Seminar on MAGNETIC MICROSPHERES Presented by Ushasri Department of Industrial pharmacy

1. Seminar on MAGNETIC MICROSPHERES Presented by Ushasri Department of Industrial pharmacy University college of pharmaceutical sciences KAKATIYA UNIVERSITY

2. CONTENTS • INTRODUCTION • DEFINITION • RATIONALE OF THE DRUG DELIVERY SYSTEM • ADVANTAGES AND DISADVANTAGES • PRINCIPLE OF MAGNETIC DRUG TARGETING • FORMULATION • PREPARATION OF MAGNETIC MICROSPHERES • CHARACTERIZATION • APPLICATIONS • CONCLUSION • REFERENCES

3. INTRODUCTION • Drug targeting:Delivery of drugs to receptors or organ or any other specific part of the body exclusively. • Various nonmagnetic micro carriers are utilized for drug targeting but they show poor site specificity and are rapidly cleared off by RES . • Magnetic particles composed of magnetite are well tolerated by body. • Magnetic fields are believed to be harmless to biological systems and adaptable to any part of the body. • Up to 60% of injected dose deposited and released in a controlled manner . • Magnetic micro carriers were developed to overcome two major problems encountered in drug targeting • RES clearance • Target site specificity.

4. DEFINITION: • Magnetic microspheres are spherical particles, composed of metallic iron and activated carbon which serves as a delivery vehicle for the site specific targeting, retention and release of pharmaceuticals. • It utilizes the physical force of magnetic field for the targeted delivery of pharmaceutical agents to specific areas of the body. • Size range -1-100µm. • ROA: • IV –NLT(5µm) • Intra arterial-NLT(1ooµm).

5. The physical force created by the magnetic field draw the magnetic targeted carrier drug compound through the arteriole wall in to the targeted disease area. This process is known as extra vasation. • Results in localization and retention of the delivered pharmaceutical agents at the delivered site even when the magnetic field has been removed.

6. This technology uses a small externally positioned magnet to create a localized magnetic field within the body.

7. Rationale of thE delivery system • Number of problems exists for successful targeting of biodegradable carriers • Rapid sequestration of IV administered drug carriers by mono nuclear phagocytes • of the RES. • Approaches to over come • By blocking RES prior to administering the drug carrier • Impart specificity to the drug carrier by incorporating the specific ligand on external • surface of the carrier.

8. 2. Vascular system • The endothelial cell lining the capillary level vasculature have severely frustrated attempt at invivo targeting • And shield the carrier from recognization • In order to circumvent the RES and provide a method of site specific drug delivery a magnetically responsive drug carrier was developed. • Retention of magnetic carrier at target site • will delay RES clearance • facilitate extravasation • prolong the systemic action of the drug .

9. ADVANTAGES • Reduces circulating concentration of free drug . • Minimizes damage to the normal tissue cells and allows an effective treatment of • regioned disease. • Delivers 60% of the dose to target tissues. • Making the drug available in controlled fashion within the tissue. • Advantage of crossing microvascular barriers . • Minimally reactive with blood components. • Clinically nontoxic from the stand points of chemical and immunological agents. • Capable of accommodating a wide variety of water soluble agents. • Magnetite is readily available from the commercial sources, individual particles are much smaller (10-20mm) occupy a fraction of the drug space.

10. DISADVANTAGES • Requires specialized microspheres and magnets . • Treatment of multiple region requires sequential targeting. • Demands new methods for noninvasive , localized monitoring term deposition • of drug levels in target tissues. • Expensive. • Tissue localization of microspheres result long term deposition of magnetite at levels of 3.5mg/kg. • Once the drug is released it is no longer attractive to the magnetic field . • Possibility of embolisation of the blood vessels due to accumulation of magnetic carriers.

