Cationic Porphyrin-Based Ionic Nanomedicines for Improved Photodynamic Therapy

Abstract

This study presents the synthesis and characterization of monosubstituted cationic porphyrin as a photodynamic therapeutic agent. Cationic porphyrin was converted into ionic materials by using a single-step ion exchange reaction. The small iodide counteranion was replaced with bulky BETI and IR783 anions to reduce aggregation and enhance the photodynamic effect of porphyrin. Carrier-free ionic nanomedicines were then prepared by using the reprecipitation method. The photophysical characterization of parent porphyrin, ionic materials, and ionic nanomaterials, including absorbance, fluorescence and phosphorescence emission, quantum yield, radiative and nonradiative rate, and lifetimes, was performed. The results revealed that the counteranion significantly affects the photophysical properties of porphyrin. The ionic nanomaterials exhibited an increase in the reactive oxygen yield and enhanced cytotoxicity toward the MCF-7 cancer cell line. Examination of results revealed that the ionic materials exhibited an enhanced photodynamic therapeutic activity with a low IC₅₀ value (nanomolar) in cancerous cells. These nanomedicines were mainly localized in the mitochondria. The improved light cytotoxicity is attributed to the enhanced photophysical properties and positive surface charge of the ionic nanomedicines that facilitate efficient cellular uptake. These results demonstrate that ionic material-based nanodrugs are promising photosensitizers for photodynamic therapy.

Graphical Abstract

1. INTRODUCTION

Cancer is currently one of the leading causes of death worldwide.¹ Traditional therapies, such as chemotherapy and radiation therapy, have been used to treat cancer for many years. These therapies are highly invasive and often cause adverse side effects to cancer patients.² Current research in this arena focuses on developing noninvasive therapies while still retaining high efficacy. Among these, photodynamic therapy (PDT) is clinically approved.^3,4 PDT involves the use of light and a photosensitizer (PS) in conjunction with molecular oxygen to generate reactive oxygen species (ROS) to cause cell death.^4–6 A PS that strongly absorbs light (high molar extinction coefficient), possesses a high singlet oxygen quantum yield, and exhibits low dark toxicity is in high demand for PDT.^7,8

Photofrin, a derivative of hematoporphyrin, was the first drug approved by the FDA for PDT. Since then, several porphyrin derivatives have been developed for PDT to improve efficiency. Porphyrin is abundant in nature, absorbs light strongly, and plays important biological roles.⁹ Despite their ability to be used as PSs, they often form aggregates in solutions due to π-π stacking between the planar porphyrin affecting their photophysical properties and decreasing their singlet oxygen generation capacity.^10–12 Therefore, different strategies have been utilized to minimize aggregation and to enhance the PDT efficacy of porphyrins through complex organic synthesis, which can sometimes be time-consuming.¹² Furthermore, mostly porphyrins absorb light in the visible region; therefore, they are mainly used for treating skin cancer by PDT. Although porphyrins are widely used as PSs for PDT, they suffer from low absorbance at longer wavelengths of electromagnetic radiation, which limit their use to treat deepseated tumors.⁵ Therefore, it is of utmost importance to implement new types of materials and synthesis schemes that are cost-effective, simple to use, produce products with high yield, and can easily tune the absorption wavelength range of the drug.

Recently, a new class of materials known as ionic materials (IMs) has gained much attention due to their high thermal and photostability, tunability, and formation of stable nanoparticles.^13–19 Unlike organic synthesis which involves multistep reactions, IMs are easily synthesized through a cost-effective and reproducible ion exchange method with high product yield.^7,20 Using the IM approach, the small spectator counterion can be replaced with a bulky ion to improve the photophysical properties by preventing aggregation in porphyrins. Several distinct IMs have been developed for a variety of applications such as cancer therapy,⁷ sensors,²¹ extraction chemistry,²² energy,²³ etc. Several anionic porphyrins have been converted into IMs.^5,7,9,11 However, to the best of our knowledge, no cationic porphyrin has been used before to develop IMs.

Nanotechnology has shown a great impact on the biomedical field, which has led to a new area known as nanomedicine. Due to their small size, they are easily uptaken by the cancerous cells and provide an enhanced permeability and retention effect.^7,24,25 Different types of nanoparticles have been used in biomedical applications such as biosensors,²⁶ drug delivery,^27,28 biomedical imaging,²⁹ cancer therapy,⁷ diagnosis,³⁰ etc. Inorganic nanoparticles, such as carbon nanotubes and quantum dots, have been widely used in cancer research. Despite their use in the biomedical field, they can exhibit side effects to the body due to their nonbiodegradability.^7,31 Organic nanoparticles with good biocompatibility and biodegradability are currently used in many research works.^32,33 This makes IMs ideal compounds since they are mainly composed of two ionic organic moieties that can easily be converted into ionic nanoparticles through the reprecipitation method. Moreover, IMs allow the synthesis of carrier-free nanomaterials, whereas previously reported nanoparticle strategies in cancer therapeutics mainly rely on nanocarriers.^11,28

In this study, for the first time, cationic porphyrin-based IMs were designed to treat cancer via PDT. Monosubstituted cationic porphyrin was synthesized. To prevent face aggregation, the small counterion (iodide) of the porphyrin cation was replaced with a bulky anion to form an IM. First, c a t i o n i c p o r p h y r i n w a s p a i r e d w i t h a b i s -(pentafluoroethanesulfonyl)imide anion (BETI⁻) to produce a [Porph][BETI] IM that enabled us to study the effect of the counterion on the photophysical properties of porphyrin, which consequently affect the PDT efficacy of the cationic porphyrin. Similarly, another IM was synthesized via the same ionic exchange reaction using the IR783 anion to broaden the absorption wavelength and to introduce the Förster resonance energy transfer (FRET) mechanism in the IMs. Heptamethine cyanine dyes such as NaIR783 absorb light in the NIR region, which is known for its anticancer properties.^34,35 Although the IR783 anion has been commonly studied as a photothermal agent (PTA), there are a few reports about the PDT activity of NIR dyes.³⁶ IR783 was utilized as a counteranion with cationic porphyrin to enhance the phototherapeutic effect of the resulting [Porph][IR783] IM. The two synthesized IMs, [Porph][BETI] and [Porph][IR783], were subsequently converted into carrier-free ionic nanomaterials (INMs) through reprecipitation in water. The IMs and INMs were characterized, and their detailed photophysical properties were investigated to evaluate their potential as PDT nanodrugs. The singlet oxygen quantum yield efficiencies were also studied using 1,3-diphenylisobenzofuran (DPBF) (in ethanol) and 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA) (in water and PBS) probes to examine the potential of these new materials for PDT applications. The cellular uptake, cytotoxicity, and subcellular localization of the carrier-free nanodrugs were investigated in vitro using the MCF-7 breast cancer cell line. All results were compared with the parent compounds to investigate the full potential of the IM approach to furnishing an effective PDT nanomedicine. Moreover, the results were also compared with the previously reported anionic porphyrin IM from our group.⁷

