What’s up in Nanomedicine?

Malaria, Nanomedicine and the Future

Due to personal interest, I was excited when I found the work of Valle-Delgado and coworkers (1). This interest is while I am involved in the DiscoGnosis project, which is a project for the development of Point-of-Care diagnosis of malaria and tropical diseases on a CD-like operating form (see also www.discognosis.eu). Valle-Delgado performed a fundamental investigation that might reopen the door to the development of a new generation of malaria medicines, which I will explain below. The investigators used single molecule force microscopy to demonstrate that heparin specifically binds to the surface of infected red blood cells (RBCs) and compared this with non-infected RBCs. RBCs infected with the parasite Plasmodium falciparum are known to bind to endothelial cells and post capillary venules of tissues. This process is called sequestration and it enables the replication of parasites while avoiding the splenic clearance. The infected RBCs are also capable to bind non-infected RBCs and form rosettes, which in their turn may at the end lead to occlusions of microvessels, which is suggested to play a lethal role in the severe cases of malaria (2–4). It is known that a specific P. falciparum protein expressed on the membranes of infected red blood cells called PfEMP1, plays a role in the cytoadherence of infected RBCs. One of the involved types of receptors in this process are the glycosaminoglycans (GAGs) (5). Unraveling the fundamentals of this binding mechanism of infected RBCs will help to develop new nanomedical strategies against malaria, such as specific competitors against the rosetting or sequestration. Valle-Delgado and coworkers investigated the binding of the GAG heparin and infected RBCs by using single molecule force microscopy. To perform this investigation, heparin molecules were cross-linked to an AFM cantilever. When retracting the heparin functionalized AFM tips, the forces of the surface and the tip were measured. Both infected and uninfected RBCs were studied, the infected RBCs obtained a binding range between 28–46 pN, whereas from the uninfected RBCs no significant binding was observed. Heparin treatments were already given in the past, however the heparin has the disadvantage of its strong anticoagulant action causing intracranial bleeding as a side effect. Interestingly, when the heparin molecules are covalently bound through its carboxylgroups, it has been shown that the anticoagulant action is dramatically reduced (6). The authors suggested that when covalently attached to nanocarriers for antimalarial drug delivery these heparin molecules could be a viable therapeutic approach and in addition, the risk of an internal bleeding might be reduced when comparing to the soluble heparin. With this they reopened the door to use heparin as an anti-malaria drug but this time covalently coupled to a nanocarrier. To read more about this, I would also like to refer to the recent editorial written by Prof. Xavier Fernàndez-Busquets head of the group of which Valle-Delgado is a member (7).

Related to the topic malaria, I would like to mention the European Malaria Reagent Repository (EMRR) developed by David Cavanagh. The aim of this repository is not only to secure the future of reagents held in the University of Edinburgh but also to maximize their utility all over the worldwide community. This huge collection includes for example parasite cell lines, rodent malaria parasites, genetically and antigenically diverse isolates, parasite proteins and antibodies. The EMRR recently provided funding by the Wellcome Trust on the basis to make it self-sustaining by 2017. As a result, reagents are provided to the community on a cost-recovery basis. For further reading see also: http://www.malariaresearch.eu/content/about-repository. Personally, I believe such initiatives are a golden key in the sense of providing materials for fundamental investigations, development of new medicines and vaccines and diagnostics.

Gold nanoparticles coated with red blood cell membranes

As most of you know, gold nanoparticles are widespread used in applications such as imaging, lateral flow assays or drug carriers. Hu and coworkers developed a method to translocate intact membranes and peripheral proteins to soft colloidal particles (8). Within the same group, Gao et al. are now applying this method to functionalize gold nanoparticles (Au-NP) (9). With this new design strategy they open the door to new nano-drug delivery systems in addition to many other biomedical applications. The investigators first studied the diameter of the nanoparticle after coating with the RBC membrane, and measured an increase from 70.1±0.3 nm to 86.5±nm, which is in agreement with the thickness of the RBC membrane (∼8 nm). The surface zeta potential was measured and changed from –42.2±1.3 mV for uncoated Au-NP to –35.1±1.1 mV after coating. Uranyl acetate staining and the visualization by using transmission electron microscopy demonstrated the successful coating with the RBC membranes by using transmission electron microscopy. The authors also investigated the interaction with thiolated ligands ((FITC)-thiol) by using electrospray mass spectrometry and measuring the quenching of the FITC emission and they further confirmed the stability and the shielding effect of the coated RBC membrane. The stability RBC-Au-NPs was also tested by suspending the particles in 1x phosphate buffered saline or 100% fetal bovine serum with a final concentration of 25 μg/mL and monitored over a timespan of 72 h. Next to the slight decrease in fluorescence intensity, which was attributed to long-range fluorescence quenching effect of Au-NPs, no further decrease in fluorescence was monitored. This confirmed the stability of the RBC-Au-NPs. The well-documented membrane protein CD47 was investigated with two distinct anti-CD47 antibodies that bind specific to the exoplasmic and cytoplasmic region of CD47. This marker is important since it is capable of inhibiting macrophage phagocytosis. After confirming that the exoplasmic region of CD47 was facing the outside of the coated Au-NPs, the uptake by J774 murine macrophage cells was investigated. Also here naked Au-NPs were used as a background control. From these results the authors of the study showed that RBC-Au-NPs had an uptake of 3.2 ng per 1000 macrophages whereas the uncoated Au-NPs had an uptake of 13.5 ng per 1000 macrophages. With these results it is confirmed that by using the RBCs membrane as a coating on the nanoparticles, the immune system can be circumvented.

