Shanghai Jiao Tong Zhou Yongfeng team: enhanced self-assembled supramolecular aggregation of vesicles photothermal conversion efficiency of 44.1%, to become the new cancer killer

Photothermal therapy (PTT) has a spatial orientation irradiation, minimally invasive and less painful advantages patient is expected to be applied to the treatment of cancer and skin diseases. Vesicles light and heat treatment agent (PTA) of the hollow structure can be multiple reflections of the incident and has excellent light trapping ability and versatility, attracted widespread attention. So far, mostly PTA vesicles as self assembled from small molecules such as liposome, poor structural stability. Thus, the development of a stable structure, high photothermal conversion efficiency of the PTA type vesicles very challenging. Polymer vesicles have a stable structure characteristics, it has great potential in the application of PTT\’s. However, the photothermal conversion efficiency of the polymer vesicles difference, it is difficult as PTA applications. This is because the fluidity of liposome membrane is much higher than the polymer vesicles, liposomes thus chromophore structure may be formed in a perfect state of the π-π stacking, is very traditional polymersomes appears powerless, Thus photothermal conversion efficiency is low.

The results presented in

Based on the above analysis, Shanghai Jiaotong University Professor Zhou Yongfeng Task Force first proposed co monovalent supramolecular polymer self-assembly and polymerization of preparing vesicles conceivable combination, called \”polymerization-enhancing supramolecular self-assembly\” (SPESA) process, and the prepared nano-based hyperbranched polymer porphyrin self-assembled capsule bubble (THPG) . Found that such rigid vesicles film structure and excellent membrane fluidity, membrane cells are stacked by directional porphyrin π-π, may form a well-structured accumulated state. At very low power density laser light (200 mW · cm-2), the solution temperature may be raised THPG vesicles from 24.1 to 46.8 deg.] C rapidly deg.] C, photothermal conversion efficiency of 44.1% staggering , and it has excellent biocompatibility. Mice after injection 24 hours, THPG vesicles gradually gathered in tumor cells, the mice formed tumors two days after eschar fall off naturally after 14 days, and no recurrence, THPG vesicles of MCF-7 tumor appears to have superior therapeutic effect. 上海交大周永丰团队: 超分子聚合增强自组装囊泡光热转化效率44.1%,成为新型癌症杀手

Characterization of the structure and a self-assembled vesicles

Characterization of FIG. 1. THPG vesicles 上海交大周永丰团队: 超分子聚合增强自组装囊泡光热转化效率44.1%,成为新型癌症杀手
. (A) SEM image of vesicles THPG, The scale of the illustration 30 nm; (b) TEM image of THPG vesicles, light green circle indicates the crushed particles of very bright opening; the STEM image and the profile elements (c) vesicle, (d) nitrogen , (e) zinc, (f) oxygen, (g) a carbon element; (H) at a concentration of 0.5 mg · mL-1 THPG vesicle solution of the SAXS spectrum; (i) THPG water, THF or UV-Vis spectra of FIG. 5% Triton X-100 detergent aqueous solution.

To prepare vesicles, the researchers to A2 diglycidyl ether monomer, tetra (4-hydroxyphenyl) porphyrin zinc (ZnTHPP) B4 is a monomer, anionic polymerization carried out prepared amphiphilic porphyrin hyperbranched polymer (THPG), a molecular weight of 6.4 kDa, the molecular weight distribution of 1.8. SEM found THPG supramolecular aggregates are spherical, the average diameter of 127 nm, THPG fluid obtained from dynamic light scattering (DLS) kinetic diameter of 176 nm. Some nm in particle failure after an internal hollow cavity, show that the polymer particles are vesicles average wall thickness of about 21 nm. In tetrahydrofuran THPG UV-Vis absorption spectra showed two strong absorption region, respectively, with B (425 nm) and the Q-band (from 545 to 626 nm) porphyrin. However, the absorption peaks in water vesicles THPG significantly widened, B band red shifted 463 nm, Q-band extending to 560 to 650 nm wavelength, which indicates that the π-π between THPG packed to form vesicles porphyrin strong J aggregates.

上海交大周永丰团队: 超分子聚合增强自组装囊泡光热转化效率44.1%,成为新型癌症杀手
Figure 2. The film structure THPG vesicles. (A) HR-TEM images of a single rupturable THPG vesicles; (BC) is an enlarged image, c, c are illustrations selected Fast Fourier Transform (FFT) as a yellow square region of the image; (d) THPG vesicles SAXS spectrum synchronization; (e) by the MD simulation THPG vesicles packing structure of porphyrins, e the red squares represent ZnTHPP portion; (f) a schematic THPG vesicles; (G) of the vesicular membrane of the enlarged schematic cross section; (h) THPG balloon structure deposited film.

