RGDS-modified ursolic acid: insights into self-assembly, nano-properties, osteoporosis inhibition and molecular mechanisms

Abstract

Herein, a novel anti-osteoporosis agent is presented, namely, RGDS (Arg–Gly–Asp–Ser)-conjugated UA (ursolic acid) (abbreviated as UA-RGDS). In ultrapure water, UA-RGDS existed as a tetramer. In the serum of ovariectomized (OVX) mice and at 10⁻⁴ M concentration, the self-assembled UA-RGDS formed nano-particles of 16–156 nm in height, suitable for transportation in blood circulation. In vivo bio-distribution data showed that UA-RGDS was only distributed in the femur of OVX mice, while null distribution was found in the blood and other organs. These observations suggested that UA-RGDS can target the femur, thereby preventing bone loss. ELISA results showed that as an anti-osteoporosis agent, UA-RGDS downregulated the expressions of IL-1β in the serum of OVX mice. Thus, serum IL-1β levels could be a biomarker to clinically detect menopause, and UA-RGDS could help prevent bone loss.

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Introduction

With the rapid acceleration of population aging, osteoporosis has become one of the most common chronic diseases and is a global health issue.^1–7 From this perspective, it is of practical significance to search novel promising anti-osteoporosis compounds. Clinically, hormone replacement therapy (HRT) is generally used for postmenopausal osteoporosis. However, long-term HRT may increase the risk of breast cancer, endometrial cancer, thromboembolism, and vaginal bleeding.^8–20

To overcome the shortcomings of HRT, some natural anti-osteoporosis agents capable of targeting programmed cell death,²¹ preventing osteoblasts from differentiation and blocking the activation of osteoclasts²² have been reported. As a triterpenoid, ursolic acid (UA) exists in the leaf, fruit and skin of various medicinal plants.²³ It may reduce fat deposition, thereby preventing or treating obesity.²⁴ Furthermore, it exhibits anti-inflammatory and anti-atherosclerosis properties,²⁵ induces chemical-induced hepatotoxicity and gentamicin-induced nephrotoxicity,²⁶ promotes the proliferation and differentiation of bone marrow mesenchymal stem cells of mice, and delays the progression of steroid-induced avascular necrosis of the femoral head.²⁷

UA can be structurally modified to form new derivatives,²⁸ and all of the structural modifications focus on its carboxyl and hydroxyl groups²⁹ to obtain dual benefits of structural and activity diversity. For this purpose, derivatives containing a nitro heterocyclic ring are prepared as anti-inflammatory agents,²⁹ 3β-ester derivatives are prepared as low density lipoprotein C inhibitors to prevent and treat cardiovascular diseases,³⁰ C-3 derivatives containing one amine group are inhibitors of leukemia cells,³¹ chalcone ursolate derivatives are acetylcholinesterase inhibitors that prevent Alzheimer's disease,³² fluorescent derivatives are anti-tumor agents,³³ derivatives containing tetrazolium fragments are anti-toxoplasma agents,³⁴ ionic derivatives inhibit HepG2 cells,³⁵ amide derivatives inhibit colistin resistant Acinetobacter baumannii (CRAB),³⁶ and the hybrid of 1,2,3-triazole on modified gallate is prepared as an antioxidant agent.³⁷

Recently, several studies have reported that structurally modified UA shows anti-osteoporosis properties.^{23,24,28,38–49} In this study, we introduced RGDS (Arg–Gly–Asp–Ser) into the carboxyl group of UA and obtained UA-RGDS. To explore the benefits of UA-RGDS, we studied its self-assembly, nano-properties, bio-distribution and osteoporosis inhibition. Furthermore, we propose that IL-1β could be a biomarker for patients with osteoporosis.

Experimental section

General

UA (Shang Hai Aladdin Biochemical Technology Co., Ltd), amino acids (Shang Hai Jier Shenghua and Beijing Bomaijie Technology Co., Ltd), and the reagents and solvents (Beijing Chemical Works) in this work were obtained commercially and used without further purification, unless otherwise specified. Thin layer chromatography silica gel (GF254) and column chromatography silica gel (200–300 mesh) were purchased from Qingdao Haiyang Chemical Co. Ltd. Isoflurane and an R530 portable small animal anesthesia machine (Shenzhen Rayward Life Technology Co., Ltd) were used for mouse anesthesia. The melting point was recorded in the micromelting point instrument (X-4, Shanghai Precision Instrument Co., Ltd), the optical rotation was recorded in the polarimeter (AUTOPOL III, RUDOLPH). ¹H (300 and 800 M Hz) and ¹³C (75 and 200 MHz) NMR spectra were recorded on a Bruker AMX-300 and AMX-800 spectrometer, while DMSO-d₆ and TMS (Sigma) were used as the solvent and the internal standard, respectively. Electrospray ionization mass spectra (ESI-MS) were recorded on a Waters ZQ 2000 mass spectrometer (USA) or a Bruker 9.4 T SolariX Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (USA) with an ESI/matrix-assisted laser desorption/ionization (MALDI) dual ion source. The purity of the compounds was determined using high-performance liquid chromatography (HPLC).

Animal

Female ICR mice (25 ± 2 g) were purchased from the Laboratory Animal Center of Capital Medical University. All evaluations were based on the protocol. The protocol was reviewed and approved by the ethics committee of Capital Medical University. The committee assured that the animal welfare was maintained in accordance with the requirements of Animal Welfare Act and NIH Guide for Care and Use of Laboratory Animals.

Statistical analyses

Statistical analyses of all biological data were carried out using one-way ANOVA. All analyses were done with SPSS 19.0 program and a P-value less than 0.05 was considered statistically significant.

