In vitro and in vivo performance of biocompatible negatively-charged salbutamol-loaded nanoparticles

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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l

In vitro and in vivo performance of biocompatible negatively-charged salbutamol-loaded nanoparticles Erik Rytting a, Michael Bur b, Regis Cartier c, Thierry Bouyssou d, Xiaoying Wang a, Michael Krüger c, Claus-Michael Lehr b, Thomas Kissel a,⁎ a

Department of Pharmaceutics and Biopharmacy, Philipps-Universität Marburg, Ketzerbach 63, D-35032 Marburg, Germany Biopharmaceutics and Pharmaceutical Technology, Im Stadtwald, Saarland University, D-66123 Saarbrücken, Germany Boehringer Ingelheim Pharma GmbH & Co. KG, Binger Strasse 173, D-55216 Ingelheim am Rhein, Germany d Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Strasse 65, D-88397 Biberach an der Riss, Germany b c

a r t i c l e

i n f o

Article history: Received 5 March 2009 Accepted 22 August 2009 Available online 29 August 2009 Keywords: Nanoparticle Salbutamol In vitro–in vivo correlation Pulmonary drug delivery Biocompatibility

a b s t r a c t The development and performance of a novel nanoparticle-based formulation for pulmonary delivery has been characterized chronologically through the particle preparation process, in vitro testing of drug release, biocompatibility, degradation, drug transport in cell culture, and in vivo bronchoprotection studies in anaesthetised guinea pigs. This study demonstrates excellent agreement of the in vitro and in vivo experiments undertaken to prove the feasibility of the design, thereby serving as an example highlighting the importance of in vitro test methods that predict in vivo performance. Nanoparticles were prepared from the newly designed negatively-charged polymer poly(vinyl sulfonate-co-vinyl alcohol)-g-poly(D,L-lactic-coglycolic acid) loaded with salbutamol free base. Average particle sizes of blank and drug-loaded nanoparticles prepared at the various stages of the investigations were between 91 and 204 nm; average zeta potential values were between − 50.1 and − 25.6 mV. Blank nanoparticles showed no significant toxicity, and no inflammatory activity was detected in Calu-3 cells. Sustained release of salbutamol from the nanoparticles was observed for 2.5 h in vitro, and a prolonged effect was observed for 120 min in vivo. These results demonstrate good agreement between in vitro and in vivo tests and also present a promising foundation for future advancement in nanomedicine strategies for pulmonary drug delivery. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The use of nanoparticles in pharmaceutical sciences has received increasing attention in recent years, due to several advantages. Pulmonary administration of nanoparticles as sustained release formulations for local drug delivery can reduce dosing frequency and improve patient compliance [1]. The pulmonary route is noninvasive and can be used for local delivery to treat lung diseases or as a means to achieve systemic delivery which avoids the first-pass effect and circumvents the extensive degradation of biotherapeutics following oral administration [2,3]. Nanoparticles can be delivered by nebulization of liquid nanosuspensions or as dry powder with nanoparticle-containing clusters having aerodynamic diameters between 1 and 5 µm; nevertheless, several factors must be taken into consideration when designing such nanocarriers, including biocompatibility, particle size and surface characteristics that influence deposition within the lung, release rates, and clearance [4].

⁎ Corresponding author. Tel.: +49 6421 2825881; fax: +49 6421 2827016. E-mail address: [email protected] (T. Kissel). 0168-3659/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.08.021

In order for further progress to be made in the field of pulmonary nanotherapeutics, it is important that the safety profile of nanoparticles in the lung be appropriately addressed, including possible immunogenicity and inflammation. Other challenges in nanomedicine include understanding how the characteristics of nanoparticles affect their clearance by macrophages in the alveolar region and their transport across airway and alveolar epithelia. When polymeric materials are employed as the carriers, then the polymer degradation rates will affect bioaccumulation, clearance of the biomaterial and its degradation products. In addition to this, the safety of the degradation products must also be considered. Another challenge is related to the stability of therapeutic nanocarriers themselves. The small diameter of nanoparticles means that only small diffusion distances separate the active agent and the nanoparticle's exterior. This leads to special difficulties in controlling nanoparticle drug loading and release rates [4]. As a strategy to improve drug loading and release rates, a new polymer class was recently brought forth. Wang synthesized a new class of negatively-charged polymers which was shown to be suitable for nanoparticle production [5]. It was proposed that the introduction of negatively-charged functional groups in the structure of this novel class of polymers would promote electrostatic interactions with

