Nanoscale Probing of Liposome Encapsulating Drug Nanocrystal Using Atomic Force Microscopy-Infrared Spectroscopy.

Use of liposomes encapsulating drug nanocrystals for the treatment of diseases like cancer and pulmonary infections is gaining attention. The potential therapeutic benefit of these engineered formulations relies on maintaining the physical integrity of the liposomes and the stability of the encapsulated drug. With the significant advancement in the microscopic and analytical techniques, analysis of the size and size distribution of these nanosized vesicles is possible. However, due to the limited spatial resolution of conventional vibrational spectroscopy techniques, the chemical composition of individual nanosized liposome cannot be resolved. To address this limitation, we applied atomic force microscopy infrared spectroscopy (AFM-IR) to assess the chemical composition of individual liposomes encapsulating ciprofloxacin in dissolved and nanocrystalline form. Spatially resolved AFM-IR spectra acquired from individual liposomes confirmed the presence of peaks related to N-H bending vibration, C-N stretching and symmetric, and asymmetric vibration of the carboxyl group present in the ciprofloxacin. Our results further demonstrated the effectiveness of AFM-IR in differentiating the liposome containing ciprofloxacin in dissolved or nanocrystalline form. Spectra acquired from dissolved ciprofloxacin had peaks related to the ionised carboxyl group, i.e., at 1576 and 1392 cm-1, which were either absent or far weaker in intensity in the spectra of liposomal sample containing ciprofloxacin nanocrystals. These findings are highly significant for pharmaceutical scientists to ascertain the stability and physicochemical composition of individual liposomes and will facilitate the design and development of liposomes with greater therapeutic benefits.

Solid State Characterization of Ciprofloxacin Liposome Nanocrystals.

Liposomes have been widely researched as drug delivery systems; however, the solid state form of drug inside the liposome, whether it is in solution or in a solid state, is often not studied. The solid state properties of the drug inside the liposomes are important, as they dictate the drug release behavior when the liposomes come into contact with physiological fluid. Recently, a new approach of making liposomal ciprofloxacin nanocrystals was proposed by the use of an additional freeze-thawing step in the liposomal preparation method. This paper aims to determine the solid state properties of ciprofloxacin inside the liposomes after this additional freeze-thawing cycle using cryo-TEM, small-angle X-ray scattering (SAXS), and cross-polarized light microscopy (CPLM). Ciprofloxacin precipitated in the ciprofloxacin hydrate crystal form with a unit cell dimension of 16.7 Å. The nanocrystals also showed a phase transition at 93 °C, which represents dehydration of the hydrate crystals to the anhydrate form of ciprofloxacin, verified by temperature-dependent SAXS measurements. Furthermore, the dependence of the solid state form of the nanocrystals on pH was investigated in situ, and it was shown that the liposomal ciprofloxacin nanocrystals retained their crystalline form at pH 6-10. Understanding the solid state attributes of nanocrystals inside liposomes provides improved understanding of drug dissolution and release as well as opening avenues to new applications where the nanosized crystals can provide a dissolution benefit.

Direct Comparison of Standard Transmission Electron Microscopy and Cryogenic-TEM in Imaging Nanocrystals Inside Liposomes.

The use of electron microscopy techniques in the understanding of shape and size of nanoparticles are commonly applied to drug nanotechnology, but the type of microscopy and suitability for the particles of interest can have a significant impact on the result. The size and shape of the nanoparticles are crucial in clinical applications; however, direct comparison of the results from standard transmission electron microscopy (TEM) and cryo-TEM have rarely been reported. As a useful case for comparison, liposomal drug nanocrystals are studied here. In this study, the effect of thawing temperature on the size and shape of the ciprofloxacin nanocrystals was determined. A quantitative standard TEM assay was developed to allow for high-throughput particle size analysis. These results were compared to size and shape information obtained using the cryo-TEM method. The results showed broad agreement between the two TEM methods and that ciprofloxacin nanocrystals formed shorter and thinner crystals inside the liposomes at higher thawing temperatures. The results provide confidence in the use of standard TEM to determine the size and shape distribution of solid nanoparticles (in this case, encapsulated inside liposomes) from aqueous media without fear of sample preparation altering the conclusions.

