Development of shape-tunabe metal-organic framework nanosystems for cancer immunotherapy

Universidade do Minho Escola de Engenharia Inês Adelaide Mota Cardoso Development of shape-tunable MetalOrganic Framework nanosystems for cancer immunotherapy Outubro de 2023 UMinho | 2023 Inês Adelaide Mota Cardoso Development of shape-tunable Metal-Organic Framework nanosystems for cancer immunotherapy Universidade do Minho Escola de Engenharia Julho de 2023 Universidade do Minho Escola de Engenharia University of Groningen University Medical Center Groningen Outubro de 2023 Inês Adelaide Mota Cardoso Development of shape-tunabe Metal-Organic Framework nanosystems for cancer immunotherapy Dissertação de Mestrado Mestrado Integrado em Engenharia Biomédica Mestrado em Engenharia Clínica Trabalho efetuado sob a orientação do Professora Doutora Lígia Raquel Marona Rodrigues Universidade do Minho Professor Doutor Hélder Almeida Santos University Medical Center Groningen ii D ireitos de A utor e C ondições de U tilização do T rabalho por T erceiros Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ iii A cknowledgments Firstly, I would like to express my deepest gratitude to my supervisor in Groningen, Professor Doctor Hélder Almeida Santos, who always provided me with exceptional mentorship. His contributions and discussions enriched my knowledge and made this research experience rewarding. Secondly, I would like to acknowledge my supervisor in Portugal, Professor Doctor Lígia Rodrigues, and the Ph.D. students in Santos’ Lab, Han Gao, and Ruoyu Cheng, for all the support and invaluable guidance throughout this journey. I am genuinely grateful for your patience, availability, and dedication. I would also like to thank the entire research group, as well as all the lab members and technicians for their valuable insights, camaraderie, and collaborative spirit. I’ll be forever grateful and proud to work with such professional persons. I want to extend my heartfelt gratitude to Maria, Micaela, and Soraia, my Erasmus family. I’m sure that we became way more than only colleagues at work, we became friends. From all the help that you gave me inside the lab to all the moments of happiness and friendship we spent outside of it, I’m forever grateful for that. Catarina, you also deserve my words of gratitude. This experience changed my life, but it would be nothing without you by my side during those six months. It was amazing to spend that time with you. Thank you for all the times that you supported and understood me, and for all the good times we spent together far away from our home, you were my rock. It’s impossible to forget the wonderful friends I have made throughout the last five years who have welcomed me into their lives and made my own a lot brighter. Margarida, Manuel, Tiago, and Pedros, this journey would not have been possible without you. Mom and Dad, I’m extremely grateful for all the love and support given to me for every decision I made so far and for providing me with everything I needed. There are no words to express how much it meant to me to have you by my side. I’m also very much thankful to my sister, Ana, for being present in all of my best and worst moments and for being a constant source of inspiration. To my lovely nephews, Manuel and José Rui, you are my inspiration to achieve greatness. Your love is what has gotten me through when I wanted to give up. Rui, thanks for your friendship, patience, comprehension, and love. You were crucial for both my academic and personal development, cheering me up when I was feeling down and celebrating each of my accomplishments. iv Thanks, Groningen, you were not just a house, you became a home. Lastly, I would like to give a special thanks to everyone else who, in any way was a part of this journey. You won’t ever be forgotten. “What we know is a drop, what we don’t know is an ocean.” − Isaac Newton v S tatement of I ntegrity I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. vi R esumo Título: Desenvolvimento de nanossistemas de Estrutura Metalo-Orgânica com forma ajustável para imunoterapia do cancro O microambiente tumoral (TME) desempenha um papel crítico enquanto nicho ecológico essencial para o desenvolvimento e progressão do tumor, essencialmente devido à alta prevalência de células imunossupressoras, sendo os macrófagos associados a tumores (TAMs) os principais mediadores envolvidos no processo patológico do cancro. Os TAMs podem ser divididos em dois estados de polarização antagónicos: os macrófagos M1, próinflamatórios, e os M2, anti-inflamatórios. De acordo com as suas funções, os TAMs presentes no TME são geralmente classificados como macrófagos do tipo fenotípico M2. Nos últimos anos, mais atenção tem sido direcionada para a reestruturação e coordenação de um TME inflamatório, e os TAMs surgiram como um paradigma para a compreensão da ligação entre a inflamação e o cancro. Uma extensão dessa perspetiva terapêutica reuniu esforços destinados a repolarizar os TAMs, induzindo a alteração do fenótipo M2 para o fenótipo M1. Em resposta a vários estímulos do microambiente, os macrófagos podem adotar diferentes fenótipos e funções. As nanopartículas de estrutura metalo-orgânicas (MOF NPs) têm sido amplamente utilizadas como sistemas controlados de libertação de fármacos e agentes terapêuticos para o cancro, devido às suas excelentes propriedades intrínsecas, tais como a alta capacidade de ajuste da sua forma. No entanto, a resposta imune celular modulada pelo ajuste da forma das NPs ainda não está clara. Neste estudo, quatro formas diferentes de MOF NPs de cobre foram sintetizadas (agulha, hexágono, quadrado e esférica) através do ajuste das condições de síntese, seguindo-se uma avaliação sistemática das suas propriedades físico-químicas, biocompatibilidade e respetiva capacidade de reprogramação imunológica. As diferentes formas das Cu MOF NPs apresentaram respostas imunes distintas. As NPs em forma de quadrado e agulha promoveram a polarização dos macrófagos para o fenótipo M1, enquanto as NPs com forma esférica e hexagonal melhoraram o perfil de expressão de biomarcadores de macrófagos tipo-M2. De um modo geral, este estudo oferece insights práticos sobre as interações entre as NPs e as células imunes, o que pode ter potenciais implicações na imunoterapia antitumoral baseada em NPs. Palavras-chave: Imunoterapia do Cancro; Estrutura Metalo-Orgânica; Nanopartículas com forma ajustável; Sistema Imunitário; Polarização de Macrófagos. vii A bstract Title: Development of shape-tunable Metal-Organic Framework nanosystems for cancer immunotherapy The tumor microenvironment (TME) plays a crucial role as an essential ecological niche for the onset and progression of cancer, the high prevalence of immunosuppressive cells, with tumor-associated macrophages (TAMs), are major contributing mediators in the pathological process of tumor. TAMs can be divided into two antagonistic polarization states: pro-inflammatory M1 and anti-inflammatory M2 macrophages. Consistent with their functions, TAMs are typically classified as M2-like macrophage phenotypes in the TME. Over the past few years, more attention has been focused on how to restructure and coordinate an inflammatory TME, and TAMs have emerged as a paradigm for understanding the link between inflammation and cancer. An extension of this therapeutic perspective has led to efforts aimed at repolarizing TAMs, shifting them from the M2-like macrophage phenotype to the M1 phenotype. In response to various microenvironmental stimuli, macrophages can adopt different phenotypes and functions. Metal-organic framework nanoparticles (MOF NPs) have been widely used as controlled drug delivery systems and cancer therapy agents due to their intrinsic superior properties, such as a highly structure-tunable capability. Yet, the cellular immune response modulated by tuning the shape of NPs remains unclear. Here, four distinct Cu MOF NPs with different shapes (needle, hexagon, square, and spherical) were synthesized by tuning the synthetic conditions, followed by systematically evaluating the physicochemical properties, biocompatibility, and immune-reprogramming capabilities. Cu MOF NPs with different shapes showed distinct immune responses. Square and needleshaped NPs promoted macrophage polarization into M1 phenotype, whereas spherically and hexagonally shaped NPs enhanced the expression profile of M2 biomarkers. Overall, this study offers practical insights into the interface interactions between NPs and immune cells, which might have potential implications for NP-based immunotherapy. Keywords: Cancer Immunotherapy; Metal-Organic Framework; Shape-tunable nanoparticles; Immune response; Macrophage Polarization. xiv FIGURE 12. TEM-EDX ANALYSIS OF CU MOF NPS WITH DIFFERENT SHAPES: (A) HEXAGON, (B) NEEDLE, (C) SQUARE, AND (D) SPHERICAL SHAPES. ....................................................................................... 38 FIGURE 13. IN VITRO CELL VIABILITY ASSAY (ALAMARBLUE) OF RAW 264.7 CELLS TREATED WITH (A) SQUARE, (B) NEEDLE, (C) SPHERICAL, AND (D) HEXAGON NPS. RAW 264.7 CELLS WERE TREATED WITH VARIOUS CONCENTRATIONS OF CU MOF NPS FOR 24 H AND 48 H. THE DASHED LINE DEFINES THE CYTOTOXICITY RANGE: NON-CYTOTOXICITY > 80%. THE DATA ARE PRESENTED AS MEANS ± SD (N=4). (*P<0.05; *P<0.01; ****P<0.0001). .................................................................................................. 39 FIGURE 14. IN VITRO CELL VIABILITY ASSAY (ALAMARBLUE) OF RAW 264.7 CELLS TREATED WITH EACH COMPONENT USED IN THE SYNTHESIS OF CU MOF NPS: (A) COPPER NITRATE AS METAL ION (B) 2-MIM AS MODULATOR (C) H4BTC AS LIGAND (D) OCTANOIC ACID AS CAPPING AGENT. THE DASHED LINE DEFINES THE CYTOTOXICITY RANGE: NON-CYTOTOXICITY > 80%. THE DATA ARE PRESENTED AS MEANS ± SD (N=4). (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). ............................................................ 41 FIGURE 15. IN VITRO CELL VIABILITY ASSAY (ALAMARBLUE) OF THP-1 CELLS TREATED WITH (A) SQUARE NPS, (B) NEEDLE NPS, (C) SPHERICAL NPS, AND (D) HEXAGON NPS. THP-1 CELLS WERE TREATED FIRST WITH PMA (100 NM) FOR 24 H AND THEN WITH VARIOUS CONCENTRATIONS OF CU MOF NPS FOR 24 H AND 48 H. THE DASHED LINE DEFINES THE CYTOTOXICITY RANGE: NON-CYTOTOXICITY > 80%. THE DATA ARE PRESENTED AS MEANS ± SD (N=4). (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). ............ 42 FIGURE 16. IN VITRO CELL VIABILITY ASSAY (ALAMARBLUE) OF HELA CELLS TREATED WITH (A) SQUARE NPS, (B) NEEDLE NPS, (C) SPHERICAL NPS, AND (D) HEXAGON NPS. HELA CELLS WERE TREATED WITH VARIOUS CONCENTRATIONS OF CU MOF NPS FOR 24 H AND 48 H. THE DASHED LINE DEFINES THE CYTOTOXICITY RANGE: NON-CYTOTOXICITY > 80%. THE DATA ARE PRESENTED AS MEANS ± SD (N=4). (**P<0.01; ***P<0.001; ****P<0.0001). ............................................................................................ 44 FIGURE 17. IN VITRO CELL VIABILITY ASSAY (ALAMARBLUE) OF HEK 293 CELLS TREATED WITH (A) SQUARE NPS, (B) NEEDLE NPS, (C) SPHERICAL NPS, AND (D) HEXAGON NPS. HEK 293 CELLS WERE TREATED WITH VARIOUS CONCENTRATIONS OF CU MOF NPS FOR 24 H AND 48 H. THE DASHED LINE DEFINES THE CYTOTOXICITY RANGE: NON-CYTOTOXICITY > 80%. THE DATA ARE PRESENTED AS MEANS ± SD (N=4). (**P<0.01; ***P<0.001; ****P<0.0001). ........................................................................... 