Hybrid Donor−Acceptor Polymer Particles with Amplified Energy Transfer for Detection and On-Demand Treatment of Breast Cancer

Abstract

Judicious combination of semiconducting polymers with alternating electron donor (D) and acceptor (A) segments created hybrid nanoparticles with amplified energy transfer and red-shifted emission, while simultaneously providing photothermal capabilities. Hybrid D−A polymer particles (H-DAPPs) passively localized within orthotopic breast tumors, serving as bright fluorescent beacons. Laser stimulation induced heat generation on par with gold nanorods, resulting in selective destruction of the tumor. H-DAPPs can also undergo multiple thermal treatments, with no loss of fluorescence intensity or photothermal potential. These results indicate that H-DAPPs provide new avenues for the synthesis of hybrid nanoparticles useful in localized detection and treatment of disease.

Graphical Abstract

Donor Acceptor (D−A) conjugated polymers were originally developed to aid in the production of more efficient organic solar cells, because photoexcitation generates a high yield of excitons, which decay into free charge carriers.¹ However, a major challenge remains reducing charge recombination, as this leads to energy losses and poor device performance.²⁻⁴ The complex mechanisms of charge recombination have been investigated by many groups, especially with regards to bulk heterojunction solar cells that comprise blends of conjugated polymers.^2,5−10 On the basis of the previous understanding of polymer blends in organic energy devices, the focus of the current work is to capitalize on the heat produced by charge recombination. Confinement of the D−A polymers into a nanoparticle minimizes interfacial regions and exciton diffusion, hence driving charge recombination, leading to generation of heat.^4,8,11 Some of the optimal D−A polymers for solar applications are designed to have strong absorption in the near-infrared (NIR), which is an ideal therapeutic window due to minimal tissue absorption, autofluorescence and scatter.¹² Nanoparticles (NPs) composed of optically tunable NIR- absorbing polymers offer flexibility in the various combinations of polymer subtypes they contain, facile synthesis, robustness, and potential for photothermal therapy (PTT).¹³⁻¹⁷ Also, NPs utilized for PTT provide greater benefits if they can be imaged to ensure their localization to a specific region or disease.

Motivation along this front has already transpired with the development of photostable D−A polymer particles (DAPPs), as nontoxic agents useful for photoacoustic and fluorescence imaging.¹⁸⁻²² Two complicating factors are that the quantum yield of conjugated polymers decreases at longer wavelengths and polymer aggregation can lead to fluorescence selfquenching.^21,23 Recently, NPs composed solely of poly[(9,9-dihexylfluorene)-co-2,1,3-benzothiadiazole-co-4,7-di(thiophen-2-yl)-2,1,3-benzothiadiazole] (PFBTDBT10), have been shown to retain their far-red and NIR fluorescence.²⁴ The aim of the current work was to evaluate specific formulations of Hybrid -DAPPs (H-DAPPs) composed of PFBTDBT10 for fluorescence imaging and poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b’]dithiophene-2,6-diyl-alt-2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe) for heat generation, for use as multimodal agents for detection and PTT of breast cancer (Figure 1a).

Figure 1.

Synthesis and characterization of PCPDTBSe and PFBTDBT10 nanoparticles. (a) Molecular structures of PCPDTBSe and PFBTDBT10, and schematic visualization of polymer chain packing in individual polymer nanoparticles. (b) Ultraviolet−visible absorption spectra (solid lines) and fluorescence spectra (dashed line) of PCPDTBSe (green) and PFBTDBT10 (red). As PCPDTBSe does not generate any fluorescent signal, one is not shown. Inset: photographic image of PCPDTBSe and PFBTDBT10 nanoparticle solutions, and corresponding TEM images. (c) Heating curve of gold nanorods (red line), gold nanoshells (blue line), and PCPDTBSe nanoparticles (green line) upon exposure to 229 J/cm² of continuous wave 800 nm light. Schematic of H-DAPPs where the polymer chains may serve as molecular spacers within the nanoparticle. (d) Absorption (solid line) and fluorescence spectra (dashed line) of H-DAPPs. The inset is a representative TEM image of nanoparticle morphology and solution color. (e) Heating curves of H-DAPPs exposed to 229.3 J/cm² (blue line) or 305.7 J/cm² (purple line) of 800 nm light.

