Biocompatible Chemically Fueled Transient Polymer Nanoparticles for Temporally Programmable in Vivo Imaging

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE10 Jun 2022Biocompatible Chemically Fueled Transient Polymer Nanoparticles for Temporally Programmable in Vivo Imaging Chunyu Pan, Jiajun Xu, Liang Wang, Yiyang Jia, Jiahui Li, Guancheng Liu, Shoujun Zhu, Bai Yang and Yunfeng Li Chunyu Pan State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Jiajun Xu State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Liang Wang State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Yiyang Jia State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Jiahui Li State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Guancheng Liu State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Shoujun Zhu State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 , Bai Yang State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 and Yunfeng Li *Corresponding author: E-mail Address: [email protected] State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012 https://doi.org/10.31635/ccschem.022.202201893 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Chemically fueled dissipative self-assembly paves the way for innovative materials with lifelike properties and functions. Achievement of nonequilibrium systems with biocompatibility and biofunctions remains an important and challenging task. Here, we present biocompatible chemically fueled transient polymer nanoparticles and their applications in temporally programmable in vivo imaging. The lifetime of the transient polymer nanoparticles can be tuned by varying the concentration of polymer, adenosine triphosphate, and phosphatase. Moreover, the transient assembly of polymer nanoparticles can be paused for storage and then subsequently restored. The transient polymer nanoparticles exhibit good biocompatibility. Notably, we implement in vivo imaging in a temporally programmable fashion by using an autonomous fluorescence modulator of transient nanoparticles assembled from polymers with a fluorescence moiety. The results in this work provide a valuable way to achieve nonequilibrium self-assembly of synthetic systems with good biocompatibility and programmable biofunctions, accelerating innovative developments of nonequilibrium soft biomaterials. Download figure Download PowerPoint Introduction Nature extensively utilizes nonequilibrium assembled structures and processes to execute the unique properties and functions by dissipating energy molecules, such as adenosine triphosphate (ATP) or guanosine triphosphate (GTP).1,2 A striking example is the GTP-driven dissipative self-assembly of microtubules that exhibits spatiotemporal control over self-assembly structures and processes, regulating important biological functions, including cell proliferation, migration, and signal transduction.1–3 Inspired by this biological scenario of self-assembly, several man-made dissipative self-assembly systems have been synthesized4,5 with assembly being driven by a variety of energy sources, including magnetic or electric fields,6–9 shear force,10 light,11–16 and chemical fuels.6,17–23 In particular, dissipative self-assembly systems mediated by chemical fuels have attracted immense interest because they can recapitulate many features of dissipative self-assembly structures and processes in the body that maintain homeostasis and biological activities.1,24–27 Chemical fuel-driven dissipative assembled systems with complex behavior can be ideal model systems for studies on the underlying fundamentals of self-assembly structures and processes in living systems.1,5 In addition, a fundamental understanding of these assemblies can instruct the design and synthesis of the next generation of materials with active, adaptive, autonomous, emergent, and intelligent behavior.1,2,5 For example, in a pioneering work by Maiti et al.,18 transient vesicles mediated by ATP with high structural complexity have been explored and were used as vesicular nanoreactors. To date, many dissipative self-assembly systems mediated by chemical fuels have been explored, including nanoreactors,18 transient hydrogels,28–34 supramolecular polymerization,35,36 structure switching,17,37–44 clustering of colloids,45–47 complex coacervates,48,49 and photonic