Synthesis

Preparing Boc-Asp(OBzl)-Ser-OBzl

To 3.60 g (11 mmol) of Boc-Asp(OBzl), 1.35 g (10 mmol) of N-hydroxybenzotriazole (HOBt) and 80 mL of anhydrous tetrahydrofuran (THF), 2.50 g (12 mmol) of dicyclohexylcarbodiimide (DCC) was added at 0 °C. The mixture was stirred for 0.5 h, then 2.00 g (10 mmol) of HCl·Ser-OBzl was added. At 0 °C, the reaction mixture was adjusted to pH 9 with N-methylmorpholine (NMM), stirred at room temperature for 4 h, and TLC (CH₂Cl₂/CH₃OH, 20/1, with one drop of acetic acid) indicated the complete disappearance of HCl·Ser-OBzl. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in 150 mL of ethyl acetate, and the solution was filtered. The filtrate was successively washed with aqueous NaHCO₃ (50 mL × 3), aqueous NaCl (50 mL × 3), aqueous KHSO₄ (50 mL × 3), aqueous NaCl (50 mL × 3), aqueous NaHCO₃ (50 mL × 3) and aqueous NaCl (50 mL × 3). The ethyl acetate phase was dried with anhydrous Na₂SO₄ for 12 h, and then filtered. The filtrate was concentrated in vacuum and the residue was purified with a silica gel column (CH₂Cl₂/CH₃OH, 40/1) to provide 4.75 g (95%) of the title compound as a colorless powder. ESI-MS (m/z): 501 [M + H]⁺.

Preparing HCl·Asp(OBzl)-Ser-OBzl

At 0 °C, 4.60 g (10 mmol) of Boc-Asp(OBzl)-Ser-OBzl was stirred in a 40 mL solution of hydrogen chloride in 4 M ethyl acetate for 4 h and TLC (CH₂Cl₂/CH₃OH, 10/1) indicated the complete disappearance of Boc-Asp(OBzl)-Ser-OBzl. The reaction mixture was concentrated under reduced pressure, the residue was dissolved in 20 mL of anhydrous ethyl acetate, and the solution was concentrated under reduced pressure. This procedure was repeated three times to completely remove excess hydrogen chloride to provide 3.80 g (95%) of the title compound as a light yellow powder. ESI-MS (m/z): 401 [M + H]⁺.

Preparing Boc-Gly-Asp(OBzl)-Ser-OBzl

Using the procedure of preparing Boc-Asp(OBzl)-Ser-OBzl from 1.75 g (10 mmol) of Boc-Gly and 4.00 g (10 mmol) of HCl·Asp(OBzl)-Ser-OBzl, 3.06 g (55%) of the title compound was obtained as a colorless powder. ESI-MS (m/z): 558[M + H]⁺.

Preparing HCl·Gly-Asp(OBzl)-Ser-OBzl

Using the procedure of preparing HCl·Asp(OBzl)-Ser-OBzl, 4.34 g (95%) of the title compound was obtained as a light yellow powder from 5.20 g (10 mmol) of Boc-Gly-Asp(OBzl)-Ser-OBzl. ESI-MS (m/z): 458 [M + H]⁺.

Preparing Boc-Arg(NO₂)-Gly-Asp(OBzl)-Ser-OBzl

Using the procedure of preparing Boc-Asp(OBzl)-Ser-OBzl, 3.79 g (50%) of the title compound was obtained as a colorless powder from 3.20 g (10 mmol) of Boc-Arg(NO₂) and 4.60 g (10 mmol) of HCl·Gly-Asp(OBzl)-Ser-OBzl after column chromatography purification (mobile phase: CH₂Cl₂/MeOH, 15/1, gradient elution). ESI-MS (m/z): 759 [M + H]⁺.

Preparing HCl·Arg(NO₂)-Gly-Asp(OBzl)-Ser-OBzl

Using the procedure of preparing HCl·Asp(OBzl)-Ser-OBzl, 6.25 g (95%) of the title compound was obtained as a light yellow powder using 5.20 g (10 mmol) of Boc-Arg(NO₂)-Gly-Asp(OBzl)-Ser-OBzl. FT-ICR-MS(m/z): 659.28030 [M + H]⁺.

Preparing UA-Arg(NO₂)-Gly-Asp(OBzl)-Ser-OBzl

At 0 °C, a solution of 548 mg (1.2 mmol) of UA, 456 mg (1.2 mmol) of 2-(7-azabenzotriazole)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) and 10 mL of N,N-dimethylformamide (DMF) was stirred for 4 h, and then 695 mg (1.0 mmol) of HCl·Arg(NO₂)-Gly-Asp(OBzl)-Ser-OBzl were added. At 0 °C, the reaction mixture was adjusted to pH 9 with N,N-diisopropylethylamine (DIEPA), stirred at room temperature for 24 h, and TLC (CH₂Cl₂/CH₃OH, 10/1) indicated the complete disappearance of HCl·Arg(NO₂)-Gly-Asp(OBzl)-Ser-OBzl. The reaction mixture was concentrated under reduced pressure, the residue was dissolved with 150 mL of ethyl acetate, and the solution was filtered. The filtrate was successively washed with aqueous NaHCO₃ (50 mL × 3), aqueous NaCl (50 mL × 3), aqueous KHSO₄ (50 mL × 3), aqueous NaCl (50 mL × 3), aqueous NaHCO₃ (50 mL × 3) and aqueous NaCl (50 mL × 3). The ethyl acetate phase was dried with anhydrous Na₂SO₄ for 12 h, filtered, the filtrate was concentrated in vacuum, and the residue was purified using column chromatography (mobile phase: CH₂Cl₂/MeOH, 15/1, gradient elution) to provide 563 mg (51%) of the title compound as a colorless powder. ESI-MS (m/z): 1098 [M + H]⁺.

