Self-Reporting Photodynamic Nanobody Conjugate for Precise and Sustainable Large-Volume Tumor Treatment

doi:10.21203/rs.3.rs-3598991/v1

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Self-Reporting Photodynamic Nanobody Conjugate for Precise and Sustainable Large-Volume Tumor Treatment

https://doi.org/10.21203/rs.3.rs-3598991/v1

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published 13 Aug, 2024

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Nanobodies (Nbs), the smallest antigen-binding fragments with high stability and affinity, bring a new opportunity to improve the specificity of photosensitizers for tumor tissue in photodynamic therapy (PDT) through an active targeting strategy. Nonetheless, the rapid metabolism of Nbs in vivo restricts the accumulation and retention of the photosensitizer at the tumor site, especially in large-volume tumors. Herein, we developed photodynamic conjugates, MNB-Pyra Nbs, through site-specific modification of 7D12 Nbs with the type I photosensitizer MNB-Pyra in a 1:2 ratio. The π − π stacking interactions of MNB-Pyra molecules within the conjugate causes fluorescence quenching, while the photosensitizer with long-term retention could be released by reactive oxygen species (ROS) cleavage, which is accompanied by fluorescence recovery for self-reporting. Moreover, the conjugation of MNB-Pyra and Nbs greatly improved the metabolism of the benzophenothiazine photosensitizer in vivo, leading to the clearance of MNB-Pyra Nbs in 24 h without illumination. Ultimately, a single dose of MNB-Pyra Nbs demonstrated highly effective tumor suppression (tumor inhibition rate > 95%) with high biosafety in the large-volume tumor model after three rounds of PDT. This self-reporting photodynamic nanobody conjugate is expected to promote the development of personalized precision PDT.

Physical sciences/Chemistry/Chemical biology/Proteins/Carrier proteins

Physical sciences/Optics and photonics/Optical materials and structures/Biomaterials/Biomaterials – proteins

Physical sciences/Chemistry/Photochemistry/Single-molecule fluorescence

Physical sciences/Chemistry/Chemical biology/Drug delivery

nanobody conjugate

photodynamic therapy

π − π stacking

self-reporting

precise long-term tumor retention

Photodynamic therapy (PDT) has emerged as a promising cancer treatment modality due to its minimal invasiveness, repeatability, negligible drug resistance, and high spatiotemporal precision.^1–3 Photosensitizers can quickly sensitize oxygen to generate reactive oxygen species (ROS) upon light irradiation, resulting in cancer cell death accompanied by destruction of the tumor vasculature and induction of an inflammatory response for tumor ablation.^4–6 Despite the progress made in the clinical application of traditional photosensitizers such as porphyrins and phthalocyanine derivatives,^{7, 8} photosensitizers are still plagued by the issues of off-target toxicity, tumor hypoxia, and inadequate accumulation or poor retention at the tumor site, which hamper the efficiency of PDT.^9–12

To achieve high tumor specificity, active targeting via antibody‒drug conjugates (ADCs) has garnered lots of attention as an approach with high biosafety.^{13, 14} Targeted therapeutics based on monoclonal antibodies (mAbs), such as cetuximab-IRdye700DX, have been extensively studied for anticancer therapy and are currently in phase III clinical trials for the treatment of recurrent head and neck cancer.¹⁵ However, the challenges associated with specific conjugation and the high production costs of mAbs have limited the development of ADCs.^{16, 17} In contrast, nanobodies (Nbs), the smallest antigen-binding fragments derived from heavy chain-only antibodies that are exclusively found in camelids (VHH) and cartilaginous fish (VNAR), have gained much interest.^{18, 19} Their single-domain nature allows their direct expression in bacterial systems, making them easier to prepare and modify as needed. Compared to conventional mAbs with a size of approximately 150 kDa, the small size of Nbs (approximately 15 kDa) enables them to reach less accessible antigens while maintaining high affinity and stability.^{20, 21} Nevertheless, due to their small size (~ 10 nm), Nbs are rapidly removed from mammals via the circulation with an elimination half-life of several hours.^{17, 22, 23}

In terms of the sustainability and phototoxic side effects of treatment, the cycling and metabolic rate of photosensitizers in PDT must be considered. Excessively fast metabolism of the photosensitizer results in insufficient drug accumulation, hindering subsequent sustained treatment, while slow metabolism leads to a long drug clearance period, potentially causing severe phototoxic side effects.^{24, 25} In this regard, accurately delivering long-term resident photosensitizers to the tumor site via Nbs may be an ideal strategy to reduce drug enrichment in normal tissues, enhance drug accumulation in tumor tissues, and enable precise and sustainable PDT, which offers the advantages of reducing the pain accompanied by multiple dosing and minimizing the potential risks of side effects associated with repeated dosing, especially for the treatment of large-volume tumors.