2. EXPERIMENTAL SECTION

2.1. Materials.

Pyrrole, benzaldehyde, propionic acid, trifluoroacetic acid, sodium nitrite, stannous chloride, hydrochloric acid, ammonium hydroxide, anhydrous magnesium sulfate, anhydrous dimethylformamide (DMF), and methyl iodide were purchased from VWR (Radnor, PA) and used as received. Meso-tetrakis(4-carboxyphenyl) porphyrin (TCPP) was purchased from the Tokyo Chemical Industry. Lithium bis(pentafluoroethanesulfonyl)imide (LiBETI), NaIR783 dye (Lot#: BCBZ9950), NaIR820 dye (Lot#: MKBZ1942 V), dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS, 1X) with pH 7.4, methylene blue (MB), ABDA, and DPBF were purchased from Sigma-Aldrich (St. Louis, MO). Triple deionized (DI) water (18.2 MΩ cm) was obtained using the Purelab Ultrapure water purification system (ELGA Woodridge, IL). Dichloromethane (DCM), hexane, methanol, and ethanol were of ACS grade and purchased from VWR (Radnor, PA). The MCF-7 cell line, a breast cancer model, was purchased from the American Type Culture Collection (ATCC, Manassas, VA). Dulbecco’s modified Eagle’s medium (DMEM), trypsin-EDTA (0.25%), penicillin, and streptomycin were purchased from Caisson Lab (Smithfield, UT). Fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Lawrenceville, GA). 4′,6-Diamidino-2-phenylindole (DAPI), primary LAMP −2 (H4B4) sc-18822 (lot no. L1720), secondary antibody mlgGk BP-CFL 488 sc-516176 (lot no. D1922), and Mito Red were purchased from Santa Cruz Biotechnology (Texas).

2.2. Synthesis of Monosubstituted Cationic Porphyrin (Scheme 1).

Scheme 1.

Synthesis Scheme of 5-(N,N,N-Trimethylanilinium-4-yl)-10,15,20-triphenylporphyrin Iodide

2.2.1. Synthesis of 5,10,15,20-Tetraphenylporphyrin.

2.0 mL of pyrrole (1 equiv) and 2.7 mL of benzaldehyde (1 equiv) were added to 100 mL of propionic acid, refluxed for 30 min, and allowed to cool to room temperature. The solution was then precipitated in an ice bath, filtered, and washed with methanol. The crude product was purified by silica gel column chromatography with DCM as the eluent. The product was recrystallized in DCM/MeOH, vacuum-filtered, and dried. The product yield was 0.60 g (9%). ¹H NMR (400 MHz) in CDCl₃ (Figure S1), δ (ppm): 8.84 (m, 8H), 8.20–8.22 (m, 8H), 7.71–7.79 (m, 12H), −2.78 (s, 2H).

2.2.2. Synthesis of 5-(4-Nitrophenyl)-10,15,20-triphenylporphyrin.

1.92 g (3 equiv) portion of tetraphenylporphyrin was dissolved in 30 mL of trifluoroacetic acid (TFA). 0.31 g (4.2 equiv) of sodium nitrite was added to the solution and stirred for 5 min. The solution was slowly poured into 100 mL ammonium hydroxide (NH₄OH) followed by extraction with DCM. The organic layer was washed with 2 × 100 mL DI water and dried with anhydrous magnesium sulfate and rotavapor to remove DCM. The crude product was purified using silica gel column chromatography with 45% DCM in hexane as the eluent. The second fraction was recrystallized in DCM/MeOH, vacuum-filtered, and dried. The product yield was 1.11 g (54%). ¹H NMR (400 MHz) in CDCl₃, δ (ppm): 8.85–8.89 (m, 6H), 8.72–8.73 (d, 2H, J = 4.4 Hz), 8.60–8.63 (d, 2H, J = 2.4 Hz), 8.37–8.39 (d, 2H, J = 8.8 Hz), 8.19–8.21 (m, 6H), 7.73–7.80 (m, 9H), −2.79 (s, 2H). The NMR and electrospray ionization mass spectrometry (ESI-MS) spectra are given in Figures S2 and S3 (Scheme 1).

2.2.3. Synthesis of 5-(4-Aminophenyl)-10,15,20-triphenylporphyrin.

One mol equivalent of 5-(4-nitrophenyl)-10,15,20-triphenylporphyrin (0.85 g) was dissolved in 25 mL of hydrochloric acid. 0.90 g of stannous chloride (3 equiv) was added to the solution and heated at 65 °C for 4 h under nitrogen. The solution was allowed to cool to room temperature, poured into 100 mL of ice-cold DI water, and neutralized with NH₄OH to a pH of 8. The product was extracted with DCM, dried over anhydrous magnesium sulfate, and the solvent was removed via rotary evaporation. The crude product was purified using silica gel column chromatography with DCM as the eluent and recrystallized in DCM/MeOH, vacuum-filtered, and dried. The product yield was 0.57 g (70%). ¹H NMR (400 MHz) in CDCl₃, δ (ppm): 8.92–8.93 (d, 2H, J = 4.8 Hz), 8.82 (m, 6H), 8.19–8.21 (m, 6H), 7.97–7.99 (d, 2H, J = 8.4 Hz), 7.72–7.78 (m, 9H), 7.04–7.06 (d, 2H, J = 8.0 Hz), 4.00 (d, 2H), −2.77 (s, 2H). The NMR and ESI-MS spectra are given in Figures S4 and S5.

2.2.4. Synthesis of 5-(N,N,N-Trimethylanilinium-4-yl)-10,15,20-triphenylporphyrin Iodide.

0.25 g of 5-(4-aminophenyl)-10,15,20-triphenylporphyrin was dissolved in 12 mL of anhydrous DMF. An excess amount of methyl iodide was added, and the solution was heated at 45 °C under nitrogen. The reaction was monitored with TLC until the product was completely formed. The mixture was poured into ice-cold DI water, vacuum-filtered, and dried in a vacuum oven. The product yield was 85%. ¹H NMR (400 MHz) in (CD₃)₂SO, δ (ppm): 8.80–8.89 (m, 8H), 8.51–8.53 (d, 2H, J = 8.0 Hz), 8.41–8.43 (d, 2H, J = 7.6 Hz), 8.23 (m, 6H), 7.84–7.89 (m, 9H), 3.92 (m, 9H), −2.93 (s, 2H). The NMR and ESI-MS spectra are given in Figures S6 and S7.

2.3. Synthesis and Characterization of IMs.

IMs were synthesized via a simple, one-step ion exchange method (as shown in Schemes 2 and S1) where the iodide counteranion of the porphyrin cation was substituted with a bulky cation to prevent aggregation between porphyrin and to increase its PDT efficacy. Two bulky anions BETI and IR783 were used for the synthesis of the IMs. LiBETI was first used, where BETI does not exhibit any photophysical properties. Another IM of porphyrin was prepared using a more biocompatible IR783 anion which not only hinders the aggregation in porphyrin but can also induce a longer wavelength absorption characteristic as well as the possibility of a FRET mechanism in the resulting compound.

Scheme 2.

Synthesis Scheme of [Porph][IR783] IMs

Both IM syntheses were performed using a similar metathesis approach reported previously.^7,19 Briefly, solution A was prepared by dissolving cationic porphyrin in DCM, and solution B was prepared by dissolving 1 mol equivalent of LiBETI or NaIR783 in water. The two solutions were combined in a round-bottom flask and stirred for 48 h at room temperature to ensure a complete ion exchange reaction. Afterward, the water layer containing lithium iodide or sodium iodide salt was discarded, and the organic layer was washed three times with water to remove any remaining salt. The organic layer was then dried via rotary evaporation to remove DCM, and the resultant IMs, [Porph][BETI] and [Porph][IR783], were collected and freeze-dried to remove any moisture. The synthesized IMs were characterized using ESI–MS, Bruker (Billerica, MA) and nuclear magnetic resonance (NMR, JEOL, 400 Hz). The mass-to-charge ratio peaks in positive and negative ion modes verified the presence of both ions in the IMs. Moreover, NMR spectra were also recorded, which also confirmed the successful synthesis of the IMs (Figures S8–S13).

