General introduction Aim and outline of this thesis · General introduction 7 3. Outline Chapter 2...
Transcript of General introduction Aim and outline of this thesis · General introduction 7 3. Outline Chapter 2...
1 General introduction
& Aim and outline of this thesis
Chapter 1
2
General introduction
3
1. General introduction
1.1. Hydrogels
Hydrogels are defined by Peppas et al. as ‘three-dimensional, hydrophilic, polymeric networks
capable of imbibing large amounts of water or biological fluids’.[1] Since the introduction of hydrogels as soft contact lenses in the 1960s,[2] their use has increased tremendously and
nowadays they are favored in a broad range of pharmaceutical and biomedical applications,[1, 3-5] of which protein delivery and tissue engineering are relevant to this thesis. The polymers that are used to create macroscopic networks can be divided into 2 groups, according to their origin:
natural or synthetic. Most common synthetic polymers, used as building blocks for hydrogels, are poly(ethylene glycol) (PEG)[6-9] and poly(hydroxyethyl methacrylate) (pHEMA).[2, 10, 11] The use of
synthetic polymers generally results in hydrogels with high mechanical strength and offers the opportunity to tailor the network properties and for instance release behavior, in a reproducible
way. Natural polymers have gained more interest the past decades because of their biocompatibility and the presence of biologically recognizable groups to support cellular activities. Two major drawbacks, however, are their generally weak mechanical properties and the batch to
batch variability. Furthermore, they can be derived from various sources and may contain impurities or pathogens as a result. Natural polymers frequently used for the preparation of
hydrogels are, amongst others, chitosan,[12-14] hyaluronic acid,[15-17] alginate,[18-20] gelatin[21] and, as will be discussed below, dextran.
1.2. Crosslinking strategies to design hydrogels
The hydrogels are crosslinked to prevent dissolution of the polymer chains when the hydrogel is in
an aqueous environment. To provide degradability of the network, degradable linkers can be incorporated.[22] A wide range of crosslinking methods has been used that can be categorized into
chemical and physical crosslinking (Fig. 1).[23] Chemical crosslinking leads to permanent covalent bonds and can be accomplished by e.g. radical,[24] high-energy[25] or enzyme-mediated
polymerization.[26] Physical crosslinking involves the introduction of non-permanent, reversible crosslinks via e.g. hydrophobic interactions,[27] hydrogen bonding,[28] stereocomplexes[29] or ionic
interactions.[30] Whereas chemical crosslinking leads to networks with relatively high mechanical strength, physical crosslinking generally results in weaker hydrogels. On the other hand, the physical crosslinks can be reversibly broken by e.g. shear forces, which may allow injectability of the
gels. Additionally, the crosslinking conditions during physical crosslinking are mild, while the use of chemical crosslinking agents might lead to denaturation of entrapped proteins or could be toxic
to cells. Depending on the specific application foreseen for the hydrogels system, the above mentioned issues need to be taken into consideration.
Chapter 1
4
1.3. In situ gelation
Hydrogels can be designed in different geometries, e.g. slabs or cylinders,[31] films,[32] microspheres[33] or nanospheres.[34] While the latter two can be administered by injection,
macroscopic cylinders need surgical administration. Therefore, nowadays, interest has shifted to hydrogels that can be administered as liquid formulation and are formed in situ, at the site of
injection.[35] In situ gelation via self-assembly can occur by physical interactions that develop in time (e.g. stereocomplexed gels,[36] ionically crosslinked gels[14]), or in response to a certain trigger
(e.g. temperature[37]). UV-polymerization can also be applied for in situ gelation if the polymer solution is injected in a cavity from where it cannot leak out before polymerization is finalized.[38] Recently, in situ gelation was accomplished via Michael-addition resulting in chemically crosslinked
hydrogels.[39-41] Currently, much research is done on ‘stimuli-responsive’ hydrogels[42] that release their content in reaction to a stimulus, such as glucose concentration,[43] pH,[44] temperature,[45, 46]
ionic strength[47] or binding to antigens.[48]
+ +
chemical crosslinkingphysical crosslinking
hydrophilicpolymer chainscrosslinks
+ +
chemical crosslinkingphysical crosslinking
hydrophilicpolymer chainscrosslinks
Figure 1: Schematic representation of physical and chemical crosslinking.
1.4. Dextran hydrogels
Dextran is one of the most promising polysaccharides of the past few decades for the preparation
of hydrogels for controlled protein release.[49] It can be produced either chemically or by bacteria
from sucrose. Bacterial dextran mainly consists of linear α-1,6-linked glucopyranose units, with
some degree of 1,3-branching (Fig. 2). Several coupling reactions have been described that
provide the opportunity to modify the numerous OH- groups along the dextran chains.
