Development of drug-loaded acetalated dextran-based nanoparticles for hear targeting and treatment of myocardial infarction

Cardiovascular diseases (CVDs), and in particular myocardial infarction (MI), represent a huge burden for society. Current therapies for MI are unfortunately unable to fully restore the lost function of the injured heart. In this regard, cell therapies and novel approaches, such as stimulation of cardiomyocytes proliferation and fibroblasts reprogramming, offer appealing alternatives, holding great potential for the regeneration of the infarcted heart. However, they suffer from degradation issues and poor pharmacokinetic properties. Nanomedicines can provide smart solutions to those challenges and have received increasing attention during the last decades. Moreover, they offer tools for minimally invasive treatments, an aspect that is very important for patient compliance and clinical translation. The ability of nanocarriers to target specific sites in the body is the most appealing feature of nanomedicines, which allows to increase the efficacy of treatments and reduce their systemic adverse effects. In the last decades, heart targeting was attempted by both passive strategies and conjugating different moieties on the surface of nanoparticles, e.g. angiotensin II type 1 (AT1) receptor and atrial natriuretic peptide (ANP). However, the constant pumping of the heart and the lack of selectivity of currently known heart targeting moieties, make heart targeting a challenging goal. Therefore, the aim of this thesis was to develop drug-loaded nanoparticles for improved targeting to the infarcted heart and its treatment. Acetalated dextran (AcDX) was chosen due to its pH-responsive properties, biocompatibility, biodegradability and ease of surface functionalization. Firstly, the heart targeting properties of ANP were improved by conjugating on the surface of putrescine modified-AcDX another peptide, lin-TT1. The conjugation of lin-TT1 peptide on the surface of the nanoparticles offered the ability to hitchhike the macrophages that are sequentially accumulating in the heart upon the onset of MI. In vitro studies showed the ability of the system to associate preferentially with M2-like macrophages, which have ant-inflammatory phenotype. Subsequent in vivo studies on a rat model of MI, confirmed the evidence of preferential accumulation of these particles in the infarcted heart after 7 days post-MI, and showed increased heart targeting ability compared to particles conjugated with only ANP peptide. Secondly, spermine-modified nanoparticles were developed and coated with a coordination complex, made of tannic acid (TA) and Fe3+ ions. TA was used for its proven heart targeting capability, which derives from its high affinity for components of the extracellular matrix, in particular collagen and elastin. In vitro studies proved the increased interaction of the system with cardiac cell cultures stimulated with transforming growth factor (TGF)-β, which induced higher collagen I production. The anti-fibrotic properties of TA were also confirmed by in vitro studies, showing the reduced expression of pro-fibrotic genes in cultured fibroblasts treated with the TA coated nanoparticles. Both systems demonstrated in vitro ability to induce cardiomyocytes proliferation due to the encapsulation of two small hydrophobic molecules, which stimulate cardiomyocytes to re-enter the cell cycle. Moreover, biocompatibility and pH-responsive release of the cargos were also evaluated. Overall, two AcDX-based nanoparticulate systems were developed for improved heart targeting and treatment of MI, bringing new insights about potential therapeutic advances in targeted delivery as a minimally invasive therapeutic approach for heart disease.Sydän- ja verisuonisairaudet, ja erityisesti sydäninfarktit, aiheuttavat