Preparing UA-RGDS

At room temperature, a suspension of 1.10 g (1.0 mmol) of UA-Arg(NO₂)-Gly-Asp(OBzl)-Ser-OBzl, 20 mL of CH₃OH and 110 mg of Pd/C were combined, H₂ was introduced for 30 h for hydrogenolysis, and TLC (CH₂Cl₂/CH₃OH, 5/1) indicated the complete disappearance of UA-Arg(NO₂)-Gly-Asp(OBzl)-Ser-OBzl. The reaction mixture was filtered, the filtrate was evaporated in vacuum, and the residue was purified using column chromatography (mobile phase: CH₂Cl₂/MeOH, 5/1, with 2‰ HCO₂H, gradient elution) to provide 546 mg (63%) of the title compound as a colorless powder. FT-ICR-MS: 872.55326 [M + H]⁺; HPLC purity: 95.3%; mp: 300–302 °C; [α]²⁵_D = −20 (c = 0.01, MeOH); IR (KBr): 3272, 2926, 2865, 1649, 1601, 1516, 1454, 1377, 1188, 1032, 997, 915, 763, 661 cm⁻¹; ¹H NMR (800 MHz, DMSO-d₆) δ/ppm = 9.96 (s, 1H), 8.61 (s, 1H), 8.33 (s, 3H), 7.26 (s, 2H), 5.21 (s, 1H), 4.36 (m, 2H), 4.27 (m, 1H), 3.90 (d, J = 6.0 Hz, 1H), 3.86 (dd, J₁ = 16.8 Hz, J₂ = 6.2 Hz, 1H), 3.53 (m, 3H), 3.15 (m, 1H), 2.99 (dd, J₁ = 11.0 Hz, J₂ = 4.8 Hz, 1H), 2.60 (dd, J₁ = 16.2 Hz, J₂ = 4.3 Hz, 1H), 2.28 (dd, J₁ = 16.2 Hz, J₂ = 8.0 Hz, 1H), 2.14 (d, J = 10.7 Hz, 1H), 1.92 (m, 2H), 1.83 (m, 2H), 1.66 (m, 5H), 1.53 (m, 1H), 1.42 (m, 9H), 1.27 (m, 3H), 1.20 (d, J = 12.1 Hz, 1H), 1.02 (s, 3H), 0.91 (d, J = 6.3 Hz, 1H), 0.89 (s, 3H), 0.85 (s, 3H), 0.83 (d, J = 6.0 Hz, 1H), 0.67 (s, 3H), 0.65 (d, J = 12.2 Hz, 1H), 0.59 (d, J = 13.8 Hz, 1H), 0.58 (s, 3H); ¹³C NMR (200 MHz, DMSO-d₆) δ/ppm = 176.71, 173.32, 173.16, 171.00, 168.87, 157.83, 138.33, 125.78, 77.31, 63.54, 62.70, 57.46, 55.68, 55.26, 53.08, 52.65, 50.26, 49.06, 47.52, 47.23, 47.15, 42.96, 42.01, 41.00, 39.26, 38.85, 38.70, 38.67, 37.97, 37.19, 36.97, 33.14, 30.84, 28.73, 27.83, 27.48, 25.10, 24.00, 23.80, 23.35, 21.57, 18.45, 17.52, 17.48, 16.91, 16.54, 15.69. The HPLC purity of the title compound was 95.3%; HPLC was performed with Agilent Technologies’ 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) on an Eclipse XDB C18 column (5 μm, 4.6 mm × 150 mm); the column temperature was 30 °C, the mobile phase consisted of water and methanol, the gradient was 0%–70% methanol (0–30 min), and the flow rate was 0.2 mL min⁻¹.

Docking and the active pockets of ALP, TNF-α, IL-1β, and IL-10

To find the molecular target, docking investigations of UA-RGDS towards the active pockets of ALP, TNF-α, IL-1β, and IL-10 were performed using Discovery Studio 2017 R2 Client software. The conformations of UA-RGDS were energy-optimized, and ten generated conformations were docked into the active sites of ALP, TNF-α, IL-1β, and IL-10, according to the standard operation of the software. ESI,† Fig. S1 shows all the interactions in detail, including the interactions between UA-RGDS and the amino acid residues of the active pockets of ALP, TNF-α, IL-1β, and IL-10.

FT-ICR-MS and qCID spectra

The spectra of the FT-ICR-MS and qCID were measured on a Bruker 9.4 T solariX FT-ICR mass spectrometer equipped with an ESI/MALDI dual ion source to reveal the molecular assembly of UA-RGDS.

NOESY 2D NMR spectrum

NOESY 2D NMR spectrum was measured on a Bruker 800 MHz spectrometer to reveal the manner of the molecular assembly of UA-RGDS.

Nano-characterization

To 8 mL of ultrapure water, 1.2 mg of UA-RGDS was added to prepare an aqueous solution (final concentration: 10⁻⁴ M, 10⁻⁵ M and 10⁻⁶ M) for determining TEM, SEM, particle size and zeta potential.

To determine the Faraday–Tyndall effect, the solution of UA-RGDS and RGDS in ultrapure water of pH 6.7 and pH 7.4 (final concentration: 10⁻⁴ M and 10⁻⁵ M) was irradiated with a laser beam of 650 nm.

To record the TEM image, a 10 μL solution of UA-RGDS in ultrapure water (pH 6.8) was dropped onto a Formvar-coated copper grid, the copper grid was dried in air and kept at 35 °C for 24 h prior to measurement. A portion of the solution was successively dropped onto three Formvar-coated copper grids to ensure the reproducibility of the measurement. Over 100 images of UA-RGDS were recorded by TEM (JEM-2100; JEOL Ltd, Tokyo, Japan) at an accelerating voltage of 80 kV using randomly selected regions on the copper grid to determine the characteristics and particle size, and each microscopy experiment was repeated three times. The TEM images were obtained on an imaging plate with 20 eV energy windows at 6000–400 000× and were digitally enlarged.

To record the SEM image, a 20 μL solution of UA-RGDS in ultrapure water (pH 6.8) was dropped onto the silicon slice, the silicon slice was dried in air and kept at 35 °C for 24 h prior to measurement. The sample was coated with 20 nm of gold-palladium at 15 kV, 30 mA and 200 mTtorr (argon) for 60 s, and imaged using SEM (S-4800; Hitachi Ltd, Tokyo, Japan). To determine the characteristics and the size of the nano-particles, more than 100 particles were examined in a randomly selected region on the SEM alloy and a triple grid test was performed for each sample. SEM images at 100–10 000× magnification were recorded using an imaging plate with a 20 eV energy window (Gatan Bioscan Camera Model 1792, Pleasanton, CA, USA).

