Patent Application
20230142745
The present disclosure belongs to the field of natural macromolecules, relates to a polypeptide monolayer, and a preparation method and application thereof, and specifically relates to a polypeptide monolayer with a low surface potential and hydrophobicity, and a preparation method and application thereof.
Collagen polypeptide is a water-soluble protein obtained by chemically thermal degradation of collagen. It is one of the most commonly used biopolymers due to its excellent biocompatibility, plasticity, viscosity, richness, and low cost. As a biodegradable and renewable resource, collagen polypeptide is widely applied to the preparation of medical materials, biomimetic materials, and packing and coating materials. Immobilized bio-coatings are usually applied to the field of biomimetic stents to achieve an aim of loading biomolecules such as enzyme and lactose, pharmaceutical molecules, synthetic macromolecules or small organic molecules, and a polypeptide monolayer prepared from collagen polypeptides has the advantage of easy and precise control of the loading amount.
However, a thickness of an immobilized bio-coating in the prior art is relatively great and difficult to control, generally greater than 100 nm. Furthermore, collagen polypeptide molecules contain a large number of polar groups such as amino groups, carboxy groups, and hydroxy groups, so that strong intermolecular hydrogen bonds are generated to form a network structure and then form a fragile thin film after dehydration. In addition, these groups and water molecules form hydrogen bonds to allow the polypeptide thin film to be susceptible to water absorption. These characteristics result in a fact that collagen polypeptide materials are fragile and soluble in water easily, which limits their application in some fields.
Secondary structures of a natural biological macromolecule can affect the exposure of functional groups in a polypeptide molecule so as to influence physicochemical properties of the surface of a monolayer, such as chemical properties, wettability, and electrical properties, and by modifying chemical properties, wettability, and electrical properties of the surface of a monolayer of an immobilized bio-coating, the immobilized bio-coating can be applied to the fields of preparation of biomimetic materials, etc.
There are a lot of studies on regulation of a conformation of polypeptide molecules on an interface with a surfactant, but due to structural complexity of natural biological macromolecules, studies on chemical properties of the surface of a polypeptide monolayer are rarely reported, and application of polypeptide molecules is limited. In addition, strengthening studies on chemical properties of the surface of a polypeptide monolayer is beneficial to further modification of polypeptide molecules, which can further make up for its shortcomings.
In order to solve the problems in the prior art, the present disclosure provides a polypeptide monolayer with a low surface potential and hydrophobicity, and a preparation method and application thereof. The present disclosure improves charges and wettability property of the surface of a monolayer by modifying the exposure of primary amino groups on the surface of the polypeptide monolayer, so that the polypeptide monolayer of the present disclosure can be used to load other target molecules and then can be applied to the field of leather manufacturing.
In the present disclosure, the exposure of primary amino groups is calculated by the following formula: the exposure of primary amino groups=molar weight of primary amino groups/collagen polypeptides (g).
In order to achieve the above objective, the present disclosure adopts the following technical solutions.
The present disclosure provides a polypeptide monolayer with a low surface potential and hydrophobicity, characterized in that the polypeptide is composed of polypeptide molecules with a molecular weight of (1.48±0.2)×105 g/mol, a thickness of the monolayer is 6.2-9.0 nm, the exposure of primary amino groups on the surface of the monolayer is 9.5-15%, a Zeta potential of the polypeptide monolayer is (−3)-(−9) mV; and a contact angle of the monolayer is (61±1°)-(84±1°).
Preferably, the polypeptide is collagen polypeptide.
Preferably, the thickness of the monolayer is (6.6±0.1)-(8.5±0.1) mm. Further preferably, the thickness of the monolayer is 6.6±0.1 mm, 7.3±0.1 mm or 8.5±0.1 mm. Much further preferably, the thickness of the monolayer is 6.6±0.1 mm.
Preferably, the polypeptide consists of 7.30±0.5% of glycine (Gly); 17.48±0.5% of valine (Vla); 36.97±0.5% of isoleucine (Ile); 13.85±0.5% of leucine (Leu); 2.68±0.5% of tyrosine (Tyr); 1.5±0.5% of phenylalanine (Phe); 4.41±0.5% of lysine (Lys); 0.45±0.5% of histidine (His); 3.45±0.5% of arginine (Arg); 5.96±0.5% of proline (Pro); and 5.95±0.5% of cysteine (Cys).