RESULTS AND DISCUSSION

NMR, UV−vis absorption, and gel permeation chromatography characterization of the synthesized D−A polymers are provided in Figure S1. Before preparing H-DAPPs, we individually synthesized water-soluble PCPDTBSe (25.8 kDa) and PFBTDBT10 (43.1 kDa) NPs using nanoprecipitation to yield green and red nanoparticle solutions, respectively (Figure 1b, inset). Both nanoparticles were spherical (Figure 1b, inset), and their hydrodynamic diameters were found to be 120 nm for PCPDTBSe and 115.3 nm for PFBTDBT10, with polydispersity indices (PDIs) of 0.301 and 0.158, respectively. The corresponding zeta potentials were −12.3 mV for PCPDTBSe and −27.9 mV for PFBTDBT10 NPs. PCPDTBSe NPs have two absorption peaks, one at 438 nm and the other at 760 nm, whereas PFBTDBT10 NPs absorb at 322 and 465 nm, respectively (Figure 1b). Upon 465 nm excitation, PFBTDBT10 NPs reveal a large Stokes shift of 191 nm, leading to a peak fluorescence emission at 656 nm with a quantum yield of 0.31; however, no fluorescence was detected when PCPDTBSe NPs were stimulated with 465 nm light. The heating efficacy of PCPDTBSe NPs was compared to two commercially available photothermal nanomaterials: gold nanorods and gold nanoshells (Figure 1c), both with dominant absorption peaks near 800 nm. On an equivalent mass basis, when exposed to 229.3 J/cm² of 800 nm light, PCPDTBSe NPs generate as much heat as gold nanorods, and significantly more than nanoshells.

To develop hybrid NPs with fluorescence and photothermal capabilities, PCPDTBSe and PFBTDBT10 were combined at different ratios prior to nanoprecipitation, yielding an orange solution (Figure 1d, inset). The D−A polymer mass ratios, absorbance, fluorescence, and size characterization of the various formulations of H-DAPPs are provided in Figure S2. The goal of developing H-DAPPs was to provide a nanoparticle that was optically detectable, aqueously stable and capable of PTT. These qualities will be described below for what was found to be the ideal polymer ratio; 95% PFBTDBT10 to 5% PCPDTBSe. As with PCPDTBSe and PFBFDBT10 NPs alone, H-DAPPs are spherical (Figure 1d, inset), and have a hydrodynamic diameter of 148.1 nm, with a PDI of 0.172, and zeta potential of −17.5 mV. The absorption spectrum of the H-DAPPs shows peaks at 322 and 464 nm indicating the presence of PFBDBT10 and a smaller peak at 760 nm, corresponding to PCPDTBSe (Figure 1d). The fluorescence spectrum of the H-DAPPs and the corresponding quantum yield, 0.055, indicates significant quenching of PFBTDBT10 fluorescence by the close association with PCPDTBSe. Amplified energy transfer, due to exciton diffusion, shifts the fluorescence emission into the NIR, with a peak at 825 nm. Similar phenomenon has been described previously for other conjugated polymer NPs.^25,26 Mild quenching was observed when aliquots of individual PCPDTBSe and PFBTDBT10 nanoparticles were mixed together (Figure S3a), compared to the H-DAPPs formulation. In addition, the 825 nm fluorescence peak due to amplified energy transfer is not observed when nanoparticles composed of either PCPDTBSe or PFBTDBT10 are mixed in the same solution. This is confirmed by the change in Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) (Figure S2c), with the appearance of two new bands around 3384 and 1060 cm⁻¹, which may be attributed to OH/NH bond stretching vibrations, and indicative of an altered interface between the two polymers and Pluronic F127.

