BRPI1104516A2 - Process for obtaining boro nitret nanotubes, so obtained nanotubes and bacterial transformation process using boro nitrete nanotubes - Google Patents

Process for obtaining boro nitret nanotubes, so obtained nanotubes and bacterial transformation process using boro nitrete nanotubes Download PDF

Info

Publication number
BRPI1104516A2
BRPI1104516A2 BRPI1104516A BRPI1104516A2 BR PI1104516 A2 BRPI1104516 A2 BR PI1104516A2 BR PI1104516 A BRPI1104516 A BR PI1104516A BR PI1104516 A2 BRPI1104516 A2 BR PI1104516A2
Authority
BR
Brazil
Prior art keywords
preferably
characterized
process according
nanotubes
step
Prior art date
Application number
Other languages
Portuguese (pt)
Inventor
Marcelo Lancellotti
Luciana Maria De Hollanda
Edesia Martins Barros De Sousa
Tiago Hilario Ferreira
Original Assignee
Unicamp
Comissao Nac Energia Nuclear
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
Application filed by Unicamp, Comissao Nac Energia Nuclear filed Critical Unicamp
Priority to BRPI1104516 priority Critical patent/BRPI1104516A2/en
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=47882465&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=BRPI1104516(A2) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Publication of BRPI1104516A2 publication Critical patent/BRPI1104516A2/en

Links

Classifications

    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00Nanotechnology for materials or surface science, e.g. nanocomposites
    • CCHEMISTRY; METALLURGY
    • C01INORGANIC CHEMISTRY
    • C01BNON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B21/00Nitrogen; Compounds thereof
    • C01B21/06Binary compounds of nitrogen with metals, with silicon, or with boron, or with carbon, i.e. nitrides; Compounds of nitrogen with more than one metal, silicon or boron
    • C01B21/064Binary compounds of nitrogen with metals, with silicon, or with boron, or with carbon, i.e. nitrides; Compounds of nitrogen with more than one metal, silicon or boron with boron
    • C01B21/0641Preparation by direct nitridation of elemental boron
    • CCHEMISTRY; METALLURGY
    • C01INORGANIC CHEMISTRY
    • C01PINDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2002/00Crystal-structural characteristics
    • C01P2002/70Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data
    • C01P2002/72Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data by d-values or two theta-values, e.g. as X-ray diagram
    • CCHEMISTRY; METALLURGY
    • C01INORGANIC CHEMISTRY
    • C01PINDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2002/00Crystal-structural characteristics
    • C01P2002/80Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70
    • C01P2002/82Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70 by IR- or Raman-data
    • CCHEMISTRY; METALLURGY
    • C01INORGANIC CHEMISTRY
    • C01PINDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00Particle morphology
    • C01P2004/01Particle morphology depicted by an image
    • C01P2004/03Particle morphology depicted by an image obtained by SEM
    • CCHEMISTRY; METALLURGY
    • C01INORGANIC CHEMISTRY
    • C01PINDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00Particle morphology
    • C01P2004/01Particle morphology depicted by an image
    • C01P2004/04Particle morphology depicted by an image obtained by TEM, STEM, STM or AFM
    • CCHEMISTRY; METALLURGY
    • C01INORGANIC CHEMISTRY
    • C01PINDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00Particle morphology
    • C01P2004/10Particle morphology extending in one dimension, e.g. needle-like
    • C01P2004/13Nanotubes

Abstract

PROCESS OF OBTAINING BORON NITRIDE NANOTUBES, NANOTUBES SO OBTAINED AND BACTERIAL TRANSFORMATION PROCESS USING BORON NITRET NANOTUBES. The present invention describes a process for obtaining boron nitride nanotubes (BNNT) and a process for bacterial transformation employing boron nitride nanotubes as facilitators. More specifically, the present invention describes a process of transformation, by thermal shock, of competent strains of Escherichia coli using boron nitride nanoparticles as a plasmid delivery system.

Description

PROCESS FOR OBTAINING BORO NITRET NANOTUBES, SO OBTAINED NANOTUBES AND BACTERIAL TRANSFORMATION PROCESS USING BORO NITRETE NANOTUBES

Field of the invention

The present invention relates to a process for obtaining boron nitride nanotubes and a process for bacterial transformation employing said nanotubes as facilitators. More specifically, the present invention describes a process of heat shock transformation of competent Escherichia coli strains using nanoparticles as carriers of genetic material.

Background of the invention Internalization of exogenous plasmid by bacteria

Competent competence is one of the main methods used in molecular biology. Through this methodology, the host bacterium expresses genes that will be translated into new proteins, not necessarily pertinent to the source cell. Unfortunately, natural bacterial transformation found in the environment is limited to special conditions and / bu occurs in small groups whose bacteria are naturally competent and capable of internalizing exogenous DNA (Chen et al., 2002a; He et al. (2002; Huang et al. 2002; Nakashima et al. 2002; Wu et al. 2002a).

To date, there are three methodologies that perform this function in vitro, sonoporation, electroporation, and end shock (Adler et al., 2002; Meadows et al., 2002).

Sonoporation, or cellular sonication, is the use of ultrasound to modify the permeability of the plasma cell membrane. Thus, this technique employs acoustic cavitation of microbubbles to increase the delivery of large exogenous DNA molecules to bacteria and cells in their transformation and transfection assays (Teng et al., 2002). The bioactivity of this technique is similar and, in some cases, superior to those found in electroporation. However, prolonged exposure to low frequency (<MHz) ultrasound demonstrated decreased cell viability. Thus, employing this technique means taking this risk (Teng et al., 2002). In addition, it becomes expensive as it needs equipment that emits sound in the range established for this methodology.