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oppositely-charged drugs and biomacromolecules. Such interactions should improve the design of nanoparticle-based drug carriers as a means of retaining the drug cargo for a longer period of time compared to typical hydrophilic drug release patterns from nanoparticles [6]. This hypothesis was tested by developing negativelycharged nanoparticles from poly(vinyl sulfonate-co-vinyl alcohol)-gpoly(D,L-lactic-co-glycolic acid) (structure shown in Fig. 1) loaded with the positively-charged drug salbutamol. Recent comments by Florence acknowledged the increasing number of investigations in the field of pharmaceutical nanotechnology, and he presented ten specific suggestions for nanoparticle research [7]. First in this list is: “The relevance to the whole animal of in vitro tests of activity, selectivity, uptake and toxicity of nanoparticulate carrier systems [7].” It is of great importance that in vitro, ex vivo, and in vivo data can be correlated in order to streamline the process of bringing advances in nanomedicine from the research lab to the pharmacy shelves. In line with this suggestion to address the “relevance to the whole animal of in vitro tests,” the work presented here takes several aspects of pharmaceutical nanoparticle development into consideration, as the salbutamol-containing nanoparticles were not only investigated with regards to their biocompatibility and in vitro attributes, but the performance of the particles was also studied in vivo and shown to have good agreement with the in vitro predictions. Highlights of this work include the demonstration of improved drug loading of the nanoparticles through the electrostatic interaction strategy realized through the use of the new poly(vinyl sulfonate-covinyl alcohol)-g-poly(D,L-lactic-co-glycolic acid) and salbutamol base. The in vitro drug release profile is also compared to the in vivo action of the nanosuspension in anaesthetised guinea pigs. Furthermore, as this is a new polymer class, this report describes important in vitro biocompatibility experiments assessing the safety of nanoparticles produced with this novel negatively-charged polymer. Although this polymer is grafted with PLGA, which has an established safety profile, it is still important to investigate the influence of the negativelycharged functional groups of this branched polymer, since some anionic polymers previously developed for medicine had resulted in severe toxicity [8]. 2. Materials and methods 2.1. Nanoparticle preparation Poly(vinyl sulfonate-co-vinyl alcohol)-g-poly(D,L-lactic-co-glycolic acid) was synthesized as described previously [5]. This polymer is hereafter abbreviated as P(VS-VA)-g-PLGA, and the two numbers appearing after the polymer name designate the ratio of vinyl sulfonate relative to vinyl acetate employed in the polymer synthesis and the PLGA grafting ratio, respectively, as described previously [5]. Salbutamol free base and salbutamol sulfate were supplied by Boehringer Ingelheim (Ingelheim am Rhein, Germany). Nanoparticles were prepared by a modified solvent displacement method, as described previously [9]. P(VS-VA)-g-PLGA and salbuta-

Fig. 1. The chemical structure of poly(vinyl sulfonate-co-vinyl alcohol)-g-poly(D,Llactic-co-glycolic acid), abbreviated as P(VS-VA)-g-PLGA.

mol were dissolved together in acetone (99.6%, Acros Organics, Geel, Belgium) at a concentration of 60 mg polymer/mL acetone and 3.16 mg drug/mL acetone, for a theoretical drug loading of 5% (mass of drug per total nanoparticle mass). One mL of this polymer-drug solution was then injected into 5 mL of purified water (conductance 0.055 µS/cm) at an injection speed of 10 mL/min through a 23-gauge 0.6 mm × 30 mm needle (Carl Roth GmbH, Karlsruhe, Germany). The injection speed was controlled with a peristaltic pump in order to improve the reproducibility of the process, and the water was stirred at 500 rpm during the injection. At the specified concentrations, the amounts of polymer, solvent, and water fall into a thermodynamically metastable location on the phase diagram referred to as the ouzo region [10], and stable polymeric nanoparticles are formed. The resulting nanosuspension was then continuously stirred at 750 rpm under a fume hood for at least 5 h to allow for complete evaporation of the acetone. In order to remove unencapsulated salbutamol, the nanosuspension was then centrifuged through Vivaspin 6 columns (100,000 MWCO, Sartorius, Göttingen, Germany) for 20 min at 1000 rcf, resuspended in 5 mL purified water, and then centrifuged and resuspended a second time. 2.2. Nanoparticle characterization Nanoparticle size and zeta potential were determined with a Zetasizer NanoZS (Malvern Instruments, Herrenberg, Germany) immediately following particle preparation. Z-average particle size and the polydispersity index (PDI) were measured at 25 °C using dynamic light scattering combined with Malvern's DTS software (v.5.02). Each measurement was performed using at least three sets of at least ten runs. Zeta potential values were measured by laser Doppler anemometry at 25 °C and calculated using DTS software, using at least three sets of at least 12 runs. 2.3. Nanoparticle degradation The degradation rate of blank and drug-loaded nanoparticles was measured by mass loss during incubations at 37 °C in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8.5 mM Na2HPO4, and 1.5 mM KH2PO4, adjusted to pH 7.4). It was observed that 1.5× PBS at pH 7.32 diluted with water to 1× PBS resulted in a pH of 7.4 after dilution, so 500 µL of aqueous nanosuspension samples were added to 1 mL of 1.5× PBS. At the specified time points (including time zero), samples were frozen at − 80 °C, thawed, immediately centrifuged for 10 min at 13,000 rcf, and the supernatant was removed carefully. Pellets were then frozen again at −80 °C before freeze drying using a Beta II freeze dryer (Martin Christ GmbH, Osterode am Harz, Germany) pre-cooled to −50 °C, with overnight lyophilization at 0.07 mbar and − 46 °C. After freeze drying, samples were allowed to equilibrate at room temperature and then the weight of the pellets was measured and corrected by subtracting the empty weight of each sample vial determined previously. In order to account for the weight of lyophilized PBS salts, samples at time zero also included pure 1× PBS and nanoparticles at identical concentrations in purified water, and these control samples underwent the same freezing, centrifuging, freeze drying, and weighing steps to make the appropriate corrections. For the samples used in the degradation studies, blank nanoparticles of P(VS-VA)-g-PLGA-6–10 were prepared at a concentration of 40 mg/mL in acetone, and the drug-loaded nanoparticles were prepared using a similar concentration of 41.5 mg/mL of this polymer, because these concentrations represent points at which the ouzo regions overlap for this polymer with and without 5% theoretical drug loading. These particles were also compared to particles prepared from poly(D,L-lactic-co-glycolic acid) (PLGA, RG502H from Boehringer Ingelheim, Ingelheim am Rhein, Germany) which was dissolved at 20.8 mg/mL in acetone, with and without 5% drug. Samples were collected in triplicate.

