Porphyrin NanoMetal-Organic Frameworks as Cancer Theranostic Agents

Metal-Organic Frameworks (MOFs) are hybrid multifunctional platforms that have found remarkable applications in cancer treatment and diagnostics. Independently, these materials can be employed in cancer treatment as intelligent drug carriers in chemotherapy, photothermal therapy, and photodynamic therapy; conversely, MOFs can further be used as diagnostic tools in fluorescence imaging, magnetic resonance imaging, computed tomography imaging, and photoacoustic imaging. One essential property of these materials is their great ability to fine-tune their composition toward a specific application by way of a judicious choice of the starting building materials (metal nodes and organic ligands). Moreover, many advancements were made concerning the preparation of these materials, including the ability to downsize the crystallites yielding nanoporous porphyrin MOFs (NMOFs) which are of great interest for clinical treatment and diagnostic theranostic tools. The usage of porphyrins as ligands allows a high degree of multifunctionality. Historically these molecules are well known for their reactive oxygen species formation and strong fluorescence characteristics, and both have proved helpful in cancer treatment and diagnostic tools. The anticipation that porphyrins in MOFs could prompt the resulting materials to multifunctional theranostic platforms is a reality nowadays with a series of remarkable and ground-breaking reports available in the literature. This is particularly remarkable in the last five years, when the scientific community witnessed rapid development in porphyrin MOFs theranostic agents through the development of imaging technologies and treatment strategies for cancer. This manuscript reviews the most relevant recent results and achievements in this particular area of interest in MOF chemistry and application.


Introduction
The development of materials with controlled structures and properties has created a plethora of prospects for disease diagnostics and therapy, particularly in oncology [1][2][3]. In recent years, much has been explored and discussed concerning the importance of the size and morphology of designed materials being highly associated with their biological performance, such as cellular uptake, cytotoxicity, biodistribution and blood circulation [4,5]. This is particularly relevant for those materials developed in the nanoscale dimension, whose unique physicochemical properties boost their biomedical applications [6][7][8][9]. Nanomaterials are fabricated into different shapes and morphologies, such as spherical nanoparticles, nanorods, nanosheets, nanodots, nanotubes, nanowires and nanocages [10][11][12][13][14][15]. This immense variety increases the biological applicability of nanomaterials where pore size and shape can embody compounds with unique abilities to treat and monitor the therapeutic effect in real-time [16][17][18].  [53].
This review highlights the most recent advances (particularly in the last five years or so) on the multifunctionality of porphyrin NMOFs as theranostic agents. The analysis intends to show that these materials are excellent candidates for simultaneous imaging/therapy applications and that many more advancements are on the horizon in this area of research. The review is further embodied with a pedagogical nature trying to introduce to the reader many concepts and known ideas already present in the various fields that come together under the umbrella of the general subject of this manuscript.

Cancer Therapy Modes
Porphyrin NMOFs are tailorable theranostic platforms for cancer diagnosis and cancer treatment, including monomodal therapeutics such as photodynamic therapy (PDT), photothermal therapy (PTT), chemotherapy and radiotherapy (RT) (Figure 2). This review highlights the most recent advances (particularly in the last five years or so) on the multifunctionality of porphyrin NMOFs as theranostic agents. The analysis intends to show that these materials are excellent candidates for simultaneous imaging/therapy applications and that many more advancements are on the horizon in this area of research. The review is further embodied with a pedagogical nature trying to introduce to the reader many concepts and known ideas already present in the various fields that come together under the umbrella of the general subject of this manuscript.
PDT is based on the action of a photosensitiser administrated intravenously (or topically) to patients, followed by local illumination to produce ROS [55,56]. This technology exploits optical fibres or LEDs with a wavelength close to the NIR (generally in the 630-660 nm wavelength) region for deeper tissue penetration. The treatment outcome depends on the nature of the cancer cells, properties, and localisation of the tumour [57]. We note that the highly localised nature of PDT has obvious advantages because it typically causes negligible damage to the surrounding normal tissues and has minor systemic effects [58]. There is no known and obvious mechanism for acquiring resistance to PDT, which makes it a promising modality for treatment where porphyrins alone are extensively used to treat skin cancers and other non-neoplastic diseases such as macular degeneration [59,60].
PTT utilises light (generally in the 808 nm wavelength) to induce hyperthermia/thermal ablation, and porphyrin PTT agents with low fluorescence quantum yield and singlet oxygen production efficiency can achieve efficient thermal events upon NIR irradiation [61,62]. Improving the intrinsic physical properties of porphyrins for an efficient PTT requires either a shorter excited state lifetime through self-assembly of porphyrin molecules into nanoformulations, or the inclusion of certain metal cations into the porphyrin core [63]. We emphasise that it is known that porphyrin materials can be joined with gold and silver nanoparticles with localised surface plasmon resonance wavelengths to improve light absorption, ultimately enhancing the photothermal effect under light irradiation [64].
MOFs have been explored in cancer treatment as drug carriers because of their high loading capacity and excellent biocompatibility [65]. Noteworthy, since drugs commonly used in cancer treatment often cause side effects, MOF modulation can provide a clever drug delivery and release systems for more effective chemotherapy [66]. The ability of porphyrin-based NMOFs to generate toxic ROS in tumour tissues, allied to drug delivery properties, permits a controlled release of chemotherapy drugs during PDT treatment. This combined treatment enhances the therapeutic efficacy, in which lower drug doses are used while obtaining the desired antitumour effect with the additional advantage of reduced toxicity for normal cells/tissues [67].
