Protein delivery from polymeric matrices
Teekamp, Naomi
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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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POLYMERIC MATRICES
From pre-formulation stabilization studies
to site-specific delivery
Naomi Teekamp
POLYMERIC MATRICES
From pre-formulation stabilization studies
to site-specific delivery
Cover artwork: Dirk Duin
Layout & cover design: Design Your Thesis, www.designyourthesis.com
Printing: Ridderprint BV, www.ridderprint.nl
ISBN: 978-94-6299-895-7
ISBN (digital): 978-94-6299-920-6
The research presented in this thesis was carried out at the Department of Pharmaceutical Technology and Biopharmacy of the University of Groningen. Financial support for printing the thesis was received from InnoCore Technologies, the University Library and the Graduate School of Science and Enigineering of the University of Groningen.
© Copyright Naomi Teekamp, 2018.
All rights reserved. No part of this publication may be reproduced , stored on a retreival system, or transmitted in any form or by any means, without permission of the author.
Cover artwork: Dirk Duin
Layout & cover design: Design Your Thesis, www.designyourthesis.com
Printing: Ridderprint BV, www.ridderprint.nl
ISBN: 978-94-6299-895-7
ISBN (digital): 978-94-6299-920-6
The research presented in this thesis was carried out at the Department of Pharmaceutical Technology and Biopharmacy of the University of Groningen. Financial support for printing the thesis was received from InnoCore Technologies, the University Library and the Graduate School of Science and Enigineering of the University of Groningen.
© Copyright Naomi Teekamp, 2018.
All rights reserved. No part of this publication may be reproduced , stored on a retreival system, or transmitted in any form or by any means, without permission of the author.
polymeric matrices
From pre-formulation stabilization studies to site-specific delivery
Proefschrift
ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen
op gezag van de
rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op vrijdag 20 april 2018 om 14.30 uur
door
Naomi Teekamp
geboren op 17 januari 1987te Zevenaar
polymeric matrices
From pre-formulation stabilization studies to site-specific delivery
Proefschrift
ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen
op gezag van de
rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op vrijdag 20 april 2018 om 14.30 uur
door
Naomi Teekamp
geboren op 17 januari 1987Prof. dr. H.W. Frijlink Copromotor Dr. W.L.J. Hinrichs Beoordelingscommissie Prof. dr. C. Foged Prof. dr. M.J.T.H. Goumans Prof. dr. G. Molema Prof. dr. H.W. Frijlink Copromotor Dr. W.L.J. Hinrichs Beoordelingscommissie Prof. dr. C. Foged Prof. dr. M.J.T.H. Goumans Prof. dr. G. Molema
7
15
Chapter 1. Introduction and scope of this thesis
Chapter 2. Production methods and stabilization strategies for polymer-based nanoparticles and microparticles for parenteral delivery of peptides and
proteins
57
79
Chapter 3. Addition of Pullulan to Trehalose Glasses Improves the Stability
of β-Galactosidase at High Moisture Conditions
Chapter 4. Protein Stability during Hot Melt Extrusion: The Effect of Extrusion Temperature, Sugar Glass Pre-stabilization and Hydrophilicity of
Polymers
101
131
Chapter 5. Polymeric Microspheres for the Sustained Release of a Protein-based Drug Carrier Targeting the
PDGFβ-Receptor in the Fibrotic Kidney
Chapter 6. Pharmacokinetics and Pharmacodynamics of a Sustained Release Formulation of PDGFβ-Receptor Directed Proteins to Target
the Fibrotic Liver
159
181
Chapter 7. General Discussion and
Future Directions
Chapter 8. Summary, Samenvatting, Curriculum Vitae, List of Publications,
Dankwoord
7
15
Chapter 1. Introduction and scope of this thesis
Chapter 2. Production methods and stabilization strategies for polymer-based nanoparticles and microparticles
for parenteral delivery of peptides and proteins
57
79
Chapter 3. Addition of Pullulan to Trehalose Glasses Improves the Stability
of β-Galactosidase at High Moisture Conditions
Chapter 4. Protein Stability during Hot Melt Extrusion: The Effect of Extrusion Temperature, Sugar Glass Pre-stabilization and Hydrophilicity of
Polymers
101
131
Chapter 5. Polymeric Microspheres for the Sustained Release of a Protein-based Drug Carrier Targeting the
PDGFβ-Receptor in the Fibrotic Kidney
Chapter 6. Pharmacokinetics and Pharmacodynamics of a Sustained Release Formulation of PDGFβ-Receptor Directed Proteins to Target
the Fibrotic Liver
159
181
Chapter 7. General Discussion and
Future Directions
Chapter 8. Summary, Samenvatting, Curriculum Vitae, List of Publications,
1
