AgNPs (⌀5 and 50 nm) were purchased from Xi’an Ruixi Biological Technology Co., Ltd. (Xi’an City, China). Triton™ X100, sodium lauryl sulfate and soy peptone were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). Agar, pepsin, and Masson’s trichrome staining reagent were purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). Tryptone was purchased from OXOID (Shanghai, China), calcein was purchased from Yeasen Biotech Co., Ltd. (Shanghai, China), and Cell Counting Kit-8 (CCK8) was obtained from APExBIO Technology, LLC (USA). Trypsin was purchased from Sigma–Aldrich (USA). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent was purchased from GlpBio (USA).
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Pig skin was harvested from fresh adult pig skin tissue collected from local slaughterhouses. The porcine skin tissue was rinsed using sterile water for 3 h and then frozen and thawed three times (between − 80 and 37 °C). The tissue was cut into 1 × 1 cm squares, and the subcutaneous tissue was removed using scissors and shaken at 120 rpm at a constant temperature (25 °C). The subcutaneous tissue was then treated with a 1% Triton™ X-100 solution for 12 h and 0.1% sodium dodecyl sulfate for 6 h. The treated tissue was rinsed extensively in phosphate buffered saline (PBS) and lyophilized. The lyophilized tissue was ground into powder. Then, 20 mg mL− 1 of the powder was digested using pepsin in a dilute hydrochloric acid solution (pH 2–3) for 10 min [21], and a ⌀5-nm Ag NP solution was added dropwise, and the mixture was stirred rapidly. Subsequently, this mixture was digested for 2 h until the gel was translucent and viscous and was stored in a refrigerator at 4 °C. PBS solution was added to adjust the osmotic pressure, and precooled 10 M NaOH was added to adjust the pH to 7–8. The gel was maintained at 37 °C for 20 min and was subsequently used to prepare the AgNP–PADM hydrogel.
The cellular and nuclear removals were assessed using hematoxylin and eosin (H&E) and 4′6-diamino-2-phenylindole (DAPI) stainings, respectively. The AgNP, PADM hydrogel, and AgNP–PADM hydrogel absorptions were investigated using ultraviolet–visible (UV–Vis) spectrophotometry. The particle size and distribution of the ⌀5-nm AgNPs were observed using transmission electron microscopy (TEM) before the AgNPs were added to the hydrogel and after they were released from the AgNP–PADM hydrogel. The spatial structures of the PADM and AgNP–PADM hydrogels prepared using the 20, 50, and 80 μg mL− 1 AgNP solutions were studied using scanning electron microscopy (SEM; JSM-6360LV, JEOL, Tokyo, Japan). After the hydrogels were fixed using 2.5% (w/v) glutaraldehyde for 1 h, the samples were washed thrice with PBS solution and then dehydrated sequentially using 30, 40, 50, 70, 80, 90, 95, and 100% CH3CH2OH. Subsequently, the samples were dehydrated using liquid carbon dioxide in a critical point dryer and were observed using SEM to structurally characterize the PADM and AgNP–PADM hydrogels. Energy-dispersive spectroscopy (EDS; X-act, OXFORD, England) was used to study the elemental composition and distribution.
The PADM and AgNP–PADM hydrogel porosities were investigated by immersion in isopropyl alcohol and SEM cross-section observation [22]. In the first method, a certain hydrogel volume was prefrozen at − 80 °C for 1 h and then placed in a freeze dryer for 12 h under negative pressure. The solid gel was weighed (W1), and the hydrogel was then immersed in an isopropyl alcohol solution until saturation. The isopropanol volume before and after hydrogel soaking in the beaker (V1 and V2, respectively) was measured. Then, the hydrogel was removed from the isopropanol, excess isopropyl alcohol was gently wiped off the gel surface with gauze, and the soaked hydrogel was reweighed (W2). The porosity was calculated as follows: Porosity = (W2 − W1)/[(V2 − V1) *𝛒], where 𝛒 is the isopropanol density. In the second method, the sample cross-section was scanned using SEM, and the hydrogel porosity was calculated using ImageJ software (National Institutes of Health, USA).
Rheological measurements were performed using a rotary rheometer (MCR302). The scanning frequency was set at 1 rad s− 1, and the Peltier probes were preheated to 37 °C. The PADM and AgNP–PADM hydrogel acid flow dynamics were adjusted between pH 7 and 8, and the gel was rapidly drawn and filled between two parallel ⌀20-mm plates exhibiting ~ 1-mm clearance. The storage modulus (G′) and loss modulus (G″) of the PADM and AGNP–PADM hydrogels were recorded over time until the fluid gel dynamically solidified.
