Preparing NANO-carriers containing curcumin and boswellic acid

Abstract

The optimized drug delivery system, with a diameter of less than 150 nm, can cross the Blood-Brain Barrier (BBB) and effectively target brain cells. This all-natural herbal formulation contains Curcumin from Turmeric and Boswellic acids from Frankincense, encapsulated in a vesicular structure. The surface of vesicular structure has been modified and optimized for maximum bioavailability. This Nanocarrier is used for prevention of Alzheimer's disease and memory enhancement. Animal studies confirmed its effectiveness, showing positive results.

Description

English Description DescriptionTitle of Invention : PREPARING NANO-CARRIERS CONTAINING CURCUMIN AND BOSWELLIC ACIDTechnical Field

[0001] The present invention relates to a method for treating an individuals at risk of or suffering from Alzheimer's disease with a method by which the drug crosses the blood-brain barrier (BBB).Background Art

[0002] In a patent published in the USPTO (Publication number: US20070160658), a delivery system is provided according to the present invention, which includes a delivery vehicle for a cargo moiety such as a diagnostic and / or therapeutic agent. The delivery vehicle is capable of crossing the blood-brain barrier and delivering a cargo moiety to the CNS. A delivery vehicle included in an embodiment of a system according to the invention includes particles capable of association with a cargo moiety for the delivery of the cargo to a target. A particle is capable of association with a cargo moiety where association does not inactivate a desired function of the cargo moiety and where the cargo moiety may be transported along with the particle to a desired location. Such particles include microspheres, nanoparticles, micelles, niosomes, and liposomes.

[0003] In a patent published in the USPTO (Publication number: US11173152), this new invention addresses the long-standing need for brain-targeting therapies for tauopathies and cancer. The invention involves a composition or method of treatment using nanoparticles containing MKT, encapsulated within biocompatible, non-toxic polymers like poly(lactide-co-glycolide). The nanoparticles are surface-coated with glutathione, which, due to its hydrophilic nature, enables MKT to effectively cross the blood-brain barrier (BBB). The composition allows for the sustained release of MKT over 72 hours, with nearly 100% release during this period. This innovation aims to reduce toxicity and enhance the effectiveness of brain-targeted therapies.

[0004] The method, formulation, and advantages of the claimed invention differ from those of the introduced patents.English Description Technical Problem

[0005] Current medications for Alzheimer's disease, which are designed to cross the blood-brain barrier (BBB), face several challenges. These drugs are produced in two forms: nano and conventional. The nano form offers high absorption but has drawbacks such as limited shelf life, the need for additives due to bad taste, difficulty in carrying because of its liquid nature, low product stability, and the requirement for refrigeration.

[0006] On the other hand, the conventional form benefits from a long shelf life, being flavorless, easy portability in capsule form, high product stability, and no need for refrigeration. However, its main drawback is low solubility and limited absorption,. In our invention, all these issues have been effectively resolved, offering a significant advancement in Alzheimer's drug delivery.Solution to Problem

[0007] The resulting optimized drug delivery system has a diameter of less than 150 nm, enabling it to traverse the Blood-Brain Barrier (BBB) and effectively target brain cells. This invention serves as a preventive against Alzheimer's disease, functions as a memory enhancement agent, and contributes to dementia prevention.

[0008] This invention is an all-natural herbal product composed of active components, including Curcumin (Cn) extracted from Turmeric and Boswellic acids from Frankincense. These active materials are encapsulated within a vesicular structure, optimized through experimental design. The vesicle structure, formed through a combination of surfactants, undergoes surface modification to maximize bioavailability for the active ingredients. The concentration of the surface modifier is fine-tuned through additional experiments.

[0009] To assess the efficacy of this invention, an animal study was conducted, confirming its ability to penetrate the BBB. The study involved administering various doses of this invention to animals with Alzheimer's induction, revealing a significant memory enhancement at the optimal dosage.

