The influence of covalently linked and free polyethylene glycol on the structural and release properties of rhBMP-2 loaded microspheres

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Journal of Controlled Release 147 (2010) 92–100

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The influence of covalently linked and free polyethylene glycol on the structural and release properties of rhBMP-2 loaded microspheres Alexander Lochmann a,b, Hagen Nitzsche a, Sabrina von Einem c, Elisabeth Schwarz c, Karsten Mäder a,b,⁎ a b c

Translational Centre for Regenerative Medicine Leipzig, Leipzig, Germany Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle, Germany Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Halle, Germany

a r t i c l e

i n f o

Article history: Received 28 April 2010 Accepted 27 June 2010 Available online 29 July 2010 Keywords: Microspheres BMP-2 Drug distribution Labelling PEG-PLGA Block-copolymer

a b s t r a c t The current clinical success of therapies with recombinant human Bone Morphogenetic Protein 2 (rhBMP-2) is limited due to inefficient delivery. The high doses applied have frequently been related to severe adverse effects such as tissue swelling, seroma, inflammatory effects and heterotopic ossification. The controlled delivery of lower doses is supposed to reduce adverse effect incidence as well as costs. In this study, novel polyethylene glycol-poly(lactic-co-glycolic acid) (PEG-PLGA) diblock copolymers were used to produce low dose controlled delivery vehicles for rhBMP-2. A method to fabricate a variety of microsphere formulations with a high encapsulation efficiency in high yields was developed. The influence of PEG as an inner phase cosolvent and linked PLGA as copolymer was investigated. Six different microsphere systems with varying PEG amounts in both core and shell were characterised thoroughly with respect to the specific properties of rhBMP-2. The particle size of the microspheres was investigated with both laser diffraction and environmental scanning electron microscopy. Higher PEG/PLGA ratios showed a tendency to increase in size and a wider distribution. Due to the low rhBMP-2 doses, a profound characterisation was very challenging. The growth factor was covalently attached to rhodamine B for the first time. Studies on drug distribution in the microspheres were performed by means of confocal laser scanning microscopy. The addition of PEG to the inner phase was found to impair the formation of spherical microdomains with localized higher growth factor concentrations. Release profiles, determined with ELISA, were linked to the structural changes that were monitored. Distinct, controlled release profiles were achieved in all formulations and showed that PEG is a versatile tool in the effective control of release rates from microspheres. Higher PEG/PLGA ratios in the polymer were shown to increase the release rate from the microspheres. In contrast, PEG administered to the inner phase decreased the release rate. The biological activity of released protein was shown in vitro in an alkaline phosphatase assay. It was demonstrated that PEG-PLGA microspheres are a promising sustained delivery system which allows a reduction of the required rhBMP-2 dose to limit both adverse effects and costs. Furthermore, the data indicated that the use of PEG as an inner phase cosolvent is not suitable for rhBMP-2 in contrast the reported beneficial effects for other growth factors. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Bone morphogenetic proteins have already made their way from bench to bedside. RhBMP-2 and rhBMP-7 (OP-1) are marketed for

Abbreviations: rhBMP-2, recombinant human Bone Morphogenetic Protein 2; ALP assay, alkaline phosphatase assay; BCA assay, bicinchoninic acid assay; SDS-PAGE, sodium dodecyl sulfate - polyacrylamide gel electrophoresis; CLSM, confocal laser scanning microscopy; ESEM, environmental scanning electron microscopy; PLGA, poly(lactic-co-glycolic) acid; PEG-PLGA, polyethylene glycol - poly(lactic-co-glycolic) acid copolymer; LD, laser diffraction. ⁎ Corresponding author. Institute of Pharmacy, Martin Luther University HalleWittenberg, Wolfgang-Langenbeck-Str. 4, D-06120 Halle, Germany. Tel.: +49 345 5525167; fax: +49 345 5527029. E-mail address: [email protected] (K. Mäder). 0168-3659/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2010.06.021

spinal fusion and long bone non-unions in tibiae, respectively [1]. Both growth factors are just adsorbed in high amounts to collagen sponges before application. Numerous studies also depicted adverse effects of the potent treatment [2–5]. Particularly high doses have directly been attributed to a higher probability of severe adverse effects [6]. Thus, there is a clear medical need for substantial improvement. Several different strategies were reported recently [7–14]. In addition to the search for alternative osteoinductive proteins [11] or more stable BMP-2 derivatives [7,8], most approaches focussed on the dose minimization and tailored release of the expensive growth factors. One strategy to do so is the controlled delivery of rhBMP-2 from microspheres. Chen and coworkers reported a synergistic effect of the combination of BMP-2 and insulin-like growth factor 1 [14] which may be used for dose

reduction. Patel et al. demonstrated the adaptability of gelatine for the controlled delivery of rhBMP-2 [14]. Furthermore, modified hyaluronic acid has recently been evaluated as vehicle for rhBMP-2 release [12]. However, besides batch-to-batch variations, natural polymers were shown to reach desirable release kinetics in microspheric delivery only after chemical modifications such as crosslinking [9] or conjugation to other natural [12] or synthetic polymers [13], which may cause difficulties on the road towards the clinic. Amongst synthetic polymers, polyesters are the most frequently used group due to their approval for parenteral application and numerous possibilities to tailor their release behaviour. There have been attempts to immobilize the growth factor on the microsphere surface by soaking particles in a protein solution [15–18], but these were accompanied with high burst rates, similar to these documented in the collagen sponge approach. Wang et al. recently compared the incorporation principles of soak-loading and incorporation into PLGA microspheres after adding hydroxyapatite. They found only slight advantages for the soak-loading group. This finding may be explained by the enormous loss of bioactivity upon lyophilization reported by the authors and with that a lower effective dose studied in the incorporation group [10]. There are only few reports on microspheric delivery systems that utilize an incorporation of the protein into PLGA [19–22] and some of them work with extremely high doses [21] or denaturing agents [22], even though the need for a well-balanced delivery of active protein is widely accepted [13,20,23]. Pegylated poly-α-hydroxy acids have been described to decrease pH drops within microspheres [24], which is why they can provide a better environment for growth factors compared to the non-pegylated polyesters. PEG itself was reported to stabilize growth factors upon microencapsulation [25,26]. To the best of our knowledge, neither encapsulation of rhBMP-2 into PEG-PLGA diblock polymers nor effects of PEG coencapsulation have been investigated for rhBMP-2 so far. Pegylated poly(lactic-co-glycolic acid) diblock copolymers have not been used for microsphere production up to now with any formulation. No commercial material has been available so far. The development of a suitable delivery system has to be accompanied by appropriate and extensive characterisation. As rhBMP-2 is a protein with unusual properties, many issues have to be taken into account in the experimental design, such as its solubility minimum at physiological pH and its tendency to form different forms of aggregates [27,28]. These aspects are rarely considered in the literature [21] and release data obtained with iodine-labelled rhBMP-2 bear no information on the state of the released protein. Here, the use of enzyme-linked immunosorbent assays can provide more information. In this study, we have developed microspheric delivery systems for rhBMP-2 based on polymers with varying PEG ratios in both core and shell, characterized them thoroughly with respect to the special properties of rhBMP-2 and compared them with microspheres prepared from PEG-free PLGA.

