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J Nanobiotechnology. 2021; 19: 352.
Anti-senescence ion-delivering nanocarrier for recovering therapeutic properties of long-term-cultured human adipose-derived stem cells
Yeong Hwan Kim
1School of Chemical Engineering, Sungkyunkwan University, Suwon, 440-746 Republic of Korea
Gwang-Bum Im
iSchool of Chemical Engineering, Sungkyunkwan University, Suwon, 440-746 Republic of Korea
Sung-Won Kim
aneSchoolhouse of Chemic Engineering science, Sungkyunkwan Academy, Suwon, 440-746 Republic of korea
Yu-Jin Kim
1School of Chemical Engineering, Sungkyunkwan University, Suwon, 440-746 South korea
Taekyung Yu
2Department of Chemical Engineering, Higher of Engineering, Kyung Hee University, Yongin, 17104 South korea
Ju-Ro Lee
3Center for Biomaterials, Biomedical Inquiry Institute, Korea Establish of Science and Technology, Hwarang-ro 14-gil 5, Seoungbuk-gu, Seoul, 02792 South korea
Soong Ho Um
1School of Chemic Engineering, Sungkyunkwan Academy, Suwon, 440-746 Republic of Korea
Yoon Ki Joung
3Center for Biomaterials, Biomedical Research Institute, Korea Found of Science and Technology, Hwarang-ro xiv-gil five, Seoungbuk-gu, Seoul, 02792 South korea
fourDivision of Bio-Medical Scientific discipline & Technology, Academy of Science and Technology, 113 Gwahangno, Yuseong-gu, Daejeon, 305-333 Democracy of Korea
Suk Ho Bhang
1Schoolhouse of Chemical Applied science, Sungkyunkwan Academy, Suwon, 440-746 Republic of Korea
Received 2022 Jul sixteen; Accepted 2022 Oct 20.
- Supplementary Materials
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Boosted file 1. TEM images of hADSCs after AINs treatment for 12 h shown every bit stepwise magnification (Northward: nucleus, ruby arrows bespeak AINs in a jail cell).
GUID: 5B022DF7-B7B0-41FD-96B2-0323B6A3D350
Abstract
Background
Human adipose-derived stem cells (hADSCs) take been used in diverse fields of tissue engineering because of their promising therapeutic efficacy. Nevertheless, the stemness of hADSCs cannot be maintained for long durations, and their therapeutic cellular functions, such as paracrine factor secretion subtract during long-term cell civilisation. To facilitate the use of long-term-cultured hADSCs (50-ADSCs), nosotros designed a novel therapeutic anti-senescence ion-delivering nanocarrier (Own) that is capable of recovering the therapeutic properties of Fifty-ADSCs. In the nowadays report, nosotros introduced a low-pH-responsive ion nanocarrier capable of delivering transition metallic ions that can enhance angiogenic paracrine gene secretion from Fifty-ADSCs. The AINs were delivered to L-ADSCs in an intracellular manner through endocytosis.
Results
Low pH conditions within the endosomes induced the release of transition metallic ions (Fe) into the L-ADSCs that in turn caused a mild acme in the levels of reactive oxygen species (ROS). This balmy meridian in ROS levels induced a downregulation of senescence-related gene expression and an upregulation of stemness-related gene expression. The angiogenic paracrine gene secretion from L-ADSCs was significantly enhanced, and this was evidenced by the observed therapeutic efficacy in response to treatment of a wound-closing mouse model with conditioned medium obtained from AIN-treated L-ADSCs that was similar to that observed in response to treatment with brusque-term-cultured adipose-derived stem cells.
Conclusions
This study suggests a novel method and strategy for cell-based tissue regeneration that can overcome the limitations of the low stemness and therapeutic efficacy of stem cells that occurs during long-term cell civilization.
Graphical Abstract
Supplementary Information
The online version contains supplementary material available at 10.1186/s12951-021-01098-7.
Keywords: Angiogenesis, Functionality restoring, Intracellular ion delivery, Ischemic disease, Senescence, Stem cell therapy
Background
Transition metal-based nanoparticles take been widely studied for use in biomedical applications, such every bit hyperthermic therapy, drug delivery, and bio-imaging [1, 2]. These nanoparticles take also been practical to stem cells to improve their therapeutic efficacy [3]. Iron (Fe), the representative element of transition element, has been used to better the therapeutic upshot of stalk prison cell based therapies [4–six]. Despite of its great therapeutic effect, Atomic number 26 cannot exist used in clinical trials due to of its potential toxicity such every bit ferroptosis. Ferroptosis is a upshot of iron-dependent oxidative cellular damage, and it tin can cause neurodegeneration [vii, 8]. To utilize the effect of transition metals on the therapeutic efficacy of stem cells without damage, there is a need to accurately command the handling concentration. In this report, to prohibit the cellular damages such as ferroptosis [9, 10] and heighten the therapeutic consequence on stalk cells, we first optimized the concentration of Atomic number 26 for cell treatment and and so evaluated the cellular functions.
Stem cell-based therapy has been highlighted as a regenerative medicine approach for vascular diseases [11, 12]. In item, human adipose-derived stem cells (hADSCs) serve as attractive tools for vascularization and skin regeneration because of their user-friendly direction, ability to differentiate into various cell-types, and secretion of angiogenic factors, such equally vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and hepatocyte growth gene [13–fifteen]. Despite the therapeutic and differentiation power of ADSCs, a limited number of clinical trials have been performed using ADSCs because of their short lifespan, quantitative limitations for cell transplantation, and required utilise at depression passage numbers [16, 17]. Cell-free conditioned media (CM) is retrieved from cultured cells and can be used to overcome the aforementioned obstacles. Recently, multiple studies have investigated the validity of the wound-healing furnishings of jail cell-free CM [18, xix]. However, long-term-cultured ADSCs (50-ADCSs) secrete low concentrations of angiogenic cytokines, and this makes it difficult to use them as an alternative to short-term-cultured hADSCs (S-ADSCs) in the context of CM awarding.
