Pulmonary disposition of vancomycin nebulized as lipid vesicles in rats
MJ de Jesu´s Valle1, J Garavı´s Gonza´lez1, F Gonza´lez Lo´pez1 and A Sa´nchez Navarro1,2
Formulation of antibiotics as inhalable products is proposed to improve their therapeutic index when intended for the treatment of pulmonary infections; as vancomycin shows reduced values of lung partition coefficient, pulmonary administration might be an interesting alternative to conventional administration routes. An experimental study has been performed to compare the pulmonary disposition of vancomycin after inhalation of the drug formulated as a solution and as lipid vesicles (conventional liposomes or liposomes modified with chitosan). Vancomycin concentrations were determined in bronchoalveolar fluid, pulmonary tissue and blood samples from 27 Wistar rats distributed in three groups subjected to nebulisation of the drug formulated as a solution, conventional liposomes or chitosomes. Statistically significant differences between the mean drug
concentrations in bronchoalveolar lavage (BALF) and lung tissue were found upon comparing the solution to lipid vesicles (116.95 lg ml —1±62.13 versus 68.34 lg ml —1±28.90 for liposomes and 65.36±22.11 lgg —1 for chitosomes in BALF; 222.74±37.15 lgg —1 versus 357.17±65.37 lgg —1 for liposomes and 378.83±85.87 lgg —1 for chitosomes in pulmonary tissue). The amount of available drug estimated by mass balance reached the highest values for chitosomes followed by
liposomes (24289.66±4795.48 lg and 20207.91±5318.29 lg, respectively) and the lowest for the solution (18971.64±4765.38 lg). The drug transport and tissue uptake processes showed to be dependent on the nebulized formulation, being facilitated by the lipid vesicles that improved drug passage from the airway space to the pulmonary tissue
and systemic circulation.
The Journal of Antibiotics (2013) 66, 447–451; doi:10.1038/ja.2013.32; published online 15 May 2013
Keywords: chitosan; inhalatory administration; lipid vesicles; pulmonary delivery; pulmonary drug disposition; vancomycin
Pulmonary delivery is currently a promising administration route to improve local effects of drugs used for treatment of respiratory diseases and this is also proposed as an alternative route for biotechnological drugs intended for systemic effects.
Among the nanotechnology-based drug delivery systems assayed for these purposes, liposomes have a relevant role and offer a feasible way of delivering drugs to the lung owing to their attractive biological properties. These spherical, self-closed, biocompatible structures allow entrapment of either hydrophilic or hydrophobic drugs and show different size, charge and surface characteristics according to the formulation procedure applied.
Several strategies based on the pulmonary administration of different types of lipid vesicles have been assayed, and important achievements related to lung targeting of antinfective and anti- asthmatic as well as the systemic delivery of biotechnological drugs have been reported in the recent literature. Amphotericin B, rifampicin, isoniazid, ciprofloxacin or gentamicin have been formu- lated as liposomes in order to improve their therapeutic index by increasing the lung/plasma concentration ratios. Chitosan or specific
ligands, such as mannan and pullulan, added to the surface of amphotericin-loaded vesicles provide sustained drug levels for longer times.1,2 Liposomes of isoniazide formulated with dipalmitoyl- phosphatidylcholine3 have been proposed with the dual aim of providing tuberculosis patients with a pulmonary formulation and also with the phospholipid material that is lacking as pulmonary surfactant in these patients. The use of anionic compounds or specific ligands such as maleylated bovine serum albumin or O-steroyl amilopectine4 has been proposed to formulate liposomes aimed at pulmonary delivery and the macrophage targeting of rifampicin, as these approaches lead to higher and more sustained drug concentrations in the lung tissue. Aerosolization of pro-liposomes is another promising strategy for the pulmonary administration of this drug.5 The formulation of ciprofloxacin6 and gentamicin7 as inhalable liposomes is also considered a good approach to improve their therapeutic index when intended for the treatment of pulmonary infections. Regarding non-antiinfective drugs, budesonide8 or vasoactive intestinal peptide included in sustained-release formulations have been proposed as a ‘dispersable drug depot’ that produces longer-lasting effects than those observed with the
1Department of Pharmacy, University of Salamanca, Salamanca, Spain and 2Institute of Biomedical Research of Salamanca, Salamanca, Spain Correspondence: Professor AS Navarro, Department of Pharmacy, University of Salamanca, Licenciado Me´ndez Nieto s/n, 37007 Salamanca, Spain. E-mail: [email protected]
Received 22 February 2013; accepted 5 March 2013; published online 15 May 2013
corresponding free drug.9 Many attempts have been made to achieve good selectivity in the targeting of tumor cells by preparing specialized carrier agents that are profitable for anticancer therapy. Among these, in the context of antitumor therapy, liposomes are the colloidal particles that to date have received the most attention.10–12 The relevance of research work on pulmonary delivery is not restricted to local effects but also for the systemic delivery of proteins and peptide drugs. Studies carried out with liposomal formulations of insulin13,14 or heparin15–17 reveal that this strategy seems to increase the serum half-life of these drugs.