2. Materials and methods 2.1. Materials All chemicals were obtained from Sigma, Taufkirchen, Germany, and were at least of reagent grade unless otherwise stated. RGPd 5055, RGPd 50105 and RG 503 (Table 1) were purchased from Boehringer, Ingelheim, Germany.

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2.2. Preparation of rhBMP-2 and labelled rhBMP-2 Deep-frozen E. coli BL 21 cells with rhBMP-2 inclusion bodies were extracted and purified as reported earlier [30]. The bioactivity of nonglycosylated, E. coli-derived rhBMP-2 has been demonstrated in both ectopic and orthotopic sites in vivo [31]. The protein was divided into aliquots, freeze-dried and stored at −20 °C until further use. For investigations of encapsulation efficiency and load distribution within the microspheres, fluorescence techniques were applied. Therefore, rhBMP-2 had to be covalently linked to a fluorescent dye. 80 mg of rhodamine B were dissolved in 2 ml of dimethyl sulphoxide. 25 mg of N, N'-dicyclohexylcarbodiimide and 25 mg of N-hydroxysuccinimide were added to the reaction vessel. The mixture was protected from light and moisture and stirred with a magnetic bar at room temperature for 2 days. After filtration, the obtained solution was diluted 24-fold with sodium acetate buffer (20 mM, pH 5.5). 20 μl of the diluted activated dye solution were added to 500 μl of a chilled rhBMP-2 solution. The mixture was shaken for 3 h at 37 °C and subsequently dialysed four times against 500 ml of sodium acetate buffer (20 mM, pH 4.5). SDS-PAGE was used for labelling control. 10 μl of the sample were mixed with 10 μl of SDS buffer, prepared without bromophenol blue. After finishing the run, the gel was subjected to fluorescence imaging. The obtained image was compared with the result of subsequent conventional staining. The labelling efficiency was determined with fluorescence spectroscopy (Perkin Elmer MPF 44). The slit widths were set to 6 nm. Excitation and emission spectra were recorded with maxima λex = 555 nm and λem = 578 nm, respectively. According to the maxima, a calibration curve was recorded. Standards and samples were corrected for the background for 40 s at each measurement with a subsequent signal determination period of 80 s. Each second, one data point was recorded. The obtained data points were corrected with the arithmetic mean of the respective background scans. All measurements were carried out threefold and the mean of all 216 data points was used as the sample value. 2.3. Production of microspheres Microspheres of two PEG-PLGA diblock-copolymers (PEP 5, PEP 10) and one PLGA copolymer (PEP 0) were fabricated by a double emulsion-solvent evaporation technique modified from Witschi and Doelker [32]. At first, 150 μl of rhBMP-2 in an aqueous buffer were homogenized with 4.0 ml methylene chloride containing 10% (w/v) polymer in a polytetrafluoroethylene beaker. The concentration of rhBMP-2 solution was 12 ± 1.5 μg/ml. An emulsion was formed by homogenization for 2 min at 10,000 rpm (Ultra turrax T 18, IKA, Staufen, Germany). Thereafter, the emulsion was immediately dropped into 65 ml of ice-cooled 2% (w/v) aqueous solution of polyvinyl alcohol. In step 3, solvent evaporation and hardening of the nascent particles was achieved by stirring with a two-blade paddle mixer for 1 h. Subsequently, residual methylene chloride was removed at a reduced pressure of 200 mbar for at least 1 h. Due to low glass transition temperatures of the pegylated polymers (TG PEP 10 = 29.7 °C; TG PEP 5 = 34.5 °C), the latter steps of production were conducted on ice. At last, the microparticles were collected, washed three times with a mixture of isopropyl alcohol and purified water and dried under vacuum. All obtained samples were stored at 4 °C in the dark. Residual methylene chloride was determined by gas chromatography and flame ionization detection based on a method modified from the European Pharmacopeia. 200 mg of blank microspheres

Table 1 Composition of the used polymers. Brand name

Abbrev.

PEG part (%)

Composition

Mw in kDa

Resomer RG 503 Resomer RGP d 5055 Resomer RGP d 50105

PEP 0 PEP 5 PEP 10

0% 5% 10%

Poly(D,L-lactic-co-glycolic acid), Lactic/glycolic acid ratio 1:1 Poly(D,L-lactic-co-glycolic acid) + PEG 5000 block, Lactic/glycolic acid ratio 1:1 Poly(D,L-lactic-co-glycolic acid) + PEG 5000 block, Lactic/glycolic acid ratio 1:1

40.6 kDa [29] 95 kDa PLGA + 5 kDa PEG 45 kDa PLGA + 5 kDa PEG

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were dissolved in 20 ml of N,N-dimethyl formamide. The samples were extracted for 15 min at 105 °C by using a headspace unit. 1 ml of the gaseous phase was withdrawn and subjected to gas chromatography. A calibration curve of standards with concentrations ranging from 100 to 600 ppm was recorded. Nine batches of microspheres, i.e. three of each polymer, were subjected to the analysis. The use of low molecular weight PEG as an inner phase cosolvent has been reported to stabilize proteins during encapsulation [25]. In one group, the protein was dissolved in sodium acetate buffer (Ac), in another group, the inner phase of the microspheres consisted of one third of PEG 300 and two thirds of sodium acetate buffer (AcPEG). The pH was readjusted accordingly. An overview of the different formulations is provided in Table 2. 2.4. Particle size, morphology and swelling behaviour The particle surface was scanned with Environmental Scanning Electron Microscopy (ESEM XL 30 FEG, Philips Electron Optics). Microspheres were fixed on a double-sided tape. Feret's diameter was measured for all particles conspicuously imaged in ESEM overview pictures (AnalySIS Auto, Olympus). A lower magnification was chosen in order to investigate more than 100 microspheres per picture. Usually, around 400 particles were measured. For comparison, laser diffraction measurements were carried out. 50 mg of microspheres were dispersed in 25 ml of 0.05% (w/v) polysorbate 20 solution and stirred for 2 h. Subsequently, 5 ml of dispersion were analyzed in quintuplicate (Mastersizer, Malvern). This step was repeated three times. For comparison with the microscopic data, the numberweighted mean was used. Refractive indices for PLGA particles were found ranging between 1.44 and 1.51–1.67 [33–35], typical absorption values were reported between 0.00 and 0.01. Measurements were made for particle sizes from 4 to 400 μm as known from the ESEM results with the assumption of a refractive index of 1.50 and an absorption of 0.001. 2.5. Encapsulation efficiency Data were acquired by fluorescence spectroscopy, using the same method as for the labelling efficiency measurements. Both the outer phase of the w/o/w-emulsion and the washing water were assessed for fluorescence intensity. Therefore, the outer phase was gathered and adjusted to a total of 100 ml. During the washing process, the rinsing water was collected and evaporated. The residue was reconstituted in 10 ml of 2% (w/v) aqueous solution of polyvinyl alcohol and subjected to the same analytic method. The addition of both values was regarded as loss during fabrication and subtracted from the total amount to yield the encapsulation efficiency. 2.6. Drug distribution Each of the six variations was manufactured twofold with rhodamine-labelled rhBMP-2 and examined by means of CLSM. The microspheres were dispersed in Prolong Gold mounting medium (Invitrogen). The distribution of the labelled protein within the microspheres was examined using a LSM 510 Duoscan (Zeiss, Jena,

