Description:1
FORM-2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE
SPECIFICATION
(See section 10 and rule 13)
Transferrin-decorated chitosan-ethylcellulose nanoparticles for controlled and targeted delivery of myricetin for diabetes mellitus
G D Goenka University, an Indian university of Sohna Gurugram Road, Sohna, Haryana, India, 122103
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED
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FIELD OF INVENTION:
The present invention relates to pharmaceutical science field, which aims at design and development of Transferrin-decorated chitosan-ethylcellulose nanoparticles for controlled and targeted delivery of myricetin for diabetes mellitus management.
BACK GROUND:
Diabetes, also known as diabetes mellitus, is a group of common endocrine diseases characterized by sustained high blood sugar levels. Diabetes is due to either the pancreas not producing enough insulin, or the cells of the body not responding properly to the insulin produced. Diabetes, if left untreated, leads to many health complications. Untreated or poorly treated diabetes accounts for approximately 1.5 million deaths per year.
There is no widely accepted cure for most cases of diabetes. The most common treatment for type 1 diabetes is insulin replacement therapy (insulin injections). Anti-diabetic medications such as metformin and semaglutide, as well as lifestyle modifications, can be used to prevent or respond to type 2 diabetes. Gestational diabetes normally resolves shortly after delivery.
Most medications used to treat diabetes act by lowering blood sugar levels through different mechanisms. There is broad consensus that when people with diabetes maintain tight glucose control – keeping the glucose levels in their blood within normal ranges – they experience fewer complications, such as kidney problems or eye problems. There is however debate as to whether this is
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appropriate and cost effective for people later in life in whom the risk of hypoglycemia may be more significant.
There are a number of different classes of anti-diabetic medications. Type 1 diabetes requires treatment with insulin, ideally using a "basal bolus" regimen that most closely matches normal insulin release: long-acting insulin for the basal rate and short-acting insulin with meals. Type 2 diabetes is generally treated with medication that is taken by mouth (e.g. metformin) although some eventually require injectable treatment with insulin or GLP-1 agonists.
Metformin is generally recommended as a first-line treatment for type 2 diabetes, as there is good evidence that it decreases mortality. It works by decreasing the liver's production of glucose, and increasing the amount of glucose stored in peripheral tissue. Several other groups of drugs, mainly oral medication, may also decrease blood sugar in type 2 diabetes. These include agents that increase insulin release (sulfonylureas), agents that decrease absorption of sugar from the intestines (acarbose), agents that inhibit the enzyme dipeptidyl peptidase-4 (DPP-4) that inactivates incretins such as GLP-1 and GIP (sitagliptin), agents that make the body more sensitive to insulin (thiazolidinedione) and agents that increase the excretion of glucose in the urine (SGLT2 inhibitors). When insulin is used in type 2 diabetes, a long-acting formulation is usually added initially, while continuing oral medications.
Some severe cases of type 2 diabetes may also be treated with insulin, which is increased gradually until glucose targets are reached.
Drugs are administered parenterally, e.g., by injection of drug solutions, for various reasons. For example, injection, rather than oral administration, is used for compounds which partially or totally degrade in the gastrointestinal tract. Injections are also preferred when a rapid response is required, i.e., where the time lag between oral administration of the drug and its action on the target site is too long. In addition, the effective use of drugs often requires continuous,
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controlled parenteral administration to achieve the desired effect. This type of prolonged parenteral administration also has been achieved by the injection of drug solutions.
The continuous parenteral delivery of drugs can be accomplished by mechanical perfusion devices that include a catheter and needle, or by sustained release compositions that typically include a drug and a carrier such as polylactide polymers that retards the release of the drug so that it is slowly dispensed over time.
Sonali 2016 et al. formulate transferrin-conjugated docetaxel (DTX)-loaded d-alpha-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or TPGS) micelles for targeted brain cancer therapy. The micelles with and without transferrin conjugation were prepared by the solvent casting method and characterized for their particle size, polydispersity, drug encapsulation efficiency, drug loading, in vitro release study and brain distribution study. Particle sizes of prepared micelles were determined at 25 °C by dynamic light scattering technique. The external surface morphology was determined by transmission electron microscopy analysis and atomic force microscopy. The encapsulation efficiency was determined by spectrophotometery. In vitro release studies of micelles and control formulations were carried out by dialysis bag diffusion method. The particle sizes of the non-targeted and targeted micelles were <20 nm. About 85% of drug encapsulation efficiency was achieved with micelles. The drug release from transferrin-conjugated micelles was sustained for >24 h with 50% of drug release. The in vivo results indicated that transferrin-targeted TPGS micelles could be a promising carrier for brain targeting due to nano-sized drug delivery, solubility enhancement and permeability which provided an improved and prolonged brain targeting of DTX in comparison to the non-targeted micelles and marketed formulation.
