Page 3 - An ultrasound-driven immune-boosting molecular machine for systemic tumor suppression

SCIENCE ADVANCES | RESEARCH ARTICLE

with an identical chemical structure to Ce6 was successfully pre- metathesis reactions. The aspartic acid side chain was introduced at
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pared from the natural spirulina powders originating in Chenghai C-15 regioselectively using 1-ethyl-3-(3-dimethylaminopropyl)carbo-
Lake in Yunnan Province, China, which was named Chenghai chlorin diimide hydrochloride (EDCI) as a condensing agent, followed by
(CHC) (25). Our early findings demonstrated that the introduction hydrolysis to yield C34 (fig. S1).
of the -extension system to the macrocyclic structure of CHC could The ultraviolet-visible (UV-vis) absorption spectra of CHC,
finely modulate its photochemical features, molecular lipophilicity, NPe6, and C34 in methanol are shown in Fig. 2B. The C34 molecule
and even ROS production ability toward favorable directions in demonstrated a considerable bathochromic shift (7 nm) in both
PDT (25, 26). On the basis of this, we suspected that the -extension Soret and Qy bands compared with CHC or NPe6. The redshift
system might be able to improve sonochemical properties, SDT ef- might be due to the -extension effect caused by the conjugated
fect, and other molecular properties of CHC as well. connection of the aromatic groups to 3-vinyl of chlorin macrocycle,
Here, we designed a “molecular machine” named DYSP-C34 which had been fully studied in our previous work (26). As similarly
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(abbreviated as C34) by introducing 3 -aryl and 15-aspartyl sub- observed in fluorescence spectra (Fig. 2C), C34 showed distinct
stituents to CHC. Our results revealed that this strategic chemical emission bands, with a closely 8-nm bathochromic shift in compari-
modification successfully endowed the resultant molecule, C34, with son to CHC or NPe6.
multiple favorable properties, such as improved lipophilic/hydrophilic To assess the acoustic responsive activity of the C34 molecule,
balance, intensified US-induced ROS production capacity, and one self-designed low-intensity US irradiation system was set up for
better cellular permeability, resulting in the excellent tumor target in vitro study (Fig. 2D). Currently, the most common US exposure
efficiency and notable SDT-mediated tumor regression. Unexpectedly,
C34 itself exhibited mild immunogenicity by stimulating APCs which the medium-containing cell culture dish is placed above the
directly. The sono/immunosynergistic effect induced by C34-mediated center of the transducer (fig. S2) (27–29). In this way, US spreads
SDT could profoundly regress tumor systemically (Fig. 1). through the bottom of the dish to the water-air boundary on the top
(30–32), resulting in the formation of standing waves (32), which
are unstable, less reproducible, and much easier for causing cellular
RESULTS mechanical damage (33). Thus, to completely eliminate standing
Preparation and characterization of C34 waves, we designed an US irradiation configuration with a 1.0-MHz
The scheme of preparation of C34 is shown in Fig. 2A. CHC was therapeutic transducer (20 mm in diameter) on the side of the tank
prepared from chlorophyll a, which was extracted from spirulina that was coated with an ultrasonic absorbent material to minimize
powders derived from Chenghai Lake in Yunnan Province, China, water-air boundary reflections (Fig. 2D). In addition, under this
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as we previously reported (25). The synthesis of 3 -(4-methoxyphenyl)- optimal free-field condition, the pressure amplitude and system
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15 -aspartyl-CHC is described in fig. S1. Briefly, (E)-3 -(4-methoxyphenyl)- temperature could be maintained to be very stable during US ex-
CHC (A15) was prepared from CHC via Ru-catalyzed olefin posure (Fig. 2E). The root mean square pressure (p RMS) amplitude

configuration is usually equipped with a transducer at its bottom, in Downloaded from https://www.science.org at Dalian University of Technology on October 20, 2021

Fig. 1. Schematic illustration of the process of the potent US-driven molecular machine, C34, for systemic tumor suppression. C34 molecular machine had pref-
erential tumor accumulation ability, potent US-triggered cytotoxicity, and intrinsic immune-boosting capacity.
Wang et al., Sci. Adv. 2021; 7 : eabj4796 20 October 2021 2 of 15