Synthesis of (2,2,5-trimethyl-1,3-dioxan-5-yl)methanol (2): The 2-(hydroxymethyl)-2-methylpropane-1,3-diol (10.10 g, 80.1 mmol) was stirred in 50 ml of acetone, 2,2-dimethoxypropane (DMP) (13.1 g, 126 mmol) and PTSA (0.79 g, 4.14 mmol) were added under room temperature. After the completion of the addition, the reaction mixture was stirred for 4 h. Then it was filtered through an amberlyst column, and the solvent was evaporated, and the residue was put under 60°C and full vacuum for 2 h. Then it was put under vacuum overnight to give (2) as a colorless liquid with 96% yield. ¹H NMR (500 MHz, CDCl₃) δ 3.67 [m, (d, s overlap J = 11.8 Hz, 2H; s, 2H)], 3.60 (d, J = 11.8 Hz, 2H), 2.55 (s, 1H), 1.44 (s, 3H), 1.40 (s, 3H), 0.83 (s, 3H).

Synthesis of (2,2,5-trimethyl-1,3-dioxan-5-yl)methyl 4-methylbenzenesulfonate (3): Compound (2) (10.9 g, 68.0 mmol) was dissolved in 34 ml of pyridine, and it was added dropwise to the stirred solution of p-toluenesulfonyl chloride (35.7 g, 187 mmol) in 48 ml of pyridine at 0°C under nitrogen. After the complete addition, the reaction mixture was stirred 48 h at room temperature. Then the reaction mixture was added dropwise to 100 ml of 40% ammonium chloride solution at 0°C. After complete addition, it was allowed to stir at room temperature for 2 h. Then it was filtered and washed with DI water until the pyridine smell was gone. Then the residue was dissolved in 25 ml of DCM and extracted with half saturated ammonium chloride and saturated NaCl solution. Yellow DCM solution was dried with anhydrous sodium sulfate. Then the solvent was evaporated, and the residue was placed under full vacuum for 12 h to give (3) as a yellow solid with 89% yield. ¹H NMR (500 MHz, CDCl₃) δ 7.86–7.79 (m, 2H), 7.38 (d, J = 7.6 Hz, 2H), 4.11 (s, 2H), 3.58 (s, 4H), 2.47 (s, 3H), 1.39 (d, J = 4.8 Hz, 3H), 1.25 (d, J = 4.7 Hz, 3H), 0.84 (s, 3H).

Synthesis of 5-(azidomethyl)-2,2,5-trimethyl-1,3-dioxane (4): Compound (3) (14.6 g, 46.4 mmol), NaN₃ (12.1 g, 186 mmol), water (10 ml), and DMF (80 ml) were stirred at 110°C for 48 h under reflux. The mixture was poured into 150 ml water and extracted four times with Et₂O (4 × 200 ml). The organic phase was dried over anhydrous MgSO_4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography with silica gel (100 g) and ethyl acetate/n-hexane (1:4) to give 7.48 g of a colorless liquid with an 87% yield. ¹H NMR (500 MHz, CDCl₃) δ 3.58 (d, J = 2.8 Hz, 4H), 3.51 (s, 2H), 1.40 (d, J = 13.8 Hz, 6H), 0.81 (d, J = 1.1 Hz, 3H).

Synthesis of 2-(azidomethyl)-2-methylpropane-1,3-diol (5): Compound (4) (7.05 g, 40.3 mmol) was dissolved in 35 ml of methanol. 7.00 g of a Dowex, acid resin was added, and the reaction mixture was stirred for 12 h at 50°C. When the reaction was complete, the Dowex was filtered off in a vacuum filter under a low vacuum and carefully washed with methanol. The methanol was evaporated to give 5.41 g of white crystals with a 93% yield. ¹H NMR (400 MHz, CDCl₃) δ 3.73–3.58 (m, 4H), 3.56–3.43 (m, 2H), 2.19 (s, 2H), 0.89 (d, J = 2.0 Hz, 3H).

