Protein delivery from polymeric matrices
Teekamp, Naomi
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Publication date: 2018
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Teekamp, N. (2018). Protein delivery from polymeric matrices: From pre-formulation stabilization studies to site-specific delivery. Rijksuniversiteit Groningen.
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04/03/2018 20:42STABILIZATION STRATEGIES FOR
POLYMER-BASED NANOPARTICLES
AND MICROPARTICLES FOR
PARENTERAL DELIVERY OF PEPTIDES
AND PROTEINS
AUTHORS Naomi Teekamp* Luisa F. Duque* Henderik W. Frijlink Wouter L.J. Hinrichs Peter Olinga*The authors contributed equally
Expert Opinion on Drug Delivery (2015) 12(8):1311-1331
STABILIZATION STRATEGIES FOR
POLYMER-BASED NANOPARTICLES
AND MICROPARTICLES FOR
PARENTERAL DELIVERY OF PEPTIDES
AND PROTEINS
AUTHORS Naomi Teekamp* Luisa F. Duque* Henderik W. Frijlink Wouter L.J. Hinrichs Peter Olinga*The authors contributed equally
Expert Opinion on Drug Delivery (2015) 12(8):1311-1331
16
ABSTRACT
Introduction: Therapeutic proteins and peptides often require parenteral administration, which compels frequent administration and patient discomfort; this ultimately decreases compliance and leads to therapy failure. Biocompatible and biodegradable polymers offer a versatile matrix for particles suitable for the parenteral delivery of these biomacromolecules, with the added possibility of long-term controlled release. During the past decade, research on polymeric micro- and nanoparticles as delivery vehicles has intensified; nevertheless, only few products have been commercialized.
Areas covered: This review discusses the different production techniques for micro- and nanoparticles suitable for peptide and protein delivery; including examples of recently developed formulations. Stability of the biomacromolecules related to these production techniques is evaluated, as it is a critical parameter to be considered during product development. Additionally, several strategies to improve stability are described in detail, providing insight and guidance for further formulation development.
Expert opinion: In the conventionally used and thoroughly investigated emulsification method stability of peptides and proteins is still a challenge. Emerging methods like solvent displacement, layer-by-layer polymer deposition, electrospraying and supercritical fluid technologies have the potential to improve stability of the protein and peptide. Nonetheless, these methods are still under development and they need critical evaluation to improve production efficiency before proceeding to in vivo efficacy studies. Improvement should be achieved by strengthening cooperation between academic research groups, pharmaceutical companies and regulatory authorities.
16
ABSTRACT
Introduction: Therapeutic proteins and peptides often require parenteral administration, which compels frequent administration and patient discomfort; this ultimately decreases compliance and leads to therapy failure. Biocompatible and biodegradable polymers offer a versatile matrix for particles suitable for the parenteral delivery of these biomacromolecules, with the added possibility of long-term controlled release. During the past decade, research on polymeric micro- and nanoparticles as delivery vehicles has intensified; nevertheless, only few products have been commercialized.
Areas covered: This review discusses the different production techniques for micro- and nanoparticles suitable for peptide and protein delivery; including examples of recently developed formulations. Stability of the biomacromolecules related to these production techniques is evaluated, as it is a critical parameter to be considered during product development. Additionally, several strategies to improve stability are described in detail, providing insight and guidance for further formulation development.
Expert opinion: In the conventionally used and thoroughly investigated emulsification method stability of peptides and proteins is still a challenge. Emerging methods like solvent displacement, layer-by-layer polymer deposition, electrospraying and supercritical fluid technologies have the potential to improve stability of the protein and peptide. Nonetheless, these methods are still under development and they need critical evaluation to improve production efficiency before proceeding to in vivo efficacy studies. Improvement should be achieved by strengthening cooperation between academic research groups, pharmaceutical companies and regulatory authorities.
17
2
INTRODUCTION
In the past decades developments in biochemistry and biology have resulted in an expansion of the number of drug candidates that have a protein or peptide structure. As the conventional oral administration route is not applicable for these compounds due to their instability and poor membrane permeability in the gastrointestinal tract and other non-invasive administration routes face similar challenges,1 these molecules are usually administered via the parenteral route.2,3 However, the rapid clearance from the system after injection impels frequent injection and consequently patient discomfort leading to poor compliance. To solve this problem several solid injectable protein and peptide formulations with a prolonged drug release have been introduced. However, many of these formulations cannot fulfill the requirement of less frequent administration due to an immediate or burst release, or poor stability preservation of the biomacromolecules. Consequently, there is a continuous search for novel formulations with wide applicability in which the biomacromolecule is stable. Formulations based on biocompatible and biodegradable polymers offer great potential for preserving protein and peptide integrity and decreasing the administration frequency. Natural and synthetic polymers offer a large versatility, as their properties can be tailored for specific applications in drug delivery. Despite the favorable properties of polymer-based formulations, the production processes employed to prepare those are often the main source of instability of peptides and proteins.
Currently, the research for parenteral drug delivery is mainly focused on nano- and micro-sized formulations. Already in 1984 the development of a peptide loaded microparticle formulation was described,4 yet only a few nano- and micro formulations have reached the market since then. In this review, we want to assess the progress, prospects and pitfalls that have been reported in literature related to protein and peptide delivery with nano- and micro-sized polymer based formulations with emphasis on the last 5 years. Recent examples of the different applied production techniques in this research field will be presented (Figure 2.1). Especially, the stability of proteins and peptides in relation to the production processes will be discussed in a separate section, along with several production process and formulation related strategies that have been recently described to improve the stability. The release characteristics of this type of formulations have recently been reviewed5 and are therefore not discussed here. Polymer based nano-, micro- and in-situ forming gels are also not described here, as these systems have been extensively described in recent reviews as well.6–8 The Expert
17
2
INTRODUCTION
In the past decades developments in biochemistry and biology have resulted in an expansion of the number of drug candidates that have a protein or peptide structure. As the conventional oral administration route is not applicable for these compounds due to their instability and poor membrane permeability in the gastrointestinal tract and other non-invasive administration routes face similar challenges,1 these molecules are usually administered via the parenteral route.2,3 However, the rapid clearance from the system after injection impels frequent injection and consequently patient discomfort leading to poor compliance. To solve this problem several solid injectable protein and peptide formulations with a prolonged drug release have been introduced. However, many of these formulations cannot fulfill the requirement of less frequent administration due to an immediate or burst release, or poor stability preservation of the biomacromolecules. Consequently, there is a continuous search for novel formulations with wide applicability in which the biomacromolecule is stable. Formulations based on biocompatible and biodegradable polymers offer great potential for preserving protein and peptide integrity and decreasing the administration frequency. Natural and synthetic polymers offer a large versatility, as their properties can be tailored for specific applications in drug delivery. Despite the favorable properties of polymer-based formulations, the production processes employed to prepare those are often the main source of instability of peptides and proteins.
Currently, the research for parenteral drug delivery is mainly focused on nano- and micro-sized formulations. Already in 1984 the development of a peptide loaded microparticle formulation was described,4 yet only a few nano- and micro formulations have reached the market since then. In this review, we want to assess the progress, prospects and pitfalls that have been reported in literature related to protein and peptide delivery with nano- and micro-sized polymer based formulations with emphasis on the last 5 years. Recent examples of the different applied production techniques in this research field will be presented (Figure 2.1). Especially, the stability of proteins and peptides in relation to the production processes will be discussed in a separate section, along with several production process and formulation related strategies that have been recently described to improve the stability. The release characteristics of this type of formulations have recently been reviewed5 and are therefore not discussed here. Polymer based nano-, micro- and in-situ forming gels are also not described here, as these systems have been extensively described in recent reviews as well.6–8 The Expert
18
Opinion will address the following issues: (i) which production techniques are suitable for preparation of nano- and/or microparticles that also decrease the risks of peptide or protein degradation; (ii) which are the perspectives of the applied approaches to improve the stability of proteins and peptides in polymer-based formulations; (iii) and whether the current nano- and microparticle formulations have potential for future success of protein and peptide parenteral delivery.