materials.50 Despite the progress in the field, the number of successful chemical fuel-driven dissipative self-assembly systems remains rather limited.4,5 Most of the reported dissipative systems studied the transient changes in self-assembly structures. However, the spatiotemporally programmable properties and functions similar to life systems have remained largely unexplored.5 A dissipative self-assembly system is very attractive in biomaterials science and the biomedical field because it can produce dynamic structures and properties that mimic those found in biological systems, such as cellular transport, self-healing, adaptability, and so on.1,2,5 For biomedical applications, a dissipative self-assembly system with good cytocompatibility and biocompatibility is necessary but difficult to achieve. Recently, the integration of stimuli-responsive fluorescence moieties with dissipative self-assembly systems led to interesting autonomous changes in the systems' fluorescence,51,52 which represents a promising approach for temporally programmable in vivo bioimaging. Although the nanoparticles with stimuli-responsive fluorescence properties have been exploited for in vivo imaging of tumors and tumor metastatic lymph nodes,53–55 the development of in vivo imaging that uses polymer nanoparticles with autonomous regulation of their fluorescence intensity in a temporally programmable fashion remains a significant challenge. Herein, we report a dissipative self-assembly system of polymer nanoparticles with good biocompatibility that enables temporally programmable in vivo imaging. The system mainly comprises three components, namely, a positively charged polymer, ATP, and a phosphatase. Through changing the concentration of these components, the lifetime of transient polymer nanoparticles was tuned from several minutes to hundreds of minutes. Up to 11 cycles of dissipative self-assembly of polymer nanoparticles were obtained by refueling the system with ATP. Moreover, this dissipative system can be paused by freezing it, and transient behavior was restored after at least 45 days of storage in a freezer. More importantly, the dissipative system of polymer nanoparticles in our work shows good cytocompatibility, which was demonstrated by in vitro cell culture of SW620 and HeLa cell lines. Moreover, the transient polymer nanoparticles temporally self-regulate their fluorescence intensity by using polymers modified by sulfo Cyanine 5 (Cy5) as building blocks. Finally, the dissipatively self-assembled polymer nanoparticles enable the in vivo imaging of lymph nodes in a temporally programmable way. Experimental Methods Dissipative self-assembly of polymer nanoparticles In a typical experiment, poly(ethylene glycol)114-block-poly(l-lysine hydrochloride)200 (PEG-PLL, MW = 38,000 g/mol) and potato apyrase (PA) were mixed to get a solution with the concentration of PEG-PLL and PA of 0.75 mg/mL and 0.2 U/mL, respectively. Subsequently, 20 μL solution of 108 mg/mL ATP was added into a 2 mL mixed solution of PEG-PLL and PA. The dissipative self-assembly of polymer nanoparticles occurred under magnetic stirring. The process of dissipative self-assembly was monitored by a camera (Nikon D7500). Turbidity measurement The turbidity was monitored by measuring the absorbance at 600 nm by using the Thermo Fisher NanoDrop OneC kinetic mode. The stir rate was set at mode 6. The interval time was dependent on the ATP or PA concentration, and the measurement points were set as 999. The measurement was performed in a 10 mm × 10 mm quartz cuvette. In a typical measurement, the interval time was set as 2 s, and the measurement points were set as 999. A 3 mL solution of 0.75 mg/mL PEG-PLL and 0.2 U/mL PA was added into a cuvette. After the data was collected for 1.7 min, 30 μL of 108 mg/mL ATP was added into the solution. The data were collected for around 25 min. Characterization of transmission electron microscopy The cryogenic transmission electron microscopy (cryo-TEM) images of transient polymer nanoparticles were performed by FEI Talos-F200C TEM. To prepare the sample for the cryo-TEM experiments, 30 μL of 108 mg/mL ATP was added into a 3 mL solution of 0.75 mg/mL PEG-PLL and 0.2 U/mL PA. Every 2 min after adding the ATP, a 3.5 μL of polymer nanoparticle solution of dissipative self-assembly