Repurposing Cyanine Photoinstability To Develop Near-Infrared Light-Activatable Nanogels for In Vivo Cargo Delivery

Abstract

The favorable properties of cyanines (e.g., near-infrared (NIR) absorbance and emission) have made this class of dyes popular for a wide variety of biomedical applications. However, many cyanines are prone to rapid photobleaching when irradiated with light. In this study, we have exploited this undesirable trait to develop NIR-nanogels for NIR light-mediated cargo delivery. NIR-nanogels feature a photolabile cyanine cross-linker (Cy780-Acryl) that can cleave via dioxetane chemistry when irradiated. This photochemical process results in the formation of two carbonyl fragments and concomitant NIR-nanogel degradation to facilitate cargo release. In contrast to studies where cyanines are utilized as photocages, our approach does not require direct chemical attachment to the cargo, thus expanding our ability to deliver molecules that cannot be covalently modified. We showcase this feature by encapsulating a palette of small-molecule chemotherapeutics that feature a structurally diverse chemical architecture. To demonstrate site-selective release in vivo, we generated a murine model of breast cancer. Relative to nonlight irradiated and drug-free controls, treatment with NIR-nanogels loaded with paclitaxel (a potent cytotoxic agent) and NIR light resulted in significant attenuation of tumor growth. Moreover, we show via histological staining of the vital organs that minimal off-target effects are observed.

Graphical Abstract

INTRODUCTION

Since the discovery of the first cyanine in the mid-1800s,¹ molecules belonging to this family of dyes have been employed as contrast agents in a diverse range of biomedical applications such as identification of blood vessels, sentinel lymph nodes, and tumors for fluorescence-guided surgery.^2,3 Additionally, cyanines have served as important scaffolds for the development of activity-based sensing probes for in vivo molecular imaging.^4–6 Some notable examples include sensors for metal ions,^7,8 nitric oxide,⁹ pH,¹⁰ and reactive oxygen species (ROS).^11,12 Beyond examples that exhibit absorbance and emission profiles in the visible and near-infrared (NIR) range, the cyanine core can be readily diversified to access congeners that extend into the SWIR (NIR-II) window (>950 nm) to enable deeper tissue penetration.^13–23 Despite their versatility and widespread use, cyanines, in general, are limited by their flat planar structures which contribute to their poor aqueous solubility; reactivity toward nucleophiles (e.g., biological thiols) that can disrupt the conjugated π-system; propensity to undergo oxidative decomposition; and instability to extended light exposure.

Exploiting the “drawbacks”, such as those mentioned above, to design new chemical tools and approaches based on aberrant cyanine chemistry is an innovative way to turn a negative trait into a favorable property. For instance, cyanines that are prone to aggregate due to poor solubility can be induced to form self-assembled particles that can be used as red-shifted imaging agents.^24,25 Likewise, the photoswitching behavior of cyanines that result from a thiol reversibly adding across the conjugated system is commonly employed for super-resolution microscopy.²⁶ Moreover, fluorogenic probes for ratiometric detection of ROS such as O₂^•−, H₂O₂, ONOO⁻, ClO⁻, etc., have been developed by linking an unstable cyanine prone to oxidative cleavage to a reference dye that is resistant to ROS-mediated decomposition.^11,12,27 Lastly, Schnermann and co-workers have leveraged the photobleaching properties of heptamethine cyanine dyes to serve as NIR photocages,^28–31 which can release an appended molecule (e.g., gabapentin) through an intramolecular cyclization event after photolysis. However, it is notable that this uncaging strategy requires covalent appendage of the cargo to the cyanine photocage through a cleavable linker. As such, this approach is not suitable for molecules lacking a convenient handle for attachment. To overcome this challenge, we report the first cyanine-based nanogel system for NIR light-mediated drug delivery, which we have named NIR-nanogels. In addition to showcasing the generalizability of our approach by releasing structurally diverse cargo in vitro, we selected paclitaxel for encapsulation to assess efficacy in a murine model of breast cancer.

RESULTS AND DISCUSSION

Design and Synthesis of Cy780-Acryl.

