2.3.1. Silica Nanoparticles
Although silica lacks PDT activity on its own, silica nanoparticles can be used to encapsulate PS in PDT due to the chemically inert, nontoxic, and optically transparent nature of silica
[53][128]. In addition, it is commonly used for drug delivery in research because it is possible to functionalize chemicals to the silica through the hydroxyl groups on the silica surface (
Figure 34)
[54][55][129,130]. Mesoporous silica nanoparticles (MSNs) have been extensively utilized to deliver PSs, typically due to their interesting features such as large surface area and pore volume as well as high chemical stability
[56][57][58][131,132,133]. One group has developed mesoporous silica-based nanoparticles to exploit continuous oxygen evolution to enhance the effectiveness of PDT treatment in hypoxic cancer environments
[57][132]. To assemble Fe
3O
4 nanocrystals on silica nanoparticles doped with mesoporous dye, the surface was treated with 3-aminopropyltriethoxysilane and functionalized with amine groups. The oleic acid-stabilized Fe
3O
4 nanocrystals synthesized in an organic medium were reacted with the amine group of 2-bromo-2-methylpropionic acid, and the resulting Fe
3O
4 nanocrystals were assembled on the MSN surface by direct nucleophilic substitution between terminal bromine groups. The synthesized biocompatible manganese ferrite nanoparticle-immobilized mesoporous silica nanoparticles alleviated the hypoxic state of tumors with only a small number of nanoparticles and improved the treatment outcome of PDT in vivo.
Figure 34. Porphyrin-containing mesoporous silica nanoparticles for PDT.
NIR light-reactive multifunctional nanoparticles are ferrocene-modified with ICG rods and β-cyclodextrin (β-CD) capping for cooperative chemo-dynamic/photothermal/photodynamic (CDT/PTT/PDT) NPs made of mesoporous silica
[56][131]. As a mechanism of chemo-dynamic therapy, ferrocene released from multifunctional nanoparticles was able to efficiently kill cancer cells by converting intracellular H
2O
2 into toxic OH through a ferrocene-mediated Fenton reaction. Moreover,
1O
2 generated by ICG from near-infrared irradiation can kill cancer cells in cooperation with PDT. The results of in vitro experiments show that the CDT/PTT/PDT collaboration significantly amplified the inhibition rate of HeLa cells.
It was reported in one study that silica nanoparticles modified with folic acid (FA) could enhance the site-specific delivery of PS chlorin e6 (Ce6)
[59][134]. By improving the efficiency of targeted drug delivery by FA, efficient generation of singlet oxygen at 670 nm irradiation was obtained, which improved the killing efficacy of NPs on MDA-MB-231 cells compared to free Ce6. Furthermore, a perfluoro hexane (PFH)-encapsulated MSN-based multifunctional nanoplatform using the PS ICG loaded into a polydopamine (PDA) layer and PEG-FA decoration was presented
[60][135]. When excited with 808 nm light irradiation, it mediates the vaporization of PFH, creating bubbles for tumor ultrasound imaging and simultaneously inducing burst drug release. The PTT effect was exerted on the PDA layer, and the loaded ICG was able to generate ROS, a PDT mechanism, while providing NIR fluorescence emission.
2.3.2. Gold Nanoparticles
Gold nanoparticles have been studied for many years for effective PDT induction as well as drug carriers due to promising properties such as high surface area, facile surface modification through gold thiol chemistry, and biocompatibility. Furthermore, gold nanoparticles are being extensively studied for diagnostic applications because of their ability to tune optical scattering and absorption via physical features such as surface plasmon resonance effects
[61][136]. Gold nanoparticles can be applied to PDT without the use of an organic PS. The first use of gold nanorods (AuNRs) alone was reported in 2014
[62][137]. Upon excitation with relatively long-wavelength NIR light (915 nm), gold nanorods were able to generate a singlet oxygen (
1O
2) and destroy B16F0 melanoma tumors in mice. Excitation of gold nanorods at a wavelength of 780 nm (λ2), at which the PTT effect can be expected after generation of
1O
2, increases the temperature around the tumor tissue, as confirmed by formation of heat shock protein (HSP 70) in which photon energy is converted into heat. By changing the activation wavelength band, the dominant phototherapeutic effect can be switched between PDT and PTT and a synergistic effect can be obtained. It was also possible to trace the distribution of gold nanorods in vivo through self-emitting single-photon-induced fluorescence.