RT is a local treatment that inflicts ionisation damage to tumour tissues in an X-ray dose-dependent method. RT efficacy is usually limited by the maximum radiation dose administered to the tumour tissues without incurring substantial injuries to the neighbouring tissues or organs [68,69]. One method to reduce X-ray doses while maintaining sufficient ionisation damage to tumours is to use heavy metals with high X-ray absorption coefficients to significantly increase the radiosensitivity difference between healthy cells and tumours [68]. This can be achieved by combining tumour-targeted porphyrin NMOFs PDT is based on the action of a photosensitiser administrated intravenously (or topically) to patients, followed by local illumination to produce ROS [55,56]. This technology exploits optical fibres or LEDs with a wavelength close to the NIR (generally in the 630-660 nm wavelength) region for deeper tissue penetration. The treatment outcome depends on the nature of the cancer cells, properties, and localisation of the tumour [57]. We note that the highly localised nature of PDT has obvious advantages because it typically causes negligible damage to the surrounding normal tissues and has minor systemic effects [58]. There is no known and obvious mechanism for acquiring resistance to PDT, which makes it a promising modality for treatment where porphyrins alone are extensively used to treat skin cancers and other non-neoplastic diseases such as macular degeneration [59,60].
PTT utilises light (generally in the 808 nm wavelength) to induce hyperthermia/thermal ablation, and porphyrin PTT agents with low fluorescence quantum yield and singlet oxygen production efficiency can achieve efficient thermal events upon NIR irradiation [61,62]. Improving the intrinsic physical properties of porphyrins for an efficient PTT requires either a shorter excited state lifetime through self-assembly of porphyrin molecules into nanoformulations, or the inclusion of certain metal cations into the porphyrin core [63]. We emphasise that it is known that porphyrin materials can be joined with gold and silver nanoparticles with localised surface plasmon resonance wavelengths to improve light absorption, ultimately enhancing the photothermal effect under light irradiation [64].
MOFs have been explored in cancer treatment as drug carriers because of their high loading capacity and excellent biocompatibility [65]. Noteworthy, since drugs commonly used in cancer treatment often cause side effects, MOF modulation can provide a clever drug delivery and release systems for more effective chemotherapy [66].
The ability of porphyrin-based NMOFs to generate toxic ROS in tumour tissues, allied to drug delivery properties, permits a controlled release of chemotherapy drugs during PDT treatment. This combined treatment enhances the therapeutic efficacy, in which lower drug doses are used while obtaining the desired antitumour effect with the additional advantage of reduced toxicity for normal cells/tissues [67].
RT is a local treatment that inflicts ionisation damage to tumour tissues in an X-ray dose-dependent method. RT efficacy is usually limited by the maximum radiation dose administered to the tumour tissues without incurring substantial injuries to the neighbouring tissues or organs [68,69]. One method to reduce X-ray doses while maintaining sufficient ionisation damage to tumours is to use heavy metals with high X-ray absorption coefficients to significantly increase the radiosensitivity difference between healthy cells and tumours [68]. This can be achieved by combining tumour-targeted porphyrin NMOFs with heavy metals such as Au and Hf that work as radio enhancers, minimising side effects to the surrounding tissues while making RT a compatible and effective treatment.

Cancer Imaging Techniques
Most diagnostic systems based on a single mode are limited. One of the most exciting advantages of using porphyrins NMOFs imaging agents is the possibility of using different imaging approaches in one platform, thereby significantly improving diagnostic accuracy. The most explored imaging modes of NMOF materials are fluorescence (FI), magnetic resonance, computed tomography (CT), and Photoacoustic (PAI) imaging ( Figure 3). with heavy metals such as Au and Hf that work as radio enhancers, minimising side effects to the surrounding tissues while making RT a compatible and effective treatment.

Cancer Imaging Techniques
Most diagnostic systems based on a single mode are limited. One of the most exciting advantages of using porphyrins NMOFs imaging agents is the possibility of using different imaging approaches in one platform, thereby significantly improving diagnostic accuracy. The most explored imaging modes of NMOF materials are fluorescence (FI), magnetic resonance, computed tomography (CT), and Photoacoustic (PAI) imaging ( Figure 3). Combining the porphyrin fluorescence and metals can lead to the generation or enhancement of contrast signals in targeted tissues. The intrinsic characteristics of porphyrin NMOFs can achieve multimode imaging-guided therapy, which can avoid the inherent limitations of single-mode imaging. This multi-mode imaging capability delivers more accurate localisation of lesions/particles and treatment guidelines. This section will present several examples of imaging methods used by porphyrin NMOFs theranostic agents.
FI is a fast, non-invasive screening method that offers high signal sensitivity based on fluorophore photon absorption and emission capabilities [70]. In vivo fluorescence imaging employs a sensitive camera to detect the fluorescence emission from fluorophores in whole-body small living animals. Fluorophores with extended emission at the NIR are generally used to avoid photon attenuation in tissues [70,71]. Porphyrins and analogues (chlorins and benzoporphyrins) are popular fluorophores and have long been incorporated into NMOFs for both in vitro and in vivo fluorescence imaging [72]. With the combination of fluorescence and photosensitivity from porphyrins, imaging-guided therapy nanosystems can be constructed based on porphyrin NMOFs to more efficiently visualise tumours [73].
