ABSTRACT
Breast cancer stem cells (CSCs) play a pivotal role in therapy resistance and tumor relapse, emphasizing the need for reliable in vitro models that recapitulate the complexity of the CSC tumor microenvironment to accelerate drug discovery. We present a bioprinted breast CSC tumor-stroma model incorporating triple-negative breast CSCs (TNB-CSCs) and stromal cells (human breast fibroblasts), within a breast-derived decellularized extracellular matrix bioink. Comparison of molecular signatures in this model with different clinical subtypes of bioprinted tumor-stroma models unveils a unique molecular profile for artificial CSC tumor models. We additionally demonstrate that the model can recapitulate the invasive potential of TNB-CSC. Surface-enhanced Raman scattering imaging allowed us to monitor the invasive potential of tumor cells in deep z-axis planes, thereby overcoming the depth-imaging limitations of confocal fluorescence microscopy. As a proof-of-concept application, we conducted high-throughput drug testing analysis to assess the efficacy of CSC-targeted therapy in combination with conventional chemotherapeutic compounds. The results highlight the usefulness of tumor-stroma models as a promising drug-screening platform, providing insights into therapeutic efficacy against CSC populations resistant to conventional therapies.
Subject(s)
Bioprinting , Neoplastic Stem Cells , Printing, Three-Dimensional , Triple Negative Breast Neoplasms , Humans , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , Neoplastic Stem Cells/metabolism , Triple Negative Breast Neoplasms/pathology , Triple Negative Breast Neoplasms/drug therapy , Female , Tumor Microenvironment/drug effects , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Stromal Cells/drug effects , Stromal Cells/pathology , Stromal Cells/metabolismABSTRACT
In this study, we developed functionalized polymeric micelles (FPMs) loaded with simvastatin (FPM-Sim) as a drug delivery system to target liver sinusoidal endothelial cells (LSECs) for preserving liver function in chronic liver disease (CLD). Polymeric micelles (PMs) were functionalized by coupling peptide ligands of LSEC membrane receptors CD32b, CD36 and ITGB3. Functionalization was confirmed via spectroscopy and electron microscopy. In vitro and in vivo FPM-Sim internalization was assessed by means of flow cytometry in LSECs, hepatocytes, Kupffer and hepatic stellate cells from healthy rats. Maximum tolerated dose assays were performed in healthy mice and efficacy studies of FPM-Sim were carried out in bile duct ligation (BDL) and thioacetamide (TAA) induction rat models of cirrhosis. Functionalization with the three peptide ligands resulted in stable formulations with a greater degree of in vivo internalization in LSECs than non-functionalized PMs. Administration of FPM-Sim in BDL rats reduced toxicity relative to free simvastatin, albeit with a moderate portal-pressure-lowering effect. In a less severe model of TAA-induced cirrhosis, treatment with FPM-CD32b-Sim nanoparticles for two weeks significantly decreased portal pressure, which was associated with a reduction in liver fibrosis, lower collagen expression as well as the stimulation of nitric oxide synthesis. In conclusion, CD32b-FPM stands out as a good nanotransporter for drug delivery, targeting LSECs, key inducers of liver injury.
ABSTRACT
Cerium oxide nanoparticles (CeO2NPs) have exceptional catalytic properties, rendering them highly effective in removing excessive reactive oxygen species (ROS) from biological environments, which is crucial in safeguarding these environments against radiation-induced damage. Additionally, the Ce atom's high Z number makes it an ideal candidate for utilisation as an X-ray imaging contrast agent. We herein show how the injection of albumin-stabilised 5 nm CeO2NPs into mice revealed substantial enhancement in X-ray contrast, reaching up to a tenfold increase at significantly lower concentrations than commercial or other proposed contrast agents. Remarkably, these NPs exhibited prolonged residence time within the target organs. Thus, upon injection into the tail vein, they exhibited efficient uptake by the liver and spleen, with 85% of the injected dose (%ID) recovered after 7 days. In the case of intratumoral administration, 99% ID of CeO2NPs remained within the tumour throughout the 7-day observation period, allowing for observation of disease dynamics. Mass spectrometry (ICP-MS) elemental analysis confirmed X-ray CT imaging observations.