Zhang, Y 2020 et al. constructed AS1411 aptamer-based gold nanoparticles with appropriate size facilitating endocytosis and actively targeted drug delivery for gastric cancer cells via the specific AS1411–nucleolin interaction. The AS1411-
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based nanoparticles showed obviously increased targeted capacity towards AGS cells compared to random ssDNA-based nanoparticles. Meanwhile, compared to L929 cells, the AS1411-based nanoparticles showed selective drug uptake and delivery for AGS cells. Importantly, the AS1411-based nanoparticles exhibited significantly stronger antitumor effects on AGS cells under laser irradiation compared to chemotherapy alone. Our nanoparticles combined targeted drug delivery and efficient antitumor effects within one single nanoplatform, which are promising to be applied as targeted nanomedicines against gastric cancer.
Diabetes mellitus (hyperglycemia), a metabolic disorder, is caused either due to lower insulin secretion by the cells or due to lower binding efficiency of insulin on their cell surface receptors resulting in high blood glucose level. Insulin and other diabetes medications, such as glucagon peptide 1, must be given subcutaneously due to the harsh environment of the gastrointestinal system, which can be uncomfortable and cause low patient compliance. Long term utilization of conventional drug results in drug toxicity in patients. Herewith, we have developed a novel strategy for diabetes management using transferrin-decorated chitosan-ethylcellulose nanoparticlescontaining myricetin. D-alpha-Tocopheryl polyethylene glycol 1000 succinate (TPGS) is a biocompatible polymer that is recommended by USFDA to use in drug delivery technology. It possesses extraordinary features such as high hydrophilic-lipophilic balance (13.5) and offers several good characteristics as an emulsifier, solubilizer, drug penetrating agent, and biocompatible surfactant. Therefore, we have synthesized a novel conjugate of both D-alpha-tocopheryl polyethylene glycol 1000 succinate-transferrin (TPGS-Tf) for targeted delivery myricetin in diabetes management.
In diabetes management, the targeed delivery of therapeutic agents is much challenging. Therefore, we have developed TPGS- TF conjugate through the carbodiimide chemistry and conjugated to the surface of chitosan-modified ethylcellulose nanoparticles for diabetes cell specific delivery of myricetin in diabetes therapy of patients.The nanoparticles were synthesized by a modified emulsion/solvent evaporation method.The prepared formulations were
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characterized for Fourier-Transform Infrared Spectroscopy, 1H Nuclear Magnetic Resonance (NMR) Spectroscopy, and Electrospray Ionisation Mass Spectrometry analysis.
OBJECTIVE OF THE INVENTION:
1. It is an object of the invention to provide a new strategy to formulate TPGS-Tf conjugated chitosan-modified ethylcellulose nanoparticles of myricetin.
2. It is another object of the invention to provide formulation is biodegradable, non-cytotoxic and biocompatible and demonstrates the controlled and sustained delivery of myricetin.
3. It is another object of the invention to develop TPGS- TF conjugate through the carbodiimide chemistry and conjugated to the surface of chitosan-modified ethylcellulose nanoparticles for diabetes cell specific delivery of myricetin in diabetes therapy of patients.
4. It is another object of the invention to develop and characterize transferrin-decorated chitosan-ethylcellulose nanoparticles for controlled and targeted delivery of myricetin for diabetes mellitus management
5. It is another object of the invention to provide a process of synthesis of transferrin-decorated chitosan-ethylcellulose nanoparticles for controlled and targeted delivery of myricetin for diabetes mellitus.
6. It is another object of the invention to provide a process of activation of D-alpha-Tocopheryl polyethylene glycol 1000 succinate (TGPS) as TGPS-COOH.
7. It is another object of the invention to provide a process of synthesis of TPGS-Tf conjugate.
SUMMARY
Diabetes mellitus (hyperglycemia), a metabolic disorder, is caused either due to lower insulin secretion by the cells or due to lower binding efficiency of
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insulin on their cell surface receptors resulting in high blood glucose level. Insulin and other diabetes medications, such as glucagon peptide 1, must be given subcutaneously due to the harsh environment of the gastrointestinal system, which can be uncomfortable and cause low patient compliance. Long term utilization of conventional drug results in drug toxicity in patients. Herewith, we have developed a novel strategy for diabetes management using transferrin-decorated chitosan-ethylcellulose nanoparticlescontaining myricetin. D-alpha-Tocopheryl polyethylene glycol 1000 succinate (TPGS) is a biocompatible polymer that is recommended by USFDA to use in drug delivery technology. It possesses extraordinary features such as high hydrophilic-lipophilic balance (13.5) and offers several good characteristics as an emulsifier, solubilizer, drug penetrating agent, and biocompatible surfactant. Therefore, we have synthesized a novel conjugate of both D-alpha-tocopheryl polyethylene glycol 1000 succinate-transferrin (TPGS-Tf) for targeted delivery myricetin in diabetes management. In diabetes management, the targeed delivery of therapeutic agents is much challenging. Therefore, we have developed TPGS- TF conjugate through the carbodiimide chemistry and conjugated to the surface of chitosan-modified ethylcellulose nanoparticles for diabetes cell specific delivery of myricetin in diabetes therapy of patients.The nanoparticles were synthesized by a modified emulsion/solvent evaporation method.The prepared formulations were characterized for Fourier-Transform Infrared Spectroscopy, 1H Nuclear Magnetic Resonance (NMR) Spectroscopy, and Electrospray Ionisation Mass Spectrometry analysis.
