1. A novel therapeutic formulation comprising: (i) an exogenous mitochondria from a healthy donor tissue; and (ii) a nanocarrier comprising a mitochondria targeting lipophilic cation and an antioxidant Coenzyme; wherein said novel therapeutic formulation capable of being used in mitochondrial dysfunction diseases.

2. The novel therapeutic formulation as claimed in claim 1, wherein said exogenous mitochondria from a healthy donor tissue obtained by differential centrifugation method.

3. The novel therapeutic formulation as claimed in claim 1, wherein said exogenous mitochondria is isolated from a healthy donor tissue such as brain, liver and other tissues.

4. The novel therapeutic formulation as claimed in claim 1, wherein said mitochondrial dysfunction disease is a neurodegenerative disease, but not limited to, Alzheimer's disease, Parkinsons disease.

5. The novel therapeutic formulation as claimed in claim 1, wherein said mitochondria targeting lipophilic cation is selected from a group of Triphenyl phosphonium (TPP), and many small molecule cations such as Tetraalkylammonium (TAA) salts, pyridinium, and cyanine derivatives, or peptides like Szeto‑Schiller (SS) peptides.

6. The novel therapeutic formulation as claimed in claim 1, wherein said antioxidant Coenzyme is selected from a group of CoQ10, Resveratrol, Ubiquinol, Alpha-Lipoic Acid (ALA), Vitamin E (Alpha-Tocopherol), curcumin, N-Acetyl Cysteine (NAC).

7. The novel therapeutic formulation as claimed in claim 1, said formulation comprising: (i) an exogenous mitochondria from a healthy donor brain tissue; and (ii) a nanocarrier comprising Triphenyl phosphonium (TPP), and CoQ10.

8. A novel therapeutic formulation comprising (i) an exogenous mitochondria from a healthy donor tissue; (ii) a nanocarrier comprising a mitochondria targeting lipophilic cation and an antioxidant Coenzyme; and (iii) at least a pharmaceutically acceptable carrier; wherein said novel therapeutic formulation capable of being used in mitochondrial dysfunction diseases.

9. A method to obtain novel therapeutic formulation of claim 1 comprising the steps: (i) obtaining an exogenous mitochondria from a healthy donor tissue by differential centrifugation method; (ii) obtaining a nanocarrier by co-solvent evaporation method comprising of: (e) a mitochondria targeting lipophilic cation; and (f) an antioxidant Coenzyme; (iii) loading said exogenous isolated mitochondria from a healthy donor tissue of step (i) with said nanocarrier of step (ii) to obtain an exogeneous mitochondria loaded with nanocarrier; wherein said novel therapeutic formulation capable of being used in a mitochondrial dysfunction diseases including but not limited to neurodegenerative diseases, Alzheimer's disease, Parkinsons disease.

10. The method to obtain novel therapeutic formulation as claimed in claim 9, wherein said exogenous mitochondria from a healthy donor tissue including but limited to brain, live and other tissues.

11. The method to obtain novel therapeutic formulation as claimed in claim 9, wherein said mitochondria targeting lipophilic cation is selected from a group of Triphenyl phosphonium (TPP), and many small molecule cations such as Tetraalkylammonium (TAA) salts, pyridinium, and cyanine derivatives, or peptides like Szeto‑Schiller (SS) peptides.

12. The method to obtain novel therapeutic formulation as claimed in claim 9, wherein said antioxidant Coenzyme is selected from a group of CoQ10, Resveratrol, Ubiquinol, Alpha-Lipoic Acid (ALA), Vitamin E (Alpha-Tocopherol), curcumin, N-Acetyl Cysteine (NAC).

Description:FIELD OF THE INVENTION:
The present invention relates to the field of medicines. Particularly, the present invention relates to a novel therapeutic formulation of exogeneous mitochondria loaded with nanocarrier comprising Triphenyl phosphonium (TPP) and an antioxidant Coenzyme, said formulation is capable of being used in mitochondrial dysfunction diseases, such as neurodegenerative diseases. The invention also relates to the method of obtaining a novel therapeutic formulation of exogeneous mitochondria loaded with nanocarrier comprising Triphenyl phosphonium (TPP) and an antioxidant Coenzyme, that is capable of being used in mitochondrial dysfunction diseases, such as neurodegenerative diseases such as Alzheimer’s disease, Parkinsons disease.

