Microcapsules of alginate cross-linked with divalent ions are the most common system for cell immobilization. In this study, we wanted to characterize the effect of different alginates and cross-linking ions on important microcapsule properties. The dimensional stability and gel strength increased for high-G alginate gels when exchanging the traditional Ca2+ ions with Ba2+. The use of Ba2+ decreased the size of alginate beads and reduced the permeability to immunoglobulin G. Strontium gave gels with characteristics lying between calcium and barium. Interestingly, high-M alginate showed an opposite behavior in combination with barium and strontium as these beads were larger than beads of calcium-alginate and tended to swell more, also resulting in increased permeability. Binding studies revealed that different block structures in the alginate bind the ions to a different extent. More specifically, Ca2+ was found to bind to G- and MG-blocks, Ba2+ to G- and M-blocks, and Sr2+ to G-blocks solely.

The availability of mannuronan and mannuronan C-5 epimerases allows the production of a strictly alternating mannuronate-guluronate (MG) polymer and the MG-enrichment of natural alginates, providing a powerful tool for the analysis of the role of such sequences in the calcium-alginate gel network. In view of the calcium binding properties of long alternating sequences revealed by circular dichroism studies which leads eventually to the formation of stable hydrogels, their direct involvement in the gel network is here suggested. In particular, 1H NMR results obtained from a mixed alginate sample containing three polymeric species, G blocks, M blocks, and MG blocks, without chemical linkages between the block structures, indicate for the first time the formation of mixed junctions between G and MG blocks. This is supported by the analysis of the Young's modulus of hydrogels from natural and epimerized samples obtained at low calcium concentrations. Furthermore, the "zipping" of long alternating sequences in secondary MG/MG junctions is suggested to account for the shrinking (syneresis) of alginate gels in view of its dependence on the length of the MG blocks. As a consequence, a partial network collapse, macroscopically revealed by a decrease in the Young's modulus, occurred as the calcium concentration in the gel was increased. The effect of such "secondary" junctions on the viscoelastic properties of alginate gels was evaluated measuring their creep compliance under uniaxial compression. The experimental curves, fitted by a model composed of a Maxwell and a Voigt element in series, revealed an increase in the frictional forces between network chains with increasing length of the alternating sequences. This suggests the presence of an ion mediated mechanism preventing the shear of the gel.

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There is an increased need for alginate materials with both enhanced and controllable mechanical properties in the fields of food, pharmaceutical and specialty applications. In the present work, well-characterized algal polymers and mannuronan were enzymatically modified using C-5 epimerases converting mannuronic acid residues to guluronic acid in the polymer chain. Composition and sequential structure of controls and epimerized alginates were analyzed by (1)H NMR spectroscopy. Mechanical properties of Ca-alginate gels were further examined giving Young's modulus, syneresis, rupture strength, and elasticity of the gels. Both mechanical strength and elasticity of hydrogels could be improved and manipulated by epimerization. In particular, alternating sequences were found to play an important role for the final mechanical properties of alginate gels, and interestingly, a pure polyalternating sample resulted in gels with extremely high syneresis and rupture strength. In conclusion, enzymatic modification was shown to be a valuable tool in modifying the mechanical properties of alginates in a highly specific manner.

Confocal laser scanning microscopy (CLSM) was used to study the distribution of polymers and cross-linking ions in alginate-poly-L-lysine (PLL) -alginate microcapsules made by fluorescent-labeled polymers. CLSM studies of Ca-alginate gel beads made in the presence and absence of non-gelling sodium ions revealed a more inhomogeneous distribution of alginate in beads formed in the absence of non-gelling ions. In the formation of alginate-PLL capsules, the polymer gradients in the preformed gel core were destabilized by the presence of non-gelling ions in the washing step and in the PLL solution. Ca-alginate gels preserved the inhomogeneous structure by exposure to ion-free solution in contrast to exposure to non-gelling ions (Na(+)). By exchanging Ca(2+) with Ba(2+) (10 mM), extremely inhomogeneous gel beads were formed that preserved their structure during the washing and exposure to PLL in saline. PLL was shown to bind at the very surface of the alginate core, forming a shell-like membrane. The thickness of the PLL-layer increased about 100% after 2 weeks of storage, but no further increase was seen after 2 years of storage. The coating alginate was shown to overlap the PLL layer. No difference in binding could be observed among coating alginates of different composition. This paper shows an easy and novel method to study the distribution of alginate and PLL in intact microcapsules. As the labeling procedures are easy to perform, the method can also be used for a variety of other polymers in other microencapsulation systems.