BRIEF DESCRIPTION OF FIGURES
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying figures in which like characters represent like parts throughout the figures, wherein:
Figure 1, illustrates a view of Schematic representation of the preparation of nanoparticles for the present invention.
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Fig. 2: illustrates a view of FTIR spectra of myricetin for the present invention.
Fig.3: illustrates a view of FTIR spectra of TPGS for the present invention.
Fig.4: illustrates a view of FTIR spectra of ethylcellulose for the present invention.
Fig: 5: illustrates a view of FTIR spectra of chitosan for the present invention.
Fig: 6: illustrates a view of FTIR spectra of TPGS-Tf conjugate for the present invention.
Fig: 7: illustrates a view of FTIR spectra of drug and polymer combination for the present invention.
Fig: 8: illustrates a view of 1HNMR spectra of TPGS- Tf conjugate for the present invention.
Fig: 9: illustrates a view of Average Particle size of CENP, MCENP, MCENP-Tf decorated chitosan ethylcellulose nanoparticles for the present invention.
Fig: 10: illustrates a view of Zeta potential of CENP, MCENP, MCENP-Tf decorated chitosan ethylcellulose nanoparticles for the present invention.
Fig: 11: illustrates a view of TEM images of (i) CENP, (ii) MCENP, and (iii) MCENP-Tf decorated chitosan-ethylcellulose nanoparticles for the present invention.
Fig: 12: illustrates a view of Encapsulation efficiency of MCENP and MCENP-Tf decorated chitosan-ethylcellulose nanoparticles for the present invention.
Fig: 13: illustrates a view of In-vitro drug release study of Myricetin, MCENP and MCENP-Tf decorated chitosan-ethylcellulose nanoparticles in phosphate buffered saline (pH 7.4) (n = 3) for the present invention.
Further, skilled artisans will appreciate that elements in the figures are illustrated for simplicity and may not have been necessarily been drawn to scale. For example, the flowcharts illustrate the method in terms of the most prominent steps involved to help to improve understanding of aspects of the present invention. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the figures by conventional symbols, and the figures may show only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the figures with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.
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DETAILED DESCRIPTION:
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or systems or elements or structures or components proceeded by "comprises... a" does not, without more constraints, preclude the existence of other devices or other systems or other elements or other structures or other components or additional devices or additional systems or additional elements or additional structures or additional components.
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Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items.
The terms “having”, “comprising”, “including”, and variations thereof signify the presence of a component.
Now the present invention will be described below in detail with reference to the following embodiment.
Example 1
Preparation of TPGS-Tf conjugate
Activation of TPGS as TPGS-COOH
In brief, 0.77 g of TPGS (equivalent to 0.5 mM), 0.10 g of succinic anhydride (equiv. to 1 mM) and 0.12 g of DMAP (equiv. 1 mM) were added in round bottom flask and then heated at near about 100 ? under inert nitrogen atmosphere for 24 h. Then the whole resultant mixture was cooled at 25 ? afterward it was dissolved in 10 ml of dichloromethane (DCM). Then the product was filtered through the Whatmann filter to remove unreacted succinic anhydride and DMAP and further, the product was precipitated in di ethyl approximately at -10? overnight. The obtained white precipitate of TPGS-COOH was filtered by filter paper (150 mm) and dried under a vacuum. The prepared TPGS-COOH intermediate was stored in freeze in an eppendorf tube till further analysis and used for micelles formations
Synthesis of TPGS- Tf conjugate
TPGS-TF conjugate was synthesized by the carbodiimide chemistry method. In brief, TPGS-COOH was reacted with EDC and NHS with a molar ratio of 1:5 (TPGS-COOH: EDC or
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NHS) in phosphate buffer saline (PBS) at pH 5.5. 200 mg of TPGS-COOH, 96 mg of EDC and 74 mg of NHS were mixed in 8 ml of PBS at pH 5.5 at 25 °C for 5-6 h and then stored in a refrigerator at 4? over 24 h. Then a 1 ml of 0.2% (w/v) TF was added into the solution and allowed to stirred at 4? over 8 h for conjugation to the TPGS-COOH. Final product was dialyzed using a dialyzing membrane (MWCO: 12 kDa) against PBS to remove the excess/unreacted TPGS-COOH, NHS, and EDC. At last, the prepared TPGS-TF conjugate product was freeze-dried, stored and used for preparation of micelles formations..