Germany). The rhodamine-BMP-2 was excited at a wavelength of 543 nm and emission was filtered by a longpass filter at 560 nm. Pictures were taken using Zeiss Axiovision LE software. 2.7. In vitro release studies Twelve batches of microspheres were prepared, two of each formulation. All batches were assessed for release data in duplicate. 50 mg of microspheres were dispersed in 1000 μl of release medium, which consisted of phosphate-buffered saline, pH 7.4, with additives including polysorbate 20, 0.05% (w/v); bovine serum albumin, 1% (w/ v); disodium EDTA, 0.15% (w/v) and sodium azide 0.02% (w/v). The samples were placed on an orbital shaker and maintained at 37 °C. At predefined times, 200 μl of the release medium were withdrawn and frozen to −20 °C for further analysis. 200 μl of fresh medium were again added to the samples. A sandwich ELISA kit was used for concentration determination (R&D Systems, Abingdon, UK). 2.8. Stability of rhBMP-2 in release medium Four concentrations, i.e., 5000, 500, 50, 5 ng/ml, of four BMP-2 standard samples were each subjected to the release conditions in absence of microspheres. Constants were calculated for the decrease of BMP-2 concentration by time in the standard samples. To elucidate the influence of microsphere degradation products on BMP-2 stability, three batches of microspheres were produced as described above, one of each polymer used in the release study. A blank sodium acetate buffer was utilized as inner phase instead of buffered rhBMP-2 solution. Samples were taken according to the same time scheme as in the release experiment, but 100 μl were frozen to −20 °C and another 100 μl were stored in the fridge. One week before the ELISA, 10 μl of rhBMP-2 solution were added to 70 μl of each sample and the storage continued the same way as indicated before. The residue of approximately 30 μl was kept for pH profiling. The concentration of the remaining rhBMP-2 was determined by using the ELISA kit according to the manufacturer's instructions. 2.9. In vitro activity of released rhBMP-2 The induction of alkaline phosphatase in C2C12 mouse myoblasts was investigated according to a modified version of a protocol reported previously [36]. Briefly, 4 × 10³ fourth passage cells were seeded in each well of a 96 well plate. Microspheres from all three polymers were tested. In a transwell plate (Corning Inc., Corning, United States), 50 mg of rhBMP-2-loaded microspheres were inserted and covered with differentiation medium. The rhBMP-2 was released and diffused through the transwell membrane into the wells with the cells below. Negative controls included medium alone, medium with cells and empty microspheres of all three polymers. As positive control, a rhBMP-2 standard solution was used. After three days, the alkaline phosphatase activity was measured. The results were normalized for the protein content of each well as determined by a bicinchoninic acid assay kit (Pierce, Rockford, United States). 3. Results and discussion 3.1. Labelling of rhBMP-2

Table 2 Composition of formulations A through F with respect to their PEG contents. PEG 300 was used in the inner phase, PEG 5000 is linked to the polymer. Details on polymer composition are given in Table 1.

PEP 0 PEP 5 PEP 10

Ac; Inner phase: 0%

AcPEG; Inner phase: 33%

A C E

B D F

Unexpectedly, all three polymers showed autofluorescence, although chromophores should be absent in the backbone. Autofluorescence was detectable in both the unprocessed substances and fabricated microspheres. All three polymers exhibited the same background fluorescence spectra. Pretests had shown an undesirably large overlap between the emission spectra of carboxyfluorescein-labelled rhBMP-2 and the autofluorescence of the polymers, disabling further differentiation

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Fig. 1. Normalized fluorescence spectra of rhBMP-2 covalently linked to fluorescein or rhodamine B and background spectra of PLGA. Local maxima at 550 + 50 x nm (x = 1, 2, 3) are artefacts due to a mode hop in the liquid crystal tunable filter of the in vivo imaging system.

between the two (Fig. 1) and an insufficient fluorescence intensity of the construct. Consequently, rhBMP-2 was linked covalently to rhodamine B. Fluorescent spots on the SDS gel were compared with subsequent Coomassie staining results, and were in excellent match with them. No significant increase in aggregates or oligomers was detected. During dialysis, the amount of unbound rhodamine was visually detectable. The labelling efficiency was found to be rather low, with values ranging from 8.8% to 9.7%. Nevertheless, the rhodamine bound to rhBMP-2 was sufficient for detection in all further experiments. The low labelling efficiency is probably due to an incomplete activation of rhodamine B and the relatively low pH value during labelling. At a higher pH, a higher percentage of free, non-protonated amino groups is available, which is necessary for the nucleophilic substitution at the carboxylic acid group of the dye. At higher pH values, however, a partial precipitation of rhBMP-2 could be observed, which is in agreement with the literature [27]. At a lower pH, a clear solution was obtained, which was stable at 4 °C for at least 3 weeks. Out of the six lysines per rhBMP-2 monomer, lysine no. 97 has the highest probability to be amidated with N-hydroxysuccinimide-activated dyes because of its ease of accessibility [37]. Due to its relatively small size compared with the protein, the dye is supposed not to alter the behaviour of rhBMP-2 during encapsulation. 3.2. Production of microspheres From all polymers, well-shaped microspheres were obtained (Fig. 2). Higher yields were achieved with no or low PEG content in the polymer, ranging around 93% for formulation A and 89% for C microspheres. Batches of formulation E yielded about 85% of microspheres. Batches with AcPEG as the inner phase (B, D, F) showed a similar pattern. A possible concern may arise from the use of organic solvents. Nevertheless, the concentration of residual methylene chloride was generally found to be below 250 ppm, less than half of the accepted maximum according to the European Pharmacopeia. The washing and evaporation steps during microsphere production appear to suffice for the removal of methylene chloride. 3.3. Size distribution The ESEM image analysis was easy to apply and is, as a direct method, more accurate than laser diffraction measurements. Its drawbacks include the lower number of investigated particles and