Epidermal growth factor receptor (EGFR) is a heavily glycosylated transmembrane receptor tyrosine kinase that is closely associated with tumorigenesis and cancer proliferation.^{26, 27} Due to its overexpression in tumor cells, EGFR is an important target in cancer therapy.^28–30 The nanobody 7D12 specifically binds to EGFR ectodomain epitopes inaccessible to mAbs, subsequently promoting EGFR aggregation and inducing receptor-mediated lysosomal endocytosis.^{22, 23} The benzophenothiazine structure of MNB has been classified as a type I photosensitizer, which efficiently produces superoxide radicals (O₂^–•)by electron transfer after photoexcitation.³¹ Then, the MNB-Pyra molecule was constructed to covalently modify the Nbs by connecting MNB with the pyrazolone (Pyra) structure through a ROS-cleavable linker.^{32, 33} The good stability and solubility of MNB-Pyra in aqueous solution indicate that this molecule has high protein compatibility, which is conducive to bioconjugation.

Herein, the MNB-Pyra Nbs conjugates were constructed by connecting the type I photosensitizer MNB-Pyra with 7D12-fGly via a Knoevenagel − Michael tandem reaction, as shown in Scheme 1, in which 7D12-fGly is a modified form of 7D12 with a C-terminus aldehyde handle for specific and quantitative conjugation.^{34, 35} MNB-Pyra Nbs exhibited significant fluorescence quenching due to the π − π stacking interactions of the planar molecule MNB-Pyra after site-specific modification of the Nbs with MNB-Pyra in a 1:2 ratio. Upon light irradiation, fluorescence recovery was observed since the monomer photosensitizer was released due to cleavage by ROS, displaying the self-reporting ability of MNB-Pyra Nbs during PDT. Moreover, the MNB-Pyra Nbs demonstrated excellent targeting ability and PDT efficiency in vitro and in vivo. Interestingly, MNB-Pyra Nbs were almost completely metabolized 24 h after intravenous injection without illumination, but the long-term retention characteristics of the benzophenothiazine compound ensured that the released monomer photosensitizer remained precisely at the tumor site for 5 days after light irradiation. Significantly, MNB-Pyra Nbs efficiently inhibited tumor growth in the large-volume tumor model after single-dose administration and continuous PDT. Consequently, this photodynamic nanobody conjugate provides a paradigm for the development of precise long-term resident photosensitizer.

MNB-Pyra dimerization leads to dimerization-caused quenching (DCQ). The route by which MNB-Pyra was synthesized is shown in Scheme S1. The MNB-Pyra dimer was synthesized by leveraging the reactivity of the pyrazolone to aldehydes, in which MNB-Pyra dimerized upon reaction with isobutyraldehyde via a Knoevenagel − Michael tandem reaction (Fig. 1a). All intermediates and products were characterized by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy in Figures S18–S27. As shown in Fig. 1b and 1c, MNB-Pyra displayed a main absorption band at 645 nm. In comparison, the MNB-Pyra dimer exhibited a blueshifted band at 600 nm and significant fluorescence quenching, with its absolute fluorescence quantum yield (Φ_f) decreasing from 4.6–0.9% (Table S1).

To explore the fluorescence quenching mechanism and gain deeper insights into the structural arrangement of the MNB-Pyra dimer, we performed a conformational search of the MNB-Pyra dimer in water using the GFNn-xTB method for molecular dynamics calculations and geometry optimization at the B97-3c density functional approximation level.^{36, 37} The lowest energy conformation of the MNB-Pyra dimer in water (99.8% abundance) presented with a π-stacked geometrical arrangement and orbital overlap with a center-to-center distance of 4.4 Å and a flip angle of θ = 55.2° (Fig. 1d and 1e). The noncovalent intramolecular interaction was analyzed using an independent gradient model, which also revealed strong interchromophoric π − π interactions (Fig. 1e). These results confirm that the MNB-Pyra dimer is a self-quenching foldamer with a dimerization-caused quenching (DCQ) mechanism. The foldamer structures of most DCQ fluorophores will open after solubilization in organic solvents, which occurs along with the abolishment of aggregation and recovery of fluorescence emission. However, the MNB-Pyra dimer maintained DCQ effects with weak fluorescence emission even in polar solvents (e.g., acetonitrile and methanol), highlighting the strong H-bonding interactions between the MNB-Pyra molecules in the dimer (Figure S1). In addition, the fluorescence quenching of the MNB-Pyra dimer remained unperturbed in biological media in the presence of serum proteins (Figure S2), indicating the stability of the self-quenching MNB-Pyra dimer structure in a biological context.

