Molecular engineering of a spheroid-penetrating phage nanovector for photodynamic treatment of colon cancer cells.

Photodynamic therapy (PDT) represents an emerging strategy to treat various malignancies, including colorectal cancer (CC), the third most common cancer type. This work presents an engineered M13 phage retargeted towards CC cells through pentavalent display of a disulfide-constrained peptide nonamer. The M13CC nanovector was conjugated with the photosensitizer Rose Bengal (RB), and the photodynamic anticancer effects of the resulting M13CC-RB bioconjugate were investigated on CC cells. We show that upon irradiation M13CC-RB is able to impair CC cell viability, and that this effect depends on i) photosensitizer concentration and ii) targeting efficiency towards CC cell lines, proving the specificity of the vector compared to unmodified M13 phage. We also demonstrate that M13CC-RB enhances generation and intracellular accumulation of reactive oxygen species (ROS) triggering CC cell death. To further investigate the anticancer potential of M13CC-RB, we performed PDT experiments on 3D CC spheroids, proving, for the first time, the ability of engineered M13 phage conjugates to deeply penetrate multicellular spheroids. Moreover, significant photodynamic effects, including spheroid disruption and cytotoxicity, were readily triggered at picomolar concentrations of the phage vector. Taken together, our results promote engineered M13 phages as promising nanovector platform for targeted photosensitization, paving the way to novel adjuvant approaches to fight CC malignancies.

Putting a "C60 Ball" and Chain to Chlorin e6 Improves Its Cellular Uptake and Photodynamic Performances.

Chlorin e6 (Ce6) and fullerene (C60) are among the most used photosensitizers (PSs) for photodynamic therapy (PDT). Through the combination of the chemical and photophysical properties of Ce6 and C60, in principle, we can obtain an "ideal" photosensitizer that is able to bypass the limitations of the two molecules alone, i.e., the low cellular uptake of Ce6 and the scarce solubility and absorption in the red region of the C60. Here, we synthesized and characterized a Ce6-C60 dyad. The UV-Vis spectrum of the dyad showed the typical absorption bands of both fullerene and Ce6, while a quenching of Ce6 fluorescence was observed. This behavior is typical in the formation of a fullerene-antenna system and is due to the intramolecular energy, or electron transfer from the antenna (Ce6) to the fullerene. Consequently, the Ce6-C60 dyad showed an enhancement in the generation of reactive oxygen species (ROS). Flow cytometry measurements demonstrated how the uptake of the Ce6 was strongly improved by the conjugation with C60. The Ce6-C60 dyad exhibited in A431 cancer cells low dark toxicity and a higher PDT efficacy than Ce6 alone, due to the enhancement of the uptake and the improvement of ROS generation.

Dissecting the Interactions between Chlorin e6 and Human Serum Albumin.

Chlorin e6 (Ce6) is among the most used sensitizers in photodynamic (PDT) and sonodynamic (SDT) therapy; its low solubility in water, however, hampers its clinical exploitation. Ce6 has a strong tendency to aggregate in physiological environments, reducing its performance as a photo/sono-sensitizer, as well as yielding poor pharmacokinetic and pharmacodynamic properties. The interaction of Ce6 with human serum albumin (HSA) (i) governs its biodistribution and (ii) can be used to improve its water solubility by encapsulation. Here, using ensemble docking and microsecond molecular dynamics simulations, we identified the two Ce6 binding pockets in HSA, i.e., the Sudlow I site and the heme binding pocket, providing an atomistic description of the binding. Comparing the photophysical and photosensitizing properties of Ce6@HSA with respect to the same properties regarding the free Ce6, it was observed that (i) a red-shift occurred in both the absorption and emission spectra, (ii) a maintaining of the fluorescence quantum yield and an increase of the excited state lifetime was detected, and (iii) a switch from the type II to the type I mechanism in a reactive oxygen species (ROS) production, upon irradiation, took place.

Identification of Blood Transport Proteins to Carry Temoporfin: A Domino Approach from Virtual Screening to Synthesis and In Vitro PDT Testing.

Temoporfin (mTHPC) is one of the most promising photosensitizers used in photodynamic therapy (PDT). Despite its clinical use, the lipophilic character of mTHPC still hampers the full exploitation of its potential. Low solubility in water, high tendency to aggregate, and low biocompatibility are the main limitations because they cause poor stability in physiological environments, dark toxicity, and ultimately reduce the generation of reactive oxygen species (ROS). Applying a reverse docking approach, here, we identified a number of blood transport proteins able to bind and disperse monomolecularly mTHPC, namely apohemoglobin, apomyoglobin, hemopexin, and afamin. We validated the computational results synthesizing the mTHPC-apomyoglobin complex (mTHPC@apoMb) and demonstrated that the protein monodisperses mTHPC in a physiological environment. The mTHPC@apoMb complex preserves the imaging properties of the molecule and improves its ability to produce ROS via both type I and type II mechanisms. The effectiveness of photodynamic treatment using the mTHPC@apoMb complex was then demonstrated in vitro. Blood transport proteins can be used as molecular "Trojan horses" in cancer cells by conferring mTHPC (i) water solubility, (ii) monodispersity, and (iii) biocompatibility, ultimately bypassing the current limitations of mTHPC.