Effective Treatment of Mycobacterium avium subsp. hominissuis and Mycobacterium abscessus Species Infections in Macrophages, Biofilm, and Mice by Using Liposomal Ciprofloxacin.

Nontuberculous mycobacteria (NTM) affect an increasing number of individuals worldwide. Infection with these organisms is more common in patients with chronic lung conditions, and treatment is challenging. Quinolones, such as ciprofloxacin, have been used to treat patients, but the results have not been encouraging. In this report, we evaluate novel formulations of liposome-encapsulated ciprofloxacin (liposomal ciprofloxacin) in vitro and in vivo Its efficacy against Mycobacterium avium and Mycobacterium abscessus was examined in macrophages, in biofilms, and in vivo using intranasal instillation mouse models. Liposomal ciprofloxacin was significantly more active than free ciprofloxacin against both pathogens in macrophages and biofilms. When evaluated in vivo, treatment with the liposomal ciprofloxacin formulations was associated with significant decreases in the bacterial loads in the lungs of animals infected with M. avium and M. abscessus In summary, topical delivery of liposomal ciprofloxacin in the lung at concentrations greater than those achieved in the serum can be effective in the treatment of NTM, and further evaluation is warranted.

Tuning Ciprofloxacin Release Profiles from Liposomally Encapsulated Nanocrystalline Drug.

PURPOSE: In order to attenuate the drug release rate, a single freeze-thaw step was previously shown to convert encapsulated drug into a single nanocrystal within each liposome vesicle. The goal of this study was to alter the nanocrystalline character, and thus the drug encapsulation state and release profile, by addition of surfactant prior to freeze-thaw. METHODS: A liposomal ciprofloxacin (CFI) formulation was modified by the addition of surfactant and frozen. After thawing, these formulations were characterized in terms of drug encapsulation by centrifugation-filtration, liposome structure by cryo-TEM imaging, vesicle size by dynamic light scattering, and in vitro release (IVR) performance. RESULTS: The addition of increasing levels of polysorbate 20 (0.05 to 0.4%) or Brij 30 (0.05 to 0.3%) to the CFI preparations followed by subsequent freeze-thaw, resulted in a greater proportion of vesicles without drug nanocrystals and reduced the extent of growth of the nanocrystals thus leading to modified release rates including an increase in the ratio of non-encapsulated to sustained release of drug. CONCLUSIONS: This study provides another lever to achieve the desired release rate profile from a liposomal formulation by addition of surfactant and subsequent freeze-thaw, and thus may provide a personalized approach to treating patients.

Aerosol performance and long-term stability of surfactant-associated liposomal ciprofloxacin formulations with modified encapsulation and release properties.

Previously, we showed that the encapsulation and release properties of a liposomal ciprofloxacin formulation could be modified post manufacture, by addition of surfactant in concert with osmotic swelling of the liposomes. This strategy may provide more flexibility and convenience than the alternative of manufacturing multiple batches of liposomes differing in composition to cover a wide range of release profiles. The goal of this study was to develop a surfactant-associated liposomal ciprofloxacin (CFI) formulation possessing good long-term stability which could be delivered as an inhaled aerosol. Preparations of 12.5 mg/ml CFI containing 0.4% polysorbate 20 were formulated between pH 4.7 and 5.5. These formulations, before and after mesh nebulization, and after refrigerated storage for up to 2 years, were characterized in terms of liposome structure by cryogenic transmission electron microscopy (cryo-TEM) imaging, vesicle size by dynamic light scattering, pH, drug encapsulation by centrifugation-filtration, and in vitro release (IVR) performance. Within the narrower pH range of 4.9 to 5.2, these formulations retained their physicochemical stability after 2-year refrigerated storage, were robust to mesh nebulization, and formed respirable aerosols with a volume mean diameter (VMD) of 3.7 µm and a geometric standard deviation (GSD) of 1.7. This study demonstrates that it may be possible to provide a range of release profiles by simple addition of surfactant to a liposomal formulation post manufacture, and that these formulations may retain their physicochemical properties after long-term refrigerated storage and following aerosolization by mesh nebulizer.

