Abstract

Chemophotothermal therapy is an emerging treatment of metastatic and drug-resistant cancer anomalies. Among various photothermal agents tested, poly(dopamine) provides an excellent biocompatible alternative that can be used to develop novel drug delivery carriers for cancer treatment. This study explores the synthesis of starch-encapsulated, poly(dopamine)-coated core-shell nanoparticles in a one-pot synthesis approach and by surfactant-free approach. The nanoparticles produced are embellished with polymeric stealth coatings and are tested for their physiologic stability, photothermal properties, and drug delivery in metastatic triple-negative breast cancer cell (TNBC) lines. Our results indicate that stealth polymer-coated nanoparticles exhibit superior colloidal stability under physiologic conditions, and are excellent photothermal agents, as determined by the increase in temperature of solution in the presence of nanoparticles, upon laser irradiation. The chemotherapeutic drug–loaded nanoparticles also showed concentration-dependent toxicities in TNBC and in a brain metastatic cell line.

SIGNIFICANCE STATEMENT This study develops, for the first time, biocompatible core-shell nanoparticles in a template-free approach that can serve as a drug delivery carrier and as photothermal agents for cancer treatment.

Introduction

Chemophotothermal therapy is an emerging and promising strategy to ensure effective drug delivery and complete ablation of residual tumors, where elevated temperatures enhance the sensitivity of cancer cells toward chemotherapeutics along with facile drug release in the tumor environment (Ho and Ding, 2013; Ball, 2018; Ambekar and Kandasubramanian, 2019; Tian and Lei, 2019; Xiong et al., 2019; Zhang et al., 2019). Among various organic and inorganic materials that have been explored for their potential as photothermal agents, poly(dopamine) (PDA) are advantageous due to their biocompatibility, biodegradability, adhesiveness, facile synthesis, and superior drug-loading capacity (Zhu and Su, 2017; Jin et al., 2020; Li et al., 2021a; Liu, et al., 2021). PDA are melanin-like structures that are prepared by the simple oxidation of 3,4-dihydroxy-L-phenylalanine (DOPA) in an alkaline aqueous environment in the presence of oxygen, and their size can easily be tuned as a function of the pH of the solution (Ho and Ding, 2013; Zhang et al., 2015a,b; Li et al., 2021a). Owing to superior adhesive properties, PDA nanoparticles are extensively studied for various biomedical applications, including drug delivery, sensors fabrication, and tissue engineering (Ball, 2018; Ambekar and Kandasubramanian, 2019).

PDA nanoparticles are especially exciting for chemotherapeutics delivery as PDA absorbs near-infrared region (NIR), converting light energy into hyperthermia, hence providing a multipronged strategy for cancer treatment (Ball, 2018). Furthermore, the highly adhesive nature of PDA enables superior drug-loading capacity via hydrophobic and Π-Π interactions and provides active surface for the functionalization of various biomolecules, including targeting agents, imaging probes, and stimuli-responsive polymer chains (Jin et al., 2020). Surface coating of PDA nanoparticles with hydrophilic biomacromolecules, including polymers, DNA, and proteins, by electrostatic interactions, surface adsorption, and covalent bonding has yielded physiologically stable and stimuli-responsive nanocarriers with superior drug delivery efficacies in various tumor models in vitro and in vivo (He et al., 2017; Li et al., 2017; Liu et al., 2022a; Siani and Di Valentin, 2022). To further improve the drug-loading capacity of PDA-based carriers, mesoporous nanoparticles are synthesized by the soft-template approach and are tested for their photothermal properties and drug delivery efficacies (Zhang et al., 2019; Chen et al., 2021).

Similarly, core-shell nanoparticles, comprised of an organic core and PDA shell, have been developed to improve the stability and chemotherapeutic potential of nanoparticles in cancer cells (Zhang et al., 2015b). The biocompatible, Food and Drug Administration–approved poly(lactide-co-glycolide) (PLGA) nanoparticles coated with PDA are further modified with stealth polymers and targeting moieties as photothermally active drug-loaded carriers (He et al., 2017). Similarly, paclitaxel (PTX)-loaded poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanoparticles prepared by the emulsion solvent evaporation method were coated with a PDA shell, and their surface was functionalized with RGD peptide to yield pH-responsive spherical nanoparticles with in vitro and in vivo antitumor efficacies in liver cancer model (Wu et al., 2021). Others have demonstrated that the encapsulation of 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000] (DSPE-PEG) micelles in a PDA shell can yield potent dual-drug delivery carriers for DOX and bortezomib delivery in breast cancer cells in vitro and in mice xenograft model (Zhang et al., 2015b). PDA-coated polymeric nanoparticles are typically prepared by a multistep approach that first requires the fabrication of a polymeric core, followed by coating and surface functionalization with a PDA shell and stealth/targeting moieties.