General introduction
5
O
O
OHO
HOHO
O
O
OH
HOHO
O
OH
HOHO
O
O
O
OHO
HO
α-1,3 linkage
α-1,6 linkage
glucopyranose unit
Figure 2: Chemical structure of dextran.
In our group, extensive research on dextran hydrogels as biodegradable protein delivery systems
has been performed. Dextran (dex) was modified with methacrylate (MA),[50] hydroxethyl methacrylate (HEMA)[51] and oligolactate[52] for the formation of both chemically and physically
crosslinked macroscopic hydrogels. Dex-MA hydrogels can be enzymatically degraded, while the other formulations are mainly degraded by chemical hydrolysis.
A novel preparation method for dex-MA and dex-HEMA microspheres was developed, without the
use of organic solvents.[53, 54] The approach is based on the immiscibility of aqueous solutions of dextran and PEG, resulting in a water-in-water emulsion. Next, radical polymerization leads to the formation of dex-MA or dex-HEMA microspheres (Fig. 3). The microspheres can be loaded with
therapeutic proteins and subsequently injected intramuscularly or subcutaneously.[55, 56] These microspheres are very promising protein delivery vehicles, but the necessity of radicals for the
polymerization reactions remains an obstacle, especially when the microspheres are loaded with oxidation-sensitive proteins.[57] To circumvent this hurdle, we are interested in physically
crosslinked, injectable, macroscopic dextran hydrogels.
dexPEG PEG
dex+
stirring radical
polymerization
water-in-water emulsion
dexPEG PEG
dex+
stirring radical
polymerization
water-in-water emulsion Figure 3: Preparation of dex-MA or dex-HEMA microspheres via a water-in-water emulsion and subsequent
radical polymerization.
Chapter 1
6
2. Aim of this thesis
The aim of this thesis was the synthesis and physicochemical characterization of self-assembling
microsphere-based networks, arising through physical crosslinking of dextran microgels, for drug delivery purposes.
Additionally, as Figure 4 illustrates, we aimed to evaluate whether proteins and/or cells can be entrapped in the pores between the individual microspheres. Two different approaches to self-
assemble the microspheres were investigated: ionic interactions and hydrophobic interactions combined with stereocomplexation.
In the first approach dex-HEMA was copolymerized with methacrylic acid (MAA) or N,N-
dimethylamino ethyl methacrylate (DMAEMA) resulting in, respectively, negatively and positively
charged microspheres at physiological pH. Because ‘opposites attract’, hydrogel formation occurred when aqueous dispersions of both types of microspheres were mixed. In the second
approach, hydrogels were obtained through hydrophobic self-assembly of and stereocomplexation between dex-HEMA microspheres, grafted with oligolactates of opposite chirality.
This thesis aimed (a) to reveal the network properties of these self-assembled microsphere-based
hydrogels through rheological analysis, (b) to develop various strategies to tailor the network properties and the degradation behavior of the matrices, (c) to evaluate the applicability of the
hydrogels as protein delivery systems by monitoring the in vitro release of proteins.
type 1 microspheres type 2 microspheres
proteins
macroscopic hydrogel
type 1 microspheres type 2 microspheres
proteins
macroscopic hydrogel
Figure 4: Schematic representation of the self-assembling microsphere-based hydrogels in which proteins are
entrapped by simply mixing the protein solution with the microsphere dispersions.
General introduction
7
3. Outline
Chapter 2 reviews the current state-of-the-art of dextran hydrogels for pharmaceutical
applications. Both chemically and physically crosslinked hydrogels are described. Special attention is given to the medical applicability of the systems.
In Chapter 3, a novel self-assembling hydrogel is reported based on ionic interactions between
oppositely charged dextran microspheres. Dex-HEMA was copolymerized with methacrylic (MAA)
acid or N,N-dimethylamino ethyl methacrylate (DMAEMA) yielding negatively and positively charged microspheres, respectively. Equal amounts of aqueous dispersions of both microsphere types were mixed and their network forming capacity was investigated. Rheological analysis was
applied to monitor the network properties.
Chapter 4 describes the mobility of model proteins (lysozyme, BSA and IgG) in the ionically self-
assembled microsphere-based dextran hydrogels. Protein release was studied and mathematical modeling of the release curves was done to obtain a better understanding of the mechanisms that are involved. Fluorescence recovery after photobleaching experiments were applied to gain
insight into the mobility of proteins in the hydrogels on a microscopic level.
