Intraperitoneaalsete kasvajate sihtmärgistatud ravi kasutades peptiididega suunatud nanoosakesi

Väitekirja elektrooniline versioon ei sisalda publikatsiooneSeedetrakti ja günekoloogiliste pahaloomuliste kasvajate puhul on kasvajarakkude levik kõhuõõnes ehk peritoneaalne kartsinomatoos (PK) üks sagedasemaid ilminguid. PK ravivõimalused on piiratud, kuna süsteemne keemiaravi on madala efektiivsusega ning patsiendile manustatavat ravimidoosi piiravad kõrvalnähud kõhuõõnevälistes kudedes. Võrreldes intravenoossete ravimitega saavutavad otse kõhuõõnde manustatud vähiravimid kasvajakoes kõrgema kontsentratsiooni ning on oluliselt efektiivsemad. Sellegipoolest põhjustavad intraperitoneaalselt manustatud tsütotoksilised ravimid kõrvaltoimeid kõhuõõne normaalsetes kudedes. Üheks võimaluseks ravimite ja kontrastainete efektiivsemaks muutmiseks ja kõrvalnähtude vähendamiseks on nende laadimine nanosakestesse. Nanoosakeste abil on võimalik parandada ravimite lahustuvust, koeselektiivsust ja vabanemist sihtmärkkoes. Mõned nanoravimid PK ravimiseks on jõudnud kliiniliste uuringuteni, kuid hetkel ei ole veel ühtegi Ravimiametite poolt heaks kiidetud nanoravimit turul, mis oleks spetsiaalselt sellise manustamisviisi (st. otse kõhuõõne manustatuna) jaoks kinnitatud. Vähiravimite ja nanoosakeste koeselektiivsuse ja efektiivsuse parandamiseks saab neid suunata keemiliselt konjugeeritud afiinsusligandidega (nt. antikehad, peptiidid, aptameerid). Meie uurimisgrupis kasutab sellel eesmärgil vähiselektiivseid peptiide, näiteks iRGD vähkipenetreerivat peptiidi (TPP). Pärast seondumist rakupinna integriinidega läbib iRGD proteolüütilise lõikamise, mis aktiveerib seondumise teise vähirakkudel üleekspresseeritud valgu, NRP-1’ga, et käivitada rakuinternalisatsiooni rada. TT1 vähkipenetreeriva peptiidi primaarne retseptor on vähirakkude pinnal ekspresseeruv valk p32, mis normaalsetes rakkudes paikneb mitokondrites. TT1 peptiid kinnitub kasvajarakkude pinnal olevale p32’le ning käivitab seejärel NRP-1’st sõltuva rakkusisenemise protsessi. Käesolev prekliiniline töö keskendus kõhuõõne vähkkasvajate (maovähk, soolevähk ja munasarjavähk) uute kuvamis- ja ravimeetodite väljatöötamisele kasutades erinevate koostisega nanoosakesi (nanoravimid) ning suunavaid vähiselektiivseid peptiide. Töös uuriti polümeeridel põhinevate ja raudoksiidi sisaldavate nanoosakeste selektiivsust kasvajakoe suhtes peale kõhuõõnde süstimist. Katses kasutati erinevatel kõhuõõne kasvajarakkudel põhinevaid hiire loommudeleid. Töö tulemus näitas, et vähiselektiivsete peptiidide konjugeerimine aitab kaasa nanoosakeste paremale akumulatsioonile kasvajakoes ja rakku sisenemisele. IP süstitud TPP-NP on võrreldes IV süstitud osakestega vähikoe suhtes selektiivsemad. IP manustatud TPP-NP akumuleeruvad kasvajakoes nii otsese seondumise teel kasvajarakkudele kui ka kaudselt, vereringe kaudu ning ekperimentaalteraapia hiiremudelil näitas et, nanoosakeste suunamine vähiselektiivsete peptiididega võimendab osakeste terapeutilist efektiivsust.Gastrointestinal and gynecological malignancies often disseminate in the peritoneal cavity and cause severe complications such as bowel obstruction and the formation of ascites- a condition known as peritoneal carcinomatosis (PC). PC results from the dissemination of the primary tumor or seeding after surgical intervention and is a cause for incurability of intra-abdominal cancers. In the treatment of peritoneal tumor lesions, intraperitoneal chemotherapy can be used to improve delivery of drugs into peritoneal tumors by providing direct contact and higher local concentration, but this approach as well is not harmless and can cause side effects in the normal organs residing in the IP cavity. IP chemotherapy is an attractive strategy to improve the outcome of PC. During the last decades, substantial amount of work has been put into improving the therapeutic outcome of PC by applying different therapeutic approaches that maximize selectivity and limit side effects. Nanoparticles in the context of direct targeting of IP tumors are actively being evaluated in preclinical studies due to their potential of increasing the retention time in the IP cavity and to target drugs specifically to the tumor site compared to the conventional drugs. A few nanoparticle formulations of chemotherapeutic drugs have reached human trials, but there are no approved drug formulations for the specific use in the IP cavity. Novel strategies such as development of precision nanomedicines to specifically target cancerous lesions and development of drugs/nanoparticles with extended residence time in the IP cavity may help to increase efficacy of IP chemotherapy. The tissue selectivity and efficacy of nanoparticles and chemotherapeutic drugs can be increased by conjugating affinity ligands (i.e antibodies, peptides, aptamers) on their surface. Our lab focuses on tumor penetrating peptides (TPP), such as iRGD and TT1. The iRGD peptide is recruited to integrins expressed on endothelial cells and other cells in tumors. After recruitment to integrins, iRGD is proteolytically cleaved to expose c-terminally CRGDK CendR motif, and the truncated peptide loses most of its integrin-binding capacity and gains affinity for neuropilin-1 (NRP-1). Binding to NRP-1 mediates penetration to cells and tissues. The primary homing receptor for TT1 is p32, a mitochondrial protein aberrantly expressed on the cell surface of activated malignant and stromal cells in solid tumors. TT1 peptide is after binding to p32 proteolytically cleaved to expose C-terminally the RGAR peptide that interacts with tissue penetration receptor NRP-1. Current preclinical study focused on intraperitoneal (IP) tumors (i.e gastric, colon and ovarian cancer) in exploring new imaging and therapeutic strategies by using different nanoparticles coated with tumor penetrating peptides. Specific focus was on polymeric and iron oxide nanoparticles and their selectivity towards IP tumor lesions following IP administration. We used different IP tumor cell lines and mouse models in animal experimentations. The results from the study show that by conjugating TPP on the surface on nanoparticles the accumulation in the tumor tissue is increased. IP administered TPP-nanoparticles accumulate in the tumor tissue by direct binding as well as indirectly via systemic circulation. Importantly, peptide targeted nanoparticles increased therapeutic efficacy of nanoparticles loaded with anticancer drugs.https://www.ester.ee/record=b519761

