- Email: [email protected]
Incorporation of biodegradable nanoparticles into human airway epithelium cells—in vitro study of the suitability as a vehicle for drug or gene delivery in pulmonary diseases
BBRC Biochemical and Biophysical Research Communications 318 (2004) 562–570 www.elsevier.com/locate/ybbrc
Incorporation of biodegradable nanoparticles into human airway epithelium cells—in vitro study of the suitability as a vehicle for drug or gene delivery in pulmonary diseases M. Brzoska,a,* K. Langer,b C. Coester,c S. Loitsch,a T.O.F. Wagner,a and C.v. Mallinckrodta a
b
Department of Pulmonology, Medical Clinic of Johann-Wolfgang-Goethe University Frankfurt am Main, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany Institute for Pharmaceutical Technology, Biocenter of Johann-Wolfgang-Goethe University, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany c Department of Pharmacy, Pharmaceutical Technology and Biopharmacy, Butenandstrasse 5-13, 81377 Munich, Germany Received 30 March 2004 Available online 22 April 2004
Abstract Purpose. Nanoparticles are able to enhance drug or DNA stability for purposes of optimised deposition to targeted tissues. Surface modifications can mediate drug targeting. The suitability of nanoparticles synthesised out of porcine gelatin, human serum albumin, and polyalkylcyanoacrylate as drug and gene carriers for pulmonary application was investigated in vitro on primary airway epithelium cells and the cell line 16HBE14o-. Methods. The uptake of nanoparticles into these cells was examined by confocal laser scan microscopy (CLSM) and flow cytometry (FACS). Further the cytotoxicity of nanoparticles was evaluated by an LDHrelease-test and the inflammatory potential of the nanoparticles was assessed by measuring IL-8 release. Results. CLSM and FACS experiments showed that the nanoparticles were incorporated into bronchial epithelial cells provoking little or no cytotoxicity and no inflammation as measured by IL-8 release. Conclusions. Based on their low cytotoxicity and the missing inflammatory potential in combination with an efficient uptake in human bronchial epithelial cells, protein-based nanoparticles are suitable drug and gene carriers for pulmonary application. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Nanoparticles; Gelatin; Human serum albumin; Polyalkylcyanoacrylates; IL-8; Airway epithelium cells; Bronchial epithelium cells; Drug delivery; Gene delivery; Particulate carrier system; Particulate delivery system
Nanoparticles as drug carrier systems are able to increase intracellular drug and gene delivery in vitro and in vivo to various tissues [1–12]. This has contributed to protection of drugs or DNA from degradation by the envelopment in or binding to a nanoparticle preparation. Additionally, nanoparticles seem to be well suited to traverse cellular membranes. Described applications range from increasing the bioavailability of antiretroviral drugs [2], over gene and oligonucleotide transfer [5,13] to desensibilisation in peanut allergy [7].
* Corresponding author. Fax: +49-69-6301-4749. E-mail address: [email protected] (M. Brzoska).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.04.067
Speaking about gene therapy, especially for diseases of the lung, viral vectors are plagued by safety concerns and non-viral agents lack the necessary in vivo efficiency so that a definitive vehicle is still in demand [14–16]. Nanoparticles have advantages over viral systems such as safety, low cost, stability, ease of manufacturing, and high flexibility concerning the size of the transgene to be delivered. Their disadvantages include reduced efficiency of transgene delivery, toxicity at higher concentrations, and interactions with negatively charged molecules in serum on the cell surface and, as a consequence, poor delivery to the targeted cells [17]. However, protein nanoparticle preparations recently seem to overcome these obstacles [11,12,18]. Especially human serum
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
albumin (HSA) appears to be a suitable agent for gene therapy, because it might avoid undesired interactions with serum that are often encountered after intravenous injection of transfection complexes [19]. The aim of this study was to evaluate a certain gelatin and HSA preparation for the use on the human airway epithelium. One aspect addressed is the incorporation of the nanoparticles into the targeted cells. So far incorporation of nanoparticles has mainly been observed in cells of mononuclear origin such as macrophages [20]. Additionally, we addressed inflammation and cytotoxicity which are known to be an obstacle especially for gene therapy. Materials and methods Particle preparation Preparation of polyalkylcyanoacrylate nanoparticles. Polyalkylcyanoacrylate nanoparticles were prepared according to a previously published method [24–26]. Briefly, 500 ll of the respective alkyl-2-cyanoacrylate monomer was added dropwise to 50.0 ml of an acidic aqueous solution (0.01 N HCl) containing dextran 70.000 or poloxamer188 as stabilisers at a concentration of 1% (m/v). As monomers nbutyl-2-cyanoacrylate (n-BCA) and n-hexyl-2-cyanoacrylate (n-HCA) (Sichel-Werke, Hannover) were used, respectively. The suspensions were stirred for 4 h at room temperature. After neutralisation of the suspension with 0.1 N NaOH, stirring was continued for 1 h followed by filtration through a glass filter (G3, Schott). Preparation of protein-based nanoparticles. Human serum albumin nanoparticles were prepared using a previously outlined desolvation technique [28,29]. HSA (200 mg) was dissolved in 2.0 ml purified water followed by desolvation of the 10% protein solution by the dropwise addition of 6.0 ml ethanol. For stabilisation of the nanoparticles 235 ll of a 8% aqueous glutaraldehyde solution was added to achieve crosslinking. The gelatin nanoparticles were prepared by a two-step desolvation method [30]. Gelatin type A (1.25 g) from porcine skin (175 Bloom; Sigma–Aldrich) was dissolved in 25 ml water under heating. For desolvation 25 ml acetone (Merck) was added. The resulting sediment was redissolved in 25 ml water under heating at pH 2.5. The gelatin was then desolvated again by dropwise addition of 50 ml acetone. After 10 min of stirring 500 ll glutaraldehyde (8%) (Sigma–Aldrich) was added for cross-linking. After additional stirring over 24 h the resulting nanoparticles, gelatin as well as HSA, were purified by 5-fold centrifugation (20,000g, 8 min) and redispersed in an equal volume of water by ultrasonication (Elma Transsonic Digital). For fluorescence labelling fluoresceinamine (Sigma–Aldrich) was added 30 min after addition of the cross-linker in order to quench
563
the remaining free aldehyde groups of the bifunctional glutaraldehyde. Determination of particle size and concentration The particle size was measured by photon correlation spectroscopy (PCS) using a BI-200 SM Goniometer Version 2 (Brookhaven Instruments Corporation). Concentration of the nanoparticle dispersions was determined by gravimetry from the difference of the dried mass of the total sample and the dried mass of the supernatant. An overview over the different nanoparticle preparations and their physicochemicalcharacteristics is given in Table 1. Isolation and culture of airway epithelium cells Human airway epithelium cells were collected from lobectomies by brushing segmental bronchi with Curaprox LS interdental brushes (Curaden AG) avoiding tumorous formations in the process. For cell culture the cells were seeded on collagen coated (Collagen IV; Promocell) tissue culture plastic. Additionally, an air–liquid interface method was used seeding the cells on a permeable membrane (TranswellClear inserts; Corning Costar) placed between the air and the liquid phase. This culture method has been described to provide a more physiological environment in airway epithelium culture [31–33]. As culture medium we used Promocell’s Bronchial Epithelial Cell Growth Medium which is a serum-free medium supplemented with growth factors and hormones [23]. Gentamicin (50 lg/ml) and amphotericin B (5 lg/ml) were further added. Cell culture was