Production techniques
Several production techniques can be employed for the production of nano- and microparticles. The size ranges that can be achieved by these different production methods are depicted in Figure 2.1. The main principles of the production methods will be discussed in this section along with recent examples of the application of these methods for the encapsulation of peptides and proteins in polymeric nano- and microparticles. More elaborate information on the production techniques and formulation performance can be found elsewhere.1,9,10
FIGURE 2.1: Overview of size range of nano- and microparticles produced with different techniques, according to the examples in this review.
18
Opinion will address the following issues: (i) which production techniques are suitable for preparation of nano- and/or microparticles that also decrease the risks of peptide or protein degradation; (ii) which are the perspectives of the applied approaches to improve the stability of proteins and peptides in polymer-based formulations; (iii) and whether the current nano- and microparticle formulations have potential for future success of protein and peptide parenteral delivery.
Production techniques
Several production techniques can be employed for the production of nano- and microparticles. The size ranges that can be achieved by these different production methods are depicted in Figure 2.1. The main principles of the production methods will be discussed in this section along with recent examples of the application of these methods for the encapsulation of peptides and proteins in polymeric nano- and microparticles. More elaborate information on the production techniques and formulation performance can be found elsewhere.1,9,10
FIGURE 2.1: Overview of size range of nano- and microparticles produced with different techniques, according to the examples in this review.
19
2
Emulsification – solvent evaporation
Emulsification-solvent evaporation is the most frequently applied process for the manufacturing of polymeric nano- and microparticles containing proteins or peptides.
Figure 1.2 depicts the different types of emulsions that can be prepared depending on the physicochemical properties of the components and process conditions. The manufacturing route typically comprises the following steps:11–13
I. Primary dispersion: incorporation of the biomacromolecule into a solution of the polymer in an organic solvent, which is immiscible with water or ethanol. The peptide or protein can be added in the solid-state (S/O dispersion), as aqueous solution (W/O dispersion), as organic solution, when the used organic solvents form a single phase (Om/O solution), as organic dispersion, when the solvent containing the protein emulsifies into the other organic solvent (O/O dispersion), as emulsion (W/O/O dispersion) or as organic suspension (S/O/O dispersion). II. Secondary dispersion: emulsification of the primary dispersion or organic solution
with the external continuous phase, which is immiscible with the dispersed phase. III. Organic solvent removal: evaporation of the organic solvent at elevated
temperature and/or reduced pressure; or extraction of solvent by the continuous phase, which ultimately results in the formation of nano- or microparticles. IV. Harvest and drying: collection of the solidified particles by filtration or
centrifugation and subsequent drying by lyophilization or evaporation at reduced pressure.
Some examples of protein and peptide loaded nano- and microparticle formulations produced by emulsion-solvent techniques are listed in Tables 2.1 and 2.2, respectively. It is worth mentioning that while literature regarding protein-loaded particles mainly focuses on approaches to obtain formulations with optimal performance with regard to encapsulation efficiency (EE), release kinetics and protein integrity, publications related to peptide-loaded particles concentrate more on the mechanistic understanding of their performance. This may be related to the fact that various peptide-loaded polymer-based formulations have been approved by regulatory authorities and are already marketed, whereas protein-loaded particles are still under development.14,15 Although the emulsification-solvent evaporation process is the most frequently used technique for the preparation of nano- and microparticle formulations, large variations between the particle properties are encountered as described in Table
19
2
Emulsification – solvent evaporation
Emulsification-solvent evaporation is the most frequently applied process for the manufacturing of polymeric nano- and microparticles containing proteins or peptides.
Figure 1.2 depicts the different types of emulsions that can be prepared depending on the physicochemical properties of the components and process conditions. The manufacturing route typically comprises the following steps:11–13
I. Primary dispersion: incorporation of the biomacromolecule into a solution of the polymer in an organic solvent, which is immiscible with water or ethanol. The peptide or protein can be added in the solid-state (S/O dispersion), as aqueous solution (W/O dispersion), as organic solution, when the used organic solvents form a single phase (Om/O solution), as organic dispersion, when the solvent containing the protein emulsifies into the other organic solvent (O/O dispersion), as emulsion (W/O/O dispersion) or as organic suspension (S/O/O dispersion). II. Secondary dispersion: emulsification of the primary dispersion or organic solution
with the external continuous phase, which is immiscible with the dispersed phase. III. Organic solvent removal: evaporation of the organic solvent at elevated
temperature and/or reduced pressure; or extraction of solvent by the continuous phase, which ultimately results in the formation of nano- or microparticles. IV. Harvest and drying: collection of the solidified particles by filtration or
centrifugation and subsequent drying by lyophilization or evaporation at reduced pressure.
Some examples of protein and peptide loaded nano- and microparticle formulations produced by emulsion-solvent techniques are listed in Tables 2.1 and 2.2, respectively. It is worth mentioning that while literature regarding protein-loaded particles mainly focuses on approaches to obtain formulations with optimal performance with regard to encapsulation efficiency (EE), release kinetics and protein integrity, publications related to peptide-loaded particles concentrate more on the mechanistic understanding of their performance. This may be related to the fact that various peptide-loaded polymer-based formulations have been approved by regulatory authorities and are already marketed, whereas protein-loaded particles are still under development.14,15 Although the emulsification-solvent evaporation process is the most frequently used technique for the preparation of nano- and microparticle formulations, large variations between the particle properties are encountered as described in Table
20
1.1 and Table 1.2. The performance of the formulation depends on many factors including: emulsion type, type of organic solvents and co-solvents, process conditions, type of polymer carrier and inclusion, amount and type of co-excipients.
FIGURE 2.2: Type of single and multiple, emulsions employed for preparation of nano-and microparticles. Performance parameters: EE -> Poor <50%; Fair: >50% & <75%; Good: >75%. Burst release -> Low <25%; Fair ≤35%; High >35%. Recovery upon release -> Poor <60%; Fair ≥60% & <85%; Good ≥85%.15,24,87,92
E, ethanolic phase; O, organic phase; Oi, organic phase immiscible with aqueous phase; Om, organic phase miscible with aqueous phase; S, solid phase; W, aqueous phase.
Spray drying and electrospraying
Spray drying is a simple technique to produce dry microparticles. Other advantages of this technique include the possibility of a continuous process, its cost-effectiveness and the controllability of the product.10 In the process of spray drying, a solution of the drug is pumped through a nozzle, producing an aerosol which is then dried by
20
1.1 and Table 1.2. The performance of the formulation depends on many factors including: emulsion type, type of organic solvents and co-solvents, process conditions, type of polymer carrier and inclusion, amount and type of co-excipients.
FIGURE 2.2: Type of single and multiple, emulsions employed for preparation of nano-and microparticles. Performance parameters: EE -> Poor <50%; Fair: >50% & <75%; Good: >75%. Burst release -> Low <25%; Fair ≤35%; High >35%. Recovery upon release -> Poor <60%; Fair ≥60% & <85%; Good ≥85%.15,24,87,92
E, ethanolic phase; O, organic phase; Oi, organic phase immiscible with aqueous phase; Om, organic phase miscible with aqueous phase; S, solid phase; W, aqueous phase.
