Executive Summary of Research Proposal (maximum 300 words)
(Please include the problem statement, objectives, research methodology, expected output/outcomes/implication, and significance of output from the research project)
The last three decades have witnessed remarkable and breathtaking advances in the field of biotechnology, biochemistry, molecular biology and peptide synthesis. These developments have facilitated the pharmaceutical industry to make remarkable progress in the development of peptide and proteins as drugs. Since proteins are known to be involved in essentially all biological processes and reactions, they represent a promising class of therapeutics. Administration of these classes of drugs to humans is formidable challenge for biotechnologist as well as pharmacist. The biggest problems lying in their governance are physical and chemical instability, poor bioavailability and lack of knowledge for delivering them. These problems can be solved by using the aquasomes. Aquasomes combines biotechnology and nanotechnology approaches. These sugar balls are the recent addition in delivery systems that have wider applications in peptide and protein delivery. Aquasomes are three-layered self-assembled nanostructures. They contain solid nanocrystalline core lile calcium phosphate coated with polyhydroxy oligomers over which peptide and protein are adsorbed. The carbohydrate coating protects the peptide from dehydration and stabilizes the active peptide molecules. Structural stability is provided by solid core. Aqausomes maintains the conformational integrity of peptide which makes it ideal carrier system for delivery of peptides. In the proposed work Aquasomes, a novel nano drug delivery system compassing of hydroxy apatite (HA) core having carbohydrate coating will be prepared. Urokinase will be immobilized on these nanostructures for thromobolytic therapy. The prepared systems will be characterized for size, shape, size distribution, enzyme loading efficiency, and in vivo performance. The in vivo performance of the formulated aquasome will be compared with standard urokinase preparation. In Aquasome the steric hindrance is provided by polyhydroxyoligomers between enzyme and blood component (Plasma protein). Later RES cells assist in removing exogenous material from blood stream .The polyhydroxy oligomers maintain three dimensional conformation of enzyme and also helps in deferring recognizition from RES cells. Therefore it is proposed that aquasome not only act as dehydroprotectant but also preserve the three dimensional conformation of enzyme in blood, which enhanced dramatically the half-life of enzyme. So it is expected that proposed system can add new dimension in delivery of urokinase through its rapid onset of action, maximal efficacy and safety
Research background including Problem Statement, Hypothesis/Research Questions, Literature Reviews, Related References and Relevance to Goverment Policy, if any.
Problem statement
Urokinase is a serine protease enzyme which is widely used as an anti-thromboembolic drug in thrombolytic therapy. Urokinase is a strong plasminogen activator. Activation of plasmin activates a proteolysis cascade which breaks down the fibrin polymers of blood clots. This makes urokinase a very important drug against vascular diseases.Urokinase has a half-life of 10-20 mins in plasma. Due to which it is needed to given patient in a short time span for treatment.2

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These problems can be solved by using the aquasomes. Aquasomes combines biotechnology and nanotechnology approaches. These sugar balls are the recent addition in delivery systems that have wider applications in peptide and protein delivery. Aquasomes are three-layered self-assembled nanostructures. They contain solid nanocrystalline core like calcium phosphate coated with polyhydroxy oligomers over which peptide and protein are adsorbed. The carbohydrate coating protects the peptide from dehydration and stabilizes the active peptide molecules. Structural stability is provided by solid core. Aqausomes maintains the conformational integrity of peptide which makes it ideal carrier system for delivery of peptides.3,4
Hypothesis
Urokinase is a thrombolytic enzyme having half life of 10-20 minutes. In the present work is an attempt is to retain the spatial properties of streptokinase i.e. three-dimensional conformation, which is a freedom internal molecular rearrangement generated by intermolecular interaction and a freedom of bulk movement. Using aquasomes a high degree of molecular preservation may be achieved by virtue of the significant degree of retained biological activity. The aquasomes also avoid the elimination of drug by reticuloendothelial system therefore sustained delivery of drug may be achieved, and a circulating bioreactor could possibly be developed which may be used as preventive measure to avoid probable vascular embolism
Research Questions

Is it possible to immobilize Urokinase on aquasomes?
Do Aquasmoes will be able to preserve the activity of Urokinase?
Is it possible to achieve sustain release of urokinase with aquasomes after PEGylation?
Is it possible to use similar platform for other peptide drugs?