Fig. 2. Environmental Scanning Electron micrographs of microspheres prepared from the different formulations A–E. Variations in morphology due to the polymer composition can be found. Differences in structure caused by the inner phase composition are most pronounced between formulation A and B.

their time-consuming analysis. Furthermore, it is not possible to observe any swelling behaviour of the microspheres, as can be done with laser diffraction. Size evaluation in LD was accomplished according to the Mie theory, which is regarded as more accurate, especially in low absorbing samples [38]. Otherwise, an overestimation of small particles can be found. For comparison with microscopic data, a change into number-weighted data is necessary [39]. All of the particles were small enough to fit through cannulae upon injection. As determined by ESEM, Feret's diameter was in the range of 5 to 185 μm for all microspheres, none of the particles was larger than 200 μm and only less than 2.5% of all particles exceeded 100 μm. Thus, an enhanced degradation rate like in very large particles [22] or a very high burst release, as obtained with very small particles, were circumvented. The size distribution of formulations A and B was rather narrow, with more than 80% of the particles smaller than 60 μm. In contrast, formulations E and F showed a broad distribution with noticeable amounts of microspheres at least between 20 and 80 μm, but the batches were rather uniform. Comparable results were obtained with laser diffraction. An overview of the results of both methods is provided in Table 3. A gradual increase of particle size with increasing PEG-polymer ratio was detectable in spite of the use of the same preparation method. This may be due to a slower microsphere hardening and thus more time for coalescence during particle formation. The presence or absence of PEG or rhBMP-2 did not alter the particle size as determined by LD. In contrast, with ESEM a gradual decrease of the particle size in AcPEG (B, D, F) samples was detected compared to the respective Ac formulations A, C and E. In general, laser diffraction tended to yield slightly higher results than ESEM with the pegylated polymers. A probable alteration in optical properties and/or swelling behaviour of the particles, which is not accessible for ESEM, can be made responsible for this effect [40].

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3.4. Encapsulation efficiency One advantage of double emulsion techniques is the usually high encapsulation efficiency into PLGA [41]. Common methods for the determination of entrapment rates include the measurement of radioactive labelled protein [20], which requires special equipment besides environmental concerns, or dissolution and extraction of the particles in organic solvent/ sodium dodecyl sulfate mixtures. Several different alkaline methods can be applied [42], but due to the low solubility of rhBMP-2, none of these were promising. Kempen et al. estimated 85% of rhBMP-2 to be encapsulated into PLGA microspheres by a radiolabelling approach [20]. Ruhé et al. found comparable results (78 ± 9%) [19], while Shi and coworkers reported an incorporation rate of more than 90% [21]. Isobe et al. found an encapsulation rate of 91% for rhBMP-2 in large PLGA microcapsules [22]. In this study, the amount of encapsulated rhBMP-2 was calculated from remaining fluorescence activities in the outer phase of the double emulsion and the washing water. The outer phase contained between 0.9 and 11.7% of rhodamine-BMP-2. In the washing water, 0.3 to 4.8% of the initial rhodamine-BMP-2 concentration were found. Mean encapsulation values reached 95.0 ± 4.7% for formulation A, and comparable results for formulations C and E (97.1 ± 1.5% and 97.5 ± 0.6%, respectively). High values, ranging from 72 to 99% have also been reported for other pegylated polyesters [43]. The obtained data are in the higher range compared to the literature. However, it has to be kept in mind that encapsulation rates determined by differential methods tend to be higher compared to extraction measurements. Sah described an underestimation of encapsulation with extraction techniques [42] because of protein loss during the extraction process and incomplete extraction steps. The propensity of rhBMP-2 to interact non-specifically with hydrophobic surfaces may also decrease the loss into a second aqueous phase and will thus favour both high encapsulation and lower extraction.

3.5. Particle morphology The obtained particles of different polymers varied in size and shape in spite of the use of one preparation technique (Fig. 2). Although the use of PEG as an inner phase cosolvent (formulations B, D, F) did not alter the particle size, it caused remarkable differences in microsphere shape and drug distribution depending on the used polyester. PEP 0, the PLGA monoblock, was affected most by the change of the inner aqueous phase upon preparation. Labelled rhBMP-2 was detected in all investigated samples, exceeding a negligible background fluorescence in the negative controls at least fivefold (Fig. 3). The inner cavities of the rhodamineBMP-loaded particles appear much brighter, indicating the localization of rhodamine-labelled rhBMP-2. A comparable distribution was

reported by Ding and Schwendeman for a 10 kDa dextran conjugated dye [44]. The low signals detected in the negative controls could be attributed to the autofluorescence of the polymer at a lower wavelength. The influence of PEG on particle morphology and drug distribution was dependent on the combination of the two application modes of PEG, either covalently bound to the polymer backbone or simply added to the primary emulsion. Covalently attached PEG in formulations C - F did not alter the inner phase morphology (Fig. 4). In contrast, microspheres with an inner phase comprised partly of PEG (B, D, F) exhibited a rather blurred distribution of the labelled growth factor within the particle. This effect is negligible in preparation F and most apparent in formulation B. Here, and to some extent also in formulation D, the use of AcPEG led to a partial loss of the spherical microdomains. They partly gave way to a rather blurred drug distribution and the particles appeared shrivelled. It is hypothesized that PEG 300 interferes with PLGA chains during emulsification and particle formation as it may also be used as a solvent for PLGA in higher concentrations [26,45,46]. In contrast, PEGPLGA microspheres did not interact the same way. Here, PEG 300 is more likely to interact with the PEG 5000 part of the polymer, interfering less with the particle formation process. Hence, no effect is seen with PEP 10 and very little is detectable with PEP 5, which has a much lower PEG ratio in its polymer chain. The loss of spherical microdomains containing rhBMP-2 will also be at least partially responsible for a lower release from the AcPEG-containing microspheres. The active ingredient is smeared between the polymers chains, making it prone to faster aggregation due to contact with the organic phase [47]. Release may be hindered or impaired under these circumstances.

