Copper sulfide perfluorocarbon nanodroplets as clinically relevant

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Copper sulfide perfluorocarbon nanodroplets as clinically relevant photoacoustic/ultrasound imaging agents Daniela Y Santiesteban, Diego S Dumani, Daniel Profili, and Stanislav Y Emelianov Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b02105 • Publication Date (Web): 19 Sep 2017 Downloaded from http://pubs.acs.org on September 19, 2017

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Copper sulfide perfluorocarbon nanodroplets as clinically relevant photoacoustic/ultrasound imaging agents

Daniela Y. Santiesteban, Diego S. Dumani, Daniel Profili, Stanislav Y. Emelianov* 1

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology

and Emory University School of Medicine, Atlanta, GA, 30332, USA. 2

School of Electrical and Computer Engineering, Georgia Institute of Technology, Atlanta, GA

30332, USA.

*Correspondence to: [email protected]

ACS Paragon Plus Environment

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Abstract:

We have developed laser-activated perfluorocarbon nanodroplets containing copper sulfide nanoparticles (CuS NPs) for contrast-enhanced ultrasound and photoacoustic imaging. As potential clinical contrast agents, CuS NPs have favorable properties including biocompatibility, biodegradability and enhance contrast in photoacoustic images at clinically relevant depths. However, CuS NPs are not efficient optical absorbers when compared to plasmonic nanoparticles and therefore, contrast enhancement with CuS NPs is limited, requiring high concentrations to generate images with sufficient signal-to-noise ratio. We have combined CuS NPs with laser-activated perfluorocarbon nanodroplets (PFCnDs) to achieve enhanced photoacoustic contrast and, more importantly, ultrasound contrast while retaining the favorable clinical characteristics of CuS NPs. The imaging characteristics of synthesized CuS-PFCnD constructs were first tested in tissue mimicking phantoms and then in in vivo murine models. The results demonstrate that CuS-PFCnDs enhance contrast in photoacoustic (PA) and ultrasound (US) imaging. Upon systemic administration in vivo, CuS-PFCnDs remain stable and their unique vaporization provides sufficient PA/US contrast that can be further exploited for contrast-


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enhanced background-free imaging. The conducted studies provide a solid foundation for further development of CuS-PFCnDs as PA/US diagnostic and eventually therapeutic agents for clinical applications.

Keywords:

Perfluorocarbon

nanodroplets,

copper

sulfide

nanoparticles,

ultrasound,

photoacoustic, imaging, contrast


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Introduction: Despite intense research efforts by the biomedical community, clinical translation of nanoparticles (NPs), particularly for diagnostic purposes, has been limited.1 NPs can be readily classified into two groups: organic (i.e. liposomes, micelles, polymeric complexes, etc.) or inorganic (i.e. metallic, semiconductor, silica, etc.). Each group has its inherent strengths and weaknesses. Organic NPs are great therapeutic carriers, biodegradable and effectively cleared, but are typically incapable of providing image contrast on their own. Thus, they are poor diagnostic imaging agents. On the other hand, the majority of inorganic NPs are superior contrast agents, but struggle as efficacious therapeutics. Additionally, most inorganic NPs have limited biodegradability and clearance, which raises concerns over their clinical potential due to questionable long-term toxicity and bioeffects.2 In the quest for ideal diagnostic and therapeutic NPs, researchers have turned to combinations of NPs.3-4 However, while combining NPs may augment the strengths of individual particles, they are often unable to overcome inherent barriers. For instance, combinations of particles may result in increased contrast, but at the expense of biocompatibility or limited clearance. We have engineered a perfluorocarbon nanodroplet (PFCnD) that synergistically combines organic (micelle) and inorganic (copper sulfide nanoparticles, CuS NPs) components for enhanced image contrast, while possessing high clinical translation potential. Our developed CuS-PFCnDs merge the increased photoacoustic and ultrasound contrast enhancement of PFCnDs with the favorable biocharacteristics of CuS NPs. Additionally, we describe an image processing algorithm which takes advantage of CuS-PFCnD’s acoustic and optical properties


