- Email: [email protected]
The effect of HPβCD on Cyclosporine A in-vitro release from PLGA nanoparticles
e40
Abstracts / Journal of Controlled Release 148 (2010) e21–e56
Fig. 3 shows that in agreement with previous studies [6] the degradation time of the microspheres depends on the copolymer composition: the higher the HMG content, the faster the degradation. It should be stressed that in contrast to our previous study [5], no insoluble residues remained. Fig. 4 shows that the molecular mass of the copolymers decreased in time which demonstrates that the microspheres degrade by bulk degradation. Combining Figs. 2 and 3, it can be concluded that the release of BSA is largely governed by the degradation of the copolymer.
The effect of HPβCD on Cyclosporine A in-vitro release from PLGA nanoparticles Kris Hermans⁎, Wim Weyenberg, Annick Ludwig Laboratory of Pharmaceutical Technology and Biopharmacy, University of Antwerp, Universiteitsplein 1, B2610 Wilrijk-Antwerp, Belgium ⁎Corresponding author. E-mail: [email protected]. Abstract summary A mixture of Cyclosporine A (CyA) and hydroxypropyl-β-cyclodextrin (HPβCD) was prepared to evaluate the effect of HPβCD on the in-vitro release of CyA from PLGA nanoparticles compared to CyA loaded nanoparticles. NMR proton spectroscopy has been used to study the inclusion of CyA into HPβCD which showed no difference compared to the spectrum of CyA, indicating that complexation did not occur. However, a small effect of HPßCD on CyA in-vitro release from PLGA nanoparticles has been observed.
Fig. 3. Relative weight decrease of the dry microspheres prepared from different PLHMGA copolymers. Ratio of HMG to D, L-lactide was 50/50 (▼),35/65 (▲) and 25/75 (■).
n
Fig. 4. Number average molecular weight (M ) decrease of PLHMGA as a function of time. Ratio of HMG to D, L-lactide was 50/50 (▼), 35/65 (▲) and 25/75 (■).
Conclusion Microspheres prepared from PLHMGA showed a sustained release of BSA for 1 week to 2 months dependent on the copolymer composition. The higher the content of HMG, the faster the release. We showed that the microspheres are fully degradable and the degradation kinetics can be tailored by the copolymer composition. The release is to a large extent governed by degradation of the copolymer.
Introduction Cyclosporine A (CyA) is a cyclic undecapeptide with a poor aqueous solubility [1]. Cyclodextrins are cyclic oligosaccharides with a hydrophilic outer surface and a lipophilic central cavity. These cyclic oligosaccharides can form inclusion complexes with lipophilic molecules by taking up the whole molecule or rather some non-polar parts in the hydrophobic cavity. Complexation of drug can result in a higher aqueous solubility compared to the free drug [2,3]. In the present study, NMR proton spectroscopy has been used to study possible complexation of CyA by hydroxypropyl-β-cyclodextrin (HPβCD). The in vitro drug release properties from poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with CyA or CyA/HPβCD mixture were evaluated. Experimental methods Materials Cyclosporine A (MW 1 203) was purchased from Roig Farma (Terrassa, Spain). The PLGA polymer employed was Resomer® RG 503 (Boehringer Ingelheim, Ingelheim am Rhein, Germany) with a molecular weight of 40 000 and a D,L-lactide/glycolide molar ratio of 52/48. Poly(vinylalcohol) (PVA) (MW 30 000–70 000) was purchased from Sigma Chemicals Co. (St Louis, United States of America). Dichloromethane (DCM) and HPβCD (MW 1 380) were obtained from Sigma-Aldrich (Steinheim, Germany), Dmannitol from Sigma-Aldrich Chemicals (Buchs, Switzerland). Phosphate buffered saline pH 7,4 (PBS pH 7,4), containing di-sodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate obtained from Merck (Darmstadt, Germany) and sodium chloride from VWR (Leuven, Belgium), was prepared. Throughout the experiments filtered (Sartorius 0.2 μm membrane filter, Goettingen, Germany) milliQ water was used (Millipore, Mollsheim, France).
