WO2023248246A1 - Compositions of calcium phosphate nano-particles with sophorolipids and process for preparation thereof - Google Patents

Compositions of calcium phosphate nano-particles with sophorolipids and process for preparation thereof Download PDF

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WO2023248246A1
WO2023248246A1 PCT/IN2023/050593 IN2023050593W WO2023248246A1 WO 2023248246 A1 WO2023248246 A1 WO 2023248246A1 IN 2023050593 W IN2023050593 W IN 2023050593W WO 2023248246 A1 WO2023248246 A1 WO 2023248246A1
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calcium phosphate
particles
sophorolipid
nano
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French (fr)
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Isha Hemant ABHYANKAR
Anuya Amol NISAL
Asmita Ashutosh Prabhune
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Council Of Scientific And Industrial Research
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    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B25/00—Phosphorus; Compounds thereof
    • C01B25/16—Oxyacids of phosphorus; Salts thereof
    • C01B25/26—Phosphates
    • C01B25/32—Phosphates of magnesium, calcium, strontium, or barium
    • C01B25/321—Methods for converting an alkaline earth metal ortho-phosphate into another ortho-phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/28—Bones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/30—Joints
    • A61F2/46—Special tools or methods for implanting or extracting artificial joints, accessories, bone grafts or substitutes, or particular adaptations therefor
    • A61F2/4644—Preparation of bone graft, bone plugs or bone dowels, e.g. grinding or milling bone material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/28—Bones
    • A61F2002/2835—Bone graft implants for filling a bony defect or an endoprosthesis cavity, e.g. by synthetic material or biological material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/30—Joints
    • A61F2/46—Special tools or methods for implanting or extracting artificial joints, accessories, bone grafts or substitutes, or particular adaptations therefor
    • A61F2/4644—Preparation of bone graft, bone plugs or bone dowels, e.g. grinding or milling bone material
    • A61F2002/4648—Means for culturing bone graft
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/60—Particles characterised by their size
    • C01P2004/64—Nanometer sized, i.e. from 1-100 nanometer

Definitions

  • the present invention relates to novel compositions of calcium phosphate nano-particles with sophorolipids. More particularly, the present invention discloses a composition of calcium phosphate nano-particles with higher content of tri calcium phosphate. Further, the present invention discloses a process of synthesis of composition of calcium phosphate nano-particles with sophorolipids.
  • Calcium phosphate compounds are highly desirable materials due to their varied applications. They play an important role in the biomedical sector due to their resemblance to natural bone. These compounds are available in many forms such as powder, sponge or putty. Chemically, these compounds can exist as mono calcium phosphate, di calcium phosphate (DCP), tri calcium phosphate (TCP), octa calcium phosphate (OCP) and hydroxyapatite (HA). HA is the main inorganic component of the bone.
  • DCP di calcium phosphate
  • TCP tri calcium phosphate
  • OCP octa calcium phosphate
  • HA hydroxyapatite
  • TCP tri calcium and di calcium phosphates.
  • DCP in monetite or brushite forms.
  • they are used as non- viral vectors in drug delivery, gene silencing and cancer therapy.
  • the preferred form of these materials in biomedical applications is a powder of nano-particles.
  • Synthesis of calcium phosphate nano-particles includes different physical, chemical and biological methods. Chemical processes for synthesis of calcium phosphate nano-particles typically require longer hours of synthesis. This hampers their large-scale production. Also, the properties of calcium phosphates are largely dependent on its composition and morphology. Therefore, tuning the right composition along with size will aid in enhanced properties, especially for biomedical applications. With this view, newer processes are now being explored so that compositions of calcium phosphate with distinct and advantageous properties within short time may be evolved. Calcium phosphate compounds as bone graft substitutes in dental and orthopedic fields have gained tremendous interest over the time.
  • HA hydroxyapatite
  • Tri calcium phosphate commonly known as TCP has gained interest owing to its good biological properties. As reported by Kucharska et al., the presence of TCP within the material enhances its biological and mechanical properties, and this is relatable to increasing concentration of TCP. [Kucharska, M.; Walenko, K.; Lewandowska-Szumiel, M.; Brynk, T.; Jaroszewicz, J.; Ciach, T. Chitosan and Composite Microsphere-Based Scaffold for Bone Tissue Engineering: Evaluation of Tricalcium Phosphate Content Influence on Physical and Biological Properties. J. Mater. Set. Mater. Med. 2015, 26 (3)].
  • Sophorolipids are a group of extracellular biosurfactants produced at relatively high yields by several non-pathogenic yeast species. Sophorolipid comprise a residue of sophorose, the disaccharide consisting of two glucose residues linked by the P-1,2' bond, and fatty acid as an aglycone (Extracellular Glycolipids of Yeats - Biodiversity , Biochemistry and Prospects 2014 pages 35-64 Chapter 4).
  • Literature discloses sophorolipids as FDA approved molecules for human consumption. Up till now sophorolipids have been used for synthesizing metallic nano-particles but the same has not been studied with calcium phosphate nano-particles.
  • the inventors of the present invention have synthesized calcium phosphate nanoparticles by using a biosurfactant.
  • the nano-particles thus formed by the efforts of the inventors of the present invention are a mixture of brushite (di calcium phosphate dihydrate) and have higher content of TCP (tri calcium phosphate) along with a sophorolipid.
  • This composition of nano-particles is more desirable as, they are biocompatible, bioresorbable and are osteoconductive and osteoinductive.
  • the process followed for synthesizing or preparing these calcium phosphate nano-particles with biosurfactants is less time consuming and economical. It requires minimum downstream processing to obtain nano-particles with specific chemical composition and appropriate physical and biological properties.
  • the present invention relates to a composition of biocompatible, bioresorbable and osteoconductive and osteoinductive calcium phosphate nano-particles comprising of a mixture of brushite (di calcium phosphate dihydrate) and a higher content of TCP (tri calcium phosphate) along with a sophorolipid.
  • sophorolipid based calcium phosphate nano-particles composition is disclosed.
  • a composition comprising: i. a sophorolipid with an amount ranging between 20-30% by weight; and ii. a calcium phosphate nano-particles with an amount ranging between 70- 80% by weight; wherein the calcium phosphate nano-particles are comprised of di-calcium phosphate dehydrate in an amount ranging from 45-60 % by weight of the total composition, and tricalcium phosphate in an amount ranging from 10-25 % by weight of the total composition.
  • the sophorolipid is synthesized using saturated or unsaturated fatty acids.
  • the sophorolipid is synthesized using different fatty acids selected from myristic acid, oleic acid, lauric acid, palmitic acid, stearic acid, linolenic acid or combinations thereof. In some embodiments, the sophorolipid is synthesized using myristic acid or oleic acid.
  • a process for the preparation of calcium phosphate nanoparticles composition comprises the following steps: a) mixing a sophorolipid in 0.01-1 M (preferably 0.1 M) of Na2HPO4 to obtain mixture A.
  • step b) sonicating the mixture A for time period of 5-20 minutes at power of 40-60 Hz with a 1-30 seconds (preferably 10 seconds) pulse having a 1-5 seconds (preferably 3 seconds) interval; c) mixing 0.01-1 M (preferably 0.1 M) of CaCh solution with the mixture A of step b) to obtain a mixture B; d) sonicating the mixture B for a time period of 10-30 minutes at 30-80 Hz for with a 1-30 seconds (preferably 10 seconds) pulse having a 1-5 seconds (preferably 3 seconds) interval to obtain a solution containing calcium phosphate nano-particles; e) centrifuging the solution of step d) at a speed in the range of 7900-8100 rpm for time period of 10-20 minutes followed by washing with water as a solvent to obtain a wet mass; and f) drying the wet mass of step e) in an oven at temperature in a range of 50-100 °C for a time period of 8-20 hours to obtain the desired composition.