MRI is a non-invasive imaging technique that rapidly differentiates tumours from other normal tissues [74]. This medical imaging technique uses a magnetic field and computer-generated radio waves to create detailed images of the organs and tissues. Contrasting with X-rays and CT, MRI does not involve hazardous ionising radiation and provides high spatial resolution and unlimited penetration depth [75]. The low MRI sensitivity can be improved by contrast agents that usually are divided into positive contrast agents (T1), which can reduce the longitudinal relaxation time and negative contrast agents (T2) that can shorten the transverse relaxation time [76]. Gadolinium and manga- Combining the porphyrin fluorescence and metals can lead to the generation or enhancement of contrast signals in targeted tissues. The intrinsic characteristics of porphyrin NMOFs can achieve multimode imaging-guided therapy, which can avoid the inherent limitations of single-mode imaging. This multi-mode imaging capability delivers more accurate localisation of lesions/particles and treatment guidelines. This section will present several examples of imaging methods used by porphyrin NMOFs theranostic agents.
FI is a fast, non-invasive screening method that offers high signal sensitivity based on fluorophore photon absorption and emission capabilities [70]. In vivo fluorescence imaging employs a sensitive camera to detect the fluorescence emission from fluorophores in wholebody small living animals. Fluorophores with extended emission at the NIR are generally used to avoid photon attenuation in tissues [70,71]. Porphyrins and analogues (chlorins and benzoporphyrins) are popular fluorophores and have long been incorporated into NMOFs for both in vitro and in vivo fluorescence imaging [72]. With the combination of fluorescence and photosensitivity from porphyrins, imaging-guided therapy nanosystems can be constructed based on porphyrin NMOFs to more efficiently visualise tumours [73].
MRI is a non-invasive imaging technique that rapidly differentiates tumours from other normal tissues [74]. This medical imaging technique uses a magnetic field and computergenerated radio waves to create detailed images of the organs and tissues. Contrasting with X-rays and CT, MRI does not involve hazardous ionising radiation and provides high spatial resolution and unlimited penetration depth [75]. The low MRI sensitivity can be improved by contrast agents that usually are divided into positive contrast agents (T 1 ), which can reduce the longitudinal relaxation time and negative contrast agents (T 2 ) that can shorten the transverse relaxation time [76]. Gadolinium and manganese-based porphyrins can be employed as T 1

contrast agents and incorporated into porphyrin NMOFs
Molecules 2022, 27, 3111 6 of 21 ultimately reducing the known toxicity of some contrast agents used in clinical diagnosis and research [77,78].
CT is a computerized imaging procedure in which X-rays are focused on a patient and quickly rotated around the body, producing signals processed by the machine's computer to generate cross-sectional images [79]. Contrast agents with high electron density are vital for CT, with gold, iodine, bismuth or gadolinium providing different densities between organs and adjunct tissues [80]. Nonetheless, the nonspecific distribution and high doses of contrast agents used to obtain decent images may induce side effects and toxicity to normal tissues [81]. A workaround for this toxicity is metal coordination into a porphyrin core, or by combining MOFs with gold nanoparticles in a composite. These approaches may diminish the overall toxicity of the contrast agent, either by lowering the administered doses or by increasing their stability and specificity.
PAI is a technology that combines optical and ultrasound imaging for the detection of endogenous or exogenous chromophores that are excited through an illumination carried out with pulsed laser light, typically in the NIR range [82]. The nonradiative release of the absorbed energy produces a micro heating and a thermoelastic expansion in the chromophore surrounding, thus generating ultrasounds [83]. The acoustic waves are detected by a transducer and transformed into a 3D-high resolution image of soft tissues. Because of the shallow penetration depth, PAI is limited to superficial organ analysis.

Porphyrin Inclusion into NMOFs
The first methodologies to prepare porphyrin MOF theranostic agents were indirect methods, with the porphyrin macrocycle included in the MOF network either by supramolecular entrapment or covalent attachment within the network with a tetrapodal porphyrin ligand. Both methods are accessible and straightforward ways to combine the advantages of porphyrins with NMOFs without the need to prepare pure porphyrin NMOFs.
The second approach focused on a photoactive chlorin (H 6 TCPC) that was incorporated into the HfUiO-66 framework by a facile mixed-component one-pot strategy ( Figure 4C) [86]. The rationale behind the structure choice lies in the inherent chemical stability of HfUiO-66, which displays high tolerability to accommodate geometrically different ligands in a single structure. Hafnium enhances the intersystem crossing of singlet-totriplet by weakening spin prohibition because of the relatively sizeable spin-orbit coupling constant, which is beneficial for PTT and X-ray attenuating ability for CT imaging. The chlorin spatial arrangements and photochemical properties within H 2 TCPC-HfUiO-66 afforded the best of both worlds with enhanced multimodal-imaging-guided MRI and PTT antitumour therapy. H 2 TCPC-HfUiO-66 has an impressive anticancer activity against H22 tumour-bearing mice in vivo, with a tumour inhibition growth rate above 90%. In addition, this nanomaterial showed a high photothermal conversion efficiency, favourable photostability, and biocompatibility, being used in trimodal CT/TI/PAI as contrast agents. The success of the implementation of this material as a theranostic agent, with stronger fluorescent MOFs and higher quantum yield when compared with H 6 TCPC, was ascribed to the periodicity and regularity of chromophore distribution within the MOF framework, which is beneficial due to the increased intramolecular distances between chromophores avoiding the self-quenching effects.
