California, USA), and the charge of the prepared microbubbles was detected using the Zeta potential analyzer (Brookhaven, MA, USA).

To confirm that the prepared microbubbles and ICAM-1 antibody were conjugated, three groups were extracted from the ICAM-1 targeted biotinylated microbubbles, the ICAM-1 targeted biotinylated microbubbles with the bFGF gene, and the biotinylated control microbubbles. Dylight 488 goat anti-rabbit IgG (H + L) (1 mL; 1:100 dilution; BOSTER Biological Technology, Ltd, Wuhan, China) was added to each group. The fluorescence distribution was observed in each of three groups using an inverted fluorescence microscope (Olympus Corporation). The experiments were performed three times.

2.2 Ultrasound imaging of targeted biotinylated cationic microbubbles (TBCMs) carrying the bFGF gene in rat livers

Twelve Sprague-Dawley rats weighing 200 ± 20 g (Medical Laboratory Animal Center, Guangdong, China) were divided into four groups and intraperitoneally injected with 0.2 mL of BCMs, TBCMs, TBCMs carrying bFGF, and SonoVue microbubbles, respectively, then followed by an injection of 0.5 mL of normal saline. The rat livers were imaged with the two-dimensional mode (Mylab90 Esaote, Indianapolis, IN, USA). The ultrasound imaging mode was adjusted (Mylab90, LA523 line probe, frequency: 7–10 MHz, MI: 0.8) until enhanced images of rat liver were achieved.

2.3 Targeting ability of targeted microbubbles carrying the ICAM-1 antibody in vitro

Umbilical vein endothelial cells were cultured in 12-well culture plates to a confluency of 70–80% and divided into control group (normal medium), targeted microbubbles group (microbubbles carrying the ICAM-1 antibody), tumor necrosis factor alpha (TNF-α) group, and TNF-α+ targeted microbubbles group (TNF-α microbubbles group). After washing, 5 µL of 1 ng/µL TNF-α liquid (ACRO Biosystems Beijing, China) was added to the TNF-α group and the TNF-α microbubble group. About 5 µL of normal medium was added to the control group and targeted microbubble group. The cells were incubated for 6 h at 37°C in 5% CO2. After washing, cells of the control group and TNF-α group were treated with 2 mL of 4% paraformaldehyde for 15 min, 0.5% Triton X-100 for 20 min at 37°C, and normal goat serum for 2 h. After washing with PBS, cells were incubated with the primary antibody ICAM-1 (1:400; MXB Biotechnologies, Fuzhou, China) at 4°C for 12 h, then stained with secondary antibody (1:400; MXB Biotechnologies, Fuzhou, China) at 37°C for 1 h in dark and finally treated with 4′,6-diamidino-2-phenylindole for 15 min in dark. The cells of targeted microbubble group and TNF-α microbubble group were washed three times. Then, the cells of all groups were observed by an inverted fluorescence microscope.

2.4 Rabbit model of Achilles tendon injury and postoperative inflammation

Twelve healthy male New Zealand rabbits weighing 2.0–3.0 kg (Medical Laboratory Animal Center, Guangdong, China) were included for animal model. All procedures in the animal experiments were conducted in accordance with the guidelines developed by the National Institutes of Health and approved by the Institutional Animal Care and Use Committee of Peking University Shenzhen Hospital (March 31, 2014, Permit No. 14-105). Rabbits were anesthetized by phenobarbital sodium injections into the ear vein (1:1 diluted; 0.3 mL/kg; Shanghai New Asia Pharmaceutical Co. Ltd, Shanghai, China). The rabbits were then fixed on the operating table (in the prone position), and the bilateral tendons were fully exposed and disinfected with an iodine solution. The skin of the Achilles tendon was cut into 2 cm longitudinal sections, and the subcutaneous tissue was incised layer by layer until the Achilles tendon was exposed. Then, the tendon was partially cut with transverse sections. The ruptured tendon was fixed with intermittent sutures, sterile saline was used to wash the wound, and the skin was sutured and bandaged with sterile gauze. Penicillin (North China Pharmaceutical Company Limited, Hebei, China) was injected intramuscularly at a dose of 4,00,000 units for the first 3 days post-surgery. The Achilles tendons of the rabbits were then used for the targeted microbubble studies.

2.5 Targeting ability and efficiency of microbubbles [7]

Twelve New Zealand rabbits were randomly divided into four groups: Group 1 was the control group that received normal saline; Group 2 was the bFGFp group that received the plasmid with bFGF gene; Group 3 was the targeted bFGFp microbubble group that received the contrast agent with the bFGFp and the ICAM-1 to help locate the target; and Group 4 was the bFGFp microbubble group that received the contrast agent with the bFGFp. Each group was injected with 1 mL of the respective fluid into the ear vein. We observed the condition of Achilles’ tendons by ultrasonography, and after 20 min, enhancement of the tendon was achieved. Observations and recordings were made after 7 days and 2 weeks, after which time all the rabbits were sacrificed and sections of the Achilles tendons from each group were taken and prepared for histopathology.

2.6 Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining

The Achilles tendon was fixed with 4% paraformaldehyde and then dehydrated with graded ethanol, dipped in wax, embedded in paraffin, and sectioned. Then, the paraffin sections were dewaxed, colored, rehydrated, transparent, and finally stained by H&E. For IHC, paraffin sections were incubated with the rabbit ICAM-1 primary antibody (Biosynthesis Biotechnology Co, Ltd, Beijing, China) and then with biotin-labeled secondary goat anti-rabbit IgG (BOSTER Biological Technology, Ltd, Wuhan, China). The sections were photographed by using an inverted microscope (Olympus, Germany).