3.6. In vitro degradation of the microspheres In vitro release data depend on a complex matrix of parameters, such as release volume, temperature, pH, sampling regimen et cetera [48]. In order to compare the results with in vitro release profiles, the studies of in vitro degradation were conducted under the same conditions. It is well accepted that acidic hydrolysis is the main mechanism of polyester degradation [44,49]. The autocatalytic process is characterised by a fast degradation onset, but also by a delayed yet even more intense particle erosion when the polymer becomes water soluble due to the decrease in its molecular weight. Interestingly, the pH profiles of the release medium exhibited a large drop in pH in both pegylated formulations and much less pronounced for the PEP 0 microspheres (Fig. 5).

Table 3 Particle sizes (number-weighted mean) as determined by laser diffraction (LD) and environmental scanning electron microscopy (ESEM). Data are given batchwise for LD data and particlewise cumulated from both batches under ESEM investigation. The abbreviations w/o and n.a. refer to “without” and “not assessed,” respectively. Formulation

LD mean ± SD [μm]

n

ESEM mean ± SD [μm]

A A w/o BMP B C C w/o BMP D E E w/o BMP F

37 ± 7 35 ± 4 32 ± 4 50 ± 9 50 ± 4 50 ± 5 64 ± 10 67 ± 5 67 ± 3

14 8 4 9 5 3 11 4 3

39 ± 21 n.a. 34 ± 16 51 ± 30 n.a. 41 ± 26 51 ± 27 n.a. 47 ± 27

n 837 1770 447 640 718 644

Fig. 3. Overview micrograph of the fluorescence intensity and drug distribution as obtained from confocal laser scanning microscopy. Microspheres with labelled rhBMP-2 according to formulation E (left). The labelled dye is well distributed in the bright microdomains throughout all particles. For comparison, the same microspheres without labelled rhBMP-2 are pictured as control (right).

Within 4 weeks, the pH of the buffered release medium had dropped to less than 4 in both PEP 5 and PEP 10, whereas the decrease to pH 5.8 in PEP 0 microspheres is in good accordance with values determined for comparable polymers by Witschi and Doelker [32]. Fluorescence techniques and ESR measurements have been utilized to monitor the pH in PLGA microspheres [24,44]. In all approaches a huge accumulation in acidic products was detected over time. The particle degradation may be accelerated in larger particles compared to smaller ones due to acidic degradation residues, even in the microsphere scale investigated in this study [50]. Pegylated polymers have been reported to yield microspheres with less intense acidification upon degradation. It is assumed that a better transport through the more hydrophilic pegylated particles may enable a faster removal of acidic degradation products. Kissel et al. reported a faster degradation onset and a slower degradation rate in vitro for microspheres of PEG-PLGA triblock polymers compared to microspheres made from PLGA of comparable molecular weight [43]. Consequently, the larger pH drop in the release medium observed with PEP 5 and PEP 10 resulted from better transport of acidic degradation products out of the microspheres. With them, the acidsoluble rhBMP-2 was also released more easily and in higher amounts.

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3.7. Stability of rhBMP-2 in release medium As reported by Schwartz and Luca et al. [27,28], the solubility profile of rhBMP-2 exhibits a minimum under physiological conditions. Interestingly, this fact is neglected in nearly all in vitro release studies [13,18,51,52], which would consequently lead to an underestimation of the burst effect and total release when the solubility is exceeded. The alteration of pH in the release media by other groups [53,54] may support the solubility of the growth factor in solution. In this work, both salt concentration and pH were not favourable in terms of rhBMP-2 stability. Although commonly used additives were applied, a continuous decrease of rhBMP-2 concentration in the release medium was observed. In the stability test, the standards exhibited the same half life, independent of the concentration used. The calculated loss constants were used for the determination of release profiles later on. Loss constants were calculated to k(e) = −0.04 h−1 for all four investigated concentrations. The unfavourable pH, the shaking stress or the elevated temperature upon release may have contributed to the instability. None of these factors was changed, because all of them reflect physiological conditions as closely as possible. The restricted volume of the release medium was also chosen to achieve a better in vitro–in vivo correlation. It was shown that rhBMP-2 is extremely prone to detrimental effects upon freezing and thawing in release buffer. After 1 week of storage at 4 °C, rhBMP-2 was still found in the same concentration range as the freshly reconstituted standard, as shown in Fig. 6. In contrast, one single freeze–thaw-cycle caused a loss of about one third in ELISA detection, probably due to adsorption to vessel walls. The freezing of samples is a common storage method during release studies [27]. It is necessary to consider the effect upon experimental design. In contrast, degradation products of microspheres such as glycolic or lactic acid as well as ethylene glycol oligomers or monomers were found not to interfere significantly with the stability of rhBMP-2 as determined by ELISA.

3.8. Release determination With respect to the facts stated above, in vitro release profiles were assessed for a period of 28 days. All release profiles appeared more or less biphasic. In an initial burst phase, about one third of the amount found during the total investigated period was released within the first 24 h. A second increase in the release rate was detectable after approximately ten days (Fig. 7).

Fig. 4. Confocal Laser Scanning micrographs of single microspheres loaded with rhodamine-labelled rhBMP-2. Particles consisted of formulations A–F. For details, see Table 2. With a lower content of PEG 5000 in the polymer matrix, the influence of PEG 300 on the inner structure increased dramatically. Almost no distribution alteration was found between formulations E and F, whereas, in opposition to formulation A, the dye in formulation B was no longer located predominantly in microdomains.

Fig. 5. Time dependence of pH in the release medium under in vitro release conditions. The pH decrease in the buffer was significantly higher with PEGylated polymers, indicating a better transport of acidic degradation products out of the microspheres into the release medium.

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The obtained profiles were comparable to findings reported by Kempen et al. [55]. Interesting differences were found between the release rates of the three polymers. A higher PEG content in the polymer matrix, i.e. 10% in PEP 10, was found to yield significantly higher release rates than lower PEG contents in the polymer matrix. PEP 5 is intermediate with regard to the slight increase in the release rates compared to PEP 0. The use of AcPEG instead of Ac as the protein solvent generally led to a decrease of protein release, especially during the first hours. The profile shapes did not differ apparently, whereas the total amount released was 30% to 60% less than without the incorporation into external PEG. Out of 225 ng rhBMP-2 possibly detectable per release batch, between 3 and 60 ng were found during release measurements. At first, this may appear rather low compared to the data reported by other groups, but it has to be taken into account that most release measurements were conducted with radioactive labelled rhBMP-2. Those measurements are less sensitive to changes of protein conformation or aggregation. Another limitation is the possible detachment of the radioactive tracer from the growth factor [20]. It has been supposed that higher molecular weight of PLGA may be one reason for lower and incomplete release [56]. Hence, higher release rates from PEP 10 may result from the combination of a shorter polymer chain and the presence of a PEG block in the polymer, both increasing the permeability of the spheres and possibly retaining a less hydrophobic environment during microsphere formation. PEP 5, due to its high molecular weight, still shows slightly higher release rates than PEP 0, supporting the idea of increased permeability in the presence of a PEG block. In contrast, the addition of PEG 300 to the inner phase during MP formation appears not favourable for the increase of rhBMP-2 stability, yielding lower release rates and less defined drug distributions within the microspheres.