Preferably, secondary structures of the collagen polypeptide monolayer include 24-30% of α-helix; 18-24% of β-sheet; 4-8% of β-turn; and 43-48% of random coil.
Preferably, the polypeptide monolayer is composed of close-packed nanoparticles, and the spherical nanoparticles have an average particle size of 30±2 nm.
Preferably, the exposure of primary amino groups on the surface of the monolayer is (9.92±0.3%)-(14.51±0.3%), and further preferably, the exposure of primary amino groups is 9.92±0.3%, 11.6±0.3% or 14.51±0.3%. Further preferably, the exposure of primary amino groups is 14.51±0.3%.
Preferably, the Zeta potential of the polypeptide monolayer is −(3.33±0.2) mV, −(8.75±0.2) mV or −(8.99±0.2) mV.
Preferably, the secondary structures of the monolayer include 29.66±0.1% of α-helix; 18.98±0.15 of β-sheet; 7.93±0.05% of β-turn; and 43.44±0.26% of random coil;
or, the secondary structures of the monolayer include 24.77±0.1% of α-helix; 20.50±0.11% of β-sheet; 7.26±0.08% of β-turn; and 47.47±0.19% of random coil;
or, the secondary structures of the monolayer include 24.28±0.1% of α-helix; 23.21±0.12% of β-sheet; 4.70±0.03% of β-turn; and 47.80±0.20% of random coil.
The present disclosure also provides a composite film containing a polypeptide monolayer, including a polyethyleneimine thin film and a polypeptide monolayer, wherein the polyethyleneimine thin film and the polypeptide monolayer are bound together via ionic bonds, a thickness of the polyethyleneimine thin film is 0.25-0.38 nm, and a thickness of the polypeptide monolayer is 6.2-9.0 nm.
The present disclosure also provides a preparation method of the above polypeptide monolayer, characterized by including the following steps:
Preferably, the temperature at step (1) and the temperature during deposition at step (4) are both 50° C.
Preferably at step (1), a concentration of the polypeptide solution is 4 wt %; a concentration of sodium tetradecyl sulfonate in the mixed solution is 2.50-7.96 mmol/L. Further preferably, the concentration of sodium tetradecyl sulfonate in the mixed solution is 2.5 mmol/L, 7.00 mmol/L or 7.96 mmol/L.
Preferably, at step (1), a preparation method of the collagen polypeptide solution includes the following steps: mixing polypeptides with deionized water, swelling at room temperature for 0.5 h, heating to 50° C., stirring for 2 h until the polypeptides are completely dissolved; and then regulating the pH to 10.00±0.02.
Preferably, at step (2), after being ground and polished by using metallographic sandpaper, the titanium sheet is ultrasonically washed with deionized water, absolute ethanol, and acetone in sequence for 15 min for each time, blow-dried with high-purity nitrogen, and dried in an oven at 60° C. for 12 h. Further preferably, a grinding and polishing method includes the following steps: grinding and polishing the titanium sheet by using metallographic sandpaper to 800, 1,500, 3,000, 5,000, and 7,000 meshes in sequence.
Preferably, at step (2), the mixed acid solution is a mixed solution of 30% H2O2 and 98% H2SO4 in a volume ratio of 1:1, and the treatment time is 1 h.
Preferably, at step (2), the titanium sheet is treated in the aqueous solution of PEI for 20-40 min.
Preferably, in the present disclosure, the polypeptide is collagen polypeptide with a regular structure obtained by dialyzing a commercially available polypeptide product.
The present disclosure also provides application of the above collagen polypeptide monolayer in the field of leather manufacturing.
The present disclosure has the following beneficial effects.
The polypeptide monolayer of the present disclosure has relatively high and controllable exposure of primary amino groups, and can be easily used to load other target molecules, so that it can be applied to the field of leather manufacturing. The monolayer can be ultrathin, with a minimum thickness of only about 6.6 nm. Its relatively low surface potential and certain hydrophobicity enable the monolayer to be applied to the field of leather manufacturing. The polypeptide monolayer of the present disclosure can also be applied to the preparation of a biosensor, which helps to increase the limit of detection. The content of primary amino groups on the surface of polypeptide monolayer of the present disclosure is controllable, which is conductive to controllability of further chemical modification and laying the foundation for achieving controllable grafting of polysiloxane and biological preparations in the later stage.