Spray drying and electrospraying
Spray drying is a simple technique to produce dry microparticles. Other advantages of this technique include the possibility of a continuous process, its cost-effectiveness and the controllability of the product.10 In the process of spray drying, a solution of the drug is pumped through a nozzle, producing an aerosol which is then dried by
21
2
a heated gas.10 The dry particles are subsequently separated from the drying gas and guided into a collection vessel e.g. using a cyclone. EE’s up to 99% can be achieved with this method.16 The size of the spray dried particles, generally between 0.5 and 5.5 μm (volume average) (see Figure 2.1), depends on several factors, including viscosity and surface tension of the solution, nozzle type, the atomizing air-flow, the flow rate during spraying and concentration of the sprayed solution.10,17 Two basic nozzles can be distinguished: the 2-fluid nozzle, with the peptide or protein dissolved or suspended in the polymer solution in one channel and atomizing air in the other, and the 3-fluid nozzle, where an aqueous peptide or protein solution is in a separate channel from the (organic) polymer solution and atomizing air in the third channel. Next to these basic nozzle types, several other types, like ultrasonic or hydraulic nozzles,18 can be used. To minimize residual solvents and increase the yield, the drying gas is usually heated to 60-120°C.
When spray drying protein and polymer with a 3-fluid nozzle, the choice of the organic solvent has a significant influence on protein encapsulation and drying kinetics. Important factors are the vapor pressure, surface tension and the miscibility of the aqueous phase with the organic solvent.10,19 In addition, the toxicity of these solvents should also be considered, as a residual amount could be still present after drying; immiscible organic solvents are generally more toxic than organic solvents miscible with the aqueous phase.
A technique to produce nano- and microparticles, which resembles conventional spray drying, is electrohydrodynamic spraying or electrospraying. With this technique a voltage of several kilovolts is applied to the nozzle, charging the solution. The high voltage overcomes the surface tension at the interface of the spraying capillary, generating a Taylor cone.20 Applying a higher voltage will cause the tip of the cone to break into small, highly charged droplets, which are directed to the collection surface or non-solvent of opposite charge. The distance to the collection surface is relevant for the size of the particles, since the particles will shrink due to evaporation of solvent during traveling through the gas phase. Next to the above mentioned factors, the size of electrosprayed particles also depends on the conductivity of the solvents. In electrospraying a coaxial nozzle, a specific type of the 3-way nozzle consisting of a core and annular fluid channel, is frequently used, which offers the possibility to produce core/shell particles more efficiently.21,22 Furthermore, effective encapsulation of protein by coaxial electrospraying has been demonstrated.23,24 Electrospraying of
21
2
a heated gas.10 The dry particles are subsequently separated from the drying gas and guided into a collection vessel e.g. using a cyclone. EE’s up to 99% can be achieved with this method.16 The size of the spray dried particles, generally between 0.5 and 5.5 μm (volume average) (see Figure 2.1), depends on several factors, including viscosity and surface tension of the solution, nozzle type, the atomizing air-flow, the flow rate during spraying and concentration of the sprayed solution.10,17 Two basic nozzles can be distinguished: the 2-fluid nozzle, with the peptide or protein dissolved or suspended in the polymer solution in one channel and atomizing air in the other, and the 3-fluid nozzle, where an aqueous peptide or protein solution is in a separate channel from the (organic) polymer solution and atomizing air in the third channel. Next to these basic nozzle types, several other types, like ultrasonic or hydraulic nozzles,18 can be used. To minimize residual solvents and increase the yield, the drying gas is usually heated to 60-120°C.
When spray drying protein and polymer with a 3-fluid nozzle, the choice of the organic solvent has a significant influence on protein encapsulation and drying kinetics. Important factors are the vapor pressure, surface tension and the miscibility of the aqueous phase with the organic solvent.10,19 In addition, the toxicity of these solvents should also be considered, as a residual amount could be still present after drying; immiscible organic solvents are generally more toxic than organic solvents miscible with the aqueous phase.
A technique to produce nano- and microparticles, which resembles conventional spray drying, is electrohydrodynamic spraying or electrospraying. With this technique a voltage of several kilovolts is applied to the nozzle, charging the solution. The high voltage overcomes the surface tension at the interface of the spraying capillary, generating a Taylor cone.20 Applying a higher voltage will cause the tip of the cone to break into small, highly charged droplets, which are directed to the collection surface or non-solvent of opposite charge. The distance to the collection surface is relevant for the size of the particles, since the particles will shrink due to evaporation of solvent during traveling through the gas phase. Next to the above mentioned factors, the size of electrosprayed particles also depends on the conductivity of the solvents. In electrospraying a coaxial nozzle, a specific type of the 3-way nozzle consisting of a core and annular fluid channel, is frequently used, which offers the possibility to produce core/shell particles more efficiently.21,22 Furthermore, effective encapsulation of protein by coaxial electrospraying has been demonstrated.23,24 Electrospraying of
22
emulsions is also suitable for core/shell particle production, provided that the polymer deposits at the oil/water interface, as was shown by Wu et al. for poly(ε-caprolactone)-polyamino-ethyl ethylene phosphate/BSA microparticles.25
Solvent displacement (nanoprecipitation)
Solvent displacement or nanoprecipitation is a simple technique to produce nanoparticles. The technique involves precipitation of a polymer, drug and, optionally, other excipients, dissolved in an organic solvent, when slowly added to a non-solvent, usually water, under continuous intense stirring.26,27
Ethanol and acetone, due to their polarity and miscibility with water, are often used to dissolve the polymer, peptide or protein, and a stabilizer (e.g. a surfactant). When the organic solution is slowly poured or injected into the intensively stirred, surfactant-stabilized aqueous phase, polymer will deposit at the interface between the aqueous and organic phase, due to the rapid solvent diffusion. The particles are obtained by evaporation of both the solvent and the non-solvent under reduced pressure.26,27 The final particle size that can be obtained is typically around 100-300 nm, with narrow size distributions.26,28 The organic solvent used, the ratio organic: aqueous solvent and the temperature are factors that can be varied to tailor the size.29
Depending on the type of polymer used, different nanoparticle systems can be formed upon precipitation. Amphiphilic copolymers will form micelles in which the biomacromolecules are embedded in the hydrophilic parts of the polymer,30 whereas polymers that are not amphiphilic will precipitate around a droplet to form a core/ shell structure, encapsulating the biomacromolecules.26
As proteins and peptides (partly) dissolve in the aqueous phase upon addition of the organic phase to the aqueous phase, the EE of the biomacromolecules is generally poor. However, some modifications to the method enable quite efficient encapsulation in polymers by solvent displacement, albeit the EE is not as high as with other methods. For example in a study by Ali et al.,31 PEG 400 was used as the organic phase for the preparation of Eudragit RL-BSA core/shell structured nanoparticles,31 which resulted in an EE of around 70%. In another study, hyperbranched poly(amine-ester) (HPAE)-co-PLA nanoparticles with BSA encapsulated were produced by dissolving the protein in the aqueous phase instead of the organic phase,32 yielding an EE of around 60%. A similar EE was found for BSA and trypsin encapsulated in lactosylated PLGA by