BACKGROUND OF THE INVENTION:
Mitochondria, which are central to metabolic processes, are responsible for producing most of the energy for a cell, essential for the maintenance of life. Mitochondrial dysfunction is a group of conditions that affects the normal functioning of the mitochondria in the human body. When a mitochondria is not able to produce enough energy, it leads to affect normal functioning of many organs and can cause a range of human diseases including cancer, diabetes, neurodegenerative diseases, Alzheimer’s Disease (AD), Parkinsons disease and aging.
Mitochondrial diseases or disorders can be caused by mutations in genes in the nuclear DNA (nDNA) and/or mitochondrial DNA (mtDNA) that encode structural mitochondrial proteins or proteins involved in mitochondrial function.
Alzheimer's disease (AD) is characterised by mitochondrial dysfunction in the brain, which affects energy production, regulation, and cellular metabolism. AD is a progressive neurodegenerative disorder that affects regions of the brain that control cognition, memory, language, speech and awareness to one’s physical surroundings. Early symptoms of AD include forgetting recent events or conversations. Over time, it can lead to serious memory loss and affects a person's ability to do everyday tasks. In advanced stages, loss of brain function can cause dehydration, poor nutrition or infection and can lead to death of the subject.
There’s no treatment available to cure AD, but certain medications and therapies can help manage symptoms temporarily. The classical method for the treatment of AD involves the removal of endogenous mtDNA, which involves long term treatment along with addition to the potential chances for unwanted side effects. Such treatment also has limitations of its application for therapeutic purposes and its clinical use. Moreover, mitochondrial transfer protocols generally involve a complete depletion of endogenous mtDNA, before transfer of exogenous mitochondria. This complete depletion of mtDNA severely hinders the ability of a cell to ingest exogenous mitochondria.
The prior art US20200054682A1discloses a method and composition for generation of mitochondria replaced cells (MirC), and therapeutic method for using such composition for treating a subject having an age-related disease or syndrome, mitochondrial disease or disorder, or otherwise in need of mitochondrial replacement. It is hypothesized that the exogenous mitochondria are able to briefly interact with the endogenous mitochondria, and transport mtDNA during the brief contacts. Then, the exogenous mitochondrial membrane complexes can be degraded in the cytosol to provide building blocks for the reconstituted mitochondria. The mitochondria of the recipient cell that receive the exogenous mitochondria are able to gradually reconstitute the mitochondrial membrane complex and demonstrate the functional recovery.
Another prior art AU2017208013B2 discloses a method of treating a subject having an ischemia-related disease, by administering a therapeutically effective amount of a composition comprising isolated mitochondria or a combined mitochondrial agent. This prior art using isolated mitochondria in their true form therefore the therapeutic efficacy was affected.
Thus, there is no effective treatment available for treatment of neurodegenerative diseases. There is a need to develop novel therapeutic formulations that enhance therapeutic outcomes leading to effective amelioration of neuronal degeneration.
OBJECTS OF THE INVENTION:
Some of the objects of the present disclosure, which at least one embodiment herein satisfy are as follows:

The main object of the present invention is to provide a novel therapeutic formulation of exogenous mitochondria for use in mitochondrial dysfunction diseases.
Another object of the present invention is to provide a novel therapeutic formulation comprising an exogenous mitochondria and a nanocarrier, for use in mitochondrial dysfunction disease such as neurodegenerative diseases including Alzheimer's disease (AD), Parkinsons disease.
Yet another object of the present invention is to provide a novel therapeutic formation comprising an exogenous mitochondria from healthy tissue and a nanocarrier, resulting in the enhance therapeutic outcomes which leads to effective amelioration of neuronal degeneration of AD.
Yet another object of the present invention is to provide a novel therapeutic formulation comprising an exogenous mitochondria and a nanocarrier, said nanocarrier comprises Triphenyl phosphonium (TPP) and an antioxidant Coenzyme.
Yet another object of the present invention is to provide a method to obtain a novel therapeutic formulation of exogenous mitochondria for use in mitochondrial dysfunction diseases such as neurodegenerative diseases including Alzheimer’s diseases, Parkinsons disease.
Other objects and advantages of the present disclosure will be more apparent from the following description when read in conjunction with the accompanying figures, which are not intended to limit the scope of the present disclosure.

SUMMARY OF THE INVENTION:
Accordingly, the present invention provides a novel therapeutic formulation comprising an exogenous mitochondria and a nanocarrier, said formulation is capable of being used in mitochondrial dysfunction diseases such as neurodegenerative diseases.
In one of the embodiments, the present invention provides a novel therapeutic formation comprising an exogenous mitochondria from healthy tissue and a nanocarrier. The formulation enhances therapeutic outcomes which leads to effective amelioration of neuronal degeneration of Alzheimer’s diseases.
The exogenous mitochondria from healthy tissue is obtained by centrifugation method including but not limited to differential centrifugation method. The exogenous mitochondria is isolated from any healthy tissue, such as brain, liver of a healthy subject.
The nanocarrier is obtained by co-solvent evaporation method comprising:
(a) mitochondria targeting lipophilic cation and
(b) an antioxidant Coenzyme.
The mitochondria targeting lipophilic cation is selected from a group of Triphenyl phosphonium (TPP), and many small molecule cations such as Tetraalkylammonium (TAA) salts, pyridinium, and cyanine derivatives, or peptides like Szeto‑Schiller (SS) peptides. The antioxidant Coenzyme is selected from a group of CoQ10, Resveratrol, Ubiquinol, Alpha-Lipoic Acid (ALA), Vitamin E (Alpha-Tocopherol), curcumin, N-Acetyl Cysteine (NAC), etc.
In another embodiment, the present invention provides a method to obtain novel therapeutic formation for use in mmitochondrial dysfunction diseases, including but not limited to, neurodegenerative diseases, Alzheimer's disease, Parkinsons disease. The method comprises:
(i) obtaining an exogenous mitochondria from a healthy donor tissue by differential centrifugation method;
(ii) obtaining a nanocarrier by co-solvent evaporation method comprising of:
(a) a mitochondria targeting lipophilic cation; and
(b) an antioxidant Coenzyme;
(iii) loading said exogenous mitochondria from a healthy donor tissue of step (i) with said nanocarrier of step (ii), to obtain an exogeneous mitochondria loaded with nanocarrier.
The exogenous isolated mitochondria from healthy tissue used in the method for preparation of a novel therapeutic formulation of the present invention is obtained by centrifugation method including but not limited to differential centrifugation method, and the exogenous isolated mitochondria is obtain from an healthy tissue such as brain, liver or any other tissue.
The nanocarrier used in the method for preparation of a novel therapeutic formulation of the present invention, is obtained by co-solvent evaporation method comprising:
(a) mitochondria targeting lipophilic cation and
(b) an antioxidant Coenzyme.
The mitochondria targeting lipophilic cation is selected from a group of Triphenyl phosphonium (TPP), and many small molecule cations such as Tetraalkylammonium (TAA) salts, pyridinium, and cyanine derivatives, or peptides like Szeto‑Schiller (SS) peptides. The antioxidant Coenzyme is selected from a group of CoQ10, Resveratrol, Ubiquinol, Alpha-Lipoic Acid (ALA), Vitamin E (Alpha-Tocopherol), curcumin, N-Acetyl Cysteine (NAC), etc.
In certain embodiments of the novel therapeutic formation provided herein, the therapeutic formulation further comprises a pharmaceutically acceptable carrier.
These and other aspects herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the invention herein without departing from the spirit thereof.

BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 depicts a flow chart of a method to obtain a novel therapeutic formulation that capable of being used in the mitochondrial dysfunction diseases.
Fig. 2 A(i)& A(ii) depicts Zeta size and Zeta potential of CoQ10-TPP Nanocarrier respectively.
Fig. 2 B(i) & B(ii) depicts Zeta size and Zeta potential of isolated mitochondria respectively.
Fig. 2 C(i) & C(ii) depicts Zeta size and Zeta potential of isolated mitochondria loaded CoQ10-TPP (Mt-CoQ10-TPP) respectively.
Fig. 3 (A), (B) and (C) depicts TEM images of CoQ10-TPP, isolated mitochondria, and Mt-CoQ10-TPP respectively.
Fig 4 depicts FT-IR of CoQ10-TPP.
Fig. 5 (A) and (B) depicts effects of Liver Mt-CoQ10-TPP and Brain Mt-CoQ10-TPP in the Y-maze and radial arm maze task in the spontaneous alternation and the reference memory errors respectively for 7 days duration in treated mice.
Fig. 6 (A) and (B) depicts effects of Liver Mt-CoQ10-TPP and Brain Mt-CoQ10-TPP in the Zero arm maze and Elevated plus maze in No. Of Entries and Time in seconds respectively for 7 days duration in treated mice.
Fig. 7 (a), (b), (c), (d) and (e) depicts Biochemical Assays for the concentration of SOD, CAT, GSH, NO and TBARs respectively in the brain tissues of indicated groups.
Fig. 8 depicts Complex Assays for concentration of Complex I (a), Complex II (b) Complex III (c) and Complex IV (d) in the brain tissues of indicated groups.
Fig. 9 depicts AChE activity of different groups in AD mice.
Fig. 10 depicts Gene expressions of neurodegenerative functional genes by Brain Mt+CoQ10+TPP.
Fig. 11 (A) (i) and (ii) depicts Histopathology images of mice brain Control group at 10µm and 50 µm respectively
Fig. 11 (B) (i) and (ii) depicts Histopathology images of mice brain STZ group at 10µm and 50 µm respectively
Fig. 11 (C) (i) and (ii) depicts Histopathology images of mice brain Donepezil group at 10µm and 50 µm respectively
Fig. 11 (D) (i) and (ii) depicts Histopathology images of mice brain Liver Mito group at 10µm and 50 µm respectively
Fig. 11 (E) (i) and (ii) depicts Histopathology images of mice brain Brain Mito group at 10µm and 50 µm respectively
Fig. 11 (F) (i) and (ii) depicts Histopathology images of mice brain Liver Mt-CoQ10-TPP group at 10µm and 50 µm respectively
Fig. 11 (G) (i) and (ii) depicts Histopathology images of mice brain Brain Mt-CoQ10-TPPgroup at 10µm and 50 µm respectively
Fig. 11 (H) (i) and (ii) depicts Histopathology images of mice brain CoQ10-TPP group at 10µm and 50 µm respectively
Fig. 12 (A), (B), (C), (D), (E), (F) (G) and (H) depicts Mitotracker tagged images of mice brain at 50 µm for Control group, STZ group, Donepezil group, Liver Mito group, Brain Mito group, Liver Mt-CoQ10-TPP group, Brain Mt-CoQ10-TPP group and CoQ10-TPP group respectively.

DETAILED DESCRIPTION OF THE INVENTION WITH NON-LIMITING EMBODIMENTS AND EXAMPLES

In the following detailed description of the invention, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be obvious to a person skilled in the art that the invention may be practiced with or without these specific details. In other instances, well known methods, procedures and components have not been described in details so as not to unnecessarily obscure aspects of the invention.

Furthermore, it will be clear that the invention is not limited to these alternatives only. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art, without parting from the scope of the invention.

In one embodiment, the present invention provides a novel therapeutic formulation comprising of an exogenous mitochondria and a nanocarrierthat capable of being used in mitochondrial dysfunction disease such as neurodegenerative diseases, Alzheimer's disease (AD), Parkinsons disease.

