Biodegradable insulin-loaded PLGA microspheres fabricated by three different emulsification techniques: Investigation for cartilage tissue engineering

Acta Biomaterialia 7 (2011) 1485–1495

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Biodegradable insulin-loaded PLGA microspheres fabricated by three different emulsification techniques: Investigation for cartilage tissue engineering Kristin Andreas a,⇑, Rolf Zehbe b, Maja Kazubek a,1, Karolina Grzeschik a, Nadine Sternberg c, Hans Bäumler c, Helmut Schubert b, Michael Sittinger a, Jochen Ringe a a Tissue Engineering Laboratory and Berlin-Brandenburg Center for Regenerative Therapies, Department of Rheumatology and Clinical Immunology, Charité-Universitätsmedizin Berlin, Föhrer Strasse 15, 13353 Berlin, Germany b Institute of Materials Science and Technologies, Technische Universität Berlin, Englische Strasse 20, 10587 Berlin, Germany c Institute of Transfusion Medicine and Berlin-Brandenburg Center for Regenerative Therapies, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany

a r t i c l e

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Article history: Received 16 August 2010 Received in revised form 17 November 2010 Accepted 13 December 2010 Available online 17 December 2010 Keywords: Insulin PLGA microspheres Controlled release Drug delivery In situ cartilage tissue engineering

a b s t r a c t Growth, differentiation and migration factors facilitate the engineering of tissues but need to be administered with defined gradients over a prolonged period of time. In this study insulin as a growth factor for cartilage tissue engineering and a biodegradable PLGA delivery device were used. The aim was to investigate comparatively three different microencapsulation techniques, solid-in-oil-in-water (s/o/w), water-in-oil-in-water (w/o/w) and oil-in-oil-in-water (o/o/w), for the fabrication of insulin-loaded PLGA microspheres with regard to protein loading efficiency, release and degradation kinetics, biological activity of the released protein and phagocytosis of the microspheres. Insulin-loaded PLGA microspheres prepared by all three emulsification techniques had smooth and spherical surfaces with a negative zeta potential. The preparation technique did not affect particle degradation nor induce phagocytosis by human leukocytes. The delivery of structurally intact and biologically active insulin from the microspheres was shown using circular dichroism spectroscopy and a MCF7 cell-based proliferation assay. However, the insulin loading efficiency (w/o/w about 80%, s/o/w 60%, and o/o/w 25%) and the insulin release kinetics were influenced by the microencapsulation technique. The results demonstrate that the w/o/w microspheres are most appropriate, providing a high encapsulation efficiency and low initial burst release, and thus these were finally used for cartilage tissue engineering. Insulin released from w/o/w PLGA microspheres stimulated the formation of cartilage considerably in chondrocyte high density pellet cultures, as determined by increased secretion of proteoglycans and collagen type II. Our results should encourage further studies applying protein-loaded PLGA microspheres in combination with cell transplants or cell-free in situ tissue engineering implants to regenerate cartilage. Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Growth, differentiation and migration factor-based tissue engineering approaches have evolved to become a powerful and innovative tool in regenerative medicine [1–4]. In general these strategies involve scaffolds that provide physical support, cells that integrate into the defect tissue site and the presence of growth, differentiation and/or migration factors that support proliferation, differentiation and recruitment of cells. The demand for tissue engineered cartilage is immense because of the tissue’s poor intrinsic healing potential; untreated degenerative and traumatic cartilage lesions often progress to degenerative arthritis [5,6]. Thus, ⇑ Corresponding author. Tel.: +49 30 450 513 188; fax: +49 30 450 513 957. E-mail address: [email protected] (K. Andreas). Present address: Chair and Department of Toxicology, Silesian Piasts University of Medicine, Traugutta 57/59, 50-417 Wrocław, Poland. 1

applications in tissue engineering that improve cartilage repair have a high clinical impact. In current cartilage tissue engineering clinical applications cell-seeded or cell-free scaffolds are transplanted to regenerate injured or diseased tissue. Future approaches will combine scaffolds with bioactive factors, for example relying on cell-free implants in combination with chemoattractants that recruit the patient’s own cells for subsequent in situ repair [7]. For tissue engineering the applied factors need to be available in bioactive form for a prolonged period of time. Hence, it is essential to control their spatial and temporal concentration, and this suggests the use of appropriate delivery devices [2,8,9]. A promising tool for controlled and sustained delivery is the entrapment of bioactive factors in biodegradable polymeric materials that release the protein locally at slow rates. In particular, biodegradable poly(D,Llactide–co-glycolide) (PLGA) microspheres have already facilitated distinct advances in growth factor delivery for numerous tissue engineering applications [10–12].

1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.12.014

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With regard to cartilage tissue engineering, selective growth factors here help to form and maintain cartilage and facilitate production of the extracellular matrix (ECM) [9,13,14]. In particular, transforming growth factor-b (TGF-b) and insulin-like growth factor-1 (IGF-1) play a significant role in promoting ECM synthesis and in inhibiting ECM breakdown [15–17], and PLGA microspheres releasing TGF-b and IGF-1 have recently been described [18,19]. Insulin has structural similarities to IGF-1, binds to the IGF-1 receptor and, thus, elicits similar effects on cartilage [20–22]. This suggests that insulin may be an appropriate and inexpensive alternative to IGF-1 as a growth factor to improve tissue engineered cartilage [23]. Recently Göpferich and colleagues established insulin-based cartilage tissue engineering approaches based on the covalent binding of insulin to polymeric scaffolds [24] and on insulin-loaded lipid implants [25,26]. In this study the microencapsulation and solvent evaporation technique was applied to prepare biodegradable insulin-loaded PLGA microspheres. This preparation method usually involves the protein in a solid or liquid form that is mixed with the polymer, which is dissolved in a water immiscible organic solvent. This protein–polymer mixture is then emulsified in an aqueous continuous phase containing an emulsifier such as polyvinyl alcohol. The organic solvent evaporates and the microspheres harden, resulting in entrapment of the protein in the PLGA microspheres [27]. Three different emulsification techniques, solid-in-oil-in-water (s/o/w), water-in-oil-in-water (w/o/w) and oil-in-oil-in-water (o/o/w) were investigated comparatively for the preparation of PLGA–insulin microspheres with respect to the characteristic properties of the microspheres and to the release of structurally intact and bioactive insulin. Using the s/o/w technique the insulin is dispersed in the polymer as a solid powder. Using the w/o/w and o/o/w preparation techniques the insulin is dissolved in an aqueous and organic solvent, respectively, before being mixed with the polymer. The most appropriate formulation technique was determined and, finally, used in cartilage tissue engineering. The aim of this work was to investigate comparatively the effect of three different emulsification techniques (s/o/w, w/o/w and o/o/w) on the loading efficiency, protein release, polymer degradation and biocompatibility of the polymeric particles, and on the structural integrity and bioactivity of the released insulin. Insulin-loaded PLGA microspheres of the most appropriate formulation were finally employed in a high density three-dimensional (3D) in vitro cartilage engineering model and the effects on the formation of cartilage-like tissue were investigated.