15346-teekamp-layout.indd 6 04/03/2018 20:42 15346-teekamp-layout.indd 6
1
04/03/2018 20:42OF THIS THESIS
AUTHOR Naomi TeekampOF THIS THESIS
AUTHOR Naomi Teekamp9
1
The therapeutic effects of insulin were first discovered almost a century ago in 1922.Although at that time insulin was an impure pancreatic extract rather than a pure therapeutic protein, the administration of this extract is widely recognized as the
first application of a proteinaceous drug in human medicine.1,2 In the following
decades, more therapeutically active proteins were discovered, such as blood factors, for which the production was based on extraction of these proteins from animal (or even human) sources. Then, in the 1970’s, genetic engineering and recombinant DNA technologies were developed enabling synthesis of recombinant (human) proteins. These developments lead to the next large breakthrough in medicine:
the large scale production of recombinant therapeutic proteins.3,4 Consequently, a
plethora of recombinant vaccines, peptides and proteins (mainly antibodies) have been commercialized ever since, reaching 166 distinct active ingredients and 212
products in 2014,5 and this novel class of drugs, the so-called biopharmaceuticals,
have revolutionized the treatment of many diseases.6
Following its discovery, insulin was thoroughly characterized,2 and during this process,
the difficulty of delivering proteins to patients became apparent.3 For example when
administered via the conventional, oral route, proteins are denatured in the highly acidic environment of the stomach or rapidly degraded by proteolytic enzymes in the gastrointestinal tract (e.g. pepsin, trypsin, and chymotrypsin). Moreover, the size of proteins hampers passage through intestinal membranes and thereby the absorption into the systemic circulation. Naturally, researchers attempted to find the ideal route of administration and formulation for insulin, though their endeavors more often failed than succeeded.
Although the pulmonary administration of insulin was already described in 1924,7 to
date, biopharmaceuticals are almost exclusively administered using aqueous solutions which provide limited shelf life. Furthermore, protein-based therapy often involves frequent injections which impair both patient comfort and compliance. Many products suffer from stability issues due to the large and complex structure of proteins, which in turn may lead to safety issues (i.e. immunological reactions). Hence, stabilization and formulation strategies for proteins continue to be a subject of investigation.
One approach to relieve the patient’s burden of frequent injections is to deliver biopharmaceuticals using systems for sustained and controlled release. In particular, biodegradable polymers are considered to be excellent matrices for such controlled
delivery systems,8,9 and release of proteins from such systems can range from hours
9
1
The therapeutic effects of insulin were first discovered almost a century ago in 1922.Although at that time insulin was an impure pancreatic extract rather than a pure therapeutic protein, the administration of this extract is widely recognized as the
first application of a proteinaceous drug in human medicine.1,2 In the following
decades, more therapeutically active proteins were discovered, such as blood factors, for which the production was based on extraction of these proteins from animal (or even human) sources. Then, in the 1970’s, genetic engineering and recombinant DNA technologies were developed enabling synthesis of recombinant (human) proteins. These developments lead to the next large breakthrough in medicine:
the large scale production of recombinant therapeutic proteins.3,4 Consequently, a
plethora of recombinant vaccines, peptides and proteins (mainly antibodies) have been commercialized ever since, reaching 166 distinct active ingredients and 212
products in 2014,5 and this novel class of drugs, the so-called biopharmaceuticals,
have revolutionized the treatment of many diseases.6
Following its discovery, insulin was thoroughly characterized,2 and during this process,
the difficulty of delivering proteins to patients became apparent.3 For example when
administered via the conventional, oral route, proteins are denatured in the highly acidic environment of the stomach or rapidly degraded by proteolytic enzymes in the gastrointestinal tract (e.g. pepsin, trypsin, and chymotrypsin). Moreover, the size of proteins hampers passage through intestinal membranes and thereby the absorption into the systemic circulation. Naturally, researchers attempted to find the ideal route of administration and formulation for insulin, though their endeavors more often failed than succeeded.