22
emulsions is also suitable for core/shell particle production, provided that the polymer deposits at the oil/water interface, as was shown by Wu et al. for poly(ε-caprolactone)-polyamino-ethyl ethylene phosphate/BSA microparticles.25
Solvent displacement (nanoprecipitation)
Solvent displacement or nanoprecipitation is a simple technique to produce nanoparticles. The technique involves precipitation of a polymer, drug and, optionally, other excipients, dissolved in an organic solvent, when slowly added to a non-solvent, usually water, under continuous intense stirring.26,27
Ethanol and acetone, due to their polarity and miscibility with water, are often used to dissolve the polymer, peptide or protein, and a stabilizer (e.g. a surfactant). When the organic solution is slowly poured or injected into the intensively stirred, surfactant-stabilized aqueous phase, polymer will deposit at the interface between the aqueous and organic phase, due to the rapid solvent diffusion. The particles are obtained by evaporation of both the solvent and the non-solvent under reduced pressure.26,27 The final particle size that can be obtained is typically around 100-300 nm, with narrow size distributions.26,28 The organic solvent used, the ratio organic: aqueous solvent and the temperature are factors that can be varied to tailor the size.29
Depending on the type of polymer used, different nanoparticle systems can be formed upon precipitation. Amphiphilic copolymers will form micelles in which the biomacromolecules are embedded in the hydrophilic parts of the polymer,30 whereas polymers that are not amphiphilic will precipitate around a droplet to form a core/ shell structure, encapsulating the biomacromolecules.26
As proteins and peptides (partly) dissolve in the aqueous phase upon addition of the organic phase to the aqueous phase, the EE of the biomacromolecules is generally poor. However, some modifications to the method enable quite efficient encapsulation in polymers by solvent displacement, albeit the EE is not as high as with other methods. For example in a study by Ali et al.,31 PEG 400 was used as the organic phase for the preparation of Eudragit RL-BSA core/shell structured nanoparticles,31 which resulted in an EE of around 70%. In another study, hyperbranched poly(amine-ester) (HPAE)-co-PLA nanoparticles with BSA encapsulated were produced by dissolving the protein in the aqueous phase instead of the organic phase,32 yielding an EE of around 60%. A similar EE was found for BSA and trypsin encapsulated in lactosylated PLGA by
23
2
a comparable process. By the addition of ε-polylysine, a cationic polyamide, to the formulation, the EE of the negatively charged BSA increased to 75%, but decreased to 30% for the positively charged trypsin probably due to electrical repulsion.33
While the solvent displacement technique can be used to produce nanoparticles with a narrow size distribution, the solubility of proteins in the aqueous phase complicates the production of a satisfactory formulation that has a controlled and reproducible drug load.
Self-assembly systems – Micelles and Polymersomes
Self-assembly systems are formed by weak non-covalent interactions between molecules of the carrier matrix (e.g. block co-polymers), which lead to a specific structural organization. Amphiphilic block copolymers are frequently used in the formation of these systems by assembling into a more organized structure spontaneously or in response to an exogenous stimulus, such as temperature or pH. Micelles and polymersomes are typical structures formed by a self-assembly process. Micelles consist of a hydrophobic inner core surrounded by an hydrophilic outer shell, whereas polymersomes are spherical vesicles that have a polymeric lamellar structure (bi-layer membrane) surrounding an aqueous core.34,35
Due to their amphiphilic characteristics, hydrophilic compounds, such as peptides and proteins, can be covalently attached to the outer surface of the polymersome, linked to specific regions of the polymer backbone or encapsulated in the core of a polymersome. These two interactions have been specially investigated for drug targeting and intracellular delivery; but since no protein sustained delivery is involved they are out of this review’s scope.36–42 Conversely, encapsulation of biomacromolecules in the core of polymersomes has been described for both drug targeting and sustained drug release.35,43–46 Proteins can be directly encapsulated within the polymersome during self-assembly by solvent-switching techniques or by rehydration of a polymer film with the protein/peptide solution.43,47,48 Properties of this system can be tailored as to modify membrane thickness, permeability and release properties or to tune response to stimuli.35 For example, Kim et al. 44 described a polymersome system based on polyboroxole block copolymers in which the release of the encapsulated insulin was triggered by presence of monosaccharide in the environment.44 Using the same trigger system, phenylboronic acid containing polymers with different compositions of the amphiphilic polymer, were used as delivery carriers for insulin.45,46 A drawback of this
23
2
a comparable process. By the addition of ε-polylysine, a cationic polyamide, to the formulation, the EE of the negatively charged BSA increased to 75%, but decreased to 30% for the positively charged trypsin probably due to electrical repulsion.33
While the solvent displacement technique can be used to produce nanoparticles with a narrow size distribution, the solubility of proteins in the aqueous phase complicates the production of a satisfactory formulation that has a controlled and reproducible drug load.
Self-assembly systems – Micelles and Polymersomes
Self-assembly systems are formed by weak non-covalent interactions between molecules of the carrier matrix (e.g. block co-polymers), which lead to a specific structural organization. Amphiphilic block copolymers are frequently used in the formation of these systems by assembling into a more organized structure spontaneously or in response to an exogenous stimulus, such as temperature or pH. Micelles and polymersomes are typical structures formed by a self-assembly process. Micelles consist of a hydrophobic inner core surrounded by an hydrophilic outer shell, whereas polymersomes are spherical vesicles that have a polymeric lamellar structure (bi-layer membrane) surrounding an aqueous core.34,35
Due to their amphiphilic characteristics, hydrophilic compounds, such as peptides and proteins, can be covalently attached to the outer surface of the polymersome, linked to specific regions of the polymer backbone or encapsulated in the core of a polymersome. These two interactions have been specially investigated for drug targeting and intracellular delivery; but since no protein sustained delivery is involved they are out of this review’s scope.36–42 Conversely, encapsulation of biomacromolecules in the core of polymersomes has been described for both drug targeting and sustained drug release.35,43–46 Proteins can be directly encapsulated within the polymersome during self-assembly by solvent-switching techniques or by rehydration of a polymer film with the protein/peptide solution.43,47,48 Properties of this system can be tailored as to modify membrane thickness, permeability and release properties or to tune response to stimuli.35 For example, Kim et al. 44 described a polymersome system based on polyboroxole block copolymers in which the release of the encapsulated insulin was triggered by presence of monosaccharide in the environment.44 Using the same trigger system, phenylboronic acid containing polymers with different compositions of the amphiphilic polymer, were used as delivery carriers for insulin.45,46 A drawback of this
24
drug delivery system is the low EE, mainly due to low diffusion of the peptide/protein to the inner core of the polymersome or poor entrapment efficiency of these molecules during self-assembly.43
A special feature of the polymersomes is their ability to co-encapsulate active compounds with different hydrophilicity due to their amphiphilic character. For example, Qiao et al.49 utilized this attribute by using an acid-responsive system that successfully entrapped lysozyme as well as doxorubicin.49 Further improvement of this type of formulation could lead to an elegant therapy where two compounds can be loaded to achieve a complementary pharmacological effect.
