Oxygen Nanoshuttles for Tumor Oxygenation and Enhanced Cancer Treatment

Abstract

Open AccessCCS ChemistryMINI REVIEW1 Aug 2019Oxygen Nanoshuttles for Tumor Oxygenation and Enhanced Cancer Treatment Liangzhu Feng†, Oshra Betzer†, Danlei Tao, Tamar Sadan, Rachela Popovtzer and Zhuang Liu Liangzhu Feng† Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), College of Nano Science & Technology (CNST), Soochow University, Suzhou, Jiangsu 215123 (China) †L. Feng and O. Betzer contributed equally to this article.Google Scholar More articles by this author , Oshra Betzer† Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), College of Nano Science & Technology (CNST), Soochow University, Suzhou, Jiangsu 215123 (China) Faculty of Engineering, Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 52900 (Israel) †L. Feng and O. Betzer contributed equally to this article.Google Scholar More articles by this author , Danlei Tao Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), College of Nano Science & Technology (CNST), Soochow University, Suzhou, Jiangsu 215123 (China) Google Scholar More articles by this author , Tamar Sadan Faculty of Engineering, Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 52900 (Israel) Google Scholar More articles by this author , Rachela Popovtzer *Corresponding authors: E-mail Address: [email protected], E-mail Address: [email protected] Faculty of Engineering, Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan 52900 (Israel) Google Scholar More articles by this author and Zhuang Liu *Corresponding authors: E-mail Address: [email protected], E-mail Address: [email protected] Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), College of Nano Science & Technology (CNST), Soochow University, Suzhou, Jiangsu 215123 (China) Google Scholar More articles by this author https://doi.org/10.31635/ccschem.019.20190010 SectionsAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Tumor hypoxia is one of the hostile tumor microenvironment characteristics occurring in solid tumors. This feature is closely related to tumor progression and can negatively impair the effectiveness of cancer therapeutics. Recently, various strategies have been developed that enable efficient tumor oxygenation, not only enhancing treatment outcome of oxygen-consuming cancer therapeutics such as radiotherapy and photodynamic therapy, but also reversing the immunosuppressive tumor microenvironment to promote cancer immunotherapy. This review focuses on the latest progress in design and fabrication of innovative tumor-targeted oxygen nanoshuttles that attenuate tumor hypoxia and enhance cancer therapy. Future perspectives for clinical translation of tumor-targeted oxygen nanoshuttles will be further discussed. Download figure Download PowerPoint Introduction Tumor hypoxia, together with low pH values, excess extracellular matrix, and immunosuppressive factors, among others, are common hostile features of the solid tumor microenvironment (TME) resulting from altered metabolic pathways and abnormal tumor vasculature.1–3 Accumulating evidence indicates that the hypoxic TME plays a pivotal role in promoting tumor growth. It also significantly attenuates response of tumor cells to various cancer treatments, including radiotherapy and chemotherapy.4–8 Moreover, severe tumor hypoxia endows solid tumors with immunosuppressive TME features that disable the host immune system from detecting of the tumor and thus reducing the effects of immunotherapy.9–11 There is thus an increased need for effective strategies that address tumor hypoxia. With the rapid advancement of nanotechnology, a variety of nanoformulations with unique physiochemical properties have recently been designed for enhanced cancer treatment by promoting tumor oxygenation or depleting tumor oxygen, since oxygen plays a pivotal role in the cancer development and treatment.12–16 Recent studies have shown that catalytic nanoformulations that reach tumor cells decompose the high-endogenous H2O2 levels and generate O2, thus effectively attenuating tumor hypoxia.17–19 Such nanoformulations enhance treatment outcomes of oxygen-consuming cancer radiotherapy and photodynamic therapy (PDT), as well as promote a therapeutic response to immune checkpoint blockade therapy by modulating the immunosuppressive TME.20–26 Other pioneering strategies have used various nanoformulations