Bioactive nanostructured materials

Transcription

1 Bioactive nanostructured materials I.Yudovin-Farber, N.Beyth, E.Wies, H.Elyahu and A.J.Domb Department of Medicinal Chemistry and Natural Products, School of Pharmacy, Faculty of Medicine, the Hebrew University of Jerusalem, Jerusalem 91120, Israel To whom correspondence should be addressed. Phone: Fax:

2 Nanoscale materials has an increasingly important impact on a number of sectors, including biotechnology, electronics, energy, and industrial products. For instance: nanoelectronic devices based on quantum dots or molecular switches will enable nextgeneration memory and logic chips, nanoparticles are being developed for the targeting and destruction of breast cancer cells and, at last, antimicrobial nanocoatings on wound dressings kill bacteria, reduce inflammation and promote healing. This report focus on nanoparticles applied on three main fields: development of antibacterial materials in dentin bonding systems, design of anti-prion agents and nonviral vectors for gene delivery. Antibacterial Nanoparticles Resin composite restorations are widely used in the dental practice. These restorations tend to accumulate bacterial biofilm forming dental plaque. Accumulation of dental plaque at the interface between the composite restorations and the tooth tissues may result in secondary caries. Cavity preparation for a dental restorative procedure does not eliminate the cariogenic bacteria in the dentinal tissues, thus in future may be a bacterial reservoir that will cause cavitations and pulpal inflammation. Due to the difficulty in achieving a hermetic seal between the restoration and the tooth tissues, bacterial microleakage should be prevented by developing restorations bearing antibacterial surface properties. A number of studies demonstrated experiments in which an antibacterial agent was incorporated into the filling materials, in order to inhibit bacterial development. However, the antibacterial activity of these materials is dependent upon release of the antibacterial agents. Following drawbacks such as influence on mechanical properties, loss of effectiveness and toxic effects can be associated with controlled release.

3 Attempts have been made to use another approach without releasing the antibacterial agent into a solution. The incorporation of methacryloxydodecyl pyridinium bromide has been reported to be effective in providing dentin bonding systems with antibacterial activity before and after curing[1, 2]. Prompted by these results, we decided to focus on incorporation of the antibacterial agent, which can be based on cationic polymer exhibits antibacterial properties in dental system. Some publications can be found about polycations possessing antibacterial properties by interacting with and disrupting bacterial cell membranes. A number of polymers that exhibit antibacterial properties were developed for this purpose including soluble and insoluble pyridinium-type polymers which are involved in surface coating[3], azidated poly(vinyl chloride)[4] which can be used to prevent bacterial adhesion of medical devices, PEG polymers that can be modified on polyurethane surfaces and also prevent initial adhesion bacteria to the biomaterial surfaces[5], and polyethyleneimine (PEI)[6] that exhibit high antibacterial and antifungal activity. High activity of polycationic agents is related to absorption of positive charged nanostructures onto negative by charged cell surfaces of the bacteria. This process is thought to be responsible for the increase of cell permeability and may disrupt the cell membranes. Hydrophobically modified PEI exhibits amphiphilic properties. In this study, crosslinked polycations were prepared as nanoparticles that were formed from PEI by crosslinking and alkylation followed by methylation in order to increase degree of amino group substitution. Because of its positive charge and hydrophobicity, PEI nanoparticles have attracted attention as possible antimicrobial agents. Studies on PEI nanostructured compounds were made to evaluate its antibacterial properties as a function of hydrophobicity, molecular weight, particle size and charge

4 that can play a significant role in antibacterial effect of the tested compound. The antibacterial activity was -evaluated against Streptoccocus mutans a cariogenic bacteria. Various PEI nanoparticles from 100nm to 1 micron in diameter were prepared having different degree of crosslinking, particle size and zeta potential that were achieved by alkylation with a bromoalkane followed by methylation. Their antibacterial effect was examined against Streptoccocus mutans in direct contact with bacteria. In a brief, a 10 l of bacterial suspension (ca.10 6 bacteria) was placed on each tested material sample in a set of 7 wells, and the plate was incubated at a vertical position for 1 hour at 37 0 C. During this incubation period, the suspension s liquid evaporated and a thin layer of bacteria was achieved ensuring direct contact between bacteria and the tested surface as demonstrated by scanning electron microscopy (Fig.1). The plate was then placed horizontally and 220 l of brain-heart infusion broth were added to each well containing the material. PEI samples were incorporated at 1%w/w ratio into the flowable composite resin. The tests were performed on the fresh samples, one week and one month aged samples. Majority of the tested compounds were very effective by immediate contact and showed complete decay of the bacteria. Data analysis was evaluated by the absorbance measurements. In a brief, the absorbance measurements were plotted resulting in bacterial growth curves for each well in the microtiter plate. The linear portion of the logarithmic growth phase was subjected to statistical analysis. Results are expressed in two parameters; the slop (a) and the constant (b) of a linear function ax+b=y derived from the ascending segment of the bacterial growth curve. The slop (a) and the constant (b) correlate with the growth rate and the initial number, respectively. The data was analyzed by one way

