RU2504403C1 - Method for preparing water suspension of biocompatible porous silicon particle - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a method for preparing a water suspension of silicon nanocrystalline particles for biomedical applications. The declared method is characterised by the fact that a porous silicon film of the thickness of 1 to 100 mcm and the porosity of 50 to 80% is formed on the surface of silicon plates. The film is formed by electrochemical etching in a solution containing 40-50% aqueous solution of fluoric acid and alcohol in ratio 1:1 to 1:5 for a period of time of 10 to 60 minutes at specific current 20 to 60 mA/cm². The prepared silicon plates coated with a porous silicon layer is washed in distilled water, dried and placed in a container with distilled water. Then, they are exposed to ultrasound for more than 10 minutes at the intensity of the silicon plate exposure in water of 100 Wt/cm² at frequency 23 kHz. This provides forming the stable suspension in distilled water from nanoparticles of the lateral dimension of 10 to 500 nm.

EFFECT: invention provides preparing the biocompaticle suspension of silicon nanoparticles that penetrate into living cells preserving its useful biological properties and luminescence.

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Description

Technical field

The invention relates to the field of inorganic nanomaterials that can be used in biomedical applications. More specifically, the present invention relates to silicon nanoparticles for biomedical applications, as well as to a method for producing such nanoparticles.

State of the art

From the current level of development of biology and medicine, technical solutions based on the use of various types of nanoparticles are well known.

Silicon is one of the most common elements on Earth, and interest in its role in human physiology and metabolism is growing steadily after the pioneering work of EM Karlisle (Carlisle E.M. "Silicon: an essential element for the chick" // Science 1972. V.178. PP.619-62; Carlisle EM "The nutritional essentiality of silicon" // Nutr. Rev. 1982. V .40. PP.193-1988) and subsequent research (Dobbie JW, Smith MJB "Silicon: its role in medicine and biology" // Scott. Med. 1982. V.27; Seaborn C. "Silicon: a nutritional beneficence for bones, brain and blood vessels "// Nutr. Today. 1993. V.28. PP.13-18; Van Dyck K." Indication of silicon essentiality in humans: serum concentrations in Belgian children and adults, including pregnant women " // Biol. Trace Elem. 2000. V.77. PP.25-32; Bisse E. "Reference values for serum silicon in adults" // Anal. Biochem. 2005. V.337. PP.130-135). It has been established that orthosilicic acid is the most rapidly absorbed and excreted form of silicon and silicates in humans (Jugdaosingh R. "Dietary silicon intake and absorption" // Am. J. Clin. Nutr. 2002. V.75. PP.887-993) .

The total silicon content in the body of a healthy adult person weighing 70 kg is normally 0.5-1.0 g, which makes silicon the third most common trace element in the human body after iron and zinc (Jugdaosingh R. "Dietary silicon intake and absorption" / (Am. J. Clin. Nutr. 2002. V.75. PP.887-93).

Commercially available silicon additives are based on silica or silicates instead of using silicon in its pure semiconductor form, and the bioavailability of such silicon forms is variable and low (Jugdaosingh R. "Dietary silicon intake and absorption" // Am. J. Clin. Nutr. 2002. V.75. PP.887-93).

