A Nucleic Acid‐Based LYTAC Plus Platform to Simultaneously Mediate Disease‐Driven Protein Downregulation

Abstract Protein degradation techniques, such as proteolysis‐targeting chimeras (PROTACs) and lysosome‐targeting chimeras (LYTACs), have emerged as promising therapeutic strategies for the treatment of diseases. However, the efficacy of current protein degradation methods still needs to be improved to address the complex mechanisms underlying diseases. Herein, a LYTAC Plus hydrogel engineered is proposed by nucleic acid self‐assembly, which integrates a gene silencing motif into a LYTAC construct to enhance its therapeutic potential. As a proof‐of‐concept study, vascular endothelial growth factor receptor (VEGFR)‐binding peptides and mannose‐6 phosphate (M6P) moieties into a self‐assembled nucleic acid hydrogel are introduced, enabling its LYTAC capability. Small interference RNAs (siRNAs) is then employed that target the angiopoietin‐2 (ANG‐2) gene as cross‐linkers for hydrogel formation, giving the final LYTAC Plus hydrogel gene silencing ability. With dual functionalities, the LYTAC Plus hydrogel demonstrated effectiveness in simultaneously reducing the levels of VEGFR‐2 and ANG‐2 both in vitro and in vivo, as well as in improving therapeutic outcomes in treating neovascular age‐related macular degeneration in a mouse model. As a general material platform, the LYTAC Plus hydrogel may possess great potential for the treatment of various diseases and warrant further investigation.

ethyl acetate was added to dissolve the product.Next, the organic solution was washed with hydrochloric acid solution (0.1 M), saturated NaHCO3, and distilled water respectively.After drying with Na2SO4 and concentrated by rotary evaporation, the residue was purified by flash chromatography on silica gel (petroleum/ethyl acetate = 1:3) to give a colorless oil (M1, 10.28 g, 95%).
2) Synthesis of M2 M1 (10.0 g, 25.6 mmol) and 3-bromo-1-propanol (4.62 g, 33.3 mmol) were successively added to 25 mL of anhydrous dichloromethane, and then 30 mL of Boron trifluoride etherate was slowly added at ice bath.After 0.5 h, the ice bath was removed and the mixture solution continue to stir for 24 h at room temperature.
Finally, the reaction was quenched by saturated NaHCO3, the organic phase was washed with saturated NaHCO3 and distilled water (twice), dried with anhydrous Na2SO4, and concentrated by a rotary evaporator.The pure product (M2) was obtained by the purification of flash column chromatography (ethyl acetate /petroleum ether = 1:3, v/v) Yield：35%.
3) Synthesis of M3 M2 (2.69 g, 5.75 mmol) was dissolved in 10 mL of dry dimethylformamide, and sodium azide (1.86 g, 28.75mmol) was added slowly and carefully.Then the mixture solution was stirred at 80˚C for 24 h.After cooling, the insoluble object was filtered and the filtrate was concentrated by a rotary evaporator, and the residue was redissolved in ethyl acetate.Then the organic phase was washed with distilled water (twice), dried with anhydrous Na2SO4, and evaporated by a rotary evaporator.The product (M3) was got without further purification.Yield：100%.

4) Synthesis of M4
M3 (0.797 g, 1.85 mmol) was dissolved in 10 mL of dry methanol, and sodium methoxide (0.12 g, 2.22 mmol) was added slowly.And then the mixture solution was stirred at 0˚C for 4 h.The pH of the solution was adjusted to 6 by Dowex 50WX2, H form, ion-exchange resin.The ion-exchange resin was filtered and the filtrate was concentrated by a rotary evaporator to afford M4 without further purification.Yield： 95 %.

5) Synthesis of M5
M4 (0.789 g, 3 mmol), trityl chloride (4.17 g. 15 mmol), and DMAP (36.3 mg, 0.3 mmol) were successively added to 10 mL of pyridine, and then the mixture solution was stirred at 55˚C for 24 h.After cooling, the solvent was removed by a rotary evaporator, the residue was redissolved in dichloromethane was washed with 0.1 M hydrochloric acid solution, saturated NaHCO3, and distilled water (twice), dried with anhydrous Na2SO4 and evaporated by a rotary evaporator.The crude product was purified using flash column chromatography (ethyl acetate /dichloromethane = 1:1, v/v) to get the yellow oil (M5).Yield：40 %.