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2.4. Biocompatibility

2.5. Drug loading and in vitro drug release

Nanoparticles from a series of six types of P(VS-VA)-g-PLGA, namely P(VS-VA)-g-PLGA-2–10, -4–5, -4–10, -4–15, -6–10, and -8–10 were investigated to establish any influence of vinyl sulfonate composition or PLGA grafting ratio on cytotoxicity. Nanoparticles from these polymers were also compared to nanoparticles from two other negatively-charged polymers, namely sulfobutylated poly(vinyl alcohol)-g-PLGA-33–10 and -41–10, synthesized as described previously [9], and abbreviated as SB-PVA-g-PLGA. For these polymers, the numbers 33 and 41 represent the degree of substitution of the sulfobutyl groups and the number 10 indicates the PLGA grafting ratio. The biocompatibility of the blank nanoparticles was investigated by IL-8, LDH, and MTT assays on Calu-3 cells. The human adenocarcinoma cell line Calu-3 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Calu-3 cells (passage numbers 41 to 46) were seeded on 96-well plates at a density of 20,000 cells/ well. Immediately after seeding, cells were grown in 200 μL apical media (Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 μg/mL streptomycin and 100 U/mL penicillin) at 37 °C in a 5% CO2 incubator. For all experiments, the cells were used 13 days after seeding. In each biocompatibility assay, cell culture medium was used as negative control, and 1% Triton-X was used as positive control in the LDH and MTT assays. Interleukin-8 concentrations, as a marker of induced inflammatory stimuli, were quantified using commercially available sandwich ELISA kits (PromoCell, Heidelberg, Germany). In brief, quantification of IL-8 took place using mouse monoclonal anti-human IL8 antibody diluted to 4.0 μg/mL in phosphate buffered saline PBS. Recombinant human IL-8, serially diluted from 5000 pg/mL was utilised as standard. Cell culture medium samples were diluted 1:20 with 0.1% bovine serum albumin, 0.05% Tween 20 in PBS, immediately before use. Secondary detection antibody was rabbit anti-human IL8 antibody. Incubation of samples and standards, and then of the secondary antibody, took place on a plate agitator for 1 h at 37 °C. Between each stage, all wells were aspirated, washed forcefully five times with wash buffer (0.05% Tween 20 in PBS), and blotted dry. Measurement of absorbance took place using wavelengths of 450 and 550 nm. All samples were analyzed in duplicate. Cytokine concentrations are expressed per volume of cell culture medium (pg/mL). The influence of the nanoparticles on cell membrane integrity was assessed by measuring the release of lactate dehydrogenase (LDH) and the mitochondrial conversion of the tetrazolium salt WST-1, respectively. The investigated nanoparticles were suspended in cell culture medium and added to Calu-3 cells seeded in 96 well plates. After 4 h incubation WST-1 was applied to the cells. The plate was incubated for 60 min, shaking the first 10 min. Thereafter, the reaction was stopped using 50 μL DMSO. The fluorescence of the generated purple formazan was measured with a microplate reader using the excitation wavelength of 560 nm and the emission wavelength of 590 nm. Percent cell membrane damage was expressed in correlation to pure cell culture medium as negative control and Triton-X as positive control. The effect of nanoparticles on the metabolic activity of Calu-3 cells in 96-well plates was assessed using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were cultured for 4 h with suspended nanoparticles (0.1 µg/mL–1000 µg/ mL). MTT solution (5 mg/mL in HBSS) was added to the cells (final concentration 0.5 mg/mL). The cells were incubated for 2.5 h at 37 °C in 5% CO2/95% humidified air. Thereafter, the medium was removed and added to blue formazan crystals dissolved in 200 μL DMSO; the absorbance was measured at 550 nm (655 nm as background) using a microplate reader.