While sonoporation uses ultrasound to permeate the

Electroporation cell membrane utilizes the electric pulse of many kV cm'1 amplitude with submicrosecond duration (Wu et al., 2002a). This methodology temporarily permeabilizes the bacterial cell membrane allowing solvated molecules such as DNA to enter the electroporated cells. However, if electrical impulses are misused, cell lysis will occur (Rojas-Chapana etal., 2005).

Another methodology employed is thermal shock. The original protocol of this methodology involves a pretreatment of E. coli with calcium ions (Ca + 2) and magnesium ions (Mg + 2) at 0 ° C. This treatment allows the formation of pores in the bacterial cell wall. The plasmid of interest is added and the subsequent heat shock at 42 ° C allows its internalization (Chen et al., 2002d; Chen et al., 2002f). Thus, this is an inexpensive methodology, since it does not require the purchase of equipment for cellular transformation. Depending on the plasmid to be inserted, the transformation rate is low because the wall is not permeable enough for entry.

Thus, to minimize the deleterious effects of the methodologies described above, several researchers have invested in the use of nanobiotechnology as facilitators of transformation rates.

Rojas-Chapana and colleagues (Rojas-Chapana et al., 2005) 25 used carbon nanotubes to transform E. coli DH5a strains with plasmid pUC19. The authors developed a technique that uses carbon nanotubes as physical vectors for electroporation, unlike other methods used, since no electrodes are required. Water-dispersed carbon nanotubes (NTCs) have an anionic surface charge and thus bind the membrane of gram-negative bacteria by electrostatic interactions (Chen et al., 2002e; Rojas-Chapana et al., 2005). On the other hand, the effect of microwaves (1000W at a frequency of 2.45Ghz) of the electromagnetic field pulse on the interaction of carbon nanotubes with bacteria leads to electropermeabilization of the cell wall. Thus, the authors have demonstrated by this technique that reversible electroporation is possible in which bacterial growth and morphology are not altered, as occurs in the process described in the present invention. However, unlike the present invention, for the material to be internalized, an electron emission source is required, which makes the methodology more expensive, and Zeng and colleagues employed silica nanoparticles,

substituted with amino groups for transformation of E. coli. The authors constructed a silica nanoparticle from TEOS (tetraethoxysilane) and AEAPS (N- (P-aminoethyl) -Yaminopropyltriethoxysilane), forming the aSiNP particle. The surface of the particle was functionalized with amidogen 15 transforming it into an electrically positive particle, which would facilitate the binding of DNA to bacterial cell surface, since both are negatively charged. The boron nanoparticle described in the present invention has a positive charge at the end of its obtaining process, without the need for another functionalization step. Thus, nanotubes present themselves as a more attractive alternative, since their obtaining is simplified, without the need for functionalization with another chemical compound.

In addition to these delivery systems, we highlight the boron nitride nanotubes (BNNTs) employed in the present invention. BNNTs are similar in structure to carbon nanotubes, with carbon atoms replaced by boron and nitrogen, exhibiting superior chemical and thermal stability (Han et al., 2002; Wu et al., 2002b). In the last five years these nanoparticles have been much studied due to their chemical properties. When subjected to ultrasound or polarized light they are able to acquire electrical charge, making them of great importance for the biomedical area (Gnjatic et al., 2002; Han et al., 2002), being suitable for the development of new nanovectors for cell therapy, controlled release of active ingredients, and other clinical applications (Han et al., 2002; Li et al., 2002).

In the last decades numerous syntheses have been made using different techniques such as chemical vapor deposition, laser ablation, arc discharge, ball mill, carbothermal reduction, hydrothermal growth among others (Chen et al, 2002b; GAN et al, 2005).

Initially the methods of arc discharge and laser ablation were the main methods for obtaining nanotubes with low defect concentration. However, these methods have some disadvantages. Among them we can highlight the high temperatures (above 3000 ° C) required for the evaporation of the target, which makes the application of systems to industrial scale very difficult. Another problem is that the sample has impurities such as metal particles and amorphous BN phases, and the formed BNNTs are usually in a highly agglomerated form, forming very cohesive beams, which makes it difficult to separate and obtain individual nanotubes for later application. (Chen et al., 1999).

In 1999, an alternative way of producing BNNTs involving the milling of elemental boron in ammonia gas (NH3) 20 flow followed by N2 or Ar flux heat treatment was reported (Chen et al, 1999). This process involves milling the reagents over a long period of time to produce significant structural changes in the formed compound. This technique is widely used to produce large-scale BNNT, however it produces significant amounts of amorphous material.

The process of obtaining nanotubes by chemical deposition by

Steam (CVD) generally occurs by heating the catalyst material to a certain temperature, which may range from 300 to 1200 ° C. The introduction of a gas stream containing the appropriate reagents occurs at a specific temperature. Five years after the first reports of 30 BNNT synthesis, it became possible to synthesize BNNT using the CVD method (Lourie et al, 2000). Other similar results were obtained by synthesizing using B2O3 as one of the reagents, in which case the synthesis yield reached hundreds of milligrams in a single experimental trial (Zhi et al., 2005).

There are some variations in the method, adapted to the type of synthesis proposed. In this work the reagents and catalysts are placed on a substrate at the beginning of the process at room temperature. As the temperature increases, the system vapor pressure increases and consequently the reactants volatilize. From there occurs the vapor phase deposition for the formation of the nanostructures. From this process it is possible to obtain BNNT efficiently, at low cost and in large quantity.

Initial studies have experimentally confirmed that BNNTs have high thermal and chemical stability, and higher than those presented by CNTs, so the use of BNNT is preferred for applications in devices that will be subjected to high temperatures and chemically active environments (Golberg et al. al., 2001).

