A method for synthesising ion doped nanobioglass by a single-phase microfluidic device

Abstract

The present invention relates to a method of synthesis of ion doped bioglass nanoparticles (BGNs) by micro emulsion assisted sol-gel technique. More particularly, the present invention provides a method of synthesizing ion doped BGNs in an efficient, safe, simple, rapid and cost- friendly manner. The ion doped BGNs synthesized are non-agglomerated, toxic free and have a homogenous well-defined spherical morphology without any post-synthesis treatment. The ion doped BGNs synthesised are applied in various fields like diagnostics, pharmaceuticals, and therapeutics.

Description

[0001] A METHOD FOR SYNTHESISING ION DOPED NANOBIOGLASS BY A SINGLEPHASE MICROFLUIDIC DEVICE

[0002] FIELD OF INVENTION:

[0003] [1] The present invention is in the field of microfluidics. More particularly, the present invention relates to a method for synthesizing ion doped bioglass nanoparticles (BGNs) by a single-phase microfluidic device. The synthesized ion doped BGNs are applied in the field of diagnostics, pharmaceuticals, therapeutics, regenerative medicine and dentistry.

[0004] BACKGROUND OF INVENTION:

[0005] [2] Over the years, there has been increasing demand for the usage of bioglass nanoparticles (BGNs) or nanobioglass in the field of biomedical science. This is due to the versatile properties like bioactivity, resorbability, antibacterial and osteoproductivity depending on its flexible compositional range. This led to the wide range of applications including bone augmentation, drug delivery, repair and regeneration of nerve endings, enhancing wound healing process, as scaffolds for regenerative medicine, etc.

[0006] [3] The bioactivity of BGN is due to the surface reactivity and composition of BGNs. It is also believed that a more physiological environment is important to improve the bioactive properties of the material. Thus, doping or incorporation of different ions into the composition of BGN has been shown to enhance its physical and therapeutic properties.

[0007] [4] Since 1985, various ions like gold, silver, zinc, magnesium, boron, lithium, copper, cerium, strontium have been doped into BGN. The conventional methods used for doping BGN are ball milling technique, melt quenching, spray pyrolysis and surface modification method. In the ball milling technique, doping with ions like titanium, boron and hydroxyl apatite have been carried out but the size of the particles was found out to be in microns. Further, this procedure requires erudite equipment. On the other hand, in melt quenching and spray pyrolysis methods, ions like zinc, yttrium, and copper ions have been doped but the yielded particles were again found to be in microns size scale.

[0008] [5] To incorporate different ions into BGN, the most common method has been the surface modification approach. In this method, the BGN is synthesized using micro emulsion assisted sol-gel technique and later the ion of interest is incorporated. Ions like silver, copper, zinc, cerium, boron etc. have been incorporated onto the synthesized BGN. The particles produced using these methods were in nanosize between 200 - 400 nm which were non-uniform in nature. The concentration of the doped ion into the BGN ranges from 2-6 mol %. Microfluidics has acquired dominance over the last years, due to their accuracy in manipulating and monitoring of the fluids on micrometric scale channels. BGNs provide a controlled environment for synthesis of nanoparticles and have several advantages over batch synthesis. They have high reproducibility, are cost efficient, have good control over particle size and mixing time and reduce the synthesis time drastically.

[0009] [6] Reference is made to IN202441014037 which discloses a method for synthesizing toxic-free spherical shaped bioglass nanoparticles (BGNs) by a microfluidic device. The synthesized BGNs are applicable in drug delivery and tissue regeneration. However, the precursors used in the device require mixing of two components that are, component A having TEOS, TEP and ammonia and component B having ethanol, deionised (DI) water and calcium nitrate that gets mixed in the device fabricated to produce homogenous nanobioglass. The method disclosed thus involves reaction of many chemicals making it lengthy, complex and cost inefficient.

[0010] [7] Reference is further made to CN113666641 A which discloses a multifunctional bioactive glass ceramics nano material and a preparation method and application thereof. The method comprises the steps of synthesizing BGNs by respectively taking tetraethoxy silane (TEOS), triethyl phosphate (TEP) and calcium nitrate tetrahydrate as a silicon source, a phosphorus source and a calcium source based on a sol-gel template method. Then, mixing BGN and molybdenum acetyl acetonate, and carrying out hydrothermal reaction to obtain the molybdenum-doped bioactive glass ceramic nano material. However, the method takes 12-15 hours to complete the process and require a lot of external equipment during the process which makes it a time consuming and complex process. Further, no microfluidic device is being used in this citation.

