DESCRIPTION
Field of the Invention
Embodiments of the present invention relate to nanoemulsion formulations and methods for delivering of an effective amount of oxygen to cells. More specifically, the embodiments of the present invention relate to nanoemulsion formulations for parenteral and non-parenteral delivery of oxygen to a subject.
Background of the Invention
Approximately there are 34 million critical care admissions recorded annually in India alone, of which 30% of victims die due to the imbalance in blood
Every human cell requires a continuous supply of oxygen to conserve cellular structure and homeostasis. This source of oxygen is mainly provided by hemoglobin, which transports inspired liquid oxygen from the pulmonary capillaries to the tissues and expire carbon dioxide from tissues to pulmonary capillaries. In conditions where a patient's lungs are incapable to transfer adequate amounts of oxygen to circulating erythrocytes, severe hypoxia results and can rapidly lead to severe organ injury and demise. Refurbishment of blood oxygen tension is chief to revival of the majority of pathophysiologic states.
Some clinical states such as lung injury, airway obstruction, asthma, pneumonia and intra-cardiac mixing, exhibit hypoxemia and desaturation refractory to medical efforts to restore levels of oxygen saturation sufficient to limit ischemic injury. Ischemic injury may take place within minutes or seconds due to inadequate oxygen supply. In such conditions the human body may lead to low oxygen tension, can cause end-organ dysfunction, failure and

mortality. The capability to elevate oxygenation on the mortality and morbidity
from acute hypoxia, in count to a number of other clinical situations.
Even during emergency, patients have to rely on oxygen mask and cannula
which are not sufficient to serve the need of liquid oxygen. Moreover,
emergency efforts to deliver oxygen to a patient are often inadequate and/or require too long to take effect, either due to lack of an adequate airway or overwhelming lung injury or psychological impact like anxiety, panickiness. This results in irreparable injury to the brain and other vital organs. To initiate rescue therapy in these patients is troublesome and time consuming. There is a need to quickly provide liquid oxygen directly to the blood of patients, thereby inhibiting or lessening irreversible injury due to hypoxemia.
Currently there are traditional attempts to reinstate oxygen levels in patients by using supportive therapy to the patient's respiratory system. Most usually by using the method mechanical ventilation or by Extracorporeal Membrane Liquid oxygenation (ECMO). However, patients with lung injury and intensive care unit patients often face difficulties in exchanging oxygen and carbon dioxide across a damaged respiratory system. This requires clinicians to upsurge ventilator pressures often, this further cause’s lung injury and systemic inflammation. Significant morbidity and mortality has been related with ventilator induced lung injury and barotrauma to the lungs which often occurs by inadequate systemic oxygen delivery. A method to non-invasively maintain even small percentages of oxygen delivery can significantly reduce the morbidity of mechanical ventilation and other complexities associated with hypoxia, perinatal asphyxia, breathlessness etc.
US patent application 20050042132 discloses an apparatus for blood oxygenation. The apparatus includes a delivery assembly including an elongated, generally tubular assembly including a central lumen and at least

one end placeable within a patient body proximate a tissue site to be treated, the end including an outlet port for the oxygenated blood. However, the apparatus not easy to use and the oxygen used is not encapsulated.
ANEMIC HYPOXIA
The arterial blood contains oxygen at its normal tension in anemic hypoxia, but there is a shortage of functioning hemoglobin. Anemic hypoxia, on the whole, is less serious than hypoxic hypoxia. However, it does affect the whole body. Anemic hypoxia may be caused by acute or chronic hemorrhage, primary or secondary anemia, alterations in the hemoglobin of the blood (caused by nitrates, chlorates, or coal tar derivatives), and carbon monoxide poisoning. STAGNANT HYPOXIA
Stagnant hypoxia is due to a decrease in the rate of flow of the circulating blood. Local regions of the body are usually involved, but it may affect the entire body. The blood is saturated normally with oxygen, and the oxygen load, as well as the tension under which it is held, also may be normal. Hypoxia is produced because the amount of oxygen reaching the tissues is inadequate. Sluggishness in the rate of the circulating blood allows the blood to stagnate and give up a greater percentage of its oxygen. This slow circulation also permits the accumulation of a greater quantity of carbon dioxide in the tissues. Stagnant hypoxia is produced by failure of the circulation, impairment of venous return, and shock. HYPOXIC HYPOXIA
In hypoxic hypoxia, there is a lack of oxygen in the arterial blood. The oxygen tension is lowered in both the lungs and the arterial blood, and the hemoglobin is not saturated with oxygen to its normal extent. This type of hypoxia affects the body as a whole and is one of the most serious forms of hypoxia. Hypoxic hypoxia is often produced by low tensions of oxygen in the inspired air as is seen in high altitudes, breathing of inert gases, and the inhalation of anesthetic

