Stretching single DNA-molecules with temperature-stabilized optical tweezers

This thesis consists of two parts; in the first part we performed a single-molecule force extension measurement with 10kb long DNA-molecules from phage-λ to validate the calibration and single-molecule capability of our optical tweezers instrument. Fitting the worm-like chain interpolation formula to the data revealed that ca. 71% of the DNA tethers featured a contour length within ±15% of the expected value (3.38 µm). Only 25% of the found DNA had a persistence length between 30 and 60 nm. The correct value should be within 40 to 60 nm. In the second part we designed and built a precise temperature controller to remove thermal fluctuations that cause drifting of the optical trap. The controller uses feed-forward and PID (proportional-integral-derivative) feedback to achieve 1.58 mK precision and 0.3 K absolute accuracy. During a 5 min test run it reduced drifting of the trap from 1.4 nm/min in open-loop to 0.6 nm/min in closed-loop.Tämä tutkielma koostuu kahdesta osasta; ensimmäisessä osassa tutkimme optinen pinsetti-laitteistomme kalibraatiota ja soveltuvuutta yksittäismolekyylikokeisiin 10kb pituisien λ-faagista peräisin olevien DNA-molekyylien voima-venytyskokeilla. Mittauksiin sovitettiin 'worm-like chain'-interpolaatio malli, joka osoitti, että n. 71%:lla löydetyistä DNA ketjuista oli pituus 15% sisällä odotetusta pituudesta (3.38 µm). Vain 25%:lla DNA:sta oli sitkeyspituus 30-60 nm odotetun arvon ollessa 40-60 nm. Tutkielman toisessa osassa rakensimme lämpötilakontrollerin, jonka tarkoituksena oli poistaa lämpötilavaihteluiden aiheuttama 'ajelehtiminen' optisilla pinseteillä kiinnipidetyn mikroskooppisen pallon paikassa. Kontrolleri käyttää 'feedforward-' ja takaisinkytkentäsilmukoita saavuttaakseen 1.58 mK sisäisen tarkkuuden ja 0.3 K ulkoisen tarkkuuden. Viiden minuutin kokeen aikana pallo ajelehti 1.4 nm/min avoimella silmukalla ja 0.6 nm/min suljetulla silmukalla

Controlled release device for oral cavity

The present invention relates to a controlled release device for oral cavity, which is attached on a hard dental surface of a tooth. The attachment is done using any adhesive layer that can attach on enamel surface of a tooth or using a holder attached to the hard dental surface, to which holder the device can be attached, clipped or fastened. The device comprises of two or more polymeric materials forming a polymer matrix or matrices. The polymer matrices incorporate one or more agent(s), which release rate in the oral cavity can be con-trolled with the polymeric materials in the matrices. The invention also relates to a method producing a controlled re-lease device for delivering an agent to the oral cavity

Kohdennetut nanolääkkeet voivat muokata tulevaisuuden terveydenhoitoa

Kohdennettu nanolääke on nanokokoisen kantajahiukkasen ja lääkeainemolekyylin yhdistelmä. Tällaisten lääkkeiden etu tavanomaisiin lääkkeisiin nähden on se, että ne voivat kuljettaa lääkkeet oikeaan kudokseen tai soluun ja vapauttaa ne vasta siellä. Ideaaliset nanolääkkeet ovat yhteensopivia kudosten kanssa (bioyhteensopivia) ja biohajoavia. Ne voivat tunkeutua syvälle syöpäkudokseen ja niitä voidaan muokata siten, että ne kiinnittyvät vain tiettyihin kohdesoluihin. Ensimmäinen nanolääkesukupolvi on jo markkinoilla, ja seuraavia tutkitaan kliinisissä kokeissa.Kohdennettu nanolääke on nanokokoisen kantajahiukkasen ja lääkeainemolekyylin yhdistelmä. Tällaisten lääkkeiden etu tavanomaisiin lääkkeisiin nähden on se, että ne voivat kuljettaa lääkkeet oikeaan kudokseen tai soluun ja vapauttaa ne vasta siellä. Ideaaliset nanolääkkeet ovat yhteensopivia kudosten kanssa (bioyhteensopivia) ja biohajoavia. Ne voivat tunkeutua syvälle syöpäkudokseen ja niitä voidaan muokata siten, että ne kiinnittyvät vain tiettyihin kohdesoluihin. Ensimmäinen nanolääkesukupolvi on jo markkinoilla, ja seuraavia tutkitaan kliinisissä kokeissa.Peer reviewe

Folic acid-mesoporous silicon nanoparticles enhance the anticancer activity of the p73-activating small molecule LEM2