2.7 Western blots

The bFGF protein expression was determined by western blot analysis. The proteins were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis in 12% (w/v) polyacrylamide gels and electrotransferred to Millipore polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked in Tris-buffered saline containing 0.05% Tween-20 and 5% nonfat dry milk for 60 min at room temperature. Then, the blots were incubated with a 1:500 dilution of rabbit bFGF primary antibody (Biosynthesis Biotechnology Co., Ltd, Beijing, China) at room temperature in a blocking solution. The membranes were washed in Tris-buffered saline and incubated with biotin-labeled secondary goat anti-rabbit IgG antibody (1:400; BOSTER Biological Technology, Ltd, Wuhan, China) in a blocking solution for 1.5 h. The blots were examined using a chemiluminescent substrate (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer’s instructions. The protein bands were normalized to glyceraldehyde 3-phosphate dehydrogenase, and the signal intensity of all the blots was quantified by Image J 1.43 software.

2.8 Statistical analysis

SPSS19.0 software was used to analyze the data, and the mean number of the groups was compared with the paired samples t-test analyses. A p-value of less than 0.05 was defined statistically significant.

3 Results

3.1 Preparation of cationic microbubbles

As shown in Figure 1, SPEI, DSPC, and DSPE–PEG 2000–Biotin were self-assembled to form BCM under a C3F8 environment. The biotin tag incorporated in the microbubble shell can further combine with ICAM-1–biotin antibodies in the suspension via the existence of avidin by biotin–avidin interaction, leading to the TBCMs. The SPEI could enhance the DNA-loading capability of the TBCM.

Figure 1 Schematic illustration of cationic microbubbles. SPEI600, DSPC, and DSPE–PEG 2000–Biotin first self-assembled to form BCM under a C3F8 environment. Then, biotinylated ICAM-1 assembled onto the shell of the TBCM by avidin–biotin interaction. Meanwhile, the plasmid bFGF can further assemble on the TBCM through electrostatic interaction and change to TBCM-bFGF.
Figure 1

Schematic illustration of cationic microbubbles. SPEI600, DSPC, and DSPE–PEG 2000–Biotin first self-assembled to form BCM under a C3F8 environment. Then, biotinylated ICAM-1 assembled onto the shell of the TBCM by avidin–biotin interaction. Meanwhile, the plasmid bFGF can further assemble on the TBCM through electrostatic interaction and change to TBCM-bFGF.

3.2 DNA loading efficiency of bFGFp onto normal microbubbles and cationic microbubbles

As shown in Figure 2, when the concentration of targeted biotinylated cation microbubbles is within a specific range, the amount of gene bound to the microbubbles is directly proportional to the DNA concentration in the solution. Finally, when the DNA concentration was 17.57 ng/µL, the saturated loading capacity for 1.1 × 108 microbubble was 6.55 ± 0.53 µg.

Figure 2 The amount of bFGFp bound onto microbubbles. The relationship between the binding rate and bFGF DNA concentration.
Figure 2

The amount of bFGFp bound onto microbubbles. The relationship between the binding rate and bFGF DNA concentration.

3.3 Physical properties of acoustic lipid-encapsulated microbubble contrast agents

The peak value of the microbubble point measured by the Zeta potential potentiometer was 0–100 mV, which is located in the positive charge range (Figure 3a).

Figure 3 The concentrations and particle sizes of three kinds of microbubbles: (a) the surface charges of the cationic acoustic microbubbles; (b) the diameter of three kinds of microbubbles; (c) the concentrations of three kinds of microbubbles; (d) particle size distribution of BCMs; (e) particle size distribution of TBCMs; and (f) particle size distribution of TBCMs with the bFGF gene (TBCM-bFGF).
Figure 3

The concentrations and particle sizes of three kinds of microbubbles: (a) the surface charges of the cationic acoustic microbubbles; (b) the diameter of three kinds of microbubbles; (c) the concentrations of three kinds of microbubbles; (d) particle size distribution of BCMs; (e) particle size distribution of TBCMs; and (f) particle size distribution of TBCMs with the bFGF gene (TBCM-bFGF).

Particle sizes and concentrations were measured using the automatic dilution particle counter. BCMs were with an average particle size of 0.89 ± 0.11 µm and an average concentration of 2.63 ± 0.40 × 109/mL (Figure 3b–d). TBCMs were with an average particle size of 0.96 ± 0.03 µm and an average concentration of 1.52 ± 0.18 × 109/mL (Figure 3b, c and e). TBCMs carrying the bFGF DNA with an average particle size of 1.08 ± 0.06 µm and an average concentration of 1.12 ± 0.11 × 108/mL (Figure 3b, c and f).

3.4 Qualitative ICAM-1 immunofluorescence detection on the surface of cationic microbubbles

ICAM-1 immunofluorescence can be seen as expressed on the surface of the TBCMs and the TBCMs carrying the bFGF gene with the Dylight488 marker. The surface of the normal biotinylated cationic acoustic microbubbles does not express the green fluorescence (Figure 4).

Figure 4 ICAM-expressing microbubbles observed using an inverted fluorescence microscope (a, c and e: white light; b, d and f: fluorescence). a and b: TBCM; c and d: TBCMs with the bFGF gene; and e and f: BCMs.
Figure 4

ICAM-expressing microbubbles observed using an inverted fluorescence microscope (a, c and e: white light; b, d and f: fluorescence). a and b: TBCM; c and d: TBCMs with the bFGF gene; and e and f: BCMs.

3.5 Ultrasound imaging of the acoustic microbubble contrast agents in rat livers

Hepatic boundaries of the rats were clearly defined using the two-dimensional ultrasound. BCMs, TBCMs, TBCMs carrying the bFGFp, and SonoVue microbubbles were injected into the ear veins of the rats. After 3 s, ultrasonic imaging of the hepatic vascular beds was started. Hepatic parenchyma began to show enhancement after about 15 s, and the enhancement was sustained for approximately 12 min (Figure 5).