Our design utilizes acrylamide-based nanogels, which are nanoparticles comprising a cross-linked hydrophilic network, as the cargo carrying system.^32–34 In addition to cross-linking with N-isopropylacrylamide (NIPAM), we developed Cy780-Acryl and incorporated it into the hydrogel backbone (Figure 1a). Specifically, Cy780-Acryl is a modified version of Cy7-Cl which features two acrylamide units on the peripheral of the molecule. Of note, a cyclohexenyl fused cyanine was chosen to serve as the cross-linker backbone, as opposed to an ICG core, owing to its enhanced stability and ease of synthetic manipulation. We hypothesize that light irradiation at 780 nm (the λ_max) will generate an excited state that can interact with molecular oxygen to form an unstable dioxetane intermediate. Subsequent collapse of the dioxetane will yield two carbonyl fragments which facilitate separation within the polymer structure, nanogel degradation, and subsequent cargo release (Figure 1b,c).

Figure 1.

(a) Chemical structures of NIPAM, Cy780-Acryl, and Cy780-Actamido. (b) Spontaneous decomposition of unstable 1,2-dioxetane cyanine intermediates yields ketone and aldehyde fragments. (c) Irradiation of intact nanogels with NIR light at 780 nm results in release of the encapsulated cargo (red circles). The Cy780-Acryl cross-link is shown in green.

In brief, the synthesis of Cy780-Acryl began with the nitration of 2,3,3-trimethylindolenine using a sulfuric acid and nitric acid mixture to afford 1 in 95% yield. The nitro intermediate was then reduced in the presence of SnCl₂ and hydrochloric acid, followed by Boc protection using Boc₂O to furnish 3 in 41% yield over two steps. Subsequently, 3 was alkylated with ethyl iodide to give 4 quantitatively. The Boc-protected cyanine 5 was obtained in 24% yield upon treatment of 3 with activated Schiff base 4 in the presence of acetic anhydride. Finally, the acrylamide units were appended by sequential Boc deprotection with TFA, followed by treatment with acryloyl chloride to afford Cy780-Acryl in 39% yield over two steps (Scheme 1). In addition to Cy780-Acryl, we also synthesized Cy780-Acetamido, a control cyanine which is incapable of cross-linking to probe the proposed mechanism of NIR light-mediated release (Figure 1a). Cy780-Acetamido was prepared in a similar manner using acetyl chloride in the final step (25% yield over two steps).

Scheme 1.

Synthesis of Cy780-Acryl and Cy780-Acetamido

Assessment of Photobleaching Properties.

With the various components in hand, we turned our attention to testing whether the amide moiety would impact the photophysical properties of the cyanine core. First, we analyzed the absorption spectrum and measured the extinction coefficient of Cy780-Acryl. Compared to the Cy7-Cl reference in DMSO, the λ_max (831 nm cf. 793 nm) and extinction coefficients (1.11 × 10⁵ M⁻¹ cm⁻¹ cf. 1.23 × 10⁵ M⁻¹ cm⁻¹) were similar. Next, we evaluated the photostability of the cross-linker. When Cy780-Acryl (as well as Cy780-Acetamido) was irradiated with an LED light source at 780 nm (300 mW power output, 47.3 μW/mm² maximum irradiance), we found complete degradation of the molecule after only 25 min based on the disappearance of the NIR absorbance band (Figure S1). This result is comparable to when Cy7-Cl is irradiated under identical conditions, demonstrating that the chemical modifications we made did not significantly alter photobleaching properties (Figure S2). Of note, when dissolved oxygen was removed prior to the initiation of photobleaching, we found the change in absorbance to be minimal, supporting the proposed dioxetane mechanism (Figure S3). Indeed, the anticipated carbonyl fragmentation products were detected via LC−MS when samples were irradiated in the presence of oxygen (Figure S4).

Fabrication of NIR-Nanogels.