agents. Abnormal lung conditions may also produce hypoxic hypoxia. Emphysema, asthma, pneumonia, or pneumothorax encourage the formation of this type of hypoxia. Mechanical obstruction of the airway by foreign objects, laryngospasm, or bronchospasm inhibits the flow of oxygen from the atmosphere into the lungs, creating a state of oxygen want. Shallow respiratory movements from any cause, with either a decrease in rate or amplitude, may cause hypoxic hypoxia. A chronic state of hypoxic hypoxia may result from a patent foramen ovale and other embryo- logical malformations of the heart and blood vessels. HISTOTOXIC HYPOXIA
As the term suggests, the tissue cells are poisoned and are unable to accept oxygen from the capillaries. In this type of hypoxia the cells are not able to utilize the oxygen, although the amount of oxygen in the blood may be normal and under normal tension. Histotoxic hypoxia is produced by cyanides. Theoretically, it may be produced by any agent which depresses cellular respiration.
FULMINATING HYPOXIA
Fulminating hypoxia is a newly recognized form of hypoxia. It is a very rapidly induced type of hypoxia caused by the inhalation of undiluted inert gases such as nitrogen, methane, or helium. In anesthesia, fulminating hypoxia may be produced by administering nitrous oxide anesthesia without the simultaneous use of oxygen.
Therefore, in order to overcome the above mentioned drawbacks, there is need to develop a composition and a method to deliver an effective amount of oxygen to a patient to assuage or prevent ischemic injury.
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.
It is an object of the invention to provide liquid oxygen nanoemulsion composition.
It is another object of the invention to provide an improved composition for delivering liquid oxygen to patients, tissues or organs.
It is yet another object of the invention to deliver oxygen alone or in combination with other molecules or biologicals to have targeted deliver, controlled release, synergy.
Accordingly, the present invention relates to a liquid oxygen
nanoencapsulation composition and a method for delivering and eliminating liquid oxygen and carbon dioxide respectively, directly to/from patient’s blood flow and thereby to tissues and organs in respective order for maintaining blood homeostasis.
The underlying object of the present invention is to propose a liquid oxygen
nanoencapsulation composition that is easy to use, has no side effects and
affordable.
The present invention relates to a liquid oxygen nanoemulsion composition
comprising liquid oxygen, an aqueous component, a lipophilic component, and
an emulsifying agent. The nanoemulsion composition is in the form of a

continuous aqueous phase and a discontinuous liquid oxygen, wherein the liquid oxygen is present in the nanoemulsion at approximately 5 - 65% w/v. The liquid oxygen emulsion is stabilized by an emulsifying agent in the amount of approximately 3.5% - 4 % w/v of the total nanoemulsion composition.
One advantage of the present invention is that to deliver sufficient oxygen through intravascular route for patient in critical condition. Another advantage is targeted delivery of oxygen to solid tumors, which can enhance the efficacy of chemotherapy and radiotherapy.
These and other advantages of the invention will become apparent when viewed in light of the accompanying drawings, examples, and detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
Other objects and advantages of the present invention will become apparent to those skilled in the art upon reading the following detailed description of the preferred embodiments, in conjunction with the accompanying drawings, wherein like reference numerals have been used to designate like elements, and wherein:
FIG. 1 depicts manufacture of liquid oxygen nanoemulsion according to an embodiment of the invention;
DETAILED DESCRIPTION
For the purposes of this invention, the following definitions are provided. These definitions are intended to be illustrative and exemplary. They are not intended

to restrictively limit the common meaning of the terms to those skilled in the art. These definitions are as follows:
Blood gas homeostasis: Blood gas homeostasis means maintaining oxygen and carbon dioxide saturation levels in the blood. Blood gas homeostasis also means maintaining balance of oxygen and carbon dioxide levels to have optimum cellular physiological functioning and metabolism.
Hypoxia: Hypoxia means deficiency in the amount of oxygen reaching the tissues.
Embodiments of the composition and the method will now be described.
The present invention relates to nanoemulsion formulations and methods for delivering of an effective amount of oxygen to cells, and in particular nanoemulsion formulations for parenteral and non-parenteral delivery of oxygen to a subject.
The present invention relates to nanoemulsion formulations and methods for delivering of an effective amount of oxygen to cells, and in particular nanoemulsion formulations for intravascular delivery of liquid oxygen to a subject.
This present invention relates to compositions and methods for oxygen perfusion of tissues, and especially delivery of an effective amount of oxygen to a patient to alleviate or prevent ischemic injury. Administering oxygen intravascular route can increase venous and arterial oxygen tensions directly or, indirectly by complimenting the conventional methods of oxygen administrations like ventilation, nasal mask or tracheal tube.