The same PADM and AgNP–PADM hydrogel masses were incubated at 37 °C, and the sample masses were measured at different time points until the masses stopped changing. The following formula was used to calculate the hydrogel water retention: water retention rate = W2/W1 × 100%, where W2 represents the hydrogel weight measured at each time point, and W1 represents the initial hydrogel weight.
To measure the hydrogel degradation under different conditions, we placed the PADM and AgNP–PADM hydrogels in a centrifuge tube, incubated them at 25, 37, or 42 °C, and observed the samples regularly to check whether the PADM and AgNP–PADM hydrogels had dissolved. The liquid in the centrifuge tube was promptly aspirated, and the remaining samples were weighed.
In addition, we solidified 500 μL of the PADM and AgNP–PADM hydrogels and placed them in a centrifuge tube containing 1 mL of PBS. Then, 7.5 mg of trypsin was added to the experimental (trypsin) group at 37 °C. When the hydrogels had been immersed for 7 days, both solution groups were refreshed once a day. The PBS and trypsin solutions were removed from the centrifuge tubes at the same time every day, and the hydrogels were rinsed using PBS. The water on the hydrogel surface was wiped off, the sample was weighed, and same solution was readded.
To determine the AgNP release from the AgNP–PADM hydrogel under different conditions, we initially prepared a series of AgNP solutions, measured their UV absorbances, and generated a standardized UV absorbance curve to calibrate the measurements. The AgNP release from the AgNP–PADM hydrogel was then measured at different temperatures. The PADM hydrogels (e.g., 500 μL of the PADM and AgNP–PADM hydrogels) were placed in a centrifuge tube and incubated at 25, 37, or 42 °C for certain times. The dissolved hydrogel solutions were removed for a period, and their optical densities (ODs) were measured at ~ 400 nm. This operation was repeated until the solution OD stopped increasing.
Trypsin is a commercial food-grade enzyme exhibiting increased proteolytic activity and is used to break down collagen [23]. The AgNP release of the PADM and AgNP–PADM hydrogels was also measured using trypsin-containing and pure PBS. A group comprising solid samples prepared using 500 μL of the PADM and AgNP–PADM hydrogels was placed in centrifuge tubes, and trypsin (7.5 mg) and PBS (1 mL) were added. PBS solution (1 mL) was added to another set of centrifuge tubes containing only two samples. Every day, 1 mL of the solution was drawn, and its OD was measured at ~ 400 nm. Then, 1 mL of the corresponding solution was added to the centrifuge tube after the measurement, and this operation was repeated until the removed solution OD stopped increasing.
Finally, we measured the AgNP release from the PADM and AgNP–PADM hydrogels using an ultrasonic crushing instrument. The solid samples prepared using 500 μL of the PADM and AgNP–PADM hydrogels were cut into small pieces and placed in a centrifuge tube. Deionized (DI) water (400 μL) was added, and an ultrasonic shatterer was used to vibrate the samples at 25 and 30 Hz. The samples were vibrated for 30 s and rested for 20 s per cycle to prevent overheating the solutions. After 24 cycles, the samples were centrifuged at 4000 g for 3 min, and 200 μL of the supernatant was removed. The OD was measured at ~ 400 nm. The supernatant was added to the sample after the test, and these operations were repeated until the solution OD stopped increasing.
The AgNP–PADM hydrogel antibacterial potential was investigated in vitro using the Oxford cup method (OCM) and the colony count method (CCM). Gram-negative bacteria (Escherichia coli) and gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) were used for the OCM experiments. First, single colonies of Escherichia coli, Enterococcus, and Staphylococcus aureus were added to the medium-containing test tubes, which were shaken at 150 rpm and 37 °C overnight. Logarithmically growing bacteria were selected for the experiment. An appropriate number (0–1.5 × 108 L− 1) of bacteria was selected, and the bacterial solution OD was adjusted to 0.1. Then, the number of bacteria was diluted to 105 L− 1, and 1/10 of the culture-medium volume was added to a noncoagulated-agar-containing culture medium, which was fully mixed and added to a Petri dish and cooled. Then, the PADM and AgNP–PADM hydrogels were added. The Petri dish was incubated at 37 °C for 12 h, and the bacteriostatic zone diameter around the samples was measured using a scale. Each experiment was repeated thrice.
