DESC:4. DESCRIPTION
Technical Field of the Invention

The present invention generally relates to a composition composed of chemotherapeutic agent or/and biological loaded in alpha lactalbumin nanoparticles.
Background of the Invention

Cardiovascular diseases are one of the major concerns predominantly in golden agers. Current therapeutics though can intervene the prognosis of the disease however cannot attain the long term results due to side effects and non-specific localization.

Over the past several decades the nano-particle based drug delivery have become increasingly popular. The nanoparticle technology has enabled in the development of wide range of novel therapeutic and diagnostic platforms. The nanoparticle delivery offers many advantages such as solubilization, of hydrophobic payloads, extended blood residence times and the ability to better target a region of interest. One of the major areas in which nanoparticles have particularly excelled is cancer treatment. The nanoparticle therapeutics generally relied on this passive targeting mechanism to improve efficacy over traditional free-drug formulations that often have severe systemic side effects.

Current treatment involves use of soluble or regulated biological, no combinations are available for treatment. Current oral treatment using soluble drug combination induces severe gastrointestinal and other toxicities, thus limiting continuation of therapy and patient adherence.

We found that lactalbumin wound serve as vehicle for heart and can be used to deliver therapeutics to intervene any heart disorders due to some structural privileges which can help its internalization in heart. Most pertinent property of lactalbumin is due to its’ affinity with calcium ions which is beneficial to maintain heart rhythm as more release of calcium in cardiomyopathy condition leads to tachycardia, this protein has calcium binding site so it might maintain the heart rhythm by binding extra calcium ion. Thus, our present invention of a nano formulation using lactalbumin would serve a mimic for target localization of API in the heart.

A few patents related to our present invention have been discussed below:
The patent CN106687110B (Purified therapeutic nanoparticles and method of making same) comprise an active ingredient and human serum albumin, wherein the weight ratio of human serum albumin and active ingredient in the therapeutic nanoparticles is from 0.01:1 to 1:1, and the nanoparticles are substantially free of free human serum albumin not contained in the nanoparticles.

The patent “New nanoparticle composition” (JP6756810B2) relates to a nanoparticle composition, wherein the plurality of nanoparticles further comprises a preparation comprising one or more hydrophilic cell components, respectively. The preparation was obtained by destroying one or more cells selected from the group consisting of plant cells, microbial cells, animal cells, human cells and combinations thereof to separate hydrophobic cell components and hydrophilic cell components.

The present invention reduces such toxicities and provides target direction of natural site of action, thus enhances efficacy. In this formulation of our present invention, biological and/ or drugs are protected from non-specific effects by lactoferrin nanoparticle that further helps in localization of biological and/or drug in blood and various tissue that harbors disease and thus providing biological and/or drug affectivity against diseases. A GFP plasmid is a plasmid that uses green fluorescent protein (GFP) to study gene expression in a non-invasive way. GFP is a protein that glows green under ultraviolet light, and is isolated from the jellyfish Aequorea victoria. GFP plasmids are used in cellular and molecular biology research for a variety of applications. pEGFP-C1 encodes a red-shifted variant of GFP that is optimized for higher expression and brighter fluorescence in mammalian cells. It contains the GFPmut1 variant, which has a double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr.

The nanoparticles are represented as: LF stands for lactoferrin; LA stands for Lactalbumin; AL for Albumin, LANP stands for Lacto Albumin Nano Particles throughout our complete specification; these challenges by offering a compact, portable, and fully automated solution.

Objective of our present invention

The main objective of our present invention is to provide a nano formulation using lactalbumin would serve a mimic for target localization of API in the heart.

Another objective of our present invention is the establishment of potent delivery vehicle might ensure stability of therapeutic molecules in the system (in-vivo) and efficacy at site of action. Delivery of therapeutic gene using these systems corroborate intervention of DCM, heart failure and injury.

Another objective of our present invention is to determine the ability loaded API in localization in specific tissue using a composition and ratio of lactalbumin and lactoferrin, thus for treatment of tissue-specific cancers, a specific composition and ratio of lactoferrin and lactalbumin would provide tissue-directed localization of API and/or nucleic acids.

Another objective of our present invention is to provide the ability of API in reaching all sites of virus reservoirs including latent reservoirs for determination, treatment of virus infection such of HIV using a composition and ratio of lactoferrin and lactalbumin.

Brief Summary of the Invention

The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

According to a first aspect of the present invention provides a DNA loaded alpha lactalbumin nanoparticles are 44 to 70 nm by FE SEM, while hydrodynamic size is 537 nm.

Another aspect of the invention establishes significant transfection efficiency which was observed in heart cells H9c2(2-1), when DNA delivered through alpha lactalbumin nanoparticles.

Significant localization of DNA and drug takes place in heart, lymph node and thymus, when delivered through alpha lactalbumin nanoparticles.

DNA and drug loaded alpha lactalbumin nanoparticles have potential in treatment of cardiomyopathy. In addition, composition containing alpha lactalbumin in combination with another protein like lactoferrin and apotransferrin would provide a broad-spectrum antiviral for delivery of drugs and biologicals to all virus replication and latent reservoirs including lymph nodes, thymus and heart.

Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, the detailed description and specific examples, while indicating preferred embodiments of the invention, will be given by way of illustration along with complete specification.