[0010] The niosome (Tablel) were prepared via the heating method according to a protocol previously reported by the current inventors. Briefly, Tween 60 (0.7-0.1English Description g) and span 60 (0.7-0.1g) were stirred for 1h in phosphate buffer (PBS, pH 7.4, autoclaved) at 25°C. In addition to surfactants, hydration of cholesterol (1 -01 g) was also done at 70-120°C in phosphate buffer and glycerol (2-10% v / v).Hydrated substances were added to the mixture of Dihexadecyl phosphate (at 40-70°C). Boswellic acids (BAs) (5-18 mg), and curcumin (Cn) (5-18 mg). The prepared suspension was stirred (at 40-70°C, 1000 rpm) on a hot plate for 60 min. Then, the sample was sonicated for 16 min. Stirring and heating were continued for 30-60 minutes. The prepared bare niosomes (BN) kept refrigerated (at 4-8°C) for applying chitosan (Figure 11 ).

[0011] Tablel :Independent Variables Dependent variablesSaSpan Tween Cholesterol Z average PDI Zeta potential EE% EE%(Cn) mple 60 60 (nm) (value) (mV) (BA)N1 0.134 0.133 0.133 121±0.34 0.20±0.005 -32±0. 8 90±0.48 89±0.38 N2 0.206 0.097 0.097 139±0.21 0.26±0.018 -25±0.2 55±0.1 60±0.2 N3 0.06 0.28 0.06 80±0.34 0.24±0.024 -21±0.1 90±0.43 89±0.33 N4 0.097 0.201 0.097 108±0.13 0.19±0.011 -25±0.01 80±0.22 85±0.32 N5 0.097 0.097 0.206 144±0.43 0.30±0.002 -27±0.3 95±0.23 95±0.32 N6 0.28 0.06 0.06 129±0.26 O.3O±O.OO3 -35±0.2 85±0.34 87±0.23 N7 0.06 0.06 0.28 137±0.45 0.15±0.001 -32±0.6 55±0.12 60±0.13

[0012] Tablel : Chitosan in acetic acid (1 -5% w / v) was stirred 24 h at 25°C. Then, Bare Niosome (BN) was added to the chitosan solution and the suspension was stirred for 30-60 min at 25°C. Finally, it was sonicated for 5 minutes to control the vesicle size.

[0013] Characterization of NPs Formulations:

[0014] Encapsulation efficiency:

[0015] Bare Niosome (BN) and Chitosan coated niosomes (ChN) suspension (5ml) were transmitted into centrifuge Amicon MPS (Millipore, USA) filtration tube, and then centrifuged (Hettich Tuttlingen centrifuge, model EBA 20, Germany) at 3460”g to separate BN and / or ChN from the supernatant. Cn and BAs values in the supernatant were determined by HPLC. Encapsulation efficiency was calculated according to equationl (Bhushan et al. 2013).EE%= (Total A- A in supernatant) / (Total A) x100 Eq. 1English Description where A is Cn or BAs content.

[0016] High performance liquid chromatography (HPLC) analysis:

[0017] The Cn and BAs determination was performed on HPLC (Waters, USA) at 40°C and 30°C, respectively. HPLC system consisted of a binary pump, a 2489 Uv-Vis detector, and an Inertsustian Swift C18 column (4.6 x 250 mm, the particle size of 5pm). The mobile phase was composed of acetonitrile solution in water (5 v / v%), buffered by 10 % ortho-phosphoric acid (90:10 v / v) to pH 2.7, at a flow rate of 1 mL / min (Tsai et al. 2011 ; Yt et al. 2014). AB, AKBBA, BBA, and Cn concentration determined by measuring the absorption at 254, 254, 210, and 420 nm, respectively.

[0018] Size, polydispersity index, and zeta potential analysis

[0019] Size, PDI, and zeta potential of diluted BN and ChN (1 :10 v / v) were measured through Zeta sizer Nano ZS (Malvern Instruments Ltd., United Kingdom) (Bhushan et al. 2013).Advantageous Effects of Invention

[0020] This invention offers significant advantages over both the conventional product and the nano-based version. Unlike the standard product, it provides high absorption, ensuring better efficacy. Additionally, it boasts a long shelf life and is flavorless, addressing some of the limitations of both the regular and nano products. In comparison to the nano-based product, it is more stable, does not require refrigeration, and comes in a convenient capsule form for easy carry. This invention of high absorption, stability, and ease of use, along with its long shelf life and no need for refrigeration, makes it a more practical and effective option.Brief Description of Drawings

[0021] Fig.1 : TEM image of bare niosome, and chitosan-coated niosome.

[0022] Fig.2: FT-IR spectra of boswellic acid, cholesterol, chitosan, span 60, tween 60.