Inhaled, dual release liposomal ciprofloxacin in non-cystic fibrosis bronchiectasis (ORBIT-2): a randomised, double-blind, placebo-controlled trial.

BACKGROUND: The delivery of antipseudomonal antibiotics by inhalation to Pseudomonas aeruginosa-infected subjects with non-cystic fibrosis (CF) bronchiectasis is a logical extension of treatment strategies successfully developed in CF bronchiectasis. Dual release ciprofloxacin for inhalation (DRCFI) contains liposomal ciprofloxacin, formulated to optimise airway antibiotic delivery. METHODS: Phase II, 24-week Australian/New Zealand multicentre, randomised, double-blind, placebo-controlled trial in 42 adult bronchiectasis subjects with ≥2 pulmonary exacerbations in the prior 12 months and ciprofloxacin-sensitive P aeruginosa at screening. Subjects received DRCFI or placebo in three treatment cycles of 28 days on/28 days off. The primary outcome was change in sputum P aeruginosa bacterial density to the end of treatment cycle 1 (day 28), analysed by modified intention to treat (mITT). Key secondary outcomes included safety and time to first pulmonary exacerbation-after reaching the pulmonary exacerbation endpoint subjects discontinued study drug although remained in the study. RESULTS: DRCFI resulted in a mean (SD) 4.2 (3.7) log10 CFU/g reduction in P aeruginosa bacterial density at day 28 (vs -0.08 (3.8) with placebo, p=0.002). DRCFI treatment delayed time to first pulmonary exacerbation (median 134 vs 58 days, p=0.057 mITT, p=0.046 per protocol). DRCFI was well tolerated with a similar incidence of systemic adverse events to the placebo group, but fewer pulmonary adverse events. CONCLUSIONS: Once-daily inhaled DRCFI demonstrated potent antipseudomonal microbiological efficacy in adults with non-CF bronchiectasis and ciprofloxacin-sensitive P aeruginosa. In this modest-sized phase II study, DRCFI was also well tolerated and delayed time to first pulmonary exacerbation in the per protocol population.

Liposomal nanoparticles control the uptake of ciprofloxacin across respiratory epithelia.

PURPOSE: Liposomal ciprofloxacin nanoparticles were developed to overcome the rapid clearance of antibiotics from the lungs. The formulation was evaluated for its release profile using an air interface Calu-3 cell model and further characterised for aerosol performance and antimicrobial activity. METHODS: Liposomal and free ciprofloxacin formulations were nebulised directly onto Calu-3 bronchial epithelial cells placed in an in vitro twin-stage impinger (TSI) to assess the kinetics of release. The aerosol performance of both the liposomal and free ciprofloxacin formulation was characterised using the next generation impactor. Minimum inhibitory and bactericidal concentrations (MICs and MBCs) were determined and compared between formulations to evaluate the antibacterial activity. RESULTS: The liposomal formulation successfully controlled the release of ciprofloxacin in the cell model and showed enhanced antibacterial activity against Pseudomonas aeruginosa. In addition, the formulation displayed a respirable aerosol fraction of 70.5 ± 2.03% of the emitted dose. CONCLUSION: Results indicate that the in vitro TSI air interface Calu-3 model is capable of evaluating the fate of nebulised liposomal nanoparticle formulations and support the potential for inhaled liposomal ciprofloxacin to provide a promising treatment for respiratory infections.

Robustness of aerosol delivery of amikacin liposome inhalation suspension using the eFlow® Technology.