In another embodiment, the present invention aims to provide a novel therapeutic formation comprising an exogenous mitochondria from healthy tissue and a nanocarrier result in the enhance therapeutic outcomes which leads to effective amelioration of neuronal degeneration of AD.
The exogenous mitochondria from a healthy tissue is obtained by centrifugation method including but not limited to differential centrifugation method. The exogenous mitochondria is obtain from healthy tissue such as brain, liver or any other tissue of a healthy subject.
The nanocarrier is obtained by co-solvent evaporation method comprising:
(a) mitochondria targeting lipophilic cation and
(b) an antioxidant Coenzyme.
The mitochondria targeting lipophilic cation is selected from a group of Triphenyl phosphonium (TPP), and many small molecule cations such as Tetraalkylammonium (TAA) salts, pyridinium, and cyanine derivatives, or peptides like Szeto‑Schiller (SS) peptides. The antioxidant Coenzyme is selected from a group of CoQ10, Resveratrol, Ubiquinol, Alpha-Lipoic Acid (ALA), Vitamin E (Alpha-Tocopherol), curcumin, N-Acetyl Cysteine (NAC), etc.
In another embodiment, the present invention provides a method to obtain a novel therapeutic formation capable of being used in a mitochondrial dysfunction disease such as neurodegenerative diseases, Alzheimer's disease, Parkinsons disease. The method comprises:
(i) obtaining an exogenous mitochondria from a healthy donor tissue by differential centrifugation method;
(ii) obtaining a nanocarrier by co-solvent evaporation method comprising of:
(c) a mitochondria targeting lipophilic cation; and
(d) an antioxidant Coenzyme;
(iii) loading said exogenous mitochondria from a healthy donor tissue of step (i) with said nanocarrier of step (ii), to obtain an exogeneous mitochondria loaded with nanocarrier.
The exogenous mitochondria from healthy tissue used in the method for preparation of a novel therapeutic formulation of the present invention is obtained by centrifugation method including but not limited to differential centrifugation method and the exogenous mitochondria is obtain from healthy tissue such as brain, liver of a healthy subject.
The nanocarrier used in the method for preparation of a novel therapeutic formulation of the present invention is obtained by co-solvent evaporation method comprising:
(a) mitochondria targeting lipophilic cation and
(b) an antioxidant Coenzyme.
The mitochondria targeting lipophilic cation is selected from a group of Triphenyl phosphonium (TPP), and many small molecule cations such as Tetraalkylammonium (TAA) salts, pyridinium, and cyanine derivatives, or peptides like Szeto‑Schiller (SS) peptides. The antioxidant Coenzyme is selected from a group of CoQ10, Resveratrol, Ubiquinol, Alpha-Lipoic Acid (ALA), Vitamin E (Alpha-Tocopherol), curcumin, N-Acetyl Cysteine (NAC), etc.
In certain embodiments of the novel therapeutic formation provided herein, the therapeutic formulation further comprises a pharmaceutically acceptable carrier.
Experiments:
Alzheimer's disease (AD) is characterised by mitochondrial dysfunction in the brain, which affects energy production, regulation, and cellular metabolism. The isolated mitochondria from healthy brain and liver tissues of mice and their combinatorial effect with mitochondria targeting lipophilic cation Triphenyl phosphonium (TPP) and antioxidant Coenzyme Q10 (CoQ10) conjugant in AD mice were examined. Although TPP+CoQ10 has been used as mitochondria targeted drugs in vivo, but they have never been loaded to isolated mitochondria. The present invention shows first time that mitochondria was isolated from healthy brain and liver tissues of mice and then loaded them with TPP+CoQ10 conjugant, then entire moiety was transferred to mice bearing dysfunctional neuronal behaviour.
The Swiss Albino mice (weight: 25-35g/ 8-10 weeks old) were obtained from Lala Lajpat Rai Veterinary and Animal Sciences (Hisar, India) and used to establish an AD model in this investigation. The mice were maintained in housing with a light/dark cycle of 12/12 hours and free access to food and drink. The Institutional Animal Ethical Committee (IAEC), Banasthali Vidyapith, obtained ethical approval for all the animal experiments.
Fig. 1 depicts a flow chart for method to obtain a novel therapeutic formulation in which an exogenous isolated mitochondria was obtained from a healthy donor i.e. Swiss Albino mice’s Brain and Liver tissue by differential centrifugation method.
Induction of AD in mice model
Mice were anaesthetized by intraperitoneal injections of ketamine (100 mg/kg) and xylazine (10 mg/kg). The lateral ventricles were injected with streptozotocin intracerebroventricular (ICV) injections to establish an Alzheimer's disease model. The stereotaxic coordinates were set with regard to Bregma: depth: -2.8 mm, lateral: -1.1 mm, and anteroposterior: -0.5 mm. Evan's blue was injected, and the anatomical accuracy of the stereotaxic approach was confirmed by cutting out the region of interest. 1.5 μL of streptozotocin (3 mg/kg) was injected into each of the lateral ventricles at a rate of 0.5 μL/min.
Mitochondria isolation and protein estimation
Fresh brain tissue (100 mg) was extracted from healthy mice and rinsed with 0.1 M NaCl. The tissue was then minced and homogenized in 10 ml of mitochondria isolation buffer before undergoing sequential centrifugation to isolate the mitochondria. After resulting pellet, the mixture was again mixed with mitochondrial suspension buffer (pH 7.4), 1.0 ml Tris (10 mM), 21.9 g mannitol, and 40 ml HEPES (20 mM). The concentration of mitochondrial protein (mg/ml) was measured by applying Lowry's technique. Through the tail vein, a single intravenous (i.v.) dose of the isolated mitochondria (0.5 mg/kg) was given. Following therapy, the outcomes were contrasted with a control group that received oral donepezil (50 mg/kg) for ten days in a row. The mice were euthanized after a 7-day behavioural study, and their tissues were kept at -80°C for further examination.
Synthesis of nanocarrier
The mitochondria-targeted delivery system for CoQ10 was developed using polymer group PEG-PCL-TPP. A poly-ethylene-glycol and poly-ε-caprolactone (PEG-PCL) acetonitrile solution was rapidly stirred at room temperature for 20 minutes. Subsequently, the solution was dialyzed against double-distilled water for four hours. After that, the nanocarrier structures were stored at 4°C. The reduced form of CoQ10 was contained inside the nanocarrier's core through self-assembly.