The drug loading is defined as the mass of drug associated with the nanoparticles relative to the total nanoparticle mass (polymer + drug). Encapsulation efficiency is defined as the percentage of drug associated with the nanoparticles relative to the total amount of drug added during the nanoparticle preparation process. For example, if 5 mg of drug were dissolved together with 95 mg of polymer in acetone, the theoretical drug loading would be 5%. Following nanoparticle preparation, if only 3 mg of drug are found in 98 mg of nanoparticles, then the actual drug loading would be 3.06%, and the encapsulation efficiency would be 61.2%. To determine drug loading, a 1 mL sample of nanosuspension was frozen at − 80 °C, thawed and immediately centrifuged for 10 min at 13,000 rcf. At this point, the supernatant was sampled to determine the amount of unencapsulated salbutamol remaining in the nanosuspension. The pellet was freeze dried as described above, weighed, and then dissolved in 450 µL of CHCl3 (Acros Organics, Geel, Belgium) with shaking. After complete dissolution, 900 µL of acidic water (adjusted to pH 2.5 with H3PO4), was added to the CHCl3 solution, and this mixture was shaken for at least 30 min and then centrifuged for at least 5 min at 13,000 rcf to extract the salbutamol located in the top (aqueous) layer. Salbutamol concentrations were determined by HPLC and compared to control standards undergoing the same treatments as the samples (chloroform extraction or dissolution in PBS, as appropriate). Chromatography was carried out using a Waters Nova-Pak C18 column (150 × 3.9 mm, 60 Å pore size, 4 µm particle size) at 35 °C, with a mobile phase flow rate of 0.6 mL/min. The mobile phase contained a mixture of 10% methanol and 90% of the following solution: 36.6 mM Na2HPO4 + 33.4 mM triethylamine (Acros Organics, Geel, Belgium), adjusted to pH 6.0 with H3PO4. Detection was with a Waters 486 UV detector at a wavelength of 277 nm. Drug release was determined by adding 500 µL of aqueous nanosuspension to 1 mL of 1.5× PBS at 37 °C. At the specified time points, samples were frozen in liquid nitrogen and then thawed and centrifuged as mentioned above. Supernatants were analyzed by HPLC to determine the amount of salbutamol released from the nanoparticles. 2.6. In vitro transport in cell culture The transport of free salbutamol across primary cultured human alveolar epithelial cells was compared to the transport of nanoparticles prepared from P(VS-VA)-g-PLGA-6–5 containing salbutamol at 5% theoretical drug loading. Human alveolar epithelial cells in primary culture are an excellent representative model for pulmonary transport studies. Fresh human type II alveolar epithelial cells were isolated from non-tumorous lung tissue which was obtained from patients undergoing lung resection. The use of human material for isolation of primary cells was reviewed and approved by the local ethical committees (Saarland State Medical Board, Germany). Isolation of primary human type II pneumocytes (AT II) was performed according to a protocol modified from those of Elbert et al. [11] and Ehrhardt et al. [12]. Briefly, finely minced lung tissue was digested for 40 min at 37 °C using a combination of trypsin and elastase. The resulting crude cell mixture was purified to get a pure pneumocyte culture, using a combination of differential cell attachment, centrifugation with a percoll density gradient, and cell sorting with magnetic beads (anti-HEA (EpCAM) MicroBeads, Miltenyi Biotec, Bergisch Gladbach, Germany). The identity and purity of type II pneumocytes cells was determined by staining cells for alkaline phosphatase. Purified AT II cells were then seeded at a density of 600,000 cells/cm2 on collagen/fibronectin coated polyester filter inserts (Transwell Clear, 6.5 mm diameter, 0.4 μm pore size, Corning, Wiesbaden, Germany) using SAGM medium (Cambrex Bio Science, Verviers,