Figure 5 Hepatic ultrasonography in rats: (a) BCMs, (b) TBCMs, (c) TBCMs carrying the bFGFp, and (d) SonoVue microbubbles.
Figure 5

Hepatic ultrasonography in rats: (a) BCMs, (b) TBCMs, (c) TBCMs carrying the bFGFp, and (d) SonoVue microbubbles.

3.6 In vitro target binding experiments

To detect the targeting ability of targeted microbubbles carrying the ICAM-1 antibody in vitro, the expression of ICAM-1 on the cell surface was stimulated by TNF-α. However, when the endothelial cells were not stimulated with TNF-α, the nuclei fluoresced blue, but the cell membranes did not fluoresce green. The green fluorescence in cells stimulated with TNF-α was accompanied by numerous targeted microbubbles binding with ICAM-1 antibody on the surface of the cells. The endothelial cells that were not stimulated with TNF-α had fewer targeted microbubbles in the field of view, and most were dispersed in the cells (Figure 6).

Figure 6 Photomicrographs from an inverted fluorescence microscope (200×). (a and b) Cells stimulated with TNF-α in white light and fluorescence model. (c and d) Cells stimulated with TNF-α and combined with targeted ultrasound microbubbles in white light and fluorescence model. (e and f) Cells without TNF-α stimulation in white and fluorescence model. (g and h) Cells without TNF-α stimulation and combined with targeted ultrasound microbubbles in white and fluorescence model.
Figure 6

Photomicrographs from an inverted fluorescence microscope (200×). (a and b) Cells stimulated with TNF-α in white light and fluorescence model. (c and d) Cells stimulated with TNF-α and combined with targeted ultrasound microbubbles in white light and fluorescence model. (e and f) Cells without TNF-α stimulation in white and fluorescence model. (g and h) Cells without TNF-α stimulation and combined with targeted ultrasound microbubbles in white and fluorescence model.

3.7 New Zealand rabbit recovery assessments after the Achilles tendon surgery and treatment

In Group 1 that received normal saline, obvious limping was observed; stitches were present in the surgical incision with swelling in the surrounding tissue. Moderate–severe adhesion was found between the Achilles tendon and the surrounding tissues, which was difficult to separate from the surrounding tissues. A few gaps were also seen in the Achilles tendon. In Group 2 that received injections of the bFGFp, moderate limping was noticed, and the wounds were mostly healed, but visible scars were present. Severe adhesions between the Achilles tendon and the surrounding tissues were present, which could not be separated from the surrounding tissues, and more scar tissues were present. In Group 3 that received targeted microbubble with bFGFp and ICAM-1 antibody, rabbits appeared normal. Mild edema was present in the surrounding tissues, but the wound healed well. Smooth Achilles tendons were seen with no obvious adhesions with the surrounding tissues. Mild swelling around the tendon was appeared, and they had good textures and elasticity. In Group 4 that received microbubbles with bFGFp, slight limping was noted with mild edema in the surrounding tissues, and the wounds were almost healed. Smooth Achilles tendons were also seen in this group with mild adhesions in the surrounding tissues. Tendon textures and elasticities were also firm compared with those of Group 3 (Figure 7).

Figure 7 Adhesions associated with the Achilles tendon in rabbits: (a) Group 1 received normal saline; (b) Group 2 received injections of the bFGFp; (c) Group 3 received targeted microbubble (the contrast agent with bFGFp and the target location factor, ICAM-1 antibody); and (d) Group 4 received microbubbles with bFGFp.
Figure 7

Adhesions associated with the Achilles tendon in rabbits: (a) Group 1 received normal saline; (b) Group 2 received injections of the bFGFp; (c) Group 3 received targeted microbubble (the contrast agent with bFGFp and the target location factor, ICAM-1 antibody); and (d) Group 4 received microbubbles with bFGFp.

3.8 In vivo target binding experiments

Using contrast-enhanced ultrasonography (CEUS), we observed the following results. Both Groups A and B had partial incisions of the Achilles tendon. However, Group A received TBCMs, whereas Group B, the control group, received non-TBCMs. Group A had significantly more enhancement on ultrasound than Group B. Group C, which received TBCMs similar to Group A, showed significant enhancement on ultrasound when compared with the control group (Group D), which did not receive incision of the Achilles tendon and received TBCMs (Figure 8).

Figure 8 Contrast-enhanced ultrasound imaging of the Achilles tendons in rabbits: (a) experimental group (partial incision of the Achilles tendon and TBCMs), (b) control group (partial incision of the Achilles tendon and non-TBCMs), (c) experimental group (partial incision of the Achilles tendon and TBCMs), and (d) control group (no-incision of the Achilles tendon and TBCMs).
Figure 8

Contrast-enhanced ultrasound imaging of the Achilles tendons in rabbits: (a) experimental group (partial incision of the Achilles tendon and TBCMs), (b) control group (partial incision of the Achilles tendon and non-TBCMs), (c) experimental group (partial incision of the Achilles tendon and TBCMs), and (d) control group (no-incision of the Achilles tendon and TBCMs).

3.9 H&E staining of the Achilles tendons in rabbits

In experimental Group 3, the collagenous fibers were normally arranged, and the cells were mostly inside the tendons than in the surrounding tissues. The loose connective tissues outside the tendon sheath are not seen within the tendon sheath, and the inner and outer boundaries of the tendon sheath were clear. In the control group (Groups 1 and 2), collagenous fiber had a more disordered arrangement, loose connective tissue was found inside the tendons, and the internal and external boundaries of the tendon sheath were not clear. In addition, the composition of cells inside and outside the tendon sheath was not different. However, in Group 2, more cells were outside the tendon than inside. The control group (Group 4) had normally arranged collagen fibers, and the cells were mostly inside the tendon than outside, but fewer cells inside the tendon were present in Group 4 than in Group 3. The internal and external boundaries of the tendon sheaths were clear (Figure 9).

