Inorganic-templated and polymeric layer-by-layer nanocapsules for the treatment of cancer

Inorganic-templated and polymeric layer-by-layer nanocapsules for the treatment of cancer Filipa Bastos UMinho | 2023 Universidade do Minho Escola de Engenharia Instituto de Investigação em Biomateriais, Biodegradáveis e Biomiméticos Filipa Isabel Rafael Bastos Inorganic-templated and polymeric layer-bylayer nanocapsules for the treatment of cancer January 2023 Filipa Isabel Rafael Bastos Inorganic-templated and polymeric layerby-layer nanocapsules for the treatment of cancer Master Thesis Master’s degree in Biomedical Engineering Dissertation supervised by: Natália Maria de Araújo Alves Iva Hristova Pashkuleva January 2023 i DECLARATION Name: Filipa Isabel Rafael Bastos Project Title: Inorganic-templated and polymeric layer-by-layer nanocapsules for the treatment of cancer Mentors: Natália Maria de Araújo Alves; Iva Hristova Pashkuleva Conclusion Year: 2023 Master Designation: Mestrado Integrado em Engenharia Biomédica Master Branch: Biomateriais, Reabilitação e Biomecânica I declare that I grant to the University of Minho and its agents a non-exclusive license to file and make available through its repository, in the conditions indicated below, my individual project, as a whole or partially, in digital support. 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Atribuição-NãoComercial-SemDerivações CC BY-NC-ND Universidade do Minho, 31/01/2023 Signature: ___________________________________ ii AGRADECIMENTOS Esta dissertação representa o fim de um ciclo. “Estes anos são viagem” e várias pessoas se cruzaram nela, fazendo desta viagem a mais incrível de sempre. Dessa forma, não podia encerrar este ciclo sem antes lhes agradecer. Em primeiro lugar, gostaria de agradecer ao Professor Rui Reis pela oportunidade e privilégio de desenvolver a minha dissertação de mestrado no Instituto de Investigação em Biomateriais, Biodegradáveis e Biomiméticos (I3Bs), na Universidade do Minho. Agradeço à minha orientadora, Professora Natália Alves, por toda a ajuda e pela disponibilidade de desenvolver esta dissertação sob sua orientação. Muito obrigada pela oportunidade de ganhar experiência na área de investigação, pela dedicação e por todo o tempo dispensado! À minha coorientadora, Iva Pashkuleva, por todo o conhecimento transmitido, pelos conselhos e por toda a ajuda ao longo desta dissertação. Muito obrigada pela disponibilidade e pela oportunidade de poder integrar o seu grupo de investigação! Ao Rui Costa e à Diana Costa, por toda a ajuda no laboratório, bem como na análise de resultados. Obrigada pela paciência, tiveram um papel bastante importante nesta etapa e sem vocês não teria conseguido. À antiga Library Crew e agora aos novos “Arrumos”, estou eternamente grata por estarem sempre presentes. Por todas as palavras de motivação, por todos os momentos divertidos e por alegrarem sempre os dias menos bons. Por sentirem este meu fim de ciclo, como se fosse o vosso. Obrigada por serem quem são, foi uma sorte ter-vos conhecido. Que isto seja apenas um até já e nunca um adeus. À Crop, por todo o apoio que me deu, por todas as palavras, por estar sempre lá quando tudo parecia difícil, nas vitórias e nas derrotas. Por todos os momentos que passámos ao longo destes 5 anos. Contigo estes anos foram uma viagem da qual eu sempre me vou lembrar. Ao Tiago, por estar sempre ao meu lado e me transmitir paz quando tudo parecia desabar. Obrigada, também à sua família por sempre me ouvirem com interesse a falar do tema, mesmo sem entenderem do que falava. Obrigada por todo o apoio e por me ajudarem a acreditar que conseguia tudo, mesmo quando eu própria não acreditava em mim! E por fim, à minha família, em especial à minha mãe, por ter sido uma fonte de inspiração durante toda esta dissertação. Por todo o esforço que fez para que nunca me faltasse nada para alcançar tudo o que alcancei até hoje. À minha avó e à minha irmã por todas as palavras de força. Também ao meu pai, que apesar de já ter partido, sei que estaria muito orgulhoso de mim. Foram 5 anos com altos e baixos, com muitas dúvidas e incertezas. É com nostalgia que hoje termino com a certeza de que este percurso não poderia ter sido melhor. Por todas as pessoas que por ele passaram, por todas as aprendizagens, por todos os obstáculos. Um muito obrigada! iii STATEMENT OF INTEGRITY 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. Universidade do Minho, 31/01/2023 Signature: ________________________________________ iv ABSTRACT Breast cancer is one of the most prevalent diseases worldwide and the most malignant in women. Conventional treatments include a radiotherapy, which kills both healthy and cancerous cells unselectively and chemotherapy, which is more selective but fails against aggressive tumors that present drug resistance. These limitations, together with their side effects and high tumor heterogeneity, are major obstacles to treatment efficacy. Therefore, it is important to develop new classes of carriers that can deliver anticancer agents to the tumor site, while minimizing the impact on healthy cells. This project proposes a drug delivery system that involves several bioactive components to endow targeting capacity against cancer cells and in situ release of anticancer drugs. To this end, we will develop core/shell nanocapsules that are composed by calcium carbonate (CaCO3) nanoparticles with capacity for loading chemotherapeutics, and a layer-by-layer (LbL) coating comprising ligands for receptors that are overexpressed by breast cancer cells. The work plan comprises: (i) synthesis of spherical CaCO3 nanoparticles loaded with a model low molecular weight molecule ( i.e. , rhodamine), (ii) coating the nanoparticles using the polycationic poly-L-lysine and the polyanionic hyaluronic acid (HA), the latter a known ligand that target CD44 receptors overexpressed in breast cancer cells, (iii) characterization of the loading efficiency and releasing profiles in an acidic buffer that simulates a tumor microenvironment, and (iv) in vitro impact on breast cancer cell lines with different metastatic potential – MDA-MB-231 and SKBR-3. We expect that this project would contribute to the development of a new drug delivery system with an improved therapeutic response. We were able to produce nanoparticles that enabled the deposition of an LbL coating that endowed CD44 targeting. Rhodamine was loaded as a model molecule to demonstrate a pH-dependent release from CaCO3 nanoparticles. The breast cancer cells MDA-MB-231 (aggressive phenotype) and SK-BR-3 (less aggressive) showed a substantial immobilization of coated nanoparticles in the ECM compared with a control of healthy cells (MCF10A cells), consistent with the interaction of the HA in the coating with the CD44 receptors in the pericellular space and reduced metabolic activity. These findings also suggest that different receptors may mediate internalization instead of immobilization in the ECM, as our experiments showed that by blocking CD44 the nanoparticles entered the cells. These results demonstrate that LbL-coated CaCO3 systems would be an excellent family of carriers for the transport of chemotherapeutics and localized delivery targeted at the tumor microenvironment. Keywords: Layer-by-layer, drug delivery, breast cancer, calcium carbonate, bioactive components. v RESUMO O cancro da mama é uma das doenças mais prevalentes em todo o mundo e a mais maligna, nas mulheres. Os tratamentos convencionais incluem a radioterapia, que mata células saudáveis e cancerígenas de forma não seletiva e a quimioterapia, que apesar de ser mais seletiva, falha contra tumores agressivos que apresentam resistência aos medicamentos. Estas limitações, juntamente com os seus efeitos secundários, constituem grandes obstáculos à eficácia do tratamento. Dessa forma, é importante desenvolver novos portadores que possam fornecer agentes anticancerígenos ao local do tumor, minimizando ao mesmo tempo o impacto nas células saudáveis. Este projeto propõe um sistema de entrega de fármacos que envolve vários componentes bioativos com capacidade seletiva contra as células cancerígenas e a libertação in situ de agentes anticancerígenos. Para este fim, desenvolveremos nanocápsulas compostas por carbonato de cálcio (CaCO3) com carregar agentes quimioterapêuticos, e um revestimento camada por camada, compreendendo ligandos para recetores que são sobreexpressos pelas células de cancro da mama. O plano de trabalho inclui: (i) síntese de nanopartículas esféricas de CaCO3 carregadas com uma molécula modelo de baixo peso molecular (rodamina), (ii) revestimento das nanopartículas utilizando poli-lisina e ácido hialurónico cujo alvo são os recetores CD44, (iii) caracterização da eficiência do encapsulamento e perfis de libertação num tampão ácido que simula um microambiente tumoral, e (iv) impacto in vitro nas linhas celulares de cancro da mama com diferentes potenciais metastáticos. Produzimos nanopartículas que permitiram o revestimento camada por camada, que interagiu com o alvo CD44. A rodamina foi encapsulada como uma molécula modelo para demonstrar a libertação dependente do pH das nanopartículas. As células de cancro da mama MDA-MB-231 (fenótipo agressivo) e SK-BR-3 (menos agressivo) mostraram uma imobilização substancial de nanopartículas revestidas, em comparação com o controlo de células saudáveis (MCF10A), consistente com a interação do HA no revestimento com os recetores CD44 no espaço pericelular, e uma atividade metabólica reduzida. Estas descobertas sugerem também que diferentes recetores podem mediar a internalização, uma vez que os nossos ensaios mostraram que, ao bloquear o CD44, as nanopartículas entraram nas células. Estes resultados demonstram que os sistemas CaCO3 revestidos seriam um excelente candidato para o transporte de agentes anticancerígenos e entrega localizada direcionada para o microambiente tumoral. Palavras-chave: camada por camada, libertação de fármacos, cancro da mama, carbonato de cálcio, componentes bioativos. vi TABLE OF CONTENTS Declaration ....................................................................................................................................... i Agradecimentos .............................................................................................................................. ii Statement of Integrity ..................................................................................................................... iii Abstract ......................................................................................................................................... iv Resumo .......................................................................................................................................... v Table of contents ............................................................................................................................ vi List of Figures ................................................................................................................................ ix List of tables ................................................................................................................................... xi List of Abbreviations and acronyms ................................................................................................ xii Chapter 1. General Information ........................................................................................................... 