2.4. Synthesis and Characterization of INMs.

INMs were prepared using the reprecipitation method as described in other papers.^9,20 Briefly, a 1 mM stock solution of IMs was prepared in DMSO and was added dropwise to a scintillation vial of deionized water/PBS in an active sonication bath. The vial was allowed to sonicate for 5 min, after which it was allowed to rest for 30 min to stabilize the particles before measurements. The hydrodynamic diameter of the nanoparticles was determined by using the dynamic light scattering (DLS) method. Zetasizer Advance Series Pro (Red Label, Malvern Panalytical Inc., WESTBOROUGH, MA) was used for the hydrodynamic diameter and ζ potential (surface charge of the nanoparticles) measurement.

2.5. Photophysical Properties.

A UV–vis absorption spectrophotometer (Agilent Cary 5000, Santa Clara, CA) and a fluorometer (Horiba Fluorolog, Kyoto, Japan) were used for the photophysical characterization of the IMs, INMs, and parent compounds. The photophysical properties included absorbance, molar absorption coefficient, fluorescence and phosphorescence emission, fluorescence quantum yield, fluorescence lifetime, photostability, and phosphorescence decay. Starna quartz cuvettes polished with two sides and four sides having a path length of 1 cm were used for the absorbance and fluorescence and phosphorescence emission measurements, respectively. Serial dilutions were prepared, and the absorbance data were used to calculate the molar extinction coefficient using Beer’s law. The phosphorescence emission was recorded using a 2 ms delay time at 77 K at a 415 nm excitation wavelength.

FRET mechanisms in IM/INMs were also investigated due to the spectral overlap between the fluorescence emission spectra of porphyrin and the absorption spectra of IR783. The details of the spectral overlap and equations used to calculate the spectral overlap integral, the theoretical Förster distance between two ions, and FRET efficiency are presented in the Supporting Information on pages S-12 and S-13.

Fluorescence quantum yield (FLQY) measurements were performed using the relative method (eq 1) where TCPP was used as the standard for experiments in both ethanol and water. From the absorbance and emission spectra, the quantum yields (Φ_F), radiative (k_rad), and nonradiative (k_nonrad) constants of parent compounds, IMs, and INMs were also calculated using eqs 2 and 3. These measurements are crucial to understanding the changes in the photophysical properties simply by modifying the counterion, which can directly impact the PDT performance of porphyrin.

$${\Phi }_{\text{un}}={\Phi }_{\text{std}}\times \frac{I_{\text{un}}}{I_{\text{std}}}\times \frac{{\text{Abs}}_{\text{std}}}{{\text{Abs}}_{\text{un}}}\times {\left(\frac{n_{\text{un}}}{n_{\text{std}}}\right)}^2$$ (1)

$$k_{\text{rad}}=2.88\times {10}^{-9}\times \frac{\int (⊽)\text{d}⊽}{\int (⊽){⊽}^{-3}}\times \int \frac{\epsilon ⊽}{⊽}\text{d}⊽$$ (2)

$${\Phi }_{\text{F}}=\frac{k_{\text{rad}}}{k_{\text{rad}}+k_{\text{non}-\text{rad}}}$$ (3)

where Φ_std is the quantum yield of the standard, Φ_un is the quantum yield of the unknown (in this case, the IMs and INMs), I is the integrated emission intensity, Abs is the absorbance at the excitation wavelength, and n is the refractive index. ⊽ is the wavenumber of light, and ε is the molar extinction coefficient.

Photostability measurement of parent compounds, IMs, and INMs was conducted using a kinetic mode over 1800 s at an excitation wavelength of 415 nm while monitoring the emission at 650 nm with a slit width of 14–14 nm. For [Porph][IR783], the photostability was also determined at the excitation (720 nm) and emission (820 nm) wavelengths of IR783. Fluorescence lifetimes in ethanol and water (solutions with absorbance less than 0.1 were prepared) were recorded using the time-correlated single photon counting (TCSPC) method with a Delta Hub controller in conjunction with a Horiba fluorometer. A NanoLED (Horiba) source with an excitation wavelength of 390 nm was used as the excitation source for the lifetime measurement. The data obtained for the lifetime were processed with DAS6 software using exponential fitting of the raw data. The phosphorescence lifetime of the drugs was recorded using the decay by delay method at an excitation wavelength of 415 nm and an emission wavelength of 775 nm. The data were fit with two exponentials.

2.6. Singlet Oxygen Quantum Yield (SOQY).

The SOQY of parent porphyrin, IMs, and INMs was investigated to determine their PDT efficiency. The experiment was conducted using two different probes in three solvents. DPBF was used as a singlet oxygen probe in ethanol, while the ABDA probe was used for INMs in water and PBS.

DPBF is yellow in solution and exhibits absorption maxima at 413 nm. In the presence of reactive oxygen species (ROS), yellow DPBF changes to colorless due to oxidation.^37,38 Therefore, a decrease in the absorbance of DPBF absorption was used to quantify the singlet oxygen quantum yield in ethanol. In this experiment, TCPP with a reported SOQY in ethanol was used as the standard compound to evaluate the SOQY of the drugs.⁶ Solutions of parent porphyrin, IMs, TCPP, and DPBF were all prepared separately in ethanol. An equal volume of 2 μM parent porphyrin/IMs/TCPP with 150 μM DPBF was mixed, and the mixture was irradiated at the wavelength maxima of the drugs (415 nm) for 1 min time intervals using a fluorescence spectrofluorometer (14 nm slit width). After each irradiation, changes in the absorbance intensity of DPBF at 413 nm were monitored using a UV/vis spectrophotometer to quantify the singlet oxygen generated by compounds.

DPBF is known not to be stable at wavelengths below 500 nm.³⁹ To prove that the decrease in the DPBF peak is due to the presence of the singlet oxygen, another experiment was performed at the Q-band of the porphyrin. Equal volumes of 4 μM parent porphyrin/IMs/TCPP with 80 μM DPBF were irradiated at 515 nm at a 1 min time interval. The absorption maximum wavelength of DPBF overlaps with that of porphyrin, resulting in their wavelength maxima adding up together. To prove that the decrease in absorption intensity was due to DPBF and not photobleaching of porphyrin or IMs, another experiment was designed. In this experiment, only parent porphyrin or IM solution without DPBF was irradiated at 415 nm.

ABDA was used as a singlet oxygen probe for nanomedicines in water and PBS solvents. Twenty μM/10 μM concentrations of the compounds were mixed with 200 μM/100 μM ABDA in water/PBS, respectively. The mixture was irradiated at 415 nm at a 2 min interval, and the decrease in ABDA absorbance at 380 nm was monitored. A control experiment with just the probes (DPBF and ABDA) in the absence of drugs was also conducted.