The same group tested the effect of PDT by comparing different types of gold nanoshells, including nanorod-in-shell, nanocage and nanoparticle-in-shell, and demonstrated that it could completely eliminate solid tumors in mice
[63][138]. They can modulate and switch the dominant roles of PDT and PTT by altering the activation wavelength that can excite the gold nanocage. As the most optimal conditions suggested by them, the nanocages mostly showed PDT effect when excited by 980 nm light, whereas 808 nm irradiation induced effective PTT. In vivo studies at 940 nm excitation, a wavelength band between 980 nm and 808 nm, demonstrate that gold nanoshells could induce dual-mode PDT/PTT for more efficient treatment of B16F0 melanoma tumors than that of doxorubicin, a clinically used drug.
Another group found that singlet oxygen could be produced when irradiated with a wide range of wavelengths (660–975 nm)
[64][139]. Even under low-intensity light irradiation of 200 mW/cm
2, the highest production of
1O
2 was observed when a wavelength overlapped with the localized surface plasmon resonance (LSPR) peak, which is a characteristic of gold nanoparticles.
Many previous studies have demonstrated the ability of metal nanoparticles to efficiently excite PS through a single-photon excitation mechanism to generate singlet oxygen, which has been applied to typical PDT therapy
[65][66][140,141]. However, one-photon excitation can cause potential photodamage to tissues adjacent to the tumor site due to the high energy provided by the comparatively short light wavelength. Therefore, two-photon excitation that precisely manipulates the therapeutic dose is preferable in this sense. To overcome this, a two-photon PDT was developed using a femtosecond laser beam capable of obtaining a high luminous flux. In one study, two-photon-induced singlet oxygen generation was observed by irradiating femtosecond laser pulses at 800 nm to aggregates of gold nanospheres and gold nanorods developed using non-agglomerated or aggregated gold nanoparticles
[67][142]. As a result, the
1O
2 generation capacity in gold nanoparticle was generally enhanced by the agglomerated state and was 8.3 times higher than that of the non-agglomerated gold nanoparticles. A similar trend was observed when the agglomerated gold nanorods were used; the singlet oxygen production efficiency was improved by 1.8 times compared to the non-agglomerated gold nanorods.
With the rapid advances in nanotechnology, there are a variety of synthetic methods available to researchers to obtain gold nanoparticles with suitable structures and features for PDT applications
[68][143]. In addition to the various physicochemical properties, the additional chemical modification potential mentioned above could improve bioavailability and usability, suggesting gold nanoparticles as a promising candidate for clinical cancer treatment.
2.3.3. Graphene Nanomaterials
Graphene-based nanomaterials, including graphene oxide (GO) and graphene quantum dots (GQD), have been widely used for cancer treatment such as anticancer drug delivery and PDT
[69][70][71][144,145,146]. GO produced through oxidation process shows more favorable properties in terms of PS transport mediation due to improved water solubility and various functionalization chemistries. Characterized by abundant oxygen-containing moieties on their surface, GO nanomaterials allow further modification by many functional molecules such as targeting agents, activators and hydrophilic macromolecules, expanding biological applications and reducing toxicity
[72][147]. Because the fluorescence quenching ability of GO nanomaterials is very high, it modulates the activity that generates ROS, further expanding the applications of PDT (
Figure 45).
Figure 45. Graphene quantum dots (GQDs)-based nanomaterials for PDT.