Conjugation of Isolated Mitochondria with nanocarrier
The CoQ10-TPP nanocarrier conjugation with isolated mitochondria by making a suspension of CoQ10-TPP nanocarrier at a concentration of 10 mg/ml in phosphate-buffered saline (PBS). A pellet of isolated mitochondria was added to this suspension in an amount of 200 µl. Incubation was carried out at 4⁰C, the mixture was centrifuged for 15 minutes at 4°C and 3000×g. Brain/Liver Mt-CoQ10-TPP pellet was obtained and reconstituted in 100 µl respiration buffer.
Structural and Functional characterization of CoQ10-TPP Nanocarrier and Mt-CoQ10-TPP
The Zeta size and Zeta potential of the CoQ10-TPP Nanocarrier, isolated mitochondria and isolated mitochondria loaded CoQ10-TPP (Mt-CoQ10-TPP) a were examined using a Malvern Zetasizer. Fig.2 A(i), A(ii), B(i), B(ii), C(i) and C(ii) shows Zeta size and Zeta potential of the CoQ10-TPP Nanocarrier, isolated mitochondria and Mt-CoQ10-TPP respectively. It was observed that the CoQ10-TPP nanocarrier's typical size ranged from 10 to 100 nm. The formulation Mt-CoQ10-TPP showed an increase in the particle size range of 100-500 nm when isolated mitochondria were attached. The conjugation of isolated mitochondria, which have a size of roughly 300–500 nm, is expected to provide this effect. Zeta potential is an important parameter that defines the distribution of electrical charges on the surface of the nanocarrier, determining the morphology and stability of particles. The zeta potential of CoQ10-TPP and Mt-CoQ10-TPP was recorded as 19.4 mV and 27.2 mV, respectively. This difference between the formulations might be because different compounds or molecules have additional superficial charges.
Fig. 3 (A), (B) and (C) shows transmission electron microscope (TEM) images of CoQ10-TPP, Isolated Mitochondria, and Mt-CoQ10-TPP for the morphological investigation using a TEM (TECNAI 200 Kv, Fei, Electron Optics). The development of small, spherical structure confirmed the consistent synthesis of CoQ10-TPP nanocarrier. Conversely, well-organized cristae were seen in spherical, oblong-shaped mitochondria, suggesting that the mitochondria are healthy (Fig. 3 (B)). CoQ10-TPP and mitochondrial structure were verified, conjugated, and subjected to further analysis. Further, (Fig. 3(C)) shows that the mitochondria seemed to have tiny dots affixed to their outer membrane, providing additional evidence that CoQ10-TPP were conjugated with mitochondria and might be employed to monitor the dispersion of intravenous mitochondrial transplantation across various tissue.
The Fourier transform infrared (FTIR) spectra of the CoQ10-TPP Nanocarrier using a Bruker Vertex V70 FTIR instrument were recorded as shown in the Fig. 4 which demonstrates a notable shift in the FT-IR peaks of the Mt-CoQ10-TPP. Observations were made of the stretching vibration of the bond -O-H at 3432 cm-1, the existence of the alcohol group, the CH bond, and the presence of alkene at 3088 cm-1, the peak at 2135 of carbonyl group, and 1626 cm-1 shows the presence of Azide bond as shown in the Fig. 4.
Behavioural studies
Y-Maze
Y maze was utilised to assess the task Spontaneous alternation behaviour for short-term memory replicates spatial working memory. The dimensions of the three arms of the Y-maze used in this study were 30 cm in length, 12 cm in height, and 5 cm in breadth. The day after treatment, mice were allowed to move freely by placing at end of the arm of maze for eight minutes. When mice's hind paws were completely contained within the arm, the arm entry was recorded. The spontaneous alternation was calculated by minus total number of arms entered by two. Then, using the formula (actual alternations/maximum alternations) 9 100, the percent of spontaneous alternation was calculated. The arms was cleaned by 10% ethanol and cloth prior for next mice. Fig. 5 (A) shows effects of Liver Mt-CoQ10-TPP and Brain Mt-CoQ10-TPP in the Y-maze task in the spontaneous alternation and the reference memory errors respectively for 7 days duration in treated mice. The percentage of spontaneous alternations in the Y-maze task indicates a substantial group impact (F(7,48) = 60.37, p<0.001) on spatial working memory. Furthermore, Tukey's post hoc analysis showed that the spontaneous alternations % varied significantly between the control and STZ groups (p<0.001), STZ and Brain Mt-CoQ10-TPP groups (p<0.001), and Liver Mt-CoQ10-TPP and Brain Mt-CoQ10-TPP groups (p<0.001).
Radial Arm Maze
The radial arm maze was utilized in this investigation reference memory error. The arms were numbered 1 through 8 (48 9 12 cm), and they prolonged from the centre that was 32 cm in diameter. The food pellet was placed at end of each arm. The animals trained to move to the end of the arms and consume the pellet, for seven days. One day after the therapy was given, each animal was positioned at the centre of maze and trained using reference memory tasks. When a mice’s four limbs were contained within an arm, the entry was counted as an arm. The reference memory errors, or entering an arm without being baited were recorded. Reference memory is considered to be a long-term memory for data was endured throughout multiple trials, such the placements of baited arms. Fig. 5 (B) shows effects of Liver Mt-CoQ10-TPP and Brain Mt-CoQ10-TPP in the radial arm maze task in the spontaneous alternation and the reference memory errors respectively for 7 days duration in treated mice. The radial arm-maze determines whether the Brain Mt-CoQ10-TPP influences the establishment of spatial memory. A significant group effect was found by ANOVA (F(7,48) = 140.5, p<0.001). Tukey's post hoc analysis also revealed a significant difference in reference memory errors between the control and STZ groups (p<0.001), STZ and Brain Mt-CoQ10-TPP groups (p<0.001), and Liver Mt-CoQ10-TPP and Brain Mt-CoQ10-TPP groups (p-0.001).
Zero arm
The zero-arm maze is a behavioural test utilized to assess anxiety-like behaviour in rodents. In the test, mice are first habituated by allowing them to explore the maze, then they are placed at the start and their movements are recorded. Behavioural measures include the time spent in the open versus enclosed arms, the number of entries into each arm, and overall activity. The size of the zero-arm maze can vary, but for mice, it is generally designed to be compact to accommodate their smaller size and encourage exploration. Platform Diameter 30-40 cm, Arm Length 10-15 cm long and Arm Width wide. Fig. 6 (A) shows effects of Liver Mt-CoQ10-TPP and Brain Mt-CoQ10-TPP in the Zero arm maze in No. of Entries and Time in Seconds respectively for 7 days duration in treated mice. A significant group effect was found by ANOVA (F(7,8) = 0.8321, p<0.001). There is no significant difference in the open and close arm entries of control group, but there is a significant difference between open and close arm entries of STZ group, the STZ mice was more present in the closed arm.