[0011] [8] Reference is also made to A. Saatchi et al. in Ceramics International, Volume 47, Issue 1, 1 January 2021, Pages 260-271, which describes fabrication of a series of electrospun chitosan / polyethylene oxide (CH / PEO / (8Ce-BG)) nanofibrous scaffolds containing different amount of cerium-doped bioactive glasses employing sol-gel method for tissue engineering applications. However, the said process takes around 10 days for completion and agglomeration of particles is seen with <10% doping of cerium ion into nanobioglass. The final product requires electrospinning with various other biopolymers like chitosan, polyethylene oxide.

[0012] [9] Reference is further made to A. Singh et al. in Nanotechnology, 2008 Jun 18;19(24):245613, which discloses the synthesis of water soluble 1 -thioglycerol- capped Mn-doped ZnS nanocrystalline semiconductor nanoparticles (TG-capped ZnS:Mn) via a microfluidic approach. However, the synthesis process requires a temperature of 80°C to be maintained at the outlet collection and the particles obtained were found to have no distinguishable shape and size. Further, agglomeration of particles is seen at different concentrations of the particles synthesized.

[0013]

[0010] The abovementioned prior arts have several shortcomings such as long and complex method of doping, requirement of a lot of external equipment, agglomeration of the synthesized particles, requirement of a particular temperature to be maintained throughout the process and no distinguishable shape and size of the synthesized particles.

[0014]

[0011] In view of above, there exists a dire need in the state of art to provide a method for synthesizing ion doped bioglass nanoparticles (BGNs) in a short period of time, less reagents and high reproducibility of the BGNs synthesized.

[0015] OBJECTS OF THE INVENTION:

[0016]

[0012] The principal object of the present invention is to provide a method of synthesis of ion doped bioglass nanoparticles (BGNs) by a single-phase microfluidic device.

[0013] Another obj ect of the present invention is to provide a method of synthesis of ion doped BGNs by surface modification technique that is efficient, simple, rapid and cost- friendly.

[0017]

[0014] Another object of the present invention is to provide various ions such as cerium, boron and copper doped into BGNs.

[0018]

[0015] Another object of the present invention is to provide ion doped BGNs having well defined spherical morphology and bioactivity.

[0019]

[0016] Yet another object of the present invention is to provide a single-phase microfluidic device that is used to dope different ions into BGNs in a time efficient manner.

[0020]

[0017] Yet another object of the present invention is to provide ion doped BGNs for applications in diagnostics, pharmaceuticals, therapeutics, regenerative medicine and dentistry.

[0021] SUMMARY OF THE INVENTION:

[0022]

[0018] In one aspect, the present invention provides a method for synthesizing ion doped bioglass nanoparticles (BGNs), comprising the steps of: (a) dissolving ions into ethanol solution to obtain an ion solution; (b) mixing BGNs into the ion solution of step (a) to obtain a solution; (c) seeding the solution of step (b) into inlet of a microfluidic device in order to attach ions to the surface of BGNs to form doped BGNs; (d) collecting the doped BGNs of step (c) in the outlet and subjecting to centrifugation followed by drying the same in a hot air oven.

[0023]

[0019] In another aspect, the present invention provides a single-phase microfluidic device for synthesizing ion doped BGNs as described herein, comprising: (i) serpentine structures; (ii) a plurality of semi-spiral structures; (iii) a whole spiral structure; (iv) an inlet; and (v) an outlet.

[0020] Therefore, the present invention provides a method for synthesizing ion doped BGNs by a single-phase microfluidic device.

[0024] DESCRIPTION OF ACCOMPANYING FIGURES:

[0025]

[0021] The accompanying drawings constitute a part of the description and are used to provide further understanding of the present invention. Such accompanying drawings illustrate the embodiments of the present invention, which are used to describe the principles of the present invention together with the description.