To record the atomic force microscopy (AFM) image, 10 mL of blood from OVX mice were collected in centrifuge tubes. The blood was centrifuged at 2500 × g for 10 min to separate the serum. The serum was diluted 40 times with pure water to prepare serum solution containing UA-RGDS (final concentration: 10⁻⁴ M, 10⁻⁵ M and 10⁻⁶ M), and 20 μL of the solution was uniformly dropped on mica sheet with pipette gun, dried in an electric oven at 37 °C for 24 h. Under atomic force microscopy, a 180–220 μm long non-conductive silicon nitride (Si₃N₄) gold-coated tip and a 0.15 N m⁻¹ stiffness cantilever were used with a scan rate of 0.5 nm and a pixel resolution of 512 × 512. AFM images were recorded on a Nano-scope 3D AFM (Veeco Metrology, Santa Barbara, CA, USA) using contact mode. The results were processed using Nano-scope analysis 1.7 software.

The measurements of nano-particle size and zeta potential of UA-RGDS were performed with a laser particle Zetasizer Nano ZS90 size analyzer (Malvern Instruments Limited, UK) at 25 °C for seven consecutive days and three times for each sample.

Anti-osteoporosis assay in vivo

ICR mice (female, 25 ± 2 g) were used. After receiving ether anesthesia, both ovaries were surgically removed from the mice, and the fallopian tubes were ligated to prove model mice. After seven days, the model mice were orally administered with UA-RGDS (100 μmol kg⁻¹ per day, successively for 28 days), or UA (300 μmol kg⁻¹ per day, 28 consecutive days, mother nucleus control-1), or UA (200 μmol kg⁻¹ per day, 28 consecutive days, mother nucleus control-2), or 5% CMC-Na (successively 28 days, negative control). Alternatively, the sham mice were orally administered with 5% CMC-Na (successively 28 days, healthy control), received ether anesthesia and the surgical operation, but no ovaries were removed and there was no fallopian tube ligation. On the day after the last administration, the mice were weighed to record the body weight, received ether anesthesia, and were sacrificed by cervical dislocation to collect the blood, followed by immediate dissection to obtain the femur and the uterus. The blood was centrifuged at 2500 × g for 10 min and the separated serum was stored at −20 °C for ELISA (enzyme-linked immunosorbent) assays, while the femur and the uterus were used for the corresponding tests.

Scanning and trabecular bone of femur

The Bruker Skyscan 1276 Micro-CT was used to scan the mouse femur. Before scanning, the femur was removed from the 4% paraformaldehyde solution, rinsed with phosphate buffered saline, and placed in the specimen tube for scanning. After scanning, the microstructure of the femoral trabecula was analyzed using SkyScan CT-Analyser software, and the region of interest of the distal femur with the same height was selected to calculate the relevant parameters of the femoral trabecula. The bone density of the femoral trabecular bone, bone volume fraction, bone trabecular number, bone trabecular thickness, bone trabecular separation and bone trabecular pattern factor of the ovaries excised from mice orally treated by UA-RGDS or UA were quantitatively analyzed. The 3D model of the mouse femoral bone trabecula was reconstructed by SkyScan CT-Analyser software and the 3D reconstructed image was processed using CT VOX software. The effects of UA-RGDS or UA on the fracture, tightness, integrity and the order of femoral trabecular structure of OVX mice were described for the 3D reconstruction of the femoral trabecular bone.

Effect of UA-RGDS on IL-1β

As mentioned above, the molecular docking (ESI,† Fig. S1 shows all the interactions in detail, including the interactions between UA-RGDS and the amino acid residues of four active pockets) suggested that the active pocket of IL-1β was the most suitable space for UA-RGDS to enter. In this case, the effect of UA-RGDS on IL-1β was addressed by ELISA. The samples from the anti-osteoporosis evaluation were used for ELISA. The IL-1β level was tested using an Interleukin-1β assay kit (Fankew, Shanghai FANKEL Industrial Co., Ltd, China) following the manufacturer's instructions. The optical density (OD) per hole was measured at 450 nm using a SpectraMax iD5 microplate reader (Molecular Devices). The sample concentration was calculated based on the OD value of the standard wells.

FT-ICR-MS for femur homogenate

The mouse femur was ground in methanol according to the standard method of the KZ-III-F ultra-low temperature tissue grinder (Servicebio), the supernatant was extracted via centrifugation and concentrated. The FT-ICR-MS spectrum of supernatant was determined.

Results and discussion

IL-1β is a potential target of UA-RGDS to prevent bone loss

The molecular docking based interactions between UA-RGDS and the active pockets of ALP, TNF-α, IL-1β and IL-10 were theoretically calculated. Among the four active pockets, IL-1β contained the most suitable binding site for UA-RGDS. This was confirmed by the ELISA result which revealed that the serum level of IL-1β (but not the serum level of ALP, TNF-α and IL-10) decreased. This meant that UA-RGDS prevented bone loss and IL-1β was the molecular target of UA-RGDS inhibiting osteoporosis by decreasing the level of IL-1β in the blood of OVX mice.

The synthesis route of UA-RGDS is feasible

UA-RGDS was prepared according to the synthetic route of Scheme 1 with a yield of 62.7%, while the HPLC purity was 95.3%. These data indicated that the synthesis route provided sufficient high-quality UA-RGDS for corresponding investigations.

Scheme 1 Synthetic route of UA-RGDS: (i) anhydrous tetrahydrofuran, dicyclohexylcarbodiimide, and 1-hydroxybenzotriazole; (ii) hydrogen chloride–ethyl acetate (4 M) solution; (iii) anhydrous N,N-dimethylformamide, 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluoro- phosphate, and N,N-diisopropylethylamine; (iv) Pd/C and H₂.