Researchers detailed study of molecular arrangement in the vesicle THPGColumn mode, using high resolution TEM observation to rupture vesicles, found that hydrophobic interaction of porphyrin unit supramolecular polymerized to form nanowires black, hydrophilic diglycidyl ether of a white portion is formed by π-π stripes, black average width of the nanowires about 9.3Å. SAXS measurements are synchronous at q = 1.72 and 1.56Å-1 found in two peaks, which is between the adjacent π-π stacking unit porphyrin formation. Researchers molecular dynamics (MD) simulations demonstrated self-assembly mechanism vesicles, found porphyrins tend to accumulate be ordered by π-π, and showed a certain helix angle, between the two porphyrin molecules units distance between 3.6 Å, due to the π-π stacking of the average energy of each porphyrin is 29.7 kCal · mol-1. After a series of structural characterization and modeling studies, researchers believe THPG in water in the hydrophobic interaction driven self-assembled film thickness of about 21 nm vesicles. This self-assembly process triggers covalent polymer molecule tightly packed porphyrin group or intermolecular promote super group porphyrin molecules are polymerized to an average width of about 9.5Å nanowires, and the π-π a small amount of accumulated driving coil is formed at J aggregates. This is the first reported the self-assembly process, inducing the formation of vesicular structures by a unique covalent and non-covalent polymer. photothermal efficiency and stability analysis of vesicles

photothermal efficiency and stability analysis THPG vesicles 上海交大周永丰团队: 超分子聚合增强自组装囊泡光热转化效率44.1%,成为新型癌症杀手
3. FIG. (A) THPG vesicles in various fluorescence spectra THF / aqueous solution and a 5% Triton X-100 detergent solution; (b) with a laser wavelength of 635 nm (200 mW · cm-2) and irradiated with thermal imager PBS imaging, 0.3 mg · mL-1THPG micelles and 0.3 mg · mL-1 THPG vesicles solution; (c) THPG negative vesicles cooling time and temperature of a linear fitting curve of the number of ([tau] is the sample time of the system constant); (d) (200 mW · cm-2) thermal stability evaluation of light irradiation of the water THPG vesicles laser of 635 nm wavelength.

THPG polymers exhibit strong fluorescence in THF effect, but this effect is gradually attenuated after addition of water, it is completely quenched in pure water. This is due to THAfter PG / THF solution was added water, the self-assembly process triggers THPG vesicles. As a result, since the π-π stacking interactions leads to polymerization of supramolecular porphyrin, nanowires formed within the vesicles, resulting in self-quenching of the fluorescence. However, after adding 5% Triton X-100 detergent to THPG vesicles, fluorescence recovery, which is due to π-π structure is disrupted, causing the decomposition of the vesicles. In order to study the performance of the photothermal THPG vesicles, researchers recorded by an infrared thermal imager temperature change after 635nm laser irradiation of an aqueous solution of vesicles. Found that even at very low power density laser (200 mW · cm-2), the temperature is still THPG vesicle solution rapidly increased from 24.1 deg.] C to 46.8 ℃, and the temperature of the PBS solution only increased by 2.1 ℃, indicating that THPG vesicles having excellent light trapping performance and converted to heat by a photothermal conversion efficiency calculated that THPG vesicles of 44.1%, far higher than most organic photothermal agent. After three heating and cooling cycles of the 635 nm laser, THPG vesicles equilibrium temperature remains at about 46.1 deg.] C, exhibit excellent photothermal stability, which with strong π-π stacking between the porphyrin intravesicular interaction, hydrophobic interaction between the multilayer film and a hydrophobic structural unit inseparable. In vitro cell vesicles photothermal therapeutic agent assessment

上海交大周永丰团队: 超分子聚合增强自组装囊泡光热转化效率44.1%,成为新型癌症杀手
FIG. 4. THPG vitro evaluation vesicles biosafety and photothermal effects. (A) MCF-7 cancer cells taken at different times CLSM image representative THPG-Rb vesicles, magenta THPG-Rb vesicles, cell nuclei blue Hoechst, the scale of 20μm; (b) various under the experimental conditions, the representative CLSM image MFC-7 cells with calcein AM and propidium iodide (PI) staining together, represent the green and red of live and dead cells, dimensions: 150μm; (c) in the presence or no laser irradiation, the relative activity of MCF-7 cells were incubated with various amounts THPG 635 nm; (d) in the case where no THPG or vesicles, relative activity of various laser irradiation power of MCF-7 cells.

Researchers by methyl thiazolyl tetrazolium (MTT) of NIH / 3T3 mouse embryo fibroblast cells, assessedTHPG vesicles potential cytotoxicity in vitro. It found even after 48 h of incubation at concentrations up to 0.2 mg · mL-1 of THPG vesicles, 3T3 relative cell viability remained at 86.0%, non-toxic and excellent vesicles described THPG of NIH / 3T3 biocompatibility. To study the effect of cancer cells on THPG vesicles, researchers MCF-7 cancer cells and the load of Rb THPG vesicles at 37 ℃ for then using confocal laser scanning microscopy (the CLSM) were observed, as found incubation time increased to 15 minutes from 2 h, the observed red fluorescence emitted THPG-Rb in cancer cells continued to increase, suggesting that THPG-Rb vesicle is efficient uptake MCF-7 cells, under laser irradiation, MCF-7 cells THPG uptake of vesicles greatly enhanced. Researchers conducted calcein -AM / propidium iodide (PI) double-stained cells Experiment 7 MCF-, direct PTT THPG vesicles in vitro performance was observed. After the discovery intake THPG, MCF-7 cells are very healthy. However, · mL-1 at a concentration of 200 mg was incubated with vesicles THPG irradiated with laser light and MCF-7 cells after 10 minutes, with the increase of the laser power, the number of dead cells increased significantly.

