Nanobody-Targeted Photodynamic Therapy Selectively Kills Viral GPCR-Expressing Glioblastoma Cells.
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ABSTRACT:
SUBMITTER: De Groof TWM
PROVIDER: S-EPMC6728091 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Systemic sclerosis (SSc) is a rare, severe, auto-immune disease characterized by inflammation, vasculopathy and fibrosis. Activated (myo)fibroblasts are crucial drivers of this fibrosis. By exploiting their expression of fibroblast activation protein (FAP) to perform targeted photodynamic therapy (tPDT), we can locoregionally deplete these pathogenic cells. In this study, we explored the use of FAP-tPDT in primary skin fibroblasts from SSc patients, both in 2D and 3D cultures. Method: The FAP targeting antibody 28H1 was conjugated with the photosensitizer IRDye700DX. Primary skin fibroblasts were obtained from lesional skin biopsies of SSc patients via spontaneous outgrowth and subsequently cultured on plastic or collagen type I. For 2D FAP-tPDT, cells were incubated in buffer with or without the antibody-photosensitizer construct, washed after 4 h and exposed to λ = 689 nm light. Cell viability was measured using CellTiter Glo®®. For 3D FAP-tPDT, cells were seeded in collagen plugs and underwent the same treatment procedure. Contraction of the plugs was followed over time to determine myofibroblast activity. Results: FAP-tPDT resulted in antibody-dose dependent cytotoxicity in primary skin fibroblasts upon light exposure. Cells not exposed to light or incubated with an irrelevant antibody-photosensitizer construct did not show this response. FAP-tPDT fully prevented contraction of collagen plugs seeded with primary SSc fibroblasts. Even incubation with a very low dose of antibody (0.4 nM) inhibited contraction in 2 out of 3 donors. Conclusions: Here we have shown, for the first time, the potential of FAP-tPDT for the treatment of fibrosis in SSc skin.
| S-EPMC8657852 | biostudies-literature
Project description:Nanobody-targeted photodynamic therapy (NB-PDT) has been recently developed as a more tumor-selective approach rather than conventional photodynamic therapy (PDT). NB-PDT uses nanobodies that bind to tumor cells with high affinity, to selectively deliver a photosensitizer, i.e., a chemical which becomes cytotoxic when excited with light of a particular wavelength. Conventional PDT has been reported to be able to induce immunogenic cell death, characterized by the exposure/release of damage-associated molecular patterns (DAMPs) from dying cells, which can lead to antitumor immunity. We explored this aspect in the context of NB-PDT, targeting the epidermal growth factor receptor (EGFR), using high and moderate EGFR-expressing cells. Here we report that, after NB-PDT, the cytoplasmic DAMP HSP70 was detected on the cell membrane of tumor cells and the nuclear DAMP HMGB1 was found in the cell cytoplasm. Furthermore, it was shown that NB-PDT induced the release of the DAMPs HSP70 and ATP, as well as the pro- inflammatory cytokines IL- 1? and IL-6. Conditioned medium from high EGFR-expressing tumor cells treated with NB-PDT led to the maturation of human dendritic cells, as indicated by the upregulation of CD86 and MHC II on their cell surface, and the increased release of IL-12p40 and IL-1?. Subsequently, these dendritic cells induced CD4+ T cell proliferation, accompanied by IFN? release. Altogether, the initial steps reported here point towards the potential of NB-PDT to stimulate the immune system, thus giving this selective-local therapy a systemic reach.
| S-EPMC7226123 | biostudies-literature
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.
| S-EPMC7140040 | biostudies-literature
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.
| S-EPMC7694906 | biostudies-literature
Project description: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.
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Project description:This contribution features a small molecule that binds TrkC (tropomyosin receptor kinase C) receptor that tends to be overexpressed in metastatic breast cancer cells but not in other breast cancer cells. A sensitizer for (1)O2 production conjugated to this structure gives 1-PDT for photodynamic therapy. Isomeric 2-PDT does not bind TrkC and was used as a control throughout; similarly, TrkC- cancer cells were used to calibrate enhanced killing of TrkC+ cells. Ex vivo, 1- and 2-PDT where only cytotoxic when illuminated, and 1-PDT, gave higher cell death for TrkC+ breast cancer cells. A 1 h administration-to-illumination delay gave optimal TrkC+/TrkC--photocytotoxicity, and distribution studies showed the same delay was appropriate in vivo. In Balb/c mice, a maximum tolerated dose of 20 mg/kg was determined for 1-PDT. 1- and 2-PDT (single, 2 or 10 mg/kg doses and one illumination, throughout) had similar effects on implanted TrkC- tumors, and like those of 2-PDT on TrkC+ tumors. In contrast, 1-PDT caused dramatic TrkC+ tumor volume reduction (96% from initial) relative to the TrkC- tumors or 2-PDT in TrkC+ models. Moreover, 71% of the mice treated with 10 mg/kg 1-PDT (n = 7) showed full tumor remission and survived until 90 days with no metastasis to key organs.