11. PRINCIPLE OF MAGNETIC DRUG TARGETING Major pathway Target tissue Magnetic carrier Circulation RES organs (liver,spleen,bonemarrow Little access

12. FORMULATION OF MAGNETIC MICROSPHERES • Components • Matrix: • It comprises cross linkable material that will cross link upon heating . • The matrix material forms the bulk micro particle structure in which the agent is dispersed orencapsulated. • The matrix material should be biodegradable. • It is either a polymer or a protein . • Ex: Albumin ,Egg albumin , Bovine serum albumin. • Thermo setting polymers include poly urethanes, phenol/formaldehyde and urea formaldehyde resins.

13. HYDROPHOBIC LIQUID PHASE • The flash point of the oil should be higher than the denaturation temparature of the protein. • If solvents are used the boiling point should be higher than the denaturation temparature. • Ex: cotton seed oil, Corn seed oil, Silicon oil. • SURFACTANTS • Used to decrease the hydrophobicity of matrix material to mitigate agglomeration of microdroplets, • Disperse the carriers more evenly • Eg: Sorbitan esters, polyoxyethylene ethers, Glycerol esters, Sorbitan sesquiolate

14. MAGNETS • Iron oxide such as magnetite (Fe3O4) or magnemite ( ץ-Fe2O3) are more stable against oxidation and are preferentially used as core material instead of iron. • Estoper M particles are super paramagnetic and used in immuno assays,cell separation and nucleic acid purification. • Ex: Silica magnet particles • Cellulose magnetic particles • Ferro fluids • LIGANDS • Incorporated in order to prevent the RES clearance. • Ex:Avidin, Biotin, Streptavidin, Lectins.

15. PREPARATION OF MAGNETIC MICROSPHERES • The magnetic microspheres are prepared by : • 1)Heat denaturation • 2)Chemical cross linking: • also prepared by stabilising the albumin matrix with aldehyde cross linking agent. • Ex: glutaraldehyde. • These have been solubilized in organic phase. • Emulsion is homogenized by sonication method for 1min at 100W at 27°c. • Sonifier with a 0.635 cm titanium probe inside a circulating attachment chamber is used.

16. Preparation involves emulsification fOllowed by heat denaturation

17. In the heat denaturation process stabilization varied with temperature and time. • This procedure generates small non cross linked microspheres. • Matrix stabilization is subsequently achieved by : • Heat denaturation at a temperature between 100-165°c • Treatment with oil soluble reagents such as 0.2M 2,3-butane dione or glutaraldehyde. • In the case of chemical cross linkers denaturation varies with exposure time • Oil and the cross linking agents are removed by multiple diethyl ether washings. • STORAGE • Magnetic micro spheres are stored in the powder form • These are stored at a temperature of 4°c.

18. CHARACTERIZATION OF MAGNETIC MICRO SPHERES DETERMINATION OF DRUG LOADING AND ENTRAPMENT EFFICIENCY Drug loaded microspheres are digested with suitable reagent at room temperature and kept for 24 hrs to precipitate the protein .The digested homogenate is centrifuged and supernatant is analyzed for drug content by measuring its absorbance. The drug loading in microspheres is estimated by using the formula . L = QM/WM*100 L = %Drug loading of micro spheres QM= Quantity of drug present WM= Weight of microspheres

19. DETERMENATION OF MAGNETITE CONTENT • The magnetite content in microspheres is estimated quantitatively by hydrolyzing an aliquot of the microspheres in concentrated HCl and the resultant hydrolysate for iron is determined by atomic absorption spectroscopy at 248nm .It has been demonstrated that microspheres containing magnetite of 20-28%w/w are effectively targeted by at the site by using the magnet of 5000-8000G. • PARTICLE SIZE ANALYSIS: • scanning electron microscopy • Laser diffraction • Zeta sizer • Confocal laser scanning microscopy

20. MAGNETIC RESPONSIVITY MEASUREMENT • Mille pore pumps air in to the flask containing normal saline . • It results in the flow of saline into the tube exposed to an electro magnet. • The pump pressure maintained was 0.5 cm/s resembles the rate blood flow in the capillaries. • 1ml microspheres dispersed in saline containing tween-80 was injected in to the injection site. • 8000G of magnetic field was applied for 15min a,samples collected every minute. • samples analysed by UV-Visible specrophotometer • at the גmax of the drug. • Higher the absorbance –greater the amount of drug • lower the magnetic retention in the tube – • lower the magnetic responsivity .

