WO2022157678A1 - Method of obtaining surfactin-stabilised poly(d,l-lactide) nanocarriers and nanocarriers produced with this method - Google Patents
Method of obtaining surfactin-stabilised poly(d,l-lactide) nanocarriers and nanocarriers produced with this method Download PDFInfo
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- WO2022157678A1 WO2022157678A1 PCT/IB2022/050506 IB2022050506W WO2022157678A1 WO 2022157678 A1 WO2022157678 A1 WO 2022157678A1 IB 2022050506 W IB2022050506 W IB 2022050506W WO 2022157678 A1 WO2022157678 A1 WO 2022157678A1
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- WIPO (PCT)
- Prior art keywords
- surfactin
- lactide
- carriers
- poly
- aqueous
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- A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/20—Compounding polymers with additives, e.g. colouring
- C08J3/205—Compounding polymers with additives, e.g. colouring in the presence of a continuous liquid phase
- C08J3/21—Compounding polymers with additives, e.g. colouring in the presence of a continuous liquid phase the polymer being premixed with a liquid phase
- C08J3/215—Compounding polymers with additives, e.g. colouring in the presence of a continuous liquid phase the polymer being premixed with a liquid phase at least one additive being also premixed with a liquid phase
Definitions
- the subject of the invention is a method of obtaining surfactin-stabilised poly(D,L-lactidc) nanocarriers and nanocarriers produced with this method.
- the invention constitutes poly(D,L- lactide) nanoparticles stabilised with surfactin, that is an anion surfactant classified into the bio-surfactant group, wherein said nanocarriers are intended for use in cosmetic and pharmaceutical formulations as the hydrophobic active substance carriers.
- compositions can comprise a carrier (e.g. polymer) and surfactant (e.g. surfactin).
- surfactants advantageously improve surface properties by, for example reducing the particle-particle interactions, and can decrease the surface of the particles less adhesive.
- surfactants may also promote solubilization (encapsulation) of a therapeutic or diagnostic substance and increase its bioavailability. This discussed document indicates that, the surfactant may stabilise a given particle, both inside and on its surface.
- the surfactant can be for example introduced to the carriers of controlled or prolonged release, such as polymeric microspheres.
- the literature discloses also the use of surfactin as the emulsion stabiliser.
- the system of drug delivery in the form of surfactin-stabilised nanoemulsion produced from Bacillus subtilis is revealed.
- the disclosed nanoemulsion comprises 50% of surfactin from Bacillus subtilis, 30% Transcutol and 20% of oil phase.
- the composition is intended for encapsulation of active substances contained in the oil phase (such as vitamin C, vitamin E, curcumin).
- the produced carriers demonstrated medium size of particles of 69-183 nm (Lewinska A, Domzal-Kedz.ia M, Jaromin A, Lukasiewicz M.
- polymer micelles constitute an attractive alternative for drug delivery carriers.
- the key feature of the micellar delivery systems which distinguishes them from the other drug carriers in the form of particles is their small size and narrow range of sizes.
- the use of polymer-based micelles drew much attention due to high diversity of polymers, their biocompatibility and presence of many function groups during conjugation.
- the new system of delivery of indomethacin (IND) with the use of PEG- stabilised polymer PDLLA micelles was proposed.
- the authors pointed out that the disclosed mPEGPDLLA micelles can be used as effective carriers of compounds that demonstrate poor solubility, undesired pharmacokinetics and low stability in physiological conditions.
- Hydrophilic coating significantly contributes to preservation of pharmaceutical polymer-based preparation by means of keeping the micelles dispersed as well as reducing the undesired interactions of drugs with cells and proteins (Ouahab A, Shen Y, Tu J. Novel oral delivery system of indomethacin by solidified mPEG-PDLLA micelles: in vivo study. Drug Delivery 2012; 19(4): 232-237).
- insufficient size of produced carriers suggests that they can be too quickly removed from the circulatory system via kidneys in effect of "renal clearance” referred to above.
- the latest reports prove harmful properties of polyethylene glycol (PEG), which stabilises these systems.
- Serious side effects of PEG use include, but are not limited to, undesired immunological response in effect of anti-PEG antibodies, that may result in accelerated blood clearance, low effectiveness of the loaded active substance, hypersensitivity, allergy, and in certain cases other lifethreatening side effects [The Importance of Poly(ethylene glycol) Alternatives for Overcoming PEG Immunogenicity in Drug Delivery and Bioconjugation, Polymers 2020, 12, 298], Therefore seeking the other carriers of relevant sizes and more biocompatible component is highly desired.
- polydispersity (or “dispersion” as recommended by IUPAC) is used to describing the degree of particle size distribution unevenness.
- PDI also referred to as the heterogeneity index, is the number calculated from two-parameter compatibility with correlation data (analysis of cumulants). This index is non-dimensional and scaled in a way that the values below 0.05 are observed mostly in highly mono-dispersive standards. Such mono-dispersive systems do not occur in nature.
- the document IN01641MU2008 A concerning the use of metal (gold and silver) nanoparticles in medicine demonstrates that in order to survive in the in vivo environment it is necessary to design the system which is soluble in water, resistant to aggregation, biocompatible and comprising no toxic chemicals.
- the document presents the new method of preparation and formulation of colloidal metal nanoparticles (in particular gold and silver) with natural rubbers (e.g. Gellan rubber), acting as a reductant and stabiliser.
- the produced nanoparticles are then closed with biosurfactant (sophorolipid), which supports stabilisation, modification of surface, enables binding with active bioparticles and absorption of particles by biological membranes, including by blood-brain barrier.
- Nanoprecipitation during which a polymer is dissolved in an organic solvent mixed with water and upon adding to aqueous surfactant solution, precipitates at the interphase boundary of organic solvent/water. Nanoprecipitation distinguishes from among the other methods of nanocarrier production in that it requires no high energy consumption, high shearing forces and temperatures, rapid mixing, sonification and use of toxic solvents.