FT-ICR-MS and the tetramer of UA-RGDS

FT-ICR-MS can accurately measure the molecular weight and explore the self-assembly of molecules. Accordingly, the FT-ICR-MS spectra of UA-RGDS gave three peaks: the peak at 870.52963 of UA-RGDS minus H (theoretical value: 870.53352), the divalent negative peak at 1306.79697 of [UA-RGDS]₃ minus H (theoretical value: 2613.6179), and the peak of the divalent negative ion at 1743.05182 of [UA-RGDS]₄ minus H (theoretical value: 3485.15974) (ESI,† Fig. S2A). To confirm the relationships between the ion peak of [UA-RGDS]₄ minus H and the ion peak of [UA-RGDS]₃ minus H as well as the ion peak of UA-RGDS minus H, the qCID of 1743.00000 was determined (ESI,† Fig. S2B), which gave an anion at 870.52898 of UA-RGDS minus H (theoretical value: 870.53352). In addition, the qCID of 1306.28784 was determined, which gave an anion peak at 870.52898 of UA-RGDS minus H (theoretical value: 870.53352) and an anion peak at 1743.02558 of [UA-RGDS]₂ minus H (theoretical value: 1742.07541) (ESI,† Fig. S2C). Thus, ESI,† Fig. S2 confirmed that under the condition of FT-ICR-MS test the trimer, the dimmer and the monomer were the cleavage products of the tetramer. Therefore, the tetramer was the largest self-assembled aggregator of UA-RGDS.

NOSEY 2D ¹H NMR and bird-like conformation of [UA-RGDS]₄

The NOSEY 2D ¹HNMR spectrum was recorded to determine the self-assembly manner of UA-RGDS forming the tetramer (ESI,† Fig. S2D). There were two meaningful crossing peaks (marked with green circles). Cross peak 1 resulted from the interactions of the COOH of the Asp residue of the first UA-RGDS with the NH₂ of the Arg residue of the second UA-RGDS and the third UA-RGDS. Cross peak 2 resulted from the interactions of the NH of the Gly residue of the fourth UA-RGDS and the NH₂ of the Arg residue of the second UA-RGDS. According to NOSEY, the distance between the mentioned group's H was less than 4 Å. To bring four UA-RGDS closer to each other, the distances of the mentioned group's H were simultaneously less than 4 Å, the conformation of the monomer of UA-RGDS was energy minimized, and the four energy-minimized conformations of UA-RGDS were manually adjusted to form the tetramer. This operation resulted in the tetramer having a bird-like conformation (ESI,† Fig. S2E). These operations could be used as a universal method for visualizing self-assembled aggregation.

Faraday–Tyndall effect of UA-RGDS in ultrapure water

The nano-property of the aqueous solution of UA-RGDS was characterized by the Faraday–Tyndall effect. Ultrapure water of pH 6.8 alone was colorless and transparent with and without the radiation of the 650 nm laser beam (Fig. 1(A) and (D)). The solution of UA-RGDS in ultrapure water of pH 6.8 (10⁻⁵ M and 10⁻⁴ M) was colorless and transparent without the radiation of the 650 nm laser beam (Fig. 1(B) and (C)), although it exhibited the Faraday–Tyndall effect under the radiation of the 650 nm laser beam (Fig. 1(E) and (F)). Therefore, the aqueous solution of UA-RGDS in ultrapure water possessed nano-properties. In contrast, UA did not dissolve in ultrapure water of pH 6.8 and was unable to receive the test of the Faraday–Tyndall effect.

Fig. 1 Tyndall effect of UA-RGDS in ultrapure water of pH 6.8 with and without the radiation of the 650 nm laser beam: (A) ultrapure water of pH 6.8 alone without the radiation of the 650 nm laser beam appears colorless and transparent; (B) the solution of UA-RGDS in ultrapure water of pH 6.8 (10⁻⁵ M) without the radiation of the 650 nm laser beam appears colorless and transparent; (C) the solution of UA-RGDS in ultrapure water of pH 6.8 (10⁻⁴ M) without the radiation of the 650 nm laser beam appears colorless and transparent; (D) ultrapure water of pH 6.8 alone with the radiation of the 650 nm laser beam appears colorless and transparent; (E) the solution of UA-RGDS in ultrapure water of pH 6.8 (10⁻⁵ M) with the radiation of the 650 nm laser beam exhibits the Tyndall effect; (F) the solution of UA-RGDS in ultrapure water of pH 6.8 (10⁻⁴ M) with the radiation of the 650 nm laser beam exhibits the Tyndall effect; (G) the solution of RGDS in ultrapure water of pH 6.8 (10⁻⁵ M) without the radiation of the 650 nm laser beam appears colorless and transparent; (H) the solution of RGDS in ultrapure water of pH 6.8 (10⁻⁴ M) without the radiation of the 650 nm laser beam appears colorless and transparent; (I) the solution of RGDS in ultrapure water of pH 6.8 (10⁻⁵ M) with the radiation of the 650 nm laser beam exhibits the Tyndall effect; (J) the solution of RGDS in ultrapure water of pH 6.8 (10⁻⁴ M) with the radiation of the 650 nm laser beam exhibits the Tyndall effect.

The observation mentioned above suggested that the modification of UA with RGDS led the solution of UA-RGDS in ultrapure water of pH 6.8 to exhibit the nano-property, and gave UA-RGDS superior water solubility. This change will definitely benefit the anti-osteoporosis activity of UA-RGDS.

On the other hand, as the peptide control RGDS exhibited very weak in ultrapure water of pH 6.8, suggesting the solution of RGDS in ultrapure water of pH 6.8 having very weak nano-property.

TEM image of UA-RGDS

The particle sizes of UA-RGDS in ultrapure water were within 34 nm to 162 nm in diameter according to TEM (Fig. 2(A)–(D)). Specifically, the diameters of the nano-particles were 71–162 nm, 67–149 nm, 44–60 nm, and 39–56 nm at 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, and 10⁻⁷ M, respectively. The diameter of the nano-particles decreased in a concentration-dependent manner. On the other hand, the particle sizes of 10⁻⁴ M UA-RGDS were 60–141 nm, 77–154 nm, and 53–139 nm at pH 7.4, 2.1 and 7.1, respectively (Fig. 2(E)–(G)). This meant that pH did not change the diameter of the nano-particles at the same concentration.