ture experiments phosphate-buffered saline (PBS), RPMI 1640 medium, fetal bovine serum (FBS), amphotericin B, penicillin, streptomycin and L-alanyl-L-glutamine were purchased from Biochrom (Berlin, Germany). All further medium supplements and reagents for quantification of hydroxyproline and glycosaminoglycans were obtained from Sigma–Aldrich. 2.1.3. Staining reagents For histological and immunohistochemical staining alcian blue 8GX was obtained from Roth (Karlsruhe, Germany), nuclear fast red from DAKO (Hamburg, Germany) and polyclonal rabbit antihuman types I and II collagen and rabbit immunoglobulin G antibodies were obtained from DPC Biermann (Bad Nauheim, Germany). 2.2. Methods 2.2.1. Preparation of microspheres by microencapsulation and solvent evaporation PLGA microspheres loaded with human insulin were prepared with s1/o2/w2, w1/o2/w2 and o1/o2/w2 emulsions and solvent evaporation (Fig. 1). For preparation of the s1/o2 primary suspension 2 mg lyophilized insulin powder (s1) were dispersed in 1 ml PLGA (o2) (100 mg ml 1 in methylene chloride) by vortex mixing at 2500 rpm for 1 min (vortex genius 3, IKA Laboratory Technology, Staufen, Germany). The w1/o2 primary emulsion was prepared by emulsifying an aqueous insulin solution (w1) (2 mg in 75 ll of 0.01 M HCl) into 1 ml PLGA (o2) (100 mg ml 1 in methylene chloride) by vortex mixing at 2500 rpm for 1 min. The 2 mg of human insulin were dissolved in 50 ll organic solvent HFIP (o1) and vortex mixed with 1 ml PLGA (o2) (100 mg ml 1 in methylene chloride) at 2500 rpm for 1 min to prepare the o1/o2 emulsion. To further prove the existence of an oil-in-oil emulsion in the o/o/w formulation, insulin was dissolved in HFIP, emulsified in methylene chloride and investigated under a light microscope. The primary s1/o2, w1/o2 and o1/o2 mixtures were emulsified in 2 ml external aqueous phase (w2) of 1% (w/v) PVA by vortex mixing at 2500 rpm for 3 min. The s1/o2/w2, w1/o2/w2 and o1/o2/w2 emulsions were then poured into 20 ml of 0.5% (w/v) PVA/450 mM NaCl solution and stirred for 4 h on a magnetic stirrer at 700 rpm to evaporate the solvent. Hardened microspheres were recovered by centrifugation (2000 rpm, 5 min), washed three times with distilled water and freeze dried (Thermo Fisher Scientific, Dreieich, Germany). FITC–insulin-loaded microspheres and blank microspheres (without insulin) were prepared accordingly.

2. Materials and methods 2.1. Materials 2.1.1. Preparation and characterization of insulin-loaded PLGA microspheres PLGA was purchased from Boehringer Ingelheim (Ingelheim, Germany) with a lactide:glycolide ratio of 50:50, a residual monomer content of 60.5% and an inherent viscosity of 0.32–0.44 dl g 1. Methylene chloride (CH2Cl2) was obtained from Merck (Darmstadt, Germany) and hexafluoroisopropanol (HFIP) was purchased from DuPont (Neu-Isenburg, Germany). Poly(vinyl alcohol) (PVA) (molecular weight 67,000), recombinant human insulin and FITC–insulin were purchased from Sigma–Aldrich (Taufkirchen, Germany). All further reagents, including NaCl, NaF and Na2HPO4, were also obtained from Sigma–Aldrich. 2.1.2. Cell culture The insulin-sensitive human breast cancer cell line MCF7 was purchased from LGC Standards (Wesel, Germany). For the cell cul-

2.2.2. Characterization of insulin-loaded PLGA microspheres 2.2.2.1. Scanning electron microscopy. Freeze dried particles were distributed homogeneously on aluminum SEM stubs with selfadhesive graphite pads, gold sputtered (Emitech K550, Röntgenanalytik Messtechnik, Taunusstein, Germany) in an argon atmosphere (10 2 mbar, 240 s, 30 lA) and inspected using a scanning electron microscope (Philips XL20, FEI, Eindhoven, The Netherlands). 2.2.2.2. Particle size distribution. Insulin-loaded PLGA microspheres were investigated for particle size distribution by laser light diffraction (Sympatec Helos Sucell 2, Sympatec GmbH, ClausthalZellerfeld, Germany). Particles were suspended in distilled water and ultrasound was applied prior to measurement (60 W, 60 s). The particle sizes were calculated with the Fraunhofer approximation and are given as volume diameter at 10% (DV,10,), 50% (DV,50) and 90% (DV,90) of the volume distribution, representing the percentage of particles smaller than the indicated value (means ± standard deviation, n = 5). The population dispersity is referred as the span and was calculated as (DV,90 DV,10)/DV,50.