In this study, we report a facile one-step method for the synthesis of biodegradable core-shell nanoparticles comprised of a starch core and PDA shell (Scheme 1). The nanoparticles synthesized are then fabricated with stealth layers of poly(ethylene glycol) (PEG) and poly(vitamin B5 analogous methacrylamide) [poly(B5AMA)] in a one-pot approach. Poly(B5AMA) is a hydrophilic polymer that has recently been studied for its polyethylene glycol (PEG)-like antifouling and stealth properties (Nazeer and Ahmed, 2019; Combita et al., 2022). Starch-encapsulated PDA nanoparticles (PDA-SNPs) embellished with PEG or poly(B5AMA); namely, PEG@PDA-SNPs and P(B5AMA)@PDA-SNPs, respectively, were loaded with chemotherapeutic drugs and were studied for their anticancer effects in primary and metastatic breast cancer cells. Our results show that stealth polymers functionalized PDA-SNPs prepared by one-step fabrication method and in the absence of any template exhibit excellent physiologic stability, photothermal properties, and in vitro anticancer activities in breast cancer cell lines.

Scheme 1.

Schematics depicting the synthesis of drug-loaded biodegradable, core-shell nanoparticles comprised of a starch core and PDA shell in a one-pot approach.

Materials and Methods

Dopamine hydrochloride, water-soluble starch, 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris base), Dulbecco’s modified Eagle’s medium (DMEM) high-glucose media, FBS, penicillin-streptomycin, (2-N-Morpholino)ethanesulfonic acid sodium salt (MES sodium salt), EDTA, sodium chloride, methanol, calcium chloride, and acetic acid were purchased from Fisher Scientific. Five-kiloDalton PEG (Thermofisher), paclitaxel 99.5%, and 5-carboxytetramethylrhodamine (TAMRA) were purchased from Sigma Aldrich. MDA-MB-231 cell line was purchased from the American Type Culture Collection, and MDA-MB-231-BR was a gift from Dr. Kata Henji, Japan. Synthesis of B5AMA was achieved according to previously established procedures (Combita et al. 2022).

Polymerization of B5AMA.

Polymerization of B5AMA was conducted using the reversible addition−fragmentation chain-transfer polymerization technique at 50°C using a deionized water/methanol solution as a solvent in a ratio of 10:1 (v/v). 4-Cyano-4-[[(ethylthio)thioxomethyl]thio]pentanoic acid (TCT-2) was used as a chain transfer agent and VA-044 as an initiator. In a typical procedure, a 25-mL two-neck flask was charged with B5AMA monomer (1290 mg, 5 mmol), TCT-2 (77 mg, 0.25 mmol, target degree of polymerization (DP) = 20), and VA-044 (27 mg, 0.083 mmol) dissolved in 5 mL of solvent. The reagent solution was degassed by three freeze-vacuum-thaw cycles and was then allowed to react at 50°C. At the end of the reaction, the polymer solution was dialyzed against deionized water for at least 48 hours. Finally, 5-kDa polymer was obtained by freeze drying and was analyzed by gel permeation chromatography (GPC) (Supplemental Fig. 1).

Synthesis of Core-Shell Nanoparticles.

A 0.05 weight/volume% (w/v%) stock solution of starch was prepared in Tris buffer of pH 8.5 and was heated at 85°C and mixed until the starch dissolved completely and was then cooled to 19–21°C. A solution containing 2 mg/mL of dopamine and 1 mg/mL PEG/P(B5AMA) was prepared in distilled water, and 1 mL of this solution was mixed with 1 mL of cooled starch solution drop-wise. The final concentration of dopamine, PEG/P(B5AMA), and starch was 1 mg/mL, 0.5 mg/mL, and 0.25 mg/mL, respectively, in the reaction solution. The solution was left stirring at room temperature and covered from light for 2.5 hours. The nanoparticles formed were centrifuged at 17,000 RPM for 10 minutes and were washed two times with filtered distilled water at 17,000 RPM for 10 minutes. For drug-loaded nanoparticles, 60 µg paclitaxel was added to 1 ml of starch solution, followed by the addition of dopamine solution, to obtain final total volume of 2 mL. The drug-loaded nanoparticles synthesis was achieved as described above.

Synthesis of PDA Nanoparticles.

A solution containing 2 mg/mL of dopamine was prepared in 1mL of distilled water and 1mL of Tris buffer (pH 8.5) was added. The solution was left stirring at room temperature covered from light for 2.5 hours. The nanoparticles formed were centrifuged at 17,000 RPM for 10 minutes and were washed 2 times with filtered distilled water at 17,000 RPM for 10 minutes.

Dynamic Light Scattering and ζ Potential Analysis.

Size, net charge and polydispersity analysis of the nanoparticles were performed using dynamic light scattering (DLS) [Nano Brook 90 Plus (Brookhaven, Holtsville, NY, USA)]. The 2.5 µg/mL nanoparticles solution was prepared in PBS buffer of pH 7.4, supplemented with 1%FBS and the nanoparticles were incubated for 24 hours. The changes in the size and polydispersity of nanoparticles were analyzed at different time points 0 hours, 1, 2, 3 and 24 hours. All the measurements were taken at room temperature at a 90° scattering angle, equilibration count of 180 seconds and count time of 300 seconds. The ζ potential of nanoparticles was analyzed at room temperature in deionized water.

Transmission Electron Microscopy.