Synthesis of A-MPA-4-AC (6): 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid was prepared using similar method mentioned in compound (2) synthesis. 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid (6.48 g, 37.2 mmol), 1,1′-carbonyldiimidazole (CDI) (9.05 g, 55.8 mmol) were dissolved in 30 ml of ethyl acetate and it was stirred 1 h at 50°C. CsF (0.75 g, 4.93 mmol), Compound (5) (1.80 g, 12.4 mmol) were dissolved in 10 ml of ethyl acetate separately, and it was slowly added to the reaction mixture under nitrogen at 50°C. It was stirred for 12 h. When the reaction was complete 200 ml DI water was added and allowed to stir for 2 h at room temperature. Then it was extracted with 1 M HCl (200 ml × 3), 1 M NaHSO₄ (200 ml × 3), 10% Na₂CO₃, saturated NaCl (200 ml), and it was dried under anhydrous MgSO₄. Ethyl acetate was evaporated to give 5.22 g of colorless oil liquid with a 92% yield. ¹H NMR (500 MHz, CDCl₃) δ 4.21 (d, J = 11.7 Hz, 4H), 4.13–4.09 (m, 4H), 3.68 (d, J = 11.7 Hz, 4H), 3.42–3.39 (m, 2H), 1.43 (d, J = 32.2 Hz, 12H), 1.18 (d, J = 1.6 Hz, 6H), 1.08 (d, J = 1.5 Hz, 3H).

Synthesis of A-MPA-4-OH (7): Compound (6) (5.00 g, 10.9 mmol) was dissolved in 20 ml of methanol. 5.00 g of a Dowex, acid resin was added, and the reaction mixture was stirred for 12 h at 50°C. When the reaction was complete the Dowex, acid resin was filtered off in a vacuum filter under a low vacuum and carefully washed with methanol. The methanol was evaporated to give 3.96 g of colorless liquid with a 96% yield. ¹H NMR (300 MHz, CDCl₃) δ 4.09 (s, 4H), 3.88 (d, J = 11.2 Hz, 4H), 3.78–3.67 (m, 4H), 3.43 (s, 4H), 3.38 (s, 2H), 1.12–1.09 (m, 6H), 1.09–1.07 (m, 3H).

Synthesis of A-MPA-4-ala (8): N-boc-alanine (3.70 g, 19.6 mmol), 1,1′-carbonyldiimidazole (CDI) (3.49 g, 21.5 mmol) were dissolved in 30 ml of dry ethyl acetate, and it was stirred for 1 h at 50°C. CsF (0.43 g, 2.81 mmol), compound (7) (1.23 g, 3.26 mmol) were dissolved in 5 ml of dry ethyl acetate and it was slowly added to the reaction mixture under nitrogen at 50°C. It was stirred for 12 h. When the reaction was complete 250 ml DI water was added and allowed to stir for 2 h at room temperature. Then it was extracted with 1 M HCl (200 ml × 3), 1 M NaHSO₄ (200 ml × 3), 10% Na₂CO₃, saturated NaCl (200 ml), and it was dried under anhydrous MgSO₄. Ethyl acetate was evaporated to give 3.15 g with a 91% yield. ¹H NMR (400 MHz, CDCl₃) δ 5.19 (s, 4H), 4.31–4.19 (m, 8H), 4.02 (d, J = 2.4 Hz, 4H), 3.38 (q, J = 6.2 Hz, 8H), 3.33 (s, 2H), 2.55 (t, J = 5.9 Hz, 8H), 1.27 (s, 6H), 1.02 (s, 3H).

Synthesis of 3,4-dichloro-1-(prop-2-yn-1-yl)-1H-pyrrole-2,5-dione (9): A total of 0.40 g of dichloromaleimide (2.40 mmol) and 2-ethylhexylbromide (0.48 ml, 2.9 mmol) and DMF (10 ml) in a 2-neck round bottom flask under nitrogen. The mixture was vigorously stirred at 140°C for 24 h. After that, the solution was quenched with 0.1 M HCl and extracted with diethyl ether. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure, and purification by silica gel column chromatography (30% DCM/Hexane) resulted in a cream-colored powder of N-(propargyl) dichloromaleimide (0.554 g, 90%). ¹H NMR (400 MHz, CDCl₃) δ 4.31–4.30(d), 2.23(s).