The IR783 anion is known to exhibit PDT as well as PTT mechanisms upon irradiation of light. Therefore, both ROS quantum yield, as well as light-to-heat conversion efficiency experiments are designed to evaluate their PDT as well as their PTT efficiency, respectively. Since NaIR783 absorbs light in the NIR region, another SOQY experiment in ethanol was designed for parent NaIR783 and [Porph][IR783] by irradiating the samples with an 808 nm laser (1 W/cm²) for 15 s time intervals. NaIR820, with a reported SOQY in ethanol, was used as the standard compound.²⁰ The singlet oxygen quantum yield of the samples was calculated using eq 4.

$${\Phi }_{\text{un}}={\Phi }_{\text{std}}\times \frac{S_{\text{un}}}{S_{\text{std}}}$$ (4)

where S is the slope obtained from the plot of the absorbance of the probe versus irradiation time, subscript “un” stands for the unknown samples analyzed, and subscript “std” is used for the standard. The experimental details as well as results of the photothermal efficiency are presented in the Supporting Information.

2.7. Cell Culture and Cellular Uptake.

Cells were grown in an incubator at 37 °C and 5% CO₂ in a complete medium. MCF-7 cells were cultured in DMEM, supplemented with FBS (10% v/v) and penicillin/streptomycin antibiotic solution (500 units/mL). When cells reached 80% confluence, they were subcultured via trypsinization, and the detached cells were stained with trypan blue exclusion dye, followed by counting using a hemacytometer.

Cellular uptake experiments were performed at three different incubation times of the drug to investigate the amount of drug internalized in the MCF-7 cells at a given time interval. 6 ×10⁵ cells per well were seeded in a six-well plate and incubated at 37 °C for 24 h. Drug samples were added to each well at a final concentration of 15 μM and incubated in the dark for different time intervals (4, 6, and 8 h). After the allotted time, the cells were washed thoroughly with PBS three times to remove any external drug. Cells were dissolved in DMSO, and the drug’s concentration was determined by using a UV–vis absorption spectrophotometer using the calibration curve obtained from solutions of known concentrations.

2.8. Cell Viability Studies.

Dark toxicity (24 h) was determined using a typical MTT assay.⁷ Briefly, cells were seeded in 96-well plates at a density of 10⁴ cells per well and incubated at 37 °C (5% CO₂) for 24 h. The cells were then treated with different concentrations of the drug for 24 h. INMs were prepared at various concentrations by diluting the stock solution in cell culture media following bath sonication while maintaining a sterile environment. DMSO used as a vehicle was at a maximum of 0.5% to avoid any cellular toxicity. Appropriate controls with complete media alone and vehicle control without drugs (DMSO) were included. Following treatment, cells were washed with PBS buffer, pH 7.4. A microplate reader (Biotek Synergy H1, Winooski, VT) was used to determine the optical density at 570 nm for the MTT assay. For in vitro studies, each experiment was performed in triplicate and repeated three times. All data that show error bars are presented as mean ± standard deviation (SD) unless otherwise mentioned. The significant difference in the mean values was determined using a two-tailed Student’s t-test. Values with significant differences are denoted as *p < 0.05, **p < 0.01, ***p < 0.005.

Light toxicity experiments were performed to determine the effect of PDT via cationic porphyrin and anionic IR783 dyes in both [Porph][BETI] and [Porph][IR783] INMs. Briefly, 10⁴ cells per well were plated in a 96-well plate and allowed to incubate for 24 h. Afterward, the cells were treated with INMs or parent molecules and incubated again for 4 h. The media containing the drug was then removed and cells were washed thrice with PBS to remove any uninternalized drug. Cell media were replaced in each well, and the plate was irradiated with either a visible lamp (0.14 W/cm²) for 10 min (to investigate the PDT activity of porphyrin) or an 808 nm laser (1 W/cm²) for 5 min (to investigate the PDT activity of IR783). A control experiment was run in tandem with identical conditions but was not exposed to light. Following irradiation, the plate was incubated for an additional 24 h, and an MTT assay was performed to determine cell viability.²⁰

2.9. Subcellular Localization.

Cell seeding density of 5 × 10⁴ cells was introduced onto a 12 mm coverslip previously introduced in each well of the 24-well plate. Cells were incubated for 24 h. Next, the cells were washed and were further incubated with 5 μM porphyrin or porphyrin-based INMs prepared in cell media for 6 h. Following 6 h of incubation, the leftover particles were aspirated, and cells were washed twice with PBS. Then, 150 nM Mito Red solution prepared in cell media was introduced for 45 min. Coverslips were washed several times and proceeded with the addition of 4% paraformaldehyde for 15 min. Next, cells were permeabilized using a blocking solution for 30 min and subsequently washed twice. LAMP primary was added at a dilution of 1:100 for 30 min, according to the manufacturer’s protocol. Cells were washed with PBS for 5 min each. Then a secondary LAMP antibody conjugated with AlexaFluor 488 was added at 1:100 dilution for 30 min. Slips were further washed three times with PBS at every 5 min interval. Cells were further treated with DAPI at a concentration of 300 nM for 5 min, and the coverslips were washed twice with PBS. Coverslips were then mounted onto a microscope slide and imaged using the laser scanning confocal microscope. A laser scanning confocal microscope (Zeiss, LSM 880), attached to an inverted microscope with an oil immersion objective lens (63x), was used for the imaging. A diode excitation source of 633 nm was utilized with the emission set at 650 nm to view the drugs.

3. RESULTS AND DISCUSSION

3.1. Characterization of IMs and INMs.

The synthesized IMs, [Porph][BETI] and [Porph][IR783], were characterized using NMR, and the NMR spectra of both IMs are presented in Figures S8 and S11, respectively. Further characterization with ESI-MS was performed to confirm the presence of both ions in the IMs. The mass-to-charge ratio peaks in the positive and negative ion modes confirmed the presence of cationic porphyrin and the BETI/IR783 anion in [Porph][BETI] and [Porph][IR783] IMs. The expected m/z⁺ peak of 672.3 calculated for [Porph] was observed in the positive ion mode of [Porph][BETI] and [Porph][IR783] with an experimental value of the m/z⁺ peak at 672.3122 and 672.3129, respectively (Figures S9 and S12). In the negative ion mode, the expected m/z⁻ peak of 380.159 calculated for BETI was observed at 379.9114 (Figure S10) for [Porph][BETI], while in [Porph][IR783], the calculated value of IR783 for 726.36 showed an experimental m/z⁻ peak for IR783 at 725.2490 (Figure S13).

The hydrodynamic diameter of the INMs in water was analyzed using DLS (Figure S14). The solvated diameter value was determined to be 42.4 ± 3.29 nm for porphyrin. [Porph][BETI] produced particles with a hydrodynamic diameter of around 151.8 ± 3.14 nm, while [Porph][IR783] produced particles with a solvated diameter of 92.9 ± 2.5 nm. The surface charges of the [Porph], [Porph][BETI], and [Porph][IR783] nanoparticles were determined to be +36.85 ± 4.6, + 28.1 ± 1.5, and +28.6 ± 1.2 mV, respectively (Figure S15). The surface charge of the cationic porphyrin-based INMs being positive means the cation was mostly at the surface. A decrease in the positive surface charge was observed for the INMs compared to the parent porphyrin, which indicates the presence of the bulky anion on the INMs’ surface. The positive zeta potential is highly desirable to attain high cellular uptake by the negatively charged cell membranes.