huomattavan tautitaakan yhteiskunnalle. Nykyiset sydäninfarktin hoitokeinot eivät kuitenkaan täysin korjaa vaurioituneen sydämen toimintakykyä. Soluterapiavalmisteet ja muut uudet terapeuttiset keinot, kuten esimerkiksi sydänlihassolujen proliferaation edistäminen ja sidekudossolujen uudelleenohjelmointi sydänlihassoluiksi, avaavat uusia mahdollisuuksia sydänlihaksen regeneraatiokyvyn parantamiseen sydäninfarktin jälkeen. Näiden terapeuttien ongelmia ovat kuitenkin muun muassa niissä käytettyjen lääkkeiden epästabiilisuus ja epätyydyttävät farmakokineettiset ominaisuudet. Näihin ongelmiin nanolääkkeet ovat älykäs ratkaisu, ja ne ovatkin saaneet lisää huomiota viime vuosikymmeninä. Nanolääkkeet tarjoavat myös mahdollisuuden vähentää hoitojen invasiivisuutta, mikä on erittäin tärkeä näkökulma potilaiden hoitomyöntyvyyden ja hoitojen käyttöönoton kannalta. Nanolääkkeet voidaan kohdistaa tiettyihin kudoksiin elimistössä, mikä lisää hoitojen tehokkuutta ja vähentää niiden haittavaikutuksia. Viime vuosikymmeninä lääkeaineiden kohdentamista sydämeen on tutkittu sekä passiivisilla että aktiivisilla kohdentamismenetelmillä, kuten päällystämällä kantaja(nano)hiukkaset erilaisilla kohdentamismolekyyleillä. Aktiivisista kohdentamismolekyyleistä esimerkkejä ovat muun muassa angiotensiini II typpi 1-reseptori (AT1) ja A-tyypin natriureettinen peptidi (ANP). Kuitenkin sykkivä sydän ja tällä hetkellä kohdentamiseen käytettyjen molekyylien puuttuvat kohdennusominaisuudet ovat keskeisiä ongelmia lääkevaikutusten sydänkohdentamisessa. Tämän väitöskirjan tavoite oli kehittää lääkkeillä kuormattuja nanohiukkasia tehostamaan lääkevaikutuksen sydänkohdentumista sydäninfarktin hoidossa. Asetyloitu dekstraani (AcDEX) valittiin pH-vasteen, biologisen yhteensopivuuden, biohajoavuuden ja yksinkertaisen pintapäällystämisen vuoksi. Ensimmäiseksi ANP:n sydämeen kohdentavia ominaisuuksia parannettiin konjugoimalla putreskiini-modifioidun AcDX:n pinnalle toinen peptidi, lin-TT1. Nanohiukkasten päällystäminen lin-TT1:llä mahdollistaa nanohiukkasten kiinnittymisen infarktialueelle kertyviin makrofageihin sydäninfarktin alkuvaiheessa. In vitro soluviljelykokeissa havaittiin nanohiukkasten kiinnittyvän ensisijaisesti M2-tyypin, anti-inflammatorisiin makrofageihin. In vivo eläinkokeet rotan sydäninfarktimallissa vahvistivat nanohiukkasten kertymisen infarktialueelle 7 päivän kuluttua infarktin jälkeen ja osoittivat paremman kohdentumisen sydämeen verrattuna nanohiukkasiin, jotka oli konjugoitu vain ANP-peptidillä. Toiseksi valmistettiin spermiini-nanohiukkasia, jotka päällystettiin kompleksilla, joka oli valmistettu tanniinihaposta (TA) ja Fe3+-ioneista. TA:ta käytettiin sen aikaisemmin havaitun sydänkohdentamisominaisuuden vuoksi, mikä johtuu sen korkeasta affiniteetista soluväliaineen komponentteihin, erityisesti kollageeniin ja elastiiniin. In vitro soluviljelytutkimukset vahvistivat nanohiukkasten lisääntyneen vuorovaikutuksen sydänlihassolujen kanssa, joita oli stimuloitu kollageenin-1 tuotantoa aktivoivalla transformoivalla kasvutekijällä (TGF)-β:llä. TA:n antifibroottiset ominaisuudet vahvistettiin in vitro soluviljelykokeilla, jotka osoittivat profibroottisten geenien ilmentymisen vähentyneen sidekudossoluissa, joita oli käsitelty TA:lla päällystetyillä nanohiukkasilla. Molempien kantajahiukkasjärjestelmien, jotka oli kuormattu kahdella pienellä hydrofobisella, solusykliin vaikuttavilla lääkemolekyyleillä, havaittiin in vitro soluviljelyolosuhteissa lisäävän sydänlihassoluja proliferaatiota. Lisäksi tutkittiin lääkekuormien bioyhteensopivuutta ja pH-herkkää vapautumista. Kokonaisuudessaan väitöskirjatutkimuksessa onnistuttiin kehittämään kaksi AcDX-nanohiukkasjärjestelmää parantamaan lääkkeiden kohdentamista sydämeen infarktin hoidossa, avaten siten uusia näkökulmia sydänsairauksien kohdennettuun ei-invasiiviseen hoitoon