to deliver compounds that inhibit oxygen consumption in tumor cells, thus increasing tumor oxygenation and leading to improved results with PDT.27,28 Another promising strategy for efficient tumor oxygenation is the increase of oxygen supply within tumors.29,30 Inspired by artificial blood cell substitutes, several groups have recently designed different types of tumor-targeted oxygen-delivery nanoshuttles.31,32 In this review, we will describe the design of these different nanoshuttles, mechanisms underlying their mode of action, their effect on tumor oxygenation, and their efficacy for improving various cancer treatments (summarized in Table 1). Table 1 | A Summary of Oxygen Nanoshuttles for Tumor Oxygenation and Cancer Therapy Enhancement Shuttle Type Fabrication Method Applications References Hemoglobin-based oxygen nanoshuttles Noncovalent encapsulation PDT [33] Chemotherapy [34] Covalent hybridization/conjugation PDT [35,33] Immunotherapy [36] PFC-based oxygen nanoshuttles Nanodroplets stabilized with lipids or HSA molecules Radiotherapy [37–39] PDT [37,40,41] Encapsulation within a red blood cell membrane “ghost” Radiotherapy [42] PDT [41] Loading with hollow nanoparticles Radiotherapy [43–45] Sonodynamic therapy [46] Fluorinated COPs PDT [47] MOF-based oxygen nanoshuttles UiO-66 MOFs PDT [48] Oxygen precursors CaO2/catalase-encapsulated alginate pellets Chemotherapy [49] H2O2-encapsulated liposomes Radioimmunotherapy [20] Approaches to Oxygen Nanoshuttles Design Hemoglobin, a protein consisting of four subunits each with a heme group and a globin chain, is the oxygen carrier within red blood cells.50 Hemoglobin-based artificial blood substitutes have been extensively explored over the past few decades,31 inspiring development of hemoglobin-based nanoformulations for tumor-targeted oxygen delivery.51 Such nanoformulations show passive tumor homing via the enhanced permeability and retention effect, leading to efficient tumor oxygenation.35,52,53 Cai and co-workers34,36,54 have encapsulated hemoglobin within either poly(lactic-co-glycolic acid) (PLGA) nanoparticles, lipids, or cancer cell membranes and have also formed intermolecular disulfide bonds of hemoglobin and reduced human serum albumin (HSA) to create hybrid hemoglobin-based oxygen nanocarriers. In addition, Zhang and co-workers55 have coated polydopamine-encapsulated hemoglobin with a red blood cell membrane “ghost,” thus achieving man-made red blood cells for tumor-targeted oxygen shuttling that enhances cancer treatment. Another type of widely explored artificial blood substitute is represented by nanoemulsions based on perfluorocarbon (PFC), owing to this compound’s high oxygen-dissolving capacity, low immunogenicity, and excellent physiochemical stability.32 Several research groups, including ours, have designed such PFC nanoemulsions, stabilized with lipids or HSA molecules.37,40 Moreover, to exploit the long circulation time of red blood cells, we and others have designed PFC nanoemulsions encapsulated within red blood cell membrane “ghosts” as stable biomimetic oxygen nanoshuttles.56,57 In addition, various mesoporous nanostructures such as hollow Bi2Se3 or mesoporous organosilica nanostructures perfused with PFC have been demonstrated to be effective in promoting tumor oxygenation for enhanced radiotherapy or sonodynamic therapy.43,44,46 Our group has also developed a type of fluorinated covalent organic polymer (COP) cross-linked with perfluorosebacic acid, thereby enabling efficient PFC loading and tumor oxygenation.47 Hu and co-workers38,58,59 have recently reported that albumin or liposomal nanoformulations loaded with a unique type of PFC, perfluorotributylamine, can inhibit tumor-associated platelets, leading to disruption of tumor vessel barriers and thereby increased red blood cell infiltration, which enables synergistic tumor oxygenation. Metal–organic frameworks (MOFs) are an emerging class of crystalline materials comprised of inorganic metal ion nodes bound via organic linkers. Owing to their characteristic mesoporous structure, MOFs have been extensively explored for gas storage, separation, and sensing, as well as controllable drug delivery in biomedical applications.60,61 Several types of MOFs have recently shown promise as sorbent materials for oxygen storage.62,63 More recently, Gao et al.48 used a MOF nanoplatform based on