The degradation behavior of both the charged dextran microspheres as well as the ionically crosslinked macroscopic gels is in focus in Chapter 5. Besides swelling experiments, confocal
imaging and rheology on degrading hydrogels were performed. For the swelling experiments, the ratio positively/negatively charged particles was varied to evaluate the charge effect on the
degradation behavior of the microspheres.
In Chapter 6 the effect of the particle size (distribution) and charge of the dextran microspheres
on the network properties of the self-assembled hydrogels is demonstrated. The ratio (MAA or
DMAEMA)/HEMA was varied to obtain microspheres with different zeta (ξ)-potentials. Different
microsphere preparation procedures were explored to alter the particle size and size distribution.
Positively charged microspheres were prepared with degradable cationic monomers based on 2-hydroxypropyl methacrylamide (HPMA), i.e. HPMA-DMAE (N,N-dimethyl 2-aminoethanol) and HPMA-DMAPr (N,N-dimethyl 3-aminopropanol) instead of DMAEMA in Chapter 7. Introductory
degradation experiments were performed to investigate the effect of charge loss in time on the network properties of the macroscopic gels.
Chapter 8 deals with a second approach to create self-gelling microsphere-based dextran
hydrogels. For this purpose, dex-HEMA microspheres were grafted with oligolactates of opposite
chirality. The potential of hydrophobic interactions or stereocomplexation between the lactate grafts on the individual microspheres to create a macroscopic network was evaluated rheologically.
Finally, in Chapter 9, potential applications and future perspectives of the self-assembling
microsphere-based dextran hydrogels are outlined, with a special focus on biocompatibility of the
materials. Some pilot cytotoxicity data are reported.
Chapter 1
8
References
1. Peppas NA, Bures P, Leobandung W, Ichikawa H. Hydrogels in pharmaceutical formulations. Eur. J. Pharm.
Biopharm. 2000;50:27-46.
2. Wichterle O, Lim D. Hydrophilic gels for biological use. Nature 1960;185:117-118.
3. Hoffman AS. Hydrogels for biomedical applications. Adv. Drug Deliver. Rev. 2002;43:3-12.
4. Kashyap N, Kumar N, Ravikumar MNV. Hydrogels for pharmaceutical and biomedical applications. Crit. Rev. Ther.
Drug 2005;22:107-150.
5. Peppas NA. Hydrogels and drug delivery. Curr. Opin. Colloid In. 1997;2:531-537.
6. Quick DJ, Anseth KS. DNA delivery from photocrosslinked PEG hydrogels: encapsulation efficiency, release profiles,
and DNA quality. J. Control. Release 2004;96:341-351.
7. Van de Wetering P, Metters AT, Schoenmakers RG, Hubbell JA. Poly(ethylene glycol) hydrogels formed by conjugate addition with controllable swelling, degradation, and release of pharmaceutically active proteins. J.
Control. Release 2005;101:619-627.
8. Mahoney MJ, Anseth KS. Three-dimensional growth and function of neural tissue in degradable polyethylene
glycol hydrogels. Biomaterials 2006;27:2265-2274.
9. Hiemstra C, Zhong ZY, Van Tomme SR, Hennink WE, Dijkstra PJ, Feijen J. Protein release from injectable stereocomplexed hydrogels based on PEG-PDLA and PEG-PLLA star block copolymers. J. Control. Release
2006;116:e19-e21.
10. Lu S, Anseth KS. Photopolymerization of multilaminated poly(HEMA) hydrogels for controlled release. J. Control.
Release 1999;57:291-300.
11. Garcia DM, Escobar JL, Noa Y, Bada N, Hernaez E, Katime I. Timolol maleate release from pH-sensible poly(2-
hydroxyethyl methacrylate-co-methacrylic acid) hydrogels. Eur. Polym. J. 2004;40:1683-1690.
12. Obara K, Ishihara M, Ishizuka T, Fujita M, Ozeki Y, Maehara T, Saito Y, Yura H, Matsui T, Hattori H. Photocrosslinkable
chitosan hydrogel containing fibroblast growth factor-2 stimulates wound healing in healing-impaired db/db
mice. Biomaterials 2003;24:3437-3444.
13. Akbuga J, Ozbas-Turan S, Erdogan N. Plasmid-DNA loaded chitosan microspheres for in vitro IL-2 expression. Eur. J.
Pharm. Biopharm. 2004;58:501-507.
14. Berger J, Reist M, Mayer JM, Felt O, Peppas NA, Gurny R. Structure and interactions in covalently and ionically
crosslinked chitosan hydrogels for biomedical applications. Eur. J. Pharm. Biopharm. 2004;57:19-34.