45 FIGURE 18. RAW 264.7 MACROPHAGES UNTREATED (A). EFFECTS OF (B) HEXAGON (C) SQUARE (D) SPHERICAL AND (E) NEEDLE MOF NPS ON RAW 264.7 MACROPHAGE CELL MORPHOLOGY AFTER 1 DAY OF INCUBATION WITH DIFFERENT CU MOF NPS. WHITE ARROWS POINT TO M1 MACROPHAGES AND RED ARROWS INDICATE M2 MACROPHAGE. IMAGES WERE OBTAINED USING AN INVERTED LIGHT MICROSCOPY xv UNDER PHASE CONTRAST. ALL THE MICROGRAPHS IN THIS FIGURE WERE TAKEN AT THE SAME MAGNIFICATION (20). .......................................................................................................... 47 FIGURE 19. RAW 264.7 MACROPHAGES UNTREATED (A). EFFECTS OF (B) HEXAGON (C) SQUARE (D) SPHERICAL AND (E) NEEDLE MOF NPS ON RAW 264.7 MACROPHAGE CELL MORPHOLOGY AFTER 2 DAYS OF INCUBATION WITH DIFFERENT CU MOF NPS. WHITE ARROWS POINT TO M1 MACROPHAGES. IMAGES WERE OBTAINED USING AN INVERTED LIGHT MICROSCOPY UNDER PHASE CONTRAST. ALL THE MICROGRAPHS IN THIS FIGURE WERE TAKEN AT THE SAME MAGNIFICATION (20). .................................................. 48 FIGURE 20. HISTOGRAMS DEPICTING FOLD CHANGE IN RAW 264.7 CELLS GENE EXPRESSION, DETERMINED BY QUANTITATIVE REAL-TIME PCR ANALYSIS, AFTER 1 (A) AND 2 (B) DAYS OF TREATMENT WITH CU MOF NPS. AN UNTREATED CONDITION (RAW 264.7 CELLS WITHOUT CU MOF NPS STIMULATION) WAS USED TO DETERMINE THE RELATIVE EXPRESSION, AND THE TRANSCRIPT EXPRESSION OF TARGET GENES (CD86, CD206, ARG1) WAS NORMALIZED TO THE EXPRESSION OF ENDOGENOUS HOUSEKEEPING GENE GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAPDH). ...................................................... 49 FIGURE 21. ELISA RESULTS OF CYTOKINE PRODUCTION BY RAW 264.7 CELLS: (A) TNF-Α AND (B) IL-12, BOTH PRO-INFLAMMATORY CYTOKINES; (C) IL-10 AND (D) IL-4 BOTH ANTI-INFLAMMATORY CYTOKINES. THE DATA ARE PRESENTED AS MEANS ± SD (N=3). (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). .......................................................................................................................................... 52 FIGURE 22. THEORETICAL PREDICTIONS OF UPTAKE EFFICIENCY OF SPHERICAL AND NON-SPHERICAL CU MOF NPS (HEXAGON, SQUARE, AND NEEDLE) DETERMINED BY THEIR SHAPE. CREATED WITH BIORENDER.COM. ..... 54 FIGURE 23. SCHEMATIC ILLUSTRATION OF THE SHAPE EFFECT OF CU MOF NPS ON IMMUNE RESPONSE. CREATED WITH BIORENDER.COM. ......................................................................................................... 55 FIGURE 24. MULTICOLOR FLOW CYTOMETRY PANEL TO IDENTIFY M1 AND M2 MACROPHAGES. FLOW CYTOMETRY ANALYSIS OF CD206 AND CD86 EXPRESSION OF RAW 264.7 CELLS TREATED WITH (A) SQUARE, (B) NEEDLE, (C) HEXAGON, AND (D) SPHERICAL CU MOF NPS AFTER 1 DAY AND 2 DAYS INCUBATION. CD206 WAS USED TO IDENTIFY M2 MACROPHAGES AND CD86 WAS USED TO IDENTIFY M1 MACROPHAGES. CELLULAR DEBRIS AND EVENTS TOO SMALL TO BE CELLS WERE EXCLUDED FROM ALL EVENTS. SAMPLES WERE INITIALLY GATED ON SINGLE CELLS USING FSC-H/FSC-A GATING, THEREBY EXCLUDING DOUBLES (DATA NOT SHOWN). FROM THE LIVE MACROPHAGE POPULATION, THE CELLS WERE FURTHER SEPARATED BASED ON CD206 AND CD86 EXPRESSION. THEN, THE PERCENTAGES OF CD206+ AND CD86+ POPULATIONS WERE CALCULATED.............................................................................................. 57 xvi L ist of T ables TABLE 1. VOLUMES OF 2-MIM AND H2O ADDED TO PREPARE THE LIGAND SOLUTION. ............................... 20 TABLE 2. VOLUMES OF CU(NO3)2 ADDED TO THE REACTION MIX ACCORDING TO THE MOLAR RATIO ESTABLISHED. .......................................................................................................................................... 21 TABLE 3. PRIMERS OF GENES USED IN THE QRT-PCR ANALYSIS. .............................................................. 28 TABLE 4. REACTION PROTOCOL USED FOR CDNA SYNTHESIS. ................................................................. 28 TABLE 5. CU MOF NPS SIZES, ZETA POTENTIAL, AND CONCENTRATIONS FROM NTA. DATA ARE PRESENTED AS MEANS ± SD......................................................................................................................... 36 1 1. I ntroduction 1.1 Context and Motivation In recent years, immunotherapy has gradually become a new standard therapy for cancer, since it can stimulate the immune system to recognize and attack tumor cells. However, the therapeutic effect of most immunotherapy strategies can be weakened as a result of the immunosuppressive tumor microenvironment (TME), which is caused by the presence of immunosuppressive cells and tumor-associated macrophages (TAMs) account for the highest proportion. TAMs usually display the tumor promoting M2 phenotype rather than the tumoricidal M1 phenotype. However, in response to various stimuli in TME, the TAM population shows a state of constant transition between these two major forms, M1 and M2-like phenotypes, that exhibit different functions. Therefore, in the last few years, more attention has been given on how to promote the polarization of TAMs to the M1 type. Current clinical trials use bacterial cell wall components, lipopolysaccharide (LPS) or muramyl dipeptide (MDP), and cytokines like interferon- (IFN-) and granulocytemacrophage colony-stimulating factor (GM-CSF) to activate macrophages towards M1 phenotype as a novel immunotherapeutic approach. However, the short duration of action of IFN-γ and MDP, primarily due to their rapid clearance, along with the challenge of systemic administration of LPS without inducing toxicity in vivo, have collectively hindered their progression as viable clinical alternatives. To overcome this hurdle, a nanoparticle (NP)- based strategy can provide a viable alternative to immunomodulatory therapies. For instance, metal-organic framework (MOF) nanoparticles provide a possible solution to overcome the above challenge. MOF nanoparticles consist of crystalline hybrid materials built up from the coordination between metal (transition metal or lanthanide metal) ions and organic ligands (carboxylates, azolates, and phosphonates). Furthermore, MOF nanoparticles have emerged as promising candidates for biomedical applications due to their low toxicity, structural flexibility, and safe biodegradation in a physiological environment. However, the biological effects of nanoparticles are largely dictated by their unique physicochemical properties, for example, chemical composition, shape, size, and surface properties. In fact, previous studies have shown an effect of NP shape on modulating macrophage phagocytosis, revealing it to be an important parameter for regulating the immune response. Thus, investigating how the shape of NPs affects macrophage 2 polarization, can aid the optimal NPs design to elicit a robust immune response and, consequently, provide new solutions for treating cancer and other types of diseases. 1.2 Aim of the Work The main objective of the present study was to investigate the impact of shape-tunable MOF NPs on modulating macrophage phenotype. For this purpose, Cu-based MOF NPs with different shapes have been fabricated, characterized, and further incubated with macrophages to evaluate their capability on polarizing macrophage phenotypes. The specific aims of this thesis were: i. To study the effect of the synthesis conditions of Cu MOF NPs on their physicalchemical properties. ii. To evaluate the biocompatibility of Cu MOF NPs by using different cell lines. iii. To evaluate the polarization effect of Cu MOF NPs on immune cells. 1.3 Project Outline This thesis includes four chapters, namely the State of the Art, Materials and Methods, Results and Discussion, and finally Main Conclusions. The State of Art (chapter 2) describes the current advances of NPs for biomedical applications and cancer immunotherapy. The Materials and Methods (chapter 3) is composed of a brief summary of methods, equipment, and analytical software used during the project development. Chapter 4 presents the results and respective discussion in this study. Finally, chapter 5, highlights the major conclusions and emphasizes limitations and future perspectives. 3 2. S tate of the A rt 2.1 Nanotechnology in Medicine Nanotechnology has introduced innovative and promising approaches for manipulating various diseases in contrast to conventional therapies. Traditionally, nanotechnology includes materials, devices, and systems with the size of 1 to 100 nm, but many nanomedicines are within the submicron size of 100-1000 nm (Pillai et al., 2013). These nanoparticles are regarded as nano-sized tools for the diagnosis, prevention, monitoring, and treatment of various diseases (Astruc, 2016). For instance, nanotechnology solutions have been shown to be beneficial for the treatment of neurodegenerative (Silva Adaya et al., 2017), cardiovascular (Karimi et al., 2016; Kratz et al., 2016), and autoimmune diseases (Gharagozloo et al., 2015), osteoporosis (Ahmad et al., 2022; Barry et al., 2016; MoraRaimundo et al., 2017) and against bacterial infections (Hajipour et al., 2021; Yeh et al., 2020). Indeed, the more prominent applications of nanotechnology in medicine are in imaging techniques, tissue-engineered constructs, implants, and drug delivery systems, particularly in the field of cancer therapy (Zhang et al., 2023). To date, a set of nanomaterials has been developed and approved for clinical use (Sim et al., 2021). 2.1.1 Nanoparticles for Biomedical Applications Nanoparticles (NPs) have been tremendously adopted in important biomedical applications such as imaging, drug delivery, and immunotherapy (Guo et al., 2021). For example, gold NPs decorated with a prostate-specific membrane antigen RNA aptamer have been shown a higher computed tomography (CT) density for prostate cancer cell imaging (Kim et al., 2010). Moreover, super-paramagnetic iron oxide nanoparticle (SPIONs) surfaces decorated with a high-affinity anti-EGFR antibody have been shown to target lung tumors by MRI imaging (Wang et al., 2017). Besides imaging, one of the most promising applications of NPs is the possibility to specifically target their content, delivering small drugs as well as macromolecules like proteins, peptides, or genes, and exhibiting improved drug bioavailability, uptake of low solubility drugs and decreased toxicity (Sim et al., 2021). For example, metallic NPs can be conjugated with a fluorescent marker for drug delivery and imaging in magnetic resonance (Khodabandehloo et al., 2016; Mirza et al., 2014). Cationic liposomal systems are also 4 employed in drug delivery because of their capacity to transport charged structures, such as DNA and RNA, via the electrostatic interactions between the positively charged phospholipids and the negatively charged nucleic acids (Sercombe et al., 2015). Other hydrophilic polymers, such as dextran-based polysaccharide, consists of hydrophilic and neutral chains at the NPs surface, repelling plasma proteins (Al-Musawi et al., 2020). For cancer immunotherapy, NPs play a vital role in activating the host’s immune system. Accordingly, different types of NPs have been and are being widely used in cancer immunotherapy, such as polymeric NPs (Li et al., 2018), liposomes (Yoshizaki et al., 2017), mesoporous silica NPs (Thakur et al., 2020), micelles (Zeng et al., 2017), and nanosized metal-organic frameworks (Ni et al., 2020) (Figure 1). Figure 1. Overview of