Next, the ROS generating ability of the molecules was evaluated with fluorescent probes. 1,3-Diphenylisobenzofuran (DPBF) was employed to detect the generation of ¹O₂, where dihydrorhodamine 123 (DHR 123) served as an indicator of superoxide (O₂^–•) with fluorescence emission at 526 nm. As anticipated, both the MNB-Pyra and MNB-Pyra dimers exhibited negligible ¹O₂ generation (Figs. 1f and S3a) and efficient O₂^–• generation (Figs. 1g and S3b), affirming their excellent efficiency as type I photosensitizers. In addition, the generation of O₂^–• by MNB-Pyra was confirmed with the indicator 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) by electron paramagnetic resonance (EPR) spectroscopy, and a characteristic paramagnetic adduct of O₂^−• was observed (Fig. 1h). Subsequently, a redshifted absorption band and remarkable fluorescence enhancement of the MNB-Pyra dimer were observed after illumination, as shown in Fig. 1i and 1j, indicating depolymerization of the dimer and disruption of the DCQ effect. The cleaved products were further analyzed by high resolution mass spectrometry (HRMS), which demonstrated that the MNB-Pyra dimer was completely consumed and that the monomer photosensitizer (m/z = 479.1568 for [M]⁺) was released (Figure S4). As a result, the MNB-Pyra dimer can self-report the occurrence of PDT by the fluorescence enhancement after the monomer photosensitizer is released.

Preparation of the Nbs conjugate via in situ MNB-Pyra dimerization. Instead of presynthesizing MNB-Pyra dimers for bioconjugation, the MNB-Pyra molecule was dimerized in situ at the C-terminus of 7D12-fGly utilizing the reactivity of the pyrazolone moiety to the aldehyde tag, which was installed using a formylglycine-generating enzyme.³¹ MNB-Pyra Nbs were formed by reacting 7D12-fGly with MNB-Pyra in 2-morpholinoethanesulfonic acid buffer (MES buffer, pH 6.5) at room temperature (Fig. 2a). Sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and high-performance liquid chromatography–high resolution mass spectrometry (HPLC–HRMS) were used to characterize the synthesized MNB-Pyra Nbs. As displayed in Fig. 2b, the conjugation product displayed a weak fluorescence band with no significant band migration compared to the original 7D12-fGly protein. HPLC–HRMS analysis of the protein (Fig. 2c) also confirmed the formation of MNB-Pyra Nbs (mass of 7D12-fGly: 17990.770; mass of MNB-Pyra Nbs: 19536.219), in which each 7D12-fGly nanobody was conjugated with two MNB-Pyra molecules with a conjugation efficiency exceeding 95% in 24 h. The transmission electron microscopy (TEM) images showed that both 7D12-fGly and MNB-Pyra Nbs had a granular morphology with a uniform size distribution of approximately 15 nm (Fig. 2d), which indicated that the particle size of the antibody did not change significantly after 7D12-fGly and MNB-Pyra were covalently bound. Importantly, the binding affinity of the 7D12-fGly ligand for EGFR remained unaffected after conjugation, as confirmed by the cell-based ELISA results (Figure S5).

Moreover, MNB-Pyra dimerization in the nanobody conjugate was expected to realize fluorescence quenching, resulting from the formation of a DCQ foldamer. Thus, the absorption and fluorescence spectra of the MNB-Pyra Nbs were investigated in phosphate-buffered saline (PBS, pH 7.4). As shown in Fig. 2e, the maximum absorption peak of MNB-Pyra Nbs exhibited a noticeable blueshift from 645 nm to 600 nm in comparison to the spectrum in MNB-Pyra. Significant fluorescence quenching was observed in Fig. 2f. The absolute fluorescence quantum yield of MNB-Pyra Nbs (0.8%) in aqueous solution closely resembled that of the MNB-Pyra Nbs dimer (Φ_f = 0.9%, Table S1), confirming the significant reduction in fluorescence emission. The dimer-like spectral features of the MNB-Pyra Nbs verified the aggregation of MNB-Pyra within the nanobody conjugates. Similarly, MNB-Pyra Nbs were able to recover their fluorescence emission upon exposure to 630 nm light (Fig. 2g), indicating that the MNB-Pyra dimer within the conjugate had depolymerized. The cleaved products were identified by HRMS analysis, and the product (m/z = 479.1566 for [M]⁺) was the same monomer photosensitizer as that detected after MNB-Pyra dimer cleavage (Figure S6). The successful fluorescence recovery of the MNB-Pyra Nbs suggested their efficient ROS generation ability under light irradiation. Therefore, this photodynamic nanobody conjugate can self-report the production of ROS and the release of monomer photosensitizers during PDT through the increasing fluorescence intensity.