Synthesis of TRPZ-Propagyl (10): A mixture of 2-aminothiophenol (0.386 g, 2.8 mmol) and N-(propargyl) dichloromaleimide (0.30 g, 1.4 mmol) was dissolved in 24 ml of acetic acid. The reaction mixture was refluxed under a nitrogen atmosphere for 24 h. After being cooled to room temperature, the mixture was quenched with brine and extracted with ethyl acetate. The organic layer was separated, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The product was purified via flash column chromatography (30% chloroform/hexane) and obtained as orange solid (0.18 g, 50%). ¹H NMR (400 MHz, chloroform-d) ¹H NMR (400 MHz, CDCl₃) δ 7.72 (d, J = 8.0 Hz, 2H), 7.34–7.26 (m, 4H), 7.18 (ddd, J = 8.3, 7.2, 1.4 Hz, 2H), 5.20 (s, 2H), 2.29 (t, J = 2.5 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 148.79, 136.97, 130.48, 128.26, 127.11, 126.57, 111.17, 77.23, 73.36, 33.08. ¹H–¹H correlation spectroscopy (COSY) spectrum is provided in the SI. HRMS (ESI/Q-TOF) m/z: [M + H]⁺ Calcd for C₁₉H₁₁N₃S₂ 345.0394; Found 345.0365 with an isotope pattern similar to the predicted pattern.

Synthesis of TRPZ-bisMPA-amine-BOC (11): This procedure was modified from previously reported procedures (Ihre et al., 1998). To the stirred solution of A-MPA-4-ala (0.60 g, 0.56 mmol), TRPZ-propagyl (0.29 g, 0.84 mmol) in 8 ml of DMF, CuBr (322 mg, 2.21 mmol) was added under nitrogen flushing. After complete addition, N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA) (388 mg, 2.30 mmol) was added and allowed to stir under nitrogen at 55°C for 48 h. The reaction mixture was precipitated to 200 ml diethyl ether. After settling, the diethyl ether layer was decanted, and the remaining product was air-dried. This precipitation, decanting, and the air-drying process was repeated twice more. The crude product was dissolved in 100 ml of DCM. It was extracted with 0.1 M EDTA solution. It was further purified using size exclusion chromatography (Sephadex LH-20) to give 0.59 g of TRPZ-bisMPA-amine-Boc with 74% yield. ¹H NMR (400 MHz, DMSO-d ₆ ) δ 8.03 (s, 1H), 7.51 (d, J = 7.9 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.32 (t, J = 7.6 Hz, 2H), 7.22 (t, J = 7.6 Hz, 2H), 6.77 (s, 4H), 5.32 (s, 2H), 4.41 (s, 2H), 4.16 (d, J = 11.9 Hz, 8H), 3.94 (s, 4H), 3.12 (d, J = 6.4 Hz, 8H), 2.41 (t, J = 7.4 Hz, 8H), 1.34 (s, 36H), 1.17 (d, J = 20.4 Hz, 6H), 0.84 (s, 3H). HRMS (ESI/Q-TOF) m/z: [M + H]+ Calcd for C₆₆H₉₀N₁₀O₂₀S₂ 1407.5852; Found 1407.5813 with an isotope pattern similar to the predicted pattern.

Synthesis of TRPZ-bisMPA (12): TRPZ-bisMPA-amine-Boc was dissolved in 5 ml of chloroform and 2 ml of trifluoroacetic acid (TFA) was slowly added and stirred for 45 min. The reaction mixture was air-dried and dissolved in 10 ml of chloroform. It was added dropwise to 500 ml of diethyl ether and stirred for 2 h. It was filtered, and the precipitation procedure was repeated three times. Finally, the resulting solid was put under the vacuum for 24 h to give 0.39 g of the pure product with 93% yield. The formation of the TRPZ-bisMPA was confirmed via the disappearance of BOC groups (1.34 ppm). ¹H NMR (400 MHz, DMSO-d ₆ ) δ 8.07 (s, 1H), 7.53 (d, J = 7.8 Hz, 2H), 7.44 (d, J = 7.8 Hz, 2H), 7.33 (t, J = 7.7 Hz, 2H), 7.24 (t, J = 7.6 Hz, 2H), 5.33 (s, 2H), 4.42 (s, 2H), 4.22 (d, J = 11.5 Hz, 8H), 3.94 (s, 4H), 3.01 (q, J = 6.2 Hz, 8H), 2.64 (d, J = 7.1 Hz, 8H), 1.17 (s, 6H), 0.85 (s, 3H).

For the nanoprecipitation method (Chandrasiri et al., 2020), 100 μL tetrahydrofuran (THF) was used as the organic solvent to dissolve 2 mg of TRPZ-bisMPA separate glass vial. The solution was added dropwise to a separate vial of Milli-Q water at pH 7.0 (2 ml) while gently stirring to obtain a 1 mg/ml final concentration. THF was allowed to evaporate under a stream of nitrogen. NP solutions were allowed to equilibrate for 12 h before further study.