Despite the efforts being made in research into pulmonary administration and the development of drug delivery systems through this route, current applications in the clinical setting are still very limited. The lung toxicity of excipients may be one of the main limitations to further advances in dosage forms able to produce efficacious pulmonary delivery. Phospholipids are natural components of the pulmonary surfactant and chitosan has shown a high biocompatibility even with the pulmonary environment;18 accordingly, formulations based on these compounds seem to be a reasonable proposal for pulmonary delivery systems of clinical usefulness. Building on this hypothesis, the present work addresses the evaluation of lipid vesicles as vehicles for the pulmonary administration of antibiotics used in respiratory infections. Conventional and chitosan-modified liposomes (chitosomes) were studied to determine the pulmonary disposition of vancomycin in laboratory animals after the nebulisation of drug-loaded vesicles. Vacomycin was chosen as the loading drug due to its characteristics of low partition coefficient value in lung together with narrow therapeutic window, both making this drug a good candidate for inhalatory administration. Comparison of results obtained for aerosolized free drug was made in order to determine the influence of the formulation on the kinetic behavior of the antibiotic after pulmonary delivery.
MATERIALS AND METHODS
Vancomycin was provided by Combino Pharm Labs (Barcelona, Spain), egg L-a- phosphatidylcholine, lanolin cholesterol, soluble low-molecular weight chitosan, Na2HPO4 2H2O and KH2PO4 were purchased from Sigma-Aldrich Quimica SA (Madrid, Spain). Acetonitrile for HPLC and Triton were provided by Merck
SA (Madrid, Spain); perchloric acid and acetic acid of analytical grade were from Panreac Quimica SA (Barcelona, Spain). Ultra-pure water was obtained with a MilliQ Millipore device (Millipore Iberica SAU, Madrid, Spain). Methanol HyperSolv Chromanorm of HPLC-gradient grade was provided by VWR International Eurolab SL (Barcelona, Spain), and trichloromethane was from Panreac Quimica SA.
Preparation of liposomes. A mixture of phosphatidylcholine and cholesterol (molar ratio 0.7) was used to prepare the vesicles, applying the method of solvent evaporation and lipid hydration.19 Briefly, the lipid mixture was dissolved in a chloroform-methanol (2:1 v/v) mixture and poured into a glass flask placed in a water bath at 40 1C connected to a rotary evaporator (60 r.p.m. and 50 mBar pressure) for 30 min for solvent evaporation. Following this, the temperature was reduced to 25 1C and a vacuum was maintained for 24 h in order to eliminate residual solvents. Then, the vancomycin solution (5 mg ml—1) and 1 g of glass beads were added to the flask containing the dried
lipids and returned to the rotary evaporator at 40 1C (60 r.p.m. for 30 min) for
lipid hydration. Next, the suspension was kept at room temperature for 10 min to complete the formation of liposomes and was subsequently subjected to sonication for 15 min. An additional step was performed to obtain chitosomes; 0.1% chitosan (w/v) dissolved in a 1% (w/v) acetic acid solution was added
dropwise to 10 ml of the previous lipid vesicle suspension at room temperature with continuous stirring.