Herein, we developed a novel and simple anti-senescence ion-delivering nanocarrier (Own) and an iron-incorporated gold nanoparticle (AuNP) to enhance the therapeutic efficacy of CM from Fifty-ADSCs. AuNP is well known as not-cytotoxic and stable material and used for bio-imaging material due to their surface plasmon resonance [20]. Merely iron ions are released from AINs under the low pH conditions present in belatedly endosomes [21] due to the difference in the reduction potentials of gold and iron [22, 23]. Iron reacts with hydrogen ions and is ionized under the acidic conditions present in late endosomes, while Au remains intact. Iron has been reported to enhance the angiogenic therapeutic efficacy of stem cells past upregulating the expression of the hypoxia-inducible factor (HIF) cistron that is associated with mitochondrial reactive oxygen species (ROS) [24, 25]. Excessive intracellular ROS levels cause the initiation of programmed cell expiry [26]; even so, mild ROS production induces the upregulation of angiogenic paracrine factor secretion and delays cellular senescence by downregulating senescence-associated gene expression [27–xxx].
The AINs used in this study can deliver an appropriate quantity of iron ions to hADSCs, thus resulting in no induction of apoptosis in these cells. As a result, atomic number 26 ions released from the AINs within endosomes generate mild ROS levels, upregulate HIF expression, raise angiogenic therapeutic efficacy, and contrary the senescence of L-ADSCs. This provides meaningful authorization to allow for the therapeutic usage of Fifty-ADSCs that until now were considered to be of no use for stem jail cell therapy. This anti-senescence strategy may expand the biomedical field by allowing for further utilization of stalk prison cell-based therapies.
Results and discussion
Senescent cells are considered to be bio-waste for stem cell therapy due to their reduced therapeutic efficacy, reduced differentiation power, and the risk of mutation [31–33]. Strategies for the restoration of the functionality of stem cells, including extracellular modifications and cell reprogramming, have been widely researched [34–37]. However, extracellular modifications are plush and can be time-/labor-consuming and involve complex processes. Likewise, several studies aimed to overcome the senescence and restore cellular functionality of stalk cells using biomaterials accept been reported [38–40]. In previous studies, most of biomaterials acted equally carriers of genes or drugs. Therefore, additional processes for enhancing gene or drug delivery efficiency were required. Additionally, not-degradable holding of biomaterials that can induce materials accumulation in body remained equally a problem to be solved. In this study, we used transition metal-based nanoparticle equally a therapeutic agent. We designed the nanoparticle to undergo deposition depends on low pH condition. In this study, nosotros developed a safe and simple nanocarrier to restore the functionality of senescent hADSCs. Intracellularly delivered AINs release iron ions in Fifty-ADSCs due to the acidic weather condition nowadays in the exosomes, and this release generates mild levels of ROS (Additional file i: Fig. S1). As a consequence, the generated balmy levels of ROS facilitate a reduction in cellular senescence and restore the functionality of Fifty-ADSCs.
Characterization of AINs
The transmission electron microscopy (TEM) image of the AINs revealed a spherical shape possessing an boilerplate diameter of iii.57 ± 0.six nm (Fig.aneA and B). Energy-dispersive X-ray spectroscopy (EDXS) analysis indicated that the AINs consisted of fe and aureate (Fig.1C). A slight summit movement (blue squares) was detected due to the different sizes of the gold and iron ions based on powder X-ray diffraction (XRD) patterns (Fig.1D). The ultraviolet/visible (UV-Vis) spectrum tiptop of the AINs at pH 7.0, was observed at 505 nm co-ordinate to plasmon resonance, while those of AuNPs and AINs at pH 4.five were observed at 529 nm and 530 nm according to plasmon resonance, respectively. This demonstrates that the presence of fe causes a red shift (Fig.1E) [41]. The atomic ratio alteration of AINs nether acidic (pH 4.5) and standard (pH 7.0) conditions are presented in Fig.aneF. No significant difference was observed in the atomic ratio (gold/iron) at pH seven.0 compared to that nether standard conditions (gilded/iron = 2.four), while the same ratio was markedly increased nether acidic conditions. The atomic ratio amending of AINs in response to unlike pH conditions highlights the potency of AINs in regard to serving every bit intracellular metal ion carriers to stalk cells.