Preparation of nanoparticles
Nanoparticles were prepared by slightly modified solvent evaporation method technique depicted in Table 1 [Shah et al., 2009]. In brief 3 mg of myricetin, 20 mg of ethylcellulose, 30 mg of TPGS was dissolved in 7 ml of DMSO. For the preparation of aqueous phase, 20 mg of chitosan was dissolved in 1% acetic acid and the pH was adjusted to 6.0 using sodium hydroxide solution.After that the organic phase was added to the aqueous phase by dropwise at continuous stirring on magnetic stirrer at 300 rpm. Then the resulting mixture was ultrasonicated (UP200Ht ultrasonic processor) for 8 min at 60% amplitude to form fine droplets of emulsion and afterwards the organic solvent was evaporated completely by stirring overnight. Formed nanoparticles were filtered using a 0.45 µm PVDF syringe filter (MCENP: Chitosan coated ethylcellulose nanoparticles containing myricetin) then centrifuged (Remi, C23 Plus, Refrigerated Centrifuge, Mumbai, India) for 40 min at 7,000 rpm at 4 0C. Wash the nanoparticles with distilled water and centrifuge the suspension. Nanoparticles was dried by rotary evaporator (Rotapaor® R100, Buchi, Switzerland) then collected in to 3 ml of PBS at pH 7.4 and in 15 ml falcon tube. Similar procedure was used for the preparation of Tf decorated chitosan coated ethylcellulose nanoparticle (MCENPs-Tf: Transferrin decorated chitosan coated ethylcellulose nanoparticles containing myricetin) 10 mg of TPGS-Tfconjugate to enhance drug loading and improve drug bioavailability in diabetic patients (Fig.1).
Table 1Formula for preparation of nanoparticles
Formulation
Formulation
Myricetin
Myricetin
(in mg)
(in mg)
TPGS
TPGS
(in mg)
(in mg)
Ethylcellulose
Ethylcellulose
(in mg)
(in mg)
Chitosan
Chitosan
(in mg)
(in mg)
Conjugate
Conjugate
(in
(in mg)mg)
Myricetin
Myricetin
3
3
-
-
-
-
-
-
-
-
12
CENP
CENP
-
30
30
20
20
20
20
-
-
MCENP
MCENP
3
3
30
30
20
20
20
20
-
-
MCENP
MCENP--TfTf
3
3
30
30
20
20
20
20
10
10
Example 2
Characterization of nanoparticles
Determination of ? max
Determination of ? max
The ?max of myricetin was determined in different solvent systems using a UV The ?max of myricetin was determined in different solvent systems using a UV spectrophotometer. In brief, a 3 mg of myricetin was accurately weighed and dissolved in 10 ml spectrophotometer. In brief, a 3 mg of myricetin was accurately weighed and dissolved in 10 ml of methanol to make a drug stock solution. The prepared stock solution was used to mof methanol to make a drug stock solution. The prepared stock solution was used to make further ake further dilution of myricetin at different concentrations (i.e., 3, 6, 9, 12, 15 µg/ml) in methanol or dilution of myricetin at different concentrations (i.e., 3, 6, 9, 12, 15 µg/ml) in methanol or phosphate buffer saline pH 7.4, separately shown in Fig.4. The volumetric flasks were vortexed phosphate buffer saline pH 7.4, separately shown in Fig.4. The volumetric flasks were vortexed for 10 min at room temperature to make homogeneous solfor 10 min at room temperature to make homogeneous solutions. The samples were filtered utions. The samples were filtered through a 0.45 µm syringe filter before being transferred into a UVthrough a 0.45 µm syringe filter before being transferred into a UV--visible spectrophotometer visible spectrophotometer cuvette. The UV absorption spectrum was recorded for all dilutions using UV range of 200cuvette. The UV absorption spectrum was recorded for all dilutions using UV range of 200--400 400 nm. All measurements were carried onm. All measurements were carried out in triplicate (n = 3). ut in triplicate (n = 3).
Characterization of drug, polymer, surfactant and conjugates by FTIR spectroscopy
a) FTIR spectroscopy of myricetin
Fig.2 shows FTIR spectroscopy of myricetin. It was performed to identify the bond linkages and functional groups associated with Myricetin. These groups are important to understand their participation in the reduction process. The results revealed four peaks at 3400.91, 1657.41, 1380.13, and 1070.44 cm-1. The absorption band at 3400.91 cm-1was assigned to the stretching vibration of ? (O-H). The absorption band at 1657.41cm-1was attributed to the stretching vibration of ? (C=C).25 The absorption band at 1380.13cm-1 was the in-plane bending vibration of d (O-H). Moreover, the band at 1070cm-1 was contributed by the skeletal C-O
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bond of glycosidic linkage. It showed the presence of the residual flavonoids on the surface of Myricetin.