  • the sophorolipid may be myristic acid-derived sophorolipid (MASL) or oleic acid-derived sophorolipid.
  • MSL myristic acid-derived sophorolipid
  • oleic acid-derived sophorolipid oleic acid-derived sophorolipid
  • the calcium phosphate nanoparticles composition was dried in oven at a temperature of 40-80 °C for 6-24 hours.
  • the calcium phosphate nanoparticles composition was dried in the oven at 60 °C for 10 hours.
  • the sonication is done for time period of 5-20 minutes wherein in that time period, repeating cycles of 10 seconds sonication/pulse followed by 3 second break.
  • a scaffold comprises the following: i. a polymer; ii. the calcium phosphate nanoparticles composition as described hereinabove.
  • the polymer is naturally occurring polymer or a synthetic polymer.
  • the naturally occurring polymers is selected from collagen, silk, chitosan, alginate, gelatin and combinations thereof and synthetic polymers is selected from polylactic acid, polyethlene oxide, polyglycolic acid, polyvinyl acetate and combinations thereof.
  • Fig 1 illustrates FTIR of short chain derived sophorolipids.
  • Fig 2 illustrates HPLC of synthesized sophorolipids.
  • Fig 3 illustrates XRD of nano-particles synthesized without using sophorolipids (CNP).
  • Fig 4 illustrates XRD of nano-particles synthesized using sophorolipids (SL-NP).
  • Fig 5 illustrates XRD of TCP composition of nano-particles (control (CNP) and sophorolipids nanoparticles (SL-NP).
  • Fig 8 illustrates SEM of sophorolipid nano-particles (SL-NP).
  • Fig 9a illustrates a graph showing different concentrations of sophorolipids (MASL).
  • Fig 9b illustrates a graph showing different concentration of sophorolipids (OASL).
  • Fig 10a illustrates a graph of the in vitro studies (proliferation assay).
  • Fig 10b illustrates a graph of the in vitro studies (ALP estimation).
  • Source of S. bombicola and silk fibroin the yeast culture used for fermentation (Starmerella bombicola, formerly known as Candida bombicola) was procured from ATCC (ATCC 22214). Bombyx mori cocoons were procured from CSRTI - Central Sericultural Research & Training Institute, Mysore, India)
  • the present invention relates to calcium phosphate nano-particles synthesized by using a biosurfactant.
  • the nano-particles, thus, formed are a mixture of brushite (di calcium phosphate dihydrate) and TCP (tri calcium phosphate), with a higher content of TCP.
  • the present invention relates to a composition comprising calcium phosphate nano-particles synthesized by using a biosurfactant.
  • This composition of calcium phosphate nano-particles is more desirable as they are biocompatible, bioresorbable, osteoconductive and osteoinductive.
  • the biosurfactant used in the present invention is sophorolipid.
  • Control nano-particles means nanoparticles synthesized without incorporation of sophorolipids (Abbreviated as CNP) and nano- particles with incorporation of sophorolipids are abbreviated as SL-NP.
  • nano-particles has been used interchangeably for calcium phosphate nano-particles and refer to calcium phosphate nano-particles unless specified otherwise.
  • the present invention provides a novel composition of calcium phosphate nano-particles comprising brushite (di calcium phosphate dihydrate) and TCP (tri calcium phosphate).
  • Scaffold is defined as a 3-dimensional material that supports cell attachment, proliferation and functioning.
  • the present invention provides calcium phosphate nanoparticles using Sophorolipids.
  • the nanoparticles have sizes ranging from 10-100 nm and these nanoparticles assemble to form aggregates > 50 micron in size.
  • the present invention provides a process for the synthesis of sophorolipid wherein S. bombicola was grown in MGYP broth for incubation. It was then transferred to a growth medium and after growth the cells were harvested by centrifugation. The pellet was then redispersed in the production medium and further incubated.
  • the sophorolipid thus synthesized was characterized by FTIR and HPLC. Please refer to figures 1 and 2.
  • the present invention provides a process for the synthesis of calcium phosphate nano-particle composition.
  • the required quantity of sophorolipid was dissolved in Na2HPO4 and sonicated.
  • To this CaCh was added dropwise and was again sonicated to form nano-particles.
  • the solution was centrifuged and washed twice with water.
  • the precipitated nano-particles were further dried in an oven. Similar protocol was followed for different concentration of MASL (Myristic acid derived Sophorolipid) and OASL (Oleic acid derived Sophorolipid).
  • a composition in one embodiment, comprises 05-30 wt% of the sophorolipid of the total weight of the composition, preferably, 5-20 wt%, or 10-20 wt%, or 15-20 wt%, or 10-15 wt%, or 5-15 wt% or 20-30 wt% of the sophorolipid of the total weight of the composition.
  • the composition comprises 70-80 wt% of the calcium phosphate nano-particles of the total weight of the composition, preferably, 72-80 wt%, or 74-80 wt%, or 75-80 wt%, or 76-80 wt%, or 70-75 wt% or 78-80 wt% of the calcium phosphate nano-particles of the total weight of the composition.
  • the composition comprises 45-60 wt% of di-calcium phosphate dehydrate of the total weight of the composition, preferably, 45-50 wt%, or 50-55 wt%, or 55-60 wt%, or 45-55 wt%, or 50-60 wt% or 50 wt% of di-calcium phosphate dehydrate of the total weight of the composition.
  • the composition comprises 10-25 wt% of tri-calcium phosphate of the total weight of the composition, preferably, 15-25 wt%, or 20-25 wt%, or 15-20 wt%, or 10-20 wt%, or 10-15 wt% or 16 wt% of tri-calcium phosphate of the total weight of the composition.
  • the calcium phosphate nano-particles are synthesized without using sophorolipids resulting in decreased concentration of TCP. (Refer to Figure 5, Examples 2 and 3).
  • the calcium phosphate nano-particles are synthesized using sophorolipids resulting in an increase in 0 TCP concentration.
  • the sophorolipds used for synthesizing the nano-particles was derived from saturated or unsaturated fatty acids.
  • the sophorolipid used for synthesizing the nano-particles was derived from a short-chain saturated fatty acid.
  • the fatty acids are selected from myristic acid, oleic acid, lauric acid, palmitic acid, stearic acid, linolenic acid and linoleic or combinations thereof.
  • Myristic acid (MASL) was used and evaluated. (Refer to Figure 9a, Examples 1, 4 and 5)
  • the sophorolipid used for synthesizing the nano-particles was derived from a long chain unsaturated fatty acid.
  • Oleic acid OASL
  • composition of the synthesized nano-particles was determined using Xpert Highscore software. Area under the peak was calculated for each sample and the percentage area of each component was calculated. Graph was plotted for control and sophorolipid - nano-particles. (Refer to Figure 3, 4, and example 3)
  • the TCP composition of nano-particles (Control and SL nano-particles) was deduced by XRD (Refer to figure 5, example 3).
  • control nano-particles nanoparticles synthesized without sophorolipid
  • sophorolipid nano-particles were studied using SEM (Refer to Figure 7, 8, example 4,5).
  • scaffolds were obtained by dispersing the synthesized nanoparticles into silk solution. All the in vitro experiments were performed on these scaffolds (Refer to Figure 10a, 10b, Example 6-9).
  • the polymer may be a naturally occurring polymer or a synthetic polymer.
  • the naturally occurring polymers is selected from collagen, silk, chitosan, alginate, gelatin and combinations thereof; and synthetic polymers is selected from polylactic acid, polyethlene oxide, polyglycolic acid, polyvinyl acetate and combinations thereof.