Concrete and chemical properties of anti-senescence ion-delivering nanocarriers (AINs). A A transmission electron microscopy (TEM) image of AINs (high power magnification at the top-left corner). B Size distribution of AINs, as measured using TEM. C Free energy-dispersive 10-ray spectroscopy (EDXS) analysis of AINs. Fe atom peaks are indicated with white arrows. D X-ray diffraction (XRD) patterns of AINs. Atomic number 26 ion patterns are emphasized using blue squares. E Ultraviolet-visible spectroscopy (UV-Vis) spectra of AINs at pH vii.0 (blue line), aureate nanoparticles (Au) at pH vii.0 (crimson line), and AINs at pH iv.five (black line). Peak shifts owing to the present of iron. F The component golden/iron ratio contour of AINs as estimated using EDXS under the unlike pH weather condition at 12 h (north = 3, *p < 0.05 versus standard group)
Optimizing the concentration and treatment time of AINs
To optimize the concentration of AINs used for L-ADSC treatment, at cell viability analysis was performed in a 24-well cell culture dish using Prison cell Counting Kit-viii (CCK-8) to verify the cytotoxicity of AINs later treatment for 24 h (Fig.ii A). AINs exhibited no cytotoxicity up to a concentration of 3 µg/mL of AINs; however, the cytotoxicity was significantly increased at Ain concentrations of greater than 4 µg/mL. Similar to the results of the CCK-viii assay, treatments with five µg/mL of AINs induced cell death in L-ADSCs, and at a concentration of 3 µg/mL, AINs exerted no harmful effects on the cell viability of Fifty-ADSCs co-ordinate to the results of the concluding deoxynucleotidyl transferase-mediated dUTP nick cease labeling (TUNEL) assay (Fig.2B) and the fluorescein diacetate/ethidium bromide (FDA/EB) staining analysis (Fig.2C). In response to handling with 5 µg/mL of AINs, L-ADSCs inverse morphologically and shrunk in size compared to the characteristics the other groups, thus indicating that a high dose of AINs induced prison cell apoptosis (Fig.2D). Every bit presented in Fig.twoEast, we measured the expression of angiogenic genes, including VEGF and FGF2, in Fifty-ADSCs at various time-points post-handling with 0, 1, 2, and iii µg/mL of AINs (Fig.2E). The cistron expression levels of VEGF and FGF2 in L-ADSCs were highest at 12 h when the cells were treated with three µg/mL of AINs. Apoptosis-related genes, including BAX and CASPASE3, were significantly upregulated in L-ADSCs that were treated with v µg/mL of AINs (Fig.iiF). Additionally, the proliferation-related genes KI-67 and PCNA were significantly downregulated in L-ADSCs that were treated with v µg/mL of AINs (Fig.2G). Nevertheless, there was no significant difference in the expression levels of both jail cell apoptosis-related and proliferation-related genes between cells treated with 0 and three µg/mL of AINs.
Optimization of the concentration of AINs used for handling of L-ADSCs. A Cell viability of L-ADSCs after treatment with AINs (north = four, **p < 0.01 versus no treatment group). B Terminal deoxynucleotidyl transferase-mediated dUTP nick terminate labeling (TUNEL) analysis examining Own treated L-ADSCs (blueish: nucleus, greenish: apoptotic cell). White arrows bespeak cells undergoing apoptosis that was induced by AINs. Scale bar: 100 μm. C Fluorescein diacetate/ethidium (FDA/EB) staining of L-ADSCs postal service-handling with AINs (light-green: live cells, red: expressionless cells). Scale bar: 100 μm. D DiI staining of L-ADSCs treated with AINs (blue: nucleus, red: cellular membrane). Scale bar: 100 μm. E Relative mRNA expression levels of VEGF and FGF2 in L-ADSCs at 3, 6, nine, and 12 h after treatment with diverse concentration of AINs (northward = 4, *p < 0.05 and **p < 0.01 versus no handling group, # p < 0.05 and ## p < 0.01 versus each group). Relative mRNA expression of F the apoptosis-related genes BAX and CASPASE3 and G the proliferation-related genes KI-67 and PCNA in L-ADSCs after treatment with various concentrations of AINs (n = 3, **p < 0.01 and ***p < 0.001 versus no treatment group)
Functionality-restoring effects of AINs
The AINs treated the cells tin be delivered to intracellular domain through endocytosis. Subsequently, the AINs release Fe ions due to low pH condition of endosome. Released Fe ions induce balmy ROS generation through affecting mitochondria which results HIF gene expression. The treatments with AINs induced the upregulation of stemness- and differentiation-related gene expression levels forth with a downregulation of senescence-relate cistron expression in Fifty-ADSCs (Fig.3A). To investigate the cellular uptake process for AINs, we observed the cytoplasm of L-ADSCs using TEM (Fig.iiiB and Additional file i: Fig. S2). Twelve hours afterward treatment, AINs were observed inside the endosomes of L-ADSCs. To quantify the amount of AINs within the L-ADSCs, the cells were analyzed using an inductively coupled plasma optical emission spectrometer (ICP-OES) (Fig.iiiC). Based on this analysis, 5.19 ± 0.33 µg of iron was detected in the 50-ADSCs. Additionally, the amounts of atomic number 26 ions in CM harvested from Fifty-ADSCs at passage 15 without AIN treatment (fifteen Due north-CM) and from Fifty-ADSCs at passage 15 with Ain treatment (15 A-CM) were analyzed (Fig.threeD). We observed that the majority of the AINs were retained within L-ADSCs, and based on this ascertainment, nosotros did not focus extensively on the possible iron effect during CM handling at the wound site. In detail, the estrus map epitome revealed downregulation of senescence-related gene expression and upregulation of stemness- and differentiation-related gene expression in AIN-treated Fifty-ADSCs compared to the expression levels in untreated L-ADSCs (Fig.3E). AINs treatment significantly decreased the gene expression of senescence-related markers, including P16, P21, and P53, and increased the gene expression of the stemness-related markers NANOG, OCT4, KLF4, and SOX2 as evaluated using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses (Fig.3F and G). As mentioned earlier, senescence- and stemness-related genes are regulated past HIF. HIF has besides been reported to regulate the expression of several differentiation-related genes [42, 43]. The expression levels of differentiation-related genes, including transforming growth gene-beta 1 (TGF-β1) and CCAAT/enhancer-binding protein α (C/EBPA) the part as chondrogenesis and adipogenesis factors, respectively, were increased in L-ADSCs that were treated with AINs compared to the levels in untreated L-ADSCs (Fig.3 H). Upon handling, the AINs were successfully delivered to the intracellular domain of L-ADSCs without whatever detectable extracellular leakage of iron, and these AINs exhibited showed meaningful senescence-overcoming potency by downregulating senescence-related gene expression and upregulating stemness- and differentiation-related gene expression.