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Fig. 1. Processing technique for insulin immobilization in PLGA microspheres according to the s/o/w, w/o/w and o/o/w formulations. Insulin immobilization (A) was carried out by (a) emulsifying water solubilized insulin in PLGA–CH2Cl2, (b) suspending insulin in PLGA–CH2Cl2 or (c) dissolving insulin in HFIP (primary oily phase) and using PLGA– CH2Cl2 as the secondary oily phase. In a second step (B) these formulations were emulsified in 1% PVA as the external phase by vortex mixing and particles were hardened in a bath of 0.5% PVA/450 mM NaCl. The displayed human insulin was generated from the 3i3z-file (www.pdb.org) using RasTop (http://www.rasmol.org/).

2.2.2.3. Zeta potential. The particle charge was quantified as the zeta potential by laser Doppler velocimetry and phase analysis light scattering using a Zetasizer nano ZS (Malvern Instruments, Malvern, UK). Samples were diluted in distilled water. The results are shown as means ± standard deviation (n = 5). 2.2.2.4. Insulin loading efficiency. Microspheres were dissolved in methylene chloride and insulin was extracted into 0.1 N HCl under vigorous shaking. The dispersion was allowed to settle and centrifuged (5000 rpm, 15 min). The protein concentration of the upper aqueous phase was determined using the bicinchoninic acid assay (BCA assay) according to the manufacturer’s instructions (Sigma– Aldrich). The loading efficiency (wt.%) was calculated as the insulin entrapped in the microspheres as a percentage of the total amount of protein used for particle preparation (n = 3). 2.2.2.5. Insulin release and polymer degradation. About 100 mg insulin-loaded microspheres were incubated in 1 ml PBS in a rotary shaker (neoLab, Heidelberg, Germany) at 3 rpm and 37 °C. At predetermined time intervals the samples were centrifuged (3000 rpm, 1 min), the supernatants withdrawn and the microspheres were resuspended in fresh PBS. The protein content of the supernatants was determined with the BCA assay. Percent cumulative release at each time was normalized to the total amount of protein released up to the last day of the study. All samples were run in triplicate and data points are shown as means ± standard deviation. For determination of particle mass loss 100 mg microspheres were centrifuged (3000 rpm, 1 min) after 3, 5, 7 and 9 weeks incubation in 1 ml PBS, freeze dried and weighed. The dry weight loss was calculated as the percentage of particle weight prior to and after incubation. The data points are shown as means ± standard deviation (n = 3). 2.2.2.6. Phagocytosis of insulin-loaded PLGA microspheres. Phagocytosis of FITC–insulin-loaded microspheres was assessed using the Phagotest Kit (Orpegen Pharma, Heidelberg, Germany), which is based on the ingestion of fluorescently labeled samples by human

leukocytes and subsequent detection of phagocytic cells by flow cytometry. Samples of human heparinized whole blood (n = 6 healthy donors) were cooled on ice, mixed with FITC–insulin PLGA microspheres and incubated for 1 h at 37 °C to analyze particle phagocytosis. FITC-labeled opsonized Escherichia coli (FITC–E. coli) served as positive controls. Simultaneously, FITC–E. coli were put on ice to reduce the phagocytic activity to a minimum and these samples were used as negative controls. Afterwards, quenching solution was added and, following two washing steps, the erythrocytes were lysed, washed out and propidium iodide was added. The percentage of leukocytes that had ingested FITC–E. coli or FITC– insulin particles was determined by flow cytometric analysis using a 488 nm argon ion laser (FACS-Canto, BD, Franklin Lakes, NJ). 2.2.3. Structural integrity and bioactivity of released insulin 2.2.3.1. Circular dichroism spectroscopy. The structural integrity of insulin released within the first 5 days from s/o/w, w/o/w and o/ o/w microspheres into a solution of 154 mM NaF/10 mM Na2HPO4 was compared with native (not released) insulin using circular dichroism (CD) spectroscopy (Jasco J-720 spectrometer, Jasco, Tokyo, Japan). The released insulin samples were filtered (0.2 lm) and measurements were performed using a 1 cm path cuvette at 100 nm min 1 scanning speed, with a step size of 0.1 nm at 25 °C and a wavelength range of 195–260 nm. Each spectrum is the average of 10 runs. The data are expressed as mean residual ellipticity (hmr). 2.2.3.2. Proliferation assay with human breast cancer cell line MCF7. Bioactivity of the released insulin was assessed using a proliferation assay with the insulin-sensitive human breast cancer cell line MCF7 [28–30]. Briefly, s/o/w, w/o/w and o/o/w insulin-loaded particles were incubated in serum-free RPMI 1640 medium supplemented with 100 U ml 1 penicillin, 100 lg ml 1 streptomycin, 2 mM L-alanyl-L-glutamine and 55 lg ml 1 transferrin in a rotary shaker for 5 days. The concentration of released insulin in the supernatant was determined using ELISA according to the manufacturer’s instructions (Mercodia, Uppsala, Sweden). MCF7 cells

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were seeded at 10,000 cells cm 2 in RPMI 1640 medium supplemented with 10% FBS, 100 U ml 1 penicillin and 100 lg ml 1 streptomycin at 37 °C in a 5% CO2 atmosphere. When they had reached 50% confluence the cells were washed with PBS, serum starved for 24 h and cultivated for 6 days with release supernatants containing 10 lg ml 1 insulin released from the s/o/w, w/o/w and o/o/w microspheres, respectively. Medium supplemented with 10 lg ml 1 native (not released) insulin served as a positive control and supernatants of blank s/o/w, w/o/w and o/o/w microspheres were used as negative controls. The medium was changed every 2 days. The assay was performed under serum-free conditions to detect the effect of insulin on MCF7 cell proliferation. Cell number was quantified using the MTT proliferation assay following the manufacturer’s instructions (Promega, Mannheim, Germany). Data points are shown as means ± standard deviation (n = 3).