Theoretical predictions and experimental results regarding the mechanical properties of BNNTs suggest their promising use as nanofibers for reinforcement in nanocomposites (Bettinger et al., 2002). Currently boron nitride nanostructures also have

aroused great interest in the biomedical field. Their configurations, dimensions and physicochemical properties influence the cellular interactions that lead to several possibilities of bioapplication. However, to date, biomedical applications for BNNTs have been little explored (Feng et al., 2002). Zhi and colleagues investigated the interaction between BNNTs and various protein species (Hung et al., 2002) and between BNNT and DNA (Xiang et al., 2002). Ciofani and colleagues were the first to test the interactions of BNNTs in cell culture. The authors demonstrated that BNNTs mixed with PEI (polyethyleneimine) are 30 soluble and when tested in vitro on SHSY5Y neuroblastomas showed no cytotoxic effect (Chen et al., 2002c). Another experiment involving the same cell line, with BNNT resuspended in PEI and conjugated with fluorescence marker, demonstrated excellent cell endocytosis (Olivier et al., 2002), demonstrating that this nanoconjugate has nanocarrier properties.

Several nanocarrier applications have been suggested for

BNNTs, among them stand out cell therapy by gene and drug delivery. A study by Ciofani and colleagues showed that for both drug delivery transfections, the physical and electronic properties of BNNTs are useful in the process of cellular electropermeabilization by electroporation (Li et al., 2002). In addition, Raffa and colleagues have shown that these nanovectors can enter the cell when they are subjected to low electric field electroporation (40-60V cm -1) (Han et al., 2002).

Although the literature already describes some works of the use of boron nitride nanoparticles as nanocarriers, the present invention advantageously deals with a bacterial transformation, never before described for this nanomaterial, besides not using an external electron emission source or ions.

In view of the information available in the prior art, the present invention becomes useful in routine molecular biology as it increases the rate of bacterial transformation by the thermal shock process. The process is simple and inexpensive. In addition, it features easy manipulation as it does not need an external electron source. Moreover, it is advantageously efficient by tripling the number of transformants relative to usual chemical transformation processes. Boron nitride nanotubes (BNNT) are primarily responsible for the efficiency of the process. Despite having a similar structure to carbon nanotubes, BNNT have different characteristics due to the character of their B-N chemical bonds, which ensure greater chemical and thermal stability.

In addition, the boron nanoparticle described in this

This invention has a positive charge at the end of its production process, without the need for another functionalization step. Thus, nanotubes are a simple and efficient alternative for the transfer of genetic material in bacterial cells.

In addition to application in the bacterial transformation process, object of the present invention, boron nitride nanotubes can also be applied as nanovectors for cell therapy, controlled release of active ingredients, bacterial transforming agent and cellular transfectant.

Brief Description of the Invention

The present invention relates to a process for obtaining

boron nitride nanotubes comprising the steps of mixing the boron, ammonium nitrate and hematite reagents, heat treatment, followed by another heat treatment step under inert nitrogen atmosphere, ammonia gas addition, cooling, ammonium nitrate addition, again 15 a heat treatment under an inert nitrogen atmosphere and a cooling, ending with the nanoparticle purification, employing an acidic solution, filtration and drying. Furthermore, the present invention relates to the use of said nanotubes.

The present invention also addresses the bacterial transformation process employing boron nitride nanotubes, following the thawing steps of competent bacterial cells, addition of boron nitride nanotubes, addition of plasmid of interest, ice incubation, hot bath and again on ice, addition of appropriate culture medium, incubation in a bacteriological oven and finally inoculation in 25 Petri dishes.

Brief Description of the Figures

The structure and operation of the present invention, together with

Additional advantages thereof may be better understood by reference to the accompanying figures and the following description:

-Figure 1 shows an infrared spectrum (FTIR) of the

BNNT sample synthesized by the process described in this invention. Figure 2 shows the X-ray diffractograms of the boron nitride sample before and after the purification process.

- Figure 3 shows the mean and standard deviation of bacterial samples transformed with 1mg / mL BNNT (column A) and samples

controls transformed under the same conditions without the use of BNNT (column F), with P <0.0017.

- Figure 4 shows the mean and standard deviation of bacterial samples transformed with 0.5 mg / mL BNNT (column A) and control samples (column F), with P <0.0087.

Short description of attachments

- Annex 1 presents the electron microscopy images of

sweep (SEM) of the BNNT sample, where in A the increase is 1μιτι and in B the increase is 10μιτι.

- Annex 2 presents the scanning and transmission electron microscopy (TEM) images of BNNT samples obtained at 950 ° C at

a heating rate of 3 ° C / min, where A is the SEM image, B is the TEM image, and C and D the larger TEM images allowing a better view of the pipe walls.

Detailed Description of the Invention

The present invention is a process for obtaining

boron nitride nanotubes. Additionally, the present invention describes the process for heat shock transformation of competent bacterial cells using boron nitride nanoparticles as carriers of genetic material for bacterial transformation.

An object of the present invention is a process for obtaining the

boron nitride nanotubes comprising the following steps:

The. Mixing of reagents;

B. Heat treatment;

ç. Heat treatment under inert nitrogen atmosphere;

d. Addition of ammonia gas; and. Cooling; f. Addition of ammonium nitrate;

g. Heat treatment under inert atmosphere of

nitrogen;

H. Cooling;

i. Purification

11. Acid solution treatment;

12. filtration;

13. Drying.

In the first step (a) boron, ammonium nitrate and hematite reagents, with a purity greater than 95%, are employed in the following weight ratio which may vary from 15: 45: 1 to 15: 15: 1 and from 15:15 to : 4 to 15: 15: 1, preferably being 15: 15: 1. The compounds are mixed and pulverized in porcelain gravel.

The obtained mixture is then subjected to heat treatment step (b) using a boat-shaped alumina crucible. The material is taken to the tubular furnace, preferably in the absence of gas flow. The heat treatment is carried out at a temperature range of 400 to 600 ° C, preferably 550 ° C, lasting 30 to 120 minutes, preferably for 60 minutes.