Although the pulmonary administration of insulin was already described in 1924,7 to
date, biopharmaceuticals are almost exclusively administered using aqueous solutions which provide limited shelf life. Furthermore, protein-based therapy often involves frequent injections which impair both patient comfort and compliance. Many products suffer from stability issues due to the large and complex structure of proteins, which in turn may lead to safety issues (i.e. immunological reactions). Hence, stabilization and formulation strategies for proteins continue to be a subject of investigation.
One approach to relieve the patient’s burden of frequent injections is to deliver biopharmaceuticals using systems for sustained and controlled release. In particular, biodegradable polymers are considered to be excellent matrices for such controlled
10
to months depending on the polymer type, size and composition.9,10 These polymers
can be processed into different formulation types such as microparticles, micro-sized implants and gels, which usually can be administrated subcutaneously thereby further reducing patient discomfort.
In these polymeric drug delivery systems, however, the stability of proteins is not guaranteed. Destabilization of proteins can occur during the production process, but also during storage or release which may be due to interactions with the polymer and its degradation products. In Chapter 2 an overview of production methods is presented for polymeric micro- and nanoparticles containing proteins and peptides as well as an overview of potentially destabilizing conditions during the production of these particles and approaches to improve the stability of the encapsulated biomacromolecules.
One widely applied strategy to improve the stability of proteins is drying them in the presence of sugars. Upon drying, a sugar glass is formed which stabilizes the protein by vitrification (i.e. immobilization; which reduces molecular mobility and thereby degradation reactions) and replacement of water in the hydration shell of proteins (i.e. the water molecules surrounding proteins by which they maintain their tertiary structure) by forming hydrogen bonds with functional groups at the protein’s
surface.11,12 Disaccharides such as trehalose are known to be excellent stabilizers due
to their ability to create a tight packing around the irregularly shaped surface of the protein. However, disaccharides are also prone to crystallization when temperature and humidity rise, and these sugars thereby lose their stabilizing properties. The onset of this crystallization process is partly determined by the glass transition temperature
(Tg) of the sugar, which is lowered in the presence of plasticizers, such as water.
Polysaccharides have a higher Tg due to their larger size, and corresponding sugar
glasses are more resistant to temperature and humidity, though generally lack the ability to tightly surround the protein. In Chapter 3, the advantages of disaccharides and polysaccharides are combined to optimize protein stabilization at non-ideal, yet clinically relevant conditions: temperatures above room temperature and high relative humidity. Several blends of trehalose and the polysaccharide pullulan are
freeze-dried to form binary glasses and are evaluated based on their Tg at different relative
humidities and their protein stabilizing abilities upon storage. In this work, we are the first to extensively investigate the polysaccharide pullulan for its stabilizing properties.
10
to months depending on the polymer type, size and composition.9,10 These polymers
can be processed into different formulation types such as microparticles, micro-sized implants and gels, which usually can be administrated subcutaneously thereby further reducing patient discomfort.
In these polymeric drug delivery systems, however, the stability of proteins is not guaranteed. Destabilization of proteins can occur during the production process, but also during storage or release which may be due to interactions with the polymer and its degradation products. In Chapter 2 an overview of production methods is presented for polymeric micro- and nanoparticles containing proteins and peptides as well as an overview of potentially destabilizing conditions during the production of these particles and approaches to improve the stability of the encapsulated biomacromolecules.