Next, we turned our attention to fabricating NIR-nanogels with the following key properties in mind. First, we targeted a size distribution between 100−200 nm to ensure efficient tumor uptake.³⁵ Second, NIR light irradiation should be the only input that triggers cargo release. Finally, we aimed to develop a robust delivery system that exhibits minimal off-target toxicity prior to light activation. The synthesis of the NIR-nanogels was accomplished through a self-assembly method using reverse micelles in aqueous solution.^36,37 We selected this approach because the size distribution can be controlled by varying the concentration of each polymeric component, as well as the micelle composition. In brief, the NIPAM monomer and the Cy780-Acryl cross-linker were rapidly stirred in Milli-Q water in the presence of SDS at a molar ratio of 0.98 and 0.02, respectively. After the removal of dissolved oxygen from the solution, polymerization was initiated by adding ammonium persulfate (APS) and TEMED. The resulting nanogels were then purified via dialysis and analyzed using dynamic light scattering (DLS) measurements. We found an average hydrodynamic diameter of 125 nm ± 41 nm, which was within our target size range and a polydispersity index (PDI) of 0.15, indicating a normal size distribution (Figure S5). To support these results, we employed scanning electron microscopy (SEM) and obtained a size distribution of 110 nm ± 35 nm (Figure S6). It is noteworthy that the particles exhibited a spherical morphology, which is consistent with other nanogel systems.^34,38,39

In Vitro Evaluation of NIR-Nanogels.

To assess responsiveness to NIR light, the particles we obtained were irradiated at 780 nm. Although the loss of NIR absorbance was slower than the free dye, the requisite photobleaching was observed (Figure 2a). DLS analysis of the dark sample showed that there was no change in size (Figure 2b) and further, we found the particles were stable for up to 3 months. In contrast, the NIR light-treated samples were degraded after 30 min and showed a size increase of 47 nm by DLS (Figure 2b). Moreover, we observed a significant change in morphology from spheres to an irregular appearance, as well as clear signs of coalescence (Figure 2c,d). In comparison, hydrogels synthesized with the control non-cross-linker (Cy780-Acetamido), displayed a size distribution of 120 nm by DLS, with no observable absorbance coming from the cyanine after dialysis purification. As anticipated, exposure to NIR light did not result in a change of the particle size, highlighting the critical role played by the Cy780-Acryl cross-linker. Furthermore, NIR-nanogels were treated with a panel of biologically relevant analytes to assess potential issues with stability when applied in vivo. Reactive oxygen and nitrogen species (O₂^•−, H₂O₂, •OH, ONOO⁻, and ClO⁻) and biological thiols (cysteine and glutathione) were among those tested. Our results indicate that in each instance, our nanogel system exhibited good stability, presumably due to the hydrogel network that can protect Cy780-Acryl from decomposition (Figure 2e). For instance, although ONOO⁻ and ClO⁻ can rapidly decompose various cyanine dyes in free solution,⁴⁰ we only observed a 16 and 10% decrease in absorbance, respectively, even at high concentrations (0.1 mM). We also assessed nanogel stability across a pH range spanning from 4.2 to 12.0. Our findings indicate the NIR-nanogels are resistant to changes in pH (Figure 2f). Stability in the presence of serum was also evaluated owing to possible interactions in vivo; however, we did not find any effect on Cy780-Acryl absorbance upon coincubation (Figure S7).

Figure 2.

(a) Normalized absorbance spectra demonstrating time-dependent photobleaching of Cy780-Acryl. Time points = 0, 15, 30, 45, 60, 90, 120, 150, and 180 min. (b) Diameter of NIR-nanogels with and without NIR light irradiation measured via DLS (left) and SEM (right). SEM images of NIR-nanogels (c) before and (d) after NIR light treatment for 30 min (n = 300 for SEM). Scale bar represents 500 nm. (e) Normalized absorbance of nanogels exposed to biologically relevant reactive species. (f) Normalized absorbance of nanogels suspended in Britton−Robinson buffer (pH 4.2 to 12.0). Statistical analysis was performed using a two-tailed Student’s t-test (α = 0.05), ****P < 0.0001.

Cargo Selection, Encapsulation, and Light-Mediated Release.

