Toluene, chloroform, tetrahydrofuran (THF), acetone and sucrose were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China). Olive oil (O108686) was provided by Aladdin (Shanghai, China). 2,5-Bis(2-octyldodecyl)-3,6-bis(5-(trimethylstannyl)thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione (DPP) and 4,8-dibromo-6-(2-ethylhexyl)-[1,2,5]thiadiazolo[3,4-f]benzotriazole (TBZ) were purchased from Derthon Optoelectronic Materials Science Technology Co., Ltd. (Guangdong, China). Tris(dibenzylideneacetone)dipalladium(0), tri(o-tolyl)phosphine and DNFB (D1529) were supplied from Merck Millipore (Darmstadt, Germany). DSPE-PEG2000, (molecular weight: 2805.5) and DSPE-PEG2000-MAL were purchased from Xi’an ruixi Biological Technology Co., Ltd. (Xi’an, China). Transactivator of transcription (TAT) peptide (296229) was purchased from GL Biochem, Ltd. (Shanghai, China). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS, 10099141), 0.25% trypsin-ethylenediaminetetraacetic acid (25200056), TRIzol™ Reagent and RevertAid First Strand cDNA Synthesis Kit (K1622) were purchased from Invitrogen (Carlsbad, CA, USA). Lenti-Pac HIV Expression Packaging Kit (LT001) and polybrene (LT010-S) were purchased from IGeneBio (Guangzhou, China). Radioimmunoprecipitation (RIPA) lysis buffer (P0013B) was purchased from Beyotime Biotech Inc. (Shanghai, China). Bovine serum albumin (BSA, A8020) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Transfer membrane, goat polyclonal anti-rabbit horseradish peroxidase (HRP) conjugated antibody (ab205718), rabbit polyclonal antibody against GFP (ab290), 3,3′-Diaminobenzidine (DAB) substrate kit (ab64238) were purchased from Abcam (Cambridge, MA, USA). Tris buffered saline with tween (TBST), PBS, crystal violet, H&E staining kit (G1005) and 4% paraformaldehyde solution (PFA, G1101) were purchased from Wuhan Servicebio Technology Co., Ltd. (Wuhan, China). Rabbit polyclonal antibody against Cxcr3 (NB100-56404), recombinant Cxcl10 protein (466-CR) were purchased from R&D systems (Minneapolis, MN, USA). Meilunbio® fg super sensitive electrochemiluminescence luminescence reagent (MA0186) was purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China). CCK-8 reagent (KGA317) was purchased from KeyGen Biotech Co., Ltd. (Nanjing, China). TB Green Premix Ex Taq II (Tli RNase H Plus) (RR820A) was purchased from Takara Bio Inc. (Otsu, Japan). All other chemicals were used as received.

Preparation of TAT-CPNPs

Conjugated polymer PTD was synthesized by alternative copolymerization between TBZ and thiophene-substituted DPP monomers via Stille coupling reaction. For imaging in vitro and in vivo, PTD molecules were processed into water dispersible conjugated polymer nanoparticles (CPNPs) via a nanoprecipitation method. The process procedure is described briefly as follows. PTD (1 mg), DSPE-PEG2000-MAL (1 mg) and DSPE-PEG2000 (1 mg) were dissolved in THF (2 mL) and dispersed in DW under ultrasonification (Ultrasonic Homogenizer SCIENTZ-IID, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China). After THF evaporation, the mixture was filtered through 450-nm-pore polyether sulfone filters. To prepare TAT-CPNPs, TAT peptide (3.3 mg) was added to the filtered mixture and stirred for 12 h. The mixture was dialyzed against DW for 24 h and concentrated to about 1 mg/mL with Amicon® Ultra Centrifugal Filters (UFC9010, Merck Millipore, Germany).

Characterization of TAT-CPNPs

The size distribution of TAT-CPNPs was determined by dynamic light scattering with Zetasizer Nano ZS system (Malvern Instruments, Malvern, UK). Transmission electron microscopy (TEM) images of TAT-CPNPs were taken with FEI TECNAI G2 20 transmission electron microscope (FEI, Hillsboro, USA) at 200 kV. Ultraviolet–visible-near infrared spectra of TAT-CPNPs and absorbance of TAT was measured with an ultraviolet–visible-near infrared spectrophotometer (PerkinElmer Inc., Waltham, USA). PA signal intensities of TAT-CPNPs at 10, 20, 100, 200, 2000 µg/mL and PA signal intensity of MSCs cultured with CPNPs and TAT-CPNPs for different time (1 h, 16 h, 24 h) were measured with a custom-built PACT at 10 mJ/cm2 as previously described in literature. Photostability was assessed by quantification of PA signal intensity of TAT-CPNPs during continuous 2000 pulses of 1064 nm laser irradiation.