Transmission electron microscope (TEM) (Hitachi 7700) was performed at 80 kV with a Lab6 filament. Samples of 3 µL were drop casted onto the 200-mesh copper TEM grids with a layer of formvar and thin film of carbon (Ted Pella) and were allowed to air dry. Histogram characterization was performed with image J software by counting over 100 nanoparticles for each sample and organizing by different number distribution ranges. The average median size was calculated to determine the sample size distribution.

UV-Vis-NIR Spectroscopy.

Absorbance of nanoparticles was performed at range of wavelength (400–1000 nm) for different concentrations (150–37.5 µg/mL) of nanoparticles, the nanoparticles were prepared by serial dilution.

Fourier Transform Infrared Spectroscopy.

Samples of poly(dopmaine) and poly(dopamine)-coated core-shell nanoparticles were prepared, as described above and were freeze dried. Fourier transform infrared (FTIR) analysis was performed on a Bruker Alpha-T spectrometer operated in transmission mode.

Starch Release and Encapsulation Efficiency.

Starch encapsulation in core-shell nanoparticles was evaluated with a starch test comprised of an iodine-KI solution (Pesek et al., 2022). The standard curve was first prepared with varying concentrations of starch (250–1.95 µg/mL) in water. To address the interference of PDA-NPs with the blue color development during starch detection using the iodine-KI solution, a second standard curve was prepared by the addition of 4% volume/volume (v/v) of PDA-NPs washes and was used as a control to evaluate the interference of PDA with a starch assay. The starch solution mixed with PDA washes was read at 600 nm to produce a standard curve. The comparison of absorbance value of the two standard curves indicated that free starch can be detected in nanoparticles wash with a sensitivity of 15.6 µg/mL. The presence of residual nanoparticles color in reaction washes interferes with starch concentrations that are lower than 15.6 µg/mL when starch is detected using the iodine-KI–based starch assay. The amount of residual starch in the supernatant of the reactions was analyzed by starch assay, and starch encapsulation efficacy in dopamine core-shell nanoparticles was calculated using the standard curves generated. The encapsulation efficiency is given by

Drug Encapsulation Efficiency

The drug encapsulation efficiency of nanoparticles was evaluated using TAMRA fluorescent dye as a probe and is given by

The synthesis of nanoparticles was performed in the presence of 300 µg of TAMRA dye as indicated above. The encapsulation efficacy of the dye was calculated by measuring the amount of residual dye post synthesis of nanoparticles in the reaction supernatant and was quantified using the calibration curve of TAMRA dye at 552 nm excitation and 578 nm emission.

Drug Release Study

The release of TAMRA dye from nanoparticles was evaluated as follows:

Nanoparticles (300 µg/mL) were suspended in PBS (pH, 7.4 and 5.5) and were irradiated with an 808-nm diode laser (Luck Laser Model 7/24, 200 mW nominal power) for 10 minutes. Samples were taken over different time periods, then centrifuged for 10 minutes at 17,000 RPM to remove nanoparticles, and the absorbance of TAMRA dye in the supernatant was measured at excitation and emission wavelengths of 552 nm and 578 nm, respectively. Fresh PBS was added to the pellet, and nanoparticles were added back to the samples for continued evaluation. The dye release was studied at different periods of time ranging from 0 to 120 hours at 37°C. The dye release was quantified using the calibration curve of TAMRA dye at 552 nm excitation and 578 nm emission.

Laser Characterization

The operating power of the laser was measured using a laser power meter (Nova II, OPHIR). The laser beam dimensions at the operating distance from the top of the sample were measured using a graded ruler system.

Temperature Studies

Nanoparticles were suspended at a concentration of 200 µg/mL in distilled water and were irradiated for 10 minutes. The temperature of the solution was evaluated at time 0 and following the 10 minutes of laser irradiation using a thermal camera (FLIR ONE Pro) positioned near the center of the sample. Deionized water was used as a control.

Cellular Uptake Studies

MDA-MB-231 and MDA-MB-231-BR cells were seeded in high-glucose DMEM containing 10% FBS and 1% antibiotic at 37°C and 5% CO2 in six-well plates on glass coverslips and were treated with TAMRA-loaded nanoparticles at a final concentration of 40 µg/mL. The nanoparticles were incubated with cells for 4 hours, were fixed with 3.7% formalin, and were stained with 4′,6-diamidino-2-phenylindole (DAPI; a nuclear stain). The glass coverslips were mounted onto the glass slides and were examined by fluorescent microscopy with the excitation and emission of the two dyes, TAMRA (excitation 552 nm and emission 578 nm) and DAPI (excitation 359 nm and emission 457 nm).

In Vitro Cytotoxicity

MDA-MB-231 and MDA-MB-231-BR were seeded in 96-well plates at a density of 2 × 104 cells per well in high-glucose DMEM containing 10% FBS and 1% antibiotic at 37°C and 5% CO2. After 24 hours, the cells were treated with 50–200 µg/mL nanoparticles in high-glucose DMEM. The cells were incubated with the nanoparticles containing media for 4 hours and were then irradiated using the 808-nm laser for 10 minutes. Cells were then incubated overnight, and an MTS assay (a colorimertic assay for cell viability) was performed according to the manufacturer’s protocol. Briefly, treated cells were washed with PBS, media containing MTS was added and incubated for 3 hours, and the absorbance of the samples was read at 490 nm. Statistical analysis was performed with the use of Graph Pad Prism version 10. An unpaired two-tailed t test with Welch correction was performed on cytotoxicity data. All asterisks (*) indicate P < 0.05; the number of asterisks indicate the groups that are significantly different to each other. Square symbols (■) indicate P < 0.1.