was dropped onto a Lacey carbon film on a copper TEM grid (Lacey Formvar/Carbon, 200 mesh, Cu; Ted Pella, Inc.) at room temperature. A thin film of polymer nanoparticle solution of dissipative self-assembly was formed under controlled conditions of temperature and humidity (100%) in a custom-built environmental chamber to prevent water evaporation. The excess solution was removed by the filter paper. The thin film on the TEM grid was rapidly vitrified by immersing the grid into liquid ethane (cooled by liquid nitrogen) at its freezing point. The grid was transferred into a Gatan 626 cryo-holder by using a cryo-transfer device and then transferred to the FEI Talos-F200C TEM for imaging. Images were taken at a temperature of approximately 98 K and an accelerating voltage of 200 kV, and the images were acquired with a Ceta 16M CMOS camera. The diameters of the polymer nanoparticles were measured using ImageJ software. Characterization of dynamic light scattering The dynamic light-scattering (DLS) of the dissipative self-assembly of PEG-PLL was performed by ALV/CGS-3 Goniometer System (ALV-GmbH) using a 632.8 nm laser. The scattering angle was kept at 90° during the experiment at room temperature. To avoid the influence of impurities, all solutions were filtered through a 0.45-μm membrane filter. The silica bottles were purchased from the manufacturer and washed with the ultrapure water and hot acetone. Before the measurement, the laser was stabilized for 30 min. 2 mL solution of 0.75 mg/mL PEG-PLL and 0.2 U/mL PA was initially added into a clean silica bottle, and then the silica bottle was wiped with an air-laid paper soaked by butanone. 20 μL of 108 mg/mL ATP was added into the silica bottle containing PEG-PLL and PA. The silica bottle was wiped with air-laid paper soaked by butanone again, and the measurement was started. The duration of each measurement was set at 10 s. The mean of count rate was the average of CR0 and CR1, which were determined by the ALV-7004 software. The hydrodynamic radius of the polymer nanoparticles was calculated by the ALV-7004 software by using the CONTIN method in the manual provided by the manufacturer. Self-regulatory fluorescence of transient polymer nanoparticles Fluorescence spectra of dissipative self-assembly of PEG-PLL-Cy5 were performed by SHIMADZU RF-6000 in a quartz cuvette. In a typical experiment of dissipative self-assembly, after 2 μL of 108 mg/mL ATP was added into a 0.2 mL mixed solution of 0.1 mg/mL PEG-PLL-Cy5 and 19.4 U/mL calf intestinal alkaline phosphatase (CIAP). The fluorescence spectrum was taken every 1 min when the ATP concentration was 0.52, 0.70, and 0.87 mg/mL. The fluorescence spectrum was taken every 2 min when the ATP concentration was 1.05 mg/mL. In vivo bioimaging All animal experiments in this work were handled under protocols approved by the Institutional Animal Care and Use Committee of Jilin University (Protocol number: 20210642). Balb/c mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. Bedding, nesting material, food, and water were provided ad libitum. Ambient temperature was controlled at 20 to 22 °C with 12-h light/12-h dark cycles. All mice were anesthetized by using isoflurane and fixed in the prone position on a black paperboard for imaging. A 0.1 mL solution of 0.1 mg/mL PEG-PLL-Cy5 and 19.4 U/mL CIAP were added in a polymerase chain reaction (PCR) tube. After 1 μL ATP solutions with different concentrations were added to the mixed solution of PEG-PLL-Cy5 and CIAP, and the solution was mixed by vortexing. Within 2 min after the addition of ATP, 25 μL suspension was injected into the foot pads of mice, and the near-infrared (NIR) images of the mice were collected by a Retiga LUMO camera (Teledyne Photometrics). The foot pads were gently pressured for 1 min, and pictures of the mice were taken. Subsequently, the images of mice were taken every 1 min in the first 20 min, and then the images of mice were taken every 5 min from 20 to 102 min. The laser was only turned on when taking the pictures to avoid quenching of the Cy5. The sensor size was 2688 × 2200 with a 785 nm long-pass filter (Thorlabs) for NIR imaging. The 655 nm laser was purchased from CNI Ltd. The 655 nm laser model was MDL-XD-655nm-5W-BJ00334. The laser was used with a power density of 3.12 mW /cm−2. The pictures