performed for 4–5 passages. Additionally, 16HBE14o-cells [34] were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 lg/ml). The number of passages ranged from 20 to 30. Nanoparticle uptake by airway epithelium cells Nanoparticle uptake into cultured airway epithelium was examined by tracing fluoresceineamine-labelled nanoparticles at various concentrations using confocal laser scanning microscopy (CLSM) and flow cytometry (FACS). For CLSM the cells were seeded on Lab-Tek chambered coverglasses (Nalge Nunc International) and incubated with fluorescencelabelled gelatin and HSA nanoparticles at a final concentration of 50 lg/ml for 6 h at 37 and 4 °C, respectively. After washing the cells with PBS the cell membrane was contrasted with a 0.0005% (m/v) solution of tetramethylrhodamine (TRITC)-labelled lectin (concanavalin A-tetramethylrhodamine conjugate, Molecular Probes Europe BV). The cells were fixed with a 4% solution of paraformaldehyde for 10 min and covered with 10% Mowiol 488 (Clariant), 2.5% 1,4-diazabicyclo[2,2,2]octane (Fluka), and 25% glycerol in 0.2 M Tris buffer. Confocal microscopy was then performed with a Leitz microscope (Leitz DM IRB) and a TCS True Confocal Scanner (TCS 4D, Leica) equipped with a krypton–argon laser. For FACS cultivated cells were incubated for 6 h with fluoresceinamine-labelled nanoparticles in culture at 37 and 4 °C and were then
Table 1 Physiochemical characteristics of nanoparticle preparations with regard to size and polydispersity index
Gelatin A nanoparticles Human serum albumin (HSA) nanoparticles Poly-n-butyl-2-cyanoacrylate nanoparticles prepared with poloxamer 188 as stabiliser Poly-n-butyl-2-cyanoacrylate nanoparticles prepared with dextran 70.000 as stabiliser Poly-n-hexyl-2-cyanoacrylate nanoparticles prepared with poloxamer 188 as stabiliser Poly-n-hexyl-2-cyanoacrylate nanoparticles prepared with dextran 70.000 as stabiliser
Diameter (nm)
Polydispersity index
277.8 235.7 141.8 208.8 112.9 238.9
0.0500 0.0966 0.0468 0.2725 0.0957 0.1124
564
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
trypsinised. Alternatively, the cells were trypsinised first followed by an incubation at various nanoparticle concentrations in suspension at 37 and 4 °C for 6 h. Nanoparticle concentrations of 5, 10, and 50 lg/ml were used. As described earlier we stained the cell membrane with a TRITC-concanavalin A conjugate. The cells were analysed with a FACScalibur flowcytometer (Becton–Dickinson). Significance was calculated from raw data with the Wilcoxon–Mann–Whitney test. Cytotoxicity assay Primary pulmonary epithelial cells and immortalised pulmonary epithelial cells (16HBE14o-) were subcultured on 96-well microtitre plates and incubated with the different preparations for 6 h at nanoparticle concentrations of 5, 10, and 50 lg/ml to allow cellular uptake of the nanoparticles. To achieve an accumulation of lactate dehydrogenase (LDH) in the supernatant the cells were incubated for a further period of 12 h after an exchange of the cell culture medium. A LDH-test (CytoTox 96; Promega) was then carried out according to the manufacturer’s instructions. For the determination of possible long term effects the cells were cultivated for further 96 h with intermittent LDH determination in the supernatant after 48 h and after 96 h. The various samples were compared using the Wilcoxon-rank-sum-test.
attached to the cell surface even after repeated washing steps (see Fig. 1). Dose and temperature dependent nanoparticle uptake by airway epithelium cells Our data collected by flow cytometric analysis show that incubation with fluorescence-labelled nanoparticles at 37 °C produces an increased FL1-signal in primary airway epithelium cells at all tested concentrations (5, 10, and 50 lg/ml). This affects all aquired cells and the extent of this shift is dose and temperature dependent (see Fig. 2). Additionally, our testing of primary cells on air–liquid culture systems demonstrates that a polarising cell culture system featuring more “nature like” conditions does not interfere with nanoparticle uptake (data not shown). Cytotoxicity
IL-8 test Interleukin 8 (IL-8) was chosen as parameter of inflammation being expressed by airway epithelium cells and reacting to bacterial, oxidative, and chemical stress [27,35]. Additionally, data on IL-8 release after inoculation of airway epithelium, especially 16HBE14o-cells, with adenovirus are available for comparison [21,22]. The concentration of IL-8 in the supernatant of the 16HBE14o-cell culture and human primary pulmonary epithelium cells was measured by ELISA after 24 h of exposure of the cells to various nanoparticle concentrations (5, 10, 50, and 100 lg/ml). As positive control IL-8 expression and release was provoked in 16HBE14o-cells by incubation with hyperosmotic cell culture medium, generated by adding sodium chloride for a final concentration of 300 mmol/L [36]. IL-8 expression in primary pulmonary epithelium cells was provoked with TNF-a at a concentration of 10 ng/ml since primary cells showed no reaction on elevated osmolarity [27]. The results were analysed using Wilcoxon’s test.
Results Intracellular tracing of nanoparticles The sections produced by the CLSM show that gelatin nanoparticles as well as HSA nanoparticles are incorporated into primary airway epithelium cells during the incubation period. Furthermore, the formation of nanoparticle aggregates is observed under cell culture conditions with HSA nanoparticles forming bigger aggregates than gelatin nanoparticles. A special scheme of intracellular distribution is not observed, also the staining is more cytoplasmatic and no nuclear targeting is detected. At an incubation temperature of 4 °C only an adhesion of the nanoparticles to the outer membrane of the cells is observed, but not a cellular uptake. It is remarkable that gelatin nanoparticles are practically eliminated by the washing procedure after the incubation period, whereas HSA nanoparticles remained
Unlike gelatin- and HSA-based nanoparticles which show no cytotoxic effect compared to the controls (i.e., Fig. 3) polybutylcyanoacrylate as well as polyhexylcyanoacrylate nanoparticles cause a release of intracellular LDH (i.e. Fig. 4). The cytotoxic effect is independent of the stabiliser (dextran 70.000 or poloxamer 188) used for the nanoparticle preparation (data not shown). Furthermore, the cytotoxicity is concentration dependent. Nanoparticles prepared of polybutylcyanoacrylates show the highest cytotoxic effect followed by nanoparticles prepared of polyhexylcyanoacrylates (see Fig. 5). Our long term observations of LDH in the supernatant after 48 and 96 h of cells incubated with gelatin- and HSA-nanoparticles show constant LDH levels equal to the controls of spontaneous LDH release whereas late LDH observation of cells incubated with polyalkylcyanoacrylate nanoparticles shows irregular LDH levels, partly elevated, partly below negative control. Those irregular LDH levels are related to cell attrition due to cell death before medium replacement and thereby reduced cell number during LDH cumulation (data not shown). Inflammation Neither gelatin nor HSA nanoparticles provoke increased interleukin 8 expression at the tested concentrations of 5, 10, 50, and 100 lg/ml in primary airway epithelium cells. 16HBE14o-cells show the same result (see Fig. 6). Regarding the effect of polyalkylcyanoacrylate nanoparticles on interleukin 8 expression in airway epithelium cells the assay was not feasible due to increasing cell detachment at higher nanoparticle concentrations.
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
565
Fig. 1. A picture obtained by confocal microscopy of primary pulmonary epithelium cells showing cross-sections through a cell layer. These sections have been directed to show the interior of a single cell as indicated by arrows in the lower right corner. The predominant section is practically showing the interior of all cells. The nanoparticle bound FITC signal is coded green and the cell membrane bound TRITC-concanavalin is coded red. Primary airway epithelium cells incubated 6 h with either gelatin or HSA nanoparticles at 37 and 4 °C, respectively.