commonly used nanoparticle types, classified as organic, inorganic or carbonbased structures. Created with BioRender.com. 2.1.1.1 Metal-Organic Framework Nanoparticles Metal-organic framework nanoparticles (MOF NPs) are one type of NPs that have gained considerable relevance in preclinical and clinical settings, including gas storage and separations (Venna et al., 2015), chemical sensing (Kreno et al., 2012), catalysis (Dhakshinamoorthy et al., 2018), drug delivery (Zhuang et al., 2014), among others. MOF NPs consist of crystalline hybrid materials built up from the coordination between metal (transition metal or lanthanide metal) ions and organic ligands (carboxylates, azolates, and phosphonates) (Wang et al., 2018) (Figure 2). MOF NPs have unique properties that cannot be found in organic or inorganic systems due to their hybrid nature, namely their consistency in physicochemical properties unrivaled by conventional structures (Burtch et al., 2018; Li et al., 2022). Indeed, MOF NPs exhibit controlled surface chemistry, unparalleled 5 surface areas, pore volumes, and reversible structural flexibility (Sun et al., 2020), allowing the efficient prediction of their physiological metabolism and functions in vivo (Li et al., 2022). Then, these unique properties of MOF NPs, including their porous structure, large surface area, flexibility of the coordination between organic ligands and nodes, and inherent photo-responsive features provide significant advantages for a wide range of biological applications, including cancer phototherapy and immunotherapy (Fernandes et al., 2023). Moreover, these properties enable the development of suitable MOF-based systems that can be modified for a variety of therapeutic purposes while ensuring good compatibility (Liu et al., 2022; Yang et al., 2020). MOF NPs can also be synthesized with functional nodes and linkers (such as peptides and fluorescence) that possess inherent antitumor and photosensitizer properties (Gao et al., 2021), acting directly as immunotherapy agents. Even without extra loading of functional drugs, the ligands or metal ions in MOF NPs can also be employed for immune activation or biological imaging (Lan et al., 2018). MOF NPs have been designed and functionalized as on-demand therapeutics that are activated by either external energy stimuli or endogenous triggers to generate reactive oxygen species (ROS), inducing immunogenic cell death of tumor cells (Ni et al., 2020). Thus, the flexible topological structure, large specific surface area, high porosity, and multiple encapsulation strategies of MOF NPs endow them with the ability to deliver antitumor drugs ensuring high drug loading capacities and controllable release rates (Luo et al., 2021;Wu et al., 2017). Drugs with different properties, such as hydrophobic and hydrophilic drugs, can be effectively loaded into MOF NPs (Cheng et al., 2022). For example, in a relatively recent study, a hollow MOF drug delivery system loaded with 5-fluorouracil (5-FU) was used in order to verify its targeted antitumor drug transport abilities, demonstrating high drug loads and sustaining its drug release behavior (Gao et al., 2016). However, challenges in employing MOF NPs for some biomedical applications remain. The main synthetic challenge in the synthesis of MOF NPs is their low stability towards water. Reducing the contact between water molecules and MOF host frameworks, particularly the coordination bonds, was considered to be an effective strategy to improve MOF stability (Lv et al., 2019). For instance, to address this issue, polytopic rigid linkers that favor strong coordination bonds are used for their construction. Aromatic phenylene or acetylene-based linkers are traditionally employed in combination with strongly coordinating carboxylate linkers and high oxidation state metal ions (Ahmed et al., 2022). Moreover, by 6 introducing hydrophobic pore surfaces or blocked metal ions, water molecules are prevented from approaching the framework. Therefore, to sustain the framework robustness in the presence of an aqueous medium, some MOF NPs have been specifically functionalized by introducing nonpolar alkyl functional, fluorinated, or methyl groups (Ahmed et al., 2022). Another existing challenge, if the unreacted precursors cannot be rapidly depleted, is that the generated nucleus will continuously grow, generating microparticles rather than NPs. These microparticles normally exhibit limited accumulation in the tumor tissues compared to the NPs administered by intravenous injection. In addition, although MOF NPs are fabricated by coordinating metal ions and organic ligands, massive ions and proteins in the circulation system will also interact with the metal ions or organic ligands of the MOF, causing degradation or aggregation. Therefore, developing a strategy to increase MOF NP accumulation in tumor tissues is necessary to achieve enhanced therapeutic effects (Cheng et al., 2022). To prepare large-scale uniform MOF crystals for practical applications, numerous synthetic schemes have been developed, including water/solvothermal synthesis (Zou et al., 2019), microwave-assisted and ultrasonic-assisted methods (Bakhshi et al., 2021), microemulsion-based synthesis (Sun et al., 2016), and direct mixing method (Wang et al., 2013). For example, the preparation of MOF crystals through water/solvothermal synthesis is straightforward and simple to perform. It involves the dissolution of the metal ions and organic ligands in solvents, followed by their incubation in a reactor. The MOF crystals are obtained after a set amount of time, using high temperature and pressure (Luo et al., 2021). Otherwise, the microwave synthesis method involves mixing the chemicals needed for the reaction and the solvent together and then putting the mixture in a microwave reactor. This method is useful because the reaction is relatively fast, taking only a few tens of minutes, compared to the longer reaction time of solvothermal synthesis. Also, the microwave method allows the formation of nanoscale MOF particles because it provides uniform heating (Luo et al., 2021). It is known that some unit operations can modify the size, structure, and morphology of MOF particles during processing (Marshall et al., 2019; Wang et al., 2013; Xin et al., 2015) (Figure 2). 7 Figure 2. Schematic representation of (a) the modular synthesis of MOF NPs, with control over size and morphology, and (b) the post-synthetic external surface functionalization with multiple conjugation strategies and types of surface ligands. Taken from Wang et al., 2018. Copyright 2018, Wiley-VCH. In fact, the potential efficiency of MOF NPs can be exceptionally improved by finely adjusting their size and morphology (Liu et al., 2018). Some of the synthesis methods mentioned before having been reported to control MOF morphologies based on varying solvents, introducing surfactant or template agents (Liao et al., 2019). In addition, the incorporation of monotopic ligands that mimic the functionality of the multitopic MOF linkers into solvothermal syntheses, specifically named coordinating modulating, has gradually become a widely used method for adjusting the chemical and physical properties of the MOF NPs by manipulating their morphology, defect rate, exposed crystal facets, surface chemistry, and other factors (Figure 3) (Forgan, 2020). This method involves introducing a modulator, usually a monotopic analog of the linker, to control the growth of MOF crystals. The modulator often contains a carboxylic acid as the linker because it can serve two functions: acting as a surface capping ligand to stop the growth of MOF crystals and modify the MOF surface and acting as an acid to adjust the rate of metal-linker complexation for crystal growth by interfering with linker deprotonation (Li et al., 2023). For example, CuBDC-NBA with different morphology and size were achieved at room temperature through coordination modulation. Indeed, with increased 4-n-Butylbenzoic Acid (NBA, modulator) addition, the morphological transformation from nanosheet to 14 2.3.2.1.1.1 Characterization of the Polarized Macrophage Phenotype Macrophages, fundamental cells of the mononuclear phagocytic system (MPS), are highly plastic and heterogeneous immune cells that serve a multitude of functions, including the elimination of pathogens, clearance of cellular debris, tissue development, and hemostasis, and regulation of inflammatory responses (Anderson et al., 2021; Miao et al., 2017). Postnatal development of macrophages is driven by the differentiation of circulating monocytes through the macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage-stimulating colony factor (GM-CSF). These monocytes have their origins in the bone marrow from myeloid-derived progenitor cells and the resulting macrophages can display a broad spectrum of phenotypic states. Without any stimulation or in the resting cells, the phenotype of the macrophage is referred to as an M0 macrophage. M0 macrophages can be induced by environmental signals toward M1 and M2 polarization, which are the two main activated states of macrophages (Miao et al., 2017). While the M1/M2 model is commonly used to classify macrophages, it is important to note that this model is oversimplified and does not fully capture the complexity of macrophage function. Macrophages are highly plastic and can exhibit overlapping functions and markers between subsets, and it may be more accurate to consider their activation as a continuum of functional states rather than discrete M1 and M2 subsets (Martinez et al., 2014; Murray et al., 2014). M1 macrophages perform a pro-inflammatory function and are polarized by lipopolysaccharide and some cytokines such as interferon-gamma (IFN-) or GM-CSF to display strong effector functions against pathogens and cancer cells (e.g., trap, phagocytose, and lyse tumor cells) (Yunna et al., 2020). Moreover, M1 macrophages not only have high phagocytic ability but also overexpress CD80, CD86, and CD16/32 and produce increased levels of pro-inflammatory cytokines, including interleukin 12 (IL-12), interleukin 23 (IL-23), and tumor necrosis factor-alpha (TNF-𝛼), which facilitate leukocyte recruitment and activation during injury (Figure 4). In addition, the enhanced tumor antigen-presenting ability of M1 promotes other leukocytes’ cytotoxic functions (Liu et al., 2021). For example, CD8+ T cells and NK cells can be strengthened by immuno-stimulatory cytokines from M1 phenotype macrophages (Dungan et al., 2014). Conversely, polarization by interleukin 4 (IL-4) and interleukin 13 (IL-13) can result in 15 alternatively activated M2 macrophages that perform anti-inflammatory functions (Sylvestre et al., 2020). The expression of arginase-1 (Arg1), mannose receptor (CD206), antiinflammatory factor (IL-10), and chemokines CCL17 and CCL22 are elevated in M2 macrophages, acting as an immune-suppressor in tumor nest (Figure 4). In fact, while M2 macrophages have a critical role in maintaining normal immune function and homeostasis, like promoting Th2 responses, combating parasites, contributing to wound healing and tissue regeneration through the clearance of debris and the release of growth factors such as transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) (Sylvestre et al., 2020), specific subsets of M2 macrophages also play a pivotal role in supporting tumor progression (Anderson et al., 2021). On one hand, due to weakened tumor antigen-presenting ability, the proinflammatory potential of M2 phenotype macrophages is dramatically descended. On the other hand, M2 phenotype macrophages are educated to be tumor promoting by releasing growth factors forming a positive feedback loop in accordance with cytokines and factors of tumor cells (IL-4, IL-6, IL-10, among others) (Liu et al., 2021). Figure 4. Macrophages that have been activated can generally be grouped into two categories: M1like and M2-like macrophages. These types of macrophages are activated by different stimuli, have different characteristics, and perform different functions. Taken from Sylvestre et al., 2020. 