Light-Enhanced Cytotoxicity of Doxorubicin by Photoactivation.

The combination of photodynamic therapy with chemotherapy (photochemotherapy, PCT) can lead to additive or synergistic antitumor effects. Usually, two different molecules, a photosensitizer (PS) and a chemotherapeutic drug are used in PCT. Doxorubicin is one of the most successful chemotherapy drugs. Despite its high efficacy, two factors limit its clinical use: severe side effects and the development of chemoresistance. Doxorubicin is a chromophore, able to absorb light in the visible range, making it a potential PS. Here, we exploited the intrinsic photosensitizing properties of doxorubicin to enhance its anticancer activity in leukemia, breast, and epidermoid carcinoma cells, upon irradiation. Light can selectively trigger the local generation of reactive oxygen species (ROS), following photophysical pathways. Doxorubicin showed a concentration-dependent ability to generate peroxides and singlet oxygen upon irradiation. The underlying mechanisms leading to the increase in its cytotoxic activity were intracellular ROS generation and the induction of necrotic cell death. The nuclear localization of doxorubicin represents an added value for its use as a PS. The use of doxorubicin in PCT, simultaneously acting as a chemotherapeutic agent and a PS, may allow (i) an increase in the anticancer effects of the drug, and (ii) a decrease in its dose, and thus, its dose-related adverse effects.

Enhanced Uptake and Phototoxicity of C60@albumin Hybrids by Folate Bioconjugation.

Fullerenes are considered excellent photosensitizers, being highly suitable for photodynamic therapy (PDT). A lack of water solubility and low biocompatibility are, in many instances, still hampering the full exploitation of their potential in nanomedicine. Here, we used human serum albumin (HSA) to disperse fullerenes by binding up to five fullerene cages inside the hydrophobic cavities. Albumin was bioconjugated with folic acid to specifically address the folate receptors that are usually overexpressed in several solid tumors. Concurrently, tetramethylrhodamine isothiocyanate, TRITC, a tag for imaging, was conjugated to C60@HSA in order to build an effective phototheranostic platform. The in vitro experiments demonstrated that: (i) HSA disperses C60 molecules in a physiological environment, (ii) HSA, upon C60 binding, maintains its biological identity and biocompatibility, (iii) the C60@HSA complex shows a significant visible-light-induced production of reactive oxygen species, and (iv) folate bioconjugation improves both the internalization and the PDT-induced phototoxicity of the C60@HSA complex in HeLa cells.

EGFR-Targeted Photodynamic Therapy.

The epidermal growth factor receptor (EGFR) plays a pivotal role in the proliferation and metastatization of cancer cells. Aberrancies in the expression and activation of EGFR are hallmarks of many human malignancies. As such, EGFR-targeted therapies hold significant potential for the cure of cancers. In recent years, photodynamic therapy (PDT) has gained increased interest as a non-invasive cancer treatment. In PDT, a photosensitizer is excited by light to produce reactive oxygen species, resulting in local cytotoxicity. One of the critical aspects of PDT is to selectively transport enough photosensitizers to the tumors environment. Accordingly, an increasing number of strategies have been devised to foster EGFR-targeted PDT. Herein, we review the recent nanobiotechnological advancements that combine the promise of PDT with EGFR-targeted molecular cancer therapy. We recapitulate the chemistry of the sensitizers and their modes of action in PDT, and summarize the advantages and pitfalls of different targeting moieties, highlighting future perspectives for EGFR-targeted photodynamic treatment of cancer.

Orthogonal nanoarchitectonics of M13 phage for receptor targeted anticancer photodynamic therapy.

Photodynamic therapy (PDT) represents a promising therapeutic modality for cancer. Here we used an orthogonal nanoarchitectonics approach (genetic/chemical) to engineer M13 bacteriophages as targeted vectors for efficient photodynamic killing of cancer cells. M13 was genetically refactored to display on the phage tip a peptide (SYPIPDT) able to bind the epidermal growth factor receptor (EGFR). The refactored M13EGFR phages demonstrated EGFR-targeted tropism and were internalized by A431 cancer cells, that overexpress EGFR. Using an orthogonal approach to the genetic display, M13EGFR phages were then chemically modified, conjugating hundreds of Rose Bengal (RB) photosensitizing molecules on the capsid surface, without affecting the selective recognition of the SYPIPDT peptides. Upon internalization, the M13EGFR-RB derivatives generated intracellularly reactive oxygen species, activated by an ultralow intensity white light irradiation. The killing activity of cancer cells is observed at picomolar concentrations of the M13EGFR phage.