5 ANOVA, and Tukey multiple comparison test. Level of statistical significance was determined at p<0.05. Strong antibacterial effect against tested bacteria was exhibited by all composite resin samples incorporated with PEI nanoparticles relative to the commercial composite resin. Although the detailed mechanism of the antibacterial effect of these materials was not yet determined, it is suggested that quaternary ammonium compounds cause lysis of the bacterial cells by binding to the cell wall components and causing leakage of the cytoplasmatic material. One important feature of the antibacterial agent is to maintain antibacterial activity over time. With this goal in mind, we examined antibacterial stability of the compounds for several weeks. However, only the PEI nanoparticle samples including long chain alkyls demonstrated high antibacterial effect against Streptoccocus mutans for more than four weeks. Nanoparticles in Gene Therapy Gene therapy can be defined as an introduction of genetic material into a cell for the production of a needed peptide or protein. Successful gene therapy depends on the efficient delivery of genetic material into the cell nucleus and its effective expression within the cell[7]. A number of techniques have been developed for DNA delivery: direct introduction of transgene using cell electroporation, microinjection of DNA and incorporation of the gene by viral[8] and synthetic vectors[9]. Viral vectors including retroviruses, adenoviruses and adeno-associated viruses impress by their high efficiency in introducing their genetic material into host cells[10], although immunogenicity, inflammatory effects and safety concerns restrict their usefulness. The advantages of non viral vectors are that they do not integrate into chromosome, can introduce DNA into nondividing cells, do not possess infective risk and they are

6 less expensive than viral vectors[11]. The major disadvantage of non viral systems is their relatively low transfection efficiency. The non viral delivery vectors should overcome intracellular barriers, such as plasma membranes, endosomes and nuclear membranes[12]. Polycations are a leading class of non viral gene delivery vectors. Their activity is effected by due to their molecular weight, polymer type, polymer-dna ratio and molecular architecture. Polycations and negatively charged nucleic acids can spontaneously form nanocomplexes by electrostatic interaction. Polycationic vector reduces the electrostatic repulsion between DNA and cells by neutralizing the negative charge and also protects it from enzymatic digestion by nucleases in serum and extracellular fluids [13-16].Cationic polymers that are commonly used in gene delivery include poly( L -lysine)[17], cationic liposomes[18], polyethyleneimine[19], dendrimers[19], poly( L -histidine)[20], polyvinylpyridine and cationic polysaccharides[21, 22]. Polycations that were mentioned earlier and are used as gene delivery systems are usually polyamines that contain primary, secondary or tertiary amines[21]. Under physiological conditions, polyamines become cationic and condense DNA into compact structures. The efficacy of the carrier usually can be attributed to the ability of the polycation to condense DNA that is dependant on the structure of the polymer, the distance of cationic charge from the backbone, charge spacing along the polymer backbone, order of charged amine and molecular weight. More than 300 different polycations were prepared in our laboratory starting from various polysaccharides and oligoamines having two to eight amino groups. Although most of these conjugates formed stable complexes with various plasmids, only the dextran-spermine was found to be active in transfecting cells in vitro[23] and in vivo[24]. We have recently studied the toxicity of dextran-spermine in mice after