Self assembly Systems: Layer-by-layer
Polymeric multilayer capsules can be produced in a simple but lengthy process known as the layer-by-layer (LbL) technique. The capsules are assembled by coating a sacrificial template particle several times, while alternating between two different polymer coating solutions. After application of 1-10 bilayers the template particle is removed.50,51
The alternating layers of the capsule can be linked by different interactions including hydrogen bonding, covalent bonding or electrostatic interactions. The latter is most common in drug delivery systems, where the layers consist of oppositely charged polyelectrolytes.50,51 Examples of charged polymers that are suitable for LbL preparation of micro- and nanocapsules are chitosan (cationic) and sodium alginate (anionic).52–54 Biomacromolecules can be incorporated by preloading the template particle or by postloading the hollow capsules. The latter is done by reversibly increasing the permeability of the capsule layers. For polyelectrolytes, a change in pH can be sufficient to make the capsule more permeable to macromolecules; (de)protonation within the layers creates electrostatic repulsion, which disturbs the layer conformation. A gradient into the capsule can be created by (electrostatic) interaction of the protein or peptide with the most inner layer51 or with excipients present in the sacrificial template.55 After restoring the pH to the initial value, the integrity of the capsules will be recovered, encapsulating the biomacromolecules.51,55 Changing the permeability by pH may seem an elegant way to postload the particles, however, drastic pH changes can also damage proteins and peptides. Moreover, postloading generally does not result in a high EE, as can be expected from a mainly diffusion controlled process. To smuggle away the
24
drug delivery system is the low EE, mainly due to low diffusion of the peptide/protein to the inner core of the polymersome or poor entrapment efficiency of these molecules during self-assembly.43
A special feature of the polymersomes is their ability to co-encapsulate active compounds with different hydrophilicity due to their amphiphilic character. For example, Qiao et al.49 utilized this attribute by using an acid-responsive system that successfully entrapped lysozyme as well as doxorubicin.49 Further improvement of this type of formulation could lead to an elegant therapy where two compounds can be loaded to achieve a complementary pharmacological effect.
Self assembly Systems: Layer-by-layer
Polymeric multilayer capsules can be produced in a simple but lengthy process known as the layer-by-layer (LbL) technique. The capsules are assembled by coating a sacrificial template particle several times, while alternating between two different polymer coating solutions. After application of 1-10 bilayers the template particle is removed.50,51
The alternating layers of the capsule can be linked by different interactions including hydrogen bonding, covalent bonding or electrostatic interactions. The latter is most common in drug delivery systems, where the layers consist of oppositely charged polyelectrolytes.50,51 Examples of charged polymers that are suitable for LbL preparation of micro- and nanocapsules are chitosan (cationic) and sodium alginate (anionic).52–54 Biomacromolecules can be incorporated by preloading the template particle or by postloading the hollow capsules. The latter is done by reversibly increasing the permeability of the capsule layers. For polyelectrolytes, a change in pH can be sufficient to make the capsule more permeable to macromolecules; (de)protonation within the layers creates electrostatic repulsion, which disturbs the layer conformation. A gradient into the capsule can be created by (electrostatic) interaction of the protein or peptide with the most inner layer51 or with excipients present in the sacrificial template.55 After restoring the pH to the initial value, the integrity of the capsules will be recovered, encapsulating the biomacromolecules.51,55 Changing the permeability by pH may seem an elegant way to postload the particles, however, drastic pH changes can also damage proteins and peptides. Moreover, postloading generally does not result in a high EE, as can be expected from a mainly diffusion controlled process. To smuggle away the
25
2
low EE’s, the loading capacity is often used as a parameter instead, making the poor production process efficacy less transparent. When the EE is provided, the inefficiency of the process becomes evident. E.g. sodium carboxymethylcellulose/poly(allylamine hydrochloride) capsules postloaded in an optimized process with BSA, had a maximal EE of only 10%.55
Preloading of proteins and peptides is preferably performed with CaCO3 template particles, since they allow for relatively mild process conditions. Loading can simply be done by co-precipitation during production of the CaCO3 particles or (less efficiently) by diffusion into the porous particle structure. Removal of CaCO3 after applying the polymer layers is accomplished by complexating Ca2+ with EDTA.51 EE's that can be achieved in CaCO3 particles by co-precipitation are >90%. However, upon application of the layers and the removal of CaCO3, a substantial amount of protein is lost from the capsule (15% to 90%) depending on process conditions, amount of layers and protein size.53,56 This loss is probably caused by competition for binding places on the surface of the sacrificial particle upon applying the first polyelectrolyte layer and the increased permeability during removal of the sacrificial particle.56 Furthermore, CaCO3 particles are less suitable for loading positively charged proteins, possibly due to electrostatic repulsion by the positively charged surface of the CaCO3 particles.56 An elegant example is given by Costa et al.;53 CaCO
3 particles preloaded with BSA were encapsulated in chitosan/elastin-like recombinamer microcapsules, which resulted in a high EE of about 75%. The authors mentioned some protein loss during application of the layers, yet it was limited by using the preloading approach.53
Another material that can be used for the sacrificial particle is mesoporous silica. However, the cytotoxicity of silica itself and the harmful solvents needed to remove it discourages its use; furthermore, the removal of both may not be complete.51
In contrast to postloading, which shows very low EE's and high burst release, preloading of biomacromolecules seems less problematic for encapsulation and release. However, the protein loss during layer application poses a problem, since these critical steps in the production process do not allow much tailoring. Moreover, little is known about the effect of the LbL process on the stability of proteins and peptides.
25
2
low EE’s, the loading capacity is often used as a parameter instead, making the poor production process efficacy less transparent. When the EE is provided, the inefficiency of the process becomes evident. E.g. sodium carboxymethylcellulose/poly(allylamine hydrochloride) capsules postloaded in an optimized process with BSA, had a maximal EE of only 10%.55
Preloading of proteins and peptides is preferably performed with CaCO3 template particles, since they allow for relatively mild process conditions. Loading can simply be done by co-precipitation during production of the CaCO3 particles or (less efficiently) by diffusion into the porous particle structure. Removal of CaCO3 after applying the polymer layers is accomplished by complexating Ca2+ with EDTA.51 EE's that can be achieved in CaCO3 particles by co-precipitation are >90%. However, upon application of the layers and the removal of CaCO3, a substantial amount of protein is lost from the capsule (15% to 90%) depending on process conditions, amount of layers and protein size.53,56 This loss is probably caused by competition for binding places on the surface of the sacrificial particle upon applying the first polyelectrolyte layer and the increased permeability during removal of the sacrificial particle.56 Furthermore, CaCO3 particles are less suitable for loading positively charged proteins, possibly due to electrostatic repulsion by the positively charged surface of the CaCO3 particles.56 An elegant example is given by Costa et al.;53 CaCO
3 particles preloaded with BSA were encapsulated in chitosan/elastin-like recombinamer microcapsules, which resulted in a high EE of about 75%. The authors mentioned some protein loss during application of the layers, yet it was limited by using the preloading approach.53
Another material that can be used for the sacrificial particle is mesoporous silica. However, the cytotoxicity of silica itself and the harmful solvents needed to remove it discourages its use; furthermore, the removal of both may not be complete.51
In contrast to postloading, which shows very low EE's and high burst release, preloading of biomacromolecules seems less problematic for encapsulation and release. However, the protein loss during layer application poses a problem, since these critical steps in the production process do not allow much tailoring. Moreover, little is known about the effect of the LbL process on the stability of proteins and peptides.