3.2. Photophysical Characterization (Absorption and Fluorescence Emission).

The absorbance and fluorescence emission spectra of the parent compounds, IMs, and INMs were recorded and compared to investigate the effect of different anions on the photophysical properties, which directly impact the PDT activity of the compound. It is also worthwhile to determine if there were any changes in the peak position of porphyrin upon conversion into IMs. The absorbance and fluorescence emission spectra of [Porph][BETI] IMs were compared to only parent porphyrin since LiBETI does not absorb light in the visible region. The [Porph][IR783] on the other hand was compared to both parent porphyrin and NaIR783. All compounds showed the characteristic spectra of porphyrins that contain a Soret band and four Q-bands (Figures 1a,c and S16a).⁴⁰ In ethanol, parent porphyrins, [Porph][BETI], and [Porph][IR783] all showed similar peak shapes and wavelength maxima, but the molar extinction coefficient of the IMs exhibited a significant increase as compared to parent porphyrin (Table 1). Also, while the BETI anion does not absorb light, conversion of parent porphyrin to [Porph][BETI] IM showed an increase in the absorption intensity of the Soret peak of porphyrin, indicating that the exchange of the small counteranion (iodide) of porphyrin with a bulky anion facilitated reduced aggregation in porphyrin. [Porph][IR783] IMs exhibited an improved molar extinction coefficient when compared to parent porphyrin as well as [Porph][BETI].

Figure 1.

(a, c) Absorption spectra and (b, d) fluorescence emission spectra of parent porphyrin and IMs in ethanol and water at an excitation wavelength of 415 nm.

Table 1.

Molar Absorptivity at Different Wavelengths for Compounds in Ethanol and Water Solvents

compound	λmax (nm)	ε (L/mol cm) × 104
[Porph]_ethanol	415, 512, 546	39.75, 1.58, 0.63
[Porph][BETI]_ethanol	415, 512, 547	47.90, 1.96, 0.91
[Porph][IR783] _ethanol	415, 512, 547, 724, 788	56.50, 2.58, 1.21, 4.94, 18.55
[NaIR783] _ethanol	724, 788	7.70, 29.45
[Porph]_water	414, 520, 553	7.90, 0.56, 0.14
[Porph][BETI]_water	419, 520, 553	6.85, 0.48, 0.14
[Porph][IR783]_water	419, 520, 553, 746, 807	15.39, 1.44, 0.63, 2.83, 4.28
[NaIR783]_water	712, 776	7.25, 19.75
[Porph]_PBS	428, 518, 554	6.38, 1.32, 0.76
[Porph][BETI]_PBS	427, 518, 554	8.64, 1.61, 0.98
[Porph][IR783]_PBS	427, 518, 554, 746, 807	11.07, 2.05, 1.21, 2.82, 4.00
[NaIR783]_PBS	712, 776	5.34, 14.30

In water, a slight bathochromic shift in the absorption maxima wavelength from 415 to 419 nm was observed for both INMs compared to parent porphyrin. Although the shift is small, it is desirable to attain a longer absorption peak for porphyrin. It is possible to further tune the absorption wavelength by controlling the morphology of INMs.³⁰ In PBS, a bathochromic shift of about 9 nm was observed in the INMs compared to that in water at the porphyrin Soret band (Figure S16a). An increase in absorbance was observed for both INMs as compared with parent porphyrin. Whereas, in water, the absorbance of porphyrin was slightly higher than that of [Porph][BETI]. No change in wavelength was observed for [Porph][IR783] and NaIR783 at the IR dye’s wavelength in both water and PBS media. The molar extinction coefficient of the INMs in water and PBS was lower compared to that of IMs in ethanol. As shown in Table 1, an increase in the molar extinction coefficient was observed in the Q-bands of porphyrin for the INMs in PBS as compared to water. Since porphyrin is in the particle form in INMs, it is quite possible that those porphyrin moieties that are present in the core of the nanoparticle are not able to absorb the light significantly. A broad Soret peak for the INMs was acquired, which also suggests that this nanomedicine can be irradiated by a wide range of wavelengths of electromagnetic radiation to activate the PDT mechanism. The presence of the IR783 absorbance peak further confirmed the presence of both ions in the [Porph][IR783] IMs and INMs. A notable bathochromic shift of the main absorbance peak of IR783 was observed from 776 to 807 nm, while the shoulder peak also shifted from 712 to 746 nm in [Porph][IR783] INMs. In addition, the absorption peak exhibited significant broadness in comparison to the parent compound and absorbed up to 875 nm wavelength light. This is a tremendous finding that suggests that the INMs can be irradiated using a longer wavelength radiation source that can penetrate deep in the body tissue to target the deeply seated tumor, which is not possible when using the parent porphyrin compound.

Comparing the results to the previous work done by Macchi et al.⁷ for tetrasubstituted anionic porphyrins, the mono cationic porphyrin synthesized in this work showed high molar absorptivity (Table S1), which further increased upon conversion to IMs. It demonstrated that a low dose of PS is sufficient to activate the light therapeutic mechanism. Thus, it validates that cationic porphyrin has better potential for PDT than tetra-anionic porphyrin due to its improved photophysical properties.

The fluorescence emission spectra of the parent compounds, IMs, and INMs are shown in Figure 1b,d. The fluorescence emission spectra were recorded at an excitation wavelength of 415 nm in ethanol and water as well. For [Porph][IR783], the fluorescence emission spectra were also recorded at the IR783 wavelength (720 nm excitation) and compared with parent NaIR783 (Figure S17). The emission spectra recorded in PBS at the excitation wavelengths of 420 and 720 nm are presented in Figure S16b,c, respectively. All of the fluorescence emission spectra at the excitation wavelength of porphyrin (415 nm) exhibited two peaks, which is consistent with the literature for free-base porphyrins.⁴⁰ A slight decrease in the fluorescence intensity was observed for the IMs in ethanol compared with parent porphyrin. A notable decrease in the fluorescence emission intensity was recorded along with a slight red shift for the compounds in water. A significant decrease in the fluorescence emission intensity was observed for [Porph][IR783] INMs in PBS (Figure S16b) when excited at 420 nm. A similar trend was observed for parent NaIR783 and [Porph][IR783] in the fluorescence emission spectra for both water and PBS. The results demonstrated a significant change in the photophysical properties of porphyrin when converted into IMs and INMs. The decrease in the fluorescence emission intensity of IMs/INMs was attributed to the increase in other nonradiative pathways including internal conversion and intersystem crossing. Therefore, the fluorescence quantum yield, radiative rate, and nonradiative rate constants were investigated to understand the excited-state decay mechanism.

3.3. Förster Resonance Energy Transfer (FRET).

Previous work by our research lab has shown the possibility of FRET mechanism in IMs/INMs upon removing spectator ions.⁴¹ Therefore, the FRET phenomenon in [Porph][IR783] IMs and INMs was investigated in three different solvents (ethanol, water, and PBS) and details of the spectra are provided in Figure S18. The high spectral overlap integral (J(λ)) value (Table 2) obtained using eq S1 in the SI supports the possibility of energy transfer from porphyrin to IR783. It is noted that the spectral overlap value increased in INMs in comparison to IMs. The theoretical Förster distances calculated using eq S2 in the SI were from 2 to 4 nm, indicating the feasibility of energy transfer (Table 2). The distance between the two ions in the INMs was shorter than the IMs, indicating that the two ions are close in the INM form (water and PBS) as compared to the IMs (ethanol). This indicates that INMs are highly suitable for FRET mechanisms. The FRET efficiency was calculated using eq S3. As shown in Table 2, the efficiency of energy transfer is significantly affected by the solvent system. It is interesting to note that the highest value of FRET efficiency was observed in the buffer media, and it is attributed to the minimum distance between the donor and acceptor in PBS media.

