1 Characterization:
Microspheres have different microstructures which determine the release and the stability of the carrier. Thus the characterization of microspheres is important in order to design a suitable carrier for drug delivery. Characterization can be done by checking the follo wing parameters:
Evaluation Parameters Characterization Methods
1. Physical Characterization
a. Particle Size and Size Distribution Scanning Electron Microscopy, Transmission electron microscopy, laser light scattering, optical microscopy, gel exclusion chromatography, sieve analysis
b. Surface morphology Scanning electron microscopy,
transmission electron microscopy, Atomic Force Microscopy, electron microscopy, Confocal laser scanning microscopy, Confocal fluorescence microscopy
c. Surface charge Free-flow electrophoresis, d. Electrical surface potential and
surface pH
Zeta sizer and pH sensitive probe
e. Density Multivolume pycnometer or hydrometer
f. Drug Loading Direct and Indirect method
g. Drug Release Dissolution apparatus , Dialysis bag 2. Che mical Characterization
a. Surface degradation Electron spectroscopy for chemical analysis, attenuated total reflectance, Fourier transform infrared spectroscopy b. Surface carboxylic acid and amino
acid residue
Liquid scintillation counter
c. pH pH meter
2
d. Compatibility study FTIR , XRD
3. Thermal Prope rties
a. Crystallanity DSC, XRD
4. Biological Characterisation
a. Sterility Aerobic or anaerobic cultures
b. Pyrogenicity Limulus amebocyte lysate test
c. Animal toxicity Monitoring survival test , histology and pathology
Physical Characterization
Particle Size and Size distribution:
Particle size and size distribution parameter is important to study because it affects the other parameters like stability of the microsphere , drug release from the microsphere, drug loading.
The various techniques used to characterize the particle size and size distribution is already listed in table. Brief explanation of the above technique is given below:
Electron Microscopy:
It includes SEM and TEM. This technique uses the beam of electron with interacts with the sample thus producing signals which is detected and provides the sample. SEM is based on scattered electron whereas TEM is based on transmitted electron. SEM provides the informatio n about the topography of the the sample surface , its composition where as TEM provides information about the internal structure of the particles with high resolution power than SEM Size exclusion chromatography:
This technique works on the basis of molecular size of components to be separated. Samples are passed through the bed of porous particles and hence by the differe ntial exclusion from the pores separation occurs . SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution results for polymers.
Optical Microscopy:
3 This technique is based on the simple ordinary microscope technique. Here an emulsion or suspension diluted or undiluted is mounted on the slide or ruled cell and placed on mechanical stage. The microscope eyepiece is fitted with a micrometer by which the size of the particle can be estimated.
Seive analysis:
Sieve analysis is performed using a nest or stack of sieves where each lower sieve has a smaller aperture size than that of the sieve above it which is allowed to shake for the particular time period. Thus the mesh size of the sieve through which the particle passes and through which it does not pass gives the rough idea of the particle size. Sieves can be referred to either by their aperture size or mesh size or sieve number.
Surface Morphology:
SEM and TEM are already discussed in the previous section.
Atomic Force Microscopy:
Atomic force microscopy (AFM) is powerful tool for surface and morphological analysis. AFM uses a sharp tip to probe the surface features by scanning, can image the surface topography with extremely high magnifications, up to 1,000,000X, comparable or even better than electronic microscopes.
Confocal Laser Scanning Microscopy:
Confocal works similarly to that of the SEM. However, it is able to provide the internal structure information provided that the particles are transparent and can be fluorescently labeled.
Surface Charge:
Surface charge plays a important role in the stability of the microspheres as well as in the circulation time in the blood thus affecting the bioavailability of the drug. Negatively charged particle are more subjected to opsonization process by RES. However for better stability in the colloidal system, charged particle are more stable as they have the tendency to repel and hence
4 no agglomeration. Surface charge can be measured by Free- flow electrophoresis or by measuring zeta potential.
