Microencapsulation Methods for Delivery of Protein Drugs

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216 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4 100 ) e (% m 75 z y o s o f ly e 50 le a s e re u la tiv Q u m 25 0 3. Release of lysozyme from PEG-PBT microspheres. Mo- Fig. weight of PEG segment was 600. Polyvinyl alcohol lecular was used as a stabilizer in the external aqueous phase. (4%) percentage of PBT in PEG-PBT block copolymer Increasing resulted in slower release of lysozyme. From reference [16]. of glycolide in PLGA polymers result in rapid deg- tents of the polymer, resulting in faster release of the radation drugs. The release profile can also be controlled loaded the processing factors, such as shear forces, since by shear forces tend to produce smaller microparticles high release drugs at a faster rate due to higher surface which area and shorter diffusion path length. Limitations 0 10 20 30 40 50 Time (days) 23% 45% solvent evaporation/extraction methods have While used widely in preparing microparticles for pep- been delivery, the methods are still not ideal and tide/protein many aspects to be improved. First, the drug have efficiency into microspheres is not high. encapsulation protein drugs can be produced in larger scale Although to development of genetic engineering technique, due cost is still high. Thus, increasing the encapsulation the is still quite important for preparing micro- efficiency Second, the solvent evaporation/extraction spheres. require use of toxic organic solvents, such as methods and ethyl acetate, as a solvent for dis- dichloromethane biodegradable polymers. To meet the regulatory solving regarding the residual solvent in the final requirement the use of toxic organic solvent should be products, or avoided. Third, in most situations, the minimized proteins are released with the initial burst release, loaded in many situations the loaded proteins are not re- and completely. The initial burst release may cause leased response in patients due to abnormally high abnormal concentration in blood. Incomplete release of the drug proteins is often related to protein stability. Pro- loaded have to maintain their 3-dimensional conformateins to keep their bioactivity. Proteins, however, are tion prone to be denatured and to form aggregates by vari- factors, including exposure to large interface beous organic and aqueous phases, shear force in the tween steps (e.g., homogenization, and sonication), processing protein-polymer interaction. Some reports showed or the loss of protein activity or denaturation/ aggre- that of protein could be minimized by using stabilizgation such as poloxamer, BSA or hydroxypropyl-βers [13,14,18]. Fourth, although continuous cyclodextrin of peptide/protein drugs is desirable, there are release where pulsatile release is preferred as in the situations of insulin delivery. Furthermore, the timing and the case amount of the released insulin need to be con- exact carefully. This type of release can not be obtrolled by biodegradable microspheres prepared by soltained vent evaporation/extraction methods. Modifications of Solvent Extraction an effort to protect proteins from denaturation In microencapsulation processes, alternative methods during been developed to minimize exposure of protein have to denaturing conditions, such as high tempera- drugs ture and water-oil interfaces [19]. Spray-desolvation involves spraying a polymer solu- Spray-desolvation onto desolvating liquid. For example, microdroplets tion made by spraying PVA aqueous solution onto ace- were bath, where the polymer solvent (water) was extone into acetone and PVA precipitated to form solid tracted [20]. This method was also applied to microparticles peptides and BSA as model drugs in PLGA. encapsulate micronized drug was suspended in a PLGA-acetone The and atomized ultrasonically into ethanol bath, solution where microspheres were precipitated [21]. Solvent Extraction Cryogenic is dissolved in an organic solvent such as di- PLGA together with protein powders. As shown chloromethane, Fig. 4, the polymer/protein mixture is atomized over in bed of frozen ethanol overlaid with liquid nitrogen, at a temperature below the freezing point of the poly- a solution. The microdroplets freeze upon mer/protein the liquid nitrogen, then sink onto the fro- contacting ethanol layer. As the ethanol layer thaws, the mizen which are still frozen sink into the ethanol. croparticles the solvent in the microparticles Dichloromethane, thaws and is slowly extracted into ethanol, result- then in hardened microparticles containing proteins and ing matrix. polymer process was used to produce a microsphere for- This for human growth hormone (hGH) [23-26]. mulation to the encapsulation process, hGH is formulated Prior zinc to produce insoluble Zn:hGH complex. The with of this complex contributed to stabilize encapsulation during the fabrication process and within the mi- hGH after hydration [23,24]. An in vivo study of crospheres formulation demonstrated a lower Cmax and an ex- this serum level for weeks, but a similar bioavailabiltended as compared with the protein in solution [26]. The ity,
Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4 217 suspension Protein polymer solution in Liquid nitrogen Frozen microspheres 4. Schematic diagram of the ProLease Fig. ® encapsulation From reference [22]. process. formulation using this method (referred to as hGH ProLease ® was approved by the FDA in December 1999, ) an injectable suspension for once- or twice-a-month as under a brand name of Nutropin depot adminitration, ® . this process, all manipulations are performed at In temperatures, therefore thermal denaturation of low drugs can be prevented. There are no aqueous protein thus the protein is not subjected to oil-water phases, where some proteins may denature. In addi- interfaces the process utilizes solvents in which most protion, are insoluble (dichloromethane as a polymer solteins ethanol as an extraction solvent), therefore a high vent, efficiency can be achieved [22,27]. How- encapsulation this system is not easy to apply to various protein ever, The use of liquid nitrogen also makes scale-up drugs. difficult. The system still utilizes toxic organic rather and thus it has similar problems associated solvents, solvent evaporation/extraction methods. with PHASE SEPARATION (COACERVATION) Methods Nozzle Extraction solvent Atomized droplets of solvent Extraction microspheres from Drug particle Release modifier PLG microsphere by phase separation is basically a Microencapsulation process: (1) phase separation of the coating three-step to form coacervate droplets; (2) adsorption of polymer coacervate droplets onto the drug surface; and (3) the of the microcapsules [28], as shown in Fig. solidification Phase separation techniques can be classified accord- 5. to the method to induce phase separation; noning addition, temperature change, incompatible solvent or salt addition, and polymer-polymer interac- polymer [29]. tion Addition Non-solvent polymer to be coated is dissolved in a good sol- The and drug may be dissolved or suspended or emulvent, in the polymer solution. Then the first nonsified (also called ‘coacervating agent’ or ‘phase insolvent which is miscible with the good solvent but ducer’) not dissolve the polymer is slowly added to the does solution system. The polymer is concen- polymer-drug as the solvent for the polymer is slowly extracted trated the first non-solvent. The polymer is then induced into by Induced Non-solvent 1. Temperature change 2. Salt / Incompatible polymer 3. 4. Polymer-polymer interaction separation Phase the coating polymer of Drug particle of Adsorption droplets coacervate of Solidification microcapsules the 5. Schematic diagram of the formation of a coacervate Fig. From reference [30]. microcapsule. phase separate and form coacervate droplets that to the drug (Fig. 6(A)). The coacervate droplet size contain be controlled by adjusting the stirring speed. At this can of the process, the coacervates are usually too soft point be collected, and thus they are usually transferred to to large body of a second non-solvent (also called ‘hard- a agent’) to harden the microparticles [31]. ening polyesters, such as PLA and PLGA, are used, When widely used solvents are dichloromethane, ethyl most and acetonitrile. The first non-solvents for the acetate, are low-molecular weight liquid polybutadi- polymers low-molecular weight liquid methacrylic polymers, ene, oil, vegetable oil, and light liquid paraffine oils. silicone hydrocarbons, such as heptane, hexane, and Aliphatic ether, have been used as the second non- petroleum solvent. Change Temperature system comprised of a polymer and a solvent exists A a single phase at all points above the phase boundary. as is dispersed in the polymer solution with stirring. Drug the temperature is decreased below the curve (Fig. As phase separation of the dissolved polymer occurs 6(B)), the form of immiscible liquid and the polymer coa- in around the dispersed drug particles, thus forming lesces Further cooling accomplishes gelation microcapsules. solidification of the coating. The microcapsules are and from the solvent by filtration, decantation, or collected techniques [32]. centrifugation Polymer Addition or Salt Addition Incompatible two chemically different polymers dissolved in When common solvent are incompatible and do not mix in a phase separation takes place. This phenome- solution, is well described by the phase diagram of a ternary non consisting of a solvent, and two polymers, X system Y (Fig. 6(C)). A drug is dispersed in a solution of and Y (point a in Fig. 6(C)) and polymer X is added polymer the system, denoted by the arrowed line. When the to boundary is passed with the further addition of phase X, polymer rich phase begins to separate to polymer immiscible droplets containing the drug, and coa,- form
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