Figure 9 Photomicrographs of H&E staining of the Achilles tendons in rabbits and microscopic observations (100×). (a) Group 1: the control group that received only normal saline; (b) Group 2: the group that received the bFGFp (the plasmid with bFGF gene); (c) Group 3 that received targeted microbubble (the contrast agent with bFGFp and the target location factor, ICAM-1 antibody); (d) Group 4: the group that received the microbubbles with the bFGFp. (Black arrows indicate the external tendon sheath. White arrows indicate the internal tendon sheath).
Figure 9

Photomicrographs of H&E staining of the Achilles tendons in rabbits and microscopic observations (100×). (a) Group 1: the control group that received only normal saline; (b) Group 2: the group that received the bFGFp (the plasmid with bFGF gene); (c) Group 3 that received targeted microbubble (the contrast agent with bFGFp and the target location factor, ICAM-1 antibody); (d) Group 4: the group that received the microbubbles with the bFGFp. (Black arrows indicate the external tendon sheath. White arrows indicate the internal tendon sheath).

3.10 IHC staining of rabbit Achilles tendons

In the experimental group (Group 3), ICAM-1 expression inside the tendon sheath is significantly greater than outside the tendon sheath. In the control groups (Groups 1, 2, and 4), ICAM-1 expression inside the tendon sheath was not significantly different from outside the tendon sheath (Figure 10).

Figure 10 ICAM-1 IHC staining of the Achilles tendons in rabbits and microscopic observation (200×). (a) Group 1: the control group that received only normal saline; (b) Group 2: the group that received the bFGFp (the plasmid with bFGF gene); (c) Group 3: the group that received targeted microbubble (the contrast agent with bFGFp and the target location factor, ICAM-1 antibody); (d) Group 4: the group that received the microbubbles with the bFGFp. (Black arrows indicate the external tendon sheath. White arrows indicate the internal tendon sheath.)
Figure 10

ICAM-1 IHC staining of the Achilles tendons in rabbits and microscopic observation (200×). (a) Group 1: the control group that received only normal saline; (b) Group 2: the group that received the bFGFp (the plasmid with bFGF gene); (c) Group 3: the group that received targeted microbubble (the contrast agent with bFGFp and the target location factor, ICAM-1 antibody); (d) Group 4: the group that received the microbubbles with the bFGFp. (Black arrows indicate the external tendon sheath. White arrows indicate the internal tendon sheath.)

3.11 Achilles tendon protein imprinting analysis

Western blot test results show that bFGF protein bands could be observed in all of the groups, and the bFGF protein expression was the highest in the experimental group (Group 3) when compared with the control groups (Groups 1, 2, and 4; Figure 11).

Figure 11 bFGF protein expression in the Achilles tendon of rabbits. Group 1: the control group that received only normal saline; Group 2: the group that received the bFGFp; Group 3: the group that received the microbubbles with the bFGFp. Group 4: the group that received targeted microbubble (the contrast agent with bFGFp and the target location factor, ICAM-1 antibody).
Figure 11

bFGF protein expression in the Achilles tendon of rabbits. Group 1: the control group that received only normal saline; Group 2: the group that received the bFGFp; Group 3: the group that received the microbubbles with the bFGFp. Group 4: the group that received targeted microbubble (the contrast agent with bFGFp and the target location factor, ICAM-1 antibody).

4 Discussion

The acoustic microbubble contrast agent is a kind of microbubble that has a diameter from 1 to 8 µm and contains gas and an outer membrane layer. With intravenous injections, acoustic microbubble contrast agents can reach the organs or tissues through the pulmonary circulation, which can be seen on ultrasound imaging. Thus, clinicians can image specific areas of interest and thereby increase the ability to diagnosis diseases. However, acoustic microbubbles do not have a special affinity for diseased tissues and do not reside long in tissues [8]. Therefore, researchers have begun to study targeted acoustic microbubbles which are refer to the microbubbles carring a specific ligand (such as peptides and antibodies). Targeted acoustic microbubble contrast agents can combine with receptors of organs and tissues through the circulatory system to enhance the ultrasonic imaging times and effects on local target tissues, which improve the specificity imaging of local target tissues and the sensitivity of early diagnosis. Because of these advantages, targeted microbubble contrast agents have become a hot topic in the field of ultrasonic research.

The preparation of the targeted microbubble contrast agents requires the necessary conditions [9]. In this study, the targeted acoustic microbubbles with the ICAM-1 antibody were prepared by using the “biotin–avidin” bridge method, which was considered having high sensitivity, stability, and affinity. Finally, the successful preparation of the targeted microbubble contrast agent with the bFGFp and the ICAM-1 antibody to prevent postoperative adhesions in Achilles tendon injuries becomes a possible choice.

Polyethyleneimine (PEI) is also known as a heterocyclic nitrogen propane and is a highly charged and dense cationic polymer that can be combined with DNA to form nanoscale particles [10]. As a cationic polymer, it protects DNA from enzymatic degradation. For instance, the PEI/DNA complexes escaped lysosomal compartments and degradation after endocytosis [11]. Compared with other cationic polymers, such as polylysine, complete dendritic polymers, and fracture dendritic polymers, PEI has a better acid–base buffering capacity [12,13]. In this experiment, we used PEG-modified phospholipids with PEI, which ensured that the surface of prepared microbubbles was positively charged. Then, bFGF gene was added into the cationic acoustic microbubbles via the mechanical oscillation method for enhancing transfections. Finally, we succeeded in creating biotinylated cationic liposome-encapsulated acoustic microbubbles that were uniform in size, and the ultrasound images of rat livers showed similar enhancing effects compared with those using the SonoVue microbubbles. Reverse transcription polymerase chain reaction showed a more than 30% greater plasmid carrying capacity of the microbubbles.