ResultsOne-Pot Synthesis of Core-Shell Nanoparticles.

In this study, dopamine-coated starch nanoparticles were synthesized in one step by dissolving different concentrations of water-soluble starch in Tris buffer (pH, 8.5), followed by the addition of dopamine that undergoes rapid oxidation under alkaline pH (Zhu and Su, 2017). The nanoparticles synthesized were optimized as a function of concentration of reactants, pH, and reaction time and were characterized by FTIR, DLS, and ζ potential equipment and by TEM for the composition, size, and surface charge of nanoparticles. The dispersion of 0.05% w/v of starch alone in Tris buffer yielded nanoparticles of ∼150 nm with a polydispersity index (PDI) 0.31. The addition of dopamine to a starch solution of 8.5 pH quickly resulted in color change from transparent to brown, and dark brown nanoparticles were obtained after 24 hours of reaction time (Supplemental Fig. 2). Poly(dopamine) nanoparticles strongly absorb at 600 nm (A600), and synthesis of nanoparticles, as a function of time, can be monitored by UV-Vis-NIR spectroscopy (Chen et al., 2021). The increase in the absorbance of reaction solution with time indicated successful formation of nanoparticles, and no significant change in the absorbance values was observed 2.5 hours post reaction time [A600 = 0.7 after 1 hour, 1.15 after 2 hours, and 1.4 after 3 hours versus 1.7 after 24 hours (without starch)], indicating the optimal time for nanoparticles formation.

The purified nanoparticles analyzed by DLS showed discrete nanoparticles of 400–600 nm, and the size of nanoparticles was dependent on the concentration of reactants in the solution (Supplemental Table 1). The PDA-SNPs prepared at low w/w ratio of starch/dopamine (0.05 and 0.1) showed large particle sizes, possibly due to the agglomeration of starch molecules on the surface of dopamine nanoparticles and presence of insufficient starch in the reaction solution to serve as a nano-template for the formation of a PDA shell. The nanoparticles obtained at a dopamine:starch w/w ratio of 0.25 and 0.5, however, showed discrete nanoparticles of 465 ± 53 nm and 470 ± 48, respectively, indicating the presence of near monodisperse PDA-SNPs. PDA-NPs prepared in the absence of starch were synthesized as a control and were 330 ± 52 nm in size (Table 1). The larger sizes of PDA-SNPs compared with PDA-NPs are attributed to the swelling capacity of starch present in the nanoparticles’ core under alkaline pH (Jivan et al., 2014; Chou et al., 2020).

TABLE 1

Characterization of size and ζ potential of nanoparticles in deionized water and phosphate-buffered saline in the presence of serum proteins

The supernatant of the nanoparticles postsynthesis was analyzed for the presence of free starch in the reaction solution by an iodine-KI assay (Pesek et al., 2022), and the encapsulation efficacy of starch in a PDA shell was evaluated by developing a starch calibration curve prepared at 600 nm (Supplemental Fig. 3). The possible interference of dopamine nanoparticles present in nanoparticle washes in the iodine-KI assay was evaluated by developing the calibration curve of the known amount of starch in the presence of bare dopamine nanoparticles washes, and sensitivity of the assay was confirmed by comparing the data with the calibration curve of starch prepared in deionized water. Our data indicated that free starch in dopamine nanoparticle washes can be detected accurately up to a concentration of 15.6 µg/mL (Supplemental Fig. 4) without any significant interference of dopamine nanoparticles with the assay absorbance. The encapsulation efficacy of starch in the dopamine core was calculated to be 75 ± 15% for PDA-SNPs.

The synthesis of PDA-SNPs was further optimized at 10 and 13 pH and at a starch:dopamine w/w ratio of 0.25. PDA-SNPs prepared at various pH levels showed pH-dependent size variations. The nanoparticles prepared in Tris buffer at 8.5 pH yielded a hydrodynamic diameter of 465 ± 53 nm. However, the increase in pH to 10.5 and 13 resulted in larger aggregates (1 to 2 µm) and poor encapsulation of starch in the PDA shell as was apparent by the presence of white starch precipitates in the reaction tube (Supplemental Table 2).