- the structures obtained in effect of this approach have adjusted sizes (100-1000 nm), unimodal distribution of carrier sizes (polydispersity index PDI even below 0.2), high degree of hydrophobic bioactive compound encapsulations (>80%) and the desired long-term colloidal stability.
- the precipitated systems are widely used in the industry, among others in the catalysis, diagnostics, household chemistry, agricultural chemistry, but also as the active substance carriers, among others, in the cosmetic, pharmaceutical industries and in agriculture (Mora- Huertas CE, Fessi H, Elaissari A. Polymer-based nanocapsules for drug delivery. Int J Pharmaceut. 385 (2010) 113-142).
- Document W02009137112 describes the method of production of non-immunogenic nanoparticles synthesized from protein compounds used for therapeutic and diagnostic purposes.
- the carriers are obtained with the nanoprecipitation method, during which the protein material is precipitated by a change of pH, which leads to formation of nanoparticles of preferred size range from 100 to 300 nm.
- US patent application no. US 2009/0099282 Al discloses the non-organic composed of SiOi, AI2O3 or mixture of SiCL i AI2O3 of sizes below 400 nm, which have covalently bound different colourants, including of cyanine type.
- the described nanoparticles are used for polymer staining.
- patent application (WO/2010/035118) relates to the systems comprising the polymer nanoparticles (built of polymer or polyelectrolyte) and used for encapsulation of compounds used in agriculture.
- Patent application presents the images taken with the atomic force microscopy (AFM) and with the use of transmission electron microscopy (TEM).
- AFM atomic force microscopy
- TEM transmission electron microscopy
- the aim of the invention was to provide the new polymer drug delivery carriers and the new method of preparation thereof.
- poly(D,L-lactide) nanocarriers also referred to as poly(D,L-lactic) nanocarriers, surf actin- stabilised PDLLA, in a form of sodium salt or carboxylic acid are not known in the art.
- surfactin is able to produce the spherical PDLLA carriers of size range of 125-200 nm in the interphase precipitation process, that are useful for encapsulation of highly hydrophobic biologically active compounds.
- the subject of the invention is a method of obtaining surfactin- stabilised poly(D,L-lactide) carriers characterized in that it comprises the following steps: a) dissolution of poly(D,L-lactide) in organic solvent; b) interphase precipitation by dropwise addition of organic phase obtained in step a) to aqueous surfactin solution within 1-5 minutes with continuous mixing of aqueous phase, wherein surfactin in aqueous solution is surfactin in the form of carboxylic acid or sodium salt; c) mixing until the polymer is precipitated; d) evaporation of organic solvent.
- organic solvent used in step a) is selected from the group comprising acetone, tetrahydrofuran, acetonitrile and ethanol.
- poly(D,L-lactide) and hydrophobic active substance is dissolved in organic solvent in step a).
- dropwise addition of organic phase to surfactin solution is made at mixer speed from 800 to 1200 rpm, preferably above 1000 rpm.
- the ratio of organic phase to aqueous phase is from 1:3 to 1:20,
- the ratio of organic phase to aqueous phase is 1:5,
- aqueous surfactin solution of concentration from 0.1 to 1% is used.
- polymeric surfactin-stabilised poly(D,L-lactide) carriers obtained with the method according to the invention, characterized in that they constitute spherical particles of hydrodynamic diameter of from 125 to 200 nm and polydispersity index PDK0.15.
- the carriers according to the invention comprise hydrophobic active substance.
- Surfactin is a biocompatible and biodegradable cyclical natural lipoheptapeptide of high surface activity produced by Bacillus subtilis. This biosurfactant is a beautiful bioparticle that is able to reduce surface tension of water from 72 to 27 mN / m at concentration as low as 10 mg / 1.
- surfactin in contrary to the commercial surfactants available on the market, demonstrates antibacterial, antifungal, antiviral and even anticancer effect, at the same time supporting the collagen production [ACS'. Shaligram and R.S. Singhal, Surfactin -A review on biosynthesis, fermentation, purification and applications. Food Technol. Biotechnol.
- fig. 1 presents surfactin A) in carboxylic form; B) in sodium form;
- fig. 2 presents the distribution of the systems size according to the invention obtained with the use of dynamic light scattering (DLS) technique for the carriers described in example 3, while fig. 3 presents the morphological image taken with the transmission electron microscopy (TEM) for the carriers described in example 3;
- fig. 4 presents the image of carriers according to the invention with incorporated cumarin (i.e. hydrophobic fluorescent colourant) made with fluorescent microscopy technique;
- fig. 5 presents the microscopic analysis of skin penetration by the carriers according to the invention carried out with the use of fluorescent microscope, where A) shows skin penetration by the carriers according to the invention, while B) shows skin penetration by a control sample.
- the DLS measurements were performed at scattering angle of 173° using the Zetasizer Nano ZS instrument (Malvern Instruments), equipped with helium neon laser (He-Ne) emitting 632.8 nm radiation and the ALV 5000 correlator. Prior to measurement, the sample was thermostated for 3 minutes in temperature of 25 °C, and measured in at least 3 repetitions to calculate the mean to be used as a final result. The results were evaluated with DTS 6.20 (Nano) software. The TEM measurements were performed using the FEI Tecnai G 2 20 X-TWIN microscope with cathode LaBg, CCD FEI Eagle 2K camera, EDS detector and STEM detector. The sample was prepared on carbon and copper meshes. Incubation with carrier suspension was carried out for 24 hours, followed by observation.
- nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering, DLS technique) is 156 nm at polydispersity index of 0.098.
- the size of nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering, DLS technique) is 126 nm at polydispersity index of 0.074. Distribution of sizes of these nanocarriers obtained with DLS technique is presented in fig. 2. Morphology of the obtained particles was analysed using the transmission electron microscopy (TEM). As presented in fig. 3, the spherical carriers are obtained. Cytotoxicity of the produced carriers according to the invention was determined by viability of human keratinocyte cells HACAT using the MTT method.
- the cell culture was grown on the Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% bovine serum and 2 mM glutamine in presence of antibiotics comprising 100 U/mL of penicillin and 100 pg/mL of streptomycin.