Collagen polypeptides used in the embodiments of the present disclosure are commercially available polypeptide products (A.R.) with a molecular weight of 5.00×104−1.80×105 g/mol, and polypeptides with a molecular weight of (1.48±0.2)×105 g/mol is obtained by dialyzing the collagen polypeptides. Unless otherwise specified, other reagents are all commercially available.
Collagen polypeptide is an amphoteric polyelectrolyte, which can agglomerate into a spherical particle at the isoelectric point. Based on the aggregation behavior of collagen polypeptide, collagen polypeptides with a lower molecular weight can pass through a semi-permeable membrane by adjusting factors such as temperature, concentration, pH, and ionic strength, so as to achieve the purpose of separating from collagen polypeptides with a higher molecular weight. Study results obtained through gel electrophoresis and a laser particle analyzer show that collagen polypeptides with a narrow molecular weight distribution can be prepared by using dialysis tubing with a molecular-weight cutoff of 50,000 kDa under the conditions that the dialysis concentration of collagen polypeptides is 2%, the dialysis temperature is 45° C., and the concentration of NaCl is 0.9 mol·L−1.
Comparison of CP, CA, MW, and the isoelectric point (IP) of collagen polypeptides before and after dialysis is shown in Table 1, and comparison of amino acid types before and after dialysis is shown in Table 2. Determination results obtained through GPC show that a weight-average molecular weight Mw of the dialyzed collagen polypeptides is 1.48×105 gmol−1, and Mw/Mn=1.43. Determination results obtained by the Kjeldahl method show that the protein content (CP) in the collagen polypeptides is 83.38%, and the amino acid content (CA) is 4.95×10−4 mol·g−1, and determination results obtained by a primary amino group quantometer at 50° C. show that the primary amino group content in the dialyzed collagen polypeptide molecules is 4.95×10−4 g·mol−1, and the molecular structure of the collagen polypeptides has no obvious change before and after dialysis. The collagen polypeptides are prepared into a 5% aqueous solution with a conductivity of 5.98 μS cm−1, a conductivity of deionized water is 2.06 μS cm−1, and the above results indicate that collagen polypeptides with a low molecular weight and inorganic salt mixed in the collagen polypeptides are dialyzed out.
A preparation method of a polypeptide monolayer included the following steps.
The obtained polypeptide monolayer was denoted as G-STSocac.
A preparation method of a polypeptide monolayer included the following steps.
The obtained polypeptide monolayer was denoted as G-STSocmc.
A preparation method of a polypeptide monolayer included the following steps.
The obtained polypeptide monolayer was denoted as G-STSo6%.
Collagen polypeptide solutions at different concentrations of 1-5 wt % were prepared: the mass of collagen polypeptides and the volume of deionized water required were calculated, collagen polypeptides were precisely weighed and placed into a 50 mL three-neck flask, deionized water was precisely weighed and poured into the three-neck flask, the polypeptides swelled at room temperature for 0.5 h, the three-neck flask was placed into a water bath at 50° C., the solution was stirred for 2 h until the collagen polypeptides were completely dissolved, and the pH of the solution was regulated with 1 mol/L sodium hydroxide to 10.00±0.02 for later use.
The above collagen polypeptide solutions at different concentrations were characterized by circular dichroism chromatography (CD), and the size of circular dichroism is usually determined based on a molar extinction coefficient difference Δε(M−1·cm−1) and a molar ellipticity θ. CD detection was carried out on a Chirascan system (Applied Photophysics Ltd., UK), the blowing rate of nitrogen was 35 mL/min. Concentrations of proteins in all the solutions were reduced to 0.16 mg/mL by dilution, the mixed sample was balanced at 50° C. for 1 h, and meanwhile, 200 μL of solution was taken and detected in a 1 mm sample pool at 50° C., and the temperature during detection was kept at 50° C. Spectra within a range of 190-260 nm were recorded, the resolution was 0.2 nm, and the samples were scanned 6 times. Data processing: the spectrum of the buffer solution was subtracted to correct the baseline, the CD spectra were normalized in units of molar ellipticity, and the content of secondary structures was calculated by the peak regression calculation method and the CONTIN fitting program. The effect of polypeptide concentration on secondary structures of polypeptide is shown in
5%
2%
2%
32%
31%
48%
As shown in Table 3 and
The difference between a preparation method of a polypeptide monolayer of the present example and that of Example 1 was that no surfactant was added in the preparation process of the monolayer, only collagen polypeptides were deposited onto a positively ionized titanium sheet, and other conditions were the same as those of Example 1.
