Sequentially Programmable and Cellularly Selective Assembly of Fluorescent Polymerized Vesicles for Monitoring Cell Apoptosis

Abstract The introduction of controlled self‐assembly into living organisms opens up desired biomedical applications in wide areas including bioimaging/assays, drug delivery, and tissue engineering. Besides the enzyme‐activated examples reported before, controlled self‐assembly under integrated stimuli, especially in the form of sequential input, is unprecedented and ultimately challenging. This study reports a programmable self‐assembling strategy in living cells under sequentially integrated control of both endogenous and exogenous stimuli. Fluorescent polymerized vesicles are constructed by using cholinesterase conversion followed by photopolymerization and thermochromism. Furthermore, as a proof‐of‐principle application, the cell apoptosis involved in the overexpression of cholinesterase in virtue of the generated fluorescence is monitored, showing potential in screening apoptosis‐inducing drugs. The approach exhibits multiple advantages for bioimaging in living cells, including specificity to cholinesterase, red emission, wash free, high signal‐to‐noise ratio.

Oxalyl chloride (0.14 mL, 1.6 mmol) in CH 2 Cl 2 (5 mL) was added to a solution of DA (500 mg, 1.33 mmol) in CH 2 Cl 2 (10 mL) at 0 C. After stirring for 2 h at room temperature, the solvent and surplus oxalyl chloride was removed in vacuo and the residue was dissolved in CH 2 Cl 2 (10 mL). Then, the solution was added N,N-dimethylethanolamine (0.13 mL, 1.33 mmol) and triethylamine (0.28 mL, 2 mmol) at 0 C. The mixture solution was stirred over night at room temperature. The mixture was washed with saturated Na 2 CO 3 , water and brine.
After drying with Na 2 SO 4 , the solvent was removed in vacuo and the residue was purified by column chromatography over silica gel with CH 2 Cl 2 /MeOH = 1/0 to 1/1 as the eluents to give diacetylene-appended dimethylethanolamine (460 mg, 77%). To the CH 2 Cl 2 solution of diacetylene-appended dimethylethanolamine (460 mg, 1.03 mmol) CH 3 I (0.096 mL, 1.5 mmol) was added, and the mixture solution was stirred over night at room temperature. The solvent was removed in vacuo and the residue was purified by column chromatography over silica gel with CH 2 Cl 2 /MeOH = 200/1 to 20/1 as the eluents to give DC (504 mg, 83%). 1 Figure S1. 1 H NMR spectrum of DC in CDCl 3 at 25 °C.

UV-Vis spectroscopy
The optical absorbance of the solutions were measured in a quartz cell (light path 10 mm) on a Shimadzu UV-3600 spectrophotometer equipped with a PTC-348WI temperature controller.

Fluorescence spectroscopy
Fluorescence spectra were recorded in a conventional quartz cell (light path 10 mm) on a Varian Cary Eclipse equipped with a Varian Cary single-cell peltier accessory to control S6 temperature.

Dynamic light scattering (DLS) measurements
Angle-dependent DLS experiments were examined on a laser light scattering spectrometer (BI-200SM) equipped with a digital correlator (TurboCorr) at 636 nm. The others were measured by NanoBrook 173 plus at scattering angle of 90°.

Cryo-electron microscopy (Cryo-EM) experiments
Aliquots of 3.5 μL sample were applied to glow-discharged Quantifoil grids, blotted for 6s in a room temperature and 100% humidity chamber, and plunged into liquid ethane cooled by liquid nitrogen in the automated EFI Vitrobot device. We performed structural analysis by cryo-electron microscopy, on the FEI Talos F200C with constant-power C-Twin objective lens which was equipped with a Gatan Model 626 cryo-transfer specimen holder, operated at 200 kV. Images were recorded on the FEI 16Megapixel Ceta CMOS camera. The electron dose for each micrograph was approximately 30 e/Å 2 .

Small-angle X-ray scattering (SAXS) measurements
SAXS experiments were performed with the high-flux small-angle X-ray scattering instrument (SAXSess, Anton Paar) equipped with a Kratky block collimation system and a Philips PW3830 sealed-tube X-ray generator (Cu Kα).

Zeta potential measurements
Zeta potential measurements were measured by NanoBrook 173 plus.
Nuclear magnetic resonance (NMR) spectroscopy 1 H NMR and 13 C NMR spectra were recorded on a Bruker AV400 spectrometer.

Mass spectroscopy (MS)
High resolution-MS measurements were recorded on a VG ZAB-HS mass spectrum.
Electrospray ionization fourier transform ion cyclotron resonance-MS measurement was recorded on a Varian 7.0T FTICR-MS.

Confocal laser scanning microscopy (CLSM)
HepG2 and PC-3 cells incubated with target molecules in petri dish were observed by a

The photopolymerization performance of DA with different PEG-doping ratios
It is a prerequisite to ascertain a molar ratio of PEG-DA and DA that ensures not only the solubility and stability but also the photopolymerization. Excessive PEG would result in assembling morphology transition as well as reduced photopolymerization because PEG-DA forms micellar assembly in aqueous media without any chromogenic property in response to UV irradiation. We examined the assembling and photopolymerization properties of PEG-DA in HEPES buffer. PEG-DA, similar to DC, forms micelles with an averaged diameter of 8 nm ( Figure S5b), and no appreciable chromogenesis was observed upon UV irradiation ( Figure   S6). We therefore screened the doping stoichiometry between PEG-DA and DA by monitoring the chromogenic performance after UV irradiation ( Figure S6a). When doping PEG-DA into DA, the hydration could be successfully operated under mild conditions, where the suspension became translucent soon after sonication. And also, the PEG-coated DA vesicles are stable enough to achieve a shelf life of several weeks at either 4 C or room temperature. However, we noticed that the photopolymerization induced chromogenesis performance of DA decreases gradually along with increasing the PEG-DA ratio ( Figure S6b).
It is reasonably acceptable because PEG-DA itself tends to form micelles without showing any chromogenesis by UV irradiation. Consequently, coating DA vesicles with as little PEG as possible should be adopted to maintain high-performance chromogenesis. We further reduced the PEG-DA ratios to 1%, 2.5%, and 5% in all three cases of which satisfactory hydration and photopolymerization were successfully achieved ( Figure S7). However, the hydration of 99% and 97.5% DA vesicles (corresponding to 1% and 2.5% PEG doping) occurs merely under a relatively harsh condition (sonication at 80 C for 30 min), which is not biocompatible, and therefore, 1% and 2.5% PEG incorporations were excluded for fabricating the designed assembling entities in living cells.

PEG-doping ratios
Another important factor that should be of concern is the colorimetric response (CR) temperature of the polymerized vesicles. CR is defined as the percentage from blue to red. In this context, B value was defined as (A blue / [A blue + A red ])  100%, where A is the peak intensity corresponding to the blue or red form. CR was then calculated according to the initial and final B values, CR = (B initial  B final ) / B initial  100%. [2] As mentioned in the main text, the blue PDA changes completely its color to red when heated at 70 C. It is ideal that the color change occurs around body temperature (37 C) to make the construction of fluorescent polymerized vesicles practically operational in living cells. A fascinating phenomenon is that PEG doping could tune the CR temperature of the PDA vesicles. [     In a reference sequence, the photopolymerization of DC by UV irradiation formed PDC, and then PDC was incubated with BChE, which is expected to produce oligomeric PDA. MS shows obvious choline signal after incubation of PDC with BChE ( Figure S17b). However, DLS results showed that both 95% PDC before and after incubation with BChE formed merely the small micellar aggregation ( Figure S16). We supposed that the oligomeric PDA,