After establishing a robust protocol to fabricate NIR-nanogels displaying the desired properties, we turned our attention to identifying the cargo to showcase its utility for in vivo applications such as the treatment of breast tumors. Cisplatin, gefitinib, and GW9662 were identified as candidates owing to the absence of a convenient covalent attachment point (e.g., carboxylate and hydroxyl) (Figure 3). In the context of mode of action, cisplatin was selected because it can trigger apoptosis of breast cancer cells (as well as other cancers) through binding to DNA base pairs to yield DNA adducts.⁴¹ Likewise, gefitinib is an epidermal growth factor receptor (EGFR) inhibitor based on an anilino quinazoline scaffold used to treat certain breast cancer types.⁴² Lastly, GW9662 is an N-phenylbenzamide analog that can selectively and irreversibly inhibit unregulated peroxisome proliferator-activated receptor-γ (PPARγ) found in breast cancer.⁴³ It is noteworthy that we also selected paclitaxel for encapsulation due to its ubiquitous use in breast cancer treatment.⁴⁴ Cargo encapsulation using standard sonication protocols proved to be challenging in our hands as we were unable to achieve consistent loading results. After extensive screening, we obtained an optimized procedure that yielded reliable encapsulation results for each drug. We discovered that slow addition (versus bolus addition) of the drug to NIR-nanogel samples was essential to maintain a homogeneous suspension of the particles in solution. Furthermore, the inclusion of at least 10% ethanol as a solubilizing cosolvent was necessary for drug encapsulation. However, beyond 25% ethanol content, we observed swelling of the hydrogels, leading to an increased hydrodynamic diameter above our targeted size distribution of 100−200 nm. After removal of the nonencapsulated drug, we established a method to assess passive leeching of the contents by dialyzing the drug-loaded sample against Milli-Q water. Small aliquots were removed at various time points and compared to the initial cargo concentration. We found that cisplatin displayed the highest degree of leeching (~50%), while gefitinib showed a concentration loss of ~6% after 3 hours of dialysis. On the contrary, no passive release of GW9662 was observed. The corresponding data for paclitaxel also demonstrates minimal leeching (Figure S9). In general, the extent of passive cargo loss was determined by placing a photobleached sample of each drug-loaded NIR-nanogel in 3 kDa MWCO dialysis tubing for dialysis against DI water. Aliquots were taken to determine the absorbance at various time points for comparison against the corresponding calibration curve.

Figure 3.

Chemical structures of the encapsulation cargo employed in this study.

Drug Delivery in a Murine Model of Breast Cancer.

Next, a murine model of breast cancer was established by inoculating BALB/c mice (6−8 weeks old) with 4T1 cells. The animals were then randomly divided into two groups. Once the tumors grew to ~150 mm³ in volume, all animals were treated with paclitaxel-loaded nanogels (NIR-nanogel (Pac)) at a dose of 2 mg/kg via systemic administration. After 30 min, the experimental group of animals were treated with NIR light from a 780 nm LED positioned 2 cm away from the tumor site. Of note, the total irradiation duration was 30 min, which was separated into two 15 min sessions with a 15 min interval. Since oxygen is required to fragment Cy780-Acryl, we hypothesized that this sequence would be critical to ensure that the tumor adequately reoxygenates. Each mouse was treated every other day for a total of three treatments. Compared to the control group of animals that did not receive light, the tumors were 44% smaller as determined by the caliper method. We confirmed these results by excising the tumors after the animals were sacrificed (Figure 4a–d). To verify that the nanogel delivery system can reduce the off-target toxicity that is typically associated with systemic administration of a drug such as paclitaxel, we harvested the vital organs (heart, kidneys, liver, muscle, and spleen) from a cohort of mice administered with paclitaxel-loaded NIR-nanogels to perform H&E staining (Figure 5). Analysis of the histological data revealed no discernable difference relative to control mice. This is contrary to histology performed on excised tumors from animals treated with paclitaxel-loaded NIR-nanogels, followed by either light irradiation or no light. Our results show massive differences in the nuclear staining pattern between the two groups, indicating cell death results from drug release (Figure S10). Finally, we performed a series of experiments to determine whether attenuation of tumor growth involved singlet oxygen-mediated cell death. First, we used esterase-activated 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) to detect ROS (including singlet oxygen) from a sample of light-irradiated NIR-nanogel. We found that upon irradiation, the signal from the DCF scavenger increased by fourfold. Next, we performed a complementary animal experiment to determine if the in vitro result translates into an in vivo setting. Tumors were treated with unloaded NIR-nanogels and then dosed with light. We hypothesized that if singlet oxygen production in vivo contributed to the observed attenuation of tumor growth, irradiation of nanogels without paclitaxel would result in smaller tumors relative to the dark controls. As shown in Figure S11, no statistical difference based on tumor volume or tumor mass measurements were noted. The discrepancy between the in vitro and in vivo experiments is likely due to a lower effective singlet oxygen concentration within the tumor.