zirconium(IV), oxygen-storing material, to deliver oxygen to the tumor site via the enhanced permeability and retention effect. Peroxides such as H2O2 can be converted to molecular oxygen in the presence of catalase.64 Thus, nanoshuttles loaded with catalase have been used to catalytically decompose excess endogenous H2O2 within solid tumors and promote tumor oxygenation.17,18 Moreover, recent studies have delivered nanoparticles loaded with catalase either separately or together with oxygen precursors (e.g., H2O2, CaO2), to serve as oxygen-generating depots at tumors.20,49 The next sections will classify currently developed nanoshuttles according to their use for enhancing various cancer treatments: PDT, radiotherapy, chemotherapy, and immunotherapy. Oxygen Nanoshuttles for Enhanced PDT Photodynamic therapy is a promising noninvasive technique for the treatment of many tumor types. This technique uses excitation light and photosensitizers to generate reactive singlet oxygen from molecular oxygen and thus induce cell death.65 However, owing to the severe tumor hypoxia of most solid tumors, current PDT treatment outcomes are still far from satisfactory.2 Early strategies for improving PDT efficacy commonly focused on enhancing tumor accumulation of photosensitizers or synthesizing new photosensitizers with increased singlet oxygen quantum yield and longer excitation wavelength.66 Later efforts have focused on improving intratumoral oxygen concentration, another key component for PDT.29 In 2015, Hu and co-workers40 prepared oxygen self-enriching nanophotosensitizers by coencapsulating the near-infrared (NIR) photosensitizer IR780 and perfluorohexane (PFH) within lipid nanodroplets. PFH has high oxygen-dissolving capacity and thus enriches molecular oxygen for photodynamic consumption by IR780; it also protects the PDT-produced singlet oxygen from decay, thus synergistically improving singlet oxygen quantum yield of IR780 and extending its action time. Following intravenous injection, the PFH nanodroplets were shown to overcome hypoxia-induced resistance to PDT and inhibit tumor growth in mice. We have recently developed fluorinated COPs synthesized via one-pot esterification by cross-linking perfluorosebacic acid and polyethylene glycol with the photosensitizer meso-5,10,15,20-tetra (4-hydroxylphenyl) porphyrin (THPP) (Figure 1).47 Due to the mesoporous structure of perfluorosebacic acid, these THPPpf-PEG nano-COPs showed efficient loading of a PFC variant, perfluoro-15-crown-5-ether (PFCE), enabling molecular oxygen together with THPP. Intravenously injected PFCE-loaded THPPpf-PEG enhanced PDT in tumor-bearing mice, probably due to nanoparticle absorbance of oxygen when passing through the lungs, and its gradual release upon tumor accumulation, which attenuated tumor hypoxia. Figure 1 | Long circulating fluorinated nano-COPs for tumor-targeted oxygen delivery and enhanced PDT. (a) Schematic illustration of the synthesis process of fluorinated COPs and subsequent PFCE loading. (b) Scheme illustrating the tumor oxygenation process, during which PFCE-loaded THPPpf-PEG would efficiently absorb oxygen when passing through the lungs and then gradually release the loaded oxygen when accumulated inside tumors to endow efficient tumor hypoxia attenuation. (c) Photoacoustic imaging of tumors upon mice that received systemic administration of plain THPPpf-PEG and PFCE-loaded THPPpf-PEG at indicated time points post injection. (d) Ex vivo immunofluorescence staining of tumor slices of tumor-bearing mice that received systemic administration of plain THPPpf-PEG and PFCE-loaded THPPpf-PEG at varying time points using external pimonidazole as the hypoxia probe. (e) In vivo tumor growth curves of mice after various treatments, as indicated. Originally published by Tao and co-workers47 and approved for reuse by John Wiley & Sons, Inc. Download figure Download PowerPoint Other types of oxygen nanoshuttles formulated with hemoglobin, or zirconium(IV)-based MOFs, have also been shown to enhance the photodynamic effects of coloaded photosynthesizers (e.g., chlorin e6 [Ce6] or indocyanine green).48,33 Gao et al.48 reported that the zirconium(IV)-based MOFs, which exhibited high loading capacity toward both oxygen and indocyanine green (ICG) molecules, showed burst release of oxygen inside tumors upon NIR light exposure, thereby significantly