16 Copyright 2020, Wiley-VCH. Tumors recruit both circulating monocytes and resident tissue macrophages to the TME and polarize them toward an M2 phenotype via a variety of soluble and mechanical factors, creating TAMs. These cells can influence both tumor progression and antitumor response through a variety of mechanisms (Crezee et al., 2020) (Figure 5). On one hand, TAMs can promote the growth and spread of cancer by promoting genetic instability, supporting the formation of new blood vessels, and releasing growth factors and enzymes that break down the extracellular matrix and basement membrane. In addition, they can stimulate cancer cell migration through paracrine signaling C-C motif ligand 18 (CCL18), as well as prepare distant metastatic sites for seeding (Williams et al., 2016). On the other hand, they can also suppress the immune system, making it harder for the body to fight cancer, through upregulation of immunosuppressive surface proteins, secretion of ROS, production of cytokines to suppress T-cell function, secretion of chemokines that recruit regulatory T cells (Anderson et al., 2021), and inhibition of B cell signaling (Guo et al., 2016; Timosenko et al., 2017). Thus, nowadays, different research approaches have sought to develop strategies to reprogram TAMs to perform an antitumor action (Anfray et al., 2020). Figure 5. TAMs contribute to the growth and spread of tumors in several ways, such as by suppressing the immune system, recruiting monocytes, and creating environments suitable for distant metastasis. They also help tumors invade surrounding tissues by releasing enzymes that dissolve the basement membrane and secreting angiogenic growth factors. Taken from Sylvestre et al., 2020. Copyright 2020, Wiley-VCH. 17 2.3.2.1.1.2 Mechanisms of Macrophage Polarization Since macrophages play an important role in regulating the body’s immune response and metabolism, the direction of macrophage polarization can be modulated to change to the desired phenotype. Macrophage polarization is defined as the dynamic process in which resting macrophages experience various functional changes to adjust to the changing of the residing milieu. For example, regulating the acidity of the TME is an effective strategy for changing the polarization phenotype of macrophages, since their phagocytic function is closely related to the pH of lysosomes (Lavine et al., 2018; Murray et al., 2014). While M2 macrophages repair damaged tissues by phagocytosis, the basic property of M1 macrophages is to promote tissue damage (Yunna et al., 2020). Therefore, altering the pH of lysosomes may be an effective approach for resetting the polarization phenotype of macrophages. Alkaline agents such as chloroquine (CQ) are known to be trapped in the lysosomal compartment, increasing lysosomal pH and allowing TAMs to be reset from M2 to the M1 phenotype (Chen et al., 2018). Additionally, due to the rapid growth of malignant tumors, there is an area of hypoxia inside the tumor (Wang et al., 2019), that is involved in the induction of epithelial-metabolism and TAMs infiltration. Hypoxia may have a deep effect on the polarization of macrophages due to the preferential accumulation of macrophages in hypoxic tumor areas and the retention of relatively immature cell types (Chen et al., 2013). The hypoxic TME stimulates macrophages to secrete exosomes rich in immunoregulatory proteins and chemokines, which promote macrophage polarization to the M2 phenotype, thereby stimulating angiogenesis, metastasize, and inhibiting host immunity (Jeong et al., 2017; Park et al., 2019). Therefore, improving the hypoxic TME is an efficient method to regulate the polarization phenotype of macrophages. Furthermore, the development of some diseases like cancer is accompanied by changes in the composition of the ECM (extracellular matrix), a highly dynamic and complex macromolecular network, and contributes to the development of the disease (Mouw et al., 2014). Among them, the ECM component in the TEM has a regulatory effect on macrophage polarization. For example, proteases and their inhibitors are key physiological regulators of ECM remodeling. The extracellular serine protease inhibitor (serpin) serpinE2 is overexpressed in various human cancers and participates in tumor progression and metastasis. By inhibiting the expression of serpinE2, M2-like macrophages can be promoted to M1-like phenotype polarization and 18 inhibit vascular invasion and tumor spread (Smirnova et al., 2016). Finally, it is well-known that nanocarriers cause macrophage polarization phenotype changes in vivo mainly through macrophage internalization, leading to changes in cellular uptake and secretion of cytokines and chemokines (Yunna et al., 2020). Remarkably, material parameters such as porosity (Sussman et al., 2014), stiffness (Blakney et al., 2012), and nanotopography (Lee et al., 2011) change the inflammatory response. Therefore, understanding how nanocarriers affect macrophage polarization and inflammatory response is an effective strategy to promote the clinical application of nanocarriers. Various engineered drug-free nanocarriers were used to reset the macrophage polarization phenotype as it can be used to induce TAM polarization from the M2 phenotype to the M1 phenotype (Pal et al., 2016; Zanganeh et al., 2016). 2.4 In vitro models Several different biological cell models are being used to explore NPs’ safety and efficacy. Recently, different cell lines have been used as models because they maintain representative functional features, that enable the clear analysis of a well-known cell response. Cell lines such as human cervical adenocarcinoma (HeLa) (Schirinzi et al., 2017) and human epithelial colorectal adenocarcinoma (Caco-2) (Wu et al., 2019) have been used as a model of anatomical barriers. Likewise, human embryonic kidney cells (HEK 293) have been widely used in biomedical research as a model system due to their high transfection efficiency and reliable growth. On the other hand, human peripheral blood monocyte cells (PBMCs), human monocytic (UP37 and THP-1), mouse macrophage (DMBM-2) (Prietl et al., 2014), and murine macrophages (RAW 264.7) have been used as immune cell models for in vitro evaluation of NPs effect (Aguilar-Guzmán et al., 2022). RAW 264.7 cells are adherent macrophage-like cells derived from tumors, inoculated with Abelson murine leukemia virus. These cells are immortalized, relatively stable, and mature and have been used for models of macrophage activation in numerous studies (Bastos, 2016). According to previous studies, the murine leukemic monocyte-macrophage cell line RAW 264.7 is a representative and versatile macrophage cell line worldwide accepted as a macrophage model. It has been an amenable in vitro model as it exhibits key characteristics representative of different macrophage types in vivo (Bordbar et al., 2012). 19 3. M aterials & M ethods 3.1 Synthesis of Cu MOF Nanoparticles with Different Shapes An overview of the different methods used in the synthesis of Cu MOF NPs with different shapes is reported in Figure 6. In this work, to produce MOF NPs with a similar composition, the metal source used was Cu2+ from copper (II) nitrate trihydrate (Cu(NO3)2.3H2O) and the ligands were benzene-1,4-dicarboxylic acid (H2BDC), benzene-1,3,5-tricarboxylic acid (H3BTC), and 1,2,4,5-benzenetetracarboxylic acid (H4BTC). Herein, BDC will be used to define benzene dicarboxylic acid and BTC to benzene trior tetra-carboxylic acids. The following sections describe each fabrication protocol in detail. 3.1.1 Preparation of Cu-H 3 BTC Crystals (Hexagon shape) To produce [Cu3(BTC)2]n crystals with hexagon shapes, the following protocol was adapted from Umemura et al. (Umemura et al., 2011). Cu(NO3)2.3H2O (2.1 mg, 0.0085 mmol) and octanoic acid (118.88 mg, 0.825 mmol) were dissolved in 10 mL of butanol. The mixed solution was heated with a heat gun until a transparent solution was obtained. H3BTC (1 mg, 0.165 mmol) was added and the mixture was heated by microwave irradiation at 413 K for 60 min. The resulting blue powder was isolated by centrifugation (Eppendorf Centrifuge 5430 R) and washed with ethanol (3x3 mL). Centrifugation was performed at 7800 rpm, at 4C for 20 min. Then, the solutions were sonicated (Sonics Vibra-Cell) at 20% amplitude for 1 min (3 sec pulses of ultrasound, with 1 sec spacing in between each pulse). 3.1.2 Preparation of Cu-H 2 BDC Crystals (Needle shape) 3.1.2.1 Preparation of ligand solution Two solutions were prepared separately by dissolving 2-Methylimidazole (2-MIM) (40 mg) in 1 mL of water (40 mg/mL) and Cu(N03)2.3H2O (10 mg) in 1 mL of water (10 mg/mL). Then, H2BDC (13 mg, 0.08 mol) was dissolved in 2-MIM, keeping in a 1:3.2 molar ratio, and H2O was added to make a final volume of 4 mL. The sonication bath (Transsonic 690) was used to promote ligand dissolution in 2-MIM/H2O. Thus, an organic ligand solution was prepared as indicated in Table 1, and the pH was measured. 20 Table 1. Volumes of 2-MIM and H2O added to prepare the ligand solution. Molar Ratio H2BDC: 2-MIM 2-MIM (L) H2O (L) pH 1:3.2 514 3486 7.84 3.1.2.2 Preparation of MOF NPs The ligand solution prepared previously was used for the production of MOF NPs. Thus, 400 L of pure ethanol and 400 L of water were first added to each Eppendorf, and then, under stirring (600 rpm), 100 L of the ligand solution and 94.5 L of the copper solution were added at room temperature, to keep the molar ratio 1:2. The reaction mixture was stirred vigorously for an additional 8 h at room temperature. The resulting blue powder was isolated by centrifugation (Eppendorf Centrifuge 5417 R) and washed with ethanol (3x3 mL). Centrifugation was performed at 14 000 rpm, at 23C for 10 min. 3.1.3 Preparation of Cu MOF NPs (Spherical and Square shape) 3.1.3.1 Preparation of ligand solutions The ligand solutions were synthesized by dissolving each of the ligands [H2BDC (16 mg, 0.096 mmol) (square shape) and H4BTC (16 mg, 0.063 mmol) (spherical shape)] in 4 mL of dimethylformamide (DMF). 