1 1.1 Motivation ........................................................................................................................... 2 1.2 Breast Cancer ..................................................................................................................... 4 1.3 Current clinical treatments .................................................................................................. 4 1.3.1 Chemotherapy ............................................................................................................. 5 1.3.2 Radiotherapy ............................................................................................................... 6 1.3.3 Endocrine Therapy ....................................................................................................... 6 1.3.4 Anti-HER2 Therapy ...................................................................................................... 7 1.3.5 PD-1/PD-L1 Inhibitors.................................................................................................. 8 1.4 Emerging designs for enhanced delivery systems targeting cancer ....................................... 9 1.4.1 Nanoparticles as drug delivery systems ........................................................................ 9 1.4.1.1. Iron Oxide Nanoparticles .................................................................................... 10 1.4.1.2. Polymers nanoparticles ...................................................................................... 12 1.4.2 Layer-by-Layer assembly and its importance in targeting ............................................. 13 1.4.3 Precision Oncology .................................................................................................... 14 1.5 Proposed strategy for the treatment of breast cancer ......................................................... 14 1.5.1 Calcium Carbonate particles ...................................................................................... 15 1.5.2 Layer-by-layer ............................................................................................................ 16 1.6 References ........................................................................................................................ 17 Chapter 2. Materials and Methods .................................................................................................... 26 2.1. Materials ........................................................................................................................... 27 2.1.1. Hyaluronic Acid...................................................................................................... 27 2.1.2. Poly-L-Lysine .......................................................................................................... 27 xiii HER2 Human epidermal growth factor receptor 2 HCl Hydrochloric acid I IONPs Iron Oxide Nanoparticles K KCl Potassium Chloride L LbL Layer-by-Layer M MRI Magnetic Resonance Imaging N Na2HPO4 Sodium phosphate dibasic NaH2 Sodium phosphate monobasic NP Nanoparticle NaHCO3 Sodium Bicarbonate Na2CO3 Sodium Carbonate NaCl Sodium Chloride P PLGA Poly(lactide-co-glycolide) PTX Paclitaxel PBS Phosphate Buffered Saline PDI Polydispersity Index PDEGMA Poly (di(ethylene glycol) methyl ether methacrylate PLL Poly-L-Lysine PD-1 Programmed Cell Death 1 PD-L1 Programmed Cell Death Ligand 1 R REDOX Oxidation-reduction RHAMM Receptor for Hyaluronic acid-mediated motility Rho Rhodamine RT Radiotherapy RNA Ribonucleic Acid rpm Rotation per Minute xiv S SEM Scanning Electron Microscope SERMs Selective Estrogen Receptor Modulator SPIONs Superparamagnetic Iron Oxide Nanoparticle T TNBC Triple Negative Breast Cancer TME Tumor Microenvironment W WHO World Health Organization xv “Enquanto não alcances Não descanses. De nenhum fruto queiras só metade.” Miguel Torga CHAPTER 1. GENERAL INFORMATION CHAPTER 1GENERAL INTRODUCTION 2 CHAPTER 1GENERAL INTRODUCTION This first chapter presents the motivation behind the dissertation as well as an overview of breast cancer, typical treatments and current research for treatments. 1.1 MOTIVATION Cancer is an uncontrolled growth of dysfunctional cells that can spread to other cells, organs and to other parts of the body. Benign tumors do not spread like malignant tumors, and usually do not require treatment or removal. Malignant tumors are treated by surgical removal, radio/chemotherapy, or combination of these. The treatment is often complicated due to development of metastases and multiple resistance, thus making cancer one of the deadliest diseases in modern societies [1]. According to Dyba et al. , there were approximately 2.7 million new cases of cancer in Europe in 2020. The different types of cancers that affect humans can be classified according to the affected anatomical part, the severity, and the metastatic potential. Four types of cancer represent about half (49.7%) of the overall burden: breast cancer (13.3%), colorectal cancer (12.7%), lung cancer (11.9%) and prostate cancer (12.5%) [2]. In 2020, according to Sung et al . there were 19.3 million new cases of cancer worldwide, where the most diagnosed type in both genders was breast cancer (11.7%), followed by lung cancer (11.4%), colorectal cancer (10%), prostate cancer (7.3%) and stomach cancer (5.6%) (Figure 1.1, left) [3]. In women, the breast cancer was most prevalent (24.5%) (Figure 2, left) and in men this was the lung cancer (14.3%) (Figure 1.2, right). According to the same study, there were estimated 9.9 million deaths caused by cancer in 2020, with lung cancer classified as the deadliest (18%) followed by colorectal (9.4%), liver (8.3%), stomach (7.7%), and female breast cancer (6.9%) (Figure 1.1, right). In 12% 10% 11% 7% 6% 54% Incidence in both sexes Breast cancer Colorectal Lung cancer Prostate Cancer Stomach Others 18% 10% 7% 7% 8% 50% Mortality in both sexes Lung Cancer Colorectal Cancer Stomach Breast Cancer Liver Others Figure 1. 1Distribution of new cases and deaths in both sexes. Adapted from [3]. CHAPTER 1GENERAL INTRODUCTION 3 women, breast cancer was also the most common cause of death (15.5%) (Figure 1.3, left), and in men the major cause of death continued to be lung cancer (21.5%) (Figure 1.3, right) [3]. Early detection and diagnosis decrease the mortality [4]. However, the symptoms are usually felt only when the tumor is already developed, i.e. it is difficult to diagnose cancer at an early stage when the available therapies are more efficient. The most common symptom is the growth of a mass or the process of ulceration. The current treatments for cancer are still limited: the effect of therapeutic agents depends on its targeted delivery through the hyperdeveloped extracellular matrix of tumor cells. A lot of effort has been put into targeting caveolin endocytic pathways [5], though due to the complex biochemistry of cancer, it is necessary to develop selective biomaterials/carriers that target multiple receptors that are overexpressed in cancer cells [6]. Therefore, in this study a drug delivery system that involves several bioactive components to endow targeting capacity against cancer cells and in situ release of anticancer drugs is proposed. Core/shell nanocapsules composed by calcium carbonate nanoparticles with capacity Figure 1. 2Distribution of new cases in women and men. Adapted from [3]. Figure 1. 3-Distribution of deaths in men and women. Adapted from [3]. CHAPTER 1GENERAL INTRODUCTION 4 for loading chemotherapeutics, and a layer-by-layer (LbL) coating comprising ligands for receptors that are overexpressed by the breast cancer cells will be developed. 1.2 BREAST CANCER Breast cancer is highly heterogeneous and metastatic. An early diagnostic of this cancer is crucial not only to prevent tumor metastasis to different organs such as liver, lung, and brain, but also to increase the good prognosis and survival rate [4], [7]. The probability of developing breast cancer is related to some factors like gene mutation, estrogen levels, family history, lifestyle, aging, and gender. Routine exams by mammography and Magnetic Resonance Imaging (MRI) are the most common preventive practices and clinical means for diagnosis [4]. At molecular level, different types of breast cancer can be identified by immunohistochemistry using the hormone receptors (estrogen receptor (ER) and progesterone receptor (PR)), human epidermal growth factor receptor (HER2) and proliferation protein Ki-67 as markers [8], [9].Triple negative breast cancer (TNBC), as the name suggests, is characterized by the absence of ER, PR and HER2 overexpression and represents 15% of breast carcinomas [10]. TNBC is considered the most aggressive type of breast cancer, where the only treatment available is chemotherapy, because cells lack the typical biomarkers that can be targeted, i.e. current endocrine and anti-HER2 therapies are ineffective [11]. Luminal A breast cancer is ER and PR positive, HER2 negative and there is low expression of Ki-67 [9]. For this type of cancer, endocrine therapy is usually applied [12]. Luminal B breast cancer is ER positive, but there are two subtypes depending on HER2 status: for HER2 negative subtype (expression of PR is low or negative and expression of Ki-67 is high [9]), cytotoxic and endocrine therapies are recommended [12], while for HER2 positive subtype (no expression of PR and Ki-67) [9], anti-HER2 therapy is recommended along with a cytotoxic endocrine therapy [12]. Despite TNBC being the more invasive breast cancer, the cancer where ER expression is positive is the most common and the one responsible for most of the deaths [13]. 