Numerous studies have been conducted to achieve tumor targeting, in vivo imaging, and improved PDT effects through functionalization on the GO surface. In one study, PEG-functionalized GO was loaded with the PS 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-alpha (HPPH) via supramolecular π-π stacking
[73][148]. HPPH radiolabeled with
64Cu enabled in vivo positron emission tomography and fluorescence imaging, resulting in improved cellular uptake of HPPH compared to free HPPH with GO-PEG-HPPH through a more aggressive endocytosis strategy. As a result, GO-PEG-HPPH exhibited enhanced phototoxicity to breast cancer cells when irradiated with light at a wavelength of 671 nm. Through in vivo experiments, mice injected with GO-PEG-HPPH showed a 16-day longer lifespan than mice treated with free HPPH. This indicates that GO-PEG-HPPH utilizing GO as a nanocarrier delivered the drug more efficiently and thereby increased long-term survival. In another study, the PS hypocrelin A (HA) and TiO
2 nanoparticles were mounted on GO surfaces to form a light-sensitive drug delivery system
[74][149]. By loading TiO
2 onto GO, ROS could be generated upon exposure to visible light, and the ability to generate ROS was improved through a mutual sensitization mechanism in which a sensitizing effect contributed by the HA-TiO
2 stable complex. The generated ROS were able to destroy GO, indicating a potential use of this drug delivery system in clinical PDT in terms of metabolism.
In another study, PS Ce6 was conjugated to GO via a redox-responsive cleavable disulfide linker (GO-SS-Ce6) to develop a form that could be released on-demand from cancer cells at significantly higher GSH concentrations compared to normal cells. Therefore, fluorescence and ROS generation were selectively activated by redox agents such as glutathione at high concentrations in tumor cells
[75][150]. On the other hand, in the absence of glutathione, the fluorescence of Ce6 bound to GO was largely quenched due to the FRET process, avoiding the nonspecific excitation and poor targeting ability of PS. The developed GO-SS-Ce6 complex has been proposed as an effective drug delivery vehicle with the strengths of GO’s high surface area and improved chemical tethering properties.
Furthermore, GQDs doped with quantum dots in graphene could provide excellent quantum yield of singlet oxygen as a PDT agent
[76][151]. It is known as a common method to synthesize GQDs using polythiophene as a carbon precursor using hydrothermal methods. The GQDs fabricated in the study were excited by visible light and showed photodynamic activity; their PDT effects were observed through apoptosis of HeLa cells and oncolysis of BALB/nude mice with breast cancer. On the other hand, more advanced studies showed that GQDs could be functionalized and doped with nitrogen and amino groups to show that the amino-N-GQDs exhibited excellent singlet oxygen generation capacity in the NIR region (800 nm)
[77][152].
2.3.4. Upconversion Nanoparticles
Upconversion nanoparticles (UCNPs) are a unique class of optical nanomaterials characterized by their ability to convert low-energy NIR light into high-energy visible/ultraviolet light using a nonlinear anti-Stokes mechanism
[78][153]. The upconversion phenomenon is based on inorganic host crystal lattices doped with trivalent lanthanide ions such as Yb
3+, Er
3+, and Tm
3+. UCNPs require the presence of two different dopant ions
[79][154]. One acts as a sensitizer to absorb NIR radiation, and the other acts as an activator to emit visible light. Two frequently used rare earth ion pairs are ytterbium-thulium (Yb
3+-Tm
3+) and ytterbium-erbium (Yb
3+-Er
3+). The Yb
3+ ions act as antennas, absorbing NIR light at about 900–1100 nm and transmitting it to the lanthanide ions, where they mutually upconvert. If this ion is Er
3+, green and red emission is observed, whereas if it is Tm
3+, the emitted light is near-ultraviolet, blue and red. In addition, the emission band of UCNP is similar to the band in which PS can be excited, which is characterized by improved ROS production efficiency
[53][128]. In this regard, UCNP may serve as a promising carrier to overcome the limitations of PDT due to the insufficient tissue penetrating ability of short wavelengths (600–850 nm) (
Figure 56).
Figure 56. Schematic diagram showing the mechanism of photodynamic therapy and bioimaging through long-wavelength to short-wavelength conversion of upconversion nanoparticles (UCNPs).
