Nanotechnology

Injectable photocrosslinking spherical hydrogel-encapsulated targeting peptide-modified engineered exosomes for osteoarthritis therapy | Journal of Nanobiotechnology


Chemicals and reagents

LRRK2-IN-1 (CAS 1234480-84-2) was purchased from Topscience (T2246; China). Recombinant mouse IL-1β cytokine (#501-RL-010) and human IL-1β cytokine (201-LB-025) were obtained from R&D Systems (USA). The primary antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; #60004-1-Ig), inducible nitric oxide synthase (iNOS; #22226-1-AP), cyclooxygenase 2 (COX2; #66351-1-Ig), type II collagen (COL2; #28459-1-AP), aggrecan (#13880-1-AP), and matrix metallopeptidase 13 (MMP13; 18165-1-AP) were acquired from Proteintech Group (China) and the primary antibodies against matrix metallopeptidase 3 (MMP3; # BM4074) and SRY-box transcription factor (SOX9; #A00177-2) from Boster Biological Technology (China). Trypsin, collagenase type II, phosphate buffer saline (PBS) buffer solution, and the secondary antibodies for western blot and immunofluorescence analyses were provided by Boster Biological Technology (China). In addition, fetal bovine serum (FBS) and Dulbecco’s modified Eagleʼs medium F12 (DMEM/F12) were provided by Gibco (USA). Safranin O solution was provided by Solarbio (#G1067; China).

Chondrocyte isolation and culture

Chondrocytes were obtained from the knee joints of sacrificed C57BL/6 mice (5 days old) by sequential enzymatic digestion as previously described [29]. Briefly, the total knee cartilage was extracted and placed in PBS. The synovium and other attachments were stripped thoroughly from the cartilage surface. Then, the separated cartilage was crumbled into miniature patches and immersed in 0.25% trypsin for digestion in a cell incubator for 30 min. After centrifugation, the sediment was transferred and regurgitated with 0.2% collagenase II for 6 h in a hybridization oven at 37 °C. Then, the chondrocyte sediment was isolated from suspension by centrifugation at 1500 r/min for 5 min and cultured in a complete medium containing 10% FBS (Gibco, USA), 1% penicillin/streptomycin, and DMEM/F12 culture medium in a cell incubator. Matured chondrocytes were collected for subsequent in vitro experiments. IL-1β (5 ng/mL) was used for the induction of OA-like chondrocyte phenotypes in vitro [4, 30].

In Vitro cytotoxicity test

In vitro cytotoxicity of LRRK2-IN-1 was measured using the cell counting kit-8 (CCK8) method. In brief, chondrocytes were cultured in 96-well plates (5000–10,000 cells/well) for 24 h, followed by LRRK2-IN-1 treatment for 24 h. The CCK8 reagent was added to the plates and the absorbance value in 450 nm wavelength was detected using a microplate reader (BioTek, USA).

Western blot analysis

Protein extraction and western blot analysis were performed as the previous studies described [31, 32]. In brief, the cells mixed with radio-immunoprecipitation assay buffer (RIPA) supplied with phosphatase inhibitors and protease inhibitors in the proportion of 100:1:1 (Boster, China) were scraped off with cell wipers and the cell lysis was collected. The cell lysis was treated using an ultrasonic crusher (Sonicator Q125; Qsonica, USA) and centrifuged at 12,000 rpm, 4 °C for 30 min. After centrifugation, the supernatant was collected and added with loading buffer in the proportion of 4:1, refrigerated for 5 min, and denatured at 95 °C for 5 min. The total protein was then subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis (8.0–12.5%) and transferred to a polyvinylidene difluoride membrane. The unbound sites on the membrane were blocked with 5% bovine serum albumin for 1 h. The targeted bands were cut out from the membrane and incubated with the specific primary antibodies at 4 °C overnight, and the membranes were rinsed thrice with tris-buffered saline with 0.1% Tween 20 for 15 min, incubated with specific secondary antibodies for 1 h at room temperature, and washed again thrice with tris-buffered saline with 0.1% Tween 20 for 15 min. Protein blots were developed using a Western ECL Substrate Kit (Thermo Fisher Scientific, USA) and Bio-Rad scanner (BioRad, USA). The intensity of bands was quantified by digital image analysis software (Bio-Rad, USA).