- DMEM Dulbecco’s modified Eagle’s medium
- the culture was grown in atmosphere comprising 95% air and 5% CC in temperature of 37 °C.
- Cells were passaged 1-2 times per week. Cells from passages 4 - 10 were used for experiments, wherein cells were seeded in 96-well plates, up to density of 4 x 10 3 cells/well, and incubated for approx. 24 hours. Prepared cultures were treated with the carrier according to the invention in appropriate concentration and incubated for 18-24 hours.
- cell viability was determined by adding 50 pl of MTT solution at concentration of 0.5 mg/ml and incubated in dark in temperature of 37 °C for 4 hours. After this time, the precipitated formazan particles were dissolved by adding 50 pl DMSO, and then the absorbance was measured at wavelength of 570 nm.
- the reference group was the cells not treated with carrier with the determined viability of 100%. The statistical analysis demonstrated no significant decrease in viability of the tested cells, which means that the carrier is not cytotoxic to the tested cells.
- the carrier according to the invention can be safely used in e.g. cosmetic preparations intended for human use.
- the method of obtaining particles according to the invention with incorporated hydrophobic active substance is presented.
- the used hydrophobic active substance is astaxanthin, however the particles according to the invention may be used as carriers for any hydrophobic active substance, both liquid and solid, dissolved in appropriate oil e.g.
- Such properties of carriers according to the invention result from surfactin properties i.e. its specific hydrophilic-hydrophobic structure enabling encapsulation of compounds demonstrating poor or very poor solubility in water, which increases their bioavailability [ Wu YS, Ngai S.C., Goh BH, Chan KG, Lee LH, Chuah LH. Anticancer activities of surfactin and potential application of nanotechnology assisted surfactin delivery. Front. Pharmacol. 8:761. doi: 10.3389/fphar.2017.00761 .
- nano-capsules (expressed as hydrodynamic diameter determined using the dynamic light scattering technique) is 187 nm at polydispersity index of 0.100.
- the devil's claw used in this invention is the extract from Harpagophytum procumbens root called "Devil's Claw", of the following iridoids as the key and active substances: harpagoside, harpagide, procumbide, procumboside, phenols (acetoside, isoacetoside, bioside) and bioflavonoids, primarily luteoline, kaempferol and quercetin.
- the complex of active substances demonstrates numerous properties such as: anti-inflammatory, analgesic, anaesthetic, detoxifying, anti-histamine, regenerative and reducing blood cholesterol level.
- Example 6 5 mg of poly(D,L-lactide) and 0.5 mg/ml of Devil's Claw (hydrophobic active substance being the devil's claw root extract) is dissolved in 1 ml of acetone. Such prepared organic phase is added dropwise within 5 minutes to 5 ml of aqueous surfactin solution (0.25% w/w) in a form of carboxylic acid. Dropwise addition is performed at mixer speed of 1000 rpm. After 5 hours of mixing, the solvent is removed on rotary evaporator at reduced pressure. The size of nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering technique) is 136 nm at polydispersity index of 0.093.
- nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering technique) is 147 nm at polydispersity index of 0.092.
- nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering technique) is 192 nm at polydispersity index of 0.123.
- Example 10 The size of nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering technique) is 192 nm at polydispersity index of 0.123.
- a microscopic analysis showing the efficiency of incorporation of active substances by the carriers according to the invention is performed.
- the surfactin-stabilised poly(D,L-lactide) carriers obtained with the method according to the invention were prepared, in which the hydrophobic active substance was replaced with cumarin (Cumarin 6, Sigma Aldrich), which is a hydrophobic fluorescent colourant known in the art.
- cumarin Cumarin 6, Sigma Aldrich
- the confocal microscope imaging was performed.
- the results presented in fig.4 confirmed effective incorporation of cumarin in the carrier according to the invention.
- the microscopic analysis of skin penetration by the carriers according to the invention The suspension of carriers with incorporated fluorescent colourant (i.e. cumarin) was applied on a pig ear skin section in controlled conditions (Franz chamber). After one hour the carrier suspension was washed out. Skin was solidified and frozen. The frozen sections were cut laterally, applied on a slide and imaged under the fluorescent microscope (fig. 5A). The reference sample is cumarin of the same concentration as in the carrier dispersed in oil phase (fig. 5B). The results confirmed higher effectiveness of penetration by starum corneum of the incorporated cumarin inside the carrier.
- fluorescent colourant i.e. cumarin
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Abstract
The subject of the invention is a method of obtaining surfactin-stabilised poly(D,L-lactide) carriers, characterized in that it comprises following steps: a) dissolution of poly(D,L-lactide) in organic solvent; b) interphase precipitation by dropwise addition of the organic phase obtained in step a) to aqueous surfactin solution within 1-5 minutes with continuous mixing of aqueous phase, wherein surfactin in aqueous solution is surfactin in the form of carboxylic acid or sodium salt; c) mixing until the polymer is precipitated; d) evaporation of organic solvent. Another subject of the invention are surfactin-stabilised poly(D,L-lactide) carriers obtained with the method according to invention, characterized in that they constitute spherical particles of hydrodynamic diameter from 125 to 200 and polydispersity index PDI<0.15.
Description
Method of obtaining surfactin-stabilised poly(D,L-lactide) nanocarriers and nanocarriers produced with this method
The subject of the invention is a method of obtaining surfactin-stabilised poly(D,L-lactidc) nanocarriers and nanocarriers produced with this method. The invention constitutes poly(D,L- lactide) nanoparticles stabilised with surfactin, that is an anion surfactant classified into the bio-surfactant group, wherein said nanocarriers are intended for use in cosmetic and pharmaceutical formulations as the hydrophobic active substance carriers.