6) Synthesis of M6
1.2 mL acetic anhydride was slowly added to 10 mL of pyridine containing M5 (0.55 g, 1.1 mmol) at 0˚C.And then the mixture solution was stirred at room temperature for 24 h.The residue was redissolved in dichloromethane following that the solvent was removed.The organic phase was washed with 0.1M hydrochloric acid solution, saturated NaHCO3, and distilled water (twice), dried with anhydrous Na2SO4, and evaporated by a rotary evaporator.The oil product (M6) was obtained by purification using flash column chromatography (elution: dichloromethane).Yield：81 %.

7) Synthesis of M7
3 mL of dichloromethane containing M6 (0.631g, 1 mmol) was slowly added into the mixture solution of formic acid and diethyl ether (1:1~3ml/3ml) and stirred at room temperature for 1.5 h.After adding 100 mL of dichloromethane, the organic phase was washed with distilled water (twice), dried with anhydrous Na2SO4, and removed by a rotary evaporator.The oil product (M7) was obtained by flash column chromatography (ethyl acetate /dichloromethane = 1:1, v/v).Yield：55 %.
After 100 mL of dichloromethane was added, the organic phase was washed with distilled water (twice), dried with anhydrous Na2SO4, and removed by a rotary evaporator.The oil product (M8) was purified using flash column chromatography by flash column chromatography (elution: ethyl acetate /petroleum ether = 1:1, v/v).Yield：40 %. 9) Synthesis of M9 M8 (0.29 g, 0.5 mmol) was dissolved in 5 mL of dry methanol, and sodium methoxide (0.32 mg, 0.6 mmol) was added slowly.And then the mixture solution was stirred at 0˚C for 4 h.The pH of the solution was adjusted to 6 by Dowex 50WX2, H form, ion-exchange resin.The ion-exchange resin was filtered and the filtrate was concentrated by a rotary evaporator to afford M4 without further purification.Yield： 90 %. 10) Synthesis of M10 M9 (0.1g, 0.22 mmol) was slowly added to the mixture solution of trifluoroacetic acid and dichloromethane (1:1~3ml/3ml) and was stirred at 0˚C for 3 h.After the reaction finished, the mixture solution was added dropwise into amounts of diethyl ether, some sediment was observed, and the solid was separated by centrifugation.

Figure S18 .
Figure S18.MALDI-TOF spectra of Y-a before and after DBCO-NHS ester modification.

Figure S22 .
Figure S22.(a) DLS and (b) ζ-potential measurements of LYTAC Plus gel (400 μM in term of component Y-motifs) after incubation with different concentrations of DNase I at 37 ℃ for 12 h.

Figure S24 .
Figure S24.10% native PAGE gel image of LYTAC Plus gel after incubation with different concentrations of RNase H for 1.0 h at 37 °C.

Figure S26 .
Figure S26.Western blot analysis of M6PR expression in HUVECs and HRMECs.

Figure S28 .
Figure S28.Cell proliferation situation of HUVECs treated with 2 μM different formations for 48 h under the stimulation of VEGF165.

Figure S29 .
Figure S29.Cell migration capability of HUVECs after treatment with different samples and analyzed by wound-healing experiment and captured images at indicated time.

Figure S30 .
Figure S30.Standard curve of human ANG-2 ELISA kit experiment used in this study.

Figure S34 .
Figure S34.A representative TUNEL staining of retina from mice with lase-induced

Figure S35 .
Figure S35.Systematic toxicity of designed pharmaceutical regimen.Histological staining of body tissues of mice (N' = 3) in each experimental group.Scale bar: 200 µm for spleen and lung; 100 µm for heart, liver and kidney.N', number of mice.

Figure S36 .
Figure S36.In vivo biodistribution of LYTAC Plus.Fluorescence signal of (a) main organs and (b) serum of mice within 7 days after intravitreal injection of LYTAC Plus.(c) Fluorescence imaging and (d) semi-quantitative analysis of fluorescence signals of different eye tissues of mice within 7 days after intravitreal injection of LYTAC Plus-1: cornea and conjunctiva; 2: sclera and choroid; 3: crystalline lens; 4: retina; 5: optic nerve.