A collagen polypeptide solution at a concentration of 4% was deposited onto a titanium sheet treated with PEI at 50° C. for 10 min, the titanium sheet was pulled 20 times, and collagen polypeptide molecules were loosely arranged, as shown in
A preparation method of a polypeptide monolayer included the following steps.
The obtained polypeptide monolayer was denoted as G-SDScac.
A preparation method of a polypeptide monolayer included the following steps.
The obtained polypeptide monolayer was denoted as G-SDS6%.
After the PEI-treated titanium sheet was deposited with collagen polypeptides, —COO− in a polypeptide molecule and —NH3+ in PEI could form a strong ionic bond. In order to verify that the collagen polypeptide molecules are bound to a substrate via ionic bonds rather than physical adsorption, fluorescence intensities corresponding to different numbers of pulling in the deposition process of the polypeptide monolayer were determined. As the number of pulling is increased (5-20 times), the polypeptides that are physically adsorbed onto the substrate are washed away while those bound via ionic bonds are firmly immobilized onto the substrate. It can be seen from
In the present disclosure, the surface morphology of the monolayer was detected by using a Multimode 8 AFM (Bruker, Germany). The polypeptide monolayer sample was placed onto a working table, and the morphology of the sample was detected in a Peak Force mode. Determination of the thickness of the monolayer: when a monolayer was prepared by a deposition method, half of a titanium sheet was wrapped with tin foil to keep it from being contaminated by the solution. During detection, a boundary of the titanium sheet was found by using a build-in auxiliary optical system of the atomic force microscope, a detection range was set to 20 μm to span the substrate and the sample area, the sample was scanned by an AFM tip along the boundary from the thickness corresponding to the monolayer substrate to the bottom of the boundary, and 3 different areas were scanned so as to obtain an average thickness of the monolayer. The scanning speed was 0.977 Hz, the scanning ranges were 20 μm, 10 μm, 5 μm, and 1 μm, respectively, and the data processing software was build-in NanoScope Analysis of AFM.
It can be found from an AFM image in
In addition, the collagen polypeptide monolayers obtained in Examples 1 to 3 are all composed of close-packed nanoparticles, and spherical nanoparticles have an average particle size of about 30 nm. However, the G-SDS6% monolayer is formed by packing of spherical nanoparticles having an average particle size of about 60 nm, and its average thickness is 14.2 nm. It can be known that the polypeptide monolayer of the present disclosure is ultrathin.
The samples obtained in Examples 1 to 3 and Comparative Example 2 were characterized by XPS, and N elements were subjected to peak separation. The binding energy for primary amines is 400.05 eV, the binding energy for amido bonds is 398.89 eV, and the binding energy for secondary amines is 398.26 eV. The XPS data can also be used to determine changes in the binding energy and local chemical state so as to achieve semiquantitative analysis of functional groups. High-resolution spectra of N is core regions (from 396 to 402 eV) and the exposure of primary amino groups are shown in
A water contact angle (CA) of the monolayer sample was determined at room temperature by using a DSA-100 optical contact angle measuring system (KRÜSS, Germany). 2 mL of deionized water was dropwise added to the sample by using an automatic assign controller, and CA was automatically determined by the Laplace-Young fitting algorithm. Five different positions on the sample were determined to obtain an average value of CA, and photos were taken by using a digital camera (SONY, Japan). A Zeta potential of the surface of the monolayer was determined by using a SurPASS electrokinetic solid surface analyzer.
1 mM Na2SO4 solution was used as an electrolyte to determine the Zeta potential of the surface of the monolayer.