1.3 CURRENT CLINICAL TREATMENTS Table 1.1 gives an overview of the current clinical therapies as well as their advantages and disadvantages. The most common therapies are described in detail in the following subsections. CHAPTER 1GENERAL INTRODUCTION 5 1.3.1 CHEMOTHERAPY Chemotherapy is the most common treatment for breast cancer and the only treatment that provides an efficient therapeutic response in TNBC. It can be also applied in HR positive breast cancers but usually only at an advanced stage [14], [15]. There are different chemotherapeutics according to the cancer stage and the patient tolerance [16]. Anthracyclines, such as doxorubicin and epirubicin, are used to treat different types of cancer [17]. Doxorubicin has the capacity to intercalate inside DNA base pairs, thus inhibiting DNA and RNA synthesis and breaking DNA strands, causing an apoptosis [18]. For adjuvant chemotherapy (after surgery), an anthracycline-taxane therapy is recommended for patients who can tolerate it (usually for high-risk patients, i.e. with tumor that has size > 5 mm) [16]. Neoadjuvant chemotherapy (before surgery) is not common, but the use of carboplatin has shown some positive results in patients who received it. Nevertheless, this treatment is accompanied by a high toxicity [19]. The main cause of chemotherapy inefficiency is the development of drug resistance [20]. The problem is caused partly by a small population of cancer stem cells (CSC), capable of self-renewal, that can differentiate into cancer cells (Figure 1.4) [20]. CSCs are chemoresistant and after treatment their population is enriched and enter in a transient inactive status that could lead into metastasis or tumor recurrence [21]. This phenomenon is more concerning in TNBC since it is the type of breast cancer that has the most CSCs [16]. Figure 1. 4-Schematic presentation of the CSCs involvement in the development of drug resistance [16]. CHAPTER 1GENERAL INTRODUCTION 6 Secondary effects are the major drawback of chemotherapy: 79% of the patients with breast cancer suffer one side effect, 9% suffered one to three side effects, 5% experienced four to five and 7% six or more side effects. Side effects included chest pain, fatigue, constipation, diarrhea, pain, rash, mucositis, dyspnea, and vomiting [22]. 1.3.2 RADIOTHERAPY Radiotherapy (RT) is another approach used in 50% of all cancer patients. RT is commonly applied in combination with chemotherapy and/or surgery and aimed to reduce the risk of recurrence [23], [24]. Usually RT uses high-energy photons (6-25 MV) focused deep in the tissue while protect the skin from the radiation [23]. The major drawback of this therapy is the radiation effect over the healthy tissue [24]: RT is nonspecific, killing not only cancer cells but also healthy cells, and thus different side effects can be observed. Some studies have already proved that breast cancer patients are experiencing more severe psychosocial side effects than the patients with other cancers [25], and RT in breast cancer patients has been associated with an increase of heart diseases [24]. 1.3.3 ENDOCRINE THERAPY Endocrine therapy (ET) is used to treat luminal breast cancer. Using selective ER modulator (SERMs) or selective ER degraders (SERDs), ET can directly target the ER. It can also work with aromatase inhibitors by blocking estrogen synthesis in premenopausal patients with ovarian function suppression or postmenopausal patients, avoiding tumor cell replication and the activation of the ER pathway signaling [13]. Tissues that produce androgens, such as ovaries, adipose tissue, breast, and others, convert androgen into estrogen by aromatase. The ER dimerizes and translocate to the nucleus, after bond to estrogen, where ER dimers bind coactivators (CoA) to form a transcription active complex ER (Figure 1.5A) [26]. With SERMs, like tamoxifen, the binding of estrogen to ER are completely inhibit. SERMbound ER dimers interact with the chromatin at estrogen response elements (ERE). Nevertheless, SERMbound ER dimers are related with corepressors (CoR), that inhibit ER transcription in the breast (Figure 1.5B) [26]. Finally, with aromatase inhibitors the production of estrogen is blocked by the inhibition of aromatization of androgens to estrogens (Figure 1.5C) [26]. CHAPTER 1GENERAL INTRODUCTION 7 Tamoxifen is the most used agent for this therapy, over all stages of luminal breast cancer. Tamoxifen showed 53% of clinical benefit rate, a global response rate of 34% [13] and belongs to the list of essential drugs for the treatment of breast cancer issued by the World Health Organization (WHO). It is used not only to treat the cancer tissue but also to prevent recurrence after surgical removal of the tumor or in patients with high-risk to develop breast cancer. Aromatase inhibitors (AI) such as anastrozole, exemestane and letrozole are also used for ET [14]. Aromatase is the enzyme responsible for the synthesis of estrogens from androgenic substrates and AI suppress estrogen levels, inhibiting these enzyme [13]. AI are used for treatment of postmenopausal women, including women with ovarian suppression or when at least one ovary was removed [14]. 1.3.4 ANTI-HER2 THERAPY This therapy is applied for patients with luminal B breast cancer (HER2 positive). Different approaches have been developed to target HER2. These contain antibodies such as trastuzumab which targets domain IV of the receptor and pretuzumab which binds to domain II and inhibits the heterodimerization of HER2 with other ErbB receptor [27]. The first antibody used in clinics and approved by FDA, was the trastuzumab in combination with chemotherapy. Trastuzumab is a murine monoclonal antibody that targets HER2 [28]. Clinical studies demonstrated that this antibody increases the benefit of chemotherapy in luminal B breast cancer. A major side effect is cardiac dysfunction, observed mostly when a combinatory therapy of anthracycline and trastuzumab was applied [28]. A B C Figure 1. 5-Scheme of endocrine therapy. (A) Mechanism without treatment. (B) Mechanism of inhibition with SERMs. (C) Mechanism with aromatase inhibitors [26]. CHAPTER 1GENERAL INTRODUCTION 14 1.4.3 PRECISION ONCOLOGY Precision oncology is a branch of precision medicine, an approach of treatment and prevention that considers individual variation of environment, genes, and lifestyle, focuses on oncological diseases [60]. Despite precision medicine is already applied during the practice of medicine, oncological diseases are the prime candidate for genomic treatment. It is important to understand the different mutations that occurs in cancers to administrate the adequate drug [61]. Recently, molecular therapeutic agents targeting specific actionable molecular modification were established with success, demonstrating that the use of molecular-based therapy to treat cancer is positive [62]. In a study conducted by Sultova et al. [63], it was discovered more than 30 mutations occurring in breast cancer, such as PIK3CA, ERBB2, KRAS and CCND1 mutations. Precision oncology is in constant evolution and different targets are being developed where different targets are being developed and respective therapies approved [63]. In 2019, FDA already approved Alpelisib, an oral α-specific PI3K inhibitor, to treat women with PIK3CA mutation, postmenopausal women with HR overexpressed and lack of amplification of HER2 and advanced or metastatic breast cancer followed with an endocrine therapy [64]. Although this therapy seems to be a good approach, the associated costs are still high for most patients. It is important to analyze the therapy cost-effectiveness in order to support their translation into clinical practice, e.g. establishing research programs and clinical trials [65]. 1.5 PROPOSED STRATEGY FOR THE TREATMENT OF BREAST CANCER The vast amount of therapies developed against cancer have been insufficient to lower the societal impact of this disease. The driving hypothesis of this project is that coating of drug-loaded nanoparticles with specific ECM-derived ligands will increase the internalization of chemotherapeutics by breast cancer cells. To this end, we will develop calcium carbonate (CaCO3) nanoparticles as carriers for chemotherapeutic substances and coat them in a multilayered fashion with HA to target the CD44 receptors overexpressed in different aggressive breast cancers. This system could mitigate several problems of current cancer treatments, such as low encapsulation efficiency and off-targeted delivery. The latter is particularly important in cases of TNBC since the receptors targeted by conventional therapies are missing. A structure-activity relationship for this system will be established by assessment of the response of cancer cells with different aggressiveness. CHAPTER 1GENERAL INTRODUCTION 15 1.5.1 CALCIUM CARBONATE PARTICLES Nanoparticles concede stability to circulating drugs and thus improve their bioavailability and efficiency [66]. CaCO3 is an inorganic material that shows pH-dependent dissolution, low toxicity, and slow biodegradability. CaCO3 nanoparticles are easy to synthesize from commercially available and cheap reagents [67]. These nanoparticles are good candidates for tumor therapy due to their pH-sensitiveness: since the tumor microenvironment tends to be more acidic than normal tissue, these particles can dissolve in the cytoplasm and release the encapsulated agent(s) intracellularly [68]. There are three major anhydrous crystalline polymorphs of CaCO3: calcite, vaterite and aragonite [69]. Under ambient temperature and pressure, rhomboidal calcite