Table 2.

FRET Efficiency, Förster Distance, and Spectral Overlap Integral of [Porph][IR783] in Different Solvents

solvent	% FRET efficiency	Förster distance (R0, nm)	spectral overlap integral × 1016 (J(λ)/M−1 cm−1 nm4)
ethanol	13.5 ± 0.01	3.66	3.25
water	26.8 ± 0.04	2.74	5.57
PBS	73.0 ± 0.02	2.61	4.37

3.4. Fluorescence Quantum Yield (FLQY) and Photophysical Rate Constant.

The FLQY was measured for the parent compounds, IMs, and INMs using the relative method, and the results were compared. TCPP with a reported quantum yield value in ethanol⁶ and water⁷ was used as a standard. The FLQY values obtained were calculated using eq 1 and all results are presented in Table 3. A decrease in the FLQY was observed for the IMs in ethanol, water, and PBS (as INMs) as compared to parent porphyrin, which suggests that there is a greater chance of other excited-state pathways including intersystem crossing to the triplet state. To enhance the PDT effect of a PS, a greater population of excited molecules should undergo intersystem crossing to populate the excited triplet state and thus decrease the FLQY.⁴²

Table 3.

Fluorescence Quantum Yield and Radiative/Nonradiative Rates of Compounds

compound	ΦF (%)	krad × 105 (s−1)	knonrad × 105 (s−1)
TCPP ethanol	4.40a	2.41	52.4
[Porph]_ethanol	4.28	1.87	61.9
[Porph][BETI]_ethanol	3.36	1.55	84.7
[Porph][IR783] _ethanol	3.31	0.94	137
TCPP_water	5.80b	13.0	211
[Porph]_water	0.44	8.89	1290
[Porph][BETI]_water	0.30	6.71	3589
[Porph][IR783]_water	0.16	5.68	6310
[Porph]_PBS	0.19	7.42	3130
[Porph][BETI]_PBS	0.14	4.00	7411
[Porph][IR783]_PBS	0.03	2.60	14100

^aThis value was obtained from ref 6.

^bThis value was taken from ref 7.

The radiative and nonradiative rate constants were investigated to further understand the results obtained for the FLQY. The radiative and nonradiative rate constants were calculated using eqs 2 and 3 and the results are tabulated in Table 3. As shown in Table 3, a decrease in the radiative rate and an increase in the nonradiative rate was observed upon conversion of parent porphyrin into IMs and INMs. INMs showed a tremendous increase in the nonradiative rate, exhibiting tremendous potential for internal conversion and intersystem crossing. An extremely high value of nonradiative rate was observed for the INMs in PBS as compared to that of water, indicating that the solvent plays a role in radiative and nonradiative pathways. The high nonradiative rate of the [Porph][IR783] INMs in PBS also correlated with the high FRET efficiency result attained in PBS. Comparing the results to the previous work in our lab using tetrasubstituted anionic porphyrin, there was a great decrease in the FLQY and radiative rate as well as an increase in the nonradiative rate of the INMs (Table S2).⁷ Detailed examination of the resultant values demonstrates the promising potential of INMs as a PDT nano drug.

3.5. Phosphorescence Emission Study.

For a compound or fluorophore to undergo the PDT mechanism, there must be the presence of a triplet excited state in the compound. When molecules from the excited singlet state undergo intersystem crossing, they reach the triplet excited state, where they react with molecular oxygen to cause cell death via radical formation. Porphyrin is well known for its triplet state. However, to answer the question of whether the population of the triplet state is increasing or not by changing the counteranion of the porphyrin cation, phosphorescence emission measurements were performed. To investigate the triplet-state population, the phosphorescence emissions of parent porphyrin and IMs in water, ethanol, and PBS were recorded at 77 K (Figures S19 and S20). It was observed that IMs/INMs showed a more intense phosphorescence emission peak in comparison to the soluble parent drug, indicating a greater population in the triplet state. Examination of these results suggests that newly synthesized nanomedicines should exhibit a better PDT performance than their respective parent compounds.

3.6. Photostability.

Adequate photostability is an important property for PSs or fluorophores that are used for biological applications. The photostability studies in ethanol and water were performed over 1800 s after excitation at 415 nm and monitoring emission at 650 nm for Porph, [Porph][BETI], and [Porph][IR783] using silt widths of 14/14 nm. A similar experiment was performed for NaIR783 and [Porph][IR783] samples at an excitation wavelength of 720 nm and an emission wavelength of 810 nm. All compounds exhibited good photostability with little to no photodegradation, but IMs were found to be more photostable as compared to parent porphyrin (Figure S21).

3.7. Fluorescence and Phosphorescence Lifetimes.

The fluorescence lifetimes of Porph, [Porph][BETI], and [Porph][IR783] in ethanol, water, and PBS were measured by using the TCSPC method, and the recorded lifetimes are shown in Table 4. The fluorescence decays in ethanol exhibited two exponential decays, while three exponential decays were observed in water and PBS. All compounds exhibited a lifetime of around 9 ns in ethanol and water. Although the longest lifetimes were mostly observed for the second population, the [Porph][BETI] INM showed a shorter lifetime in that population. The lifetime is slightly increased in [Porph][IR783] INMs, which is desirable to increase the chances of intersystem crossing. In PBS, a lifetime of about 7 ns was observed for [Porph] and [Porph][BETI], whereas no fluorescence lifetime was obtained for [Porph][IR783] due to the high nonradiative rate and FRET mechanism.

Table 4.

Fluorescence Lifetimes of Compounds in Ethanol and Water Recorded at a 390 nm Excitation/650 nm Emission Wavelength

compound	τ1 (ns)	α 1	τ2 (ns)	α 2	τ3 (ns)	α 3	χ 2
[Porph]_ethanol	1.33	0.0174	9.68	0.9826			1.05
[Porph][BETI]_ethanol	1.75	0.0344	9.75	0.9656			1.04
[Porph][IR783] _ethanol	1.89	0.0228	9.67	0.9772			1.02
[Porph]_water	2.84	0.0371	9.54	0.8633	0.12	0.0996	1.03
[Porph][BETI]_water	1.87	0.1702	0.276	0.7867	9.13	0.0431	1.10
[Porph][IR783]_water	6.55	0.0793	9.66	0.8651	0.16	0.0556	1.05
[Porph]_PBS	0.346	0.4669	1.91	0.4875	7.30	0.0456	1.12
[Porph][BETI]_PBS	0.345	0.5703	1.73	0.3881	7.59	0.0416	1.11
[Porph][IR783]_PBS

The molecules in the triplet state usually show a longer lifetime (up to tens of microseconds), allowing them to have enough time to react with molecular oxygen to produce singlet oxygen to cause cell death.⁴² The phosphorescence lifetime was also investigated in both ethanol, water, and PBS at 77 K and the decay was fitted using a second exponential fitting (Figures S22 and S23). The IMs/INMs’ longest lifetime was in the range of 0.2–0.5 ms, which indicates the possibility of the excited molecules having enough time to react with molecular oxygen before relaxing back to the ground state via phosphorescence. An increase in phosphorescence lifetime was observed for the INMs in water and PBS compared to parent porphyrin.