Brief Summary of the Drawings

The invention will be further understood from the following detailed description of a preferred embodiment taken in conjunction with an appended drawing, in which:

Fig. 1 illustrates the diagram of the docking between bovine alpha lactalbumin and p53 DNA was done using Haddock server we found the best fitted interacting structure chosen by z score (lowest the z score more reliable the model), in accordance to an exemplary embodiment of the present invention;

Fig. 2 illustrates the Agarose Gel image representing pEFGP-C1 and Alpha lactalbumin interaction, in accordance to an exemplary embodiment of the present invention;

Fig 3: Diagram showing the Agarose Gel image represent increasing amount of alpha lactalbumin keeping GFP-p53 DNA amount constant, in accordance to an exemplary embodiment of the present invention;

Fig 4: Diagram showing the: (A) shows Sonication of GFP-p53plasmid only with Variation in amplitude (increasing Amplitude); (B). shows Sonication of GFP-p53 plasmid with alpha lactalbumin with variation in amplitude (Increasing amplitude), in accordance to an exemplary embodiment of the present invention;

Fig 5 shows Lactalbumin nanoparticles (LANP) were successfully made using sol oil method, in accordance to an exemplary embodiment of the present invention;

Fig 6 shows Alpha lactalbumin loaded DNA nanoparticles shows hydrodynamic diameter of 141.68 nm (Panel A) and zeta potential of -14.3 mV (Panel B), in accordance to an exemplary embodiment of the present invention;

Fig 7 shows The Lactalbumin nanoparticles (LANP) which showed a DNA loading efficiency of 65+10%, in accordance to an exemplary embodiment of the present invention;

Fig. 8. Panel A. Image shows transfected H9C2(2-1) heart cells with pEGFP-C1 plasmid using lipofectamine loaded DNA as positive control, lactoferrin-loaded DNA nanoparticles as negative control (as lactoferrin receptors are lower expression on heart cells), GFP-p53 plasmid DNA using alpha-lactalbumin nanoparticles showed in the experiment;

Panel B. Quantitative analysis of transfection efficiency of pEGFP-C1 delivered via lipofectamine (left) (control), Lactoferrin (middle) (Control), and alpha-lactalbumin (right) nanoparticles using Image J software, in accordance to an exemplary embodiment of the present invention;

Fig 9 shows Localization of GFP-p53 DNA in rat organs when administered intravenous route using nanoparticles (LF lactoferrin; LA: Lactalbumin; AL: Albumin), in accordance to an exemplary embodiment of the present invention;

Fig 10 (A) shows Western Blot analysis for the expression of GFP after delivery of pEGFP-C1 plasmid using variant of albumin, in accordance to an exemplary embodiment of the present invention;

Fig 10 (B) shows the Ponceau image of the blot to check the transfer and equal loading, in accordance to an exemplary embodiment of the present invention;

Fig. 11 illustrates the lactalbumin possess significant affinity to Carvedilol with binding energy of -7.0 to -8.1, in accordance to an exemplary embodiment of the present invention;

Fig 12 shows TEM analysis of Carvedilol loaded lactalbumin loaded nanoparticles, in accordance to an exemplary embodiment of the present invention;

Fig 13 (A) shows Dynamic Light Scattering DLS analysis of Carvedilol loaded lactalbumin loaded nanoparticles (size), in accordance to an exemplary embodiment of the present invention;

Fig 13 (B) shows DLS analysis of Carvedilol loaded lactalbumin loaded nanoparticles (Zeta potential), in accordance to an exemplary embodiment of the present invention;

Fig 14 shows FTIR analysis of Carvedilol loaded lactalbumin loaded nanoparticles, in accordance to an exemplary embodiment of the present invention;

Fig 15 panel A shows Dynamics of drug localization, in accordance to an exemplary embodiment of the present invention;

Fig 15 panel B. shows Densitometric analyses of the above fluorescence images using image J, in accordance to an exemplary embodiment of the present invention;

Fig 16 protocol employed for induction of short term and long term dilated cardiomyopathy in rats using doxorubicin, in accordance to an exemplary embodiment of the present invention;

Fig 17 shows effect of Doxorubicin Induced Short term cardiomyopathy on Heart Rate, in accordance to an exemplary embodiment of the present invention;

Fig.18 (A) shows Effect on mean heart rate in individual rats, in accordance to an exemplary embodiment of the present invention;

Fig.18 (B) shows Effect on heart rate over time interval;
(C) shows Effect on mean R-R interval in individual rats;
(D) shows Effect on mean R-R interval (population) over time interval, in accordance to an exemplary embodiment of the present invention;

Fig. 19 (A) illustrates the Panel A. Western blot image represents levels of troponin I in heart, in accordance to an exemplary embodiment of the present invention;

Fig.19 (B) the Panel B. Ponceau image of the blot to check the transfer and equal loading, in accordance to an exemplary embodiment of the present invention;

Fig.19C shows Effect on mean R-R interval in individual rats, in accordance to an exemplary embodiment of the present invention;

Fig.19D shows a decrease in R-R interval after the completion of four doses of doxorubicin, in accordance to an exemplary embodiment of the present invention;

Fig. 20 shows Panel A. Western blot image represents levels of troponin I in blood, in accordance with our present invention.