is the stable phase, while spherical-vaterite and needle-like aragonite are the metastable forms that easily convert into rhomboidal calcite [68], [69]. The reaction conditions, e.g. concentrations of the reagents, use of additives and temperature [68] can be adjusted to obtain different forms. Calcite is thermodynamically stable and has been combined with different polymers to develop sustained and targeted drug release systems for cancer treatment [70]. Aragonite is the densest phase of CaCO3 and has been mostly used for bone repair [68], [71]. Vaterite is a good candidate for a controlled drug delivery system due to its larger pores, large surface area and quick decomposition under mild conditions [68], [70]. CaCO3, can also be obtained in the amorphous state but it is typically unstable in aqueous environment, limiting its bioapplications [72]. Other limitation of amorphous CaCO3 nanoparticles is their difficult synthesis that requires harsh conditions, such as high pressure, doping materials or other additives [73]. However, it has been shown that it is possible to synthesize a stable amorphous CaCO3, for example, when it is doped due to electrostatics interactions, increasing the interest on these nanoparticles as a drug delivery system [74]. In previous studies, these nanoparticles have already shown promising results as a drug carrier for different types of cancer treatment. According to Kamba et al . [75], CaCO3 nanocrystals loaded with doxorubicin demonstrated to be a good proposal as a delivery system for the treatment of osteosarcoma bone cancer, not only inhibiting MG 63 cell growth, but also showing faster release in an acidic environment (pH=4.8) than at physiological pH (7.4) [75]. In other study performed by Peng et al. [76], CaCO3 nanospheres loaded with etoposide demonstrated to be more effective than free etoposide and CaCO3. With etoposide loaded, they revealed to be cytotoxic against human gastric cancer cell line (SGC-7901) by suppressing tumor growth. Against normal cells, they showed 80% survival rate. Also, this pH-sensitive controlled release system released CHAPTER 1GENERAL INTRODUCTION 16 80% of etoposide in acidic environment and about 30% at normal pH. These results suggested that it could be a promising approach to overpass the difficulties of current cancer treatments [76]. We intend to move forward with these nanoparticles to offer a selective therapy against breast cancer. Because the tumor microenvironment of breast cancer is more acidic than normal tissue and the reported results with these NPs demonstrated the pH-selectiveness, it is indicative of a promising proposal for drug delivery in distinct types of cancer. 1.5.2 LAYER-BY-LAYER The primary goal of drug delivery systems is to deliver drugs at the proper concentration to the specific target site. The use of thin films in drug delivery has recently gained attention due to its capacity to properly load drugs and release them in a regulated way, which increases therapeutic effectiveness [77]. Techniques for creating ultrathin film devices include Langmuir–Blodgett method, self-assembled monolayer techniques and layer-by-layer (LbL) assembly [78]. Due to the lack of limits on the size or form of the substrate and the absence of high temperatures or pressure, LbL assembly is ideal for the creation of films used for drug delivery. The LbL assembly procedure involves the alternate adsorption of the interacting components, which results in the deposition of multilayer films onto the substrate's surface [79]. Its composition can be strikingly versatile: virtually any charged material can be used as a building in LbL, from inorganic particles to bioactive polymers, and these can adsorb to a chosen substrate of any shape [80]–[82]. The assembly procedure is tipically based on the alternate immersion of the substrate in polyelectrolyte solutions of opposite charge (Figure 1.6). While electrostatic interactions are commonly used to drive LbL assembly – using polyelectrolytes as building blocks – other interactions have been explored, such as hydrophobic interactions, hydrogen bonding, coordination chemistry, stereo complexation, among others [80], [83]. Depending on the materials employed in the specific multilayer film carrying the drug, these features enable the regulated release of the drug. As a result, the LbL process may be regarded as the best technique for creating nanomultilayer films containing therapeutic compounds [77]. Some examples of polyelectrolytes used in LbL are the cationic poly-L-lysine (PLL) and the anionic HA. These materials have been selected to conduct this project based on their properties. PLL is a cationic polypeptide thanks to the presence of amino groups in its structure. It is commonly used in promoting cell adhesion to solid substrates, and the attachment of bioactive molecules [84], thus assuming not a bioactive role but a structural one. HA is a linear glycosaminoglycan composed of one N -acetylated CHAPTER 1GENERAL INTRODUCTION 17 glucosamine and one glucuronic acid. The latter presents the carboxyl groups that render HA negatively charged and has a pKa of around 3.5 [85]. As previously mentioned, HA is indispensable to target the overexpressed CD44 receptors by establishing monovalent bonds with CD44, thus avoiding receptor clustering [86], [87]. Despite CD44 being the primary binding receptor for HA, that bonding depends on the type of cell involved since the expression of CD44 may vary according to the aggressiveness and metastatic potential of the cells[88]. Because of that, CD44 can be used to discriminate between normal and cancer tissues in a targeted therapy [89]. 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Additionally, rhodamine was encapsulated. Scanning electron microscopy (SEM) was used to confirm the geometry of the nanoparticles and dynamic light scattering (DLS) allowed to measure the nanoparticle dimensions and aspect ratio. Fluorescence and confocal microscopy were used to show the fluorescent molecules immobilized in the interior (rhodamine is fluorescent in the far-red spectrum). The surface charge of CaCO3 was assessed by zeta potential measurements. 2.2.1.1. PURE CACO3 To make pure CaCO3, 5 M CaCl2 and 1 M Na2CO3 solutions were first produced in distilled water. The reaction was then initiated by adding 650 µL CaCl2 to 125 µL distilled water, followed by 2.5 mL Na2CO3. This mixture was regularly stirred at 650 rpm. 5 mL of distilled water was added after approximately 16 hours and stirring was stopped after 5 minutes. Figure 2. 6Methods of preparation of CaCO3 particles. In methods A and B, H2O is used as solvent. H2O or a solution of Hep are placed in the beaker where the coprecipitation is induced. CaCl2 and Na2CO3 are added subsequently in this order. In method C, a mixture of 1:5 H2O/EG is used as solvent. Na2CO3 is prepared first, onto which CaCl2 is poured. CaCO3 particles are obtained from all methods. CHAPTER 2MATERIALS AND METHODS 31 2.2.1.2. HEP-CACO3 The above-described method was modified by replacing the 125 µL of distilled water by Hep solutions with concentrations ranging from 0.038 to 4.77 mg∙mL-1. Hep-CaCO3 loaded with Rho were produced by adding Rho to the Hep solution at a concentration of 100 µg∙mL-1. 2.2.1.3. EG-CACO3 1 M Na2CO3 and 0.4 M CaCl2 were first prepared in distilled water. For each solution, 1 mL was added to 10 mL of 1:5 H2O:EG mixtures. Then, the mixture containing CaCl2 was poured into the Na2CO3 mixture under agitation (600 rpm) and stopped after 30 s, 1 h, 1 h 30 min, 2 h, 3 h, 4 h and 24 h. EG-CaCO3 was further loaded with Rho by adding 1 mL of 100 gmL-1 Rho to the Na2CO3 mixture. 2.2.2. MULTILAYER COATING OF THE NANOPARTICLES The CaCO3 nanoparticles were suspended successively in polyelectrolyte solutions – PLL and HA (2 mg/mL in 0.15 M NaCl) under mild agitation (250 rpm). After each deposition step, the nanoparticles were isolated from the solution by low-speed centrifugation (150 rcf, 3 min). The supernatant was removed with the aid of a micropipette and replaced by the next coating solution. The incubation with the polyelectrolytes was intercalated with NaCl for washing and removing loosely bonded polyelectrolytes. The procedure was repeated until 1, 2 and 3 bilayers were assembled. The coated nanoparticles were designated as "nanocapsules". The assembly of the coating was followed by measuring the zeta potential after each deposited layer, which showed a charge reversal corresponding to the sign of the last polyelectrolyte. 2.2.3. DRUG RELEASE In vitro release profiles of Rho were examined at the same conditions of buffer, pH and temperature. After the EG-CaCO3 synthesis, the NPs were stored in acidic PBS (6.3) and normal pH PBS (7.4). To produce this acidic PBS, the following reagents were added in the corresponding order and dissolved in 200mL of water gradually. First, we added 1.64g of NaCl and dissolved in 150mL of water. Then, we added 44.73mg of KCl, 60.76 mg of sodium phosphate dibasic (Na2HPO4) and in the last 188.61 mg of sodium phosphate monobasic (NaH2). At the end, 50mL were added to make up the 200mL. CHAPTER 2MATERIALS AND METHODS 32 At 0h, 1h, 3h, 1 day, 3 days and 7 days, three 400µL aliquot were removed for quantification and dissolved in HCl. The fluorescence of the aliquots was measured to determine the remaining Rho. For this purpose, a fluorescence spectrometer was used to identify the maximum excitation/emission wavelengths at maximum peak. At all time-points, the leached mass of Rho was quantified, and the release profile compared to known drug release models: zero order (independent of the drug concentration), first order (directly proportional to the drug concentration), Higuchi (pure drug diffusion from a homogeneous matrix) and Korsmeyer-Peppas (dependent on diffusion and dynamic relaxation/swelling mechanisms), represented by Equations 1 to 4, respectively [25]. Zero-order release: 𝑄𝑡= 𝑄0+ 𝐾0𝑡 (1) where Qt is the cumulative amount of protein released at time t , Q0 is the initial amount of protein and K0 is the rate constant for the zero-order model. First-order release: log(𝑄𝑡) = log(𝑄0) + 𝐾𝑡 2.303 (2) where Qt is the cumulative amount of protein released at time t , Q0 is the