The purpose of these studies was to understand the effect on product performance of batch-to-batch variability in both the amikacin liposome inhalation suspension (ALIS) formulation and its delivery device, the Lamira® nebulizer system, designed and manufactured by PARI (PARI Pharma GmbH, Munich, Germany). Three batches of ALIS spanning a range of lipid concentrations (43, 48 and 54 mg/mL) were tested with nine PARI inhalation devices that varied within the production process of the vibrating membrane with respect to hole geometry. Three hole geometry clusters were built including a geometry close to the mean geometry (median) and two geometries deviating from the mean geometry with smaller (smaller) and larger (larger) holes. The output parameters included the nebulization rate, the aerosol droplet size distribution, the liposome vesicle size post-nebulization, and the fraction of amikacin that remained encapsulated post-nebulization. Across the 27 experimental combinations of three formulation batches and nine devices, the nebulization time varied between 12 and 15 min with the fastest nebulization rate occurring with the combination of low lipid concentration and larger hole geometry (0.68 g/min) and the slowest nebulization rate occurring with the combination of high lipid concentration and the smaller hole geometry (0.59 g/min). The mean liposome vesicle size post-nebulization ranged from 269 to 296 nm across all experimental combinations which was unchanged from the control samples (276-292 nm). While all three batches contained > 99% encapsulated amikacin prior to nebulization, the nebulization process resulted in a consistent generation of ~ 35% unencapsulated amikacin (range: 33.8% to 37.6%). There was no statistically significant difference in the generated aerosol particle size distributions. The mass median aerodynamic diameters (MMAD) ranged from 4.78 µm to 4.98 µm, the geometric standard deviations (GSD) ranged from 1.61 to 1.66, and the aerosol fine particle fraction (FPF < 5 µm) ranged from 50.3 to 53.5%. The emitted dose (ED) of amikacin ranged from 473 to 523 mg (80.2 to 89.3% of loaded dose (LD)) and the fine particle dose (FPD < 5 µm) ranged from 244 to 278 mg (41.4 to 47.1% of label claim (LC)). In conclusion, while variations in the lipid concentration of the ALIS formulation and the device hole geometry had a small but significant impact on nebulization time, the critical aerosol performance parameters were maintained and remained within acceptable limits.

Storage stability of inhalable, controlled-release powder formulations of ciprofloxacin nanocrystal-containing liposomes.

Novel inhalable and controlled release powder formulations of ciprofloxacin nanocrystals inside liposomes (CNL) were recently developed. In the present study, the storage stability of CNL powders consisting of lyoprotectant (i.e. sucrose or lactose), lipids, ciprofloxacin (CIP), and magnesium stearate or isoleucine was investigated. These powders were produced by spray drying, collected in a dry box at <15% relative humidity (RH), then stored at room temperature and either 4 or 20 %RH. Liposomal integrity, CIP encapsulation efficiency (EE), in vitro drug release (IVR), aerosol performance, and solid-state properties were examined over six months. Sucrose CNL powder exhibited consistent liposomal integrity, aerosol performance, and controlled release of CIP over six months of storage at 4 %RH. However, storage of the powder at 20 %RH for the same period caused sucrose crystallization and consequently a significant drop in EE and aerosol performance (p-values < 0.05), along with the IVR of CIP becoming similar to that of the non-crystalline CIP liposomal dispersions (f2 > 50). Lactose CNL maintained superior aerosol performance over the six months irrespective of the storage RH. However, liposomal instability occurred at both RHs within the first month of storage with a significant drop in EE and an increase in liposome size (p-values < 0.05). Moreover, the IVR assay of CIP from lactose CNL showed a less controlled release and a substantial difference (f2 < 50) from its initial value after six months regardless of the storage RHs. In conclusion, dry powder inhalers of CNL were physiochemically stable in sucrose lyoprotectant when stored below 4 %RH at room temperature for six months.

Modeling of a spray drying method to produce ciprofloxacin nanocrystals inside the liposomes utilizing a response surface methodology: Box-Behnken experimental design.

