RGD-Functionalized Melanin Nanoparticles for Intraoperative Photoacoustic Imaging-Guided Breast Cancer Surgery


 Purpose: Obtaining tumour-free margins is critical for avoiding re-excision and 35 reducing local recurrence following breast-conserving surgery (BCS); however, it 36 remains challenging. Imaging-guided surgery provides precise detection of residual 37 lesions and assists surgical resection. Herein, we describe water-soluble melanin 38 nanoparticles (MNPs) conjugated with cyclic Arg-Gly-Asp (cRGD) peptides for breast 39 cancer photoacoustic imaging (PAI) and surgery navigation. 40Methods: cRGD-MNPs was synthesized and characterized for morphology, 41 photoacoustic characteristics and stability. Tumour targeting and toxicity were 42 determined by cells and tumour-bearing mice. PAI was used to locate the tumour and 43 guide surgical resection in MDA-MB-231 tumour-bearing mice. 44Results: The cRGD-MNPs exhibited excellent tumour-targeting in vitro and in vivo, 45 with low toxicity. Intravenous administration of cRGD-MNPs to MDA-MB-231 46 tumour-bearing mice showed an approximately 2.1-fold enhancement in photoacoustic 47 (PA) intensity at 2 h, and the ratio of the PA intensity at the tumour site compared to 48 that in the surrounding normal tissue was 3.2 ± 0.1, which was much higher than that 49 using MNPs alone (1.7 ± 0.3). Similarly, the PA signal in the mammary glands 50 containing spontaneous breast cancer was enhanced (2.5 ± 0.3-fold) in MMTV-PyVT 51 transgenic murine model. Preoperative screening by PAI could assess tumour volume 52 and offer a three-dimensional (3D) reconstruction image for accurate surgical planning. 53 Surgical resection following real-time PAI on the tumour bed showed high consistency 54 with histopathological analysis. 55Conclusion: These results highlight that cRGD-MNPs combined with PAI provide 56 a powerful tool for breast cancer imaging and precise tumour resection. cRGD-MNPs 57 with good PA properties have great potential for clinical translation.

Recently, photoacoustic imaging (PAI) has been developed as a novel imaging 89 technology for biomedical applications. PA I detects optical absorption contrast 90 acoustically via the photoacoustic (PA) effect, a physical phenomenon that converts absorbed optical energy into acoustic energy [11]. Based on endogenous contrast 92 molecules (e.g., oxyhaemoglobin, deoxyhaemoglobin, lipid, or DNA-RNA), PAI has 93 been used in the clinic trial to demonstrate its highly desirable capabilities for breast 94 cancer imaging in vivo and ex vivo, particularly for assessing tumour margins 95 macroscopically and microscopically [12][13][14]. However, intrinsic chromophores 96 provide access to only a limited range of biological processes but low tumour-imaging 97 contrast. Hence, molecular PAI for breast cancer still requires a targeted contrast agent 98 that can selectively bind to surface receptors on cancer cells or respond to the tumour 99 microenvironment [11,15].

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Natural melanin is a group of biopigments with multifunctionality (i.e., ultraviolet 101 protection, radical scavenging, and photothermal conversion) [16]. Due to its good 102 intrinsic biocompatibility, natural melanin or synthetic melanin-like nanomaterials have 103 been successfully developed as novel nano-bioplatforms in bioimaging, therapy, 104 theranostics, and biosensing [17,18]. As an endogenous PA contrasting agent, melanin 105 was used to detect the metastatic status of ex vivo human melanoma sentinel lymph 106 nodes by multispectral optoacoustic imaging [19]. The results showed an excellent 107 correlation with the histological assessment of melanoma cell infiltration with 100% 108 sensitivity and 62% specificity [19].

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The cRGD-MNPs were obtained as previously described [20]. Briefly, melanin (2 144 mg/mL) was dissolved in NaOH aqueous solution (0.1 N) followed by the rapid 145 addition of HCl aqueous solution (0.1 N) to a pH of 7.0 under sonication. The 146 neutralised solution was centrifuged and washed with deionized water several times.

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The black solid of MNPs was obtained by freeze-drying. The MNPs aqueous solution

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After vigorous stirring for 12 h, the PEG-modified MNPs were retrieved by 150 centrifugation and washed several times to remove the unreacted NH2-PEG5000-NH2. MNPs or cRGD-MNPs aqueous solution was dropped onto a carbon-coated copper grid 162 and air-dried. The 1 H NMR spectra were recorded at 20°C on a 400 MHz NMR 163 spectrometer (Bruker) using D2O as the solvent. Zeta potentials were measured using a 164 laser particle size analyser system (Malvern, Zetasizer Nano ZS90). The absorption 165 spectra were obtained using a Multiskan Spectrum Microplate Spectrophotometer 166 (Thermo, USA).

PAI system
168 Both PA and US images were recorded using the Vevo LAZR-X photoacoustic 169 imaging system (VisualSonics, FujiFilm, Japan). A tunable laser (680- MHz was used to obtain single-plane, full-spectrum and 3D PA images. The 3D 172 scanning was controlled using an electric motor with a step length of 0.14 mm. The     signals. Data are presented as the mean ± standard error of the mean. Significant 293 differences between or among the groups are indicated as follows: ns for no significant 294 difference, * for P < 0.05, ** for P < 0.01, and *** for P < 0.001.