One widely applied strategy to improve the stability of proteins is drying them in the presence of sugars. Upon drying, a sugar glass is formed which stabilizes the protein by vitrification (i.e. immobilization; which reduces molecular mobility and thereby degradation reactions) and replacement of water in the hydration shell of proteins (i.e. the water molecules surrounding proteins by which they maintain their tertiary structure) by forming hydrogen bonds with functional groups at the protein’s
surface.11,12 Disaccharides such as trehalose are known to be excellent stabilizers due
to their ability to create a tight packing around the irregularly shaped surface of the protein. However, disaccharides are also prone to crystallization when temperature and humidity rise, and these sugars thereby lose their stabilizing properties. The onset of this crystallization process is partly determined by the glass transition temperature
(Tg) of the sugar, which is lowered in the presence of plasticizers, such as water.
Polysaccharides have a higher Tg due to their larger size, and corresponding sugar
glasses are more resistant to temperature and humidity, though generally lack the ability to tightly surround the protein. In Chapter 3, the advantages of disaccharides and polysaccharides are combined to optimize protein stabilization at non-ideal, yet clinically relevant conditions: temperatures above room temperature and high relative humidity. Several blends of trehalose and the polysaccharide pullulan are
freeze-dried to form binary glasses and are evaluated based on their Tg at different relative
humidities and their protein stabilizing abilities upon storage. In this work, we are the first to extensively investigate the polysaccharide pullulan for its stabilizing properties.
11
1
An alternative to binary glasses is the application of a flexible oligosaccharide such asinulin. Because of its flexible backbone, inulin can provide a tight packing around
proteins while its larger molecular mass ensures resistance to higher temperatures.13,14
This sugar is applied in Chapter 4 where we investigate whether proteins can be successfully incorporated in polymeric implants produced by hot melt extrusion (HME). Because of the high temperatures that are usually required for the HME production process, proteins are inclined to denature. Therefore, HME is generally not considered to be a suitable production process for protein-containing formulations. Moreover, many biodegradable polymers have a hydrophobic nature which might induce unfolding of proteins and thereby destabilize them. The use of engineered phase-separated, multi-block copolymers which contain hydrophilic polyethylene glycol (PEG) blocks may reduce protein denaturation. In Chapter 4 we hypothesize that several approaches could improve the stability of proteins during HME, namely lowering the extrusion temperature, the use of inulin pre-stabilized protein, and the use of hydrophilic polymers. This study was performed using two model proteins and six (co)polymers with different properties.
Research and development of formulations containing proteins is often performed using model proteins due to practical or financial reasons. Such model proteins are usually therapeutically inactive proteins, and are typically similar to the active protein in size or charge. Admittedly, it is unlikely that such a model is a perfect predictor for the behavior of a specific therapeutically active protein. Therefore, progress in formulation development of protein drugs might benefit from already using the active protein in early stages of development. In Chapters 5 and 6 we put this thought into practice and developed a polymeric microsphere formulation for a protein drug carrier targeted to fibrotic tissue. Fibrosis is a progressive disease that can develop in various organs. Currently, no effective treatment is available for this disease15 which is partly due to the systemic side effects of many potential antifibrotic drugs. Thus, specific delivery of drugs to the diseased tissue – targeting – would enhance drug efficacy and may decrease side effects. As a target for our local drug delivery efforts, the platelet-derived growth factor β receptor (PDGFβR) was selected because this receptor is specifically upregulated in fibrotic tissue. Previously, a cyclic peptide referred to as pPB has been developed to target this receptor.16 This peptide binds to the PDGFβR without activating the intracellular downstream pathway. Furthermore, pPB was coupled to human serum albumin (HSA) to prolong its half-life.17 In Chapter 5, we aimed to develop a controlled sustained release formulation for the carrier pPB-HSA.