Elevated plus maze
In each age group, the animals' anxiety-like behaviour was assessed using the EPM test. After that, each animal was graciously placed on the EPM apparatus's central platform and then, for five minutes, inside the centre of the box. The EPM device comprised of two arms measuring 30 × 5 cm each, one closed and one open, positioned in opposing directions and connected at a central platform measuring 5 × 5 cm. Open arms (OA) have no walls, while closed arms (CA) have walls that are 17 cm high. Fig. 6 (B) shows effects of Liver Mt-CoQ10-TPP and Brain Mt-CoQ10-TPP in the Elevated plus maze in No. of Entries and Time in Seconds respectively for 7 days duration in treated mice. A significant group effect was found by ANOVA (F(7,8) = 0.02173, p<0.001). There is significant difference in the time spent in the arms of control group, also same significant difference was observed of STZ group, the STZ mice spent more time in the closed arm and vice versa activity was observed in control mice.
Biochemical analysis
Brain tissue was homogenized and adequately prepared as outlined in previous protocols. A standard curve was generated using the established standard, which was then utilized to compare and quantify the sample readings. The TBARs (thiobarbituric acid reactive substances) method quantifies lipid peroxidation by measuring MDA, the end product, which reacts with thiobarbituric acid to form TBARs. The colour development, caused by the breakdown of peroxides through heat and acid, was analysed spectrophotometrically at 532 nm. Superoxide dismutase (SOD) activity was assessed using the NBT assay, where formazan crystals are formed and measured at 560 nm. Nitric oxide (NO) levels were determined using the Griess reagent, consisting of sulphanilic acid and (1-naphthyl) ethylenediamine in glacial acetic acid, which reacts with nitrite to produce a purple azo dye, measurable at 545 nm (Noureen et al., 2017). For glutathione reductase (GSH) analysis, the sulfhydryl group of GSH reacts with DTNB (Ellman's reagent) to yield a yellow TNB compound, with absorbance recorded at 412 nm. Catalase (CAT) activity was measured by the reduction of dichromate in acetic acid to chromic acetate in the presence of hydrogen peroxide under heat, forming perchloric acid, which was detected at 570 nm. Fig. 7 (a), (b), (c), (d) and (e) shows Biochemical Assays for the concentration of SOD, CAT, GSH, NO and TBARs respectively in the brain tissues of indicated groups.
A significant group effect for SOD was shown by the biochemical assay results ANOVA (F(7,40) = 130.2, p <0.001). The SOD level was observed to be considerably lower in the STZ group (P < 0.001) compared to the control group, according to Tukey's post hoc analysis. The Brain Mt-CoQ10-TPP group shows a significant increase (P<0.001) in comparison to the STZ group, and further Liver Mt-CoQ10-TPP and Brain Mt-CoQ10-TPP groups also show significant difference (P<0.001The brain's CAT activity ANOVA results showed a significant group effect (F (7,40) = 64.62, p <0.001), and Tukey's post hoc analysis showed that the STZ group's brain activity had significantly decreased (P < 0.001) in comparison to the control group. In contrast to the STZ group, the Brain Mt-CoQ10-TPP group exhibits a substantial increase (P<0.001), and as shown in Fig., the Liver Mt-CoQ10-TPP and Brain Mt-CoQ10-TPP groups also exhibit a marginally significant difference (P- 0.003). The GSH level of the brain demonstrated that ANOVA shows significant group effect (F (7,40) = 169.4, p <0.001), and Tukey’s post hoc analysis showed a significant decrease in the STZ group (P < 0.001) as compared to the control group. The Brain Mt-CoQ10-TPP group shows a significant increase (P<0.001) in comparison to the STZ group, and the Liver Mt-CoQ10-TPP and Brain Mt-CoQ10-TPP groups also indicate significant difference (P<0.001) as shown. The ANOVA of NO (F (7,40) = 274.6, p <0.001) and TBARs (F (7,40) = 418.6, p <0.001) revealed a significant group effect. Tukey’s post hoc analysis revealed NO and TBARs result show a considerable increase in the STZ group (P<0.001) as compared to control group. The Brain Mt-CoQ10-TPP group shows a significant decrease (P<0.001) in comparison to the STZ group, and further Liver Mt-CoQ10-TPP and Brain Mt-CoQ10-TPP groups also show a significant difference
Mitochondrial complex enzyme activities
The mitochondrial complex enzyme assay involves using 100 mg of brain tissue from each group to extract mitochondria, with the resulting pellet re-suspended in a mitochondrial respiration buffer. Equal amounts of protein were used for all tissue samples. Enzyme activities for mitochondrial complexes I, II, III, and IV were measured in nmol/min*mg protein, following established protocols. Enzyme activity was calculated using the formula: (nmol/min*mg protein) = (∆ Abs/min × 1,000). Fig. 8 shows Complex Assays for concentration of Complex I (a), Complex II (b) Complex III (c) and Complex IV (d) in the brain tissues of indicated groups.
In the brain, mitochondrial complex I, II, III and IV activities results has shown that ANOVA has a significant group effect of complex I (F (7,40) = 480.8, p <0.001), Complex II (F (7,40) = 160.2, p <0.001), Complex III (F (7,40) = 375.6, p <0.001), and Complex IV (F (7,40) = 401.1, p <0.001). Tukey’s post hoc analysis revealed that the STZ group showed a significant decrease for the STZ group (P < 0.001) in comparison to the control group in all complexes. Further, Complex I Tukey’s post hoc analysis shows a significant increase in Brain Mt-CoQ10-TPP (P < 0.001) in comparison to the STZ group, and Liver Mt-CoQ10-TPP and Brain Mt-CoQ10-TPP groups also show a significant difference (P < 0.001) when compared to each other.
Acetylcholine Assay