Zheng and collaborators used a similar rationale to design a multifunctional platform with comparable therapy and imaging capabilities to the previous work [87]. While similar structural resemblances can be found in the starting materials (HfUiO-66 and the tetraaminophenyl chlorin, H2TAPC), the group employed a different approach, including HfUiO-66 within a chlorin porous organic polymer (POP) ( Figure 4D). The strong extinction coefficient of chlorin compared to porphyrins at the lowest-energy Q band promoted an efficient light-harvesting ability. Consequently, both ROS and heat generation achieved a much higher antitumoural activity (tumour inhibition rate up to 88.4%) evaluated in U14 tumour-bearing Kunming strain mouse. The HfUiO-66@POP system displays good stability, biocompatibility, and high photothermal conversion efficiency. These systems showed that porphyrins could be included in MOFs using innovative and straightforward methods for the preparation of theranostic agents with outstanding performances. Zheng and collaborators used a similar rationale to design a multifunctional platform with comparable therapy and imaging capabilities to the previous work [87]. While similar structural resemblances can be found in the starting materials (HfUiO-66 and the tetraaminophenyl chlorin, H 2 TAPC), the group employed a different approach, including HfUiO-66 within a chlorin porous organic polymer (POP) ( Figure 4D). The strong extinction coefficient of chlorin compared to porphyrins at the lowest-energy Q band promoted an efficient light-harvesting ability. Consequently, both ROS and heat generation achieved a much higher antitumoural activity (tumour inhibition rate up to 88.4%) evaluated in U14 tumour-bearing Kunming strain mouse. The HfUiO-66@POP system displays good stability, biocompatibility, and high photothermal conversion efficiency.
These systems showed that porphyrins could be included in MOFs using innovative and straightforward methods for the preparation of theranostic agents with outstanding performances.

Porphyrin NMOFs
The most iconic and commonly used porphyrin porous coordination networks (PCNs) are PCN-222, PCN-223 and PCN-224 ( Figure 5). These are the most prolific theranostic NMOF platforms prepared to date: these materials can be integrated into image-guided therapy systems with tunable compositions for bioimaging, suitable sizes and shapes for bio transfer, proper channels and pores for drug loading, and are intrinsically biocompatible. For instance, a biocompatible nanoscale version of ZrPCN-222 was prepared from a microemulsion [88]. The high porphyrin content (59.8%) allows the use of concurrent efficient fluorescent imaging and PDT. The onedimensional channel of the NMOF provides a high doxorubicin loading and pH-response smart release for chemotherapy. The fluorescence guiding of the chemotherapy-and-PDT dual system is confirmed by the concentration of NMOF at cancer sites after laser irradiation and doxorubicin release, while low toxicity is observed in normal tissues. Using the same MOF Liu and collaborators employed a phosphate-terminal aptamer modified with 5-carboxytetramethylrhodamine (TAMRA) and anchored to the ZrPCN-222 particles through strong coordination between the phosphate and zirconium ( Figure 6) [89]. These materials ZrPCN-222-P-DNA were tested for target-induced bioimaging and photodynamic therapy. These are the most prolific theranostic NMOF platforms prepared to date: these materials can be integrated into image-guided therapy systems with tunable compositions for bioimaging, suitable sizes and shapes for bio transfer, proper channels and pores for drug loading, and are intrinsically biocompatible. For instance, a biocompatible nanoscale version of ZrPCN-222 was prepared from a microemulsion [88]. The high porphyrin content (59.8%) allows the use of concurrent efficient fluorescent imaging and PDT. The onedimensional channel of the NMOF provides a high doxorubicin loading and pH-response smart release for chemotherapy. The fluorescence guiding of the chemotherapy-and-PDT dual system is confirmed by the concentration of NMOF at cancer sites after laser irradiation and doxorubicin release, while low toxicity is observed in normal tissues. Using the same MOF Liu and collaborators employed a phosphate-terminal aptamer modified with 5carboxytetramethylrhodamine (TAMRA) and anchored to the ZrPCN-222 particles through strong coordination between the phosphate and zirconium ( Figure 6) [89]. These materials ZrPCN-222-P-DNA were tested for target-induced bioimaging and photodynamic therapy.  [89].
The target-induced imaging was achieved because of the structural change of the aptamer upon binding with the complementary DNA (c-DNA) and hybridisation to form a double-stranded DNA target that enhances TAMRA fluorescence ( Figure 6). This system exhibited an excellent target-induced imaging ability when incubated with HeLa cells. A similar system built with a random DNA sequence showed no target ability towards HeLa cells. On the other hand, the ZrPCN-222-P-DNA specificity towards HeLa cells increased The target-induced imaging was achieved because of the structural change of the aptamer upon binding with the complementary DNA (c-DNA) and hybridisation to form a double-stranded DNA target that enhances TAMRA fluorescence ( Figure 6). This system exhibited an excellent target-induced imaging ability when incubated with HeLa cells. A similar system built with a random DNA sequence showed no target ability towards HeLa cells. On the other hand, the ZrPCN-222-P-DNA specificity towards HeLa cells increased the PDT efficiency significantly reducing cell viability to 48% and 17% when treated at concentrations of 100 µg mL −1 and 200 µg mL −1 .
In 2018, gadolinium-porphyrin spherical MOF nanoparticles were conjugated with folic acid (FA), and their theranostic capabilities were tested against HepG2 cells and embryonic and larval zebrafish [90]. The photodynamic therapeutic effects of FA-MOF nanoparticles were explored in transgenic zebrafish with doxycycline-induced hepatocellular carcinoma. As described before the FA-derived MOF nanoparticles target specific cancer cells that over-express folate receptors (HeLa cells) enhancing their internalisation via folate receptor-mediated endocytosis. These nanomaterials showed low biotoxicity, emitted bright red fluorescence, and exhibited an excellent MRI contrast and PDT treatment capabilities both in vitro and in vivo.