Pulmonary administration. Twenty-seven Wistar male rats from Charles River (Barcelona, Spain) with a mean body weight of 247.41±20.40 g were included in the study. Twelve hours prior to the experiments, the animals were isolated in cages and allowed access to tap water ad libitum. The housing and
experimental treatment of the animals were in accordance with the corresponding Guide from the Institute for Laboratory Animal Research (ILAR 1996). The experiments complied with current Spanish legislation and adhered to the ‘Principles of Laboratory Animal Care’. After being weighed, the animals were anesthetized with 80 mg kg—1 sodium thiopental (intraperitoneal route) and 1000 IU sodium heparin was injected by the same route to avoid clotting. Then, tracheotomy and tracheal cannulation were performed with the
animals on their backs and the cannula was connected to the respiratory equipment, which consists of a nebulizer (Ultrasonic Aerosol Generator 700700-UV system TSE, Technical & Scientific Equipment GmbH, Bad Homburg, Germany) and an artificial ventilator (7025 Rodent Ventilator, Ugo Basile, Comerio VA, Italy). The latter was set at 60 respirations per min and 2 ml of tidal volume was delivered to the rat lungs. All animals were subjected to 20 min of nebulization of the drug formulated as an aqueous dissolution (Group I), a liposome suspension (Group II), or a chitosome suspension (Group III). At the end of this period, samples were collected as follows: blood by direct puncture of the left ventricle, bronchoalveolar fluid by bronchoalveolar lavage (BALF) using 0.3 ml of 0.9% saline solution, and pulmonary tissue by lung excision. The excised tissue was weighed and a 1-g
aliquot was separated for processing by homogenization with 50 mM phosphate buffer, pH¼ 7.4 and 0.1% Triton X-100.
Blood samples were split into two aliquots: one was used to quantify vancomycin concentrations in plasma and the other to determine concentra- tions in whole blood. In all samples, drug concentrations were determined with an HPLC technique validated and described previously.20 The actual nebulized volume was determined from the initial and final amounts of the formulation in the nebulizer equipment.
Data analysis. The amount of drug available (Dav) was estimated by mass balance according to the disposition model shown in Figure 1 and the following equations:
Dav ¼ Qbal þ QL þ Qb þ Qt þ Qel ð1Þ
Qbal ¼ Vbal×Cbal ð2Þ
Qel ¼ CLr×Cp× 20 min ð6Þ
where, Qbal, QL, Qb, and Qt represent the amount of drug in the BALF, lung tissue, blood and rat body, respectively; Qel is the estimated amount of drug excreted in urine; Vbal is the bronchoalveolar fluid volume; Cbal is the bronchoalveolar fluid concentration; WL is lung weight; CL is the lung concentration; Cb is the blood concentration; Vb is the blood volume; R is the tissue/plasma partition coefficient; Cp is the plasma concentration; BW is body weight, and CLr is the renal drug clearance.
Vbal, Cbal, WL, CL, Cb and Cp and BW were determined experimentally. The Vb, R and CLr values (Vb 60 ml kg—1; R 0.5 and CLr 3.2 ml min) were obtained from the literature.21–23
Comparison of the results obtained for vancomycin in the three groups was performed by statistical analysis (analysis of variance, ANOVA)24 using GraphPad Prism, version 4, package (GraphPad Software, San Diego, CA, USA; www.graphpad.com).
Table 1 shows the mean values of the available dose (Dav) estimated from equation 1, together with the amounts of vancomycin in
BALF, lung tissue, blood, and the rest of body at the end of drug nebulisation for all three formulations assayed (solution,
Table 2 Vancomycin concentrations after pulmonary delivery
liposomes and chitosomes). Dav
values obtained for lipid vesicles
were higher than observed for solution; in particular, chitosomes showed the highest value (24.29±4.79 mg) followed by liposomes (20.21±5.32 mg) and dissolution (18.97±4.76 mg), although the statistical comparison failed to reveal significant differences among the three formulations (P ¼ 0.13). On the contrary, the amount of drug remaining at the BALF was higher for solution, while the amounts in the rest of the sampled spaces were lower than the observed for both types of vesicles. As the drug concentration, rather than the amount of drug, is responsible for drug effects, a comparison of the vancomycin concentrations achieved in the different body spaces was performed. Table 2 shows the mean drug concentration values achieved in the BALF, lung tissue and plasma at the end of the nebulization period. Statistical comparison revealed significant differences between the solution and lipid vesicles for concentrations in the BALF and lung tissue (P ¼ 1.15 × 10 —4 and P ¼ 5.80 × 10 —3, respectively) but no statistically significant differences were detected for plasma (P ¼ 0.07).
Figure 2 Vancomycin mean concentrations in BALF, lung tissue and plasma samples after drug nebulization as a solution or as lipid vesicles (***Po0.001 and **Po0.01).
Figure 1 Schematic representation of the physiologically based pharmacokinetic (PBPK) model considered.