References [1] R.C. Mehta, R. Jeyanthi, S. Calls, B.C. Thanoo, K.W. Burton, P.P. DeLuca, Biodegradable microspheres as depot system for patenteral delivery of peptide drugs, J. Control. Release 29 (3) (1994) 375–384. [2] M. van de Weert, W.E. Hennink, W. Jiskoot, Protein instability in poly(lactic-coglycolic acid) microparticles, Pharm. Res. 17 (10) (2000) 1159–1167. [3] Y. Zhang, A. Sophocleous, S. Schwendeman, Inhibition of peptide acylation in PLGA microspheres with water-soluble divalent cationic salts, Pharm. Res. 26 (8) (2009) 1986. [4] M. Leemhuis, C.F. van Nostrum, J.A.W. Kruijtzer, Z.Y. Zhong, M.R. ten Breteler, P.J. Dijkstra, J. Feijen, W.E. Hennink, Functionalized Poly(a-hydroxy acid)s via RingOpening Polymerization: Toward Hydrophilic Polyesters with Pendant Hydroxyl Groups, Macromolecules 39 (10) (2006) 3500–3508. [5] A.H. Ghassemi, M.J. van Steenbergen, H. Talsma, C.F. van Nostrum, W. Jiskoot, D.J.A. Crommelin, W.E. Hennink, Preparation and characterization of protein loaded microspheres based on a hydroxylated aliphatic polyester, poly(lactic-co-hydroxymethyl glycolic acid), J. Control. Release 138 (1) (2009) 57. [6] M. Leemhuis, J.A.W. Kruijtzer, C.F. van Nostrum, W.E. Hennink, In vitro hydrolytic degradation of hydroxyl-functionalized poly(alpha-hydroxy acid), Biomacromolecules 8 (2007) 2943–2949.
doi:10.1016/j.jconrel.2010.07.047
Methods CyA loaded nanoparticles were prepared by the o/w solvent evaporation method [4]. Briefly, an amount of 100 mg CyA and 500 mg PLGA were dissolved in 10 ml DCM. This solution was emulsified in 25 ml of a 1% (w/v) aqueous PVA solution by ultrasonication. This emulsion was then pushed through a high-pressure homogenisator at 500 bar for 1 cycle (M-110L, Microfluidics, Newton, United States of America) in order to reduce the droplet size and to obtain a more narrow size distribution. Afterwards this emulsion was diluted in 120 ml of a 0.3% (w/v) aqueous PVA solution. The solvent was allowed to evaporate under magnetic stirring at room temperature for 4 h. The resulting suspension was then stored at −18 °C and subsequently freeze-dried (FreeZone 1 l Benchtop, Kansas City, United States of America) in the presence of 5% (w/v) mannitol. CyA/HPβCD loaded nanoparticles were prepared by a similar o/w solvent evaporation method. First a mixture (molar ratio 1/10) was prepared by thoroughly mixing 300 mg CyA and 3.45 g HPβCD with a
Abstracts / Journal of Controlled Release 148 (2010) e21–e56
pestle in a mortar for 30 min. Nanoparticles were then prepared using an amount of mixture containing 100 mg CyA. NMR spectra of a solution of the CyA/HPβCD mixture in CD3OD (99.8%D) were recorded at 30 °C on a Bruker DRX 400 NMR spectrometer operating at 400 MHz. 64 scans were accumulated. In order to study the release of CyA, 40 mg of freeze-dried nanoparticles were accurately weighed and transferred to a glass vial containing 10 ml PBS pH 7.4. The vials were oscillated in a water bath at 32 °C. To avoid water evaporation, the vials were covered with rubber caps. Throughout the experiment 4 ml sample was withdrawn at specified time intervals and replaced by an equal volume of fresh buffer solution. The sample was centrifuged for 3 h (4000 rpm, 32 °C) and the CyA concentration of the supernatant was determined by a validated HPLC method. Result and discussion In a CyA/HPβCD inclusion complex it is expected that the lipophilic side chains of CyA will enter the hydrophobic cavity of HPβCD and that shielding of methyl groups will be observed. Therefore the aliphatic region (0.7–1.7 ppm) of the NMR spectrum of the CyA/HβPCD sample was compared in detail to the spectrum of a CyA solution [5]. No substantial differences (<0.003 ppm) could be detected hence there is no evidence for complexation. Considering the large structure of CyA containing 8 lipophilic side chains, a possible explanation may be that a dynamic equilibrium, whereby all different lipophilic side chains only enter the hydrophobic cavity for a relatively short time (i.e. on the average 1/8 of the total time), leads to a proportional reduction of the shielding effect. The cumulative amount of CyA released from the nanoparticles was determined. The results are reported as mean ± SD of three experiments in Table 1. In Fig. 1 the release curves are presented. An initial burst effect followed by a slower release rate was observed. At each time interval, the amount of CyA released is about 5% higher in the presence of HPβCD resulting in a cumulative release of 63% and 81% after 27 h from nanoparticles loaded by respectively CyA and CyA/HPβCD mixture. Table 1 The cumulative percentage of CyA release from CyA and CyA/HPβCD loaded nanoparticles. Time (min)
240 360 540 1620
CyA release (%) ± SD n = 3 CyA loaded nanoparticles
CyA/HPβCD loaded nanoparticles
37 ± 1 51 ± 1 60 ± 0 63 ± 1
42 ± 3 61 ± 1 71 ± 2 81 ± 2
Taking into account the NMR results, the improvement of CyA release in the presence of HPβCD should not be due to formation of a complex with higher aqueous solubility, but to the formation of pores inside the PLGA nanoparticles.