Spray drying was previously used to modify the physical form of the encapsulated ciprofloxacin drug to produce ciprofloxacin nanocrystals inside the liposomes (CNL). The purpose of the present study was to optimize CNL powder production by evaluating the response surface via design of experiments (DoE). Using the Box-Behnken (BB) design, the study independent variables were the protectant type (sucrose, trehalose or lactose), protectant amount, drying temperature, and spray gas flow. Individual spray drying experiments were performed at various set points for each variable followed by characterization of the produced powders. Liposomal particle size, drug encapsulation efficiency (EE%), liposomal surface zeta potential, and nanocrystal dimensions were the design dependant variables. By applying the least square regression method on the experimental data, mathematical models were developed using the mathematical software package MATLAB R2018b. Model reliability and the significance of the model's factors were estimated using analysis of variance (ANOVA). The generated CNL powders showed spherical to elliptical liposomal vesicles with particle sizes ranging from 98 to 159 nm. The EE (%) ranged from 30 to 95% w/w while the zeta potential varied between -3.5 and -10.5 mV. The encapsulated ciprofloxacin nanocrystals were elongated cylindrical structures with an aspect ratio of 4.0-7.8. Coefficients of determination (R2 > 0.9) revealed a good agreement between the predicted and experimental values for all responses except for the nanocrystal dimensions. Sucrose and lactose were superior to trehalose in protecting the liposomes during spray drying. The amount of sugar significantly affected the characteristics of the CNL powders (p-value < 0.05). In conclusion, the DoE approach using BB design has efficiently modelled the generation of CNL by spray drying. The optimum processing conditions which produced high drug encapsulation (90%) after formation of nanocrystals and a vesicle size of ~125 nm utilized 57% (w/w) sucrose, an 80 °C inlet temperature, and an atomization rate of 742 L/hr.

Formation of ciprofloxacin nanocrystals within liposomes by spray drying for controlled release via inhalation.

The present study was conducted to harness spray drying technology as a novel method of producing Ciprofloxacin nanocrystals inside liposomes (CNL) for inhalation delivery. Liposomal ciprofloxacin dispersions were spray dried with sucrose as a lyoprotectant in different mass ratios (0.5:1, 1:1 and 2:1 sucrose to lipids), along with 2% w/w magnesium stearate and 5% w/w isoleucine as aerosolization enhancers. Spray drying conditions were: inlet air temperature 50 °C, outlet air temperature 33-35 °C, atomizer rate 742 L/h and aspirator 35 m3/h. After spray drying, the formation of ciprofloxacin nanocrystals inside the liposomes was confirmed by cryo- transmission electron microscopy. The physiochemical characteristics of the spray dried powder (particle size, morphology, crystallinity, moisture content, drug encapsulation efficiency (EE), in vitro aerosolization performance and drug release) were determined. The EE of the liposomes was found to vary between 44 and 87% w/w as the sucrose content was increased from 25 to 57% w/w. The powders contained partially crystalline particles with a volume median diameter of ~1 µm. The powders had low water content (~2% wt.) and were stable at high relative humidity. Aerosol delivery using the Osmohaler® inhaler at a flow rate of 100 L/min produced an aerosol fine particle fraction (% wt. <5 µm) of 58-64%. The formulation with the highest sucrose content (2:1 w/w sucrose to lipid) demonstrated extended ciprofloxacin release from liposomes (80% released within 7 h) in comparison to the original liquid formulation (80% released within 2 h). In conclusion, a stable and inhalable CNL powder with controlled drug release was successfully prepared by spray drying.

Ciprofloxacin nanocrystals liposomal powders for controlled drug release via inhalation.

This study was conducted to evaluate the feasibility of developing inhalable dry powders of liposomal encapsulated ciprofloxacin nanocrystals (LECN) for controlled drug release. Dry powders of LECN were produced by freeze-thaw followed by spray drying. The formulations contained sucrose as a lyoprotectant in different weight ratios (0.75:1, 1:1 and 2:1 sucrose to lipids), along with 2% magnesium stearate and 5% isoleucine as aerosolization enhancers. The powder physical properties (particle size, morphology, crystallinity, moisture content), in vitro aerosolization performance, drug encapsulation efficiency and in vitro drug release were investigated. The spray dried powders were comprised of spherical particles with a median diameter of ∼1 µm, partially crystalline, with a low water content (∼2% mass) and did not undergo recrystallization at high relative humidity. When dispersed by an Osmohaler® inhaler at 100 L/min, the powders showed a high aerosol performance with a fine particle fraction (% wt. <5 µm) of 66-70%. After reconstitution of the powders in saline, ciprofloxacin nanocrystals were confirmed by cryo-electron microscopy. The drug encapsulation efficiency of the reconstituted liposomes was 71-79% compared with the stock liquid formulation. Of the three formulations, the one containing a sucrose to lipids wt. ratio of 2:1 demonstrated a prolonged release of ciprofloxacin from the liposomes. In conclusion, ciprofloxacin nanocrystal liposomal powders were prepared that were suitable for inhalation aerosol delivery and controlled drug release.