上海交大周永丰团队: 超分子聚合增强自组装囊泡光热转化效率44.1%,成为新型癌症杀手
5. THPG vesicles FIG pharmacokinetics, and biological research distribution imaging. (A) in rats (n = 3 independent biological rats) after tail vein injection THPG vesicles, blood clearance curve with the curve (ID behalf of injected dose) time; (b) were injected intravenously THPG- Cy7.5 Cy vesicles and after 7.5, MCF-7 tumors in mice with fluorescence images at different times (Ex: 745 nm, Em: 820 nm; mode: fluorescence), the white arrows indicate the location of the tumor; (c) after injection. THPG-Cy7.5 vesicles and Cy 7.5, isolated organs and tumors in vitro for 24 hours image; (d) at 1,4 and 24 h after injection, THPG biodistribution. .

In order to study THPG vesicle retention capacity in the blood, researchers conducted a pharmacokinetic study. We found THPG vesicles blood circulation half-life of 29.5 h, a longer blood circulation capability, due to the good junction vesiclesDue to the structural stability. After 72 hours after the injection, the blood concentration of low THPG, vesicles may indicate THPG removed from the rats. Researchers to intravenous vesicles THPG-Cy7.5 (Cy7.5 using near infrared dye encapsulated THPG vesicles) mice with MCF-7 tumors, the animals were fluorescence imaging experiments. Found 24 hours after injection, in vivo Cy7.5 fluorescence signal is weaker than the free THPG-Cy7.5 vesicles, indicating that vesicles THPG-Cy7.5 longer retention time in the body. In the 24 hours after injection, Cy7.5 does not accumulate in the tumor tissue, in contrast, THPG-Cy7.5 fluorescence signal in the vesicles still evident after 24 hours at the tumor site. The researchers further studied the accumulation in tumor THPG and other major organs. It found 24 hours after injection, THPG content decreased in most organs of mice, but no significant decrease in the content of the tumor, indicating THPG vesicles gradually gathered to the tumor tissue after 24 hours. vesicles in vivo animal photothermal therapeutic agent assessment

上海交大周永丰团队: 超分子聚合增强自组装囊泡光热转化效率44.1%,成为新型癌症杀手
FIG 6. THPG vivo evaluation vesicles photothermolysis. Injection of PBS (a) and THPG solution (b) and the thermal image laser at 635 nm is irradiated at a power of 200 mW · cm-2 in tumor-bearing mice with MCF-7 after 10 minutes; (c) by monitoring the laser Imager irradiating the tumor area during temperature change quantitative analysis (n = 5 two biologically independent experiments; photograph of mice (d) PTT process; tumors (e) different groups of mice with MCF-7 tumor growth inhibition profiles; (f) MCF-7 tumor-bearing mice in different treatment body weight curve (n = 3 independent biological mice).

researchers further investigated THPG vivo efficacy PTT vesicles found THPG vesicles after injection, 60 seconds after the laser irradiation, the temperature was increased from 35.1 mouse tumors deg.] C to 55.9 deg.] C, while the temperature of control mice the tumor only a slight increase 35.3 deg.] C to 38.9 deg.] C, indicating that the balloon THPG after the light bulb heat transfer efficiency is very high. THPG vesicles researchers injected mice 4 hours, tumors were irradiated with laser for 10 minutes, to study the evolution of the tumor. tumor was found in mice injected with PBSContinues to increase, THPG mice injected tumors two days after the formation of eschar, which fall off naturally after 14 days, and no recurrence.

Summary

To prepare a stable structure PPT photothermal therapeutic agent, Prof. Zhouyong Feng Shanghai Jiaotong based research group \”supramolecular self-assembly of the polymerization-enhancing\” the process for building efficient photothermal hyperbranched porphyrin vesicle therapy. It found that the polymer prepared vesicle average diameter of 176 nm, having excellent biocompatibility, structural stability and membrane fluidity. Driven by π-π stacking interactions, vesicles porphyrin unit may further self-assemble into supramolecular polymer nano-filament, fluorescence self-quenching rate up to 1100, the photothermal conversion efficiency of 44.1% staggering, in extremely low power density laser (200 mW · cm-2), the solution temperature may be raised THPG vesicles rapidly from 24.1 deg.] C to 46.8 ℃. THPG vesicles after injection and 60 seconds of laser irradiation, the temperature was increased from 35.1 mouse tumors deg.] C to 55.9 deg.] C, two days after the formation of tumors in eschar fall off naturally for 14 days, no recurrence, the polymer vesicles prepared of MCF-7 tumor cells in mice exhibited excellent therapeutic efficacy. Description link

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