were taken by Ocular. The exposure time was set as 100 ms. All pictures were finally analyzed by ImageJ to obtain the brightness of the lymph nodes and the normal tissues. The fluorescence ratio between lymph nodes and normal tissue (LN to tissue ratio) was calculated by using the following equation: LN to tissue ratio = ( lymph node brightness − background ) / ( tissue brightness − background ) . The cutoff of the fluorescence ratio between lymph nodes and normal tissues was calculated by two standard deviations above the average LN to normal tissue ratio from 92 to 102 min. Results and Discussion ATP-fueled dissipative self-assembly of polymer nanoparticles Figure 1a shows the typical system of dissipative self-assembly of polymer nanoparticles driven by ATP, which is known as the energy currency of cells, to maintain their biological activities.1 This dissipative system mainly comprises a polymer, PEG-PLL, ATP, and a phosphatase, PA. The cycle starts with PEG-PLL (pKa=9.4) that carries positively charged PLL segments in an aqueous solution56 (N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), 10 mM, pH 7.0). Upon the addition of negatively charged ATP, PEG-PLL and ATP form polymer nanoparticles by electrostatic interactions between ATP molecules and the PLL segments.57 In the presence of a phosphatase,18 PA, ATP is hydrolyzed to a single-charged phosphate, adenosine monophosphate (AMP), and two molecules of orthophosphate Pi. After ATP depletion, there are not enough electrostatic interactions to support the assembled polymer nanoparticles, leading to the dissociation of polymer nanoparticles, restoring them to the initial state of free polymers in the solution. Technically, the successive cycles of dissipative self-assembly can be achieved by sequential addition of the fuel, ATP, in the system. Figure 1 | Transient polymer nanoparticles formed by PEG-PLL and ATP. (a) Schematic representation of the dissipative self-assembly of polymer nanoparticles driven by ATP. (b) Absorbance at 600 nm as a measure of solution turbidity as a function of time. ATP was added at 1.7 min for turbidity measurement. (c) Variation in hydrodynamic radius of the polymer nanoparticles after the addition of ATP. (d) DLS count rates of the solution as a function of time. (e–h) Cryo-TEM images of transient polymer nanoparticles after adding ATP at 4 (e), 8 (f), 14 (g), and 18 min (h), scale bar, 500 nm. Experimental conditions in all cases: HEPES buffer (10 mM, pH 7), [Ca2+] = 1 mM, PEG-PLL, 0.74 mg/mL, ATP, 1.07 mg/mL, and PA, 0.2 U/mL. Download figure Download PowerPoint The ATP-driven dissipative self-assembly of PEG-PLL was studied by a variety of analytical techniques. After the addition of 20 μL of 108 mg/mL ATP into a mixed solution of 2 mL of 0.75 mg/mL PEG-PLL and 0.2 U/mL PA, the clear solution rapidly turned into a turbid solution, and thus turbidity is an indication of assemblies formed by PEG-PLL and ATP by electrostatic interactions ( Supporting Information Figures S1a and S1b). After ∼25.0 min upon adding the ATP, the turbid solutions autonomously turned into transparent solutions under the hydrolysis of ATP by PA ( Supporting Information Figure S1c). The transient turbidity in the dissipative self-assembly system was quantified through measuring the absorbance at 600 nm by using a UV–vis spectroscopy. The turbidity of a solution with PEG-PLL and PA simultaneously increased after the addition of ATP (Figure 1b). Relatively steady turbidity was maintained from ∼3.2 to ∼17.0 min, which implicated the steady state in the process of dissipative assembly. The turbidity of the solution autonomously decreased and rapidly returned to a value similar to the initial solution after ∼22.5 min. In this dissipative self-assembly system, we used a lifetime, τ, to name the duration that was needed to complete one cycle. DLS was used to further evaluate the transient self-assembly of PEG-PLL nanoparticles. Size distribution analysis by DLS revealed that the hydrodynamic radius, Rh, of the free PEG-PLL at the start was ∼3.3 nm (Figure 1c). Subsequently, the Rh of nanoparticles increased to ∼20 nm (hydrodynamic diameter, ∼40 nm) at 3.5 min, and then was stable at ∼60 nm (hydrodynamic diameter, ∼120 nm) from 4 to 15 min, and finally autonomously reduced to ∼3.0 nm after 20.7 