11
1
An alternative to binary glasses is the application of a flexible oligosaccharide such asinulin. Because of its flexible backbone, inulin can provide a tight packing around
proteins while its larger molecular mass ensures resistance to higher temperatures.13,14
This sugar is applied in Chapter 4 where we investigate whether proteins can be successfully incorporated in polymeric implants produced by hot melt extrusion (HME). Because of the high temperatures that are usually required for the HME production process, proteins are inclined to denature. Therefore, HME is generally not considered to be a suitable production process for protein-containing formulations. Moreover, many biodegradable polymers have a hydrophobic nature which might induce unfolding of proteins and thereby destabilize them. The use of engineered phase-separated, multi-block copolymers which contain hydrophilic polyethylene glycol (PEG) blocks may reduce protein denaturation. In Chapter 4 we hypothesize that several approaches could improve the stability of proteins during HME, namely lowering the extrusion temperature, the use of inulin pre-stabilized protein, and the use of hydrophilic polymers. This study was performed using two model proteins and six (co)polymers with different properties.
Research and development of formulations containing proteins is often performed using model proteins due to practical or financial reasons. Such model proteins are usually therapeutically inactive proteins, and are typically similar to the active protein in size or charge. Admittedly, it is unlikely that such a model is a perfect predictor for the behavior of a specific therapeutically active protein. Therefore, progress in formulation development of protein drugs might benefit from already using the active protein in early stages of development. In Chapters 5 and 6 we put this thought into practice and developed a polymeric microsphere formulation for a protein drug carrier targeted to fibrotic tissue. Fibrosis is a progressive disease that can develop in various organs. Currently, no effective treatment is available for this disease15 which is partly due to the systemic side effects of many potential antifibrotic drugs. Thus, specific delivery of drugs to the diseased tissue – targeting – would enhance drug efficacy and may decrease side effects. As a target for our local drug delivery efforts, the platelet-derived growth factor β receptor (PDGFβR) was selected because this receptor is specifically upregulated in fibrotic tissue. Previously, a cyclic peptide referred to as pPB has been developed to target this receptor.16 This peptide binds to the PDGFβR without activating the intracellular downstream pathway. Furthermore, pPB was coupled to human serum albumin (HSA) to prolong its half-life.17 In Chapter 5, we aimed to develop a controlled sustained release formulation for the carrier pPB-HSA.
12
To obtain the desired release profile, two phase-separated, multi-block copolymers were mixed in different ratios. In addition, differential scanning calorimetry was performed to gain more insight in the release mechanisms of pPB-HSA from our polymeric microspheres. The production process was optimized ensuring maximum robustness, and the resulting formulation was used for a proof of concept in vivo study using a mouse model for renal fibrosis.
In continuation of the proof of concept in vivo study, the in vivo pharmacokinetics of
pPB-HSA were investigated in Chapter 6 in two murine models for fibrosis: the CCl4
model of acute fibrosis, as well as in the Mdr2 knockout model for advanced biliary (liver) fibrosis, using the same formulation as in the previous chapter.
The administration of proteins from different species, in this case human serum albumin to mice, can potentially induce an immunological response and thereby impair the efficacy of the delivery and targeting. Therefore, we also aimed to improve the delivery of the PDGFβR-targeted construct by using microspheres containing pPB coupled to mouse serum albumin (pPB-MSA). In addition, this chapter describes the extension of our protein targeting and delivery endeavors to a therapeutically active protein by coupling the antifibrotic rho-kinase inhibitor Y27632 to pPB-MSA and subsequent evaluation of this construct in Mdr2 knockout mice.
Chapter 7 gives an overview of all findings in this thesis, as well as some final remarks on how to get controlled release protein therapeutics to the clinic, how to apply the sugar glass technology in polymeric formulations and the potential of delivering targeted proteins from controlled release formulations.
12
To obtain the desired release profile, two phase-separated, multi-block copolymers were mixed in different ratios. In addition, differential scanning calorimetry was performed to gain more insight in the release mechanisms of pPB-HSA from our polymeric microspheres. The production process was optimized ensuring maximum robustness, and the resulting formulation was used for a proof of concept in vivo study using a mouse model for renal fibrosis.