3.1.3.2 Preparation of MOF NPs with different molar ratios between the ligand and the metal A copper solution was prepared by dissolving Cu(N03)2.3H2O (50 mg) in 5 mL of DMF (10 mg/mL). First, 3 mL of DMF and 1 mL of each ligand solution prepared previously were mixed, and then different amounts of 0.04 M Cu(N03)2.3H2O solution were added under stirring (Table 2). To explore the effect of the concentration of the metal ion, the molar ratios between the Cu (II) salt and each ligand (BDC2-, BTC4-) were fixed at 1:1 and 2:1, respectively. The reaction solution was stirred vigorously overnight at 80C using an oil bath. The resulting blue power was isolated by centrifugation (Eppendorf Centrifuge 5430 R) and washed 3 times (3x3 mL), first with DMF and then with ethanol. Centrifugation was performed at 7800 rpm, at 4C for 20 min. 21 Table 2. Volumes of Cu(NO3)2 added to the reaction mix according to the molar ratio established. Molar Ratio Cu(NO3)2: BDC/BTC Cu(NO3)2 to H2BDC (L) Cu(NO3)2 to H4BTC (L) 1:1 2:1 604 1208* 386** 773 Represent the final volume values used to synthesize the *square and **spherical Cu MOF NPs, after testing both molar ratios. Figure 6. Schematic description of the methods used in the synthesis of Cu MOF NPs with different shapes. Created with BioRender.com. 3.2 Dynamic Light Scattering and Nanoparticle Tracking Analysis Dynamic light scattering (DLS) is a non-invasive, well-established technique for measuring the size, size distribution, and zeta potential of molecules and particles dispersed or dissolved in a liquid, typically in the submicron region, and with the latest technology, lower than 1 nm (Falke et al., 2019). This approach involves the measurement of light 22 interference based on the Brownian motion of particles or molecules in suspension, and on the correlation of particle velocity with their size using the Stokes-Einstein relationship (Joudeh et al., 2022). Nanoparticle Tracking Analysis (NTA) utilizes the properties of both light scattering and Brownian motion to obtain the hydrodynamic particle size, zeta potential, concentration, fluorescence, and colocalization of samples in liquid suspension. Each individual particle is counted and tracked in short video clips, creating accurate concentration calculations and particle size distributions. Moreover, these measurements are combined with microelectrophoresis, for the determination of zeta potential (Joudeh et al., 2022; Malloy et al., 2006). The properties of synthesized NPs were evaluated by DLS (Malvern ZetaSizer SZ) and NTA (ZetaView® PMX-420 QUATT equipped with the software version 8.05.19 SP3). For each assay, 3 runs were performed. NP size measurements by DLS were done at 25C with a scattering angle of 173º and using disposable polystyrene cuvettes (Sarstedt Ag & Co., DE), while zeta potential measurements were conducted at 25C with a period stabilization of 120 sec using a disposable folded capillary cell (DTS1070, Malvern, U.K.). NTA and DLS measurements were conducted in pure deionized water. 3.3 Transmission Electron Microscopy Transmission Electron Microscopy (TEM) is a microscopy technique capable of providing very high-resolution images down to the scale of several Angstroms (~0.19 nm), in which a beam of electrons passes through a thin sample to produce an image. The electron beam is impacted by the sample’s thickness/density, composition, and, in some cases, crystallinity. By monitoring what has happened to the beam of electrons in transit it is possible to image the fine structure (by contrast) of the sample as well as describe physical and chemical characteristics (Winey et al., 2014). TEM was used to observe the shape of the synthesized Cu MOF NPs. Then, for TEM analysis, 10 L of samples were mounted in a specific grid and left standing for 30 sec. The liquid in excess was removed with filter paper. The analysis was performed on a microscope from Thermo Scientific, USA and the model is Talos F200i. The grids used are Formvar/Carbon 200 Mesh, Copper (FCF200-CU-50, 50, pk) from Electron Microscopy Sciences. 23 3.4 Transmission Electron Microscopy and Energy Dispersive X-ray Analysis Energy dispersive X-ray spectroscopy (EDX) is a standard method for identifying and quantifying elemental compositions in a very small sample of material. Energy dispersive Xray systems are typically used in conjunction with electron microscopy techniques such as transmission electron microscopy or scanning electron microscopy (Bergström, 2015). EDX analysis is based on the emission of characteristic X-rays from a specimen when it is bombarded with a beam of high-energy charged particles, such as electrons or protons. When an electron from a higher binding energy level falls into a core hole, an X-ray is emitted with an energy equal to the difference in binding energies of the two levels. This process generates a spectrum that shows peaks corresponding to the elemental composition of the sample being analyzed. In addition, the elemental mapping of a sample can be created with this characterization method (Bergström, 2015; Colpan et al., 2018; Raval et al., 2019). EDX analysis was also done with transmission electron microscopy (Thermo Scientific Talos F200) to ensure the composition mapping of Cu MOF NPs. The Super-X Detection System features 4 SDDs and Velox Software were used for the elemental analysis. 3.5 Scanning Electron Microscopy Scanning electron microscopy (SEM) is another technique where a small amount of material may be used to determine particle size, shape, and texture. In SEM a fine beam of electrons scans across the prepared sample in a series of parallel tracks. Therefore, the electrons interact with the sample and produce several different signals which can be detected and displayed on the screen of a cathode ray tube. Moreover, since the depth of focus is so much greater than that of the light microscope, information on particle surface texture can be generated (Ferreira et al., 2018). For SEM analysis, 10 L of the light-blue solids stored in ethanol were dispersed and deposited on carbon adhesive tabs (77825-12 EMS) or silicon wafer chips (G3391 Agar Scientific) and then coated with chrome120 for measurement using a high vacuum sputter coater (Leica EM ACE200). The images were obtained from a ZEISS Model Supra 55 SEM system operating at 3.0 kV. 30 antibody that is linked to a reporter enzyme. Detection is achieved by measuring the activity of the reporter enzyme after it has been incubated with a substrate that produces a measurable product. The most crucial element of an ELISA is a highly specific antibodyantigen interaction (Engvall, 2010; Shah et al., 2016). RAW 264.7 macrophage cells were incubated with the four different types of MOF NPs at 75 μg/mL for 24 h. After 24 h, the culture media were collected and stored at −80C, until further used to evaluate differences in cytokine release by cells exposed to the differently shaped NPs. The supernatants were tested for the production of pro-inflammatory cytokines, TNF-α and IL-12, and anti-inflammatory cytokines, IL-4 and IL-10, using an ELISA kit (PeproTech TMB ELISA Buffer Kit), following the manufacturer’s instructions. The optical density (OD) at 450 nm was determined on a plate reader. 3.11.1 Plate Preparation The sandwich ELISA starts with a capture antibody coated onto the wells of the plate. It is termed a “sandwich” because the antigens are sandwiched between two layers of antibodies (capture and detection antibodies). Hence, the capture antibody, specific for each cytokine, was first diluted with PBS, according to the manufacturer’s instructions, and 100 μL of the diluted solution was added immediately to each ELISA plate well. After adding the capture antibody to the plates, the plates were covered and incubated overnight at room temperature. Once the step was completed, the plates were washed 4 times of any potential unbound antibodies. Each wash consisted of adding 300 μL wash buffer per well, followed by aspiration. After the last wash, the plate was inverted to remove the residual buffer and blot it on a paper towel. Afterward, a 300 μL of blocking buffer was added to each well and the plate was incubated for at least 1 hour at room temperature. Finally, the plate was rewashed 4 times with PBS before the addition of the antigen. 3.11.2 Elisa Protocol 3.11.2.1 Standard/Sample After washing, 100 μL of standard solutions of each cytokine kit or of the cell culture supernatant samples were added to each well, in triplicate, of the antibody-coated ELISA plates, and incubated for at least 2 h at room temperature, to allow cytokines to bind to the capture antibody. 31 3.11.2.2 Detection The plate was rewashed 4 times to remove unbound reactants. Afterwards, 100 μL of the primary detection antibody was immediately added to each well and incubated for another 2 h at room temperature to allow the binding to the captured cytokines, followed by a buffer wash. 3.11.2.3 Avidin-HRP Conjugate Then, the secondary enzyme-conjugated antibody was added to each well. For that, Avidin-HRP conjugate was diluted 1:2000 in diluent and 100 μL was added per well, followed by incubation for another 30 minutes at room temperature. 3.11.2.4 ABTS Liquid Substrate The plate was rewashed another 4 times, and 100 μL of the substrate solution (ELISA Colorimetric 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) reagent) was added to each well, to produce a color change, and incubated at room temperature for 20 minutes, protected from light. During this process, a colored product is formed which allows to indirectly determine the cytokine concentration of each analyzed sample. The color development was monitored with an ELISA plate reader (BioTek Synergy H1 Plate Reader) at 450 nm with a wavelength set at 620 nm. 3.12 Statistical Analysis Between one and three independent assays were performed for each experiment with three or more replicates per condition. The graphs and statistical tests were executed in GraphPad Prism (version 9.4.0, GraphPad Software) and all results presented in graphs include means ± standard deviations (SD). The statistical significance of differences between the means of individual groups was calculated using a one-way or two-way analysis of variance (ANOVA) with Dunnett’s post hoc test. A p-value of ≤0.05 was considered statistically significant. The significant differences between the groups are represented in the graphs by the asterisk (*), where *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. 