For the histological analyses pellets were embedded in TissueTec (Sakura, Zoeterwoude, The Netherlands) and cryosections were mounted on aminoalkyl silane-coated slides. Proteoglycans were stained with alcian blue 8GX at pH 2.5 and counterstained with nuclear fast red. Immunohistochemical collagen staining and quantification of glycosaminoglycans and hydroxyproline were performed as described previously [31–33]. In brief, the cartilage-specific content of glycosaminoglycans per pellet was determined spectrophotometrically at 525 nm following papain digestion and reaction with dimethylmethylene blue. The amount of hydroxyproline, a major component of collagen fibrils, per pellet was measured spectrophotometrically at 595 nm following papain digestion, acid hydrolysis and reaction with chloramine-T and pdimethylaminobenzaldehyde. 3. Results

2.2.3.3. Three-dimensional cartilage engineering pellet culture. Cartilage was harvested from porcine femurs obtained from a local slaughterhouse, therefore ethical committee consent was unnecessary. Porcine chondrocytes were isolated and expanded in monolayer culture as described previously [31]. For the formation of high density pellet cultures 0.6  106 chondrocytes (passage 2) per 200 ll were placed in the wells of a 96-well tissue culture plate and cultivated in RPMI 1640 medium supplemented with 100 U ml 1 penicillin, 100 lg ml 1 streptomycin, 100 ng ml 1 amphotericin B, 50 lg ml 1 L-ascorbic acid 2-phosphate, 5.5 lg ml 1 transferrin, 5 ng ml 1 selenium, 0.5 mg ml 1 bovine serum albumin, 4.7 lg ml 1 linoleic acid and 10 lg ml 1 insulin released from w/o/w microspheres. Supernatants of blank w/o/w microspheres served as negative controls. The medium was changed every day and pellets were harvested after 1 week of serumfree culture. Serum-free cultivation was performed to detect the effect of insulin on chondrocyte ECM synthesis. All experiments were run in triplicate.

3.1. Characteristics of insulin-loaded PLGA microspheres Morphological examination using scanning electron microscopy showed that microparticles of all three encapsulation techniques had smooth and spherical surfaces without aggregation or visible pores at the surface (Fig. 2, day 0). The size distribution of the PLGA–insulin microspheres was rather narrow for preparation with a vortex mixer: 10% of the microspheres were smaller than 5.8 ± 0.9 lm for s/o/w, 5.6 ± 0.4 lm for w/o/w and 5.5 ± 0.2 lm for o/o/w and none of the particles was larger than 50 lm (Table 1 and Fig. 3A). The span, which describes the extent of the particle size distribution was about 1.2 for all particle formulations. The average median particle size (DV,50) was slightly decreased for microspheres prepared by o/o/w (18.2 ± 0.3 lm) compared with s/o/w (20.9 ± 0.1 lm) and w/o/w (21.5 ± 0.2 lm) particles. Zeta potential analysis revealed that the insulin-loaded microspheres made by all preparation techniques had negative surface charges

Fig. 2. SEM analysis of insulin-loaded PLGA microspheres prepared by the three techniques s/o/w, w/o/w and o/o/w over time. Directly after preparation on day 0 the microspheres were round with smooth surfaces. During incubation in phosphate buffer at 37 °C the particles degraded, developed pores at the surface, disintegrated and finally the particle fragments coalesced.

Table 1 Particle size distribution, zeta potential and encapsulation efficiency of insulin-loaded PLGA microspheres prepared by the techniques s/o/w, w/o/w and o/o/w. Formulation

Particle size distribution (lm) DV,10

DV,50

DV,90

s/o/w w/o/w o/o/w

5.8 ± 0.9 5.6 ± 0.4 5.5 ± 0.2

20.9 ± 0.1 21.5 ± 0.2 18.2 ± 0.3

29.8 ± 0.1 31.9 ± 0.3 26.5 ± 0.3

Zeta potential (mV)

12.3 ± 1.3 13.6 ± 1.4 14.1 ± 1.6

Encapsulation efficiency (wt.%)

59.0 ± 1.2 79.9 ± 2.3 25.2 ± 1.1

The particle size distribution is presented as the diameter at 10% (DV,10,), 50% (DV,50) and 90% (DV,90) of the volume distribution. The data are shown as means ± SD for at least three determinations.

K. Andreas et al. / Acta Biomaterialia 7 (2011) 1485–1495

differential volume (%)

A

s/o/w w/o/w

22 1,5 1.5

11 0,5 0.5

cumulative release (%)

10 particle size ( m)

100

10

100

100 100

8080 6060 4040 s/o/w 2020 00

w/o/w o/o/w 0 0

C

3.2. Release of insulin and degradation of microspheres

o/o/w

00 1 1

B

complex in methylene chloride with a droplet diameter of approximately 1–10 lm (data not shown).

33 2,5 2.5

7 7

14 21 28 35 42 time (days) 14

21

28

35

42

56 49 56 49

63 63

100 100

The cumulative in vitro release profiles of insulin from the s/o/w, w/o/w and o/o/w microparticles are shown in Fig. 3B. Release from microspheres prepared by s/o/w microencapsulation showed a triphasic profile. Here a high initial insulin burst release of about 32% was observed within the first day, which is normally attributed to the presence of protein at or near the surface of the microspheres. Afterwards a period of slightly increasing insulin release occurred up to day 21, followed by an elevated protein release phase for several weeks. In contrast, the w/o/w and o/o/w PLGA–insulin microspheres showed only a minimal initial burst release (0.3% for w/o/w, 3% for o/o/w) that was also followed by a period of slightly increasing release up to day 21, and then by a period of high protein release for several weeks. Particle degradation was investigated by SEM (Fig. 2) and by particle mass loss (Fig. 3C) and was closely correlated with the insulin release profiles. Up to the end of the third week the insulin-loaded microspheres remained intact and the dry weight remained constant, resulting in slow insulin release rates during the first 3 weeks. As can be seen in the SEM images and the particle mass loss profiles, degradation started rapidly around day 21, which corresponds to high insulin release rates, due to erosion of the polymeric matrix. Degradation of the microspheres was independent of the microencapsulation technique that was used for particle preparation. During degradation the smooth and spherical surfaces of the microspheres developed numerous pores, lost their spherical shape and, finally, showed increased coalescence. After 9 weeks the particles had turned into an unstructured mass, disintegrated into irregular shaped fragments and the dry weight decreased to about 15% of the initial value. 3.3. Insulin-loaded particles were not phagocytosed