The interest in silicon nanoparticles at the initial stage of the study was caused by the possibilities of their use in optoelectronics and the work of L. Kenama (Canham LT "Nanoscale semiconducting silicon as a nutritional food additive" // Nanotechnology. 2007. V.18. P.185704), which demonstrated intense red photoluminescence of this material. However, even more surprising was the discovered property of biocompatibility and biodegradation of nanosilicon (Seaborn C. "Silicon: a nutritional beneficence for bones, brain and blood vessels" // Nutr. Today. 1993. V.28 PP.13-18; Van Dyck K. "Indication of silicon essentiality in humans: serum concentrations in Belgian children and adults, including pregnant women" // Biol. Trace Elem. Res. 2000. V.77. PP.25-32). The material behaves so friendly to living tissue that it promotes the growth of living cells on its surface (neurons, bone tissue, etc.). This discovery marked a breakthrough in the biomedical applications of nanosilicon, allowing us to offer innovative methods and materials for drug delivery that can absorb orthopedic materials, biocompatible tissues, etc. In the work of E. Bisset (Bisse E. "Reference values for serum silicon in adults" // Anal. Biochem. 2005. V.337. PP.130-135.) It was established that nanosilicon can become an important biomaterial, and suggests that Based on it, biomedical engineering and biosensor technology can be further developed. Biomaterial is an inanimate material that is used in biomedicine to interact with biological systems. Biomaterials can be “bioinert”, “bioactive” and “bio-soluble”. Bioactive - these are materials that, when they enter a living object (in-vivo), form bonds between this material and living tissue (Bisse E. "Reference values for serum silicon in adults" // Anal. Biochem. 2005. V.337. PP.130-135).

One of the first methods for producing silicon nanoparticles was the method of electrochemical formation of the so-called porous silicon (PC) during its electrochemical etching of single-crystal silicon (c-Si), proposed back in 1956 by a group of scientists led by A. Uhlir, and then studied by many researchers. A review of such works is available, for example, in the work of R. Yugdaosain (Jugdaosingh R. "Dietary silicon intake and absorption" // Am. J. Clin. Nutr. 2002. V.75. PP.887-93). Concentrated hydrofluoric acid (HF) or its water-alcohol mixtures HF: H ₂ O: C ₂ H ₅ OH in various proportions, depending on the experimental conditions, are usually used as electrolytes. This method of obtaining films of PC, consisting of nanocrystalline silicon particles, is still one of the most practical and affordable.

Numerous methods are also known for the production of PCs using not electrochemical, but purely chemical etching of silicon. For this, along with HF, a strong oxidizing agent, for example, KNO ₂ , HNO ₃ , etc., is introduced into the solution (Jugdaosingh R. "Dietary silicon intake and absorption" // Am. J. Clin. Nutr. 2002. V. 75 .PP. 887-93). Such porous silicon in comparison with the formed electrochemical method has a limited layer thickness (from 100 nm to units of microns). There is also a method of creating silicon nanoparticles by grinding (grinding) silicon powders in planetary mills (Seaborn C. "Silicon: a nutritional beneficence for bones, brain and blood vessels" // Nutr. Today 1993. V. 28. PP. 13- 18., Canham LT "Nanoscale semiconducting silicon as a nutritional food additive" // Nanotechnology. 2007. V.18. P. 185704.). To obtain silicon nanopowders, laser ablation from silicon targets is also used (Canham LT, Reeves CL, King DO, Branfield PJ, Crabb J., Ward MCL // Adv Mater. 1996. V.8. P. 850; Canham LT // Adv Mater. 1995. V.7. No 12. P.1033).

All of the above indicates that the problem of obtaining silicon nanopowders is still in the process of discussion, and the corresponding theories of obtaining silicon nanoparticles are complex, and in some cases contradictory. It is also obvious that understanding the mechanisms of the formation of silicon nanoparticles, as well as the resulting biocompatibility and bioactivity of silicon nanoparticles, is impossible without taking into account the specific chemical properties of silicon.

Known 1999 patent US 5,914,183, which describes the layers of porous silicon and disclosed a method for their manufacture using liquid electrochemical etching. Also known patent US 6,666,214, as well as patents US 7,186,267 and US 7,332,339, where it is shown that some types of silicon nanostructures and nanoparticles are biocompatible and bioactive.