3.9. In vitro activity of released rhBMP-2 In all investigated samples, bioactive rhBMP-2 was successfully encapsulated. Interestingly, the amount of bioactive rhBMP-2 released from microspheres differed not as much as determined by ELISA. Again, PEP 10 yielded better results than PEP 5, causing an ALP induction of 0.85 dEmin−1 μg−1 at an equivalent concentration of 2.6 nM compared to 0.52 dEmin−1 μg−1. The ALP induction with rhBMP-2 released from PEP 0 microspheres (0.96 dEmin−1 μg−1) did not differ significantly from the PEP 10 value. The induction of alkaline phosphatase was in the expected range for the pegylated microspheres, but surprisingly high for the non-pegylated polyester PEP 0.

Fig. 6. Recovery of rhBMP-2 in release buffer after 1 week of storage at 5 °C (checked columns) and at −20 °C (plain columns) as determined by ELISA. About one third of detectable concentration was lost for ELISA detection in a single freeze-thaw cycle.

Fig. 7. Release profiles of microspheres as determined by ELISA and corrected for loss the constant of rhBMP-2 in the release medium. Error bars represent standard deviation.

Due to the instability of rhBMP-2 in the release medium and the loss upon freezing and thawing, the obtained equivalent concentration for PEP 0 is slightly higher in the cell assay than the calculation from the release study. It is hypothesized, that the microspheres made of pegylated polymers may, to some extent, release rhBMP-2 which is still detectable by ELISA in spite of not being bioactive anymore. However, a full answer of bioactivity shall be given by subsequent in vivo investigations. 4. Conclusions We have developed and characterized microspheric delivery systems that provide a controlled release for rhBMP-2 based on the first commercially available PEG-PLGA copolymers. By labelling rhBMP-2 with rhodamine B for the first time, we were able to investigate the influence of a cosolvent, PEG 300, on particle properties. Results were compared with data from unmodified PLGA microspheres. The addition of PEG 300 was shown to alter the inner morphology, drug distribution and the release profile from the microspheres. Lower release rates in particles containing PEG 300 could be explained by the drug distribution. The impact of PEG 300 on structure alteration is related to the type of polymer used to produce the microspheres. PLGA microspheres were more prone to structural changes by PEG 300 than PEG-PLGA particles. The use of PEG 300 as an additive to the inner phase was found to decrease the burst release as well as the overall release of the expensive protein and is therefore regarded as unfavourable for the investigated system. The stabilizing effect of PEG addition described for Nerve Growth Factor and other protein preparations was not detected. Alkaline phosphatase activity data draw an ambivalent picture. Increased activity of the early differentiation marker was detected three days after microsphere addition. The highest BMP-2 expression levels can usually be found during the early phase of fracture healing, but BMP-2 is also found in several other stages of bone regeneration [57]. It may be assumed that the effects of BMP-2 are needed for an extended period of time in critical size defects. However, the kind of defect and its inherent regeneration potential may determine whether the growth factor needs to be administered during the whole period or whether a shorter initiation phase is sufficient. Here, the final conclusion needs to be drawn from in vivo data. In this work, it was shown that the formulation of rhBMP-2 with PEG-PLGA diblock polymers is a promising approach to combine the various advantages of increased polymer hydrophilicity and the versatility of microspheres with the effective bone formation potential of rhBMP-2. An in vivo evaluation of the formulations should follow to confirm the results.

Conflict of interest statement No conflict of interest has to be stated in conjunction with this work.

[21]

[22]

Acknowledgements [23]

