MICROSPHERE DISPERSIONS: METHODS AND CHARACTERIZATION

2. MICROSPHERE DISPERSIONS: METHODS AND CHARACTERIZATION

2.1. Formulation=Composition

2.1.1. Typical Polymers Table 1 shows a list of polymers employed in microsphere

manufacture and their intended purpose. These consist of natural and synthetic polymers with a variety of properties. Among the properties of interest are the rate of degradation and nature of the erosion process. In this regard, PLGA microparticles may be contrasted with polyanhydride. PLGA erodes from every available wettable surface and after an initial surface erosion develops a porosity which results in the final structural collapse of the particle. Polyanhydride in contrast erodes from the surface in a manner dictated by the particle geometry and as such offers predictable degrada- tion throughout its lifespan.

Particle Engineering Each method of manufacture involves a number of pro-

cessing variables that can be adjusted to achieve objectives of drug load, particle size, and distribution, porosity, tortuos- ity, and surface area. Since each method involves different variables, it is sufficient to note that key physicochemical

Microspheres: Design and Manufacturing 313 Table 1 Selected Papers Reflecting Use of Polymers for

Parenteral Administration (1995–2003) Component

(matrix = Literature encapsulator)

Application source PLA

Chemoembolization 28, 29

Testosterone delivery system

30 Adjuvants for adsorbed influenza

virus Encapsulation of 5-fluorouracil for

treatment of liver cancer Encapsulation of cisplatin for direct

33 intratumoral injection with reduction of acute renal toxicity

PLA =PLGA Antigen entrapment for diphtheria 34, 35

and tetanus vaccines

PLA =PGA Long-lasting ivermectin delivery 36 system for control of livestock pests (larval horn flies, hematobia irritants)

PLGA Intraocular delivery of guanosine 37 DNA vaccine and encapsulation

Intracerebral treatment of

malignant glioma Encapsulation of ultrasonographic

41 contrast agents for differentiation of coagulation necrosis in adenocarcinoma tumors

Microencapsulation of an influenza 42 antigen for a single dose vaccine delivery system

Pulsatile single immunization for HIV

Poly-(caprolactone) Potential drug delivery system 44, 45 Polyanhydride

46 (II) citrate, an anti-tumor agents Poly(anhydride-

Encapsulation of rhodium

47 co-imides)

Controlled delivery of vaccine

antigens

Chitosan Device for gadolinium 48

neutron-capture therapy by intratumoral injection

Delivery system for steroids

(progesterone)

(Continued)

314 Burgess and Hickey Table 1 Selected Papers Reflecting Use of Polymers for

Parenteral Administration (1995–2003) (Continued ) Component

(matrix = Literature encapsulator)

Application source

Delivery system for antibiotic

agents Encapulation of bisphosphonates

51 delivered by local implantation or injection for site-specific therapy in pathological conditions associated with bone destruction

52 Controlled and localized delivery

system of endothelial cell growth factor for stimulation of vascularization

Gelatin Carrier matrix of basic fibroblast 53

growth factor (bFGF) to enhance the vascularization

54 Gelatin =chondroitin

Microspheres for implantation 55 6-sulfate

as an embolization material Microspheres as immunological

adjuvant Intraarticular delivery system of

therapeutic proteins

Gelatin =alginate Sustained-release microparticles 57 for delivery of interferon-a Alginate

Delivery of TGF-b to inhibit

fibrosis of the corpus cavernosum

Alginate =polylysine

59 Magnetic dextran

Intrahepatic implantations

Targeted drug delivery system 60

for brain tumors

Hydroxyethyl-starch Potential drug delivery system 61, 62 Imaging

factors include starting concentrations of drug and polymer, partitioning, and solubility; and that processing variables include the presence of surfactant (for emulsion formula- tions), the presence of polymerizing agent (initiator or ionic species for synthetic or natural polymer cross-linking)

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stirring conditions, vessel geometry, nozzle geometry, and feed conditions (spray drying, spray freezing, and supercriti- cal fluid manufacture) and in general, fluid flow, heat and mass transfer.