Fig 21 shows a mild decrease in troponin I levels compared to control rat heart, in accordance to an exemplary embodiment of the present invention;

Fig. 22 shows Panel A. Western blot image represents levels of troponin I in blood, in accordance to an exemplary embodiment of the present invention;

Fig 23 shows ECG Data is representative image of treatment groups suggests a significant increase in QRS complex, in accordance to an exemplary embodiment of the present invention;

Fig 24 shows suggest a significant difference in QRS complex in case of nano formulated after 14 days of treatment of plasmid, in accordance to an exemplary embodiment of the present invention;

Fig 25 shows analysis of heart tissue in DNA-loaded lactalbumin nanoparticles treated rats, in accordance to an exemplary embodiment of the present invention;

Fig 26 shows the collected heart was sectioned (T.S) and H&E staining was performed to observe tissue morphology as compared to control, in accordance to an exemplary embodiment of the present invention;

Detailed Description of the Invention

It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. In addition, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. 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. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

The principles of operation, design configurations and evaluation values in these non-limiting examples can be varied and are merely cited to illustrate at least one embodiment of the invention, without limiting the scope thereof.
The embodiments disclosed herein can be expressed in different forms and should not be considered as limited to the listed embodiments in the disclosed invention. The various embodiments outlined in the subsequent sections are constructed such that it provides a complete and a thorough understanding of the disclosed invention, by clearly describing the scope of the invention, for those skilled in the art.
Throughout this specification various indications have been given as to preferred and alternative embodiments of the invention. It should be understood that it is the appended claims, including all equivalents, which are intended to define the spirit and scope of this invention.
The present invention relates to a novel nanoparticle formulation composed of chemotherapeutic agent or/and biological loaded in alpha lactalbumin nanoparticles. DNA Loaded alpha lactalbumin nanoparticles are characterized using DLS and FE-SEM and an average size of 537nm and 55 nm nanoparticles are produced respectively.

In-vitro localization of pEGFP-C1 plasmid using alpha lactalbumin nanoparticles in rat heart cardio myoblasts is more as compared to BSANP and LFNP. In-Vivo study suggest localization of pEGFP-C1 plasmid using alpha lactalbumin nanoparticles in Thymus, heart and lymph node and Brain.

Establishment of potent delivery vehicle might ensure stability of therapeutic molecules in the system (in-vivo) and efficacy at site of action. Delivery of therapeutic gene using these systems corroborate intervention of DCM, heart failure and injury.

Furthermore, a composition and ratio of lactalbumin and lactoferrin determine the ability loaded API in localization in specific tissue, thus for treatment of tissue-specific cancers.

The method of preparation and the various studies and tests conducted using our formulation have been explained in detail below:
Preparation of GFP-p53 DNA-loaded lactoferrin and alpha lactalbumin nanoparticle. The preparation of Lactoferrin nanoparticles was carried out using the sol-oil method. 1: 10 molar ratio of DNA: Alpha lactalbumin was taken and incubated overnight at 4?C. With an amplitude of 40 and 30 sec on & off pulse for 5 mins, sonication (ultrasonic homogenizer 300 V/T, Biologics Inc., Manassas, Virginia, USA) of the above mixture has done on ice; the above pattern was repeated three times then immediately snap frozen in liquid nitrogen for 10 mins and later on thawed on ice. The nanoparticles were pelleted down at 6000 RPM for 10mins at 4?C and washed with ether till the removal of oil traces. The nanoparticles were re-suspended in 0.1 M PBS buffer pH-7.4. In case of drug, the drug was incubated for period of saturation as per spectrophotometric analysis.

Nanoparticle composition with Lactoferrin and Lactalbumin at ratio of 80:20 and 60:40: Weighed rhodamine 6G 1mg and dissolved in water and lactoferrin and lactalbumin in the ratio of 80:20 and 60:40percent (i.e.,3.2mg and 0.8mg and 2.4mg and 1.6mg respectively) and dissolved in 1X PBS(pH – 7.4). Mixed and incubated on ice for 1hr. After 1h added 2ml of olive oil while vortexing slowly. Sonicated for 15mins at 40Hz amplitude with 5mins time interval. Next snap freezed in liquid nitrogen for 10min. Thawed on ice for 3hrs. Centrifuged at 6000rpm for 10mins. Removed supernatant and washed the pellet with diethyl ether. Airdried pellets and dissolved in 1X PBS(pH – 7.4).

Example 1. Protein & DNA docking –
Before preparation of DNA loaded protein nanoparticles interaction of DNA with Lactoferrin and alpha lactalbumin need to be scrutinized, docking studies of DNA with both the proteins were done. PDB structure of B- type DNA and both the protein was procured from RCSB. PDB website in .pdb format, then submitted to Haddock 2.4 server for blind docking. The results obtained from server can be visualized in pymol application.

Results- Docking study suggests interaction between target DNA Molecule and alpha lactalbumin at Lysine, Isoleucine, Asparagine and Glutamine

Example 2. Gel Binding Assay to analyse the interaction of GFP-p53 plasmid DNA and Alpha lactalbumin Protein : To perform binding analysis, we calculated the copy number of 1 microgram of GFP-p53 plasmid DNA based on that copy number of the alpha lactalbumin protein was calculated and then converted to molarity. Table 1. shows the molarity of the protein used to encapsulate 0.327 pM of DNA i.e. 1 microgram of DNA.