improving the photodynamic efficacy of ICG in the hypoxic regions. Therefore, elevating oxygen concentrations inside tumors is a promising strategy for enhancing PDT treatment outcomes. Oxygen Nanoshuttles for Enhanced Radiotherapy Radiotherapy, either by external radiation (e.g., via X-rays, γ-rays, electrons) or by internal radiation with implantable radioactive isotopes (brachytherapy), is a well-known cancer treatment modality generally used alongside surgery and chemotherapy.67 Molecular oxygen, an extremely electron-affinic molecule, fixates the DNA damage induced by radiation, which leads to tumor cell apoptosis.68 Thus, tumor oxygen concentrations have a crucial role in the therapeutic outcome of radiotherapies.69 Over the past decades, clinical trials have made different attempts to address resistance to radiotherapy caused by tumor hypoxia, for example, via hyperbaric oxygen breathing.70 More recently, we prepared HSA-stabilized PFC nanodroplets for attenuating tumor hypoxia and thus enhanced radiotherapy (Figure 2a).37 Following intravenous injection of the PFC nanodroplets, tumors in mice under hyperbaric oxygen breathing presented significantly improved oxygenation, as visualized with photoacoustic imaging. Simultaneously, tumor-localized ultrasound stimulation was performed to boost release of the loaded oxygen and of oxygen adsorbed by the nanodroplets when circulating through the lungs. This approach led to efficient tumor hypoxia attenuation, enhancing therapeutic efficacy of both X-ray-based radiotherapy and Ce6-mediated PDT, in different tumor xenograft models. Figure 2 | PFC-based oxygen nanoshuttles for stimuli-responsive tumor oxygenation and enhanced PDT and radiotherapy. (a) Schematic illustration showing tumor-bearing mice that received intravenous injection of HSA-stabilized PFC nanodroplets followed by hyperbaric oxygen breathing and tumor-localized ultrasound stimulation. Treatments attenuated tumor hypoxia and enhanced response to both PDT and radiotherapy. The figure was originally published by Song et al.37 and was approved for reuse by ACS Publishing group. (b) Preparation of PFC-loaded hollow Bi2Se3 nanoparticles for NIR light-responsive tumor oxygenation and enhanced cancer radiotherapy. The figure was originally published by Song et al.44 and was approved for reuse by John Wiley & Sons, Inc. Download figure Download PowerPoint Another factor that severely impairs radiotherapy treatment outcome is limited absorption cross-section of radiation beams.62 Therefore, an alternative radiosensitization strategy is to deposit high Z-element-containing materials (e.g., Au) within solid tumors.17,69 Our group has prepared hollow Bi2Se3 nanostructures, via a facile one-pot cation exchange method and Kirkendall effect using small MnSe nanoparticles as a template, and loaded with PFH under reduced pressure, to serve as oxygen nanoshuttles.44 Following intratumoral injection of the particles to tumor-bearing mice and treatment with a 808 nm laser, the strong NIR absorbance of Bi2Se3 elevated the local temperature over the evaporation point of PFH, thereby modulating the oxygen release rate and promoting tumor oxygenation (Figure 2b). This remotely controllable NIR-induced tumor oxygenation, together with the capacity of Bi as a high Z element for radiosensitization, yielded a synergistically enhanced antitumor effect. Long, circulating PFC-based nanoshuttles, such as red blood cell membrane “ghosts” enveloping [email protected] nanoparticles,42 were shown to attenuate tumor hypoxia in mice due to efficient tumor accumulation, and without requiring external stimulation, ultimately enhancing cancer radiotherapy. These studies reveal that tumor oxygenation is a promising strategy for radiotherapy sensitization. Oxygen Nanoshuttles for Enhanced Chemotherapy The treatment outcome of chemotherapy, another widely used treatment in the clinic, also correlates with intratumoral oxygen concentrations.71 Tumor hypoxia has a detrimental effect on delivery of chemodrugs to distant hypoxic zones and on their subsequent cellular uptake, and also impairs the generation of reactive oxygen species from molecular oxygen by several chemo drugs (e.g., doxorubicin [DOX], oxaliplatin), which hinders their ability to induce cell death.72 Moreover, tumor hypoxia induces proteomic and genomic adaptations causing tumor cells