2.2. Desired Performance Characteristics and Relative Performance Measurements

Desirable performance characteristics must be considered in developing a formulation for a therapeutic agent. The first consideration is the target disease state or desired therapeu- tic intervention. This will dictate relevant routes of adminis- tration, dosage, and period and =or frequency of delivery. Thus, therapeutic, pharmacokinetic, and pharmacodynamic considerations may be used as criteria to judge the merits of dosage forms following characterization of relevant physico- chemical properties.

The goal in preparing any drug delivery system is to maximize the therapeutic effect while minimizing toxicity. Contemporary concepts in drug delivery and disposition focus on specific delivery to the site of action in the absence of effects at any other site. This may be achieved by direct or indirect local delivery. Indirect targeted delivery can be brought about by using the capacity of the dosage form to preferentially localize in certain organs or tissues or by molecular modifications to the drug to achieve high recep- tor =enzyme=protein affinities.

Matrix or encapsulating components and processing parameters should be selected to produce particles with the desired drug load, particle size, surface area, surface and dissolution properties to achieve acceptable in vitro and ultimately in vivo dose delivery.

2.2.1. Release Factors governing release from microsphere systems have

been discussed by Burgess and Hickey (1). Mechanisms of release are from the microsphere surface, through pores in the microspheres, diffusion from swollen microspheres, and

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following erosion and =or bulk degradation. Polymer type is of importance, hydrophilic polymers tend to release drug much more rapidly than hydrophobic polymers as a result of pene- tration of aqueous media into the microspheres. In order to achieve extended release profiles of weeks or months, it is usually necessary to use hydrophobic polymers, such as poly- lactic and polyglycolic acid. Amorphous polymer structures facilitate absorption of water compared to crystalline poly- mers and therefore amorphous polymers and polymers with

a high proportion of amorphous regions tend to have faster release rates compared to crystalline polymers or polymers with a high proportion of crystalline regions. Crystalline regions decrease the diffusion rate of drug molecules by increasing the diffusional path length. Crystalline regions of polymers have a higher density and a lower specific volume than amorphous regions (64). Polylactic acid is an example of a polymer with a high proportion of crystalline regions, whereas polyglycolic acid has a high proportion of amorphous regions. These two polymers are usually used in combination to achieve desired release rates.

Other factors affecting release rates include the drug solubility, diffusivity, molecular weight, and particle size; the microsphere particle size, the percentage loading of the drug, the dispersion of the drug in the microspheres, any drug =polymer interactions, the rate of biodegradation of the polymer and the stabilities of the polymer matrix and the drug in the polymer matrix before and after injection. The lar- ger the microsphere particle size, the longer the diffusional path length and the smaller the surface to volume ratio; therefore, release rates of larger microspheres are usually slower. For large drug molecules, the release rate is usually dominated by the polymer properties. If the microspheres have low porosity, then the polymer must degrade in order to create channels for drug release.

PLGA microspheres have been successfully used to achieve extended release profiles. Controlled release from PLGA microspheres will be discussed since this system has been widely investigated; there are five products on the mar- ket and several are in clinical trials. Although PLGA might

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not be the most appropriate polymer for all applications, this system is very attractive since it has been used in sutures for many years. Investigators have used a number of meth- ods to increase and decrease release rates from this polymer system (65). Release can be diffusion and =or degradation controlled. The more crystalline the PLGA co-polymer, the higher the molecular weights of both the polymer and the drug and the less porous the microspheres, the slower the release rate. The slower the diffusion rate of the drug, the greater the contribution that polymer degradation will make to release (64). Porosity can be controlled by adding water soluble excipients such as NaCl which rapidly dissolves and diffuses away from the microspheres, leaving pores. High concen- trations of albumin have been added to increase release rates of high molecular weight drugs such as protein therapeutics. The albumin releases and leaves behind large pores and the protein therapeutic agent can then diffuse out along these pores (64). Release is also ionic strength dependent. High ionic strength inside the microspheres results in the influx of water. Similarly, decreased release rates elevate the ionic strength of the medium (66). The addition of plasticizers should give enhanced release due to less resistance to diffusion. However, the use of plasticizers can result in a less porous microsphere matrix and therefore drug release can be reduced (64). Since PLGA degradation is acid catalyzed, the addition of salts such as calcium carbonate and magnesium hydroxide confers an alkaline pH and reduces PLGA degrada- tion rates (67).

