Inorganic Nanoparticles for Cancer Treatment

The application of porphyrins and their derivatives have been investigated extensively over the past years for phototherapy cancer treatment. Phototherapeutic Porphyrins have the ability to generate high levels of reactive oxygen with a low dark toxicity and these properties have made them robust photosensitizing agents. In recent years, Porphyrins have been combined with various nanomaterials in order to improve their bio-distribution. These combinations allow for nanoparticles to enhance photodynamic therapy (PDT) cancer treatment and adding additional nanotheranostics (photothermal therapy—PTT) as well as enhance photodiagnosis (PDD) to the reaction. This review examines various porphyrin-based inorganic nanoparticles developed for phototherapy nanotheranostic cancer treatment over the last three years (2017 to 2020). Furthermore, current challenges in the development and future perspectives of porphyrin-based nanomedicines for cancer treatment are also highlighted.

These types of porphyrins are important PS agents in cancer phototherapy due to their structural presence of N-H groups and nitrogen atoms, whereby they can be further functionalized by having either nanoparticles, metal ion chelates or polymers conjugated onto their surfaces . Since first-and second-generation porphyrins have a high ability to produce ROS in phototherapy cancer applications, they are lipophilic in nature , have a low dark toxicity and low water solubility, as well as bio-distribution and their overall emission quantum yields are greatly affected . Furthermore, the low passive selectivity of porphyrins for tumor tissue can cause remarkable off-target and unwanted normal tissue damage . Thus, more studies are now focusing on porphyrin-NP conjugated systems for higher phototherapy efficiency, in relation to concentrated tumor cell uptake and so overall improved ROS production for PDT and PTT cancer treatments .
Noble metallic NPs such as gold, silver, and platinum have unique properties such as ease of functionalization due to their different chemical moieties and broad optical properties . Additionally, noble metallic NPs can be fine-tuned to The application of porphyrins and their derivatives have been investigated extensively over the past years for phototherapy cancer treatment. Phototherapeutic Porphyrins have the ability to generate high levels of reactive oxygen with a low dark toxicity and these properties have made them robust photosensitizing agents. In recent years, Porphyrins have been combined with various nanomaterials in order to improve their bio-distribution. These combinations allow for nanoparticles to enhance photodynamic therapy (PDT) cancer treatment and adding additional nanotheranostics (photothermal therapy-PTT) as well as enhance photodiagnosis (PDD) to the reaction. This review examines various porphyrin-based inorganic nanoparticles developed for phototherapy nanotheranostic cancer treatment over the last three years (2017 to 2020). Furthermore, current challenges in the development and future perspectives of porphyrin-based nanomedicines for cancer treatment are also highlighted.
Within studies performed by Penon et al. (2017), AuNPs conjugates were synthesized using biphasic and monophasic methods to investigate the optimal method of synthesis . These synthesized AuNPs were then conjugated to a porphyrin PS derivative containing an alkanethiol (5-[4-(11-mercaptoundecyloxy) phenyl]-10,15,20-triphenylporphyrin, PR-SH) and a thiolated carboxyl terminated polyethylene glycol (PEG) counterpart . The results from these studies reported that the monophasic method of AuNPs produced the highest amount of singlet oxygen, and so was utilized for PDT treatment assays on in vitro cultured SK-BR-3 human breast cancer . Additionally, the porphyrin-based monophasic AuNPs were also covalently functionalized with a specific breast cancer targeting antibody (Ab) anti-erbB2, to enhance cellular uptake . Overall, a higher cellular uptake was achieved when using the porphyrin-based monophasic-AuNP-PEG-Ab conjugate and a more significant cellular membrane damage was attained after PDT irradiation, when compared to controls .
Metalloporphyrins which contain Zinc (Zn) also provide a high PDT efficiency when compared to porphyrins alone, due to their metallic photothermal contribution . However, due to their low solubility and distribution, they are often found conjugated to various vehicles like NPs in order to overcome these shortfalls . Within studies by Alea-Reyes et al.