ABSTRACT
Kirsten rat sarcoma (KRAS) is a small GTPase which acts as a molecular switch to regulate several cell biological processes including cell survival, proliferation, and differentiation. Alterations in KRAS have been found in 25% of all human cancers, with pancreatic cancer (90%), colorectal cancer (45%), and lung cancer (35%) being the types of cancer with the highest mutation rates. KRAS oncogenic mutations are not only responsible for malignant cell transformation and tumor development but also related to poor prognosis, low survival rate, and resistance to chemotherapy. Although different strategies have been developed to specifically target this oncoprotein over the last few decades, almost all of them have failed, relying on the current therapeutic solutions to target proteins involved in the KRAS pathway using chemical or gene therapy. Nanomedicine can certainly bring a solution for the lack of specificity and effectiveness of anti-KRAS therapy. Therefore, nanoparticles of different natures are being developed to improve the therapeutic index of drugs, genetic material, and/or biomolecules and to allow their delivery specifically into the cells of interest. The present work aims to summarize the most recent advances related to the use of nanotechnology for the development of new therapeutic strategies against KRAS-mutated cancers.
ABSTRACT
Despite all the advances seen in recent years, the severe adverse effects and low specificity of conventional chemotherapy are still challenging problems regarding cancer treatment. Nanotechnology has helped to address these questions, making important contributions in the oncological field. The use of nanoparticles has allowed the improvement of the therapeutic index of several conventional drugs and facilitates the tumoral accumulation and intracellular delivery of complex biomolecules, such as genetic material. Among the wide range of nanotechnology-based drug delivery systems (nanoDDS), solid lipid nanoparticles (SLNs) have emerged as promising systems for delivering different types of cargo. Their solid lipid core, at room and body temperature, provides SLNs with higher stability than other formulations. Moreover, SLNs offer other important features, namely the possibility to perform active targeting, sustained and controlled release, and multifunctional therapy. Furthermore, with the possibility to use biocompatible and physiologic materials and easy scale-up and low-cost production methods, SLNs meet the principal requirements of an ideal nanoDDS. The present work aims to summarize the main aspects related to SLNs, including composition, production methods, and administration routes, as well as to show the most recent studies about the use of SLNs for cancer treatment.
ABSTRACT
The Kirsten rat sarcoma viral oncogene (KRAS) is one of the most well-known proto-oncogenes, frequently mutated in pancreatic and colorectal cancers, among others. We hypothesized that the intracellular delivery of anti-KRAS antibodies (KRAS-Ab) with biodegradable polymeric micelles (PM) would block the overactivation of the KRAS-associated cascades and revert the effect of its mutation. To this end, PM-containing KRAS-Ab (PM-KRAS) were obtained using Pluronic F127. The feasibility of using PM for antibody encapsulation as well as the conformational change of the polymer and its intermolecular interactions with the antibodies was studied, for the first time, using in silico modeling. In vitro, encapsulation of KRAS-Ab allowed their intracellular delivery in different pancreatic and colorectal cancer cell lines. Interestingly, PM-KRAS promoted a high proliferation impairment in regular cultures of KRAS-mutated HCT116 and MIA PaCa-2 cells, whereas the effect was neglectable in non-mutated or KRAS-independent HCT-8 and PANC-1 cancer cells, respectively. Additionally, PM-KRAS induced a remarkable inhibition of the colony formation ability in low-attachment conditions in KRAS-mutated cells. In vivo, when compared with the vehicle, the intravenous administration of PM-KRAS significantly reduced tumor volume growth in HCT116 subcutaneous tumor-bearing mice. Analysis of the KRAS-mediated cascade in cell cultures and tumor samples showed that the effect of PM-KRAS was mediated by a significant reduction of the ERK phosphorylation and a decrease in expression in the stemness-related genes. Altogether, these results unprecedently demonstrate that the delivery of KRAS-Ab mediated by PM can safely and effectively reduce the tumorigenicity and the stemness properties of KRAS-dependent cells, thus bringing up new possibilities to reach undruggable intracellular targets.