Cellular uptake and furnishings of AINs in Fifty-ADSCs. A Schematic representation of the functionality-restoring effects of AINs. B TEM images of L-ADSCs after handling with AINs for 12 h (Due north: nucleus, red arrow: intracellular AINs). C Mass detection of iron in L-ADSCs with or without AIN treatment equally quantified using inductively coupled plasma optical emission spectrometer (ICP-OES; n = 4). D Mass detection of atomic number 26 ions in CM harvested from Fifty-ADSCs at passage 15 without AIN treatment (15 N-CM) and from 50-ADSCs at passage 15 with AIN treatment (xv A-CM) as quantified by ICP-OES (n = iv). E Heat map image of the expression levels of senescence-related and stemness-related genes. F Relative mRNA expression of the senescence-related genes P21, P53, and P16 in L-ADSCs with or without AINs treatment (north = four). G Relative mRNA expression of the stemness-related genes NANOG, OCT4, KLF4, and SOX2 in L-ADSCs with or without Ain handling (n = 4). H Relative mRNA expression of chondrogenesis-related and adipogenesis-related genes in Fifty-ADSCs with or without AIN treatment (n = 4). (# p < 0.05, ##p < 0.01, and ### p < 0.001 versus each grouping)
Restoration of angiogenic functionality by AINs
To quantify the angiogenic paracrine cistron secretion from 50-ADSCs post-AINs treatment, VEGF and FGF2, both of which are representative angiogenic factors, were analyzed in CM harvested from S-ADSCs at passage seven without AIN treatment (7 Northward-CM, 15 N-CM, and fifteen A-CM) using an enzyme-linked immunosorbent assay (ELISA) (Fig.fourA). The concentrations of VEGF and FGF2 in fifteen A-CM were significantly higher than those in 15 N-CM, and the concentration in 15 Due north-CM was similar to that in 7 N-CM. Additionally, the profiles of angiogenesis-related proteins, including TGF-β1, endocrine gland-derived vascular endothelial growth factor (EG-VEGF), VEGF, and FGF2, indicated that the concentrations of these proteins were higher in 15 A-CM than they were in 15 N-CM (Fig.4B). As presented in Fig.4C, the gene expression of C-X-C motif chemokine 12 (CXCL-12) that is related to prison cell migration was markedly increased in L-ADSCs that were treated with AINs compared to levels in the untreated Fifty-ADSCs. As senescence is known to decrease jail cell migration ability [44], we performed scratch wound assays using seven N-CM, 15 North-CM, and 15 A-CM treatments in hADSCs (Fig.4D). The fifteen A-CM significantly enhanced the migration of hADSCs in a manner that was like to that of 7 North-CM.
Enhanced angiogenic potency of AINs-treated Fifty-ADSCs. A Quantification of human VEGF and FGF2 protein secretion within CM harvested from South-ADSCs at passage 7 without AIN treatment (7 N-CM, fifteen N-CM, and 15 A-CM) as evaluated using enzyme-linked immunosorbent assay (ELISA; n = four, **p < 0.01 versus no treatment group, # p < 0.01 versus each group). B Representative profiles of angiogenesis-related proteins secreted from seven N-CM, 15 N-CM, and 15 A-CM as evaluated using a man angiogenesis antibody array. C Relative mRNA expression of CXCL12 in South-ADSCs and in L-ADSCs with or without AINs handling. as quantified using qRT-PCR after handling with AINs for 12 h (n = 4, **p < 0.01 versus no treatment grouping). D Representative images of scratched expanse and relative jail cell migration area after treatment with vii N-CM, 15 N-CM, and 15 A-CM as analyzed using a scratch wound assay (red area indicates the scratched surface area, n = 4, **p < 0.01 versus no treatment group)
Enhanced therapeutic ability of fifteen A-CM
To verify the therapeutic efficacy of CM, the closed wound sizes, degrees of regenerated musculus, and complete formation of dermis and epidermis were evaluated at 14 d after surgery. Each CM was injected at a volume of 200 µL daily for the initial iv d period. At 11 d later on the last CM injection, the wounds of the 7 Northward-CM- and xv A-CM-injected groups were healed by approximately 99% compared to the size initial wound area, while the wounds of the 15 N-CM-injected group were healed past approximately 88% (Fig.fiveA). The degree of regenerated muscle was evaluated by measuring the altitude between the edges of the defect. The distance between regenerated muscles for the 15 A-CM-injected group was approximately 3,417 μm on an average, and this was significantly shorter compared to that of the fifteen Northward-CM-injected group (Fig.5B). Hematoxylin and eosin (H&Due east) staining and Masson's trichrome (MT) staining of skin tissue at solar day 14 revealed complete germination of dermis and epidermis with reduced fibrosis and inflammation in the 15 A-CM-injected group (Fig.5C and D). The xv Due north-CM-injected group clearly exhibited less of dermis and epidermis germination and a greater degree of fibrosis and inflammation compared to these values in the 7 N-CM- and 15 A-CM-injected groups.