Density:
For the determination of density different techniques can be used. Densities can be mainly of different types: True Density, Granule density
True density:
It can be determined by helium densitometer or can use liquid displacement technique with the help of pycnometer. However, helium is able to penetrate the smaller pore and cervices so helium method gives the closest approximation to true density.
Granule density:
It uses the technique similar to liquid displacement technique. However mercury is used to fill the void spaces and fails to penetrate the internal pores of the particles.
Flow Properties:
Flow properties play important role in designing the tablet, capsule dosage form. Similarly when microspores are used to prepare tablet or capsule dosage form flow property need to be measured. Angle of repose is one of the parameter which can be measured to check flow ability of microsphere powder. Rougher and more irregular the surface of the microsphere more is the angle of repose with poor flow.
Drug Loading :
Drug can be loaded into the microsphere at the time of preparation of microsphere or a fter the preparation of placebo microsphere. Drug loading can be checked either by dissolving the formed microsphere in suitable solvent and then calculating the amount of drug present or by checking the amount of drug left in supernatant after centrifuging ( especially for incubation method of drug loading ).
Drug loading can be calculated as:
5 Amount of drug in microspheres (mg)/amount of microsphere (mg recovered)*100
Entrapment Efficiency:
The entrapment efficiency can be determined by measuring the amount of drug that is encapsulated in microsphere
Entrapment efficiency%= (amount of drug in microsphere (mg)/ initial amount of drug(mg))*100
Drug Release:
Drug release can be determined either by the use of Dissolution apparatus or by the use of dialysis bag. Dissolution apparatus basically uses paddle and is carried in similar ways as for tablet or capsule dosage form. For Dialysis, first the activation of dialysis bag is necessary by treating with different chemical reagent and then the drug loaded microsphere suspension is placed within the dialysis bag and the bag placed in the receptor compartment which is under stirring and set at particular temperature. Sink condition is maintained. Aliquots are withdrawn at different time intervals and are analyzed quantitatively with suitable analytical method. Amount of drug released at different time interval is plotted to fit different release models like zero order release, first order release, Higuchi model, Hixson Crowell model, Korsmeyer–Peppas model and others.
Che mical Characterization:
Surface Carboxylic Acid residue and surface Amino Acid residue:
Surface Carboxylic acid residue is measured by using radioactive glycine. The radioactive is conjugated with the microsphere by using a linker and the radioactivity of the conjugate is measured by using liquid scintillation counter from which carboxylic acid residue is correlated.
Similarly for surface amino acid residue is determined by radioactive 14C – acetic acid .this is indirect method of determination. Initially carboxylic acid residue is measured through scintillation counter and then amino acid residue is linked indirectly.
Compatibility Studies:
6 It can be checked by many techniques. Most commonly used techniques in the research are by FTIR study and DSC study. Comparing the FTIR spectrum DSC curves of the drug, Microsphere carrier and microsphere loaded drug , one can indentify changes in the drug molecule due to interaction with carrier molecule.
Thermal Prope rties Analysis:
Thermal property analysis is especially important to check for the drug loaded microsphere.
Because of the technique used in the preparation of microsphere sometimes there is change in the internal properties of drug like change in crystallanity. Drugs may change from crystalline to the amorphous form thus enhancing bioavailability. Thus these parameters can be checked from Differential Scanning calorimetry or X-ray Diffraction ( XRD) pattern. For the amorphous, DSC shows no peak whereas for crystalline structure there is the sharp peak due to the phase transition with increase in temperature.
Biological Characterization:
Since the microspheres intended for parenteral administration should be sterile, pyrogen free and should not produce any kinds of toxicity in human beings. Thus the produced microsperes should be tested for sterility by incubating in suitable culture media for aerobic and anerobic bacteria and checking for the growth of the microorganism. For pyrogen testing LAL test is commonly used.
Application of Microsphere
Microspheres encapsulate or entrap the drug molecules into the polymer thus enabling controlled and sustained release, protection of drug molecules or protein and peptide from degradation, targeting drug to the site of action especially for the anticancer drug delivery by coating with suitable ligands, antibody to the surface of microsphere. Nowadays there have been developments of bio-adhesive microspheres for nasal delivery system, mucosal drug delivery
7 system , vaginal drug delivery system especially for the drug that undergo hepatic metabolism , poorly stable drug in GI track thus enhancing bioavailability of the drug. These points are described in detail in the following section.