26
Supercritical fluids
Super critical carbon dioxide (scCO2) has the ability to dissolve certain polymers. Because of the relative low critical temperature and critical pressure of CO2, i.e. 31.1°C at 73.8 bar, respectively, mild process conditions can be employed. This is the main reason to use supercritical fluid technology with CO2 instead of processes that involve organic solvents during the preparation of microparticles.57–59 However, only few examples have been reported on the use of this technology for the preparation of protein and peptide loaded microparticles.60–62 Generally, rapid expansion of supercritical solution (RESS) is used for particle formation. In this process fine particles are formed using the supercritical fluid as a solvent.57,63 First the polymer and peptide/protein are dissolved in scCO2, followed by atomization and depressurization of the mixture through a nozzle into a lower pressure environment. Thus, particles are formed due to the rapid decompression of the solution.57,60 Process parameters, such as temperature, pressure, nozzle diameter, and composition of the polymeric mixture affect the formulation performance characteristics.57 Recombinant human Growth Hormone (rhGH) and tetanous toxoid have been encapsulated in poly(lactic-co-glycolic acid)/ poly(lactic acid) (PLGA/PLA) and PLA matrices, respectively. Particles with overall good encapsulation efficiency (>78%), preserved protein integrity and bioactivity, showed the suitability of this process for encapsulation of biomacromolecules.60,61 Also other process employing scCO2 such as the so-called gas antisolvent process and supercritical antisolvent process have been evaluated for their suitability in the drug delivery field.58 However, the use of co-solvents that may affect protein integrity and processabilty of the polymer during manufacturing with these processes could hamper the further development of these techniques.57,58 Advances on understanding the mechanisms of particle formation during these methods could serve as base for further successful formulation development as recently reported by Montes et al..63
26
Supercritical fluids
Super critical carbon dioxide (scCO2) has the ability to dissolve certain polymers. Because of the relative low critical temperature and critical pressure of CO2, i.e. 31.1°C at 73.8 bar, respectively, mild process conditions can be employed. This is the main reason to use supercritical fluid technology with CO2 instead of processes that involve organic solvents during the preparation of microparticles.57–59 However, only few examples have been reported on the use of this technology for the preparation of protein and peptide loaded microparticles.60–62 Generally, rapid expansion of supercritical solution (RESS) is used for particle formation. In this process fine particles are formed using the supercritical fluid as a solvent.57,63 First the polymer and peptide/protein are dissolved in scCO2, followed by atomization and depressurization of the mixture through a nozzle into a lower pressure environment. Thus, particles are formed due to the rapid decompression of the solution.57,60 Process parameters, such as temperature, pressure, nozzle diameter, and composition of the polymeric mixture affect the formulation performance characteristics.57 Recombinant human Growth Hormone (rhGH) and tetanous toxoid have been encapsulated in poly(lactic-co-glycolic acid)/ poly(lactic acid) (PLGA/PLA) and PLA matrices, respectively. Particles with overall good encapsulation efficiency (>78%), preserved protein integrity and bioactivity, showed the suitability of this process for encapsulation of biomacromolecules.60,61 Also other process employing scCO2 such as the so-called gas antisolvent process and supercritical antisolvent process have been evaluated for their suitability in the drug delivery field.58 However, the use of co-solvents that may affect protein integrity and processabilty of the polymer during manufacturing with these processes could hamper the further development of these techniques.57,58 Advances on understanding the mechanisms of particle formation during these methods could serve as base for further successful formulation development as recently reported by Montes et al..63
27
2
TA BL E 2.1: Over view of protein-loaded nano- and micr
opar
ticles pr
epar
ed via emulsification-solvent evapor
ation Emulsion type Pr otein [loading] Polymer [composition; MW] A ver age par ticle size (μm) EE (%) Burst release (%) In vitr o
release time investigated
Amount of pr
otein
released during time of in
vestigation (%) Refer ence Micropar ticles W/O/W BSA [1 2.1 5%] rhEGF [0.02%] PL GA [LA:GA 50:50, fr ee
carboxylic acid end; 1
3.6 kDa] 6 6 83 97 16 13 3 weeks 3 weeks 40 36 10 0 W/O/W rhGH [6%] mPEG-PLA copolymer [2 0 kDa -2 0 kDa] (1:9) 6 81 <25 66 day s 80 91 W/O/W Bee venom 50 mg/mL PL GA (fr
ee carboxylic acid end;
34 kDa) 17 75 <30 70 day s 10 0 101 W/O/W PEG-TRAIL [0.1%] TRAIL [0.1%] PL GA [LA:GA 50:50, fr ee
carboxylic acid end; IV 0.4
dl/g] 19 14 84 40 <20 45 4 weeks 14 day s 80 50 94 W/O/W HBsAg 2.2 4mg/ mL + T rehalose (1:2 weight r atio) PCL [65 kDa] 5 55 <10 6 months 84 96 W/O/W Ly s VEGF Fg F Mixtur e of PL GA [LA:GA 50:50; ester end-capped; 5 1 kDa], HDS, MgCO 3 , and tr ehalose (9:1 , w/w) NR 97 73 87 <30 <30 ND 55 day s 42 day s ND 80 72 ND 84 W/O/W Etaner cept MPEG-PCL -MPEG copolymer
[MPEG 2 kDa; PCL 65 KDa]
5 76 <15 90 day s ~70 10 3 S/O/W IFN α-2b (incorpor ated in 5 µm gelatin micr opar ticles) [2 00000IU] Mixtur e of PEGT+PBT [7 0/30;
PEG; 600 Da] and PL
GA [LA:GA 50:50; 1 0 kDa] (9:1 , w/w) 30 86 17 23 day s 83 71 S/O/W Spr ay -dried IgG:mannitol (1:1 2) [5%] PL
GA [LA:GA 50:50, ester
end-capped; IV 0.4 dg/l] 92 50 52 10 weeks 10 0 86 27
2
TA BL E 2.1: Over view of protein-loaded nano- and micr
opar