32 4. R esults & D iscussion 4.1 Nanoparticle Preparation and Characterization The physicochemical characterization of NPs intended for immunology research is important as it helps to explain the observed immunological effects. More importantly, it relates the physicochemical and immunological properties to draw meaningful conclusions. Moreover, some of those properties can also influence the biocompatibility, biodistribution, clearance, and immunotoxicity of the nanosystems. Therefore, the physicochemical parameters like size, surface charge (zeta potential), compositional analysis, and morphology are commonly measured to characterize NPs (Clogston, 2021). In this work, to get differently shaped MOF NPs, some reaction conditions and synthesis methods were modified as described in Chapter 3 (Materials and Methods), while the metal source and the carboxylate ligands remained unchanged. Consequently, Cu MOF NPs properties depend on different parameters like reaction time, temperature, type of solvent, agitation speed, molar ratios between the Cu (II) salt and each carboxylate ligand, and the addition of modulators/capping agents, among others. Thus, four differently shaped MOF NPs, exhibiting hexagon (Figure 8A), needle (Figure 8B), square (Figure 8C), and spherical (Figure 8D) shapes, were obtained. As the magnified TEM images show, the synthesized Cu MOF NPs were highly dispersed, and each revealed a uniform shape with a controlled size (from approximately 250 to 500 nm). Figure 8. TEM images of different Cu MOF NPs, including (A) Hexagon, (B) Needle, (C) Square, and (D) (A) (C) (B) (D) (A) (C) (B) (D) 33 Spherical shapes (scale bars: 500 nm and 1 μm). The synthesis of shape-controlled MOF crystals usually depends on the specific control over MOF nucleation and growth (Guo et al., 2018). The reaction of copper nitrate with a carboxylate ligand gives slow nucleation and then faster crystal growth, which inherently results in [(Cu)n(BDC/BTC)2]n with a better-defined shape (Umemura et al., 2011). The synthesized hexagon and needle Cu MOF NPs involved the addition of different modulators to get their shapes. In the case of hexagon MOF NPs, the shape was achieved using octanoic acid as an additive (modulator), which perturbs the coordination between Cu-BTC, thus leading to the anisotropic crystal growth (Figure 9). Previous studies suggest that the competitive interaction brought by the monocarboxylic additive leads to the alteration of the nucleation process (Umemura et al., 2011). In fact, when adding octanoic acid as a modulator of the growth process, the attachment of the growth unit is perturbed due to the competition between the modulator and one of the carboxylates in the BTC ligand at the attachment event. At the concentration used of octanoic acid, it seems that all the edges equally grow, i.e., the growth rate on each edge appears to be constant throughout the growth, resulting in hexagonal 2D MOF NPs. A different modulator (2-methylilimidazole) was incorporated into the needle MOF synthesis process. 2-MIM not only can act as a base to promote the rapid growth of crystals, by accelerating the deprotonation of H2BDC, but also can serve as a competitive ligand to prevent further growth of the crystal (Guo et al., 2018). Thus, in the presence of 2-MIM, more deprotonated H2BDC ions were available for coordination with metal ions, resulting in nanosized crystals with a limited and regulated rate of framework extension (or faceselective modulation, Figure 9), provided by the competitive effect, which culminates with needle-shaped crystals. In both cases, the use of modulators also improves the crystallinity of MOF NPs by enhancing the reversibility of MOF formation (Chen et al., 2022), a fact that is comported by TEM images for these two shapes of Cu MOF NPs that appear darker due to the fewer electrons transmitted through these samples, which means higher crystallinity. In addition, modulators may also physically prevent crystal’s aggregation, which leads to anisotropic growth (Łuczak et al., 2023). 34 Figure 9. The role of chemical modulators for fabricating MOF nanocrystals. Taken from Tsuruoka et al., 2009. Copyright 2009, Wiley-VCH. Regarding the synthesis process of spherical and square Cu MOF NPs, without any modulators involved, the only existing differences were the molar ratio between the cupric ions and the ligand and the number of carboxylic groups of the BTC/BDC ligand used in each one, a dicarboxylic ligand in the case of square shape and a tetracarboxylic linker for spherical shape. Considering the synthesized square Cu MOF NPs, as shown in Figure 10A, the copper (II) ions are joined by benzene dicarboxylate ligands, resulting in a two-dimensional square network structure. Changing the organic linker from linear BDC to quadrangular BTC resulted in a change in Cu MOF shape, which exhibits a spherical-like shape. The use of linker H4BTC is relatively less reported, probably because, for steric reasons, all of the four carboxyl groups are unlikely to engage in coordination with metal ions (Cao et al., 2002). Yet, through carefully manipulating the reaction conditions (e.g., the molar ratio between the metal ion and the linker), H4BTC was shown as a versatile building block for the construction of metal-organic complexes through complete or partial deprotonation of its carboxyl groups (Figure 10B). Indeed, spherically shaped NPs were obtained by adjusting the metal-ligand molar ratio to 1:1, which differs from that used for square-shaped NPs (2:1). 35 Figure 10. The proposed structure for the synthesized (A) square (“Synthesis, Characterization and Comparative Study of Copper and Zinc Metal-Organic Frameworks,” 2013) and (B) spherical (R. Cao et al., 2002) Cu MOF NPs. The properties of Cu MOF NPs obtained were characterized by NTA revealing a size range from 200 to 400 nm (Table 5), which was consistent with TEM results. Moreover, all four Cu MOF NPs were negatively charged when dispersed in water and there were no significant differences in particle concentration between the differently shaped NPs (around 1011 particles/mL) when using the same mass concentration of stock solutions, thus offering an ideal system to address the sole impact of Cu MOF NPs shape on macrophage phenotype modulation. DLS was also selected to measure the size and the zeta potential of spherical NPs, 230.9 nm and −14.30 mV, respectively, confirming the measured values by NTA. While spherical particles are described by a single-size parameter, for non-spherical NPs, several dimensions are needed to fully report their dimensions. Although the DLS and NTA methods in their pure form are not suitable for measuring such NPs, since quite often these instruments only measure at one or two angles (Arenas-Guerrero et al., 2018; Doncom et al., 2017), they allow an indirect measurement of the characteristics of liquid dispersions (Monakhova et al., 2023). Thus, in addition to allowing the observation of NPs shape, TEM is an instrument more sophisticated and able to measure in different ranges of angles, which can give advanced information, such as better analysis of multi-modal particle size distributions and information on the interaction between the particles (Monakhova et al., 2023). (B) (A) 36 Table 5. Cu MOF NPs sizes, zeta potential, and concentrations from NTA. Data are presented as means ± SD. Group Size (nm) 𝜻 Potential (mV) Concentration* (particles/mL) Hexagon 392.8 ± 50.3 −23.9 ± 0.4 1.7 × 1011 Needle 307.5 ± 48.4 −18.8 ± 0.2 2.3 × 1011 Square 218.1 ± 23.5 −14.4 ± 0.0 2.1 × 1011 Spherical 170.7 ± 44.7 −11.8 ± 0.7 0.6 ± 1011 * For all the four different types of MOF NPs, the concentration in terms of the number of particles per mL was calculated from a stock solution with a concentration of 75 μg/mL. The morphological and textural structures of these Cu MOF NPs are further confirmed by comparing their SEM images with those of TEM. Since the main objective of performing SEM analysis was to give a 3D overview of the synthesized MOF NPs, the SEM images of spherical and square Cu MOF NPs were not included here as it was not possible to detect individual NPs and to image them. Only images of NP aggregates were collected, despite the use of sonication to disrupt them, not allowing access to their 3D structure. These aggregates can be related to the synthesis method since these two types of Cu MOF NPs were synthesized by the solvothermal method and without the use of modulators that, as previously mentioned, are well known to improve the crystallinity of materials and to prevent their aggregation. Both low and high-magnification SEM images (Figure 11) show that the samples are highly homogeneous. As could be seen from the Figure 11A, the prepared hexagon MOF NPs possessed a truncated octahedron 3D structure, exhibiting faces with hexagonal shapes with the length of each edge around 500 nm, and a clearly very smooth surface, indicating their high crystallinity. Accordingly, the high magnification of Figure 11B shows that the prepared needle Cu MOF NPs presented typical lengths ranging from 300 to 500 nm with a smooth surface, very low thickness, and sharp vertices. 37 Figure 11. SEM images of different Cu MOF NPs, revealing (A) Hexagon and (B) Needle shapes (scale bars: 2 and 10 μm). Then, TEM-EDX analysis was employed to analyze the chemical compositions of the asprepared Cu MOF NPs. Figure 12 shows the mapping results for each component elemental, confirming the composition of the material. Indeed, several key elements including Cu (Copper), C (Carbon), O (Oxygen), and S (Sulfur) were all identified in the spectrum. The presence of Mo (Molybdenum) in all the samples is derived from the molybdenum grids used for characterization. These findings suggest the successful formation of Cu MOF NPs in agreement with previous studies (Lee et al., 2022; Yusuff et al., 2019). The EDX spectrum shows the high purity of the synthesized Cu MOF NPs along with oxygen content which indicates the formation of copper particles. The absence of other elements in the EDX spectrum also confirms the purity of Cu MOF NPs. (B) (A) (A) (B) 38 Figure 12. TEM-EDX analysis of Cu MOF NPs with different shapes: (A) Hexagon, (B) Needle, (C) Square, and (D) Spherical shapes. These results indicated that these four Cu MOF NPs exhibited very similar physicochemical properties except shape and thus would be ideally suited for studies aimed at assessing the impact of shape on a given biological activity. 4.2 Cell Viability Studies In vitro effects of Cu-based MOF NPs were studied by AlamarBlue assay, in a wide range of concentrations (from 10 to 200 μg/mL), using four distinct cell lines, namely a cancerous cell line (HeLa), a human embryonic kidney cell line (HEK 293) usually used as a normal cell line, a human monocytic cell line (THP-1) following differentiation using PMA, the most widely used model for primary human monocytes/macrophages, and a macrophage-like, leukemia virus-transformed murine macrophage cell-line (RAW 264.7) that is commonly used as the model of mouse macrophages (Li et al., 2021). All types of Cu MOF NPs were very well dispersed in an aqueous solution – a complete culture medium. 