[0026]

[0022] Figure 1 illustrates complete design of the microfluidic device in part (a); and enlarged designs used in developing this microfluidic device along with their dimensions in parts (b, c and d), in accordance with an implementation of the present invention.

[0027]

[0023] Figure 2 illustrates the steps (i to vi) in fabrication of proposed microfluidic device by photolithography technique in part (a); the image of the patterned design in silicon wafer in part (b); the polydimethylsiloxane (PDMS) microfluidic image in part (c); and the PDMS device bonded onto a clean glass substrate using plasma bonding technique in part (d), in accordance with an implementation of the present invention.

[0028]

[0024] Figure 3 illustrates transmission electron microscopy (TEM) image of different doped BGN along with enlarged single particle, in accordance with an implementation of the present invention.

[0029]

[0025] Figure 4 illustrates X-Ray diffraction (XRD) analysis of the particles in parts (i to iii), in accordance with an implementation of the present invention.

[0030]

[0026] Figure 5 illustrates scanning electron microscope (SEM) and energy-dispersive X-ray (ED Ax) analysis of the differently doped BGN in parts (i to iii), in accordance with an implementation of the present invention.

[0031]

[0027] Figure 6 illustrates fourier transform infrared (FTIR) spectra analysis of different doped BGN in parts (i to iii), in accordance with an implementation of the present invention.

[0028] Figure 7 illustrates scanning electron microscope (SEM) and energy dispersive X-ray (ED Ax) analysis before immersion of particles in parts (i to iii); and after immersion of particles in simulated body fluid (SBF) for 10 days in parts (iv to vi), in accordance with an implementation of the present invention.

[0032] DETAILED DESCRIPTION OF THE INVENTION:

[0033]

[0029] While the invention is susceptible to various modifications and alternative forms, specific embodiment thereof will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the scope of the invention as defined by the appended claims.

[0034]

[0030] Although one or more features and / or elements may be described herein in the context of only a single embodiment, or alternatively in the context of more than one embodiment, or further alternatively in the context of all embodiments, the features and / or elements may instead be provided separately or in any appropriate combination or not at all. Conversely, any features and / or elements described in the context of separate embodiments may alternatively be realized as existing together in the context of a single embodiment.

[0035]

[0031] The terminology used herein is for the purpose of describing particular various embodiments only and is not intended to be limiting of various embodiments. As used herein, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0036]

[0032] As discussed in the background section of the present invention, the existing methods reported in the literature are long and complex, require a lot of external equipment, synthesize particles that are agglomerated in nature, require a particular temperature to be maintained throughout the process and synthesize no distinguishable shape and size of the particles.

[0037]

[0033] Therefore, to overcome the existing problems in the art, the present invention provides a method of synthesizing ion doped BGNs by a single-phase microfluidic device. The invention provides homogenous spherical ion doped BGNs which are synthesized in a time efficient manner with a good yield.

[0038]

[0034] Overall, the present invention is different and technically advance over the conventional prior art(s) in view of the following advantages: a) Simple and time efficient method of synthesis: The present invention provides a simple and efficient method for synthesizing ion doped BGNs in a 6-10 minutes without any requirement of any varying temperature range during the entire process. b) Biomedical applications of the synthesized ion doped BGNs: The present invention provides ion doped BGNs that show applicability in therapeutics, pharmaceuticals, regenerative medicine and dentistry. Due to the same, the ion doped BGNs have vast biomedical applications. c) High reproducibility of the synthesized ion doped BGNs: The present invention provides a method of synthesis of ion doped BGNs without the requirement of complex equipment and reagents, thereby increasing the rate of reproducibility and cost efficiency of the ion doped BGNs. d) Toxic free and well-defined morphology of the synthesized ion doped BGNs: The present invention provides ion doped BGNs having homogenous well-defined spherical morphology that are toxic free.

[0039]

[0035] In an embodiment, the present invention provides a method for synthesizing ion doped bioglass nanoparticles (BGNs), comprising the steps of: (a) dissolving ions into ethanol solution to obtain an ion solution; (b) mixing BGNs into the ion solution of step (a) to obtain a solution; (c) seeding the solution of step (b) into inlet of a microfluidic device in order to attach ions to the surface of BGNs to form doped BGNs; (d) collecting the doped BGNs of step (c) in the outlet and subjecting to centrifugation followed by drying the same in a hot air oven.