(2017) AuNPs were synthesized with thiol-PEG to promote water solubility and stabilized with amphiphilic gemini-type pyridinium salt . Onto these AuNPs an anionic molecule of Zn(II)meso-tetrakis(4-carboxyphenyl) porphyrin (Zn-TCPP) was incorporated . The synthesized Zn-TCPP loaded AuNPs generated remarkable amounts of singlet oxygen for the PDT treatment of in vitro human breast cancer cell line (SKBR-3), when compared to normal epithelium cells (MCF-10A) .
Over the past few years, researchers have shown that the combination of PDT and PTT has higher treatment efficiency than when compared to these treatments in singular form . In a study, by Zhang et. al. (2019) they integrated synergistic PDT and PTT treatment of in vitro A549 cells and in vivo lung cancer mouse models, using 660 and 808 nm laser irradiations . They fabricated 4-carboxyphenyl porphyrin conjugated silica-coated gold nanorods ( AuNR@SiO -TCPP), in which the AuNRs acted as the photothermal conversion agent for PTT, while the TCPP porphyrin PS produced ROS for effective PDT treatment . This study reported that the coating of AuNRs with mesoporous silica, improved PS loading capability and overall drug biocompatibility. In vitro experiments post phototherapy treatment noted a significant cell death of 79% of cultured lung cancer cells . Within in vivo AuNR@SiO -TCPP phototherapy treated mouse models, a remarkable inhibition of tumor volumes was found when compared to control mouse model groups which did not receive treatment. In addition, treated mice with PDT/PTT and AuNR@SiO2-TCPP showed a significant reduction tumor volumes, while AuNRs/PTT and PDT/ AuNR@SiO -TCPP treatments alone produced minimal effect .
Since AuNPs exhibit a high tendency to aggregate, researchers have overcome this issue by coating them hydrophilic polymers such as chitosan polyethylene glycol (PEG) or hyaluronic acid in order to promote stability, as well as prevent reduction of heat conversion properties . Additionally, these polymers not only enhance stability and solubility AuNPs, but also reduce their overall biotoxicity . In this regard, studies performed by Zeng et al. (2018) AuNPs were modified with chitosan (QCS-SH) via ligand exchange and then were attached to a PS porphyrin derivative, known as mesotetrakis (4-sulphonatophenyl) porphyrin (TPPS) for dual PDT and PTT therapy ( Figure 1) . This nanohybrid (TPPS/QCS-SH/AuNPs) produced high singlet oxygen and noted high elevated temperature of 56°C applications when compared to AuNPs or TPPS treatment alone, suggesting that this drug has potential for applications in tumor phototherapy fields .
During characterization experiments the PEG-AuNPs-CP-TPP nanospheres demonstrated high surface plasmon resonance in the infrared (NIR) region for PDT applications and high localized temperature under laser irradiation for PTT assays . Overall, the in vitro cytotoxicity results from this study showed that under sequential irradiation at 630 nm and 808 nm, the cervical cancer cells viability decreased to 10%, when the highest concentration of the drug conjugate was applied and so confirmed the effectiveness of combined PDT and PTT for enhanced cancer therapeutics .
Nonselective activation or universal aggregation-caused quenching (ACQ) has greatly decreased the efficiency of conventional PSs for PDT clinical applications . ACQ is the main setback in conventional PSs, since they have extended π-conjugation chains, when used at high concentrations and so their fluorescence is reduced PSs remarkably.
When a PS aggregates, strong intermolecular π-π stacking interactions occur leading to quenched emission via nonradiative pathways. Thus, conventional PSs can only be utilized at low concentrations, however this affects their photostability . Furthermore, it has been reported that nonselective activation or nonspecific drug leakage of conventional PSs can occur during blood circulation or diffusion into normal tissues which exert unwanted therapy-related side effects, such as toxicity and drug resistance .