Wettability of the surface can be directly reflected by a water contact angle, as shown in
In the vibration process of the amide groups, Raman peaks of amide I and amide III bands are very sensitive to conformational changes of protein backbone. In amide III band, four secondary structures, i.e. α-helix, β-sheet, β-turn, and random coil, are located at 1265-1300 cm−1, 1230-1240 cm−1, 1305 cm−1, and 1240-1260 cm−1, respectively. SAMs of G-SDS mounted on the surface of Ti were characterized by Raman spectra, and a Raman spectrum of amide III band reveals surface-sensitive information on secondary structures of the collagen polypeptide monolayer. Content of the secondary structures of the surface of the polypeptide monolayer was determined by using a confocal Raman spectrometer, and a determination method included: a vibrational Raman spectrum of the sample was recorded by using a LabRAM HR800 Raman spectrometer (Horiba Jobin Yvon, France) equipped with a He—Ne laser (632.8 nm) and 600 groove mm−1 grating. The measurement accuracy of Raman intensity was about 1.2 cm−1. A Raman reference spectrum of the sample was obtained under the conditions of a laser power of 1.1 mw, an irradiation time of 1 s, and 30 accumulations. Raman spectra of the PEI-modified sample and the collagen polypeptide-covered sample were obtained under the conditions of a laser power of ˜0.06 mW, an irradiation time of 1 s, and 10 scans. In all Raman experiments, the orientation of a platform was carefully controlled to allow a polarizer to which a laser was input to be parallel to a bow-tie shaft. The spectra were processed on PeakFit of Systat software. A baseline was determined, and the position of each sub-peak was determined with reference to a deconvolution spectrum and a third derivative spectrum. It helps to resolve overlapping sub-peaks and distinguish interference from noise peaks. Percentage of the secondary structures was obtained by the curve-fitting method. Then, the peak height of each sub-peak, a peak width at half height, the Gaussian content were changed to minimize a root-mean-square of curve-fitting, and the root-mean-square of curve-fitting was characterized with the secondary peak area. Amide III band in the original spectrum was analyzed by the curve-fitting method. In the region of amide III band, typical absorption peaks of α-helix, β-sheet, β-turn, and random coil structures appear at 1265-1300 cm−1, 1230-1240 cm−1, 1305 cm−1, and 1240-1260 cm−1, respectively.
The content of the secondary structures of the surface of the polypeptide monolayer are shown in Table 4, and by adding STSo at different concentrations, the content of α-helix, β-sheet, β-turn, and random coil in the monolayer is changed. As the concentration of STSo is increased from CAC to 6 wt %, the total content of β-sheet and random coil is increased from 62% to 71%. In addition, the content of α-helix in the collagen polypeptide monolayer containing STSo changes slightly, which indicates that the secondary structures of the collagen polypeptide are stabilized with the STSo.
Probe synthesis: a fluorescent probe molecule tetraphenylethylene-isothiocyanate (TPE-ITC) responsive to primary amino groups was synthesized to visually characterize the distribution of primary amino groups on the surface of the polypeptide monolayer. Specifically, the probe was 1-[4-(methyl isothiocyanate)phenyl]-1,2,2-triphenylethylene (TPE-ITC), which was an adduct of tetraphenylethylene (TPE) and isothiocyanate (ITC).
As shown in Formula (1) above, a synthesis method specifically included 5 steps. {circle around (1)} In a 250 mL two-neck round-bottomed flask, 5.05 g (30 mmol) of diphenylmethane was dissolved in 100 mL of distilled tetrahydrofuran in the presence of N2. After the mixture was cooled to 0° C., 15 mL of n-butyllithium (2.5 M hexane solution, 37.5 mmol) was slowly added by using a syringe. The mixture was stirred at 0° C. for 1 h. Then, 4.91 g (25 mmol) of 4-methylbenzophenone was added to the reaction mixture. The mixture was heated to room temperature and stirred for 6 h. A compound 3 was synthesized.
{circle around (2)} The reaction mixture was quenched with a saturated ammonium chloride solution, and extracted with dichlorocarbene. An organic layer was collected and concentrated. The crude product and 0.20 g of p-toluenesulfonic acid were dissolved in 100 mL of toluene. The mixture was subjected to heating reflux for 4 h. After being cooled to room temperature, the reaction mixture was extracted with dichlorocarbene. An organic layer was collected and concentrated. The crude product was purified by silica gel column chromatography in which hexane was used as an eluent to obtain a white solid 4.