Brändimine Eesti ettevõtetes majandussurutise tingimustes reklaamiekspertide hinnangul

The aim of this study was to analyse how advertising experts assess the changes in branding in the current economic recession. In using the assessment of the advertising experts, it was important that they could analyse the general tendency of the market, without concentrating on any certain segment. There have yet to be any similar research done in Estonia and also there have not been any comprehensive studies available in studies abroad that emanate from the opinions of people who work in advertising agencies. Half of the volume of this thesis is dedicated to the theory, which compares the economical crises of Asia and Finland in the 1990’es to the current situation in Estonia. An overview is given about economical crises and branding. Also the general analysis of branding in the current crisis that started in 2008. The empirical part of the study consists of the conclusions of half-structured expert interviews conducted with nine advertising experts. The questions in the study were designed to analyse the current economic situation as well as to give the experts an opportunity to speculate about the future and to give organisations suggestions how to improve marketing communication during the recession. The most evident proved to be the advertising expert’s abilities to define changes in their work. Seven out of nine people pointed out that change in their daily work was evident because of the recession. Two of the most mentioned answers during the study were that people tend to focus on reduced prices in advertisements and also that customers of the advertising agencies are more price sensitive regarding projects. During the analysis of the study, it became clear that major advertising companies will continue with their current brand communication or increase its proportion. Regarding smaller companies, their situation will deteriorate and they may lose their current position in the market or in worst case scenario, disappear from the market all together. At the same time it was concluded that all parties in the advertising market are feeling insecure at the moment. The most popular suggestions, made by the experts, how to promote brands and better marketing communication during the recession, were to analyse the brand and continue communication even in the event that the situation of the company worsens. The main justification for these was that in doing so, consumers will not forget the company and that people wouldn’t gravitate away from the basic values of the brand. Regarding the changes in channels of marketing communication, an increase in the percentage of internet, television and public relation and a reduction of printed media was noted. It was based upon the theories of foreign countries, general noticeable tendencies in the market and also the relative cost of the fore mentioned channels. It was also found that it was not possible to analyse how much the changes in advertisement have happened as a result of the recession. The most important resources for an organization are most commonly considered money, motivation of leaders and the values of the organization. The most popular answer was in fact money, but it was always followed by a „but“. Money became vital only when it was combined with another resource, like skills or motivation of leaders. In terms, it can be concluded that regarding the success of organizations, it can only be achieved by the right combination of resources. The main general conclusions about this study would be that despite the uncertainty of the economic situation a calm and rational way of thinking should be preserved and each decision weighed thoroughly before any actions are made. Also a way to display unique qualities to consumers should be found and the core values of the brand should not be sacrificed by focusing on solely price promotions.http://tartu.ester.ee/record=b2450118~S1*es

