Fourth Winner of the SPECTROstar Nano Promotion:
Dr. Marco G. Casteleijn, University of Helsinki

"We at The Centre for Drug Research, at the Faculty of Pharmacy in the University of Helsinki are thrilled our proposal was chosen from so many excellent proposals. Our facility acts as an incubator to develop novel drug technologies by combining recent advances in biosciences and materials sciences. However, it is a challenge to keep implementing novel techniques for screening and evaluating novel drug formulation and drug delivery. We, at the research group of Prof. and CDR Director, Arto Urtti, where I just started as a post doctoral researcher; we would like to thank BMG LABTECH for the opportunity provided.

When I heard from a collaborator about the competition I was immediately very interested. Mainly since building a flexible high throughput platform for protein engineering and evaluating nanoparticle formulations requires various components of high quality that need to be integrated together. Another parameter is fast readout to avoid drift and non- robust assays. It was clear from the specifications of the SPECTROstar Nano, that if we could somehow attain one, we could get a head start on implementing at least two vital methods needed on the larger theme of nanoparticle drug delivery to selected tissues (e.g. ocular drug delivery). " 

Automated Evaluations of Biologically Inspired Nanoparticles and its Immune Response

Abstract

Biologically inspired nano-particles and related recombinant protein engineering libraries are in need for automated methods evaluation. Our goals to achieve this are: (1) set-up of a flexible platform for liquid handling, which includes an automated spectrometer, (2) implement the ELISA based Complement Activation Assay, and (3) implement an Automated Protein Production method. These goals seem feasible, and are planned for 2011 -2012.

Background

Drug delivery is a major challenge in modern drug development. There are several reasons for this development. Firstly, biotechnological drugs, based on the body’s own components, are emerging: plasmid DNA, oligonucleotides (siRNA, miRNA, antagomirs), and proteins. Secondly, some important target sites are only poorly accessible even for small molecular weight drugs, e.g. the brain and retina. Thirdly, drug delivery to tumors is relatively inefficient, and thus high doses of anti-cancer drugs produce severe off-target effects. Fourthly, the quest for high affinity ligands in drug discovery has resulted in complex molecules with so poor water solubility that is hampers their use. Finally, efficient genetic engineering of the cells (e.g. induced pluripotent stem cells, IPSC; cell array generation) with nano-particles would benefit cell biology, cell therapy and drug development. It is obvious that solving the drug delivery problems would have enormous significance in biomedical science.

In the Centre for Drug Research several projects are geared towards solving these problems systematically in a High Throughput (HTP) approach. Our main objectives in this project are:

To build an unprecedented systematic discovery platform for pharmaceutical nanoparticles encompassing delivery of small molecules, siRNA and DNA.The specific aims are to:

• Generate novel nano-particles
• Build systematic understanding between the nano-particle structure and drug delivery based on high throughput evaluation
• Reveal the delivery mechanisms of nano-particles
• Optimize the pharmaceutical nano-particles towards:
• (Long term) drug delivery to the retina and choroid of the eye

A discovery platform for pharmaceutical nano-particles relies on modern methods. It will open important new horizons and it is expected to fundamentally renew the field of drug delivery.

Methods and Materials

In order to achieve our research goals, several experiments and techniques which fall out of scope with this application will be applied or implemented. However, a few specific experiments could benefit greatly by the use to the SPECTROstar Nano due to its fast reading times, robotic interface mode, low volume measurements, plate reading capabilities,  shaking and incubating capabilities, and full spectrum readout. In addition the gas vent for atmospheric control could be useful for other applications in the future, in case of automated growth and control of mammalian cell lines.

Below are two planned experiments, based on already existing in-house methods, to be used to evaluate aspects of nano-particle libraries (Fig. 1), namely: the Complement Activation Assay and Automated Protein Production for the evaluation of Protein Engineered Targets.