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Belgium) supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), and 1% fetal calf serum. Transport experiments were conducted using hAEpC monolayers from day 9 after differentiation to type I like cells with rigid barrier properties. Both sides of the monolayers were washed twice with preequilibrated Krebs–Ringer's solution (KRB; 15 mM HEPES, 116.4 mM NaCl, 5.4 mM KCl, 0.78 mM NaH2PO4, 25 mM NaHCO3, 1.8 mM CaCl2, 0.81 mM MgSO4, and 5.55 mM glucose, pH 7.4). Monolayers were then placed in new 24-well cluster plates containing 800 µl per well of KRB, pre-warmed to 37 °C. After 60 min of equilibration, transport experiments were initiated (i.e., t = 0) by replacing the donor fluid with 200 µl of KRB containing the salbutamol-loaded nanoparticles and free salbutamol, respectively. Receiver samples (100 µl) were drawn serially from the basolateral compartment at t = 2, 4, 18, and 24 h. After each sampling, fresh transport buffer of an equal volume was returned to the receiver side to maintain a constant volume. Each transport experiment was performed using 4–6 monolayers (obtained from two cell preparations). In order to assess the integrity of monolayers during the flux studies, transepithelial electrical resistance (TEER) was measured before and after each transport experiment. These TEER values are included with this manuscript as Supplementary information. To investigate whether the nanosuspension exhibited a retarded release effect compared to free salbutamol at an identical concentration, transport across the cell layer was characterized by plotting the increase of the drug concentration in the acceptor compartment over the time and monitoring the area under the curve, the maximum concentration, and the mean permeation time, which considers the slope of the curve and represents the point at which half of the drug is transported. 2.7. In vivo bronchoprotection studies The in vivo activity of the salbutamol-containing nanoparticles was determined by measuring its bronchoprotective action against acetylcholine-induced bronchospasm in anaesthetised guinea pigs according to the Konzett–Rößler model. All animal experiments were carried out according to the German law of protection of animal life and approved by an external review committee for laboratory animal care. After anaesthesia, the animal was connected to the air pressure apparatus and the lung resistance was recorded. Acetylcholine (9 µg/kg) was injected intravenously, and then 10 min later the salbutamol (as either a nanosuspension or as a free drug solution) was administered by nebulization during the breathing process with the Respimat device. Prior to the experiments with 6 animals, the drug content of the nanoparticles delivered from the Respimat device were verified in order to match the doses given. Suspensions of P(VS-VA)g-PLGA-6–10 salbutamol-loaded nanoparticles or free salbutamol were each administered at concentrations of 6 µg drug/kg. Acetylcholine was then injected intravenously every 10 min for a recording period of 2 h. Bronchoprotection of the drug was expressed as a percentage of inhibition of acetylcholine bronchospasm preceding drug inhalation. 2.8. Statistical analysis Data were compared by a two-tailed Student's t-test for differences between two groups; at least 3 replicates were analyzed in each case, and differences were deemed statistically significant if p < 0.05. 3. Results and discussion 3.1. Nanoparticle degradation Fig. 2 shows that the nanoparticles of both P(VS-VA)-g-PLGA-6–10 and PLGA degraded at similar rates. After 7 days, the remaining mass

Fig. 2. Nanoparticle degradation as mass loss. Nanoparticles prepared from PLGA with (black triangles) or without (white triangles) salbutamol or prepared from P(VS-VA)-gPLGA-6–10 with (black circles) or without (white circles) salbutamol were incubated in PBS at 37 °C.

of drug-loaded P(VS-VA)-g-PLGA-6–10 nanoparticles was significantly lower than the mass of the empty nanoparticles (p < 0.05). The addition of drug had the opposite effect on the degradation of the PLGA nanoparticles, however. In this case, the remaining mass of drug-containing PLGA particles was significantly higher than the remaining amount of empty PLGA particles after 7 days (p < 0.05). In both cases, the drug-containing nanoparticles were smaller than the blank particles. The Z-average diameter of the blank PLGA nanoparticles was 147.5 ± 0.6 nm (PDI = 0.10 ± 0.02) and 113.4 ± 2.2 nm (PDI = 0.18 ± 0.03) for the PLGA particles containing salbutamol. The blank P(VS-VA)-g-PLGA-6–10 particles were 203.7 ± 1.9 nm (PDI = 0.14 ± 0.02), compared to 148.7 ± 3.3 nm (PDI = 0.13 ± 0.01) for the drug-loaded particles. In both cases, the zeta potential (ζ) increased upon drug loading, from − 42.8 ± 0.7 mV to − 38.4 ± 1.6 mV for PLGA and from −45.9 ± 0.4 mV to − 43.7 ± 0.7 mV for P(VS-VA)g-PLGA-6–10. The similarities in the overall degradation rates of both particle types can be explained by the similarities in the chemical makeup of the polymers, as the amount of PLGA grafted on the P(VS-VA)-gPLGA-6–10 polymer (a 10 to 1 ratio) represents a significant fraction of this polymer's composition. The observed differences between the effects of salbutamol on the particle degradation rates may be attributed to the differences in the encapsulation efficiencies of the two polymers. The encapsulation efficiency of the P(VS-VA)-g-PLGA6–10 nanoparticles prepared for the degradation experiments was twice as high as the encapsulation efficiency of the PLGA nanoparticles (19.1 ± 0.5% compared to 9.2 ± 0.9%). The degradation rate of PLGA depends on its molecular weight and composition (ratio of lactic acid relative to glycolic acid) [13]. The type of PLGA investigated in this work was of relatively low molecular weight (Mn = 12.2 kDa, as measured by Dirk Grafahrend, DWI, Aachen, Germany), and the molecular weight of P(VS-VA)-g-PLGA6–10 was slightly higher (Mn = 14.4 kDa) [5], suggesting little influence of molecular weight upon the comparative blank nanoparticle degradation rates. However, the P(VS-VA)-g-PLGA-6–10 nanoparticles were larger in size, due to the higher polymer concentrations necessary to form stable particles in the ouzo region compared to the PLGA particles. One might suppose that the greater relative surface area of the smaller PLGA particles might provide a greater opportunity for hydrolysis and explain the faster degradation rate of the PLGA particles. This was investigated by measuring the degradation rate of a new batch of blank P(VS-VA)-g-PLGA-6–10 nanoparticles of similar size (146.1 ± 0.9 nm, PDI = 0.12 ± 0.01, ζ = −45.1 ± 1.4 mV). These mass loss of these particles (not shown) was almost identical to that of the larger P(VS-VA)-g-PLGA-6–10 nanoparticles, which suggests that the differences in the degradation rates of the blank PLGA