The next step is heat treatment under an inert nitrogen atmosphere (c), where the material is heated to a temperature range of from 1100 to 1500 ° C, preferably from 1300 ° C.

After temperature stabilization ammonia gas (d) is added to the reaction medium for 1 to 5 hours, preferably 2 hours. Then, in step (e), the inert atmosphere is restored until the material has completely cooled. The formation of boron nitride from the process described in the present invention is represented in reactions 1 and 2:

10B (s) + 4NH4NO3 (s) + 2F62O3 (s) -> 4BN (s) + 3B2O3 (g) + 4Fe (S) + 4NO3 (gj + 4H2 (g) (1)

3B2O3 (g) + 6NH3 (g) -> 6BN (s) + 9H2O (g) (2)

The next step in the process of synthesis of boron nitride nanotubes is the addition of ammonium nitrate (f), in a molar ratio ranging from 2: 2 to 4: 2, preferably 3: 2. After this step the material obtained is heat treated under an inert atmosphere of nitrogen (g) at a heating rate of 1 to 10 ° C / min, preferably 3 ° C / min, to a preferably controllable temperature of 950 ° C. 0C for 1 to 5 hours, preferably 2 hours.

In the cooling step (h) the material is subjected to a flow of

nitrogen.

The purification step (i) for the removal of impurities is started by treating the material with an acidic solution (i1), preferably HCl solution (3 M), at a temperature range of 40 to 90 ° C. preferably 90 ° C for 10 minutes to 3 hours, preferably 10 minutes. After treatment the material goes through a simple filtration (i2) and then a drying process (i3) at a temperature range of 40 to 60 ° C, preferably 40 ° C.

Another object of protection of the present invention is the process of

bacterial transformation with boron nitride nanotubes which

comprises the following steps: a. Thawing of competent bacterial cells; B. Addition of boron nitride nanotubes; ç. Addition of the plasmid of interest; d. Incubation; and. Addition of culture medium; f. Incubation; g. Inoculation. In the first step (a), the competent bacterial cells, previously frozen at -80 ° C, are completely thawed on ice.

Then, in step (b), from 50 to 300 μΙ_, preferably 200μΙ_ of boron nitride nanotubes are added to the bacterial cell solution of step (a). The solution is gently mixed with the aid of a micropipette. In step (c) it is added from 0.5 μΙ_ to 15 μΙ_, preferably 5μΙ_ of the plasmid of interest and again the solution is gently homogenized with the aid of a micropipette.

After addition of the facilitator and plasmid of interest, the mixture is incubated (d). At this stage the microtube containing the solution is incubated on ice for preferably 30 minutes. After this time, the microtudo is placed in a preheated bath at a preferably controllable temperature of 42 ° C for exactly 90 seconds. Again the microtubes are incubated on ice for preferably 2 minutes. In the next step (e) the addition of 300 to 800 μΙ_ occurs.

preferably 600μΙ of a preheated culture medium to preferably 37 ° C.

The tubes are again incubated (f), now in a bacteriological oven, for 1 to 4 hours, preferably 3 hours, at 37 ° C.

Finally, the solution is inoculated (g) by spreading on the

Petri dish surface containing the appropriate inoculum medium and an antibiotic on which the inserted plasmid is resistant.

Example 1: Characterization of boron nitride nanotubes (BNNT)

After the BNNT synthesis process, the materials are submitted to different characterization methods in order to confirm the formation of hexagonal boron nitride, which is the base compound to obtain BNNT, and to verify the structural characteristics of the formed nanotubes.

With the analysis made by the Fourrier Transform Infrared Spectroscopy (FTIR) technique 25 it was possible to characterize the typical functional groups of boron nitride, as well as possible impurities present. IR spectra spanning the 4000-400 cm-1 region were obtained on a Galaxy-Matson model 3020 FTIR spectrophotometer. The spectra were obtained at room temperature on solid powder pellets with KBR.

The samples were also analyzed by X-ray diffraction.

(XRD) for identification and semi-quantitative evaluation of crystalline phases present. An Rigaku X-ray diffractometer, model Geigerflex was used. The identification of crystalline phases was obtained by comparing the X-ray diffractogram of the samples with the ICDD - International Center for Diffraction Data / Joint Committee on Powder Diffraction Standards - JCPDS database (Sets 01 - 50; 2000).

For a better morphological characterization of the samples, images were obtained by scanning electron microscopy (SEM) and transmission (TEM). The images were taken at the Microscopy Center of the Federal University of Minas Gerais (UFMG) with the Quanta 200 FEG (FEI) and Tecnai-G2-20-FEI equipment, respectively.

In the infrared spectrum (FTIR) of the BNNT sample synthesized by the process described in this invention the most important feature presented is the strong asymmetric band centered at 1380 cm -1, which corresponds to the stretching of the BN bond along with another less intense band 15. at 780 cm -1, assigned to BNB bonds. Therefore only the presence of characteristic vibrations for hexagonal boron nitride was verified. Absorbances around 3430 cm -1 can be attributed to the adsorbed water molecules on the sample surface (Figure 1).

It is important to highlight that this technique is very relevant for characterizing h-BN, since it is possible to distinguish the sp2 bonds from the hexagonal phase and sp3 from the cubic phase. According to studies presented by Rao and co-workers, h-BN sp2-type bonds are thermodynamically stable under the synthesis conditions of this work, whereas for the formation of typical c-BN sp3-bonds there is a kinetic barrier of 25 formation. . Peaks between 1096 and 1166 cm -1 are attributed to the formation of the cBN phase. Therefore, only the presence of characteristic vibrations for h-BN was verified. This result is very important for the proposed objective, since only h-BN has the necessary characteristics for the formation of desired BN nanostructures.