Advanced photodynamic therapy with an engineered M13 phage targeting EGFR: Mitochondrial localization and autophagy induction in ovarian cancer cell lines.

Photodynamic therapy (PDT) is a potential synergistic approach to chemotherapy for treating ovarian cancer, the most lethal gynecologic malignancy. Here we used M13 bacteriophage as a targeted vector for the efficient photodynamic killing of SKOV3 and COV362 cells. The M13 phage was refactored (M13r) to display an EGFR binding peptide in its tip that is frequently overexpressed in ovarian cancer. The refactored phage was conjugated with chlorin e6 (Ce6), one of the most widely used photosensitizers (M13r-Ce6). The new platform, upon irradiation, generated ROS by type I mechanism and showed activity in killing SKOV3 and COV362 cells even at concentrations in which Ce6 alone was ineffective. A microscopy analysis demonstrated an enhanced cellular uptake of M13r-Ce6 compared to free Ce6 and its mitochondrial localization. Western blot analysis revealed significant downregulation in the expression of EGFR in cells exposed to M13r-Ce6 after PDT. Following PDT treatment, autophagy induction was supported by an increased expression of LC3II, along with a raised autophagic fluorescent signal, as observed by fluorescence microscopy analysis for autophagosome visualization. As a conclusion we have herein proposed a bacteriophage-based receptor targeted photodynamic therapy for EGFR-positive ovarian cancer.

Carrying Temoporfin with Human Serum Albumin: A New Perspective for Photodynamic Application in Head and Neck Cancer.

Temoporfin (mTHPC) is approved in Europe for the photodynamic treatment of head and neck squamous cell carcinoma (HNSCC). Although it has a promising profile, its lipophilic character hampers the full exploitation of its potential due to high tendency of aggregation and a reduced ROS generation that compromise photodynamic therapy (PDT) efficacy. Moreover, for its clinical administration, mTHPC requires the presence of ethanol and propylene glycol as solvents, often causing adverse effects in the site of injection. In this paper we explored the efficiency of a new mTHPC formulation that uses human serum albumin (HSA) to disperse the photosensitizer in solution (mTHPC@HSA), investigating its anticancer potential in two HNSCC cell lines. Through a comprehensive characterization, we demonstrated that mTHPC@HSA is stable in physiological environment, does not aggregate, and is extremely efficient in PDT performance, due to its high singlet oxygen generation and the high dispersion as monomolecular form in HSA. This is supported by the computational identification of the specific binding pocket of mTHPC in HSA. Moreover, mTHPC@HSA-PDT induces cytotoxicity in both HNSCC cell lines, increasing intracellular ROS generation and the number of γ-H2AX foci, a cellular event involved in the global response to cellular stress. Taken together these results highlight the promising phototoxic profile of the complex, prompting further studies to assess its clinical potential.

Human Serum Albumin-Oligothiophene Bioconjugate: A Phototheranostic Platform for Localized Killing of Cancer Cells by Precise Light Activation.

The electronic, optical, and redox properties of thiophene-based materials have made them pivotal in nanoscience and nanotechnology. However, the exploitation of oligothiophenes in photodynamic therapy is hindered by their intrinsic hydrophobicity that lowers their biocompatibility and availability in water environments. Here, we developed human serum albumin (HSA)-oligothiophene bioconjugates that afford the use of insoluble oligothiophenes in physiological environments. UV-vis and electrophoresis proved the conjugation of the oligothiophene sensitizers to the protein. The bioconjugate is water-soluble and biocompatible, does not have any "dark toxicity", and preserves HSA in the physiological monomeric form, as confirmed by dynamic light scattering and circular dichroism measurements. In contrast, upon irradiation with ultralow light doses, the bioconjugate efficiently produces reactive oxygen species (ROS) and leads to the complete eradication of cancer cells. Real-time monitoring of the photokilling activity of the HSA-oligothiophene bioconjugate shows that living cells "explode" upon irradiation. Photodependent and dose-dependent apoptosis is one of the primary mechanisms of cell death activated by bioconjugate irradiation. The bioconjugate is a novel theranostic platform able to generate ROS intracellularly and provide imaging through the fluorescence of the oligothiophene. It is also a real-time self-reporting system able to monitor the apoptotic process. The induced phototoxicity is strongly confined to the irradiated region, showing localized killing of cancer cells by precise light activation of the bioconjugate.

Spiky Gold Nanoparticles for the Photothermal Eradication of Colon Cancer Cells.