Discussion Gelatin nanoparticles as well as nanoparticles based on HSA were incorporated into airway epithelium cells in a concentration and temperature dependent manner. Temperature dependency of nanoparticle uptake suggests that nanoparticle incorporation is an active, endocytosis like process, and not diffusion through the cellular membrane. This makes an accumulation of the carrier system in endolysosomes most likely even though our investigations with CLSM and FACS do not allow statements about the accumulation of the carrier system in certain cellular organelles. The aspect has already been addressed in other studies by adding certain lysosomolytic agents to nanoparticle formulations, as for instance polyethyleneimine, to enhance plasmid expression in 293-cells [8]. We therefore suggest that additional studies tracing the intracellular trafficking of nanoparticles could indicate further approaches to enhance nanoparticle efficiency.
Previous studies have shown in a rat model that a majority of systemically applied nanoparticles are accumulating in tissues belonging to the reticuloendothelial system [37], also cellular nanoparticle uptake has mainly been described for macrophages [2,20]. In contrast to that our results indicate that colloidal carrier systems can be used for different cellular targets, in this case epithelial cells. In principle, these cells can easily be targeted by inhaling a nanoparticle formulation containing a desired drug or gene, thus circumventing systemic application. Gelatin and HSA nanoparticles showed only little or no cytotoxicity in primary pulmonary epithelium cells and 16HBE14o-cells and in this aspect are highly suitable as drug carriers or for gene therapy on the human airway epithelium. On the other hand, polyalkylcyanoacrylate nanoparticles are highly toxic for airway epithelium cells in contrast to previous studies performed with macrophages [20]. Further, we ascertained that polylacylcyanoacrylate toxicity is dependent on alkyl
566
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
Fig. 2. Primary human airway epithelial cells incubated with FITC stained gelatin and HSA nanoparticles have been analysed by FACS. The figure shows the frequency distributions of FITC emission strength within the cell population at different nanoparticle concentrations at 37 °C compared to incubation at 4 °C. The two columns show our results with gelatin (left) and HSA (right) nanoparticles. The control population is displayed in a blue interlaced line and the sample in continuous red. All the displayed increases of fluorescence in the cell populations incubated with FITC stained nanoparticles are significant with a 95% confidence as verified with Wilcoxon’s test.
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
567
Fig. 3. Cytotoxicity of gelatin nanoparticles on primary pulmonary epithelium cells after 6 h of incubation. Significance was calculated using Wilcoxon’s test. Gelatin nanoparticles act here as a representative for peptide-based formulations with HSA nanoparticles showing similar cytotoxicity.
Fig. 4. LDH release of primary airway epithelium cells after incubation with polybutylcyanoacrylate nanoparticles (short butyl sidechain) prepared with dextran 70,000 as stabiliser. Similar results were observed using poloxamer 188 as stabiliser.
sidechain length with short sidechains being more cytotoxic than longer ones. This result is in good agreement with earlier work of Lherm et al. [38]. The stabiliser used in the nanoparticle formulation had little
influence producing similar result in our test of dextran and poloxamer stabilised cyanoacrylate nanoparticles, indicating that cytotoxicity in fact depends on the polymer itself rather than on the stabilising agent.
568
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
Fig. 5. LDH release after incubation with cyanoacrylate nanoparticles with dextran as stabiliser. This figure compares polyalkylcyanoacrylates of different sidechain length, here with long hexyl and short butyl sidechain.
Fig. 6. Interleukin 8 levels in the supernatant of primary airway epithelium cells after 6 h of incubation with gelatin nanoparticles. A positive control was performed by IL-8 stimulation with TNF-a. Significance was calculated with Wilcoxon’s test. HSA nanoparticles showed similar results.
Gene transfer and gene therapy studies, especially those performed with adenovirus, have shown so far that gene transfer to the airways in vivo induces inflammation. This inflammatory response is eliminating the transgene expression quickly [16]. Two studies have
addressed this problem performing interleukin 8 quantification after inoculation of cell cultures with adenovirus [21,22]. The peptide nanoparticles tested in our study provoked no inflammation corresponding to cytokine cascades involving interleukin 8.
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
Meeting requirements such as low cytotoxicity, low inflammation, and significant uptake into the targeted cells, protein-based nanoparticles remain promising candidates for an efficient in vivo drug or gene transfer in the future. Potential applications in drug targeting include antiviral therapy, for instance using ganciclovirloaded nanoparticles in cytomegalovirus infection [39], or treatment of facultative intracellular microorganisms with antibiotics bound to nanoparticles [40]. Nanoparticle bound formulations might improve drug deposition and activity while reducing systemic toxicity and adverse events. Especially antisense oligonucleotides would benefit from stabilisation and protection from enzymatic digestion when bound in a colloidal drug carrier [41]. To optimise the cellular uptake characteristics of a nanoparticulate system the peptide-based nanoparticles can easily be surface modified by the introduction of functional groups or proteins. For example, the possibility of preparing protein nanoparticles followed by covalent linkage of avidin was recently investigated [42,43]. Other proteins such as transferrin can be used to enable receptor mediated endocytosis [18].
Acknowledgments This work was supported in part by Theodor-Stern Stiftung and Heinz Riese Stiftung. We thank D.C. Gruenert, Cardiovascular Research Institute, University of California, San Francisco, CA, for the gift of 16HBE14o-cells; and Dr. A. Doermer and Dr. M. Zegelmann from Surgical Clinic Nordwest, Frankfurt, for material support.
References [1] J. Kreuter, Evaluation of nanoparticles as drug-delivery systems II: comparison of the body distibution of nanoparticles with the body distribution of microspheres (diameter >1 lm), liposomes, and emulsions, Pharm. Acta Helv. 58 (1983) 217–226. [2] A.R. Bender, H. Von Briesen, J. Kreuter, I.B. Duncan, H. R€ ubsamen-Waigmann, Efficiency of nanoparticles as a carrier system for antiviral agents in human immunodeficiency virusinfected human monocytes/macrophages in vitro, Antimicrob. Agents Chemother. 40 (1996) 1467–1471. [3] R. Gaspar, F.R. Opperdoes, V. Preat, R. Roland, Drug targeting with polyalkylcyanoacrylate nanoparticles: in vitro activity of primaquine-loaded nanoparticles against intracellular Leishmania donovani, Ann. Trop. Med. Parasitol. 86 (1992) 41–49. [4] U. Bodner, Antisense phosphorothioat-oligonukleotide: inhibitoren der translation in vivo, Biotechnol. Appl. Biochem. 2 (1991) 51–54. [5] C. Chavany, D. Trung Le, P. Couvreuer, F. Puisieux, C. Helene, Polyalkylcyanoacrylate nanoparticles as polymeric carriers for antisense oligonucleotides, Pharm. Res. 9 (1992) 441–449. [6] M.F. Moffatt, W.O.C. Cookson, Gene therapy for peanut allergy, Nat. Med. 5 (1999) 380–380. [7] K. Roy, H.-Q. Mao, S.-K. Huang, K.W. Leong, Oral gene delivery with chitosan-DNA nanoparticles generates immunologic protection in a murine model of peanut allergy, Nat. Med. 5 (1999) 387–391.
