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Dual Targeting of Endothelial and Cancer Cells Potentiates In Vitro Nanobody-Targeted Photodynamic Therapy.

ABSTRACT: Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer, although sub-optimal tumor specificity and side effects have hindered its clinical application. We introduced a new strategy named nanobody-targeted PDT in which photosensitizers are delivered to tumor cells by means of nanobodies. As efficacy of targeted PDT can be hampered by heterogeneity of target expression and/or moderate/low target expression levels, we explored the possibility of combined targeting of endothelial and cancer cells in vitro. We developed nanobodies binding to the mouse VEGFR2, which is overexpressed on tumor vasculature, and combined these with nanobodies specific for the cancer cell target EGFR. The nanobodies were conjugated to the photosensitizer IRDye700DX and specificity of the newly developed nanobodies was verified using several endothelial cell lines. The cytotoxicity of these conjugates was assessed in monocultures and in co-cultures with cancer cells, after illumination with an appropriate laser. The results show that the anti-VEGFR2 conjugates are specific and potent PDT agents. Nanobody-targeted PDT on co-culture of endothelial and cancer cells showed improved efficacy, when VEGFR2 and EGFR targeting nanobodies were applied simultaneously. Altogether, dual targeting of endothelial and cancer cells is a promising novel therapeutic strategy for more effective nanobody-targeted PDT.

SUBMITTER: Mashayekhi V

PROVIDER: S-EPMC7650791 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Dual Targeting of Endothelial and Cancer Cells Potentiates In Vitro Nanobody-Targeted Photodynamic Therapy.

Mashayekhi Vida V Xenaki Katerina T KT van Bergen En Henegouwen Paul M P PMP Oliveira Sabrina S

Cancers 20200923 10

Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer, although sub-optimal tumor specificity and side effects have hindered its clinical application. We introduced a new strategy named nanobody-targeted PDT in which photosensitizers are delivered to tumor cells by means of nanobodies. As efficacy of targeted PDT can be hampered by heterogeneity of target expression and/or moderate/low target expression levels, we explored the possibility of combined t ...[more]

PMID: 32977602

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Project description:meta-Tetra(hydroxyphenyl)chlorin (mTHPC) is one of the most potent second-generation photosensitizers, clinically used for photodynamic therapy (PDT) of head and neck squamous cell carcinomas. However, improvements are still required concerning its present formulation (i.e., Foscan, a solution of mTHPC in ethanol/propylene glycol (40:60 w/w)), as mTHPC has the tendency to aggregate in aqueous media, e.g., biological fluids, and it has limited tumor specificity. In the present study, polymeric micelles with three different diameters (17, 24, and 45 nm) based on benzyl-poly(?-caprolactone)-b-poly(ethylene glycol) (PCLn-PEG; n = 9, 15, or 23) were prepared with mTHPC loadings ranging from 0.5 to 10 wt % using a film-hydration method as advanced nanoformulations for this photosensitizer. To favor the uptake of the micelles by cancer cells that overexpress the epidermal growth factor receptor (EGFR), the micelles were decorated with an EGFR-targeted nanobody (named EGa1) through maleimide-thiol chemistry. The enhanced binding of the EGFR-targeted micelles at 4 °C to EGFR-overexpressing A431 cells, compared to low-EGFR-expressing HeLa cells, confirmed the specificity of the micelles. In addition, an enhanced uptake of mTHPC-loaded micelles by A431 cells was observed when these were decorated with the EGa1 nanobody, compared to nontargeted micelles. Both binding and uptake of targeted micelles were blocked by an excess of free EGa1 nanobody, demonstrating that these processes occur through EGFR. In line with this, mTHPC loaded in EGa1-conjugated PCL23-PEG (EGa1-P23) micelles demonstrated 4 times higher photocytotoxicity on A431 cells, compared to micelles lacking the nanobody. Importantly, EGa1-P23 micelles also showed selective PDT against A431 cells compared to the low-EGFR-expressing HeLa cells. Finally, an in vivo pharmacokinetic study shows that after intravenous injection, mTHPC incorporated in the P23 micelles displayed prolonged blood circulation kinetics, compared to free mTHPC, independently of the presence of EGa1. Thus, these results make these micelles a promising nanomedicine formulation for selective therapy.