Figure 3 Lung tissue/BALF and plasma/BALF partition coefficient values of vancomycin after nebulization of the solution and lipid vesicles (***Po0.001 and **Po0.01).
Table 1 Vancomycin amounts in different body spaces after pulmonary delivery
Q blood (mg) Q BALF (mg) Q lung tissue (mg) Q rat body (mg) Qel (mg) Dav (mg)
Solution 942.57±248.46 35.08±18.64 261.09±54.63 11782.07±3105.80 5950.83±1337.84 18971.64±4765.38
Liposomes 992.28±278.69 20.50±8.67 433.62±98.39 12403.53±3483.61 6357.97±1448.93 20207.91±5318.29
Chitosomes 1182.00±243.04 19.61±6.63 478.18±134.09 14774.96±3037.94 7804.91±1373.78 24289.66±4795.48
Abbreviations: BALF, bronchoalveolar lavage fluid; Dav, total available drug; Qel, eliminated drug amount estimated.
Q blood, Q BALF, Q lung tissue, Q rat body: drug amounts in each space at the end of nebulization period.
Figure 4 Lung tissue/BALF and plasma/BALF partition coefficient values of vancomycin after nebulization of liposomes and chitosomes.
Figures 2 and 3 illustrate differences in pulmonary drug disposition between the solution and lipid vesicle formulations (data from liposomes and chitosomes were pooled and represented in these figures in order to illustrate and highlight the influence of lipid formulation). As shown in Figure 3, lung tissue/BALF concentration ratio takes a value of 1.9 for drug dissolution and about 5.5 for lipid vesicles; the plasma/BALF ratio also increased from 0.8 to 1.6, the differences being statistically significant in both cases (P ¼ 5.48 × 10 —4 and P ¼ 8.36 × 10 —3, respectively).
Conventional liposomes and chitosomes were also compared with
each other and slight but interesting differences were found between both types of vesicles. Both lung tissue/BALF and plasma/BALF ratios were higher for chitosomes compared with liposomes. Figure 4 illustrates these results. Although no statistically significant differences
were found for the BALF (P ¼ 0.81) and pulmonary tissue concen- trations (P ¼ 0.55), statistically significant differences were detected for plasma (P ¼ 0.04).
Data on the available dose estimated from pharmacokinetic model assumed (Figure 1) reveal that a higher amount of drug is able to access the body compartments when nebulized vancomycin is included in lipid vesicles as compared with the solution. Only at the BALF, the remaining drug amount was higher for solution than that observed for both types of vesicles, while in the rest of the sampled spaces these were lower for the solution nebulization. These results indicate that drug transport from the air space to the pulmonary tissue, and subsequently to the systemic blood is facilitated by the lipid formulations. Differences in pulmonary drug disposition between the solution and lipid vesicle formulations are evident in this study (Figures 2 and 3), leading to a pulmonary tissue/BALF concentration ratio of 5.5 when estimated from both lipid formulations (liposomes and chitosomes) versus 1.9 when calculated from solution. This finding means that transport and tissue uptake of aerosolized vancomycin in the respiratory system is dependent on the delivery formulation used, being facilitated by lipid
and pulmonary tissue concentrations, but statistically significant differences were detected for plasma. These results are in accordance with those recently reported by Al-Quadi et al.,25 who found a prolonged hypoglycaemic effect of dry powders containing insulin- loaded chitosan nanoparticles administered through the intratracheal route to rats. The mucoadhesive properties of chitosan might minimize particle mucociliary clearance, as well as being responsible for enhancing drug absorption by opening the epithelial tight junctions, facilitating paracellular transport. This has been proposed previously26 and observed upon incubation with cell lines (Cacu-3 and 16HBE14o) representative of the bronchial epithelium.27,28 Low toxicity, biodegradability, biocompatibility, mucoadhesivity and permeation enhancement are all desirable characteristics for pulmonary delivery formulations, making chitosan one of the most interesting and promising materials currently under investigation for inhalation of different type of drugs even for gene delivery.29
It is interesting to notice that for pulmonary infection treatments, antibiotic delivery by inhalation is proposed for local targeting and that drug access to the systemic circulation may not be desirable in those cases, particularly when the antiinfective agent shows a narrow therapeutic window. Vancomycin nebulization for the treatment of pulmonary infections is an example of this situation, because the drugs showing low lung tissue/plasma partition coefficient but high plasma levels are not desirable due to related toxic effects.30 Many other antibiotics including aminoglycosides or linezolid show the same peculiarity; passive drug targeting at the respiratory system instead of improved transport to the systemic circulation would be desired for those cases. According to the results of the present study, the nebulization of conventional liposomes would be the best of the three assayed options for pulmonary antibiotic delivery, from both the therapeutic and toxicological points of view, because uptake and permanence in pulmonary spaces instead of systemic access is aimed. Chitosomes would be a better option when systemic effect is pursued and drug solution when BALF is the target.