nanoparticles compared to the blank P(VS-VA)-g-PLGA-6–10 nanoparticles is most likely due to the chemical differences, i.e., the presence of the negatively-charged sulfonate functional groups in the P(VS-VA)-g-PLGA polymers. 3.2. Biocompatibility No significant toxic effects were observed from the investigation of blank nanoparticles in IL-8, LDH, or MTT assays (Fig. 3A, B, and C, respectively) compared to control. Although high concentrations of nanoparticles from the polymer SB-PVA-g-PLGA-33–10 display

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slightly higher values in the IL-8 and LDH assays compared to the other polymers, these increases were not statistically significant. The biocompatibility of PLGA is well documented [14]. As the negatively-charged polymers investigated in this work are grafted with large ratios of PLGA (5:1, 10:1, or 15:1), it is not surprising that these polymers show nontoxic profiles in Calu-3 airway epithelial cells. Sivadas et al. recently reported that PLGA microspheres having a geometric diameter of 5.5 ± 0.4 µm showed 98% cell viability in Calu-3 cells, as measured by MTT assay; however, they did observe a slight increase in the release of the inflammatory cytokine IL-8 after an hour exposure to 2 mg/mL of PLGA microspheres prepared from RG504H [15]. One cannot directly compare that report to this present work, however. This is the first investigation of biocompatibility with these negatively-charged polymers. Although they do contain large amounts of PLGA, the functional groups and smaller particle size may account for some differences in comparison to the previously reported biocompatibility profile of PLGA microparticles in Calu-3 cells [15]. 3.3. Drug loading and in vitro drug release P(VS-VA)-g-PLGA-6–10 nanoparticles prepared at a concentration of 50 mg/mL polymer in acetone with a theoretical drug loading of 5% resulted in particles 156 ± 6 nm in size, with a PDI of 0.25 ± 0.0 and ζ = −54.8 ± 2.5 mV. The actual drug loading after cleaning with the Vivaspin 6 columns was 2.7%, corresponding to 55.0 ± 0.4% encapsulation efficiency. The encapsulation of a hydrophilic drug in polymeric nanoparticles validates the strategy to achieve the entrapment of salbutamol by electrostatic interactions with the negatively-charged functional groups of the P(VS-VA)-g-PLGA polymer chain. Further evidence for the success of this strategy is provided from a separate comparison of nanoparticles prepared from polymer concentrations of 10 mg/mL in acetone with approximately 10% theoretical drug loading of salbutamol free base or salbutamol sulfate. Table 1 demonstrates that polymers with less negative charge, namely PLGA and PVA-g-PLGA-10 [16], resulted in much lower encapsulation efficiencies for salbutamol, whereas the negatively-charged polymers P(VS-VA)-g-PLGA-6–10 and SB-PVA-g-PLGA-41–10 resulted in higher encapsulation efficiencies. A positively-charged polymer, DEAPAPVA-g-PLGA-33–10 [16], did not result in successful particle formation under the same conditions. Table 1 also shows the advantage of using salbutamol free base with the negatively-charged polymers rather than salbutamol sulfate. Low encapsulation efficiencies were observed using salbutamol sulfate because the positive charge of the salbutamol molecule is tightly bound to the sulfate component of the salt; however, the free base form of salbutamol has greater access for interaction with the negatively-charged groups of the P(VS-VA)-g-PLGA polymer. The solubility of salbutamol free base in acetone is higher than that of salbutamol sulfate (measured by HPLC after shaking for 24 h to be 1.99 mg/mL compared to 0.016 mg/mL), but even higher amounts of salbutamol free base could be successfully put in solution in acetone because the P(VS-VA)-g-PLGA polymer acted as a co-solute. Nevertheless, the solubility of salbutamol in acetone did limit the total