15. Peattie RA, Nayate AP, Firpo MA, Shelby J, Fisher RJ, Prestwich GD. Stimulation of in vivo angiogenesis by cytokine-
loaded hyaluronic acid hydrogel implants. Biomaterials 2004;25:2789-2798.
16. Masters KS, Shah DN, Leinwand LA, Anseth KS. Crosslinked hyaluronan scaffolds as a biologically active carrier for
valvular interstitial cells. Biomaterials 2005;26:2517-2525.
17. Shu XZ, Liu Y, Palumbo FS, Luo Y, Prestwich GD. In situ crosslinkable hyaluronan hydrogels for tissue engineering.
Biomaterials 2004;25:1339-1348.
18. Gombotz WR, Wee SF. Protein release from alginate matrices. Adv. Drug Deliver. Rev. 1998;31:267-285.
19. Rowley JA, Madlambayan G, Mooney DJ. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials
1999;20:45-53.
20. Leonard M, De Boisseson MR, Hubert P, Dalencon F, Dellacherie E. Hydrophobically modified alginate hydrogels as
protein carriers with specific controlled release properties. J. Control. Release 2004;98:395-405.
21. Sutter M, Siepmann J, Hennink WE, Jiskoot W. Recombinant gelatin hydrogels for the sustained release of proteins.
J. Control. Release 2007;119:301-312.
22. Peppas NA, Hilt JZ, Khademhosseini A, Langer R. Hydrogels In biology and medicine: from molecular principles to
biotechnology. Adv. Mater. 2006;18:1345-1360.
23. Hennink WE, van Nostrum CF. Novel crosslinking methods to design hydrogels. Adv. Drug Deliver. Rev. 2002;54:13-36.
24. Chung JT, Vlugt-Wensink KDF, Hennink WE, Zhang Z. Effect of polymerization conditions on the network
properties of dex-HEMA microspheres and macro-hydrogels. Int. J. Pharm. 2005;288:51-61.
25. Leach JB, Schmidt CE. Characterization of protein release from photocrosslinkable hyaluronic acid-polyethylene
glycol hydrogel tissue engineering scaffolds. Biomaterials 2005;26:125-135.
26. Ferreira L, Gil MH, Cabrita AMS, Dordick JS. Biocatalytic synthesis of highly ordered degradable dextran-based
hydrogels. Biomaterials 2005;26:4707-4716.
27. Li J, Li X, Ni X, Wang X, Li H, Leong KW. Self-assembled supramolecular hydrogels formed by biodegradable PEO-
PHB-PEO triblock copolymers and [alpha]-cyclodextrin for controlled drug delivery. Biomaterials 2006;27:4132-4140.
General introduction
9
28. Noro A, Nagata Y, Takano A, Matsushita Y. Diblock-type supramacromolecule via biocomplementary hydrogen
bonding. Biomacromolecules 2006;7:1696-1699.
29. Li S. Bioresorbable hydrogels prepared through stereocomplexation between poly(L-lactide) and poly(D-lactide)
blocks attached to poly(ethylene glycol). Macromol. Biosci. 2003;3:657-661.
30. Kuo CK, Ma PX. Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: Part 1. Structure,
gelation rate and mechanical properties. Biomaterials 2001;22:511-521.
31. Franssen O, Vandervennet L, Roders P, Hennink WE. Degradable dextran hydrogels: controlled release of a model
protein from cylinders and microspheres. J. Control. Release 1999;60:211-221.
32. Draye J-P, Delaey B, Van de Voorde A, Van Den Bulcke A, Bogdanov B, Schacht E. In vitro release characteristics of
bioactive molecules from dextran dialdehyde cross-linked gelatin hydrogel films. Biomaterials 1998;19:99-107.
33. Sinha VR, Trehan A. Biodegradable microspheres for protein delivery. J. Control. Release 2003;90:261-280.
34. Van Thienen TG, Lucas B, Demeester J, De Smedt SC. On the synthesis and characterization of biodegradable
dextran nanogels with tunable degradation properties. J. Control. Release 2006;116:e12-e13.
35. Hatefi A, Amsden B. Biodegradable injectable in situ forming drug delivery systems. J. Control. Release 2002;80:9-
28.
36. Bos GW, Jacobs JJL, Koten JW, Van Tomme SR, Veldhuis TFJ, van Nostrum CF, Den Otter W, Hennink WE. In situ crosslinked biodegradable hydrogels loaded with IL-2 are effective tools for local IL-2 therapy. Eur. J. Pharm. Sci.
2004;21:561-567.
37. Ruel-Gariepy E, Leroux J-C. In situ-forming hydrogels--review of temperature-sensitive systems. Eur. J. Pharm.
Biopharm. 2004;58:409-426.
