Phage-Templated Synthesis of Targeted Photoactive 1D-Thiophene Nanoparticles.

Thiophene-based nanoparticles (TNPs) are promising therapeutic and imaging agents. Here, using an innovative phage-templated synthesis, a strategy able to bypass the current limitations of TNPs in nanomedicine applications is proposed. The phage capsid is decorated with oligothiophene derivatives, transforming the virus in a 1D-thiophene nanoparticle (1D-TNP). A precise control of the shape/size of the nanoparticles is obtained exploiting the well-defined morphology of a refactored filamentous M13 phage, engineered by phage display to selectively recognize the Epidermal Growth Factor Receptor (EGFR). The tropism of the phage is maintained also after the bioconjugation of the thiophene molecules on its capsid. Moreover, the 1D-TNP proved highly fluorescent and photoactive, generating reactive oxygen species through both type I and type II mechanisms. The phototheranostic properties of this platform are investigated on biosystems presenting increasing complexity levels, from in vitro cancer cells in 2D and 3D architectures, to the in vivo tissue-like model organism Hydra vulgaris. The phage-templated 1D-TNP showed photocytotoxicity at picomolar concentrations, and the ability to deeply penetrate 3D spheroids and Hydra tissues. Collectively the results indicate that phage-templated synthesis of organic nanoparticles represents a general strategy, exploitable in many diagnostic and therapeutic fields based on targeted imaging and light mediated cell ablation.

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.

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.

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.