  • HPLC was used as a tool to confirm formation of sophorolipid in Figure 2. Further, the composition of acidic: lactonic components present in synthesized myristic acid derived sophorolipid (MASL) were deduced by using HPLC. The composition of nano-particles synthesized without using sophorolipids (CNP) was deduced by XRD in Figure 3. Control samples (nano-particles synthesized without using sophorolipid - CNP) contained higher brushite and low TCP.
  • composition of nano-particles synthesized using sophorolipids was deduced by XRD in Figure 4.
  • Control samples nano-particles synthesized without using sophorolipid-SL-NP contained low brushite and higher content of TCP.
  • the graph in Figure 6 summarizes the compositional changes in % TCP at different time intervals.
  • the data includes both samples CNP and SL-NP (Sophorolipid nano-particles and Control nano-particles).
  • the present invention discloses a composition, comprising: i). a sophorolipid in an amount ranging between 20-30% by weight; and ii).a calcium phosphate nano-particles in an amount ranging between 70-80% by weight; wherein the calcium phosphate nano-particles are comprised of di-calcium phosphate dehydrate in an amount ranging from 45-60 % by weight of the total composition, and tri- calcium phosphate in an amount ranging from 10-25 % by weight of the total composition.
  • the sophorolipid is selected from saturated or unsaturated fatty acids.
  • the saturated or unsaturated fatty acids are selected from myristic acid, oleic acid, lauric acid, palmitic acid, stearic acid, linolenic acid or combinations thereof.
  • the sophorolipid is myristic acid-derived sophorolipid (MASL) or oleic acid-derived sophorolipid.
  • the present invention discloses a process for preparation of a composition
  • a sophorolipid in an amount ranging between 20-30% by weight; and a calcium phosphate nano-particles in an amount ranging between 70-80% by weight; wherein the calcium phosphate nano-particles are comprised of di-calcium phosphate dehydrate in an amount ranging from 45-60 % by weight of the total composition, and tri-calcium phosphate in an amount ranging from 10-25 % by weight of the total composition
  • the process comprising the steps of: a) mixing the sophorolipid in 0.01-1 M Na2HPO4 to obtain mixture A; b) sonicating the mixture A for time period of 5-20 minutes at power of 40-60 Hz with a 1-30 seconds pulse having an interval of 1-5 seconds; c) mixing 0.01-1 M of CaCh solution with the mixture A of step b) to obtain a mixture B; d) sonicating the mixture B for a time period of 10-30 minutes at 30-80
  • the sophorolipid is myristic acid-derived sophorolipid (MASL) or oleic acid-derived sophorolipid.
  • the calcium phosphate nanoparticles composition is dried in oven at a temperature of 40-80 °C for 6-24 hours.
  • the present invention discloses a scaffold comprising: a polymer; and a composition comprising a sophorolipid in an amount ranging between 20-30% by weight; and a calcium phosphate nano-particles in an amount ranging between 70-80% by weight; wherein the calcium phosphate nanoparticles are comprised of di-calcium phosphate dehydrate in an amount ranging from 45- 60 % by weight of the total composition, and tri-calcium phosphate in an amount ranging from 10-25 % by weight of the total composition.
  • the polymer is naturally occurring polymer or a synthetic polymer.
  • the naturally occurring polymer is selected from collagen, silk, chitosan, alginate, gelatin and combinations thereof; and the synthetic polymer is selected from polylactic acid, polyethlene oxide, polyglycolic acid, polyvinyl acetate and combinations thereof.
  • MASL was synthesized and characterized as per the protocol described in Abhyankar et al ACS Omega 2021, 6, 1273-1279. Briefly, MASL was synthesized using the resting cell method. S. bombicola was grown in 10 mL of Malt extract, Glucose, Yeast extract, Peptone (MGYP) broth for 24 hours incubation (28°C and 180 rpm). Later it was transferred to 90 mL of growth medium (GM) [in (g/L) glucose-20, yeast extract- 1, MgSO 4 7H 2 O-0.3, Na 2 HPO 4 -2, NaH 2 PO 4 -7, and (NH 4 ) 2 SO 4 -1 at 28°C] with mild shaking conditions of 180 rpm.
  • GM growth medium
  • the MASL synthesised was characterized by Fourier Transform Infrared Spectroscopy (FTIR) and High-Performance Liquid Chromatography (HPLC).
  • FTIR Fourier Transform Infrared Spectroscopy
  • HPLC High-Performance Liquid Chromatography
  • the chemical composition of the synthesized Myristic acid derived sophorolipid (MASL) was analyzed using Fourier transform infrared spectroscopy (FTIR). All measurements were obtained in the ATR mode on a Bruker TENSOR II instrument, and 32 runs scans in the 400-4000 cm -1 wavenumber were recorded. Myristic acid and glucose were also done for reference.
  • FTIR Fourier transform infrared spectroscopy
  • the HPLC ( Figure 2) confirms the conversion of substrates into SL and also shows the acidic and lactonic ratio of 80:20 present in synthesized sophorolipid.
  • Calcium phosphate nano-particles were synthesized with/without sophorolipid.
  • MASL 0.5mg/ml
  • 10 ml (0.1 M) Na2HPO4 10 ml
  • the solution was sonicated for 10 minutes at 50-60 Hz for with a 10 second pulse having a 3 second interval.
  • 10 ml of (0.1 M) CaCh was added dropwise. It was again sonicated for 20 minutes at 50-60 Hz for with a 10 second pulse having a 3 second interval.
  • the nano-particles were centrifuged at 8000 rpm for 15 minutes and washed twice with water. The nano-particles were dried in the oven at 60 °C for 10 hours.
  • Figures 3 and 4 depict the XRD of nano-particles synthesized without / with sophorolipids respectively.
  • Example 3 Method followed for evaluating the composition of synthesized nanoparticles
  • Powder X-ray diffraction (PXRD) patterns were recorded on a Rigaku Micromax-007HF equipment with a high-intensity Microfocus rotating anode X-ray generator. All the samples were coated (10 mg/ per sample) on an aluminum holder, and the data was collected using a Rigaku R axis IV ++ detector. Data was collected from 10°- 70° (2 Theta).
  • composition of the synthesized nano-particles was evaluated through Powder X-ray diffraction (PXRD) patterns which have been depicted in Figure 5.
  • PXRD Powder X-ray diffraction
  • Figure 7 and Figure 8 depicts SEM images of CNP (control nano-particles) and SEM images of SL-NP (sophorolipid nano-particles) respectively.
  • Nano-particles with sophorolipids assemble into a defined morphology as compared to control nano-particles, which are seen mostly as individual particles.
  • MASL 0.5 mg/ml, 2.5 mg/ml and 5 mg/ml
  • OASL 2.5 mg/ml and 5 mg/ml
  • a comparative graph with reference to the control was plotted for %TCP and different concentrations of MASL and OASL.
  • FIG. 9a and 9b depicts the effect of concentration of sophorolipids (MASL and OASL) on the composition of nano-particles.
  • the synthesized nano-particles were dispersed into silk solution and scaffolds were obtained. All the in vitro experiments were performed on these scaffolds.
  • Bombyx mori cocoons (Procured from CSRTL Central Sericultural research & Training Institute, Mysore) were boiled in 0.5 w/v% of NaHCCh solution twice for 30 minutes each for sericin removal. Collected fibroin was vacuum dried at 60°C followed by dissolution in 9.3 M lithium bromide (LiBr) at 60°C for 4 hours. This solution was dialyzed extensively against water and the resultant regenerated silk fibroin solution (RSF) was used for further experiments.
  • LiBr lithium bromide
  • the nano-particles were dispersed into distilled water and sonicated for 2 minutes. 3% RSF + NP solution was mixed, and probe sonicated (30 seconds pulse with 3 sec interval). The resultant solution was added into 96 well plate, kept at 37°C. After gelation, the plate was lyophilized and the scaffolds were removed.