Nuts and Bolts : Microfluidics for the Production of Biomaterials

Nanotechnology holds the promise of bringing revolutionary therapeutic strategies into the clinic. However, an enormous fraction of the currently proposed nanotechnology-based therapies suffers from lack of reproducibility, complexity, high costs, and scale-up-related issues. For these reasons, the research community is moving toward the miniaturization of biomaterials and fabrication methods. Customizable microfluidic-based products have gained tremendous relevance in the development of biomedical technologies. This review provides an overview of different materials that can be used for the fabrication of microfluidic devices, as well as the other parameters influencing the production of biomaterials and biosensors. Moreover, several advanced microfluidic-based technologies that are designed to overcome the current challenges of cancer, immunotherapy, and diabetes therapy, among others are described. Then, the pros and cons of microfluidics as alternative to conventional preparation methods, and the challenges of translating this technique to an industrial context are highlighted. Overall, microfluidic technologies and their accessibility to the research community offer a set of exciting opportunities to bridge the development of innovative therapies and their commercialization in the foreseeable future.Peer reviewe

Regeneration of Rogoredo railway: a combined approach using multi-criteria and financial analysis

I vuoti urbani, quali ex aree industriali ed ex scali ferroviari, rappresentano oggi un’importante occasione di riconversione delle città, nell’ottica di uno sviluppo in chiave sostenibile, resiliente e circolare. Tuttavia, gli interventi di trasformazione e rigenerazione urbana sono caratterizzati da un elevato grado di complessità e dinamicità, così come da un’elevata interazione tra le diverse componenti urbane, quali gli aspetti economici, ambientali, sociali e tra i diversi attori coinvolti nel processo. In questo contesto, l’approccio metodologico proposto nel presente contributo combina le Analisi-Multicriteri (AMC) con l’analisi Analisi Finanziaria (AF). Questo modello permette di analizzare e supportare il processo decisionale nella sua complessità, considerando sia gli aspetti qualitativi (sociali e ambientali) sia quelli quantitativi (economico- finanziari). Il caso degli scali ferroviari dell’area di Rogoredo a Milano (Italia) rap- presenta un caso emblematico. La stessa città di Milano, già all’interno del Piano di Governo del Territorio (PGT), propone interventi volti alla riconnessione di questo nodo infrastrutturale per renderlo un polo attrattivo e inclusivo. L’obiettivo di questo contributo è quello di applicare le AMC con l’AF per la valutazione di scenari alternativi, volti alla riqualificazione dell’ex scalo ferroviario di Rogoredo. La valutazione diventa, quindi, parte integrante dell’intero processo decisionale, supportandone tutte le fasi, da quella iniziale fino alla definizione dello scenario più idoneo agli obiettivi prefissati e agli interessi degli stakeholder coinvolti. Il valore aggiunto fornito dalla presente applicazione è rappresentato proprio dalla possibilità di considerare sia il punto di vista degli investitori, attraverso l’AF, sia la più ampia prospettiva pubblica, attraverso il supporto delle AMC. In questo modo è stato possibile costruire e valutare scenari di trasformazione in grado di attrarre possibili investitori e al tempo stesso capaci di promuovere modelli di mobilita sostenibile, forme di inclusione sociale, sviluppo eco-sostenibile, miglioramento della qualità ambientale, attraverso la progettazione di nuove aree pubbliche, spazi verdi e servizi per i cittadini. In questo pro- cesso, la valutazione assume un ruolo essenziale in quanto consente di mettere in luce i diversi obiettivi perseguiti dall’intervento di rigenerazione e le loro eventuali conflittualità. Inoltre, la loro identificazione può supportare la definizione di scenari alternativi di sviluppo, rendendo partecipati sia il processo progettuale sia quello decisionale.Abandoned areas such as neglected railways and urban voids represent a suitable opportunity for the regeneration and requalification of cities, according to the paradigms of sustainability and resilience. Urban transformation and urban regeneration processes are characterized by a high level of complexity, a dynamic behavior over time and interactions between the various actors involved in the process. Within this context, the present paper proposes the application of a combined evaluation framework, based on the integration of Multi-Criteria Decision Analysis (MCDA) with a Financial Analysis (FA) to assess different strategic scenarios for the regeneration of the Rogoredo railways area (Milan, Italy). The purpose of this framework is to take into account the complexity of the decision-making process, considering both the qualitative (social and environmental) and quantitative (economic-financial) aspects. In detail, the railway yards in the Rogoredo area in Milan (Italy) represent an emblematic case. The city of Milan, within the Territory Governance Plan (PGT), has already proposed interventions in this site aimed at reconnecting the infrastructural node and making it an attractive and inclusive pole. The present paper demonstrates the usefulness of evaluation procedures in supporting the entire decision-making process and defining the most suitable scenario considering the initial objective and the stakeholders’ interests. The innovative value provided by this application is represented precisely by the possibility of considering both the developer point of view through FA and the broader public perspective through the support of MCDA. This approach allowed to build and evaluate transformation scenarios capable of both attracting potential investors and promoting sustainable mobility models, social inclusion, eco-sustainable development, improvement of environmental quality through the design of new public areas, green spaces, and services for citizens