Many drugs with anticancer potential fail in their translation to the clinics due to problems related to pharmacokinetics. LEM2 is a new dual inhibitor of MDM2/mutp53-TAp73 interactions with interesting in vitro anticancer activity, which opens new hopes as an unconventional anticancer therapeutic strategy against cancers lacking p53 or with impaired p53 pathways. As others xanthone derivatives, LEM2 has limited aqueous solubility, posing problems to pursue in vivo assays, and therefore limiting its potential clinical translation. In this work, a mesopomus silicon (PSi)-based nanodelivery system was developed with folate functionalization (APTES-TCPSi-PEG-FA) for targeted delivery, which successfully increased LEM2 solubility when compared to bulk LEM2, evidenced in payload release study. Such effect was reflected on the increase of LEM2 cytotoxicity in HCT116 and MDA-MB-231 cancer cells when treated with LEM2-loaded APTES-TCPSi-PEG-FA, by reducing cell viability lower than 50% in comparison with bulk LEM2. Despite the reduced LEM2 loading degree, which still limits its application in further in vivo assays, the results obtained herein recognize PSi-based nanodelivery systems as a promising strategy to improve LEM2 anticancer activity and bioavailability, which will be relevant for the potential use of this potent TAp73 activator in anticancer therapy.Peer reviewe

Double-Layered Polyvinylpyrrolidone–Poly(Methyl Vinyl Ether-Alt-Maleic Acid) based Microneedles to Deliver Meloxicam:An In Vitro, In Vivo and Short-Term Stability Evaluation Study

This study aims to explore the use of polymeric microneedles (MNs) for the transdermal delivery of drugs, a non-invasive and convenient method that avoids first-pass metabolism and gastrointestinal complications. Specifically, we develop a double-layered MN formulation using polyvinylpyrrolidone and cross-linked poly(methyl vinyl ether-alt-maleic acid), comprising a dissolvable layer and a hydrogel-forming layer. Meloxicam serves as the model drug, and no organic solvents are employed in the manufacturing process to reduce toxicity. Coherent Anti-Stokes Raman Spectroscopy (CARS) is utilized to confirm that the manufacturing process does not alter the drug's physical properties. In vitro and ex vivo studies demonstrate that the double-layered MN formulation exhibits faster drug release in the first few hours, followed by a slower release. This results in extended bioavailability in vivo compared to the commercial oral formulation of meloxicam. Preliminary results indicate that the MN formulation is also effective in pain relief and inflammation reduction. The short-term stability of the MNs formulation is also confirmed, including its mechanical properties, sustained skin permeability, drug physical properties, and distribution within MNs using CARS microscopy. Overall, these results suggest that the double-layered MN formulation holds significant potential for transdermal drug delivery, offering a safer and more effective alternative to traditional oral administration

One-step microfluidics production of enzyme-loaded liposomes for the treatment of inflammatory diseases

The biopharmaceuticals market is constantly growing. Despite their advantages over the conventional drugs, biopharmaceuticals have short biological half-lifes, which can be increased using liposomes. However, the common bulk methods to produce biopharmaceuticals-loaded liposomes result in lost of encapsulation efficiency (E.E.), resulting in an expensive process. Herein, the encapsulation of a therapeutic enzyme in liposomes is proposed, using a glass-capillary microfluidic technique. Cu,Zn- Superoxide dismutase (SOD) is successfully encapsulated into liposomes (SOD@Liposomes). SOD@Liposomes with a mean size of 135 ± 41 nm, a polydispersity index of 0.13 ± 0.01, an E.E. of 59 ± 6 % and an enzyme activity of 82 ± 3 % are obtained. In vivo experiments show, through an ear edema model, that SOD@Liposomes administered by the intravenous route enable an edema inhibition of 65 % ± 8 %, over the 20 % ± 13 % of SOD in its free form. The histopathological analyses show a higher inflammatory cell accumulation on the ear treated with SOD in its free form, than treated with SOD@Liposomes. Overall, this work highlights the potential of microfluidics for the production of enzyme-loaded liposomes with high encapsulation efficiency, with the intrinsic advantages of the low time-consuming and easily upscaling microfluidic assembly method.Peer reviewe

Hybrid red blood cell membrane coated porous silicon nanoparticles functionalized with cancer antigen induce depletion of T cells

Erythrocyte-based drug delivery systems have been investigated for their biocompatibility, long circulation time, and capability to transport cargo all around the body, thus presenting enormous potential in medical applications. In this study, we investigated hybrid nanoparticles consisting of nano-sized autologous or allogeneic red blood cell (RBC) membranes encapsulating porous silicon nanoparticles (PSi NPs). These NPs were functionalized with a model cancer antigen TRP2, which was either expressed on the surface of the RBCs by a cell membrane-mimicking block copolymer polydimethylsiloxane-b-poly-2-methyl-2-oxazoline, or attached on the PSi NPs, thus hidden within the encapsulation. When in the presence of peripheral blood immune cells, these NPs resulted in apoptotic cell death of T cells, where the NPs having TRP2 within the encapsulation led to a stronger T cell deletion. The deletion of the T cells did not change the relative proportion of CD4+ and cytotoxic CD8+ T cells. Overall, this work shows the combination of nano-sized RBCs, PSi, and antigenic peptides may have use in the treatment of autoimmune diseases.Peer reviewe