TNF-α is a pro-inflammatory cytokine produced by the activated macrophages and is involved in the mediation of early inflammation. During inflammation, endothelial cells are damaged by inflammatory cytokines such as interleukin-1beta (IL-1β) and TNF-α, which can cause vascular endothelial cell dysfunction and induce expression of several cell growth factors, such as ICAM-1 and vascular cell adhesion molecule-1 [14]. Research has shown that TNF-α can induce ICAM-1 antigen expression on the surface of umbilical vein endothelial cells causing vascular endothelial dysfunction. Moreover, studies have shown that ICAM-1 is not expressed on the surface of normal endothelial cells [15]. Therefore, ICAM-1 can be used to target genes to inflammatory sites for ultrasound imaging studies. In this experiment, we stimulated human umbilical vein endothelial cells with TNF-α and found that the endothelial cell surfaces fluoresced green after stimulation, which was not seen in the endothelial cells that were not stimulated with TNF-α. These results suggested that TNF-α was stimulating ICAM-1 expression on the surface of the endothelial cells. We then incubated the umbilical vein endothelial cells expressing ICAM-1 with the microbubbles carrying the ICAM-1 antibody and found that the florescence of the targeted microbubbles surrounding the cells was greater than that of the non-targeted microbubbles using fluorescence microscopy. These results indicated that the targeted ability of the interaction of ICAM-1 of the endothelial cells with the ICAM1-1 antibody on the targeted microbubbles in vitro.

In this rabbit Achilles tendon study, we demonstrated that the group that received targeted microbubbles containing ICAM-1 antibody and partial incision was the only group to show significant enhancement, using CEUS, as opposed to the group that received microbubbles without ICAM-1 or non-partial incision, which did not show enhancement. This study indicated that the targeted ultrasound microbubbles work in vivo and that targeted ultrasound could promote the bFGF gene expression in tendon injuries.

Tendon injuries are common in clinical surgery clinics and can be caused by external causes, such as direct trauma (car accidents), impacts from landing on feet from great heights, strenuous physical activity, iatrogenic factors, systemic diseases, and chronic degenerative changes [16]. Currently, two main treatments for tendon injuries are nonsurgical and surgical methods. Non-surgical methods include physical therapy, injections, traction therapy, systemic drugs, and adjuvant therapies [17]. Surgical treatments are important in Achilles tendon injuries, and although surgical methods continue to improve, fiber adhesions from the damaged tendons and surrounding tissues remain an obstacle to the functional recovery of patients with Achilles tendon injury.

The tendon healing process includes exogenous healing and endogenous healing [18]. Endogenous healing involves tendon cell proliferation in the tendon sheath and collagen production by tendon fibroblasts to form normal tendon collagen fibers. However, due to the distance between the tendon sheath and surrounding tissues, adhesions with the surrounding tissues would not likely occur. Exogenous healing is caused by angiogenesis and fibrous hyperplasia outside the tendon sheath, allowing both sides of the tendon to be healed. However, granulation tissue can interfere with healing as it promotes adhesion between the tendon and surrounding tissues. Therefore, therapies that support endogenous healing and reduce the exogenous healing are needed. Studies have shown that the growth factors associated with tendon healing are bFGF, epithelial growth factor (EGF), platelet-derived growth factor (PDGF), IGF-1, TGF-β, and vascular endothelial growth factor (VEGF). These growth factors might have specific regulatory functions in the healing of tendon injuries.

bFGF is a heparin-binding growth factor and polypeptide composed of 146 amino acids [19]. The main biological effects of bFGF are as follows: (1) angiogenesis, (2) fibroblast proliferation for wound healing and tissue repair, (3) regeneration of tissues, and (4) nerve regeneration [20]. Alkaline phosphatase can significantly improve type I collagen fiber expression and promote DNA synthesis of tendon fibroblasts [21]. bFGF has been shown to encourage endogenous healing of tendons by promoting collagen secretion and of tenocyte proliferation [22]. It has also been shown to have a dose correlation with tenocyte growth [23,24]. Wang et al. placed bFGF composite membranes in the joints of rat with tendon injuries and found that bFGF promoted endogenous healing of the tendon that was faster than that of the surrounding tissues [25]. This study suggested that bFGF not only accelerates tendon injury healing but also prevents tendon adhesions [26]. However, other studies show that bFGF promotes type III collagen fiber formation and angiogenesis outside the tendons, causing increased external healing of the tendons [26]. Therefore, we wanted to use bFGF to promote endogenous healing of tendons and reduce exogenous healing to prevent adhesions. To perform these experiments, we used the ICAM-1 antibody with targeted ultrasound microbubbles as the bFGF DNA vector to mediate transfections and showed that the tendon healing and adhesions of rabbits with Achilles tendon injuries were significantly better in the experimental groups when compared with the control groups. H&E and IHC staining showed that the cellularity inside the tendon sheaths was significantly greater in the experimental group when compared with that of the control group, suggesting that endogenous healing was faster than exogenous healing in the tendons of the experimental group. Preliminary conclusions of these experiments are that the targeted ultrasound microbubbles can significantly improve bFGF transfection rates and thereby promote endogenous healing of injured Achilles tendons and can effectively prevent adhesions caused by exogenous healing.

5 Conclusion

In this study, we prepared BCMs with PEI and used the ICAM-1 antibody to localize the transfection to a specific target. The bFGF target gene was attached to the targeted ultrasound microbubble surfaces via the charge effect. TNF-α stimulation induced the ICAM-1 surface expression on human endothelial cells. The results show that the targeted biotinylated microbubbles successfully target endothelial cell surfaces in vivo and in vitro. Thus, the targeted biotinylated microbubbles with bFGF and ICAM-1 could prevent adhesions after the tendon injury and provide a new pathway to study treatments to prevent adhesion after tendon injuries.

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Acknowledgments

This study was supported by the Natural Science Foundation of China (Grant no. U1804187), the Science Technology and Innovation Commission of Shenzhen Municipality in China (Grant no. JCYJ20170413100222613, JCYJ20160429090753103, and JCYJ20170306154931588), and the Health and Family Planning Commission of Shenzhen Municipality in China (Grant no. SZSM201512026).