Subject(s)
Colorectal Neoplasms , Neoplasms , Animals , Mice , Carcinogenesis , Cell Proliferation , Colorectal Neoplasms/pathology , Micelles , Mutation , Polymers/pharmacology , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Proto-Oncogene Proteins p21(ras)/pharmacology , Intracellular SpaceABSTRACT
Tumor secreted extracellular vesicles (EVs) are potent intercellular signaling platforms. They are responsible for the accommodation of the premetastatic niche (PMN) to support cancer cell engraftment and metastatic growth. However, complex cancer cell composition within the tumor increases also the heterogeneity among cancer secreted EVs subsets, a functional diversity that has been poorly explored. This phenomenon is particularly relevant in highly plastic and heterogenous triple-negative breast cancer (TNBC), in which a significant representation of malignant cancer stem cells (CSCs) is displayed. Herein, we selectively isolated and characterized EVs from CSC or differentiated cancer cells (DCC; EVsCSC and EVsDCC , respectively) from the MDA-MB-231 TNBC cell line. Our results showed that EVsCSC and EVsDCC contain distinct bioactive cargos and therefore elicit a differential effect on stromal cells in the TME. Specifically, EVsDCC activated secretory cancer associated fibroblasts (CAFs), triggering IL-6/IL-8 signaling and sustaining CSC phenotype maintenance. Complementarily, EVsCSC promoted the activation of α-SMA+ myofibroblastic CAFs subpopulations and increased the endothelial remodeling, enhancing the invasive potential of TNBC cells in vitro and in vivo. In addition, solely the EVsCSC mediated signaling prompted the transformation of healthy lungs into receptive niches able to support metastatic growth of breast cancer cells.
Subject(s)
Extracellular Vesicles , Triple Negative Breast Neoplasms , Humans , Triple Negative Breast Neoplasms/pathology , Cell Line, Tumor , Extracellular Vesicles/pathology , Neoplastic Stem Cells/metabolism , Lung/pathology , Tumor MicroenvironmentABSTRACT
Cancer maintenance, metastatic dissemination and drug resistance are sustained by cancer stem cells (CSCs). Triple negative breast cancer (TNBC) is the breast cancer subtype with the highest number of CSCs and the poorest prognosis. Here, we aimed to identify potential drugs targeting CSCs to be further employed in combination with standard chemotherapy in TNBC treatment. The anti-CSC efficacy of up to 17 small drugs was tested in TNBC cell lines using cell viability assays on differentiated cancer cells and CSCs. Then, the effect of 2 selected drugs (8-quinolinol -8Q- and niclosamide -NCS-) in the cancer stemness features were evaluated using mammosphere growth, cell invasion, migration and anchorage-independent growth assays. Changes in the expression of stemness genes after 8Q or NCS treatment were also evaluated. Moreover, the potential synergism of 8Q and NCS with PTX on CSC proliferation and stemness-related signaling pathways was evaluated using TNBC cell lines, CSC-reporter sublines, and CSC-enriched mammospheres. Finally, the efficacy of NCS in combination with PTX was analyzed in vivo using an orthotopic mouse model of MDA-MB-231 cells. Among all tested drug candidates, 8Q and NCS showed remarkable specific anti-CSC activity in terms of CSC viability, migration, invasion and anchorage independent growth reduction in vitro. Moreover, specific 8Q/PTX and NCS/PTX ratios at which both drugs displayed a synergistic effect in different TNBC cell lines were identified. The sole use of PTX increased the relative presence of CSCs in TNBC cells, whereas the combination of 8Q and NCS counteracted this pro-CSC activity of PTX while significantly reducing cell viability. In vivo, the combination of NCS with PTX reduced tumor growth and limited the dissemination of the disease by reducing circulating tumor cells and the incidence of lung metastasis. The combination of 8Q and NCS with PTX at established ratios inhibits both the proliferation of differentiated cancer cells and the viability of CSCs, paving the way for more efficacious TNBC treatments.
Subject(s)
Triple Negative Breast Neoplasms , Animals , Cell Line, Tumor , Cell Proliferation , Humans , Mice , Neoplastic Stem Cells/metabolism , Niclosamide/pharmacology , Niclosamide/therapeutic use , Oxyquinoline , Triple Negative Breast Neoplasms/pathologyABSTRACT
Local cancer treatment by in situ injections of thermo-responsive hydrogels (HG) offers several advantages over conventional systemic anti-cancer treatments. In this work, a biodegradable and multicompartmental HG composed of N-isopropylacrylamide, cellulose, citric acid, and ceric ammonium nitrate was developed for the controlled release of hydrophilic (doxorubicin) and hydrophobic (niclosamide) drugs. The formulation presented ideal properties regarding thermo-responsiveness, rheological behavior, drug release profile, biocompatibility, and biological activity in colon and ovarian cancer cells. Cellulose was found to retard drugs release rate, being only 4 % of doxorubicin and 30 % of niclosamide released after 1 week. This low release was sufficient to cause cell death in both cell lines. Moreover, HG demonstrated a proper injectability, in situ prevalence, and safety profile in vivo. Overall, the HG properties, together with its natural and eco-friendly composition, create a safe and efficient platform for the local treatment of non-resectable tumors or tumors requiring pre-surgical adjuvant therapy.