Immunofluorescence staining

Chondrocytes were seeded at 2 × 104 per dish and cultured on dishes for 24 h. These were then stimulated with IL-1β (5 ng/mL) for 24 h with or without LRRK2-IN-1 (5 µM) for 24 h. Next, the plates were fixed with 4% paraformaldehyde and rinsed with 0.2% Triton X-100 for 15 min. Then, the unbound sites on the plates were blocked with 1% bovine serum albumin for 30 min at normal temperature. Subsequently, they were treated with primary antibodies against aggrecan, iNOS, and MMP13 at 4°C overnight. After washing with PBS twice, the chondrocytes were incubated with Cy3-conjugated goat anti-rabbit secondary antibody for 1 h at room temperature in the dark. Finally, the nuclei of chondrocytes were dyed using 4ʹ,6-diamidino-2-phenylindole (DAPI) for 10 min. Screening and photography were performed using a fluorescence microscope (Nikon, USA).

Isolation of exosomes

Rat bone marrow mesenchymal stromal cells (BMSCs) were harvested and cultured with DMEM medium containing 10% FBS as previously described [33]. When the cell confluency reached 30–50%, the original medium was discarded and DMEM basic medium without FBS was added after the cells were washed with PBS three times. After 48–72 h, the cell culture supernatant was collected and underwent ultracentrifugation to collect exosomes. The detailed steps are as follows: centrifuge at 300 g for 10 min, take the supernatant, and remove the pellets to remove the cells; centrifuge at 2000 g for 10 min, take the supernatant, and discard precipitate to remove dead cells; centrifuge at 10,000 g for 30 min to remove the cell fragments in the sediment; centrifuge at 100,000 g for 70 min by ultracentrifugation; resuspend pellets at the bottom of the centrifuge tubes with a small amount of PBS. The concentration was quantified with a BCA reagent kit (#23225, Thermo Fisher Scientific, USA).

Drug loading in exosomes

LRRK2-IN-1 and exosomes were mixed in a 1:1 mass ratio. With the probe set as 0.25 mm and the amplitude set as 20%, the mixed solution was treated sequentially by 3 min ultrasound, 30 s ultrasound, and 2 min pause on ice, and these steps were repeated 6 times. After the recovery of the exosome membrane at 37 ℃ for 1 h, the mixed solution of exosomes and LRRK2-IN-1 was centrifuged at 100,000 g for 70 min to collect LRRK2-IN-1 loaded exosomes (Exo-L). To measure drug concentration, Exo-L were destroyed using an ultrasonic cleaning machine for 30 min, and centrifuged for 20 min at 4 ℃, 15,000 g. After diluting the supernatant with deionized water, the drug content in the exosomes was determined by liquid chromatography. The 20 µL supernatant of the test sample was injected into the HPLC column to detect the elution of the drug. The drug encapsulation efficiency was calculated according to the following formula (Additional file 1: Table S1): The drug encapsulation efficiency = Wb/W× 100%. Note: Wa is the mass of the drug loaded, and Wb is the initial system mass of the drug. The drug loading rate was calculated according to the following formula (Additional file 1: Table S2): Drug loading rate = W1/W0 × 100%. Note: W1 is the mass of the drug loaded, and W0 is the total mass of the drug loaded exosomes.

Target peptide-modification of exosomes

Drug-loaded exosomes were dissolved in 15 mL HEPES buffer solution (#15630080, Gibco, USA). 2 µL methyl acrylic anhydride (276685, Sigma, Germany) was added into the mixed solution which was then stirred for 24 h. The mixed solution was filtered by a 0.22 μm filter to remove the precipitated acrylic acid. Methacrylic anhydride was acylated with the amino groups on the surface of exosomes to form olefin double bond modified exosomes. The exosomes were collected again by ultracentrifugation, dissolved in 15 mL HEPES buffer, added with 1 mg peptide WYRGRL (Additional file 1: Fig. S1), mixed well, placed at 0 ℃, and stirred for 24 h. Alkene double bond modified exosomes underwent acylation reactions with amino groups in targeted peptides, achieving the binding of peptides to exosomes. The peptide segments were combined with the exosome membrane. The superfluous unconnected peptide segments were removed by ultrafiltration tube, and the peptide-modified exosomes (W-Exo-L) were collected by ultracentrifugation. The formulation of resulted peptide-modified exosomes was resuspended in phosphate buffer for quantification and freezing.