3.8. ROS/Singlet Oxygen Quantum Yield (PDT Activity).

The PDT efficacy is mainly dependent on the drug’s ability to produce singlet oxygen (SO) to damage cancer cells. The performance of a PDT drug can be determined by calculating the reactive oxygen species (ROS)/singlet oxygen quantum yield (SOQY) values. The results obtained from the photophysical characterization show significant improvement in the properties of the IMs in comparison with parent porphyrin. The SOQY of the parent compounds and IMs was investigated in ethanol using a DPBF probe. The experiment was also conducted in water and PBS using an ABDA probe. The absorbance of drugs with DPBF and ABDA probes at different irradiation times is presented in Figures S24–S27. From the figures, a decrease in the probe peak was observed with increasing irradiation time. This decrease in the absorbance of the probe is attributed to the presence of ROS generated by the samples.

Since the absorbance of the sample overlaps with the probe, the absorbance spectra of just the samples in the absence of the probe under light irradiation were investigated and the results are shown in Figure S28. The result confirmed that the decrease in intensity was not due to porphyrin’s degradation, thus further supporting that the porphyrin compounds are highly stable. A control experiment for the DPBF or ABDA probe in the absence of the drugs was also performed, which did not exhibit any decrease in the probe’s absorbance. Thus, all of these control experiments proved that the decrease in absorption of the probe’s peak in the presence of drugs was due to the generation of singlet oxygen or ROS under light irradiation (Figure S29). The SOQY was calculated using eq 4 and the slope for this equation was calculated from the graph (Figure 2). The results obtained for the 515 and 808 nm excitation wavelengths are shown in Figure S30. The calculated SOQY is listed in Table 5. The results indicate that the SOQY is significantly affected by solvents. Parent porphyrin showed a higher SOQY than the IM in ethanol and water. Notably, parent porphyrin was also prepared as nanoparticles in water and PBS. A slight change in the SOQY was observed for the samples in ethanol at 415 and 515 nm excitation. This confirms that the decrease of the DPBF peak is not because of the degradation of DPBF but rather due to the presence of reactive oxygen species. In PBS, the SOQY for [Porph][BETI] was higher than that of [Porph][IR783], but a reverse result was seen in the water solvent. This is attributed to the FRET mechanism present in [Porph][IR783] in PBS. A decrease in the SOQY was observed for [Porph][IR783] IMs compared to the parent IR783 anion upon laser irradiation at 808 nm with values of 6.2 and 2.5%, respectively, which is attributed to the low molar extinction coefficient of the IMs. The results obtained correlate well with the improved absorbance, decreased fluorescence, low quantum yield, high nonradiative rate constant, and good photostability of the INMs.

Figure 2.

Photodegradation of (a) DPBF in ethanol, (b) ABDA in water, and (c) ABDA in PBS at 415 nm excitation.

Table 5.

ROS/Singlet Oxygen Quantum Yield Values Obtained in Ethanol Using a DPBF Probe at 414, 515, and 808 nm Excitation Wavelengths

	TCPP	[Porph]	[Porph][BETI]	[Porph][IR783]
415 nm excitation (ethanol)	14.7a	14.2 ± 0.03	13.6 ± 0.02	11.6 ± 0.005
415 nm excitation (water)	10.0a	25.0 ± 0.001	18.7 ± 0.001	19.5 ± 0.007
415 nm excitation (PBS)	10.0a	22.6 ± 0.007	24.3 ± 0.012	15.3 ± 0.004
515 nm excitation (ethanol)	14.7a	15.7 ± 0.03	12.1 ± 0.02	11.0 ± 0.005

^aThis value was obtained from ref 6.

3.9. Light-to-Heat Conversion Efficiency (Photothermal Activity).

Since NIR dyes are known for their potential in photothermal therapy (PTT), light-to-heat conversion experiments were conducted in aqueous and PBS environments. The experiment was performed at two different concentrations and heating and cooling times to investigate if concentration and time play a role in photothermal efficiency. The change in temperature data in water and PBS is displayed in Figures S31 and S32. The increase in the heating and cooling times did not have a significant effect on the temperature change of the samples, as shown in Figures S31, S32, and Table S3. However, with increasing concentration, an increase in temperature was observed. The light-to-heat conversion efficiency of the samples was investigated for the 5 min heating using eqs S4–S6 and the results are shown in Table S4. Detailed analyses of the results revealed that the parent NaIR783 dye exhibited a higher light-to-heat efficiency as compared to the [Porph][IR783] INM. These findings demonstrated that the photothermal therapy activity of the NIR dye was diminished when converted from NaIR783 to the [Porph][IR783] INM.

Thus, the SOQY and light-to-heat conversion efficiency results recommend that the PDT mechanism of IR783 was enhanced, while PTT activity decreased upon converting from NaIR783 to the [Porph][IR783] INM. Despite that, both PTT and PDT mechanisms are present in the IM when irradiated with 808 nm laser light.

3.10. Cellular Uptake.

The uptake of drugs by cells is crucial for analyzing the drug efficacy. The cellular uptake was quantified by comparing the absorbance of cells treated with drugs to that of untreated cells as blank, as shown in Figure 3. Examination of the data revealed that the cellular uptake was significantly affected by the porphyrin’s counterion. The results revealed that [Porph][BETI] INMs had the highest cellular uptake, whereas a slight change in the uptake of parent porphyrin and [Porph][IR783] INMs was observed. This is attributed to the varying mechanisms of drug internalization or differences in the sizes of the nanoparticles. With the increase in incubation time from 4 to 6 h, an increase in drug internalization by the cells was observed, which decreased over time at 8 h incubation. From the results, it can be concluded that the optimum uptake of the drugs by the cell occurs at 6 h.

Figure 3.

Cellular uptake of parent porphyrin and INMs after 4, 6, and 8 h of incubation of 15 nmol of drug with MCF-7 cancer cells. Data are presented as the mean SD (n = 3).

3.11. In Vitro Cellular Toxicity of INMs.

MCF-7, a human breast cancer cell line, was used to investigate the cytotoxicity of the drugs in vitro. The half-maximum inhibition concentrations (IC₅₀) of the drugs were calculated and compared with the controls. A dose-dependent relationship was exhibited for all of the drugs used in this study (Figure S33). The cell viability in the dark for each drug as a function of concentration in MCF-7 for 24 h is shown in Figure 4a. The calculated IC₅₀ values are presented in Table 6. A lower IC₅₀ value was obtained for [Porph][IR783] INMs as compared to parent porphyrin, but a significant decrease was recorded for [Porph][BETI] INMs. The significantly lower IC₅₀ of [Porph][BETI] INMs is attributed to the nanoparticle size and the high cellular uptake of the BETI anion. No IC₅₀ value was determined in the dark for parent LiBETI and NaIR783 at the tested concentration range (Figure S34). Thus, it indicates that both compounds are not toxic in the dark. NaIR783 is known as a phototherapeutic agent, and it is expected to be nontoxic since there is no light irradiation. Dark studies were also performed at 4 and 6 h and the data are shown in Figures S35 and Table S5. All drugs exhibited high cytotoxicity at 6 h in the dark due to high cellular uptake.