The use of surfactin as a surfactant in pharmaceutical compositions is well known in the art. For example, document W02009050726 A2 relates to a pharmaceutical composition of micronized bupropion having controlled particle size in the range of 1-60 pm, wherein median particle size is less than 40 pm. In preferable variant, composition can comprise a carrier (e.g. polymer) and surfactant (e.g. surfactin). The surfactants advantageously improve surface properties by, for example reducing the particle-particle interactions, and can decrease the surface of the particles less adhesive. Surfactants may also promote solubilization (encapsulation) of a therapeutic or diagnostic substance and increase its bioavailability. This discussed document indicates that, the surfactant may stabilise a given particle, both inside and on its surface. The surfactant can be for example introduced to the carriers of controlled or prolonged release, such as polymeric microspheres.
Unfortunately, the literature suggests that the systems in micro size, above 5pm, may block capillaries, therefore it is recommended to produce the delivery systems much below this size [R.H. Muller, C. Jacobs, O. Kayser, Adv Drug Deliv Rev 47 (2001) 3-19}. In addition, it is known that kidneys are able to filtrate particles below 30 nm from blood (so called renal clearance), while the endothelial cells in spleen blood vessels or in bone marrow may demonstrate discontinuities of even up to 100 nm. On the other hand, size of nanocarriers above 200 nm promotes their uptake by hepatic macrophage cells (so called Browicz-Kupffer cells constituting approx. 15-20% of all hepatic cells). It was demonstrated however that the structures below 150 nm are the most effectively internalised by target neoplastic cells. Considering the above limitations, it can be concluded that the most optimum nanocarrier size that would enable penetration into the pathological neoplastic tissues sparing the normal ones and reduce the negative uptake by the macrophage system, ranges between 125-200 nm [C. He, Y. Hu, L. Yin, C. Tang, C. Yin, Biomaterials 31 (2010) 3657-3666; M. Gaumet, A.
Vargas, R. Gurny, F. Delie, Eur J Pharm Biopharm 69 (2008) 1-9; S. Acharya, K. Sahoo, Adv Drug Deliv Rev 68 (2011 ) 70-183].
The literature discloses also the use of surfactin as the emulsion stabiliser. In the publication by Lewinska et al., the system of drug delivery in the form of surfactin-stabilised nanoemulsion produced from Bacillus subtilis is revealed. The disclosed nanoemulsion comprises 50% of surfactin from Bacillus subtilis, 30% Transcutol and 20% of oil phase. The composition is intended for encapsulation of active substances contained in the oil phase (such as vitamin C, vitamin E, curcumin). The produced carriers demonstrated medium size of particles of 69-183 nm (Lewinska A, Domzal-Kedz.ia M, Jaromin A, Lukasiewicz M. Nanoemulsion Stabilized by Safe Surfactin from Bacillus subtilis as a Multifunctional, Custom-Designed Smart Delivery System. Pharmaceutics. 2020 Oct; 12(10): 953. DOI: 10.3390/pharmaceuticsl 2100953 ) .
According to the literature, polymer micelles constitute an attractive alternative for drug delivery carriers. In this context, the key feature of the micellar delivery systems which distinguishes them from the other drug carriers in the form of particles is their small size and narrow range of sizes. The use of polymer-based micelles drew much attention due to high diversity of polymers, their biocompatibility and presence of many function groups during conjugation. In the study by Ouahab et al. the new system of delivery of indomethacin (IND) with the use of PEG- stabilised polymer PDLLA micelles was proposed. The authors pointed out that the disclosed mPEGPDLLA micelles can be used as effective carriers of compounds that demonstrate poor solubility, undesired pharmacokinetics and low stability in physiological conditions. Hydrophilic coating significantly contributes to preservation of pharmaceutical polymer-based preparation by means of keeping the micelles dispersed as well as reducing the undesired interactions of drugs with cells and proteins (Ouahab A, Shen Y, Tu J. Novel oral delivery system of indomethacin by solidified mPEG-PDLLA micelles: in vivo study. Drug Delivery 2012; 19(4): 232-237). However insufficient size of produced carriers (DH<20 nm) suggests that they can be too quickly removed from the circulatory system via kidneys in effect of "renal clearance" referred to above. In addition, the latest reports prove harmful properties of polyethylene glycol (PEG), which stabilises these systems. Serious side effects of PEG use include, but are not limited to, undesired immunological response in effect of anti-PEG antibodies, that may result in accelerated blood clearance, low effectiveness of the loaded active substance, hypersensitivity, allergy, and in certain cases other lifethreatening side effects [The Importance of Poly(ethylene glycol) Alternatives for Overcoming
PEG Immunogenicity in Drug Delivery and Bioconjugation, Polymers 2020, 12, 298], Therefore seeking the other carriers of relevant sizes and more biocompatible component is highly desired.
Apart from the size and biocompatibility, low polydispersity of the produced carriers is also an aspect of high importance. The term "polydispersity" (or "dispersion" as recommended by IUPAC) is used to describing the degree of particle size distribution unevenness. PDI, also referred to as the heterogeneity index, is the number calculated from two-parameter compatibility with correlation data (analysis of cumulants). This index is non-dimensional and scaled in a way that the values below 0.05 are observed mostly in highly mono-dispersive standards. Such mono-dispersive systems do not occur in nature. Therefore carriers of active substances of the PDI ranges <0.2 are traditionally determined as carriers of good quality, while these of PDI < 0.15 belong to the most desired ones in the application studies, since these parameters prove their long stability [Pharmaceutics 2018, 70(2), 57],
The document IN01641MU2008 A concerning the use of metal (gold and silver) nanoparticles in medicine demonstrates that in order to survive in the in vivo environment it is necessary to design the system which is soluble in water, resistant to aggregation, biocompatible and comprising no toxic chemicals. The document presents the new method of preparation and formulation of colloidal metal nanoparticles (in particular gold and silver) with natural rubbers (e.g. Gellan rubber), acting as a reductant and stabiliser. The produced nanoparticles are then closed with biosurfactant (sophorolipid), which supports stabilisation, modification of surface, enables binding with active bioparticles and absorption of particles by biological membranes, including by blood-brain barrier.