In summary, the pulmonary disposition of vancomycin after nebulisation is influenced by the type of drug formulation administered. Lipid vesicles improve drug transport from the airways to lung tissue and the systemic circulation, leading to higher available doses as well as higher drug concentrations in pulmonary tissue and plasma. Our results confirm the properties of chitosan as a permea- tion enhancer in the respiratory system and suggest that, among systems ML162 assayed here, conventional liposomes would be the best option for vancomycin nebulization.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
The authors acknowledge the Consejerı´a de Educacio´n de la Junta de Castilla y Leo´n for financial support. (Research Project SA011A08)
vesicles, which showed an ability to increase the drug passage to deeper lung spaces. Regarding comparison between conventional
liposomes and chitosomes, slight but interesting differences were found as both lung tissue/BALF and plasma/BALF ratios were higher for chitosomes compared to liposomes (Figure 4). It seems that the inclusion of chitosan in the vesicles produces a more efficient drug transport from the airways to the body spaces, because the concen- trations in pulmonary tissue and plasma as well as the available dose (Dav) were the highest for the chitosomes. As indicated in the Results section, no statistically significant differences were found for the BALF
1 Albasarah, Y. Y., Somavarapu, S., Stapleton, P. & Taylor, K. M. G. Chitosan-coated
antifungal formulations for nebulisation. J. Pharm. Pharmacol. 62, 821–828 (2010).
2 Vyas, S. P., Quraishi, S., Gupta, S. & Jaganathan, K. S. Aerosolized liposome-based delivery of amphotericin B to alveolar macrophages. Int. J. Pharm. 296, 12–25 (2005).
3 Chimote, G. & Banerjee, R. In vitro evaluation of inhalable isoniazid-loaded surfactant liposomes as adjunct therapy in pulmonary tuberculosis. J. Biomed. Mater. Res. B Appl. Biomater 94B (1), 1–10 (2010).
4 Vyas, S. P., Kannan, M. E., Jain, S., Mishra, V. & Singh, P. Design of liposomal aerosol for improves delivery of rifampicin to alveolar macrophages. Int. J. Pharm. 269, 37–49 (2004).
5 Gaur, P. K. et al. In-situ formation of liposome of rifampicin: better availability for better treatment. Curr. Drug Deliv. 6, 461–468 (2009).
6 Chono, S., Tanino, T., Seki, T. & Morimoto, K. Efficient drug targeting to rat alveolar macrophages by pulmonary administration of ciprofloxacin incorporated into manno- sylated liposomes for treatment of respiratory intracellular parasitic infections. J. Control Release 127, 50–58 (2008).
7 Pinto-Alphandary, H., Andremont, A. & Couvreur, P. Targeted delivery of antibiotics using liposomes and nanoparticles: research and applications. Int. J. Antimicrob. Agents 13, 155–168 (2000).
8 Parmar, J. J. et al. Development and evaluation of inhalational liposomal system of budesonide for better management of asthma. Indian J. Pharm. Sci. 72 (4), 442–448 (2010).
9 Stark, B., Debbage, P., Andreae, F., Masgoeller, W. & Prassl, R. Association of vasoactive peptide with polymer-graftes liposomes: structural aspects for pulmonary delivery. Biochim. Biophys. Acta. 1768, 705–714 (2007).
10 Cattel, L., Ceruti, M. & Dosio, F. From conventional to stealth liposomes: a new frontier in cancer chemotherapy. Tumori 89 (3), 237–249 (2003).
11 Zhao, L., Ye, Y., Li, J. & Wei, Y. Preparation and the in-vivo evaluation of paclitaxel liposomes for lung targeting delivery in dogs. J. Pharm. Pharmacol. 63, 80–86 (2011). 12 Li, P. et al. A novel cationic liposome formulation for efficient gene delivery via
pulmonary route. Nanotechnology 22, 1–10 (2011).