The table summarizes the application of microspheres in different fields:
Application of Microsphere
Types of microsphere Research Done
Controlled release and enhanced bioavaibility
Bioadhesive microsphere – Nasal Drug Delivery
- Ocular Drug Delivery
Studies on nasal drug delivery- Wang et al.
evaluated gelatin microspheres for the nasal delivery of insulin and reported a significant hypoglycemic effect in rats Studies on the nasal delivery of carbamazepine using chitosan microspheres , carvedilol - by alginate microspheres and many others which confirm the huge potential of these systems in nasal route of administration Similarly, Van der Lubben etal. confirmed that there is considerable enhancement for systemic and local immune response against dip htheria toxoid after nasal administration in mice.
Studies on Ocular drug Delivery- Different drug like Methyl prednisolone , ganciclovir, ofloxacin, vancomycin have been studied . In study by Gavini E etal. PLGA microspheres as the carrier were used for ocular delivery of Vancomycin(VA). It was observed that high and prolonged VA concentration and increased AUC values with respect to the aqueous solution of the drug.
8 - Buccal drug delivery
- Floating Microsphere-
- Polymeric microsphere
Different studies for the controlled drug delivery of lidocaine, nystatin, venlafaxine.
Polymers generally used are Chitosan, alginate, linseed mucilage, Carbopol, Poloxamer. The main reason being the intention to avoid hepatic first-pass metabolism and local drug delivery.
Different studies targeting gastric disease like Gastric ulcer by H pylori , microspheres of riboflavin were developed with the intention of site-specific gastroretentive system to prolong the residence time of drug in the stomach and, consequently, to enhance the bioavailability.
Natural and synthetic polymer studied as carriers for drugs like antiviral, antifungal and antibiotic agents, non-steroidal anti inflammatory drugs. For
Vaccine delivery: Hepatitis, Influenza , Diptheria toxoid . For Oral drug delivery, Gene therapy with DNA plasmids; delivery of insulin, LHRH Controlled drug delivery after local application : Release of proteins, hormones and peptides over extended times
Targeting Magnetic Microsphere Magnetic microspheres has been expensively researched for targeting brain tumors as well as other solid tumors. Recently it potential for catheter-directed drug delivery to liver tumors
9
for reduced systemic toxicity and superior therapeutic outcomes. Magnetic resonance imaging (MRI)-visible amonafide-eluting alginate microspheres were developed for targeted arterial- infusion chemotherapys to deliver these microspheres to liver and to visualize the intra-hepatic delivery of these microspheres to both liver tumor and normal tissues with MRI immediately after infusion.
Diagnostic purpose and therapeutic purpose
( as a reagent or as element of diagnostic device)
Radioactive microsphere Diagnostic radioembolization:
99mTc- macroaggregated human serum albumin (MAA) , Thrombus imaging in deep vein thrombosis : 99mTc-sulfur colloid
For therapeutic purpose - Radioembolization of liver and spleen tumours:90Ymicrospheres, Local radiotherapy: 212Pb-sulfur colloid
Microspheres for controlled and sustained drug delivery system:
Polymeric microspheres are the ideal vehicles for many controlled drug delivery application due to their ability to encapsulate a variety of drugs, biocompatibility, high bioavailability and sustained drug release characteristics. There are many factors that determine the release properties of the drug like nature of polymer that allows for bulk erosion or surface erosion, molecular weight of the polymer as with increase in molecular weight there is decrease in release as there is decrease in diffusion of the solvent into the polymer and hence decreasing the drug release, Size of the microsphere with the decrease in size of microsphere there is increase in surface area and hence increase in solubility of the drug hence enhancing release.