Characterization of target peptide-modified exosomes

The total protein of BMSCs and exosomes was extracted using RIPA lysis (#89900, Thermo Fisher Scientific, USA). In brief, BMSCs and exosomes were added with RIPA lysate containing PMSF (#36978, Thermo Fisher Scientific, USA), incubated on ice for 30 min at 4 ℃, and centrifuged at 8000 g for 10 min. The supernatant was taken and the total protein concentration was detected with the BCA kit. After 20 µg protein was dissolved in the loading buffer and incubated in a 100 ℃ metal bath for 5 min, the protein samples were analyzed by western blot analysis to detect the expression of the exosome marker proteins including TSG101 (#28283-1-AP, Proteintech, China), CD81 (#27855-1-AP, Proteintech, China), and CD9 (#20597-1-AP, Proteintech, China). The isolated and purified exosomes were quickly taken out, dropped onto the copper mesh, and fixed in 5% glutaraldehyde with protection from light. The exosomes were negatively stained with 2% phosphotungstic acid in 0.1 M phosphate buffer at 4 ℃, and analyzed by transmission electron microscope (TEM, HT-7700, Hitachi, Japan) at 100 KV. 10 µg exosomes were dissolved in 1 mL PBS, swirled and shaken, and then mixed well. Then, the particle size distribution and surface charge of exosomes were directly observed and measured with a Nanoparticle Tracking Analyzer.

Photocrosslinking spherical hydrogel encapsulation of targeting peptide-modified exosomes

The methacrylate gelatin (GelMA) (#900629, Sigma, Germany) solution with a concentration of 20% was prepared, and 300 µg/mL exosomes modified with targeted peptides were added to 1 g GelMA and mixed uniformly at 40 ℃. The liquid paraffin span-80 (10:1) (8.40123, Sigma, Germany) was added to the round bottom flask and stirred at 350 r/min for 10 min to obtain a well-mixed emulsion, which was used as the oil phase for standby. The mixed solution of GelMA and exosomes were slowly added to the oil phase which was then stirred and emulsified at a certain speed for 10 min. Then the mixed solution was stirred in a ~ 4 ℃ ice water bath and added with 200 µL LAP photoinitiator (#900889, Sigma, Germany) for photocrosslinking. After crosslinking, the mixed solution was added with isopropanol to generate flocculation, which was further filtered, washed by isopropanol for 3 times to remove the un-encapsulated exosomes, and dried to obtain the exosomes encapsulated with GelMA microspheres (W-Exo-L@GelMA).

Encapsulation efficiency and loading capacity of exosomes into GelMA

The exosomes loaded GelMA prepared using the above method were destroyed using an ultrasonic cleaning machine for 30 min, and centrifuged for 20 min at 4 ℃, 15,000 g. After diluting the supernatant with deionized water, the protein content was determined through the BCA protein quantification kit (Thermo Fisher Scientific). Briefly, 25 µL standard and diluted test samples were added to the 96 well plate and 200 µL of BCA working solution was added to a 96 well plate and incubated at 37 ℃ for 30 min. The sample was measured by a multifunctional enzyme-linked immunosorbent assay under the absorbance conditions of A562. According to the measured values of the standard sample, the standard curve is drawn and the protein concentration of the sample to be tested is calculated using the standard curve. The exosomes encapsulation efficiency was calculated according to the following formula (Additional file 1: Table S3): The exosome encapsulation efficiency = Wb/W× 100%. Note: Wa is the mass of the exosome loaded, and Wb is the initial system mass of the exosome. The exosome loading rate was calculated according to the following formula (Additional file 1: Table S4): Drug loading rate = W1/W0 × 100%. Note: W1 is the mass of the exosome loaded, and W0 is the total mass of the exosome-loaded GelMA.

Characterization of spherical hydrogel

The spherical hydrogel was observed under an inverted fluorescent microscope (Olympus IX71, Olympus, Japan). The chemical composition was analyzed by Fourier-transform infrared spectroscopy (FT-IR) (Nicolet 6700, Thermo Fisher Scientific, USA) in the 400–4000 cm−1 range. The morphology of the hydrogel microsphere was observed by scanning electron microscopy (SEM) (Zeiss, Gemini300, Germany). The hydrogel microsphere was added to the PBS solution and stirred at 37 ℃. 100 µL test samples were taken out from the dialysis bag at each time point, followed by 100 µL PBS solution supplement. The test samples were heated at 75 ℃, vortexed, and centrifuged at 13,000 rpm for 10 min. After filtration, 20 µL supernatant of the test sample was injected into the HPLC column to detect the elution of the drug. The release of LRRK2-IN-1 was expressed as a percentage of the total amount of LRRK2-IN-1. In vitro cytotoxicity of W-Exo-L@GelMA treatment for 48 h was measured using the CCK8 method.