All of the above factors can be manipulated to achieve the desired release rates. Mixed populations of microspheres may also be used to this end (137,138). As a consequence of the manufacturing process, microspheres often have surface- associated drug which can contribute to a burst release effect. Microspheres can be washed with water before release testing to determine surface associated drug. It is also possible to use predegraded microspheres to avoid the initial burst release effect (137,138).

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2.3. In Vitro Release Testing The reader is referred to the chapter in this book by

Clark et al., for additional information on in vitro release testing.

In vitro testing of microsphere release rates is often con- ducted by suspending the microspheres in the release media under conditions of mild stirring. The samples must be pro- cessed to remove any suspended microspheres prior to analy- sis to avoid interference with the analytical method and so that the microspheres are returned to the release media to continue the release process. This is usually achieved by fil- tration or centrifugation. This in vitro release methodology is referred to as sample and separate. The continuous flow method (USP apparatus 4) has also been used for micro- spheres. Here the microspheres are maintained in a compart- ment and the release media is continuously circulated through this compartment (closed system) or fresh media is continu- ously pumped through (open system). Filters are used to iso- late the microspheres in the compartment. Glass beads can

be added to avoid microsphere aggregation and to alter the flow pattern so as to avoid the production of flow channels within the microsphere bed that would lead to inaccurate release pro- files due to uneven contact between individual microspheres and the release media. The USP 4 flow-through method has been recommended for microspheres as it can avoid problems of microsphere aggregation and sink conditions can be easily maintained (134). Problems may arise due to filter blockage and this should be monitored periodically.

USP apparatus 1 has been used to test in vitro release rates from microspheres. Typically microspheres are sus- pended in 900 mL of release medium placed in dissolution apparatus and stirred using an overhead stirrer at 100 rpm (68). The amount of microspheres used is dependent on micro- sphere drug loading and sink conditions. The relatively slow stirring rates used can be problematic as the microspheres may settle in the large USP vessels under slow stirring. The volumes used are another problem particularly for expensive biotech drugs. In addition, large volumes are not representa-

Microspheres: Design and Manufacturing 319

tive of the parenteral situation with the exception of the i.v. route and as previously discussed this route is generally not applicable for microspheres.

Release media can be standard, pH 7.4, phosphate buffer or other suitable media depending on the drug. Since microspheres usually have hydrophobic surfaces, dispersing agents (such as Tween surfactants) are often added to assure dispersion of the microspheres in the media. Ethanolic phosphate media has been used for PLGA microspheres as this causes plastization of the polymer, simulating the plasti- zation effect of lipids in vivo. Non-ethanolic degradation of PLGA microspheres in vitro has been reported, in some cases, to be two to six times slower than occurs in plasma.

Several variations on miniaturized release methods have been reported for systems intended for parenteral use. These include: a miniaturized version of the standard USP appara- tus 1 method in a scaled down beaker using 50–100 mL of media; a dialysis sac method using volumes in the order of 50–100 mL; magnetic stirring in place of overhead stirring that is used in the USP method; small sample vial method, using 1–10 mL volumes and shaking or rotating rather than stirring. If a sufficiently miniaturized method is used then each vial can be used for a single sample. The vial is centri- fuged and the whole supernatant is taken for the sample. The pellet may then be discarded or analyzed for polymer degradation or percent drug remaining.

A problem that can arise during in vitro release testing of microspheres is floating of microspheres composed of hydro- phobic materials (such as, PLGA) due to difficulty in wetting. To overcome this problem, Tween 80 or similar surfactants can be added to the dissolution media. Care should be taken as surfactants will solubilize hydrophobic drugs at concentra- tions above the surfactant CMC. This can be used as a method of obtaining sink conditions without having to dilute the sam- ple too much and therefore can help in analysis to obtain concentrations within the detectable range. Microsphere hydrophobicity can also result in aggregation of the particles at the bottom of the dissolution vessel. This can result in irreproducible data. The surface area exposed and hence the

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release rates may be reduced as a consequence of aggregation, again resulting in irreproducible data.