Controlling the size and shape of liposomal ciprofloxacin nanocrystals by varying the lipid bilayer composition and drug to lipid ratio.

Drug nanocrystals precipitated inside liposomes are of increasing interest in liposomal drug delivery. For liposomal nanocrystal formulations, the size and shape of the drug nanocrystals can influence the apparent drug release properties, providing opportunities for developing tailored liposomal drug release systems. Small angle X-ray scattering (SAXS) and quantitative transmission electron microscopy (TEM) can be used to analyse the size distributions of the nanoparticles. In this study, by changing the fluidity of the membrane through the use of different membrane phospholipids with varying cholesterol content, the impact of lipid phase, fluidity and permeability on the size distribution of ciprofloxacin nanocrystals were investigated using standard TEM and SAXS as orthogonal techniques. The results show that the phospholipid phase behaviour has a direct effect on the nanocrystal size distribution, where shorter and thinner nanocrystals were formed in liposomes made from hydrogenated soy phosphatidylcholine (HSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) phospholipids with higher phase transition temperatures than 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with lower transition temperatures. This is mainly due to the phase behaviour of the liposome during nanocrystal formation. The addition of cholesterol that reduces fluidity and permeability of the DOPC liposomes was also shown to restrict the growth of the ciprofloxacin nanocrystals. Moreover, increasing the drug loading of the liposomes made from HSPC and DPPC produced longer and wider nanocrystals. The findings open new opportunities to tailor nanocrystal size distributions, as well as the aspect ratio of the enclosing liposomes with potential to alter drug release and in vivo behaviour.

Drug nanocrystallisation within liposomes.

Liposomes are phospholipid bilayer vesicles that have been explored in pharmaceutical research as drug delivery systems for >50 years. Despite being important to their morphology and drug release pattern, the physical state of the drug within liposomes (liquid, solid, crystalline form) is often overlooked. This review focuses on precipitation of drug within liposomes, which can result in the formation of confined nanocrystals, and consequent changes in liposome morphology and drug release patterns. The type of drugs that form nanocrystals within liposomes, preparation and characterisation of liposomal drug nanocrystals, and the in vitro drug release behaviour from these systems are communicated, with a discussion of their potential as drug delivery systems.

Aerosol Performance and Stability of Liposomes Containing Ciprofloxacin Nanocrystals.

BACKGROUND: Previously we showed that the release properties of a liposomal ciprofloxacin (CFI) formulation could be attenuated by incorporation of drug nanocrystals within the vesicles. Rather than forming these drug nanocrystals during drug loading, they were created post manufacture simply by freezing and thawing the formulation. The addition of surfactant to CFI, either polysorbate 20 or Brij 30, provided an additional means to modify the release profile or incorporate an immediate-release or 'burst' component as well. The goal of this study was to develop a CFI formulation that retained its nanocrystalline morphology and attenuated release profile after delivery as an inhaled aerosol. METHODS: Preparations of 12.5 mg/mL CFI containing 90 mg/mL sucrose and 0.1% polysorbate 20 were formulated between pH 4.6 to 5.9, stored frozen, and thawed prior to use. These thawed formulations, before and after mesh nebulization, and after subsequent refrigerated storage for up to 6 weeks, were characterized in terms of liposome structure by cryogenic transmission electron microscopy (cryo-TEM) imaging, vesicle size by dynamic light scattering, pH, drug encapsulation by centrifugation-filtration, and in vitro release (IVR) performance. RESULTS: Within the narrower pH range of 4.9 to 5.3, these 12.5 mg/mL liposomal ciprofloxacin formulations containing 90 mg/mL sucrose and 0.1% polysorbate 20 retained their physicochemical stability for an additional 3 months refrigerated storage post freeze-thaw, were robust to mesh nebulization maintaining their vesicular form containing nanocrystalline drug and an associated slower release profile, and formed respirable aerosols with a mass median aerodynamic diameter (MMAD) of ∼3.9 µm and a geometric standard deviation (GSD) of ∼1.5. CONCLUSIONS: This study demonstrates that an attenuated release liposomal ciprofloxacin formulation can be created through incorporation of drug nanocrystals in response to freeze-thaw, and the formulation retains its physicochemical properties after aerosolization by mesh nebulizer.