Specificity of MNB-Pyra Nbs for EGFR-positive cells. We reasoned that attaching MNB-Pyra to an anti-EGFR nanobody (7D12-fGly) would endow the molecule with high selectivity for EGFR-overexpressing cells. Therefore, four different cell lines with different EGFR expression levels were employed for cell experiments, including epidermoid carcinoma (A431), cervical carcinoma (HeLa), lung cancer (A549) and embryonic cells (NIH-3T3). Western blot assays confirmed that A431 and HeLa cells exhibited high EGFR expression, while A549 and NIH-3T3 cells had almost negligible EGFR expression (Fig. 3a). First, the cytocompatibility of the MNB-Pyra Nbs was investigated. More than 90% of each type of cell survived after incubation with increasing concentrations of MNB-Pyra Nbs for 24 h without light irradiation (Fig. 3b), manifesting the excellent biocompatibility of the conjugate in vitro. Next, the uptake efficiency of MNB-Pyra Nbs by different cells was evaluated using confocal laser scanning microscopy (CLSM) and flow cytometry. EGFR-positive A431 and HeLa cells exhibited significant cellular uptake, with increasing fluorescence signals in the cells over time (Figure S7 and 3c). In contrast, negligible fluorescence signals were observed in A549 and NIH-3T3 cells, indicating minimal cellular uptake of MNB-Pyra Nbs. Flow cytometry quantification of MNB-Pyra Nbs (0.2 µM) uptake further supported the significantly higher fluorescence signals in A431 and HeLa cells (Fig. 3d). These results confirmed the high selectivity of MNB-Pyra Nbs for EGFR-overexpressing cells. Notably, neither the MNB-Pyra molecule nor the MNB-Pyra dimer exhibited any cell selectivity, as there were no observable differences in cellular fluorescence after various cell types were treated with these molecules (Figure S8). Therefore, modifying MNB-Pyra with the 7D12-fGly nanobody enables MNB-Pyra Nbs to specifically target EGFR-overexpressing cancer cells, thereby minimizing potential side effects during cancer therapy.

Moreover, the intracellular localization of MNB-Pyra Nbs was investigated using commercial dyes. As shown in Figure S9, the intracellular fluorescence of MNB-Pyra Nbs overlapped well with the green fluorescence of LysoTracker Green with a colocalization coefficient (Pearson's correlation) of 0.871, which was ascribed to receptor-mediated endocytosis to the lysosomes.

Self-reporting PDT effect of MNB-Pyra Nbs in vitro. ROS production by the MNB-Pyra Nbs in cells after illumination was confirmed with fluorescent probes, including the reactive oxygen indicator 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and O₂^–• indicator dihydroethidium (DHE). As shown in Fig. 3e, bright fluorescence signals from the probes in cells were collected under both normoxia and hypoxia conditions, demonstrating efficient ROS generation. Moreover, remarkable DHE fluorescence, indicating the presence of O₂^−•, was also detected in the cells, implying that MNB-Pyra Nbs might have satisfactory PDT activity under hypoxia.

Subsequently, the PDT effect of MNB-Pyra Nbs was investigated. As presented in Fig. 3g, MNB-Pyra Nbs exhibited potent phototoxicity toward A431 cells under normoxia conditions with a half-maximal inhibitory concentration (IC₅₀) of 0.2 µM. Furthermore, cell viability was evaluated by simulating the hypoxia microenvironment of tumors (2% O₂). Although hypoxia conditions slightly overwhelmed the PDT outcome, treatment with MNB-Pyra Nbs still successfully led to severe cell disruption with an IC₅₀ of 0.4 µM (Fig. 3g), confirming their efficient PDT effect under hypoxia conditions. Additionally, a calcein AM-PI kit was used for live/dead cell imaging, where calcein AM labels live cells with green fluorescence and PI labels dead cells with red fluorescence.³⁸ A431 cells presented green fluorescence only after incubation with MNB-Pyra Nbs without light irradiation, suggesting the biosafety of the MNB-Pyra Nbs (Figure S10). In contrast, strong red fluorescence from PI was observed in cells under both normoxia and hypoxia conditions, validating the effective cell killing capability of MNB-Pyra Nbs after PDT.

Notably, MNB-Pyra Nbs had high selectivity for EGFR-positive cells during PDT. As presented in Fig. 3h, MNB-Pyra Nbs showed potent effects on A431 and HeLa cell killing with IC₅₀ values of 0.2 and 0.3 µM, respectively. However, the cell killing efficiency of MNB-Pyra Nbs in A549 and NIH-3T3 cells was negligible due to the low expression of EGFR in these cells. Live/dead cell images of different cell lines were also collected after PDT or without PDT. As shown in Figure S10, all nonilluminated cells exhibited green fluorescence only. Moreover, strong red fluorescence from PI was observed in A431 and HeLa cells after PDT, whereas A549 and NIH-3T3 cells retained bright green fluorescence. These results confirmed the high specificity of MNB-Pyra Nbs for EGFR-overexpressing cells. In comparison, the differences in phototoxicity of the MNB-Pyra dimer was negligible among the different cell lines (Fig. 3i), suggesting that the dimer does not have cell specificity.