For the acetylcholinesterase (AChE) experiment, 50 μl of tissue homogenized, 3 ml of 0.01 M PBS (pH 8.0), 100 μl of acetylthiocholine iodide as the substrate, and 100 μl of DTNB were combined. A UV-visible spectrophotometer was used to measure the absorbance at 412 nm for two minutes, taking readings every thirty seconds. The absorbance was recorded immediately. The results were calculated by the number of nanomoles of substrate hydrolysed each minute per milligram of protein, and were computed using the chromophore's molar extinction coefficient of 1.36 × 10⁴ M−1 cm−1.To measure AchE activity in brain ANOVA has a significant group effect (F (7,40) = 109.1, p <0.001) was determined by Tukey’s post hoc analysis revealed that STZ group (P < 0.001) showed a significant increase in comparison to control group, and Brain Mt-CoQ10-TPP group shows a significant decrease (P<0.001) in comparison to the STZ group, and further Liver Mt-CoQ10-TPP and Brain Mt-CoQ10-TPP groups also show a significant difference, where Liver Mt-CoQ10-TPP shows significant decrease in comparison to Brain Mt-CoQ10-TPP (P<0.001) as shown in Fig. 9.
RNA Isolation, cDNA preparation and Real Time PCR
The brain tissue were removed from mice and quickly snap-frozen in liquid nitrogen to preserve the mRNA. Later, TRIzol was used to separate the entire mRNA in an RNAse-free environment. A NanoDrop 2000 spectrophotometer was used to measure the amount of RNA present. In the T100 Thermal Cycler, cDNA was generated using the Verso cDNA synthesis kit from Thermo-Fisher Scientific, USA. The quantification of cDNA transcript level was done by RT-PCR using thermos Fisher Scientific's SYBR Green Master Mix (USA). The data were analysed using the manufacturer's protocol using the CFX96 C1000 Touch Real-Time PCR Detection System (USA) and CFX Manager TM Software.Arginase1, MAP 2, and β2-microglobulin gene expression was measured as shown in the Fig. 10. As per manufacturer's instructions, the gene expression of Arginase1, MAP 2, and β2-microglobulin was measured using the 2Δct method and normalized to theβ-actin gene. To assess gene expression, primer pairs for Arginase1, MAP 2, and β2-microglobulin were utilized, and the housekeeping gene, β-actin, was used to standardize the data. The 2∆∆ ct technique was used to calculate gene expression by comparing it to nuclear gene expression.
The effect of Brain Mt+CoQ10+TPP and Liver Mt+CoQ10+TPP on the overall gene expression levels of brain proteins and, mitochondrial biogenesis, detected by the expression of Arginase 1, MAP 2 and β2-microglobulin the overall impact on the gene expression. STZ treatment reduced the normal brain functions shown by a reduction in gene expression of MAP 2 and vice versa expression by gene Arginase 1 and β2-microglobulin as shown in Fig. 10. The delivery of Brain Mt+CoQ10+TPP significantly restored gene expression of brain. However, a significant reduction in the neurodegenerative genes was noted after treatment with Brain Mt+CoQ10+TPP.
Histology assessment
Histopathological evaluation of brain tissue was conducted in specific stages of the process. Tissue sections were stained using hematoxylin and eosin (H&E) and examined under a Nikon Eclipse Ni-U microscope. The H&E-stained brain histopathological analysis was used to examine of the hippocampal region of brain sections of control mice reveals that the hippocampal structure was normal, containing dense layers of small and few large pyramidal cells in addition to vesicular nuclei. Conversely, the mice treated with STZ exhibit ineffectiveness or loss of some pyramidal cells along with the shrinkage of large pyramidal cells, nuclei that have darkened, and disfigured neuronal cells with nuclei that are fractured, hyperchromatic, or vacuolated, as well as a darker shade that indicates neurodegeneration and dead cells. While mice treated with Brain Mt-CoQ10-TPP showed a protection of minor pyramidal cells with more of a decrease in the dysfunction of cells and vacuolation. On the other hand, mice treated Liver Mt-CoQ10-TPP group did not produce profound improvement as compared to Brain Mt-CoQ10-TPP as shown in Fig. 11.
Ex vivo-mitochondria fluorescent analysis
Male mice were utilized to isolate mitochondria from brain and liver tissues, which were then re-suspended in a respiration buffer and pre-labelled with 100 μg of MitoTracker Green (ThermoFisher Scientific). After intravenous injection of the pre-labelled mitochondria, animals were sacrificed 7 days later. Brain tissue was -6+subsequently harvested and processed for histological evaluation. Fig. 12 (A), (B), (C), (D), (E), (F) (G) and (H) shows Mitotracker tagged images of mice brain at 50 µm for Control group, STZ group, Donepezil group, Liver Mito group, Brain Mito group, Liver Mt-CoQ10-TPP group, Brain Mt-CoQ10-TPP group and CoQ10-TPP group respectively.
Statical analysis
ANOVA was used to evaluate the gathered data, and findings were shown as mean ± standard deviation (S.D.). Significant p-values were figured using the Windows program GraphPad Prism Version 8.0, which was used for statistical analysis. A p-value of less than 0.05 was deemed statistically significant.
Therefore, in view of the above experimental details the present invention discloses that Fresh mitochondria loaded with CoQ10+TPP has shown increased therapeutic potential as compared to the treatment of isolated mitochondria alone in AD mice. Exogeneous isolated mitochondria loaded with CoQ10+TPP demonstrated better cognitive performance on behaviour assays as compared to both non-loaded mitochondria-treated and untreated AD mice.
Therefore, the present invention provides a novel therapeutic formation and a method thereof to effectively address AD via loading exogenous isolated mitochondria with CoQ10+TPP nanocarrier to increase their therapeutic outcome. The present invention shows the augmented effectiveness of isolated mitochondria when loaded with CoQ10+TPP compared to use of non-modified isolated mitochondria The present invention also discloses the safety of therapeutic formation (Mt+CoQ10+TPP) in vitro and demonstrated its therapeutic potential in in vivo Alzheimer's mice model by showing promising results in improving cognitive functions using behavioural assays (Y maze, zero arm maze, radial arm maze, elevated plus maze) in AD mice and through various histopathological, biochemical assays and gene expression studies. In conclusion, the novel therapeutic formulation of the present invention shows the augmented therapeutic effects and proves that capable of being used in the various mitochondrial dysfunctional diseases such as neurodegenerative diseases.