[0023] Fig.3: Cumulative release of boswellic acid in chitosan-coated niosomes containing curcumin and uncoated niosomes containing curcumin.English Description

[0024] Fig.4: Cumulative release of boswellic acid in chitosan-coated and uncoated niosomes containing boswellic acid

[0025] Fig.5: The concentration of curcumin and boswellic acid in niosomes with and without a chitosan coating in different parts of the central nervous system, plasma, and liver.

[0026] Fig.6: Latency time and the number of entries into the dark chamber in the shuttle test.

[0027] Fig.7: Time spent in the dark chamber in the shuttle box test.

[0028] Fig.8: The number of platform entries and time spent in the platform area by laboratory rats in different groups.

[0029] Fig.9: Swimming speed and distance traveled in the platform area in different groups.

[0030] Fig.10: Total distance traveled in different groups.Description of Embodiments

[0031] Further details on the characteristics of NPs formulations:

[0032] Transmission Electron Microscopy (TEM) Analysis:

[0033] The samples were diluted 10 times using deionized water to reduce the vesicle concentration. The diluted samples were added with a volume ratio equal to 2% uranyl acetate solution. After 2 min, the mixture was sonicated (1 min) in an ultrasound bath. Then, one drop of the obtained solution was placed on a copper mesh for 2 min, with the residual solution removed by filter papers, and then air-dried at room temperature. Finally, the sample was examined using a Leo 912 OMEGA TEM (Germany)(Mandal et al. 2013). TEM image (Figure 1) of bare niosome (a), and chitosan-coated niosome (b).

[0034] Fourier-transform infrared spectroscopy (FT-IR)

[0035] FT-IR (Thermo Nicolet, AVATAR, 370 FT-IR, USA) was used to examine the absorption bands of functional groups in the BA and ChN. The samples were scanned with a resolution of 4 cm-1between 4000 cm"1and 400 cm-1. FT-IREnglish Description spectra of boswellic acid (a), cholesterol (b), chitosan (c), span 60 (d), and tween 60 (e) are shown in Figure2.

[0036] Assessment of the stability of BN and ChN in the simulated digestivesystem

[0037] To assess the effect of the chitosan layer on the digestion process and the bioavailability of Cn and BAs, BN and ChN were placed into the simulated mouth fluid, gastric fluid (SGF), and intestinal fluid (SIF)(Cuomo et al. 2018).

[0038] Release studies:

[0039] The release of Cn was accomplished using the dialysis technique (Le and Kim 2019), (Xu et al. 2016). Sample (5 mL) were wrapped in a dialysis bag (D0666, Sigma). The dialysis bag was immersed in 50 mL of a simulated gastric fluid(SGF, HCI solution 0.1 N, pH = 1.2) and simulated intestinal fluid (SIF, PBS, pH 7.4) for 2h and 6 h, respectively in a digital shaker incubator (Hanyang, SI-64A, 50 rpm, 37°C).

[0040] The charts of cumulative release of boswellic acid in chitosan-coatedniosomes containing curcumin and uncoated niosomes containing curcumin(Figure 3), as well as in chitosan-coated and uncoated niosomes containingboswellic acid (Figure 4), can be seen.

[0041] Table2:

[0042] Table2: Different letters in a column indicate a statistically significantdifference between the mean values (P < 0.05). The data are presented as the mean ± standard deviation of three replicates.English DescriptionExamples

[0043] Animal study:

[0044] Wistar rats (12 male individuals, 190 g ±10g in body weight) were obtained from house breeding colonies at the Neuroscience Department of Mashhad, University of Medical Sciences and were kept under standard conditions (12h light and 12h dark cycle, 23± 1 °C, with easy access to food and water sources). Animal handling and all the other related procedures were approved by Medical Sciences, Ethical Committee Acts (Mashhad, Iran, IR. mums. Rec.1399.287). The minimum number of animals was used to respect animal rights.

[0045] Drug administration and accessing its bioavailability:

[0046] The rats were randomly divided into the following three groups (4 rats in each group): treated with free Cn (group I), treated with BN (group II), and treated with ChN (group III). An aqueous solution of carboxymethylcellulose (CMC, 0.5%w / v) was then applied for preparing free Cn solution(Hoppe et al. 2013). Subsequently, the 50 mg / kg of freshly Cn suspension, BN, and ChN were administered by intraperitoneal injection (i.p.) to the rats(Tsai et al. 2011). After 15 minutes from the injection, the experimental rats were anesthetized by a mixture containing 10% ketamine (100 mg / kg body weight) and 2% xylazine (30 mg / kg body weight) (Tiwari et al. 2014).