In addition, a wound healing assay was conducted with MNB-Pyra Nbs to visualize the suppression of cell proliferation after PDT. MNB-Pyra Nbs (0.5 µM) were added when the cells in the dishes reached a density of approximately 80% and the cells were then exposed to light irradiation (630 nm, 30 mW/cm², 20 min) after incubation. Bright field images of different cells were captured at 0 h and 24 h after treatment. It was found that the proliferation of A431 and HeLa cells was significantly inhibited, with wound healing rates of 17% and 13%, respectively, while the wounds in A549 and NIH-3T3 cells healed completely (Figs. 3j and S12). This further confirmed the specificity of MNB-Pyra Nbs to inhibit the proliferation and invasion of EGFR-overexpressing cells.

Besides, the self-reporting capability of MNB-Pyra Nbs during PDT was observed in cells. A431 and HeLa cells were exposed to 630 nm light irradiation (30 mW/cm², 10 min) after incubation with 0.2 µM MNB-Pyra Nbs for 2 h, and significantly enhanced fluorescence signals were detected in these cells (Fig. 3k and 3l). Additionally, flow cytometry was used to quantify the intracellular fluorescence, which was notably increased after light irradiation (Fig. 3m). Thus, it was suggested that the MNB-Pyra Nbs were cleaved in the cells by ROS, leading to the release of the monomer photosensitizer with increased fluorescence intensity to monitor the occurrence of PDT, as illustrated in Fig. 3n.

The mechanism of cell death was investigated using an Annexin V-FITC/PI kit. During apoptosis, Annexin V-FITC specifically binds to phosphatidylserine on the outer surface of apoptotic cells, while PI enters the dead cells and emits red fluorescence upon binding to DNA.³² Cells were incubated with the Annexin V-FITC/PI dyes for 30 min after various treatments. The control groups treated with MNB-Pyra Nbs only exhibited no fluorescence signals from either FITC or PI. However, bright green fluorescence from Annexin V-FITC and red fluorescence from PI were observed in the PDT group (Fig. 3f), indicating that MNB-Pyra Nbs induced apoptosis after light irradiation (630 nm, 30 mW/cm², 20 min). The apoptosis process was also identified with Annexin V-FITC/PI using flow cytometry (Figure S13). The cells in the PDT group were mainly found in the third quadrant, which represents apoptosis.³⁹

Targeting and precise long-time retention of MNB-Pyra Nbs in vivo. Encouraged by the impressive specificity of MNB-Pyra Nbs for EGFR-overexpressing cells, the targeting ability of the conjugate was evaluated in tumor-bearing nude mice. An A431 tumor-bearing mouse model was constructed by subcutaneous injection of A431 cells into Nu/J female mice. As shown in Fig. 4a and S13a, there was no discernible fluorescence signal at the tumor site over time following intravenous injection of the MNB-Pyra dimer (0.3 µM, 100 µL). In stark contrast, mice injected with MNB-Pyra Nbs (0.3 µM, 100 µL) via the tail vein displayed a progressive increase in fluorescence at the tumor site, reaching its peak at 4 h (Fig. 4b and S14b). Quantitative analysis corroborated the efficient tumor targeting of the MNB-Pyra Nbs in vivo (Fig. 4c). This result clearly indicated that conjugating the Nbs with the MNB-Pyra molecule endowed the molecule with excellent targeting capability in vivo. Upon dissection at 24 h postinjection, the MNB-Pyra dimer was still mainly distributed in the liver, while the fluorescence of the MNB-Pyra Nbs in the organs was exceedingly weak, revealing the effective clearance of the conjugate. Additionally, a bilateral tumor model was constructed, and MNB-Pyra Nbs were intravenously injected (0.3 µM, 200 µL). As shown in Fig. 5d, obvious fluorescence signals from the MNB-Pyra Nbs were collected at the tumor sites after 4 h. Then, the right tumor was exposed to 630 nm light irradiation (30 mW/cm²_, 20 min), and the left tumor without any treatment was used as a control. As a result, a remarkable increase in fluorescence intensity was observed at the right tumor site (Fig. 5d and 5e), which indicated that the MNB-Pyra Nbs could self-report monomer release and the occurrence of PDT by the increase in fluorescence signal intensity.