Increased wound-healing effect of 15 A-CM at 14 d after treatment. A Images of wounds and wound-endmost percentages at 14 d subsequently treatment with CM (n = 5, ##p < 0.01 versus other groups). B Representative images and quantification of muscle regeneration at 14 d afterward treatment with CM (due north = three, ###p < 0.001 versus other groups). Representative histological images of C hematoxylin and eosin (H&E) and D Masson's trichrome (MT) staining of wounds at 14 d after handling with CM
To determine the extent of angiogenesis and dermal regeneration within the wound tissues, blood vessels and dermal formation-related markers in the defect areas were analyzed using qRT-PCR and immunostaining. The fifteen A-CM-injected group expressed CD31 to a greater degree and possessed more singled-out microvessels that were immunostained with anti-CD31 antibodies compared to these values in the 15 N-CM-injected group (Fig.half dozenA). Similar to the observations regarding CD31, the 15 A-CM-injected group exhibited significantly college gene expression and more obvious detection of laminin and involucrin (major protein components of the dermis and substrate protein, respectively) inside the keratinocytes of the epidermis (Fig.6B and C).
Improved angiogenesis and dermal regeneration at 14 d afterward 15 A-CM treatment within the wound tissue. A Analysis of a vascular marker (CD31) using qRT-PCR and immunostaining (n = iii, blue: nucleus, green: CD31). B Assay of a representative dermis marking (laminin) using qRT-PCR and immunostaining (n = 3, bluish: nucleus, green: laminin). C Assay of a representative epidermis marker (involucrin) using qRT-PCR and immunostaining (north = 3, bluish: nucleus, green: involurin) (# p < 0.05, ## p < 0.01, and ### p < 0.001 versus each group)
Conclusions
In the present study, we designed a novel anti-senescence nanomaterial that can answer to low pH weather condition and release iron ions in an intracellular way to recover the therapeutic properties of Fifty-ADSCs. Iron ions released from AINs in response to the depression pH condition nowadays inside the endosomes induce mild ROS generation in L-ADSCs and upregulate HIF-related pathways that can enhance angiogenic paracrine factor secretion. Interestingly, 50-ADSCs treated with AINs exhibited downregulated senescence-related gene expression and upregulated stemness- and differentiation-related gene expression without the occurrence of extracellular iron leakage. The secretion of angiogenic paracrine factors past L-ADSCs treated with AINs was significantly enhanced to levels that were like to those observed in Southward-ADSCs. As a consequence of the enhanced secretion of angiogenic paracrine factors, CM harvested from 50-ADSCs that were treated with AINs displayed drastically enhanced therapeutic results compared to those observed in response to CM harvested from conventional L-ADSCs. Consequently, L-ADSCs that were previously considered to be of no use tin can now exist utilized based on treatment of these cells with AINs. Thus, the AINs described in this study may serve as a safe, economical, and progressive strategy for promoting stem cell-based therapy and senescence studies.
Methods
Materials
All materials used for the synthesis of AIN, including polyvinylpyrrolidone (PVP), NaBHiv, HAuCl4∙xH2O (99.995%), and FeClthree (98%), were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Synthesis of AINs
To synthesize AINs, a PVP solution (9 mL of deionized [DI] water + 100 mg of PVP) was prepared one day prior to synthesis under magnetic stirring (600 rpm) at approximately 25 °C. Prior to synthesis, 4 mg of NaBHfour, 2 mg of FeCl3 and 4 mg of HAuCl4∙xH2O, were dissolved in 1 mL of DI water. Subsequently, 1 mL of NaBH4 solution was added to 9 mL of the previously prepared PVP solution and stirred at 600 rpm and at approximately 25 °C for ten min. Thereafter, the prepared FeCl3 (1 mL) and HAuCl4∙xH2O solutions (1 mL) were injected drop-wise using a micro pipette into the reaction solution. The solution (12 mL) was allowed to react under magnetic stirring (600 rpm) at approximately 25 °C for an boosted fifteen min. The reacted solution was subjected to centrifugation (8000 rpm, 10 min) to precipitate the AINs, and so washed alternately with DI water and acetone twice. The final AINs were dispersed into l mL of DI h2o.
Characterization
An emission electron microscope (JEM-2100 F, JEOL, Tokyo, Nippon) was used to obtain TEM and EDXS images (200 kV). The TEM images were analyzed to determine the size distribution of the AINs. An Ten-ray diffractometer (D-MAX/A, Rigaku, Tokyo, Japan) was used to capture pulverisation XRD patterns at 35 kV and 35 mA. The UV-Vis spectrum in the range of 250–850 nm was analyzed in a crystal cube using a UV–Vis spectrophotometer (Cary lx UV–Vis, Agilent Technologies, Santa Clara, CA, Us). The elemental amounts of gold and iron were direct measured using an inductively coupled plasma (ICP) spectrometer (Direct Reading Echelle ICP, Teledyne Leeman Labs, Hudson, NH, USA).