Project description:Background: To investigate the efficacy of a PLGA-based nanobody complex in photodynamic therapy (PDT) and NIR-II imaging in A549 tumor hypoxic model.Method: IR1048-MZ was firstly synthesized by conjugating a nitro imidazole group to IR1048. IR1048-MZ and Cat were then encapsulated in PLGA-SH solution. Anti-EGFR-Nanobody was also expressed and purified, and finally Anti-EGFR-Nanobody@PLGA-IR1048MZ-Cat (Nb@IC-NPs) nanobody complex was obtained based on the formation of desulfide bond between PLGA-SH and Anti-EGFR-Nanobody. Size distribution and morphology were characterized by TEM and DLS. Spectrum of Nb@IC-NPs towards NTR was measured by UV and fluorescence, while the particle's selective response was studied using fluorescence. The uptake of Nb@IC-NPs in A549 cells was observed by flow cytometry and CLSM. In the meantime, its' catalytic ability that decomposes H2O2 both extra-and intra-cellular was observed by fluorescence and CLSM. In vitro photodynamic toxicity of Nb@IC-NPs was examined by MTT, Live/Dead Cell Staining, Flow Cytometry and Apoptosis Assay. Tumor-bearing model was constructed to observe a semi-quantitative fluorescent distribution and the possibility of NIR-II fluorescence/photoacoustic (PA) imaging. Effect of Nb@IC-NPs on enhancing A549 tumor hypoxia and expression profile of HIF-1? was investigated in the presence of NIR. An A549 tumor metastasis model was also constructed to confirm the complex' potential to destroy primary tumor, inhibit lung metastasis, and prolong mice' survival. Lastly, impact of Nb@IC-NPs on mice' main organs and blood indices was observed.Results: Nb@IC-NPs was successfully fabricated with good homogeneity. The fluorescent absorbance of Nb@IC-NPs showed a linear relationship with the concentration of NTR, and a higher concentration of NTR corresponded to a stronger photoacoustic signal. In addition, Nb@IC-NPs showed a stable selectivity toward NTR. Our results also suggested a high efficient uptake of Nb@IC-NPs in A549 cells, which was more efficient than IC-NPs and IR1048-MZ alone. In vitro assays confirmed the effects of Nb@IC-NPs on catalytic O2 generation even in hypoxic cells. The cell viability was upregulated with the nanocomplex at the absence of the laser, whereas it was dramatically declined with laser treatment that excited at 980 nm. Nb@IC-NPs achieved tumor hypoxia NIR-II/PA imaging through assisting A549 gathering. When NIR was applied, Nb@IC-NPs can significantly relieve A549 cellular/tumor hypoxia by generating more reactive oxygen species (ROS), which in turn helps lower the expression level of HIF-1?. In summary, Nb@IC-NPs based PDT can efficiently decimate A549 primary tumor, inhibit metastatic lung cancer, and prolong the lifespan of the mice under tolerable dosage. At last, in vivo toxicity tests of the nanocomplex showed its biosafety to the main organs and normal blood indices values.Conclusion: Nb@IC-NPs improves tumor hypoxia through catalytic reaction and lowers the expression level of HIF-1?. It achieves tumor PA imaging through intensified NIR-II fluorescence signal that caused by response of the complex to the lesion's nitroreductase (NTR). Nb@IC-NPs based PDT can efficiently kill A549 primary tumor, inhibit a lung metastasis, as well as prolong mice' survival cycle.

Project description:The objective of this study was to develop a ligand-targeted photodynamic therapy (tPDT) by conjugating factor VII (fVII) protein with photosensitiser verteporfin in order to overcome the poor selectivity and enhance the effect of non-targeted PDT (ntPDT) for cancer. fVII is a natural ligand for receptor tissue factor (TF) with high affinity and specificity. The reason for targeting receptor TF for the development of tPDT is that TF is a common but specific target on angiogenic tumour vascular endothelial cells (VEC) and many types of tumour cells, including solid tumours and leukaemia.Murine factor VII protein (mfVII) containing a mutation (Lys341Ala) was covalently conjugated via a cross linker EDC with Veterporfin (VP) that was extracted from liposomal Visudyne, and then free VP was separated by Sephadex G50 spin columns. fVII-tPDT using mfVII-VP conjugate, compared to ntPDT, was tested in vitro for the killing of breast cancer cells and VEGF-stimulated VEC and in vivo for inhibiting the tumour growth of breast tumours in a mouse xenograft model.We showed that: (i) fVII protein could be conjugated with VP without affecting its binding activity; (ii) fVII-tPDT could selectively kill TF-expressing breast cancer cells and VEGF-stimulated angiogenic HUVECs but had no side effects on non-TF expressing unstimulated HUVEC, CHO-K1 and 293 cells; (iii) fVII targeting enhanced the effect of VP PDT by three to four fold; (iii) fVII-tPDT induced significantly stronger levels of apoptosis and necrosis than ntPDT; and (iv) fVII-tPDT had a significantly stronger effect on inhibiting breast tumour growth in mice than ntPDT.We conclude that the fVII-targeted VP PDT that we report here is a novel and effective therapeutic with improved selectivity for the treatment of breast cancer. Since TF is expressed on many types of cancer cells including leukaemic cells and selectively on angiogenic tumour VECs, fVII-tPDT could have broad therapeutic applications for other solid cancers and leukaemia.

Dataset Information

Dual Targeting of Endothelial and Cancer Cells Potentiates In Vitro Nanobody-Targeted Photodynamic Therapy.

Publications

Dual Targeting of Endothelial and Cancer Cells Potentiates In Vitro Nanobody-Targeted Photodynamic Therapy.

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