Furthermore, within clinical trials it had been noted that PDT patients sometimes become photosensitive and so are required to stay away from light to prevent unwanted PS activation until it is completely absorbed by tumor cells, in order to prevent unwanted damage to normal tissues . In this sense, smart PSs have opened a growing research field of PSs. Smart PSs generally remain in the "Off" state during the absorption period and are only selectively activated or turned "On" once they have been fully internalized by cancer tumor cells . Recently, a new phototherapy strategy based on NIR smart PS platforms was proposed by Huang and co-workers (2019) to evaluate its in vitro and in vivo PDT efficiency in breast cancer (4T1) cells and murine tumor induced models . They integrated porphyrin PS units into upper critical solution temperature (UCST) block copolymer decorated gold nanorods (AuNR-P(AAm-co-AN-co-TPP)-b-PEG) . The AuNRs acted as a NIR-manipulated PDT smart PS, as well as a fluorescence quencher of the porphyrin PS and photothermal producer . Results noted that during blood circulation, the UCST block of the copolymer formed a collapsed-core and so caused aggregation of porphyrin PS units, subsequently leading to its "Off" state . Upon [16] [30] [30] [30] [31][32] [33] [30] [34] [35] [36] [35] [37] [38] [39] [39] [39] [39] internalization of the smart PS nanoplatform into cancer cells and NIR irradiation at 808 nm, the π−π stacking between the porphyrin units broke, activating the PS via a phase transition of UCST polymers from a collapsed to an extended state, causing the porphyrin PS unit to turn "On" (Figure 2) . In this "On" state and localized state within tumor cells, the porphyrin PS then received 650 nm PDT laser irradiation to more effectively generate ROS and singlet oxygen, in order boost the phototherapy efficacy of this smart PDT treatment . Furthermore, the NIR irradiation allowed for a photothermal heating reaction to occur within the AuNRs, which were contained within this drug conjugate and so added to the overall phototherapy efficacy of this smart PDT PS treatment . Overall, this smart PDT "Off/On" state process could be well manipulated using hybrid nanoplatforms with UCST block copolymers and AuNRs, and so could open new prospects for clinical-based PDT treatments . Regarding these in vivo studies, although the tumor growth in the mice injected with the nanoplatforms under 808 and 650 nm laser were remarkably increased, the therapeutic effect was similar to the mice treated with the nanoplatform under 650 nm laser only . Generally, phototherapeutic agents are mainly locate in adjacent regions of a tumor, due to their abnormal vasculature nature and high interstitial fluid pressure (IFP), thus large parts of the tumor remain unaffected, since they do not adequately absorb PSs . However, evidence has shown that many types of important cells associated with tumor initiation and progression are those fed by the defective blood vessels which supply solid tumors and research must focus on phototherapy research to target these cells, which in turn can prevent tumor growth and metastases .
Various strategies have focused on adjacent cells, while those distant from blood vessels remain untouched .
Therefore, the development of novel strategies to improve the penetration and uptake properties of PSs still remains an obstacle in nanotherapeutics.
A new treatment modality known as sonodynamic therapy (SDT) was proposed by Liang et al. (2019). SDT is used to excite and so activate PSs to produce high enough ROS levels for effective cancer therapy . Furthermore, as already mentioned, photothermal absorbers or photothermal transduction agents (PTAs) are adopted in PTT to harvest light energy and generate hyperthermia . These PTAs are categorized into semiconductor nanocrystals (NCs) , inorganic materials , and organic dyes . It has been reported that platinum or palladium-based photothermal transduction agents (PTAs) as inorganic PTAs have a higher photothermal stability and better catalytic properties than when compared to Au-based PTAs . Furthermore, studies have noted that copper sulfide semiconductor NCs provide very high LSPR within the NIR region .
In this regard, studies developed platinum-copper sulfide Janus nanoparticles conjugated to tetra-(4-aminophenyl) porphyrin (TAPP) to overcome the low penetration depth of PDT . In fact, they integrated semiconductor NCs and noble platinum metal to form their PTAs . The noble platinum metal promoted the photothermal conversion efficiency under 808 nm laser irradiation by changing the electron transport pathway and the large space of the hollow copper sulfide NPs interior facilitated a high loading capacity of the TAPP PSs . The synthesized nanohybrid was further coated with a temperature-sensitive polymer consisting of (poly (oligo (ethylene oxide) methacrylate-co-2-(2methoxyethoxy) ethyl methacrylate) to increase the biocompatibility and temperature triggered drug controlled release . Various studies have noted that metal and nitrogen co-doped carbon nanospheres could produce porphyrin-like metal

Porphyrin-Based Silica Nanoparticles
[82] [83] which facilitates the use of various methods of surface modifications for diagnostic and therapeutic applications . They also enable the fabrication of different silica nanoplatforms with various morphologies, sizes and porosity such as hollow or mesoporous silica NPs .