Literature review
Current status of research and development in the subject
Kossovsky et al.5( 1995) reported first synthesis of aquasomes for delivery of protein antigen and mussel adhesive protein. After that around fifteen research publications were appeared in scientific community utilizing aquasomes for peptide and drug delivery. Recently Aquasomes were used in delivery of peptide and drugs like insulin6 and indomethacin.7 Vyas et al.8 also used aquasomes for hepatitis antigen delivery.
The relevance and expected outcome of the proposed study
Venous thromboembolism (VTE) is a common and potentially life threatening condition which is still under diagnosed and undertreated.VTE treatment is full of risk as patient requires precise dosing of drugs with careful monitoring.9 Due to these problems in last decade lot of studies were done for developing novel antithrombotic agents. Urokinase is a serine protease (EC 3.4.21.73) enzyme which is also called urokinase-type plasminogen activator (uPA). It is a thromobolytic agent. It was originally isolated from human urine, but it is also found in blood stream and the extracellular matrix. Urokinase directly activates conversion of plasminogen to plasmin which is a primary protein accountable for fibrinolysis.10 Urokianse has a half-life of 10-20 mins due to which it is not available in body for longer time. There is urgent need of a carrier which can carry the urokinase for longer time. Aquasomes is an answer for this need. As it carry the peptide with full retention of therapeutic activity for longer time. So there is a need of developing a drug delivery system for delivery of Urokinase in sustain manner11
It is projected that propose system can add new dimension in delivery of urokianse through its rapid onset of action, maximal efficacy and safety.
References

Degim IT, Celebi N. Controlled delivery of peptides and proteins. Curr Pharm Des 2007;13:99-117
Erdogan S, Ozer AY, and Bilgili H. In vivo behaviour of vesicular urokinase. Int. J. Pharm.2005 295: 1–6
Juliano RL. Microparticulate drug carriers: liposomes, microspheres and cells. In: Robinson JR, Lee VHL, editors. Controlled drug delivery. 2nd ed. New York: Marcel Dekker, Inc.; 2005. p. 555-80.
Rawat M, Singh D, Saraf S, Saraf S. Nanocarriers: promising vehicles for bioactive drugs. Biol Pharm Bull 2006; 29:1790-8.
Kossovsky N, Gelman A, Rajguru S, Nguyan R, Sponsler E, Hnatyszyn CK, et al. Control of molecular polymorphism by a structured carbohydrate/ceramic delivery vehicle-aquasomes. J Control Release 1996; 39:383-8.
Cherian AK, Rana AC, Jain SK. Self-assembled carbohydrate-stabilized ceramic nanoparticles for the parenteral delivery of insulin. Drug Dev Ind Pharm 2000;26:459-63.
Oviedo RI, Lopez SAD, Gasga RJ, Barreda CTQ. Elaboration and structural analysis of aquasomes loaded with indomethecin. Eur J Pharm Sci 2007; 32:223-30.
Vyas SP, Goyal AK, Rawat A, Mahor S, Gupta PN, Khatri K.Nanodecoy system: a novel approach to design hepatitis B vaccine for immunopotentiation. Int J Pharm 2006; 309:227-33.
Agarwal S, Lee AD, Raju RS, Stephen E. Venous thromboembolism: A problem in the Indian/Asian population? Indian J Urol 2009; 25:11-6.
Agarwal Y.K, Vaidya H, Bhatt H, Manna K, Brahmkshatriya P Recent Advances in the Treatment of Thromboembolic Diseases: Venous Thromboembolism Medicinal Research Reviews, 2007 ; 27:891-914,
Kaur K,Kush P,Pandey RS,Madan J,Jain UK,Katare OP Stealth lipid coated aquasomes bearing recombinant human interferon-α-2b offered prolonged release and enhanced cytotoxicity in ovarian cancer cells.2015; 59 :267–276