Table 1: the molar ratio of DNA and variant of albumin used for binding assay.

Fig 2 shows the Agarose Gel image representing pEFGP-C1 and variant of albumin interaction, Gel retardation assay was performed with increasing concentration of variant of albumin (32.7,65.4, 98.1,130.8, 163.5, 196.2,228.9, 261.6, 294.3,327 nM) along with fixed concentration of pEGFP (327 pM) , the DNA/Protein mixture were made upto 30 µl using 1x PBS pH-7,4 and incubated for 12 hrs at 4 °C. The above samples were loaded on to gel using 6x Loading Dye (Thermoscientific). Lane 1 – 10 Kb ladder , Lane 2 – variant of albumin Control (342pM) , Lane 3- pEGFP-C1 ( 327pM) , Lane 4- 11 DNA/ Protein mixture, The gel retardation assay was performed with a fixed concentration of DNA (pEGFP-C1) and an increasing concentration of a variant of albumin. The above mixture was incubated o/n at 4 degrees, then loaded onto 1% agarose gel, and the gel was stained with EtBr and visualized under Chemidoc (Bio-Rad). (Fig:2). The gel was then stained with sypro-ruby to counter-stain the protein (Fig 3). Sypro-Ruby Staining- To show the increasing amount of variant of albumin, the above gel was stained with sypro-ruby (protein binding fluorescence dye) for 30 mins in dark later washed with double distilled water. The image was taken in Chemidoc (Bio-Rad).
Fig 3 shows Agarose Gel image represent increasing amount of variant of albumin interaction, the above gel was counter stained with sypro-ruby. Lane 4-11 increasing concentration of variant of albumin ( 32.7,65.4, 98.1,130.8, 163.5, 196.2,228.9, 261.6, 294.3,327 nM) along with fixed concentration of pEGFP (327 pM) , the DNA/Protein mixture were made upto 30 µl using 1x PBS pH-7,4 and incubated for 12 hrs at 4 °C. The above samples were loaded on to gel using 6x Loading Dye (Thermoscientific). Lane 1 – 10 Kb ladder , Lane 2 – variant of albumin Control (342pM) , Lane 3- pEGFP-C1 ( 327pM) , Lane 4- 11 DNA/ Protein mixture.

The results in Fig 2 and 3 suggest at a molar ratio of 1: 5x105 variant of albumin masks the plasmid completely hence the above ratio is suitable for the preparation of nanoparticles. Though the gel binding assay does not give proper ratio due to smaller size of protein , to check the binding sensitivity Microscale thermophoresis was performed.

Example 3. Stability of DNA and lactalbumin complexes analyzed by Microscale Thermophoresis –
Microscale thermophoresis is a method to evaluate the degree of interaction between two molecules by accessing differential fluorescence signal as an outcome of Infrared laser-based temperature variation, certainly measurement of the movement of fluorescent labelled molecules with the change in temperature. Fluorescence signal also change on the basis of fluorescent target and ligand binding. Before performing MST, target molecule needed to be labelled with fluorescent tag hence GFP-p53 plasmid DNA was tagged with cy5.5, the fixed amount of tagged DNA was then incubated with increasing concentration of protein separately at 37 degrees, the above samples were then loaded in capillaries and then subjected to run in Nano temper Monolith.

Example 4. Sonication Optimization for Preparation of DNA loaded Protein nanoparticles -

GFP-p53 plasmid DNA was incubated with Protein (Lactalbumin separately), ratio of DNA: protein used is similar which was obtained from Microscale thermophoresis. The reaction was prepared in three different microcentrifuge tubes for each protein, the reaction was then sonicated at increasing amplitude (20%, 30% &40% respectively.) for 15 mins to check the integrity of DNA during sonication process used for the preparation of nanoparticles. The above samples were then run on 1% agarose gel, and image was taken using Gel doc (Bio-Rad). The results in figure show the DNA is protected by alpha lactalbumin under sonication.

Example 5. Preparation of DNA loaded lactalbumin nanoparticles -
GFP-p53 plasmid DNA was incubated with lactalbumin separately at molar ratio of 1:10 at 37 degree C for 1 hr. The above mixture was slowly mixed with twice volume of olive oil and vortex, then sonicated for 15 mins total, in pulsative manner (5 second on and Off) at 40% amplitude continuously for 5 mins then incubated on ice for 2 mins. The sonicated mixture was snap frozen with liquid nitrogen and thawed on ice, later centrifuged at 10,000 RPM for 15 mins at 4-degree C. Supernatant was removed and pellet was washed with Di ethyl ether thrice to remove traces of olive oil, finally dissolved in 0.1 M PBS buffer (pH-7.4).

Characterization of Protein nanoparticles using TEM and FE-SEM –
For transmission electron microscopy samples carbon coated copper grids of 200 mesh size were used, Nanoparticle samples were diluted then added on a parafilm, grid was placed on sample for 10 mins extra samples were then removed from sides with the help of tissue paper. 2 % uranyl acetate was used to stained the sample for 30 sec and removed with the tissue from the corners. After sample preparation, grids were subjected to imaging.
For Field emission scanning electron microscopy samples were added to a sticky carbon disc which increases the conductivity that carbon disc was later placed on metal stub for imaging.
Electron microscopy (TEM & FEM) images Suggests the particles sizes are in 100 nm range. Left Panel : blank Alpha lactalbumin nanoparticles size ranges from 30-40nm while DNA loaded particles are in range of 45-77nm.