4.2.1 The effect of Cu MOF NPs on RAW 264.7 cells The cell viability of macrophages is significantly impacted by the materials, sizes, and surface modifications of inorganic NPs as the result of differences in cellular uptake and physiological functions (Cheng et al., 2019). To investigate the cytotoxicity, a resazurin assay was performed to assess the cell viability of RAW 264.7 macrophages upon exposure to Cu A B C D 39 MOF NPs for 24 h and 48 h with a wide dose range of 0 - 200 μg/mL. Overall, as shown in Figure 13, all four differently shaped Cu MOF NPs reduced the cell viability of RAW 264.7 macrophage cells at higher doses for both time points. Figure 13. In vitro cell viability assay (AlamarBlue) of RAW 264.7 cells treated with (A) Square, (B) Needle, (C) Spherical, and (D) Hexagon NPs. RAW 264.7 cells were treated with various concentrations of Cu MOF NPs for 24 h and 48 h. The dashed line defines the cytotoxicity range: noncytotoxicity > 80%. The data are presented as means ± SD (n=4). (*p<0.05; *p<0.01; ****p<0.0001). Regardless of the broad spectrum of Cu MOF shapes, increasing NPs concentrations caused only a statistically significant decrease in RAW 264.7 cell viability in a range of concentrations above 75 μg/mL. These findings indicated that, until this concentration value, the exposure to NPs did not affect the cell’s metabolism based on the quantification of resorufin, which is proportional to the number of viable cells, when compared with the control cells. Therefore, 75 μg/mL is the maximum and “safe” concentration to be used of all four differently shaped NPs that was selected to proceed with further experiments. Additionally, as shown in Figure 13B, cells treated with needle-shaped MOF NPs retained high cell viability (above 80%) under a dosage of 75 μg/mL, but it dramatically fell to lower than 50% at 100 μg/mL both at 24 h and 48 h. In contrast, there was no such evident cytotoxicity of hexagon Cu MOF NPs in RAW 264.7 cells when exposed at concentrations higher than 75 μg/mL, demonstrating that the hexagon shape can significantly reduce the (A) (B) (C) (D) 46 cytotoxicity than star-, flowerand prism-shaped AuNPs due to a higher uptake (Woźniak et al., 2017). Given that spherical NPs are known to have higher cellular uptake in HEK 293 cells (Awashra et al., 2023; Woźniak et al., 2017), a possible interpretation for this trend is that, after a long-time interval of exposure at high concentrations, a higher cellular uptake of spherical and hexagon NPs compared with needle and square shapes can led to a greater cytotoxicity (Awashra et al., 2023). Yet, short-term exposure at lower concentrations of needle-shaped MOF NPs showed higher toxic effects than incubation with spherical and hexagon NPs. Further studies, e.g., confocal microscopy analysis of cellular uptake of NPs, are required to understand this phenomenon. An additional critical result is that, after treatment for 48 h, the Cu MOF NPs were almost three-fold toxic towards HEK 293 cells in comparison to the HeLa cells. It is worth noting that various types of cell lines can react differently after incubation with NPs (Woźniak et al., 2017). As previously reported by Woźniak et al., the HEK 293 cell line is more sensitive than HeLa cell line, and its viability depends on the concentration and time interval of incubation with NPs (Woźniak et al., 2017). In this experiment, Cu MOF NPs increased early cell death in HeLa cells, but only late cell death in HEK 293 cells, which is in agreement with previous studies for other types of NPs (Yang et al., 2020). Since it was found that RAW 264.7 cells exposure to 75 μg/mL of MOF NPs for both 24 h and 48 h did not potently affect the cells, for this next series of experiences, this concentration was used as the highest safe concentration tested. 4.3 Comparison of effects of different Cu MOF NPs shapes on RAW 264.7 cell morphology and proliferation Because the M2-like macrophages and M1-like macrophages exhibit distinct morphologies, the phenotypic transformation of macrophages can be indicated by an obvious change in cell morphology (Rodell et al., 2018). Figures 18 and 19 show the cell morphology of the macrophage RAW 264.7 cells under treatment with Cu MOF NPs at 75 μg/mL. The cells were monitored under an optical microscope (20) after 24 h and 48 h incubation. Striking differences were observed among non-NP-treated cells and Cu MOF NP-treated cells. In the untreated cells (M0) (Figure 18A), the cell morphology generally showed a round form with a cortical ring of actin filaments, dense cytoplasm mostly in the peri-nuclear areas, 47 and limited cytoplasmic extensions or spreading (Dabare et al., 2023). Also, vacuoles were absent in the majority of unstimulated cells. However, most of RAW 264.7 cells treated with different shapes of Cu MOF NPs had changed to an irregular form with accelerated spreading and forming pseudopodia, and a few becoming elongated with a spindle-shaped morphology. In fact, in contrast to cells cultured in medium alone, cells incubated with Cu MOF NPs generally presented the following features: flattened stellar morphologies associated with cells presenting three or more short cytoplasmic projections or lamellipodial extensions, together with a granular cytoplasm with several vacuolations visible in many cells (Sigola et al., 2016). This morphological behavior is consistent with a pro-inflammatory phenotype (M1) (Sigola et al., 2016) and was mostly remarked at 24 h incubation (Figure 18) with needle-shaped Cu MOF NPs (Figure 18E). Indeed, these cells exhibited podosomes that have been described as zones of close contact to the substratum, which combine several key abilities including cell adhesion, matrix degradation, and mechanosensing (Neacsu et al., 2014). This suggests that the needle-like shape of NPs is prone to provoke a proinflammatory response, probably because it presents a challenge to macrophages attempting to engulf and clear these long and straight structures (Nel, 2023). Additionally, these qualitative morphological results indicated that, after treatment for 24 h, the number of M2 cone-shaped cells also increased, but apparently much less, in cells treated with the differently shaped Cu MOF NPs compared with the control group. This M2 macrophage morphological behavior, characterized by an elongated morphology with only two or fewer pseudopods (Yang et al., 2021), looked more obvious in the group treated with spherical Cu MOF NPs (Figure 18D). Figure 18. RAW 264.7 macrophages untreated (A). Effects of (B) Hexagon (C) Square (D) Spherical D E B A C 48 and (E) Needle MOF NPs on RAW 264.7 macrophage cell morphology after 1 day of incubation with different Cu MOF NPs. White arrows point to M1 macrophages and red arrows indicate M2 macrophage. Images were obtained using an inverted light microscopy under phase contrast. All the micrographs in this figure were taken at the same magnification (20). Similarly, after 48 h, the majority of RAW 264.7 cells in the control group (without stimulation by Cu MOF NPs) were small and round (Figure 19A). However, more significantly in the square and needle groups, the cells showed a flat and pancake-like morphology with many synaptic structures (Yang et al., 2021) (Figures 19C and 19E). On the contrary, spherical-shaped Cu MOF NPs, after 2 days of incubation, did not induce morphological changes in RAW 264.7 cells when compared with the control group (Figure 19D). In conclusion, these results showed that square and needle-shaped MOF NPs induced a higher percentage of M1 macrophages and suggested that this trend was more significant after two days of culture. Figure 19. RAW 264.7 macrophages untreated (A). Effects of (B) Hexagon (C) Square (D) Spherical and (E) Needle MOF NPs on RAW 264.7 macrophage cell morphology after 2 days of incubation with different Cu MOF NPs. White arrows point to M1 macrophages. Images were obtained using an inverted light microscopy under phase contrast. All the micrographs in this figure were taken at the same magnification (20). The morphological results were qualitative, as macrophages presented a continuous spectrum between the different phenotypes. To quantify these observations, gene expression, cytokine production, and flow cytometry in vitro were further explored. D E B A C 49 4.4 Real-Time PCR Analysis of gene expression in RAW 264.7 cells The gene expression levels of the two macrophage phenotype markers in RAW 264.7 cells, determined by RT-qPCR after 24 h and 48 h of incubation with 75 μg/mL of Cu-based MOF NPs, are depicted in Figure 20. M1 and M2 macrophages express common or exclusive marker genes (Zhang et al., 2020). Among the top distinct genes in M1 and M2 macrophages, several studies have shown that CD86 is expressed in and used as a M1 marker, and CD206 and Arg1 as M2 markers, in both mouse and humans (Orecchioni et al., 2019; Taciak et al., 2018). Thus, these set of genes was selected to evaluate the phenotypic profile of RAW 264.7 macrophages post-incubation with Cu MOF NPs. The results are expressed as the fold change compared with the untreated control cells, i.e., the ratio of gene expression between each treated group and the control group, and those genes altered at least 2-fold (p<0.05) were statistically different expressed (Mccarthy et al., 2009). The concentration and purity ratio (A260/280) of extracted RNA are presented in Appendix 1. The ratio of absorbance at 260 nm and 280 nm for all RNA samples was ~ 2.0, providing a rough indication of purity and considered suitable for use in cDNA synthesis. Figure 20. Histograms depicting fold change in RAW 264.7 cells gene expression, determined by quantitative real-time PCR analysis, after 1 (A) and 2 (B) days of treatment with Cu MOF NPs. An untreated condition (RAW 264.7 cells without Cu MOF NPs stimulation) was used to determine the relative expression, and the transcript expression of target genes (CD86, CD206, Arg1) was normalized to the expression of endogenous housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The gene expression of CD86, representing M1 macrophages, was significantly upregulated in the group treated with square-shaped Cu MOF NPs both at days 1 and 2 of culture compared to the control group (mean fold change: 3.32 and 6.80, respectively), Needle Square Spherical Hexagon 0 2 4 6 8 Fold change CD86 Day 1 CD206 Arg Needle Square Spherical Hexagon 0 2 4 6 8 Fold change CD86 Day 2 CD206 Arg (A) (B) 50 while much lower levels of Arg1/CD206 gene expression (M2 macrophage markers) were induced at both time points. Likewise, compared with the untreated group, both needle and hexagon Cu MOF NPs induced relatively high levels of CD86 gene expression but only on day 2 of treatment (mean fold change: 4.32 and 3.03, respectively). Even though the expression of M2 marker genes was upregulated (around 2-fold or less) in response to needle-like Cu MOF NPs, there was not a statistically significant difference, so in cells treated with this group for 2 days was highly induced the M1-associated gene. However, after 2 days of incubation, RAW 264.7 cells treated with hexagon-shaped NPs also expressed higher levels of CD206 (mean fold change: 4.27) compared with the non-treated cells, thus coexisting the two macrophage phenotypes. By contrast, for the spherical-like Cu MOF NPs, the CD86 gene expression levels were almost consistent over time, and not statically significant (approximately 2-fold increase), lower than the expression of M2 macrophage marker genes, Arg1 at 1 day of culture, and CD206 after 2 days of treatment, that reached 3.68-fold increase. Therefore, the above results indicated not only that RAW 264.7 cells treatment with square and needle Cu MOF NPs for 2 days clearly induced the immune response toward M1 polarization, but also that the treatment with spherical MOF NPs just promotes, in a significant way, an M2 polarization phenotype, thus revealing that a variation in shape could alter the ability of Cu MOF NPs to elicit macrophage polarization. Additionally, comparing these results with cytotoxicity analysis on RAW 264.7, it seems that the cytotoxicity of the four MOF NPs largely correlated with their respective ability to induce macrophage polarization, since it has been observed that needle-shaped Cu MOF NPs were more cytotoxic than hexagon and spherical ones. In fact, previous studies proposed that the cytotoxicity of different inorganic NPs is closely related to their impact on immunological response (Miao et al., 2017). For instance, Nishanth et al. reported that Au and Ag NPs with smaller sizes had a more potent effect on inducing M1 polarization than their larger counterparts, as determined by a higher expression of TNF-α and IL-6 in RAW 264.7 and J774A.1 macrophages. Also, they found that NPs cytotoxicity increased as their size decreased, which could be a reason for their size-dependent macrophage polarization (Nishanth et al., 2011; Yen et al., 2009). Thus, the degree of induced macrophage polarization toward the M1 subtype by NPs has previously been related to their greater cytotoxicity. 