Hõbekuulid vähiteraapias: teel suunatud vähiravi poole

Vähiuuringute üheks keskseks eesmärgiks on vähirakke normaalsetest rakkudest eristavate ravimite väljatöötamine. Kliinilises kasutuses olevate vähiravimite puuduseks on nende vähene selektiivsus ja sellest tingitud kõrvalmõjud normaalsetes kudedes. Artiklis on antud ülevaade suunatud vähiravimite valjatöötamisest ja suundumustest. Suunatud vähiravi põhineb molekulaarsetel erinevustel vähikoe ja normaalsete veresoonte vahel. In vivo faagidisplei meetodi abil on võimalik veresoonte haigusspetsiifilisi molekulaarseid mustreid kaardistada. Selle tulemuseks on peptiidid, mis seostuvad selektiivselt vähiveresoontega. Need kullerpeptiidid nagu ka teised kasvaja veresoontega selektiivselt seonduvad molekulid (antikehad, aptameerid) võimaldavad kasvajasse viia vähiravimeid ja kontrastaineid. Hiljuti avastatud kasvajakude penetreerivad peptiidid põhjustavad vähiveresoonte selektiivset lekkimist. Koos ravimitega manustatuna põhjustavad need peptiidid ravimi väljumist kasvaja veresoontest ja tungimist kasvajakoesse – tulemuseks on ravimi terapeutilise indeksi paranemine.Eesti Arst 2015; 94(5):281–28

Peritoneal Carcinomatosis Targeting with Tumor Homing Peptides

Over recent decades multiple therapeutic approaches have been explored for improved management of peritoneally disseminated malignancies—a grim condition known as peritoneal carcinomatosis (PC). Intraperitoneal (IP) administration can be used to achieve elevated local concentration and extended half-life of the drugs in the peritoneal cavity to improve their anticancer efficacy. However, IP-administered chemotherapeutics have a short residence time in the IP space, and are not tumor selective. An increasing body of work suggests that functionalization of drugs and nanoparticles with targeting peptides increases their peritoneal retention and provides a robust and specific tumor binding and penetration that translates into improved therapeutic response. Here we review the progress in affinity targeting of intraperitoneal anticancer compounds, imaging agents and nanoparticles with tumor-homing peptides. We review classes of tumor-homing peptides relevant for PC targeting, payloads for peptide-guided precision delivery, applications for targeted compounds, and the effects of nanoformulation of drugs and imaging agents on affinity-based tumor delivery

A novel oral iron-complex formulation: Encapsulation of hemin in polymeric micelles and its in vitro absorption

Anemia resulting from iron deficiency is one of the most prevalent diseases in the world. As iron has important roles in several biological processes such as oxygen transport, DNA synthesis and cell growth, there is a high need for iron therapies that result in high iron bioavailability with minimal toxic effects to treat patients suffering from anemia. This study aims to develop a novel oral iron-complex formulation based on hemin-loaded polymeric micelles composed of the biodegradable and thermosensitive polymer methoxy-poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl)methacrylamide-dilactate], abbreviated as mPEG-b-p(HPMAm-Lac2). Hemin-loaded micelles were prepared by addition of hemin dissolved in DMSO:DMF (1:9, one volume) to an aqueous polymer solution (nine volumes) of mPEG-b-p(HPMAm-Lac2) followed by rapidly heating the mixture at 50 °C to form hemin-loaded micelles that remain intact at room and physiological temperature. The highest loading capacity for hemin in mPEG-b-p(HPMAm-Lac2) micelles was 3.9%. The average particle diameter of the hemin-micelles ranged from 75 to 140 nm, depending on the concentration of hemin solution that was used to prepare the micelles. The hemin-loaded micelles were stable at pH 2 for at least 3 h which covers the residence time of the formulation in the stomach after oral administration and up to 17 h at pH 7.4 which is sufficient time for uptake of the micelles by the enterocytes. Importantly, incubation of Caco-2 cells with hemin-micelles for 24 h at 37 °C resulted in ferritin levels of 2500 ng/mg protein which is about 10-fold higher than levels observed in cells incubated with iron sulfate under the same conditions. The hemin formulation also demonstrated superior cell viability compared to iron sulfate with and without ascorbic acid. The study presented here demonstrates the development of a promising novel iron complex for oral delivery