The NaYF
4: Yb
3+/ Er
3+, the first UCNPs used in PDT studies, showed strong emission spectrum in the visible region around 537 and 635 nm when excited by an infrared light source of 974 nm
[80][155]. During the silica coating procedure in the UNCP synthesis, the PS molecule merocyanine 540 (MC-540) was mounted on the nanoparticle. However, the activation wavelength of these PSs is under 700 nm, which is a range in which endogenous molecules such as hemoglobin have strong absorption, a great limitation in their use in PDT. A study successfully detected the generation of singlet oxygen mediated by UCNPs coated with MC-540 with NIR excitation by measuring the decrease in the fluorescence band of the
1O
2 sensor 9,10-anthracenedipropionic acid. Moreover, the first application of UCNP-mediated PDT for in vivo tumor therapy is NaYF
4:Yb/Er nanoparticles coated with mesoporous silica as nano-transducers and carriers of two different PSs such as MC-540 and ZnPc
[81][156]. Another study found that UCNPs synthesized using dual PS had higher PDT efficacy than using single PS, with improved ROS production capacity and enhanced cytotoxicity. In the tumor-bearing mice, both intratumoral injection of UCNP or intravenous injection of FA and PEG-modified UCNPs (FA-PEG-UCNP) into tumor resulted in tumor growth inhibition at 980 nm excitation. In addition, the tumor-targeting ability and circulating lifespan of UCNP were improved by FA and PEG, respectively, indicating a greater PDT effect when administered intravenously.
One research team prepared NaYF
4:Er/Yb/Gd upconversion nanocrystals by doping NaYF
4:Yb/Er UCNP with gadolinium ions and loading them with PS drugs to use as a carrier
[82][157]. Through a water-in-oil inverse microemulsion strategy, methylene blue (MB), a hydrophilic PS drug, was efficiently conjugated to UCNPs in a silica matrix to provide UCNP/MB nanocomposites with a particle size less than 50 nm. The obtained UCNP/MB-based PDT drug successfully generated singlet oxygen at 980 nm excitation, whereas no signal was observed with free MB solution alone or with NaYF
4:Er/Yb/Gd under the same conditions. Furthermore, polymer-coated NaYF
4:Yb/Er nanoparticles were used as transport mediators of PS Ce6 to form UCNP-Ce6 supramolecular complexes
[83][158]. Because this UCNP-Ce6 nanosystem showed two emission bands at 550 nm and 660 nm with 980 nm irradiation, PDT performance was improved in that the 660 nm emission wavelength overlapped the absorption band of Ce6, and singlet oxygen production was increased under NIR light irradiation. In particular, there were few observations of UCNPs administered to mice after 1–2 months, demonstrating their nontoxicity to the treated animals.
Although it is common to form NaYF
4 crystals with a host co-doped with Yb
3+/Er
3+ in UCNP-based PDT, doping NaYF
4 with a Yb
3+/Tm
3+ couple shows a similar phenomenon. In one study, NaYF
4:Yb/Tm UCNPs were coated with a nanometer silica layer, which was further modified with (3-aminopropyl)triethoxysilane APTES using the Stöber method
[84][159]. After that, the UCNPs were covalently bound to PS Ce6 via the amino group of the silica layer. A low concentration (50 μg/mL) of this UCNP-Ce6 nanocomposite was used to kill 50% of CF-7 human breast adenocarcinoma cells at a low dose (7 mW/cm
2) of 980 nm light for 10 min. Furthermore, they achieved a cell viability greater than 90% under the same conditions without light irradiation, indicating low toxicity of this UCNP-Ce6 nanosystem in the effective concentration range. Alternatively, LiYF
4:Tm
3+/Yb
3+-UCNPs prepared using m-THPC with PS modified with 4-(bromomethyl)benzoic acid performed better when activated with 980 nm NIR irradiation compared to conventional NaYF
4UCNPs. They emitted an intense blue color and produced a larger amount of singlet oxygen
[49][85][86][124,160,161].