to develop into more malignant phenotypes, which can lead to more severe chemoresistance.72,73 A recent pioneering study showed that improved tumor oxygenation with hyperbaric oxygen breathing increases sensitization of hypoxic cells to DOX and other chemodrugs.74 In a recent work by Sung and co-workers49, the authors prepared an implantable oxygen-generating depot by adding an alginate solution containing the oxygen precursor, CaO2, and catalase to a CaCl2 bath, which yielded Ca2+-cross-linked microencapsulated pellets. Upon implantation close to the tumor region, the CaO2 reacts with water that infiltrates the pellet from interstitial tissues, forming calcium hydroxide and H2O2; the latter, via coencapsulated catalase, decomposes to produce molecular oxygen. As a result, these tumor-localized, oxygen-generating depots increase response to intravenously administered DOX. For more effective and tumor-targeted oxygen delivery, Cai and co-workers75 recently constructed tumor-homologous targeting oxygen nanoshuttles, by loading PLGA-encapsulated hemoglobin and DOX within tumor cell membranes (Figure 3). Due to the tumor cell adhesion molecules upon their surface, these nanoshuttles increased tumor homing efficiency following intravenous injection, leading to effective tumor hypoxia attenuation. This, in turn, led to downregulation of hypoxia-inducible factor-1α, multidrug resistance gene 1, and P-glycoprotein, important factors involved in exocytosis of DOX; thus, these nanoshuttles increased the antitumor efficacy of chemotherapy in mice. Figure 3 | Hemoglobin-based oxygen nanoshuttles for tumor oxygenation and enhanced chemotherapy. (a) Scheme showing preparation of DOX/Hb loaded PLGA-cancer cell membrane nanoparticles (DHCNPs). (b) Scheme showing utilization of the DHCNPs for effective tumor oxygenation via homologous targeting and subsequent downregulation of HIF-1α, MDR1, and P-gp for enhanced chemotherapeutic efficacy of DOX. (c) Ex vivo immunofluorescence hypoxia staining of tumor slices collected from mice at 24 h post intravenous injection of DHCNPs. (d) P-gp expression levels of tumors with or without oxygenation treatment, evaluated by Western blotting assay. (e) and (f) Tumor growth curves and survival rates of tumor-bearing mice post various treatments as indicated. The figure was originally published by Tian et al.75 and has been approved for reuse by John Wiley & Sons, Inc. Download figure Download PowerPoint Thus, oxygen nanoshuttles that attenuate tumor hypoxia facilitate safe and efficient chemotherapy of intrinsically chemoresistant solid tumors. Oxygen Nanoshuttles for Enhanced Cancer Immunotherapy Immunotherapy is a promising cancer treatment modality, utilizing the host immune system for potent systemic attack of metastatic tumors.76 Over the past several years, we have witnessed the success of cancer immunotherapies, including chimeric antigen receptor T-cell immunotherapy, as well as immune checkpoint blockade using anti-PD-1/PD-L1 and anti-CTLA-4 antibodies, for the treatment of a wide range of cancers.76–78 However, accumulating clinical data show that chimeric antigen receptor T-cell immunotherapy has limited therapeutic efficacy for treatment of solid tumors, and that checkpoint blockade also shows an average objective response rate of ∼20% in most solid tumors.79 Preclinical and clinical evidence indicate that tumor hypoxia may induce an immunosuppressive TME that limits T-cell infiltration within the hypoxic zones. In addition, the immunosuppressive TME features in remarkably elevated populations of regulatory T cells, M2 macrophages, myeloid-derived suppressor cells, and myofibroblasts, which prevent tumor cells attack by natural killer and T cells during tumor progression and after chemotherapy or immunotherapy.80 Recently, various nanoparticle-based strategies have also been examined for modulating and diminishing the immunosuppressive TME, and thus enhance immunotherapy.24,25,81 Photodynamic therapy induces release or surface exposure of damage-associated molecular patterns by dying tumor cells, such as ATP, calreticulin, and high-mobility group protein B1.82 These patterns prime the host immune system to generate a systemic attack against residual tumor cells, by promoting maturation of dendritic cells, and subsequent activation of effector T cells and natural killer cells.83 However, this