[0047] Then, the cardiac puncture was utilized to collect 400 pl of blood with a heparinized syringe. Thereafter, the rats were perfused with normal saline to remove their brain and liver. Moreover, to evaluate crossing the BBB, the cerebral cortex, hippocampus, cerebellum, and striatum were collected.

[0048] Afterward, the samples were stored in microtubes at -80°C. Cn was extracted from plasma, and different regions of the brain and liver according to the protein precipitation technique proposed by Dalvi (2018) with some modifications(Dalvi et al. 2018). Briefly, 400 pL methanol was added to 90 pL Plasma. The mixture was then vortexed for 2 min, and the samples were centrifuged at 14000g for 20 min at 4°C. The supernatant was dried under N2 gas at 40 °C. Finally, 100 pL mobile phase was added for reconstitution, which was then analyzed by HPLC (WatersEnglish Description equipped with 1525 binary pump along with UV 2485 and FLD2475 detector, USA).

[0049] Different regions of brain and liver tissues were homogenized using a probe sonicator for 1min (25 s on 15 s off) at 4°C in PBS (1 :4 W / V, pH: 7.4). Then, the mixture was centrifuged at 14000 g for 20 min at 4°C. The supernatant (100 pL) was moved to a microtube, and 200 pL methanol was added. After 2 minutes of vortexing, the sample followed the producer mentioned for the plasma samples.

[0050] Animals and experimental groups:

[0051] Wistar rats (12 male individuals, 190 g ±10g in body weight) were obtained from house breeding colonies at the Neuroscience Department of Mashhad, University of Medical Sciences and were kept under standard conditions (12h light and 12h dark cycle, 23± 1 °C).

[0052] Stereotaxic surgery, behavior, and cognitive tests:

[0053] The animals were deeply anesthetized with 10% ketamine (100 mg / kg body weight) and 2% xylazine (30 mg / kg body weight), placed on a stereotaxic frame, and the skull was exposed. The stereotaxic coordinates for the lateral ventricles were measured as anteroposterior(AP): - 0.8 mm from the bregma, mediolateral (ML): ±1.5 mm from the midline and dorsoventral (DV): - 3.8 mm from the skull (Paxinos and Watson, 1997). Through a skull hole, a 28-gauge stainless steel needle was lowered manually into each lateral ventricle.

[0054] The control of the flow of the injections was made by using an electronic pump (Insight, Brazil) at a rate of 1.0 pL / min, for 4.5 min, followed by 2 min with the needle in the injection site to avoid reflux. The lesioned group received bilateral icv injections of STZ (total of 3 mg / kg in sterile saline, 4.5 pL / injection site) into the lateral ventricles. Sham operations followed the same procedure, but the same volume of sterile saline (0.9 %) was injected instead of STZ (Bassani et al., 2017; Bassani et al., 2018; Moura et al., 2020). The drug STZ was dissolved in cold sterile saline, protected from light and kept on ice until the time of injection in order to prevent its degradation.

[0055] All rats received Pentabiotico® (0.1 mL intramuscular) to avoid infections.After surgery, the animals took approximately 2-4 h to recover from anesthesia.English Description In the meanwhile, they were kept in a heated and wellventilated room. Food and water was placed inside the cage for 7-10 days so that the animals could easily access it without physical trauma due to head injury.

[0056] Drug administration and memory parameter:

[0057] The rats were randomly divided into the following four groups (8 rats in each group): control (group I), sham (group II), Alzheimer (group III), and treated with ChN (group IV). The 2.5 mg / kg of freshly ChN were administered by intraperitoneal injection (i.p.) to the rats for 21 days.

[0058] Behavioral tests:

[0059] Water maze and shuttle box Tests were performed 21 days after STZ infusion, to analyze the memory activity of the animals. The charts related to the test results are as follows.