[0040]

[0036] In another embodiment, the present invention provides a method as described herein, wherein said ions are selected from metallic and non-metallic ions.

[0041]

[0037] In another embodiment, the present invention provides a method as described herein, wherein said metallic ions are selected from a group consisting of silver, copper, zinc, cerium and boron.

[0038] In another embodiment, the present invention provides a method as described herein, wherein said ions and ethanol in step (a) are present in an amount ranging from 0.65 to 2 mg and 3 to 5 ml, respectively.

[0042]

[0039] In another embodiment, the present invention provides a method as described herein, wherein said centrifugation in step (d) is carried out at a speed in a range of 7800-7900 rpm for a time period ranging from 3-8 minutes.

[0043]

[0040] In another embodiment, the present invention provides a method as described herein, wherein said drying in step (d) is carried out at a temperature ranging from 40 to 50°C.

[0044]

[0041] In another embodiment, the present invention provides a method as described herein, wherein said BGNs are synthesized through micro emulsion assisted sol-gel technique.

[0045]

[0042] In another embodiment, the present invention provides a method as described herein, wherein said ions are doped into the BGNs that are present in an amount ranging from 6 - 8 wt% of ions.

[0046]

[0043] In another embodiment, the present invention provides a single-phase microfluidic device for synthesizing ion doped BGNs as described herein, comprising: (i) serpentine structures (1); (ii) a plurality of semi-spiral structures (2); (iii) a whole spiral structure (3); (iv) an inlet (4); and (v) an outlet (5).

[0047]

[0044] In another embodiment, the present invention provides a device as described herein, wherein said inlet (4) is connected to serpentine structures (1) including 15 rows followed by 5 sets of said semi-spiral structures (2) that is connected to said whole spiral structure (3) and said outlet (5) at the end.

[0048]

[0045] In another embodiment, the present invention provides a device as described herein, wherein said serpentine structures (1) have an outer diameter ranging from 0.1 - 0.3 mm, an inner diameter ranging from 0.05 - 0.15 mm and each row with a length ranging from 56 - 60 mm.

[0049]

[0046] In another embodiment, the present invention provides a device as described herein, wherein said inlet (4) and said outlet (5) have a dimension ranging from 1 - 3 mm diameter, each semi-spiral structure (2) with a width in a range of 2 mm between each turn, and a single spiral structure (3) with 4 turns and a distance ranging from 2 mm between each turn.

[0050]

[0047] In another embodiment, the present invention provides a device as described herein, wherein said BGNs are applied in diagnostics, pharmaceuticals, therapeutics, regenerative medicine and dentistry.

[0051]

[0048] In another embodiment, the present invention provides a device as described herein, wherein said BGNs are toxic free, spherical sphere shape having a diameter ranging from 200 to 300 nm without any agglomeration.

[0052]

[0049] The present invention is illustrated hereunder in greater detail in relation to non-limiting exemplary embodiments as per the following examples:

[0053] EXAMPLES

[0054]

[0050] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and the description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all and only experiments performed. The methodology of preparing few of the preferred embodiments shall become clearer with working examples provided below.

[0055] CHEMICALS AND INSTRUMENTS USED:

[0056]

[0051] Bioglass nanoparticles (BGNs) are procured from the microfluidic device fabricated in patent application IN202441014037 and used in the form of powder to dope various metallic and non-metallic ions to form ion doped BGNs.

[0057]

[0052] Ethanol and metallic salts are procured from Sigma Aldrich chemicals and used as such.

[0058] Example 1: Method of synthesis of ion doped bioglass nanoparticles (BGNs):

[0053] The single-phase microfluidic system has a continuous laminar flow through single or multiple inlets that provides a homogenous environment for the reaction to take place. Mixing of reagents occurs primarily through diffusion. The different geometries of the microfluidic device were used to control the mixing resulting in desirable shape and size of the nanoparticle.