Figure 2 shows the diffractogram of the boron nitride sample

before and after purification. In both samples it is possible to clearly identify the presence of typical peaks of the hexagonal phase of boron nitride at 2Θ = 26.75 °, 2Θ = 41.58 °, 2Θ = 50.16 ° and 2Θ = 75.86 °, which are perfectly aligned with the h-BN standard of the JCPDS database, no. 9-12. However, only in the unpurified sample (black curve) is it possible to observe the lowest intensity peaks for metallic iron at 2Θ = 44.67 ° and 2Θ = 65.02 ° (JCPDS, No. 12-62) and, at boron oxide B2O3 at 2Θ = 14.55 ° and 2Θ = 27.76 ° (JCPDS, No. 41-624) remaining in the final product. This result indicates that boron nitride is being formed satisfactorily and that with the The purification process employed was able to remove most of the impurities present in the sample.

Annex 1 presents SEM (Scanning Electron Microscopy) images of the sample, in which the formation of NTBs can be observed. The nanotubes present in this sample have an approximate diameter of 60 nm. Annex 1B has a smaller increase and because of this it can be observed that the formation of BNNT occurs in appreciable quantity.

Annex 2 shows some nanotubes about 3 μιτι in length and 30 nm in diameter. These SEM micrographs (Annex 2A) are related to some TEM (Transmission Electron Microscopy) images shown in Annex 2B, 2C and 2D. In Annex 2C and 20 2D, which were obtained with greater magnification, it is possible to observe two distinct nanotubes that have similar characteristics. Nanotubes have walls about 10 nm thick and approximately 30 layers that are relatively evenly arranged. The inside of the tubes is about 14 nm in diameter.

Example 2: Transformation of Escherichia coli by thermal shock using boron nitride nanotubes as facilitating particle

For bacterial transformation the Escherichia coli (MiguIa) Castellani and Chalmers DH5a cell line (ATCC No. 53868) was thawed in 5 ml of Luria-Bertani medium (10 grams of Tryptone; 5 grams of Yeast Extract; 10 grams of NaCI, Milli-Q water (1000 ml) and incubated at 37 ° C for 16 to 20 hours. The bacteria were then inoculated into Petri dishes containing the same culture medium as above by isolation streaks and incubated for 20 hours at 37 ° C.

After time, 5 isolated colonies were inoculated into erlemayers containing 100 mL Luria-Bertani medium. Next, 5 were incubated in a shaker at 37 ° C at a speed of 150 rpm for 3 hours (obtaining at the end of the process the optical density of 108 bacteria / mL). The volume was divided into two 50mL tubes previously chilled on ice and incubated at the same temperature for 10 minutes. After this process, the tubes were centrifuged at 2700 g (approximately 5,000 rpm) for 10 minutes at 4 ° C. The supernatant was discarded, the tube was inverted on paper towels for 1 minute to drain any excess media. Each pellet from each tube was resuspended in 30 mL of the cold MgCl2-CaCb solution (80 mM MgCl2 and 20mM CaCl2). Again the tubes were centrifuged at 2700 g (5000 rpm) for 10 min at 4 ° C. The supernatant was discarded again and to the pellet was added 2mL of 100mM CaCl2 with 30% Glycerol. The tubes were incubated for 16 hours on ice in the refrigerator. After this period the cells were aliquoted into 1.5mL microtubes. Each microtube was placed 200 μί of the competent cell.

In the present example, for bacterial transformation the plasmid of interest tested was GFP plasmid, which in addition to emitting fluorescence when subjected to ultraviolet light, is also resistant to the antibiotic ampicillin.

All experiments were performed in triplicate and tubes A were transformed with GFP plus 1mg / mL BNNT whereas tubes B were transformed with GFP plus 0.5 mg / mL BNNT.

The DH5a competent cells previously frozen at -80 ° C were completely thawed on ice, then 200 µl BNNT was added and the solution was gently homogenized with the aid of the micropipette. Then 5µg of GFP plasmid was added and again the solution was gently homogenized with the aid of the micropipette. The mixture was incubated on ice for 30 minutes. After time, it was placed in a preheated bath at 42 ° C for exactly 90 seconds. Again the microtubes were incubated on ice for a further 2 minutes. 600 µL of preheated Luria-Bertani medium was added at 37 ° C. The tubes were incubated in a bacteriological oven for 3 hours at 37 ° C and then inoculated in Petri dishes containing Luria-Bertani medium and 50mg / mL ampicillin antibiotic.

After 24 hours of growth the bacterial colonies were counted as follows, the plate was divided into 8 equal parts and 3 parts were counted, then the average was calculated and multiplied by 8, giving the total number of colonies from each plate. The amplitude ratio was then calculated by dividing the number of colonies of each plate containing BNNT by the number of colonies of the plate without BNNT.

Microtubes containing 1mg / mL BNNT showed the results shown in Table 1. In this table we observed that the bacterial samples transformed with BNNT had 4155 cfu 15 (colony forming units), whereas the bacterial samples transformed without BNNT (positive control). presented 1141 cfu (colony forming units). As expected, the negative control (bacteria without plasmid and without BNNT) showed no growth, since the plasmid shows resistance to the ampicillin antibiotic placed on the plate. In this way 20 we demonstrated that the bacteria and plaques were not contaminated. We also observed that if we divide the value of bacteria transformed with BNNT by bacteria transformed without BNNT (positive control), we observed a ratio of 3.64 times, showing the efficiency of the transformation process using BNNT.

Table 1 - Colony forming units (cfu) in the plates transformed with 1 mg / ml BNNT and in the plates of the control groups.