min (Figure 1c). A rapid increase in the DLS count rates was observed upon the addition of ATP into the solution with PEG-PLL and PA. A plateau of DLS count rates was formed from ∼3 to ∼12 min after adding ATP in the system (Figure 1d). After 21.6 min, the DLS count rates autonomously returned to the initial value. The transient DLS count rates of polymer nanoparticles exhibited a similarly temporal behavior in comparison with their transient size change. The transient size and count rates by the DLS analysis indicated the presence of self-assembled polymer nanoparticles, which were identified as spherical polymer nanoparticles by the cryo-TEM (Figures 1e–1h). The spherical polymer nanoparticles with an average diameter of 63.6 ± 29.2 nm formed after 4 min upon adding the ATP to the system (Figure 1e). The nanoparticle number in the solution was low, which was in agreement with the results of turbidity and DLS. A gradual increase in size and number of the polymer nanoparticles was observed over time. At 8 min, the polymer nanoparticles grew up to 140.2 ± 58.3 nm determined by the cryo-TEM (Figure 1f and Supporting Information Figure S1d). Subsequently, the size and number of the polymer nanoparticles gradually became smaller, resulting in 66.6 ± 30.1 nm at 14 min (Figure 1g). Polymer nanoparticles were hardly observed under the cryo-TEM at 18 min (Figure 1h), indicating the disintegration of polymer nanoparticles to free polymers. Temporal control of transient polymer nanoparticles Next, we studied the temporal behavior of the dissipative self-assembly of PEG-PLL by varying the concentration of PEG-PLL, ATP, and PA by using turbidity measurements. We initially investigated the lifetime, τ, of our system by varying the concentrations of ATP from 0.36 to 1.78 mg/mL at a constant concentration of PEG-PLL and PA. Figure 2a shows the normalized turbidity of solutions as a function of time. In all cases, the addition of ATP resulted in a rapid increase in the solution turbidity. Because of the hydrolysis of ATP through Michaelis–Menten kinetics,18 more ATP molecules took a longer time to hydrolyze at the constant concentration of PA. The longer steady state of dissipative self-assembly was maintained upon increasing the ATP concentration in the system (Figure 2a). Subsequently, an autonomous decrease of the solution turbidity to the value of the initial polymer solution was observed in all cases. The lifetime in our system can be tuned from 4.8 ± 0.2 to 117.0 ± 5.7 min by increasing the concentration of ATP from 0.36 to 1.78 mg/mL (Figure 2b). Figure 2 | Temporal control of transient polymer nanoparticles and their pause, storage, and restoring. (a) Normalized solution turbidity as a function of time at a constant concentration of PA (0.2 U/mL) and PEG-PLL (0.74 mg/mL). ATP was added at 1.7 min. (b) The lifetimes of the transient polymer nanoparticles as a function of the ATP concentration determined by the turbidity. (c) Normalized solution turbidity as a function of time at a constant concentration of ATP (1.07 mg/mL) and PEG-PLL (0.74 mg/mL). PA varied from 0.05 to 0.4 U/mL in the system. ATP was added at 1.7 min. (d) The lifetimes of the transient polymer nanoparticles determined by the turbidity as a function of the PA concentration. (e) The lifetimes of the transient polymer nanoparticles determined by the turbidity as a function of the concentration of PEG-PLL at a constant concentration of ATP (1.07 mg/mL) and PA (0.2 U/mL). Experimental conditions in (a–e): HEPES buffer (10 mM, pH 7), [Ca2+] = 1 mM. (f) Reversible turbidity changes of the system over time following repeated additions of ATP (30 μL of 108 mg/mL ATP). Experimental conditions: HEPES buffer (50 mM, pH 7), [Ca2+] = 1 mM, PEG-PLL, 0.74 mg/mL, and PA, 0.2 U/mL. (g) Solution turbidity before storage (red symbols) and restoring after lyophilization (black symbols) as a function of time. (h) The photograph of the white powder of transient polymer nanoparticles after lyophilization. (i) The representative SEM image of transient polymer nanoparticles after scale bar, 500 nm. The cryo-TEM image of transient polymer nanoparticles after 2 min upon the white powder by scale bar, 500 nm. Experimental conditions in HEPES buffer (10 mM, pH 7), [Ca2+] = 1 mM, PEG-PLL, 0.74 mg/mL, ATP, 1.07 mg/mL, and PA, 0.2 