Colorectal cancer (CRC) is a widespread and lethal disease. Relapses of the disease and metastasis are very common in instances of CRC, so adjuvant therapies have a crucial role in its treatment. Systemic toxic effects and the development of resistance during therapy limit the long-term efficacy of existing adjuvant therapeutic approaches. Consequently, the search for alternative strategies is necessary. Photothermal therapy (PTT) represents an innovative treatment for cancer with great potential. Here, we synthesize branched gold nanoparticles (BGNPs) as attractive agents for the photothermal eradication of colon cancer cells. By controlling the NP growth process, large absorption in the first NIR biological window was obtained. The FBS dispersed BGNPs are stable in physiological-like environments and show an extremely efficient light-to-heat conversion capability when irradiated with an 808-nm laser. Sequential cycles of heating and cooling do not affect the BGNP stability. The uptake of BGNPs in colon cancer cells was confirmed using flow cytometry and confocal microscopy, exploiting their intrinsic optical properties. In dark conditions, BGNPs are fully biocompatible and do not compromise cell viability, while an almost complete eradication of colon cancer cells was observed upon incubation with BGNPs and irradiation with an 808-nm laser source. The PTT treatment is characterized by an extremely rapid onset of action that leads to cell membrane rupture by induced hyperthermia, which is the trigger that promotes cancer cell death.

Stable and Biocompatible Monodispersion of C60 in Water by Peptides.

The lack of solubility in water and the formation of aggregates hamper many opportunities for technological exploitation of C60. Here, different peptides were designed and synthesized with the aim of monomolecular dispersion of C60 in water. Phenylalanines were used as recognizing moieties, able to interact with C60 through π-π stacking, while a varying number of glycines were used as spacers, to connect the two terminal phenylalanines. The best performance in the dispersion of C60 was obtained with the FGGGF peptidic nanotweezer at a pH of 12. A full characterization of this adduct was carried out. The peptides disperse C60 in water with high efficiency, and the solutions are stable for months both in pure water and in physiological environments. NMR measurements demonstrated the ability of the peptides to interact with C60. AFM measurements showed that C60 is monodispersed. Electrospray ionization mass spectrometry determined a stoichiometry of C60@(FGGGF)4. Molecular dynamics simulations showed that the peptides assemble around the C60 cage, like a candy in its paper wrapper, creating a supramolecular host able to accept C60 in the cavity. The peptide-wrapped C60 is fully biocompatible and the C60 "dark toxicity" is eliminated. C60@(FGGGF)4 shows visible light-induced reactive oxygen species (ROS) generation at physiological saline concentrations and reduction of the HeLa cell viability in response to visible light irradiation.

Delivery systems for agriculture: Fe-EDDHSA/CaCO3 hybrid crystals as adjuvants for prevention of iron chlorosis.

Fe-EDDHSA/CaCO3 hybrid crystals are synthesized and tested in vitro to determine their effect in treating iron chlorosis in kiwifruit plants, used as a proof of concept. Under the alkaline conditions provided by the calcareous substrate, plants release protons that dissolve the hybrids and trigger Fe uptake. These CaCO3 hybrids represent a new system for active molecule delivery in agriculture.

Pseudopeptide-Based Hydrogels Trapping Methylene Blue and Eosin†Y.

We present herein the preparation of four different hydrogels based on the pseudopeptide gelator Fmoc-l-Phe-d-Oxd-OH (Fmoc=fluorenylmethyloxycarbonyl), either by changing the gelator concentration or adding graphene oxide (GO) to the water solution. The hydrogels have been analysed by rheological studies that demonstrated that pure hydrogels are slightly stronger compared to GO-loaded hydrogels. Then the hydrogels efficiency to trap the cationic methylene blue (MB) and anionic eosin Y (EY) dyes has been analyzed. MB is efficiently trapped by both the pure hydrogel and the GO-loaded hydrogel through π-π interactions and electrostatic interactions. In contrast, the removal of the anionic EY is achieved in less satisfactory yields, due to the unfavourable electrostatic interactions between the dye, the gelator and GO.

Calcite Single Crystals as Hosts for Atomic-Scale Entrapment and Slow Release of Drugs.

Doxorubicin (DOX)/CaCO3 single crystals act as pH responsive drug carrier. A biomimetic approach demonstrates that calcite single crystals are able, during their growth in the presence of doxorubicin, to entrap drug molecules inside their lattice along specific crystallographic directions. Alterations in lattice dimensions and microstructural parameters are determined by means of high-resolution synchrotron powder diffraction measurements. Confocal microscopy confirms that doxorubicin is uniformly embedded in the crystal and is not simply adsorbed on the crystal surface. A slow release of DOX was obtained preferentially in the proximity of the crystals, targeting cancer cells.