The nanoparticles synthesized at a starch:dopamine w/w ratio of 0.25 in Tris buffer (pH, 8.5) and after 2.5 hours of reaction time were further characterized by FTIR spectroscopy (Fig. 1). FTIR spectra of PDA-NPs, starch, and PDA-SNPs were compared to confirm the encapsulation of starch in PDA core. FTIR spectra of PDA-NPs revealed a broad peak from 3200–2500 cm−1, consistent with OH stretching of poly(dopamine). The peak at 1587 cm−1 was due to stretching vibration of N-H bonds of dihydroxyindole moiety. The characteristic peaks at 1045, 1124, 1199, 1257, and 1488 cm−1 are due to CH2 bending vibrations, C-O-H bending, C-O symmetry vibration, and C-C stretching mode (Batul et al., 2020). FTIR spectra of water-soluble starch showed pronounced peaks at 3700–3000 cm−1, indicating OH stretching, and strong absorption bands in the region of 1000–1200 cm−1, arising from C-O-C and C-O-H stretching and C-O-H bending (Supplemental Fig. 5) (Warren et al., 2016). In contrast, PDA-SNPs showed an overall dampening and shifting of peaks to 1013, 1340, and 1620 cm−1, indicating the interactions between starch core and PDA shell.

Fig. 1.

FTIR spectra of PDA-NPs and PDA-SNPs.

Stealth Layer–Coated Core-Shell Nanoparticles.

PDA-SNPs were modified with stealth polymeric coatings to aid physiologic stability and to reduce the aggregation of nanoparticles. PDA-SNPs were modified with the stealth layer of PEG-SH and P(B5AMA) of similar molecular mass (5k Da) and were compared for their physiologic stability in situ. PEG@PDA-SNPs and P(B5AMA)@PDA-SNPs produced under identical synthesis condition (pH, 8.5; starch:dopamine w/w ratio of 0.25; 2.5-hour reaction time) were purified and redispersed in deionized water and PBS (supplemented with 1% FBS) and were analyzed for their hydrodynamic size and net charge by DLS and ζpotential. The stealth layer–coated nanoparticles showed sizes ranging from 250 to 470 nm and net negative surface potential of −13 to −21 mV in deionized water. Interestingly, nanoparticles suspended in the presence of serum proteins showed relatively smaller sizes (240–330 nm).

The stability of nanoparticles under physiologic conditions was evaluated as a function of time and aggregation propensity of PEG@PDA-SNPs, and P(B5AMA)@PDA-SNPs were compared with PDA-SNPs in the presence of serum proteins. As expected, PDA-SNPs showed time-dependent aggregation, and the size of nanoparticles increased from 330 ± 34 nm to >500 nm after 3 hours of incubation in serum containing PBS (Supplemental Fig. 6). PEG@PDA-SNPs and P(B5AMA)@PDA-SNPs showed sizes of 290 ± 53 and 240 ± 41 nm, respectively, under physiologic solution, and negligible changes in the size and PDI of nanoparticles were observed after 24 hours as measured by the DLS analysis, suggesting that both PEG and P(B5AMA) have the potential to serve as excellent stealth coatings for PDA-SNPs.

Nanoparticle morphology and size was further analyzed by TEM for stealth layer–coated nanoparticles (Fig. 2). TEM images showed diameters of 97 ± 27 nm and 65 ± 13 nm for PEG@PDA-SNPs and P(B5AMA)@PDA-SNPs, respectively. The size of nanoparticles was quantified by image J, and the histograms demonstrated a near normal size distribution. The larger sizes of core-shell nanoparticles by DLS (∼250–300 nm) when compared with TEM (60–100 nm) are attributed to the hydration capacity of nanoparticles when analyzed in aqueous solution by DLS (Jivan et al., 2014; Chou et al., 2020).

Fig. 2.

TEM images and histogram analysis of core-shell nanoparticles. (A) and (C) PEG@PDA-SNPs (n = 147) and (B) and (D) P(B5AMA)@PDA-SNP (n = 207). Scale bar, 500 nm.

Photothermal Properties and Laser Parameters.

The absorbance intensity of the nanoparticles is a major determinant of their photothermal capability. PEG@PDA-SNPs, P(B5AMA)@PDA-SNPs, and PDA-SNPs were analyzed and were compared with PDA-NPs for the change in absorbance as a function of concentration (Fig. 3). As seen from the UV-vis-NIR spectra in Fig. 3, the intensity of absorbance of core-shell nanoparticles is directly correlated with the concentration of nanoparticles and is comparable with PDA-NPs of similar concentration, indicating that encapsulation of starch in a PDA core does not change the absorption capacity of PDA-NPs. The surface coating of PDA-SNPs with PEG yielded absorption spectra similar to PDA-NPs and PDA-SNPs; however, P(B5AMA)@PDA-SNPs showed a significant reduction in absorption spectra when compared with the other core-shell nanoparticles.

Fig. 3.

UV-vis-NIR spectra of nanoparticles in deionized water. (A) PDA-NPs, (B) PDA-SNPs, (C) PEG@PDA-SNPs, and (D) P(B5AMA) @PDA-SNPs.

The laser power was measured to be 162 ± 4 mW. The resulting laser power density at the surface of the samples was calculated to be 1.4 W/cm2 based on the output collimated rectangular beam, 6.0 mm by 2.0 mm, as viewed on a NIR laser card (Fig. 4A).

Fig. 4.

(A) Demonstration of laser setup used for the photothermal experiments: the laser positioned above a sample well, and an 808-nm laser beam illuminating an infrared sensor card. (B) Measurement of temperature change in response to 808-nm laser irradiation in the presence of PEG @PDA-SNPs, P(B5AMA)@PDA-SNPs, and PDA-NPs and water (as a control) as measured by thermal camera. The experiments on the temperature change study were repeated three times, and the mean is presented here.