Non-viral nanoparticles for RNA interference : Principles of design and practical guidelines

Ribonucleic acid interference (RNAi) is an innovative treatment strategy for a myriad of indications. Non-viral synthetic nanoparticles (NPs) have drawn extensive attention as vectors for RNAi due to their potential advantages, including improved safety, high delivery efficiency and economic feasibility. However, the complex natural process of RNAi and the susceptible nature of oligonucleotides render the NPs subject to particular design principles and requirements for practical fabrication. Here, we summarize the requirements and obstacles for fabricating non-viral nano-vectors for efficient RNAi. To address the delivery challenges, we discuss practical guidelines for materials selection and NP synthesis in order to maximize RNA encapsulation efficiency and protection against degradation, and to facilitate the cytosolic release of oligonucleotides. The current status of clinical translation of RNAi-based therapies and further perspectives for reducing the potential side effects are also reviewed. (c) 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Peer reviewe

LinTT1 peptide-functionalized liposomes for targeted breast cancer therapy

Breast cancer, with around 2 million new cases in 2019, is the second most common cancer worldwide and the second leading cause of cancer death among females. The aim of this work is to prepare a targeting nanoparticle through the conjugation of LinTT1 peptide, a specific molecule targeting p32 protein overexpressed by breast cancer and cancer associated cells, on liposomes' surface. This approach increases the cytotoxic effects of doxorubicin (DOX) and sorafenib (SRF) co-loaded in therapeutic liposomes on both 2D and 3D breast cancer cellular models. The liposome functionalization leads to a higher interaction with 3D breast cancer spheroids than bare ones. Moreover, interaction studies between LinTT1-functionalized liposomes and M2 primary human macrophages show an internalization of 50% of the total nanovesicles that interact with these cells, while the other 50% results only associated to cell surface. This finding suggests the possibility to use the amount of associated liposomes to enrich the hypoxic tumor area, exploiting the ability of M2 macrophages to accumulate in the central core of tumor mass. These promising results highlight the potential use of DOX and SRF co-loaded LinTT1-functionalized liposomes as nanomedicines for the treatment of breast cancer, especially in triple negative cancer cells.Peer reviewe

Peptide-guided resiquimod-loaded lignin nanoparticles convert tumor-associated macrophages from M2 to M1 phenotype for enhanced chemotherapy