Subsequently, we selected Staphylococcus aureus for CCM detection. First, the PADM and AgNP–PADM hydrogels were cut into 3-mm pieces and sterilized for 2 h under UV light. Then, 2 mL of the Staphylococcus aureus (1 × 108 mL− 1) solution was added to a test tube and shaken at 150 rpm and 37 °C to culture the bacteria. The bacterial suspension (100 μL) and EP tubes were centrifuged at 2, 4, and 12 h, the supernatant was discarded, and the bacteria were resuspended in normal saline. The bacterial suspension (20 μL) was evenly coated on nutrient agar plates, and the bacteria were cultured for 24 h at 37 °C and then counted.
The hydrogel prepared using the 80 g mL− 1 AgNP solution was placed in a centrifuge tube, and was ultrasonically shocked at 25 Hz first for 30 s and then for 20 s. The AgNP release solution (100 μL) obtained at 60, 100, and 140 min was added to an Oxford cup that had been prepared previously as follows. First, an appropriate Escherichia coli solution concentration was used, its OD was adjusted to 0.1, and it was diluted to 106 L− 1. Then, 1/10 of the culture medium volume was mixed into the nonsolidified agar medium. The culture medium was poured into the Petri dish and cooled, the Oxford cup was placed, and an appropriate volume of the nonsolidified agar medium was readded. After the upper medium solidified, the prepared AgNP release solution was added, and the Oxford cup was removed 12 h later to observe the bacterial removal status at the bottom.
The hydrogel (100 μL) was added to a 24-well plate and sterilized using 25 kGy of γ radiation. HeLa cervical epithelioid carcinoma cells were seeded at 4 × 104 well− 1 in blank Petri dishes and on PADM and AgNP–PADM hydrogels [24]. Dulbecco’s modified eagle medium (DMEM; Hyclone) was added to 10% (v/v) fetal bovine serum (FBS), penicillin (100 μg mL− 1), and streptomycin (100 μg mL− 1). The cells were cocultured in a humid 5% carbon dioxide environment at 37 °C for 48 h, stained using a calcium lutein staining kit, and observed using fluorescence microscopy at 515 nm.
Cytotoxicity was investigated using CCK8. The PADM and AgNP–PADM hydrogels were immersed in DMEM (hydrogel/medium = 1:5 v/v). The supernatant was collected as a 100% leaching solution and mixed with a particular proportion of DMEM medium to obtain culture solutions containing 0, 20, 50, and 100% leaching solutions. For further experiments, HeLa cells were seeded in 96-well plates at 2000 cells well− 1 and were cultured at 37 °C for 24 h. Then, the supernatant medium was removed from each well, and 100 μL of each medium containing different proportions of leaching solution were added to the DMEM medium. The leaching-solution-free DMEM medium was the control. Then, 10 μL of the CCK8 reagent was added to each well on days 1, 3, and 5. Meanwhile, the cells were incubated at 37 °C for 1 h. Absorbance at ~ 450 nm was measured using a spectrophotometer.
The PADM and AgNP–PADM hydrogel free-radical scavengabilities were measured using the DPPH free-radical scavenging assay [9], in which the ascorbic acid clearance rate was used as the reference standard. Then, the ascorbic acid, the PADM hydrogel, and the AgNP–PADM hydrogel prepared using the 80-μg mL− 1 AgNP solution were added to a 0.4 mM DPPH anhydrous ethanol solution, and the solution was stored in the dark for 10 min. An appropriate supernatant volume was obtained from each sample, and its absorbance was measured at 517 nm. The test sample content in the DPPH-containing anhydrous ethanol solution was adjusted until the DPPH anhydrous ethanol solution absorbance corresponding to the AgNP–PADM hydrogel sample stopped changing. The sample free-radical scavengability at a particular concentration was calculated using the formula Scavengability (%) = C2/C1*100%, where C1 represents the difference between the maximum and minimum absorbances of the ascorbic-acid-containing DPPH solution, and C2 represents the difference between the absorbances of the DPPH solution at this sample concentration and of the pure DPPH solution. The 50% inhibiting concentration (IC50) was used to record the median sample concentration required when the oxygen radical clearance rate was 100%.
