Polymeric capsules are used in medicine and pharmacy for the storage and delivery of proteins and peptides [1, 2], oligonucleotides [3, 4], hormones , growth factors [6, 7] and other biologically active substances. At the same time, they act as the independent agents and functional elements of the diagnostic systems. Microencapsulation, that is the conclusion of microscopic amounts of solid, liquid or gaseous products in the protective shell, is a modern perspective technology [8, 9, 10].
The mechanical properties of microcapsules have by now been poorly studied. It is known that the mechanical stability of polymer structures increases with their increased molecular weight and during the transition from the linear structures to the network and branched ones. Thus stereoregular structures have greater strength than polymers with a disordered structure.
It is important to increase the mechanical strength of microcapsules in order to preserve their contents during the targeted drug delivery and storage of biological objects in a microencapsulated form. There is a vital task to increase the mechanical stability of polyelectrolyte structures used in the construction of biosensors based on polymeric microcapsules. Information about mechanical properties is important in the development of various microscopic objects based on polyelectrolyte capsules.
Below are the outcomes of a study of the mechanical strength of polyelectrolyte microcapsules obtained in the Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences.
Formation of capsules by the LbL adsorption of polyelectrolytes
There is a variety of techniques for preparing microcapsules with chemical, physical and physical-chemical methods. The studied capsules were prepared by the layer-by-layer adsorption technology (LbL) based on the sequential adsorption of polyanions and polycations on a charged substrate. The decisive factor for the creation of a multilayer structure is the electrostatic interaction and the change in sign of the surface charge after each stage of polyelectrolyte adsorption.
The used method for obtaining CaCO3 microparticles allows growing porous spherical particles with a diameter of 3 to 12 µm (with a spread in values from 4% to 20%) with a similar inner structure . Particles virtually do not aggregate between themselves, and they can be stored for an indefinite period at room temperature in dry form. The pattern of creation of these particles is shown in fig.1.
Currently, the basic properties of polyelectrolyte microcapsules are actively studied. The polyelectrolyte shell of a capsule is impervious to high-molecular compounds while low-molecular substances and inorganic ions can penetrate into their walls [12–13]. The shells of polyelectrolyte capsules are sensitive to environmental conditions, i.e. pH and the ionic strength of solutions, solvents, temperatures and other factors. These factors have a great impact on the structure of the polyelectrolyte complexes and therefore on the permeability of capsules [14–17].
For the mechanical characterisation of these items often use nanohardness testers or atomic force microscopes [18-22], in which for the mechanical impact on a particle the tip of the cantilever or a special microscopic bead attached to it  are used. Also used are nanoindenters in the form of a cylindrical die with a flat tip [18–21]. It should be noted that this method is widely used for biological objects, colorant particles in the toner of printers and testing of the abrasive finely-divided material [18,19].
The study of mechanical strength of the microcapsules was carried out using the NanoScan-4D nanohardness tester. A general view of the device is presented in fig.2. This device makes it possible to carry out tests by the sclerometry measurement method, to measure hardness and elastic modulus by instrumental indentation as well as to implement a number of atomic force microscopy methods. The indentation head of the device allows to apply the load in the range from a few microNewton to several Newton, and to measure displacements in the range from a few nanometers to a millimeter.
The device can be equipped with different types of tips, which include pyramidal Berkovich and Vickers indenters as well as tips in the form of a flat die. To determine the limit load withstood by microcapsules a diamond flat die was used (fig.3), the diameter of the working area of which was 100 microns.
To determine the position of the studied particles, an optical microscope was used, which is part of the NanoScan-4D device . This microscope can be used to measure the particle size. Movements between the optical head and the indentation head are implemented using the linear translators equipped with encoders that provide accurate relative positioning of the stamp and particles better than 1 micron.
Measuring particle strength
The strength of particles was measured by compressing it with an indenter having the shape of a flat die. During compression the load-displacement diagram was recorded, which was then used to determine the nature of the destruction as well as the bearable particle load limit.
The device allows measurements in both the liquid medium and the air. In this study, a few drops of a solution containing microcapsules were placed on a glass substrate. Measurements were carried out for several hours after the evaporation of the liquid while the capsules remained on the substrate. To determine the strength, the particles, which are at a sufficient distance from other objects, were selected with an optical microscope (fig.4), so that only one capsule was under the stamp during compression.
The typical size of the particle shown in fig.4 is about 10 microns. Both loading and unloading are performed while maintaining a constant scanning speed by force. Fig.5 shows a typical diagram for the case of loading a single capsule. For comparison, fig.5b shows an "incorrect" diagram with several surges in strength that indicating slippage or contact with a few particles. Diagrams like those shown in fig.5b weren't used to measure the mechanical properties.
On the presented diagram (fig.5), the load limit, at which there was a destruction of the capsule unit, was 25 mN, the capsule was thereby compressed by the value of 1.1 microns. The obtained data on the maximum strength and size of particles allow measuring the mechanical strength. For this purpose it is possible to use the methods of numerical simulation using a priori information about the object properties.
It is possible to measure the particle material strength by using the formula obtained for the case of compression of a spherical body with two dotty forces. After a number of simplifying assumptions, a formula  was obtained, which allows to link the strength during destruction Fc, the distance between the points of application of force dp during destruction and tensile strength of the particle material σ:
Based on the data obtained by indentation, the dp value can be taken as the difference between the value of displacement of the indenter, at which the force starts to increase after the destruction of the capsule (z2≈7,5 microns, see fig.5), and size of the deepening in which a break occurs (z1 = 1 micron, see fig.5a). Accordingly, the distance value is determined as the difference dp=z2 – z1. Thus, using the values Fc=25 mN and dp≈6.5, we obtain the value σ≈0.5 GPa. Naturally, this is the upper estimate because the capsules under study are not solid. The particles in question are a heterogeneous objects, and any interaction with the stamp and substrate takes place at a significant part of its surface, so more accurate information on the mechanical properties of the material of the capsule can be obtained by adding numerical simulation to the experimental data.
The study demonstrates the generic nature of the instrumental indentation method and the wide measurement scope of the NanoScan devices capable of jointly precise measurements of force and displacement. The present study has clearly shown the possibility of rapid control of their mechanical properties, and it can be used for optimising their shape, size and wall thickness.
Special emphasis should be put on important feature of the NanoScan-4D allowing not only to measure the hardness and elastic modulus of homogeneous materials but also to conduct precision indentation in the area specified in an integrated optical microscope. Measurements of the dependence of force and displacement on time allow to obtain the numerical evaluation of the mechanical properties of various objects including microcapsules that are of a natural or artificial origin. Nanoindentation can be used for the characterisation of different mechanical properties of particles including for study of their strength, toughness and elasticity. ■
In studies the equipment of the Centre for Common Use of FSBI TISNCM was used.