In vitro and in vivo behavior of nanoparticles (NPs) is often studied by tracing the NPs with fluorescent dyes. This requires stable incorporation of dyes within the NPs, as dye leakage may give a wrong interpretation of NP biodistribution, cellular uptake and intracellular distribution. Furthermore, NP labeling with trace amounts of dye should not alter NP properties such as interactions with cells or tissues.To allow for versatile NP studies with a variety of fluorescence-based assays, labeling of NPs with different dyes is desirable. Hence, when new dyes are introduced, simple and fast screening methods to assess labeling stability and NP-cell interactions are needed. For this purpose we have used a previously described generic flow cytometry assay; incubation of cells with NPs at 4°C and 37°C. Cell-NP interaction is confirmed by cellular fluorescence after 37°C incubation, and NP dye retention is confirmed when no cellular fluorescence is detected at 4°C.Three different NP-platforms labeled with six different dyes were screened, and a great variability in dye retention was observed. Surprisingly, incorporation of trace amounts of certain dyes was found to reduce, or even inhibit NP uptake. This work highlights the importance of thoroughly evaluating every dye-NP combination before pursuing fluorescent NP applications.3

Background Poly(alkyl cyanoacrylate) (PACA) nanoparticles have shown promise as drug carriers both to solid tumors and across the blood–brain barrier. Efficient drug delivery requires both high cellular uptake of the nanoparticles and release of the drug from the nanoparticles. Release of hydrophobic drugs from PACA nanoparticles is primarily governed by nanoparticle degradation, and this process has been poorly studied at the cellular level. Here we use the hydrophobic model drug Nile Red 668 (NR668) to investigate intracellular degradation of PACA nanoparticles by measuring changes in NR668 fluorescence emission and lifetime, as the spectral properties of NR668 depend on the hydrophobicity of the dye environment. We also assess the potential of poly(butyl cyanoacrylate) (PBCA) and poly(octyl cyanoacrylate) (POCA) nanoparticles for intracellular drug delivery in the prostate cancer cell line PC3 and rat brain endothelial cell line RBE4 and the role of endocytosis pathways in PACA nanoparticle uptake in those cell lines.ResultsFluorescence lifetime imaging, emission spectra analysis and Förster resonance energy transfer indicated that the intracellular degradation was in line with the degradation found by direct methods such as gas chromatography and scanning electron microscopy, showing that PBCA has a faster degradation rate compared to POCA. The combined P(BCA/OCA) nanoparticles had an intermediate degradation rate. The uptake of POCA and PBCA nanoparticles was much higher in RBE4 than in PC3 cells. Endocytosis inhibition studies showed that both clathrin- and caveolin-mediated endocytosis were involved in PACA nanoparticle uptake, and that the former played a predominant role, particularly in PC3 cells.ConclusionsIn the present study, we used three different optical techniques to show that within a 24-hour period PBCA nanoparticles degraded significantly inside cells, releasing their payload into the cytosol, while POCA nanoparticles remained intact. This indicates that it is possible to tune the intracellular drug release rate by choosing appropriate monomers from the PACA family or by using hybrid PACA nanoparticles containing different monomers. In addition, we showed that the uptake of PACA nanoparticles depends not only on the monomer material, but also on the cell type, and that different cell lines can use different internalization pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-015-0156-7) contains supplementary material, which is available to authorized users.

Compared with conventional chemotherapy, encapsulation of drugs in nanoparticles can improve efficacy and reduce toxicity. However, delivery of nanoparticles is often insufficient and heterogeneous because of various biological barriers and uneven tumor perfusion. We investigated a unique multifunctional drug delivery system consisting of microbubbles stabilized by polymeric nanoparticles (NPMBs), enabling ultrasound-mediated drug delivery. The aim was to examine mechanisms of ultrasound-mediated delivery and to determine if increased tumor uptake had a therapeutic benefit. Cellular uptake and toxicity, circulation and biodistribution were characterized. After intravenous injection of NPMBs into mice, tumors were treated with ultrasound of various pressures and pulse lengths, and distribution of nanoparticles was imaged on tumor sections. No effects of low pressures were observed, whereas complete bubble destruction at higher pressures improved tumor uptake 2.3 times, without tissue damage. An enhanced therapeutic effect was illustrated in a promising proof-of-concept study, in which all tumors exhibited regression into complete remission.