Fig. 2 TEM images of UA-RGDS in ultrapure water: (A) 10⁻⁴ M concentration forming nano-particles of 71–162 nm in size (pH = 6.8); (B) 10⁻⁵ M concentration forming nano-particles of 67–149 nm in size (pH = 6.8); (C) 10⁻⁶ M concentration forming nano-particles of 44–60 nm in size (pH = 6.8); (D) 10⁻⁷ M concentration UA-RGDS forming nano-particles of 39–56 nm in size (pH = 6.8); (E) 10⁻⁴ M concentration forming nano-particles of 60–141 nm in size (pH = 7.4); (F) 10⁻⁴ M concentration forming nano-particles of 77–154 nm in size (pH = 2.1); (G) 10⁻⁴ M concentration forming nano-particles of 53–139 nm in size (pH = 7.1).

SEM images of UA-RGDS

The particle size of the solution of UA-RGDS in ultra-pure water were within 34 nm to 167 nm in diameter according to SEM (Fig. 3). Specifically, UA-RGDS existed as dispersed nano-particles with diameters of 62–167 nm, 54–149 nm 46–144 nm, and 41–138 nm from 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, and 10⁻⁷ M aqueous solution, respectively. The diameter of the nano-particles decreased in a concentration-dependent manner.

Fig. 3 SEM images of solid state UA-RGDS from aqueous solution of different concentrations: (A) 10⁻⁴ M (dispersed nano-particles of 62–167 nm in size); (B) 10⁻⁵ M (dispersed nano-particles of 54–149 nm in size); (C) 10⁻⁶ M (dispersed nano-particles of 46–144 nm in size); (D) 10⁻⁷ M (dispersed nano-particles of 41–138 nm in size).

AFM image of UA-RGDS

The nano-properties of UA-RGDS in the serum of OVX mice was characterized by AFM (ESI,† Fig. S3). UA-RGDS existed as dispersed nano-particles of 16–156 nm 16–137 nm, and 26–158 nm in height at 10⁻⁴ M, 10⁻⁵ M, and 10⁻⁶ M, respectively in the serum of OVX mice. Therefore, the nano-particles of UA-RGDS were maintained within a stable range. It is well known that nano-particles with this height range are suitable for transport in blood.

Particle size of UA-RGDS in ultrapure water

The change of the nano-particles size of UA-RGDS in ultrapure water of pH 6.8 was measured for seven consecutive days. The average size of the nano-particles gradually decreased as the concentration decreased from 10⁻⁴ M to 10⁻⁵ M and to 10⁻⁶ M (Fig. 4(A)). Therefore, the size of the nano-particles changed within an appropriate range within 7 days, and the powders of UA-RGDS can be used to prepare suitable aqueous solution at any time within 7 days.

Fig. 4 Particle size and zeta potential of UA-RGDS in ultrapure water (10⁻⁴ M, 10⁻⁵ M and 10⁻⁶ M) for seven consecutive days: (A) nano-particle diameter at pH 6.8; (B) zeta potential at pH 6.8; (C) zeta potential at pH 7.4.

The zeta potential of UA-RGDS in ultrapure water

The change of the zeta potential of UA-RGDS in ultrapure water of pH 6.8 and pH 7.4 were measured for 7 consecutive days. The zeta potential changed irregularly within as the concentration decreasing from 10⁻⁴ M to 10⁻⁵ M and to 10⁻⁶ M (Fig. 4(B) and (C)). These data suggest that the zeta potential of the nano-particles is within a stable range and the powders of UA-RGDS can be used to prepare aqueous solution at any time within 7 days. The significant negative charge indicates potential protein adsorption on their surface, which leads to the formation of dispersed UA-RGDS nano-particles.

UA-RGDS blocking bone loss of OVX mice

The bilateral ovariectomized mouse model can simulate postmenopausal osteoporosis. The estrogen levels in ovariectomized mice were rapidly reduced. Estrogen deficiency leads to enhanced bone absorption and impaired osteoblast function, resulting in osteoporosis.⁵⁰ Therefore, OVX mice served as negative controls.

High-resolution micro-CT is a faster, more accurate and effective inspection method that does not damage the internal structure of the sample. It can determine the three-dimensional microstructure of bone with a micron resolution, especially in the research field of small animal specimens and large animal specimens in vitro. Micro-CT has become the “gold standard” for evaluating bone morphology and bone microstructure.⁵¹

UA-RGDS (100 μmol kg⁻¹ per day, 28 consecutive days) blocking bone loss was shown by the increase of the bone mineral density (BMD), the increase of the bone volume fraction, the increase of the bone trabeculae number, the decrease of the trabecular thickness and the decrease of the trabecular pattern factor of the treated OVX mice.

The BMD of OVX mice treated by UA (300 μmol kg⁻¹ per day, 28 consecutive days, mother nucleus control-1) was significantly higher than that of the OVX mice treated by CMC-Na (negative control, p = 0.0387) (Fig. 5(A)). The BMD of the OVX mice treated by UA-RGDS was significantly higher than that of the OVX mice treated by CMC-Na (p = 0.0020), while the BMD of OVX mice treated by UA (200 μmol kg⁻¹ per day, 28 consecutive days, mother nucleus control-2) was equal to that of the OVX mice treated by CMC-Na (p = 0.0694). The BMD of the OVX mice treated by UA-RGDS was equal to that of the sham mice (p = 0.4698), and was significantly higher than that of the OVX mice treated by UA (mother nucleus control-1, p = 0.0463) (Fig. 5). These data suggested that the anti-osteoporosis activity of UA-RGDS was significantly higher than that of UA.

Fig. 5 UA-RGDS blocking bone loss in OVX mice: (A) UA-RGDS and UA (300 μmol kg⁻¹ per day) increasing BMD of the treated OVX mice; (B) UA-RGDS and UA (300 μmol kg⁻¹ per day) increasing the bone volume fraction of the treated OVX mice; (C) UA-RGDS and UA (300 μmol kg⁻¹ per day) increasing the number of bone trabeculae in the treated OVX mice; (D) UA-RGDS and UA (300 μmol kg⁻¹ per day) increasing the trabecular thickness in the treated OVX mice; (E) UA-RGDS and UA (300 μmol kg⁻¹ per day) decreasing the trabecular pattern factor in the treated OVX mice; n = 10. OVX mice: Ovariectomized mice.