569
[8] S. Rhaese, H.v. Briesen, H. R€ ubsamen-Waigmann, J. Kreuter, K. Langer, Human serum albumin–polyethylenimine nanoparticles for gene delivery, J. Control. Release 92 (2003) 199–208. [9] D. Luo, W.M. Saltzman, Enhancement of transfection by physical concentration of DNA at the cell surface, Biotechnology 18 (2000) 893–895. [10] H. Cohen, R.J. Levy, J. Gao, I. Fishbein, V. Kousaev, S. Sosnowski, G. Slomkowski, G. Golomb, Sustained delivery and expression of DNA encapsulated in polymeric nanoparticles, Gene Therapy 7 (2000) 1896–1905. [11] F.M. Orson, B.M. Kinsey, P.J. Hua, B.S. Bhogal, C.L. Densmore, M.A. Barry, Genetic immunization with lung-targeting macroaggregated polyethyleneimine–albumin conjugates elicits combined systemic an mucosal immune responses, J. Immunol. 164 (2000) 6313–6321. [12] F.M. Orson, L. Song, A. Gautam, C.L. Densmore, B.S. Bhogal, B.M. Kinsey, Gene delivery to the lung using protein/polyethyleneimine/plasmid complexes, Gene Therapy 9 (2002) 463–471. [13] V.L. Truong-Le, J.T. August, K.W. Leong, Controlled gene delivery by DNA-gelatin nanospheres, Human Gene Therapy 9 (1998) 1709–1717. [14] I.M. Verma, N. Somia, Gene therapy—promises, problems and prospects, Nature 389 (1997) 239–242. [15] R.C. Boucher, Status of gene therapy for cystic fibrosis lung disease, J. Clin. Invest. 103 (1999) 441–445. [16] S.M. Albelda, R. Wiewrodt, J.B. Zuckermann, Gene therapy for lung disease: hype or hope?, Ann. Intern. Med. 132 (2000) 649– 660. [17] C. Plank, K. Mechtler, F.C. Szoka, E. Wagner, Activation of the complement system by synthetic DNA complexes: a potential barrier for intravenous gene delivery, Human Gene Therapy 7 (1996) 1437–1446. [18] V.L. Truong-Le, S.M. Walsh, E. Schweibert, H.-Q. Mao, W.B. Guggino, J.T. August, K.W. Leong, Gene transfer by DNA gelatin nanospheres, Arch. Biochem. Biophys. 361 (1998) 47–56. [19] S. Simoes, V. Slepushkin, P. Pires, R. Gaspar, M.C. Pedroso de Lima, N. Duzgunes, Human serum albumin enhances DNA transfection by lipoplexes and confers resistance to inhibition by serum, Biochim. Biophys. Acta 1463 (2004) 459–469. [20] A.R. Bender, Thesis: Nanopartikel und Liposomen als Tr€agersysteme f€ ur antivirale Wirkstoffe gegen HIV: Entwicklung und pr€aklinische Testung in der Zellkultur, 1997. [21] T.L. Noah, I.A. Wortmann, P.-C. Hu, M.W. Leigh, R.C. oucher, Cytokine production by cultured human bronchial epithelial cells infected with a replication-deficient adenoviral gene transfer vector or wild-type adenovirus type 5, Am. J. Respir. Cell. Mol. Biol. 14 (1996) 417–424. [22] J.T. Bruder, I. Kovesdi, Adenovirus infection stimulates the Raf/ MAPK signaling pathway and induces interleukin-8 expression, J. Virol. 71 (1997) 398–404. [23] J.F. Lechner, M.A. Lechner, L. Veck, A serum free method for culturing normal human bronchial epithelial cells at clonal density, J. Tissue Cult. Methods 9 (1985) 43–48. [24] Courveur, Patrick, Speiser, P.P., patent number 4.329.332 (1982). [25] P. Courveur, C. Dubernet, F. Puiseux, Controlled drug delivery with nanoparticles: current possibilities and future trends, Eur. J. Pharm. Biopharm. 41 (1995) 2–13. [26] P. Courveur, B. Kante, L. Grislain, P.P. Speiser, Toxicity of polyalkylcyanoacrylate nanoparticles: II. Doxorubicin-loaded nanoparticles, J. Pharm. Sci. 71 (1982) 790–792. [27] H.R. Black, J.R. Yankaskas, L.G. Johnson, T.L. Noah, Interleukin-8 production by cystic fibrosis nasal epithelial cells after tumor necrosis factor-alpha and respiratory syncytial virus stimulation, Am. J. Respir. Cell. Mol. Biol. 19 (1998) 210–215. [28] C. Weber, J. Kreuter, K. Langer, Desolvation process and surface characterization of protein nanoparticles, Int. J. Pharm. 194 (2000) 91–102.
570
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
[29] C. Weber, J. Kreuter, K. Langer, Desolvation process and surface characteristics of HSA-nanoparticles, Int. J. Pharm. 196 (2000) 197–200. [30] C.J. Coester, K. Langer, H.v. Briesen, J. Kreuter, Gelatin nanoparticles by two step desolvation—a new preparation method, surface modifications and cell uptake, J. Microencapsulat. 17 (2000) 187–193. [31] D. Jackson, C.F. Rayner, A. Dewar, P.J. Cole, R. Wilson, A human respiratory-tissue organ culture incorporating an air interface, Am. J. Respir. Cell. Mol. Biol. 153 (1996) 1130–1135. [32] M. Yamaya, W.E. Finkbeiner, S.Y. Chun, J.H. Widdicombe, Differentiated structure and function of cultures from human tracheal epithelium, Am. J. Physiol. 262 (1992) L713–L724. [33] M. Jorissen, B.V.d. Schueren, H.V.d. Berghe, J.J. Cassiman, Contribution of in vitro culture methods for respiratory epithelial cells to the study of the physiology of the respiratory tract, Eur. Respir. J. 4 (1991) 210–217. [34] A.L. Cozens, M.J. Yezzi, K. Kunzelmann, T. Ohrui, L. Chin, K. Eng, W.E. Finkbeiner, J.H. Widdicombe, D.C. Gruenert, CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells, Am. J. Respir. Cell. Mol. Biol. 10 (1994) 38–47. [35] O.A. Khair, R.J. Davies, J.L. Devalia, Bacterial-induced release of inflammatory mediators by bronchial epithelial cells, Eur. Respir. J. 9 (1996) 1913–1922. [36] S. Loitsch, C.v. Mallinckrodt, S. Kippenberger, D. Steinhilber, T.O.F. Wagner, J. Bargon, Reactive oxygen intermediates are
[37]
[38]
[39]
[40]
[41]
[42]
[43]
involved in IL-8 production induced by hyperosmotic stress in human bronchial epithelial cells, Biochem. Biophys. Res. Commun. 276 (2000) 571–578. J. Kreuter, Evaluation of nanoparticles as drug-delivery systems iii: materials, stability, toxicity, possibilities of targeting, and use, Pharm. Acta Helv. 58 (1983) 242–250. C. Lherm, R.H. M€ uller, F. Pusieux, P. Courveur, Alkylcyanoacrylate drug carriers: II. Cytotoxicity of cyanoacrylate nanoparticles with different alkyl chain length, Int. J. Pharm. 84 (1992) 13–22. M. Merodio, M.S. Espuelas, M. Mirshahi, A. Arnedo, J.M. Irache, Efficacy of ganciclovir-loaded nanoparticles in human cytomegalovirus (HCMV)-infected cells, J. Drug Target. 10 (3) (2002) 231–238. P. Courveur, E. Fattal, A. Andremont, Liposomes and nanoparticles in the treatment of intracellular bacterial infections, Pharm. Res. 8 (9) (1991) 1079–1086. A. Zimmer, Antisense oligonucleotide delivery with polyhexylcyanoacrylate nanoparticles as carriers, Methods 18 (3) (1999) 286– 295, see also 322. K. Langer, C. Coester, C. Weber, H. Von Briesen, J. Kreuter, Preparation of avidin-labeled protein nanoparticles as carriers for biotinylated peptide nucleic acid, Eur. J. Pharm. Biopharm. 49 (2000) 303–307. C. Coester, J. Kreuter, H. Von Briesen, K. Langer, Preparation of avidin-labelled gelatin nanoparticles as carriers for biotinylated peptide nucleic acid (PNA), Int. J. Pharm. 196 (2000) 147–149.