The scientific and patent literature describes the methods of production and use of different types of polymer carriers designed of among others biodegradable polymers, polyelectrolytes and co-polymers. One of the most preferred methods of their production is nanoprecipitation, during which a polymer is dissolved in an organic solvent mixed with water and upon adding to aqueous surfactant solution, precipitates at the interphase boundary of organic solvent/water. Nanoprecipitation distinguishes from among the other methods of nanocarrier production in that it requires no high energy consumption, high shearing forces and temperatures, rapid mixing, sonification and use of toxic solvents. The structures obtained in effect of this approach have adjusted sizes (100-1000 nm), unimodal distribution of carrier sizes (polydispersity index PDI even below 0.2), high degree of hydrophobic bioactive
compound encapsulations (>80%) and the desired long-term colloidal stability. This is why the precipitated systems are widely used in the industry, among others in the catalysis, diagnostics, household chemistry, agricultural chemistry, but also as the active substance carriers, among others, in the cosmetic, pharmaceutical industries and in agriculture (Mora- Huertas CE, Fessi H, Elaissari A. Polymer-based nanocapsules for drug delivery. Int J Pharmaceut. 385 (2010) 113-142). Unfortunately, vast majority of the performed trials concerns the polymer nanocarriers which are stabilised with non-ionic surfactants (primarily from the polyoxyethylene group) of relatively low biocompatibility and biodegradation. Study by Bazylinska et al. (Colloid Surf. A: Physicochem. Eng. Aspects 442 (2014) 42- 49), describes a synthesis of precipitated carriers composed of poly(D,L-lactide), PDLLA or polyprolactone, PCL, stabilised with polyethoxylated castor oil (Cremophor EL) intended for encapsulation of hydrophobic dyes from indocyanine group. The study by Zila et al. (Int. J. Pharm. 294 (2005) 261-267) discloses the method of poly(s-caprolactone) nanoparticles production with the use of polyoxyethylene-stabilised nanoprecipitation approach (Tween 80 and Span 80), which comprise griseofulvin. In addition, the study by Ricci-Junior et al. (Int. J. Pharm. 310 (2006) 187-195) describes the use of lactide and glycolide co-polymer nanoparticles for encapsulation of phthalocyanine comprising zinc in its structure, additionally stabilised with polyvinyl alcohol (PVA).
The description of international patent application W02006052285 discloses the other polymer nanoparticles synthesized of acrylic acid or acrylamides, which can be used for encapsulation, extraction and release of various bioactive components (e.g. drugs, antioxidants). The nano-particles are obtained using the reverse microemulsion method, which allows for obtaining of spherical particles of polyacrylic acid or polyacrylamide of size range from 50 to 80 nm. The other example of nanoparticles that can be used as drug carriers is disclosed in the patent description no. US7867984, which discloses the bioactive nanoparticles composed of chitosan or poly(glutamic acid) and comprising of cation appetitesuppressing agent. The particles can be used for oral drug administration. The other particles useful in obtaining the vaccine carriers are disclosed in document W02008115641, which discloses the compositions comprising, among others, the polymer nanoparticles, antigens, substances affecting the immunological response of the organism.
Document W02009137112 describes the method of production of non-immunogenic nanoparticles synthesized from protein compounds used for therapeutic and diagnostic purposes. The carriers are obtained with the nanoprecipitation method, during which the protein
material is precipitated by a change of pH, which leads to formation of nanoparticles of preferred size range from 100 to 300 nm. The description of US patent application no. US 2009/0099282 Al discloses the non-organic composed of SiOi, AI2O3 or mixture of SiCL i AI2O3 of sizes below 400 nm, which have covalently bound different colourants, including of cyanine type. The described nanoparticles are used for polymer staining. On the other hand, the patent application (WO/2010/035118) relates to the systems comprising the polymer nanoparticles (built of polymer or polyelectrolyte) and used for encapsulation of compounds used in agriculture. Patent application presents the images taken with the atomic force microscopy (AFM) and with the use of transmission electron microscopy (TEM).
The aim of the invention was to provide the new polymer drug delivery carriers and the new method of preparation thereof.
To this date, the methods of obtaining the polymer poly(D,L-lactide) nanocarriers, also referred to as poly(D,L-lactic) nanocarriers, surf actin- stabilised PDLLA, in a form of sodium salt or carboxylic acid are not known in the art.
Unexpectedly, the authors of the invention discovered that surfactin is able to produce the spherical PDLLA carriers of size range of 125-200 nm in the interphase precipitation process, that are useful for encapsulation of highly hydrophobic biologically active compounds.
The subject of the invention is a method of obtaining surfactin- stabilised poly(D,L-lactide) carriers characterized in that it comprises the following steps: a) dissolution of poly(D,L-lactide) in organic solvent; b) interphase precipitation by dropwise addition of organic phase obtained in step a) to aqueous surfactin solution within 1-5 minutes with continuous mixing of aqueous phase, wherein surfactin in aqueous solution is surfactin in the form of carboxylic acid or sodium salt; c) mixing until the polymer is precipitated; d) evaporation of organic solvent.
Preferably, organic solvent used in step a) is selected from the group comprising acetone, tetrahydrofuran, acetonitrile and ethanol.
Preferably, poly(D,L-lactide) and hydrophobic active substance is dissolved in organic solvent in step a).
Preferably, dropwise addition of organic phase to surfactin solution is made at mixer speed from 800 to 1200 rpm, preferably above 1000 rpm.
Preferably, the ratio of organic phase to aqueous phase is from 1:3 to 1:20,
Preferably, the ratio of organic phase to aqueous phase is 1:5,
Preferably, aqueous surfactin solution of concentration from 0.1 to 1% is used.
Another subject of the invention are polymeric surfactin-stabilised poly(D,L-lactide) carriers obtained with the method according to the invention, characterized in that they constitute spherical particles of hydrodynamic diameter of from 125 to 200 nm and polydispersity index PDK0.15.
Preferably, the carriers according to the invention comprise hydrophobic active substance.