Quantification of TAT linked to CPNPs

To quantify the TAT being linked to CPNPs, the free TAT concentration in CPNPs suspension was measured, and the linked amount of TAT was calculated according to the free TAT concentration. The free TAT concentration in CPNPs suspension was measured as follows. After preparing CPNPs with PTD (0.36 mg), DSPE-PEG2000-MAL (0.36 mg) and DSPE-PEG2000 (0.36 mg), TAT (1.19 mg) was added to the CPNPs. After stirring for 12 h, the mixture was collected and freeze-dried. 50 µL of DW was added to freeze-dried TAT-CPNPs to obtain the aqueous solution containing free TAT. Using bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China), the free TAT in the solution was estimated to be 1.04 mg based on the absorbance-TAT concentration linear fit equation in Additional file 1: Fig. S1. The TAT linked to CPNPs was calculated to be 0.16 mg (0.05 µmol). Based on the 0.36 mg of DSPE-PEG2000-MAL at the feed, the available maleimide was 0.12 µmol. Therefore, 45% of maleimide on the surface of TAT-CPNPs was linked to TAT [28].

Lentiviral vectors and lentivirus production

Entry vectors were generated according to previous literature. The lentiviral expression vector with the coding sequence of Cxcr3 (pLV/puro-EF1a-CXCR3-IRES-EGFP) was generated using a lentiviral vector that expressed eGFP (pLV/ puro-EF1a-EGFP).

For lentivirus production, HEK293T cells were transiently cotransfected with lentiviral expression vector (pLV/puro-EF1a-EGFP or pLV/puro-EF1a-CXCR3-IRES-EGFP) together with packaging plasmids from the Lenti-Pac HIV Expression Packaging Kit. The supernatant was collected 36 h and 72 h after transfection and then filtered through 0.45-µm-pore polyether sulfone filters. The lentiviral particles were concentrated by ultracentrifugation (2 h at 50,000×g) and resuspended in serum-free medium (SFM).

Cell culture

MSCs were isolated from human bone marrow samples obtained from healthy human donors with informed consent as described in previous literature [8]. MSCs were routinely cultured in DMEM complete medium (CM) containing 1 g/L glucose and 10% FBS. HEK293T were routinely cultured in DMEM CM containing 4.5 g/L glucose and 10% FBS. Cells were cultured at 37 ℃ and 5% carbon dioxide (CO2) in a CO2 incubator.

MSCs stably overexpressing Cxcr3 (MSCCxcr3), and its matched control MSCeGFP were established using lentiviral transduction. After reaching a confluence of approximately 60%, MSCs were cultured in CM containing lentivirus and 8 µg/mL polybrene for 12 h. The eGFP-expressing cells were observed under a fluorescence microscope (Olympus IX73 microscope, Olympus, Inc., Hamburg, Germany) or subjected to flow cytometry (CytoFLEX, Beckman Coulter, Inc., CA, USA) to assess the transduction efficiency.

In vitro cytotoxicity assay

MSCs were resuspended in CM at 1.5 × 105 cells/mL. In a 96-well plate, 100 µL of cell suspension was added to each well. After incubation at 37 ℃ and 5% CO2 for 24 h, MSCs were cultured with serum-free DMEM medium containing CPNPs or TAT-CPNPs at different concentrations (0, 3.25, 7.5, 15, 30, 60, 120 µg/mL). After 12 h, cell viability was measured with CCK-8 assay according to the manufacturer’s protocol.


BALB/c male mice (6 weeks) and DBA/2J male mice (8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal studies were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.

In vivo toxicity assay

100 µL of PBS containing TAT-CPNPs at 0 or 500 µg/mL was injected intravenously into mice. After 14 days, major organs (heart, liver, spleen, lungs, and kidneys) were collected from the treated mice for hematoxylin and eosin (H&E) staining. Serum was collected for biochemistry analysis with a Chemray-240 automated chemistry analyzer (Rayto Life and Analytical Sciences Co., Ltd., Guangdong, China). Parameters including AST, ALT, TBIL, Cr and BUN for liver and kidney function assessment were analyzed.

Establishment of the CHS model and ear thickness measurement

The CHS model was established as described in literature [40]. Acetone and olive oil at a ratio of 4:1 was prepared to dilute DNFB. 0.5% DNFB was applied to shaved mice back for sensitization. After 5 days, 0.2% DNFB was applied to the right ears in inflamed group, vehicle was applied to left ears in control group. The ear thickness of both ears was measured before and 24 h and 72 h after 0.2% DNFB or vehicle application.