FTIR spectroscopy of TPGS
Fig.3 shows FTIR spectra of the TPGS, The characteristic peak of carbonyl band –C=O of TPGS appears at 1646 cm-1. The alkyl –CH stretch of TPGS was observed at 2923.27 cm-1. The peaks at 1151.52, 1301.34 and 1401.98 cm-1 were observed for (–C–O–) stretching of TPGS.
FTIR spectroscopy of ethylcellulose Fig.4 shows the FT-IR spectrum of EC displayed distinct peaks at 3,486 cm-1 (O-H stretching), 2875.17 cm-1 (C-H stretching), 1402.15 cm-1 (-CH3 bending), and 1064.83 cm-1 (C-O stretching in the cyclic ether). A shift of EC band from 3402.71 to 3658.9 cm-1 may be attributed to hydrogen bond formation.
FTIR spectroscopy of chitosan
Fig.5 shows the FTIR spectroscopy of chitosan.Amine deformation vibrations usually produce strong to very strong bands in the 1523.14-1627.24 cm-1region. We therefore propose that the band at 1523.14cm-1 is the N-H bending vibration overlapping the amide II vibration and that the 1627.24 cm-1band is the amide I vibration. These positions correspond to the amide I, amide II, and N-H bending vibrations, respectively. In addition, C-N stretching vibrations occur in the 1154-1026cm-1 region and overlap the vibrations from the carbohydrate ring. N-H stretching also occurs in the 3401 cm-1region overlapping the OH stretch from the carbohydrate ring.
FTIR spectroscopy of TPGS-Tf conjugate
Fig.6 Shows the FTIR spectroscopy of the prepared conjugate. In transferrin-conjugated TPGS, a characteristic peak at 1643.97 cm-1 was appeared which corresponds to the bonded amide stretching vibrations, thus confirming the successful conjugation of transferrin from TPGS-COOH. Peak at 3433.76 cm-1was observed for terminal hydroxyl group. The above results indicate the formation of TPGS-Tf conjugate .
FTIR spectra of drug polymer combination
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Fig.7 shows the FTIR spectroscopy of myricetin, ethyl cellulose,chitosan and TPGS combination A shift of EC band from 3402.71 to 3658.9 cm-1 may be attributed to hydrogen bond formation in the combination of ftir. The band at 1070 cm-1 is contributed by the skeletal C-O bond of glycosidic linkage, it showed the presence of the residual flavonoids on the surface of Myricetin.
Example 3
Characterization of polymer, surfactant and conjugates by 1HNMR spectroscopy
a) 1HNMR spectra of TPGS
NMR spectra of TPGS and TPGS-Tf represented the characteristic peaks which confirmed the of Tf conjugate from TPGS. In 1 H NMR spectra of TPGS, the triplet signals at 3.697 ppm were observed which belongs to –CH2 protons (poly ethyleneoxide) part of TPGS, respectively. The results confirmed the formation of TPGS-Tf. The 1 H NMR spectra of TPGS-Tf demonstrated a signal at 4.722 ppm which confirms the conjugation between free amino group of Tf and carboxylic group of TPGS-COOH.
b) 1HNMR spectra of ethyl cellulose
In 1H NMR spectrum of EC sample, characteristic peaks are as follows: a triplet at 4.73 ppm (HC-I), a peak at 4.8 (HC-III), 4.869.
c) 1HNMR spectra of chitosan
The protons of H-1 [GlcN (H-1D) and GlcNAc (H-1A)] resonate at 4.693 and 4.8 ppm, respectively. The signals of the latter protons overlap with HOD signals of the solvent (D2O/ CD3COOD) at 4.691 ppm. The chemical shifts for residual protons of the solvents have been reported to be [D20 (d = 4.730 ppm).
d) 1HNMR spectra of TPGS-Tf conjugate
TPGS-Tf represented the characteristic peaks which confirmed the synthesis of TPGS-Tf (activated TPGS) and conjugation of Tf to TPGS-COOH. In 1 H NMR spectra of TPGS-Tf, the triplet signals at 3.697 ppm were observed which belongs to –CH2 protons (poly ethyleneoxide) part of TPGS-COOH, respectively. The results confirmed the formation of TPGS-Tf. The
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1HNMR spectra of TPGS-Tf demonstrated a signal at 4.722 ppm which confirms the conjugation between free amino group of Tf and carboxylic group of TPGS-COOH.