ANEMIC HYPOXIA
The arterial blood contains oxygen at its normal tension in anemic hypoxia, but there is a shortage of functioning hemoglobin. Anemic hypoxia, on the whole, is less serious than hypoxic hypoxia. However, it does affect the whole body. Anemic hypoxia may be caused by acute or chronic hemorrhage, primary or secondary anemia, alterations in the hemoglobin of the blood (caused by nitrates, chlorates, or coal tar derivatives), and carbon monoxide poisoning. STAGNANT HYPOXIA
Stagnant hypoxia is due to a decrease in the rate of flow of the circulating blood. Local regions of the body are usually involved, but it may affect the entire body. The blood is saturated normally with oxygen, and the oxygen load, as well as the tension under which it is held, also may be normal. Hypoxia is produced because the amount of oxygen reaching the tissues is inadequate. Sluggishness in the rate of the circulating blood allows the blood to stagnate and give up a greater percentage of its oxygen. This slow circulation also permits the accumulation of a greater quantity of carbon dioxide in the tissues. Stagnant hypoxia is produced by failure of the circulation, impairment of venous return, and shock. HYPOXIC HYPOXIA
In hypoxic hypoxia, there is a lack of oxygen in the arterial blood. The oxygen tension is lowered in both the lungs and the arterial blood, and the hemoglobin is not saturated with oxygen to its normal extent. This type of hypoxia affects the body as a whole and is one of the most serious forms of hypoxia. Hypoxic hypoxia is often produced by low tensions of oxygen in the inspired air as is seen in high altitudes, breathing of inert gases, and the inhalation of anesthetic agents. Abnormal lung conditions may also produce hypoxic hypoxia. Emphysema, asthma, pneumonia, or pneumothorax encourage the formation of this type of hypoxia. Mechanical obstruction of the airway by foreign objects, laryngospasm, or bronchospasm inhibits the flow of oxygen from the

atmosphere into the lungs, creating a state of oxygen want. Shallow respiratory movements from any cause, with either a decrease in rate or amplitude, may cause hypoxic hypoxia. A chronic state of hypoxic hypoxia may result from a patent foramen ovale and other embryo- logical malformations of the heart and blood vessels. HISTOTOXIC HYPOXIA
As the term suggests, the tissue cells are poisoned and are unable to accept oxygen from the capillaries. In this type of hypoxia the cells are not able to utilize the oxygen, although the amount of oxygen in the blood may be normal and under normal tension. Histotoxic hypoxia is produced by cyanides. Theoretically, it may be produced by any agent which depresses cellular respiration.
FULMINATING HYPOXIA
Fulminating hypoxia is a newly recognized form of hypoxia. It is a very rapidly induced type of hypoxia caused by the inhalation of undiluted inert gases such as nitrogen, methane, or helium. In anesthesia, fulminating hypoxia may be produced by administering nitrous oxide anesthesia without the simultaneous use of oxygen.
FORMATION OF NANOEMULSIONS
Processes and compositions for the formation of liquid oxygen nanoemulsions, particularly self-assembled nanoemulsions are provided herein. A composition suitable for forming liquid oxygen nanoemulsion composition can include a lipophilic component, liquid oxygen, a hydrophilic component, and a surfactant characterized by a Phase Inversion Technique with respect to the lipophilic and hydrophilic components. The liquid oxygen is preferably more soluble in the lipophilic component than the hydrophilic component. These compositions can include up to about 5 wt % of the lipophilic component and have the surfactant and the lipophilic component present in an initial weight ratio of 3:1