Analysis of gene expression

Gene expression was analyzed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting.

For mRNA analysis, total RNA was extracted with TRIzol™ Reagent according to manufacturer’s protocol. Reverse transcription was carried out with a RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s protocol. qRT-PCR was carried out with TB Green Premix Ex Taq II (Tli RNase H Plus) using qTower 3 (Analytik Jena AG, Thuringia, Germany). qRT-PCR was conducted in triplicate for each sample. Primers for qRT-PCR were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and are listed as follows. Mouse Cxcr3, forward, 5′-TCAGCCAACTACGATCAGCG-3′; reverse, 5′-TAGCTGCAGTACACGCAGAG-3′. Human GAPDH, forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′; reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′. Mouse Cxcl10, forward, 5′-CCAAGTGCTGCCGTCATTTTC-3′; reverse, 5′-GGCTCGCAGGGATGATTTCAA-3′. Mouse GAPDH, forward, 5′-AGGTCGGTGTGAACGGATTTG-3′; reverse, 5′-TGTAGACCATGTAGTTGAGGTCA-3′.

For western blotting, MSCs were lysed with RIPA lysis buffer. Proteins were separated with sodium dodecyl-sulfate polyacrylamide gel electrophoresis and detected with specific antibodies. Blots were detected with the Meilunbio® fg supersensitive electrochemiluminescence luminescence reagent in a BLT GelView 6000 Pro (Biolight Biotechnology Co., Ltd., Guangzhou, China) system.

Transwell migration assay

Migration was assessed with a 24-well transwell chamber system (353097, Corning Costar, Cambridge, MA, USA) with 8 µm pores. To evaluate the chemotactic response to Cxcl10, MSCeGFP and MSCCxcr3 were resuspended in serum-free DMEM medium at 1 × 105 cells/mL. Then, 150 µL of cell suspension was added to each upper chamber insert. The lower chamber contained CM with or without murine Cxcl10 (100 ng/mL). To investigate the effect of TAT-CPNPs on chemotactic migration, MSCCxcr3 were resuspended in serum-free DMEM medium containing 15 µg/mL of TAT-CPNPs or an equivalent amount of vehicle (PBS) at 1 × 105 cells/mL, and 200 µL of cell suspension was added to each upper chamber insert. The lower chamber contained CM with murine Cxcl10 (100 ng/mL). For both experiments, cells were allowed to migrate to the lower chamber at 37 ℃ and 5% CO2 for 24 h. After that, the insert was fixed with ice-cold methanol, the upper surface was scraped clean and the lower surface was stained with 0.1% crystal violet. Images of migrated cells were captured with an Olympus IX73 microscope (Olympus, Inc., Hamburg, Germany). The number of migrated cells in each chamber was determined from images of 5 nonrepeating random fields.

Labeling of MSCs

When MSCCxcr3 reached a confluence of 70%, cells were cultured with SFM containing 15 µg/mL of TAT-CPNPs for 16 h. To compare the uptake of CPNPs and TAT-CPNPs, MSCs were cultured with SFM containing 15 µg/mL of CPNPs or TAT-CPNPs for different amount of time (1 h, 16 h or 24 h). After washing with PBS for 3 times, cells were trypsinized either for subsequent PA imaging or subculture.

PA imaging of MSCs in vitro

After incubating with TAT-CPNPs, the culture plate was washed with PBS and the culture medium was changed to normal complete medium. Labeled-MSCCxcr3 were cultured and passaged as usual for 0 day, 10 days and 20 days, respectively. Then, MSCCxcr3 resuspended in PBS at 2 × 106 cells/mL were loaded into a tube phantom and PA imaging was performed with the PACT system. The amounts of NPs initially internalized by MSCs and remained in cells after different time were quantified based on the PA amplitude-concentration linear fit equation in Fig. 2f.

To compare the penetration performance of PA imaging at first and second near-infrared windows, the cell-containing tube phantoms were placed underneath approximately 5 mm, 8 mm or 12 mm of chicken breast tissue before PA imaging. Both ultrasonic and PA signals were coupled and detected by a 15 MHz transducer. PA imaging was performed at 10 mJ/cm2 under 800 nm or 1064 nm laser excitation [41].