Fig. 1. Release curves of CyA from PLGA nanoparticles (mean ± SD, n = 3).
e41
Conclusion NMR spectroscopy of the prepared CyA/HPβCD mixture showed no evidence for complexation of CyA with HPβCD. Nevertheless, an improvement of CyA release from PLGA nanoparticles containing the CyA/HPβCD mixture compared to CyA loaded nanoparticles is obtained. Acknowledgement The authors are grateful to Prof. Dr. L. Pieters for the performance of NMR spectroscopy (Department of Pharmaceutical Sciences, University of Antwerp, Belgium). References [1] G. Ismailos, C. Reppas, J.B. Dressman, P. Macheras, Unusual solubility behaviour of cyclosporine A in aqueous mediaJ. Pharm. Pharmacol. 43 (1990) 287–289. [2] B. Malaekeh-Nikouei, H. Nassirli, N. Davies, Enhancement of cyclosporine aqueous solubility using α-and hydroxypropyl β-cyclodextrin mixtures, J. Inclusion Phenom. Macrocyclic Chem. 59 (2007) 245–250. [3] Y. Ran, L. Zhao, Q. Xu, S.H. Yalkowsky, Solubilization of Cyclosporine A, Pharm. Sci. Tech. 2 (1) (2001) article 2. [4] J. Jaiswal, S.K. Gupta, J. Kreuter, Preparation of biodegradable cyclosporine nanoparticles by high-pressure emulsification-solvent evaporation process, J. Control. Release 96 (2004) 169–178. [5] H. Kessler, H.R. Loosli, H. Oschkinat, Assignment of the 1H, 13C and 15N-NMR spectra of Cyclosporine A in CDCL3 and C6D6 by a combination of homo-and heteronuclear twodimensional techniques, Helv. Chim. Acta 68 (1985) 661–681.
doi:10.1016/j.jconrel.2010.07.048
Rapid gelation of injectable hydrogels based on hyaluronic acid and poly(ethylene glycol) via Michael-type addition Rong Jin, Pieter J. Dijkstra, Jan Feijen⁎ MIRA Institute for Biomedical Technology and Technical Medicine, Department of Polymer Chemistry and Biomaterials, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands ⁎Corresponding author. E-mail: [email protected]. Abstract summary Injectable hydrogels were formed by a Michael-type addition reaction between thiolated poly(ethylene glycol) (PEG) and maleimide-functionalized hyaluronic acid (HA). Depending on the polymer concentration and the degree of substitution (DS) of HA with maleimide groups, the gelation time and storage moduli of the hydrogels can be varied. The gelation time ranged from less than 5 s to about 0.5 min and storage moduli ranged from 39 to 375 Pa. Moreover, frequency and strain sweeps demonstrated the elastic nature of these hydrogels. Introduction Injectable hydrogels are generally formed by mixing two precursor solutions that gelate in situ by crosslinking. They are attractive for biomedical applications because a minimally invasive procedure is employed and cells and bioactive molecules can be easily and homogeneously incorporated prior to gelation. Michael-type addition is a commonly used crosslinking method to obtain injectable hydrogels. In this study, maleimide-functionalized hyaluronic acids (HA-Mal) with different degree of substitution (DS) were crosslinked with thiolated 4-arm poly(ethylene glycol) (PEG-4SH, MW = 10 kg/ mol) via Michael-type addition. The influences of the DS and polymer concentration on the gelation time and modulus were studied.