TRPZ-127 NPs was prepared and modified according to the previously reported procedure (Wu et al., 2017). First, TRPZ-PG (2 mg) was dissolved under rapid sonication in THF (2 ml). Then, a THF solution (1 ml) containing TRPZ-PG (1 mg/ml) and Pluronic F-127 (5 mg/ml) was used to prepare TRPZ-127 NPs by rapidly injecting the solution into deionized water (1 ml) under continuous sonication using a bath sonicator. After sonication for an additional 1 min, THF was evaporated under a nitrogen atmosphere.

Aggregate sizes and ζ-potentials measurements were carried out on a Malvern Instrument Zetasizer Nano ZS using a He–Ne laser with a 633 nm wavelength, a detector angle of 173^o at 25°C. The size measurements were performed in triplicate for each sample at 0.5 mg/ml concentration to ensure consistency. The morphological study of the aggregates formed from the NPs was carried out by TEM using a JEOL 1230 TEM operated at 100 kV to collect the TEM images using a Gatan Orius 831 bottom mounted CCD camera.

The absorption measurements were done on a Varian Cary-5000 spectrometer (Dorval, QC, Canada). While the fluorescence studies were performed on Horiba Quantamaster fluorimeter with a xenon lamp and PMT detector using glass cuvettes. Fluorescence quantum yields were measured with samples of low sample concentration (10^–5 M) and excited close to their maximum absorption. The spectroscopic energy gap ( E g o p t ) was calculated from the onset of absorbance (Li et al., 2012).

Human embryonic kidney (HEK293) cells were used for the assay. HEK cells were grown under standard conditions (37°C, 5% CO₂, DMEM media with 10% FBS). Nanoparticles were added to tissue culture media and allowed 24 h incubation period in cytotoxicity studies. Cytotoxicity of the nanoparticles was evaluated with a CyQUANT LDH Cytotoxicity Assay Kit (Invitrogen) using a microplate reader (BioTek Synergy H1). Following manufacturer protocols, both negative and positive controls are used in the assay. Experimental values are transformed based on two values: zero-cytotoxicity value (background) and 100% cytotoxicity value (cells treated with lysis buffer based on manufacturer protocol). Each experiment is represented by relative values based on the control values. Imaging of particle distribution in cells was done 30 min after the addition of 10 μg/ml NPs to culture media. TRPZ fluorescence in HEK cells was observed with a Leica Stellaris STED confocal microscope using both conventional and STED modes.

TRPZ, first discovered by Dimroth and Reicheneder (1969), possesses a simple yet fascinating molecular structure. Its pi-framework and redox properties have made it beneficial in the field of organic electronics. TRPZ, being planar in structure, can undergo efficient crystalline packing via intermolecular sulfur–sulfur interactions and hydrogen bonding between pyrrole hydrogens and thiazine nitrogens (Hong et al., 2008; Hong et al., 2009). The molecule has multiple reactive sites which can be functionalized to increase its solubility and be further developed using electron-withdrawing and electron-donating groups to afford donor-acceptor oligomers with exceptional optical properties.

Based on its unique properties, we aimed to expand the application of this unique emissive building block towards bioimaging. However, due to its hydrophobic nature, we sought to modify TRPZ via N-substitution of the central pyrrole to produce a self-assembling amphiphile. To induce amphiphilicity and promote self-assembly, bisMPA was selected as the dendritic segment. A hydrophilic moiety, its synthetic accessibility allows esterification under mild reaction conditions and provides a level of biodegradability and biocompatibility (Feliu et al., 2012). Additionally, it was capped with alanine to afford a cationic surface to increase colloidal stability, cellular uptake and better its water solubility (Gessner et al., 2002; Lipfert et al., 2014; Dragoman et al., 2017). This amphiphilic property is expected to make TRPZ capable of forming NPs independently, TRPZ-bisMPA NPs.

The resulting NP morphology is anticipated to be that of a micelle structure in which the dendron extends towards the hydrophilic water surface protecting the hydrophobic TRPZ portion within the core. The positively charged terminal ends of the amphiphile would effectively stabilize the NPs via electrostatic interactions.