ticles pr
epar
ed via emulsification-solvent evapor
ation Emulsion type Pr otein [loading] Polymer [composition; MW] A ver age par ticle size (μm) EE (%) Burst release (%) In vitr o
release time investigated
Amount of pr
otein
released during time of in
vestigation (%) Refer ence Micropar ticles W/O/W BSA [1 2.1 5%] rhEGF [0.02%] PL GA [LA:GA 50:50, fr ee
carboxylic acid end; 1
3.6 kDa] 6 6 83 97 16 13 3 weeks 3 weeks 40 36 10 0 W/O/W rhGH [6%] mPEG-PLA copolymer [2 0 kDa -2 0 kDa] (1:9) 6 81 <25 66 day s 80 91 W/O/W Bee venom 50 mg/mL PL GA (fr
ee carboxylic acid end;
34 kDa) 17 75 <30 70 day s 10 0 101 W/O/W PEG-TRAIL [0.1%] TRAIL [0.1%] PL GA [LA:GA 50:50, fr ee
carboxylic acid end; IV 0.4
dl/g] 19 14 84 40 <20 45 4 weeks 14 day s 80 50 94 W/O/W HBsAg 2.2 4mg/ mL + T rehalose (1:2 weight r atio) PCL [65 kDa] 5 55 <10 6 months 84 96 W/O/W Ly s VEGF Fg F Mixtur e of PL GA [LA:GA 50:50; ester end-capped; 5 1 kDa], HDS, MgCO 3 , and tr ehalose (9:1 , w/w) NR 97 73 87 <30 <30 ND 55 day s 42 day s ND 80 72 ND 84 W/O/W Etaner cept MPEG-PCL -MPEG copolymer
[MPEG 2 kDa; PCL 65 KDa]
5 76 <15 90 day s ~70 10 3 S/O/W IFN α-2b (incorpor ated in 5 µm gelatin micr opar ticles) [2 00000IU] Mixtur e of PEGT+PBT [7 0/30;
PEG; 600 Da] and PL
GA [LA:GA 50:50; 1 0 kDa] (9:1 , w/w) 30 86 17 23 day s 83 71 S/O/W Spr ay -dried IgG:mannitol (1:1 2) [5%] PL
GA [LA:GA 50:50, ester
end-capped; IV 0.4 dg/l] 92 50 52 10 weeks 10 0 86
28 Emulsion type Pr otein [loading] Polymer [composition; MW] A ver age par ticle size (μm) EE (%) Burst release (%) In vitr o
release time investigated
Amount of pr
otein
released during time of in
vestigation (%) Refer ence Micropar ticles S/O/W rh-EPO:HSA [0.02 2% and 0.2 2] PL GA [LA:GA 7 5:2 5, fr ee
carboxylic acid end; 2
3kDa] 63 85 20 32 day s >90 85 S/O/W Ly s:P oloxamer 188 (1:1 0) (af ter pr epar ation of solid par ticles) [0.6%] PL GA-PEG-PL GA copolymer [LA:GA 2 5:50; PEG 9%] 60 73 10 45 day s <50 92 S/O/ O/Wh BSA (incorpor ated in dextr an micr opar ticles) [5%] Mixtur e of PL GA [LA:GA 50:50,
free carboxylic acid end; 40-7
5 kDa] and PLA [40-7 5 kDa] (40:60) 67 66 <15 60 day s >95 88,8 9 S/O/ O/Eh BSA (incorpor ated in dextr an micr opar ticles) [1 0%] Mixtur e of PL GA [LA:GA 50:50,
free carboxylic acid end; 40-7
5 kDa] and PLA [40-7 5 kDa] (40:60) 67 92 <20 65 day s >80 87, 89 W/O/ O/W O VA [1 .2 5%] Mixtur e of PL GA [LA:GA 50:50,
ester end-capped; IV 0.2 dg/l] and PL
GA [LA:GA 8 5/1 5, ester end-capped] (1:2) 34 101 12 63 day s >70 11 0 Misc. (W/O/W + gel) VEGF [5 µg/mL] PL GA [LA:GA 50:50, fr ee
carboxylic acid end; 1
0 kDa]
2.7
92
<20 (>30 in combination with Ang-1)
28 day s 100 (>90 in combination with Ang-1) 10 4 Ang-1 [5 µg/mL] 3 88 <15 (<1 0 in
combination with VEGF)
28 day
s
<90 (>90 in
combination with VEGF)
TA BL E 2.1: (continued) 28 Emulsion type Pr otein [loading] Polymer [composition; MW] A ver age par ticle size (μm) EE (%) Burst release (%) In vitr o
release time investigated
Amount of pr
otein
released during time of in
vestigation (%) Refer ence Micropar ticles S/O/W rh-EPO:HSA [0.02 2% and 0.2 2] PL GA [LA:GA 7 5:2 5, fr ee
carboxylic acid end; 2
3kDa] 63 85 20 32 day s >90 85 S/O/W Ly s:P oloxamer 188 (1:1 0) (af ter pr epar ation of solid par ticles) [0.6%] PL GA-PEG-PL GA copolymer [LA:GA 2 5:50; PEG 9%] 60 73 10 45 day s <50 92 S/O/ O/Wh BSA (incorpor ated in dextr an micr opar ticles) [5%] Mixtur e of PL GA [LA:GA 50:50,
free carboxylic acid end; 40-7
5 kDa] and PLA [40-7 5 kDa] (40:60) 67 66 <15 60 day s >95 88,8 9 S/O/ O/Eh BSA (incorpor ated in dextr an micr opar ticles) [1 0%] Mixtur e of PL GA [LA:GA 50:50,
free carboxylic acid end; 40-7
5 kDa] and PLA [40-7 5 kDa] (40:60) 67 92 <20 65 day s >80 87, 89 W/O/ O/W O VA [1 .2 5%] Mixtur e of PL GA [LA:GA 50:50,
ester end-capped; IV 0.2 dg/l] and PL
GA [LA:GA 8 5/1 5, ester end-capped] (1:2) 34 101 12 63 day s >70 11 0 Misc. (W/O/W + gel) VEGF [5 µg/mL] PL GA [LA:GA 50:50, fr ee
carboxylic acid end; 1
0 kDa]
2.7
92
<20 (>30 in combination with Ang-1)
28 day s 100 (>90 in combination with Ang-1) 10 4 Ang-1 [5 µg/mL] 3 88 <15 (<1 0 in
combination with VEGF)
28 day
s
<90 (>90 in
combination with VEGF)
TA
BL
E 2.1:
29
2
Emulsion type Pr otein [loading] Polymer [composition; MW] A ver age par ticle size (μm) EE (%) Burst release (%) In vitr orelease time investigated
Amount of pr
otein
released during time of in
vestigation (%) Refer ence Nanoparticles W/O/W BSA [1 0%] PL GA [LA:GA 50:50, fr ee
carboxylic acid end; 8 kDa]
0.1 70 90 NR NR NR 111 W/O/W BSA [9%] HP AE-co-PLA copolymer [5 1 kDa and 3 3 kDa] (7 :1) 0.1 50 98 40 14 day s 82 32 Ang-1
, angiotensin-1; BSA, bovine serum albumin; E, ethanolic phase; EE, encapsulation efficiency; F
gF
, fibr
oblast gr
owth factor; GA, glycolic acid; HBsAg, r
ecombinant h
epatitis B sur
face antigen;
HDS, high molecular weight dextr
an sulfate; HP
AE, hyperbr
anch
ed poly(amine
-ester); HSA, human serum albumin; IFNa-2b, inter
fer
on alpha 2b; IgG, polyclonal bovine immunoglobulin; IV
, intrinsic
viscosit
y; LA, lactic acid; L
ys, ly
so
zyme; MPEG-PCL
-MPEG, meth
oxypoly (ethylene glycol)-poly
-(
ε-capr
olactone)-meth
oxypoly (ethylene glycol); mPEG-PLA, poly (monometh
oxy polyethylene
glycol-co-D,L
-lactide); MW
, molecular weight
; ND, not determined; NR, not r
epor ted; O, or ganic phase; O h , hydr ophilic or ganic phase; O i , or
ganic phase immiscible with aqueous phase; O
m
,
or
ganic phase miscible with aqueous phase; O
VA, ovalbumin; PBT
, poly(but
ylene ter
ephthalate); PCL, poly(
ε-capr
olactone); PEG, polyethylene glycol; PEGT
, poly(ethylene glycol) ter
ephthalate; PLA, poly(lactic acid); PL GA, poly(lactic-co-glycolic acid); rhEGF , r ecombinant human epidermal gr owth factor; rh-EPO, recombinant human er ythr opoietin; rhGH, R ecombinant human Gr owth
Hormone; S, solid phase; TRAIL, TNF
-r