New concept of bio-adhesive microspheres has emerged in the field of application of microsphere. These types of microspheres have a huge potential in drug delivery system. They enable to provide targeted drug delivery system through localized action of bio adhesion i.e local targeting along with it also provides the controlled drug release of the drug which is ultimately
10 governed by the degradation profile of the polymer, dissolution and diffusion of drug molecule from the polymer. Furthermore, since the drug avoids degradation from the GI enzymes and hepatic metabolism thus enhancing bioavailability. The application of the bio adhesive microspheres can be best explained by the diagram below:
Application of Bio adhesive microsphere in drug delivery
Other recently developed applied field for the controlled drug delivery system is
11 Controlled release vaccines:
In recent years, research on microsphere based vaccine delivery vehicles for single administration has got much attention. Microsphere are thought as the potential alternative in the development of single shot vaccine which can provide the necessary booster dose at the specified time after administration thus eliminating the need for the patient to return for boosters and would reduce the risk associated with the reuse of disposable needles and syringes. Also, the ability to more precisely control the time course of vaccine deliver may lead to more effective vaccination with current antigens and may allow utilization of antigens that were previous ly ineffective.
Microspheres for targeted drug delivery:
Targeting techniques of microsphere is basically based on two ways i.e Active Targeting and Passive Targeting. In case of active targeting the surface of microsphere is linked to the specific molecular group that interacts to the specific receptor in the target tissue. While, the passive targeting of the microsphere is based on the physical and the chemical properties of the microsphere- especially size charge and surface characteristic ( hydrophobicity ) so that the particles reach the desired site of action. Like microspheres can be passively targeted to the antigen presenting cells of immune system, the most common method being the uptake by microphages. The ability of the microphages to phagocyte particles is dependent on the particle size. Following iv injections, microspheres with diameters > 7 µm are generally cleared by entrapment or filtration and microspheres with size < 100 nm can escape phagosytosis . However microspheres with 1- 10 µm diameter microsphere are optimally taken up by microphages in a number of tissues.
Microsphere targeting for Cancer Therapy:
Microspheres have been employed extensively with cytotoxic drugs, the chief example being doxorubicin, mitomycin C, cisplatin and 5-fluorouracil. Cancer therapy demands specificity as to avoid damage to the healthy cells thus targeting with microspheres is being exploited recently to achieve the demand. Targeting to the cancer cells can be achieved as discussed earlier or by the utilization of changes in the tumor environment like change in pH, accumulation of particular factor or receptors in the site of tumor. Magnetic microspheres, radio microsphere has
12 revolutionized the field of anticancer drug delivery. Ultrafine particles of magnetite (Fe3O4) have been incorporated into both albumin and cellulose microspheres containing the anticancer drugs doxorubicin (Dox, Adriamycin) and Didox . The movement of particles can be controlled after intra-arterial administration by an externally applied magnetic field. Up to 70% of the administered dose of Dox can be directed to the target site by this mechanism, which has shown success in inhibiting the growth of Subcutaneous tumour
A recent patent from Japan describes new technology in the form of porous silica microspheres coated with ferromagnetic ferrite. The inventors claim uses of the technology for ultrasonic diagnosis and thermatology, in particular the treatment of solid tumours utilizing heat generated through converged ultrasonic waves. Therapeutic efficacy was demonstrated after direct injection of microspheres into the liver of rats inoculated with tumor cells. Tumors were irradiated with ultrasonic waves (2 MHz, 10 W) for 3 min and temperature was raised by 5°C resulting in tumor cell death.
Diagnostic purpose
Microspheres are also useful for the targeting purpose in which various cells, tissues and organ, cell line, can be imaged by radio labeled microspheres. Particle size analysis of microspheres is a main factor in determining the imaging of particular sites. For example: The particles injected intravenously will become entrapped in the capillary bed of the lungs. This is used in imaging of the tumor masses in lungs using labeled human serum albumin microspheres.
The adequate expression of cell-surface receptors and antigens is an important requirement for the functional ability of living cells. A lot of different methods for cell surface marker tracing have been described; however, most of these techniques have disadvantages limiting their wide- scale utilization in routine laboratory and clinical practice. The most recent technique for these purposes, approaching near the ideal one, is based on the use of synthetic microsphere particles made of polymers which are formed mainly by emulsion or radiation polymerization of a variety of monomers. The resulting spherical particles bear hydroxyl, carboxyl, amino, or other functional groups capable of covalent binding of proteins, dyes, or chemotherapeutic agents.