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that are drastically different from the tissue and, therefore, allows for background-free ultrasound and photoacoustic images, ideal for diagnostic applications. PFCnDs have a long history in medical applications and are becoming increasingly studied contrast agents for various modalities.5-9 Most commonly, PFCnDs are used in ultrasound (US) imaging, which is one of the most widely used diagnostic imaging modalities due to its low-cost, portability, and ability to safely provide real-time structural information with sub-millimeter spatial resolutions.10-11 PFCnDs are the sub-micrometer, liquid version of the ubiquitously used ultrasound contrast agent, PFC microbubbles (MBs). Compared to MBs, PFCnDs offer several advantages including increased circulation lifetimes and the ability to extravasate hyperpermeable vasculature for directly diagnosing and treating diseased tissue.12 Furthermore, PFCnDs can be phase-changed into MBs, via acoustic or laser irradiation, thus acting as enhanced ultrasound contrast agents. Of the two PFCnD subsets, those that are lasertriggered are advantageous for several reasons.13-14 Laser-triggered PFCnDs are safe, the activation event is well-controlled, and they are more comprehensive imaging agents because they supplement ultrasound contrast with photoacoustic (PA) contrast.6 Photoacoustic imaging is a novel imaging technique that works synergistically with ultrasonography and can provide molecularly specific contrast at the spatial resolution of ultrasound imaging.15 The PFCnD vaporization event generates an intense PA signal, which contains information that would likely be lost to interference with the ambient high intensity ultrasound field if PFCnDs were acoustically triggered. Once properly processed, the unique vaporization event of laser-activated PFCnDs allows for background-free ultrasound and photoacoustic imaging. However, to initiate the vaporization event, a photoabsorber is required. If laser-activated PFCnDs are to be used clinically, the photoabsorber used must be efficient, biocompatible and nontoxic.


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Laser-activated PFCnDs are made with various photoabsorbers ranging from organic dyes to inorganic nanoparticles.6,

13, 16-18

The ideal photoabsorber would absorb at long

wavelengths for increased penetration resulting in a higher signal-to-noise ratio, have good photothermal stability and efficiency, and be biodegradable with good clearance properties. Thus far, photoabsorbers used as PFCnD triggers have struggled to fulfill these requirements. For instance, gold nanorods (AuNRs) can be synthesized to absorb within desirable wavelength range including 1064 nm; however, they are neither biodegradable nor effectively cleared, provoking concerns over long-term cytotoxicity and safety.18 Also, upon pulsed laser irradiation, anisotropic gold nanoparticles are prone to melting and morphing shape, altering their spectrum and thus limiting long-term applications (Figure S1).19 Finally, many dyes have been utilized as triggers, but the majority absorb in the NIR or at shorter wavelengths, resulting in reduced penetration depths. In addition, dyes are susceptible to photobleaching, once again limiting longterm applications.13,

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Engineering laser-activated PFCnDs with better photoabsorber

characteristics is crucial in developing effective and clinically relevant diagnostic imaging and eventual therapeutic agents. Copper sulfide (CuS) NPs are inorganic, semi-conductor nanoparticles primarily used as photothermal agents due to their photothermal stability.20-21 CuS NPs are non-cytotoxic, biodegradable, and are cleared within a reasonable time frame.22 They absorb within the second optical imaging window (1000 nm to 1350 nm), ideal for achieving clinically relevant penetration depths with increased signal-to-noise ratio.23 One drawback of CuS NPs is their lower extinction coefficient and photothermal efficiency compared to gold nanoparticles, thereby requiring significant CuS NP concentrations for enhanced photoacoustic contrast.24 Fortunately, this limitation is trivial when using CuS NPs in laser-activated PFCnDs. A modest amount of


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CuS NPs is enough to vaporize PFCnDs. The resulting phase-change gives an intense PA signal and a gaseous PFC microbubble which provides enhanced ultrasound contrast.