Martínez-Carmona et al. (2017) proposed a visible light-responsive nano-drug delivery which comprised of silica NPs
(MSN) decorated with porphyrin-caps to deliver topotecan (TOP) . The authors noted higher tissue penetration of visible light when compared to UV light for the PDT treatment of in vitro HOS osteosarcoma cancer cells. In the presence of visible light, the porphyrin-nanocaps produce singlet oxygen molecules which broke the sensitive-linker and triggered pore uncapping, allowing the release of the entrapped TOP . This nano-drug system was non-toxic and the greater penetration capacity of visible radiation noted a double antitumor effect due TOP release and porphyrin ROS production . Furthermore, in vitro assays revealed that TOP was released in controlled fashion inside HOS osteosarcoma cancer cells, via drug endosome escape to reach the cytoplasm . This research work opened up promising expectations for new alternative drug delivery systems for cancer treatment .
Chemotherapeutic drugs such as gemcitabine hydrochloride are noted to have a short in vivo half-life and poor membrane permeability due to its hydrophilic nature . Thus, high amounts of this drug is required to be administered to patients in order to effectively eradicate cancer tumor cells, however this induces adverse unwanted side effects . In this sense, a nanodelivery system was proposed by Aggad and co-workers (2018) for gemcitabine cancer therapy in order to overcome the hydrophilic limitations of chemotherapeutic drugs . In this study, they synthesized ethylenebased periodic mesoporous organosilica NPs (PMOs) for 2P-PDT and in order to enhance the delivery of gemcitabine within in vitro cultured MCF-7 breast cancer cells . A tetrasilylated porphyrin (PS1) PS was then attached to the ethylene-based PMOs, which caused J-aggregation inside the meso-structure of NPs leading to a two-photon PDT effect . Generally, PSs aggregate with absorption bands shifted to a longer wavelength, than when compared to monomer bands known as J-aggregates, which enhance the two-photon absorption properties . The synergistic effect of the two-photon irradiation with gemcitabine delivery and PS1-EPMOs noted more significant cancer cell death than when compared to control cells which did not receive irradiation .
Ultra-small hollow silica nanocarriers (HSdots) (~10 nm) were fabricated within studies performed by Dam et al.   [90] [92][93] [94] [94] [95] [95] [95] [95] [95] [95] Rare earth-based upconversion nanoparticles (UCNPs) have emerged recently in research as a way to circumvent the low tissue penetration depth limitation of PSs . Generally, the conversion of NIR light to a shorter wavelength of light is known as upconversion, which is an anti-Stokes process . Upconversion luminescence is a nonlinear process whereby successive lower energy photons absorb luminescence and so emit higher energy photons . This process has the advantage of low light scattering and autofluorescence background, as well as high tissue penetration depth, since this excitation occurs in the NIR region which is located within phototherapeutic window. This also consequently decreases any photo damage biological tissues might experience .
In a study by Sun et al. (2019), protoporphyrin IX PS was modified with jeffamine (PJ) to improve its hydrophilicity and MPPa/UCNP-DEVD-DOX/Crgd . Upon NIR irradiation, energy from the UCNPs was transferred to the PS which generated ROS for PDT treatment and simultaneously activated caspase-3 to initiate apoptotic cell death ( Figure 6) .  Reprinted with permission from . Copyright 2017 Elsevier.
Within PDT applied UCNPs, multiple low-energy exciting photons are used to emit a higher energy photon instead of the excitation of a single photon alone . Therefore, UCNPs can achieve improved sensitivity with a low autofluorescence . Furthermore, since UCNP PSs are activated with NIR, the PDT light penetration depth is increased, since NIR can achieve a skin penetration depth of up to 3 mm . Additionally, UCNP can be excited/activated by X-rays and so are ideal candidates for photodiagnostic detection of deep-seated tumors .