In continuation of the proof of concept in vivo study, the in vivo pharmacokinetics of
pPB-HSA were investigated in Chapter 6 in two murine models for fibrosis: the CCl4
model of acute fibrosis, as well as in the Mdr2 knockout model for advanced biliary (liver) fibrosis, using the same formulation as in the previous chapter.
The administration of proteins from different species, in this case human serum albumin to mice, can potentially induce an immunological response and thereby impair the efficacy of the delivery and targeting. Therefore, we also aimed to improve the delivery of the PDGFβR-targeted construct by using microspheres containing pPB coupled to mouse serum albumin (pPB-MSA). In addition, this chapter describes the extension of our protein targeting and delivery endeavors to a therapeutically active protein by coupling the antifibrotic rho-kinase inhibitor Y27632 to pPB-MSA and subsequent evaluation of this construct in Mdr2 knockout mice.
Chapter 7 gives an overview of all findings in this thesis, as well as some final remarks on how to get controlled release protein therapeutics to the clinic, how to apply the sugar glass technology in polymeric formulations and the potential of delivering targeted proteins from controlled release formulations.
13
1
REFERENCES
1. Banting F, Best C. The internal secretion of the pancreas. J Laboratoty Clin Med. 1922;8(5):1-16. 2. Rosenfeld L. Insulin: discovery and controversy. Clin Chem. 2002;48(12):2270-2288.
3. Brown LR. Commercial challenges of protein drug delivery. Expert Opin Drug Deliv. 2005;2(1):29-42.
4. Walsh G. Biopharmaceutical benchmarks. Nat Biotechnol. 2000;18(1):831-833. 5. Walsh G. Biopharmaceutical benchmarks 2014. Nat Biotechnol. 2014;32(10):992-1000.
6. Sanchez-Garcia L, Martín L, Mangues R, Ferrer-Miralles N, Vázquez E, Villaverde A. Recombinant pharmaceuticals from microbial cells: a 2015 update. Microb Cell Fact. 2016;15(1):33.
7. Heubner W, De Jongh S, Laquer E. Uber Inhalation von Insulin. Klin Wochenschr. 1924;3(51):2342-2343.
8. Wu F, Jin T. Polymer-based sustained-release dosage forms for protein drugs, challenges, and recent advances. AAPS PharmSciTech. 2008;9(4):1218-1229.
9. Prajapati VD, Jani GK, Kapadia JR. Current knowledge on biodegradable microspheres in drug delivery. Expert Opin Drug Deliv. 2015;12(8):1283-1299.
10. Pagels RF, Prud’homme RK. Polymeric nanoparticles and microparticles for the delivery of peptides, biologics, and soluble therapeutics. J Control Release. 2015;219:519-535.
11. Chang LL, Pikal MJ. Mechanisms of Protein Stabilization in the Solid State. J Pharm Sci. 2009;98(9):2886-2908.
12. Mensink MA, Frijlink HW, van der Voort Maarschalk K, Hinrichs WLJ. How sugars protect proteins in the solid state and during drying (review): Mechanisms of stabilization in relation to stress conditions. Eur J Pharm Biopharm. 2017;114:288-295.
13. Tonnis WF, Mensink MA, de Jager A, van der Voort Maarschalk K, Frijlink HW, Hinrichs WLJ. Size and Molecular Flexibility of Sugars Determine the Storage Stability of Freeze-Dried Proteins.
Mol Pharm. 2015;12(3):684-694.
14. Hinrichs WL, Prinsen MG, Frijlink HW. Inulin glasses for the stabilization of therapeutic proteins.
Int J Pharm. 2001;215(1-2):163-174.
15. Friedman SL, Sheppard D, Duffield JS, Violette S. Therapy for Fibrotic Diseases: Nearing the Starting Line. Sci Transl Med. 2013;5(167).