Experimental methods Polymer synthesis Hyaluronic acid (HA) (MW = 45 kg/mol, measured by viscosity) was functionalized with maleimide moieties according to previously
Abstracts / Journal of Controlled Release 148 (2010) e21–e56
Fig. 3 shows that in agreement with previous studies [6] the degradation time of the microspheres depends on the copolymer composition: the higher the HMG content, the faster the degradation. It should be stressed that in contrast to our previous study [5], no insoluble residues remained. Fig. 4 shows that the molecular mass of the copolymers decreased in time which demonstrates that the microspheres degrade by bulk degradation. Combining Figs. 2 and 3, it can be concluded that the release of BSA is largely governed by the degradation of the copolymer.
The effect of HPβCD on Cyclosporine A in-vitro release from PLGA nanoparticles Kris Hermans⁎, Wim Weyenberg, Annick Ludwig Laboratory of Pharmaceutical Technology and Biopharmacy, University of Antwerp, Universiteitsplein 1, B2610 Wilrijk-Antwerp, Belgium ⁎Corresponding author. E-mail: [email protected]. Abstract summary A mixture of Cyclosporine A (CyA) and hydroxypropyl-β-cyclodextrin (HPβCD) was prepared to evaluate the effect of HPβCD on the in-vitro release of CyA from PLGA nanoparticles compared to CyA loaded nanoparticles. NMR proton spectroscopy has been used to study the inclusion of CyA into HPβCD which showed no difference compared to the spectrum of CyA, indicating that complexation did not occur. However, a small effect of HPßCD on CyA in-vitro release from PLGA nanoparticles has been observed.
Fig. 3. Relative weight decrease of the dry microspheres prepared from different PLHMGA copolymers. Ratio of HMG to D, L-lactide was 50/50 (▼),35/65 (▲) and 25/75 (■).
n
Fig. 4. Number average molecular weight (M ) decrease of PLHMGA as a function of time. Ratio of HMG to D, L-lactide was 50/50 (▼), 35/65 (▲) and 25/75 (■).
Conclusion Microspheres prepared from PLHMGA showed a sustained release of BSA for 1 week to 2 months dependent on the copolymer composition. The higher the content of HMG, the faster the release. We showed that the microspheres are fully degradable and the degradation kinetics can be tailored by the copolymer composition. The release is to a large extent governed by degradation of the copolymer.
Introduction Cyclosporine A (CyA) is a cyclic undecapeptide with a poor aqueous solubility [1]. Cyclodextrins are cyclic oligosaccharides with a hydrophilic outer surface and a lipophilic central cavity. These cyclic oligosaccharides can form inclusion complexes with lipophilic molecules by taking up the whole molecule or rather some non-polar parts in the hydrophobic cavity. Complexation of drug can result in a higher aqueous solubility compared to the free drug [2,3]. In the present study, NMR proton spectroscopy has been used to study possible complexation of CyA by hydroxypropyl-β-cyclodextrin (HPβCD). The in vitro drug release properties from poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with CyA or CyA/HPβCD mixture were evaluated. Experimental methods Materials Cyclosporine A (MW 1 203) was purchased from Roig Farma (Terrassa, Spain). The PLGA polymer employed was Resomer® RG 503 (Boehringer Ingelheim, Ingelheim am Rhein, Germany) with a molecular weight of 40 000 and a D,L-lactide/glycolide molar ratio of 52/48. Poly(vinylalcohol) (PVA) (MW 30 000–70 000) was purchased from Sigma Chemicals Co. (St Louis, United States of America). Dichloromethane (DCM) and HPβCD (MW 1 380) were obtained from Sigma-Aldrich (Steinheim, Germany), Dmannitol from Sigma-Aldrich Chemicals (Buchs, Switzerland). Phosphate buffered saline pH 7,4 (PBS pH 7,4), containing di-sodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate obtained from Merck (Darmstadt, Germany) and sodium chloride from VWR (Leuven, Belgium), was prepared. Throughout the experiments filtered (Sartorius 0.2 μm membrane filter, Goettingen, Germany) milliQ water was used (Millipore, Mollsheim, France).