  • Cell proliferation was determined by Alamar blue assay. Before seeding the cells, scaffolds were incubated in complete media at 37°C with 5% CO2 for 12 hours. MG 63 cells were seeded onto the scaffolds at a density of 5 x 10 3 cells per scaffold in 20 pL of complete media. The plate was incubated at 37°C with 5% CO2 for 24 hours. During incubation, on the, 4 th , 7 th , 11 th and 14 th day, the media was replaced with 10 % Alamar blue prepared in ⁇ Dulbecco’s Modified Eagle medium (DMEM) and further incubated for 6 hours at 37°C with 5% CO2. Post incubation, the media was removed and transferred into another plate and fresh DMEM media was added to scaffolds and incubated. The reading of the collected media at 570 and 600 nm was noted. Assay was performed in triplicates.
  • DMEM Modified Eagle medium
  • Figure 10a depicts graph of the in vitro studies (proliferation assay) Alamar Blue Assay
  • Alkaline phosphatase (ALP) activity was assayed using colorimetric ALP kit (Abeam, U.K.). Briefly, MG 63 cells were seeded on scaffolds at a density of 5 x 10 4 cells per scaffold in 50 pL of complete media. The cells were allowed to be settled for 5-7 minutes at 37°C with 5% CO2. The additional 90 pL media for cells was then added and the cell culture plate was then incubated at 37°C with 5% CO2 for 7 days.
  • Figure 10b depicts graph of the in vitro studies (ALP estimation).
  • SL nano-particles As it may observed from the above in vitro studies (proliferation assay and ALP estimation) SL nano-particles (SL-NP) have better biological properties as compared to (CNP) control nano-particles.
  • Figure 10a and 10b reveals that the proliferation and ALP activity of cells increases in presence of sophorolipids over period of 14 days.
  • ALP is an osteogenic marker; elevated activity indicates good proliferation of bone cells.
  • the present disclosure provides sophorolipid based calcium phosphate nanoparticles composition and process to prepare the same.
  • the present disclosure provides the composition with right size, which aids in enhanced biological properties.
  • compositions of nano-particles which are more desirable as they are biocompatible, bioresorbable, osteoconductive and osteoinductive.
  • the present disclosure provides synthesis of the composition which is simple with minimal downstream processing.
  • the present disclosure provides the biosurfactant (sophorolipids) which may be used in the composition both as a reducing and capping agent thereby minimizing the downstream processing of nano-particles.

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Abstract

The present invention relates to sophorolipid based calcium phosphate nano-particles composition and a process for preparing the same. The invention thus provides a composition of calcium phosphate nano-particles having enhanced biocompatible, bioresorbable, osteoconductive and osteoinductive properties.

Description

COMPOSITIONS OF CALCIUM PHOSPHATE NANO-PARTICLES WITH SOPHOROLIPIDS AND PROCESS FOR PREPARATION THEREOF
FIELD OF THE INVENTION
The present invention relates to novel compositions of calcium phosphate nano-particles with sophorolipids. More particularly, the present invention discloses a composition of calcium phosphate nano-particles with higher content of tri calcium phosphate. Further, the present invention discloses a process of synthesis of composition of calcium phosphate nano-particles with sophorolipids.
BACKGROUND AND PRIOR ART OF THE INVENTION
Calcium phosphate compounds are highly desirable materials due to their varied applications. They play an important role in the biomedical sector due to their resemblance to natural bone. These compounds are available in many forms such as powder, sponge or putty. Chemically, these compounds can exist as mono calcium phosphate, di calcium phosphate (DCP), tri calcium phosphate (TCP), octa calcium phosphate (OCP) and hydroxyapatite (HA). HA is the main inorganic component of the bone.
Recent biomedical literature shows that the most preferred forms of these compounds are tri calcium and di calcium phosphates. TCP is available in its a or 0 forms and DCP in monetite or brushite forms. In addition to bone filling applications, they are used as non- viral vectors in drug delivery, gene silencing and cancer therapy.
Further, the preferred form of these materials in biomedical applications is a powder of nano-particles. Synthesis of calcium phosphate nano-particles includes different physical, chemical and biological methods. Chemical processes for synthesis of calcium phosphate nano-particles typically require longer hours of synthesis. This hampers their large-scale production. Also, the properties of calcium phosphates are largely dependent on its composition and morphology. Therefore, tuning the right composition along with size will aid in enhanced properties, especially for biomedical applications. With this view, newer processes are now being explored so that compositions of calcium phosphate with distinct and advantageous properties within short time may be evolved. Calcium phosphate compounds as bone graft substitutes in dental and orthopedic fields have gained tremendous interest over the time. There are different types of calcium cements available like mono, di, tri, octa calcium phosphates and hydroxyapatite (HA). HA being the inorganic component of the bone was studied extensively. However, the drawbacks of the HA such as high cost, brittleness, low biodegradability and slow resorption into the body, limits its applications.
HA are now being substituted by other forms of calcium phosphates. Tri calcium phosphate, commonly known as TCP has gained interest owing to its good biological properties. As reported by Kucharska et al., the presence of TCP within the material enhances its biological and mechanical properties, and this is relatable to increasing concentration of TCP. [Kucharska, M.; Walenko, K.; Lewandowska-Szumiel, M.; Brynk, T.; Jaroszewicz, J.; Ciach, T. Chitosan and Composite Microsphere-Based Scaffold for Bone Tissue Engineering: Evaluation of Tricalcium Phosphate Content Influence on Physical and Biological Properties. J. Mater. Set. Mater. Med. 2015, 26 (3)]. Similarly, Cama, G et al reported that di calcium phosphates, known as brushite possessing good resorbability and biological attributes are also widely studied. Owing to these properties, these forms of calcium phosphates are more beneficial for biomedical applications. [Cama, G.; Barberis, F.; Capurro, M.; Di Silvio, L.; Deb, S. Tailoring Brushite for in Situ Setting Bone Cements. Mater. Chem. Phys. 2011, 130 (3), 1139— 1145],
Felix et al. in Biomaterials 2005 July 26(21):4383-94, reported that biocompatibility and resorption of a brushite calcium phosphate cement provides a hydraulic calcium phosphate cement with beta-tricalcium phosphate (TCP) granules embedded in a matrix of dicalcium phosphate dihydrate (DCPD), which was implanted in sheep.
Fowler et. al. in Journal of Materials Chemistry Issue 32, 2005, discloses that a model for dental enamel formation provides novel calcium phosphate materials synthesized from solutions containing the surfactant bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT), water and oil. A range of morphologies was obtained by varying the relative concentrations of the solution components.
Singh, Sanjay; L. V. Prasad, B.; Ramana, C. V.; Prabhune, A. A.; Kasture, Manasi; Joy, P. A.; et al. (2016): Multiutility Sophorolipids as Nanoparticle Capping Agents: Synthesis of Stable and Water Dispersible Co nano-particles, demonstrates the application of sophorolipids obtained from oleic acid as a capping agent for Co nano-particles. Upon capping, the sugar moiety of these sophorolipids is exposed to the solvent environment, making the nano-particles stable and water-redispersible.
Sophorolipids (SLs) are a group of extracellular biosurfactants produced at relatively high yields by several non-pathogenic yeast species. Sophorolipid comprise a residue of sophorose, the disaccharide consisting of two glucose residues linked by the P-1,2' bond, and fatty acid as an aglycone (Extracellular Glycolipids of Yeats - Biodiversity , Biochemistry and Prospects 2014 pages 35-64 Chapter 4).
Literature discloses sophorolipids as FDA approved molecules for human consumption. Up till now sophorolipids have been used for synthesizing metallic nano-particles but the same has not been studied with calcium phosphate nano-particles.