16. Beljaars L, Weert B, Geerts A, Meijer DKF, Poelstra K. The preferential homing of a platelet derived growth factor receptor-recognizing macromolecule to fibroblast-like cells in fibrotic tissue. Biochem
Pharmacol. 2003;66(7):1307-1317.
17. van Dijk F, Olinga P, Poelstra K, Beljaars L. Targeted Therapies in Liver Fibrosis: Combining the Best Parts of Platelet-Derived Growth Factor BB and Interferon Gamma. Front Med. 2015;2:72.
13
1
REFERENCES
1. Banting F, Best C. The internal secretion of the pancreas. J Laboratoty Clin Med. 1922;8(5):1-16. 2. Rosenfeld L. Insulin: discovery and controversy. Clin Chem. 2002;48(12):2270-2288.
3. Brown LR. Commercial challenges of protein drug delivery. Expert Opin Drug Deliv. 2005;2(1):29-42.
4. Walsh G. Biopharmaceutical benchmarks. Nat Biotechnol. 2000;18(1):831-833. 5. Walsh G. Biopharmaceutical benchmarks 2014. Nat Biotechnol. 2014;32(10):992-1000.
6. Sanchez-Garcia L, Martín L, Mangues R, Ferrer-Miralles N, Vázquez E, Villaverde A. Recombinant pharmaceuticals from microbial cells: a 2015 update. Microb Cell Fact. 2016;15(1):33.
7. Heubner W, De Jongh S, Laquer E. Uber Inhalation von Insulin. Klin Wochenschr. 1924;3(51):2342-2343.
8. Wu F, Jin T. Polymer-based sustained-release dosage forms for protein drugs, challenges, and recent advances. AAPS PharmSciTech. 2008;9(4):1218-1229.
9. Prajapati VD, Jani GK, Kapadia JR. Current knowledge on biodegradable microspheres in drug delivery. Expert Opin Drug Deliv. 2015;12(8):1283-1299.
10. Pagels RF, Prud’homme RK. Polymeric nanoparticles and microparticles for the delivery of peptides, biologics, and soluble therapeutics. J Control Release. 2015;219:519-535.
11. Chang LL, Pikal MJ. Mechanisms of Protein Stabilization in the Solid State. J Pharm Sci. 2009;98(9):2886-2908.
12. Mensink MA, Frijlink HW, van der Voort Maarschalk K, Hinrichs WLJ. How sugars protect proteins in the solid state and during drying (review): Mechanisms of stabilization in relation to stress conditions. Eur J Pharm Biopharm. 2017;114:288-295.
13. Tonnis WF, Mensink MA, de Jager A, van der Voort Maarschalk K, Frijlink HW, Hinrichs WLJ. Size and Molecular Flexibility of Sugars Determine the Storage Stability of Freeze-Dried Proteins.
Mol Pharm. 2015;12(3):684-694.
14. Hinrichs WL, Prinsen MG, Frijlink HW. Inulin glasses for the stabilization of therapeutic proteins.
Int J Pharm. 2001;215(1-2):163-174.
15. Friedman SL, Sheppard D, Duffield JS, Violette S. Therapy for Fibrotic Diseases: Nearing the Starting Line. Sci Transl Med. 2013;5(167).
16. Beljaars L, Weert B, Geerts A, Meijer DKF, Poelstra K. The preferential homing of a platelet derived growth factor receptor-recognizing macromolecule to fibroblast-like cells in fibrotic tissue. Biochem
Pharmacol. 2003;66(7):1307-1317.
17. van Dijk F, Olinga P, Poelstra K, Beljaars L. Targeted Therapies in Liver Fibrosis: Combining the Best Parts of Platelet-Derived Growth Factor BB and Interferon Gamma. Front Med. 2015;2:72.
2
15346-teekamp-layout.indd 14 04/03/2018 20:42 15346-teekamp-layout.indd 14
2
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
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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
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.
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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
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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
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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
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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
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%.
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a comparable process. By the addition of ε-polylysine, a cationic polyamide, to theformulation, 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
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a comparable process. By the addition of ε-polylysine, a cationic polyamide, to theformulation, 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