Hence,

combining CuS NPs with PFCnDs synergistically overcomes limitations associated with CuS NPs and the aforementioned laser-activated PFCnD challenges. Results and Discussion: We have synthesized copper sulfide PFCnDs (CuS-PFCnDs) that exhibit enhanced optical and acoustic properties needed for background-free PA/US imaging capabilities. To construct the CuS-PFCnDs, CuS NPs were first synthesized according to a previously described protocol (see Supplemental Material).20 Next, CuS NPs underwent a two-step coating process to become fluorinated and ensure their solubility in perfluorocarbons. As CuS NP absorbance depends on d-d transition, fluorination of CuS NPs did not significantly alter their characteristic UV-Vis spectrum, ability to absorb at 1064 nm or their structure (Figure 1a and 1b). However, differences in surface charge were observed after fluorination (Figure 1c). Following successful surface coating, CuS NPs became soluble in perfluorocarbon solutions for future inclusion in nanodroplets (Figure 1d). CuS-PFCnDs can be synthesized in a variety of ways. The stabilizing shell (e.g. lipid, protein, surfactant) and perfluorocarbon core can be altered depending on the intended functionality and desired image contrast, respectively. For preliminary CuS-PFCnD studies, we used a Zonyl FSO fluorosurfactant shell and perfluoropentane (boiling point = 29 °C) core. CuSPFCnDs were monodisperse (polydispersity index 100 nm), where the majority remain entrapped at the injection site and are unable to directly enter the lymphatics.35-37 CuS-PFCnDs have potential applications in other diseases as well. We demonstrated that CuS-PFCnDs naturally accumulate in the red pulp tissue of the spleen where they are eventually endocytosed by resident red pulp macrophages (RPMs). Hence,


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CuS-PFCnDs could be used to deliver therapeutics and influence RPM activity, this is significant as RPMs have a role in tissue homeostasis and immune response.38 Lastly, we developed a method to form background-free ultrasound and photoacoustic images using CuS-PFCnDs. Although this feature is not specific to CuS-PFCnDs, it has not been fully explored in other laser-activated nanodroplets. Future work will consist of optimizing CuS-PFCnD composition for desired bioactivity (i.e. shell composition, targeting moieties) and studying their utility in cancer and other applications. Conclusion: The unique combination of CuS NPs and PFCnDs helps to overcome current laseractivated PFCnD limitations. By using CuS NPs, which absorb in the second optical window, as the photoabsorber that initiates the phase-change, PFCnDs can be activated to provide contrast at increased penetration depths. Unlike other metallic particles that absorb in this region, CuS NPs have been shown to be biodegradable, and can be synthesized at small sizes for renal clearance, increasing their biocompatibility over comparable photoabsorbers. Additionally, we have demonstrated a unique signal processing platform that allows PFCnDs to form background-free ultrasound and photoacoustic images in vivo. Overall, the research outlined above brings laseractivated PFCnDs closer to clinical-relevance.


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Supporting Information. Detailed methods, Figures S1-S5 (PDF), Movie S1 (AVI)

Corresponding Author *[email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. D.Y.S. synthesized samples and performed characterization studies, with assistance from D.P.. D.S.D carried out TEM experiments. D.Y.S. and D.S.D performed in vivo imaging studies. D.S.D, and D.Y.S. contributed to developing the imaging process algorithm and producing in vivo images. D.Y.S and D.S.D and S.Y.E. prepared and edited the manuscript.

Notes The authors declare no competing financial interest.