Quantum dots (QDs) are small nanocrystals which range from 2 to 10 nm in size, as well as have unique chemical and physical properties . The surface of the QDs can be modified with thiol ligands or amphiphilic copolymers to facilitate bioconjugation of antibodies or small drugs, to improve the solubility and specificity of tumor targeting porphyrin-based PS delivery . [104] [104] [104] [104] [104] [104] [97] [97] [97] [71]

Porphyrin-Based Quantum Dots
[105] [105] [106] [106] [106] [106] [106] [71] Despite the great properties QDs possess in terms of high tunability with high quantum yields , there are limited studies regarding their application in porphyrin-based PDT cancer treatments. The reason behind the limitation of QD porphyrin-based PDT applications is due to the high toxicity they possess, since most consist of toxic heavy metals like cadmium ions . The application of cadmium free QDs such as zinc and indium-based QDs or the conjugation of QDs to the surface or incorporation into polymeric NPs are a promising area of research, since this enhances their overall application in clinical trials in order to investigate their overall effectiveness for cancer diagnosis and treatment . However, due to the limited penetration of visible light, the utilization of both Cd containing and Cd-free QDs is restrained to superficial tumors for diagnosis of cancer only . Nevertheless, QDs possess a high two-photon absorption (TPA) cross-section (σ~10 GM) , which can be employed in two-photon bioimaging . Furthermore, polymer-encapsulated QDs (P-QDs) have shown a promising biocompatibility improvement, with minimal cytotoxicity in cells and animals, when compared to standard QDs .
Cancer is a dreaded disease causing a vast number of deaths worldwide. Despite ongoing, in-depth research into ways of improving cancer treatment, novel treatment modalities, which obliterate cancer specific tumors, without affecting normal tissues, is still in high demand. The arrival of nanotechnology and engineered nanomedicines have provided a novel opportunity to try and diagnose, as well as treat cancer, by promoting enhanced drug delivery.
Phototherapy is a relatively new unconventional treatment modality that is being investigated for cancer treatment, since it exhibits limited side effects. This review highlights the recent investigations of porphyrin-based nanomedicines for their phototherapeutic and diagnostic applications for cancer over the last 3 years (2017 to 2020). The focus of this review was on the inorganic-based nanoparticles incorporated with porphyrin PSs for phototherapy treatment of cancer.
Various inorganic NPs have been scrutinized for porphyrin delivery. Former porphyrin loaded inorganic NPs in cancer treatment were evaluated by Zhou et al. (2016Zhou et al. ( ) (2016 and studies by Xue et al. (2019) thoroughly reviewed porphyrin-based organic NPs for cancer therapy . Therefore, this review serves as a current update from these previous reviews, however with a core focus on inorganic porphyrin-based nanomedicines within PDD, PTT, and PDT application for cancer research.
Porphyrin PSs are the next generation of PSs which can be conjugated to various NP moieties, for improved multiple phototherapy and diagnostic functions in relation to cancer treatment regimes. However, despite its merits, the greatest drawback for porphyrin-based nanomedicines remain the limited penetration of light to deep seated tumors. This remains the greatest challenge when using porphyrin-based nanomedicines for PDT and PTT. Thus, porphyrin-inorganic NPbased PSs, for now, can only be applied for light accessible tumors, such as skin or bladder cancer . However, recent studies have reported the use of UCNPs, 2P-PDT, and X-rays for cancer phototherapy, where inorganic porphyrin-based NPs can absorb energy at much higher wavelengths, releasing a larger fluorescence signal, as well as allow for a higher wavelength of light which improves penetration of light in tissue and so largely expands their therapeutic and diagnostic applications . Furthermore, the treatment of cancer metastases is also challenging with porphyrin-based nanomedicines, due to the lack of specific cellular localization. To tackle this limitation, phototherapy using porphyrinbased nanomedicines is often integrated with immunotherapy or targeting ligands and antibodies, which enhance the active uptake of PSs via overexpressed cancer cell receptors . Lastly, it is important to note that more clinical based in vivo cancer research studies need to be performed in order to completely understand the pharmacological benefits porphyrin-based nanomedicines have .
Future directives using inorganic porphyrin NP include the use of PSs in combination with other chemotherapeutic drugs, as well as developing specific targeting nanoplatforms to boost the therapeutic uptake of drugs, limiting off-target uptake to prevent detrimental side effects and so enhancing cancer treatment.