1st Count 2nd Count 3rd Count Average Average x 8 Ratio (cfu) (cfu) (cfu) (Total cfu) A BNNT 542 516 500 519 4155 3.64 B Control 179 96 153 143 1141 1 (+) C Control 0 0 0 0 0 0 (-) Control (+) - DH5a strain with GFP plasmid without BNNT Control (-) - DH5a strain without GFP plasmid without BNNT Ratio - Total number of cfu with BNNT / Total number of cfu of Control (+)

While microtubes transformed with 0.5 mg / mL of

BNNT presented the results shown in Table 2. In this table we observed that the bacterial samples transformed with BNNT presented 2019 cfu, while the transformed bacterial samples without BNNT (positive control) presented 1352 cfu. As expected, the negative control (bacterium without plasmid and without BNNT) showed no growth, since the plasmid shows resistance to the ampicillin antibiotic placed on the plate. In this way we demonstrated that the bacteria and plaques were not contaminated. We also observed that if we divide the value of bacteria transformed with BNNT by bacteria transformed without BNNT 15 (positive control), we observe a ratio of 1.49 times. This means that BNNT increased the transformation rate by this observed proportion, demonstrating the efficiency of the process.

Table 2 - Colony forming units (cfu) in the plates transformed with 0.5 mg / ml BNNT and in the plates of the control groups.

1st Count 2nd Count 3rd Count Average Average x 8 Ratio (cfu) (cfu) (cfu) (Total cfu) A BNNT 542 516 500 519 4155 3.64 B Control 179 96 153 143 1141 1 (+) C Control 0 0 0 0 0 0 (-) Control (+) - DH5a strain with GFP plasmid without BNNT Control (-) - DH5a strain without GFP plasmid without BNNT Ratio - Total number of cfu with BNNT / Total number of cfu Control without BNNT In addition, the statistical test was performed by the GraphPad Instat 3.06 (2003) program by the paired t test, considering significant results, P £ 0.05. Figure 3 shows the results for the transformations performed with 1 mg / ml BNNT (column A) and control of bacterial transformation under the same conditions without the use of BNNT (column F). Thus, we demonstrated that the sample transformed with BNNT presented 3.64 times more transformed colonies than the control used without BNNT. Figure 4 shows the results for the transformations performed with 0.5 mg / ml BNNT (column A) and control of bacterial transformation under the same conditions only without the use of BNNT (column F). Thus, we demonstrate that the sample with BNNT presented 1.49 times more viable colonies transformed with the plasmid used than the control without BNNT.

references

Adler C., Ahammed, Z., Allgower, C., et al. (2002). "Anisotropy azimuthal of K (O) (S) and Lambda + Lambda production at midrapidity from Au + Au collisions at sqrt [s (NN)] = 130 GeV." Phys Rev Lett 89 (13): 132301.

Bettinger, H.F., Dumitrica, T., Scuseria, G.E. and Yakobson, B.I. (2002). "Mechanically induced defects and strength of BN nanotubes." Physical Review: 65.

Chen, J., Chen, Y., Niu, Y.L., Fu, H. and Zhao, Y.F. (2002a). "Carbonyl oxygen migration in electrospray ionization mass spectrometry and its application in differentiating alpha- and beta-alanyl peptides." J Mass Spectrom 37 (9): 934-939.

Chen, Y., Chadderton, L.T., Fitzgerald, J., Williams, J.S. and Bulcock, S. (1999). "A solid-state process for formation of boron nitride nanotubes." Applied Physics Letters 74: 2960-2962.

Chen, Y., Conway, M. and Williams, J. S. (2002b). "Large-quantity production of high-yield boron nitride nanotubes." J. Mater. Res. 17 (8): 1896-1899. Chen, Y., Dabovic, B., Annes, J. P. and Rifkin, D. B. (2002c). "Latent TGF-beta binding protein-3 (LTBP-3) requires binding to TGF-beta for secretion." FEBS Lett 517 (1-3): 277-280.

Chen, Y., Kamat, V., Dougherty, E.R., et al. (2002d). "Ratio statistics of light gene expression and applications to microarray data analysis." Bioinformatics 18 (9): 1207-1215.

Chen Y., Meyer, J. D., Song, J., McDonald, J. C. and Cherry, N. (2002e). "Reliability assessment of a coding scheme for the physical risk factors of workrelated musculoskeletal disorders." Scand J Work Environ Health 28 (4): 232-233.

Chen, Y.W., Jeng, Y.M., Yeh, S.H. and Chen, P.J. (2002f). "P53 gene and Wnt signaling in benign neoplasms: beta-catenin mutations in hepatic adenoma but not in focal nodular hyperplasia." Hepatology 36 (4 Pt 1): 927-935.

Feng, L.X., Chen, Y., Dettin, L., et al. (2002). "Generation and in vitro differentiation of a spermatogonial cell line." Science 297 (5580): 392-395.

GAN, W. Z., DING, X.X., HUANG1 Z.X., et al. (2005). "Growth of boron nitride nanotube film in situ." Applied Physics A 81: 527-529.

Gnjatic1 S., Jager, E., Chen, W., et al. (2002). "CD8 (+) T cell responses against a dominant cryptic HLA-A2 epitope after NY-ESO-1 peptide immunization of cancer patients." Proc Natl Acad Sci S A 99 (18): 11813-11818.

Golberg, D. and Bando Y. (2001). "Unique morphology of boron nitride nanotubes." Applied Physics Letters 79: 415-417.

Han J. Z., Lin, W. Lou, S. J., Qiu J. and Chen, Y. Z. (2002). "A rapid, nongenomic action of glucocorticoids in rat B103 neuroblastoma cells." Biochim Biophys Acta 1591 (1-3): 21-27.

He, M.L., Chen, Y., Peng, Y., et al. (2002). "Induction of apoptosis and inhibition of cell growth by developmental regulator hTBX5." Biochem Biophys Res Commun 297 (2): 185-192.