8080 dry weight (%)

1489

6060 4040 s/o/w 20

w/o/w

20

o/o/w

00 0 0

7 7

14 21 28 14

21

28

35 42 35

42

49 56 49

56

63 63

time (days) Fig. 3. Particle size distribution (A), cumulative insulin release profiles (B) and particle mass loss (C) of PLGA microspheres. The s/o/w, w/o/w and o/o/w formulations of insulin-loaded microspheres showed similar volume distributions with an average median particle size (DV,50) of 18.2 ± 0.3 lm (o/o/w), 20.9 ± 0.1 lm (s/o/w) and 21.5 ± 0.2 lm (w/o/w). High initial insulin burst release only occurred from s/o/w microspheres. After a period of 3 weeks of slow protein release particles prepared by all three techniques released high amounts of insulin for several weeks. Accordingly, the dry weight of the microspheres decreased rapidly between weeks 3 and 7. Particles prepared by the different techniques s/o/w, w/o/w and o/o/w showed very similar mass loss kinetics.

( 12.3 ± 1.3 mV for s/o/w, 13.6 ± 1.4 mV for w/o/w, 14.1 ± 1.6 mV for o/o/w, Table 1). Particles prepared by w/o/w had the highest loading efficiency (79.9 ± 2.3%) compared with those prepared by s/o/w (59.0 ± 1.2%) and o/o/w (25.2 ± 1.1%) (Table 1). For the o/o/w formulation light microscopy clearly demonstrated the existence of an emulsified oily phase of the supposed insulin–HFIP

When human leukocytes were exposed to opsonized FITC–E. coli (positive control), the phagocytotic activity was considerably elevated and the positive control samples were highly phagocytosed by human granulocytes (95.6 ± 2.8%) and monocytes (62.5 ± 16.3%) (Fig. 4). In contrast, exposure of human granulocytes and monocytes to FITC–insulin-loaded microspheres elicited very little cell-mediated immune response, irrespective of the particle preparation technique. Thus the cellular inflammatory response to the insulin-loaded microspheres prepared by all three techniques was very low and similar to the negative control. 3.4. Released insulin showed structural integrity and bioactivity CD spectroscopical analyses were performed to analyze changes in the secondary structure of burst released insulin from s/o/w, w/o/w and o/o/w microspheres in comparison with the native (not released) protein (Fig. 5). The CD spectra of insulin released within the first 5 days from particles prepared by all three techniques were similar to the standard insulin spectrum, indicating no alterations in the secondary protein structure of the released insulin. The CD spectra showed two minima around 208 and 223 nm that are typical of predominantly a-helical proteins like insulin. To determine the biological activity of the insulin released from particles prepared by the three microencapsulation techniques its proliferative effect on the insulin-sensitive human breast cancer cell line MCF7 was analyzed in comparison with the effect of the same concentration of native (not released) insulin. As shown in Fig. 6, the supplementation of serum-free medium with insulin released from all tested particle formulations stimulated the growth

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100

human granulocytes

phagocytosis (%)

80

human monocytes

60

40 10

0

positive control

negative control

s/o/w particles

w/o/w particles

o/o/w particles

x 103 (deg cm2 dmol-1)

30

mr]

Fig. 4. Phagocytosis of PLGA microspheres by human leukocytes. FITC-labeled opsonized Escherichia coli (positive control) and FITC–insulin PLGA microspheres prepared by the three emulsification techniques s/o/w, w/o/w and o/o/w were incubated with human leukocytes (n = 6 donors) at 37 °C. Negative control samples with FITC-labeled opsonized E. coli were incubated at 4 °C to reduce phagocytosis to a minimum. Positive control samples were highly phagocytosed by human granulocytes (95.6 ± 2.8%) and monocytes (62.5 ± 16.3%). FITC–insulin-loaded microspheres prepared by the tested techniques s/o/w, w/o/w and o/o/w did not show elevated phagocytosis by human granulocytes and monocytes compared with the negative control.

-10

native insulin s/o/w insulin w/o/w insulin o/o/w insulin

20 10

[

0

210

220 (nm)

230

240

Fig. 5. Far-UV circular dichroism (CD) spectra of insulin released from PLGA microspheres prepared by the three microencapsulation techniques s/o/w, w/o/w and o/o/w and native (not released) insulin. The CD spectra of insulin released from the microspheres were very similar to the spectrum of native (not released) insulin, indicating no alterations in the protein structure of the insulin after release.

of MCF7 cells. Cultivation of MCF7 cells for 6 days with medium supplemented with released insulin resulted in an increase in cell number compared with the blank controls (Fig. 6A) and in MCF7 cell adherence to the cell culture plate and the typical cell morphology (Fig. 6B). The proliferative effects of the released insulin for all three microencapsulation techniques on MCF7 cells were similar to the effect of the same concentration of native (not released) insulin, indicating that the released insulin was fully biologically active. MCF7 cells that were cultivated without insulin (blank controls) showed a decreased cell number and a round morphology with limited adherence potential. 3.5. Insulin release positively effects cartilage formation Finally in this study, the effect of insulin released from w/o/w microspheres on the in vitro formation of ECM by chondrocyte pel-

let cultures was investigated. The w/o/w formulation was chosen for the cartilage tissue engineering studies because this microencapsulation technique resulted in the highest insulin loading efficiency, lowest initial burst release and appropriate release kinetics. Similarly to both other tested formulation techniques, the insulin released from w/o/w microspheres possessed structural integrity and bioactivity. Chondrocyte pellets cultured in serum-free medium supplemented with insulin released from w/o/w microspheres increased in size, thickness and dry weight over time compared with control pellets that received no insulin supplementation (data not shown). Cryosections of chondrocyte pellets were stained with alcian blue for proteoglycans and with specific antibodies for collagen types I and II. Furthermore, the pellet hydroxyproline content, one major component of collagen fibrils, and of sulfated glycosaminoglycans, which are covalently linked to core proteins to form proteoglycans, were quantified. Cultivation of chondrocyte pellets with insulin released from w/o/w microspheres led to strong secretion of cartilage-specific proteoglycans and collagen type II, as shown by histological and immunohistochemical staining (Fig. 7), indicating the formation of cartilage-like ECM. Proteoglycan and collagen type II secretion was also evident for the blank control pellets, although to a far lesser extent. Insulin and the control pellets stained slightly positive for collagen type I. Consistent with the histological and immunohistochemical staining, pellets cultivated with released insulin showed considerably higher contents of glycosaminoglycans and hydroxyproline compared with the blank controls (Fig. 8).