NIR absorption capacity of nanoparticles and conversion of light energy into heat in the presence of nanoparticles was then studied in situ (Fig. 4B; Supplemental Fig. 7). After laser irradiation, the three nanoparticle solutions were prepared, all at a concentration of 200 µg/mL, and showed a temperature increase from 15 to 18°C by thermal camera (Fig. 4B). The temperature increase for water alone was 5°C. Interestingly, the temperature change of 15–18°C, as measured by thermal camera, was consistent for all the nanoparticles synthesized; PDA-NPs prepared at similar concentration (200 µg/mL) were used as a control and showed an increase of 18°C in temperature upon irradiation with 1.4 W/cm2 laser power density.

Drug Encapsulation Efficiency and Release Studies.

The drug-loading capacity of the stealth layer–coated core-shell nanoparticles was studied using TAMRA fluorophore as a cargo molecule and was compared with PDA nanoparticles. The fluorescent dye was encapsulated in a starch core during nanoparticles synthesis, and TAMRA loading capacity of the nanoparticles was analyzed by measuring the presence of residual dye in the supernatant of reaction by the microplate reader using excitation and emission wavelengths of 552 nm and 578 nm, respectively (Supplemental Fig. 8). PEG@PDA-SNPs and P(B5AMA)@PDA-SNPs showed TAMRA encapsulation efficacies of 66%–72%, (∼45 µg of dye/300 µg of nanoparticles), and PDA-NPs used as a control showed comparable dye encapsulation efficacies (70%) (Fig. 5).

Fig. 5.

Drug release profile of PEG@PDA-SNPs and P(B5AMA) @PDA-SNPs at pH 5.5 and 7.4 over a period of 5 days. The experiments were performed in triplicates. r.t., room temperature.

The release profile of TAMRA-loaded nanoparticles was then studied at various pH levels (5.5 and 7.4) and as a function of time at 37°C as shown in Fig. 5. PEG- and P(B5AMA)-coated core-shell nanoparticles showed sustained release of TAMRA dye over a period of 5 days at all studied pH levels. Interestingly, the release of TAMRA dye at a physiologic pH (7.4) was ∼20% after 24 hours, whereas only a ∼5% release of the dye was observed at pH 5.5 for all nanoparticles studied. Similarly, the release profile of the dye from nanoparticles was ∼40% at pH 7.4, whereas only ∼20% of the dye was released at pH 5.5 after 5 days of incubation (Fig. 5).

Cellular Uptake and Toxicity Studies.

Cellular uptake of TAMRA-loaded core-shell nanoparticles was then studied in triple-negative breast cancer (TNBC) cell lines (Supplemental Fig. 9). As shown from the fluorescent microscope images, P(B5AMA)- and PEG-coated core-shell nanoparticles are well uptaken and tend to localize in the cytoplasm of triple-negative breast cancer cells, indicating their potential usage as a drug delivery carrier.

The toxicity of bare core-shell nanoparticles was then studied in primary and metastatic triple-negative breast cancer cell lines. The stealth polymers functionalized PDA-SNP showed essentially nontoxic profile at 50 and 100 µg/mL, -while cell viabilities of >60% were calculated at high concentrations (200 µg/mL) of nanoparticles for both MDA-MB231 and MDA-MB 231-BR cell lines. Irradiation of bare nanoparticles-treated cells with an 808-nm laser did not show significant hyperthermia-induced cell death at all studied concentrations (50–200 µg/mL) in both primary and metastatic TNBCs (Fig. 6; Supplemental Figs. 9 and 10), possibly due to the low power density of the laser used for the study (Liu et al., 2022b).

Fig. 6.

Cytotoxicity of (A) empty nanoparticles in MDA-MB231 cells, (B) PTX-loaded nanoparticles in MDA-MB231 cells, (C) PTX-loaded nanoparticles in MDA-MB231-BR cells, (D) PTX-loaded and empty nanoparticles at 200 µg/mL in MDA-MB231 cells, and (E) drug-loaded and empty nanoparticles at 200 µg/mL in MDA-MB231-BR cells as determined by MTS assay. The brackets indicate different groups of treatments compared for their cytotoxicity. *P < 0.05, two-tailed Welch correction t test.

PTX-loaded core-shell nanoparticles were then synthesized using a previously established procedure optimized for TAMRA encapsulation and were tested for their cytotoxicity in MDA-MB231 and in a metastatic brain cell line (MDA-MB231-BR) in the presence and absence of laser irradiation. In this study, PTX-treated TNBCs showed ∼50% cell viability at a 100-nM concentration (Supplemental Fig. 11). The toxicity of core-shell nanoparticles in MDA-MB231 cells at 200 µg/mL nanoparticle concentrations were reduced from 80% to 45% and from 60% to 22% for PEG@PDA-SNPs and P(B5AMA)@PDA-SNPs, respectively (Fig. 6B). Interestingly, MDA-MB231-BR cells showed relatively higher susceptibility to the nanoparticles treatment at all studied concentrations (100 and 200 µg/mL), and cell viabilities were 18% and 2% for PTX-loaded PEG@PDA-SNPs and p(B5AMA)@PDA-SNPs, respectively (Fig. 6C). The irradiation of drug-loaded nanoparticles-treated cells with an 808-nm laser source showed limited effect on cell viabilities, and the toxicity profile of nanoparticles was mainly dominated by the presence of chemotherapeutics.