Huang C. C., Chen C. Y. L., Liang C. Y. and Hsu K. K. (2002). "Scroll for cAMP and protein phosphatase in the presynaptic expression of mouse hippocampal mossy fiber depotentiation." J Physiol 543 (Pt 3): 767-778. Hung, S.L., Wang, Y.H., Chen, H.W., Lee, P.L. and Chen, Y.T. (2002). "Analysis of herpes simplex virus entering cells of oral origin." Virus Res 86 (1-2): 59-69.

Li, C.S., Lin, W.H., Yang1 Y.Y., etal. (2002). "Impairment of temporal attention in patients with schizophrenia." Neuroreport 13 (11): 1427-1430.

Lourie, O.R., Jones, C.R., Bartlett, B.M., etal. (2000). "CVD growth of boron nitride nanotubes." Chemical Materials 12 (1808-1810).

Meadows, L.S., Chen, Y.H., Powell, A.J., Clare, J.J. and Ragsdale, D.S. (2002). "Functional modulation of human brain Nav1.3 sodium channels, expressed in mammalian cells, by auxiliary beta 1, beta 2 and beta 3 subunits." Neuroscience 114 (3): 745-753.

Nakashima, Y., Chen, Y.X., Kinukawa, N. and Sueishi, K. (2002). "Distributions of diffuse intimai thickening in human arteries: preferential expression in atherosclerosis-prone arteries from an early age." Virchows Arch 441 (3): 279-288.

Olivier, M., Chuang, L.M., Chang, M.S., et al. (2002). "High-throughput genotyping of single nucleotide polymorphisms using new biplex invader technology." Nucleic Acids Res 30 (12): e53.

Rojas-Chapana, J., Troszczynska, J., Firkowska, I., Morsczeck, C. and Giersig, M. (2005). "Multi-walled carbon nanotubes for plasmid delivery into Escherichia coli cells." Lab Chip 5 (5): 536-539.

Teng, Y.N., Tsai, W.H., Wu, C.J., et al. (2002). "Referral diagnosis of PraderWilli syndrome and Angelman syndrome based on methylation-specific polymerase chain reaction." J Formos Med Assoc 101 (7): 488-494.

Wu, Q., Chen, Y. Q., Chen, Z.M., Chen, F. and Su, W.J. (2002a). "Effects of retinoic acid on metastasis and its related proteins in gastric cancer cells in vivo and in vitro." Acta Pharmacol Sin 23 (9): 835-841.

Wu, Q., Zhang, M., Liu, S., Chen, Y. and Su, W. (2002b). "Retinoic acid beta receptor is required for anti-activator protein-1 activity by retinoic acid in gastric cancer cells." Chin Med J (EngI) 115 (6): 810-814. Xiang 1 C. C., Kozhich 1 O. A., Chen 1 M., et al. (2002). "Amine-modified random primers to DNA probes for DNA microarrays." Nat Biotechnol 20 (7): 738-742. Zhi1 C., Bando1 Y., Tang1 C., et al. (2005). "Perfectly dissolved boron nitride nanotubes due to polymer wrapping." J Am Chem Soc. 127 (46): 15996-15997. Annex 1

Annex 2

Claims (20)

  1. Process for obtaining boron nitride nanotubes comprising the following steps: (a) mixing of reagents; b) heat treatment; c) Heat treatment under inert nitrogen atmosphere; d) Addition of ammonia gas; e) cooling; f) Addition of ammonium nitrate; g) Heat treatment under inert nitrogen atmosphere; h) cooling; (i) Purification i1) Acid solution treatment; i2) filtration; i3) Drying.
  2. Process according to Claim 1, characterized in that step (a) comprises the mixture of boron, ammonium nitrate and hematite in the weight ratio of from 15: 45: 1 to 15: 15: 1 and 15:15: 4 to 15: 15: 1, preferably being 15: 15: 1.
  3. Process according to Claim 1, characterized in that the heat treatment step (b) is preferably carried out in the absence of gas flow at a temperature range of 400 to 600 ° C, preferably 550 ° C for 30 minutes. at 120 hours, preferably 60 minutes.
  4. Process according to Claim 1, characterized in that the heat treatment step under inert nitrogen atmosphere (c) is carried out at a temperature range of 1100 to 1500 ° C, preferably 1,300 ° C.
  5. Process according to Claim 1, characterized in that the step of adding ammonia gas (d) takes place for 1 to 5 hours, preferably 2 hours.
  6. Process according to Claim 1, characterized in that the step of adding ammonium nitrate (f) in a molar ratio of 2: 2 to 4: 2, preferably 3: 2.
  7. Process according to Claim 1, characterized in that the heat treatment step under nitrogen inert atmosphere (g) is carried out at a heating rate of 1 to 10 ° C / min, preferably 3 ° C / min, to controllable temperature and preferably 950 ° C for 1 to 5 hours, preferably 2 hours.
  8. Process according to Claim 1, characterized in that the purification step comprises treating an acid solution (i1), preferably HCl solution (3 M), at a temperature range of 40 to 90 ° C, preferably 90 ° C. for 10 minutes to 3 hours, preferably 10 minutes.
  9. Process according to Claim 1, characterized in that the purification step comprises drying (13) of the material at a temperature range of 40 to 60 ° C, preferably 40 ° C.
  10. Boron nitride nanotube characterized in that it is obtained according to the steps described in claims 1 to 9.
  11. Use of the boron nitride nanotube described in claim 10 for use as a facilitator in bacterial transformations, as nanovectors for cell therapy, in the controlled release of active ingredients, as a bacterial transformant and a cellular transfectant.
  12. Bacterial transformation process characterized by employing boron nitride nanotubes described in claim 10.
  13. 13. Bacterial transformation process comprising the following steps: (a) thawing of the competent bacterial cells; (b) Addition of boron nitride nanotubes; c) Addition of the plasmid of interest; d) incubation; e) Addition of culture medium; f) incubation; g) Inoculation.
  14. Process according to Claim 13, characterized in that step (a) comprises thawing bacterial cells on ice.
  15. Process according to Claim 13, characterized by the addition of 50 to 300 μΙ_, preferably 200μί of boron nitride nanotubes in step (b).
  16. Process according to claim 13, characterized in that step (c) comprises adding 0.5 μ _ to 15 μΙ_, preferably 5μΙ_ of the plasmid of interest.
  17. Process according to Claim 13, characterized in that it comprises an incubation step (d) in ice for preferably 30 minutes, followed by a preheated bath at a controllable and preferably temperature of 42 ° C for exactly 90 seconds and again. on ice for preferably 2 minutes.
  18. Process according to Claim 13, characterized by the addition of 300 to 800 μι, preferably 600μ Ι from a preheated culture medium to preferably 37 ° C in step (e).
  19. Process according to Claim 13, characterized in that the incubation step (f) is carried out in a bacteriological oven for 1 to 4 hours, preferably 3 hours at 37 ° C.
  20. Process according to Claim 13, characterized in that the inoculation step (g) is spread by spreading on Petri dishes containing the appropriate inoculum medium and an antibiotic.
BRPI1104516 2011-09-16 2011-09-16 Process for obtaining boro nitret nanotubes, so obtained nanotubes and bacterial transformation process using boro nitrete nanotubes BRPI1104516A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
BRPI1104516 BRPI1104516A2 (en) 2011-09-16 2011-09-16 Process for obtaining boro nitret nanotubes, so obtained nanotubes and bacterial transformation process using boro nitrete nanotubes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
BRPI1104516 BRPI1104516A2 (en) 2011-09-16 2011-09-16 Process for obtaining boro nitret nanotubes, so obtained nanotubes and bacterial transformation process using boro nitrete nanotubes
PCT/BR2011/000462 WO2013037019A1 (en) 2011-09-16 2011-12-07 Process for producing boron nitride nanotubes, boron nitride nanotube, use of the boron nitride nanotube, and method for transforming bacteria