13 Bi, R., Shao, W., Wang, Q. & Zhang, N. Spray-freeze-dried dry powder inhalation of insulin-loaded liposomes for enhanced pulmonary delivery. J. Drug Target 16 (9), 639–648 (2008).
14 Chono, S., Fukuchi, R., Seki, T. & Morimoto, K. Aerosolized liposomes with dipalmitoyl phosphatidylcholine enhance pulmonary insulin delivery. J. Control Release 137, 104–108 (2009).
15 Bai, S. & Ahsan, F. Inhalable liposomes of low molecular weight heparin for the treatment of venous thromboempolism. J. Pharm. Sci. 99 (11), 4554–4564 (2010).
16 Bai, S., Gupta, V. & Ahsan, F. Cationic liposomes as carries for aerosolized formulations of an anionic drug: safety and efficacy study. Eur. J. Pharm. Sci. 38, 165–171 (2009). 17 Oyarzun-Ampuero, F. A., Brea, J., Loza, M. I., Torres, D. & Alonso, M. J. Chitosan- hyaluronic acid nanoparticles loaded with heparin for the treatment of asthma. Int. J.
Pharm. 381, 122–129 (2009).
18 De Jesu´s Valle, M. J., Dinis-Oliveira, R. J., Carvalho, F., Bastos, M. L. & Sa´nchez, N. A. Toxicological evaluation of lactose and chitosan delivered by inhalation. J. Biomater. Sci. Polym. Ed. 19 (3), 387–397 (2008a).
19 Bangham, A. D., Standishand, M. M. & Watkins, J. C. Diffusion of univalent ions across the lamellae of swollen phospholipids. J. Mol. Biol. 13, 238–252 (1965).
20 De Jesu´s Valle, M. J., Lo´pez, F. G. & Navarro, A. S. Development and validation of an HPLC method for vancomycin and its application to a pharmacokinetic study. J. Pharm. Biomed. Anal. 48 (3), 838–839 (2008b).
21 Lee, H. B. & Blaufox, M. D. Blood volume in the rat. J. Nucl. Med. 25, 72–76 (1985). 22 Beckmann, J. et al. Tissue concentrations of vancomycin and moxifloxacin in
periprosthetic infection in rats. Acta. Orthop. 78 (6), 766–773 (2007).
23 Engineer, M. S., Ho, D. H. & Bodey, G. P. Comparison of vancomycin disposition in rats with normal and abnormal renal functions. Antimicrob. Agents Chemother. 20 (6), 718–722 (1981).
24 Milton, S. Estadı´stica para biolog´ıa y ciencias de la salud 3th edn (McGraw Hill, Interamericana, Madrid, 2007).
25 Al-Quadi, S., Grenha, A., Carrio´n-Recio, D., Seijo, B. & Remun˜an-Lo´pez, C. Microencapsulated chitosan nanoparticles for pulmonary protein delivery: in vivo evaluation of insulin-loaded formulations. J. Control Release 157, 383–390 (2012).
26 Yamamoto, H., Kuno, Y., Sugimoto, S., Takeuchi, H. & Kawashima, Y. Surface modified PLG nanosphere with chitosan improved pulmonary delivery of calcitonin by mucoadhesion and opening of the intercellular tight junctions. J. Control Release 102, 373–381 (2005).
27 Lim, S. T., Forbes, B., Martin, G. P. & Brown, M. B. In vitro and in vivo characterization of nobel microparticulates based on hyaluronan and chitosan hydroglutamate. AAPS Pharm. Sci. Tech. 2, 14 (2001).
28 Florea, B. I., Thanou, H. E., Junginger, G. & Borchard, G. Enhancement of bronchial ocreotide absorption by chitosan and N-trimethylchitosan shows linear in vitro/in vivo correlation. J. Control Release 110, 353–361 (2006).
29 Conti, D. S., Bharatwaj, B., Brewer, D. & Da Rocha, S. R. P. Propellant-based inhalers for the non-invasive delivery of genes via oral inhalation. J. Control Release 157, 406–417 (2012).
30 Elyasi, S., Khalili, H., Dashti-Khavidaki, S. & Mohammadpour, A. Vancomycin-induced nephrotoxicity: mechanism, incidence, risk factors and special populations. A literature review. Eur. J. Clin. Pharmacol. 68, 1243–1255 (2012).