or greater (e.g., 5:1-7:1 or greater) to provide liquid oxygen nanoemulsions with reduced droplet sizes of 10 to 5000 nm.
The emulsifying agent is selected from Sorbitan monostearate, sorbitan
monopalmitate and sorbitan 20 EO tristearate, Cetyl alcohol , disodium salt of
N-stearoyl-L-glutamic acid, Tween 20, 40, 60 and 80, propylene glycol
monostearate, glycerol monoleate, glycerol monostearate, acetylated
monoglycerides (stearate), sorbitan monooleate (Span 80), propylene glycol
monolaurate, sorbitan monostearate, and glycerol monolaurate,
polyoxyethylene POE (20) sorbitan monostearate, sucrose monolaurate, POE (20) sorbitan monooleate (Tween 80), and POE (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate, sucrose monolaurate, POE (20) sorbitan monooleate (Tween 80), POE (16) lanolin alcohols, and acetylated POE (9) lanolin, Caprylic capric glycerides, polyethylene glycol, Hexadecenyl succinate 18 EO, hexadecenyl succinate 45 EO, dihexadecenyl succinate 18 EO, dihexadecenyl succinate of glucose 10 EO, dihexadecenyl succinate of glucose 20 EO, dioctadecenyl succinate of methylglucose 20 EO, Sorbitan oleate, Polysorbate 80, Ceteareth 5 , Ceteareth 20 , Steareth-2, Sorbitan monooleate, polysorbate 80
The liquid oxygen can be present in the droplets of the lipophilic component of the liquid oxygen nanoemulsions composition. The droplet or particle size of a lipophilic component in the nanoemulsion can be decreased by decreasing the Ros ratio in the first composition (defined as the weight ratio of the total lipophilic component to the total weight of the lipophilic component and the surfactant) to about 0.500 and below (e.g., Ros ratios of 0.125-0.500). The liquid oxygen nanoemulsions compositions can also be characterized by one or more of the following weight ratios: an oil/(oil+surfactant) ratio of about 0.500 or less (including 0.250, 0.167 or 0.125); water/(water+oil) weight ratios

of about 0.980 or less (including ratios of 0.979, 0.978 and 0.977); oil/(oil+water) weight ratios of about 0.023 or less (including 0.022, 0.021, and 0.020); and/or water/(water+surfactant) weight ratios of 0.143 or less (including 0.102, 0.061 or 0.020). For example, liquid oxygen nanoemulsions compositions having average droplet sizes of less than 5000 nm, preferably 100 nm, 50 nm or 25 nm can be obtained using compositions with a surfactant and lipophilic component (e.g., a non-toxic oil) in a weight ratio of 3:1 or 5:1 (“S/O ratio”).
In a composition having up to about 5 wt % of the lipophilic component containing the liquid oxygen, increasing the weight ratio of the surfactant to the lipophilic component above about 3:1 can provide a reduction in the droplet size of the lipophilic component in the liquid oxygen nanoemulsions composition where the Ros ratio in the first composition (defined as the weight ratio of the total lipophilic component to the total weight of the lipophilic component and the surfactant) is about 0.500 and below (e.g., Ros ratios of 0.125-0.500). Increasing the S/O ratio in an initial formulation from 1:1 to 3:1 can result in a reduction (e.g., about an 30-97% reduction) in the average droplet size (e.g., from about 1,000 nm down to about 30-619 nm) of the lipophilic oil component in the resulting liquid oxygen nanoemulsions composition formed from the initial formulation; further increasing the S/O ratio in an initial formulation from 3:1 to 5:1 can result in about an additional reduction (e.g., a 70-83% reduction) of the average droplet size (e.g., down to about 21-121 nm) in a resulting liquid oxygen nanoemulsions composition formed from the initial formulation according to the processes described herein. Further average droplet size reductions can be obtained in a liquid oxygen nanoemulsions composition by increasing the S/O ratio from 5:1 to 7:1, which can result in an average droplet size of from about 15 to about 25 nm.

A nanoemulsion can be formed by homogenizeing a mixture of a lipophilic component and a liquid oxygen to dissolve the liquid oxygen in the lipophilic component. Next, the solution of the lipophilic component and the liquid oxygen can be homogenized and mixed with a hydrophilic component and a surfactant characterized by a temperature dependent phase inversion between the lipophilic component and the hydrophilic component at or above a phase inversion temperature (“PIT”) of the surfactant, followed by rapidly cooling the mixture to below the PIT to form the nanoemulsion (e.g., cooling the mixture in a homogenize-conducting vessel placed in an ice bath until the contents of the vessel are at room temperature or about 25 degrees C.)
Incorporation of Liquid oxygen:
The liquid oxygen can be incorporated in the nanoemulsion, for example by dissolving the liquid oxygen in the lipophilic component prior to or during step (a) above to form a bioactive liquid oxygen nanoemulsion composition. The bioactive liquid oxygen nanoemulsion compositions including one or more liquid oxygen can be made according to a method including any number of the following steps:
(a) Weigh a specified amount of a lipophilic component (e.g., 1 g, 2 wt %).
The type of lipophilic component (e.g., oil) can depend on the liquid oxygen
(e.g., nutrient/drug) being formulated (e.g., the solubility of the liquid oxygen
and desired use of the composition). Specific examples of combinations of oils
and liquid oxygen are provided herein.
(b) Add the desired amount of liquid oxygen of interest to the lipophilic
component to form a lipophilic mixture or to dissolve the liquid oxygen in the
lipophilic component.
(c) Homogenize and stir the liquid oxygen into the lipophilic component for a
suitable period of time (e.g., 5minutes) in a desired manner (e.g., using a