Thus, the inventors of the present invention have synthesized calcium phosphate nanoparticles by using a biosurfactant. The nano-particles thus formed by the efforts of the inventors of the present invention are a mixture of brushite (di calcium phosphate dihydrate) and have higher content of TCP (tri calcium phosphate) along with a sophorolipid. This composition of nano-particles is more desirable as, they are biocompatible, bioresorbable and are osteoconductive and osteoinductive. The process followed for synthesizing or preparing these calcium phosphate nano-particles with biosurfactants is less time consuming and economical. It requires minimum downstream processing to obtain nano-particles with specific chemical composition and appropriate physical and biological properties.
OBJECTS OF THE INVENTION
It is an object of the present invention to provide a novel composition of calcium phosphate nano-particles.
It is another object of the present invention to provide a composition of calcium phosphate nano-particles using a biosurfactant sophorolipid.
It is yet another object of the present invention to provide a specific composition of calcium phosphate nano-particles synthesized using sophorolipids, comprising of a mixture of brushite (di calcium phosphate dihydrate) and higher content of TCP (tri calcium phosphate)
It is still another object of the present invention to provide a composition of biocompatible, bioresorbable, osteoconductive and osteoinductive nano-particles of calcium phosphate.
It is further object of the present invention to provide a less time consuming and economical process for preparing calcium phosphate nano-particles using biosurfactants.
It is a yet further object of the present invention to provide a process for preparing calcium phosphate nano-particles requiring minimal downstream processing of nanoparticles.
SUMMARY OF THE INVENTION
The present invention relates to a composition of biocompatible, bioresorbable and osteoconductive and osteoinductive calcium phosphate nano-particles comprising of a mixture of brushite (di calcium phosphate dihydrate) and a higher content of TCP (tri calcium phosphate) along with a sophorolipid.
According to one aspect of the present disclosure a sophorolipid based calcium phosphate nano-particles composition is disclosed.
A composition, comprising: i. a sophorolipid with an amount ranging between 20-30% by weight; and ii. a calcium phosphate nano-particles with an amount ranging between 70- 80% by weight; wherein the calcium phosphate nano-particles are comprised of di-calcium phosphate dehydrate in an amount ranging from 45-60 % by weight of the total composition, and tricalcium phosphate in an amount ranging from 10-25 % by weight of the total composition.
In some embodiments, the sophorolipid is synthesized using saturated or unsaturated fatty acids.
In some embodiments, the sophorolipid is synthesized using different fatty acids selected from myristic acid, oleic acid, lauric acid, palmitic acid, stearic acid, linolenic acid or combinations thereof. In some embodiments, the sophorolipid is synthesized using myristic acid or oleic acid.
According to another aspect of the present disclosure a process for the preparation of calcium phosphate nanoparticles composition is disclosed. The method comprises the following steps: a) mixing a sophorolipid in 0.01-1 M (preferably 0.1 M) of Na2HPO4 to obtain mixture A. b) sonicating the mixture A for time period of 5-20 minutes at power of 40-60 Hz with a 1-30 seconds (preferably 10 seconds) pulse having a 1-5 seconds (preferably 3 seconds) interval; c) mixing 0.01-1 M (preferably 0.1 M) of CaCh solution with the mixture A of step b) to obtain a mixture B; d) sonicating the mixture B for a time period of 10-30 minutes at 30-80 Hz for with a 1-30 seconds (preferably 10 seconds) pulse having a 1-5 seconds (preferably 3 seconds) interval to obtain a solution containing calcium phosphate nano-particles; e) centrifuging the solution of step d) at a speed in the range of 7900-8100 rpm for time period of 10-20 minutes followed by washing with water as a solvent to obtain a wet mass; and f) drying the wet mass of step e) in an oven at temperature in a range of 50-100 °C for a time period of 8-20 hours to obtain the desired composition.
In some embodiments, the sophorolipid may be myristic acid-derived sophorolipid (MASL) or oleic acid-derived sophorolipid.
In some embodiments, the calcium phosphate nanoparticles composition was dried in oven at a temperature of 40-80 °C for 6-24 hours.
In some embodiments, the calcium phosphate nanoparticles composition was dried in the oven at 60 °C for 10 hours.
The sonication is done for time period of 5-20 minutes wherein in that time period, repeating cycles of 10 seconds sonication/pulse followed by 3 second break.
According to another aspect of the present disclosure, a scaffold is disclosed. It comprises the following: i. a polymer; ii. the calcium phosphate nanoparticles composition as described hereinabove. In some embodiments, the polymer is naturally occurring polymer or a synthetic polymer. In some embodiments, the naturally occurring polymers is selected from collagen, silk, chitosan, alginate, gelatin and combinations thereof and synthetic polymers is selected from polylactic acid, polyethlene oxide, polyglycolic acid, polyvinyl acetate and combinations thereof.
BRIEF DESCRIPTION OF THE DRAWINGS:
Fig 1 illustrates FTIR of short chain derived sophorolipids.
Fig 2 illustrates HPLC of synthesized sophorolipids.
Fig 3 illustrates XRD of nano-particles synthesized without using sophorolipids (CNP).
Fig 4 illustrates XRD of nano-particles synthesized using sophorolipids (SL-NP).
Fig 5 illustrates XRD of TCP composition of nano-particles (control (CNP) and sophorolipids nanoparticles (SL-NP).
Fig 8 illustrates SEM of sophorolipid nano-particles (SL-NP).
Fig 9a illustrates a graph showing different concentrations of sophorolipids (MASL). Fig 9b illustrates a graph showing different concentration of sophorolipids (OASL).
Fig 10a illustrates a graph of the in vitro studies (proliferation assay).
Fig 10b illustrates a graph of the in vitro studies (ALP estimation).
DETAILED DESCRIPTION OF THE INVENTION:
Source of S. bombicola and silk fibroin: the yeast culture used for fermentation (Starmerella bombicola, formerly known as Candida bombicola) was procured from ATCC (ATCC 22214). Bombyx mori cocoons were procured from CSRTI - Central Sericultural Research & Training Institute, Mysore, India)
The present invention relates to calcium phosphate nano-particles synthesized by using a biosurfactant. The nano-particles, thus, formed are a mixture of brushite (di calcium phosphate dihydrate) and TCP (tri calcium phosphate), with a higher content of TCP. More specifically, the present invention relates to a composition comprising calcium phosphate nano-particles synthesized by using a biosurfactant. This composition of calcium phosphate nano-particles is more desirable as they are biocompatible, bioresorbable, osteoconductive and osteoinductive. The biosurfactant used in the present invention is sophorolipid. By using a biosurfactant, (a sophorolipid), a mixture with higher TCP and brushite nano-particles is obtained. The calcium phosphate nano-particles thus formed have enhanced structural, compositional and biological properties as confirmed by XRD, SEM and In vitro studies. Please see Figures 6, 7, 8, & 10.
Within the purview of the present invention ‘Control nano-particles’ means nanoparticles synthesized without incorporation of sophorolipids (Abbreviated as CNP) and nano- particles with incorporation of sophorolipids are abbreviated as SL-NP.
As it would be apparent to a person skilled in the art, the term ‘nano-particles’ has been used interchangeably for calcium phosphate nano-particles and refer to calcium phosphate nano-particles unless specified otherwise.
Accordingly, in an embodiment, the present invention provides a novel composition of calcium phosphate nano-particles comprising brushite (di calcium phosphate dihydrate) and TCP (tri calcium phosphate).
Scaffold is defined as a 3-dimensional material that supports cell attachment, proliferation and functioning.
In an exemplary embodiment, the present invention provides calcium phosphate nanoparticles using Sophorolipids.
In an embodiment, the nanoparticles have sizes ranging from 10-100 nm and these nanoparticles assemble to form aggregates > 50 micron in size.