Characterization of protein nanoparticles using Dynamic light scattering -
Dynamic light scattering is a technique which measures the variation in scattered light intensity due to movement of particles size ranges from (5nm-5µm). Rate of diffusion of particles in solvents is directly proportional to rate of scattered light fluctuation which is also directly proportional to hydrodynamic radius of the particles. Nanoparticles samples were dissolved and diluted in PBS buffer filtered with 0.2 µm syringe filter, samples were added to cuvette and cuvette was placed in zeta sizer to calculate hydrodynamic radius.

Evaluation of Loading efficiency -
Percentage of GFP-p53 plasmid (cy5.5 tagged) DNA and carvedilol Drug loaded in the protein nanoparticles (Alpha lactalbumin) were evaluated by digesting of DNA loaded protein nanoparticles using proteinase K at 60 degree C, while drug loaded nanoparticles by precipitating the protein nanoparticles with acetonitrile, the above mixtures were centrifuged at 12,000 RPM for 20 mins at 4 degree C , supernatant was collected and absorbance of both DNA (cy5.5 tagged DNA) and Carvedilol drug was taken using spectrofluorometer before loading and after releasing. Formula used to calculate loading efficiency is
Loading Efficiency (%) = amount of Drug and DNA released from nanoparticles * 100 Amount of drug and DNA used for nanoparticles preparation.

Example 5
Comparison of Transfection efficiency of GFP-p53 plasmid using different nanoparticles –
H9C2(2-1) rat cardiomyobalst cell lines were maintained in 5% co2 condition in CO2 incubator at 37-degree C. The cells were seeded in 6 well plates, after attaining 70 % confluency, GFP-p53 plasmid was transfected in serum free environment using Lipofectamine 3000, Lactoferrin nanoparticles and alpha lactalbumin nanoparticles in each wells respectively. The experiment was performed in triplicates and incubated for 24 hrs, media was replenished and after 48 hrs of transfection protein expression was observed in fluorescence microscope as plasmid vector contains GFP gene cassette. Images obtained was further quantified using Image J software. The results in Fig 9 show significant localization of DNA and transfection efficiency using lactalbumin nanoparticles in heart cells.

Fig. 8. Panel A. Image shows transfected H9C2(2-1) heart cells with pEGFP-C1 plasmid using lipofectamine loaded DNA as positive control, lactoferrin-loaded DNA nanoparticles as negative control (as lactoferrin receptors are lower expression on heart cells), pEGFP-C1 plasmid DNA using alpha-lactalbumin nanoparticles showed in the experiment. Results suggest significantly higher expression of GFP in DNA loaded lactalbumin nanoparticles compared to lipofectamine and lactoferrin nanoparticles. Panel B. Quantitative analysis of transfection efficiency of pEGFP-C1 delivered via lipofectamine (left) (control), Lactoferrin (middle) (Control), and alpha-lactalbumin (right) nanoparticles using Image J software. Thus, in-vivo localization of DNA using Lipofectamine and protein nanoparticles, fluorescence microscopy images suggest Higher localization of DNA in H9C2(2-1) using LANP as compare to LFNP and Lipofectamine.

Example 6:
Localization of different GFP-p53 plasmid DNA loaded protein nanoparticles in different organs –
For localization studies, approval of institution animal ethics committee of University of Hyderabad was taken and animals were kept in animal house properly caged with water and fodder in air-conditioned environment. Three different protein nanoparticles were prepared and administered in Wistar rats weighted 150-200 gms through intravenous route each dose was given to 3 rats (group size n=3), after 2 days animals were sacrificed and organs were harvested and DNA and protein was isolated, For DNA: primers were designed against GFP and PCR was performed using organs DNA as template, amplified product was run on 2% agarose gel and image was taken using gel doc. For Protein: Protein was isolated using RIPA buffer was run on 12% SDS PAGE and western was performed using GFP antibody.

Agarose gel image after polymerase chain reaction in Fig 10 suggests localization of pEGFP-C1 DNA in rat organs Via different Protein nanoparticles (Albumin, Lactoferrin, Lactalbumin). Significant localization is found in the Heart while plasmid DNA was delivered using Alpha Lactalbumin. The expression of GFP after delivery of plasmid. Protein was isolated from the rat organs and SDS PAGE was performed followed by Western blot. The blot was probed with using anti-GFP antibody.

Fig 10 A shows Western Blot analysis for the expression of GFP after delivery of pEGFP-C1 plasmid using variant of albumin (1-11) represents L-Ladder, Bp- pEGFP without vehicle in Brain, LP - pEGFP without vehicle in Liver, Hp - pEGFP without vehicle in Heart, Kp - pEGFP without vehicle in Kidney, Sp- pEGFP without vehicle in Spleen,Ba- pEGFP with variant of albumin vehicle Brain., Ha- pEGFP with variant of albumin vehicle Heart.,La - pEGFP with variant of albumin vehicle Liver, Ka - pEGFP with variant of albumin vehicle Kidney. Sa - pEGFP with variant of albumin vehicle Spleen respectively.