Novel nanomedicines

Mucins, the major components in all mucous secretions, are a heteronegeous group of large glycoproteins. In the ocular area, they act as lubricants, as stabilizers and as a physical barrier to pathogen penetrance (10). The production of mucin can be affected by inflammation (11). The highly glycosylated mucin MUC5AC is a very important clinically relevant mucin and is secreted by specialized epithelial cells of the conjunctiva (the goblet cells). This mucin plays a key role in tear homeostasis (12) and the expression is decreased in several ocular diseases. Contreras-Ruiz and coworkers manufactured cationized gelatin-based nanoparticles (NP), which were loaded with a plasmid coding for a modified MUC5AC protein (12). These NPs were instilled in experimental dry eye mice and healthy mice. The expression of the protein, clinical signs, production of tears and corneal fluorescein staining were investigated in both mouse types. Note fluorescein staining is a routine clinical test used to determine corneal integrity in patients. Histopathologic evaluation of the ocular specimen included goblet cell count and CD4 immuno staining. In healthy mice the NPs had no effect on fluorescein staining or tear production. In the affected mice, the expression of modified MUC5A was successfully induced, which led to a reduction of the inflammation and an improvement of the clinical parameters such as fluorescein staining and tear production. Thus, with this investigation the authors presented a proof-of-concept of a safe topical transfection of ocular surface cells by using pMUC5AC-NPs and suggested this as a new therapeutic manner for the treatment of dry eye disease.

Another novel potential nanomedicine is presented by Zeng and coworkers (13). They developed cholic acid functionalized nanoparticles of star shaped poly (lactide-co-glycolide) (or PLGA)-d-α-tocopheryl polyethylene glycol succinate (or vitamin E TPGS) copolymer for docetaxel (DTX) delivery to cervical cancer. The core first method was applied to synthesize the star shaped copolymer of the three branch arms of cholic acid (CA) PLGA-b-TPGS. The synthesized structure was confirmed by nuclear magnetic resonance, gel permeation chromatography and thermogravimetric analysis. The DTX loaded CA-PLGA-b-TPGS nanoparticles were developed using a nanoprecipitation method. Of these NPs, the size and zeta potential were measured and the morphology was examined by using a field emission scanning electron microscope. The NPs were further investigated on: (I) drug loading and encapsulation efficiency, (II) the physical status of docetaxel inside the NPs, and (III) the in vitro drug release was studied. The model fluorescent molecule used was coumarin 6, which can be incorporated in the different NPs investigated here (PLGA-NPs, PLGA-b-TPGS-NPS and CA-PLGA-b-TPGS). Coumarin 6 enables the qualitative and quantitative investigation on cellular uptake of the NPs by cancer cells. Both the cellular uptake and the cytotoxicity of DTX loaded NPs was done by using HeLa cells. The capacity of the NPs in vivo was studied by using female severe combined immuno deficient mice. From this investigation it can be concluded that the newly developed system demonstrated a superior performance on anti-tumor effect in vitro and in vivo when compared with the linear PLGA-b-TPGS copolymer NPs and the drug loaded PLGA. The authors suggested that their presented star-shaped CA-PLGA-b-TPGS NPs might also be applicable to other (highly hydrophobic) drugs.

Upcoming events

An interesting event organized from 26–28th of September is the International Conference on Nanotheranostics (ICoN 2013). The aim of this conference is to provide the optimal venue to expand the research in this multidisciplinary field and to bring the key researchers in the nanotheranostics field together. Examples of included thematic areas are: (I) nanoscience technologies for theranostics, (II) cancer nano-theranostics, and (III) toxicology, regulatory aspects and ethics. For more information see also: http://cyprusconferences.org/ICoN2013/index.php.

Prior to the above-mentioned ICoN 2013 conference, the Marie Curie Industry-Academia Partnerships and Pathways (IAPP) NANORESISTANCE consortium is organizing a summer school for young researchers in both academic and entrepreneurial sectors. The NANORESITANCE consortium exist of two companies and three academic laboratories located in Cyprus, Great Britain, Greece and France. The project is focused on the management of resistance to tyrokinase inhibitors using nanometric platforms for drug delivery. For further reading about this project, see: http://www.epos-iasis.com/IAPP. The goal of this summer school is to overcome fragmented and disseminated expertise of academic and industrial groups involved in this new research domain and to stimulate the transfer of technology. Examples of included topics are: nanochemistry, cancer biology signal transduction, tissue mechanics and molecular imaging. For more details about this summer school, see: http://cyprusconferences.org/ICoN2013/school.php.

Finally I would like to inform you that after the successful CLINAM conference of 2013, I am happy to announce that also next year this conference will be organized. It will be held from 22–25th of June 2014 in Basel, Switzerland. Of course I will highlight this more extensively when this event approaches.