Patent applications US2011204489 and US201215441 describe similar methods for creating silicon nanostructures that are easily detachable from a silicon substrate by chemically depositing metal ions on an oxidized silicon surface. Metal ions create on the surface of the nanostructure, which serve as a mask during the etching of silicon. After chemical etching and metal removal, ensembles of silicon nanostructures remain. In the patent application US 2007104888 a method for creating nucleation centers and subsequent growth of nanostructures on them is disclosed. To create nucleation centers, the substrate is irradiated with a beam of silicon ions. International application WO / 2011/073666 discloses a method for creating nanostructures on a silicon surface (i.e., porous silicon) by chemical etching in the presence of metal ions.

Known methods of grinding solids. So in patents US 6,537,672 and US 6,500,534 in particular, a method is disclosed for grinding a suspension of inorganic particles, for example, boehmite, to particle sizes from 1 to 100 nm using ultrasonic treatment. International application WO / 2003/099262 discloses a method for creating biodegradable polymer nanoparticles in an oil-water emulsion using ultrasound. In the international application publication WO / 2001/003670 describes the preparation of suspensions from finished (pre-created) nanoparticles using ultrasound. US 7,671,113 describes a method for preparing a nano-emulsion of an elastic material, for example styrene-butadiene rubber with a narrow particle size distribution. For this, a fluid with particles of elastic material ranging in size from 600 to 1000 nm is passed through a cavitation device. Cavitation is caused by the passage of ultrasound through a liquid with a frequency of 16 to 100 kHz.

The closest analogue to this invention is US patent 6,585,947 "Method for producing silicon nanoparticles", which discloses a method for producing silicon nanocrystals with a size of 1 nm with a very narrow distribution of particle sizes. Silicon nanoparticles are obtained by electrochemical etching of a silicon substrate in a solution of HF and H ₂ O ₂ . Then, by ultrasonic treatment in acetone, ethanol or methanol, the nanoparticles are separated from the silicon substrate after etching. It is proposed to use the obtained nanoparticles for subsequent processing or the creation of nanocomposites, including the preparation of aqueous suspensions for biomedical applications.

SUMMARY OF THE INVENTION

The main problems in the development of methods for creating suspensions of silicon nanoparticles for biomedical applications are: 1) the creation of nanoparticles with the required sizes (10 to 500 nm) that preserve the crystalline structure that could form stable suspensions; 2) obtaining suspensions of such particles with a concentration of 1 g / l or more, so that they could be used in biomedical applications. Existing methods for producing silicon nanoparticles do not make it possible to obtain their stable aqueous suspensions. Available methods make it possible to produce nanoparticles with either a large number of defects or insufficient size. Aqueous suspensions of such nanoparticles are degrading too fast to be used in biomedical applications. In addition, the chemical composition of the surface of such particles may not be well controlled to make them biocompatible.

The objective of the invention is to provide a method for producing biocompatible aqueous suspensions of silicon nanoparticles mainly for use in biomedical purposes, including the diagnosis and treatment of various diseases.

The technical result of the invention is to obtain a biocompatible suspension of silicon nanoparticles by grinding films of porous silicon in water by ultrasonic radiation of a certain intensity while maintaining the porous and nanocrystalline structure of nanoparticles with sizes from 10 to 500 nm. When grinding porous silicon films weighing 1 g in a volume of 1 l of water, a suspension of silicon nanoparticles with a concentration of 1 g / l is obtained. The resulting nanoparticles penetrate into living cells, while maintaining their useful biological properties and luminescence.

The originality of the method used consists in the selection of porous silicon films of a certain porosity as the starting material, which determines the biocompatibility and biodegradability of the material, and their subsequent grinding with ultrasound of a certain intensity, which ensured the production of an aqueous suspension of porous silicon nanoparticles, which retain their useful biocompatibility and biodegradability properties, as well as capable penetrate into living cells.