Table 1 Encapsulation efficiencies for nanoparticles prepared from polymers with approximately 10% theoretical loading of salbutamol sulfate or salbutamol free base. Fig. 3. Biocompatibility of blank nanoparticles as measured by IL-8 production assay (A), LDH assay (B), and by MTT assay (C). In the IL-8 assay, blank nanoparticles were exposed to Calu-3 cells at a concentration of 1 mg/mL and compared to cell culture medium as control. In the LDH and MTT assays, various concentrations of nanoparticles were applied to the Calu-3 cells and compared to cell culture medium as negative control and to 1% Triton-X as positive control. In (B), the control value for Triton-X was 100 ± 5.6%, and in (C), the control value for cell culture medium was 100 ± 5.0%, and the control value for Triton-X was 0 ± 6.8%.

Polymer

PLGA PVA-g-PLGA-10 DEAPA-PVA-g-PLGA-33–10 SB-PVA-g-PLGA-41–10 P(VS-VA)-g-PLGA-6–10

Encapsulation efficiency Salbutamol sulfate

Salbutamol free base

<1% <1% <1% <1% <1%

17% 3% No particles formed 35% 43%

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amount of drug that could be loaded with the P(VS-VA)-g-PLGA particles, resulting in lower encapsulation efficiencies as the theoretical drug loading increased above this solubility limit, which was between 5 and 7.5%. Helle et al. also report a lack of success in entrapping salbutamol sulfate with poly(lactic acid) nanoparticles prepared by a similar procedure [17]. Hadinoto et al., on the other hand, report encapsulation efficiencies as high as 63% for salbutamol sulfate entrapment in poly(methyl methacrylate)-methoxy(polyethylene glycol)methacrylate nanoparticles [18]. However, their definition of drug entrapment efficiency is the amount of drug present in a freeze dried sample relative to the amount of drug added during the preparation, which would include the amount of unentrapped drug dried along with the nanoparticles during lyophilization, and therefore their reported value more accurately represents a process yield rather than a true drug entrapment efficiency. The release of salbutamol from P(VS-VA)-g-PLGA-6–10 nanoparticles was investigated following two methods to remove unencapsulated salbutamol from the nanosuspensions. The first method involved centrifugation of the nanosuspension for 5 min at 13,000 rcf and at 4 °C, removal of the supernatant, and resuspension in purified water, repeated three times. This was compared to centrifugation with Vivaspin 6 columns, details for which are provided in the Materials and methods section. The release of salbutamol following these cleaning procedures is shown in Fig. 4. In both cases, a sustained release of salbutamol from the nanoparticles is observed over 2–3 h. The centrifugation/resuspension method removes more of the unentrapped salbutamol compared to the Vivaspin column method. However, many nanoparticles are lost during the centrifugation/ resuspension process, resulting in a particle yield approximately 60% lower than that observed after the Vivaspin column procedure. Furthermore, the Vivaspin column method resulted in no significant changes in nanoparticle size, PDI, or zeta potential. Centrifugation and resuspension of the nanoparticles, on the other hand, resulted in significant increases in particle size (by about 40 nm) and zeta potential (about 10 mV). Despite its welcome decrease in PDI, the substantially reduced yield resulting from the centrifugation/resuspension prompted the decision to continue in vitro and in vivo studies with the nanosuspensions cleaned by the Vivaspin column method. 3.4. In vitro transport in cell culture Nanoparticles prepared from P(VS-VA)-g-PLGA-6–5 with 5% theoretically loaded salbutamol were administered to monolayers of primary cultured human alveolar epithelial cells and compared to a salbutamol solution of identical drug concentration in order to

Fig. 4. In vitro release of salbutamol from nanoparticles prepared from P(VS-VA)-gPLGA-6–10. Drug release is presented as percent relative to the amount of drug released after 24 h and is presented for two cases: nanoparticles cleaned with Vivaspin 6 columns (open circles) or nanoparticles cleaned by centrifugation and resuspension (black diamonds). Drug release was measured in PBS buffer (pH 7.4) at 37 °C and quantified by HPLC.