Modifying the release properties of liposomes toward personalized medicine.

Surfactant-liposome interactions have historically been investigated as a simplified model of solubilization and breakdown of biological membranes by surfactants. In contrast, our goal was to utilize surfactants to modify the encapsulation and release properties of liposomes. The ability to manufacture one liposomal formulation, which could be modified by the addition of a surfactant to support a wide range of release profiles, would provide greater flexibility than manufacturing multiple batches of liposomes, each differing in composition and with its own specific release profile. A liposomal ciprofloxacin formulation was modified by the addition of various surfactants. These formulations were characterized in terms of liposome structure by cryo-TEM imaging, vesicle size by dynamic light scattering, drug encapsulation by centrifugation-filtration, and in vitro release (IVR) performance. The addition of polysorbate 20 or polysorbate 80 to liposomal ciprofloxacin, in a hypotonic environment, resulted in a concentration-dependent loss of encapsulated drug, and above 0.4% polysorbate 20, or 0.2% polysorbate 80, a modified IVR profile as well. This study demonstrates that the encapsulation and release properties of a liposomal formulation can be modified postmanufacture by the addition of judiciously chosen surfactants in combination with osmotic swelling of the liposomes and may support a personalized approach to treating patients.

In vitro and ex vivo methods predict the enhanced lung residence time of liposomal ciprofloxacin formulations for nebulisation.

Liposomal ciprofloxacin formulations have been developed with the aim of enhancing lung residence time, thereby reducing the burden of inhaled antimicrobial therapy which requires multiple daily administration due to rapid absorptive clearance of antibiotics from the lungs. However, there is a lack of a predictive methodology available to assess controlled release inhalation delivery systems and their effect on drug disposition. In this study, three ciprofloxacin formulations were evaluated: a liposomal formulation, a solution formulation and a 1:1 combination of the two (mixture formulation). Different methodologies were utilised to study the release profiles of ciprofloxacin from these formulations: (i) membrane diffusion, (ii) air interface Calu-3 cells and (iii) isolated perfused rat lungs. The data from these models were compared to the performance of the formulations in vivo. The solution formulation provided the highest rate of absorptive transport followed by the mixture formulation, with the liposomal formulation providing substantially slower drug release. The rank order of drug release/transport from the different formulations was consistent across the in vitro and ex vivo methods, and this was predictive of the profiles in vivo. The use of complimentary in vitro and ex vivo methodologies provided a robust analysis of formulation behaviour, including mechanistic insights, and predicted in vivo pharmacokinetics.

Liposomal formulations for inhalation.

No marketed inhaled products currently use sustained release formulations such as liposomes to enhance drug disposition in the lung, but that may soon change. This review focuses on the interaction between liposomal formulations and the inhalation technology used to deliver them as aerosols. There have been a number of dated reviews evaluating nebulization of liposomes. While the information they shared is still accurate, this paper incorporates data from more recent publications to review the factors that affect aerosol performance. Recent reviews have comprehensively covered the development of dry powder liposomes for aerosolization and only the key aspects of those technologies will be summarized. There are now at least two inhaled liposomal products in late-stage clinical development: ARIKACE(®) (Insmed, NJ, USA), a liposomal amikacin, and Pulmaquin™ (Aradigm Corp., CA, USA), a liposomal ciprofloxacin, both of which treat a variety of patient populations with lung infections. This review also highlights the safety of inhaled liposomes and summarizes the clinical experience with liposomal formulations for pulmonary application.