Heterogeneous EPR in tumour

The enhanced permeability and retention (EPR) effect of tumours is a topic of debate in nanomedicine community. To address this, we established a single-vessel quantitative analysis method (nano-ISML) to reveal heterogeneity of tumour vascular permeability.

Since the discovery of enhanced permeability and retention (EPR) phenomenon in 19861, it has been regarded as the principle for the design of nanomedicines. Doxil, PEGylated liposomal doxorubicin, was the first nanomedicine approved by FDA in 1995, which approved the concept that prolonged systemic circulation time increased EPR-based tumour accumulation. Ten years later, an albumin-bound paclitaxel, named Abraxane, has been used in clinical for metastatic breast cancer, whose approval confirmed the concept that active targeting strategies enhanced the EPR-based nanomedicine delivery. However, the primary advantage of these FDA-approved nanomedicines was the reduced side effects, such as cardiotoxicity, the therapeutic efficacy was still unsatisfactory. Many strategies have been attempted to increase the therapeutic efficiency of nanomedicines based on the EPR effect. Although the successful application of nanomedicines for cancer treatment in animal models, the clinical outcomes of nanomedicines were still far below expectations. Recently, Chan’s group demonstrated that up to 97% of nanoparticles enter tumours through an active trans-endothelial transport mechanism instead of the EPR effect2. Although the conclusion was controversial with the well-accepted mechanism for nanomedicine delivery, it redirected the researchers to exploit the causes for the failure of nanomedicines in clinical therapy. The high heterogeneity of tumour, such as tumour types, stages and sizes, has been considered as the potential reason of their poor delivery efficiency and the failure in clinical trials of nanomedicines. However, these are several issues remaining unclear: i) How to quantitatively analyzed the heterogeneity of vascular permeability? ii) What is the mechanism of the heterogeneity of nanoparticle entry into tumours? iii) How to rational design of nanoparticles to modulate their vascular permeability? In this issue of Nature Nanotechnology, we described a single-vessel analysis approach on the basis of protein-based nanoprobes and image segmentation-based ML technology, named Nano-ISML, to illustrate the potential mechanism of the heterogeneity of vascular permeability

We chose genetically recombinant human ferritin nanocages (FTn) as model nanoparticles due to its homogenous size. As shown in Figure 1a, after i.v. administrated into tumour-bearing mice, the vascular permeability of Cy5-labelled FTn in individual vessels was analyzed by machine learning (ML) based-image segmentation, which may provide a potent manipulation to illustrate the heterogeneity of vascular permeability. Using the Nano-ISML, we first extracted four features from the images, the total FTn coverage area for each vessel (Amagenta), the coverage area of each vessel (Agreen), the total Cy5 fluorescence intensity for each vessel (Fmagenta), and the Cy5 fluorescence intensity in each vessel lumen (Imagenta). Based on the four features, nine indices were defined for single-vessel quantitative analysis. After analyzing over 67,000 individual vessels from 32 types of tumour models, the nine indices were normalized and displayed as heatmap, and the results demonstrated that the blood vessel permeability across different tumour model types was highly heterogeneous, and the highest vessel FTn penetration area ratio (PR) was over 100-fold greater than the lowest vessel PR (Figure 1b). The representative individual vessels and their corresponding FTn penetration are shown in Figure 1c.

Furthermore, we used Zombie model (Figure 2a) and TEM images (Figure 2b) to investigate the penetration mechanism of FTn by assessing the extent of the contribution of passive extravasation (EPR effect) and active trans-endothelial transport (transcytosis) in different types of tumours. The results implied that FTn penetration in high permeable (HP)-tumours was mainly dependent on passive extravasation via inter-endothelial gaps and vesiculo-vacuolar organelle (VVO), whereas active trans-endothelial transport, mainly by pinocytosis, played a leading role in low permeable (LP)-tumours (Figure 2c).

Furthermore, to improve the drug delivery efficacy of FTn in LP-tumours by modulation the vascular permeability, we genetically modified FTn to improve active trans-endothelial transport by reducing its lysosome trapping. An endosomolytic (H2E)9 peptide fused FTn, and human serum albumin (HSA) bound FTn were genetically engineered (Figure 3a). Then, the vascular permeability of FTn and its variants was quantitatively evaluated in HP-tumours and LP-tumours using the Nano-ISML. The vascular permeability of FTn variants was significantly improved in LP-tumours, but not in HP-tumours. Finally, the in vivo anti-tumour efficacy was evaluated by loading a chemotherapeutic drug, doxorubicin (Dox), into the cavity of FTn and its variants. As expected, the FTn variants showed a synergistic therapeutic action in LP-tumours (Figure 3b). These results revealed that improving vascular permeability of nanoparticles in LP-tumours correlated with the therapeutic outcomes.

Herein, we created a ML-based single-vessel analysis method which was capable of assessment vascular permeability quantitatively, and demonstrated that vascular permeability was highly heterogeneous among different tumour types, and the vascular permeability of nanoparticles can be modulated by improving trans-endothelial transport which is an ideal design principle for enhancing tumour penetration and drug delivery efficacy of LP-tumours. This innovative Nano-ISML technology will advance the development of vascular biology, and can be extended from basic to applied research, such as molecular mechanism of vascular biology, drug screening, drug delivery, nanomedicine design, etc. The penetration mechanism and design strategy of nanomedicine revealed in this study will provide theoretical basis and design principles for the next generation of personalized nanomedicine. This work which deeply integrates nanotechnology, artificial intelligence and synthetic biology, also provides a new paradigm to solve fundamental scientific questions through interdisciplinary.

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  1. Matsumura, Y. & Maeda, H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res 46, 6387-6392 (1986).
  2. Sindhwani, S. et al. The entry of nanoparticles into solid tumours. Nat Mater 19, 566-575 (2020).


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