  1. Conflict of Interest: The authors declare no conflict of interest.

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Received: 2020-02-06
Revised: 2020-04-12
Accepted: 2020-04-22
Published Online: 2020-06-13

© 2020 Yuzhou Shen et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/chem-2020-0110
Keywords for this article
microbubbles; ICAM-1; bFGF; tendon adhesion; gene therapy
Creative Commons
BY 4.0

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  11. Design, Synthesis and Characterization of Novel Isoxazole Tagged Indole Hybrid Compounds
  12. Comparison of kinetic and enzymatic properties of intracellular phosphoserine aminotransferases from alkaliphilic and neutralophilic bacteria
  13. Green Organic Solvent-Free Oxidation of Alkylarenes with tert-Butyl Hydroperoxide Catalyzed by Water-Soluble Copper Complex
  14. Ducrosia ismaelis Asch. essential oil: chemical composition profile and anticancer, antimicrobial and antioxidant potential assessment
  15. DFT calculations as an efficient tool for prediction of Raman and infra-red spectra and activities of newly synthesized cathinones
  16. Influence of Chemical Osmosis on Solute Transport and Fluid Velocity in Clay Soils
  17. A New fatty acid and some triterpenoids from propolis of Nkambe (North-West Region, Cameroon) and evaluation of the antiradical scavenging activity of their extracts
  18. Antiplasmodial Activity of Stigmastane Steroids from Dryobalanops oblongifolia Stem Bark
  19. Rapid identification of direct-acting pancreatic protectants from Cyclocarya paliurus leaves tea by the method of serum pharmacochemistry combined with target cell extraction
  20. Immobilization of Pseudomonas aeruginosa static biomass on eggshell powder for on-line preconcentration and determination of Cr (VI)
  21. Assessment of methyl 2-({[(4,6-dimethoxypyrimidin-2-yl)carbamoyl] sulfamoyl}methyl)benzoate through biotic and abiotic degradation modes
  22. Stability of natural polyphenol fisetin in eye drops Stability of fisetin in eye drops
  23. Production of a bioflocculant by using activated sludge and its application in Pb(II) removal from aqueous solution
  24. Molecular Properties of Carbon Crystal Cubic Structures
  25. Synthesis and characterization of calcium carbonate whisker from yellow phosphorus slag
  26. Study on the interaction between catechin and cholesterol by the density functional theory
  27. Analysis of some pharmaceuticals in the presence of their synthetic impurities by applying hybrid micelle liquid chromatography
  28. Two mixed-ligand coordination polymers based on 2,5-thiophenedicarboxylic acid and flexible N-donor ligands: the protective effect on periodontitis via reducing the release of IL-1β and TNF-α
  29. Incorporation of silver stearate nanoparticles in methacrylate polymeric monoliths for hemeprotein isolation
  30. Development of ultrasound-assisted dispersive solid-phase microextraction based on mesoporous carbon coated with silica@iron oxide nanocomposite for preconcentration of Te and Tl in natural water systems
  31. N,N′-Bis[2-hydroxynaphthylidene]/[2-methoxybenzylidene]amino]oxamides and their divalent manganese complexes: Isolation, spectral characterization, morphology, antibacterial and cytotoxicity against leukemia cells
  32. Determination of the content of selected trace elements in Polish commercial fruit juices and health risk assessment
  33. Diorganotin(iv) benzyldithiocarbamate complexes: synthesis, characterization, and thermal and cytotoxicity study
  34. Keratin 17 is induced in prurigo nodularis lesions
  35. Anticancer, antioxidant, and acute toxicity studies of a Saudi polyherbal formulation, PHF5
  36. LaCoO3 perovskite-type catalysts in syngas conversion
  37. Comparative studies of two vegetal extracts from Stokesia laevis and Geranium pratense: polyphenol profile, cytotoxic effect and antiproliferative activity
  38. Fragmentation pattern of certain isatin–indole antiproliferative conjugates with application to identify their in vitro metabolic profiles in rat liver microsomes by liquid chromatography tandem mass spectrometry
  39. Investigation of polyphenol profile, antioxidant activity and hepatoprotective potential of Aconogonon alpinum (All.) Schur roots
  40. Lead discovery of a guanidinyl tryptophan derivative on amyloid cascade inhibition
  41. Physicochemical evaluation of the fruit pulp of Opuntia spp growing in the Mediterranean area under hard climate conditions
  42. Electronic structural properties of amino/hydroxyl functionalized imidazolium-based bromide ionic liquids
  43. New Schiff bases of 2-(quinolin-8-yloxy)acetohydrazide and their Cu(ii), and Zn(ii) metal complexes: their in vitro antimicrobial potentials and in silico physicochemical and pharmacokinetics properties
  44. Treatment of adhesions after Achilles tendon injury using focused ultrasound with targeted bFGF plasmid-loaded cationic microbubbles
  45. Synthesis of orotic acid derivatives and their effects on stem cell proliferation
  46. Chirality of β2-agonists. An overview of pharmacological activity, stereoselective analysis, and synthesis
  47. Fe3O4@urea/HITh-SO3H as an efficient and reusable catalyst for the solvent-free synthesis of 7-aryl-8H-benzo[h]indeno[1,2-b]quinoline-8-one and indeno[2′,1′:5,6]pyrido[2,3-d]pyrimidine derivatives
  48. Adsorption kinetic characteristics of molybdenum in yellow-brown soil in response to pH and phosphate
  49. Enhancement of thermal properties of bio-based microcapsules intended for textile applications
  50. Exploring the effect of khat (Catha edulis) chewing on the pharmacokinetics of the antiplatelet drug clopidogrel in rats using the newly developed LC-MS/MS technique
  51. A green strategy for obtaining anthraquinones from Rheum tanguticum by subcritical water
  52. Cadmium (Cd) chloride affects the nutrient uptake and Cd-resistant bacterium reduces the adsorption of Cd in muskmelon plants
  53. Removal of H2S by vermicompost biofilter and analysis on bacterial community
  54. Structural cytotoxicity relationship of 2-phenoxy(thiomethyl)pyridotriazolopyrimidines: Quantum chemical calculations and statistical analysis