Fe ion leakage from acid-treated AINs used to mimic endosome conditions
A pH of 4.five was selected to assess Atomic number 26 ion release from AINs [nineteen]. The AINs were exposed to dissimilar pH conditions (pH 4.five and 7.0) and dispersed in phosphate-buffered saline solution (PBS; Gibco BRL, Gaithersburg, Md, United states of america) that was adjusted using hydrochloric acid (HCl 37%, Sigma-Aldrich). The AINs were exposed for 12 h, every bit this time betoken was similar to that used for the angiogenic gene expression data. The remaining AINs in PBS were extracted by centrifugation (8000 rpm, 10 min). The corporeality of Fe ions that was released from the AINs was determined using a UV-Vis spectrophotometer and an ICP spectrometer by analyzing the contradistinct Au/Iron ratio in the AINs.
hADSC civilization
hADSCs were purchased from Lonza (Bazel, Switzerland) and cultured in Dulbecco's Modified Hawkeye's Medium (DMEM; Gibco BRL) supplemented with ten% (five/v) fetal bovine serum (Gibco BRL), and 1% (v/5) penicillin/streptomycin (Gibco BRL). The cells were cultured at 37 °C in a humidified incubator with 5% (v/five) CO2. The medium was inverse every two d. To treat the hADSCs with AINs, a serum-free medium was used to eliminate potential disturbance in the surface charges of the AINs that can be affected by the proteins in the serum [45].
Jail cell viability
Cell viability was determined using the CCK-8 assay (Dojindo Molecular Technologies Inc., Rockville, MD, The states) that measures the corporeality of formazan dye that is reduced by the intracellular dehydrogenase activity. The number of living cells is proportional to the amount of formazan dye. Briefly, h-ADSCs (1 × xiv cells/well) were cultured for 24 h in 24-well plates with various concentrations of AINs, and this was followed by three consecutive washes using PBS. The CCK-viii solution was mixed with fresh serum-free medium and added into each well. After 2 h of incubation, the samples were measured using a plate reader (Space F50, Tecan, Zurich, Switzerland) at an absorbance of 450 nm. Cell viability was calculated as the percent of the viable cells in the groups treated with AINs relative to that in the untreated groups (due north = 4 per group).
Cell morphology
Cell morphology was evaluated using one,one′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Thermo Fisher Scientific, Waltham, MA, USA) staining. Afterward treating the cells with various concentrations of AINs for 24 h, the cells were incubated for 2 h at 37 °C with DiI (six.25 µM) and and then done twice with PBS. The cells were fixed with 4% paraformaldehyde solution for 10 min and washed over again with PBS. Afterwards four,six-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) staining, DiI fluorescence was measured using a fluorescence microscope (DMi8, Leica, Wetzlar, Germany).
Concluding deoxynucleotidyl transferase dUTP nick cease labeling analysis
To discover the apoptotic activeness of L-ADSCs that were treated with AINs for 24 h, the Fluorescein in situ Apoptosis Detection Kit (Merck Millipore, Darmstadt, Germany) was used co-ordinate to the manufacturer's instructions. The last fluorescence images were captured using a fluorescence microscope (DMi8).
Harvest of CM
hADSCs (ane.six × tenvi cells) were seeded into a 150 mm jail cell culture plate (Corning Inc., Corning, New York, NY, United states) and incubated overnight. The medium was replaced with medium containing AINs afterward incubation for 24 h, and the cells were and so incubated for an additional 12 h. After incubation, the cells were washed three times with PBS and cultured for 2 d in serum-free DMEM. To remove whatever remaining cell fragments and contaminants, the harvested CM was subjected to centrifugation (1500 rpm, 5 min) and filtration.
Cellular uptake and leakage of AINs
Cellular uptake and leakage of AINs in L-ADSCs (1.0 × 10v cells) were analyzed past quantifying the amount of gold and iron ions inside the cells and CM. After uptake of AINs (12 h), the cells were washed and lysed in nitric acid hydrochloride (a mixture of nitric acid and hydrochloric acid in a molar ratio of 1:iii) to deliquesce all of the components, including AINs. Ionized samples were diluted in DI water (i:4 [five/5]). The gold and iron concentrations were determined using an ICP-OES (Varian, Palo Alto, CA, USA). To observe the uptake of AINs, L-ADSCs were seeded into a 6-well plate and incubated with AINs. Ultrathin sections of the cells were analyzed using TEM (Talos L120C, Thermo Fisher Scientific) at 120 kV to discover the distribution of AINs. Briefly, Ain-treated cells were washed three times with PBS to eliminate the unbound AINs. The cells were treated with trypsin, washed again three times with PBS, and fixed with Karnovsky's fixative (5% glutaraldehyde [Sigma-Aldrich] + four% formaldehyde in 0.1 M cacodylate buffer [Sigma-Aldrich] + fifty mg CaCltwo [Sigma-Aldrich]/100 mL H2O) for two h. The fixed cells were done iii times with 0.05 Thou sodium cacodylate buffer. Post-fixation staining was performed using 2% osmium tetroxide (Sigma-Aldrich) in 0.1 Thousand cacodylate buffer for 2 h at four °C. The samples were dehydrated in alcohol (30%, fifty%, lxx%, 80%, 90%, and 100% ethanol), treated twice with propylene oxide (Sigma-Aldrich) for x min, and then treated with propylene oxide and Spurr depression-viscosity resin for 2 h. The samples were further treated for 24 h with pure resin and then embedded in molds. The resin blocks were polymerized at seventy °C for 2 d and then cut into ultrathin sections (70 nm) using an ultramicrotome (Reichert-Jung Ultracut E, Leica). The sections were stained with one% pb citrate and 0.v% uranyl acetate and so analyzed. Cellular uptake fourth dimension was determined based on the gene expression results.