Surfactin is a biocompatible and biodegradable cyclical natural lipoheptapeptide of high surface activity produced by Bacillus subtilis. This biosurfactant is a fascinating bioparticle that is able to reduce surface tension of water from 72 to 27 mN / m at concentration as low as 10 mg / 1. In addition, surfactin, in contrary to the commercial surfactants available on the market, demonstrates antibacterial, antifungal, antiviral and even anticancer effect, at the same time supporting the collagen production [ACS'. Shaligram and R.S. Singhal, Surfactin -A review on biosynthesis, fermentation, purification and applications. Food Technol. Biotechnol. 48 (2010) 119-134 ;Vollenbrich D, Ozel M, Vater J, Kamp RM, Pauli G. Mechanism of Inactivation of Enveloped Viruses by the Biosurfactant Surfactin from Bacillus subtilis. Biologicals, 1997; 25: 289-297; Meena KR, Kanwar SS. Lipopeptides as the Antifungal and Antibacterial Agents: Applications in Food Safety and Therapeutics. BioMed Research International 2015; Doi: 10.1155/2015/473050; Vollenbroich D, Pauli G, Ozel M, Vater J. Antimycoplasma Properties and Application in Cell Culture of Surfactin, a Lipopeptide Antibiotic from Bacillus subtilis. Appl Environ Microbiol 1997; 63(1): 44-49; Desmyttere H, Deweer C, Muchembled J, Sahmer K, Jacquin J, Coutte F, Jacquest P. Antifungal activities of Bacillus subtilis lipopeptides to two Venturia inaequalis strains possessing different tebuconazole sensitivity. Front. Microbiol. 10:2327. doi: 10.3389/fmicb.2019.02327]. This unique feature, combined with excellent emulsifying properties and stabilisation capacity, makes surfactin a biocompatible and unique stabiliser for the new active substance carriers.
Main advantages coming from the invention lie in the possibility of obtaining of polymer- based, biodegradable and biocompatible carriers of size range of 125-200 nm and spherical shape comprising up to 0.5 mg of encapsulated active substance. The obtained carriers demonstrate PDI <0.15, which demonstrates their long stability. The size of obtained carriers ranges within 125-200 nm, which is the size enabling penetration into target tissues with simultaneous reduction of negative uptake of carrier by the macrophage system. Nanosystems made of surfactin- stabilised PDLLA according to the invention, can be for example used in cosmetic industry as the biocompatible and biodegradable carriers transferring bioactive substances.
The object of the invention is presented in the following embodiments of new polymer nanocarriers comprising the hydrophobic bioactive substance and in the drawing, in which fig. 1 presents surfactin A) in carboxylic form; B) in sodium form; fig. 2 presents the distribution of the systems size according to the invention obtained with the use of dynamic light scattering (DLS) technique for the carriers described in example 3, while fig. 3 presents the morphological image taken with the transmission electron microscopy (TEM) for the carriers described in example 3; fig. 4 presents the image of carriers according to the invention with incorporated cumarin (i.e. hydrophobic fluorescent colourant) made with fluorescent microscopy technique; fig. 5 presents the microscopic analysis of skin penetration by the carriers according to the invention carried out with the use of fluorescent microscope, where A) shows skin penetration by the carriers according to the invention, while B) shows skin penetration by a control sample.
Please note that, if not clearly stated otherwise, all experimental tests and procedures described below were performed with the use of commercially available kits, reagents and instruments, following the recommendations of their used. All test parameters were measured with the use of standard and well-established methods known in the field of the invention.
The DLS measurements were performed at scattering angle of 173° using the Zetasizer Nano ZS instrument (Malvern Instruments), equipped with helium neon laser (He-Ne) emitting 632.8 nm radiation and the ALV 5000 correlator. Prior to measurement, the sample was thermostated for 3 minutes in temperature of 25 °C, and measured in at least 3 repetitions to calculate the mean to be used as a final result. The results were evaluated with DTS 6.20 (Nano) software.
The TEM measurements were performed using the FEI Tecnai G220 X-TWIN microscope with cathode LaBg, CCD FEI Eagle 2K camera, EDS detector and STEM detector. The sample was prepared on carbon and copper meshes. Incubation with carrier suspension was carried out for 24 hours, followed by observation.
Example 1.
5 mg of poly(D,L-lactide) is dissolved in 1 ml of acetone. Such prepared organic phase is added dropwise within 5 minutes to 5 ml of aqueous surfactin solution (1% w/w) in a form of sodium salt (fig. 1). Dropwise addition is performed at mixer speed of 1000 rpm. After 5 hours of mixing, the organic solvent is removed on rotary evaporator at reduced pressure.
The size of nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering, DLS technique) is 156 nm at polydispersity index of 0.098.
Example 2.
5 mg of poly(D,L-lactide) is dissolved in 1 ml of acetone. Such prepared organic phase is added dropwise within 5 minutes to 5 ml of aqueous surfactin solution (0.5% w/w) in a form of sodium salt. Dropwise addition is performed at mixer speed of 1000 rpm. After 5 hours of mixing, the organic solvent is removed on rotary evaporator at reduced pressure. The size of nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering, DLS technique) is 139 nm at polydispersity index of 0.085.
Example 3.
5 mg of tetrahydrofuran is dissolved in 1 ml of acetone. Such prepared organic phase is added dropwise within 5 minutes to 5 ml of aqueous surfactin solution (0.25% w/w) in a form of sodium salt. Dropwise addition is performed at mixer speed of 1000 rpm.
After 5 hours of mixing, the solvent is removed on rotary evaporator at reduced pressure. The size of nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering, DLS technique) is 126 nm at polydispersity index of 0.074. Distribution of sizes of these nanocarriers obtained with DLS technique is presented in fig. 2. Morphology of the obtained particles was analysed using the transmission electron microscopy (TEM). As presented in fig. 3, the spherical carriers are obtained.
