Friday, April 5, 2019
Synthesis of Aquasomes With Different Compositions
Synthesis of Aquasomes With Different CompositionsExecutive abridgment of interrogation Proposal (maximum 300 words)(Please include the problem statement, objectives, inquiry methodology, anticipate payoff/outcomes/implication, and significance of output from the question project)The last three decades ease up witnessed remarkable and breathtaking advances in the field of biotechnology, biochemistry, molecular biota 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 be known to be involved in essentially all biological processes and reactions, they represent a lustrous 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 atomic number 18 physical and chemical instability, poor bioavailability and lack of knowled ge for delivering them. These problems can be figure out by using the aquasomes. Aquasomes combines biotechnology and nanotechnology approaches. These sugar balls are the modern addition in pitch shot administrations that have wider applications in peptide and protein economy. Aquasomes are three-layered self-assembled nanostructures. They contain solid nanocrystalline core lile calcium phosphate surface with polyhydroxy oligomers over which peptide and protein are adsorbed. The lettuce coating protects the peptide from dehydration and stabilizes the active peptide molecules. morphologic stability is provided by solid core. Aqausomes maintains the conformational integrity of peptide which makes it deification carrier system for voice communication of peptides. In the proposed work Aquasomes, a novel nano drug delivery system compassing of hydroxy apatite (HA) core having carbohydrate coating bequeath be prepared. fibrinolysinogen activator allow for be immobilized on these nanostructures for thromobolytic therapy. The prepared systems go forth be characterized for size, shape, size distri scarceion, enzyme loading efficiency, and in vivo performance. The in vivo performance of the formulated aquasome go forth be compared with standard urokinase preparation. In Aquasome the steric hindrance is provided by polyhydroxyoligomers between enzyme and blood component (Plasma protein). ulterior 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 scarcely 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 safetyResearch bac kground 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 use 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 duration 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 manage calcium pho sphate 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.3,4HypothesisUrokinase 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 independence subjective molecular rearrangement generated by intermolecular interaction and a freedom of bulk movement. Using aquasomes a game 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 thitherfore raiseed delivery of drug may be achieved, and a circulating bioreactor could perhaps be developed which may be used as preventive measure to avoid apparent vascular embolismResearch QuestionsIs it possible to immobilize Urokinase on aquasomes?Do Aquasmoes will be able to preserve the activity of Urokinase?Is it possible to achieve sustain drop of urokinase with aquasomes afterwards 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) reported first synthesis of aquasomes for delivery of protein antigen and mussel adhesive protein. After that near 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 harsh 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 ontogenesis 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 womb-to-tomb 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. Controlled delivery of peptides and proteins. Curr Pharm Des 20071399-117Erdogan S, Ozer AY, and Bilgili H. In vivo behavior 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. 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 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-as sembled 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) target area (s) of the ResearchUrokinase is an unstable (half-life of 10-20 mins) enzyme. Pharmacist plays and important role in their stabilization, formulation and effective delivery. all over all aim of this study is to develop urokinase immobilized aquasome. Aquasome will protect urokinase from humiliation and dehydration. It will also enhance and sustain its thrombolytic activity with reduced expression 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. Characterization of these nanoparticulte system will done using Transmission negatron microscopy, scan 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 opt imisation 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 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 determination of sugars1.3. preparation of calibration switch off 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) an d Plasma(2) Preparation and 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. proof of poly hydroxyl Oligomers coating by Zeta Potential 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 formulati on 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 expedient 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.
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