Fig 10 b shows the Ponceau image of the blot to check the transfer and equal loading the results show that the expression of GFP was observed in rats after 48 hrs of its administration while deliver using various means. A. Expression was observed in Lungs, Kidney, Liver & Brain while delivered using LFNP. B. Expression was observed in Liver, Heart, Brain and spleen while delivered using LANP. C. Comparison between expression in Heart indicated band only in Heart LANP i.e. pEGFP-C1 delivered using LANP.

Example 7. Carvedilol and Alpha lactalbumin Docking to analyze interaction before nanoparticles preparation –
Carvedilol is a FDI approved beta adrenergic receptor blocker used for the intervention of DCM and hypertension. Docking was performed to check the drug and protein interaction for the preparation of the Nano formulation. Docking of carvedilol and alpha lactlbumin was performed using Auto dock vina software while setting energy range at 4 and exhaustiveness at 8. which utilizes MGL tools to get files in. pdbqt format. The completion of Docking gives Affinity of binding of protein with the drug. Values in negative represents the binding energy. Greater negative value suggests strong binding between drug and ligand.

Docking data in Fig 12 and Table 3 suggest lactalbumin possess significant affinity to Carvedilol with binding energy of -7.0 to -8.1 which indicates higher affinity and strong binding of drug and protein. Data suggests that drug can encapsulate in the protein as it has higher affinity towards the protein. Carvedilol binds to IL-59, TRP-60, ASN-66, GLU-49, ALA-106, LEU-105 , VAL-99, TYR-103 and TRP-1044 residue of alpha Lactalbumin. Binding energy suggest higher affinity of drug towards the protein.

Example 8.
Characterization of Carvedilol loaded lactalbumin nanoparticles using TEM–
For transmission electron microscopy samples carbon coated copper grids of 200 mesh size were used, Nanoparticle samples were diluted then added on a parafilm, grid was placed on sample for 10 mins extra samples were then removed from sides with the help of tissue paper. 2 % uranyl acetate was used to stained the sample for 30 sec and removed with the tissue from the corners. After sample preparation, grids were subjected to imaging. For Field emission scanning electron microscopy samples were added to a sticky carbon disc which increases the conductivity that carbon disc was later placed on metal stub for imaging. Particle size of 70-98nm of Carvedilol loaded LA nanoparticles has been observed in TEM image.

Characterization of protein nanoparticles using Dynamic light scattering
Dynamic light scattering is a technique which measures the variation in scattered light intensity due to movement of particles size ranges from (5nm-5µm). Rate of diffusion of particles in solvents is directly proportional to rate of scattered light fluctuation which is also directly proportional to hydrodynamic radius of the particles. Nanoparticles samples were dissolved and diluted in PBS buffer filtered with 0.2 µm syringe filter, samples were added to cuvette and cuvette was placed in zeta sizer to calculate hydrodynamic radius. The results in Fig 12 show that the Carvedilol loaded lactalbumin loaded nanoparticles exhibit 70-98nm has been observed in TEM images however, the hydrodynamic size is 187.63nm (Fig 13A) with zeta potential of -25mV (Fig 13B).

FTIR analysis of carvedilol loaded LANP -
for checking integrity of the drug in nano formulated form, nanoparticles particles were lyophilized and subjected for FTIR FTIR analysis in Fig 14 and Table 4 shows the functional groups of the drug is unchanged in nano formulation when compared with only drug and also the cross verified with the literatures which suggests integrity of the carvedilol drug in nano formulated form.

Example 9
Time dependent drug uptake in H9C2 (2-1): Curcumin loaded alpha lactalbumin nanoparticles were prepared and 1 micrograms curcumin loaded lactalbumin nanoparticles were added to H9C2 (2-1) cells and incubated at different time points (0hrs, 4hrs, 6hrs, 8hrs) respectively, soluble curcumin (control) was added and incubated for 4 hrs. The results in Fig 16 show that the significant localization of drug observed at 4 hours reaching high t 6 hours and decreases from 8th hours onwards.

Fig 15A shows Dynamics of drug localization. Fig 15 Panel A- Fluorescence microscopy suggests at 4 hrs (Panel B) green flurescence of curcumin was observed in the cells, maximum fluorescence intensity was observed at 6 hrs (panel C) and started reducing from 8 hrs (panel D) while at 12 hrsd (panel E) time point very less signal intensity was observed. Panel B. Densitometric analyses of the above fluorescence images using image J.
Example 10. Efficacy of lactalbumin nanoparticle in treatment of cardiomyopathy.
Cardiomyopathy Model Development:
Doxorubicin Induced Short term cardiomyopathy– The protocol as per Figure 16 was employed for induction of short term and long term dilated cardiomyopathy in rats using doxorubicin, the model development is monitored based the electrical activity of heart using ECG.
Short Term dilated cardiomyopathy -
At the time of model development, ECG recordings were taken at 3 time points, namely (1) before doxorubicin administration, (2) after three doses of doxorubicin, (3) after 6 doses of doxorubicin in case of short term (fig.17) dilated cardiomyopathy model. Parameters like heart rate and R-R interval were recorded and the values obtained were plotted against time interval and also status of HR (heart rate) and R-R interval in individual rats were recorded.