 Endocytosis of Dil-labeled W-Exo-L@GelMA by chondrocytes

The extracted Exo were diluted with PBS, blended in Dil staining solution (500:1, D3911, Invitrogen, USA), and incubated at 37 ℃ away from light for 15 min. Then, the samples were centrifuged at 100,000 g for 60 min to discard the supernatant and cleaned with PBS 3 times to remove the redundant staining solution. The labeled Exo was used to prepare W-Exo-L@GelMA and then were co-cultured with chondrocytes for 48 h. The chondrocytes were immersed 3 times in PBS, and fixed by 4% paraformaldehyde (Gibco, USA) for 10 min. After washing 3 times with PBS, the fixed cells are immersed in phalloidin liquid (A12379, Alexa Fluo 488, Invitrogen, USA) for 15 min. Subsequently, 4ʹ, 6-diamidino-2-phenylindole (DAPI) (D1306, Invitrogen, USA) was added into the fixed cells. Eventually, images were observed and collected with a fluorescence microscope (Olympus Optical Co., Ltd, Tokyo, Japan).

RNA sequencing and bioinformatic analysis

Chondrocytes were treated with IL-1β (5 ng/mL) and with or without W-Exo-L@GelMA loaded with 5 µM LRRK2-IN-1 for 48 h. Total RNA was isolated using Trizol (Invitrogen, USA). In the design of the RNA sequencing (RNA-seq) study, two biological replicates were included in each group. After a quality check, 500 ng RNA was used to construct cDNA libraries, which were subsequently sequenced using the Illumina HiSeq 4000 platform following standard protocols [34]. After trimming adapters and removing reads with low-quality bases (Q ≤ 20) using the fastp tool (v0.18.0) [35], paired-end clean reads were obtained and mapped to the reference genome of Mus musculus GENCODE Release M31 (GRCm39) using HISAT2 (v2. 2.4) [36] with default parameters. The mapped reads were assembled by using StringTie (v1.3.1) [37]. The FPKM (fragment per kilobase of transcript per million mapped reads) value was calculated to quantify the expression abundance of genes using RSEM software (v 1.2.31) [38]. Principal component analysis (PCA) was employed to determine the overall difference in transcriptome between each group. The differential expressed genes (DEGs) were identified by the criteria of FDR < 0.05 using the R package DESeq2 (v1.38.2) [39]. To identify the robustly-regulated genes, expression variance was defined by the absolute value of the difference in normalized gene expression (FPKM) between the two groups. To identify the rescued effects of LRRK2-IN-1 in gene expression, the up- or down-regulated genes were sorted in descending order of expression variance values in the comparisons between the IL-1β and VEH groups or between the W-Exo-L@GelMA/L-1β and IL-1β groups and normalized rescue scores were defined by the estimates using the R package RobustRankAggreg (v1.1) [40] to define the polarized genes in both comparisons. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the R package clusterProfiler (v3.11) [41]. Data visualizations were conducted by R package pheatmap (v1.0.12) and ggplot2 (v 3.4.0) [42, 43].

RT-qPCR

Trizol reagent (Takara, Japan) was used to extract RNA from cultured cells and then 1 µg total RNA was converted to cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China). RT-qPCR was performed using primers and templates mixed with the Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific, USA) via a CFX Connect RT-qPCR detection system (Bio-Rad, USA). The mRNA levels of genes were normalized to Gapdh mRNA in the same sample, and the relative expression of the genes of interest was determined using the formula of Livak and Schmittgen [44]. The primers used for RT-qPCR in this study are listed in Table 1.