Meie and collaborators prepared ZrPCN-222 with MnTCPP, ZrPCN-222(Mn), having an excellent dispersibility of manganese in the open framework while the high water affinity of the channel embodied the material with a high longitudinal relaxivity [91]. This feature allied to the excellent catalytic conversion of endogenous hydrogen peroxide into oxygen improved tumour hypoxia PDT and MRI guidance ( Figure 7A,B). The intravenous injection of ZrPCN-222(Mn) into tumour-bearing mice provided good tumour contrast and growth inhibition upon single-laser irradiation (660 nm laser) ( Figure 7C,D). These findings demonstrate that the simple addition of Mn to the porphyrin core in ZrPCN-222 can be a practical approach to construct an O 2 self-supplementing PDT nanoplatform with excellent T 1 -weighted MRI effective diagnosis and treatment of hypoxic tumours.
Zhang and collaborators prepared a manganese ZrPCN-223 and loaded S-nitrosothiol (SNO) into ZrPCN-223(Mn) nanoparticles to generate nitric oxide by heating [92]. The insertion of Mn ions into the porphyrin core rendered the NMOFs with strong T 1 -weighted MR contrast capacity and high photothermal conversion ( Figure 7E). SNO is a type of heat-unstable nitric oxide donor, that upon PPT initiation and temperature increase decomposes to release NO. Tumour growth in mice injected with the SNO@ZrPCN-223(Mn) was inhibited upon NIR irradiation, validating the efficiency of in vivo nitric oxide release and photothermal therapy. Moreover, SNO@ZrPCN-223(Mn) PTT treatment followed by DOX chemotherapy exhibited higher tumour inhibition efficiency than DOX chemotherapy without the PTT treatment ( Figure 7F). The role of NO in tumour therapy depends on the concentration and duration of its action within the tumour cells: in the right concentrations, it affects cancer initiation and enhances cell progression, tumour blood flow, angiogenesis, metastasis, cell death, and tumour suppression [93]. Zhou and co-workers developed a porphyrin palladium NMOF (PdNMOF) with a high dispersion of the metals and with enhanced hydrogen loading for tumour-targeted (PAI)-guided hydrogenothermal therapy [94]. The porphyrin used in this system was tetrakis(4-pyridyl)-porphyrin (H 2 TPyP), and structural characterisation of the NMOF was scarce and elusive, essentially because of the nanometer profile that does not allow single-crystal characterisation. The hydrogenated PdMOF (PdHMOF) showed, however, a sustained hydrogen release profile over five days and a high PTT effect combined with PAI performance.
Manganese porphyrins versatility promoted the development of high-performance theranostic platforms that find application in three main imaging modes, MRI/CT/PAI, and two treatment ones, PTT and RT. This was achieved with the hafnium version of HfPCN-222 coated with FA to enhance the cancer-targeting efficacy [95]. Significant tumour growth inhibition was achieved in mouse cancer models without damage to other organs. oxygen improved tumour hypoxia PDT and MRI guidance ( Figure 7A,B). The intravenous injection of ZrPCN-222(Mn) into tumour-bearing mice provided good tumour contrast and growth inhibition upon single-laser irradiation (660 nm laser) ( Figure 7C,D). These findings demonstrate that the simple addition of Mn to the porphyrin core in ZrPCN-222 can be a practical approach to construct an O2 self-supplementing PDT nanoplatform with excellent T1-weighted MRI effective diagnosis and treatment of hypoxic tumours. Zhang and collaborators prepared a manganese ZrPCN-223 and loaded Snitrosothiol (SNO) into ZrPCN-223(Mn) nanoparticles to generate nitric oxide by heating [92]. The insertion of Mn ions into the porphyrin core rendered the NMOFs with strong T1-weighted MR contrast capacity and high photothermal conversion ( Figure 7E). SNO is a type of heat-unstable nitric oxide donor, that upon PPT initiation and temperature increase decomposes to release NO. Tumour growth in mice injected with the Over time, these systems progressed in applicability and complexity with engineered structures with defects to include multiple ligands into the same network integrating the co-ligand functionality into the final porphyrin NMOF. This was performed by combining cypate (Cyp, Figure 8A) and porphyrin within the same NMOF structure to perform FI/PAI/PTI trimodal imaging-guided and PDT/PTT therapy modes in a single material [96]. The engineered Cyp-ZrPCN-224 was coated with FA to increase tumour tissue targeting and multimodal cancer phototheranostics. The PTT effect was concentrationdependent, and a lower cell survival rate was observed for the dual-laser treatment, which had an IC 50 of 45.9 µg mL −1 . Mice bearing 4T1 tumours were studied in terms of tumour volume after Cyp-ZrPCN-224@FA administration and combined 660 nm (0.2 W cm −2 , 5 min.)/808 nm (1 W cm −2 , 5 min.) laser irradiation. A total tumour suppression was observed with an inhibition rate of up to ∼97.15%, expressing the remarkable efficacy of PTT/PDT combined therapies ( Figure 8B,C).  [96,97]. (Data are presented as the mean ± SD (n = 4). **, p < 0.01; ***, p < 0.001).