initial amount of protein and K is the rate constant for the first-order model. Higuchi model release: 𝑄 = 𝐾𝐻𝑡12 ⁄ (3) where Q is the cumulative amount of protein released at time t and KH is the rate constant for the Higuchi model. Korsmeyer-Peppas model release: 𝑀𝑡𝑀∞= 𝐾𝑡𝑛 ⁄ (4) where 𝑀𝑡𝑀∞ ⁄is the fraction of protein released at time t , K is the rate constant for the Korsmeyer model, and 𝑛 is the exponent that characterizes the release mechanism. Approximation of Rho release to these models is expected to reveal the phenomena governing the eventual coating defoliation or core dissolution. CHAPTER 2MATERIALS AND METHODS 33 2.2.4. IN VITRO CULTURE CELLS Studies of cellular response were performed with the aim of evaluating the effect of the nanoparticles in two breast cancer cell lines: MDA-MB-231 and SK-BR-3 and a non-tumorigenic MCF10A breast epithelial cell line. These two cancer cell lines were selected due to their different aggressiveness: MDA-MB-231 have an aggressive phenotype, whereas SK-BR-3 are not aggressive. Cells were seeded on tissue culture polystyrene (15 000 cells/cm2) and growth in Dulbecco's Modified Eagle Medium (DMEM) high glucose culture medium with 10% fetal bovine serum (FBS) and 1% antibiotics/antimycotics at 37°C and 5% CO2. The medium was replaced every 2 or 3 days with fresh DMEM. When cell reach 80% of confluency, they were trypsinized ( 0.05% trypsin/0.53 mM EDTA (37 °C, 5% CO2, 5 min) and cultured in contact with CaCO3 nanoparticles ( NPs suspension in culture media). Cells were seeded in 48 well plates (15000 cells∙cm-2). After 24 h, approximately 4×108 uncoated or LbL-coated EG-CaCO3 nanoparticles dispersed in 2.5 µL of PBS (pH 7.4) were added to each well containing 500 µL of medium, and incubated for 1, 2 and 3 days at 37 °C and 5% CO2. All procedures from the synthesis of nanoparticles to their coating were performed in sterile conditions inside a laminar flow chamber and using filtered (cut off 0.22 µm) solutions. 2.2.5. METABOLIC ACTIVITY AND CELL VIABILITY Metabolic activity and Live/Dead assays were performed after pre-determined culture periods (1, 2, and 3 days). To access cell metabolic activity, the AlamarBlueTM testwhich contains the active agent resazurin - was performed by reading the fluorescence of this reagent in black 96well plates (λex=560 nm; λem=590 nm) using a microplate reader (Synergy HT, Bio-TEK, USA), after 4 h incubation. Three separate experiments were carried out, and each condition was assessed in triplicate. Statistical significance between groups was determined by one-way ANOVA and compared using the Shapiro-Wilks test (GraphPad Prism version 8.0.1, San Diego, CA). The levels of significance for statistical differences were set to p<0.05(*), p<0.01(**), and p<0.001(***). Live/Dead assays showed the number of living and dead cells by staining with calcein AM (live, green) and propidium iodide (red, dead). Stained cells were observed by a confocal laser scanning microscope (CLSM, Zeiss AiryScan 2 model LSM 980, Germany). CHAPTER 2MATERIALS AND METHODS 34 2.2.6. CACO3 INTERNALIZATION WITH AND WITHOUT CD44 RECEPTOR BLOCKAGE The internalization of uncoated and LbL-coated EG-CaCO3 nanoparticles was evaluated 24 h after the nanoparticles were added to the culture. (CLSM, Zeiss AiryScan 2 model LSM 980, Germany). Because Rho fluoresces in the red spectrum, it was possible to see the nanoparticles and identify where they were located. To probe if the internalization is CD44-dependent, the CD44 receptors were blocked by CD44 blocking antibody (KM201, Abcam). Twenty-four hours after seeding (15000 cells∙cm-2) cell monolayers were incubated with 200 µL of complete DMEM supplemented with the CD44 blocking antibody (10 µg∙mL-1) at 37 °C in a 5% CO2 atmosphere. After 30 min, the cells were washed with PBS and nanoparticles were added, as previously described. Because HA plays important roles in TME, we also assessed the effect of the nanoparticles on HA expression (staining with labelled lectin FITC-WGA, green) and visualized the particles embedment within pericellular HA (CLSM, Zeiss AiryScan 2 model LSM 980, Germany). To confirm the pericellular HA coating, MDA-MB-231 and SK-BR-3 cells were seeded on TCPS. After 48h, which corresponds to the time for analysis of WGA staining (after incubation with NPs), cells were fixed with 10% buffered formalin (1 h, 4 °C), and stained with biotinylated HA binding protein from bovine nasal cartilage (1 μg/mL, 1 h at room temperature, Millipore) followed by incubation with streptavidin-AlexaFluor® 488 conjugate (1 μg/mL, 10 min room temperature, Molecular Probes). Images were acquired using an Inverted confocal microscope (TCS SP8, Leica). 2.3. CHARACTERIZATION TECHNIQUES Throughout this project, some characterization techniques were used. This section focuses on a brief description of these techniques. 2.3.1. DYNAMIC LASER SCATTERING (DLS) DLS uses a laser on dissolved solutions to analyze the scattered light to reveal their size- (Figure 2.7) [26]. By the measurement of Brownian motion, DLS relates this with size distribution. To the movement of particles due to random impact with molecules of a liquid that encloses the particle we called Brownian motion. An important detail about Brownian motion is that large particles tend to move slowly while small particles move quickly [27]. Through the Stokes–Einstein equation, which relates the size of a particle and its speed, it is possible to calculate the size of a particle [28]. CHAPTER 2MATERIALS AND METHODS 35 2.3.2. SCANNING ELECTRON MICROSCOPE (SEM) SEM is known for its large magnification reaching 300000x, where details and complexity that are invisible by light microscopy can be shown through this technique [29]. It depends on electron emission, produced by an electron gun [29], [30]. Then, the anode plate accelerates the electrons and magnetic lens focuses them. The scanning coils force the electron beam to rapidly scan over an area of the specimen. Finally, the sample can be viewed in Backscattered or secondary mode via a monitor [31]. SEM is an expensive technique, however it is useful because it provides a detailed grey-scale image of the sample analyzed for a wide range of materials. 2.3.3. FLUORESCENCE MICROSCOPY In this technique, the sample intended to be examined is excited through a light with a specific wavelength, usually ultraviolet (UV) or blue. Through the barrier filter that absorbs the short wavelength light used for illumination and transmits the fluorescence, the sample is analyzed, seen as a glow through the black background. Due to the observation of fluorescence in a dark background, it can also be seen where the fluorescent components are [32], [33]. Figure 2. 7Scheme of Dynamic Light Scattering [27]. Figure 2. 8-Scheme of Scanning Electron Microscope [31]. CHAPTER 2MATERIALS AND METHODS 36 The main use is to treat samples with unique fluorescent reagents to examine them. These reagents can absorb light at a specific wavelength and emit light at different wavelengths, more towards the red spectrum area. For example, when the blue light is absorbed, green light will be emitted, green will be emitted yellow, yellow will be red and invisible UV will emit visible blue light [34]. 2.3.4. CONFOCAL MICROSCOPY In confocal microscopy, a light source, often a laser, is reflected by a dichroic mirror or beam splitter and focused by an objective lens at the "Plane of Focus" level [35]. A confocal pinhole helps the microscope reject fluorescent light that is out of focus. The principle of this mechanism is that the image derives from a thin section and, when it scans various thin sections through the sample, it provides a very sharp three-dimensional image of the sample analyzed [36]. Due to its quick and inexpensive ability to acquire large tissue samples, confocal microscopy has enabled a significant advancement in biological imaging. This often includes fluorescence imaging, which is increasingly employed as a fundamental technique in biological research. Additionally, compared to pictures created by standard light microscopes, they can often have higher sensitivity, contrast, and resolution [35]. Figure 2. 9-Scheme of fluorescence microscope [34]. CHAPTER 2MATERIALS AND METHODS 37 2.3.5. ALAMAR BLUE ASSAYS AlamarBlue is a fluorescent dye that contains an oxidation-reduction (REDOX) indicator that fluoresces and changes color in response to the chemical reduction of growth medium caused by cell growth (Figure 2.11). The alamarBlue test is developed to quantitatively evaluate the proliferation of diverse human and animal cell lines, bacteria and fungusby colorimetric and/or fluorometric readingsand qualitative—a noticeable shift in color indicating the presence or absence of live cells [37], [38]. It is straightforward to execute since the indicator is water soluble, avoiding the washing/fixing and extraction procedures necessary in other frequently used cell proliferation assays [39]. Compared to standard cell viability/metabolic activity tests, the alamarBlue Assay has a number of benefits: due to its colorimetric and fluorescent properties, it provides a variety of detection techniques, being the most sensitive technique. Additionally, because there are fewer phases in the process, it can save time and it is simple to adapt [39]. Figure 2. 10Scheme of confocal microscopy [36]. Figure 2. 