  55. A self-breaking supramolecular plugging system as lost circulation material in oilfield
  56. Synthesis, characterization, and pharmacological evaluation of thiourea derivatives
  57. Application of drug–metal ion interaction principle in conductometric determination of imatinib, sorafenib, gefitinib and bosutinib
  58. Synthesis and characterization of a novel chitosan-grafted-polyorthoethylaniline biocomposite and utilization for dye removal from water
  59. Optimisation of urine sample preparation for shotgun proteomics
  60. DFT investigations on arylsulphonyl pyrazole derivatives as potential ligands of selected kinases
  61. Treatment of Parkinson’s disease using focused ultrasound with GDNF retrovirus-loaded microbubbles to open the blood–brain barrier
  62. New derivatives of a natural nordentatin
  63. Fluorescence biomarkers of malignant melanoma detectable in urine
  64. Study of the remediation effects of passivation materials on Pb-contaminated soil
  65. Saliva proteomic analysis reveals possible biomarkers of renal cell carcinoma
  66. Withania frutescens: Chemical characterization, analgesic, anti-inflammatory, and healing activities
  67. Design, synthesis and pharmacological profile of (−)-verbenone hydrazones
  68. Synthesis of magnesium carbonate hydrate from natural talc
  69. Stability-indicating HPLC-DAD assay for simultaneous quantification of hydrocortisone 21 acetate, dexamethasone, and fluocinolone acetonide in cosmetics
  70. A novel lactose biosensor based on electrochemically synthesized 3,4-ethylenedioxythiophene/thiophene (EDOT/Th) copolymer
  71. Citrullus colocynthis (L.) Schrad: Chemical characterization, scavenging and cytotoxic activities
  72. Development and validation of a high performance liquid chromatography/diode array detection method for estrogen determination: Application to residual analysis in meat products
  73. PCSK9 concentrations in different stages of subclinical atherosclerosis and their relationship with inflammation
  74. Development of trace analysis for alkyl methanesulfonates in the delgocitinib drug substance using GC-FID and liquid–liquid extraction with ionic liquid
  75. Electrochemical evaluation of the antioxidant capacity of natural compounds on glassy carbon electrode modified with guanine-, polythionine-, and nitrogen-doped graphene
  76. A Dy(iii)–organic framework as a fluorescent probe for highly selective detection of picric acid and treatment activity on human lung cancer cells
  77. A Zn(ii)–organic cage with semirigid ligand for solvent-free cyanosilylation and inhibitory effect on ovarian cancer cell migration and invasion ability via regulating mi-RNA16 expression
  78. Polyphenol content and antioxidant activities of Prunus padus L. and Prunus serotina L. leaves: Electrochemical and spectrophotometric approach and their antimicrobial properties
  79. The combined use of GC, PDSC and FT-IR techniques to characterize fat extracted from commercial complete dry pet food for adult cats
  80. MALDI-TOF MS profiling in the discovery and identification of salivary proteomic patterns of temporomandibular joint disorders
  81. Concentrations of dioxins, furans and dioxin-like PCBs in natural animal feed additives
  82. Structure and some physicochemical and functional properties of water treated under ammonia with low-temperature low-pressure glow plasma of low frequency
  83. Mesoscale nanoparticles encapsulated with emodin for targeting antifibrosis in animal models
  84. Amine-functionalized magnetic activated carbon as an adsorbent for preconcentration and determination of acidic drugs in environmental water samples using HPLC-DAD
  85. Antioxidant activity as a response to cadmium pollution in three durum wheat genotypes differing in salt-tolerance
  86. A promising naphthoquinone [8-hydroxy-2-(2-thienylcarbonyl)naphtho[2,3-b]thiophene-4,9-dione] exerts anti-colorectal cancer activity through ferroptosis and inhibition of MAPK signaling pathway based on RNA sequencing
  87. Synthesis and efficacy of herbicidal ionic liquids with chlorsulfuron as the anion
  88. Effect of isovalent substitution on the crystal structure and properties of two-slab indates BaLa2−xSmxIn2O7
  89. Synthesis, spectral and thermo-kinetics explorations of Schiff-base derived metal complexes
  90. An improved reduction method for phase stability testing in the single-phase region
  91. Comparative analysis of chemical composition of some commercially important fishes with an emphasis on various Malaysian diets
  92. Development of a solventless stir bar sorptive extraction/thermal desorption large volume injection capillary gas chromatographic-mass spectrometric method for ultra-trace determination of pyrethroids pesticides in river and tap water samples
  93. A turbidity sensor development based on NL-PI observers: Experimental application to the control of a Sinaloa’s River Spirulina maxima cultivation
  94. Deep desulfurization of sintering flue gas in iron and steel works based on low-temperature oxidation
  95. Investigations of metallic elements and phenolics in Chinese medicinal plants
  96. Influence of site-classification approach on geochemical background values
  97. Effects of ageing on the surface characteristics and Cu(ii) adsorption behaviour of rice husk biochar in soil
  98. Adsorption and sugarcane-bagasse-derived activated carbon-based mitigation of 1-[2-(2-chloroethoxy)phenyl]sulfonyl-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl) urea-contaminated soils
  99. Antimicrobial and antifungal activities of bifunctional cooper(ii) complexes with non-steroidal anti-inflammatory drugs, flufenamic, mefenamic and tolfenamic acids and 1,10-phenanthroline
  100. Application of selenium and silicon to alleviate short-term drought stress in French marigold (Tagetes patula L.) as a model plant species
  101. Screening and analysis of xanthine oxidase inhibitors in jute leaves and their protective effects against hydrogen peroxide-induced oxidative stress in cells
  102. Synthesis and physicochemical studies of a series of mixed-ligand transition metal complexes and their molecular docking investigations against Coronavirus main protease