Quantitative reverse transcription polymerase chain reaction
qRT-PCR was used to quantify the relative expression levels of genes encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), VEGF, FGF2, Bcl-2-associated 10 poly peptide (BAX), caspase3, ki-67, proliferating cell nuclear antigen (PCNA), P21, P53, P16, NANOG, octamer-binding transcription factor iv (OCT4), kruppel-like factor 4 (KLF4), SOX2, C-X-C chemokine receptor type 4 (CXCR4), β-actin, cluster of differentiation 31 (CD31), laminin, and involucrin. The samples were lysed in TRIzol™ reagent (Invitrogen, Carlsbad, CA, U.s.), and total RNA was extracted with chloroform and precipitated with isopropanol. After the supernatant was removed, the RNA pellet was washed with 75% (v/v) ethanol, air-dried, and dissolved in 0.i% (v/v) diethyl pyrocarbonate (DEPC)-treated water. For qRT-PCR, the SsoAdvanced™ Universal SYBR Green Supermix Kit (Bio-Rad, Hercules, CA, USA) and the CFX Connect™ existent-time PCR Detection System (Bio-Rad) were used. The primers used for qRT-PCR are listed in Tabular array1.
Table 1
Sequences of qRT-PCR primers
| Primer | Sequence | |
|---|---|---|
| Human GAPDH | Frontward | 5′-GTC GGA GTC AAC GGA TTT GG-3′ |
| Contrary | 5′-GGG TGG AAT CAA TTG GAA CAT-iii′ | |
| Homo VEGF | Forward | five′-GAG GGC AGA ATC ATC ACG AAG T-3′ |
| Reverse | v′-CAC CAG GGT CTC GAT TGG AT-3′ | |
| Human FGF2 | Frontward | 5′-GAC GGC AGA GTT GAC GG-iii′ |
| Reverse | five′-CTC TCT CTT CTG CTT GAA GTT-three′ | |
| Human BAX | Forward | 5′-GCA ACT TCA ACT GGG GCC GGG-3′ |
| Reverse | 5′- GAT CCA GCC CAA CAG CCG CTC-3′ | |
| Human CASPASE3 | Forrad | v′-CCT GGT TAT TAT TCT TGG CGA AA-three′ |
| Contrary | 5′-GCA CAA AGC GAC TGG ATG AA-3′ | |
| Homo KI-67 | Frontwards | 5′-CCA CAC TGT GTC GTC GTT TG-three′ |
| Opposite | five′-CCG TGC GCT TAT CCA TTC A-iii′ | |
| Human PCNA | Forward | five′-CCT GCT GGG ATA TTA GCT CCA-3′ |
| Reverse | 5′-CAG CGG TAG GTG TCG AAG C-iii′ | |
| Homo P21 | Forwards | v'-TGA GCC GCG ACT GTG ATG-3' |
| Reverse | v′-GTC TCG GTG ACA AAG TCG AAG TT-3′ | |
| Human P53 | Forrad | five′-CCT CAG Cat CTT ATC CGA GTG G-3′ |
| Contrary | 5′-TGG ATG GTG GTA CAG TCA GAG C-iii′ | |
| Man P16 | Forward | 5′-ACT TCA GGG GTG CCA Cat TC-iii′ |
| Reverse | 5′-CGA CCC TGT CCC TCA AAT CC-iii′ | |
| Human NANOG | Forward | 5′-AGT CCC AAA GGC AAA CAA CCC ACT TC-3′ |
| Contrary | five′-TGC TGG AGG CTG AGG TAT TTC TGT CTC-three′ | |
| Man OCT4 | Frontwards | 5′-CTG GGT TGA TCC TCG GAC CT-3′ |
| Reverse | 5′-CAC AGA ACT Cat ACG GCG GG -iii′ | |
| Man KLF4 | Forward | 5′-TCT CAA GGC AGA CCT GCG AA-iii′ |
| Reverse | 5′-TAG TGC CTG GTC AGT TCA TC-3′ | |
| Human SOX2 | Forrad | 5′-TGA TGG AGA CGG AGC TGA A-iii′ |
| Opposite | 5′-GGG CTG TTT TTC TGG TTG C-3′ | |
| Human TGF-β1 | Forward | 5′-CCC AGC ATC TGC AAA GCT C-3′ |
| Reverse | v′-GTC AAT GTA CAG CTG CCG CA-3′ | |
| Human being C/EBPA | Forward | 5′-TTG TGC CTT GGA AAT GCA AAC-iii′ |
| Reverse | 5′-TCG GGA AGG AGG CAG GAA AC-3′ | |
| Human CXCR12 | Forrard | 5′-TGC CAG AGC CAA CGT CAA G-iii′ |
| Reverse | 5′-CAG CCG GGC TAC AAT CTG AA-3′ | |
| Mouse β-actin | Frontwards | 5′-GGC TGT ATT CCC CTC CAT CG-3′ |
| Opposite | 5′-CCA GTT GGT AAC AAT GCC ATG T-3′ | |
| Mouse CD31 | Forward | 5′-CAA ACA GAA ACC CGT GGA GAT G-3′ |
| Opposite | 5′-ACC GTA ATG GCT GTT GGC TTC-iii′ | |
| Mouse Laminin | Forward | 5′-GGA CGG GAA TTC CGT TAG GG-3′ |
| Reverse | five′-CAG GTC CAA GGA CTG CAC TT-iii′ | |
| Mouse Involucrin | Forward | 5′-CCT GTG AGT TTG TTT GGT CTA CA-iii′ |
| Opposite | 5′-GAA CCA CAG CTG GAA CAG TC-three′ | |
CM component analysis
The protein components of seven N-CM, 15 N-CM, and 15 A-CM were identified using a human angiogenesis array kit (R&D Systems, Inc., Minneapolis, MN, The states). Ane mL of CM was loaded onto the man angiogenesis array kit. After blocking the array membrane with blocking buffer for 1 h and subsequent membrane washing, each CM and array detection antibody cocktail was mixed and added to the blocked membrane. This was followed by overnight incubation at four °C with shaking. After washing, streptavidin-horseradish peroxidase (HRP) buffer was added to the membrane, and the membrane was incubated for 30 min. Following another wash, Chemi Reagent Mixture was added to the membrane for reaction at room temperature, and the point was measured using the LAS-3000 system (Fujifilm, Tokyo, Japan).