The authors would like to thank Sylke Meyer (Biochemistry Department of MLU Halle) for her assistance with the confocal laser scanning microscope, Frank Syrowatka at the IZM Halle for the ESEM pictures. This work was also supported by Hans-Herrmann Rüttinger (encapsulation efficiency), Ahmed Besheer (growth factor labelling) and Hendrik Metz (half life calculation), all of the Institute of Pharmacy, MLU Halle. This work was made possible in part by funding from the German Federal Ministry of Education and Research (BMBF, PtJ-Bio, 0313909) and funding from the DFG grant SCHW 375/5-1. References [1] P.C. Bessa, M. Casal, R.L. Reis, Bone morphogenetic proteins in tissue engineering: the road from the laboratory to the clinic, part I (basic concepts), J. Tissue Eng. Regen. Med. 2 (1) (2008) 1–13. [2] K. Shahlaie, K.D. Kim, Occipitocervical fusion using recombinant human bone morphogenetic protein-2: adverse effects due to tissue swelling and seroma, Spine 33 (21) (2008) 2361–2366. [3] D. Benglis, M.Y. Wang, A.D. Levi, A comprehensive review of the safety profile of bone morphogenetic protein in spine surgery, Neurosurgery 62 (5 Suppl 2) (2008) ONS423–ONS431. [4] M. Boakye, P. Mummaneni, V.M. Garrett, G. Rodts, R. Haid, Anterior cervical discectomy and fusion involving a polyetheretherketone spacer and bone morphogenetic protein, J. Neurosurg. Spine 2 (5) (2005) 521–525. [5] R.S. Brower, N.M. Vickroy, A case of psoas ossification from the use of BMP-2 for posterolateral fusion at L4-L5, Spine 33 (18) (2008) E653–E655. [6] L.M. Tumialan, J. Pan, G.E. Rodts, P.V. Mummaneni, The safety and efficacy of anterior cervical discectomy and fusion with polyetheretherketone spacer and recombinant human bone morphogenetic protein-2: a review of 200 patients, J. Neurosurg. Spine 8 (6) (2008) 529–535. [7] Z.Y. Lin, Z.X. Duan, X.D. Guo, J.F. Li, H.W. Lu, Q.X. Zheng, D.P. Quan, S.H. Yang, Bone induction by biomimetic PLGA-(PEG-ASP)n copolymer loaded with a novel synthetic BMP-2-related peptide in vitro and in vivo, J. Control. Release 144 (2) (2010) 190–195. [8] X. Niu, Q. Feng, M. Wang, X. Guo, Q. Zheng, Porous nano-HA/collagen/PLLA scaffold containing chitosan microspheres for controlled delivery of synthetic peptide derived from BMP-2, J. Control. Release 134 (2) (2009) 111–117. [9] Z.S. Patel, M. Yamamoto, H. Ueda, Y. Tabata, A.G. Mikos, Biodegradable gelatin microparticles as delivery systems for the controlled release of bone morphogenetic protein-2, Acta Biomater. 4 (5) (2008) 1126–1138. [10] C.K. Wang, M.L. Ho, G.J. Wang, J.K. Chang, C.H. Chen, Y.C. Fu, H.H. Fu, Controlledrelease of rhBMP-2 carriers in the regeneration of osteonecrotic bone, Biomaterials 30 (25) (2009) 4178–4186. [11] M. Lee, W. Li, R.K. Siu, J. Whang, X. Zhang, C. Soo, K. Ting, B.M. Wu, Biomimetic apatite-coated alginate/chitosan microparticles as osteogenic protein carriers, Biomaterials 30 (30) (2009) 6094–6101. [12] A.K. Jha, W. Yang, C.B. Kirn-Safran, M.C. Farach-Carson, X. Jia, Perlecan domain I-conjugated, hyaluronic acid-based hydrogel particles for enhanced chondrogenic differentiation via BMP-2 release, Biomaterials 30 (36) (2009) 6964–6975. [13] E.R. Balmayor, G.A. Feichtinger, H.S. Azevedo, M. van Griensven, R.L. Reis, Starchpoly-epsilon-caprolactone microparticles reduce the needed amount of BMP-2, Clin. Orthop. Relat. Res. 467 (12) (2009) 3138–3148. [14] F. Chen, R. Chen, X. Wang, H. Sun, Z. Wu, In vitro cellular responses to scaffolds containing two microencapulated growth factors, Biomaterials 30 (28) (2009) 5215–5224. [15] B.H. Woo, B.F. Fink, R. Page, J.A. Schrier, Y.W. Jo, G. Jiang, M. DeLuca, H.C. Vasconez, P.P. DeLuca, Enhancement of bone growth by sustained delivery of recombinant human bone morphogenetic protein-2 in a polymeric matrix, Pharm. Res. 18 (12) (2001) 1747–1753. [16] E. Bergeron, M.E. Marquis, I. Chretien, N. Faucheux, Differentiation of preosteoblasts using a delivery system with BMPs and bioactive glass microspheres, J. Mater. Sci. Mater. Med. 18 (2) (2007) 255–263. [17] J.A. Schrier, B.F. Fink, J.B. Rodgers, H.C. Vasconez, P.P. DeLuca, Effect of a freezedried CMC/PLGA microsphere matrix of rhBMP-2 on bone healing, AAPS PharmSciTech 2 (3) (2001) E18–E25. [18] H. Shen, X. Hu, F. Yang, J. Bei, S. Wang, An injectable scaffold: rhBMP-2-loaded poly(lactide-co-glycolide)/hydroxyapatite composite microspheres, Acta Biomater. 6 (2) (2010) 455–465. [19] P.Q. Ruhé, E.L. Hedberg, N.T. Padron, H.M. Spauwen Paul, J.A. Jansen, A.G. Mikos, rhBMP-2 release from injectable poly(DL-lactic-co-glycolic acid)/calcium-phosphate cement composites, J. Bone Joint Surg. Am. 85-A (Suppl 3) (2003) 75–81. [20] D.H.R. Kempen, M.J. Yaszemski, A. Heijink, T.E. Hefferan, L.B. Creemers, J. Britson, A. Maran, K.L. Classic, W.J.A. Dhert, L. Lu, Non-invasive monitoring of BMP-2

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33] [34]

[35]

[36]

[37] [38] [39] [40]

[41] [42] [43]

[44]

[45] [46]

[47] [48] [49]