From the features as applied to various alternatives, it can be understood that various omissions, substitutions, and changes in the form and details of the present disclosure can be made without departing from the scope of the disclosure. As can be recognized, certain alternatives described herein can be embodied within a form that does not provide all of the features and benefits set forth herein, as some features can be used or practiced separately from others.
, C , Claims:I/We claim:
1. A novel therapeutic formulation comprising:
(i) an exogenous mitochondria from a healthy donor tissue; and
(ii) a nanocarrier comprising a mitochondria targeting lipophilic cation and an antioxidant Coenzyme;
wherein said novel therapeutic formulation capable of being used in mitochondrial dysfunction diseases.

2. The novel therapeutic formulation as claimed in claim 1, wherein said exogenous mitochondria from a healthy donor tissue obtained by differential centrifugation method.

3. The novel therapeutic formulation as claimed in claim 1, wherein said exogenous mitochondria is isolated from a healthy donor tissue such as brain, liver and other tissues.

4. The novel therapeutic formulation as claimed in claim 1, wherein said mitochondrial dysfunction disease is a neurodegenerative disease, but not limited to, Alzheimer's disease, Parkinsons disease.

5. The novel therapeutic formulation as claimed in claim 1, wherein said mitochondria targeting lipophilic cation is selected from a group of Triphenyl phosphonium (TPP), and many small molecule cations such as Tetraalkylammonium (TAA) salts, pyridinium, and cyanine derivatives, or peptides like Szeto‑Schiller (SS) peptides.

6. The novel therapeutic formulation as claimed in claim 1, wherein said antioxidant Coenzyme is selected from a group of CoQ10, Resveratrol, Ubiquinol, Alpha-Lipoic Acid (ALA), Vitamin E (Alpha-Tocopherol), curcumin, N-Acetyl Cysteine (NAC).
7. The novel therapeutic formulation as claimed in claim 1, said formulation comprising:
(i) an exogenous mitochondria from a healthy donor brain tissue; and
(ii) a nanocarrier comprising Triphenyl phosphonium (TPP), and CoQ10.

8. A novel therapeutic formulation comprising
(i) an exogenous mitochondria from a healthy donor tissue;
(ii) a nanocarrier comprising a mitochondria targeting lipophilic cation and an antioxidant Coenzyme; and
(iii) at least a pharmaceutically acceptable carrier;
wherein said novel therapeutic formulation capable of being used in mitochondrial dysfunction diseases.

9. A method to obtain novel therapeutic formulation of claim 1 comprising the steps:
(i) obtaining an exogenous mitochondria from a healthy donor tissue by differential centrifugation method;
(ii) obtaining a nanocarrier by co-solvent evaporation method comprising of:
(e) a mitochondria targeting lipophilic cation; and
(f) an antioxidant Coenzyme;
(iii) loading said exogenous isolated mitochondria from a healthy donor tissue of step (i) with said nanocarrier of step (ii) to obtain an exogeneous mitochondria loaded with nanocarrier;
wherein said novel therapeutic formulation capable of being used in a mitochondrial dysfunction diseases including but not limited to neurodegenerative diseases, Alzheimer's disease, Parkinsons disease.
10. The method to obtain novel therapeutic formulation as claimed in claim 9, wherein said exogenous mitochondria from a healthy donor tissue including but limited to brain, live and other tissues.

11. The method to obtain novel therapeutic formulation as claimed in claim 9, wherein said mitochondria targeting lipophilic cation is selected from a group of Triphenyl phosphonium (TPP), and many small molecule cations such as Tetraalkylammonium (TAA) salts, pyridinium, and cyanine derivatives, or peptides like Szeto‑Schiller (SS) peptides.

12. The method to obtain novel therapeutic formulation as claimed in claim 9, wherein said antioxidant Coenzyme is selected from a group of CoQ10, Resveratrol, Ubiquinol, Alpha-Lipoic Acid (ALA), Vitamin E (Alpha-Tocopherol), curcumin, N-Acetyl Cysteine (NAC).