4. Discussion Encapsulation of proteins in PLGA microspheres by the emulsification and solvent evaporation technique has been investigated over the past 25 years and insulin is probably one of the most widely studied proteins for drug delivery, in particular for the treatment of diabetes. Nevertheless, to our knowledge this is the first comprehensive and comparative study to investigate the influence of three different insulin microencapsulation techniques,

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Fig. 6. Effect of released insulin on the proliferation of the insulin-sensitive human breast cancer cell line MCF7. Insulin released from s/o/w, w/o/w and o/o/w microspheres into the culture medium was sampled and adjusted to 10 lg ml 1. The supernatant of blank microspheres served as a control. MCF7 cells were expanded to 50% confluence, serum starved for 24 h and cultivated with the release supernatants for 6 days. (A) Insulin released from all three formulations s/o/w, w/o/w and o/o/w stimulated the proliferation of MCF7 breast cancer cells compared with the blank controls. The proliferative effect of the released insulin was similar to the effect of the same concentration of native (not released) insulin. The values are shown as means ± SD. (B) Supplementation of serum-free cell culture medium with the released insulin had positive effects on MCF7 cell adherence and morphology. Medium supplementation with 10 lg ml 1 (a) native (not released) insulin and insulin released from (b) s/o/w, (c) w/o/w and (d) o/o/w microspheres and (e–h) the respective blank controls.

s/o/w, w/o/w and o/o/w, on the microsphere properties, protein release, particle degradation and phagocytosis and on the structural integrity and bioactivity of the released insulin. Finally, the most appropriate formulation method was applied in cartilage tissue engineering. Several studies have dealt with the sustained release of proteins from PLGA microspheres due to the high biocompatibility and biodegradability of this co-polymer. Protein release from the PLGA microspheres occurs via diffusion and polymer erosion due to hydrolysis of the ester bonds in the polymer backbone. Different microencapsulation and solvent evaporation techniques for the encapsulation of proteins in PLGA microspheres can be applied. Our results show that the techniques s/o/w, w/o/w and o/o/w had no significant effect on particle morphology and surface charge, on particle phagocytosis by human leukocytes and on the structural integrity and bioactivity of the released insulin. Even the use of the polar organic solvent HFIP with strong hydrogen bonding properties in the o/o/w formulation did not show any adverse effects on the released insulin. Both organic solvents (methylene chloride and HFIP) employed in this study are potentially hazardous. These solvents may be retained in the microspheres as a residual organic volatile impurity and, thus, determination of the residual solvent content in the microspheres is required. According to the United States Pharmacopeia the residual solvent limit for methylene chloride is 600 ppm [34,35]. We have no data for the residual solvent content in this study, but for similarly sized PLGA microspheres (14.1 lm diameter) prepared as a w/o/w formulation by solvent evaporation

a residual methylene chloride content of 14.4 ppm was found for the dried microspheres [36]. Other groups have also determined a residual methylene chloride content in PLGA microspheres prepared by emulsification and solvent evaporation well below the regulatory limit [37–39]. As the HFIP concentration used was in the ratio 1:200 relative to that of methylene chloride, the amount of residual HFIP should be even lower, and, furthermore, HFIP is miscible with the external aqueous phase. Insulin-loaded microspheres prepared by the three formulation techniques have a negative surface charge, most likely attributable to the adsorption of anions from the PVA and to the ionization of carboxylic end groups in the polymer. The zeta potential is an important physico-chemical characteristic that describes the electrostatic stability of particles in suspensions. In brief, microspheres with a minimum zeta potential of ±30 mV are physically stable in suspension because electrostatic repulsion between particles with highly negative or highly positive zeta potentials prevents aggregation and thereby stabilizes the particles in suspension [40]. This indicates an incipient instability of the prepared PLGA–insulin microspheres having a zeta potential of only about 13 mV. For this reason the insulin-loaded particles should be stored as a lyophilized powder and resuspended upon need. The zeta potential of PLGA microspheres prepared by the emulsification and solvent evaporation method can also be modified by either altering the PVA concentration or using an alternative stabilizer during the preparation procedure. For example, PLGA–insulin microspheres with a more pronounced negative zeta potential can be prepared using low concentrations of poloxamer or carbopol instead of

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Fig. 7. Histological and immunohistochemical stainings of chondrocyte pellet cultures. (a–c) Porcine chondrocyte pellets were cultivated under serum-free conditions with medium supplemented with insulin released from w/o/w microspheres. (d–f) Release supernatants of blank w/o/w microspheres were used as controls. The released insulin led to a strong staining of (a) cartilage-specific proteoglycans and (b) collagen type II compared with the (d, e) control pellets. (c, f) Insulin and control pellets both stained slightly for collagen type I.

B 1515

PLGA - Insulin PLGA - Blank 1010

55

00

0,2 0.2

hydroxyproline (µg)

glycosaminoglycan (µg)

A

PLGA - Insulin PLGA - Blank

0,1 0.1

00

Fig. 8. Effect of insulin released from w/o/w microspheres on the cartilage-specific content of (A) glycosaminoglycans and (B) hydroxyproline in chondrocyte pellets. Porcine pellets were cultivated under serum-free conditions with medium supplemented with insulin released from w/o/w PLGA microspheres. The release supernatant of blank w/o/ w PLGA microspheres served as a control. Cultivation of the chondrocyte pellets with released insulin resulted in a higher content of (A) cartilage-specific glycosaminoglycans and (B) hydroxyproline compared with the blank controls. The values are shown as means ± SD.