magnetic stirrer on a hot plate) until the liquid oxygen visually appear to have dissolved in oil (e.g., homogenize to 50°-60° C.).
(d) Add a specified amount of a surfactant (e.g., an ethoxylated nonionic surfactant) in a desired weight ratio with the lipophilic component (e.g., 5 g, 10 wt %). The PIT (also referred to as HLB temperature) depends on the surfactant chemical structure, and can vary according to the HLB number (Hydrophile-Lipophile balance) of the surfactant. In general, PIT increases with increasing HLB number.
(e) Homogenize and stir the composition including the surfactant, lipophilic component, and the liquid oxygen for a desired period (e.g., 5 minutes) until the three components form a homogeneous mixture.
(f) Add distilled water to the composition including the surfactant, lipophilic
component, and the liquid oxygen and continue to mix and homogenize until
an liquid oxygen nanoemulsion forms. The water can be homogenized to the
temperature of the mixture of the surfactant, lipophilic component, and the
liquid oxygen. For example, the liquid oxygen nanoemulsion can have a total
volume of the emulsion of about 50 ml.
(g) The liquid oxygen nanoemulsion is cooled rapidly (e.g., by placing vessel containing the W/O emulsion in ice water until the temperature is reduced to room temperature or about 25° C.) below the PIT, for example to room temperature (e.g., 25-30° C.) to obtain the O/W nanoemulsion (a kinetically stable liquid oxygen nanoemulsions composition)
Certain methods of forming a nanoemulsion include preparing nanoemulsion compositions with less than 3.5 wt % (e.g., less than 3.0%) of a nonionic surfactant. Other examples of nanoemulsions are characterized by a weight ratio of oil/(water+oil) that is less than 0.2 (e.g., 0.02-0.20, including 0.02-0.03, 0.02-0.05, 0.02-0.10, and 0.02-0.15). The nanoemulsions can also have an oil/(oil+surfactant) weight ratio (“Ros value”) of less than 0.67, including ratios of 0.50 or less (e.g., 0.10-0.5, 0.2-0.5, 0.3-0.5 and 0.4-0.5). In contrast,

Morales et al. report formation of nanoemulsions from compositions of mineral oil, water, and a nonionic surfactant (hexaethylene glycol monohexadecyl ether) with an oil/(water+oil) weight ratio of 0.2 by homogenizeing the mixture above the phase inversion temperature of the surfactant (Morales et al., “A Study of the Relation Between Bicontinuous Microemulsions and O/Wr Nanoemulsion Formation,” Langmuir 19, 7196-7200 (2003)). However, the droplet size of these emulsions steadily increased as a when the ratio of oil/ (oil+surfactant) was increased or decreased below 0.67 (Morales et al., id. at page 7199). For example, higher droplet sizes of about 75 nm were reported in compositions with a lower Ros value of 0.4 (id. at page 7199). These studies describe the formulation of nanoemulsions with at least about 20 wt % oil and oil-to-surfactant weight ratios that are less than 1.0.
The emulsions of the present invention can be manufactured according to the following procedure. The aqueous solution is prepared by adding preweighed quantities of sodium phosphate monobasic monohydrate USP, sodium chloride USP, and edetate calcium disodium to the required quantity of hot water for injection (WFI) at 5 to 10°C in the aqueous tank. The aqueous tank is liquid nitrogen surrounded and temperature adjusted to 5 to 10°C (+/-5°C). The liquid oxygen tank is then sparged with nitrogen to displace dissolved oxygen and then held under vacuum to degas the liquid. The required quantities of liquid oxygen is added to the liquid oxygen tank and maintained at -300C +/-5°. The liquid oxygen tank is maintained under vacuum to -300C +/- 5°
The required quantities of egg yolk phospholipid and d-α-tocopherol USP are added to a pre-mix tank (Tank #2) and is purged with nitrogen. Under nitrogen pressure, the aqueous solution of Tank #1 is transferred through an in-line 0.40-µm filter housing to the pre-mix tank (Tank #2) and then recirculated through a dispersing mixer. The contents of the pre-mix tank are mixed with an