Subject(s)
Hydrogels , Neoplasms , Acrylamides , Cellulose/pharmacology , Doxorubicin/chemistry , Doxorubicin/pharmacology , Humans , Hydrogels/chemistry , Niclosamide , TemperatureABSTRACT
The clinical implementation of magnetic hyperthermia has experienced little progress since the first clinical trial was completed in 2005. Some of the hurdles to overcome are the reliable production of magnetic nanoparticles with controlled properties and the control of the temperature at the target tissue in vivo. Here, forty samples of iron oxide superparamagnetic nanoparticles were prepared by similar methods and thoroughly characterized in terms of size, aggregation degree, and heating response. Selected samples were intratumorally administered in animals with subcutaneous xenografts of human pancreatic cancer. In vivo experiments showed that it is possible to control the rise in temperature by modulating the field intensity during in vivo magnetic hyperthermia protocols. The procedure does not require sophisticated materials and it can be easily implemented by researchers or practitioners working in magnetic hyperthermia therapies.
ABSTRACT
Prostate cancer (PCa), one of the leading causes of cancer-related deaths, currently lacks effective treatment for advanced-stage disease. Paclitaxel (PTX) is a highly active chemotherapeutic drug and the first-line treatment for PCa; however, conventional PTX formulation causes severe hypersensitivity reactions and limits PTX use at high concentrations. In the pursuit of high molecular weight, biodegradable, and pH-responsive polymeric carriers, one conjugates PTX to a polyacetal-based nanocarrier to yield a tert-Ser-PTX polyacetal conjugate. tert-Ser-PTX conjugate provides sustained release of PTX over 2 weeks in a pH-responsive manner while also obtaining a degree of epimerization of PTX to 7-epi-PTX. Serum proteins stabilize tert-Ser-PTX, with enhanced stability in human serum versus PBS (pH 7.4). In vitro efficacy assessments in PCa cells demonstrate IC50 values above those for the free form of PTX due to the differential cell trafficking modes; however, in vivo tolerability assays demonstrate that tert-Ser-PTX significantly reduces the systemic toxicities associated with free PTX treatment. tert-Ser-PTX also effectively inhibits primary tumor growth and hematologic, lymphatic, and coelomic dissemination, as confirmed by in vivo and ex vivo bioluminescence imaging and histopathological evaluations in mice carrying orthotopic LNCaP tumors. Overall, the results suggest the application of tert-Ser-PTX as a robust antitumor/antimetastatic treatment for PCa.
Subject(s)
Antineoplastic Agents, Phytogenic , Prostatic Neoplasms , Acetals , Animals , Antineoplastic Agents, Phytogenic/therapeutic use , Cell Line, Tumor , Drug Carriers/chemistry , Humans , Male , Mice , Mice, Inbred BALB C , Paclitaxel/chemistry , Paclitaxel/pharmacology , Paclitaxel/therapeutic use , Polymers/chemistry , Prostatic Neoplasms/drug therapyABSTRACT
A rational design accurate based on the use of Statistical Design of the Experiments (DoE) and Molecular Dynamics Simulations Studies allows the prediction and the understanding of thermo-responsive hydrogels prepared regarding their gelation temperature and anti-cancer drug release rate. N-isopropylacrilamide (NIPAM) modified with specific co-monomers and crosslinkers, can be used to prepare "on-demand" thermo-responsive hydrogels with the ideal properties for clinical applications in which local sustained release of drugs is crucial. Two preferential formulations resulting from the predictive studies of DoE and In Silico methods were synthesized by radical polymerization, fully characterized, and loaded with the anticancer drug Doxorubicin (Dox). The hydrogel formulations were characterized by swelling rate, turbidity, FTIR, 1H NMR, SEM, gelation time, rheology, and biocompatibility assays. Both formulations demonstrated adequate morphologic, rheological, and biocompatibility properties; however, important differences in terms of drug retention were detected. As demonstrated by a Dox cumulative release study and posteriorly confirmed by an efficacy assay in an in vitro colorectal cancer model, the formulation composed by NIPAM and 4-penten-1-ol crosslinked with poly(ethylene glycol) diacrylate (PEGDA) (PNiPenPH) present a slow release over the time, presenting ideal properties to become and ideal depot system for the local sustained release of anticancer drugs as adjuvant therapy or in the case of non-resectable tumors.