In an embodiment, the present invention provides a process for the synthesis of sophorolipid wherein S. bombicola was grown in MGYP broth for incubation. It was then transferred to a growth medium and after growth the cells were harvested by centrifugation. The pellet was then redispersed in the production medium and further incubated. The sophorolipid thus synthesized was characterized by FTIR and HPLC. Please refer to figures 1 and 2.
In another embodiment the present invention provides a process for the synthesis of calcium phosphate nano-particle composition. The required quantity of sophorolipid was dissolved in Na2HPO4 and sonicated. To this CaCh was added dropwise and was again sonicated to form nano-particles. The solution was centrifuged and washed twice with water. The precipitated nano-particles were further dried in an oven. Similar protocol was followed for different concentration of MASL (Myristic acid derived Sophorolipid) and OASL (Oleic acid derived Sophorolipid).
In one embodiment of the present disclosure, a composition is provided. In some embodiments the composition comprises 05-30 wt% of the sophorolipid of the total weight of the composition, preferably, 5-20 wt%, or 10-20 wt%, or 15-20 wt%, or 10-15 wt%, or 5-15 wt% or 20-30 wt% of the sophorolipid of the total weight of the composition.
In some embodiments, the composition comprises 70-80 wt% of the calcium phosphate nano-particles of the total weight of the composition, preferably, 72-80 wt%, or 74-80 wt%, or 75-80 wt%, or 76-80 wt%, or 70-75 wt% or 78-80 wt% of the calcium phosphate nano-particles of the total weight of the composition.
In some embodiments, the composition comprises 45-60 wt% of di-calcium phosphate dehydrate of the total weight of the composition, preferably, 45-50 wt%, or 50-55 wt%, or 55-60 wt%, or 45-55 wt%, or 50-60 wt% or 50 wt% of di-calcium phosphate dehydrate of the total weight of the composition.
In some embodiments, the composition comprises 10-25 wt% of tri-calcium phosphate of the total weight of the composition, preferably, 15-25 wt%, or 20-25 wt%, or 15-20 wt%, or 10-20 wt%, or 10-15 wt% or 16 wt% of tri-calcium phosphate of the total weight of the composition.
In an embodiment, the calcium phosphate nano-particles are synthesized without using sophorolipids resulting in decreased concentration of TCP. (Refer to Figure 5, Examples 2 and 3).
In another embodiment, the calcium phosphate nano-particles are synthesized using sophorolipids resulting in an increase in 0 TCP concentration. (Refer to Figure 5, Examples 1, 2 and 3)
In some embodiments, the sophorolipds used for synthesizing the nano-particles was derived from saturated or unsaturated fatty acids. In yet another embodiment, the sophorolipid used for synthesizing the nano-particles was derived from a short-chain saturated fatty acid. In some embodiments the fatty acids are selected from myristic acid, oleic acid, lauric acid, palmitic acid, stearic acid, linolenic acid and linoleic or combinations thereof. In some embodiment, Myristic acid (MASL) was used and evaluated. (Refer to Figure 9a, Examples 1, 4 and 5)
In yet another embodiment, the sophorolipid used for synthesizing the nano-particles was derived from a long chain unsaturated fatty acid. Oleic acid (OASL) was used and evaluated. (Refer to Figure 9b, Examples 1, 4 and 5)
In another embodiment, the composition of the synthesized nano-particles was determined using Xpert Highscore software. Area under the peak was calculated for each sample and the percentage area of each component was calculated. Graph was plotted for control and sophorolipid - nano-particles. (Refer to Figure 3, 4, and example 3)
In yet another embodiment, the TCP composition of nano-particles (Control and SL nano-particles) was deduced by XRD (Refer to figure 5, example 3).
In a further embodiment, the morphological changes of the Control nano-particles (nanoparticles synthesized without sophorolipid) and the sophorolipid nano-particles were studied using SEM (Refer to Figure 7, 8, example 4,5).
In exemplary embodiment, scaffolds were obtained by dispersing the synthesized nanoparticles into silk solution. All the in vitro experiments were performed on these scaffolds (Refer to Figure 10a, 10b, Example 6-9).
In some embodiments, the polymer may be a naturally occurring polymer or a synthetic polymer.
In yet another embodiment, the naturally occurring polymers is selected from collagen, silk, chitosan, alginate, gelatin and combinations thereof; and synthetic polymers is selected from polylactic acid, polyethlene oxide, polyglycolic acid, polyvinyl acetate and combinations thereof.
Successful synthesis of short chain derived sophorolipid was confirmed by FTIR in Figure 1, wherein the incorporation of parent moieties (Glucose, fatty acid) were analyzed, along with synthesized sophorolipid.
HPLC was used as a tool to confirm formation of sophorolipid in Figure 2. Further, the composition of acidic: lactonic components present in synthesized myristic acid derived sophorolipid (MASL) were deduced by using HPLC. The composition of nano-particles synthesized without using sophorolipids (CNP) was deduced by XRD in Figure 3. Control samples (nano-particles synthesized without using sophorolipid - CNP) contained higher brushite and low TCP.
The composition of nano-particles synthesized using sophorolipids (SL-NP) was deduced by XRD in Figure 4. Control samples (nano-particles synthesized without using sophorolipid-SL-NP) contained low brushite and higher content of TCP.
In Figure 5 XRD of TCP composition of nano-particles (control (CNP) and sophorolipids nanoparticles (SL-NP) has been illustrated. Optimization of the process time was done for both the SL-NP (sophorolipid- nano-particles) and the CNP (control samples).
The graph in Figure 6 summarizes the compositional changes in % TCP at different time intervals.
Percentage of brushite and TCP at the optimized process time (30 min) illustrated in Figure 6. The data includes both samples CNP and SL-NP (Sophorolipid nano-particles and Control nano-particles).
Structural orientation of CNP nano-particles as observed by SEM in Figure 7.
Structural orientation of SL-NP nano-particles as observed by SEM in Figure 8.
Effect of short chain substrate (Myristic acid) concentration on composition of TCP was assessed using XRD in Figure 9a.
Effects of long chain substrate (Oleic acid) concentration on composition of TCP in nanoparticles were assessed using XRD in Figure 9b.
In vitro studies (proliferation assay) depicted in Figure 10a. Scaffolds incorporating the nanopartciles (CNP, SL-NP) were used to study the proliferation (Alamar blue assay) of bone cells (MG63) for a period of 14 days.
In vitro studies (ALP estimation) depicted in Figure 10b. Scaffolds incorporating the nanopartciles (CNP, SL-NP) were used to evaluate the osteogenic marker released by proliferative bone cells (MG63) for a period of 14 days.
In as aspect of the present invention, the present invention discloses a composition, comprising: i). a sophorolipid in an amount ranging between 20-30% by weight; and ii).a calcium phosphate nano-particles in an amount ranging between 70-80% by weight; wherein the calcium phosphate nano-particles are comprised of di-calcium phosphate dehydrate in an amount ranging from 45-60 % by weight of the total composition, and tri- calcium phosphate in an amount ranging from 10-25 % by weight of the total composition.
In a feature of the present invention, the sophorolipid is selected from saturated or unsaturated fatty acids.
In a feature of the present invention, the saturated or unsaturated fatty acids are selected from myristic acid, oleic acid, lauric acid, palmitic acid, stearic acid, linolenic acid or combinations thereof.
In a feature of the present invention, the sophorolipid is myristic acid-derived sophorolipid (MASL) or oleic acid-derived sophorolipid.