51 4.5 Cytokine Production Measure by ELISA To assess the capability of differently shaped Cu MOF NPs to induce macrophage polarization, the cytokine release by RAW 264.7 cells was evaluated after treatment with MOF NPs at 75 μg/mL for 24 h and 48 h. The levels of pro-inflammatory cytokines (TNF-α and IL-12) and anti-inflammatory cytokines (IL-4 and IL-10) are presented in Figure 21. Standard curves were generated for each cytokine, by plotting the mean absorption against cytokine concentration (Appendix 2), allowing to determine cytokine concentrations for each experimental condition. Cytokine production was roughly in line with gene expression data. As shown in Figure 21A, the pro-inflammatory cytokine, TNF-α, was significantly upregulated in cells treated with the square and needle Cu MOF NPs, when compared with the non-treated group, both at days 1 and 2. Likewise, the spherical Cu MOF NPs-treated group expressed higher concentrations of TNF-α compared with the control group, albeit the difference was only statistically significant at 2 days of culture. By contrast, there were no statistically significant differences in TNF-α concentration between the hexagon-shaped Cu MOF NPs-treated group and the control group, either at day 1 or day 2. Additionally, quantified results using ELISA revealed that all four MOF NPs induced significant release of IL-12 (Figure 21B) at both time points, with the needle, spherical and square shapes exhibiting a stronger effect than the hexagon shape. In fact, the IL-12 levels secreted by hexagon Cu MOF NPs-treated cells demonstrated a time-dependent reduction, therefore suggesting the absence of a persistent inflammatory response. The anti-inflammatory cytokine, IL-10 at day 1 was downregulated in all the cells treated with differently shaped Cu MOF NPs except on cells incubated with hexagon MOF NPs, where there was a statistically significant increase (Figure 21C). The decrease in IL-10 was more obvious and there was a statistically significant difference between the square-shaped Cu MOF NPs-treated group and the untreated group at 2 days after treatment. The concentration of another anti-inflammatory cytokine, IL-4, was also measured (Figure 21D) but, in general, the concentrations were quite low, close to the recommended detection range (8 pg/mL according to the ELISA kit). Note that the maximum value of the yaxis is 20 pg/mL, unlike the other represented graphs. One possible explanation for this is that IL-4 is mainly secreted by Th2 cells as reported by many previous studies (Bao et al., 52 2015). Despite that, the lowest amount of cytokine was secreted at day 1 by the RAW 264.7 cells incubated with square and spherical MOF NPs. No other statistically significant differences were observed. Figure 21. ELISA results of cytokine production by RAW 264.7 cells: (A) TNF-α and (B) IL-12, both proinflammatory cytokines; (C) IL-10 and (D) IL-4 both anti-inflammatory cytokines. The data are presented as means ± SD (n=3). (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Altogether, these data were consistent with previous observations and indicated that the treatment with square and needle Cu MOF NPs increased the secretion of two proinflammatory cytokines (TNF-α and IL-12) and, in the case of square-shaped NPs, significantly decreased the secretion of anti-inflammatory cytokine IL-10, which is characteristic of an upregulation of inflammation and shift toward M1 macrophage activation (Ding et al., 2023). In contrast, treatment with the hexagon-shaped Cu MOF NPs for 24 h increased anti-inflammatory cytokine secretion as compared to non-treated cells, whereas after 2 days, RAW 264.7 cells exhibited lower expression levels of antiinflammatory cytokines. (A) (C) (B) (D) 53 Although the exact mechanism requires further investigation, it was hypothesized that the differential ability of the four Cu MOF NPs to modulate macrophage polarization could be not only due to the differences in cellular internalization but also to the intracellular mechanisms, including ROS generation and lysosomal damage, as reported in previous studies for other types of NPs (Baranov et al., 2021; Ding et al., 2023). Activation of cellular signal and immune response, in turn, likely depends on the amount of NP intracellular accumulation, that is determined by NPs uptake and endocytosis by macrophage (Andrade et al., 2021). As discussed above, it was hypothesized that Cu MOF NPs shape could affect its ability to enter cells or, in other words, the endocytic uptake of NPs. For example, Li et al. have found that spherical glycol-nanoparticles (GNPs) were internalized more by RAW 264.7 macrophages than cylindrical GNPs. Besides that, they discovered that the differences in the endocytosis pathways between spherical and cylindrical GNPs could explain this observation (Li et al., 2016). Endocytosis is usually subdivided into two internalization mechanisms, namely phagocytosis and pinocytosis. Pinocytosis occurs in all cells through four main engulfment mechanisms, specifically macropinocytosis, clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrinand caveolin-independent endocytosis (Hadji et al., 2022). According to the literature, particles >0.5μm are ingested by phagocytosis, while smaller particles are internalized by endocytosis which can be clathrin or caveolin-mediated (Baranov et al., 2021). Since some of the shapes of the Cu MOF NPs synthesized (e.g., needle-shaped NPs) can reach this size threshold, these three endocytic pathways (phagocytosis and clathrin or caveolin-mediated endocytosis) were hypothesized as possible routes for NPs to enter the cells. Also, Cu MOF NPs can be described by their aspect ratio (AR), which is the ratio between their length axis (axis a, see Figure 22) over their diameter axis (axis b) (e.g., spherical-like particles AR=1). The AR is commonly used to compare different shapes of NPs (Awashra et al., 2023). 54 Figure 22. Theoretical predictions of uptake efficiency of spherical and non-spherical Cu MOF NPs (hexagon, square, and needle) determined by their shape. Created with BioRender.com. During nonphagocytic endocytosis, the energy of uptake depends on the bending properties of the membrane and the strength of receptor interactions, and overall endocytosis is more efficient (lower free energy requirement) with a stronger adhesiveness and a larger contact area (Agudo-Canalejo et al., 2015). Because of this, spherical NPs are predicted to be engulfed more efficiently than needle and square NPs (Figure 22), since they have an optimal contact area and therefore are engulfed faster (Baranov et al., 2021). In contrast, particles with extreme shapes, characterized by either high or low ARs, probably possessed hindered uptake because of the reduced contact area, which leads to less ligandreceptor interactions, and due to the greater membrane wrapping time required for the extended NPs (Baranov et al., 2021; Li et al., 2016). Similarly, NPs with high or low ARs are expected to exhibit negligible phagocytosis compared to spherical NPs mainly due to differences in the actin structure formed when the NP interacted with the macrophage surface. In fact, when the NP attaches to the cell, actin polymerizes at points of contact but fails to create the adequate actin structure necessary to engulf the NP (Hadji et al., 2022). As a result of this frustrated uptake, it has been observed that macrophages engulfing needleshaped crystals and fibers can end up being pierced by the needle-like structures and, consequently, start inflammation (Ernst et al., 2021). For instance, as reviewed elsewhere (Baranov et al., 2021), needle-like particles can result in inflammasome activation, as is wellunderstood for needle-shaped particles of titanium reticule, poly(ethylene oxide), gold and carbon, and other materials (Lebre et al., 2017; Palomäki et al., 2011). The activation of the inflammasome by needle-shaped particles depends on ROS production and cathepsin B, and although the precise mechanism and the contribution of membrane destabilization in this process are less clear, it likely relates to frustrated endocytosis/phagocytosis and the leakage 55 of ROS and lytic enzymes in the extracellular environment (Rabolli et al., 2016). Several studies revealed that macrophages displayed M1-like phenotypic characteristics when the inflammatory free radical levels of ROS were upregulated (Rakshe, 2018). In future investigations, to determine the uptake mechanisms of various Cu MOF NPs, specific inhibitors can be selected to treat the cells before incubation with different Cu MOF NPs. Still, RAW 264.7 macrophages are known to register higher levels of particle uptake due to the phagocytic nature of these cells (Zhao et al., 2013). Thus, if internalized, previous studies indicated that the shape of NPs also influences the trafficking of nanomaterial inside the cell (Panariti et al., 2012). Nanoparticles’ internalization, transportation, and degradation (Figure 23) greatly depend on the intracellular membrane structure-based organelle system such as endosomes, autophagosomes, and lysosomes. After their cell uptake, most NPs are confined in early endosomes, which mature into late endosomes and then lysosomes before exocytosis (Hadji et al., 2022). 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Samples 24 h incubation 48 h incubation Concentration (μg/ul) 𝐀𝟐𝟔𝟎/𝟐𝟖𝟎 Concentration (μg/ul) 𝐀𝟐𝟔𝟎/𝟐𝟖𝟎 Positive Control -1 313.59 2.13 944.57 2.14 Positive Control -2 204.37 2.15 1294.81 2.11 Positive Control -3 382.98 2.11 1284.69 2.13 Needle -1 258.72 2.12 358.79 2.05 Needle -2 309.83 2.13 316.93 2.07 Needle -3 223.06 2.14 734.09 2.08 Square -1 278.91 2.14 917.06 2.12 Square -2 337.56 2.13 1372.81 2.11 Square -3 300.18 2.12 426.23 2.07 Spherical -1 375.49 2.13 1170.90 2.09 Spherical -2 413.08 2.12 1105.22 2.11 Spherical -3 254.89 2.15 928.68 2.12 Hexagon -1 292.03 2.11 856.36 2.13 Hexagon -2 330.65 2.12 1067.02 2.13 Hexagon -3 269.57 2.15 1032.28 2.11 80 Appendix 2. Calibration curves for ELISA assay Calibration graph Concentration range (μg/mL) Calibration curve (y=mx+b) r2 [0;250] m=0.0017 b=0.0018 0.9989 [0;1000] m=0.0009 b=0.0030 0.9976 [0;1000] m=0.0010 b=0.0247 0.9988 [0;1000] m=0.0008 b=0.0028 0.9988 Figure A1. Calibration curves for ELISA assay. 050 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0.5 Murine IL-4 (pg/mL) Optical Density 405-650 nm Murine IL-4 R2 = 0.9989 Y = 0.001734*X + 0.001799 0250 500 750 1000 0.0 0.2 0.4 0.6 0.8 1.0 Murine TNFa (pg/mL) Optical Density 405-650 nm Murine TNFaY = 0.0008539*X + 0.003020 R2 = 0.9976 0250 500 750 1000 0.0 0.5 1.0 1.5 Murine IL-12 (pg/mL) Optical Density 405-650 nm Murine IL-12 Y = 0.001046*X + 0.02467 R2 = 0.9988 0250 500 750 1000 0.0 0.2 0.4 0.6 0.8 1.0 Murine IL-10 (pg/mL) Optical Density 405-650 nm Murine IL-10 Y = 0.0007919*X + 0.002808 R2 = 0.9988