[0060] Fig.5: Chart shows the concentration of curcumin and boswellic acid in chitosan-coated and uncoated niosomes across different regions of the central nervous system, plasma, and liver 15 minutes after i.p. administration. A statistically significant difference is observed between the mean values (P<0.05). Data are presented as mean ± standard deviation of three replicates.

[0061] Fig.6: The charts show the latency time and the number of entries into the dark chamber in the shuttle test, with data showing a statistically significant difference (P<0.05). Results are expressed as the mean ± standard deviation of three replicates.

[0062] Fig.7: The charts show the time spent in the dark chamber in the shuttle box test, with data showing a statistically significant difference (P<0.05). Results are expressed as the mean ± standard deviation of three replicates.

[0063] Fig.8: The number of times the rat accessed the platform and the time spent in the platform area across different groups (statistically significant differences were observed among the data, P<0.05). The data are presented as the mean ± standard deviation of three repetitions.

[0064] Fig.9: The charts illustrate the swimming speed, and the distance traveled in the platform area across different groups, with data showing a statisticallyEnglish Description significant difference (P<0.05). Results are expressed as the mean ± standard deviation of three replicates.

[0065] Fig.10: The charts show the total distance traveled across different groups, with data showing a statistically significant difference (P<0.05). Results are expressed as the mean ± standard deviation of three replicates.

Claims

English Claims Claims

1. A process for preparing nanocarriers containing curcumin and boswellic acid, which comprising:a. Polysorbate 60 (Tween 60, C64H126026);b. Sorbitan Monostearate (span 60, C24H46O6);c. Dihexadecyl Phosphate (CH3(CH2)15O]2P(O)OH);d. Cholesterol (C27H46O);e. Boswellic Acid (C30H48O3);f. Curcumin (C21H20O6); andg. Chitosan (C12H24N2O9).

2. The process for preparing nanocarriers containing curcumin and boswellic acid nanoparticles of claim 1 , which comprising:The weight ratio of the Tween 60 to the Nanocarrier about 0.01 :1 to about 0.07:1 ;The ratio of the Span 60 to the Nanocarrier is about 0.01 :1 to about 0.07:1 ; The weight ratio of the Cholesterol to the Nanocarrier is about 0.1 :1 to about 0.001 :1 ;The weight ratio of the Glycerol to the Nanocarrier is about 0.02:1 to about 0.10:1 ;The weight ratio of the Boswellic Acid to the Nanocarrier is about 0.0005:1 to about 0.0018:1 ;The weight ratio of the Curcumin to the Nanocarrier is about 0.0005:1 to about 0.0018:1 ;The molar ratio of the Dihexadecyl Phosphate to the surfactants is about 0.01 :1 to about 0.15:1 ; andThe weight ratio of the Chitosan to the Nanocarrier is between about 0.01 :1 to about 0.05:1.English Claims

3. The process for preparing nanocarriers containing curcumin and boswellic acid nanoparticles of claim 1 , which comprising:forming a first mixture by mixing Tween 60 and Span 60 in Phosphate Buffer;forming a second mixture based upon mixing Cholesterol, Glycerol, and Surfactants in Phosphate Buffer;forming a third mixture based upon mixing Boswellic Acid and Curcumin and Dihexadecyl Phosphate ;forming a fourth mixture based upon mixing Chitosan and Acetic Acid (CH3COOH); andcombining the first mixture, the second mixture, the third mixture and the fourth mixture to produce the product.

4. The method of claim 3, wherein the combining of the product comprising:The first mixture is stirred for 1h in Phosphate Buffer (PBS, pH 7.4, autoclaved) at 25°C;The second mixture is heated to 70-120°C in Phosphate Buffer; The second mixture (Hydrated substances) is added to the Dihexadecyl Phosphate 40-70°C;The third mixture is stirred at 40-70°C and 1000 rpm for 60 minutes;The first mixture, second mixture and third mixture are Sonicated for 16 minutes, stirring and heating are continued for 30-60 minutes, the prepared Bare Niosomes (BN) are kept refrigerated 4-8°C for applying chitosan;The fourth mixture is stirred for 24 hours at 25°C, after which the first mixture and the second mixture, to which Dihexadecyl Phosphate has been added, are combined with the third mixture, to which Bare Niosomes (BN) have been added to the Chitosan solution, and the resulting suspension is stirred for 30-60 minutes at 25°C and sonicated for 5 minutes to control the vesicle size.

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