In parallel, we compared NPs formed from TRPZ-bisMPA with conventional nanostructures in which a surfactant polymer is employed to encapsulate hydrophobic dyes. We encapsulated TRPZ-PG into a water-soluble polymer surfactant (Pluronic F-127), TRPZ-127 NPs. Although frequently employed, the encapsulation strategy has significant drawbacks (e.g., poor loading) that limit its application. Often the nature of the molecule being encapsulated has varying physiochemical properties that contrast to the solubilizing block of the amphiphilic polymer being employed. This, of course, leads to lower encapsulation efficiencies and can destabilize the resulting nanostructure (Jeong et al., 2020). By assessing the two NP systems, TRPZ-bisMPA NPs and TRPZ-127 NPs, we aim to explore the former as a versatile molecular bioimaging probe suitable for numerous biomedical applications.

The synthesis of the dendron segment, A-MPA-4-ala (8), is shown in Scheme 1, which consists of a seven-step synthetic route. In the first step, the 1,3-diol moiety of bisMPA (1) was protected to afford compound 2 by reaction of bisMPA with 2,2-dimethoxypropane and a catalytic amount of p-toluenesulfonic acid (TsOH) in dry acetone. In order to make the hydroxyl group a better leaving group, compound 2 was reacted with p-toluenesulfonyl chloride (TsCl) in pyridine to obtain compound 3. The next step, the azido group, was substituted using sodium azide (NaN₃) and DMF to obtain compound 4. The following step was carried out to deprotect dimethoxy moiety using an anion exchange resin-Dowex to obtain compound 5. Before proceeding to the next step, 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid was synthesized using a similar method to compound 2. Then, 2,2,5-trimethyl-1,3-dioxane-5-carboxylic acid was employed in a Malkoch esterification (García-Gallego et al., 2015) with compound 5, using 1,1′-carbonyldiimidazole (CDI) to activate the carbonyl group and cesium fluoride (CsF) as a catalyst to obtain compound 6. Dowex deprotection was done in the next step to obtain compound 7. The final step was esterification between beta-alanine and compound 7 to obtain A-MPA-4-ala.

The synthesis of TRPZ-bisMPA (12) consists of four synthetic steps (Scheme 2). First, dichloromaleimide was propargylated at the nitrogen position to obtain compound 9. TRPZ-PG was synthesized via a condensation method between 2-aminothiophenol and N-(propargyl) dichloromaleimide (compound 10). Then TRPZ-PG (10) and A-MPA-4-ala (8) were reacted using click-chemistry to obtain TRPZ-bisMPA-amine-Boc (compound 11). Finally, acid was used for deprotection of the tert-butyl carbamates (BOC) protected amine groups to afford a cationic terminated TRPZ-bisMPA amphiphile (compound 12).

The impact of the NPs on cell viability was tested by LDH assay (Figure 4A). Even at very high concentrations, a negligible effect was seen on cell viability. Analysis by Tukey ANOVA found no significant difference between conditions. In vivo testing will be needed to further assess the effect of the NPs on physiology; however, these results suggest our NPs will be well-tolerated in biological environments.

Confocal microscopy found that both TRPZ-bisMPA NPs and TRPZ-127 NPs accumulate most significantly in endosomes (Figures 4B–G). The cationic surface charge of TRPZ-bisMPA NPs leads to apparent cellular uptake via electrostatic interactions with anionic cell membranes. Similar results have been observed with other dendrimer-based NPs modified with anime groups (Morris et al., 2017; Ray et al., 2018; Ingle et al., 2020). Likely owing to the polar groups on the MPA dendron, TRPZ fluorescence is observed throughout the interior of the lysosome (Figure 4D).

TRPZ-127 NPs show not only accumulation in the lysosome but also labeling of various cellular membranes, including nuclear membranes (Figures 4E,F). Staining of the nuclear membrane indicates that TRPZ fluorescence has spread throughout the endomembrane system, trafficking all the way to the endoplasmic reticulum (ER). Stimulated emission depletion microscopy (STED) images of TRPZ-127 NPs and TRPZ-bisMPA NPs aid in comparing cellular uptake where fluorescence labeling is localized to the membrane of the organelle and not the interior, as observed with TRPZ-bisMPA NPs (Figures 4D,G; Supplementary Figure S17). It appears that TRPZ-127 is much more aggressively loading into cells; however, this is not the case. We speculate that the encapsulated dye may have escaped its NPs and, due to its hydrophobic nature, became embedded into membrane interiors. The unloaded dye being hydrophobic actively segregates into cells while TRPZ-bisMPA NPs remain intact and localized in the lysosome.