99

retention and bone formation in composites for bone tissue engineering using SPECT/CT and scintillation probes, J. Control. Release 134 (3) (2009) 169–176. S. Shi, X. Cheng, J. Wang, W. Zhang, L. Peng, Y. Zhang, RhBMP-2 microspheresloaded chitosan/collagen scaffold enhanced osseointegration: an experimental in dog, J. Biomater. Appl. 23 (4) (2009) 331–346. M. Isobe, Y. Yamazaki, S.I. Oida, K. Ishihara, N. Nakabayashi, T. Amagasa, Bone morphogenetic protein encapsulated with a biodegradable and biocompatible polymer, J. Biomed. Mater. Res. 32 (3) (1996) 433–438. P.C. Bessa, M. Casal, R.L. Reis, Bone morphogenetic proteins in tissue engineering: the road from laboratory to clinic, part II (BMP delivery), J. Tissue Eng. Regen. Med. 2 (2–3) (2008) 81–96. K. Mäder, B. Bittner, Y. Li, W. Wohlauf, T. Kissel, Monitoring microviscosity and microacidity of the albumin microenvironment inside degrading microparticles from poly(lactide-co-glycolide) (PLG) or ABA-triblock polymers containing hydrophobic poly(lactide-co-glycolide) A blocks and hydrophilic poly(ethylene oxide) B blocks, Pharm. Res. 15 (5) (1998) 787–793. J.M. Péan, F. Boury, M.C. Venier-Julienne, P. Menei, J.E. Proust, J.P. Benoit, Why does PEG 400 co-encapsulation improve NGF stability and release from PLGA biodegradable microspheres? Pharm. Res. 16 (8) (1999) 1294–1299. W. Al-Azzam, E.A. Pastrana, B. King, J. Mendez, K. Griebenow, Effect of the covalent modification of horseradish peroxidase with poly(ethylene glycol) on the activity and stability upon encapsulation in polyester microspheres, J. Pharm. Sci. 94 (8) (2005) 1808–1819. D. Schwartz, Development of an Aqueous Suspension of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2), PhD thesis, LMU Munich, Germany, (2005). L. Luca, M.A.H. Capelle, G. Machaidze, T. Arvinte, O. Jordan, R. Gurny, Physical instability, aggregation and conformational changes of recombinant human bone morphogenetic protein-2 (rhBMP-2), Int. J. Pharm. 391 (1–2) (2010) 48–54. C. Augsten, M.A. Kiselev, R. Gehrke, G. Hause, K. Mäder, A detailed analysis of biodegradable nanospheres by different techniques-A combined approach to detect particle sizes and size distributions, J. Pharm. Biomed. Anal. 47 (1) (2008) 95–102. F. Hillger, G. Herr, R. Rudolph, E. Schwarz, Biophysical comparison of BMP-2, ProBMP-2, and the free pro-peptide reveals stabilization of the pro-peptide by the mature growth factor, J. Biol. Chem. 280 (15) (2005) 14974–14980. J.H. Lee, C.S. Kim, K.H. Choi, U.W. Jung, J.H. Yun, S.H. Choi, K.S. Cho, The induction of bone formation in rat calvarial defects and subcutaneous tissues by recombinant human BMP-2, produced in Escherichia coli, Biomaterials 31 (13) (2010) 3512–3519. C. Witschi, E. Doelker, Influence of the microencapsulation method and peptide loading on poly(lactic acid) and poly(lactic-co-glycolic acid) degradation during in vitro testing, J. Control. Release 51 (2,3) (1998) 327–341. C. Wischke, Protein loaded microparticles with modified surfaces for the targeting of dendritic cells, PhD thesis, FU Berlin, Germany, (2006). J. Patterson, P.S. Stayton, X. Li, In situ characterization of the degradation of PLGA microspheres in hyaluronic acid hydrogels by optical coherence tomography, IEEE Trans. Med. Imaging 28 (1) (2009) 74–81. T. Musumeci, C.A. Ventura, I. Giannone, B. Ruozi, L. Montenegro, R. Pignatello, G. Puglisi, PLA/PLGA nanoparticles for sustained release of docetaxel, Int. J. Pharm. 325 (1–2) (2006) 172–179. J. Berger, M. Reist, A. Chenite, O. Felt-Baeyens, J.M. Mayer, R. Gurny, Pseudothermosetting chitosan hydrogels for biomedical application, Int. J. Pharm. 288 (2) (2005) 197–206. C. Scheufler, W. Sebald, M. Hülsmeyer, Crystal structure of human bone morphogenetic protein-2 at 2.7 Å resolution, J. Mol. Biol. 287 (1) (1999) 103–115. ISO 13320:2009, Particle size analysis—laser diffraction methods, , 2009. A. Rawle, P. Kippax, Setting New Standards for Laser Diffraction Particle Analysis, www.iscpubs.com/Media/PublishingTitles/Rawle-layout-1.pdf (2010). J.H. Jeong, D.W. Lim, D.K. Han, T.G. Park, Synthesis, characterization and protein adsorption behaviors of PLGA/PEG di-block co-polymer blend films, Colloids Surf. B Biointerfaces 18 (3,4) (2000) 371–379. J.H. Park, M. Ye, K. Park, Biodegradable polymers for microencapsulation of drugs, Molecules 10 (1) (2005) 146–161. H. Sah, A new strategy to determine the actual protein content of poly(lactide-coglycolide) microspheres, J. Pharm. Sci. 86 (11) (1997) 1315–1318. T. Kissel, Y. Li, F. Unger, ABA-triblock copolymers from biodegradable polyester Ablocks and hydrophilic poly(ethylene oxide) B-blocks as a candidate for in situ forming hydrogel delivery systems for proteins, Adv. Drug Deliv. Rev. 54 (1) (2002) 99–134. A.G. Ding, S.P. Schwendeman, Acidic microclimate pH distribution in PLGA microspheres monitored by confocal laser scanning microscopy, Pharm. Res. 25 (9) (2008) 2041–2052. F. Kang, J. Singh, Effect of additives on the release of a model protein from PLGA microspheres, AAPS PharmSciTech 2 (4) (2001) 30–36. S. Kempe, H. Metz, P.G.C. Pereira, K. Mäder, Non-invasive in vivo evaluation of in situ forming PLGA implants by benchtop magnetic resonance imaging (BT-MRI) and EPR spectroscopy, Eur. J. Pharm. Biopharm. 74 (1) (2010) 102–108. H. Sah, Stabilization of proteins against methylene chloride/water interfaceinduced denaturation and aggregation, J. Control. Release 58 (2) (1999) 143–151. S.S. D'Souza, P.P. DeLuca, Methods to Assess in Vitro Drug Release from Injectable Polymeric Particulate Systems, Pharm. Res. 23 (3) (2006) 460–474. C. Witt, K. Mäder, T. Kissel, The degradation, swelling and erosion properties of biodegradable implants prepared by extrusion or compression moulding of poly (lactide-co-glycolide) and ABA triblock copolymers, Biomaterials 21 (9) (2000) 931–938.

GENE DELIVERY

A. Lochmann et al. / Journal of Controlled Release 147 (2010) 92–100

GENE DELIVERY

100

A. Lochmann et al. / Journal of Controlled Release 147 (2010) 92–100

[50] N.K. Varde, D.W. Pack, Microspheres for controlled release drug delivery, Expert Opin. Biol. Ther. 4 (1) (2004) 35–51. [51] D.S. Keskin, A. Tezcaner, P. Korkusuz, F. Korkusuz, V. Hasirci, Collagen-chondroitin sulfate-based PLLA-SAIB-coated rhBMP-2 delivery system for bone repair, Biomaterials 26 (18) (2005) 4023–4034. [52] O. Jeon, S.J. Song, S.W. Kang, A.J. Putnam, B.S. Kim, Enhancement of ectopic bone formation by bone morphogenetic protein-2 released from a heparin-conjugated poly(-lactic-co-glycolic acid) scaffold, Biomaterials 28 (17) (2007) 2763–2771. [53] H. Maeda, A. Sano, K. Fujioka, Controlled release of rhBMP-2 from collagen minipellet and the relationship between release profile and ectopic bone formation, Int. J. Pharm. 275 (1–2) (2004) 109–122. [54] F.M. Chen, Y.M. Zhao, H.H. Sun, T. Jin, Q.T. Wang, W. Zhou, Z.F. Wu, Y. Jin, Novel glycidyl methacrylated dextran (Dex-GMA)/gelatin hydrogel scaffolds containing

microspheres loaded with bone morphogenetic proteins: formulation and characteristics, J. Control. Release 118 (1) (2007) 65–77. [55] D.H.R. Kempen, L. Lu, T.E. Hefferan, L.B. Creemers, A. Maran, K.L. Classic, W.J.A. Dhert, M.J. Yaszemski, Retention of in vitro and in vivo BMP-2 bioactivities in sustained delivery vehicles for bone tissue engineering, Biomaterials 29 (22) (2008) 3245–3252. [56] P.Q. Ruhé, O.C. Boerman, F.G.M. Russel, P.H.M. Spauwen, A.G. Mikos, J.A. Jansen, Controlled release of rhBMP-2 loaded poly(DL-lactic-co-glycolic acid)/calcium phosphate cement composites in vivo, J. Control. Release 106 (1–2) (2005) 162–171. [57] F. Bronner, M.C. Farach-Carson, A.G. Mikos, Engineering of functional skeletal tissues, first ed.Springer, London, 2007.