Discussion

Starch is the most abundant polysaccharide that is nontoxic, biodegradable, and has been extensively studied to develop drug delivery carriers (El-Naggar et al., 2015; Li et al., 2016; Ismail and Gopinath, 2017; Qi et al., 2017; Odeniyi et al., 2018). The water-soluble starch-loaded drug nanoparticles are well explored for the capability to encapsulate hydrophobic cargo molecules and for their improved antibacterial properties (Li et al., 2016). Poly(dopamine)-coated starch nanoparticles were optimized as a function of concentration of starch and dopamine, pH of the reaction and reaction time. The near monodisperse nanoparticles obtained at a starch:dopamine w/w ratio of 0.25 pH 8.5 and after 2.5 hours of reaction time showed complete encapsulation of starch in nanoparticles core as analyzed by I2-KI assay and by FTIR analysis. Previous studies indicated that at high pH, dopamine undergoes rapid oxidation into dopamine quinone and leukodopaminechrome that subsequently forms 5,6-dihydroxyindole (DHI), hence yielding smaller nanoparticles (Ho and Ding, 2013). The faster conversion of dopamine into PDA nanoparticles during PDA-SNPs synthesis, however, led to poor encapsulation of starch in core-shell nanoparticles as seen by the presence of white precipitates of starch in the reaction mixture post nanoparticles synthesis.

The nanoparticles synthesized were modified with a stealth layer to improve the colloidal stability of the nanoparticles. PEG is the most studied stealth polymer that is well documented to improve the physiologic stability of nanocarriers (Pelosi et al., 2021). In addition to PEG, hydrophilic polymers such as poly(sulfobetaine methacrylate) p(SBMA) and poly(carboxybetaine methacrylate) p(CBMA) and poly(2-methacryloxyethyl phosphorylcholine) p(MPC) have received much attention as alternative polymeric coatings (Schlenoff, 2014; Bekale et al., 2015; Jensen et al., 2021). Recently, we have developed poly(B5AMA) of predetermine molecular mass by reversible addition−fragmentation chain-transfer polymerization approach and have documented their antifouling and nontoxic behavior (Nazeer and Ahmed, 2019; Combita et al., 2022). Thiol- and amine-terminated PEG chains are well documented to undergo Michael addition/Schiff base reaction under alkaline conditions with the amino group of PDA (He et al. 2017; Liu et al. 2022). Poly(B5AMA), however, is hydroxyl groups rich telechelic polymer, and interacts with PDA shell via hydrogen bonding and hydrophobic interactions (El Yakhlifi and Ball, 2018). P(B5AMA)@PDA-SNPs and PEG@PDA-SNPs showed superior physiologic stability in the presence of serum proteins and net negative surface charge, and the results obtained were similar to a recent study where PHBV-embedded core-shell PDA nanoparticles showed a size of ∼250 nm with a net negative surface potential of −20 mV (Wu et al., 2021). The reduced size of nanoparticles in the presence of serum proteins in comparison with deionized water has been reported earlier and was attributed to the compact corona of nanoparticles in the presence of serum proteins (Ahmed and Narain, 2011).

Evaluation of UV-vis NIR spectra in the NIR region of poly(dopamine) nanoparticles as a function of concentration is well documented to demonstrate photothermal properties (Tian and Lei, 2019). PDA-coated core-shell starch nanoparticles showed absorption spectra similar to PDA nanoparticles; however, lower absorption capacity of P(B5AMA)@PDA-SNPs, when compared with other nanoparticles, may be related to their smaller sizes (analyzed by both DLS and TEM), possibly due to reduced thickness of the PDA core in the presence of the poly(B5AMA) stealth layer. The reduction in absorption potential of poly(methacrylate) functionalized PDA nanoparticles in comparison with bare PDA nanoparticles was reported by others and was attributed to the reduced thickness of the PDA shell in the case of polymer-coated nanoparticles (Tian and Lei, 2019). The core-shell nanoparticles prepared in this study showed absorbance in the NIR region (800 nm to 1000 nm), indicating potential photothermal properties of nanoparticles when irradiated with a near-infrared 808-nm laser.

Photothermal therapy is a minimally invasive treatment in which NIR laser light is absorbed by a photothermal agent, converting optical energy into thermal energy, which results in tissue destruction (Tian and Lei, 2019). The laser power, beam dimension(s), and irradiation time are the key irradiation parameters for determining the resultant heating of the photothermal agents, such as poly(dopamine) nanoparticles (Zhang et al., 2015a,b; Liu et al., 2021, 2022a,b). The nanoparticles irradiated with a 1.4 W/cm2 laser showed similar increase in temperature, indicating that complex nanoparticles morphology in the case of stealth layer–coated core-shell nanoparticles did not compromise the photothermal conversion capabilities of PDA-NPs. Liu et al. (2021, 2022a,b) and others have studied laser power density–dependent change in temperature in the presence of PDA-NPs and showed that a laser power density of 0.125 W/cm2 to 1 W/cm2 resulted in a temperature change from 0°C to 25°C, indicating a power density–dependent increase in temperature (Zhang et al., 2015a).