In another aspect of the present invention, the present invention discloses a process for preparation of a composition comprising a sophorolipid in an amount ranging between 20-30% by weight; and a calcium phosphate nano-particles in an amount ranging between 70-80% by weight; wherein the calcium phosphate nano-particles are comprised of di-calcium phosphate dehydrate in an amount ranging from 45-60 % by weight of the total composition, and tri-calcium phosphate in an amount ranging from 10-25 % by weight of the total composition, the process comprising the steps of: a) mixing the sophorolipid in 0.01-1 M Na2HPO4 to obtain mixture A; b) sonicating the mixture A for time period of 5-20 minutes at power of 40-60 Hz with a 1-30 seconds pulse having an interval of 1-5 seconds; c) mixing 0.01-1 M of CaCh solution with the mixture A of step b) to obtain a mixture B; d) sonicating the mixture B for a time period of 10-30 minutes at 30-80 Hz using a pulse of 1-30 seconds with an interval of 1-5 seconds to obtain a solution containing calcium phosphate nano-particles; e) centrifuging the solution of step d) at a speed in a range of 7900-8100 rpm for time period of 10-20 minutes followed by washing with water as solvent to obtain a wet mass; and f) drying the wet mass of step e) in an oven at temperature in a range of 50-100 °C for time period of 8-20 hours to obtain the composition.
In a feature of the present invention, the sophorolipid is myristic acid-derived sophorolipid (MASL) or oleic acid-derived sophorolipid. In a feature of the present invention, the calcium phosphate nanoparticles composition is dried in oven at a temperature of 40-80 °C for 6-24 hours.
In one another aspect of the present invention, the present invention discloses a scaffold comprising: a polymer; and a composition comprising a sophorolipid in an amount ranging between 20-30% by weight; and a calcium phosphate nano-particles in an amount ranging between 70-80% by weight; wherein the calcium phosphate nanoparticles are comprised of di-calcium phosphate dehydrate in an amount ranging from 45- 60 % by weight of the total composition, and tri-calcium phosphate in an amount ranging from 10-25 % by weight of the total composition.
In a feature of the present invention, the polymer is naturally occurring polymer or a synthetic polymer.
In a feature of the present invention, the naturally occurring polymer is selected from collagen, silk, chitosan, alginate, gelatin and combinations thereof; and the synthetic polymer is selected from polylactic acid, polyethlene oxide, polyglycolic acid, polyvinyl acetate and combinations thereof.
EXAMPLES
The present disclosure is further explained in the form of the following examples. However, it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.
Example 1: Synthesis of Sophorolipid
MASL was synthesized and characterized as per the protocol described in Abhyankar et al ACS Omega 2021, 6, 1273-1279. Briefly, MASL was synthesized using the resting cell method. S. bombicola was grown in 10 mL of Malt extract, Glucose, Yeast extract, Peptone (MGYP) broth for 24 hours incubation (28°C and 180 rpm). Later it was transferred to 90 mL of growth medium (GM) [in (g/L) glucose-20, yeast extract- 1, MgSO4 7H2O-0.3, Na2HPO4-2, NaH2PO4-7, and (NH4)2SO4-1 at 28°C] with mild shaking conditions of 180 rpm. After 48 hours of growth, cells were harvested by centrifugation at 5000 rpm for 20 minutes. The pellet was then redispersed in the production medium (PM) [in (g/L); glucose-100, yeast extract-1, MgSCU 7H2O-0.3, Na2HPO4-2, NaH2PO4-7, and (NP ^SC -l] supplemented with 1% (v/v) myristic acid and incubated at 28°C at 180 rpm shaking for 8 days. The sophorolipid, thus, synthesized was characterized by FTIR and HPLC, which have been depicted through Figures 1 and 2 respectively.
Characterization of MASL:
The MASL synthesised was characterized by Fourier Transform Infrared Spectroscopy (FTIR) and High-Performance Liquid Chromatography (HPLC).
FTIR: Fourier Transform Infrared Spectroscopy:
The chemical composition of the synthesized Myristic acid derived sophorolipid (MASL) was analyzed using Fourier transform infrared spectroscopy (FTIR). All measurements were obtained in the ATR mode on a Bruker TENSOR II instrument, and 32 runs scans in the 400-4000 cm-1 wavenumber were recorded. Myristic acid and glucose were also done for reference.
HPLC: High Performance Liquid Chromatography
The different ratios of acidic to lactonic components present in MASL were assessed using HPLC (Water 515 System, Cl 8 Column, UV detector-2489). The solvent system consisted of Acetonitrile/Water (70:30). The injection volume was 50 pL and the retention time was 35 minutes. Flow rate of the sample was maintained to 0.7 ml min-1.
The FTIR spectra (Figure 1) confirms the successful incorporation of parent moieties (Glucose, myristic acid) into sophorolipid, validating synthesis of short chain derived sophorolipid. Also, the presence of ether linkage at 1030 confirms the formation of sophorolipid.
The HPLC (Figure 2) confirms the conversion of substrates into SL and also shows the acidic and lactonic ratio of 80:20 present in synthesized sophorolipid.
Example 2: Synthesis of calcium phosphate nano-particles
Calcium phosphate nano-particles were synthesized with/without sophorolipid. MASL (0.5mg/ml) was dissolved in 10 ml (0.1 M) Na2HPO4. The solution was sonicated for 10 minutes at 50-60 Hz for with a 10 second pulse having a 3 second interval. To this 10 ml of (0.1 M) CaCh was added dropwise. It was again sonicated for 20 minutes at 50-60 Hz for with a 10 second pulse having a 3 second interval. The nano-particles were centrifuged at 8000 rpm for 15 minutes and washed twice with water. The nano-particles were dried in the oven at 60 °C for 10 hours.
Similar protocol was followed for different concentration of MASL (2.5 mg/ml, 5mg/ml) and Oleic acid derived sophorolipid (OASL). The above protocol was carried out for 30 minutes process time.
Figures 3 and 4 depict the XRD of nano-particles synthesized without / with sophorolipids respectively.
Example 3: Method followed for evaluating the composition of synthesized nanoparticles
Powder X-ray diffraction (PXRD) patterns were recorded on a Rigaku Micromax-007HF equipment with a high-intensity Microfocus rotating anode X-ray generator. All the samples were coated (10 mg/ per sample) on an aluminum holder, and the data was collected using a Rigaku R axis IV ++ detector. Data was collected from 10°- 70° (2 Theta).
The composition of the synthesized nano-particles was evaluated through Powder X-ray diffraction (PXRD) patterns which have been depicted in Figure 5.
Example 4: Determination of the Morphological changes of the nano-particles
0.2 mg/ml of nano-particles were added in DI (Deionized water) water and bath sonicated for 2 minutes. 10 uL sample was loaded onto silicon wafer. The sample was dried completely and analyzed using Quanta 200 3D scanning electron microscope (SEM). Prior to imaging, all samples were sputter coated with 5 nm gold coating using a polaron SC6420 sputters coater.
Figure 7 and Figure 8 depicts SEM images of CNP (control nano-particles) and SEM images of SL-NP (sophorolipid nano-particles) respectively.
XRD revealed changes in the composition of synthesized nano-particles by using sophorolipids. Similarly, morphological changes in presence of sophorolipids are observed through SEM. Nano-particles with sophorolipids assemble into a defined morphology as compared to control nano-particles, which are seen mostly as individual particles.
Example 5:
Evaluation and determination of the composition of the synthesized nano-particles.
Different concentration of MASL (0.5 mg/ml, 2.5 mg/ml and 5 mg/ml) and OASL (2.5 mg/ml and 5 mg/ml) were used in the synthesis of different calcium phosphate nanoparticles and the composition and morphology of the synthesized nano-particles was determined. Briefly, MASL (0.5mg/ml) was dissolved in 10 ml (0.1 M) Na2HPO4. Sonicated for 10 minutes at 50-60 Hz for with a 10 second pulse having a 3 second interval. To this 10 ml of (0.1 M) CaCh was added dropwise. It was again sonicated for 20 minutes at 50-60 Hz for 10 sec/ 3 sec interval. The nano-particles were centrifuged at 8000 rpm for 15 minutes and washed twice with water. The nano-particles were dried in oven at 60°C for 6-8 hours.