Bi-specific tenascin-C and fibronectin targeted peptide for solid tumor delivery

Oncofetal fibronectin (FN-EDB) and tenascin-C C domain (TNC-C) are nearly absent in extracellular matrix of normal adult tissues but upregulated in malignant tissues. Both FN-EDB and TNC-C are developed as targets of antibody-based therapies. Here we used peptide phage biopanning to identify a novel targeting peptide (PL1, sequence: PPRRGLIKLKTS) that interacts with both FN-EDB and TNC-C. Systemic PL1-functionalized model nanoscale payloads [iron oxide nanoworms (NWs) and metallic silver nanoparticles] homed to glioblastoma (GBM) and prostate carcinoma xenografts, and to non-malignant angiogenic neovessels induced by VEGF-overexpression. Antibody blockage experiments demonstrated that PL1 tumor homing involved interactions with both receptor proteins. Treatment of GBM mice with PL1-targeted model therapeutic nanocarrier (NWs loaded with a proapoptotic peptide) resulted in reduced tumor growth and increased survival, whereas treatment with untargeted particles had no effect. PL1 peptide may have applications as an affinity ligand for delivery of diagnostic and therapeutic compounds to microenvironment of solid tumors

In Vivo Molecular MRI of ICAM-1 Expression on Endothelium and Leukocytes from Subacute to Chronic Stages After Experimental Stroke

Molecular MRI allows in vivo detection of vascular cell adhesion molecules expressed on inflamed endothelium, which enables detection of specific targets for anti-neuroinflammatory treatment. We explored to what extent MR contrast agent targeted to intercellular adhesion molecule-1 (ICAM-1) could detect endothelial- and leukocyte-associated ICAM-1 expression at different stages after experimental stroke. Furthermore, we assessed potential interfering effects of ICAM-1-targeted contrast agent on post-stroke lesion growth. Micron-sized particles of iron oxide (MPIO) functionalized with control IgG (IgG-MPIO) or anti-ICAM-1 antibody (αICAM-1-MPIO) were administrated at 1, 2, 3, 7, and 21 days after unilateral transient middle cerebral artery occlusion in mice, followed by in vivo MRI and postmortem immunohistochemistry. αICAM-1-MPIO induced significant contrast effects in the lesion core on post-stroke days 1, 2, and 3, and in the lesion borderzone and contralesional tissue on post-stroke day 2. αICAM-1-MPIO were confined to ICAM-1-positive vessels and occasionally co-localized with leukocytes. On post-stroke day 21, abundant leukocyte-associated αICAM-1-MPIO was immunohistochemically detected in the lesion core. However, MRI-based detection of αICAM-1-MPIO-labeled leukocytes was confounded by pre-contrast MRI hypointensities, presumably caused by phagocytosed blood remains. IgG-MPIO did not induce significant MRI contrast effects at 1 h after injection. Lesion development was not affected by injection of αICAM-1-MPIO or IgG-MPIO. αICAM-1-MPIO are suitable for in vivo MRI of ICAM-1 expression on vascular endothelium and leukocytes at different stages after stroke. Development of clinically applicable MPIO may offer unique opportunities for MRI-based diagnosis of neuroinflammation and identification of anti-inflammatory targets in acute stroke patients

In Vivo Molecular MRI of ICAM-1 Expression on Endothelium and Leukocytes from Subacute to Chronic Stages After Experimental Stroke

Molecular MRI allows in vivo detection of vascular cell adhesion molecules expressed on inflamed endothelium, which enables detection of specific targets for anti-neuroinflammatory treatment. We explored to what extent MR contrast agent targeted to intercellular adhesion molecule-1 (ICAM-1) could detect endothelial- and leukocyte-associated ICAM-1 expression at different stages after experimental stroke. Furthermore, we assessed potential interfering effects of ICAM-1-targeted contrast agent on post-stroke lesion growth. Micron-sized particles of iron oxide (MPIO) functionalized with control IgG (IgG-MPIO) or anti-ICAM-1 antibody (αICAM-1-MPIO) were administrated at 1, 2, 3, 7, and 21 days after unilateral transient middle cerebral artery occlusion in mice, followed by in vivo MRI and postmortem immunohistochemistry. αICAM-1-MPIO induced significant contrast effects in the lesion core on post-stroke days 1, 2, and 3, and in the lesion borderzone and contralesional tissue on post-stroke day 2. αICAM-1-MPIO were confined to ICAM-1-positive vessels and occasionally co-localized with leukocytes. On post-stroke day 21, abundant leukocyte-associated αICAM-1-MPIO was immunohistochemically detected in the lesion core. However, MRI-based detection of αICAM-1-MPIO-labeled leukocytes was confounded by pre-contrast MRI hypointensities, presumably caused by phagocytosed blood remains. IgG-MPIO did not induce significant MRI contrast effects at 1 h after injection. Lesion development was not affected by injection of αICAM-1-MPIO or IgG-MPIO. αICAM-1-MPIO are suitable for in vivo MRI of ICAM-1 expression on vascular endothelium and leukocytes at different stages after stroke. Development of clinically applicable MPIO may offer unique opportunities for MRI-based diagnosis of neuroinflammation and identification of anti-inflammatory targets in acute stroke patients