The H-DAPPs generate heat when stimulated by 800 nm light (Figure 1e), and have the capacity to reproducibly generate heat over multiple cycles (Figure S3b). Additionally, photothermally- induced heat cycles do not significantly affect the absorption or fluorescence spectra of H-DAPPs (Figure S3c,d). Prior to their evaluation in a biological system the H-DAPPs required sterilization, which for nanoparticles is most often performed using filtration techniques. Since polymer solar cells are often annealed to improve their crystalline fraction and enhance device performance, we evaluated the potential for steam sterilization (autoclaving) of H-DAPPs.^2,27 H-DAPPs were incredibly robust, with no precipitation or aggregation incurred by autoclaving.²⁸ There was a 27 nm decrease in the hydrodynamic diameter, resulting in the autoclaved H-DAPPs having a size of 121.1 nm, and a PDI of 0.218. One possibility for the reduction in nanoparticle size may be attributed to reorganization and closer proximity of the polymer chains in the nanoparticle due to the increased temperature and pressure applied during autoclaving. The Zeta potential increased slightly, from −17.5 to −16.8 mV, which may be attributed to dissociation of the Pluronic F127 surfactant at the surface of the nanoparticle. A minor hyperchromic shift was observed near 450 nm following thermal sterilization (Figure S3e), further alluding to the potential for closer proximity of the polymer chains within the nanoparticles. There was a corresponding increase in fluorescence near 650 nm, due to the hyperchromic shift induced by autoclaving (Figure S3f).

The H-DAPPs exhibited good colloidal stability, as well as uniform absorption, and fluorescence spectra after a 30-day exposure to ambient light in a variety of solutions (Figure S4). The hydrodynamic diameter and zeta potential varied little at pH 4 or 10; however, phosphate buffered saline (PBS) resulted in a mild positive shift in the zeta potential (from −15.4 to −3.8 mV). Incubation in 10% fetal bovine serum resulted in a strong negative shift (from −15.4 to −35.6 mV), which was accompanied by a slight increase in hydrodynamic diameter, indicative of serum protein adsorption.

After 24 h of incubation in the absence of 800 nm light, HDAPPs did not exhibit any reduction in the viability of BALB/c CL.7 (noncancerous breast epithelial), EO771, or 4T1 (murine breast cancers) cells (Figure S5a). Prolonged exposure (7−10 days) of the cells to H-DAPPS also did not have a significant impact on reducing cell viability (Figure S5b). Upon stimulation with 800 nm laser light at fluences of either 229.3 J/cm² or 305.7 J/cm², the onset of cell death was observed at 50 or 80 μg/mL, with 90−100% ablation of the cells using HDAPPs concentrations above 80 μg/mL (Figure S5c, d).

The fluorescence imaging and photothermal ablation capabilities of H-DAPPs were evaluated in vivo using bioluminescent 4T1 breast cancer cells for developing a tumor in the mammary fat pad of mice. The mice were separated into 4 treatment groups: (1) PBS (n = 3), (2) PBS + NIR irradiation (n = 3), (3) H-DAPPs (n = 5), and (4) H-DAPPs + NIR irradiation (n = 5). H-DAPPs were readily detectable using fluorescence imaging following intratumoral injection, and laser treatment groups were irradiated with 229.3 J/cm² of 800 nm light. A statistically significant delay in tumor growth was observed in mice treated with both H-DAPPs and NIR light (Figure 2a), and this correlated with prolonged animal survival (Figure 2b). In an animal that received intratumoral injection of H-DAPPS (no NIR light), the fluorescent signal was still observed 15 days postinjection (Figure 2c). Analysis of the organs revealed that nanoparticles had prolonged retention in the tumor, and no other organs had evidence of H-DAPPs, as shown in Figure 2d. This indicates that intratumoral H-DAPPs do not appear to elicit an immune response that would facilitate their immediate clearance from the tumor, in the absence of PTT.