Incorporation of biodegradable nanoparticles into human airway epithelium cells—in vitro study of the suitability as a vehicle for drug or gene delivery in pulmonary diseases M. Brzoska,a,* K. Langer,b C. Coester,c S. Loitsch,a T.O.F. Wagner,a and C.v. Mallinckrodta a
b
Department of Pulmonology, Medical Clinic of Johann-Wolfgang-Goethe University Frankfurt am Main, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany Institute for Pharmaceutical Technology, Biocenter of Johann-Wolfgang-Goethe University, Marie-Curie-Strasse 9, 60439 Frankfurt, Germany c Department of Pharmacy, Pharmaceutical Technology and Biopharmacy, Butenandstrasse 5-13, 81377 Munich, Germany Received 30 March 2004 Available online 22 April 2004
Abstract Purpose. Nanoparticles are able to enhance drug or DNA stability for purposes of optimised deposition to targeted tissues. Surface modifications can mediate drug targeting. The suitability of nanoparticles synthesised out of porcine gelatin, human serum albumin, and polyalkylcyanoacrylate as drug and gene carriers for pulmonary application was investigated in vitro on primary airway epithelium cells and the cell line 16HBE14o-. Methods. The uptake of nanoparticles into these cells was examined by confocal laser scan microscopy (CLSM) and flow cytometry (FACS). Further the cytotoxicity of nanoparticles was evaluated by an LDHrelease-test and the inflammatory potential of the nanoparticles was assessed by measuring IL-8 release. Results. CLSM and FACS experiments showed that the nanoparticles were incorporated into bronchial epithelial cells provoking little or no cytotoxicity and no inflammation as measured by IL-8 release. Conclusions. Based on their low cytotoxicity and the missing inflammatory potential in combination with an efficient uptake in human bronchial epithelial cells, protein-based nanoparticles are suitable drug and gene carriers for pulmonary application. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Nanoparticles; Gelatin; Human serum albumin; Polyalkylcyanoacrylates; IL-8; Airway epithelium cells; Bronchial epithelium cells; Drug delivery; Gene delivery; Particulate carrier system; Particulate delivery system
Nanoparticles as drug carrier systems are able to increase intracellular drug and gene delivery in vitro and in vivo to various tissues [1–12]. This has contributed to protection of drugs or DNA from degradation by the envelopment in or binding to a nanoparticle preparation. Additionally, nanoparticles seem to be well suited to traverse cellular membranes. Described applications range from increasing the bioavailability of antiretroviral drugs [2], over gene and oligonucleotide transfer [5,13] to desensibilisation in peanut allergy [7].
* Corresponding author. Fax: +49-69-6301-4749. E-mail address: [email protected] (M. Brzoska).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.04.067
Speaking about gene therapy, especially for diseases of the lung, viral vectors are plagued by safety concerns and non-viral agents lack the necessary in vivo efficiency so that a definitive vehicle is still in demand [14–16]. Nanoparticles have advantages over viral systems such as safety, low cost, stability, ease of manufacturing, and high flexibility concerning the size of the transgene to be delivered. Their disadvantages include reduced efficiency of transgene delivery, toxicity at higher concentrations, and interactions with negatively charged molecules in serum on the cell surface and, as a consequence, poor delivery to the targeted cells [17]. However, protein nanoparticle preparations recently seem to overcome these obstacles [11,12,18]. Especially human serum
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
albumin (HSA) appears to be a suitable agent for gene therapy, because it might avoid undesired interactions with serum that are often encountered after intravenous injection of transfection complexes [19]. The aim of this study was to evaluate a certain gelatin and HSA preparation for the use on the human airway epithelium. One aspect addressed is the incorporation of the nanoparticles into the targeted cells. So far incorporation of nanoparticles has mainly been observed in cells of mononuclear origin such as macrophages [20]. Additionally, we addressed inflammation and cytotoxicity which are known to be an obstacle especially for gene therapy. Materials and methods Particle preparation Preparation of polyalkylcyanoacrylate nanoparticles. Polyalkylcyanoacrylate nanoparticles were prepared according to a previously published method [24–26]. Briefly, 500 ll of the respective alkyl-2-cyanoacrylate monomer was added dropwise to 50.0 ml of an acidic aqueous solution (0.01 N HCl) containing dextran 70.000 or poloxamer188 as stabilisers at a concentration of 1% (m/v). As monomers nbutyl-2-cyanoacrylate (n-BCA) and n-hexyl-2-cyanoacrylate (n-HCA) (Sichel-Werke, Hannover) were used, respectively. The suspensions were stirred for 4 h at room temperature. After neutralisation of the suspension with 0.1 N NaOH, stirring was continued for 1 h followed by filtration through a glass filter (G3, Schott). Preparation of protein-based nanoparticles. Human serum albumin nanoparticles were prepared using a previously outlined desolvation technique [28,29]. HSA (200 mg) was dissolved in 2.0 ml purified water followed by desolvation of the 10% protein solution by the dropwise addition of 6.0 ml ethanol. For stabilisation of the nanoparticles 235 ll of a 8% aqueous glutaraldehyde solution was added to achieve crosslinking. The gelatin nanoparticles were prepared by a two-step desolvation method [30]. Gelatin type A (1.25 g) from porcine skin (175 Bloom; Sigma–Aldrich) was dissolved in 25 ml water under heating. For desolvation 25 ml acetone (Merck) was added. The resulting sediment was redissolved in 25 ml water under heating at pH 2.5. The gelatin was then desolvated again by dropwise addition of 50 ml acetone. After 10 min of stirring 500 ll glutaraldehyde (8%) (Sigma–Aldrich) was added for cross-linking. After additional stirring over 24 h the resulting nanoparticles, gelatin as well as HSA, were purified by 5-fold centrifugation (20,000g, 8 min) and redispersed in an equal volume of water by ultrasonication (Elma Transsonic Digital). For fluorescence labelling fluoresceinamine (Sigma–Aldrich) was added 30 min after addition of the cross-linker in order to quench
563
the remaining free aldehyde groups of the bifunctional glutaraldehyde. Determination of particle size and concentration The particle size was measured by photon correlation spectroscopy (PCS) using a BI-200 SM Goniometer Version 2 (Brookhaven Instruments Corporation). Concentration of the nanoparticle dispersions was determined by gravimetry from the difference of the dried mass of the total sample and the dried mass of the supernatant. An overview over the different nanoparticle preparations and their physicochemicalcharacteristics is given in Table 1. Isolation and culture of airway epithelium cells Human airway epithelium cells were collected from lobectomies by brushing segmental bronchi with Curaprox LS interdental brushes (Curaden AG) avoiding tumorous formations in the process. For cell culture the cells were seeded on collagen coated (Collagen IV; Promocell) tissue culture plastic. Additionally, an air–liquid interface method was used seeding the cells on a permeable membrane (TranswellClear inserts; Corning Costar) placed between the air and the liquid phase. This culture method has been described to provide a more physiological environment in airway epithelium culture [31–33]. As culture medium we used Promocell’s Bronchial Epithelial Cell Growth Medium which is a serum-free medium supplemented with growth factors and hormones [23]. Gentamicin (50 lg/ml) and amphotericin B (5 lg/ml) were further added. Cell culture was performed for 4–5 passages. Additionally, 16HBE14o-cells [34] were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 lg/ml). The number of passages ranged from 