Another problem is drug degradation during release. For microspheres designed to release drug over periods of 1 month or more, this can be a significant problem for drug released early and then left in the dissolution media during the study. Degradation can be accounted for mathematically, if the degra- dation rate in the dissolution media is first calculated (69).

It is also important to determine if any drug remains at the end of the release study. Samples can be filtered, the poly- mer dissolved and the drug extracted as appropriate (refer Sec. 2.6 ). In vitro release studies reported in the literature usually are incomplete and this may be due to drug degrada- tion or to incomplete release form the microsphere system.

In vitro–in vivo correlation (IVIVC) is the ultimate goal and therefore in vitro dissolution methods should reflect the in vivo situation as much as possible. It should be possible to establish guidelines for IVIVC for controlled release parent- erals through a systematic evaluation of in vivo and delivery system factors. The exact in vivo situation need not be reproduced in vitro, but a situation that results in the same outcome. For example, in vivo release from microspheres may be enhanced compared to in vitro release as a result of enzymatic degradation of the polymer. Polymer degradation could be enhanced to the same degree in vitro by alteration of pH or some other variable that affects polymer degradation (e.g., PLGA degradation is enhanced by reduction in pH). Until recently, there have only been rank order correlations between in vitro and in vivo drug release from microsphere systems (14,68). However, in the last 2 years, there have been IVIVC reports for controlled release parenteral microsphere systems and the reader is referred to the chapters in this book by Clark et al., by Young, and by Chen for more information.

2.4. In Vivo Release Testing In vivo release rates are usually determined indirectly from

drug plasma levels. Animal studies have been conducted where release has been measured directly through serial

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sacrifice experiments where the tissue is excised, homogenized and the amount remaining at the site is determined (13,139). These animal studies are useful in determining the release mechanism and in vivo factors that affect release rates.

2.4.1. Polymer Degradation Gel permeation chromatography (GPC) has been utilized to

determine the molecular weight distribution of PLA and PLGA before and after different incubation times (70). Scan- ning electron microscopy (SEM) has been used to assess the extent of microsphere degradation.

2.5. Particle Size Particle size is dependent on the method of microsphere

manufacture. For the commonly used emulsion methods, microsphere particle size can be altered by decreasing the concentration of polymer, decreasing the volume fraction of the dispersed phase, increasing the rate of agitation, increas- ing the surfactant concentration, and changing the type of surfactant. For methods that involve atomization, such as spray drying, the particle size is dependent on the atomiza- tion pressure, the orifice size, as well as the viscosity of the polymer solution or suspension and the flow rate.

Microspheres are in the micron size range and, therefore, both resistance and light blockage methods are appropriate particle sizing methods (e.g., HIAC, accusizer particle sizing systems, and coulter counter), refer to Chapter 3 for dispersed system characterization. Problems in particle size analysis can arise due to the tendency of microspheres to aggregate in that two or more particles could be counted as one. Size can be determined directly by microscopy, although this method is more time consuming and is subjective.

2.6. Drug Loading Drug loading in hydrophobic polymers such as PLGA can be

up to approximately 50% for insoluble materials such as ster- oids, but typically is closer to 20% to obtain satisfactory