A novel oral iron-complex formulation: Encapsulation of hemin in polymeric micelles and its in vitro absorption

Anemia resulting from iron deficiency is one of the most prevalent diseases in the world. As iron has important roles in several biological processes such as oxygen transport, DNA synthesis and cell growth, there is a high need for iron therapies that result in high iron bioavailability with minimal toxic effects to treat patients suffering from anemia. This study aims to develop a novel oral iron-complex formulation based on hemin-loaded polymeric micelles composed of the biodegradable and thermosensitive polymer methoxy-poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl)methacrylamide-dilactate], abbreviated as mPEG-b-p(HPMAm-Lac2). Hemin-loaded micelles were prepared by addition of hemin dissolved in DMSO:DMF (1:9, one volume) to an aqueous polymer solution (nine volumes) of mPEG-b-p(HPMAm-Lac2) followed by rapidly heating the mixture at 50 °C to form hemin-loaded micelles that remain intact at room and physiological temperature. The highest loading capacity for hemin in mPEG-b-p(HPMAm-Lac2) micelles was 3.9%. The average particle diameter of the hemin-micelles ranged from 75 to 140 nm, depending on the concentration of hemin solution that was used to prepare the micelles. The hemin-loaded micelles were stable at pH 2 for at least 3 h which covers the residence time of the formulation in the stomach after oral administration and up to 17 h at pH 7.4 which is sufficient time for uptake of the micelles by the enterocytes. Importantly, incubation of Caco-2 cells with hemin-micelles for 24 h at 37 °C resulted in ferritin levels of 2500 ng/mg protein which is about 10-fold higher than levels observed in cells incubated with iron sulfate under the same conditions. The hemin formulation also demonstrated superior cell viability compared to iron sulfate with and without ascorbic acid. The study presented here demonstrates the development of a promising novel iron complex for oral delivery

Paclitaxel-Loaded Polymersomes for Enhanced Intraperitoneal Chemotherapy

Peritoneal carcinomatosis is present in more than 60% of gastric cancer, 40% of ovarian cancer, and 35% of colon cancer patients. It is the second most common cause of cancer-related mortality, with a median survival of 1 to 3 months. Cytoreductive surgery combined with intraperitoneal chemotherapy is the current clinical treatment, but achieving curative drug accumulation and penetration in peritoneal carcinomatosis lesions remains an unresolved challenge. Here, we used flexible and pH-sensitive polymersomes for payload delivery to peritoneal gastric (MKN-45P) and colon (CT26) carcinoma in mice. Polymersomes were loaded with paclitaxel and in vitro drug release was studied as a function of pH and time. Paclitaxel-loaded polymersomes remained stable in aqueous solution at neutral pH for up to 4 months. In cell viability assay on cultured cancer cell lines (MKN-45P, SKOV3, CT26), paclitaxel-loaded polymersomes were more toxic than free drug or albumin-bound paclitaxel (Abraxane). Intraperitoneally administered fluorescent polymersomes accumulated in malignant lesions, and immunofluorescence revealed an intense signal inside tumors with no detectable signal in control organs. A dual targeting of tumors was observed: direct (circulation-independent) penetration, and systemic, blood vessel-associated accumulation. Finally, we evaluated preclinical antitumor efficacy of paclitaxel-polymersomes in the treatment of MKN-45P disseminated gastric carcinoma using a total dose of 7 mg/kg. Experimental therapy with paclitaxel-polymersomes improved the therapeutic index of drug over free paclitaxel and Abraxane, as evaluated by intraperitoneal tumor burden and number of metastatic nodules. Our findings underline the potential utility of the polymersome platform for delivery of drugs and imaging agents to peritoneal carcinomatosis lesions