immune priming process is disabled by the hostile TME.83 In recent work by Cai and co-workers,36 a hybrid oxygen nanoshuttle was prepared by cross-linking hemoglobin and reduced HSA, encapsulating Ce6—a photosensitizer that produces singlet oxygen—and subsequently oxygenating the nanoparticles. These Ce6 and oxygen-carrying nanoshuttles enhanced PDT against primary tumors and improved the antitumor immune response against distant tumors and lung metastasis, via enhanced maturation of dendritic cells and activation of effector T cells and natural killer cells. Radiotherapy also induces immunogenic cell death following treatment of tumors.82 We recently found that sequential administration of liposomal catalase and liposomal H2O2 promotes tumor oxygenation of murine breast and colon xenografts, and a patient-derived prostate cancer xenograft20 (Figure 4a–e). Tumor oxygenation reversed the immunosuppressive TME by reducing the M2 macrophage population and improving CD8+ T-cell infiltration. Combining systemic administration of anti-CTLA-4 further enhanced the efficacy of tumor-oxygenation-boosted radiotherapy (Figure 4c–e). Figure 4 | Self-supplied tumor oxygenation with sequential injection of liposomal catalase and H2O2 for enhanced radioimmunotherapy. (a) Scheme showing preparation and mechanism of sequentially injected liposomal catalase and H2O2 for tumor hypoxia attenuation and reversal of immunosuppressive TME, resulting in enhanced cancer radioimmunotherapy. (b) Therapeutic timeline of radioimmunotherapy via sequential injection of liposomal catalase and H2O2, X-ray exposure, and injection of anti-CTLA-4 at indicated time points. (c)–(e) Tumor growth curves, tumor photographs, and average tumor weights after various treatments as indicated. The figure was originally published by Song et al.20 and has been approved for reuse by ACS Publishing group. Download figure Download PowerPoint Therefore, tumor oxygenation is a promising strategy for reversing the immunosuppressive TME and enhancing cancer immunotherapy. Conclusion and Perspectives In this review, we summarized the latest progress in design and fabrication of innovative tumor-targeted oxygen-delivery nanoshuttles. Such dedicated oxygen nanoshuttles were able to efficiently attenuate tumor hypoxia in mouse tumor models, leading to enhanced treatment outcome of oxygen-consuming cancer therapeutics. Moreover, treatment with such oxygen nanoshuttles could reverse the immunosuppressive TME and boost the immune system for enhanced cancer immunotherapy. Thus, oxygen nanoshuttles to promote cancer treatment hold great potential for future clinical translation. However, similar to preceding versions of artificial blood substitutes, concerns regarding biocompatibility of oxygen nanoshuttles remain a prominent obstacle to clinical translation. Circulation half-life of current oxygen nanoshuttles, for sustained oxygenation of primary tumors and the TME, is another key parameter that requires improvement. Tumor hypoxia is associated with tumor acidity, dense extracellular matrix, and high interstitial pressure, which collectively promote the immunosuppressive TME that disables tumor immune surveillance. Therefore, although tumor oxygenation enhances response to therapy, full reversal of therapeutic resistance requires concurrent modulation of other hostile features. Developing multifunctional oxygen nanoshuttles to enable multifaceted TME modulation is a promising future direction for promoting precision cancer nanomedicine. Acknowledgment This work was partially supported by the National Research Programs from Ministry of Science and Technology (MOST) of China (2016YFA0201200), the National Natural Science Foundation of China (51525203, 51761145041, and 51802209), the Natural Science Foundation of Jiangsu Province (BK20180848), the China Postdoctoral Science Foundation (2017M610348 and 2018T110545), Collaborative Innovation Center of Suzhou Nano Science and Technology, the 111 Program from the Ministry of Education of China, a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, the Joint NSFC-ISF Research Grant no. 2533/17, by the council for higher education postdoctoral fellowship for outstanding woman in Science for Oshra Betzer, and the industry-academy postdoctoral scholarship (3-15677) from the Ministry of Science, Technology & Space Israel for Oshra Betzer.