Fig. 5. Transport of salbutamol across primary cultured human alveolar epithelial cells as free drug solution (open squares) or as a suspension of salbutamol-containing P(VS-VA)-g-PLGA-6–5 nanoparticles (closed triangles); ⁎ indicates p < 0.05.

ascertain whether a retarded drug release effect could also be observed in cell culture experiments. Fig. 5 indicates that the salbutamol nanosuspension indeed displays a delayed transport of salbutamol across the alveolar epithelial cells compared to the free salbutamol. The lower AUC and Cmax values of the nanosuspension confirms that there is a retarded effect, meaning that after an initial burst release, the drug associated with the nanoparticles is gradually released and subsequently transported across the cell layer. 3.5. In vivo bronchoprotection A suspension of P(VS-VA)-g-PLGA-6–10 nanoparticles with 5% theoretically loaded salbutamol was administered to 6 guinea pigs at a dose of 6 µg salbutamol/kg and compared to free salbutamol solution. The average size of these particles was 116 ± 20 nm (PDI = 0.39 ± 0.08); ζ = −25.6 ± 1.0 mV. Fig. 6 shows that the nanoparticles had a slightly lower initial bronchoprotective effect against acetylcholineinduced bronchospasm compared to 6 µg/kg of free drug, but the bronchoprotection from the nanoparticle formulation lasted until 120 min while the effect of the free drug wore off at least 30 min earlier. The lower initial bronchoprotective effect of the nanosuspension corresponds to the initial burst release of salbutamol from the nanoparticles as had been observed in the in vitro experiments (see Fig. 4). The in vitro drug release showed an initial burst release of approximately 80%, and the in vivo results show an initial pharmacologic effect corresponding to about 75%. The initial release of salbutamol from the nanoparticles acts like a bolus dose followed by a slower controlled release of drug over the next couple hours. This retarded release observed in vitro is in excellent agreement with the extended bronchoprotection seen in vivo over 120 min

Fig. 6. In vivo bronchoprotection action of salbutamol as a free solution (grey squares) or as a suspension of salbutamol-containing P(VS-VA)-g-PLGA-6–10 nanoparticles (black triangles), presented as percent protection over time against acetylcholineinduced bronchospasm in anaesthetised guinea pigs (n = 4). Acetylcholine was injected intravenously at a dose of 9 µg/kg every 10 min; after the first 10 min, salbutamol was administered once by nebulization at a dose of 6 µg/kg as either free drug solution or as nanoparticles; ⁎ indicates p < 0.05.

(compare Figs. 4 and 6). Correlation of in vitro and in vivo results is essential to promote progress in nanomedicine. Other investigators have also observed in vivo nanoparticle performances that corroborate in vitro tests. For example, Danhier et al. recently report that paclitaxel-loaded nanoparticles prepared with PEGylated PLGA, PCLPEG, and PLGA inhibited tumor growth more efficiently than the Taxol® formulation, which was in agreement with cell culture results [19]. Howard et al. have also demonstrated knockdown of TNF-α both in vitro and in vivo using chitosan/siRNA nanoparticles [20]. These examples are essential to provide a foundation of understanding and a platform for launching more advanced projects involving nanomedicine in clinical trials. The results from this work highlight the advantages of a controlled release drug delivery system in providing a sustained pharmacologic effect after the effect of the single dose had already vanished. Although significant progress in this field of research must continue in order to optimize an appropriate drug delivery system exhibiting more enhanced features such as further improvements in drug loading, controlled release, carrier biodegradation, and biocompatibility, this work serves as an important proof-of-concept study to encourage future advancements in the pulmonary applications of nanomedicine. 4. Conclusions This study highlights the agreement of in vitro and in vivo tests during the development and analysis of a novel nanoparticle formulation containing salbutamol. Nanoparticles from the newlysynthesized P(VS-VA)-g-PLGA polymer were followed chronologically from nanoparticle characterization, degradation, biocompatibility, and in vitro release to in vivo performance. Using the free base form of salbutamol, increased drug entrapment was achieved compared to salbutamol sulfate, confirming the successful strategy to prepare nanoformulations of a hydrophilic drug with electrostatic interactions between the polymer's functional groups. Sustained release of salbutamol from the biocompatible nanoparticles was observed over 2.5 h in vitro, and the bronchoprotective effect of the salbutamolcontaining nanoparticles likewise extended over 120 min. Stronger evidence for the capabilities of the in vitro tests in predicting the in vivo action of these nanoparticles could be provided in future studies comparing a series of nanoparticles showing a broader range of drug release rates, together with in vivo biocompatibility data. Nevertheless, the in vivo validation of these in vitro results is a promising foundation for future advancement in nanomedicine strategies for pulmonary drug delivery. Acknowledgements The authors wish to express thanks to the German Ministry of Education and Research (BMBF) for financial support of the NanoInhale project (13N8888) and to Frank Morell and Tobias Lebhardt for technical assistance.

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Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version, at doi: 10.1016/j.jconrel.2009.08.021.

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