Effect of Doxorubicin Induced Short term cardiomyopathy on Heart Rate shown in Fig 18. Fig 18A shows Effect on mean heart rate in individual rats. Fig 18B shows Effect on heart rate (population) over time interval. Fig. 18C shows Effect on mean R-R interval in individual rats. Fig.18D shows Effect on R-R interval (population)over time interval.

The results in Fig. 18 C and D, represents R-R interval prior to treatment (in blue bar), after 3 doses of doxorubicin (2.5 mg/kg body weight/ day) (Orange bar) and after 6 doses of doxorubicin (2.5 mg/kg body weight/ day) (Green bar), the treatment was terminated after 6 doses of doxorubicin. The results shows that show that R-R interval decreased after 3 doses followed by significant increase after completion of six doses of treatment, thus the results of decrease in heart rate and increase in RR interval confirms establishment of bradycardic condition.

Long term cardiomyopathy: During the development of long term dilated cardiomyopathy model, the ECG recordings were taken at four time points: (1) prior to doxorubicin administration, (2) after three doses of doxorubicin (2 mg/kg bodyweight/ day), (3) after 6 doses of doxorubicin (2 mg/kg bodyweight/ day), (4) after 9 doses doxorubicin (2 mg/kg bodyweight/ day). The parameters namely heart rate and R-R interval were recorded and the values obtained were plotted against time interval and also status of HR (heart rate) and R-R interval in individual rats were recorded.

Effect of Doxorubicin Induced Long term cardiomyopathy on Heart rate-
Fig.19A shows Effect on mean heart rate in individual rats. Fig.19B shows Effect on heart rate over time interval. The results in Fig.19A and B shows significant increase in heart rate at 3 dose treatment followed by decrease in heart rate over 6 and 9 dose administration of doxorubicin, thus suggesting establishment of tachycardia condition.

Effect of Doxorubicin Induced Long term cardiomyopathy on R-R Interval
Fig.19C shows Effect on mean R-R interval in individual rats. Effect on mean R-R interval (population) over time interval. The results in Fig .19 C and D shows a decrease in R-R interval after the completion of four doses of doxorubicin, thus the decrease R-R interval confirms establishment of tachycardic condition.

Analysis of dilated cardiomyopathy using Western blot: Troponin I is a biomarker for cardiac injury and heart failure. Troponin I is very specific to cardiomyocyte as it is one of cytoskeletal component that is released in the blood upon cardiac injury, hence detecting Troponin I in blood indicate possible cardiomyocyte damage. Troponin I was analysed in serum samples of the rats treated with doxorubicin in the short-term and long-term cardiomyopathy, the results of Western blot analysis presented in Fig 15.

Fig.20 shows Western blot image represents levels of troponin I in heart: lane 1 –FH heart isolated from female DCM model, Lane 2- MH heart isolated from male DCM model, FB – Blood drawn from female DCM model after completion of all the doses, MB- blood drawn from male DCM model after completion of all doses, CH- Control rat heart.

The results in Fig 10, show that the levels of troponin I started appearing in blood in both male and female in short-term DCM, suggesting a lower damage induction in these rats. We are repeating these studies for further confirmation. Cardiac damage was studied in both short-term and long-term DCM models in 3 male rats by analysis of Troponin I, the results in Fig 21 show a mild decrease in troponin I levels compared to control rat heart, while in long term-DCM rats there is significant decrease in troponin I indicates damage in heart tissue in long-term DCM rats model is higher when compared to short-term DCM. Further, we observed a variability in Troponin I levels in rat, one rat showing very low, thus doxorubicin sensitivity is variable among the rats in long-term DCM model.

Level of Troponin I in Blood with progression of Doxorubicin dosage: During the model development we have collected the blood samples at different time points: before doxorubicin administration, after third dose, after sixth dose and after ninth dose in case of long term. Isolated plasma from blood used for western and probed against troponin I antibody.

Fig. 22 shows Panel A. Western blot image represents levels of troponin I in blood: 0 dose: without doxorubicin treatment, 3 dose: After 3 doses of Doxorubicin, 6 dose: After 6 doses of Doxorubicin, 9 dose: After 9 doses of Doxorubicin. In rats before treatment no bands of troponin I was observed while after completion of three doses, troponin I started appearing and bands intensifies at the completion of doses i.e. after 9 doses.

Panel B. Ponceau image of the blot to check the transfer and equal loading. The results in Fig 22 a show that levels of troponin I started appearing in blood after completion of three doses of doxorubicin and increases after 6 doses and reaches maximum after completion of nine doses, indicates an enhanced damage in heart tissue leading to long-term DCM.

Analysis of Dilated cardiomyopathy model development by H & E staining
After completion of the doxorubicin treatment for model generation, heart of the rats were collected and subjected for sectioning. Anatomical observation were recorded after H & E staining.
Fig.22 shows Sectioning images represents: upper panel- control rat heart sections, Middle panel – short term rat 1 tissue samples, last panel – short term rat 2 tissue sample. After the completion of model development procedure heart from the rats were harvested and sectioned, as compare to upper panel middle and last panel shows vacuolar degeneration. Comparative histochemical analysis of heart tissue sections showed that short term model for DCM shows vacuolar degeneration characterized by the gradual vacuolization of muscle fibers (Fig 22), resulting in a nucleus within a sarcolemma tube that otherwise appears empty which is a sign of chemotherapeutics induced cardiotoxicity.