7 intramuscular and subcutaneous administration and found little if any toxicity[25]. The reason for the transfection efficiency of this conjugate is probably due to unique complexation properties formed between the DNA helices and the grafted spermine moieties, which probably play a significant role in cell transfection. Various cationic polysaccharide derivatives having multiple amine functionalities of different oligoamines were tested for their transfection efficiencies in a wide range of cell lines. Although most of these conjugates formed stable complexes with plasmid as determined by the ethidium bromide quenching assay[26](fig.2), only the dextranspermine conjugate was found to be active in cell transfection (Fig.3). Polyplexes based on dextran-spermine conjugates were also characterized by size measurements in serum and without serum, and their nanosize evaluation is given in Fig. 4. High in vitro transfection efficiency was achieved with dextran-spermine based conjugates using different cell lines and plasmids including NIH3T3 and HEK293 cell lines applying p-luc and -Gal encoding plasmid DNAs (data not shown). Based on these results, dextran-spermine was selected for further studies. Dextran-spermine conjugates were modified with increasing amounts of polyethylene glycol chains (PEG) (i.e. 1%, 3% and 5% mol/mol of PEG relative to primary amine of the spermine) and their transfection properties were evaluated in 10% fetal-calf serum (FCS) and showed high gene expression. Intravenous administration of the polyplexes, based on PEGylated-dextran-spermine derivatives and psv-lacz, was the last step of this study. As control, the non- PEGylated dextran-spermine-psv-lacz complex was also applied by intravenous and intramuscular injection to mice and its efficacy in gene transfection was also examined. As expected, no gene expression was detected for the non-pegylated dextran-spermine-psv-lacz complex. On the contrary, intravenous administration of

8 the PEGylated dextran-spermine-psv-lacz complex resulted in a high gene expression in the liver (Table 1). Conclusions Various nanoparticles based on different polycations were prepared. Although the majority of the tested compounds were found to be effective in different fields including antibacterial effect and gene transport, only some of them showed high activity. These results raised the possibility that nanotechnology can be used as a prospective strategy for preparation of the various therapeutic reagents for bacterial decay and genetic disorders.

9 Figure legends Figure 1. Scanning electron microscopy pictures of the bacteria surface before treatment. Figure 2. Condensation of pluc-dna with cationic dextran conjugated with spermine, spermidine, N,N-Bis(3-aminopropyl)-1,3-propanediamine, N,N-Bis(3- aminopropyl)-ethylenediamine and N,N-Bis(2-aminoethyl)-1,3-propanediamine where 20mM HBS (ph 7.4) was used as the condensation buffer. Figure 3. Inverted fluorescent microscope of p-cmv-gfp transfected HEK-293 cells. Calcium phosphate and Transfast controls represent precipitating technique. Dextran-spermine, an optimal conjugate, complexed at 0.1 charge ratio (C, -/+). The charge content of the conjugate was determined according to elemental analysis of %N. Figure 4. Physical characterization of polycations and their polyplexes. Size measurements of D(1:1) SPM and its 1:5.0 and 1:2.5 DNA P + :NH 3 polyplexes with or without serum.

10 Figure 1 Figure 2 % Free DNA Spermine Spermidine Tetramine [3:3:3] Tetramine [3:2:3] Tetramine [2:3:2] Charge ratio (+/-)

11 Figure 3 Calcium phosphate Transfast TM Dextran-spermine Figure 4

12 Table 1. Gene expression at different organs 2 days after intravenous injection of 5% PEGylated-dextran-spermine-pSV-LacZ complex and other agents. Specific β-gal activity (mu/mg protein) Organ PBS a PEGylated-D- SPM c Free plasmid DNA d PEGylated-D- SPM-pSV-LacZ Complex e Blood 6.22 ± 0.82 b 6.42 ± ± ± 1.56 Heart 7.23 ± ± ± ± 1.10 Lung 6.94 ± ± ± ± 0.14 Liver 7.45 ± ± ± ± 3.38 Spleen 5.87 ± ± ± ± 1.45 Kidney 6.98 ± ± ± ± 1.15 Gastrointestinal tract 7.14 ± ± ± ± 1.02 Carcass 8.72 ± ± ± ± 1.08 Excretion 7.94 ± ± ± ± 3.34 a Phosphate buffer saline control (200 µl per single injection). b Mean ± SD. c PEGylated-dextran-spermine based conjugate (250 µg in 200 µl PBS per mouse). d Free plasmid DNA (50 µg in 200 µl PBS per mouse). e 5% PEGylated-dextranspermine-pSV-LacZ at weight-mixing ratio of 5 (polycation/dna) and 50 µg/mouse of the plasmid in 200 µl PBS.

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