Example 4
Particle size, PDI and ? analysis of nanoparticles:
The mean particle size and polydispersity index and ? potential values of prepared nanoparticles CENP, MCENP, MCENP-Tf are listed in Table 2, and Fig 9. The average hydrodynamic particles size of CENP, MCENP, MCENP-Tf nanoparticles was found to be 91.38 ±3.5 nm, 181.43±4.3 nm, 194.71±3.8 nm, respectively. This result may be influenced by loading of myricetin in polymerric core which have increased the size of nanoparticles. A similar trend was also observed for MCENP-Tf, it may also influence due to the incorporation of targeting ligand TPGS- Tf on the surface nanoparticles nanoparticles. PDI of all nanoparticles formulations has shown quite narrow size distribution closure to 0.1–0.3. The observed PDI results can be taken into account as they indicate a homogenous population of chitosan nanoparticles in prepared formulations.
Table 2:Particle size, polydispersity, zeta potential and encapsulation efficiency of nanoparticles
Batches
Batches
Particle size
Particle size (nm)(nm)
(mean ± S.D
(mean ± S.D**))
Polydispersity
Polydispersity (mean ± S.D.(mean ± S.D.**))
Zeta
Zeta PotentialPotential
(mV)
(mV)
Encapsulation
Encapsulation Efficiency(%) Efficiency(%) (mean ±S.D.*(mean ±S.D.*
CENP
CENP
91.38±3.51
91.38±3.51
16.2±2.5
16.2±2.5
26.3±1.59
26.3±1.59
-
-
MCENP
MCENP
185.39±4.30
185.39±4.30
24.5±4.6
24.5±4.6
24.3±3.56
24.3±3.56
74.42±3.07
74.42±3.07
MCENP
MCENP--TfTf
194.71±3.80
194.71±3.80
24.9±3.8
24.9±3.8
26.4±2.3
26.4±2.3
66.94±2.88
66.94±2.88
CENP: Chitosan Ethylcellulose nanoparticles
MCENP: Chitosan coated ethylcellulose nanoparticles containing myricetin
MCENP-Tf: Transferrin decorated chitosan coated ethylcellulose nanoparticles containing myricetin
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? potential value is an important characteristic of nanoparticles which affects its physical stability. The ? potential measurement is feasible tool which conforms the colloid electrostatic behavior of prepared nanoparticles with normal and diabetic cells. The ? values of CENP, MCENP and MCENP –Tf nanoparticles were found (+)26.3, (+)24.3, (+)26.4 mV, respectively, shown in Fig.10.
Transmission Electron Microscopy (TEM)
Morphological of CENP, MCENP, MCENP-TF nanoparticles were investigated by TEM analysis (Fig.11). The study revealed that CENP, MCENP and MCENP - Tf loaded chitosan nanoparticles were spherical with a size upto 100–200 nm. In the next experiment, the morphology of prepared nanoparticles was confirmed using TEM equipment. After loading of myricetin in nanoparticles formulation, the sizes of nanoparticles was increased and have shown spherical shape of the nanoparticles. While MCENP-Tf nanoparticles have shown larger size due to conjugation of TPGS-Tf on the surface of nanoparticles.
Encapsulation efficiency of nanoparticles
Drug encapsulation efficiency is one of the critical parameters for evaluating the drug holding capacity at the selected concentrations of polymer and surfactant. The drug encapsulation efficiency of prepared chitosan nanoparticles are summarized in Table2 and Fig.12. MCENP nanoparticles have shown good drug encapsulation efficiency up to 77.06±3.07% which conforms the suitability of chosen concentrations of chitosan and TPGS for nanoparticles fabrication. The drug encapsulation efficiencies of MCENP-Tf nanoparticles were found 66.94±2.88 %. In MCENP-Tf nanoparticle encapsulation of drug was less because some amount of drug may be loss during stirring. These results can be explained by the fact that simultaneous loading of Ethyl cellulose and TPGS in nanoparticles, the stronger hydrophobic interaction in the core of chitosan nanoparticles.
In-vitro drug release studies
The cumulative drug release profile of myricetin from chitosan nanoparticles formulations such as MCENP, MCENP-Tf compared with myricetin powder in PBS (pH 7.4) under sink conditions are shown in Fig. 13. As seen in this figure, the amount of myricetin
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released from the coarse powder was 43.68% at 2 h, 68.93% at 4 h and 99.58% at 6 h. While the drug release from MCENP nanoparticles was about 32% in 24 h and only 57% of the drug was released in 72 h. Furthermore, the TPGS-Tf conjugated nanoparticles showed only 52% of drug release in 72 h. Thereafter the layer of surfactant and TF conjugate coating played a major role in the release that went sustained after 72 h. Therefore, it can be concluded that the improved sustained release efficiency of the prepared chitosan nanoparticles was highly dependent on the amount of surfactant and further modification with TPGS-COOH-Tf on the surface of polymer nanoparticles. It is noteworthy that the drug was released by polymer degradation and its solubilization by a diffusion mechanism through eroded nanochannels of polymeric core.