11Example of a plate from an AlamarBlue trial. Image from Oz Biosciences. CHAPTER 2MATERIALS AND METHODS 38 2.4. REFERENCES [1] C. E. Schanté, G. Zuber, C. Herlin, and T. F. Vandamme, “Chemical modifications of hyaluronic acid for the synthesis of derivatives for a broad range of biomedical applications,” Carbohydr. Polym. , vol. 85, no. 3, pp. 469–489, 2011. [2] N. M. Salwowska, K. A. Bebenek, D. A. Żądło, and D. L. Wcisło-Dziadecka, “Physiochemical properties and application of hyaluronic acid: a systematic review,” J. Cosmet. Dermatol. , vol. 15, no. 4, pp. 520–526, 2016. [3] A. M. Vasi, M. I. Popa, M. Butnaru, G. Dodi, and L. Verestiuc, “Chemical functionalization of hyaluronic acid for drug delivery applications,” Mater. Sci. Eng. C , vol. 38, no. 1, pp. 177–185, 2014. [4] I. Caon et al. , “Revisiting the hallmarks of cancer: The role of hyaluronan,” Semin. Cancer Biol. , vol. 62, no. July, pp. 9–19, 2020. [5] E. Karousou et al. , “Roles and targeting of the HAS/hyaluronan/CD44 molecular system in cancer,” Matrix Biol. , vol. 59, pp. 3–22, 2017. [6] G. E. S. Chaudhry, A. Akim, M. N. Zafar, N. Safdar, Y. Y. Sung, and T. S. T. Muhammad, “Understanding hyaluronan receptor (CD44) interaction, HA-CD44 activated potential targets in cancer therapeutics,” Adv. Pharm. Bull. , vol. 11, no. 3, pp. 426–438, 2021. [7] I. S. Bayer, “Hyaluronic acid and controlled release: A review,” Molecules , vol. 25, no. 11, 2020. [8] H. Zhu, R. Liu, Y. Shang, and L. Sun, “Polylysine complexes and their biomedical applications,” Eng. Regen. , vol. 4, no. 1, pp. 20–27, 2023. [9] M. Zheng et al. , “Poly(α-L-lysine)-based nanomaterials for versatile biomedical applications: Current advances and perspectives,” Bioact. Mater. , vol. 6, no. 7, pp. 1878–1909, 2021. [10] I. C. Lee, Y. C. Wu, E. M. Cheng, and W. T. Yang, “Biomimetic niche for neural stem cell differentiation using poly-L-lysine/hyaluronic acid multilayer films,” J. Biomater. Appl. , vol. 29, no. 10, pp. 1418–1427, 2015. [11] M. S. Niepel, B. K. Ekambaram, C. E. H. Schmelzer, and T. Groth, “Polyelectrolyte CHAPTER 2MATERIALS AND METHODS 39 multilayers of poly (l-lysine) and hyaluronic acid on nanostructured surfaces affect stem cell response,” Nanoscale , vol. 11, no. 6, pp. 2878–2891, 2019. [12] V. Z. Prokopovic, A. S. Vikulina, D. Sustr, E. M. Shchukina, D. G. Shchukin, and D. V. Volodkin, “Binding Mechanism of the Model Charged Dye Carboxyfluorescein to Hyaluronan/Polylysine Multilayers,” ACS Appl. Mater. Interfaces , vol. 9, no. 44, pp. 38908–38918, 2017. [13] G. Amato et al. , “Hyaluronan/poly-l-lysine/berberine nanogels for impaired wound healing,” Pharmaceutics , vol. 13, no. 1, pp. 1–11, 2021. [14] H. Yue, Y. Zhao, X. Ma, and J. Gong, “Ethylene glycol: Properties, synthesis, and applications,” Chem. Soc. Rev. , vol. 41, no. 11, pp. 4218–4244, 2012. [15] R. K. Ibrahim, M. Hayyan, M. A. AlSaadi, S. Ibrahim, A. Hayyan, and M. A. Hashim, “Physical properties of ethylene glycol-based deep eutectic solvents,” J. Mol. Liq. , vol. 276, pp. 794–800, 2019. [16] B. V. Parakhonskiy, A. Haase, and R. Antolini, “Sub-micrometer vaterite containers: Synthesis, substance loading, and release,” Angew. Chemie - Int. Ed. , vol. 51, no. 5, pp. 1195–1197, 2012. [17] E. M. Flaten, M. Seiersten, and J. P. Andreassen, “Polymorphism and morphology of calcium carbonate precipitated in mixed solvents of ethylene glycol and water,” J. Cryst. Growth , vol. 311, no. 13, pp. 3533–3538, 2009. [18] D. B. Trushina, T. V. Bukreeva, M. V. Kovalchuk, and M. N. Antipina, “CaCO3 vaterite microparticles for biomedical and personal care applications,” Mater. Sci. Eng. C , vol. 45, pp. 644–658, 2014. [19] B. Casu, “Structure and Biological Activity of Heparin,” Adv. Carbohydr. Chem. Biochem. , vol. 43, no. C, pp. 51–134, 1985. [20] R. J. Linhardt, “2003 Claude S. Hudson award address in carbohydrate chemistry. Heparin: Structure and activity,” J. Med. Chem. , vol. 46, no. 13, pp. 2551–2564, 2003. [21] A. Onishi, K. St Ange, J. S. Dordick, and R. J. Linhardt, “Heparin and anticoagulation,” Front. Biosci. - Landmark , vol. 21, no. 7, pp. 1372–1392, 2016. CHAPTER 3LAYER-BY-LAYER COATED CALCIUM CARBONATE NANOPARTICLES FOR THE TARGETING OF BREAST CANCER CELLS 46 1.5.3. DIMENSIONS AND MORPHOLOGY OF CALCIUM CARBONATE PARTICLES The diameters of CaCO3, Hep-CaCO3, and EG-CaCO3 particles were determined in wet state by dynamic light scattering (DLS, Malvern Nano-ZS equipment with a He–Ne laser at an angle of 173°) using aliquots with 1 mL loaded in polystyrene disposable cuvettes. The polydispersity index (PDI), the size distribution and z-average were determined by fitting the correlation function with the cumulant method (Zetasizer Nano v7.10 software). The presented data are average values from 35 measurements. Dry CaCO3 particles were observed by a scanning electron microscope (SEM, JEOL-JSM-6010LV, Japan). After preparation, the particles were centrifuged for 3 min at 1500 rpm and washed in ultrapure water. A 100 µL drop of each CaCO3 formulation was dispensed on glass, placed on SEM holder, and left drying for 24 h at 37 °C. The particles were then sputtered with gold and placed in the SEM sample chamber. 1.5.4. LAYER-BY-LAYER COATING OF CALCIUM CARBONATE NANOPARTICLES EG-CaCO3 nanoparticles were washed with 0.15 M NaCl and suspended alternately in 2 mg∙mL-1 solutions of PLL and HA prepared in 0.15 M NaCl under mild agitation (250 rpm) for 10 min. Each deposition step was intercalated with a washing step in 0.15 M NaCl, and for each deposition/washing step the nanoparticles were retrieved from suspension by low-speed centrifugation (1500 rpm, 3 min). The supernatant was removed with the aid of a micropipette and replaced by the next coating/washing solution. The procedure was repeated until 3 PLL/HA bilayers were assembled. The deposition of each polyion was followed by measuring the zeta (ζ)-potential of the nanoparticles using folded capillary cuvettes and the Nano-ZS equipment (Malvern, UK). 1.5.5. IN VITRO RELEASE OF RHODAMINE The in vitro release of Rho from nanoparticles (uncoated and coated with one PLL/HA bilayer) was followed at 37 °C in PBS solution with pH 6.3 (140 mM NaCl, 3 mM KCl, 2 mM Na2HPO4, 8 mM NaH2PO4). The nanoparticles were suspended in 12 mL of PBS and aliquots of 400 µL were taken at predetermined time-points: 0 h, 1 h, 3 h, 1 day, 3 days, and 7 days. The CaCO3 in the aliquots was dissolved with 3 mL of 1 M HCl to release the remaining ( i.e. , not released) Rho. The fluorescence was measured in a fluorescence spectrophotometer (JASCO model FP-8500, Japan) using an excitation wavelength of 564 nm (determined from the maximum absorbance peak of a 50 µg∙mL-1 Rho solution (Appendix III)) and emission interval between 575 nm and 650 nm. The intensity of the emitted fluorescence was read CHAPTER 3LAYER-BY-LAYER COATED CALCIUM CARBONATE NANOPARTICLES FOR THE TARGETING OF BREAST CANCER CELLS 47 at 585 nm and compared to a calibration curve of known Rho concentrations (Appendix IV). The cumulative release was represented as a percentage of the total encapsulated mass of Rho, measured at 0 h. The release profile was fitted to several known models: zero-order, first-order, Higuchi, and Korsmeyer-Peppas models to determine the mass transport mechanism involved in Rho release. 1.5.6. IN VITRO CELL CULTURE MDA-MB-231 and SK-BR-3 epithelial breast cancer cell lines and a non-tumorigenic MCF10A breast epithelial cell line were obtained from the American Type Culture Collection (ATCC). For expansion, cells were routinely cultured on 75 cm2 tissue culture polystyrene flasks with high glucose DMEM supplemented with 10% FBS and 1% antibiotics/antimycotics in an incubator at 37 °C and a humidified air atmosphere with 5% CO2. The medium was replaced every 2 or 3 days with fresh DMEM. When cells reached 80% of confluence, they were detached with 0.05% trypsin/0.53 mM EDTA (37 °C, 5% CO2, 5 min). The trypsin was inactivated by adding complete medium, and the cells were centrifuged at 300 g for 5 min. Cells were resuspended in DMEM for a new expansion or transferred to well plates for incubation experiments with CaCO3 nanoparticles. In the latter case, cells were first seeded in 48 well plates (15000 cells∙cm-2). After 24 h, approximately 4×108 uncoated or LbL-coated EG-CaCO3 nanoparticles dispersed in 2.5 µL of PBS (pH 7.4) were added to each well containing 500 µL of medium, and incubated for 1, 2 and 3 days at 37 °C and 5% CO2. All procedures from the synthesis of nanoparticles to their coating were performed in sterile conditions inside a laminar flow chamber and using filtered (cut off 0.22 µm) solutions. 1.5.7. METABOLIC ACTIVITY AND CELL VIABILITY Metabolic activity and Live/Dead assays were performed after pre-determined culture periods (1, 2, and 3 days). Metabolic activity was assessed by the AlamarBlueTM test – which contains the active agent resazurin – following the supplier’s instructions. Resazurin is reduced to the highly fluorescent resofurin in living cells. After 4 h incubation, the fluorescence was measured in black 96-well plates (λex=560 nm; λem=590 nm) using a microplate reader (Synergy HT, Bio-TEK, USA). Three independent experiments were performed and each condition was measured in triplicate. Statistical significance between groups was determined by one-way ANOVA and compared using the Shapiro-Wilks test (GraphPad Prism version 8.0.1, San Diego, CA). The levels of significance for statistical differences were set to p<0.05(*), p<0.01(**), and p<0.001(***). CHAPTER 3LAYER-BY-LAYER COATED CALCIUM CARBONATE NANOPARTICLES FOR THE TARGETING OF BREAST CANCER CELLS 48 Live/Dead assays were performed by staining living and dead cells with calcein AM (green) and propidium iodide (red), respectively. Stained cells were observed by a confocal laser scanning microscope (CLSM, Zeiss AiryScan 2 model LSM 980, Germany). 1.5.8. CACO3 INTERNALIZATION WITH AND WITHOUT CD44 RECEPTOR BLOCKAGE The internalization of uncoated and LbL-coated EG-CaCO3 nanoparticles was assessed 24 h after the addition of the nanoparticles to the culture (CLSM, Zeiss AiryScan 2 model LSM 980, Germany). Rho is fluorescent in the red spectrum and allowed visualization of the nanoparticles and determination of their localization. To probe if the internalization is CD44-dependent, the CD44 receptors were blocked by CD44 blocking antibody (KM201, Abcam). Twenty-four hours after seeding (15000 cells∙cm-2) cell monolayers were incubated with 200 µL of complete DMEM supplemented with the CD44 blocking antibody (10 µg∙mL-1) at 37 °C in a 5% CO2 atmosphere. After 30 min, the cells were washed with PBS and nanoparticles were added, as previously described. Because HA plays important roles in TME, we also assessed the effect of the nanoparticles on HA expression (staining with labelled lectin FITC-WGA, green) and visualized the particles embedment within pericellular HA (CLSM, Zeiss AiryScan 2 model LSM 980, Germany). To confirm the pericellular HA coating, MDA-MB-231 and SK-BR-3 cells were seeded on TCPS. After 48h, which corresponds to the time for analysis of WGA staining (after incubation with NPs), cells were fixed with 10% buffered formalin (1 h, 4 °C), and stained with biotinylated HA binding protein from bovine nasal cartilage (1 μg/mL, 1 h at room temperature, Millipore) followed by incubation with streptavidin-AlexaFluor® 488 conjugate (1 μg/mL, 10 min room temperature, Molecular Probes). Images were acquired using an Inverted confocal microscope (TCS SP8, Leica). 