Treatment strategy for DCM using nano formulation of Drug and DNA –
A total 36 healthy rats were administered with doxorubicin to induced DCM after confirmation of the model development using ECG and blood marker analysis (as described above), rats were categorized into 4 DCM group and one control group, different treatment were given to each group of rats : Group 1 – Control (without Doxorubicin), Group 2 – PBS treated DCM, Group 3 – Blank LANP treated DCM rats , Group 4 Carvedilol treated , Group 5- Carvedilol LANP treated, Group 6 - GFP-p53 alone treated DCM rats, Group 7 – GFP-p53 LANP treated DCM rats, Carvedilol is a FDI approved beta adrenergic receptor blocker used for the intervention of DCM and hypertension Here p53 used as a therapeutic gene, many literature suggest the role of p53 in intervention of Chemotherapeutics induced DCM. Fig. 24. Suggest a significant difference in QRS complex in case of nano formulated after 14 days of treatment of plasmid GFP-p53 shows almost recovery of ventricular depolarization in rats as compare to GFP-p53 alone, Blank LANP and PBS treated rats while improvement was also observed in carvedilol LANP treated rats as compare to other groups. Restoration of QRS complex height majorly improves rat heart condition while treated with nanoformulations. One way Anova shows p value is < 0.001.
Fig 23 shows ECG Data is representative image of treatment groups suggests a significant increase in QRS complex in 24A/C, after treatment with Carvedilol LFNP (24D) restore back compared to Carvedilol alone (24B). Similarly DCM rats (24E/G), after treatment with GFP-p53 LANP (24H) R height seems to restore back while GFP-p53 alone (24F) shows no improvement in R height Which indicates intervention in ventricular function in case of GFP-p53 LANP treatment.

Analysis of heart tissue in DNA-loaded lactalbumin nanoparticles treated rats
As per methodology the rats were sacrificed on 25 th day after 21 days for treatment and the collected heart was sectioned (T.S) and H&E staining was performed to observe tissue morphology as compared to control Figure 25A, PBS treated rat; (Panel 2) and 25B: GFP-p53 rats (panel 3) were showing abnormal morphology of tissues, 25C: tissues isolated from GFP-p53 LANP (rightmost panel) treated rats showed restoration to to normal morphology which suggests nano formulation of GFP-p53 enhances localization of plasmid DNA and p53 expression thus restore the function of the Heart.

Analysis of heart tissue in drug-loaded nanoparticles treated rats
As per methodology the rats were sacrificed on 25th day after 21 days for treatment and the collected heart was sectioned (T.S) and H&E staining was performed to observe tissue morphology as compared to control Figure 26A, PBS treated rat; (Panel 2) and 26B: Carvedilol treated rats (panel 3 ) were showing abnormal morphology of tissues, 26C: tissues isolated from Carvedilol LANP (rightmost panel) treated rats showed restoration to normal morphology which suggests nano formulation of Carvedilol enhances localization of drug thus restore the function of the Heart
,CLAIMS:5. CLAIMS
I/We Claim:
1. A composition of DNA loaded alpha lactalbumin nanoparticles, wherein the method of preparation is given by:
a. Preparation of GFP-p53 DNA-loaded lactoferrin and alpha lactalbumin nanoparticle:
i. The preparation of Lactoferrin nanoparticles was carried out using the sol-oil method;
ii. 1: 10 molar ratio of DNA: Alpha lactalbumin was taken and incubated overnight at 4?C.
iii. With an amplitude of 40 and 30 sec on & off pulse for 5 mins, sonication of the above mixture has done on ice; the above pattern was repeated three times then immediately snap frozen in liquid nitrogen for 10 mins and later on thawed on ice.
iv. The nanoparticles were pelleted down at 6000 RPM for 10mins at 4?C and washed with ether till the removal of oil traces.
v. The nanoparticles were re-suspended in 0.1 M PBS buffer pH-7.4.
vi. In case of drug, the drug was incubated for period of saturation as per spectrophotometric analysis.
b. Nanoparticle composition with Lactoferrin and Lactalbumin at ratio of 80:20 and 60:40:
i. Weighed rhodamine 6G 1mg and dissolved in water and lactoferrin and lactalbumin in the ratio of 80:20 and 60:40percent (i.e.,3.2mg and 0.8mg and 2.4mg and 1.6mg respectively) and dissolved in 1X PBS(pH – 7.4). Mixed and incubated on ice for 1hr.
ii. After 1h added 2ml of olive oil while vortexing slowly.
iii. Sonicated for 15mins at 40Hz amplitude with 5mins time interval.
iv. Next snap freezed in liquid nitrogen for 10min. Thawed on ice for 3hrs. Centrifuged at 6000rpm for 10mins.
v. Removed supernatant and washed the pellet with diethyl ether.
vi. Airdried pellets and dissolved in 1X PBS (pH – 7.4).

2. The composition of DNA loaded alpha lactalbumin nanoparticles, as claimed in claim 1, wherein the DNA loaded alpha lactalbumin nanoparticles are 44 to 70 nm by FE SEM, while hydrodynamic size is 537 nm.