Complement Activation Assay
Nano-particles are usually recognized in the body as non-self components by the complement system, a biochemical cascade that is part of the innate immune system. Nano-particle structure influences its ability to activate the complement system, but the underlying structural features are not known. Ideal nano-particles would be inert towards the complement system. Complement activation by any one of the three main pathways results in cleavage of the C3 component and production of the C3a anaphylatoxin.
Human and mouse plasma and ocular vitreous (bovine, rabbit) will be exposed to test nano-particles. Incubation with the biological media, centrifugation and dilutions are done in 96 well plates engineered to bind low amounts of proteins. The C3a anaphylatoxin produced by complement activation is measured by ELISA, a well-characterized and sensitive immunological assay. The screening assay allows automation and testing at a higher throughput in 96 well plates allowing up to 104 determinations. Human and mouse plasma results will be compared. This is important in the light of later in vivo experiments in mice.
The SPECTROstar Nano would be place in-line with our pipetting robot outside the deck to incubate, shake and read the ELISA assay part of the Complement Activation Assay.

Automated Protein Production
Therapeutic polypeptides and proteins, and protein targets for nano-particle formulations will be produced as recombinant proteins in our emerging Protein Engineering Facilities. Recombinant proteins will be expressed and purified using cell free expression, with E. coli, insect cells and mammalian cells as backup systems. Proteins that show promise in terms of functionality (e.g. complement evasion of the conjugated nano-particles, as assayed with the Complement Activation Assay) will be then genetically engineered by directed evolution to generate mutant libraries that will be evaluated for their efficacy in conjugation with the nano-particles.

Cell growth and protein production in cell-free systems will be automated where possible. Protein production can take place in a 24, 48 or 96 well format depending on expression levels, downstream efficiency and amount needed. The automated fashion of the experiments is only possible if the Optical Density (OD) of the cultures is monitored with an automated spectrometer in-line with our pipetting robot outside the deck. At intermediate intervals small sub-samples are taken to evaluate the OD and eventual read-out of a protein evaluation assay (protein case depended and choice of tags). Since the production plates are waiting for further action (i.e. prolonged incubation, induction of protein production, or termination of program and harvest) the readouts need to be fast. Also feed-back loops of the electronically read-out data needs to evaluated by the software in order to initiate sub-programs.

The SPECTROstar Nano would be an excellent candidate for a readout device since it possesses all wanted properties for these experiments.

Additional use
In addition, model drug concentrations (Fig. 1) such as calcein, small lipophilic molecules (e.g. rhodamine B), siRNA and pDNA (incl. fluorescent derivatives of both) can be evaluated by the SPECTROstar nano (i.e. full spectrum, small volumes of 2 µl) during the formulation process.

Expected outcomes and future outlook
For the full spectrum of intermediate goals of the project we are setting up test methods with existing nano-particle materials.

The two proposed methods in the application are crucial throughout for nano-particle evaluations the since feedback related experiments in other projects will generate novel nano-particles to be evaluated with the Complement Activation Assay, and induces new Protein Engineering experiments, leading to large volumes of recombinant Protein Production.

The future outlook is that at the end of the project we have a High Throughput platform for the production, but more importantly, a detailed understanding of nano-particle formulations. We also expect to attract several research topics (and generate applications) to the Automated Protein Engineering facilities, which we are currently building up.    

Conclusions
The Complement Activation Assay is a functioning in-house assay which is in need to scaling up in a 96 well plate format. Preliminary results are very promising, since the ELISA is quite robust. The next step of automation depends on the implementation of the High Throughput facilities focused on Automated Protein Production. Although this sounds challenging, the applicant has in his previous work set up a fully automated E. coli Protein Production system at the University of Oulu (Hamilton Star pipetting deck, Hotel/incubator shaker, Victor3 plate reader, and various in-deck stations for evaluation/incubation/storage). For further reading see literature [#12].