The problem is solved in that the method of producing aqueous suspensions of silicon nanocrystalline particles for biomedical applications involves the formation on the surface of silicon wafers of a film of porous silicon (thickness from 1 to 100 μm and porosity from 50 to 80%) by electrochemical etching in a solution containing hydrofluoric acid ( an aqueous solution with an acid concentration of 40 to 50% by volume) and ethyl, methyl, propyl, isopropyl alcohol or other similar alcohols in a ratio of 1: 1 to 1: 5 over a period of time a volume of 10 to 60 minutes with a current density of 20 to 60 mA / cm ² , after which the obtained silicon wafers coated with a layer of porous silicon are washed in distilled water, dried, then placed in a container with distilled water and for a period of not less than 10 minutes are subjected to sonication with an intensity of exposure to silicon wafers in water of 10-100 W / cm ² with a frequency of 23 kHz or more to obtain a stable suspension of nanoparticles with transverse sizes from 10 to 500 nm in distilled water.

The invention is illustrated by drawings, in which Fig. 1 shows a general view of a porous silicon film in a scanning electron microscope, Fig. 2, an image of the obtained silicon nanoparticles in a transmission electron microscope, Fig. 3 is an electron diffraction pattern of the obtained silicon nanoparticles, which shows their nanocrystalline structure, Fig. 4 - results of an experiment on the use of the obtained aqueous suspensions of silicon nanoparticles as luminescent labels for staining mammalian cells, on which presented photographs dog thymocyte cells CF2Th, taken through a red filter, in which bright white points correspond silicon nanoparticles infiltrated into the cell.

The present invention proposes to use a two-stage method for the production of aqueous suspensions of nanocrystalline silicon particles for biomedical applications.

At the first stage of the process, porous silicon films are formed on the surface of silicon wafers by electrochemical etching in a solution containing hydrofluoric acid (an aqueous solution with an acid concentration of 40 to 50% by volume) and ethyl, methyl, propyl, isopropyl alcohol or other similar alcohol in the ratio from 1: 1 to 1: 5, over a period of time from 10 to 60 minutes with a current density of 20 to 60 mA / cm ² . As a result of this process, layers of porous silicon with a thickness of 1 to 100 μm and a porosity of 50 to 80% are formed on the surface of silicon wafers. The obtained silicon wafers coated with a layer of porous silicon are thoroughly washed in distilled water and dried, which provides a controlled chemical composition of their surface coating.

In the second stage of the process, silicon wafers coated with a layer of porous silicon are placed in a container with distilled water and subjected to a high-intensity ultrasonic treatment for 10 to 100 W / cm ² on a silicon wafer with a frequency of about 23 kHz or more. As a result of ultrasonic treatment, layers of porous silicon are separated from silicon wafers and destroyed into individual nanoparticles with sizes from 10 to 500 nm. In this case, the destruction occurs in the region of the most stressed and defective material. As a result, the resulting nanoparticles remain crystalline and poorly defective. These nanoparticles form a suspension in distilled water that can be introduced into a living organism.

The novelty of the proposed method for the formation of suspensions of silicon nanoparticles for biomedical applications is the use of silicon films with a high porosity of at least 50% as starting material, which can be crushed in water using ultrasound of relatively low intensity, which simultaneously preserves the nanocrystalline structure of the nanoparticles and their porosity. Such nanoparticles exist in the form of aqueous suspensions, which are biocompatible and allow the introduction into living systems and subsequent use in diagnostics and therapy.

The proposed method for the manufacture of silicon nanoparticles allows to obtain aqueous suspensions with known sizes of nanoparticles from 10 to 500 nm and a high concentration of up to 10 g / l. These suspensions can be stored in the dark at room temperature for at least one month. In this case, the sizes of nanoparticles do not change significantly. Suspension nanoparticles can be introduced into cell cultures and biological systems, namely, blood, connective and muscle tissues for use as a diagnostic and therapeutic agent.

These suspensions can be introduced into biological systems, namely, cell and tissue cultures, for subsequent activation by light, ultrasound and other physical influences to achieve the necessary diagnostic or therapeutic effect.