Figure 4.

(a) Dark cytotoxicity, (b) light toxicity at 4 h, and (c) light cytotoxicity at 6 h of MCF-7 cells upon dosage with varying concentrations of parent compounds and INMs for 24 and 4 h, respectively (*p < 0.05, **p < 0.01, ***p < 0.005).

Table 6.

Calculated IC₅₀ Concentrations (μM) of Drugs in MCF-7 Cells after 24 h (Dark) and 4 h (Light)

drug (INMs)	dark studies (24 h)	light studies (4 h) visible	light studies (6 h) visible	light studies (4 h) 808 nm
LiBETI	ND.	ND	ND	ND
[Porph]	20.5 ± 4.8	0.742 ± 0.01	0.592 ± 0.02	ND
[Porph][BETI]	8.51 ± 2.4	0.640 ± 0.02	0.510 ± 0.02	ND
[Porph][IR783]	18.5 ± 0.78	0.350 ± 0.01	0.349 ± 0.01	2.13 ± 0.21
[NaIR783]	ND	ND	ND	8.26 ± 0.61

To evaluate the PDT activity of the drugs, cell viability experiments were performed under two different light irradiation conditions. The two sources of light are a visible lamp (for porphyrin excitation) and an IR 808 nm laser (for IR783 irradiation). Parent NaIR783 did not display any toxicity under the concentration range used when irradiated with visible light. It is expected since NaIR783 does not absorb the visible wavelength of electromagnetic radiation, and this visible light source is unable to activate the phototherapeutic mechanisms in NaIR783. The IC₅₀ value drastically dropped under visible light irradiation in comparison to the dark for porphyrin-based compounds. A significant decrease in the IC₅₀ value (in the nanomolar range) (Figure 4b) was observed due to the strong absorption of light by the porphyrin moiety, thus indicating that these compounds are very promising as PDT agents. Moreover, the [Porph][IR783] INMs showed the greatest toxicity, almost half the IC₅₀ value, as compared to parent porphyrin under visible light irradiation. However, [Porph][BETI] exhibited a slight decrease in toxicity compared to parent porphyrin. The enhanced light toxicity of [Porph][IR783] INMs is attributed to the presence of the FRET mechanism in this compound. The visible light absorbed by porphyrin also activated the IR783 anion in the INMs and initiated PDT as well as PTT mechanisms, thus greatly decreasing the IC₅₀ value in the nanomolar range. Given that the INMs are almost twice as potent (IC₅₀ in the nM range) as the parent porphyrin compound, the lightinduced cytotoxicity of porphyrin-based drugs can be finely tailored by enhancing the FRET efficiency simply by tuning the anion in IMs. With the highest uptake of the drug achieved at 6 h, the light studies of the drugs were performed at 6 h using the visible lamp (Figure 4c). As shown in Table 6, it was observed that with increasing irradiation time, the PDT activity of parent porphyrin and [Porph][BETI] increased with a lower IC₅₀, while no significant change was observed for the [Porph][IR783] INMs. The improved photophysical properties and singlet oxygen quantum yield which results in an enhanced PDT effect may be the reasons for the observed high cell cytotoxicity of the INMs upon light irradiation.

A similar experiment was performed for NaIR783 and [Porph][IR783] INMs using the IR 808 nm laser to investigate the PDT effect by the IR783 anion. The result is shown in Figure S36. The [Porph][IR783] INMs showed greater cytotoxicity compared to the parent NaIR783 dye with almost four times enhanced potency when irradiated with 808 nm laser light. Since the 808 nm laser irradiation diminishes the light-to-heat conversion efficiency of the IR783 anion in INMs, the enhanced light cytotoxicity of [Porph][IR783] INMs could be mainly attributed to the enhanced ROS production of [Porph][IR783]. Despite [Porph][IR783] being somewhat less internalized by the cells than parent porphyrin, the drug was highly efficacious in killing cancer cells, possibly due to the enhanced photophysical properties. In the future, the cellular uptake of the nanomedicine could further be optimized by controlling the size and FRET efficiency of the nanoparticles. Overall, the INMs exhibit good toxicity in the presence of light and are promising drugs for photodynamic therapy for cancer cells. The dark toxicity is significantly higher (more than 10 times) as compared to light.

3.12. Subcellular Localization.

The localization of porphyrin-based INMs was monitored through confocal microscopy based on the fluorescence properties of the drugs. MCF-7 cells were subjected to post-treatment with DAPI, LAMP, and Mito Red tracker dyes to determine the localization of the drugs in the cell. Confocal imaging (Figure 5) revealed that [Porph] was localized within the mitochondria, which is evident through the overlap of [Porph]’s emission with Mito Red fluorescence. These results are well correlated with the previously published studies by Lei et al.⁴³ Similarly, [Porph][IR783] exhibited mitochondrial targeting, as affirmed by the overlapping emission of the drug with Mito Red. Furthermore, the emission of [Porph][BETI] also significantly overlaps with Mito Red as well as the lysosome tracker, as manifested by the magenta coloration observed in the merged image. The changes in the localization of BETI containing INMs in the cells aid in understanding their high toxicity in the dark as compared to the other two compounds. This suggests that the location of the drug at the subcellular level could be altered simply by changing the counterion that can also tune the cytotoxicity of the drug.

Figure 5.

Subcellular localization of 5 μM porphyrin or porphyrin-based ionic nanomedicines on MCF-7 breast cancer cells for 6 h. Scale bar = 10 μm.

4. CONCLUSIONS

Monosubstituted cationic porphyrin was synthesized to produce cationic porphyrin IMs and INMs with a positive zeta potential. The synthesized cationic porphyrin was then converted to ionic materials (IMs) through a simple one-step ion exchange reaction with LiBETI and NaIR783. IMs were further transformed into carrier-free ionic nanomedicines (INMs) by using the reprecipitation method in water. Photophysical properties, as well as singlet oxygen generation and light-to-heat conversion efficiency, of the parent compounds, IMs, and INMs, were characterized. The IMs/INMs showed a significant increase in the molar absorption coefficient, nonradiative rate, and singlet oxygen quantum yield in comparison to their respective parent compounds. The [Porph][IR783] INMs exhibited an enhanced FRET efficiency in PBS media. Therefore, changes in the light-to-heat conversion efficiency were also observed. These results indicate an improved photodynamic efficiency compared to that of parent porphyrin. The [Porph][BETI] INMs exhibited enhanced dark and light toxicity toward MCF-7 cancer cells due to the high cellular uptake of the positively charged nanomedicine. In in vitro light studies, a low IC₅₀ value (nanomolar) was recorded for INMs in MCF-7. The newly synthesized cationic porphyrin as well as the IMs showed an enhanced PDT effect with high efficiency observed for the INMs. Organelle tracking results reveal that the location of the drugs can be altered by simply changing the counterions. Collectively, these observations suggest that the PDT efficiency of a drug can be further improved by using a simple INM strategy that allows tailoring of the photophysical properties as well as cellular uptake by tuning the size of the nanoparticles. Moreover, the comparison of the current results with previously published INMs with anionic porphyrin also demonstrated that porphyrin cation charge is significantly important to attain an enhanced nonradiative rate and an extremely low IC₅₀.