322 Burgess and Hickey

spheres with the desired release characteristics. For hydro- phobic drugs, loading is dependent on the relative solubility of the drug in the organic solvent. Drugs with lower solubili- ties compared to the polymer may precipitate out of the polymer solvent system during solvent evaporation, resulting in relatively low loadings (71). For water-soluble materials, loadings are usually not more than 10%, since these drugs are rapidly lost to the external aqueous phase during manu- facture by the O =W emulsification technique through parti- tioning into the external aqueous phase. Loading of water soluble drugs into PLGA microspheres can be enhanced using W =O=W, W=O=O, and solid S=O=W techniques (72–74). In addition, drug loading of microspheres prepared by the W =O=W method can be increased by improving the stabiliza- tion of the primary emulsion (75). This helps to prevent loss of drug to the external phase during solvent evaporation. Higher internal aqueous phase, cool temperatures, and short proces- sing times have also been used to increase drug loading using the W =O=W method of manufacture (76). Other methods to increase loading of water-soluble drugs are to complex the drug with a more hydrophobic macromolecule or with the poly- mer itself (77). PLGA COO binds with positively charged drugs such as peptides. Chemical modification of hydrophilic materials may also be employed as a means to enhance loading as well as to modify release rates (70). It has been reported that the higher the PLGA concentration the greater the ent- rapment efficiency (78). Increase in drug concentration can decrease entrapment efficiency (79). In addition, high drug concentrations can result in fragmented microspheres (79).

Drug loading can be detected directly by disrup- tion =dissolution of the polymer and subsequent release of the drug. A solvent for the polymer (e.g., methylene chloride for PLGA) is added and the mixture is ultracentrifuged to separate any precipitated polymer. Drug levels are determined in the supernatant. In some cases, direct determi- nation is difficult. Then, loading can be calculated based on the percentage of drug in the supernatant fluid following col- lection of the microspheres at the end of the manufacturing process.

Microspheres: Design and Manufacturing 323

It is important to ensure that all of the loaded drug is determined. Several extraction methods have been reported for PLGA microspheres. These include shaking the micro- spheres overnight in 0.1 m NaOH with 5% SDS and meas- uring the released drug (80). A combination of polymer solubilization and drug extraction has also been employed for PLGA microspheres loaded with protein (66). The polymer was dissolved in methylene chloride and extracted into pH 4 acetate buffer to remove the protein. Exhaustive extraction in distilled water has also been utilized (81). DMSO has been used as an alternative to methylene chloride to dissolve PLGA (78). Drug loading of albumin microspheres has been deter- mined by digestion with 0.5% acetic acid, followed by centrifu- gation and extraction of hydrophobic drugs using appropriate organic solvents (68).

2.7. Porosity Large pores, or megaporosity (10–75 mm), can be measured by

air permeability applying the Kozeny–Carmen equation (82). Macroporosity (smaller than 10 mm) can be measured by mer- cury porosimetry to obtain total porous volume, specific sur- face area, average pore radius, and pore size distribution. The conventional method of surface area determination is based on the Brunauer–Emmett–Teller equation (83) that takes into account lateral interaction energies, multilayer formation and condensation in pores and conforms to one of the five isotherms described by Brunauer (83). The porosity of particles plays a role in the dissolution and release of drug and in the erosion of the polymer matrix (84).

2.8. Sterility Testing Sterilization of microspheres is usually achieved by aseptic

processing since the final product may not be able to undergo terminal sterilization. Manufacturing methods that are single step and can be performed in an enclosed chamber, such as spray drying, are ideally suited to aseptic processing. Term- inal sterilization at high temperature is likely to melt the polymer, cause alteration of drug release rates and may

324 Burgess and Hickey

destroy any targeting moiety that may be attached. The glass transition temperature of PLGA is around 44

C (79) and therefore PLGA microspheres would melt and agglomerate during autoclaving for terminal sterilization. Entrapped drug may also be degraded at sterilization temperatures. Sterility assurance is another problem, as, although it is relatively easy to determine whether the exterior of the microspheres is sterile using conventional plating methodology, it is difficult to determine whether the interior of the microspheres are sterile. Methods that can be used to break or dissolve the microspheres such as crushing, grinding, or the use of organic solvents may introduce false-positive or false-negative results.

A method of determining the presence of viable organisms in the interior of microspheres without breaking or dissol- ving microspheres was developed (85). This method involves detecting organism metabolism.