Nanomedicines represent innovative and promising alternative technologies to improve the therapeutic effects of different drugs for cancer ablation. Targeting M2-like tumor-associated macrophages (TAMs) has emerged as a favorable therapeutic approach to fight against cancer through the modulation of the tumor microenvironment. However, the immunomodulatory molecules used for this purpose present side effects upon systemic administration, which limits their clinical translation. Here, the biocompatible lignin polymer is used to prepare lignin nanoparticles (LNPs) that carry a dual agonist of the toll-like receptors TLR7/8 (resiquimod, R848). These LNPs are targeted to the CD206-positive M2-like TAMs using the “mUNO” peptide, in order to revert their pro-tumor phenotype into anti-tumor M1-like macrophages in the tumor microenvironment of an aggressive triple-negative in vivo model of breast cancer. Overall, we show that targeting the resiquimod (R848)-loaded LNPs to the M2-like macrophages, using very low doses of R848, induces a profound shift in the immune cells in the tumor microenvironment towards an anti-tumor immune state, by increasing the representation of M1-like macrophages, cytotoxic T cells, and activated dendritic cells. This effect consequently enhances the anticancer effect of the vinblastine (Vin) when co-administered with R848-loaded LNPs to the M2-like macrophages, using very low doses of R848, induces a profound shift in the immune cells in the tumor microenvironment towards an anti-tumor immune state, by increasing the representation of M1-like macrophages, cytotoxic T cells, and activated dendritic cells. This effect consequently enhances the anticancer effect of the vinblastine (Vin) when co-administered with R848-loaded LNPs. Statement of significance Lignin-based nanoparticles (LNPs) were successfully developed to target a potent TLR7/8 agonist (R848) of the tumor microenvironment (TME). This was achieved by targeting the mannose receptor (CD206) on the tumor supportive (M2-like) macrophages with the "mUNO" peptide, to reprogram them into an antitumor (M1-like) phenotype for enhanced chemotherapy. LNPs modified the biodistribution of the R848, and enhanced its accumulation and efficacy in shifting the immunological profile of the cells in the TME, which was not achieved by systemic administration of free R848. Moreover, a reduction in the tumor volumes was observed at lower equivalent doses of R848 compared with other studies. Therefore, the co-administration of R848@LNPs is a promising chemotherapeutic application in aggressive tumors, such as the triple-negative breast cancer. (c) 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.Peer reviewe

Design, Synthesis and Characterization of a PEGylated Stanozolol for Potential Therapeutic Applications

Stanozolol (STZ) is a drug used to treat serious disorders like aplastic anemia and hereditary angioedema. It is also indicated as an adjunct therapy for the treatment of vascular disorders and growth failures. Encouraging results obtained using animal models demonstrated that STZ increases bone formation and mineralization, thus improving both density and biomechanical properties. Like natural androgens, such as TST and 5α-dihydrotestosterone (5α-DHT), STZ binds androgen receptor (AR) to activate AR-mediated signalling. Despite its therapeutic effects, this synthetic anabolic-androgenic steroid (AAS), or 5α-DHT derivative, due to its high lipophilicity, is poor soluble in water. Thus, to increase the water solubility and stability of STZ, as well as its bioavailability and efficacy, an innovative PEGylated STZ (STZ conjugated with (MeO-PEG-NH2)10kDa, (MeO-PEG-NH)10kDa-STZ) was synthesized. As confirmed by chromatography (RP-HPLC) and spectrometry (ATR-FTIR, 1H-NMR, elemental CHNS(O) analysis, MALDI-TOF/TOF) analyses, a very pure, stable and soluble compound was obtained. Acetylcholinesterase (AChE) competitive ELISA kit demonstrated that the resulting PEGylated STZ competes against biological TST, especially at lower concentrations. Cytotoxicity of increasing concentrations (1, 10, 25 or 50 µM) of STZ and/or (MeO-PEG-NH)10kDa-STZ was also evaluated for up 80 h by performing the MTT assay on human osteosarcoma Saos-2 cells, which express AR and are responsive to STZ. PEGylation mitigated cytotoxicity of STZ, by increasing the cell viability values, especially at higher drug concentrations. Furthermore, these results suggest that (MeO-PEG-NH)10kDa-STZ is a promising and reliable drug to be used in clinical conditions in which TST is required.Peer reviewe