Cell migration
To evaluate the migration ability of each CM, hADSCs (ane × xv cells) were seeded into 6-well cell culture dishes (Corning Inc.). The cells were incubated for 24 h and then scratched. The migration area was measured at 0, 12, and 24 h after scratching. The relative migration area was calculated based on comparison to the initial scratch areas.
Mouse pare wound model
4-week-old female athymic mice (BALB/c-nu, xx–25 yard body weight; Orient, Seongnam, Gyeonggi, Korea) were anesthetized with xylazine (ten mg/kg, Bayer, Seoul, Korea) and ketamine (100 mg/kg, Yuhan, Seoul, Korea). Each group included six, randomly selected mice. The full-thickness skin was excised in a square shape (2 × 2 cm) and ligated using a 6-0 silk suture (AILEE, Busan, Korea) at 2 sites on each border. CM (200 µL) was injected into the muscles on each side using the same book every day for the initial 4 d menses. Based on this, 800 µL of CM was injected into each mouse. The wound was covered with sterile gauze after the CM injections. The wound-covering films were changed at 3, 7, and 10 d to acquire photographs of the wounds. All animals were sacrificed at 14 d after anesthesia to harvest the wound tissue. All creature treatments and experimental procedures were approved by the Institutional Animal Care and Apply Committee of Sungkyunkwan University (no. SKKUIACUC2017-05-03).
Histological examination
The wound tissue that was retrieved at xiv d postal service-surgery was embedded in optimal cutting temperature chemical compound (SciGen Scientific Inc., Gardenas, California, U.s.a.). The samples were frozen at − 20 °C and sliced into x μm-thick sections using a microtome (Cryostat, Leica). The sliced samples were stained with H&Eastward to verify tissue regeneration and with MT staining to appraise inflammation.
Immunohistochemistry
The sliced samples were stained with primary antibodies targeting anti-CD31, anti-laminin, and anti-involucrin antibodies (Abcam, Cambridge, U.k.). Afterwards staining with the primary antibodies, fluorescein isothiocyanate-conjugated anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used to detect the signals. The immunohistochemically stained samples were counterstained with DAPI, and the images were obtained using a fluorescence microscope (DMi8).
Statistical analyses
All quantitative data are expressed as mean ± standard deviation. For statistical analysis, 1-fashion analysis of variance was performed with the Bonferroni correction using SigmaPlot (version 12.5, Systat software, San Jose, CA, United states of america). Statistical significance was ready at p < 0.05.
Supplementary Information
Acknowledgements
We would like to thank Editage (www.editage.co.kr) for English language editing.
Abbreviations
| AIN | Anti-senescence ion-delivering nanocarrier |
| AuNP | Gold nanoparticle |
| CCK-8 | Cell Counting Kit-8 |
| CM | Conditioned media |
| EDXS | Energy-dispersive X-ray spectroscopy |
| ELISA | Enzyme-linked immunosorbent assay |
| FDA/EB | Fluorescein diacetate/ethidium |
| FGF | Fibroblast growth factor |
| hADSC | Human adipose-derived stem cell |
| HIF | Hypoxia-inducible cistron |
| H&Eastward | Hematoxylin and eosin |
| ICP-OES | Inductively coupled plasma optical emission spectrometer |
| L-ADSCs | Long-term-cultured hADSCs |
| MT | Masson's trichrome |
| ROS | Reactive oxygen species |
| S-ADSCs | Curt-term-cultured hADSCs |
| TEM | Transmission electron microscopy |
| TUNEL | Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling |
| UV–Vis | Ultraviolet–visible spectroscopy |
| VEGF | Vascular endothelial growth factor |
| XRD | X-ray diffraction |
Authors' contributions
This study was conceptualized by YHK, GBI, TY, and SHB. Nanocarriers were synthesized and characterized by TY. In vitro stem cell experiments were performed by YHK. In vivo wound modeling and analyses were performed past YHK, GBI, SWK, and YJK. The original draft was written by YHK and GBI. All authors discussed the data and reviewed the manuscript. All authors read and canonical the last manuscript.
Funding
This work was supported by the National Research Foundation of Korea (NRF) and the Ministry of Science and ICT (MSIT) (2019R1C1C1007384, and 2018M3A9E2023255). This research was as well supported by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry building of Scientific discipline and ICT, the Ministry of Merchandise, Manufacture and Free energy, the Ministry building of Health & Welfare, and the Ministry of Food and Drug Safety, South korea, Projection Number: 202011B31). This work is partially funded by a grant from the Basic Science Research Programme through the National Research Foundation of Korea funded past the Ministry of Scientific discipline, ICT, and Time to come Planning (Grant No. 2019R1A2C2002390). This piece of work was supported by the National Research Foundation of Korea (NRF) grant funded past the Korea government (MSIT) (No. 2021R1A4A1032782).
Declarations
Competing interests
The authors declare that they have no competing interests.
Footnotes
Publisher'southward Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Yeong Hwan Kim and Gwang-Bum Im contributed equally to this piece of work
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