agitator. After flow is established at not less than 150L/min, the liquid oxygen is added into the head of the dispersing mixer while the aqueous solution is recirculating. The liquid oxygen is emulsified by adding the liquid oxygen to the surfactant (EYP) and aqueous salts dispersion while employing a high-speed, high-shear mixer. This results in optimum surfactant coating of the liquid oxygen particles. Nitrogen purging in the tank is continued during pre-mixing. After the liquid oxygen has been added and emulsified in the aqueous phase, the mixture is recirculated through the dispersing mixer for 1670 +/-70L mass flow at 280 +/- 20L/min.
Following completion of pre-mixing, the emulsion prepared above is circulated from Tank #2 through an in-line 10µm filter into two homogenizer tanks H1 and H2, to a heat exchanger, and back to Tank #2 at 60 +/-5°C for a total mass flow of 1170 +/- 5-kg. Homogenization is then conducted first at 60+/- 3°C and then at 11+/-3°C at controlled pressure to achieve optimum homogenization. In the final step of homogenization, the emulsion is transferred through the homogenizers to Tank #3 instead of returning to Tank #2. This final step of homogenization is described as a "discrete pass" and ensures that all product transferred to the filling tank has been passed through homogenizers.
A nitrogen-sparred buffer solution of sodium phosphate dibasic heptahydrate USP, sodium phosphate monobasic monohydrate USP and sodium chloride USP, is prepared in Tank #1 (11 +/- 5°C) and is transferred to Tank #3 containing the homogenized emulsion through a 0.45 µm filter. The emulsion is agitated for at least 5 minutes prior to circulation through the fill loop at a flow rate of 10 +/- 1L/min. The filling temperature is controlled at 11 +/- 3°C. The emulsion is either passed through a 10 µm filter housing immediately prior to filling or recirculates back to Tank #3. The emulsion is filled into 100-mL bottles (USP Type I glass) and stoppered with 28-mm prewashed, presiliconized grey butyl rubber stoppers under a laminar flow (Class 100)

hood. Prior to receiving the stopper the headspace of each bottle is purged with nitrogen. 28-mm aluminum lacquered three-piece overseals are placed on stoppered bottles and manually crimped.
The product is then terminally steam sterilized in a steam overpressure autoclave through the injection of a clean air/clean steam mixture, inspected, and after sterilization, stored at 2 to 8°C. Samples selected from the beginning and end of the filling process are analyzed for conformance to final product specifications.
Sonication Method:
Two ultrasonic experimental set-ups were utilised. Batch experiments employed a Sonifier of nominal power 400 W and frequency 20 kHz with a 19 mm diameter tip horn. This was placed in a custom-built cylindrical glass cell of internal diameter 60 mm with in-built cooling jacket. Chilled water at 3.5 °C was passed continuously through this jacket. For each experiment, emulsion samples of either 30, 50 or 75 mL total volume were prepared and pre-mixed at 13,500 rpm with an Ultra-turrax mixer for 2 min. The droplet size after premixing was very broad, exhibiting three modes at droplet sizes Two ultrasonic experimental set-ups were utilised. Batch experiments employed a Branson Sonifier of nominal power 400 W and frequency 20 kHz with a 19 mm diameter tip horn. This was placed in a custom-built cylindrical glass cell of internal diameter 60 mm with in-built cooling jacket. Chilled water at 3.5 °C was passed continuously through this jacket. For each experiment, emulsion samples of either 30, 50 or 75 mL total volume were prepared and pre-mixed at 13,500 rpm with an Ultra-turrax mixer for 2 min. The droplet size after premixing was very broad, exhibiting four modes at droplet sizes 0.05, 0.13, 1.0 and 5.0 μm, and a volumetric mean size of 0.4 ± 0.5 μm. The samples