1.6. RESULTS AND DISCUSSION 1.6.1. SYNTHESIS AND CHARACTERIZATION OF CALCIUM CARBONATE NANOPARTICLES The properties of CaCO3 obtained by coprecipitation depend on the reaction conditions. Particles with different dimensions, shapes and crystallinity can be obtained from subtle changes in the time and velocity of agitation, Ca2+/CO32ratios, and the addition of doping agents, among others [33]. When using a simple coprecipitation of CaCl2 and Na2CO3 in water ( i.e., without any additives), CaCO3 particles with diameters between 2–10 µm are obtained [34]–[36]. Recent studies suggest that CaCO3 precipitation is CHAPTER 3LAYER-BY-LAYER COATED CALCIUM CARBONATE NANOPARTICLES FOR THE TARGETING OF BREAST CANCER CELLS 49 slower when Ca2+ is in excess compared to CO32- [37], which may result in formation of smaller particles. We used a Ca2+:CO32ratio of 5:1 and SEM showed formation of CaCO3 crystals with different dimensions, most of them around 700 nm (Figure 3.1A1). However, in aqueous media these particles had diameter of around 3.6 µm, which demonstrates a tendency to aggregate (Figure 3.1A2). Figure 3. 1SEM micrographs (A1, B1, C1) and representative size distribution of CaCO3, Hep-CaCO3 and EG-CaCO3 determined by DLS (A2, B2, C2). The values shown in the DLS graphs correspond to the diameter (d) and PDI of the distribution peak. To avoid aggregation, we added a charged polymer to the reaction to induce electrostatic repulsion between the particles. We chose Hep because it is the natural polyelectrolyte with highest negative charge and already used in clinics [38], [39]. First, we determined the influence of Hep on the size of the particles within a wide range of concentrations (Appendix I). As expected, the increasing quantities of Hep led to a systematic decrease of the particles diameter (from 2.7 µm for 0.038 mg∙mL-1 Hep to 835 nm for 4.77 mg∙mL-1 Hep) and PDI, which showed avoided aggregation (Figures 3.1B1, 3.1B2). Most of the obtained nanoparticles were rhombohedral. This shape is typically found in calcite particles, one of the most stable CaCO3 polymorphs [40], demonstrating that Hep has a stabilizing structural effect. (A2) CHAPTER 3LAYER-BY-LAYER COATED CALCIUM CARBONATE NANOPARTICLES FOR THE TARGETING OF BREAST CANCER CELLS 50 We also tested EG as an additive because previous studies have shown that EG can slow down the reaction and crystal growth [32]. To optimize the reaction time, we monitored the particles size during 24 h (Appendix II). Within 2 h, we obtained monodisperse particles with a diameter around 564 nm and an ellipsoid shape (Figures 3.1C1, 3.1C2), i.e. , EG-CaCO3 nanoparticles were smaller than CaCO3 and Hep-CaCO3 particles. Particles with a diameter below 1 µm (submicron scale) can be internalized by breast cancer cells [41]. The capacity of Hep-CaCO3 and EG-CaCO3 particles to carry low molecular weight therapeutics was investigated by encapsulating the model fluorophore Rho (≈500 Da) during the coprecipitation. The amount of the encapsulated Rho in each CaCO3 system was quantified by dissolving the particles in 1 M HCl: we obtained 50.4 µg of Rho per mole of CaCO3 in Hep-CaCO3 and 130.5 µg per mole of CaCO3 in EG-CaCO3, i.e. , the amount of Rho was 3-fold higher in EG-CaCO3 particles. The effect of Rho on the size of Hep-CaCO3 and EG-CaCO3 was also assessed and the DLS analysis showed a significant increase of Hep-CaCO3 diameter (Figure 3.2A, only 36% of the Rho-loaded particles were in the submicron scale), while the size of EG-CaCO3 was not altered significantly (Figure 3.2B, 100% all particles had a diameter below 1 µm). We therefore selected EG-CaCO3 nanoparticles for the following experiments. Figure 3. 2Size distribution of (A) Hep-CaCO3 and (B) EG-CaCO3 loaded with Rho. The main graphs show one representative distribution graph. The insets show the average distribution calculated from 35 measurements. CHAPTER 3LAYER-BY-LAYER COATED CALCIUM CARBONATE NANOPARTICLES FOR THE TARGETING OF BREAST CANCER CELLS 51 1.6.2. LAYER-BY-LAYER COATING AND RHODAMINE RELEASE We used Rho-loaded EG-CaCO3 nanoparticles (ζ-potential about -6 mV) as templates to assemble a LbL coating that endows CD44 targeting and control the release rate. Up to three bilayers of PLL and HA were assembled (Figure 3.3A). The polyanionic HA was the bioactive building block in this composition, whereas the polycationic PLL was employed as polycation that allowed electrostatic stabilization of the LbL build-up. The deposition of PLL and HA was confirmed by variation of the particles charge. Our results agree with previous data showing partial compensation of PLL charge by HA (ζpotential increase upon PLL deposition but does not become positive) and involvement not only of electrostatic interactions but also of other supramolecular interactions ( e.g. , hydrogen bonds) in the assembly of this polyelectrolyte pair [42]. Figure 3. 3A) Zeta-potential of the surface of Rho-loaded EG-CaCO3 nanoparticles. The cartoons represent nanoparticles coated with incremental numbers of layers. Three individual measurements are represented per layer. The line is a visual guide for the variation of the average zeta-potential values. (B) Cumulative release of Rho from uncoated (∆, orange) and LbL-coated EG-CaCO3 ( □ , blue) nanoparticles at pH 6.3 for 7 days. The curves are fittings to first-order (uncoated) and Korsmeyer-Peppas (coated) release models. Data are means ± one standard deviation. Some error bars are too small to be seen. The release of Rho from uncoated and LbL-coated nanoparticles was studied in an aqueous environment that mimics the acidic pH of the TME (pH=6.3). We used nanoparticles coated with only one PLL/HA bilayer because of the long LbL deposition times. By choosing a low number of layers, the coating time is shortened and a premature release due to long processing times is reduced. The data showed that uncoated EG-CaCO3 released 65% of Rho within 1 day, while only 17% of Rho was released from the CHAPTER 3LAYER-BY-LAYER COATED CALCIUM CARBONATE NANOPARTICLES FOR THE TARGETING OF BREAST CANCER CELLS 52 LbL-coated nanoparticles for the same period (Figure 3.3B). Within 3 days, these values increased to 70% and 30%, respectively, and did not change substantially until 7 days. Such a substantial difference (more than 2-fold) confirms that one PLL/HA bilayer was sufficient to slow the release. It is likely that the coating worked not only as a physical barrier between the nanoparticle and the medium but also as a buffer protecting the nanoparticle against dissolution. Of note, the release at neutral pH (pH=7.4) was significantly lower than at acidic conditions: only 10% of Rho were released from the coated nanoparticles after 3 days, evidencing the expected pH response from CaCO3 systems (Appendix V). To better understand the Rho release, the data were fitted to different drug release models (Appendix VI). Data for uncoated EG-CaCO3 fitted well to a first-order model of release, i.e. , Rho release decreased with time and was only dependent on the concentration of Rho within the nanoparticle. This is the expected behavior from water-soluble drugs released from inert porous matrices. For the LbL-coated nanoparticles, we found a better approximation to a Korsmeyer-Peppas model, which describes the release from dynamic polymeric matrices such as hydrogels. This model confirms the role of LbL in controlling the release of encapsulated molecules and agrees, with the data obtained for other LbL-coated CaCO3 particles [43]. 1.6.3. INTERACTION OF EG-CACO3 NANOPARTICLES WITH BREAST CANCER CELLS The biological impact of uncoated and LbL-coated EG-CaCO3 nanoparticles was investigated on epithelial breast cancer cells with different expression of CD44. MDA-MB-231 cells are aggressive and invasive phenotype that overexpress CD44, while SK-BR-3 cells have a basal expression of this receptor and are considered non-invasive [44]–[47]. MCF10A cells are used as a model for normal human mammary epithelial cells and have a low CD44 expression mainly detected in the cytoplasm [48]. We assessed the cytotoxicity of EG-CaCO3 with and without LbL coating and found no cytotoxic effect for the tested cell lines (Figure 3.4, Appendix VII). CHAPTER 3LAYER-BY-LAYER COATED CALCIUM CARBONATE NANOPARTICLES FOR THE TARGETING OF BREAST CANCER CELLS 53 Figure 3. 4Live/Dead assay of (A) MDA-MB-231, (B) SK-BR-3, and (C) MCF10A cell lines incubated with uncoated and LbL-coated EGCaCO3 nanoparticles for 3 days (green: live; red: dead). The small red dots correspond to the nanoparticles loaded with Rho. Despite the cells being alive, their metabolism was affected by the nanoparticles (Figure 3.5). The metabolic activity of MDA-MB-231 and SK-BR-3 cells significantly decreased with culture time when compared to the control. After 2 days of incubation with the uncoated EG-CaCO3 nanoparticles the metabolic activity of MDA-MB-231 (70.4%) and SK-BR-3 cells (81.2%) decreased whereas MCF10A cells (88.7%) were not affected significantly. At day 3, the decrease in the metabolic activity of MDA-MB-231 and SK-BR-3 cells decreased further (64.4% and 73.4%, respectively), showing that these nanoparticles affect both phenotypes. The LbL coating vanished partially this effect at short culture time (1 day). At longer culture times (2 and 3 days), we observed a decrease of the metabolic activity for the breast cancer cell lines, but not for the normal cell line, MCF10A, i.e. , the effect was similar to the uncoated particles. This delay in the response can be due to different interactions between cells and the coated nanoparticles that triggers different internalization mechanisms when compared with uncoated particles and/or different rate of the particles dissolution in the lysosome as shown by the Rho release data. CHAPTER 3LAYER-BY-LAYER COATED CALCIUM CARBONATE NANOPARTICLES FOR THE TARGETING OF BREAST CANCER CELLS 54 Figure 3. 