| S-EPMC4291778 | biostudies-literature
Project description:Despite combined treatments, glioblastoma outcome remains poor with frequent local recurrences, indicating that a more efficient and local therapy is needed. In this way, vascular-targeted photodynamic therapy (VTP) could help tumor eradication by destroying its neovessels. In this study, we designed a polysiloxane-based nanoparticle (NP) combining a magnetic resonance imaging (MRI) contrast agent, a photosensitizer (PS) and a new ligand peptide motif (KDKPPR) targeting neuropilin-1 (NRP-1), a receptor overexpressed by angiogenic endothelial cells of the tumor vasculature. This structure achieves the detection of the tumor tissue and its proliferating part by MRI analysis, followed by its treatment by VTP. The photophysical properties of the PS and the peptide affinity for NRP-1 recombinant protein were preserved after the functionalization of NPs. Cellular uptake of NPs by human umbilical vein endothelial cells (HUVEC) was increased twice compared to NPs without the KDKPPR peptide moiety or conjugated with a scramble peptide. NPs induced no cytotoxicity without light exposure but conferred a photocytotoxic effect to cells after photodynamic therapy (PDT). The in vivo selectivity, evaluated using a skinfold chamber model in mice, confirms that the functionalized NPs with KDKPPR peptide moiety were localized in the tumor vessel wall.
| S-EPMC5627731 | biostudies-literature
Project description:This study describes the employment of gold nanorods (AuNRs), known for their good reputation in hyperthermia-based cancer therapy, in a hybrid combination of photosensitizers (PS) and peptides (PP). We report here, the design and the synthesis of this nanosystem and its application as a vehicle for the selective drug delivery and the efficient photodynamic therapy (PDT). AuNRs were functionalized by polyethylene glycol, phototoxic pyropheophorbide-a (Pyro) PS, and a "KDKPPR" peptide moiety to target neuropilin-1 receptor (NRP-1). The physicochemical characteristics of AuNRs, the synthesized peptide and the intermediate PP-PS conjugates were investigated. The photophysical properties of the hybrid AuNRs revealed that upon conjugation, the AuNRs acquired the characteristic properties of Pyro concerning the extension of the absorption profile and the capability to fluoresce (?f = 0.3) and emit singlet oxygen (?? = 0.4) when excited at 412 nm. Even after being conjugated onto the surface of the AuNRs, the molecular affinity of "KDKPPR" for NRP-1 was preserved. Under irradiation at 652 nm, in vitro assays were conducted on glioblastoma U87 cells incubated with different PS concentrations of free Pyro, intermediate PP-PS conjugate and hybrid AuNRs. The AuNRs showed no cytotoxicity in the absence of light even at high PS concentrations. However, they efficiently decreased the cell viability by 67% under light exposure. This nanosystem possesses good efficiency in PDT and an expected potential effect in a combined photodynamic/photothermal therapy guided by NIR fluorescence imaging of the tumors due to the presence of both the hyperthermic agent, AuNRs, and the fluorescent active phototoxic PS.
| S-EPMC6947424 | biostudies-literature
Project description:Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer (PS) and has been used to treat head and neck cancers. Yet, common PS lack tumor specificity, which leads to collateral damage to normal tissues. Targeted delivery of PS via antibodies has pre-clinically improved tumor selectivity. However, antibodies have long half-lives and relatively poor tissue penetration, which could limit therapeutic efficacy and lead to long photosensitivity. Here, in this feasibility study, we evaluate at the pre-clinical level a recently introduced format of targeted PDT, which employs nanobodies as targeting agents and a water-soluble PS (IRDye700DX) that is traceable through optical imaging. In vitro, the PS solely binds to cells and induces phototoxicity on cells overexpressing the epidermal growth factor receptor (EGFR), when conjugated to the EGFR targeted nanobodies. To investigate whether this new format of targeted PDT is capable of inducing selective tumor cell death in vivo, PDT was applied on an orthotopic mouse tumor model with illumination at 1h post-injection of the nanobody-PS conjugates, as selected from quantitative fluorescence spectroscopy measurements. In parallel, and as a reference, PDT was applied with an antibody-PS conjugate, with illumination performed 24h post-injection. Importantly, EGFR targeted nanobody-PS conjugates led to extensive tumor necrosis (approx. 90%) and almost no toxicity in healthy tissues, as observed through histology 24h after PDT. Overall, results show that these EGFR targeted nanobody-PS conjugates are selective and able to induce tumor cell death in vivo. Additional studies are now needed to assess the full potential of this approach to improving PDT.
| S-EPMC7116242 | biostudies-literature
Project description:PurposeHexokinase II (HK2) protein expression is elevated in glioblastoma (GBM), and we have shown that HK2 could serve as an effective therapeutic target for GBM. Here, we interrogated compounds that target HK2 effectively and restrict tumor growth in cell lines, patient-derived glioma stem cells (GSCs), and mouse models of GBM.Experimental Design: We performed a screen using a set of 15 drugs that were predicted to inhibit the HK2-associated gene signature. We next determined the EC50 of the compounds by treating glioma cell lines and GSCs. Selected compounds showing significant impact in vitro were used to treat mice and examine their effect on survival and tumor characteristics. The effect of compounds on the metabolic activity in glioma cells was also assessed in vitro.ResultsThis screen identified the azole class of antifungals as inhibitors of tumor metabolism. Among the compounds tested, ketoconazole and posaconazole displayed the greatest inhibitory effect on GBM both in vitro and in vivo. Treatment of mice bearing GBM with ketoconazole and posaconazole increased their survival, reduced tumor cell proliferation, and decreased tumor metabolism. In addition, treatment with azoles resulted in increased proportion of apoptotic cells.ConclusionsOverall, we provide evidence that azoles exert their effect by targeting genes and pathways regulated by HK2. These findings shed light on the action of azoles in GBM. Combined with existing literature and preclinical results, these data support the value of repurposing azoles in GBM clinical trials.
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