Table 1 The primers used for RT-qPCR in this study

Animal study

The animal study was conducted following the instructions and approval of the Ethics and Animal Research Committee of Huazhong University of Science and Technology. 120 C57BL/6J mice (8-week-old, male) were supplied by the Experimental Animal Centre of Tongji Hospital and fed in the SPF animal laboratory. The mice were randomly assigned into six groups (Sham, DMM, DMM + L@GelMA, DMM + Exo@GelMA, DMM + Exo-L@GelMA, and DMM + W-Exo-L@GelMA). After inhalation-induced anesthesia with isoflurane, the OA model was established by the surgical destabilization of the medial meniscus (DMM) and the Sham group underwent an opening of the joint cavity and excision of only the anterior fat pad. The concentration of LRRK2-in-1 interfering with mouse chondrocytes is 5 µM in vitro, the drug concentration usually injected into mouse joints is 10–30 times than that of in vitro cell experiments, i.e. 50–150 µM, equivalent to 28.535–85.605 µg/mL. According to the sustained release curve of the drug, the cumulative release of the drug (800 µg/mL) within 15 days is 84% (672 µg/mL), an average of 45 µg/mL per day, which is always at the effective concentration. When the total amount of drug (10 µL×800 µg/mL = 8 µg) were determine based on the encapsulation efficiency of exosomes into GelMA (Additional file 1:  Table S4), we could calculate that the total amount of microspheres coated with drug exosomes (47.84 µg). Therefore, on the third day after the surgery, 10 µL L@GelMA (800 µg/mL LRRK2-IN-1), Exo@GelMA, Exo-L@GelMA (800 µg/mL LRRK2-IN-1), or W-Exo-L@GelMA (800 µg/mL LRRK2-IN-1) were injected into the joint cavity, respectively, every two weeks for 8 weeks. Simultaneously, mice in the Sham and DMM groups received 10 µL saline solution. After completing the intra-articular injection course, all the mice were sacrificed and the right knee joints were collected for further evaluation.

Micro-computed tomography scanning

Knee joints were fixed with 4% paraformaldehyde before scanning. Scanning was conducted using Scanco vivaCT 40 micro-computed tomography (µCT) instrument (Scanco Medical, Switzerland). Parameters were set for calcified tissue visualization as below: 100 kV of source voltage, 98 µA of current, 10 μm voxel size, and 300 ms integration time. Subchondral bone parameters of the tibias including trabecular bone volume fraction (BV/TV), thickness (Tb. Th), spacing (Tb. Sp), and number (Tb. N) were measured as previously described [45].

Histological staining and immunohistochemistry analysis

After the fixation in 4% paraformaldehyde for 24 h, the knee joints were decalcified in 10% ethylenediaminetetraacetic acid for 30 days, embedded in paraffin, and sliced into 5 μm-thick sections for further tissue staining and immunohistological analysis. Safranin O/fast green staining was used for the histological analysis of cartilage lesions. The degree of cartilage damage was assessed according to the Osteoarthritis Research Society International (OARSI) scoring guidelines [45]. Moreover, after the sections were subjected to deparaffinization and blocked with 5% bovine serum albumin for 1 h, they were incubated with the primary antibodies against anabolic factor aggrecan (13880-1-AP, 1:200) and catabolic factor MMP13 (18165-1-AP, 1:200) at 4 °C overnight. The sections were incubated with secondary antibodies, developed, and observed under a microscope. The human OA cartilage samples were subjected to similar protocols of immunohistochemistry analyses to analyze the expression of aggrecan and MMP13.

Human cartilage treatment

Human cartilage harvest and treatment were approved by the Human Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) (approval number: TJ-IRB20220947). Three OA patients (mean age, 69 years; range, 61–75 years; male: 2, female: 1) without other systemic diseases were included, who accepted total knee arthroplasty (TKA) at the Department of Orthopedics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology in 2022. Written informed consent of the human tissue harvest was acquired from each patient. Briefly, the medial femoral condyles were isolated during surgery and transformed on ice to the orthopedics laboratory.

The soft tissue clearance in the cartilage surface was completely removed after being washed three times using cold PBS. The cartilage explants sized in 5 × 5 × 5 mm3 were excised using a surgical saw and cultured in an incubator containing 5% CO2 at 37 °C with DMEM/F12 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and human IL-1β (10 ng/mL). The cartilage explants from the same patient were treated with or without W-Exo-L@GelMA loaded with 5 µM LRRK2-IN-1 for 72 h, fixed in 4% paraformaldehyde at 4 °C for 24 h, and decalcified in 10% EDTA for 30 days for immunohistochemistry analysis.

Statistical analysis

The experimental data were analyzed using GraphPad Prism v.8.4.0 software (GraphPad, USA). The results are shown as the mean ± SD. The differences between any two groups were determined by the student’s t-test. One-way analysis of variance (ANOVA) followed by Tukey’s test was used to compare differences among two or more groups. A P < 0.05 was recognized as significant. All data were repeated independently at least three times.