Six-month-old rats weighing 200–300 g were purchased from the Zhejiang Academy of Medical Sciences. The rats were reared in separate cages, fed standard feed and tap water, and the cages were maintained at 25 °C and 55% humidity. Day and night were alternated for 12 h. According to the National Institute of Health publication No. 18–23, 1985, which is typically referenced for the care and use of laboratory animals, great care should be taken with rats. The rats were anesthetized using ketamine (30.0 mg kg− 1), their dorsal hair was removed, and their skin was rinsed using 70% ethanol. A scalpel and tweezers were used to create three 1-cm2 skin wounds on the midline side, 30 μL of a Staphylococcus aureus (1 × 108 CFU L− 1) solution was dropped into each wound, and the bacteria were given 2 h to fully infiltrate the wounds, which were divided into three groups as follows. Group I was the control group and did not receive any treatment (e.g., naked wound); group II was treated with the PADM hydrogel; group III was treated with the AgNP–PADM hydrogel and covered with gauze, and the PADM and AgNP–PADM hydrogels were refreshed on days 3, 5, 7, 9, and 11.
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The wound area reduction (e.g., wound contraction) was used to indicate the therapeutic outcome. The wound contractions were recorded on days 0, 7, and 14. The wound healing area was expressed as a percentage. The wound-shrinkage percentage was estimated using the formula wound-healing ratio (%) = (C1 − C2)/C1 × 100%, where C1 represents the initial wound area, and C2 represents the wound area at each measurement time.
On days 7 and 14, half the rats in groups I, II, and III were randomly anesthetized and sacrificed, and the nonwounded skin was biopsied from approximately 5 mm around the wound. The skin tissue was immobilized in buffered formalin (4%) for 2–3 days before tissue processing and paraffin embedding. Tissue sections (5 μm thick) were prepared using a sectioning mechanism and stained with hematoxylin and eosin (H&E). The neovascularization, epidermis, scarring, and granulation tissue were observed and photographed.
Masson’s trichrome staining can be used to dye early and mature collagens in light and dark blues, respectively to assess wound healing. On days 7 and 14, the wound paraffin sections were stained using Masson’s trichrome reagent, and the tissue section collagen content was analyzed under a microscope. Masson’s trichrome stain was designed to distinguish smooth muscle cells from collagen. Additionally, paraffin sections were immunohistochemically stained to measure the CD68 and CD34 expressions in the samples to evaluate the changes in the macrophage and tissue microvessel contents, respectively, during wound healing.
Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, Inc., USA). The results were expressed as mean ± standard deviation. One- and two-way analysis of variance (ANOVA) were used to determine whether the results were significantly different, and P < 0.05 was considered statistically significant.
Nanotechnology is a rapidly growing science of producing and utilizing nano-sized particles that measure in nanometers (1nm = 1 billionth of a meter). One nanomaterial that is having an early impact in healthcare product is nano-silver.
Silver has been used for the treatment of medical ailments for over 100 years due to its natural antibacterial and antifungal properties. The nano silver particles typically measure 25nm. They have extremely large relatively surface area, increasing their contact with bacteria or fungi, and vastly improving its bactericidal and fungicidal effectiveness.
The nano silver when in contact with bacteria and fungus will adversely affect cellular metabolism and inhibit cell growth. The nano silver suppresses respiration, basal metabolism of electron transfer system, and transport of substrate in the microbial cell membrane. The nano silver inhibits multiplication and growth of those bacteria and fungi which cause infection, odor, itchiness and sores.
Nano Silver can be applied to range of other healthcare products such as dressings for burns, scald, skin donor and recipient sites; acne and cavity wounds; and female hygiene products – panty liners, sanitary towels and pants.
Even though many people know that Silver has superior antibacterial and antibiotic characteristics, there has been limitation on the application in real life because of darkening at high temperature and high cost. Nanotechnology solved all these two problems at the same time.
Nanotechnology is that of processing the material down to several nano meter range (1nm=10-9mm, 1/10,000 of hair thickness).
Nano Silver will combine with the cell walls of pathogenic bacteria, will then directly get inside the bacteria and quickly combine with sulphydryl (-SH) of oxygenic metabolic enzyme to deactivate them, to block inhalation and metabolism and suffocate the bacteria.
Nowadays, Nano Silver technology is used in coating the surface of electronics, carbons or carbon blocks, textiles, metals, woods, ceramics, glass, papers, mixing to the plastics resin, using in fiber and many others.
The core technology of Nano Silvver is the ability to produce particle as small as possible and to distribute these particle very uniformly. When the nano particles are coated on the surface of any material, the surface area is increasing several million times than the normal silver foil.
The maximized surface enables strong antibacterial effect with small amount of silver. The most important thing in Nano Silver is to control the purity of silver particle. In case 0.01% of Nickel impurity is added to the Nano Silver particle, it is very harmful to human body. Many researchers are focusing on the possibility to be used in conquering the “SUPER BACTERIA” which is threatening human life in the future.
In addition, Nano Silver will not expire. Light, heat and radiations will now affects its healing qualities.