21. MEASUREMENT OF ELECTROPHORETIC MOBILITY BY USING • OPTICAL MICROSCOPY • Measurement of electrophoretic mobility • helps in the prediction of magnetic • microspheres behaviour in the • patients blood circulation. • ϰ = magnetic susceptibility. • Vm =volume of the magnetic component • The movement of magnetic microspheres • is determined by the Magnetic propertites of • the particle, magnetic component volume, • magnetic susceptibility ,viscosity,diameter.

22. A piece of acrylic was placed on the micro scope table. • Glass capillary was filled with microsphere suspension using 1ml syringe. • During filling the magnet was placed18mm away from the capillary • The microscope was manually focused ,the colour video camera connected to computer ,displays a clear picture. • The magnet was now closely moved to capillary image acquisition was started. • The images were saved per sample at an interval of 10 frame per second. • The difference in two successive images results in differential picture of only the moving particles. • From the pictures individual parameters for each moving microsphere were automatically determined. • Limitation • Particle diameter lessthan1.3µm can’t be detected.

23. DENSITY DETERMENATION • Measured by using multi volume Pycnometer. • Accurately weighed sample was placed In multivolume pycnometer. • Helium was introduced at a constant pressure into the chamber. • Allowed to expand this expansion results in decrease in pressure within the chamber • Two consecutive readings of reduction in pressure were noted from this the volume and density of the carrier was determined.

24. SURFACE HYDROPHOBICITY: • Determined by measuring angle of contact. • The surface hydrophobicity of microspheres has an important influence on the interaction of colloidal particles with the biological environment . • It regulates the extent and type of hydrophobic interactions of microparticles with blood components.

25. INVITRO RELEASE STUDIES • Same as the normal dissolution procedure. Done by using USP paddle apparatus . • Initial release is due to the release of surface drug and small microspheres with increased surface area & the presence of water soluble drugs. • The drug partition coefficient effects the release rate . • Higher the partition coefficient –faster the release because it results in the migration of drug to the outer organic phase, during preparation • IN VIVO RELEASE STUDIES : • Micro spheres are tested in vivo to assess the validity of drug targeting concept .The subcutaneous tissue in the tail of rats is chosen as the initial target site for microsphere localization. • The ventral caudal artery is selected for microsphere infusion as it supplies the majority of the blood to tail.

26. APPLICATIONS • 1).Hyperthermic treatment of cancer • Hyperthermia involves heating and destroying the target tissue between temperature 42-46°C. • 20-30µm of ferromagnetic microspheres are used to induce hyperthermia in cancers, for tumors located deep inside the body. • 2.DETECTION OF GLYCATED HAEMOGLOBIN • The determination of glycated haemoglobin using PVA magnetic microspheres • It is an alternative to currently used methods • Used in blood sugar diagnosis.

27. Preparation of magnetic microspheres encapsulated with diclofenac sodiuM • Diclofenac has short half life ,it should be given frequently to maintain therapeutic activity and is often associated with GIT bleeding ,gastric ulcers ,blood dyscrasias and anaphylaxis. • To over come this toxicity and prolong the therapeutic effect at the target site magnetic albumin microspheres of diclofenac were prepared. • Using magnetic field to repair damaged retinas: • Conventional way to repair damaged retinas. • Inject silicone fluid or a special gas directly in to the eye to push the retina back into place. • This makes the repair easier and more precise by allowing the fluid to be removed to the areas of the eye that are hard to reach.

28. Magnetic immuno assay of acute myocardial infARction: • Magnetic immunoassay was developed to test the 2 markers of AMI • Myoglobin • Fatty acid binding protein • These markers shows elevated serum levels soon after infarction . • Immuno assay follows a standard solid phase ELISA consisting of the formation of a complex /sandwich by attaching two different Ab to different epitopes. • Albumin magnetic micro spheres are prepared and coupled with avidin. • Ab are monoclonal mouse anti-human cardiac myoglobin.