Xie and co-workers used a similar approach to include a co-ligand into the ZrPCN-224 network to boost both the PDT effect and the near-infrared (NIR) O2 concentration ratiometric imaging. This approach addressed a number of limitations associated with short detection wavelengths and the influence of self-concentrations. This was possible by doping ZrPCN-224 with an extended π-conjugated palladium porphyrin (Pd-tetrakis(4methoxycarbonylphenyl)-tetrabenzoporphyrin) (PdPTP, Figure 8D) [97]. 4T1 cell membrane extraction and PdPTP-ZrPCN-224 coating yielded a cloaked nanomaterial (PdPTP-ZrPCN-224@4T1) capable of targeting in vivo solid tumours with high efficiency, and effectively suppressing tumour growth via PDT. The increased PDT effect of PdPTP mediated a strong immune activation by inducing cancer immunogenic cell death ( Figure 8E).  [96,97]. (Data are presented as the mean ± SD (n = 4). **, p < 0.01; ***, p < 0.001).
Xie and co-workers used a similar approach to include a co-ligand into the ZrPCN-224 network to boost both the PDT effect and the near-infrared (NIR) O 2 concentration ratiometric imaging. This approach addressed a number of limitations associated with short detection wavelengths and the influence of self-concentrations. This was possible by doping ZrPCN-224 with an extended π-conjugated palladium porphyrin (Pd-tetrakis(4methoxycarbonylphenyl)-tetrabenzoporphyrin) (PdPTP, Figure 8D) [97]. 4T1 cell membrane extraction and PdPTP-ZrPCN-224 coating yielded a cloaked nanomaterial (PdPTP-ZrPCN-224@4T1) capable of targeting in vivo solid tumours with high efficiency, and effectively suppressing tumour growth via PDT. The increased PDT effect of PdPTP mediated a strong immune activation by inducing cancer immunogenic cell death ( Figure 8E).
Porphyrin NMOFs guarantee a high photosensitiser loading capacity and prevent the self-quenching effect observed for porphyrin molecules. There are still obstacles associated with the high glutathione (GSH) content in tumour tissues that depletes 1 O 2 and reduces the PDT efficiency. GSH is the most abundant antioxidant in human bodies, which can eliminate ROS and protect cells from acute toxicity, as well as repair damaged biomolecules [98]. The GSH concentration in cancer cells is often higher than that in normal cells making it one of the main obstacles for ROS-induced cell killing.
To overcome this, the excess acid and GSH in the tumour microenvironment can be surpassed with a redox reaction between Mn(III) and GSH, with the concomitant formation of glutathione disulphide and Mn(II). The consumption of GSH and in situ formation of Mn(II) avoids the antioxidant nature of GSH and the consequent 1 O 2 depletion. This concept was employed as two distinct approaches. One uses a custom-made Mn(III)TCPP NMOF prepared by way of a one-pot method ( Figure 9A,B), being inert in ROS generation and exhibiting an absence of fluorescence [99]. However, the presence of glutathione (GSH) inside the cells promoted the destruction of the network into in Mn(II) and Mn(III)TCPP, enhancing the MRI and the fluorescent imaging capabilities ( Figure 9C-F). More importantly, the GSH-regulated Mn(III)TCPP release could allow a controlled ROS generation under irradiation, which avoided side effects such as inflammation and damage to normal tissues. Porphyrin NMOFs guarantee a high photosensitiser loading capacity and prevent the self-quenching effect observed for porphyrin molecules. There are still obstacles associated with the high glutathione (GSH) content in tumour tissues that depletes 1 O2 and reduces the PDT efficiency. GSH is the most abundant antioxidant in human bodies, which can eliminate ROS and protect cells from acute toxicity, as well as repair damaged biomolecules [98]. The GSH concentration in cancer cells is often higher than that in normal cells making it one of the main obstacles for ROS-induced cell killing.
To overcome this, the excess acid and GSH in the tumour microenvironment can be surpassed with a redox reaction between Mn(III) and GSH, with the concomitant formation of glutathione disulphide and Mn(II). The consumption of GSH and in situ formation of Mn(II) avoids the antioxidant nature of GSH and the consequent 1 O2 depletion. This concept was employed as two distinct approaches. One uses a custom-made Mn(III)TCPP NMOF prepared by way of a one-pot method ( Figure 9A,B), being inert in ROS generation and exhibiting an absence of fluorescence [99]. However, the presence of glutathione (GSH) inside the cells promoted the destruction of the network into in Mn(II) and Mn(III)TCPP, enhancing the MRI and the fluorescent imaging capabilities (Figure 9C-F). More importantly, the GSH-regulated Mn(III)TCPP release could allow a controlled ROS generation under irradiation, which avoided side effects such as inflammation and damage to normal tissues. The second approach combined MnO2 nanoparticles with PCN-222 to construct an MnO2-coated porphyrin MOF with GSH-activated MRI-guided enhanced photodynamicand chemotherapies [100]. The preparation of PCN-222 (72 nm particles) combined with The second approach combined MnO 2 nanoparticles with PCN-222 to construct an MnO 2 -coated porphyrin MOF with GSH-activated MRI-guided enhanced photodynamicand chemotherapies [100]. The preparation of PCN-222 (72 nm particles) combined with poly(allylamine hydrochloride) and MnO 2 uniform layers around the NMOF allowed the development of a theranostic nanoplatform with high biocompatibility (no cytotoxicity at concentrations of 400 µg mL −1 ). As for the previous material, the PDT enhancement is attributed to the GSH elimination effect by the MnO 2 layer, which simultaneously permitted GSH-responsive T 1 -MRI and the controlled release of DOX.