In vitro Evaluation of the Therapeutic Effects of Dual-Drug Loaded Spermine-Acetalated Dextran Nanoparticles Coated with Tannic Acid for Cardiac Applications

Myocardial infarction results in a massive loss of cardiomyocytes (CMs). Unfortunately, current therapies are unsuccessful in replacing lost CMs, and thus, there is an urgent need for innovative approaches. Here, a nanosystem based on spermine-acetalated dextran (AcDXSp) and encapsulating two drug compounds able to stimulate in vitro CMs proliferation is developed. The nanosystem is coated by deposition of a film constituted by tannic acid (TA) and Fe3+ ions. The coating with TA increases the retention of the nanocarrier in cell co-cultures of CMs and fibroblasts stimulated with transforming growth factor (TGF)-β, due to the high affinity of TA for components of the cardiac extracellular matrix. The system exhibits biocompatibility toward primary CMs and induces their proliferation, as indicated by the two-fold increase of CMs in the active cell cycle. At the same time, the presence of TA synergistically helps contrasting fibrosis by reducing profibrotic genes expression, such as collagen 1 and osteopontin, by approximately 80% compared to the control. Overall, the developed nanosystem demonstrates the capability to stimulate CMs proliferation and reduce fibrosis, showing potential benefits for future in vivo applications.Peer reviewe

Development of Surface Chemical Strategies for Synthesizing Redox-Responsive Diatomite Nanoparticles as a Green Platform for On-Demand Intracellular Release of an Antisense Peptide Nucleic Acid Anticancer Agent

Redox-responsive silica drug delivery systems are synthesized by aeco-friendly diatomite source to achieve on-demand release of peptide nucleic acid (PNA) in tumor reducing microenvironment, aiming to inhibit the immune check-point programmed cell death 1 receptor/programmed cell death receptor ligand 1 (PD-1/PD-L1) in cancer cells. The nanoparticles (NPs) are coated with polyethylene glycol chains as gatekeepers to improve their physicochemical properties and control drug release through the cleavable disulfide bonds (S-S) in a reductive environment. This study describes different chemical conditions to achieve the highest NPs' surface functionalization yield, exploring both multistep and one-pot chemical functionalization strategies. The best formulation is used for covalent PNA conjugation via the S-S bond reaching a loading degree of 306 +/- 25 mu g (PNA) mg(DNPs)(-1). These systems are used for in vitro studies to evaluate the kinetic release, biocompatibility, cellular uptake, and activity on different cancer cells expressing high levels of PD-L1. The obtained results prove the safety of the NPs up to 200 mu g mL(-1) and their advantage for controlling and enhancing the PNA intracellular release as well as antitumor activity. Moreover, the downregulation of PD-L1 observed only with MDA-MB-231 cancer cells paves the way for targeted immunotherapy.Peer reviewe

Dual-Peptide Functionalized Acetalated Dextran-Based Nanoparticles for Sequential Targeting of Macrophages during Myocardial Infarction

The advent of nanomedicine has recently started to innovate the treatment of cardiovascular diseases, in particular myocardial infarction. Although current approaches are very promising, there is still an urgent need for advanced targeting strategies. In this work, the exploitation of macrophage recruitment is proposed as a novel and synergistic approach to improve the addressability of the infarcted myocardium achieved by current peptide-based heart targeting strategies. For this purpose, an acetalated dextran-based nanosystem is designed and successfully functionalized with two different peptides, atrial natriuretic peptide (ANP) and linTT1, which target, respectively, cardiac cells and macrophages associated with atherosclerotic plaques. The biocompatibility of the nanocarrier is screened on both macrophage cell lines and primary macrophages, showing high safety, in particular after functionalization of the nanoparticles' surface. Furthermore, the system shows higher association versus uptake ratio towards M2-like macrophages (approximately 2-fold and 6-fold increase in murine and human primary M2-like macrophages, respectively, compared to M1-like). Overall, the results demonstrate that the nanosystem has potential to exploit the "hitchhike" effect on M2-like macrophages and potentially improve, in a dual targeting strategy, the ability of the ANP peptide to target infarcted heart.Peer reviewe