20 to 30. Nanoparticle uptake by airway epithelium cells Nanoparticle uptake into cultured airway epithelium was examined by tracing fluoresceineamine-labelled nanoparticles at various concentrations using confocal laser scanning microscopy (CLSM) and flow cytometry (FACS). For CLSM the cells were seeded on Lab-Tek chambered coverglasses (Nalge Nunc International) and incubated with fluorescencelabelled gelatin and HSA nanoparticles at a final concentration of 50 lg/ml for 6 h at 37 and 4 °C, respectively. After washing the cells with PBS the cell membrane was contrasted with a 0.0005% (m/v) solution of tetramethylrhodamine (TRITC)-labelled lectin (concanavalin A-tetramethylrhodamine conjugate, Molecular Probes Europe BV). The cells were fixed with a 4% solution of paraformaldehyde for 10 min and covered with 10% Mowiol 488 (Clariant), 2.5% 1,4-diazabicyclo[2,2,2]octane (Fluka), and 25% glycerol in 0.2 M Tris buffer. Confocal microscopy was then performed with a Leitz microscope (Leitz DM IRB) and a TCS True Confocal Scanner (TCS 4D, Leica) equipped with a krypton–argon laser. For FACS cultivated cells were incubated for 6 h with fluoresceinamine-labelled nanoparticles in culture at 37 and 4 °C and were then
Table 1 Physiochemical characteristics of nanoparticle preparations with regard to size and polydispersity index
Gelatin A nanoparticles Human serum albumin (HSA) nanoparticles Poly-n-butyl-2-cyanoacrylate nanoparticles prepared with poloxamer 188 as stabiliser Poly-n-butyl-2-cyanoacrylate nanoparticles prepared with dextran 70.000 as stabiliser Poly-n-hexyl-2-cyanoacrylate nanoparticles prepared with poloxamer 188 as stabiliser Poly-n-hexyl-2-cyanoacrylate nanoparticles prepared with dextran 70.000 as stabiliser
Diameter (nm)
Polydispersity index
277.8 235.7 141.8 208.8 112.9 238.9
0.0500 0.0966 0.0468 0.2725 0.0957 0.1124
564
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
trypsinised. Alternatively, the cells were trypsinised first followed by an incubation at various nanoparticle concentrations in suspension at 37 and 4 °C for 6 h. Nanoparticle concentrations of 5, 10, and 50 lg/ml were used. As described earlier we stained the cell membrane with a TRITC-concanavalin A conjugate. The cells were analysed with a FACScalibur flowcytometer (Becton–Dickinson). Significance was calculated from raw data with the Wilcoxon–Mann–Whitney test. Cytotoxicity assay Primary pulmonary epithelial cells and immortalised pulmonary epithelial cells (16HBE14o-) were subcultured on 96-well microtitre plates and incubated with the different preparations for 6 h at nanoparticle concentrations of 5, 10, and 50 lg/ml to allow cellular uptake of the nanoparticles. To achieve an accumulation of lactate dehydrogenase (LDH) in the supernatant the cells were incubated for a further period of 12 h after an exchange of the cell culture medium. A LDH-test (CytoTox 96; Promega) was then carried out according to the manufacturer’s instructions. For the determination of possible long term effects the cells were cultivated for further 96 h with intermittent LDH determination in the supernatant after 48 h and after 96 h. The various samples were compared using the Wilcoxon-rank-sum-test.
attached to the cell surface even after repeated washing steps (see Fig. 1). Dose and temperature dependent nanoparticle uptake by airway epithelium cells Our data collected by flow cytometric analysis show that incubation with fluorescence-labelled nanoparticles at 37 °C produces an increased FL1-signal in primary airway epithelium cells at all tested concentrations (5, 10, and 50 lg/ml). This affects all aquired cells and the extent of this shift is dose and temperature dependent (see Fig. 2). Additionally, our testing of primary cells on air–liquid culture systems demonstrates that a polarising cell culture system featuring more “nature like” conditions does not interfere with nanoparticle uptake (data not shown). Cytotoxicity
IL-8 test Interleukin 8 (IL-8) was chosen as parameter of inflammation being expressed by airway epithelium cells and reacting to bacterial, oxidative, and chemical stress [27,35]. Additionally, data on IL-8 release after inoculation of airway epithelium, especially 16HBE14o-cells, with adenovirus are available for comparison [21,22]. The concentration of IL-8 in the supernatant of the 16HBE14o-cell culture and human primary pulmonary epithelium cells was measured by ELISA after 24 h of exposure of the cells to various nanoparticle concentrations (5, 10, 50, and 100 lg/ml). As positive control IL-8 expression and release was provoked in 16HBE14o-cells by incubation with hyperosmotic cell culture medium, generated by adding sodium chloride for a final concentration of 300 mmol/L [36]. IL-8 expression in primary pulmonary epithelium cells was provoked with TNF-a at a concentration of 10 ng/ml since primary cells showed no reaction on elevated osmolarity [27]. The results were analysed using Wilcoxon’s test.
Results Intracellular tracing of nanoparticles The sections produced by the CLSM show that gelatin nanoparticles as well as HSA nanoparticles are incorporated into primary airway epithelium cells during the incubation period. Furthermore, the formation of nanoparticle aggregates is observed under cell culture conditions with HSA nanoparticles forming bigger aggregates than gelatin nanoparticles. A special scheme of intracellular distribution is not observed, also the staining is more cytoplasmatic and no nuclear targeting is detected. At an incubation temperature of 4 °C only an adhesion of the nanoparticles to the outer membrane of the cells is observed, but not a cellular uptake. It is remarkable that gelatin nanoparticles are practically eliminated by the washing procedure after the incubation period, whereas HSA nanoparticles remained
Unlike gelatin- and HSA-based nanoparticles which show no cytotoxic effect compared to the controls (i.e., Fig. 3) polybutylcyanoacrylate as well as polyhexylcyanoacrylate nanoparticles cause a release of intracellular LDH (i.e. Fig. 4). The cytotoxic effect is independent of the stabiliser (dextran 70.000 or poloxamer 188) used for the nanoparticle preparation (data not shown). Furthermore, the cytotoxicity is concentration dependent. Nanoparticles prepared of polybutylcyanoacrylates show the highest cytotoxic effect followed by nanoparticles prepared of polyhexylcyanoacrylates (see Fig. 5). Our long term observations of LDH in the supernatant after 48 and 96 h of cells incubated with gelatin- and HSA-nanoparticles show constant LDH levels equal to the controls of spontaneous LDH release whereas late LDH observation of cells incubated with polyalkylcyanoacrylate nanoparticles shows irregular LDH levels, partly elevated, partly below negative control. Those irregular LDH levels are related to cell attrition due to cell death before medium replacement and thereby reduced cell number during LDH cumulation (data not shown). Inflammation Neither gelatin nor HSA nanoparticles provoke increased interleukin 8 expression at the tested concentrations of 5, 10, 50, and 100 lg/ml in primary airway epithelium cells. 16HBE14o-cells show the same result (see Fig. 6). Regarding the effect of polyalkylcyanoacrylate nanoparticles on interleukin 8 expression in airway epithelium cells the assay was not feasible due to increasing cell detachment at higher nanoparticle concentrations.
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
565
Fig. 1. A picture obtained by confocal microscopy of primary pulmonary epithelium cells showing cross-sections through a cell layer. These sections have been directed to show the interior of a single cell as indicated by arrows in the lower right corner. The predominant section is practically showing the interior of all cells. The nanoparticle bound FITC signal is coded green and the cell membrane bound TRITC-concanavalin is coded red. Primary airway epithelium cells incubated 6 h with either gelatin or HSA nanoparticles at 37 and 4 °C, respectively.