Figure 4.

Photographs of 4T1 tumors excised from BALB/c mice after systemic treatment with NIR-nanogel (pac) (a) without or (b) with NIR light irradiation. Scale bar represents 1 cm. (c) Tumor volume and (d) tumor mass measurements of the tumors shown in (a) and (b). Statistical analysis was performed using a two-tailed Student’s t-test (α = 0.05), *P < 0.05.

Figure 5.

Representative H&E staining of the heart, kidney, liver, spleen, and muscle tissue harvested from mice with and without NIR-nanogel (pac) treatment. Scale bar represents 50 μm.

CONCLUSIONS

In closing, we have developed the first NIR light-activatable nanogel delivery system that utilizes cyanine photobleaching chemistry. Light, especially at wavelengths within the NIR window (650 to 900 nm), is one of the most powerful triggers because it does not generally harm tissue (minimal photo-toxicity) and can penetrate deep within the body because there are fewer endogenous chromophores present to absorb or scatter incident light. Moreover, light can be focused onto a region of interest to afford remarkable spatiotemporal control. This is especially the case in the context of fluorescent- and photoacoustic-based imaging studies,^45–48 optogenetic stimulation,^49,50 light-activatable analyte donor systems,^51–53 and photodynamic therapy (PDT)⁵⁴ or photothermal therapy (PTT).⁵⁵ With regards to the latter two applications, we envision existing laser and LED systems used in humans can also be compatible with the NIR-nanogel technology, highlighting its translational potential. As shown in the current work, NIR-nanogels can encapsulate a diverse range of cargo, including chemotherapeutics that do not contain a site for covalent modification. The ability to keep a drug encased while the NIR-nanogels are in systemic circulation spares the rest of the body from undesirable off-target toxicity and offers an exciting complimentary approach to conventional prodrug design strategies. Through targeted irradiation, we were able to significantly attenuate tumor growth (via local paclitaxel release), while avoiding collateral damage of healthy tissue as demonstrated by histological staining experiments. Despite these excellent in vivo results obtained from this pilot study, we anticipate further tuning of the amount of cargo loaded, as well as optimization of the precise time and duration of light treatment will improve NIR-nanogel performance. In addition to such efforts, which are currently underway, we are also exploring the encapsulation of other cargo for a diverse range of biomedical applications.

EXPERIMENTAL DETAILS

Synthetic Procedure.

2,3,3-Trimethyl-5-nitro-3H-indole (Compound 1).

2,3,3-Trimethylindolenine (1.0 g, 6.3 mmol, 1 equiv) was cooled to 0 °C and mixed with sodium nitrate (0.59 g, 6.9 mmol, 1.1 equiv). While stirring, concentrated sulfuric acid (16.0 mL, 300 mmol, 48 equiv) was added dropwise over 10 min. The solution was stirred at the same temperature for 1 h. The mixture was then neutralized with solid NaOH at 0 °C. The material was then dissolved with EtOAc and washed three times with water (20 mL). The organics were collected, dried over sodium sulfate, filtered, and concentrated under reduced pressure resulting in 1.2 g of the titled compound as a red/orange solid in 95% yield, which was used without further purification. ¹H NMR (500 MHz, CDCl₃) δ 8.26 (dd, J = 8.5, 2.3 Hz, 1H), 8.16 (d, J = 2.3 Hz, 1H), 7.61 (d, J = 8.5 Hz, 1H), 2.35 (s, 3H), 1.37 (s, 6H). ¹³C NMR (125 MHz, CDCl3) δ 16.11, 22.88, 54.65, 117.31, 120.20, 124.70, 145.81, 146.83, 159.09, 194.27.

2,3,3-Trimethyl-3H-indol-5-amine (Compound 2).