[0059]

[0054] The chemical synthesis was changed into microfluidic technology by altering the concentration and volume requirements along with the flow rate. Through some calculations, the experimental flow rates were determined to avoid backflow or any leakage issues with the device. The doped BGNs used the surface modification strategy here. In this procedure, the synthesized BGNs were mixed into the desirable ion solution, which were prepared by dissolving ions to be incorporated into ethanol solution. The solution was seeded into the inlet of the device where the desired ions get attached to the surface of BGN. Figure 1 represents an AutoCAD design of the device with inlets and outlets. The doped BGNs produced were collected in the outlet and subjected to centrifugation at 7830 rpm for 5 minutes. The particles were then dried in a hot air oven at 45°C overnight.

[0060]

[0055] Particularly, 0.2 ml of cerium nitrate was added into 1 ml of ethanol and mixed till the particles dissolve. Later, 10 ml of nanobioglass powder was mixed to the ethanol mixture containing cerium nitrate. The mixing undergoes surface modification on the nanobioglass powder. The cerium ions displaced the calcium ions present in the nanobioglass to form the cerium-silica-oxygen network / cerium doped nanobioglass. Displacement of calcium ions was determined by the concentration as well as the flow rate of the microfluidic device. The resulting nanoparticles were then dried overnight to obtain doped nanobioglass powder.

[0061]

[0056] A similar method was used for boron doped nanobioglass except that instead of cerium nitrate, boric acid was added. In case of copper doped nanobioglass, copper sulphate was added in place of cerium nitrate. Finally, the resulting particles were collected, dried overnight and calcined to form doped nanobioglass.

[0062] Example 2: Designing the microfluidic device

[0057] The design for the device was made using the AutoCAD software 2019 version. The design consists of 15 rows of serpentine structures (1) followed by 5 sets of semi-spiral structures (2) connected to a single whole spiral structure (3) at the end. The device consists of a single inlet (4) and outlet (5). The dimensions were chosen assuming a laminar flow of the fluid inside the device. The inlet (4) and outlet (5) have a dimension of 2 mm diameter, each semi-spiral structure (2) with a width of 2 mm between each turn, and a single spiral structure (3) with 4 turns and a 2 mm distance between each turn. There were 15 rows of serpentine structures (1) that have an outer diameter of 0.2 mm and an inner diameter of 0.1 mm. Each row had a length of 58.4 mm as shown in Figure 1.

[0063] Example 3: Fabrication of the device

[0064]

[0058] This device was fabricated using lithography technique. The steps are shown in part (a) of Figure 2. First, a silicon wafer was piranha cleaned and SU-8 photoresist (2035 series) was spin coated at optimized parameters to obtain a desirable height ranging from 80 - 100 microns. Prebaking was done for 17 minutes after the spin coating followed by exposure of the wafer to UV using a chromium mask (designed using AutoCAD) and post exposure baking was carried out for 5 minutes. Then, to obtain the SU-8 pattern on the wafer, the pattern was developed using appropriate developer and cleaned well using acetone and isopropyl alcohol. The patterned SU-8 devices were seen in part (b) of Figure 2. Later, polydimethylsiloxane (PDMS) with a ratio of 1 : 10 was mixed and poured over the pattern and kept for curing at 60°C for 5 hours to obtain the desired inlet (4), outlet (5) and micro channels. Finally, the PDMS layer was pulled out from the SU-8 structure as shown in part (c) of Figure 2 and bonded on a glass substrate after plasma oxygen treatment in part (d) of Figure 2.

[0065] Example 4: Characterisation of the synthesised ion doped BGNs

[0066] 4.1 Transmission electron microscopy (TEM) analysis

[0067]

[0059] The transmission emission microscope (TEM) analysis that uses the principle of imaging samples through a beam of electrons transmitted through a specimen. This method revealed that cerium, boron, and copper doped BGNs were well-defined spherical spheres with a diameter of less than 300 nm and forming a monodisperse layer. In lower magnification at 5nm, a lattice-like structure was appreciated indicating a metallic ion incorporation into the BGNs as shown in Figure 3.

[0068] 4.2 X-Ray diffraction (XRD) analysis

[0069]

[0060] In part (i) of Figure 4, cerium-doped BGN exhibits prominent peaks at 35.68 and 52.7 along with peaks at 27.8 and 33.2 indicating the presence of cerium nanoparticles and calcium silicate compounds respectively. In copper-doped BGN particles, part (iii) of Figure 4, peaks were seen at 56 and 47, indicating copper nanoparticles along with calcium silicate particles.