Long-term retention of the benzophenothiazine compound in vivo was reported in our previous work, in which the fluorescence of photosensitizers persisted at the tumor site over 120 h.⁴⁰ Herein, the similarly long-term retention of the MNB-Pyra molecule (0.3 µM, 100 µL) in vivo was demonstrated as shown in Figure S15, where the fluorescence at the tumor site was retained for a week without attenuation and never spread to other sites after intratumoral injection. In fact, the prolonged retention of the photosensitizers suggests that it is difficult to clear from the body, which brings about potential phototoxicity to organs during treatment and the risk of side effects caused by the overaccumulation of drugs. Nevertheless, the clearance of MNB-Pyra Nbs within 24 h without illumination (Fig. 4b) suggested that the metabolism of MNB-Pyra was improved after its conjugation with 7D12-fGly Nbs. As a proof of concept, the tumor site was exposed to 630 nm light irradiation (30 mW/cm², 20 min) after the effective enrichment of MNB-Pyra Nbs (0.3 µM, 100 µL) administered via tail vein injection (Fig. 4f). In this case, significant fluorescence enhancement was observed at the tumor site, and the fluorescence signal persisted in the tumor for 5 days (Fig. 4g). Dissection analysis performed after 120 h revealed a concentrated fluorescence signal in the tumor tissue and negligible fluorescence in other organs, demonstrating that the released monomer photosensitizer achieved precise long-term retention at the tumor site after illumination.

MNB-Pyra Nbs for large-volume tumor treatment in vivo. Single-dose PDT can generally achieve effective small tumor inhibition, whereas multiple treatments are needed for the suppression of large-volume tumors. Photosensitizers with long-term retention characteristics are suitable for the treatment of large-volume tumors, as they enable sustained treatment after injection of a single dose, reducing the pain and inconvenience of multiple dosing. However, conventional photosensitizers with long-term retention also accumulate and are retained in organs, which brings about the risk of phototoxic side effects during subsequent continuous PDT. Therefore, the precise release of photosensitizers with long-term retention at the tumor site, along with improved photosensitizer metabolism in organs, is essential to achieve sustainable PDT and minimize side effects during the ablation of large-volume tumors.

Motivated by the excellent tumor targeting and precise long-term retention of MNB-Pyra Nbs, the therapeutic effect of MNB-Pyra Nbs was investigated in mice bearing large tumors (initial volume ≈ 350 mm³), as depicted in Fig. 5a. Mice in different groups treated with only PBS, light, or MNB-Pyra Nbs were regarded as control groups (n = 5). As shown in Fig. 5b, in mice administered a single PDT treatment (630 nm, 50 mW/cm², 20 min), tumor growth was not efficiently suppressed, with a tumor growth inhibition rate of only 18% (MNB-Pyra Nbs + L (1) group). However, the long-term retained fluorescence signal at the tumor site after light irradiation encouraged us to conduct continuous PDT on days 1, 2 and 3 (MNB-Pyra Nbs + L (3) group). Remarkably, the average tumor inhibition rate in this group was over 95%, and some tumors eventually disappeared after the mice were treated with three rounds of PDT. The tumor weights (Fig. 5c) and pictures of the tumors (Figure S16) from different groups at the end of the experiment also verified the highly effective inhibition provided by the MNB-Pyra Nbs after multiple PDT. These results clearly demonstrated that the MNB-Pyra Nbs successfully realized efficient tumor inhibition in a large-volume tumor model via sustained PDT after the administration of a single dose due to the precise tumor retention effect.

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was employed to label apoptotic cells. As anticipated, a significantly higher percentage of TUNEL-positive cells was found in the MNB-Pyra Nbs + L (3) group than in the other groups, indicating that the therapeutic efficacy in the MNB-Pyra Nbs + L (3) group was the highest due to the effective induction of apoptosis (Fig. 5e). Hematoxylin and eosin (H&E) staining was used to assess the morphology of the cells in tumor tissues. As depicted in Fig. 5f, a substantial number of tight stromal cells were observed in the control groups, and the intact cell nuclei indicated that the cells were in good condition. However, almost no cells with a complete morphology were observed in the MNB-Pyra Nbs + L (3) group, and the cell nuclei were shrunken or broken. Immunohistochemical (IHC) analysis also demonstrated the suppression of tumor proliferation with downregulated Ki-67 expression in the MNB-Pyra Nbs + L (3) group (Fig. 5g), whereas extensive cell damage was not observed in the other treatment groups.

The physiological safety of the MNB-Pyra Nbs in vivo was evaluated by monitoring the body weights of the mice and analyzing the health of the organs with H&E staining. Notably, none of the mice displayed any abnormal changes in body weight during the treatment period (Fig. 5d). Moreover, no noticeable cell necrosis or inflammatory lesions were observed in any of the major organs in all groups (Figure S17). These findings underscore the biocompatibility and applicability of MNB-Pyra Nbs in vivo.