Discussion Gelatin nanoparticles as well as nanoparticles based on HSA were incorporated into airway epithelium cells in a concentration and temperature dependent manner. Temperature dependency of nanoparticle uptake suggests that nanoparticle incorporation is an active, endocytosis like process, and not diffusion through the cellular membrane. This makes an accumulation of the carrier system in endolysosomes most likely even though our investigations with CLSM and FACS do not allow statements about the accumulation of the carrier system in certain cellular organelles. The aspect has already been addressed in other studies by adding certain lysosomolytic agents to nanoparticle formulations, as for instance polyethyleneimine, to enhance plasmid expression in 293-cells [8]. We therefore suggest that additional studies tracing the intracellular trafficking of nanoparticles could indicate further approaches to enhance nanoparticle efficiency.
Previous studies have shown in a rat model that a majority of systemically applied nanoparticles are accumulating in tissues belonging to the reticuloendothelial system [37], also cellular nanoparticle uptake has mainly been described for macrophages [2,20]. In contrast to that our results indicate that colloidal carrier systems can be used for different cellular targets, in this case epithelial cells. In principle, these cells can easily be targeted by inhaling a nanoparticle formulation containing a desired drug or gene, thus circumventing systemic application. Gelatin and HSA nanoparticles showed only little or no cytotoxicity in primary pulmonary epithelium cells and 16HBE14o-cells and in this aspect are highly suitable as drug carriers or for gene therapy on the human airway epithelium. On the other hand, polyalkylcyanoacrylate nanoparticles are highly toxic for airway epithelium cells in contrast to previous studies performed with macrophages [20]. Further, we ascertained that polylacylcyanoacrylate toxicity is dependent on alkyl
566
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
Fig. 2. Primary human airway epithelial cells incubated with FITC stained gelatin and HSA nanoparticles have been analysed by FACS. The figure shows the frequency distributions of FITC emission strength within the cell population at different nanoparticle concentrations at 37 °C compared to incubation at 4 °C. The two columns show our results with gelatin (left) and HSA (right) nanoparticles. The control population is displayed in a blue interlaced line and the sample in continuous red. All the displayed increases of fluorescence in the cell populations incubated with FITC stained nanoparticles are significant with a 95% confidence as verified with Wilcoxon’s test.
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
567
Fig. 3. Cytotoxicity of gelatin nanoparticles on primary pulmonary epithelium cells after 6 h of incubation. Significance was calculated using Wilcoxon’s test. Gelatin nanoparticles act here as a representative for peptide-based formulations with HSA nanoparticles showing similar cytotoxicity.
Fig. 4. LDH release of primary airway epithelium cells after incubation with polybutylcyanoacrylate nanoparticles (short butyl sidechain) prepared with dextran 70,000 as stabiliser. Similar results were observed using poloxamer 188 as stabiliser.
sidechain length with short sidechains being more cytotoxic than longer ones. This result is in good agreement with earlier work of Lherm et al. [38]. The stabiliser used in the nanoparticle formulation had little
influence producing similar result in our test of dextran and poloxamer stabilised cyanoacrylate nanoparticles, indicating that cytotoxicity in fact depends on the polymer itself rather than on the stabilising agent.
568
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
Fig. 5. LDH release after incubation with cyanoacrylate nanoparticles with dextran as stabiliser. This figure compares polyalkylcyanoacrylates of different sidechain length, here with long hexyl and short butyl sidechain.
Fig. 6. Interleukin 8 levels in the supernatant of primary airway epithelium cells after 6 h of incubation with gelatin nanoparticles. A positive control was performed by IL-8 stimulation with TNF-a. Significance was calculated with Wilcoxon’s test. HSA nanoparticles showed similar results.
Gene transfer and gene therapy studies, especially those performed with adenovirus, have shown so far that gene transfer to the airways in vivo induces inflammation. This inflammatory response is eliminating the transgene expression quickly [16]. Two studies have
addressed this problem performing interleukin 8 quantification after inoculation of cell cultures with adenovirus [21,22]. The peptide nanoparticles tested in our study provoked no inflammation corresponding to cytokine cascades involving interleukin 8.
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
Meeting requirements such as low cytotoxicity, low inflammation, and significant uptake into the targeted cells, protein-based nanoparticles remain promising candidates for an efficient in vivo drug or gene transfer in the future. Potential applications in drug targeting include antiviral therapy, for instance using ganciclovirloaded nanoparticles in cytomegalovirus infection [39], or treatment of facultative intracellular microorganisms with antibiotics bound to nanoparticles [40]. Nanoparticle bound formulations might improve drug deposition and activity while reducing systemic toxicity and adverse events. Especially antisense oligonucleotides would benefit from stabilisation and protection from enzymatic digestion when bound in a colloidal drug carrier [41]. To optimise the cellular uptake characteristics of a nanoparticulate system the peptide-based nanoparticles can easily be surface modified by the introduction of functional groups or proteins. For example, the possibility of preparing protein nanoparticles followed by covalent linkage of avidin was recently investigated [42,43]. Other proteins such as transferrin can be used to enable receptor mediated endocytosis [18].
Acknowledgments This work was supported in part by Theodor-Stern Stiftung and Heinz Riese Stiftung. We thank D.C. Gruenert, Cardiovascular Research Institute, University of California, San Francisco, CA, for the gift of 16HBE14o-cells; and Dr. A. Doermer and Dr. M. Zegelmann from Surgical Clinic Nordwest, Frankfurt, for material support.
References [1] J. Kreuter, Evaluation of nanoparticles as drug-delivery systems II: comparison of the body distibution of nanoparticles with the body distribution of microspheres (diameter >1 lm), liposomes, and emulsions, Pharm. Acta Helv. 58 (1983) 217–226. [2] A.R. Bender, H. Von Briesen, J. Kreuter, I.B. Duncan, H. R€ ubsamen-Waigmann, Efficiency of nanoparticles as a carrier system for antiviral agents in human immunodeficiency virusinfected human monocytes/macrophages in vitro, Antimicrob. Agents Chemother. 40 (1996) 1467–1471. [3] R. Gaspar, F.R. Opperdoes, V. Preat, R. Roland, Drug targeting with polyalkylcyanoacrylate nanoparticles: in vitro activity of primaquine-loaded nanoparticles against intracellular Leishmania donovani, Ann. Trop. Med. Parasitol. 86 (1992) 41–49. [4] U. Bodner, Antisense phosphorothioat-oligonukleotide: inhibitoren der translation in vivo, Biotechnol. Appl. Biochem. 2 (1991) 51–54. [5] C. Chavany, D. Trung Le, P. Couvreuer, F. Puisieux, C. Helene, Polyalkylcyanoacrylate nanoparticles as polymeric carriers for antisense oligonucleotides, Pharm. Res. 9 (1992) 441–449. [6] M.F. Moffatt, W.O.C. Cookson, Gene therapy for peanut allergy, Nat. Med. 5 (1999) 380–380. [7] K. Roy, H.-Q. Mao, S.-K. Huang, K.W. Leong, Oral gene delivery with chitosan-DNA nanoparticles generates immunologic protection in a murine model of peanut allergy, Nat. Med. 5 (1999) 387–391.