PVA [41]. However, cartilage is negatively charged due to the fixed charge groups of the macromolecules in the ECM, primarily the proteoglycans chondroitin sulfate and keratan sulfate that are ionized under physiological conditions [42]. Here again, the zeta potential of the insulin-loaded microspheres has to be considered when estimating their electrostatic interaction with the cartilage ECM and/or with chondrocytes; a more pronounced negatively charged particle surface than 13 mV may lead in vivo to electrostatic repulsion between the microparticles and the rather negatively charged ECM and chondrocytes. Regarding the co-

administration of PLGA–insulin microspheres with autologous chondrocytes implantation, positively charged insulin-releasing microspheres might be favorable because they stimulate both chondrocyte re-differentiation due to the release of insulin and cell adhesion of the chondrocytes to the microspheres, permitting the formation of 3D complexes producing a microenvironment that favors cell–cell and cell–ECM interactions. PLGA microspheres with high positive surface charges can, for example, be prepared using a blend of PVA and chitosan as the stabilizer during the emulsification and solvent evaporation procedure [43]. Furthermore, insulin-

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loaded microspheres were not phagocytosed by human leukocytes irrespective of the preparation procedure. This is of considerable interest because phagocytosis of particles by inflammatory cells as a foreign body host response may lead to undesired local inflammation and rapid clearance of the insulin-releasing microspheres. Uptake by phagocytic cells is mainly affected by the particle size and surface charge, with elevated phagocytosis of particles with a high negative or high positive surface charge and a size range between 1 and 3 lm [44–46]. Hence, we suppose that the prepared insulin-loaded PLGA microspheres were too large and/or had a too low charge to be susceptible to phagocytosis. Since size and surface charge were not appreciably different between the s/o/w, w/o/w and o/o/w insulin-loaded microspheres there were no significant differences in the inflammatory cellular responses. Consistent with these results, Horisawa and colleagues reported that drug-loaded PLGA microspheres of similar size (26.5 lm) were not phagocytosed and did not induce an in vivo inflammatory response, in contrast to particles on the nanoscale [47]. During preparation of the insulin-loaded microspheres the protein is exposed to a range of damaging conditions that frequently cause alterations in the protein structure and a loss of bioactivity, including high shear forces during vortex mixing, organic solvents, organic–aqueous interfaces, hydrophobic contacts between the protein and the polymer and protein dehydration during freeze drying [48–50]. In particular, the w/o/w encapsulation technique would be expected to damage the protein structure because of the high interfacial area during the first w/o emulsification step [51,52]. Nevertheless, the w/o/w double emulsion was actually an adequate emulsification procedure for the encapsulation of water-soluble proteins such as insulin. The s/o/w and o/o/w formulations can be used as alternatives to avoid the w/o interface during the first preparation step. In this study structural integrity and bioactivity were determined for the insulin that was released within the first 5 days in order to elucidate the impact of particle preparation, storage and burst release on protein stability. The insulin released from microspheres prepared by all the investigated techniques retained its structural integrity and bioactivity, as determined by CD spectroscopical analyses and MCF7 proliferation assay. CD analyses provide substantial information about protein conformation and the obtained insulin spectra were in close agreement with the spectra documented by others [53,54]. Insulin elicits mitogenic responses in MCF7 human breast cancer cells [28–30]. Thus, the biological activity of the released insulin was shown by its proliferative effect on MCF7 cells under serum-free conditions. In fact, maintenance of the structural integrity and bioactivity of the insulin after encapsulation and release was not expected, because of the damaging conditions during particle preparation mentioned above. In this study a large amount of protein (2 mg insulin per 100 mg polymer) was loaded into the PLGA microspheres, which has been described as having a self-protecting effect on protein structure during the first emulsification step [48]. With respect to the second o/w emulsion step, the rapidity of polymer hardening may have prevented insulin denaturation at the external organic–aqueous interface, as has already been shown for other proteins, which is probably related to the small size of the microspheres [49]. Furthermore, freeze drying-induced protein degradation has been shown often not to be relevant, because the amorphous character of PLGA helps to stabilize the protein [49]. However, the insulin may also have been denatured during the microsphere loading procedure and then have recovered the native protein structure within the 5 days prior to CD analysis and MCF7 proliferation assay. Further studies on the insulin structure inside the particles may ensure maintenance of the full structural integrity and bioactivity of the encapsulated protein. A further limitation of this study is that the impact of acidification due to polymer degradation on the stability of insulin has not been con-

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sidered. Studies on the structure and biological activity have been performed for insulin released within the first 5 days, however, polymer degradation and a decrease in pH began later. Ibrahim and colleagues investigated the structure of bovine insulin during PLGA degradation and determined structural changes due to protein deamination. They also showed that insulin has a minimal reactivity under acidic conditions, as occur during PLGA degradation in the interior of the microsphere and in the release medium [55]. To counteract acid-induced denaturation of insulin a basic excipient such as sodium bicarbonate can be co-encapsulated in the PLGA microspheres [39]. The results furthermore showed that the microencapsulation technique has an effect on the insulin loading efficiency and on the release kinetics. The insulin loading efficiency was highest for w/o/w microspheres, while s/o/w particles showed the highest initial burst release. Consistent with our results, the w/o/w microencapsulation technique has been described as resulting in the efficient loading of PLGA microspheres with water-soluble proteins [56]. In particular, the addition of sodium chloride at high concentrations to the external continuous phase to increase the osmolarity considerably improved insulin w/o/w entrapment (data not shown). The addition of sodium chloride to the continuous phase has been described as increasing the protein loading efficiency and reducing the initial burst from w/o/w microspheres [57,58]. The size distribution differed only marginally between the microspheres prepared by the three techniques and is thus not considered as resulting in considerable differences in protein loading and burst release. The lyophilized insulin particles employed for preparation of the s/o/w microspheres were rather big and polydisperse; 10% of the protein crystals were smaller than 3.9 lm (DV,10) and 90% of the crystals were smaller than 17.3 lm (DV,90). The average median particle size (DV,50) was 10.6 lm and the span of the particle size distribution was 1.2 (data not shown). This is most likely the reason for the low encapsulation efficiency of and the high initial burst release from the s/o/w PLGA microspheres, because the large insulin crystals were probably not homogeneously dispersed in the organic phase, which might result in its leakage into the external aqueous phase during emulsification. Consistent with this, the encapsulation of comparatively large protein crystals has been shown to result in an inhomogeneous protein distribution within the microspheres with high amounts of protein at the particle surface that can be easily released [34,59,60]. However, the use of solid proteins is a highly promising strategy for protein loading and release from PLGA microspheres because solid proteins are less prone to denaturation at interfaces and loss of bioactivity compared with dissolved proteins [52,61]. However, to achieve good dispersion of the protein particles in the organic phase and thus to achieve high encapsulation efficiencies and a low initial burst release the solid protein particles employed need to exhibit a small and narrow size distribution, preferably under 5 lm [61]. Thus, for future studies it would be favorable to reduce the size of the lyophilized insulin powder to the micron scale, as shown by others [62,63]. The low insulin loading efficiency of o/o/w particles is probably attributable to the solubility of the organic solvent HFIP in the external aqueous phase, resulting in leakage of the protein. Furthermore, the tendency of water-soluble insulin to partition into the aqueous continuous phase may also result in low loading efficiencies for o/o/w formulations [64]. Biodegradable delivery devices that release bioactive insulin in a sustained manner are essential for the engineering of cartilage because insulin has a low half-life in vivo and is unstable in the presence of cartilage [65,66]. This study has shown that the w/o/w emulsification technique is most appropriate for the preparation of insulin-loaded PLGA microspheres due to having the highest insulin loading efficiency, lowest initial burst and appropriate release kinetics. Different strategies are feasible for the