2.8.1. Chemical Stability and Protection from Degradation

Protein drugs are susceptible to breakdown by peptides at the local site and hence their in vivo half-lives are generally very short. Encapsulation in a microsphere system can protect these molecules from degradation until release at the site. This effectively extends the in vivo half-life for elimination and thus the dosing frequency can be reduced. The microen- vironment within the polymer may also cause degradation of the drug. Microspheres composed of polyesters of glycolic and lactic acid present a problem for drugs that are not stable under acidic conditions as these polymers degrade to glycolic and lactic acid and present an acidic microenvironment dur- ing dissolution. The degradation of proteins and peptides can be increased in the presence of polyesters. This problem may not be serious if the release rates are much faster than the degradation rates.

Low pH environments may be deleterious to many proteins, nucleic acids, and cells. Consequently, the selection of matrix or coating materials that are chemically compatible with these molecules is important.

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3. TISSUE TARGETING Targeting of the RES of the liver has been achieved by select-

ing dispersed systems of an appropriate size ( <5 mm) for uptake by the Kuppfer cells. This has been achieved utilizing particulates (86), liposomes (87), and emulsion systems. Particles larger than approximately 7 mm will occlude the capillaries of the lungs (86). Surface modifications have been adopted to target dispersed systems to bone and kidney.

Particle engineering has been used for targeted delivery: size, surface charge, surface hydrophobicity, and steric stabi- lization can all be manipulated to this end. Size and surface characteristics can be used to target the lung, liver, spleen, general circulation, and bone marrow following i.v. adminis- tration. Mechanical filtration by size is used to target the lung. Recognition of the particle surface by the RES is used to target the liver. Subcutaneous and peritoneal administra- tion are used to target the regional lymph nodes. Particles below 100 nm delivered by the s.c. route are taken up by the regional lymph nodes. This is useful for anti-cancer and immunomodulating agents. Hydrophilic coatings (e.g., polox- omer) are used to keep microspheres within the systemic cir- culation. The presence of the hydrophilic layer minimizes uptake of opsonic factors, reduces particle–cell interaction and therefore prevents uptake by phagocytic cells. Specific polymers can target specific sites, e.g., poloxamer 407 can tar- get the bone marrow (88). This is speculated to occur via a receptor-mediated pathway. Both particle size and surface characteristics are critical for bone marrow uptake (size range 60–150 nm). Magnetic targeting is used to overcome clearance by RES (89) and achieve target site specificity. Coated parti- cles avoid uptake by the liver Kuppfer cells and are captured by the spleen via filtration.

Different methods can be used to achieve targeting of microspheres to specific sites in the body. The theory behind this has been discussed by (1). Some parenteral targeting methods that have been proposed are not practical, e.g., block- age of the RES to avoid RES uptake and distribution accord- ing to particle size following i.v. injection since this may lead

326 Burgess and Hickey

to undesirable blockage of vessels and as discussed above microspheres are not recommended for i.v. administration. The use of antibodies to direct microspheres has met with lim- ited success. Success has been achieved in targeting the lymph system according to size and surface characteristics. Direct injection at the site, e.g., intra-articular injection, to treat arthritic joints and i.m. or s.c. injections at the local site to treat localized infection and inflammation, have been successful. Targeting using an external magnetic field to localize microspheres has also been successful but this approach does not appear to be practical.

4. TARGETING DISEASES Many diseases are characterized by anatomically localized

abnormalities. In general, these are most suited to targeted con- trolled drug delivery. Diseases of a more diffuse and systemic nature are more readily treated with conventional dosage forms. Some cancers, infectious and hereditary diseases, may

be considered suitable for treatment with dispersed systems.

Pharmaceutical journals are replete with examples of the ‘‘next wave’’ of targeted drugs. As biological molecules such as peptides (90), proteins (56), and nucleic acids (38), with highly specific mechanisms of action, are discovered and their site of action and role in disease identified, they may act at the mole- cular level as immunological, endocrine or neurological med- iators or influence replication, translation, transcription or expression of genes. Development of new therapies depends upon delivery systems compatible with the drug and the purpose for which it is intended. Hence there are new delivery systems for antagonists for inflammatory mediators, cytokines, oligonucleotides, and vaccines (31,42).