were then placed in the glass cell. The sonifier tip horn was adjusted until it was 2 cm below the surface of a 75 mL sample or 1 cm below the surface of a 30 or 50 mL sample.
EXAMPLE V: Studies of Changes in the Particle Size Distribution on Liquid Oxygen Emulsions in Ex-Vivo Blood:
Figure 7 is a plot of the particle size distributions observed in ex vivo rat blood following intravenous administration of a 60/30 % (w/v) ratio of liquid oxygen stabilized by 6% w/v EYP at a dose of 10 ml liquid oxygen/kg. There is a slight increase in droplet size for the ex vivo emulsion immediately after injection relative to the control (0.19 µm vs. 0.15 µm). After 24 hr., a significant fraction of the large droplets have been preferentially removed. The inset in Fig. 7 shows that little change is noted in the mode diameter at initial times.
EXAMPLE VI
A liquid oxygen emulsion is made according to the teachings of the present
application to formulate a 58% w/v liquid oxygen in a continuous aqueous
phase. Its components are:
Liquid Oxygen Upto 60% w/v
Egg Yolk Phospholipid 3.6% w/v
d, α-tocopherol 1.0018 % w/v
NaCl 0.36 % w/v
NaH2PO4 H2O 0.069% w/v
Na2HPO4•7H2O 0.474% w/v
EDTA 0.02% w/v
EXAMPLE VII Long term and accelerated testing of the liquid oxygen emulsion made according to the teachings of Example VI.

For determining the long term shelf life of the emulsion made according to the teachings of Example VI, stability data was generated at defined time points over a 24-month window with a Horiba CAPA-700 particle size analyser and statistically analysed with a commercially available stability software system (SCIENTEK™, Tustin, California). The emulsion was made and then stored in inverted orientation for both long-term conditions (5°C) for up to 24 months and accelerated storage conditions (25°C) for up to 6 months. The emulsion made according to Example VI was measured both on accelerated testing (6 months) and long-term test (24
EXAMPLE IX Pharmacokinetics and Tissue Distribution of Oxygen in Rats Following a Intravenous Administration of the Emulsion of Ex. VI
This study was designed to assess the pharmokinetics and tissue distribution of liquid oxygen emulsion following a single intravenous administration of the emulsion at various time intervals at dose levels of 1.8 and 3.6 ml liquid oxygen /k in male and female Sprague Daley rats, aged 7 - 9 weeks, weight between 190 - 267 grams. For pharmacokinetics, 160 rats were divided into 20 groups (N=4 rats/sex/dose/time interval) and for tissue distribution, 156 rats were divided into 26 groups (n=3 rats/sex/dose/time interval). The time intervals for pharmacokinetics blood sampling were 0.083, 0.5, 1, 3, 6, 12, 36, 48, 72 and 96 hours. The time intervals for tissue sampling was predose, 24 hours, 1, 2, 4, 8, 13, 7, 21, 26, 39, 52 and 72 weeks. Pharmacokinetics
Following administration, no adverse clinical signs were observed in the rats. Six pharmacokinetic parameters were determined following a single intravenous dose of 1.8 mg oxygen/kg or 3.6 g liquid oxygen/kg in both the male and female rats as shown in the tables below. The average terminal tl/2 of liquid oxygen for male and female rats dosed with 1.8 g liquid oxygen/kg

was ∼ 73 hours compared to an average terminal tl/2 of 181 hours for animals
dosed with 3.6 g liquid oxygen/kg. By comparison, the terminal tl/2 was several
orders of magnitude greater : ∼25,130 hours (male, 1.8 ml liquid oxygen /kg),
∼4800 hours (female, 1.8 ml liquid oxygen /kg), ∼12,550 hours (male 3.6 g
liquid oxygen/kg) and ∼7,600 hours (female, 3.6 g liquid oxygen/kg).
• [0090]
The volume of distribution (Vd) for liquid oxygen was from ∼1200 to 2200 g/kg
for both sexes and both doses.
Table V. Pharmacokinetic parameters of liquid oxygen in blood of male and
female Sprague Dawley rats after a single intravenous administration
Liquid oxygen (1.8 ml/kg)
Parameter Male Female
AUC(o-∞) (mg-h/g) 122.69 126.89
AUC(o-last) (mg-h/g) 122.65 126.86
T½(h)* 68.18 78.46
Vd(g/kg)** 1443.18 1605.67
MRT(0-last) (h) 14.67 14.19
Cl (g/h/kg) 6.01 5.41
*T1/2 was calculated using the last 3 or 4 data points ** based on the terminal phase
Tissue Distribution
26 groups (N=3 rats/sex/group). Prior to euthanasia, body weights were recorded. At designated times (see below), rats were anesthetized and exsanguinated via the abdominal artery. Blood was collected into syringes containing EDTA and transferred into sealed plastic containers for storage at approximately -20°C until analyzed. Resultant data were included in the determination of pharmacokinetic parameters. The following tissues were

collected from each rat and placed in sealed containers and stored frozen at -20° C. Prior to freezing, blood and tissue samples were processed for analysis. Oxygen is quantified in blood and tissues using a GC headspace method. The method was validated in the liver and then cross validated to all other tissues, blood, and serum. The tissues collected and analyzed include heart, lungs, abdominal fat, spleen, liver, mesenteric lymph nodes, kidneys, adrenals, testes, ovaries, uterus, whole brain, eyes, skeletal muscle, femoral bone marrow, and gastrointestinal tract.
The concentration of liquid oxygen was determined in the tissues in both sexes at 12 time points. For the males, this was a total of 360 individual results including blood. There were 24 additional results for the female.
List of reference numerals:
1-Sonicator 2-Stirrer/homogenizer 3-Raw material inlet 4-Emulsion chamber 5-Homogenised mixture 6-Product outlet 7-Liquid oxygen 8-Liquid oxygen inlet 9-Vaccum pump.