It is critical that photosensitizers have good targeting ability and improved tumor retention for accurate and effective PDT in vivo. In particular, photosensitizers demonstrating prolonged retention properties are well suited for the treatment of large-volume tumors, offering the advantages of reducing the pain caused by multiple dosing and minimizing the potential risks of side effects associated with repeated dosing. However, the drawback of prolonged retention is the challenge of clearing the photosensitizer from the body, which may lead to phototoxicity in organs during treatment. Therefore, a more strategic approach to mitigate this limitation is to precisely release long-term resident photosensitizers exclusively at the tumor site, facilitating sustainable PDT. Combining rapidly metabolized Nbs with long-term retention photosensitizers offers the potential to enhance both the targeting capability of the molecules and the metabolism of the long-term retention photosensitizers.

In summary, the photodynamic conjugates, MNB-Pyra Nbs were constructed by conjugating an EGFR-specific nanobody with the type I photosensitizer MNB-Pyra for sustainable PDT with remarkable targeting ability. MNB-Pyra Nbs demonstrated potent toxicity to EGFR-positive cells under both normoxia and hypoxia conditions after PDT. Moreover, the specific modification of 7D12-fGly Nbs with MNB-Pyra in a 1:2 ratio realized significant fluorescence quenching, while the significant increase in fluorescence intensity after illumination could indicate the occurrence of PDT, making these modified Nbs self-reporting conjugates. Additionally, the covalent bond greatly improved the metabolism of the benzophenothiazine photosensitizer in vivo, leading to the clearance of MNB-Pyra Nbs in 24 h without illumination. MNB-Pyra Nbs displayed precise long-term tumor retention due to the release of the monomer photosensitizer at the tumor site after light irradiation. This accurate accumulation and long-term retention of the photosensitizer was especially suitable for the ablation of large-volume tumors through sustained PDT after injection of a single dose. Finally, MNB-Pyra Nbs exhibited excellent tumor targeting and high efficiency PDT with a tumor inhibition rate over 95% in vivo. This versatile photodynamic nanobody conjugate could provide a new perspective for the development of precise and personalized PDT.

Materials

All chemical reagents used in this study were purchased from Energy Chemical Co., unless otherwise specified, and all solvents were of analytical grade. DHR 123 (dihydrorhodamine 123), DHE (Dihydroethidium), MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide), Hoechst 33342, LysoTracker Green, Calcein-AM/propidium iodide (PI) Detection Kit, and Annexin V-FITC/PI Kit were purchased from Beyotime Biotechnology (Shanghai, China). Dulbecco’s modified Eagle medium (DMEM) with L-glutamine was purchased from Solarbio. Penicillin–streptomycin (10000 U/mL), Trypsin–EDTA (0.05%), and fetal bovine serum (FBS) were purchased from Gibco. Silica gel (200–300 mesh) was used for flash-column chromatography.

Computational details

The 2000 initial structures of MNB-Pyra dimer were generated using Genmer. The structures were then optimized using Molclus combined with GFN2-xTB. The redundant structures were removed with an energy threshold of 0.5 kcal/mol and structure threshold of 0.5 Å. Finally, structure with lowest energy (DE < = 3 kcal/mol) were kept for next optimization steps. Geometry optimization was carried out with B97-3c density functional method using Orca 5.0 software(Software update: The ORCA program system—Version 5.0). For MNB-Pyra dimer, the structures were first optimized using Molclus combined with orca 5.0 software. The redundant structures were then removed with an energy threshold of 0.5 kcal/mol and structure threshold of 0.5 Å. The single point energy of the structures remained were then calculated at the RI-wB97M-V/def2-TZVP level with SMD solvation model. The ratio of different structure was calculated according to Boltzmann distribution at 298.15 K.

Cell incubation

Human epidermal cancer cells (A431 cells), human cervical carcinoma cells (HeLa cells), human lung cancer cells (A549 cells) and mouse embryonic cells (NIH-3T3 cells) were incubated at 37°C under a humidified atmosphere containing 5% CO₂, which were purchased from Institute of Basic Medical Sciences (IBMS) of the Chinese Academy of Medical Sciences. The DMEM used for cell incubation was supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Western blot

Cells (5×10⁶) were digested and washed with PBS three times, and were resuspended in lysis buffer (50 mM Tris, 150 mM NaCl, 0.2% Triton X-100, 10 µg/ml PMSF, pH 8.0). After 20 min of lysis on ice, the protein concentration in the supernatant was determined using the BCA assay. 20 µg of total proteins were loaded on 10% SDS-PAGE. The proteins were then transferred to PVDF membranes, followed by blocking with 5% skim milk powder for 1 h. The membranes were then incubated with primary antibodies (1: 20000 in 2% skim milk) (mouse EGFR monoclonal antibody, Proteintech) overnight at 4°C, followed by washing with TBST buffer thrice. The membranes were futher incubated with horseradish peroxide-conjugated secondary antibody (1:20000, goat anti-mouse IgG, proteintech) for 1 h at room temperature, and then were washed with TBST buffer thrice. The protein bands were visualized using ECL reagent under ChemiDoc XR + system (Bio-rad).