{circle around (3)} In a 250 mL round-bottomed flask, 5.20 g (15.0 mmol) of white solid 4, 2.94 g (16.0 mmol) of N-bromosuccinimide, and 0.036 g of benzoyl peroxide were subjected to reflux in 80 mL of carbon tetrachloride solution for 12 h. After the reaction was completed, the mixture was extracted with dichloromethane and water. Organic layers were combined and dried with anhydrous magnesium sulfate. The crude product was purified by silica gel column chromatography in which hexane was used as an eluent to obtain a white solid 5.
{circle around (4)} In a 250 mL two-neck round-bottomed flask, 1.70 g (4 mmol) of white solid 5 and 0.39 g (6 mmol) of sodium azide were dissolved in dimethyl sulfoxide in the presence of Na. The mixture was stirred at room temperature overnight (25° C., 48 h). Then, a large amount (100 mL) of water was added, and the solution was extracted 3 times with diethyl ether. Organic layers were combined and dried with anhydrous magnesium sulfate. The crude product was purified by silica gel column chromatography in which hexane/chloroform (v/v=3:1) was used as an eluent to obtain a white solid 6.
{circle around (5)} Tetraphenylethylene (the white solid 6, 0.330 g, 0.852 mmol) containing functionalized azide groups and triphenylphosphine (0.112 g, 0.426 mmol) were added into a two-neck flask, evacuated under vacuum, and washed 3 times with dry nitrogen. Carbon disulfide (0.55 g, 7.242 mmol) and distilled dichloromethane (50 mL) were added into the flask, and the mixture was stirred. The obtained reaction mixture was subjected to reflux overnight, and the solvent was removed under reduced pressure. The crude product was precipitated with cold diethyl ether (250 mL), and precipitates were filtered and washed 3 times. Finally, the product was dried under vacuum to obtain a white solid TPE-ITC.
First, the synthetic product (tetraphenylethylene-isothiocyanate (TPE-ITC)) was characterized by H-nuclear magnetic resonance spectroscopy. The 1H NMR of the product was obtained by using an AVANCE II 400 NMR spectrometer (Bruker, Germany). The sample to be detected with a size of ˜0.5 cm was placed in a nuclear magnetic resonance tube, and 0.6 mL of deuterated chloroform was added to dissolve it completely, tetramethylsilane (TMS) was used as the internal standard, and the sample was detected by manual shimming at room temperature, and the number of scans was 64, the obtained 1H NMR spectrum was processed by using MestReNova software, and results are shown in
The above results indicate that a TPE-ITC molecular probe for imaging and functionalizing primary amino groups is synthesized, in which the reactive ITC group has a sensitive response to the primary amino groups. Therefore, TPE-ITC is a typical fluorescent molecule having an aggregation-induced emission (AIE) property. The AIE property of TPE-ITC enables a TPE-polypeptide bioconjugate to fluoresce strongly by attaching a large number of AIE labels to collagen polypeptide chains. The fluorescence output of the bioconjugate can be greatly enhanced (up to 2 orders of magnitude) by simply increasing its degree of labelling (DL). The AIE probe strategy is an efficient method for real-time observation of primary amino groups. It has the advantages of simple operation, low cost, and high efficiency. In addition, further regulation of the structure of the AIE fluorophore will be still useful for the development of specific probes for surface functional group detection.
The primary amino groups on the surface of the collagen polypeptide monolayer were labelled with the synthetic TPE-ITC, and the labelling procedure is shown in Formula (2).
Specifically, the labelling steps were as follows: 0.8 mg/mL TPE-ITC/DMSO solution was prepared, 0.5 mL of above solution was taken by using a 1 mL syringe, 9 drops were added to 5 mL of Na2CO3/NaHCO3 buffer solution, and the mixed solution was subjected to ultrasonic treatment for 10 min until TPE-ITC was uniformly dispersed. The polypeptide monolayer was placed into a deposition box, the mixed solution subjected to ultrasonic treatment was slowly poured into the deposition box, the polypeptide monolayer was reacted at 50° C. for 2 h, and after the reaction was completed, the polypeptide monolayer was pulled 10 times in DMSO to remove unlabeled TPE-ITC, blow-dried with high-purity nitrogen, and stored in nitrogen.