The encapsulation and release of cargo molecules from dopamine nanoparticles was studied, and all of the nanoparticles synthesized showed similar encapsulation and release efficacies of TAMRA dye. The encapsulation of drugs in nanoparticles is variable (in the range of 25%–85%) and is dependent on the morphology of the nanoparticles and on the type of drug molecule studied (Zhu and Su, 2017; Li et al., 2021b; Liu et al., 2022a). TAMRA is a zwitterionic highly stable dye that was chosen for its excellent photostability and quantum yield that is essentially unaffected at acidic pH (Christie et al., 2009; Herce et al., 2014). PDA-NPs, when compared with PEG@PDA-SNPs and P(B5AMA)@PDA-SNPs, showed no significant difference in the release of cargo at all studied pH levels, indicating that a polymeric stealth layer does not interfere with the release of encapsulated molecules from the core of nanoparticles. The pH-dependent drug release efficacies of nanoparticles observed in this study are consistent with others, where slow release of cargo from dopamine nanoparticles was attributed to the zwitterionic property of PDA that increases the binding of anionic cargo molecules with the nanoparticles′ surface at acidic pH (Poinard et al., 2018; Pan et al., 2020). The pKa of TAMRA is <5 as carboxyl groups of molecules are fully protonated under acidic pH levels that mediate stronger interactions with anionic PDA-NPs, hence demonstrating reduced drug release at acidic pH (Christie et al., 2009; Herce et al., 2014). A handful of studies demonstrated pH-dependent disintegration and release of cargo drugs from PDA-NPs (Tian and Lei, 2019; Li et al., 2021a; Wu et al., 2021; Liu et al., 2022b). The acidic pH was shown to perturb Π-Π interactions between the drug and PDA moieties, showing faster drug release at low pH. To evaluate the degradation of PDA core-shell nanoparticles and release of starch under acidic pH, an iodine-KI assay was performed 48 hours post incubation of nanoparticles at different pH levels. The analysis of nanoparticles as a function of pH and time demonstrated less than 5% release of starch from PEG@PDA-SNPs and poly(B5AMA)@PDA-SNPs over a 48-hour time period at a pH of 5.5 and 7.4, indicating the stability of core-shell nanoparticles at different pH levels (Zhao et al., 2019; Pan et al., 2020).

PTX is a natural broad-spectrum antineoplastic drug that is capable of mediating tubulin polymerization, resulting in abnormal arrangement of cell bundles and causing cell death, and is extensively used in clinics for TNBC treatment (Wu et al., 2021). PTX is documented for its inhibitory concentration (IC50 values) in TNBCs at concentrations ranging from 1.5 to 500 nM in vitro (Kenicer et al., 2014). PTX-loaded core-shell nanoparticles showed concentration-dependent cytotoxicity, and a significant decrease in cell viabilities was observed compared with bare nanoparticles at all studied concentrations and for both cell lines.

Conclusions and Future Directions

PDA-coated starch nanoparticles synthesized in a one-pot, template-free approach and coated with poly(B5AMA) and PEG stealth layers yielded near monodisperse nanoparticles of 300–400 nm. The core-shell nanoparticles showed excellent absorption capacities in the NIR region and exhibited photothermal properties upon NIR laser irradiation. The photothermal properties of core-shell nanoparticles were comparable to PDA-NPs, indicating that the presence of a starch core and polymeric shell do not interfere with their photothermal properties. P(B5AMA)@PDA-SNPs and PEG@PDA-SNPs also exhibited excellent colloidal stability under physiologic conditions; however PDA-SNPs, prepared in the absence of a stealth coating showed rapid aggregation in the presence of serum proteins. The nanoparticles demonstrated cellular uptake in TNBC cell lines and exhibited concentration-dependent toxic effects in MDA-MB231 and MDA-MB231-BR cells. Although limited effect of photothermal properties of nanoparticles on cell viabilities was observed, possibly due to the low power of the laser used for the study, the nanoparticles exhibited excellent drug-loading capacity and concentration-dependent toxicity in TNBC cell lines. Our study demonstrates that PDA-coated starch nanoparticles have the potential to develop as delivery carriers to improve TNBC therapy.

Acknowledgments

The authors would like to thank Canadian Cancer Society Emerging Scholar Award to M.A. and Canadian Cancer Society’s JD Irving, Limited – Excellence in Cancer Research Fund to M.S. for the funding of this project.

Data Availability

The authors declare that all the data supporting the findings of this study are contained within the paper.

Authorship Contributions

Conducted experiments: Steeves, Combita, Whelan.

Contributed new reagents or analytic tools: Whelan, Ahmed.

Performed data analysis: Steeves, Combita, Whelan, Ahmed.

Wrote or contributed to the writing of the manuscript: Steeves, Combita, Whelan, Ahmed.

Footnotes