Synthesis of Aquasomes With Different Compositions

Synthesis of Aquasomes With Different CompositionsExecutive Summary of Research Proposal (maximum 300 words)(Please include the problem statement, objectives, research methodology, evaluate output/outcomes/implication, and implication of output from the research project)The last three decades draw witnessed remarkable and breathtaking advances in the field of biotechnology, biochemistry, molecular biology and peptide synthesis. These developments have facilitated the pharmaceutical industry to stumble 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 chemic instability, poor bioavailability and lack of knowledge for delivering them. T hese problems can be solved by using the aquasomes. Aquasomes combines biotechnology and nanotechnology approaches. These net balls are the recent addition in oral communication systems that have wider applications in peptide and protein oral communication. Aquasomes are three-layered self-assembled nanostructures. They contain solid nanocrystalline core lile calcium phosphate cover with polyhydroxy oligomers over which peptide and protein are adsorbed. The carbohydrate coating protects the peptide from dehydration and stabilizes the alert 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 clip Aquasomes, a novel nano drug delivery system compassing of hydroxy apatite (HA) core having carbohydrate coating will be prepared. plasminogen activator will be immobilized on these nanostructures for thromobolytic therapy. The prepare d 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 plasminogen activator preparation. In Aquasome the steric hindrance is provided by polyhydroxyoligomers between enzyme and telephone circuit theatrical role (Plasma protein). Later reticuloendothelial system 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- deportment of enzyme. So it is expected that proposed system can add new dimension in delivery of plasminogen activator through its rapid onset of action, maximum expertness and safetyResearch backgrou nd 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 plasminogen activator 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 misfortunate time span for treatment.2These 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 ca lcium phosphate coated with polyhydroxy oligomers over which peptide and protein are adsorbed. The carbohydrate coating protects the peptide from dehydration and stabilizes the dynamical 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.3,4HypothesisUrokinase 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 kept up(p) biological activity. The aquasomes also avoid the excretion of drug by reticuloendothelial system therefore sustained delivery of drug may be achieved, and a go bior eactor could possibly be developed which may be used as preventive measure to avoid probable vascular embolismResearch QuestionsIs it potential 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 reviewCurrent status of research and development in the subjectKossovsky et al.5( 1995) inform 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 studyVenous thromboembolism (VTE) is a common and potentia lly life threatening check into which is still under diagnosed and undertreated.VTE treatment is dependable of risk as patient requires precise dosing of drugs with careful monitoring.9 Due to these problems in last decade lot of studies were through 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 manner11It is projected that propose system can add new dimension in delivery of urokianse through its rapid onset of action, maximal efficacy and safety.ReferencesDegim IT, Celebi N. bindled delivery of peptides and proteins. Curr Pharm Des 20071399-117Erdogan S, Ozer AY, and Bilgili H. In vivo behaviour of vesicular urokinase. Int. J. Pharm.2005 295 16Juliano RL. Microparticulate drug carriers liposomes, microspheres and cells. In Robinson JR, Lee VHL, editors. Controlled drug delivery. 2nd ed. recent 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 291790-8.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 39383-8.Cherian AK, Rana AC, Jain SK. Self-assem bled carbohydrate-stabilized ceramic nanoparticles for the parenteral delivery of insulin. Drug Dev Ind Pharm 200026459-63.Oviedo RI, Lopez SAD, Gasga RJ, Barreda CTQ. Elaboration and structural analysis of aquasomes loaded with indomethecin. Eur J Pharm Sci 2007 32223-30.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 309227-33.Agarwal S, Lee AD, Raju RS, Stephen E. Venous thromboembolism A problem in the Indian/Asian population? Indian J Urol 2009 2511-6.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 27891-914,Kaur K,Kush P,Pandey RS,Madan J,Jain UK,Katare OP Stealth lipid coated aquasomes bearing recombinant human interferon--2b offered prolonged release and enhanced cytotoxicity in ovarian cancer cells.2015 59 267276(b) Objective (s) of the ResearchUrokinase is an unstable (half-life of 10-20 mins) enzyme. chemist 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 adulteration and dehydration. It will also enhance and sustain its thrombolytic activity with reduced side effects. Specific objectives will be1. 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. depicting 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 phospholipidsOptimization of aquasome f ormulations 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 Zetasizer3. In-Vitro evaluation of aquasomal formulationsAquasomal formulations will evaluated for protein (Urokinase) release.(c) MethodologyThe envisaged work shall be undertaken on the following lines(1) Preformulation studies1.1. Identification test for proteinsIR spectroscopySDS PAGE1.2. Identification Test of Formulation Adjuvants (Sugars)Molish TestMoores TestPolarimetric stopping point of sugars1.3. preparedness of calibration curve of adjuvants (Trehalose and Cellobiose)1.4. Preparation of calibration curve of Enzyme as a Protein1.5 Preparation of Calibration curve of Enzyme in PBS (pH 7.4) and Plasma(2) Preparation an d Characterization of Hydroxy Apatite2.1 Optimization of the method for the preparation of Hydroxy Apatite2.1.1 Characterization of Hydroxy Apatite prepared by self-precipitationSize and Shape e.g TEM and SEMCrystal properties e.g XRD2.1.2 Preparation and Characterization of Aquasome2.1.3 Optimization of the poly hydroxyl Oligomers concentration on Hydroxy Apatite2.1.4 Optimization of drying condition2.1.5 Optimization of Protein concentration2.16 Characterization of Optimized Aquasome formulation.Confirmation of poly hydroxyl Oligomers coating by Zeta possible measurementDetermination of loading efficiency of various Aquasome formulationsIn-vitro release rate studiesAssessment of Biodegradability of Different FormulationRetention of Enzyme ActivityReaction Kinetics of Aquasome adsorbed UrokinaseUrokinase specific Antibody Detection(3) Stability studies of prepared formulationSDS-PAGEStorage StabilityExpected Results/BenefitIt is expected that proposed formulation will retain the s patial 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.