Subject(s)
Antineoplastic Agents , Colonic Neoplasms , Antineoplastic Agents/pharmacology , Colonic Neoplasms/drug therapy , Delayed-Action Preparations , Doxorubicin/pharmacology , Drug Liberation , Humans , Hydrogels , TemperatureABSTRACT
Fabry disease is a lysosomal storage disease arising from a deficiency of the enzyme α-galactosidase A (GLA). The enzyme deficiency results in an accumulation of glycolipids, which over time, leads to cardiovascular, cerebrovascular, and renal disease, ultimately leading to death in the fourth or fifth decade of life. Currently, lysosomal storage disorders are treated by enzyme replacement therapy (ERT) through the direct administration of the missing enzyme to the patients. In view of their advantages as drug delivery systems, liposomes are increasingly being researched and utilized in the pharmaceutical, food and cosmetic industries, but one of the main barriers to market is their scalability. Depressurization of an Expanded Liquid Organic Solution into aqueous solution (DELOS-susp) is a compressed fluid-based method that allows the reproducible and scalable production of nanovesicular systems with remarkable physicochemical characteristics, in terms of homogeneity, morphology, and particle size. The objective of this work was to optimize and reach a suitable formulation for in vivo preclinical studies by implementing a Quality by Design (QbD) approach, a methodology recommended by the FDA and the EMA to develop robust drug manufacturing and control methods, to the preparation of α-galactosidase-loaded nanoliposomes (nanoGLA) for the treatment of Fabry disease. Through a risk analysis and a Design of Experiments (DoE), we obtained the Design Space in which GLA concentration and lipid concentration were found as critical parameters for achieving a stable nanoformulation. This Design Space allowed the optimization of the process to produce a nanoformulation suitable for in vivo preclinical testing.
ABSTRACT
Despite the enormous efforts done by the scientific community in the last decades, advanced cancer is still considered an incurable disease. New formulations are continuously under investigation to improve drugs therapeutic index, i.e., increase chemotherapeutic efficacy and reduce adverse effects. In this context, hydrogels-based systems for drug local sustained/controlled release have been proposed to reduce off-target effects caused by the repeated administration of systemic/oral anticancer drugs and improve their therapeutic effectiveness. Moreover, it increases the patient welfare by reducing the number of administrations needed. Among the several types of existing hydrogels, the thermo-responsive ones, which are able to change their physical state from liquid at 25 °C to a gel at the body temperature, i.e., 37 °C, gained special attention as in situ sustained drug release depot-systems in cancer treatment. To date, several thermo-responsive hydrogels have been used for drugs and/or genetic material delivery, yielding promising results both at preclinical and clinical evaluation stages. This culminates in the market authorization of Jelmyto® for the treatment of urothelial cancer. Here are summarized and discussed the last 10 years advances regarding the application of thermo-responsive hydrogels in local cancer treatment.
Subject(s)
Hydrogels , Neoplasms , Drug Compounding , Drug Delivery Systems , Drug Liberation , Humans , Neoplasms/drug therapy , TemperatureABSTRACT
Aim: Improving the stability and anti-cancer stem cell (CSC) activity of citral, a natural ALDH1A inhibitor. Materials & methods: Citral-loaded micelles (CLM) were obtained using Pluronic® F127 and its efficacy tested on the growth of four breast cancer cell lines. The impact of the CLM on the growth and functional hallmarks of breast CSCs were also evaluated using mammosphere and CSC reporter cell lines. Results: CLM improved the stability and growth inhibitory effects of citral. Importantly, CLM fully blocking the stemness features of CSCs (self-renewal, differentiation and migration) and in combination with paclitaxel CLM sensitized breast cancer cells to the chemotherapy. Conclusion: Targeting CSCs with CLM could improve the treatment of advanced breast cancer in combination with the standard chemotherapy.