The bone volume fraction of the OVX mice treated by UA (300 μmol kg⁻¹ per day) was significantly higher than that of the OVX mice treated by CMC-Na (p = 0.0139) (Fig. 5(B)). The bone volume fraction of the OVX mice treated by UA-RGDS was significantly higher than that of the OVX mice treated by CMC-Na (p = 0.0020), while the bone volume fraction of the OVX mice treated by UA (200 μmol kg⁻¹ per day) was equal to that of the OVX mice treated by CMC-Na (p = 0.0820). The bone volume fraction of the OVX mice treated by UA-RGDS was equal to that of the sham mice (p = 0.0555), and was significantly higher than that of the OVX mice treated by UA (300 μmol kg⁻¹ per day, p = 0.0442) (Fig. 5(B)). These data suggested that the anti-osteoporosis activity of UA-RGDS was significantly higher than that of UA.

The number of bone trabeculae of the OVX mice treated by UA (300 μmol kg⁻¹ per day) was significantly higher than that of the OVX mice treated by CMC-Na (p = 0.0317) (Fig. 5(C)). The number of bone trabeculae of the OVX mice treated by UA-RGDS was significantly higher than that of the OVX mice treated by CMC-Na (p = 0.0022), while the number of bone trabeculae of the OVX mice treated by UA (200 μmol kg⁻¹ per day) was equal to that of the OVX mice treated by CMC-Na (p = 0.2632). The number of bone trabeculae of the OVX mice treated by UA-RGDS was equal to that of the sham mice (p = 0.0640), and was significantly higher than that of the OVX mice treated by UA (300 μmol kg⁻¹ per day, p = 0.0346) (Fig. 5(C)). These data suggested that the anti-osteoporosis activity of UA-RGDS was significantly higher than that of UA.

The trabecular thickness of the OVX mice treated by UA-RGDS and UA (300 μmol kg⁻¹ per day) was significantly higher than that of the OVX mice treated by CMC-Na and UA (200 μmol kg⁻¹ per day, p = 0.0197) (Fig. 5(D)). The trabecular thickness of the OVX mice treated by UA-RGDS was equal to that of the sham mice (p = 0.6148) (Fig. 5(D)). These data suggested that the anti-osteoporosis activity of UA-RGDS was significantly higher than that of UA.

The trabecular pattern factor of the OVX mice treated by UA-RGDS was significantly lower than that of the OVX mice treated by UA (300 μmol kg⁻¹ per day, p = 0.0331), and was significantly lower than that of the OVX mice treated by CMC-Na (p = 0.0022) (Fig. 5(E)). Meanwhile, the trabecular pattern factor of the OVX mice treated by UA (200 μmol kg⁻¹ per day) was equal to that of the OVX mice treated by CMC-Na (p = 0.1197). The trabecular pattern factor of the OVX mice treated by UA-RGDS was equal to that of the sham mice (p = 0.0679) (Fig. 5(E)). These data suggested that the anti-osteoporosis activity of UA-RGDS was significantly higher than that of UA.

Micro-CT image of UA-RGDS treated OVX mice

UA-RGDS (100 μmol kg⁻¹ per day, 28 consecutive days) blocking bone loss was shown by the micro-CT image of the treated OVX mice, sham mice, the OVX mice treated by CMC-Na (negative control), and OVX mice treated by UA (300 μmol kg⁻¹ per day, 28 consecutive days, mother nucleus control).

The structure of the femoral trabecular network of sham mice was complete and arranged in an orderly manner (Fig. 6(A)). However, the structure of the femoral trabecular network of the OVX mice treated by CMC-Na was obviously different from that of sham mice. For example, the structure of the femoral trabecular network was incomplete: the structure of the femoral trabecular network became missing and disordered. The structure of the femoral trabecular network of the OVX mice treated with UA was the same as that of the OVX mice treated by CMC-Na, indicating that it exhibited no improvement (Fig. 6(A)). In contrast, the structure of the femoral trabecular network of the OVX mice treated by UA-RGDS was the same as that of sham mice. These images suggested that the anti-osteoporosis activity of UA-RGDS was significantly higher than that of UA.

Fig. 6 Micro-CT image of UA-RGDS and UA treated OVX mice: (A) the cancellous bone in the marrow cavity of sham mice and OVX mice treated with CMC-Na, UA-RGDS and UA; (B) the cortical bone of sham mice, as well as the cortical bone of the OVX mice treated with CMC-Na, UA-RGDS and UA (yellow oval); (C) the inner edge of the cortical bone of sham mice, as well as the inner edge of the cortical bone of the OVX mice treated with CMC-Na, UA-RGDS and UA; (D) the outer edge of the cortical bone of sham mice, as well as the outer edge of the cortical bone of the OVX mice treated with CMC-Na, UA-RGDS and UA.

The cortical bone of sham mice was complete and continuous, while the cortical bone of OVX mice treated by CMC-Na and UA was inserted by cancellous bone, thereby had no continuity (Fig. 6(B), yellow circle). The cortical bone of OVX mice treated by UA-RGDS was the same as that of sham mice (Fig. 6(B)). These images suggested that the anti-osteoporosis activity of UA-RGDS was significantly higher than that of UA.

The inner edge of cortical bone of sham mice was smooth, while the cortical bone of the OVX mice treated by CMC-Na was uneven along the inner edge (Fig. 6(C), green arrow). The cortical bone of the OVX mice treated by UA-RGDS, but not the cortical bone of the OVX mice treated by UA, had smooth inner bone edges (Fig. 6(C), green arrow). These images suggested that the anti-osteoporosis activity of UA-RGDS was significantly higher than that of UA.

The outer edge of cortical bone of sham mice is smooth, while the OVX mice treated by CMC-Na had uneven outer edges of the cortical bone (Fig. 6(D), red arrow). The OVX mice treated by UA-RGDS, but not the cortical bone of the OVX mice treated by UA, had smooth outer edge of the cortical bone (Fig. 6(D), red arrow). These images suggested that the anti-osteoporosis activity of UA-RGDS was significantly higher than that of UA.