5. Claims I/We Claim
1. A liquid oxygen nanoemulsion composition comprising liquid oxygen, an
aqueous component, a lipophilic component, and an emulsifying agent.
2. The nanoemulsion composition as claimed in claim 1, wherein
nanoemulsion composition is in the form of a continuous aqueous phase and a
discontinuous liquid oxygen, wherein the liquid oxygen is present in the
nanoemulsion at approximately 5 - 65% w/v.
3. The nanoemulsion composition as claimed in claim 1, wherein, the liquid
oxygen emulsion is stabilized by an emulsifying agent in the amount of
approximately 3.5% - 4 % w/v of the total nanoemulsion composition.
4. The nanoemulsion composition as claimed in claim 1, wherein the
emulsifying agent is selected from Sorbitan monostearate, sorbitan
monopalmitate and sorbitan 20 EO tristearate, Cetyl alcohol , disodium salt of
N-stearoyl-L-glutamic acid, Tween 20, 40, 60 and 80, propylene glycol
monostearate, glycerol monoleate, glycerol monostearate, acetylated
monoglycerides (stearate), sorbitan monooleate (Span 80), propylene glycol
monolaurate, sorbitan monostearate, and glycerol monolaurate,
polyoxyethylene POE (20) sorbitan monostearate, sucrose monolaurate, POE
(20) sorbitan monooleate (Tween 80), and POE (20) sorbitan monopalmitate,
polyoxyethylene (20) sorbitan monostearate, sucrose monolaurate, POE (20)
sorbitan monooleate (Tween 80), POE (16) lanolin alcohols, and acetylated
POE (9) lanolin, Caprylic capric glycerides, polyethylene glycol, Hexadecenyl
succinate 18 EO, hexadecenyl succinate 45 EO, dihexadecenyl succinate 18
EO, dihexadecenyl succinate of glucose 10 EO, dihexadecenyl succinate of

glucose 20 EO, dioctadecenyl succinate of methylglucose 20 EO, Sorbitan oleate, Polysorbate 80, Ceteareth 5 , Ceteareth 20 , Steareth-2, Sorbitan monooleate, polysorbate 80.
5. The nanoemulsion composition as claimed in claim 1, wherein the
nanoemulsion composition further comprises NaCl, NaH2PO4 and EDTA.
6. A method for preparing a liquid oxygen nanoemulsion composition, the
method comprising the steps of:
(a) providing a mixture comprising liquid oxygen, an emulsifier, water, and optionally a preservative; and
(b) mixing the mixture with a high shear mixer to obtain the nanoemulsion wherein the mixture has a viscosity of 2800 cp at 10 s-1 to 50,000 cp at 10 s-1 during at least a part of the mixing; and wherein the nanoemulsion has a particle size of the liquid oxygen from 10 nm to 2000 nm.

7. The method of claim 6, wherein the mixture is mixed at a shear rate of 30,000 s-1 to 250,000 s-1.
8. The method of claim 6, wherein the nanoemulsion has a particle size of liquid oxygen from 10 nm to 5000 nm.
9. The method of claim 5, wherein the mixture has a viscosity of 2800 cp at 10
s-1 to 30,000 cp at 10 s-1 during at least a part of the mixing.
10. The method of claim 5 further comprising adding water to the
nanoemulsion to obtain a diluted nanoemulsion wherein the diluted
nanoemulsion contains 4 wt % to 10 wt % of the liquid oxygen, and wherein
the diluted nanoemulsion has a particle size from 10 nm to 5000 nm.

11. The liquid oxygen nanoemulsion composition as claimed in claim 1,
wherein the composition is used for intravascular oxygen delivery.
12. The liquid oxygen nanoemulsion composition of any of claims 1 to 10 for
use in the treatment of hypoxia, sickle cell anemia, of carbon monoxide
poisoning, traumatic brain injury or of stroke, cancer, Alzheimer’s disease.