(b) Objective (s) of the Research
Urokinase is an unstable (half-life of 10-20 mins) enzyme. Pharmacist plays and important role in their stabilization, formulation and effective delivery. Over all aim of this study is to develop urokinase immobilized aquasome. Aquasome will protect urokinase from degradation and dehydration. It will also enhance and sustain its thrombolytic activity with reduced side effects. Specific objectives will be
1. To synthesize aquasomes having different compositions.
Aquasomes with different sugar coating will be synthesized. Sucrose, Trehalose , Lactose and Pyrodoxial-5-phospahte will be used for sugar coating. Characterization of these nanoparticulte system will done using Transmission electron microscopy, Scanning electron microscopy, Zetasizer and X-ray powder diffractometry (XRPD). Determination of particle morphology and distribution size analysis of nanoparticles will be performed.
2 To immobilize urokinase on aquasomes and coating of PEGylated phospholipids
Optimization of aquasome formulations for maximum loading of enzyme will be performed. Enzyme activity will be measured for immobilized enzyme and later they will be coated with PEGylated phospholipids for sustain release.
3. Characterization of these nanoparticulte systems after immobilization will be performed using Transmission electron microscopy, Scanning electron microscopy and Zetasizer
3. In-Vitro evaluation of aquasomal formulations
Aquasomal formulations will evaluated for protein (Urokinase) release.
(c) Methodology
The envisaged work shall be undertaken on the following lines
(1) Preformulation studies
1.1. Identification test for proteins

IR spectroscopy
SDS PAGE

1.2. Identification Test of Formulation Adjuvants (Sugars)

Molish Test
Moore’s Test
Polarimetric determination of sugars

1.3. Preparation of calibration curve of adjuvants (Trehalose and Cellobiose)
1.4. Preparation of calibration curve of Enzyme as a Protein
1.5 Preparation of Calibration curve of Enzyme in PBS (pH 7.4) and Plasma
(2) Preparation and Characterization of Hydroxy Apatite
2.1 Optimization of the method for the preparation of Hydroxy Apatite
2.1.1 Characterization of Hydroxy Apatite prepared by self-precipitation

Size and Shape e.g TEM and SEM
Crystal properties e.g XRD

2.1.2 Preparation and Characterization of Aquasome
2.1.3 Optimization of the poly hydroxyl Oligomers concentration on Hydroxy Apatite
2.1.4 Optimization of drying condition
2.1.5 Optimization of Protein concentration
2.16 Characterization of Optimized Aquasome formulation.

Confirmation of poly hydroxyl Oligomers coating by Zeta Potential measurement
Determination of loading efficiency of various Aquasome formulations
In-vitro release rate studies
Assessment of Biodegradability of Different Formulation
Retention of Enzyme Activity
Reaction Kinetics of Aquasome adsorbed Urokinase
Urokinase specific Antibody Detection

(3) Stability studies of prepared formulation

SDS-PAGE
Storage Stability

Expected Results/Benefit
It is expected that proposed formulation will retain the spatial properties of urokinase i.e. three-dimensional conformation, which is basically achieved by freedom of internal molecular rearrangement for intermolecular interaction and without any bulk movement. Using carbohydrate based aquasomes a high degree of molecular preservation may be achieved by virtue of the significant degree of retained biological activity. The aquasomes also avoid the elimination of drug by reticuloendothelial system therefore sustained and controlled delivery of drug may be achieved.
Therefore, it is aim to develop an Aquasome system being streptokinase to protect drug from degradation and dehydration as well as to enhance and sustain its biological activity with reduced side effects.
It will help us in getting preliminary results which will be very useful in writing big research project grants to other funding agencies. It is also expected that this research work will allows us to publish quality publications.