29. Enzyme is alkaline phosphate coupled with streptavidin. • Biotin serves as abridging link between microspheres -antibody – alkaline phosphate. • Results in the formation of complex. • These complexes are separated by using external magnetic field. • To this add chemical reagent Nitro-phenol phosphate –produces an yellow substrate. • Intensity of the colour was measured by UV visible spectro photometer at 450nm. Higher the intensity – greater the myoglobin concentration.

30. detox regime : magnetic microspheres also helps to treat after a poison gas attack. The poison process involves injection of magnetic particles that had been functionalized to bind with the foreign toxin. An external magnetic field would then be used to draw the toxin tagged particles out of the body. Efficient detoxification will require particles with a reasonably high magnetic moment that can be pulled together with any attached toxin molecules across a magnetic field gradient.

31. Cell separatiOn: • Polymeric microspheres conjugated to antibodies and lectins have been used previously as cell surface markers for the detection and localization of antigens and lectin receptors using scanning electron microscopy (SEM) . • A new, more versatile cell surface probe consisting of iron-containing polymeric microspheres tagged with fluorescent dyes and chemically coupled to antibodies or lectins. • Magnetic and fluorescent properties of microspheres have been utilised in the magnetic separation of red blood cells (RBC) and lymphoid cells.

32. Magnetic control of pharmaco kinetic parameters and drug release The magnetite was embedded in to the drug filled polymer matrix and showed that they activate or increase the release of drug from polymer by moving a magnet over it or by applying oscillating magnet The micro movement within the crack produce a micro crack and thus made the influx of liquid , dissolution and efflux of the drug is possible. USE OF MECHANICAL PROPERTIES OF MAGNETITE IN THERAPY: This is possible by filling an magnetic component in to the capsule or tablet, speed of travel can be slow down at specific position by an external magnet ,thus changing the drug absorption and increases the drug levels in blood.

33. OTHER APPLICATIONS • Determination of trace hydrazine by differential pulse voltametry using magnetic microspheres. • Pulsed delivery of insulin in diabetic patients. • Used to deliver therapeutic radio isotopes. • Delivery of fibrinolytic agents in the treatment of deep venous thromobosis and pulmonary embolism.

34. Magnetism has application in numerous fields like • Diagnostics, • Tumour targeting • Molecular biology • Cell isolation • Cell purification • Radioimmunoassay • Detection of immunoglobulin (Ig) receptors • wheat germ agglutinin (WGA) receptors on lymphocytes • by SEM and fluorescent microscopy..

35. CONCLUSION: • Magnetic micro carriers have been investigated for targeted drug delivery especially magnetic targeted chemotherapy due to their better tumor targeting, therapeutic efficacy, lower toxicity and flexibility . • In spite of certain drawbacks, such as strong magnetic field requires for the • ferro fluid and deposition of magnetite the magnetic microcarriers still play an important role in the selective targeting, and the controlled delivery of various drugs. • It is a challenging area for future research in the drug targeting so more researches, long term toxicity study, and characterization will ensure the improvement of magnetic drug delivery system. • The future holds lot of promises in magnetic micro carriers and by further study this will be developed as novel and efficient approach for  targeted drug delivery system.

36. References • Methods of dug delivery ,International encyclopedia of pharmacology and therapeutics section 120 by G.M. Ihler PG.NO-39-56. • Characterization of magnetic particles and their magneto phoretic mobility using a digital microscopy by u.o. hafell,european cells and materials, vol-3 suplliment 2 ,2004,pg.n-24-27. • ,”magnetically modulated drug delivery “, targeted and controlled drug delivery by S.p.vyas and khar,1st edition ,2002,pg.no-417-458. • Microspheres technology and applications “, Encyclopedia of pharmaceutical technology, 10th edition ,pg.no – 1-29. • ,” magnetically modulated therapeutic systems”, international journal of pharmaceutics, U.O. Hafel , 277(2004) pg.n :19-24. • www.Magnetic magzine.com • www.magnetic microspheres.com • www.pharmainfo net.com • www.materials today.com • www.wikipedia.com

37. THANK U