Variations and modifications of the foregoing are within the scope of the present invention. Accordingly, many variations of these embodiments are envisaged within the scope of the present invention.
The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching.
The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, and to thereby enable others skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omissions and substitutions of equivalents are contemplated as circumstances may suggest or render expedient, but such omissions and substitutions are intended to cover the application or implementation without departing from the spirit or scope of the present invention.
Acknowledgement
Inventor Rahul Pratap Singh acknowledges the fincial support from Science and Engineeting Research Board (SERB), New Delhi, for providing fincial support (EEQ/2019/000218) under the scheme of Empowerment and Equity Opportunities for Excellence in Science.
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We claim,
1. A process of synthesis of transferrin-decorated chitosan-ethylcellulose nanoparticles for controlled and targeted delivery of myricetin for diabetes mellitus comprising the steps of:
I. Activation of D-alpha-Tocopheryl polyethylene glycol 1000 succinate (TGPS) as TGPS-COOH
i.) 0.77 g of TPGS (equivalent to 0.5 mM), 0.10 g of succinic anhydride (equiv. to 1 mM) and 0.12 g of DMAP (equiv. 1 mM) are added in round bottom flask and then heated at near about 100 ? under inert nitrogen atmosphere for 24 h,
ii.) Cooling the resulted mixture at 25 ? after the termination of reaction,
iii.) Dissolving cooled mixture in 10 ml of dichloromethane (DCM),
iv.) Filtering the resultant product through Whatman filter to remove unreacted succinic anhydride and DMAP,
v.) Precipitating filtered product in diethyl ether at -10 0C for overnight,
vi.) Filtering the obtained white TPGS-COOH intermediate precipitate and dried in vacuum.
II. Synthesis of TPGS-Tf conjugate
19
i) 200 mg of TPGS-COOH, 96 mg of EDC and 74 mg of NHS are mixed in 8 ml of PBS at pH 5.5 at 25 °C for 5-6 h and then stored in a refrigerator at 4? over 24 h,
ii) Adding 1 ml of 0.2% (w/v) TF into the above solution and allowed to stirred at 4? over 8 h for conjugation to the TPGS-COOH,
iii) Final product is dialyzed using a dialyzing membrane (MWCO: 12 kDa) against PBS to remove the excess/unreacted TPGS-COOH, NHS, and EDC,
iv) At last, the prepared TPGS-TF conjugate product is freeze-dried, stored and used for preparation of micelles formations.
III. Synthesis of TPGS-Tf conjugate
i) 3 mg of myricetin, 20 mg of ethylcellulose, 30 mg of TPGS is dissolved in 7 ml of DMSO,
ii) For the preparation of aqueous phase, 20 mg of chitosan is dissolved in 1% acetic acid and the pH is adjusted to 6.0 using sodium hydroxide solution,
iii) After that the organic phase is added to the aqueous phase by dropwise at continuous stirring on magnetic stirrer at 300 rpm,
iv) Then the resulting mixture is ultrasonicated for 8 min at 60% amplitude to form fine droplets of emulsion and afterwards the organic solvent is evaporated completely by stirring overnight,
v) Formed nanoparticles are filtered using a 0.45 µm PVDF syringe filter (MCENP: Chitosan coated ethylcellulose nanoparticles
20
containing myricetin) then centrifuged for 40 min at 7,000 rpm at 40C,
vi) Washing the nanoparticles with distilled water and centrifuge the suspension,
vii) Nanoparticles are dried by rotary evaporator then collected in to 3 ml of PBS at pH 7.4 and in 15 ml falcon tube,
viii) Adding 10 mg of TPGS-Tf conjugate to enhance drug loading and improve drug bioavailability in diabetic patients.
2. The process of synthesis of transferrin-decorated chitosan-ethylcellulose nanoparticles as claimed in claim 1, wherein the size of nanoparticles is 194.71±3.80nm.
3. The process of synthesis of transferrin-decorated chitosan-ethylcellulose nanoparticles as claimed in claim 1, wherein the encapsulation efficiency of nanoparticles is 66.94±2.88%.
4. The process of synthesis of transferrin-decorated chitosan-ethylcellulose nanoparticles as claimed in claim 1, wherein the In-vitro drug release of nanoparticles is 52% of drug release in 72 h.