38. Anseth KS, Metters AT, Bryant SJ, Martens PJ, Elisseef JH, Bowman CN. In situ forming degradable networks and
their application in tissue engineering and drug delivery. J. Control. Release 2002;78:199-209.
39. Elbert DL, Pratt AB, Lutolf MP, Halstenberg S, Hubbell JA. Protein delivery from materials formed by self-selective
conjugate addition reactions. J. Control. Release 2001;76:11-25.
40. Lutolf MP, Raeber GP, Zisch AH, Tirelli N, Hubbell JA. Cell-responsive synthetic hydrogels. Adv. Mater. 2003;15:888-892.
41. Hiemstra C, vanderAa LJ, Zhong Z, Dijkstra PJ, Feijen J. Rapidly in situ-forming degradable hydrogels from dextran
thiols through Michael Addition. Biomacromolecules 2007;8:1548-1556.
42. Miyata T, Uragami T, Nakamae K. Biomolecule-sensitive hydrogels. Adv. Drug Deliver. Rev. 2002;54:79-98.
43. Tanna S, Joan Taylor M, Sahota TS, Sawicka K. Glucose-responsive UV polymerised dextran-concanavalin A acrylic
derivatised mixtures for closed-loop insulin delivery. Biomaterials 2006;27:1586-1597.
44. Gupta P, Vermani K, Garg S. Hydrogels: from controlled release to pH-responsive drug delivery. Drug Discov. Today
2002;7:569-579.
45. Huang X, Lowe TL. Biodegradable thermoresponsive hydrogels for aqueous encapsulation and controlled release
of hydrophilic model drugs. Biomacromolecules 2005;6:2131-2139.
46. Vermonden T, Besseling NAM, van Steenbergen MJ, Hennink WE. Rheological studies of thermosensitive triblock
copolymer hydrogels. Langmuir 2006;22:10180-10184.
47. Zhang R, Tang M, Bowyer A, Eisenthal R, Hubble J. A novel pH- and ionic-strength-sensitive carboxy methyl
dextran hydrogel. Biomaterials 2005;26:4677-4683.
48. Lu Z, Kopeckova P, Kopecek J. Antigen responsive hydrogels based on polymerizable antibody Fab' fragment.
Macromol. Biosci. 2003;3:296-300.
49. Coviello T, Matricardi P, Marianecci C, Alhaique F. Polysaccharide hydrogels for modified release formulations. J.
Control. Release 2007;119:5-24.
50. Van Dijk-Wolthuis WNE, Franssen O, Talsma H, van Steenbergen MJ, Kettenes-van den Bosch JJ, Hennink WE.
Synthesis, characterization and polymerization of glycidyl methacrylate derivatized dextran. Macromolecules
1995;28:6317-6322.
51. Van Dijk-Wolthuis WNE, Tsang SKY, Kettenes-van den Bosch JJ, Hennink WE. A new class of polymerizable dextrans with hydrolyzable groups: hydroxyethyl methacrylated dextran with and without oligolactate spacer. Polymer
1997;38:6235-6242.
52. De Jong SJ, De Smedt SC, Demeester J, van Nostrum CF, Kettenes-van den Bosch JJ, Hennink WE. Biodegradable hydrogels based on stereocomplex formation between lactic acid oligomers grafted to dextran. J. Control. Release
2001;72:47-56.
53. Franssen O, Hennink WE. A novel preparation method for polymeric microparticles without the use of organic
solvents. Int. J. Pharm. 1998;168:1-7.
Chapter 1
10
54. Stenekes RJH, Franssen O, van Bommel EMG, Crommelin DJA, Hennink WE. The use of aqueous PEG/dextran
phase separation for the preparation of dextran microspheres. Int. J. Pharm. 1999;183:29-32.
55. De Groot CJ, Cadée JA, Koten JW, Hennink WE, Den Otter W. Therapeutic efficacy of IL-2-loaded hydrogels in a
mouse tumor model. Int. J. Cancer 2002;98:134-140.
56. Vlugt-Wensink KDF, de Vrueh R, Gresnigt MG, Hoogerbrugge CM, van Buul-Offers SC, de Leede LGJ, Sterkman
LGW, Crommelin DJ, Hennink WE, Verrijk R. Preclinical and clinical in vitro in vivo correlation of an hGH dextran
microsphere formulation. 2007 submitted.
57. Cadée JA, van Steenbergen MJ, Versluis C, Heck AJR, Underberg WJM, den Otter W, Jiskoot W, Hennink WE.
Oxidation of recombinant human interleukin-2 by potassium peroxodisulfate. Pharm. Res. 2001;18:1461-1467.
General introduction
11