elated apoptosis-inducing ligand; VEGF
, vascular endoth elial gr owth factor; W , aqueous phase. TA BL E 2.1: (continued) 29
2
Emulsion type Pr otein [loading] Polymer [composition; MW] A ver age par ticle size (μm) EE (%) Burst release (%) In vitr orelease time investigated
Amount of pr
otein
released during time of in
vestigation (%) Refer ence Nanoparticles W/O/W BSA [1 0%] PL GA [LA:GA 50:50, fr ee
carboxylic acid end; 8 kDa]
0.1 70 90 NR NR NR 111 W/O/W BSA [9%] HP AE-co-PLA copolymer [5 1 kDa and 3 3 kDa] (7 :1) 0.1 50 98 40 14 day s 82 32 Ang-1
, angiotensin-1; BSA, bovine serum albumin; E, ethanolic phase; EE, encapsulation efficiency; F
gF
, fibr
oblast gr
owth factor; GA, glycolic acid; HBsAg, r
ecombinant h
epatitis B sur
face antigen;
HDS, high molecular weight dextr
an sulfate; HP
AE, hyperbr
anch
ed poly(amine
-ester); HSA, human serum albumin; IFNa-2b, inter
fer
on alpha 2b; IgG, polyclonal bovine immunoglobulin; IV
, intrinsic
viscosit
y; LA, lactic acid; L
ys, ly
so
zyme; MPEG-PCL
-MPEG, meth
oxypoly (ethylene glycol)-poly
-(
ε-capr
olactone)-meth
oxypoly (ethylene glycol); mPEG-PLA, poly (monometh
oxy polyethylene
glycol-co-D,L
-lactide); MW
, molecular weight
; ND, not determined; NR, not r
epor ted; O, or ganic phase; O h , hydr ophilic or ganic phase; O i , or
ganic phase immiscible with aqueous phase; O
m
,
or
ganic phase miscible with aqueous phase; O
VA, ovalbumin; PBT
, poly(but
ylene ter
ephthalate); PCL, poly(
ε-capr
olactone); PEG, polyethylene glycol; PEGT
, poly(ethylene glycol) ter
ephthalate; PLA, poly(lactic acid); PL GA, poly(lactic-co-glycolic acid); rhEGF , r ecombinant human epidermal gr owth factor; rh-EPO, recombinant human er ythr opoietin; rhGH, R ecombinant human Gr owth
Hormone; S, solid phase; TRAIL, TNF
-r
elated apoptosis-inducing ligand; VEGF
, vascular endoth elial gr owth factor; W , aqueous phase. TA BL E 2.1: (continued)
30 TA BL E 2.2: Over view of peptide
-loaded nano- and micr
opar
ticles pr
epar
ed via emulsification-solvent evapor
ation meth od. Emulsion type Peptide [loading] Polymer [composition; MW] A ver age par ticle size (μm) EE (%) Burst release (%) In vitr o
release time investigated Amount of peptide released during time of in
vestigation (%) Refer ence Micropar ticles W/O/W Exenatide PEGylated (5 kDa) Exenatide PL GA [LA:GA 50:50] 11 11 16 46 44 11 18 day s 90 94 11 2 W/O/W Octr eotide [4.5%] PLHMGA [65:3 5; 45 kDa] 16 57 21 60 day s 82 90
W/O/W S/O/W O/O/Wi
Insulin [2%] PL GA [LA:GA 50:50; IV 0.3 2-0.44 dg/l] 22 21 18 80 59 25 3 32 0.3 63 day s 63 day s 63 day s >98 >98 >98 68 Om /O/W Exenatide [5%] Mixtur e of PL GA [LA:GA 50:50, fr ee carboxylic acid end; IV 0.2 dg/l] 63 98 ND ND ND 69 Om /O/W Octr eotide [3.5%] sCT [2.1%] hPTH [2.1%] gluc-PL GA [LA:GA 50:50, D-glucose cor e; 50 kDa] and CaCl 2 or MnCl 2 as co-excipients NR 10 6 76 25 NA 28 day s 21 day s 21 day s 90 98 84 98 W/O/O Endostatin [5%] PL GA [LA:GA 50:50; 4 7 kDa] 33 93 <10 28 day s 39 10 2 Nanoparticles W/O/W Goser elin:tr ehalose (1:2) [7%]
mPEG-PCL copolymer [PEG 4
kDa; PCL 1 14 Da] 0.1 74 44 10 % 28 day s 85 97 W/O/W CTL epitope of O VA (2 4 amino acids) [2.7%] PL GA [LA:GA 50:50, fr ee
carboxylic acid end; IV 0.2
dg/l] 0.3 28 38 30 24 h 30 11 3
CTL, cytotoxic T-cell; EE, encapsulation efficiency GA, glycolic acid; hPTH, human par
athyr
oid h
ormone; IV
, intrinsic viscosit
y; LA, lactic acid; mPEG-PCL, poly (monometh
oxy polyethylene
glycol)-co-poly
-(
ε-capr
olactone); MW
, molecular weight
; NA, not applicable; ND, not determined; NR, not r
epor
ted; O, or
ganic phase; O
i
, or
ganic phase immiscible with aqueous phase; O
m , or ganic phase miscible with aqueous phase; O VA, ovalbumin; PEG, polyethylene glycol; PL GA, poly(lactic-co-glycolic acid); PLHMGA, poly(D,L -lactide -co-hydr oxy -methyl glycolide); S, solid phase; sCT , salmon calcitonin; W , aqueous phase. 30 TA BL E 2.2: Over view of peptide
-loaded nano- and micr
opar
ticles pr
epar
ed via emulsification-solvent evapor
ation meth od. Emulsion type Peptide [loading] Polymer [composition; MW] A ver age par ticle size (μm) EE (%) Burst release (%) In vitr o
release time investigated Amount of peptide released during time of in
vestigation (%) Refer ence Micropar ticles W/O/W Exenatide PEGylated (5 kDa) Exenatide PL GA [LA:GA 50:50] 11 11 16 46 44 11 18 day s 90 94 11 2 W/O/W Octr eotide [4.5%] PLHMGA [65:3 5; 45 kDa] 16 57 21 60 day s 82 90
W/O/W S/O/W O/O/Wi
Insulin [2%] PL GA [LA:GA 50:50; IV 0.3 2-0.44 dg/l] 22 21 18 80 59 25 3 32 0.3 63 day s 63 day s 63 day s >98 >98 >98 68 Om /O/W Exenatide [5%] Mixtur e of PL GA [LA:GA 50:50, fr ee carboxylic acid end; IV 0.2 dg/l] 63 98 ND ND ND 69 Om /O/W Octr eotide [3.5%] sCT [2.1%] hPTH [2.1%] gluc-PL GA [LA:GA 50:50, D-glucose cor e; 50 kDa] and CaCl 2 or MnCl 2 as co-excipients NR 10 6 76 25 NA 28 day s 21 day s 21 day s 90 98 84 98 W/O/O Endostatin [5%] PL GA [LA:GA 50:50; 4 7 kDa] 33 93 <10 28 day s 39 10 2 Nanoparticles W/O/W Goser elin:tr ehalose (1:2) [7%]
mPEG-PCL copolymer [PEG 4
kDa; PCL 1 14 Da] 0.1 74 44 10 % 28 day s 85 97 W/O/W CTL epitope of O VA (2 4 amino acids) [2.7%] PL GA [LA:GA 50:50, fr ee
carboxylic acid end; IV 0.2
dg/l] 0.3 28 38 30 24 h 30 11 3
CTL, cytotoxic T-cell; EE, encapsulation efficiency GA, glycolic acid; hPTH, human par
athyr
oid h
ormone; IV
, intrinsic viscosit
y; LA, lactic acid; mPEG-PCL, poly (monometh
oxy polyethylene
glycol)-co-poly
-(
ε-capr
olactone); MW
, molecular weight
; NA, not applicable; ND, not determined; NR, not r
epor
ted; O, or
ganic phase; O
i
, or
ganic phase immiscible with aqueous phase; O
m , or ganic phase miscible with aqueous phase; O VA, ovalbumin; PEG, polyethylene glycol; PL GA, poly(lactic-co-glycolic acid); PLHMGA, poly(D,L -lactide -co-hydr oxy -methyl glycolide); S, solid phase; sCT , salmon calcitonin; W , aqueous phase.