In vivo PA imaging of CHS model

To investigate the recruitment of MSCs to inflamed ears in vivo, a custom-built OR-PAM system was employed under 532 nm and 1064 nm laser excitation. 24 h after application of 0.2% DNFB, CHS mice were randomly assigned to 2 groups. Mice were intravenously injected with TAT-CPNPs labeled MSCeGFP (2 × 106 cells, n = 3) and TAT-CPNPs labeled MSCCxcr3 (2 × 106 cells, n = 3), respectively. Imaging of the inflamed ears of another 3 mice injected with PBS, unlabeled MSCeGFP or unlabeled MSCCxcr3 group and imaging of the non-inflamed ear of another mouse injected with labeled MSCCxcr3 were included as the control group. Each mouse was anesthetized with 2% isoflurane in oxygen and then sedated in prone position. During imaging, the mouse body temperature was maintained at 37 °C using a temperature-controlled heating pad (RWD Life Science, Shenzhen, China). PA images of inflamed ears were captured at a wavelength of 532 nm first to reveal the vasculature. Then, PA images at a wavelength of 1064 nm were captured before, 15 min, 3 h and 7 h after injection of MSCeGFP or MSCCxcr3. During the experiment, the fluence of each PA imaging on tissue surface of each mouse was kept at approximately 15 mJ/cm2 at a wavelength of 532 nm and 64 mJ/cm2 at a wavelength of 1064 nm by monitoring using a power sensor (S121C, Thorlabs, New Jersey, USA). Fluences applied at both wavelengths were well below the maximum permissible exposure standard (20 mJ/cm2 at a wavelength of 532 nm and 100 mJ/cm2 at a wavelength of 1064 nm) allowed by the American National Standard Institute (ANSI Z136. 1-2014: American National Standard for Safe Use of Lasers).

The raw 3D volume data for each animal at each time point acquired by PA imaging were processed using Matlab (R2021a) to show the maximum amplitude projection (MAP) image. The detailed imaging processing algorithm was according to previous literature from our group. For each ear, images captured at a wavelength of 1064 nm were aligned to image captured at 532 nm using the TurboReg plugin in Image J (Software version 1.8.0_112) before further processing. After alignment, the PA signal of TAT-CPNPs labeled MSCs was extracted by subtracting the pre- from postinjection PA images under 1064 nm using Matlab (R2021a). Finally, the subtracted PA image of each time point was merged with image captured at 532 nm to reveal the position of MSCs relative to blood vessels. Quantification analysis of PA images captured in vivo was performed using Image J. Layer-by-layer images and xz projected images were processed using Matlab and analyzed using Image J.

Establishment and in vivo PA imaging of RA mouse model

The RA mouse model was established following the booster immunization protocol from Chondrex, Inc,.(CA, USA). Briefly, an emulsion of collagen and complete Freund’s adjuvant with a final concentration of 0.5 mg/mL of M. tuberculosis was prepared with a homogenizer (T 10 basic ULTRA-TURRAX, IKA, Staufen, Germany). 100 µl of emulsion was subcutaneously injected into the base of the tail of DBA/2J mice. 21 days later, a booster injection was performed with an emulsion of collagen and Incomplete Freund’s Adjuvant. Arthritis was scored following the scoring system reported by D. Brand [42]. Approximately 15 days after the booster immunization, in vivo PA/US imaging was performed on the hindlimb with a severity score of 3–4 using the PACT system. Briefly, mice were sedated by continuous isoflurane inhalation. PA/US imaging was performed before and 15 min, 2 h and 4 h after injection of TAT-CPNPs labeled MSCeGFP or MSCCxcr3 under 1064 nm laser excitation. B-scan images of PA imaging were processed by subtracting the preinjection images from the postinjection images using Matlab. Subtraction images of PA imaging were overlaid with US images of related postinjection time points.

Histological staining

IHC staining was performed to detect MSCeGFP and MSCCxcr3 in inflamed ears. After PA imaging, the ears were collected, fixed in 4% PFA for 12 h and dehydrated in 30% sucrose for 72 h to prepare cryosections. Sections were blocked with goat serum for 1 h at room temperature (RT) and then incubated with rabbit anti-GFP antibody (1:500 dilution) at 4 ℃ overnight, followed by incubation with goat anti-rabbit HRP conjugated antibody (0.1 µg/mL) for 1 h at RT. After washing with PBS, DAB was applied and sections were counterstained with hematoxylin. Photographs were captured with a KEYENCE BZ-X800 Microscope (Keyence Corporation, Osaka, Japan). Quantification of GFP positive cells in each mouse ear was from measurement of 5 non-repeating random fields.

Statistical analysis

Data are expressed as means ± SEM and were analyzed by t tests and one-way analysis of variance (ANOVA) with Tukey’s posttest using Prism 7 software (GraphPad Software, Inc., CA, USA). Values of P < 0.05 were considered statistically significant.

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