Acknowledgments We would like to thank Steven Yarmoska and Donald VanderLaan of the Georgia Institute of Technology for helpful discussions. The work was supported by the National Institutes of Health


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under grants CA158598, EB008101 and CA149740, and the Breast Cancer Research Foundation grant. D.Y.S acknowledges fellowship funding from the National Institutes of Health (T32 EB007507) and the National Science Foundation Graduate Research Fellowship Program. References 1. Kiessling, F.; Mertens, M. E.; Grimm, J.; Lammers, T., Radiology 2014, 273 (1), 10-28. 2. Anselmo, A. C.; Mitragotri, S., The AAPS journal 2015, 17 (5), 1041-1054. 3. Cheon, J.; Lee, J.-H., Acc. Chem. Res. 2008, 41 (12), 1630-1640. 4. Huynh, E.; Rajora, M. A.; Zheng, G., Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol. 2016. 5. Santiesteban, D. Y.; Kubelick, K.; Dhada, K. S.; Dumani, D.; Suggs, L.; Emelianov, S., Ann. Biomed. Eng. 2016, 44 (3), 750-772. 6. Wilson, K.; Homan, K.; Emelianov, S., Nat. Commun. 2012, 3, 618. 7. Sheeran, P. S.; Luois, S.; Dayton, P. A.; Matsunaga, T. O., Langmuir 2011, 27 (17), 10412-10420. 8. Kripfgans, O. D.; Fowlkes, J. B.; Miller, D. L.; Eldevik, O. P.; Carson, P. L., Ultrasound Biol. Med, 2000, 26 (7), 1177-1189. 9. Partlow, K. C.; Chen, J.; Brant, J. A.; Neubauer, A. M.; Meyerrose, T. E.; Creer, M. H.; Nolta, J. A.; Caruthers, S. D.; Lanza, G. M.; Wickline, S. A., FASEB J. 2007, 21 (8), 1647-1654. 10. Klibanov, A. L., Adv. Drug Delivery Rev. 1999, 37 (1), 139-157. 11. Foster, F. S.; Burns, P. N.; Simpson, D. H.; Wilson, S. R.; Christopher, D. A.; Goertz, D. E., Cancer Metastasis Rev. 2000, 19 (1-2), 131-138. 12. Rapoport, N., Drug-Loaded Perfluorocarbon Nanodroplets for Ultrasound-Mediated Drug Delivery. In Therapeutic Ultrasound, 2016; pp 221-241. 13. Hannah, A.; Luke, G.; Wilson, K.; Homan, K.; Emelianov, S., ACS nano 2013, 8 (1), 250-259. 14. Giesecke, T.; Hynynen, K., Ultrasound Biol. Med, 2003, 29 (9), 1359-1365. 15. Mallidi, S.; Luke, G. P.; Emelianov, S., Trends Biotechnol. 2011, 29 (5), 213-221. 16. Wei, C.-w.; Lombardo, M.; Larson-Smith, K.; Pelivanov, I.; Perez, C.; Xia, J.; Matula, T.; Pozzo, D.; O'Donnell, M., Appl. Phys. Lett. 2014, 104 (3), 033701. 17. Paproski, R. J.; Forbrich, A.; Huynh, E.; Chen, J.; Lewis, J. D.; Zheng, G.; Zemp, R. J., Small 2016, 12 (3), 371-380. 18. Hannah, A. S.; VanderLaan, D.; Chen, Y.-S.; Emelianov, S. Y., Biomed. Opt. Express 2014, 5 (9), 3042-3052. 19. Chen, Y.-S.; Frey, W.; Kim, S.; Homan, K.; Kruizinga, P.; Sokolov, K.; Emelianov, S., Opt. Express 2010, 18 (9), 8867-8878. 20. Zhou, M.; Zhang, R.; Huang, M.; Lu, W.; Song, S.; Melancon, M. P.; Tian, M.; Liang, D.; Li, C., J. Am. Chem. Soc. 2010, 132 (43), 15351-15358. 21. Li, Y.; Lu, W.; Huang, Q.; Li, C.; Chen, W., Nanomedicine 2010, 5 (8), 1161-1171. 22. Guo, L.; Panderi, I.; Yan, D. D.; Szulak, K.; Li, Y.; Chen, Y.-T.; Ma, H.; Niesen, D. B.; Seeram, N.; Ahmed, A., ACS nano 2013, 7 (10), 8780-8793. 23. Smith, A. M.; Mancini, M. C.; Nie, S., Nat Nano 2009, 4 (11), 710-711.


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