Figure 2.

Intratumoral Injection of H-DAPPs in vivo. (a) Tumor volume progression over time. Treatment occurred on Day 0. Error bars are standard deviation. * denotes a significance p < 0.05. (b) Kaplan−Meier survival curve. ** denotes a significance of p < 0.01 with respect to all other groups. (c) Fluorescent images taken on IVIS of one individual mouse treated with H-DAPPs only (no NIR). (d) Representative photograph and fluorescence image of organs from an animal treated with intratumoral H-DAPPs only (no NIR). Organs top row: lungs, liver, spleen. Organs bottom row: kidneys, heart, tumor. Scale bar is 2 cm.

The H-DAPPs were evaluated for their potential to reach the breast tumors passively following intravenous delivery in the same orthotopic mammary fat pad tumor model. The mice were assigned to 1 of 3 treatment groups with n = 7 for all groups, including: (1) PBS + NIR irradiation, (2) H-DAPPs, or (3) H-DAPPs + NIR irradiation. An optimal treatment time for NIR irradiation following H-DAPPs accumulation in the tumor was determined by imaging mice immediately, 6, and 24 h after injection. It was found that H-DAPPs had good accumulation in the tumor at 24 h (Figure 3a), and passive accumulation was calculated to be 3.32 ± 0.5 μg, or 1.66% of the total systemic dose, possibly due to the enhanced permeability and retention (EPR) effect.^29,30 Based on this result, groups (1) and (3) were irradiated with 305.7 J/cm² of 800 nm continuous wave light 24 h after tail vein administration of PBS and H-DAPPs, respectively. Similar to direct injection, H-DAPPs combined with NIR light results in a statistically significant delay in tumor growth (Figure 3b), which corresponded to increased overall survival (Figure 3c), and decreased the bioluminescence signal from the breast cancer cells (Figure 3d). Fluorescence analyses of organs from animals in group 2 demonstrate that H-DAPPS could be detected 18 days after systemic delivery and the concentrations in liver, spleen, and tumor were calculated to be 3.3 ± 0.4 μg, 1.6 ± 0.4 μg, and 1.4 ± 0.7 μg, respectively (Figure S6). These mice also exhibited normal kidney, lung, heart, spleen and bone marrow tissue morphologies. Those mice treated with laser and saline had extramedullary hematopoiesis in the liver due to the impact of the tumor burden. However, two of the mice treated with H-DAPPs and exposed to NIR irradiation who responded well to therapy showed normal liver morphology, concluding that photothermal treatment using H-DAPPs was beneficial for aiding in the return of normal liver pathology by resolving the tumor.

Figure 3.

Systemic Injection of H-DAPPs in vivo. (a) Fluorescence images taken 0, 6, and 24 h after tail vein injection of H-DAPPs. (b) Tumor volume plotted versus time. Laser irradiation occurred on day 0. Error bars are standard error of the mean. * denotes a significance of p < 0.05. (c) Kaplan−Meier survival curves. * denotes a significance of p < 0.05 with respect to HDAPP alone. (d) Luminescent signal of luciferase transfected 4T1 cells across groups at days 0 and 11.

In summary, we have demonstrated that H-DAPPs have bright fluorescence for in vivo detection. They are colloidally and optically stable, retaining their fluorescence and absorption spectra through autoclaving, multiple laser excitations, and over 30 days in ambient light. The H-DAPPs were found to exhibit minimal cytotoxicity and are capable of generating efficient photothermal ablation. The H-DAPPs were utilized by both direct tumoral injection and systemic administration in an orthotopic murine breast cancer model and, in combination with NIR laser irradiation, reduced tumor burden and prolonged animal survival. We have also observed that H-DAPPs did not induce significant toxicity to lungs, liver, spleen, kidneys, or heart upon systemic administration. In conclusion, H-DAPPs are a promising theranostic nanoparticle for in vivo photothermal ablation and fluorescence imaging of breast cancer.