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The water-soluble MNPs were approximately 4 nm in size based on transmission 299 electron microscopy (Fig. 1a). Polyethylene glycol (PEG) chains were introduced into 300 the MNPs for further biomodification, which was confirmed by 1 H NMR spectra (Fig.   301 S1a). This can reduce the liver accumulation because of the enhanced water solubility.  This phenomenon could be blocked by excessive unlabelled RGD peptide (Fig. 2e).

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Finally, analysis of cell viability suggested low cytotoxicity for the MNPs, even at a 339 high concentration up to 10 μM (Fig. 2f). MNPs into tumour-bearing mice using MNPs as a control) (Fig. 3a, b). We showed that 350 tumour sites exhibited a higher PA signal 1 to 2 h after intravenous administration of 351 cRGD-MNPs than MNPs in vivo. Moreover, the cRGD-MNPs group had a significantly 352 higher signal-to-background ratio (tumour versus surrounding normal tissue) than the MNPs group two hours post-injection (3.2 ± 0.1 versus 1.7 ± 0.3, P < 0.05) (Fig. 3c).

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In addition, the PA signal increased approximately 2.1-fold two hours post-injection 355 compared to pre-injection (Fig. S2b). These results indicated that cRGD-MNPs 356 accumulated more in tumours than normal tissue and provided clearer tumour contrast. 357 We also used fluorescence imaging to track the biodistribution of Rho-cRGD-MNPs 358 and Rho-MNPs two hours post-injection (Fig. 3d). The ex vivo fluorescence images and 359 intensities of the major organs and tumours suggested more effective accumulation of 360 Rho-cRGD-MNPs into tumour tissue than Rho-MNPs (Fig. 3d, e).  (Fig. 4a). Compared to the imaging signal before administration, the PA signal 366 intensities of the mammary glands containing tumour increased 2.5 ± 0.3-fold (Fig.   367 4b). In contrast, the PA signal intensities of the normal mammary glands did not 368 increase post-injection (0.9 ± 0.1-fold, Fig. 4b). The pathological status of the 369 mammary glands was confirmed by H&E staining (Fig. 4c). Furthermore, melanin 370 staining confirmed the presence of the MNPs in the tumour tissue after injection of the 371 probe (Fig. 4d). These results indicate that cRGD-MNPs provide a high signal intensity 372 at the tumour site and could distinguish between normal mammary glands and breast 373 tumours.

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In order to further assess the feasibility of using the cRGD-MNPs to detect a tumour, 375 segmented tumour PAI was performed in MMTV-PyVT transgenic mice with breast 376 cancer (Fig. 4e). The fourth and fifth mammary glands of the transgenic mice were 377 divided into four segments (P1-4), and PA imaging showed complete and intense 378 enhancement of each part of the tumour. Ex vivo tissues were imaged using the PAI 379 system, and the tissue signal distribution was consistent with that observed in vivo and 380 correlated with the pathological examination (Fig. S3a). The PA images demonstrated 381 an improved contrast profile for breast cancer detection with the cRGD-MNPs. 383 We evaluated cRGD-MNPs-based PAI for tumour detection, delineation, and vivo tissue PA signal distribution was consistent with that obtained in vivo (Fig. S3b).

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Pathological examination showed that the tumour had been completely removed, and 402 the tissue with a negative PA signal on the tumour bed was muscle tissue (Fig. 5c). To 403 detect the depth of PAI, we covered the tumours with chicken breasts of various 404 thicknesses and found that the maximum imaging depth was up to 5 mm (Fig. 5d).

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Taken together, PAI can effectively detect tumours, identify residual masses, and guide 406 surgical resection.

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In vivo cRGD-MNPs biosafety 408 The in vivo biocompatibility was further evaluated in mice for prolonged durations 409 (1, 7 or 30 days) after intravenous administration of cRGD-MNPs. Saline served as a 410 negative control. No considerable body weight loss was observed for the cRGD-MNPs 411 and control groups over 30 days, indicating that cRGD-MNPs had no significant side 412 effects in mice (Fig. 6a). The serum biochemistry analysis, which included liver 413 function (ALT, AST) and kidney function (BUN, CR) markers, showed negligible 414 variations between the different groups, indicating no detectable toxicity shortly after 415 or a relatively long time after exposure (Fig. 6b). Although cRGD-MNPs also 416 accumulated in the liver and kidneys, negligible liver and kidney damage was induced.  clinical translation of these agents is prohibitive because of biosafety issues, poor 476 biodegradability, low photostability and unclear biocompatibility [37,38]. In the 477 present study, the biocompatibility and biosafety of cRGD-MNPs were systematically 478 evaluated both in vitro and in vivo. As reported previously, the strengths of melanin-479 like nanomaterials include good biocompatibility and long-term photostability [39,40], 480 prompting us to explore their biomedical applications, particularly for in vivo imaging.

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Our findings demonstrated that cRGD-MNPs represent a promising contrast agent for 482 further clinical translation. 483 However, this study has a few limitations. First, only PAI alone was applied to 484 surgical navigation. As a previous study reported [20], MNPs are an active platform to 485 simplify the assembling of different imaging moieties, such as positron emission 486 tomography and magnetic resonance imaging. Thus, complementary use of 487 multimodality imaging is promising not only for accurate tumour imaging but also for 488 guiding tumour resection. Further research efforts should be devoted to precise, targeted 489 tumour multimodality imaging. Second, the current methodology using MNPs as a 490 contrast agent is unsuitable for deep-tissue imaging in the human body. Therefore, 491 further improvements of the imaging agents are needed to increase the imaging depth.