Investigation of the efficiency of this PCN-222 composite on 4T1 tumour-bearing mice showed a very high T 1 -MRI relaxation time after one hour of administration, exhibiting a fast depletion of GSH by MnO 2 . Notably, the groups treated with PDT and chemotherapy presented the most effective inhibition of tumour growth. Both systems proved to be efficient in PDT by combining controlled ROS generation and GSH depletion after precise MRI imaging. Interestingly, the first approach shows a much simpler system in terms of preparation, further achieving dual-mode imaging. On the other hand, the second system only vehiculates to a single-mode imaging system with a controlled release chemotherapy treatment component.
Other materials can be combined with porphyrin NMOFs to increase MRI and PTT treatments (besides just the use of manganese and hafnium). For instance, Zhang and co-workers fabricated a core-shell gold nanorod@PCN-224 nanocomposite (AuNR@MOF) in which porphyrin NMOFs were grown on the surface of gold nanorods, and a large amount of camptothecin (CPT) was loaded into the shell of AuNR@MOF [101]. When AuNR@MOF@CPT was irradiated with an 808 nm laser, the nanocomposites exhibited the PTT effect, and the temperature of the AuNR@PCN-224@CPT aqueous solution increased to 51.8 • C in around 2 min., and up to 73.4 • C in just 10 min. (Figure 10A,B). The increased temperature promoted the controlled release of CPT. When irradiated with a 660 nm laser, the therapeutic effect was mainly associated with PDT arising from the porphyrin ligands present in the MOF structure. The composite PDT effect was much stronger than that of pure porphyrin, with the results indicating that the gold nanorod core played a decisive role in the absorption of excited light and the electromagnetic field, thus enhancing the PDT effect. Combined with the chemotherapy effect of the loaded CPT, the AuNR@MOFs exhibited a synergistic antitumour effect of PDT and PTT under irradiation with lights of two different wavelengths, and the tumours of the treated mice almost disappeared within 50 days of treatment ( Figure 10C). Reprinted/adapted with permission from Ref. [101,102].
Integrated systems with gold nanoparticles and porphyrin NMOF show tremendous promise in the theranostic field. The fabrication of low-cost materials within a single highefficiency theranostic platform remains, however, a challenge [103]. The PDT oxygen dependency makes it less efficient under hypoxic conditions on solid tumours, and the generation of ROS has a narrow effective function range. PTT with gold, manganese and other nanomaterials such as graphene, graphene oxide and polypyrroles have shown, con- Integrated systems with gold nanoparticles and porphyrin NMOF show tremendous promise in the theranostic field. The fabrication of low-cost materials within a single high-efficiency theranostic platform remains, however, a challenge [103]. The PDT oxygen dependency makes it less efficient under hypoxic conditions on solid tumours, and the generation of ROS has a narrow effective function range. PTT with gold, manganese and other nanomaterials such as graphene, graphene oxide and polypyrroles have shown, conversely, promising results in this field [104]. In general, incorporating MRI and PTT modes in a single material increases the cost and the overall complexity. A simple workaround replaces the gold nanorod core described above with a cluster of Fe 3 O 4 nanoparticles encapsulated in a carbon shell (Fe 3 O 4 @C). Zhang and collaborators employed this approach using superparamagnetic iron oxide nanoparticles (SPIONs) [102]. These are efficient imaging agents because they shorten the transverse relaxation (higher T 2 -MRI efficiency, Figure 10D) and have excellent biocompatibility. The composite was prepared in situ with PCN-222 growth over Fe 3 O 4 @C. The T 2 -weighted MRI/fluorescence imaging-guided PTT and PDT dual-therapy was tested on tumour-bearing mice demonstrating a high tumour accumulation and efficient ablation with similar laser irradiation at 808 and 655 nm.
A nanoscale iron porphyrin NMOF was prepared with the same concept in mind, bearing the same T 2 -weighted MRI/fluorescence imaging-guided and PTT and PDT dualtherapy [105]. The nanoparticles were coated with BSA, using carbodiimide crosslinker chemistry methods (EDC/NHS) to achieve a higher accumulation at the tumour sites and an in vivo increasing circulation time for active hypoxic tumour targeting. In vitro, ROS detection and photothermal temperature increase revealed that these nanoplatforms could exhibit a great PDT effect in tumour cells under hypoxic conditions. Unfortunately, the structure and molecular formula of the iron porphyrin MOF remain elusive and, therefore, it is highly complex to conclude anything about the structure-affinity of these nanoparticles.

Porphyrin NMOF Sheets
Nanoscale MOFs with porous structures and inherent biodegradability are attractive nanomedicine platforms. In addition to conventional particulate NMOFs, two-dimensional (2D) NMOFs are emerging as a unique type of material for nanomedicine applications. Their preparation was developed in 2015 with a surfactant-assisted synthesis (polyvinylpyrrolidone, PVP) with different metals (Zn, Cu, Cd, or Co and H 6 TCPP [106]. The surfactant plays a key role in the controlled growth of the MOF crystals, leading to anisotropic growth and the formation of ultrathin MOF nanosheets ( Figure 11). the structure and molecular formula of the iron porphyrin MOF remain elusive and, therefore, it is highly complex to conclude anything about the structure-affinity of these nanoparticles.

Porphyrin NMOF Sheets
Nanoscale MOFs with porous structures and inherent biodegradability are attractive nanomedicine platforms. In addition to conventional particulate NMOFs, two-dimensional (2D) NMOFs are emerging as a unique type of material for nanomedicine applications. Their preparation was developed in 2015 with a surfactant-assisted synthesis (polyvinylpyrrolidone, PVP) with different metals (Zn, Cu, Cd, or Co and H6TCPP [106]. The surfactant plays a key role in the controlled growth of the MOF crystals, leading to anisotropic growth and the formation of ultrathin MOF nanosheets ( Figure 11). Figure 11. NMOF sheets general preparation method and PEG coating. Reprinted/adapted with permission from Ref. [106].