References [1] R.C. Mehta, R. Jeyanthi, S. Calls, B.C. Thanoo, K.W. Burton, P.P. DeLuca, Biodegradable microspheres as depot system for patenteral delivery of peptide drugs, J. Control. Release 29 (3) (1994) 375–384. [2] M. van de Weert, W.E. Hennink, W. Jiskoot, Protein instability in poly(lactic-coglycolic acid) microparticles, Pharm. Res. 17 (10) (2000) 1159–1167. [3] Y. Zhang, A. Sophocleous, S. Schwendeman, Inhibition of peptide acylation in PLGA microspheres with water-soluble divalent cationic salts, Pharm. Res. 26 (8) (2009) 1986. [4] M. Leemhuis, C.F. van Nostrum, J.A.W. Kruijtzer, Z.Y. Zhong, M.R. ten Breteler, P.J. Dijkstra, J. Feijen, W.E. Hennink, Functionalized Poly(a-hydroxy acid)s via RingOpening Polymerization: Toward Hydrophilic Polyesters with Pendant Hydroxyl Groups, Macromolecules 39 (10) (2006) 3500–3508. [5] A.H. Ghassemi, M.J. van Steenbergen, H. Talsma, C.F. van Nostrum, W. Jiskoot, D.J.A. Crommelin, W.E. Hennink, Preparation and characterization of protein loaded microspheres based on a hydroxylated aliphatic polyester, poly(lactic-co-hydroxymethyl glycolic acid), J. Control. Release 138 (1) (2009) 57. [6] M. Leemhuis, J.A.W. Kruijtzer, C.F. van Nostrum, W.E. Hennink, In vitro hydrolytic degradation of hydroxyl-functionalized poly(alpha-hydroxy acid), Biomacromolecules 8 (2007) 2943–2949.
doi:10.1016/j.jconrel.2010.07.047
Methods CyA loaded nanoparticles were prepared by the o/w solvent evaporation method [4]. Briefly, an amount of 100 mg CyA and 500 mg PLGA were dissolved in 10 ml DCM. This solution was emulsified in 25 ml of a 1% (w/v) aqueous PVA solution by ultrasonication. This emulsion was then pushed through a high-pressure homogenisator at 500 bar for 1 cycle (M-110L, Microfluidics, Newton, United States of America) in order to reduce the droplet size and to obtain a more narrow size distribution. Afterwards this emulsion was diluted in 120 ml of a 0.3% (w/v) aqueous PVA solution. The solvent was allowed to evaporate under magnetic stirring at room temperature for 4 h. The resulting suspension was then stored at −18 °C and subsequently freeze-dried (FreeZone 1 l Benchtop, Kansas City, United States of America) in the presence of 5% (w/v) mannitol. CyA/HPβCD loaded nanoparticles were prepared by a similar o/w solvent evaporation method. First a mixture (molar ratio 1/10) was prepared by thoroughly mixing 300 mg CyA and 3.45 g HPβCD with a
Abstracts / Journal of Controlled Release 148 (2010) e21–e56
pestle in a mortar for 30 min. Nanoparticles were then prepared using an amount of mixture containing 100 mg CyA. NMR spectra of a solution of the CyA/HPβCD mixture in CD3OD (99.8%D) were recorded at 30 °C on a Bruker DRX 400 NMR spectrometer operating at 400 MHz. 64 scans were accumulated. In order to study the release of CyA, 40 mg of freeze-dried nanoparticles were accurately weighed and transferred to a glass vial containing 10 ml PBS pH 7.4. The vials were oscillated in a water bath at 32 °C. To avoid water evaporation, the vials were covered with rubber caps. Throughout the experiment 4 ml sample was withdrawn at specified time intervals and replaced by an equal volume of fresh buffer solution. The sample was centrifuged for 3 h (4000 rpm, 32 °C) and the CyA concentration of the supernatant was determined by a validated HPLC method. Result and discussion In a CyA/HPβCD inclusion complex it is expected that the lipophilic side chains of CyA will enter the hydrophobic cavity of HPβCD and that shielding of methyl groups will be observed. Therefore the aliphatic region (0.7–1.7 ppm) of the NMR spectrum of the CyA/HβPCD sample was compared in detail to the spectrum of a CyA solution [5]. No substantial differences (<0.003 ppm) could be detected hence there is no evidence for complexation. Considering the large structure of CyA containing 8 lipophilic side chains, a possible explanation may be that a dynamic equilibrium, whereby all different lipophilic side chains only enter the hydrophobic cavity for a relatively short time (i.e. on the average 1/8 of the total time), leads to a proportional reduction of the shielding effect. The cumulative amount of CyA released from the nanoparticles was determined. The results are reported as mean ± SD of three experiments in Table 1. In Fig. 1 the release curves are presented. An initial burst effect followed by a slower release rate was observed. At each time interval, the amount of CyA released is about 5% higher in the presence of HPβCD resulting in a cumulative release of 63% and 81% after 27 h from nanoparticles loaded by respectively CyA and CyA/HPβCD mixture. Table 1 The cumulative percentage of CyA release from CyA and CyA/HPβCD loaded nanoparticles. Time (min)
240 360 540 1620
CyA release (%) ± SD n = 3 CyA loaded nanoparticles
CyA/HPβCD loaded nanoparticles
37 ± 1 51 ± 1 60 ± 0 63 ± 1
42 ± 3 61 ± 1 71 ± 2 81 ± 2
Taking into account the NMR results, the improvement of CyA release in the presence of HPβCD should not be due to formation of a complex with higher aqueous solubility, but to the formation of pores inside the PLGA nanoparticles.