U/mL. Download figure Download PowerPoint The lifetime of dissipative self-assembly in our system is dependent on the hydrolysis rate of ATP under the of PA. Figure shows the normalized turbidity of solutions with PA concentration varying from 0.05 to 0.4 U/mL after adding the of ATP into the solution with a constant concentration of polymer, as a function of time. In all cases, a rapid increase in the solution a steady state of the and an autonomous decrease in the turbidity were observed in a sequential the of ATP hydrolysis by the concentration from 0.05 to 0.4 U/mL, the lifetime of dissipative self-assembly in our system can be varied from ± to ± min (Figure Moreover, the lifetime of dissipative self-assembly in our system can be tuned by changing the concentration of PEG-PLL at a constant concentration of ATP and PA. The of the transient lifetime can be into two which are in Figure In the first the lifetime was constant in the of ± 0.4 to ± min, as the concentration of polymer increased from to 0.74 mg/mL. In the the lifetime was tuned from ± to ± min when the concentration of polymer increased from 0.74 to mg/mL. In our the of PEG-PLL and ATP are to form stable polymer nanoparticles. We explored the of that can form the stable polymer nanoparticles by measuring the size through using a We found that assembly of polymer nanoparticles occurred when the ratio of ATP and PEG-PLL was 0.36 ( Supporting Information Figure However, assembly of polymer nanoparticles was observed when the ratio of ATP and PEG-PLL was The ratio of ATP and PEG-PLL for the assembly of nanoparticles was between and the PEG-PLL concentration increased from 0.74 to mg/mL, of ATP and PEG-PLL decreased from to At the concentration of PA, it took a time to the ratio of ATP and PEG-PLL for the sample with a PEG-PLL concentration. the lifetime decreased as the PEG-PLL concentration increased from 0.74 to mg/mL. The lifetime of dissipative self-assembly in our work can be controlled by the HEPES concentration. Supporting Information Figure shows the lifetime of the dissipative self-assembly of PEG-PLL as a function of the HEPES concentration. the HEPES concentration increased from 10 to in the solution, the lifetime decreased from ± to ± min. the of PA, ATP was hydrolyzed into and two molecules of orthophosphate leading to a in pH of the solution. After hydrolysis of the of ATP, the pH became ± to ± in the solution with the HEPES concentration increasing from 10 to ( Supporting Information Figure In this pH as the pH the PA to which led to the hydrolysis of ATP and thus a The of the dissipative self-assembly in our system was explored by adding of ATP into the Figure shows the turbidity changes over time following repeated additions of ATP. The transient assembly of nanoparticles performed up to 11 cycles when the HEPES concentration was mM, with the signal returned to the value (Figure The number of cycles was dependent on the HEPES concentration. The number of cycles increased from 3 to 11 with the HEPES concentration increasing from 10 to (Figure and Supporting Information Figures To further the properties of the dissipative self-assembly, we explored the of system pH and on the dissipative In the system with the HEPES of 30 mM, as the cycles of dissipative self-assembly increased from 1 to the pH of the system decreased from ± to ± ( Supporting Information Figure This decrease in pH resulted in a of and thus the lifetime in each cycle to increase with cycle number cycles of dissipative self-assembly led to a pH of in the system. Dissipative self-assembly of PEG-PLL in the solution with pH of was not observed upon adding ATP in the of the experiment ( Supporting Information Figure because the of PA was in this In control experiment, we explored the of concentration on the lifetime of the dissipative The lifetime was constant when the concentration was mg/mL. the concentration increased to mg/mL, the lifetime increased to ± min ( Supporting Information Figure results that the of in the reaction system decrease the of ATP in nanoparticle the dissipative self-assembly of the nanoparticles by ATP a in the of turbidity (Figure and Supporting Information Figures which is in agreement with the results of a reported This was by the of which the pH of the system after each cycle of nanoparticle