Several research papers reported the usage of these methods to prepare 2D porphyrin NMOF sheets for various applications [31,50,[107][108][109][110]. Their use in theranostic increased exponentially. A recent example shows the comparison of a 2D NMOF composed of Zn 2+ and H6TCPP to their particulate counterparts. The 2D NMOFs exhibited an increased DOX loading capacity and enhanced light-triggered singlet oxygen production [111,112]. In addition, the drug-loaded 2D NMOFs could be easily labelled with 99m Tc for singlephoton emission computer tomography imaging (SPECT), being an indication of an efficient tumour homing of these materials upon intravenous injection. This system arises as a remarkable tool for the combined in vivo antitumour effect and real-time imaging, with the material further showing efficient biodegradation and rapid renal clearance. This re- Figure 11. NMOF sheets general preparation method and PEG coating. Reprinted/adapted with permission from Ref. [106].
Several research papers reported the usage of these methods to prepare 2D porphyrin NMOF sheets for various applications [31,50,[107][108][109][110]. Their use in theranostic increased exponentially. A recent example shows the comparison of a 2D NMOF composed of Zn 2+ and H 6 TCPP to their particulate counterparts. The 2D NMOFs exhibited an increased DOX loading capacity and enhanced light-triggered singlet oxygen production [111,112]. In addition, the drug-loaded 2D NMOFs could be easily labelled with 99m Tc for single-photon emission computer tomography imaging (SPECT), being an indication of an efficient tumour homing of these materials upon intravenous injection. This system arises as a remarkable tool for the combined in vivo antitumour effect and real-time imaging, with the material further showing efficient biodegradation and rapid renal clearance. This research clearly shows the advantages of 2D NMOF sheets in singlet oxygen generation. Nonetheless, using these materials with different metals permits the modulation of their therapeutic effect. For instance, replacing zinc with copper in the preparation of the nanosheets improves the NIR absorption because of the d-d energy band transition of Cu 2+ and the ultrathin characteristic translating into excellent photothermal performance and MRI applications [68]. The singlet oxygen generation inherent to H 6 TCPP is maintained, thus extending the treatment possibilities in a single nanomaterial. The 2D NMOF sheets were prepared with a mean thickness of 5.1 nm and were employed in imaging-guided phototherapy. Interestingly only a marginal inhibition of tumour growth was observed with one single treatment, PTT or PDT, in nude mice bearing Saos-2 tumours. In contrast, other mice groups treated with the combination of PTT and PDT (PTT: 808 nm laser (1.0 W cm −2 for 10 min) or the PDT: 660 nm laser (10 mW cm −2 for 30 min)) had their tumours retrogressed. This work showed how the synergy between the multitude of the porphyrins' benefits could introduce into these materials, permitting the use of simultaneous PDT, PTT and external magnetic fields for MR imaging of cancers in vivo.

Conclusions
Much remains to be undertaken to transpose porphyrin NMOFs into clinical practice. Looking into the bigger picture, NMOF materials as theranostic agents are a subject of research in their infancy. However, the potential of theranostic nanoparticles is progressively gaining the attention of major clinical players and biomedicine in general.
Porphyrin NMOF probes have several advantages when compared to conventional approaches, where diagnostic and treatment procedures are frequently separated, and while there are many challenges to overcome the synthetic processes, material structures and corresponding functional modifications are described in detail, highlighting porphyrin NMOFs as promising theranostic tools for cancer treatment and diagnostic with good performances. In fact, NMOFs, show unique properties, such as accelerated adsorption/desorption kinetics, fast accessibility to the internal active sites and above all suitable sizes for biomedical applications where the nanometric size allows NMOF particles to enter living cells via the cells' endocytic mechanisms or alternatively by passive penetration of the lipid bilayer.
With further understanding of the synthetic mechanism of NMOFs preparation, a few methods were established to command the size of NMOFs by carefully isolating the nucleation and growth process during the synthesis. In this regard, the microemulsion methods offer the greatest possibility of achieving success, nonetheless, most attempts to pursue one method to prepare uniform NMOFs with all kinds of structures are still elusive. While the synthetic preparation of these materials, especially with porphyrin ligands, remains a challenging aspect to overcome in years to come, some examples of NMOF theranostic agents described in the literature also bypass critical steps in thoroughly examining the relationship between their intrinsic properties and the final applications. There are still a few critical aspects that need to be fully addressed in this subject. Issues ranging from toxicity to efficacy and precision therapy to efficient biomarkers are still unclear and require focused research and attention. Furthermore, the structure affinity relating to size, shape, and structure and how these characteristics influence the therapeutic efficacy in vivo also needs attention in years to come. The wide variety of modalities that can be combined into a single material is undoubtedly a major advantage of these materials and preparation methods are becoming more efficient streamlining the obtention of new porphyrin NMOFs capable of application as theranostic agents. Despite all these challenges and considering that most of the developments around porphyrin NMOF theranostic agents have occurred in the last 5 years, we anticipate most of these limitations to be exceeded in years to come. Theranostics have unparalleled value in diagnosing and treating cancers and the constant evolution of techniques engineering, and porphyrin NMOFs can have a tremendous impact on cancer theranostic treatment and diagnosis.
Author Contributions: Conceptualization, writing-original draft preparation, data curation F.F.; writing-review and editing, resources, funding acquisition, F.A.A.P. and J.P.C.T. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.