569
[8] S. Rhaese, H.v. Briesen, H. R€ ubsamen-Waigmann, J. Kreuter, K. Langer, Human serum albumin–polyethylenimine nanoparticles for gene delivery, J. Control. Release 92 (2003) 199–208. [9] D. Luo, W.M. Saltzman, Enhancement of transfection by physical concentration of DNA at the cell surface, Biotechnology 18 (2000) 893–895. [10] H. Cohen, R.J. Levy, J. Gao, I. Fishbein, V. Kousaev, S. Sosnowski, G. Slomkowski, G. Golomb, Sustained delivery and expression of DNA encapsulated in polymeric nanoparticles, Gene Therapy 7 (2000) 1896–1905. [11] F.M. Orson, B.M. Kinsey, P.J. Hua, B.S. Bhogal, C.L. Densmore, M.A. Barry, Genetic immunization with lung-targeting macroaggregated polyethyleneimine–albumin conjugates elicits combined systemic an mucosal immune responses, J. Immunol. 164 (2000) 6313–6321. [12] F.M. Orson, L. Song, A. Gautam, C.L. Densmore, B.S. Bhogal, B.M. Kinsey, Gene delivery to the lung using protein/polyethyleneimine/plasmid complexes, Gene Therapy 9 (2002) 463–471. [13] V.L. Truong-Le, J.T. August, K.W. Leong, Controlled gene delivery by DNA-gelatin nanospheres, Human Gene Therapy 9 (1998) 1709–1717. [14] I.M. Verma, N. Somia, Gene therapy—promises, problems and prospects, Nature 389 (1997) 239–242. [15] R.C. Boucher, Status of gene therapy for cystic fibrosis lung disease, J. Clin. Invest. 103 (1999) 441–445. [16] S.M. Albelda, R. Wiewrodt, J.B. Zuckermann, Gene therapy for lung disease: hype or hope?, Ann. Intern. Med. 132 (2000) 649– 660. [17] C. Plank, K. Mechtler, F.C. Szoka, E. Wagner, Activation of the complement system by synthetic DNA complexes: a potential barrier for intravenous gene delivery, Human Gene Therapy 7 (1996) 1437–1446. [18] V.L. Truong-Le, S.M. Walsh, E. Schweibert, H.-Q. Mao, W.B. Guggino, J.T. August, K.W. Leong, Gene transfer by DNA gelatin nanospheres, Arch. Biochem. Biophys. 361 (1998) 47–56. [19] S. Simoes, V. Slepushkin, P. Pires, R. Gaspar, M.C. Pedroso de Lima, N. Duzgunes, Human serum albumin enhances DNA transfection by lipoplexes and confers resistance to inhibition by serum, Biochim. Biophys. Acta 1463 (2004) 459–469. [20] A.R. Bender, Thesis: Nanopartikel und Liposomen als Tr€agersysteme f€ ur antivirale Wirkstoffe gegen HIV: Entwicklung und pr€aklinische Testung in der Zellkultur, 1997. [21] T.L. Noah, I.A. Wortmann, P.-C. Hu, M.W. Leigh, R.C. oucher, Cytokine production by cultured human bronchial epithelial cells infected with a replication-deficient adenoviral gene transfer vector or wild-type adenovirus type 5, Am. J. Respir. Cell. Mol. Biol. 14 (1996) 417–424. [22] J.T. Bruder, I. Kovesdi, Adenovirus infection stimulates the Raf/ MAPK signaling pathway and induces interleukin-8 expression, J. Virol. 71 (1997) 398–404. [23] J.F. Lechner, M.A. Lechner, L. Veck, A serum free method for culturing normal human bronchial epithelial cells at clonal density, J. Tissue Cult. Methods 9 (1985) 43–48. [24] Courveur, Patrick, Speiser, P.P., patent number 4.329.332 (1982). [25] P. Courveur, C. Dubernet, F. Puiseux, Controlled drug delivery with nanoparticles: current possibilities and future trends, Eur. J. Pharm. Biopharm. 41 (1995) 2–13. [26] P. Courveur, B. Kante, L. Grislain, P.P. Speiser, Toxicity of polyalkylcyanoacrylate nanoparticles: II. Doxorubicin-loaded nanoparticles, J. Pharm. Sci. 71 (1982) 790–792. [27] H.R. Black, J.R. Yankaskas, L.G. Johnson, T.L. Noah, Interleukin-8 production by cystic fibrosis nasal epithelial cells after tumor necrosis factor-alpha and respiratory syncytial virus stimulation, Am. J. Respir. Cell. Mol. Biol. 19 (1998) 210–215. [28] C. Weber, J. Kreuter, K. Langer, Desolvation process and surface characterization of protein nanoparticles, Int. J. Pharm. 194 (2000) 91–102.
570
M. Brzoska et al. / Biochemical and Biophysical Research Communications 318 (2004) 562–570
[29] C. Weber, J. Kreuter, K. Langer, Desolvation process and surface characteristics of HSA-nanoparticles, Int. J. Pharm. 196 (2000) 197–200. [30] C.J. Coester, K. Langer, H.v. Briesen, J. Kreuter, Gelatin nanoparticles by two step desolvation—a new preparation method, surface modifications and cell uptake, J. Microencapsulat. 17 (2000) 187–193. [31] D. Jackson, C.F. Rayner, A. Dewar, P.J. Cole, R. Wilson, A human respiratory-tissue organ culture incorporating an air interface, Am. J. Respir. Cell. Mol. Biol. 153 (1996) 1130–1135. [32] M. Yamaya, W.E. Finkbeiner, S.Y. Chun, J.H. Widdicombe, Differentiated structure and function of cultures from human tracheal epithelium, Am. J. Physiol. 262 (1992) L713–L724. [33] M. Jorissen, B.V.d. Schueren, H.V.d. Berghe, J.J. Cassiman, Contribution of in vitro culture methods for respiratory epithelial cells to the study of the physiology of the respiratory tract, Eur. Respir. J. 4 (1991) 210–217. [34] A.L. Cozens, M.J. Yezzi, K. Kunzelmann, T. Ohrui, L. Chin, K. Eng, W.E. Finkbeiner, J.H. Widdicombe, D.C. Gruenert, CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells, Am. J. Respir. Cell. Mol. Biol. 10 (1994) 38–47. [35] O.A. Khair, R.J. Davies, J.L. Devalia, Bacterial-induced release of inflammatory mediators by bronchial epithelial cells, Eur. Respir. J. 9 (1996) 1913–1922. [36] S. Loitsch, C.v. Mallinckrodt, S. Kippenberger, D. Steinhilber, T.O.F. Wagner, J. Bargon, Reactive oxygen intermediates are
[37]
[38]
[39]
[40]
[41]
[42]
[43]
involved in IL-8 production induced by hyperosmotic stress in human bronchial epithelial cells, Biochem. Biophys. Res. Commun. 276 (2000) 571–578. J. Kreuter, Evaluation of nanoparticles as drug-delivery systems iii: materials, stability, toxicity, possibilities of targeting, and use, Pharm. Acta Helv. 58 (1983) 242–250. C. Lherm, R.H. M€ uller, F. Pusieux, P. Courveur, Alkylcyanoacrylate drug carriers: II. Cytotoxicity of cyanoacrylate nanoparticles with different alkyl chain length, Int. J. Pharm. 84 (1992) 13–22. M. Merodio, M.S. Espuelas, M. Mirshahi, A. Arnedo, J.M. Irache, Efficacy of ganciclovir-loaded nanoparticles in human cytomegalovirus (HCMV)-infected cells, J. Drug Target. 10 (3) (2002) 231–238. P. Courveur, E. Fattal, A. Andremont, Liposomes and nanoparticles in the treatment of intracellular bacterial infections, Pharm. Res. 8 (9) (1991) 1079–1086. A. Zimmer, Antisense oligonucleotide delivery with polyhexylcyanoacrylate nanoparticles as carriers, Methods 18 (3) (1999) 286– 295, see also 322. K. Langer, C. Coester, C. Weber, H. Von Briesen, J. Kreuter, Preparation of avidin-labeled protein nanoparticles as carriers for biotinylated peptide nucleic acid, Eur. J. Pharm. Biopharm. 49 (2000) 303–307. C. Coester, J. Kreuter, H. Von Briesen, K. Langer, Preparation of avidin-labelled gelatin nanoparticles as carriers for biotinylated peptide nucleic acid (PNA), Int. J. Pharm. 196 (2000) 147–149.