We therefore think that these methods will be fully implemented in the years 2011 – 2012, and our funding efforts are geared to these applications. Some funding has been acquired already. For both of these methods a fully dedicated, automated, well rounded spectrometer is a must and, during evaluation of various plate readers found, the SPECTROstar Nano is one of the most promising candidates.

Literature for further reading

1. Ruponen M, Rönkkö S, Honkakoski P, Pelkonen J, Urtti A: Influence of extracellular glycosaminoglycans on intracellular trafficking of lipoplexes and polyplexes. J Biol Chem. 276: 33875-80, 2001
2. Pitkänen L, Ranta VP, Moilanen H, Urtti A: Permeability of retinal pigment epithelium: effect of permeant molecular weight and lipophilicity. Invest Ophthalmol Vis Sci 46: 641-646, 2005
3. Ranta VP and Urtti A: Transscleral drug delivery to the posterior eye: Prospects of pharmacokinetic modeling. Adv Drug Deliv Res 58: 1164-1181, 2006
4. M Männistö, M Reinisalo, M Ruponen, P Honkakoski, M Tammi, A Urtti: Polyplex-mediated gene transfer and cell cycle: effect of carrier on cellular uptake and intracellular kinetics, and significance of
   glycosaminoglycans. J Gene Med 9: 479-486, 2007
5. Paasonen L, Laaksonen T, Johans C, Yliperttula M, Kontturi K, Urtti A: Gold nanoparticles enable selective light induced drug release from liposomes. J Control Rel 122: 86-93, 2007
6. Lehtinen J, Hyvönen Z, Bunjes H, Subrizi A, Urtti A: Glycosaminoglycan resistant and pH-sensitive lipidcoated DNA-complexes produced by detergent dialysis method. J Control Rel 131: 145-149, 2008
7. E. Vuorimaa, A Urtti, R. Seppänen, H. Lemmetyinen,, M. Yliperttula: Time-Resolved Fluorescence Spectroscopy Reveals Differences in Structural Organisation of Cationic Polymer - DNA Complexes. J Am Chem Soc 130: 11695-11700, 2008
8. Wikström J, Elomaa M, Syväjärvi H, Kuokkanen J, Yliperttula M, Honkakoski P & Urtti A: Alginate Based Microencapsulation of Retinal Pigment Epithelial Cell Line for Cell Therapy. Biomaterials 29: 869-876, 2008
9. Hornof M, Fuente M, Hallikainen M, Tammi R, Urtti A: Low molecular weight hyaluronan shielding of DNA/PEI polyplexes facilitates CD44 receptor mediated uptake in human corneal epithelial cells J Gene Med, 10: 70-80, 2008
10. A Huhtala, S Rönkkö, M Teräsvirta, T Puustjärvi, R Sihvola, A Laukkanen, J Anttila, A Urtti, T Pohjonen, and H Uusitalo: The effects of 5-fluorouracil on ocular tissues in vitro and in vivo after controlled release from a multifunctional implant, Invest Ophthalmol Vis Sci 50(5):2216-23, 2009. Epub 2009 Jan 1 2009
11. J Panula-Pärälä, J Šiurkus, A Vasala, R Wilmanowski, MG Casteleijn, and P Neubauer: EnBase: Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks, Micro Cell Fact., 18; 7:31, 2008
12. MG Casteleijn: Towards new enzymes: protein engineering versus bioinformatic studies. (PhD thesis, Monograph). Acta Universitatis Ouluensis Technica C 349, 2010 (ISBN 978-951-42-6098-8/ISSN 0355-3213: http://herkules.oulu.fi/isbn9789514260995/)
    

For more informaiton on their research visit: http://www.ddtc.helsinki.fi/research/group_urtti.htm#nano

Marco G. Casteleijn, PhD (h.2065)
University of Helsinki
Centre for Drug Research, Faculty of Pharmacy
PO Box 56
FI-00014 Helsinki

marco.casteleijn@helsinki.fi

See the proposals of the other winners here.