Compound 1 (1.0 g, 4.9 mmol, 1 equiv) was suspended in a 6 M HCl solution (27 mL). Tin(II) chloride dihydrate (6.1 g, 27 mmol, 5.5 equiv) was then added to the reaction and heated and stirred at 100 °C for 2 h. The mixture was cooled to room temperature and then neutralized with aqueous NaOH. The material was extracted three times with EtOAc (20 mL), the organics were collected, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude material was then purified via silica flash column chromatography using a gradient of 0−100% EtOAc/hexanes (v/v) resulting in 0.77 g of the titled compound as a yellow solid in 90% yield. ¹H NMR (500 MHz, CDCl₃) δ 7.29 (d, J = 8.1 Hz, 1H), 6.61 (d, J = 2.3 Hz, 1H), 6.59−6.56 (m, 1H), 3.47 (s, 2H), 2.20 (s, 3H), 1.24 (s, 6H). ¹³C NMR (125 MHz, CDCl₃) δ 15.25, 23.36, 53.47, 109.02, 113.95, 120.26, 144.38, 146.09, 147.39, 184.27.

tert-Butyl (2,3,3-Trimethyl-3H-indol-5-yl)carbamate (Compound 3).

Compound 2 (750 mg, 4.3 mmol, 1 equiv) was dissolved in 1,4-dioxane (5.2 mL, 60.3 mmol). Di-tert-butyl dicarbonate (1.13 g, 5.2 mmol, 1.2 equiv) was added. The reaction was heated and stirred at 100 °C for 2.5 h. The reaction was cooled to room temperature and the volatiles were removed under reduced pressure. The resulting crude material was then purified via silica flash column chromatography using 1:1 EtOAc/hexanes (v/v) resulting in 936 mg of the titled compound as an orange solid in 79% yield. ¹H NMR (500 MHz, CDCl3) δ 7.61 (s, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.00 (dd, J = 8.2, 2.3 Hz, 1H), 6.54 (s, 1H), 2.25 (s, 3H), 1.52 (s, 9H), 1.29 (s, 6H). ¹³C NMR (125 MHz, CDCl3) δ 15.49, 23.24, 28.52, 54.05, 80.62, 112.58, 117.92, 119.93, 136.07, 146.86, 153.01, 187.20.

5-((tert-Butoxycarbonyl)amino)-1-ethyl-2,3,3-trimethyl-3H-indol-1-ium (Compound 4).

Compound 3 (830 mg, 3.03 mmol, 1 equiv) was dissolved in MeCN (15 mL). Iodoethane (1.22 mL, 15.1 mmol, 5 equiv) was added and the reaction was refluxed at 85 °C for 12 h. The reaction was cooled to room temperature, dissolved with a minimal amount of acetone and slowly dropped into a flask containing chilled diethyl ether. The resulting precipitate that formed was collected via vacuum filtration and dried under vacuum to obtain compound 4 in quantitative yields, which was used without further purification. ¹H NMR (500 MHz, CDCl₃) δ 8.01 (d, J = 5.9 Hz, 1H), 7.84 (s, 1H), 7.58 (d, J = 8.6 Hz, 1H), 4.55 (q, 2H), 2.94 (dd, J = 5.5, 2.4 Hz, 3H), 2.16−2.08 (m, 2H), 1.52 (s, 9H), 1.48−1.41 (m, 9H). ¹³C NMR (125 MHz, CDCl₃) δ 13.86, 16.54, 23.24, 23.34, 28.47, 28.58, 31.24, 45.42, 54.69, 113.48, 113.55, 115.75, 119.33, 135.05, 141.60, 142.98, 153.01.

Cy780-Acryl.