5Effect of uncoated and LbL-coated EG-CaCO3 nanoparticles (NPs) on metabolic activity of MDA-MB-231, SK-BR-3 and MCF10A cell lines after 1, 2 and 3 days of incubation. Data are presented as a percentage of control (i.e., cells cultured without nanoparticles, dashed line corresponds to the control). Statistical differences are represented between each sample and its control for the different days with n=3. Significant differences are indicated (*p<0.05; **p<0.01; ***p<0.001). To analyze the nanoparticles’ distribution in the pericellular space and in the cells’ cytoplasm we observed the cultures by CLSM. After 1 day, uncoated EG-CaCO3 were found mainly around the cells with very few internalized (Appendix VIII). At days 2 and 3, internalization was observed for all studied cell types (Figure 3.6), regardless of the CD44 expression level. The results for the LbL-coated EG-CaCO3 nanoparticles were different - most of the particles remained attached to MDA-MB-231 and SK-BR-3 cancer cells, even after 3 days, with only few being internalized. Because we did not observe differences between MDA-MB-231 and SK-BR-3 cells (different expression of CD44), we hypothesized that the endogenous HA creates a protective pericellular coat that engages the expressed CD44 and retains the coated nanoparticles [49]. Lectin binding experiments showed higher HA deposition in the pericellular matrix of MDA-MB-231 than of SK-BR-3 cells and mainly intracellular HA in the case of MCF10A cells (Appendix IX). Indeed, we observed more nanoparticles around MDA-MB-231 than around SK-BR-3 cells, which agrees with the ticker HA coat around MDA-MB-231 when compared to SK-BR-3 (Appendix X) [47]. CHAPTER 3LAYER-BY-LAYER COATED CALCIUM CARBONATE NANOPARTICLES FOR THE TARGETING OF BREAST CANCER CELLS 55 Figure 3. 6Distribution of uncoated and LbL-coated EG-CaCO3 nanoparticles after 3 days of incubation with (A) MDA-MB-231, (B) SK-BR-3, and (C) MCF10A cell lines (red: Rho). To understand if CD44 was involved in the internationalization of the nanoparticles, we blocked this receptor before incubation of the cells with the nanoparticles. Upon CD44 blocking, we observed enhanced internalization of uncoated and coated nanoparticles by all studied cell types after 24 h (Figure 3.7). Interestingly, this enhanced internalization was concomitant with different distribution of the noninternalized coated nanoparticles in the pericellular space of cancer cells - they were organized in discrete clusters (Figure 3.7A2-ii, 3.7B2-ii) but not homogeneously distributed as observed in cells without CD44 blocking (Figure 3.7A4-ii, 3.7B4-ii). This difference was very pronounced for MDA-MB-231 cells. On one hand, CD44 blocking can affect the HA turnover and compromise the HA coat around the cells. On the other hand, we have recently demonstrated that the receptor for hyaluronan mediated motility (RHAMM) can compensate the blocked CD44 and make cells more sensitive to exogenous supplemented HA [46]. These results show that although CD44 receptors are not directly involved in the internalization of the nanoparticles, their presence is important because of their interactions with endogenous HA. CHAPTER 3LAYER-BY-LAYER COATED CALCIUM CARBONATE NANOPARTICLES FOR THE TARGETING OF BREAST CANCER CELLS 62 [43] R. R. Costa, C. A. Custódio, F. J. Arias, J. C. Rodríguez-Cabello, and J. F. Mano, “Nanostructured and thermoresponsive recombinant biopolymer-based microcapsules for the delivery of active molecules,” Nanomedicine Nanotechnology, Biol. Med. , vol. 9, no. 7, pp. 895– 902, 2013, doi: 10.1016/j.nano.2013.01.013. [44] C. Sheridan et al. , “CD44+/CD24-Breast cancer cells exhibit enhanced invase properties: An early step necessary for metastasis,” Breast Cancer Res. , vol. 8, no. 5, pp. 1–13, 2006, doi: 10.1186/bcr1610. [45] N. Sun, H. N. Xu, Q. Luo, and L. Z. Li, “Potential indexing of the invasiveness of breast cancer cells by mitochondrial redox ratios,” Adv. Exp. Med. Biol. , vol. 923, pp. 121–127, 2016, doi: 10.1007/978-3-319-38810-6_16. [46] A. M. Carvalho, D. Soares da Costa, R. L. Reis, and I. Pashkuleva, “RHAMM expression tunes the response of breast cancer cell lines to hyaluronan,” Acta Biomater. , vol. 146, pp. 187–196, 2022, doi: 10.1016/j.actbio.2022.05.013. [47] A. M. Carvalho, D. Soares da Costa, R. L. Reis, and I. Pashkuleva, “Influence of Hyaluronan Density on the Behavior of Breast Cancer Cells with Different CD44 Expression,” Adv. Healthc. Mater. , vol. 11, no. 4, pp. 1–9, 2022, doi: 10.1002/adhm.202101309. [48] J. Puleo and K. Polyak, “The MCF10 model of breast tumor progression,” Cancer Res. , vol. 81, no. 16, pp. 4183–4185, 2021, doi: 10.1158/0008-5472.CAN-21-1939. [49] E. A. Turley, D. K. Wood, and J. B. McCarthy, “Carcinoma Cell Hyaluronan as a ‘Portable’ Cancerized ProMetastatic Microenvironment,” Cancer Res., vol. 76, no. 9, pp. 2507–2512, 2016, doi: 10.1158/0008-5472.CAN-15-3114. CHAPTER 4. GENERAL CONCLUSIONS CHAPTER 4GENERAL CONCLUSIONS 64 4.1. CONCLUSIONS AND FUTURE WORK In this thesis, different procedures for CaCO3 particle generation were employed and evaluated for as new localized breast cancer therapy systems. Since particles obtained by conventional precipitation procedures have a tendency to aggregate and are often too large to explore targeting and cell internalization ( i.e. , in the order of few micrometers), the addition of Hep and EG helped mitigating the aggregation of the nanoparticles. Herein, more profound studies were conducted with EG-CaCO3 nanoparticles since they demonstrated higher encapsulation efficiency than Hep-CaCO3 nanoparticles and the dimensions of the systems was preserved in the submicrometric scale after loading with Rho. Crucial for this work was the demonstration that EG-CaCO3 nanoparticles are suitable templates for depositing PLL and the CD44 targeting HA using the LbL technique. This technique was a convenient method for generating nanoparticle coatings thanks to the electrostatic attraction – and to some extent, hydrogen bonding – between the used polyelectrolytes and endow targeting ability. The pH responsiveness of CaCO3 was confirmed: release of the encapsulated Rho was faster in acidic medium, useful to control the release of drugs in the acidic TME. Moreover, the LbL coating helped in slowing down the release, which opens the possibility of tuning the release further in the future. Finally, it was shown that cancer cells with different metastatic potential were inhibited by the presence of EGCaCO3. Thanks to the surface-exposed HA, the nanoparticles interacted with the CD44 of the cells’ ECM and accumulated in the pericellular region. On the other hand, blocking the CD44 receptors allowed the nanoparticles to enter the cells, leading to the conclusion that internalization is CD44-independent. These achievements are a good indicator of the feasibility of this project. Furthermore, since this work also demonstrates that healthy cells are largely unaffected by LbL-coated CaCO3 nanoparticles, the potential therapeutic benefits for localized cancer treatments is evidenced. This project opens the possibility to future work with chemotherapeutic substances already used in clinics and using more complex in vitro cancer models. One such possibility is the encapsulation of doxorubicin, a well-known cancer drug. The effect of this drug not only in 2D systems but also 3D cellular arrangements, i.e. , spheroids, is expected to further validate the use of these inorganic systems and the TME targeting properties of the LbL coating. Finally, observing that coated nanoparticles are immobilized in the pericellular space enables the local delivery of drugs to the TME, but raises the question about the mechanism of internalization. Other less studied receptors, such as RHAMM, may mediate internalization and should be investigated in greater detail so that nanoparticle systems can be modified to not only the ECM but also the intracellular environment. CHAPTER 5. APPENDIX CHAPTER 5APPENDIX 66 APPENDIX I Figure S1. Size distribution of Hep-CaCO3 with different concentrations of Hep. Results are the average of 35 independent measurements. Table S1. Diameters and PDI of Hep-CaCO3 obtained with different concentrations of Hep. Results are the average of 35 independent measurements. Hep concentration (mg/ml) PDI diameter (nm) 0.038 mg/mL 0.40 ± 0.337 2771 ± 1064 0.95 mg/mL 0.35 ± 0.309 2329 ± 477 2.86 mg/mL 0.18 ± 0.098 1539 ± 276 3.82 mg/mL 0.19 ± 0.121 1514 ± 382 4.77 mg/mL 0.23 ± 0.178 835 ± 196 CHAPTER 5APPENDIX 67 APPENDIX II Figure S2. Size distributions of EG-CaCO3 with different stirring times. Each curve represents the average of 5 independent measurements. Table S2. Diameters and PDI of EG-CaCO3 obtained with different stirring times of EG-CaCO3. Results are the average of 5 independent measurements. time PDI diameter (nm) 30 s 0.64 ± 0.314 3976 ± 968 10 min 0.45 ± 0.243 904 ± 745 30 min 0.34 ± 0.081 830 ± 555 1 h 0.20 ± 0.148 517 ± 74 1 h 30 min 0.23 ± 0.168 905 ± 766 2 h 0.27 ± 0.201 564 ± 108 3 h 0.28 ± 0.174 554 ± 87 4 h 0.25 ± 0.137 506 ± 129 24 h 0.37 ± 0.105 491 ± 145 CHAPTER 5APPENDIX 68 APPENDIX III Figure S3. Absorbance spectrum of 50 µLg∙mL-1 Rho in 1 M HCl. The maximum peak intensity was found at 564 nm and this value was selected as the excitation wavelength of fluorescence measurements. CHAPTER 5APPENDIX 69 APPENDIX IV Figure S4. Fluorescence calibration curve of Rho in 1 M HCl. Samples were excited at 564 nm and the emitted intensity was read at 585 nm. The points were fit to a linear approximation. CHAPTER 5APPENDIX 70 APPENDIX V Figure S5. Cumulative release of Rho from uncoated ( □ ) and LbL-coated EG-CaCO3 ( ○ ) nanoparticles at pH 7.4 for 3 days. Data are means ± one standard deviation. Some error bars are too small to see. CHAPTER 5APPENDIX 71 APPENDIX VI Figure S6. Analysis of the Rho release data from uncoated and coated EG-CaCO3 nanoparticles approximated to the release models: zeroorder (cumulative release vs time), first-order (log remaining amount vs time), Higuchi (cumulative release vs square root of time), and Korsmeyer-Peppas (log cumulative release vs log time). Fitting equations and coefficients of determination (r2) are shown.