METHODS

Synthesis of Hybrid, PFBTDBT10, and PCPDTBSe Nanoparticles.

PFBTDBT10 and PCPDTBSe Nanoparticles.

PFBTDBT10 or PCPDTBSe (1 mL, 2 mg/mL in tetrahydrofuran (THF)) were injected under continuous horn sonication (Branson Digital Sonifier, 1 min, 20% amplitude) into 8 mL of water containing PluronicF127 (1 mg/mL).

Hybrid.

PFBTDBT10 and PCPDTBSe were combined in various ratios (1, 4, 8, 10, 20, and 40 to 1) of a 2 mg/mL polymer concentration in THF. One milliliter of the premixed PFBTDBT10 and PCPDTBSE polymer was injected under continuous horn sonication into 8 mL of water containing PluronicF127 (1 mg/mL).

Nanoparticle solutions were autoclaved to sterilize them, before being centrifuged (30 min, 7,500 rpm) to remove large nanoparticles and aggregates. The resulting supernatant was centrifuged (4 h, 14 000 rpm) to collect nanoparticles. Lyophilized NP solution was used to measure the mass, then serially diluted and absorption spectra were taken on a Beckman Coulter DU 730 Life Sciences UV−vis spectrophotometer using λ_max = 760 nm of H-DAPPs to generate an absorption-concentration calibration curve.

Development of Orthotopic Murine Mammary Fat Pad Model.

Female BALB/c mice were purchased from Charles River Laboratories and maintained in a vivarium on a 12-h light/dark schedule, at a temperature of 23 °C and a relative humidity of 50%. Food and water were available ad libitum. Mice were anesthetized with 2.5% isoflurane before receiving a 50 μL injection of 40,000 luciferase transfected 4T1 cells into the mammary fat pad of the left fourth nipple. Tumor growth was monitored by caliper measurements of the length and width, and luminescence. Tumor volumes were calculated using the volume of an ellipsoid V = (4/3)π (L)²(W). Luminescent and fluorescent images were obtained using a PerkinElmer Lumina LT In Vivo Imaging System (IVIS).

In Vivo Photothermal Ablation of 4T1 tumors in Mammary Fat Pad—Direct Injection.

Mice were divided into treatment groups, and received intratumoral injections of: (1) PBS injection, (2) PBS injection + laser exposure, (3) H-DAPPs injection, (4) H-DAPPs injection + laser exposure. Average tumor volumes were calculated using the volume of an ellipsoid V = (4/3)π (L)²(W), and had an average of 42 ± 14.7 mm³ on the day of treatment. Groups (3) and (4) received an intratumoral injection of 50 μL of 100 μg/mL of H-DAPPs (total mass of 5 μg) suspended in PBS. Groups (1) and (2) received an intratumoral injection of 50 μL of PBS. Immediately after injection, groups (2) and (4) were exposed to 229.3 J/cm², 800 nm light. Fluorescent and luminescent images of mice were taken before and after treatment and for the remainder of the study every 2 to 3 days.

In Vivo Photothermal Ablation of 4T1 Tumors in Mammary Fat Pad—Systemic Administration.

Mice were divided into treatment groups: (1) PBS + laser exposure, (2) H-DAPPs, (3) H-DAPPs + laser exposure. All groups were treated 14 days after injection of the tumor cells, and had an average tumor volume of 88 ± 11.2 mm³ on the day of treatment. Groups (2) and (3) received tail vein injection of 100 μL of 2 mg/mL of H-DAPPs (total mass of 200 μg) suspended in PBS. Group (1) received an injection of 100 μL of PBS. Twenty-4 h after injection, groups (1) and (3) were exposed to 305.7 J/cm², 800 nm light. Fluorescent and luminescent images of mice were taken before and after treatment and for the remainder of the study every 2 to 3 days.