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application of w/o/w PLGA–insulin microspheres in cartilage tissue engineering. Insulin-loaded microspheres can be used for ex vivo approaches in which cartilage-like tissue is generated outside the body and subsequently implanted into the defect site. On the one hand, insulin-releasing PLGA microspheres can function as microcarriers for the 3D cultivation and expansion of autologous chondrocytes in a bioreactor and/or can be injected together with the cells into the cartilage defect, which is then covered with a periostal flap or collagen membrane. 3D cell expansion in a bioreactor preserves the chondrocyte phenotype while allowing extensive cell expansion under controlled cultivation conditions [67,68]. Chondrocyte adhesion can be assured by producing microspheres with a positive surface charge (see above) and by distinct surface modifications [69]. As injectable scaffolds cell–microsphere composites offer minimal surgical invasiveness during transplantation and assemble in situ into a 3D scaffold to induce tissue regeneration [70]. On the other hand, insulin-releasing microspheres can also be incorporated into 3D PLGA scaffolds or biodegradable hydrogels, seeded with autologous chondrocytes and then implanted into small sized cartilage defects. 3D chondrocyte cultivation in a scaffold or hydrogel supports cell–cell and cell–ECM interactions and promotes chondrocyte re-differentiation [5]. For future in situ cartilage tissue engineering approaches PLGA microspheres can be loaded with chemotactic molecules and differentiation factors that enhance intrinsic repair processes by recruiting mesenchymal stem cells and progenitor cells from the subchondral bone, bone marrow and surrounding tissues, with subsequent differentiation and in situ tissue repair [71]. In this study, we have shown that the released insulin had positive effects on ECM component synthesis by high density chondrocyte pellet cultures, stimulating the secretion of cartilage-specific proteoglycans and collagen type II. Cai and colleagues have even shown that insulin not only stimulates ECM component synthesis by chondrocytes but also inhibits further ECM degradation through inhibition of aggrecanase activity [66]. Insulin at a concentration of 10 lg ml 1 activates the IGF-1 receptor and thus elicits the cartilage-protective effects of IGF-1 [72]. Even lower insulin concentrations have been shown to decrease ECM breakdown and stimulate ECM synthesis by chondrocytes without interacting with the IGF-1 receptor [66]. In inflammatory joint diseases like rheumatoid arthritis high levels of interleukin-1 (IL-1) shift cartilage homeostasis towards catabolism and enhance ECM degradation and suppress ECM synthesis [73,74]. Insulin antagonizes the deleterious effects of IL-1 and thus the application of insulinloaded PLGA microspheres may yield both stimulation of cartilage regeneration and inhibition of inflammatory mediators [66]. 5. Conclusion In this study, we have shown that all three different microencapsulation techniques (s/o/w, w/o/w and o/o/w) are applicable for the preparation of PLGA microspheres as sustained release devices for human insulin. However, due to having the highest insulin loading efficiency and lowest initial insulin burst release, the w/o/w emulsification technique is considered to be most appropriate. PLGA microspheres prepared by the w/o/w procedure showed sustained release of structurally intact and biologically active insulin that promoted the formation of cartilage-specific ECM and thus represent a potent delivery device for application in cartilage tissue engineering. Conflict of interest M.S. is a shareholder of CellServe GmbH (Berlin, Germany) and BioRetis GmbH (Berlin, Germany) and works as consultant for

BioTissue Technologies GmbH (Freiburg, Germany) that develops autologous tissue transplants for the regeneration of bone and cartilage. The other authors have no competing interests. Acknowledgements The authors are very grateful to Dr. Margitta Dathe and Heike Nikolenko from the Leibniz Institute of Molecular Pharmacology in Berlin for their kind assistance in the CD spectroscopical analyses. The authors would also like to express their gratitude to Dr. Radostina Georgieva and Ines Peifer (Institute of Transfusion Medicine, Charité-Universitätsmedizin Berlin) for critical discussions and helping with the ZetaSizer (f potential measurement) and the FACS (phagocytosis assay) analyses. Furthermore, the authors gratefully acknowledge the technical assistance of Anja Wachtel and Johanna Golla. This study was supported by the Bundesministerium für Bildung und Forschung (Grant no. BMBF 0313911) and by the Deutsche Forschungsgemeinschaft (Grant no. DFG SI 569/71). The grant sponsors had no influence on the study design or the collection, analysis and interpretation of data, the writing of the manuscript and on the decision to submit the manuscript to Acta Biomaterialia.

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