Subject(s)
Breast Neoplasms , Micelles , Acyclic Monoterpenes , Breast Neoplasms/drug therapy , Cell Line, Tumor , Female , Humans , Neoplastic Stem Cells , PoloxamerABSTRACT
Cytotoxic chemotherapy continues to be the main therapeutic option for patients with metastatic breast cancer. Several studies have reported a significant association between chronic inflammation, carcinogenesis and the presence of cancer stem cells (CSC). We hypothesized that the use of non-steroidal anti-inflammatory drugs targeted to the CSC population could help reducing tumor progression and dissemination in otherwise hard to treat metastatic breast cancer. Within this study cationic naproxen (NAP)-bearing polymeric nanoparticles (NPs) were obtained by self-assembly and they were coated with hyaluronic acid (HA) via electrostatic interaction. HA-coated and uncoated NAP-bearing NPs with different sizes were produced by changing the ionic strength of the aqueous preparation solutions (i.e. 300 and 350 nm or 100 and 130 nm in diameter, respectively). HA-NPs were fully characterized in terms of physicochemical parameters and biological response in cancer cells, macrophages and endothelial cells. Our results revealed that HA-coating of NPs provided a better control in NAP release and improved their hemocompatibility, while ensuring a strong CSC-targeting in MCF-7 breast cancer cells. Furthermore, the best polymeric NPs formulation significantly (p < 0.001) reduced MCF-7 cells viability when compared to free drug (i.e. 45 ± 6% for S-HA-NPs and 87 ± 10% for free NAP) by p53-dependent induction of apoptosis; and the migration of these cell line was also significantly (p < 0.01) reduced by the nano-formulated NAP (i.e. 76.4% of open wound for S-HA-NPs and 61.6% of open wound for NAP). This increased anti-cancer activity of HA-NAP-NPs might be related to the induction of apoptosis through alterations of the GSK-3ß-related COX-independent pathway. Overall, these findings suggest that the HA-NAP-NPs have the potential to improve the treatment of advanced breast cancer by increasing the anti-proliferative effect of NAP within the CSC subpopulation.
Subject(s)
Antineoplastic Agents , Breast Neoplasms , Nanoparticles , Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Cell Line, Tumor , Endothelial Cells , Glycogen Synthase Kinase 3 beta , Humans , Hyaluronan Receptors , Hyaluronic Acid , Naproxen/pharmacology , Neoplastic Stem CellsABSTRACT
Diamond nanoparticles (nanodiamonds) can transport active drugs in cultured cells as well as in vivo. However, in the latter case, methods allowing the determination of their bioavailability accurately are still lacking. A nanodiamond can be made fluorescent with a perfectly stable emission and a lifetime ten times longer than that of tissue autofluorescence. Taking advantage of these properties, we present an automated quantification method of fluorescent nanodiamonds (FND) in histological sections of mouse organs and tumors, after systemic injection. We use a home-made time-delayed fluorescence microscope comprising a custom pulsed laser source synchronized on the master clock of a gated intensified array detector. This setup allows ultra-high-resolution images (120 Mpixels in size) of whole mouse organ sections to be obtained, with subcellular resolution and single-particle sensitivity. As a proof-of-principle experiment, we quantified the biodistribution and aggregation state of new cationic FNDs capable of transporting small interfering RNA inhibiting the oncogene responsible for Ewing sarcoma. Image analysis showed a low yield of nanodiamonds in the tumor after intravenous injection. Thus, for the in vivo efficacy assay, we injected the nanomedicine into the tumor. We achieved a 28-fold inhibition of the oncogene. This method can readily be applied to other nanoemitters with ≈100 ns lifetime.
Subject(s)
Nanodiamonds , Neoplasms , Animals , Fluorescence , Mice , RNA, Small Interfering , Tissue DistributionABSTRACT
In the present study the use of extracellular vesicles (EVs) as vehicles for therapeutic enzymes in lysosomal storage disorders was explored. EVs were isolated from mammalian cells overexpressing alpha-galactosidase A (GLA) or N-sulfoglucosamine sulfohydrolase (SGSH) enzymes, defective in Fabry and Sanfilippo A diseases, respectively. Direct purification of EVs from cell supernatants was found to be a simple and efficient method to obtain highly active GLA and SGSH proteins, even after EV lyophilization. Likewise, EVs carrying GLA (EV-GLA) were rapidly uptaken and reached the lysosomes in cellular models of Fabry disease, restoring lysosomal functionality much more efficiently than the recombinant enzyme in clinical use. In vivo, EVs were well tolerated and distributed among all main organs, including the brain. DiR-labelled EVs were localized in brain parenchyma 1 h after intra-arterial (internal carotid artery) or intravenous (tail vein) administrations. Moreover, a single intravenous administration of EV-GLA was able to reduce globotriaosylceramide (Gb3) substrate levels in clinically relevant tissues, such kidneys and brain. Overall, our results demonstrate that EVs from cells overexpressing lysosomal enzymes act as natural protein delivery systems, improving the activity and the efficacy of the recombinant proteins and facilitating their access to organs neglected by conventional enzyme replacement therapies.