Synthesis of Cy780-Acryl was performed over three steps. First, compound 3 (1.0 g, 2.955 mmol, 2 equiv) was mixed with (E)-2-chloro-3-(hydroxymethylene)cyclohex-1-ene-1-carbaldehyde (255 mg, 1.477 mmol, 1 equiv). The solids were then dissolved in acetic anhydride (5 mL) under a nitrogen atmosphere. Sodium acetate (242 mg, 2.96 mmol, 2 equiv) was added and the reaction was heated to 130 °C for 1 hour. The reaction was allowed to cool to room temperature and the resulting precipitate that formed was collected via vacuum filtration and washed sequentially with diethyl ether and an aqueous solution of potassium iodide. The solid was collected and dried under vacuum and further purified via silica gel flash column chromatography using 7:93 MeOH/CH₂Cl₂ (v/v). The intermediate (105 mg) was dissolved in CH₂Cl₂ (515 μL) and treated with 2,2,2-trifluoroacetic acid (197 μL, 18 equiv) and stirred at room temperature for 12 h. The reaction was neutralized using a saturated aqueous solution of sodium bicarbonate and extracted with CH₂Cl₂. The organic fractions were combined and washed with a solution of potassium iodide. The organic layer was collected, dried over sodium sulfate, and concentrated under reduced pressure. The material was used without further purification. This material (67 mg) was dissolved in MeCN (1.25 mL) under a nitrogen atmosphere, cooled to 0 °C, and treated with sodium carbonate (65.8 mg, 621 μmol, 5 equiv). A solution of acryloyl chloride (37.15 μL, 457 μmol, 3.7 equiv) in MeCN (100 μL) was added to the reaction dropwise. The reaction was allowed to warm to room temperature, then potassium iodide was added (246 mg, 1.48 mmol, 12 equiv). The mixture was allowed to stir for 12 h. The crude material was diluted with CH₂Cl₂ and washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was then purified via silica gel flash column chromatography initially using 1:19 MeOH/CH₂Cl₂ (v/v) and then 3:17 MeOH/CH₂Cl₂ (v/v) to give Cy780-Acryl (19 mg) as a green metallic solid in 25% yield. ¹H NMR (500 MHz, CD₃OD) δ 8.42 (d, J = 14.1 Hz, 2H), 7.96 (d, J = 1.9 Hz, 2H), 7.61 (dd, J = 8.6, 1.9 Hz, 2H), 7.32 (d, J = 8.6 Hz, 2H), 6.50−6.36 (m, 4H), 6.28 (d, J = 14.1 Hz, 2H), 5.81 (dd, J = 9.6, 2.2 Hz, 2H), 4.21 (q, J = 7.2 Hz, 4H) 2.74 (t, J = 6.0 Hz, 4H), 1.97 (p, J = 6.1 Hz, 2H), 1.74 (s, 12H), 1.42 (t, J = 7.2 Hz, 6H). ¹³C NMR (125 MHz, CD₃OD) δ 173.10, 166.03, 150.63, 144.93, 143.50, 139.46, 137.86, 132.27, 128.12, 127.90, 121.64, 115.83, 112.27, 102.07, 71.53, 50.73, 40.44, 28.27, 27.34, 22.16, 12.51.

NIR-Nanogel Synthesis.

NIR-nanogels were prepared by mixing 200 μL of a 20 mg/mL SDS solution in a 50 mL round-bottom flask equipped with a magnetic stir bar. Subsequently, 1.44 mL of a 10 mg/mL NIPAM solution was added followed by 17.76 mL of Milli-Q water. The flask was placed on an ice bath and the mixture was stirred on a magnetic stir plate (500 rpm). Cy780-Acryl (1.6 mg) was then dissolved in MeOH (600 μL) and this was added to the reaction vessel resulting in a total combined concentration of 6.5 mM monomer and cross-linker. The mixture was allowed to stir at the same temperature while dry nitrogen gas bubbled through the mixture. After 30 min, 46 μL of a 60 mg/mL solution of APS was added slowly, followed by 44 μL of a 10% v/v TEMED solution in Milli-Q water. The mixture was allowed to stir at 0 °C and slowly warmed to room temperature for at least 12 h. The sample was dialyzed using a 3.5 kDa MWCO dialysis tubing against DI water for 2 days with water changes twice a day.

Paclitaxel Loading.

To encapsulate paclitaxel, 4.5 mL of the above nanogel solution was placed in a 20 mL vial equipped with a magnetic stir bar. EtOH (71 μL) was then added, and the solution was stirred at 600 rpm on a magnetic stir plate. A 1.75 mM solution of paclitaxel in EtOH (429 μL) was added dropwise while stirring. The solution was allowed to stir overnight in the dark. Excess drug was removed by centrifuging the nanogels at 10,000× g for 20 min, removing the supernatant and washing the nanogels with DI water three times and finally resuspending in PBS buffer for next experiments.

ABBREVIATIONS

SDS

sodium dodecyl sulfate

TEMED

N′-tetramethyl ethylenediamine

SWIR

short-wave infrared

UV−Vis−NIR

ultraviolet−visible−near-infrared

LED

light-emitting diode

LC−MS

liquid chromatography−mass spectrometry

MTT

3-(4,5-dimethylthiazole-2-yl)-2,5-diphenylte-trazolium bromide

H&E

hematoxylin and eosin