Development and characterization of lipid nanocarriers as a therapeutic tool against pancreatic cancer

Abstract

Pancreatic cancer (PC) is recognized as the seventh cause of cancer-related death, with an extremely poor 5-year survival rate. Over 80% of patients have advanced disease at the time they are diagnosed, which usually does not allow the surgical resection of the tumour. Despite the progresses in cancer knowledge and the developments of new treatments, the current therapies against pancreatic cancer are inefficient. The contributing factors for PC lethality include the lack of early symptoms, lack of accurate biomarkers for early diagnosis, and rapid propensity for metastasis to the lymphatic system and distant organs. It has been recently established that these properties may be in part attributed to Cancer Stem Cells (CSCs), a small subpopulation within tumours cells responsible for tumour initiation, growth, recurrence, and resistance to chemotherapy. Cancer treatments are often toxic to normal cells and they fail to selectively kill CSCs, which can survive treatment and give rise to new malignant cells. Thus, CSCs are the underlying cause of tumour recurrence and metastasis. Moreover, these cells can be inherently resistant to chemotherapy and radiation giving as a result more aggressive tumour. The presence of high metastatic and drug-resistant pancreatic cancer stem cells (PCSCs) contributes to the treatments failure resulting in high mortality of patients diagnosed with PC. Therefore, it is important to elucidate the molecular mechanisms underlying drug resistance and metastasis with respect to PCSCs to push forward the development of novel therapeutic strategies for targeting PCSCs, which would result in increasing drug sensitivity and inhibition of invasion and metastasis. Drug-loaded nanocarriers (NCs) provide several advantages as drug delivery systems, such as increase of the solubility of low water-soluble compounds, protection of the drug from degradation, reduction of side effects, sustained drug release, or selective drug release. But one of the most relevant advantages that NCs offers is the specific drug release in the tumour. This can be achieved thanks to a passive targeting. This takes advantage of the abnormal physiology in the tumour and the consequent enhanced permeation and retention effect to achieve a preferential retention of the NCs (_ 20-200 nm) in the tumour. However, the active targeting of the NCs to the tumour is also possible. In this case, the shell composition of the NC includes ligands that specifically binds to overexpressed receptors in the target cell. For instance, hyaluronic acid (HA) has been used for decorating NCs to target CSCs, since HA specifically binds to the CD44 receptor. This receptor is overexpressed in CSCs populations found in pancreatic cancer, but also in CSCs from others neoplasms. In any case, passive and active targeting mechanisms allow the drug accumulation in the target area. This reduces the side effect, which, unfortunately, are so frequent with the current chemotherapies. In addition, it will allow the reduction of the administered dose, since the drug will not be lost in off target tissues. On the other hand, the drug protection, sustained drug release, the solubility improvement that NCs confer to the drug, as well as their possible uptake and transport through the intestinal mucosa, make NCs interesting systems for the oral administration of some compounds. Looking at the patients’ quality of life, the oral route has gained major focus as compared to the parenteral route. However, oral delivery of anticancer drugs is still a great challenge owing to the peculiar physicochemical properties and physiological barriers that NCs have to overcome along the gastrointestinal tract (GIT). In the GIT, the NCs have to deal with great pH changes, a high ionic strength, the action of digestive enzymes and surface active molecules, and the peristaltic movements, which suppose a threaten to their colloidal stability. One of the limiting factors for drugs and NCs absorption at intestinal level is the mucus layer. The mucus layer is mainly composed of mucins and water, and it conforms a gel mesh which acts as a filter for the particles. So, all the particles have to spread through the mucus layer to reach the intestinal epithelium and be absorbed. There is a great variety of NCs, but in this thesis we focus in two different lipid based NCs as drug delivery systems for the oral administration of drugs against pancreatic cancer. We have studied liquid lipid nanocapsules (LLNs) with an olive oil core and an albumin and/or HA shell. On the other hand, we have studied solid lipid nanoparticles (SLNs) with a maslinic acid core and a polymeric shell of Poloxamer 407 or dicarboxylic acid Poloxamer 407. Moreover, SLNs have also been functionalized with the mucoadhesive molecule HA. These so different colloidal systems have been evaluated in terms of colloidal stability, digestibility, ability to encapsulate anticancer drugs, in vitro cytotoxic effects in the pancreatic cancer cell line BxPC3, and in vivo toxicity in mice. This involved several knowledge areas and required a multidisciplinary approach to bring together medical and materials sciences. The first part of the thesis focus in the design and characterization of olive oil LLNs. The shell of these LLNs was composed of seric albumin (human, HSA, or bovine, BSA), seric albumin + HA, or HA. We evaluated the colloidal stability of the systems, their interaction with mucin, and their stability and release of encapsulated curcumin (Cur) under simulated gastrointestinal conditions. Moreover, we studied the adsorption of BSA, HSA, BSA+HA, HSA+HA, and HA to the olive oil/water interface and the hydrolysis of these interfacial layers by gastrointestinal digestive enzymes. We found that the shell composition defined the stability of the system and the Cur release profile under gastrointestinal digestion, as well as the interaction of LLNs with mucin. The presence of HA in the shell enhanced the interaction of LLNs with mucin and protected the interfacial protein from the enzymatic hydrolysis. HA-BSA LLNs retained better the encapsulated Cur under gastric in vitro digestion conditions. The second part of the work introduces maslinic acid (MA) based SLNs. MA is a pentacyclic triterpenoid that has antitumour activity and many other interesting biological activities potentially good for health. This compound is found in olive oil and is especially abundant in olive leaves and pomace. However, the bioaccessibility and bioavailability of MA are restricted by the low water solubility of this compound. In this work, we have produced SLNs with a MA core stabilized with Poloxamer 407 or dicarboxylic acid-Poloxamer 407. We started by isolating MA from olive pomace, then, we produced MA SLNs and studied their colloidal characteristics, as well as their capacity to encapsulate other hydrophobic compounds, like Cur or paclitaxel (PTX), their ability to enter tumour cells, and their toxicity in tumour cells, in healthy cells and in mice. In addition, we functionalized the system with HA and studied the in vitro digestibility and the intestinal permeability of MA SLNs in in vitro epithelial models (Caco-2 and the mucus-producing Caco- 2/HT29-MTX). The main conclusions of these studies were that SLNs improved the solubility of MA, which was translated in a great increase of the bioaccessibility of the compound after in vitro gastrointestinal digestion. SLNs improved the intestinal permeability of MA in vitro. MA SLNs had cytotoxic activity against BxPC3 cancer cells and CSCs in the same range of concentration. We found synergistic effect in BxPC3 cancer cells and CSCs between MA and PTX and we produced PTX-loaded MA SLNs. The encapsulated MA and PTX had the same cytotoxic effect in BxPC3 cancer cells and CSCs than the free compounds. On the other hand, the oral or intravenous administration of MA SLNs to healthy mice did not report any toxicity, which probed their safety. The in vivo absorption of orally administered MA SLNs could not be confirmed, but the biodistribution assays in mice showed that, when intravenously administered, they were distributed homogeneously in the animal. This thesis presents two different types of lipid based NCs with different characteristics and behaviour in relation to the digestive process. The characterization of both systems brings some light to the better understanding of the interaction of these kinds of systems with the gastrointestinal milieu, which can help in the rational design of NCs as oral drug delivery systems.