UA-RGDS down-regulates IL-1β

The blocking of bone loss by UA-RGDS (100 μmol kg⁻¹ per day, 28 consecutive days) was attributed to the decrease of serum IL-1β and uterus IL-1β of the treated OVX mice.

The serum level of IL-1β of the OVX mice treated by CMC-Na (negative control) and UA (300 μmol kg⁻¹ per day, 28 consecutive days) was significantly higher than that of sham mice (p = 0.0025) (Fig. 7(A)). The serum level of IL-1β of the OVX mice treated by UA-RGDS was equal to that of sham mice (p = 0.2593) (Fig. 7(A)). These data emphasized that UA-RGDS, but not UA, inhibited the inflammatory response by down-regulating IL-1β in the blood, thereby blocking bone loss.

Fig. 7 UA-RGDS affecting mouse IL-1β and uterus weight: (A) UA-RGDS decreasing serum IL-1β; (B) UA-RGDS decreasing uterus IL-1β; (C) UA-RGDS blocking the atrophy of uterus; owing to the low water solubility and low bioavailability, the dosage of UA was considerably much higher than the dosage of UA-RGDS; n = 10.

The uterus IL-1β level of the OVX mice treated by CMC-Na and UA was significantly higher than that of sham mice (p = 0.0002) (Fig. 7(B)). The uterus IL-1β level of the OVX mice treated by UA-RGDS was equal to that of sham mice (p = 0.1803) (Fig. 7(B)). These data emphasized that UA-RGDS, but not UA, blocked the atrophy of uterus of the treated OVX mice by decreasing the uterus IL-1β level. This was supported by the data of Fig. 7(C), wherein the uterine weight of the OVX mice treated by CMC-Na was significantly lower than those of sham mice and the OVX mice treated by UA-RGDS (p = 0.0026). Therefore, the molecular mechanism of UA-RGDS, but not UA, preventing uterus atrophy involves decreasing the uterus IL-1β level.

In vivo bio-distribution of UA-RGDS

To support UA-RGDS blocking bone loss of OVX mice resulting from the ability to target the femur, the homogenate extract of the femur, the serum, the heart, the liver, the spleen, the kidney, the lung and the brain of UA-RGDS treated OVX mice were prepared and tested using FT-ICR-MS (ESI,† Fig. S7–S14). The FT-ICR-MS of the femur homogenate, but not the blood and the other organs, of the OVX mice treated by UA-RGDS (100 μmol kg⁻¹ per day, 28 consecutive days) gave a peak at 870.52096 (the negative ion of UA-RGDS minus H, theoretical value: 870.53407) and a peak at 432.17703 (the negative ion of Arg–Gly–Asp–Ser minus H, theoretical value: 432.18429). In addition, no similar peaks were detected in the femur homogenate of the OVX mice treated by UA (300 μmol kg⁻¹ per day, 28 consecutive days) and CMC-Na (negative control, 28 consecutive days). Thus, the FT-ICR-MS tests of the homogenate extract showed that UA-RGDS, but not UA, targeted OVX the mouse femur, wherein it released UA to selectively exhibit anti-osteoporosis action.

Previous structural modification of UA involving no RGDS

The structural modification sites of UA are 28 carboxyl groups and 3 hydroxyl groups, which leads to UA to having diverse derivatives, such as the derivatives containing the nitro heterocyclic ring,²⁹ the 3β-ester derivatives,³⁰ the heterocyclic ring,³¹ the chalcone derivatives,³² the indolequinone derivatives,⁵² the fluorescent derivatives,³³ the derivatives containing tetrazolium fragments,³⁴ the ionic derivatives,³⁵ the 2α,3β,6β,19α-23-pentahydroxy-12-ene derivative, the 2α,3β,6β,19α-23-tetrahydroxy-12-ene derivative⁵³ and the derivatives extracted from witch hazel (LH).⁵⁴ It is worthy to emphasize that all of these derivatives involve no RGDS.

In contrast to the derivatives mentioned above, UA-RGDS provided a novel strategy for modifying the carboxyl group of UA, and led to a series of benefits. For example, UA-RGDS existed as the tetramer in ultrapure water, the self-assembly of UA-RGDS in the serum of OVX mice led to the formation of 16–156 nm nano-particles in height. UA-RGDS was only distributed in the femur of OVX mice in vivo, blocking bone loss by down regulating the expression of IL-1β. Thereby IL-1β is a potential biomarker of osteoporosis.

Conclusions

UA-RGDS was prepared to improve the poor water solubility and low bio-availability of UA. Unexpectedly, the introduction of RGDS improves the poor water solubility, enhances the low bio-availability of UA, and gives UA-RGDS self-assembly properties and a bone-targeting property. UA-RGDS forms nano-particles with a size that is suitable for transport in blood circulation. It selectively enters the bone and releases UA, thereby blocking bone loss. In addition, UA-RGDS decreases the level of IL-1β in the blood and in the uterus. Mechanistically, simultaneously decreasing the level of IL-1β in the blood and in the uterus indicates that UA-RGDS inhibits bone loss and alleviates uterine atrophy. From the perspective of clinical application, UA-RGDS provides a linker for bone loss through down regulation of the expression of IL-1β, thereby showing that monitoring the level of IL-1β in the blood is a potential biomarker for patients with osteoporosis.

Author contributions

Xiaoxiao Yuan: writing – original draft, investigation, formal analysis, methodology, and data curation. Xiaoyi Zhang: writing – review & editing, visualization, formal analysis, methodology, and data curation. Yifan Yang: visualization and investigation. Yaonan Wang: methodology, software, and visualization. Shurui Zhao: methodology, software, and investigation. Qiqi Feng: data curation and methodology. Jianhui Wu: investigation and software. Shiqi Peng and Ming Zhao: writing – review & editing, conceptualization, supervision, funding acquisition, conceptualization, and project administration.

Data availability

Data supporting the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This study was funded by National Major Scientific and Technological Special Project for “Significant New Drug” (2018ZX097201003), National Natural Science Foundation of China (81703332). The authors also extent their appreciation to the Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China for its financial support.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4tb02447h

‡ These authors contributed equally.

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