Confocal laser scanning microscopy (CLSM) images of the samples were obtained by using a TCS SP8 STED 3× confocal laser scanning microscope (Leica Camera AG, Germany) equipped with an argon-ion laser and two photomultiplier tubes. The resonance scanner was used together with an ultra-sensitive HyD™ detector. The samples were excited with a laser of 405 nm, and fluorescence was detected at 430-493 nm. The CLSM images are shown in
Cytocompatibility of the monolayer sample was tested by using cholecystokinin octapeptide (CCK-8) and methyl thiazolyl tetrazolium (MTT). A material to be tested was prepared in the same size as wells in a 12-well cell culture plate. The pure Ti and G-STSo6% monolayer samples were placed into the wells, and three parallel wells were used for each sample. Human umbilical vein endothelial cells (HUVECs, 5×105 cells/mL) were inoculated into each well and cultured in an RPMI 1640 medium containing 10% fetal bovine serum (FBS) at 37° C. and 5% CO2 for 24 h. Then, the cells were washed twice with a serum-free minimum essential medium (MEM) Eagle, and 15 μL of CCK-8 solution was added to each well containing 100 μL of serum-free MEM. After the cells were incubated at 37° C. and 5% CO2 for 1 h, 100 μL of mixture was transferred to another 12-well plate, as residual G-STSo6% monolayer would affect absorbance at 450 nm. With absorbance at 655 nm as reference, the absorbance of the mixed solution at 450 nm was measured by using an iMark microplate reader, and the wells containing only the cells and the medium served as a control. The cell viability was calculated by the following formula:
ViabilityCCK-8=(Sample abs450-655 nm/Positive control abs450-655 nm)×100
In addition to the CCK-8 assay, the cell viability of HUVECs was tested by an MTT assay. The cell viability was calculated by the following formula. The cells incubated without the monolayer served as a control.
ViabilityMTT=(Sample abs570-655 nm/control abs570-655 nm)×100
Results of the CCK-8 assay indicate that compared with the control group, G-STSo6% serving as a modifying surface has no effect on cell viability and growth (
Cell cloning experiment: MCF-7 cells were cultured in a 60 mm culture dish, and incubated in DMEM at 37° C. and 5% CO2 for 24 h, and then the cells were treated at 3 different steps: a blank control group and G-STSo6% monolayer groups. 8 h later, the cells were washed 3 times with PBS (10 mM, pH=7.4). Then, the cells were cultured in fresh DMEM at 37° C. and 5% CO2 for another 10 d, immobilized with 4% paraformaldehyde, and stained with 0.2% crystal violet. Colonies composed of more than 50 cells were counted. An average surviving fraction was obtained from three parallel experiments.
Surviving Fraction=(Number of colonies formed by cell clones)/(Number of inoculated cells×Inoculation efficiency)
The cells were treated differently (the control group and the G-STSo6% group which was repeated twice), and 8 h later, cell colonies were counted (
Stability of the collagen polypeptide monolayer was tested by using a DMI3000B inverted fluorescence microscope (Leica, Germany) equipped with a Leica DFC 450C CCD. After being immersed in normal saline at room temperature for 7 d, the different samples were blow-dried with high-purity nitrogen for later use. G-STSo6% was placed in a biochemical incubator at 40° C. for immersion for another 15 d, and blow-dried with high-purity nitrogen for later use. Before observation, it is necessary to turn on a fluorescence module, and the machine was preheated for 15 min before use. A glass slide was cleaned, the sample to be tested was placed onto the clean glass slide, the glass slide was fixed on an objective table, the height of the objective table was roughly adjusted, then the focus was fine-tuned, the clearest sample details were found with a bright field and observed by using the fluorescence module, the distribution of fluorescent spots was observed under 50× magnification, the magnification was enlarged in sequence to observe the distribution of fluorescent spots, and stability can be analyzed visually by comparison of the distribution of fluorescent spots before and after the immersion of collagen polypeptide monolayer. Results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010753400.5 | Jul 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/132334 | 11/27/2020 | WO |