Cytotoxicity of the produced carriers according to the invention was determined by viability of human keratinocyte cells HACAT using the MTT method. The cell culture was grown on the Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% bovine serum and 2 mM glutamine in presence of antibiotics comprising 100 U/mL of penicillin and 100 pg/mL of streptomycin. The culture was grown in atmosphere comprising 95% air and 5% CC in temperature of 37 °C. Cells were passaged 1-2 times per week. Cells from passages 4 - 10 were used for experiments, wherein cells were seeded in 96-well plates, up to density of 4 x 103 cells/well, and incubated for approx. 24 hours. Prepared cultures were treated with the carrier according to the invention in appropriate concentration and incubated for 18-24 hours. Next, cell viability was determined by adding 50 pl of MTT solution at concentration of 0.5 mg/ml and incubated in dark in temperature of 37 °C for 4 hours. After this time, the precipitated formazan particles were dissolved by adding 50 pl DMSO, and then the absorbance was measured at wavelength of 570 nm. The reference group was the cells not treated with carrier with the determined viability of 100%. The statistical analysis demonstrated no significant decrease in viability of the tested cells, which means that the carrier is not cytotoxic to the tested cells. Thus, the carrier according to the invention can be safely used in e.g. cosmetic preparations intended for human use.
Example 4.
In this non-limiting embodiment, the method of obtaining particles according to the invention with incorporated hydrophobic active substance is presented. The used hydrophobic active substance is astaxanthin, however the particles according to the invention may be used as carriers for any hydrophobic active substance, both liquid and solid, dissolved in appropriate oil e.g. arbutin, devil's claw, allantoin, astaxanthin, feluric acid, raspberry ketone glucoside, lypoglicyne, vitamin A, vitamin D, vitamin E, vitamin K, avocado oil, buriti seed oil, bakuchiol, vitamin C (hydrophobic form), ethyl esters of fatty acids, tsubaki oil, muru muru butter, argan oil, sandthom oil, cinnamon oil, tea tree oil, buckwheat seed oil, green tea seed oil, apple seed oil, strawberry seed oil, blue tansy oil, canopy oil, cananga tree oil, vervain oil, camelina oil, pomegranate oil, passiflora oil, sesame oil, rape oil, apricot seed oil, tamanu oil, Chaulmoogra oil, carrot seed oil, cactus pear oil, olive oil, meadowfoam seed oil, plant olein, lipoglycin, lidolcaine, curcumin, vinblastine and its derivatives, corticosteroids, naproxen, argining, diclofenac, quinine, chloroquine, fendrimetrasine, ibuprofen, carbamazepine, thiopental and zoplicone.
Such properties of carriers according to the invention result from surfactin properties i.e. its specific hydrophilic-hydrophobic structure enabling encapsulation of compounds demonstrating poor or very poor solubility in water, which increases their bioavailability [ Wu YS, Ngai S.C., Goh BH, Chan KG, Lee LH, Chuah LH. Anticancer activities of surfactin and potential application of nanotechnology assisted surfactin delivery. Front. Pharmacol. 8:761. doi: 10.3389/fphar.2017.00761 .
In this example, 5 mg of poly(D,L-lactide) and 0.5 mg/ml of astaxanthin (hydrophobic active substance) were dissolved in 1 ml of acetone. Such prepared organic phase is added dropwise during 5 minutes to 5 ml of aqueous surfactin solution (0.25% w/w) in a form of sodium salt. Dropwise addition is performed at mixer speed of 1000 rpm.
After 5 hours of mixing, the solvent is removed on rotary evaporator at reduced pressure. The size of nano-capsules (expressed as hydrodynamic diameter determined using the dynamic light scattering technique) is 187 nm at polydispersity index of 0.100.
Example 5.
5 mg of poly(D,L-lactide) and 0.5 mg/ml of devil's claw (hydrophobic active substance) is dissolved in 1 ml of acetone. Such prepared organic phase is added dropwise within 5 minutes to 5 ml of aqueous surfactin solution (0.25% w/w) in a form of sodium salt. Dropwise addition is performed at mixer speed of 1000 rpm. After 5 hours of mixing, the solvent is removed on rotary evaporator at reduced pressure. The size of nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering technique) is 138 nm at polydispersity index of 0.015.
Provided that the devil's claw used in this invention is the extract from Harpagophytum procumbens root called "Devil's Claw", of the following iridoids as the key and active substances: harpagoside, harpagide, procumbide, procumboside, phenols (acetoside, isoacetoside, bioside) and bioflavonoids, primarily luteoline, kaempferol and quercetin. The complex of active substances demonstrates numerous properties such as: anti-inflammatory, analgesic, anaesthetic, detoxifying, anti-histamine, regenerative and reducing blood cholesterol level.
Example 6.
5 mg of poly(D,L-lactide) and 0.5 mg/ml of Devil's Claw (hydrophobic active substance being the devil's claw root extract) is dissolved in 1 ml of acetone. Such prepared organic phase is added dropwise within 5 minutes to 5 ml of aqueous surfactin solution (0.25% w/w) in a form of carboxylic acid. Dropwise addition is performed at mixer speed of 1000 rpm. After 5 hours of mixing, the solvent is removed on rotary evaporator at reduced pressure. The size of nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering technique) is 136 nm at polydispersity index of 0.093.
Example 7.
5 mg of poly(D,L-lactide) and 0.5 mg/ml of astaxanthin (hydrophobic active substance) is dissolved in 1 ml of acetone. Such prepared organic phase is added dropwise within 5 minutes to 5 ml of aqueous surfactin solution (0.25% w/w) in a form of carboxylic acid. Dropwise addition is performed at mixer speed of 1000 rpm. After 5 hours of mixing, the solvent is removed on rotary evaporator at reduced pressure. The size of nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering technique) is 139 nm at polydispersity index of 0.115.
Example 8.
5 mg of poly(D,L-lactide) and 0.5 mg/ml of Devil's Claw (hydrophobic active substance being the devil's claw root extract) is dissolved in 1 ml of acetone. Such prepared organic phase is added dropwise within 5 minutes to 5 ml of aqueous surfactin solution (0.5% w/w) in a form of carboxylic acid. Dropwise addition is performed at mixer speed of 1000 rpm. After 5 hours of mixing, the solvent is removed on rotary evaporator at reduced pressure.