Toxicological safety studies of nanoparticles in biomedical research are receiving increased attention (Garnett M.S., Kallinteri P. "Nanomedicines and nanotoxicology. Some physiological principles" // Occup.Med. 2006. V.56, PP.307-311; Durnev A. D., Solomina A.S. et al. "Study of the genotoxic and teratogenic activity of silicon nanocrystals" // Bulletin of experimental biology and medicine. 2010. V.148. No. 4. S.429-433).

In the work (Durnev A.D., Solomina A.S. et al. "Study of the genotoxic and teratogenic activity of silicon nanocrystals" // Bulletin of Experimental Biology and Medicine. 2010. V.148. No. 4. S.429-433), It is described that when nanoparticles enter the bloodstream, their transport through the whole body and accumulation in specific organs is possible. It is also noted here that if biosoluble silicon enters the body, it is easily excreted in the form of orthosilicic acid.

An example of a suspension

Nanostructured silicon surfaces were obtained by the standard method of electrochemical etching of crystalline silicon (c-Si) p-type wafers with a surface orientation of (100) and a resistivity of 10 Ohm-cm in a solution of hydrofluoric acid and ethyl alcohol (HF (50%): C ₂ H ₅ OH) at an etch current density of 50 mA / cm ² for 60 minutes. This made it possible to form so-called porous silicon layers with a porosity of 50% on a c-Si substrate. The thickness of the porous silicon layer was about 100 μm (a longer etching time provided a larger layer thickness). As shown by studies in an atomic force microscope, nanostructures in the form of protrusions and depressions 10-100 nm high were present on the surface of the layer (Figure 1). After obtaining the porous layer, the silicon wafers were thoroughly washed and dried, which ensured a controlled composition of the surface coating of silicon nanoparticles. To obtain aqueous suspensions of silicon nanoparticles, silicon wafers with a porous film or peeled films of porous silicon were processed by ultrasonic vibrations at a frequency of 23 kHz and an intensity of 60 W / cm ² in distilled water for 15 min. When this film of porous silicon was destroyed with the formation of an aqueous suspension of silicon nanoparticles with a size of 200-400 nanometers, preserving the porous structure (Figure 2). The concentration of the resulting suspensions was 1 g / l, which was determined by the proportion of the mass of the used films of porous silicon (1 g) and water (1 l). The nanocrystalline structure of the obtained nanoparticles was confirmed by electron diffraction data (Figure 3).

The biocompatibility of the obtained aqueous suspensions was checked in animal experiments, during which the suspension was administered intraperitoneally to rats at doses of 5, 25 and 50 mg / kg. The test material did not show cytogenetic activity in the bone marrow cells of the test animals after 24-hour, 7 and 14 days exposure with a single injection.

Thus, the study did not reveal the cytogenetic effects of an aqueous suspension of silicon nanoparticles in vivo, their insignificant genotoxic activity in bone marrow and brain cells is shown. The presented results characterize the obtained aqueous suspensions of silicon nanoparticles as a biocompatible nanomaterial.

Claims (1)

A method of obtaining an aqueous suspension of silicon nanocrystalline particles for biomedical applications, characterized in that on the surface of the silicon wafers a porous silicon film is formed with a thickness of 1 to 100 μm and a porosity of 50 to 80% by electrochemical etching in a solution containing hydrofluoric acid in the form of an aqueous solution with concentration from 40 to 50% by volume, and ethyl or other alcohol in a ratio of 1: 1 to 1: 5, for a period of time from 10 to 60 minutes with a current density of 20 to 60 mA / cm ² , after which the resulting pl silicon wafers coated with a layer of porous silicon are washed in distilled water, dried, then placed in a container with distilled water and subjected to an ultrasound treatment with an intensity of exposure to silicon wafers of 100 W / cm ² with a frequency of 23 for at least 10 minutes kHz with obtaining in distilled water a stable suspension of nanoparticles with transverse sizes from 10 to 500 nm.

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