Dated this 28/07/2023 G D Goenka University, Sohna Gurugram Road, Sohna, Haryana, India, 122103
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ABSTRACT
Transferrin-decorated chitosan-ethylcellulose nanoparticles for controlled and targeted delivery of myricetin for diabetes mellitus
Diabetes mellitus, a metabolic disorder, is caused either due to lower insulin secretion by the cells or due to lower binding efficiency of insulin on their cell surface receptors resulting in high blood glucose level. Insulin and other diabetes medications, such as glucagon peptide 1, must be given subcutaneously due to the harsh environment of the gastrointestinal system, which can be uncomfortable and cause low patient compliance. Long term utilization of conventional drug results in drug toxicity in patients. Herewith, we have developed a novel strategy for diabetes management using transferrin-decorated chitosan-ethylcellulose nanoparticlescontaining myricetin. D-alpha-Tocopheryl polyethylene glycol 1000 succinate (TPGS) is a biocompatible polymer. It possesses extraordinary features such as high hydrophilic-lipophilic balance (13.5) and offers several good characteristics as an emulsifier, solubilizer, drug penetrating agent, and biocompatible surfactant. Therefore, a novel conjugate of both D-alpha-tocopheryl polyethylene glycol 1000 succinate-transferrin (TPGS-Tf) for targeted delivery myricetin in diabetes management is synthesized. , Claims:We claim,
1. A process of synthesis of transferrin-decorated chitosan-ethylcellulose nanoparticles for controlled and targeted delivery of myricetin for diabetes mellitus comprising the steps of:
I. Activation of D-alpha-Tocopheryl polyethylene glycol 1000 succinate (TGPS) as TGPS-COOH
i.) 0.77 g of TPGS (equivalent to 0.5 mM), 0.10 g of succinic anhydride (equiv. to 1 mM) and 0.12 g of DMAP (equiv. 1 mM) are added in round bottom flask and then heated at near about 100 ? under inert nitrogen atmosphere for 24 h,
ii.) Cooling the resulted mixture at 25 ? after the termination of reaction,
iii.) Dissolving cooled mixture in 10 ml of dichloromethane (DCM),
iv.) Filtering the resultant product through Whatman filter to remove unreacted succinic anhydride and DMAP,
v.) Precipitating filtered product in diethyl ether at -10 0C for overnight,
vi.) Filtering the obtained white TPGS-COOH intermediate precipitate and dried in vacuum.
II. Synthesis of TPGS-Tf conjugate
19
i) 200 mg of TPGS-COOH, 96 mg of EDC and 74 mg of NHS are mixed in 8 ml of PBS at pH 5.5 at 25 °C for 5-6 h and then stored in a refrigerator at 4? over 24 h,
ii) Adding 1 ml of 0.2% (w/v) TF into the above solution and allowed to stirred at 4? over 8 h for conjugation to the TPGS-COOH,
iii) Final product is dialyzed using a dialyzing membrane (MWCO: 12 kDa) against PBS to remove the excess/unreacted TPGS-COOH, NHS, and EDC,
iv) At last, the prepared TPGS-TF conjugate product is freeze-dried, stored and used for preparation of micelles formations.
III. Synthesis of TPGS-Tf conjugate
i) 3 mg of myricetin, 20 mg of ethylcellulose, 30 mg of TPGS is dissolved in 7 ml of DMSO,
ii) For the preparation of aqueous phase, 20 mg of chitosan is dissolved in 1% acetic acid and the pH is adjusted to 6.0 using sodium hydroxide solution,
iii) After that the organic phase is added to the aqueous phase by dropwise at continuous stirring on magnetic stirrer at 300 rpm,
iv) Then the resulting mixture is ultrasonicated for 8 min at 60% amplitude to form fine droplets of emulsion and afterwards the organic solvent is evaporated completely by stirring overnight,
v) Formed nanoparticles are filtered using a 0.45 µm PVDF syringe filter (MCENP: Chitosan coated ethylcellulose nanoparticles
20
containing myricetin) then centrifuged for 40 min at 7,000 rpm at 40C,
vi) Washing the nanoparticles with distilled water and centrifuge the suspension,
vii) Nanoparticles are dried by rotary evaporator then collected in to 3 ml of PBS at pH 7.4 and in 15 ml falcon tube,
viii) Adding 10 mg of TPGS-Tf conjugate to enhance drug loading and improve drug bioavailability in diabetic patients.
2. The process of synthesis of transferrin-decorated chitosan-ethylcellulose nanoparticles as claimed in claim 1, wherein the size of nanoparticles is 194.71±3.80nm.
3. The process of synthesis of transferrin-decorated chitosan-ethylcellulose nanoparticles as claimed in claim 1, wherein the encapsulation efficiency of nanoparticles is 66.94±2.88%.
4. The process of synthesis of transferrin-decorated chitosan-ethylcellulose nanoparticles as claimed in claim 1, wherein the In-vitro drug release of nanoparticles is 52% of drug release in 72 h.