  103. A study of in vitro metabolism and cytotoxicity of mephedrone and methoxetamine in human and pig liver models using GC/MS and LC/MS analyses
  104. A new phenyl alkyl ester and a new combretin triterpene derivative from Combretum fragrans F. Hoffm (Combretaceae) and antiproliferative activity
  105. Erratum
  106. Erratum to: A one-step incubation ELISA kit for rapid determination of dibutyl phthalate in water, beverage and liquor
  107. Review Articles
  108. Sinoporphyrin sodium, a novel sensitizer for photodynamic and sonodynamic therapy
  109. Natural products isolated from Casimiroa
  110. Plant description, phytochemical constituents and bioactivities of Syzygium genus: A review
  111. Evaluation of elastomeric heat shielding materials as insulators for solid propellant rocket motors: A short review
  112. Special Issue on Applied Biochemistry and Biotechnology 2019
  113. An overview of Monascus fermentation processes for monacolin K production
  114. Study on online soft sensor method of total sugar content in chlorotetracycline fermentation tank
  115. Studies on the Anti-Gouty Arthritis and Anti-hyperuricemia Properties of Astilbin in Animal Models
  116. Effects of organic fertilizer on water use, photosynthetic characteristics, and fruit quality of pear jujube in northern Shaanxi
  117. Characteristics of the root exudate release system of typical plants in plateau lakeside wetland under phosphorus stress conditions
  118. Characterization of soil water by the means of hydrogen and oxygen isotope ratio at dry-wet season under different soil layers in the dry-hot valley of Jinsha River
  119. Composition and diurnal variation of floral scent emission in Rosa rugosa Thunb. and Tulipa gesneriana L.
  120. Preparation of a novel ginkgolide B niosomal composite drug
  121. The degradation, biodegradability and toxicity evaluation of sulfamethazine antibiotics by gamma radiation
  122. Special issue on Monitoring, Risk Assessment and Sustainable Management for the Exposure to Environmental Toxins
  123. Insight into the cadmium and zinc binding potential of humic acids derived from composts by EEM spectra combined with PARAFAC analysis
  124. Source apportionment of soil contamination based on multivariate receptor and robust geostatistics in a typical rural–urban area, Wuhan city, middle China
  125. Special Issue on 13th JCC 2018
  126. The Role of H2C2O4 and Na2CO3 as Precipitating Agents on The Physichochemical Properties and Photocatalytic Activity of Bismuth Oxide
  127. Preparation of magnetite-silica–cetyltrimethylammonium for phenol removal based on adsolubilization
  128. Topical Issue on Agriculture
  129. Size-dependent growth kinetics of struvite crystals in wastewater with calcium ions
  130. The effect of silica-calcite sedimentary rock contained in the chicken broiler diet on the overall quality of chicken muscles
  131. Physicochemical properties of selected herbicidal products containing nicosulfuron as an active ingredient
  132. Lycopene in tomatoes and tomato products
  133. Fluorescence in the assessment of the share of a key component in the mixing of feed
  134. Sulfur application alleviates chromium stress in maize and wheat
  135. Effectiveness of removal of sulphur compounds from the air after 3 years of biofiltration with a mixture of compost soil, peat, coconut fibre and oak bark
  136. Special Issue on the 4th Green Chemistry 2018
  137. Study and fire test of banana fibre reinforced composites with flame retardance properties
  138. Special Issue on the International conference CosCI 2018
  139. Disintegration, In vitro Dissolution, and Drug Release Kinetics Profiles of k-Carrageenan-based Nutraceutical Hard-shell Capsules Containing Salicylamide
  140. Synthesis of amorphous aluminosilicate from impure Indonesian kaolin
  141. Special Issue on the International Conf on Science, Applied Science, Teaching and Education 2019
  142. Functionalization of Congo red dye as a light harvester on solar cell
  143. The effect of nitrite food preservatives added to se’i meat on the expression of wild-type p53 protein
  144. Biocompatibility and osteoconductivity of scaffold porous composite collagen–hydroxyapatite based coral for bone regeneration
  145. Special Issue on the Joint Science Congress of Materials and Polymers (ISCMP 2019)
  146. Effect of natural boron mineral use on the essential oil ratio and components of Musk Sage (Salvia sclarea L.)
  147. A theoretical and experimental study of the adsorptive removal of hexavalent chromium ions using graphene oxide as an adsorbent
  148. A study on the bacterial adhesion of Streptococcus mutans in various dental ceramics: In vitro study
  149. Corrosion study of copper in aqueous sulfuric acid solution in the presence of (2E,5E)-2,5-dibenzylidenecyclopentanone and (2E,5E)-bis[(4-dimethylamino)benzylidene]cyclopentanone: Experimental and theoretical study
  150. Special Issue on Chemistry Today for Tomorrow 2019
  151. Diabetes mellitus type 2: Exploratory data analysis based on clinical reading
  152. Multivariate analysis for the classification of copper–lead and copper–zinc glasses
  153. Special Issue on Advances in Chemistry and Polymers
  154. The spatial and temporal distribution of cationic and anionic radicals in early embryo implantation
  155. Special Issue on 3rd IC3PE 2020
  156. Magnetic iron oxide/clay nanocomposites for adsorption and catalytic oxidation in water treatment applications
  157. Special Issue on IC3PE 2018/2019 Conference
  158. Exergy analysis of conventional and hydrothermal liquefaction–esterification processes of microalgae for biodiesel production
  159. Advancing biodiesel production from microalgae Spirulina sp. by a simultaneous extraction–transesterification process using palm oil as a co-solvent of methanol
  160. Topical Issue on Applications of Mathematics in Chemistry
  161. Omega and the related counting polynomials of some chemical structures
  162. M-polynomial and topological indices of zigzag edge coronoid fused by starphene
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