Fluorescent, radioactive or haptenic labels may be introduced already during the polymerization, too. There are three main fields of application of such specific labeled microspheres
13 in cell biology: (1) detection of cell surface markers; (2) studies of phagocytosis mediated via cell surface markers; and (3) cell separation according to cell surface markers.
Microsphere with different functional groups like hydroxyl, carbonyl and aldehyde can be obtained by the polymerization technigues. These functionally activated microsphere can be used for adsorption and covalent immobilization of of human and animal proteins–human serum albumin (HSA), gamma globulins (γG; human, rabbit and goat), human fibrinogen (Fb), and enzymes: glucose oxidase (GOD) and urease (Urs).
Bioavailability Enhancement:
The above applications like targeting, bio adhesive, sustained release property is directly or indirectly liked with the enhancement of the bioavailability of the drug . Targeting enhances the availability to the site of action; bio adhesive like mucoadhesive eliminates the harsh condition of GIT and hence enhances bioavailability. Protection of drug by the encapsulation of the drug also ultimately enhances the bioavailab ility. Floating microspheres enhances the residence time of the formulation on to the stomach sustaining the drug release and thus improving the bioavailability . Also acts for targeting disease related to stomach and colon.
It was reported by Jong Soo Woo etal. For the enhanced oral bioavailability of poorly soluble drug Cyclosporin A by formulating it in combination with hyaluronic acid into microsphere. Oral bioavailability was enhanced because of enhanced dissolution rate and bioadhesive property of hyaluronic acid.
Polymer microspheres can also be used to deliver of the proteins. This is a very important class of drug delivery as the release of protein from such formulations can be well controlled, thus protecting the encapsulated proteins from enzyma tic degradation. A large body of literature exists which shows that small (generally less than 2 mm) thermoplastic microspheres delivered orally are able to cross the intestinal epithelium, either through the Payer’s patch and/or the regular absorptive epithelium and travel systemically to other sites including the liver, spleen and other tissues. These observations have led to the proposition of three major mechanisms of action for microspheres for improving oral delivery of proteins: (1) the spheres can protect proteins from proteolysis, (2) the spheres cross over the intestinal mucosa and (3) the microspheres change the intertissular distribution of protein throughout the organism. The last proposed
14 mechanism can be extremely important for drugs like insulin as microspheres are expected to build up in the liver where the loaded insulin can be used to limit the abnormally elevated hepatic glucose alone, without the use of encapsulated formulations production present in diabetic patients.
Available Marketed Products Marketed
formulation
Drug Route Manufacturer
VIVITROL® (Naltrexone for extended- release injectable suspension – For treatment of alcohol dependence
Intramuscular injection
Alkermes, Inc
Lupron Depot Leuprolide acetate (GnRH or LH-RH) for prostate cancer
Intramuscular injection
Abbott Laboratories
Nutropin Depot r_ Somatropin recombinant human growth hormone (rhGH)
Subcutaneous Injection
Genentech, Inc
Arestin Minocycline HCl Oral powder for
dental purpose
OraPharma, a division
of Valeant
Pharmaceuticals Retin- A-Micro Tretinoin gel For topical treatment
of acne vulgaris
Ortho-McNeil Pharmaceutical, Inc.
Carac fluorouracil Cream
Fluorouracil cream Topical
dermatological cream
VALEANT PHARM.
COSMOL™ Lipstick with
KOBOGUARD®
Lipstick Kobo Products, Inc.
Somatuline LA Lanreotide acetate- For acromegaly
Intramuscular injection
Ipsen Pharma Biotech
15
Bydureon Exenatide extended-
release for injectable suspension)-For type II diabetes
Subcutaneous Injection
AstraZeneca
Zmax Azithromycin extended
release) - Antimicrobial
Oral suspension Pfizer