[0070] 4.3 Scanning electron microscopy (SEM) analysis

[0071]

[0061] Parts (i-iii) of Figure 5 shows the surface image of the particles through SEM and the percentage of different ions present in each ion doped BGN.

[0072] 4.4 Fourier transform infrared (FTIR) analysis

[0073]

[0062] Parts (i-iii) of Figure 6 depicts FTIR spectra where when the sample was kept in infrared range, the rays entered the sample and transmitted energy was absorbed and finally a spectrum was generated. This spectrum represents the bond between the metallic ions and nanobioglass complex indicated as wavenumbers.

[0074] 4.5 Bioactivity assay

[0075]

[0063] To assess the bioactivity of the BGNs, the synthesized particles were immersed in simulated body fluid (SBF) for 10 days and SEM was used to image the surface morphology of the particles before and after immersion. Figure 7 shows the topographical image of the particles before and after immersion in SBF for 10 days using SEM. Figure 7 (i) & (iv) represents the deposition of calcium phosphate particles in cerium-doped bioglass. Similarly, parts (ii and v) of Figure 7 represent the deposition of cerium particles before and after immersion, respectively. Parts (iii and vi) of Figure 7 represent an increase in calcium deposition along with a decrease in silica was seen indicating an increased bioactivity of the synthesized particles.

[0076] ADVANTAGES OF PRESENT INVENTION:

[0077]

[0064] The present invention provides a method for synthesising ion doped BGNs in an efficient, cost-friendly, rapid and toxic-free manner.

[0078]

[0065] The advantages of the method of the present invention are: a) The present invention provides a method of synthesis of ion doped BGNs in a rapid, simple, efficient and cost friendly manner. b) The present invention provides ion doped BGNs having vast biomedical applications in the fields of therapeutics, pharmaceuticals, regenerative medicine, and dentistry. c) The present invention allows synthesis of ion doped BGNs in a few minutes without any requirement of varying temperature range. d) The present invention allows synthesis of ion doped BGNs having homogenous well-defined spherical morphology and toxic-free nature.

Claims

WE CLAIM:

1. A method for synthesizing ion doped bioglass nanoparticles (BGNs), comprising the steps of: a) dissolving ions into ethanol solution to obtain an ion solution; b) mixing BGNs powder into the ion solution of step (a) to obtain a solution; c) seeding the solution of step (b) into inlet of a microfluidic device in order to attach ions to the surface of BGNs to form doped BGNs; d) collecting the doped BGNs of step (c) in the outlet and subjecting to centrifugation followed by drying the same in a hot air oven.

2. The method as claimed in claim 1, wherein said ions in step (a) are selected from metallic and non-metallic ions.

3. The method as claimed in claims 1 and 2, wherein said metallic ions are selected from a group consisting of silver, copper, zinc, cerium and boron.

4. The method as claimed in claim 1, wherein said ions and ethanol in step (a) are present in an amount ranging from 0.6 to 2 mg and 3 to 5 ml, respectively.

5. The method as claimed in claim 1, wherein said centrifugation in step (d) is carried out at a speed in a range of 7800 - 7900 rpm for a time period ranging from 3 - 8 minutes.

6. The method as claimed in claim 1, wherein said drying in step (d) is carried out at a temperature ranging from 40 to 50°C.

7. The method as claimed in claim 1, wherein said BGNs are synthesized through micro emulsion assisted sol-gel technique.

8. A single-phase microfluidic device for synthesizing ion doped BGNs as claimed in claim 1, comprising: i) serpentine structures; (1)ii) a plurality of semi-spiral structures; (2) iii) a whole spiral structure; (3) iv) an inlet (4); and v) an outlet (5).

9. The device as claimed in claim 8, wherein said inlet (4) is connected to serpentine structures (1) including 15 rows followed by 5 sets of said semi-spiral structures (2) that is connected to said whole spiral structure (3) and said outlet (5) at the end.

10. The method as claimed in claim 1, wherein said ion doped BGNs are applied in diagnostics, pharmaceuticals, therapeutics, regenerative medicine and dentistry.

11. The method as claimed in claim 1, wherein said ion doped BGNs are toxic free and spherical sphere in shape having a diameter ranging from 200 to 300 nm without any agglomeration.

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