Cell Imaging

Cells (5 × 10⁴) were seeded into cell dishes containing DMEM supplemented with 1% penicillin-streptomycin and 10% FBS and cultured overnight. Various molecules were directly added to the cell culturing medium and followed by imaging using a confocal laser scanning microscope (Olympus FV3000) without washing (λ_ex = 640 nm, λ_em = 690 − 720 nm).

Flow cytometry

Cells (1×10⁵) were incubated 100 nM of MNB-Pyra Nbs for 2 h, followed by digestion and washing with PBS. Cells were then resuspended in PBS (200 µl) and analyzed using Attune™ NxT flow cytometer (ThermoFisher Scientific).

Cell Viability Assays In Vitro

Cell viability was measured by reducing of 3-(4, 5)-dimethylthiahiazo(-2-yl)-3,5-diphenytetrazoliumromide (MTT) to formazan crystals under mitochondrial dehydrogenases activity in living cells. All types of cells were seeded in 96-well microplates and each well had a density of 1 × 10⁵ cells per well in a 100 µL DMEM medium containing 10% FBS. After 24 h of cell growth, 100 µL different concentrations of molecule drugs from 0 to 2 µm in DMEM medium were added to the wells. The dark groups were continuously cultured for 12 h whereas the irradiation groups were irradiated with 630 nm light (30 mW/cm², 20 min) after 2 h of incubation with drugs and then cultured for another 12 h. Next, the MTT-containing medium was carefully poured out, and the generated formazan crystals were dissolved with 200 µL DMSO for each well. The microplate reader was taken to detect the absorbance of solutions at 490 nm and cell viability was calculated using the following equation:

Cell viability (%) = [(OD_Ps − OD_{blank control}) ∕ (OD_control − OD_{blank control})] × 100% (1)

Tumor Suppression Experiments In Vivo

All animal experiments were approved by the Dalian University of Technology Animal Care and Use Committee (DUT20230816). The Nu/J female mice inoculated with A431 cells in armpit were divided into five groups randomly (n = 5) when their tumor volume reached about 350 mm³. The control groups were treated with saline, light or MNB-Pyra Nbs only. The fourth group was injected with MNB-Pyra Nbs (0.5 µM, 100 µL) treated with light (630 nm, 50 mW/cm², 20 min) only once, and the last group was injected with MNB-Pyra Nbs (0.5 µM, 100 µL) treated with light (630 nm, 50 mW/cm², 20 min) three time at the 1st, 2nd, and 3rd day respectively. The injection of saline or drugs was in an intravenous way and exposed under light irradiation after 4 h. Subsequently, the weight of the mice and the volume of primary and distant tumors were monitored every day for 15 days. The volume of the tumor was calculated using the following formula:

V = L × D × D∕2 (2)

where V is the tumor volume, L is the longest diameter of the tumor, and D is the diameter perpendicular to L.

Statistical analysis

Origin software (Origin 2021 Corporation, U.S.) was used for the graph plot. The data were the mean values of the three experiments, expressed as the means ± the standard deviations (SD). The statistical analysis was performed using students’ t-test, and ***P < 0.001 were used to show statistical significance.

Data availability

All the data supporting the findings of this study are available within the article and its supplementary information files and from the corresponding author upon reasonable request.

Acknowledgment

This work was financially supported by National Natural Science Foundation of China (21925802, 22338005), Liaoning Binhai Laboratory (LBLB-2023-03) and the Fundamental Research Funds for the Central Universities (DUT22LAB601).

Competing interests

The authors declare no competing interests.

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Scheme 1 is available in the Supplementary Files section.

There is NO Competing Interest.

ESI.docx
Self-Reporting Photodynamic Nanobody Conjugate for Precise and Sustainable Large-Volume Tumor Treatment
rs.pdf
Reporting Summary
Scheme1.tif
Scheme 1. Construction of the self-reporting photodynamic nanobody conjugate to realize self-reporting and precise long-term retention in tumor tissue for sustainable PDT treatments.

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Self-Reporting Photodynamic Nanobody Conjugate for Precise and Sustainable Large-Volume Tumor Treatment

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