The size of nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering technique) is 147 nm at polydispersity index of 0.092.
Example 9.
5 mg of poly(D,L-lactide) is dissolved in 1 ml of ethanol. Such prepared organic phase is added dropwise within 1 minute to 5 ml of aqueous surfactin solution (0.1% w/w) in a form of sodium salt. Dropwise addition is performed at mixer speed of 1200 rpm. After 5 hours of mixing, the solvent is removed on rotary evaporator at reduced pressure.
The size of nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering technique) is 192 nm at polydispersity index of 0.123.
Example 10.
1 mg of poly(D,L-lactide) is dissolved in 5 ml of acetonitrile. Such prepared organic phase is added dropwise within 3 minutes to 5 ml of aqueous surfactin solution (0.1% w/w) in a form of sodium salt. Dropwise addition is performed at mixer speed of 800 rpm. After 5 hours of mixing, the solvent is removed on rotary evaporator at reduced pressure. The size of nanocarriers (expressed as hydrodynamic diameter determined using the dynamic light scattering technique) is 200 nm at polydispersity index of 0.148.
Example 11.
A microscopic analysis showing the efficiency of incorporation of active substances by the carriers according to the invention is performed. The surfactin-stabilised poly(D,L-lactide) carriers obtained with the method according to the invention were prepared, in which the hydrophobic active substance was replaced with cumarin (Cumarin 6, Sigma Aldrich), which is a hydrophobic fluorescent colourant known in the art. Next, the confocal microscope imaging was performed. The results presented in fig.4 confirmed effective incorporation of cumarin in the carrier according to the invention.
Example 12.
The microscopic analysis of skin penetration by the carriers according to the invention. The suspension of carriers with incorporated fluorescent colourant (i.e. cumarin) was applied on a pig ear skin section in controlled conditions (Franz chamber). After one hour the carrier suspension was washed out. Skin was solidified and frozen. The frozen sections were cut laterally, applied on a slide and imaged under the fluorescent microscope (fig. 5A). The reference sample is cumarin of the same concentration as in the carrier dispersed in oil phase (fig. 5B). The results confirmed higher effectiveness of penetration by starum corneum of the incorporated cumarin inside the carrier.
Claims
Claims A method of obtaining surfactin-stabilised poly(D.L-lactidc) carriers, characterized in that it comprises following steps: a) dissolution of poly(D,L-lactide) in organic solvent; b) interphase precipitation by dropwise addition of organic phase obtained in step a) to aqueous surfactin solution within 1-5 minutes with continuous mixing of aqueous phase, wherein surfactin in aqueous solution is surfactin in the form of carboxylic acid or sodium salt; c) mixing until the polymer is precipitated; d) evaporation of organic solvent. The method according to claim 1, characterized in that the organic solvent used in step a) is selected from the group comprising acetone, tetrahydrofuran, acetonitrile and ethanol. The method according to claim 1 or 2, characterized in that poly(D,L-lactide) and hydrophobic active substance is dissolved in organic solvent in step a). The method according to any of the previous claims from 1 to 3, characterized in that the dropwise addition of organic phase to surfactin solution is made at mixer speed from 800 to 1200 rpm, preferably above 1000 rpm. The method according to any of the previous claims from 1 to 4, characterized in that the ratio of organic phase to aqueous phase is from 1:3 to 1:20, The method according to claim 5, characterized in that the ratio of organic phase to aqueous phase is 1:5. The method according to any of the previous claims from 1 to 6, characterized in that the aqueous solution of surfactin of concentration from 0.1 to 1% is used. Surfactin-stabilised poly(D,L-lactide) carriers obtained with the method according to any of the previous claims from 1 to 7, characterized in that they constitute spherical particles of hydrodynamic diameter from 125 to 200 and polydispersity index PDK0.15. Polymeric carriers according to claim 8, characterized in that they comprise a hydrophobic active substance.
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Non-Patent Citations (4)
Title |
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BAZYLINSKA URSZULA ET AL: "Polymeric nanocapsules and nanospheres for encapsulation and long sustained release of hydrophobic cyanine-type photosensitizer", COLLOIDS AND SURFACES A: PHYSIOCHEMICAL AND ENGINEERING ASPECTS, ELSEVIER, AMSTERDAM, NL, vol. 442, 1 March 2013 (2013-03-01), pages 42 - 49, XP028828470, ISSN: 0927-7757, DOI: 10.1016/J.COLSURFA.2013.02.023 * |
DROZDEK SLAWOMIR ET AL: "Biocompatible oil core nanocapsules as potential co-carriers of paclitaxel and fluorescent markers: preparation, characterization, and bioimaging", COLLOID & POLYMER SCIENCE, SPRINGER VERLAG, HEIDELBERG, DE, vol. 294, no. 1, 18 September 2015 (2015-09-18), pages 225 - 237, XP035878383, ISSN: 0303-402X, [retrieved on 20150918], DOI: 10.1007/S00396-015-3767-5 * |
HAZRA CHINMAY ET AL: "Poly(methyl methacrylate) (core)-biosurfactant (shell) nanoparticles: Size controlled sub-100nm synthesis, characterization, antibacterial activity, cytotoxicity and sustained drug release beha", COLLOIDS AND SURFACES A: PHYSIOCHEMICAL AND ENGINEERING ASPECTS, ELSEVIER, AMSTERDAM, NL, vol. 449, 1 March 2014 (2014-03-01), pages 96 - 113, XP028833758, ISSN: 0927-7757, DOI: 10.1016/J.COLSURFA.2014.02.051 * |
WU YUAN-SENG ET AL: "Anticancer Activities of Surfactin and Potential Application of Nanotechnology Assisted Surfactin Delivery", vol. 8, 26 October 2017 (2017-10-26), pages 1 - 22, XP009535367, ISSN: 1663-9812, Retrieved from the Internet <URL:http://journal.frontiersin.org/article/10.3389/fphar.2017.00761/full> [retrieved on 20171026], DOI: 10.3389/FPHAR.2017.00761 * |
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