A comparative graph with reference to the control was plotted for %TCP and different concentrations of MASL and OASL.
It can be seen from figures 9a and 9b that use of SL caused changes in the composition and morphology of the synthesized calcium phosphate nano-particles. Figures 9a and 9b depicts the effect of concentration of sophorolipids (MASL and OASL) on the composition of nano-particles.
In vitro studies
For in vitro experiments, the synthesized nano-particles were dispersed into silk solution and scaffolds were obtained. All the in vitro experiments were performed on these scaffolds.
Example 6: Preparation of Silk Fibroin Solution (RSF)
Bombyx mori cocoons (Procured from CSRTL Central Sericultural research & Training Institute, Mysore) were boiled in 0.5 w/v% of NaHCCh solution twice for 30 minutes each for sericin removal. Collected fibroin was vacuum dried at 60°C followed by dissolution in 9.3 M lithium bromide (LiBr) at 60°C for 4 hours. This solution was dialyzed extensively against water and the resultant regenerated silk fibroin solution (RSF) was used for further experiments.
Example 7: Preparation of Silk scaffolds
The nano-particles were dispersed into distilled water and sonicated for 2 minutes. 3% RSF + NP solution was mixed, and probe sonicated (30 seconds pulse with 3 sec interval). The resultant solution was added into 96 well plate, kept at 37°C. After gelation, the plate was lyophilized and the scaffolds were removed.
Example 8: Alamar blue assay
Cell proliferation was determined by Alamar blue assay. Before seeding the cells, scaffolds were incubated in complete media at 37°C with 5% CO2 for 12 hours. MG 63 cells were seeded onto the scaffolds at a density of 5 x 103 cells per scaffold in 20 pL of complete media. The plate was incubated at 37°C with 5% CO2 for 24 hours. During incubation, on the, 4 th, 7th, 11th and 14th day, the media was replaced with 10 % Alamar blue prepared in \Dulbecco’s Modified Eagle medium (DMEM) and further incubated for 6 hours at 37°C with 5% CO2. Post incubation, the media was removed and transferred into another plate and fresh DMEM media was added to scaffolds and incubated. The reading of the collected media at 570 and 600 nm was noted. Assay was performed in triplicates.
Figure 10a depicts graph of the in vitro studies (proliferation assay) Alamar Blue Assay
Example 9: Alkaline phosphatase (ALP) estimation
Alkaline phosphatase (ALP) activity was assayed using colorimetric ALP kit (Abeam, U.K.). Briefly, MG 63 cells were seeded on scaffolds at a density of 5 x 104 cells per scaffold in 50 pL of complete media. The cells were allowed to be settled for 5-7 minutes at 37°C with 5% CO2. The additional 90 pL media for cells was then added and the cell culture plate was then incubated at 37°C with 5% CO2 for 7 days.
On the second day of the experiment, seeding media was replaced with osteogenic differentiation media (Invitrogen). During incubation, on the 4th, 7th, 11th and 14th day, spent media from cell seeded scaffolds was collected and stored. Post incubation, 20 pL of the spent media and 60 pL of assay buffer was incubated with 50 pL of p-nitrophenyl phosphate (5 mM) solution at room temperature for 1 hour in the dark. At the end of the incubation, enzyme activity was stopped by adding 20 pL of stop solution. Simultaneously, standard curve was plotted as per manufacturer's instruction. The amount of p-nitro-phenol produced was measured by measuring absorbance at 405 nm.
Figure 10b depicts graph of the in vitro studies (ALP estimation).
As it may observed from the above in vitro studies (proliferation assay and ALP estimation) SL nano-particles (SL-NP) have better biological properties as compared to (CNP) control nano-particles.
Figure 10a and 10b reveals that the proliferation and ALP activity of cells increases in presence of sophorolipids over period of 14 days. ALP is an osteogenic marker; elevated activity indicates good proliferation of bone cells.
ADVANTAGES OF THE INVENTION
1. The present disclosure provides sophorolipid based calcium phosphate nanoparticles composition and process to prepare the same.
2. The present disclosure provides the composition with right size, which aids in enhanced biological properties.
3. The present disclosure provides compositions of nano-particles which are more desirable as they are biocompatible, bioresorbable, osteoconductive and osteoinductive.
4. The present disclosure provides synthesis of the composition which is simple with minimal downstream processing.
5. The present disclosure provides the biosurfactant (sophorolipids) which may be used in the composition both as a reducing and capping agent thereby minimizing the downstream processing of nano-particles.

Claims

We Claim:
1. A composition, comprising: i. a sophorolipid in an amount ranging between 20-30% by weight; and ii. a calcium phosphate nano-particles in an amount ranging between 70-80% by weight; wherein the calcium phosphate nano-particles are comprised of di-calcium phosphate dehydrate in an amount ranging from 45-60 % by weight of the total composition, and tri-calcium phosphate in an amount ranging from 10-25 % by weight of the total composition.
2. The composition as claimed in claim 1, wherein the sophorolipid is selected from saturated or unsaturated fatty acids.
3. The composition as claimed in claim 2, wherein the saturated or unsaturated fatty acids are selected from myristic acid, oleic acid, lauric acid, palmitic acid, stearic acid, linolenic acid or combinations thereof.
4. The composition as claimed in claim 1, wherein the sophorolipid is myristic acid- derived sophorolipid (MASL) or oleic acid-derived sophorolipid.
5. A process for preparation of a composition, comprising the steps of: a) mixing a sophorolipid in 0.01-1 M Na2HPO4 to obtain mixture A; b) sonicating the mixture A for time period of 5-20 minutes at power of 40-60 Hz with a 1-30 seconds pulse having an interval of 1-5 seconds; c) mixing 0.01-1 M of CaCh solution with the mixture A of step b) to obtain a mixture B; d) sonicating the mixture B for a time period of 10-30 minutes at 30-80 Hz using a pulse of 1-30 seconds with an interval of 1-5 seconds to obtain a solution containing calcium phosphate nano-particles; e) centrifuging the solution of step d) at a speed in a range of 7900-8100 rpm for time period of 10-20 minutes followed by washing with water as solvent to obtain a wet mass; and f) drying the wet mass of step e) in an oven at temperature in a range of 50-100 °C for time period of 8-20 hours to obtain the composition.
6. The process as claimed in claim 5, wherein the sophorolipid is myristic acid-derived sophorolipid (MASL) or oleic acid-derived sophorolipid.
7. The process as claimed in claim 5, wherein the calcium phosphate nanoparticles composition is dried in oven at a temperature of 40-80 °C for 6-24 hours.
8. A scaffold comprising: i. a polymer; and ii. the composition as claimed in claim 1.
9. The scaffold as claimed in claim 8, wherein the polymer is naturally occurring polymer or a synthetic polymer.
10. The scaffold as claimed in claim 8, wherein the naturally occurring polymer is selected from collagen, silk, chitosan, alginate, gelatin and combinations thereof; and the synthetic polymer is selected from polylactic acid, polyethlene oxide, polyglycolic acid, polyvinyl acetate and combinations thereof.
PCT/IN2023/050593 2022-06-21 2023-06-21 Compositions of calcium phosphate nano-particles with sophorolipids and process for preparation thereof WO2023248246A1 (en)

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ABHYANKAR ISHA, SEVI GANESH, PRABHUNE ASMITA A., NISAL ANUYA, BAYATIGERI SANTHAKUMARI: "Myristic Acid Derived Sophorolipid: Efficient Synthesis and Enhanced Antibacterial Activity", ACS OMEGA, ACS PUBLICATIONS, US, vol. 6, no. 2, 19 January 2021 (2021-01-19), US , pages 1273 - 1279, XP093122206, ISSN: 2470-1343, DOI: 10.1021/acsomega.0c04683 *

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