Fig. 1. Release curves of CyA from PLGA nanoparticles (mean ± SD, n = 3).
e41
Conclusion NMR spectroscopy of the prepared CyA/HPβCD mixture showed no evidence for complexation of CyA with HPβCD. Nevertheless, an improvement of CyA release from PLGA nanoparticles containing the CyA/HPβCD mixture compared to CyA loaded nanoparticles is obtained. Acknowledgement The authors are grateful to Prof. Dr. L. Pieters for the performance of NMR spectroscopy (Department of Pharmaceutical Sciences, University of Antwerp, Belgium). References [1] G. Ismailos, C. Reppas, J.B. Dressman, P. Macheras, Unusual solubility behaviour of cyclosporine A in aqueous mediaJ. Pharm. Pharmacol. 43 (1990) 287–289. [2] B. Malaekeh-Nikouei, H. Nassirli, N. Davies, Enhancement of cyclosporine aqueous solubility using α-and hydroxypropyl β-cyclodextrin mixtures, J. Inclusion Phenom. Macrocyclic Chem. 59 (2007) 245–250. [3] Y. Ran, L. Zhao, Q. Xu, S.H. Yalkowsky, Solubilization of Cyclosporine A, Pharm. Sci. Tech. 2 (1) (2001) article 2. [4] J. Jaiswal, S.K. Gupta, J. Kreuter, Preparation of biodegradable cyclosporine nanoparticles by high-pressure emulsification-solvent evaporation process, J. Control. Release 96 (2004) 169–178. [5] H. Kessler, H.R. Loosli, H. Oschkinat, Assignment of the 1H, 13C and 15N-NMR spectra of Cyclosporine A in CDCL3 and C6D6 by a combination of homo-and heteronuclear twodimensional techniques, Helv. Chim. Acta 68 (1985) 661–681.
doi:10.1016/j.jconrel.2010.07.048
Rapid gelation of injectable hydrogels based on hyaluronic acid and poly(ethylene glycol) via Michael-type addition Rong Jin, Pieter J. Dijkstra, Jan Feijen⁎ MIRA Institute for Biomedical Technology and Technical Medicine, Department of Polymer Chemistry and Biomaterials, Faculty of Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands ⁎Corresponding author. E-mail: [email protected]. Abstract summary Injectable hydrogels were formed by a Michael-type addition reaction between thiolated poly(ethylene glycol) (PEG) and maleimide-functionalized hyaluronic acid (HA). Depending on the polymer concentration and the degree of substitution (DS) of HA with maleimide groups, the gelation time and storage moduli of the hydrogels can be varied. The gelation time ranged from less than 5 s to about 0.5 min and storage moduli ranged from 39 to 375 Pa. Moreover, frequency and strain sweeps demonstrated the elastic nature of these hydrogels. Introduction Injectable hydrogels are generally formed by mixing two precursor solutions that gelate in situ by crosslinking. They are attractive for biomedical applications because a minimally invasive procedure is employed and cells and bioactive molecules can be easily and homogeneously incorporated prior to gelation. Michael-type addition is a commonly used crosslinking method to obtain injectable hydrogels. In this study, maleimide-functionalized hyaluronic acids (HA-Mal) with different degree of substitution (DS) were crosslinked with thiolated 4-arm poly(ethylene glycol) (PEG-4SH, MW = 10 kg/ mol) via Michael-type addition. The influences of the DS and polymer concentration on the gelation time and modulus were studied.
Experimental methods Polymer synthesis Hyaluronic acid (HA) (MW = 45 kg/mol, measured by viscosity) was functionalized with maleimide moieties according to previously