Publications (1)

Publication Number Publication Date
BRPI1104516A2 true BRPI1104516A2 (en) 2014-07-08

Family

ID=47882465

Family Applications (1)

Application Number Title Priority Date Filing Date
BRPI1104516 BRPI1104516A2 (en) 2011-09-16 2011-09-16 Process for obtaining boro nitret nanotubes, so obtained nanotubes and bacterial transformation process using boro nitrete nanotubes

Country Status (2)

Country Link
BR (1) BRPI1104516A2 (en)
WO (1) WO2013037019A1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2877060C (en) 2013-04-18 2015-07-28 National Research Council Of Canada Boron nitride nanotubes and process for production thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040023372A1 (en) * 2002-05-28 2004-02-05 The Trustees Of The University Of Pennsylvania Tubular nanostructures
US20110177154A1 (en) * 2008-09-15 2011-07-21 Searete Llc, A Limited Liability Corporation Of The State Of Delaware Tubular nanostructure targeted to cell membrane

Also Published As

Publication number Publication date
WO2013037019A1 (en) 2013-03-21

Similar Documents

Publication Publication Date Title
Jiang et al. Recent progress on fabrications and applications of boron nitride nanomaterials: a review
Kalay et al. Synthesis of boron nitride nanotubes and their applications
RU2591942C2 (en) Obtaining graphene carbon particles using hydrocarbon precursor materials
Levchenko et al. Scalable graphene production: perspectives and challenges of plasma applications
Cantaert et al. Think positive: phase separation enables a positively charged additive to induce dramatic changes in calcium carbonate morphology
Whitener Jr et al. Graphene synthesis
Zhang et al. Large-scale production of high-quality graphene using glucose and ferric chloride
Cai et al. Methods of graphite exfoliation
Musso et al. Influence of carbon nanotubes structure on the mechanical behavior of cement composites
Perez-Cabero et al. Characterization of carbon nanotubes and carbon nanofibers prepared by catalytic decomposition of acetylene in a fluidized bed reactor
Nasibulin et al. A novel approach to composite preparation by direct synthesis of carbon nanomaterial on matrix or filler particles
Zheng et al. Plasma‐Assisted Approaches in Inorganic Nanostructure Fabrication
Kim et al. Sol–gel synthesis and characterization of nanostructured hydroxyapatite powder
US20170216923A1 (en) Porous materials comprising two-dimensional nanomaterials
Li et al. Exfoliation of hexagonal boron nitride by molten hydroxides
Malesevic et al. Synthesis of few-layer graphene via microwave plasma-enhanced chemical vapour deposition
Li et al. In situ synthesis and biocompatibility of nano hydroxyapatite on pristine and chitosan functionalized graphene oxide
Kröger et al. Bioenabled synthesis of rutile (TiO2) at ambient temperature and neutral pH
Liu et al. Preparation of few-layer and single-layer graphene by exfoliation of expandable graphite in supercritical N, N-dimethylformamide
Palkar et al. Reactivity differences between carbon nano onions (CNOs) prepared by different methods
Xue et al. Solvothermal synthesis and photoluminescence properties of BiPO4 nano-cocoons and nanorods with different phases
Zhao et al. Large‐scale synthesis of nitrogen‐rich carbon nitride microfibers by using graphitic carbon nitride as precursor
Mao et al. Virus-based toolkit for the directed synthesis of magnetic and semiconducting nanowires
Zhou Reversed crystal growth: implications for crystal engineering
Li et al. Clean double-walled carbon nanotubes synthesized by CVD

Legal Events

Date Code Title Description
B03A Publication of an application: publication of a patent application or of a certificate of addition of invention
B06F Objections, documents and/or translations needed after an examination request according art. 34 industrial property law
B06T Formal requirements before examination
B07A Technical examination (opinion): publication of technical examination (opinion)