Subject(s)
Extracellular Vesicles/metabolism , Extracellular Vesicles/transplantation , Lysosomal Storage Diseases/therapy , Pharmaceutical Vehicles , Animals , Brain/metabolism , CHO Cells , Cloning, Molecular , Cricetulus , Fabry Disease/enzymology , Fabry Disease/therapy , HEK293 Cells , Humans , Hydrolases/metabolism , Lysosomal Storage Diseases/enzymology , Lysosomes , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Pharmaceutical Vehicles/metabolism , Recombinant Proteins/administration & dosage , Recombinant Proteins/metabolism , Recombinant Proteins/therapeutic use , Trihexosylceramides/metabolism , alpha-Galactosidase/metabolismABSTRACT
Fabry disease is a rare lysosomal storage disorder characterized by a deficiency of α-galactosidase A (GLA), a lysosomal hydrolase. The enzyme replacement therapy administering naked GLA shows several drawbacks including poor biodistribution, limited efficacy, and relatively high immunogenicity in Fabry patients. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating the enzyme. Nanoliposomes functionalized with RGD peptide have already emerged as a good platform to protect and deliver GLA to endothelial cells. However, low colloidal stability and limited enzyme entrapment efficiency could hinder the further pharmaceutical development and the clinical translation of these nanoformulations. Herein, the incorporation of the cationic miristalkonium chloride (MKC) surfactant to RGD nanovesicles is explored, comparing two different nanosystems-quatsomes and hybrid liposomes. In both systems, the positive surface charge introduced by MKC promotes electrostatic interactions between the enzyme and the nanovesicles, improving the loading capacity and colloidal stability. The presence of high MKC content in quatsomes practically abolishes GLA enzymatic activity, while low concentrations of the surfactant in hybrid liposomes stabilize the enzyme without compromising its activity. Moreover, hybrid liposomes show improved efficacy in cell cultures and a good in vitro/in vivo safety profile, ensuring their future preclinical and clinical development.
Subject(s)
Enzyme Replacement Therapy , Fabry Disease/therapy , Nanostructures/chemistry , alpha-Galactosidase/metabolism , Fabry Disease/enzymology , Humans , Oligopeptides/chemistry , Particle Size , Surface Properties , Surface-Active Agents/chemistryABSTRACT
Colorectal cancer (CRC) is a highly prevalent disease worldwide. Patient survival is hampered by tumor relapse and the appearance of drug-resistant metastases, which are sustained by the presence of cancer stem cells (CSC). Specific delivery of anti-CSC chemotherapeutic drugs to tumors by using targeted drug delivery systems that can also target CSC sub-population might substantially improve current clinical outcomes. CD44v6 is a robust biomarker for advanced CRC and CSC, due to its functional role in tumorigenesis and cancer initiation process. Here, we show that CD44v6-targeted polymeric micelles (PM) loaded with niclosamide (NCS), a drug against CSC, is a good therapeutic strategy against colorectal CSC and circulating tumor cells (CTC) in vivo. HCT116 cells were sorted according to their CD44v6 receptor expression into CD44v6+ (high) and CDv44v6- (low) subpopulations. Accordingly, CD44v6+ cells presented stemness properties, such as overexpression of defined stemness markers (ALDH1A1, CD44v3 and CXCR4) and high capacity to form colonspheres in low attachment conditions. NCS-loaded PM functionalized with an antibody fragment against CD44v6 (Fab-CD44v6) presented adequate size, charge, and encapsulation efficiency. In addition, Fab-CD44v6 significantly increased PM internalization in CD44v6+ cells. Further, encapsulation of NCS improved its effectiveness in vitro, particularly against colonspheres, and allowed to increase its intravenous dosage in vivo by increasing the amount of NCS able to be administered without causing toxicity. Remarkably, functionalized PM accumulate in tumors and significantly reduce CTC in vivo. In conclusion, CD44v6 targeted PM meet the essential conditions to become an efficient anti-CSC therapy.